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Studies on Arabidopsis MYB transcription factor genes : potential regulators of the phenylpropanoid pathway… Wang, Qing 2003

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Studies on Arabidopsis M Y B Transcription Factor Genes: Potential Regulators of the Phenylpropanoid Pathway and Analysis of the Root Preferentially Expressed Gene  AtMYB68  By Qing Wang Master of Agronomy, Beijing Agriculture University, Beijing, China, 1991 A THESIS S U B M I T T E D IN P A R T I A L F U L F I L L M E N T OF T H E R E Q U I R E M E N T S FOR T H E D E G R E E OF DOCTOR OF PHILOSOPHY in  T H E F A C U L T Y OF G R A D U A T E STUDIES (Department of Botany)  We accept this thesis as conforming to the required standard  T H E UNIVERSITY OF BRITISH C O L U M B I A April, 2003 © Qing Wang, 2003  In  presenting  degree freely  this  at the  thesis  in  partial  University  of  British  available for  copying  of  department publication  this or of  reference  thesis by  this  his  for  scholarly  or  her  thesis for  of  B>o  The University of British Columbi Vancouver, Canada  DE-6  (2/88)  the  requirements that the  I further agree  purposes  may  representatives.  financial  (~Lt^  of  Columbia, I agree  and study.  permission.  Department  fulfilment  It  gain shall not  be is  that  Library  an  advanced  shall make  permission for  granted  by  understood be  for  allowed  the that  without  it  extensive  head  of  my  copying  or  my  written  Abstract  Genes encoding M Y B transcription factors constitute a large family in Arabidopsis. With completion of the Arabidopsis genome sequence, over 130 MYB genes have been annotated in Arabidopsis. However functions of most of these genes are unknown. To attempt to identify MYB genes involved in the regulation of the phenylpropanoid pathway, in particular, the lignin biosynthesis pathway, we took advantage of the ongoing Arabidopsis EST project at the time I started my thesis, and collected all the available 25 MYB EST clones. These clones represented 21 unique MYB genes. Expression patterns of these genes were determined by northern blot analyses to search for those that were expressed coordinately with At4CLl, a gene encoding one of the major enzymes in the general phenylpropanoid pathway and used as a marker for this search. Accumulation of transcripts was not detectable in the seedling and mature organs for 11 out of 21 genes. None of detected expression patterns of the MYB genes was similar to that of At4CLl. MYB cDNAs were then cloned from Arabidopsis bolting stems where At4CLl was highly expressed. A novel MYB gene, STM31, was identified. The transcript of STM31 was accumulated ubiquitously in the seedling and all organs tested. Thus STM31 did not appear to be a good candidate to regulate At4CLl transcription either. By searching Arabidopsis T-DNA tagged populations, one T - D N A line that had the insertion in the promoter region of AtMYB68 was identified in the Douglas laboratory. AtMYB68 was further characterized by expression profiling. Both northern blot and RT-PCR analyses showed that the transcript of AtMYB68 was predominantly accumulated in the root but was detectable in the seedling shoot. At the tissue and cellular level, GUS activity driven by the AtMYB68 promoter was detected in the stele tissue of the root. A root cross-section revealed that GUS staining was more intense in the pericycle cells near the two xylem poles in the root. In the shoot, GUS staining was detected in the stomata guard cells of young leaves. GUS activity driven by the AtMYB68 promoter was also examined in the woodenleg mutant background, in which the xylem was the only component in the vascular tissue of the primary root. GUS  staining was strong in the whole layer of pericycle cells and expanded to the endodermis layer in this mutant background. This result suggests that the activity of the AtMYB68 promoter is induced by a signal from the root xylem. The level of the endogenous AtMYB68 transcript was induced by the plant hormone I A A ~2-fold, and by A B A ~3-fold. In order to determine AtMYB68 function, transgenic lines overexpressing and underexpressing AtMYB68 were generated in Arabidopsis. In addition, by searching several TD N A tagged populations, one knockout line for AtMYB68 was identified. In this line, the level of the AtMYB68 transcript was very low. Phenotypes of both the overexpression line and the knockout line were examined. No morphological changes were found in either mutant line, and no phenotypic changes in response to external stimuli examined were observed. Expression patterns of the members in the AtMYB68 phylogenetic subgroup were determined by RT-PCR. At least one more member, AtMYB36, had the organ-specific expression pattern very similar to that of AtMYB68. These results suggest that functional redundancy may exist in this subgroup. Taken together, the data of expression profiling suggest that AtMYB68 may play a role in the pericycle cell identity adjacent to the xylem pole. In the future, atmyb68 double or triple mutants with closely related members might reveal a precise role of AtMYB68. In addition, characterization of AtMYB68 downstream target genes can be done by using the microarray analysis.  Table of Contents  Abstract Table of Contents List of Tables List of Figures Abbreviations Acknowledgments  Chapter 1 General Introduction 1.1 The Phenylpropanoid Pathway 1.2 Plant Transcription Factors 1.3 M Y B Transcription Factors: Structure 1.4 Plant M Y B Transcription Factors: Functions 1.4.1 Plant M Y B Factors with One Repeat 1.4.2 Plant M Y B Factors with Three Repeats 1.4.3 R2R3-Type Plant M Y B Factors  1.5 Combinatorial Control: Protein-Protein Interactions 1.6 The Way Forward in Plant MYB Gene Functional Studies  1.7 Research Objectives and Approaches  17  Chapter 2 Materials and Methods  18  2.1 Plant Material Preparation  18  2.1.1 Plant Growth Conditions  18  2.1.2 Plant Treatments  18  2.1.3 Plant Transformation  20  2.2 General Molecular Methods  20  2.2.1 Plasmid D N A Preparation and D N A Sequencing  20  2.2.2 Plant Genomic D N A Isolation  20  2.2.3 Total R N A and Poly(A) R N A Isolation  21  +  2.2.4 Northern and Southern Blot Analysis  ,  2.2.5 Quantitative R T - P C R Analysis  22  2.3 Cloning  24  2.3.1 Cloning of MYB cDNAs 2.3.2 Generation of the AtMYB68.:GUS  21  24 Construct  2.3.3 Cloning of the AtMYB68 c D N A into  E. coli  Expression Vectors  2.3.4 Generation of Constructions to Sense and Antisense AtMYB68  2.4 Protein Analysis  24 25 25  26  2.4.1 Protein Extraction  26  2.4.2 Generation of Anti-Serum against the AtMYB68 Recombinant Protein  29  2.4.3 AtMYB68 Anti-Serum Purification  29  2.4.4 Western Blot Analysis  30  2.5 Hi stology and Hi stochemi stry  31  2.5.1 Histochemical G U S Staining  31  2.5.2 Root Section Preparation  31  2.6 Primers Used in Screening T-DNA Tagged Populations  31  Chapter 3 Searching for Arabidopsis MYB Genes Involved in Regulation of the Phenylpropanoid Pathway  33  3.1 Introduction  33  3.2 Expression of Arabidopsis 4CL and 4CL-Like Genes  34  3.3 Expression Patterns of Arabidopsis MYB Genes  35  3.4 Cloning MYB cDNAs from Arabidopsis Bolting Stems  42  3.5 Discussion  45  Chapter 4 AtMYB68: a Root Preferentially Expressed MYB Gene — Expression Profile Studies  49  4.1 Introduction  49  4.2 Organ and Cell-Type Specific Expression of AtMYB68  54  4.3 Expression of AtMYB68 under the woodenleg  (wol)  Mutant Background  4.4 Expression of AtMYB68 in Response to External Stimuli  61 65  4.5 Expressing AtMYB68 Recombinant Protein and Raising A t M Y B 6 8 Antiserum 68 4.6 Discussion  74  Chapter 5 AtMYB68: a Root Preferentially Expressed MYB Gene— Functional Studies 81 5.1 Introduction  81  5.2 Overexpression of AtMYB68 in the Control of the CaMV 35S Promoter  81  5.3 Repression of AtMYB68 Expression by Antisense R N A  84  5.4 Screen for an AtMYB68 Knockout Mutation in T-DNA Tagged Populations  85  5.5 Phenotypic Analysis of AtMYB68 Mutant Lines  97  5.6 Expression Patterns of the Members in A t M Y B Phylogenetic Subgroup 14  101  5.7 Discussion  105  Bibliography  108  List of Tables Table 1-1. Major Gene Families of Arabidopsis Transcription Factors in Comparison to Other Species (According to Riechmann and Ratcliffe, 2000) Table 1-2. Genes Encoding M Y B 3 R - and R2R3-Type M Y B Transcription Factors in Arabidopsis (According to Stracke et al., 2001) Table 2-1. Primers Used in Quantitative RT-PCR Analyses  4 6 23  '  Table 2-2. AtMYB68 Primers Used for Screening T - D N A Tagged Populations  32  Table 3-1. Conserved Cis-Elements in Promoters of Phenylpropanoid Genes and M Y B P Binding Site (According to Douglas, 1996)  33  Table 3-2. Examined Arabidopsis MYB EST Clones Table 3-3. Expression Patterns of Examined Arabidopsis MYB Genes  38 39  Table 5-1. Summary of Screens for a T-DNA Insertion in AtMYB68  87  Table 5-2. Growth of AtMYB68 Mutant Seedlings on Plates* Table 5-3. Seedling growth under A B A treatment*  99 100  Table 5-4. Seedling lateral root number under I A A treatment (No./L (cm)" )* 1  Table 5-5. Summary of Phenotypic Examinations of AtMYB68 Mutant Lines  100 101  List of Figures Figure 1-1. Reactions of General Phenylpropanoid Metabolism (according to Hahlbrock and Scheel et al., 1989) Figure 2-1. Determination of the Linear Range for RT-PCR  2 23  Figure 2-2. Plasmids Generated for Various AtMYB68 Constructs  28  Figure 3-1. Northern Blot Analysis of Expression Patterns of Arabidopsis 4CL Genes. 36 Figure 3-2. Alignment of A t 4 C L l and the 4CL-Like Protein Encoded by EST 139B10T7  37  Figure 3-3. Northern Blot Analysis of Expression Patterns of Arabidopsis MYB Genes: 42 Figure 3-4. Nucleotide and Deduced Amino Acid Sequences of Arabidopsis MYB STM31 (AtMYB73)  44  Figure 3-5. Northern Blot Analysis of 57M5iExpression  45  Figure 4-1. Schematic View of Root Anatomy  50  Figure 4-2. Schematic of a T - D N A Insertion within AtMYB68 in Line T-14  53  Figure 4-3. Northern Blot Analysis of AtMYB68 Expression in Arabidopsis  55  Figure 4-4. RT-PCR Analysis of AtMYB68 Expression in Arabidopsis Seedlings.  55  Figure 4-5. Whole-Mount Histochemical Staining of GUS Driven by the AtMYB68 Promoter during Seedling Development in Line 12-2G Figure 4-6. Root Cross-Sections of the AtMYB68::GUS Line 12-2G  59 60  Figure 4-7. Whole-Mount Histochemical Staining of GUS Driven by the AtMYB68 Promoter in the woodenleg Mutant Background  62  Figure 4-8. Root Cross-Sections of the woodenleg Seedling Containing the AtMYB68::GUS fusion  64  Figure 4-9. RT-PCR Analysis of AtMYB68 Expression in Response to External Stimuli. 67 Figure 4-10. SDS-PAGE Analysis of AtMYB68 Recombinant Protein Expressed in E. coli and Recombinant Protein Purification  70  Figure 4-11. Western Blot Analysis of Protein Extracts from E. coli and Arabidopsis Seedlings. 72 Figure 4-12. Western Blot Analysis of Protein Extracts from the atmyb68 Knockout Line and Wild Type Seedlings  74  Figure 5-1. Northern Blot Analysis of AtMYB68 Expression in 35S::AtMYB68 Transgenic Lines. 83 Figure 5-2. RT-PCR Analysis of AtMYB68 Expression in Homozygous Transgenic Lines Harboring the 35S::AtMYB68 Antisense Transgene  85  Figure 5-3. Northern Blot Analysis of AtMYB68 Expression in the T - D N A Insertion Line T-14. 86  Figure 5-4. Southern Blot Analysis of PCR Amplification for a T - D N A Insertion within AtMYB68 Using the Wisconsin Collection  89  Figure 5-5. Second Round of PCR Amplification for a T - D N A Insertion within AtMYB68 Using the Wisconsin Collection  90  Figure 5-6. Re-Amplification of an AtMYB68 -T-DNA Allele from Sub-Super Pool 61 of the Wisconsin Collection  91  Figure 5-7. Southern Blot Analysis of PCR Amplification for an AtMYB68-T-DNA Allele in 9Line Pools of the Wisconsin Collection  92  Figure 5-8. PCR Amplification of a T - D N A Insertion within AtMYB68 in the Garlic 94B Line Using the T M R I Collection  93  Figure 5-9. Schematic of the T - D N A Insertion within AtMYB68 from the Garlic 94B Line. 94 Figure 5-10. PCR Screening for Homozygous Insertion Lines Derived from Garlic 94B. 95 Figure 5-11. RT-PCR Analysis of AtMYB68 Expression in the T - D N A Insertion Line 94B-18. 96 Figure 5-12. Morphology of AtMYB68 Mutant Plants  97  Figure 5-13. Re-Drawing of A t M Y B Phylogenetic Subgroup 14 (According to Stracke et al., 2001)  102  Figure 5-14. Alignment of AtMYB68 with the other Two Members of Phylogenetic Sub-Group 14  103  Figure 5-15. RT-PCR Analysis of Expression Patterns of Three Members in the A t M Y B Phylogenetic Sub-Group 14  104  Abbreviations 4CL  4-coumarate:CoA ligase  ABA  abscisic acid  ABRC ABREs  Arabidopsis Biological Resource Center ABA-responsive elements  ACC  1 -arhinocyclopropane-1 -carboxylie acid alcohol dehydrogenase  ADH Am AN AP2  Antirrhirum majus  AS1 ASA  ASYMMETRICAL LEAVES 1 anthranilate synthase oc-subunit  At  Arabidopsis thaliana  ATR1  ALTERED TYPTOPHAN REGULATION 1  Bl  BOOSTER 1  BAN  BSA  BANYULS 6-benzylaminopurine helix-loop-helix bovine serum albumin  CI  COLORLESS 1  C4H  cinnamate 4-hydroxylase  CAD  cinnamyl alcohol dehydrogenase  CaMV  35S cauliflower mosaic virus 35S promoter  CCA1  O R C A D I A N C L O C K ASSOCIATED 1  CHS CNBr  chalcone synthase  Col  Arabidopsis Columbia ecotype  CPC  CAPRICE  DFR  DIHYDROFLAVONOL-4-REDUCTASE  DIC  Differential Interference Contrast  EDTA EREBP  ethylenediaminetetra-acetic acid ethylene-responsive-binding element protein  EST  Expressed Sequence Tag  GA3  gibberelic acid  GL1  GLABROUS 1  GUS  (^-glucuronidase  BAP bHLH  ANTHOCYANIN APETALA2  cyanogen bromide  HR  hypersensitive response  IAA  indole-3-acetic acid  ICK  interactors of Cdc2 kinase  IPTG  isopropyl-(3-D-thiogalactopyranoside  Kan  kanamycin  Kb  kilobase  KDa  kiloDalton  KNOX  knotted 1-like homeobox  LC-MS LHY MES  liquid chromatography-mass spectrometer LATE ELOGATED HYPOCOTYL  MOPS  3- [N-Morpholino]propanesulfonic acid  MPSS  Massively Parallel Signature Sequencing  MS  Murashige and Skoog  MSA  M-specific activator  MYB3R  M Y B homologue with three M Y B repeats  MYBR1  M Y B homologue with a single M Y B repeat  ORF  Open Reading Frame  PI  PURPLE L E A F  PAGE  polyacrylamide gel electrophoresis  PAL  phenylalanine ammonia-lyase  PAP  PRODUCTION OF A N T H O C Y A N I N PIGMENT  PBS  phosphate-buffered saline  Pc  Petroselinum crispum  PCR  Polymerase Chain Reaction  PEG  polyethylene glycol  Ph  Petunia hybrida  PHAN  PHANTASTICA  PMSF  phenylmethylsulfonyl fluoride  PVDF  polyvinylidene difuoride  RTBP1  RICE TELOMERE-BINDING PROTEIN 1  Rl  RED1  RACE  Rapid Amplification of cDNA Ends  RNAi  R N A interference  RS2  R O U G H SHEATH2  RT  reverse transcription  SD  standard deviation  SDS  sodium dodecyl sulphate  shr  short-root  SOS  Salt Overly Sensitive  SSC  standard saline citrate buffer  2[N-Morpholino]ethansulfonic Acid  STM  SHOOT MERISTEMLES S  T-DNA  transferred-DNA  TILLING  Targeting Induced Local Lesions in Genomes  TMRI  Torrey Mesa Research Institute  T0C1  TIMING OF C A B 1 T R A N S P A R E N T TESTA  TT TTG WER wol WS Zm  T R A N S P A R E N T TESTA G L A B R O U S WEREWOLF woodenleg Arabidopsis Wassilewskija ecotype Zea mays  Acknowledgements  I would like to thank my supervisor Dr. Carl Douglas for his supervision, patience and encouragement throughout the duration of my PhD study, and for accepting me as a student first of all. I would like to thank my committee members Dr. B . Green, Dr. G. Haughn and Dr. J. Kronstad for their guidance and suggestion. Thank you, the Douglas lab former and current members for a co-operative and friendly environment, and members in the Haughn and Kunst labs. Dr. A. Pri-Hadash initiated the second project presented in my thesis. Dr. J. Ehlting is always willing to help, and Dr. D.K. Ro is so supportive during my thesis writing. Thank you, my mom and dad. Although they are far away from me, they have been trying to give me a hand financially and mentally as always. During my study, I hardly got a chance going back to see them. I feel sorry for this. Special thanks to my husband Jing for being fundamental in helping me to achieve my degree. He experienced his mother and sister lost in last few years, but he showed me to be optimistic and open-minded to difficulty. During my study, I gave birth to my daughter Angie. She is a really great gift. Definitely she adds more work on me, but she is the light during these years' frustration. From knowing nothing about Arabidopsis, to my thesis is full of Arabidopsis, I went through a hard journey. 'No pain, no gain', I wish it's true to me.  Chapter 1 General Introduction  1.1 The Phenylpropanoid Pathway  The phenylpropanoid pathway is required for the biosynthesis of a series of natural products based on a phenylpropane skeleton derived from L-phenylalanine. These phenylpropanoid products play important roles in higher plants. They serve as low molecular weight pigments, antibiotics (phytoalexins), U V protectors, insect repellents, and signal molecules in plant defense. They also function as polymers in surface and support structures, such as suberin, lignin (Hahlbrock and Scheel, 1989; Dixon and Paiva, 1995). The flow of carbon from primary metabolism into an array of secondary phenylpropanoid products is through the general phenylpropanoid pathway (Figure 1-1). The specific branch pathways derive their basic phenylpropanoid unit from this core reaction (Hahlbrock and Scheel, 1989; Douglas, et al., 1992). This pathway is composed of three enzymes, phenylalanine ammonia-lyase (PAL), cinnamate 4-hydroxylase (C4H) and 4-coumarate:CoA ligase (4CL). P A L catalyzes the first step, the de-amination of L-phenylalanine to produce cinnamic acid. Cinnamic acid is then hydroxylated at the para-position by C4H, a cytochrome P450 monooxygenase. The product is further modified by other hydroxylases and O-methyltransferase. The enzyme 4CL catalyzes the formation of CoA esters of cinnamic acids, and these activated intermediates serve as the substrates for specific branch pathways, such as those leading to the synthesis of flavonoids and lignin. With the large structural and functional diversity of these phenylpropanoid compounds, their temporal and spatial distributions are very different during development. For example, biosynthesis and deposition of lignin are developmentally regulated, and occur in tracheary elements during xylem differentiation. The flavonoid pigment deposition in plant organs also occurs in a cell type specific manner, such as in the epidermal cells of petals and in the aleurone  COUMARIN  FLAVONOID  S O L U B L E ESTER  General Phenylpropanoid Metabolism COOH  f ^ N H 3  I  H  Phenylalanine  SUBERIN  COQH  COQH  r•  ts  +  r  • lr^ »  +  1  fl1  Cinnamic a d d  LIGNIN  —  •  CPR  L ^j  Q - S C o A  4CL —  p-coumaric acid  •  J s .  r  [  4-Coumaroyl C o A  OTHER W A L L - B O U N D PHENOLICS  Figure 1-1. Reactions of General Phenylpropanoid Metabolism (according to Hahlbrock and Scheel et al., 1989). Thicker arrows indicate branch pathways emanating from the general pathway. P A L , phenylalanine ammonia-lyase; C4H, cinnamate 4-hydroxylase; 4CL, 4-coumarate:CoA ligase.  layer of maize kernels. The biosynthesis of phenylpropanoid compounds is also induced upon environmental stimuli such as wounding, pathogen infection, and U V irradiation (Hahlbrock and Scheel, 1989; Douglas, et al., 1992; Dixon and Paiva, 1995). Genes encoding the enzymes in the general phenylpropanoid pathway and some branch pathways have been isolated from many plant species (Dixon and Harrison, 1990). In many cases it has been shown that the gene transcripts accumulate in a tissue/cell type specific manner during plant development, and that expression is activated in response to different environmental stimuli (Hahlbrock and Scheel, 1989; Dangl, 1991; Douglas et al., 1992; Dixon and Paiva, 1995). This point can be illustrated using the example of 4CL genes. 4CL genes have been cloned from Arabidopsis and other plant species (Lee et al., 1995; Douglas et al., 1992). In all these plants, 4CLs are encoded by a small gene family. In Arabidopsis, in addition to A t 4 C L l ,  two more 4CL genes, At4CL2 and At4CL3, have been identified. At4CLl and At4CL2 cDNAs share 80% identity (Ehlting et al., 1999). It has been shown that expression of At4CLl is correlated with lignin deposition during seedling development. In the mature plant, At4CLl is strongly expressed in bolting stems compared to leaves and flowers. The expression of At4CLl is also induced by wounding and bacterial pathogen infection (Lee et al., 1995). It has been demonstrated that expression of genes encoding enzymes in the phenylpropanoid pathway is coordinately regulated. In Arabidopsis, expression of PAL, C4H and 4CL, the genes encoding enzymes in the general phenylpropanoid pathway, is coordinately induced by wounding (Lee et al., 1997). Similar results are also obtained upon wounding, elicitor or U V irradiation in other plant species (Fahrendorf et al., 1995; Gorlach et al., 1995; Logemann et al., 1995). It is believed that the phenylpropanoid pathway is regulated primarily through control of transcription initiation. The presence or activity of transcription factors is modulated by developmental and environmental signals, and these factors in turn regulate the expression of sets of genes encoding enzymes in the phenylpropanoid pathway, and thus the activity of the pathway (Hahlbrock and Scheel, 1989; Douglas et al., 1992). In vivo footprinting and deletion analyses have defined putative cis-elements, which are conserved in the promoters of genes encoding enzymes in the phenylpropanoid pathway. In particular, the conserved motifs, P- and L-boxes have been found in the promoters of PAL, 4CL and other structural genes in the phenylpropanoid pathway (Lois et al., 1989; Logemann et al., 1995; Douglas, 1996). These common cis-elements provide a basis for the coordinate regulation of the transcription of structural genes in the phenylpropanoid pathway.  1.2 Plant Transcription Factors  A 'transcription factor' is a protein that regulates the transcription frequency of a target gene (Stracke et al., 2001). A transcription factor usually displays a modular structure, and can  be a transcription activator, repressor, or both. Transcription factor genes comprise a substantial fraction of all eukaryotic genomes. On the basis of similarity in the sequence of the D N A binding domain, the majority of transcription factors can be classified into a limited number of different families (Pabo and Sauer, 1992; Riechmann and Ratcliffe, 2000. Table 1-1).  Table 1-1. Major Gene Families of Arabidopsis Transcription Factors in Comparison to Other Species (According to Riechmann and Ratcliffe, 2000) Gene Family  Predicted Gene Number in Genomes Arabidopsis  Drosophila  MYB  -130  35  16  19  AP2/EREBP  150  0  0  0  NAC  105  0  0  0  bHLH/MYC  100  61  38  8  bZIP  100  24  18  15  HB  90  113  88  10  Z-C2H2  85  352  138  47  MADS  80  2  2  4  WRKY  75  0  0  0  ARF  42  0  0  0  Dof  41  0  0  0  C. elegans  S. cerevisiae  ARF, auxin response factor; E R E B P , ethylene-responsive-binding element protein; H B , homeobox; Z - C H , zinc2  2  finger protein of the C H type. 2  2  With the completion of the Arabidopsis thaliana genome sequence, it is estimated that there might be more than 1,600 transcription factor genes in Arabidopsis, which represents about 6% of the total -26,000 genes in this plant species (Riechmann and Ratcliffe, 2000), as compared to 700 in Drosophila and 500 in the C. elegans genome, which respectively represent 5% and 2.7% of the total genes in each genome (Rubin et al., 2000). These numbers suggest that Arabidopsis is comparable to Drosophila in terms of gene regulatory networks. It has been noted that many families of transcription factors exhibit great variation in numbers among different eukaryotes, and some families are organism-specific (Rubin et al., 2000). From Table 1-1, it can be seen that some classes of transcription factors are plant-specific, such as AP2/EREBP and N A C . For some classes of transcription factors, such as the M Y B and MADS-box classes, the  numbers are significantly higher in plants. Among these transcription factors, MYB genes comprise one of the largest gene families in Arabidopsis.  1.3 MYB Transcription Factors: Structure  The first MYB gene identified was v-myb, a chicken oncogene derived from the avian myeloblastosis virus, which causes acute myeloblastic leukemia (Klempnauer et al., 1982). vmyb is a truncated version of the wild type gene c-myb. c-myb is well conserved in all vertebrates examined to date. A-Myb and B-Myb are two homologs of c-myb in vertebrates. The proteins encoded by these genes contain the conserved M Y B DNA-binding domain and share limited homology in other regions (Weston, 1998). Deletion analysis of these members has revealed three functional domains: an amino-terminal DNA-binding domain, a central transcription activation domain and a carboxyl-terminal negative regulatory domain (Thompson and Ramsay, 1995). The DNA-binding domain is generally composed of three imperfect repeats of 51-52 amino acids. Each repeat includes three regularly spaced tryptophan residues, and forms three alpha helical bundles around hydrophobic cores of the conserved tryptophan residues (Thompson and Ramsay, 1995). The three repeats in c-Myb are referred as R I , R2 and R3 (Thompson and Ramsay, 1995; Weston, 1998). Proteins containing the M Y B DNA-binding domain are also found in plants, including Arabidopsis, maize, Anthirrhinum and rice (Romero et al, 1998; Rabinowicz et al., 1999; Jackson et al., 1991; Locatelli et al., 2000). Compared to M Y B factors in vertebrates, most plant M Y B homologs contain two repeats in the DNA-binding domain, corresponding to the R2 and R3 repeats in c-Myb (R2R3-type M Y B ) . Genes encoding the conserved M Y B DNA-binding domain usually constitute large gene families in plants. In Aradidopsis, there are a total of 125 R2R3-type MYB genes annotated on the basis of the completed genome sequence (Stracke et al., 2001. Table 1-2). The large size of the MYB gene family is also confirmed in maize, in which 97 R2R3-type MYB genes have been  identified so far (Rabinowicz et al., 1999). According to the similarity of amino acid sequences outside the M Y B DNA-binding domain, Arabidopsis M Y B factors have been categorized into 22 phylogenetic subgroups (Kranz et al., 1998; Stracke et al., 2001). In contrast to intensive sequence information available, functions of most of these genes are not known yet.  Table 1-2. Genes Encoding M Y B 3 R - and R2R3-Type M Y B Transcription Factors in Arabidopsis (According to Stracke et al., 2001) Gene Name  Gene Number  AtMYB3R genes:  5  AtMYB3Rl to AtMYB3R5  R2R3-type genes:  125  AtMYBO to AtMYB124  'Unusual' MYB genes:  2  AtMYBCDC5 AtMYB4Rl  1.4 Plant MYB Transcription Factors: Functions  1.4.1 Plant MYB Factors with One Repeat  Gene encoding proteins with only one M Y B repeat (MYB1R) have been identified from several plant species, including potato, Arabidopsis, parsley and rice (Baranowskij et al., 1994; Kirik and Baumlein, 1996; Feldbrugge et al., 1997; L u et al., 2002). This group of MYB-like proteins is fairly divergent, and has low identity (30%-40%) to either the second (R2) or the third repeat (R3) of the M Y B DNA-binding domain in the human c-Myb. Over 9 genes encoding M Y B 1 R proteins are detected in the Arabidopsis genome (Carre and Kim, 2002). Arabidopsis M Y B 1 R proteins L H Y and CCA1 have been found to be related to the circadian clock. Accumulation of LHY and CCA1 transcripts is closely associated with the  circadian rhythm (Schaffer et al., 1998; Wang et al., 1997). Overexpression of either gene abolishes rhythmic expression of a set of circadian genes. These two genes are also able to regulate negatively their own transcription. L H Y and CCA1 share high similarity at the amino acid level and might be functionally redundant (Wang and Tobin, 1998; Schaffer et al., 1998). They have been proposed to interact with the other regulator TOC1 as components of the Arabidopsis circadian oscillator (Alabadi et al., 2001). M Y B R 1 proteins have also been found to be involved in other developmental processes. For instance, P c M Y B l from parsley interacts with a light-regulatory cis-element (Feldbrugge et al., 1997), and Arabidopsis CPC is a positive regulator of root hair formation (Wada et al., 1997). T R I P T Y C H O N , a homolog of CPC, regulates trichome formation in the leaf epidermis in Arabidopsis (Schellmann et al., 2002). Three cDNAs encoding the single M Y B repeat have been identified from rice. Their products bind to the conserved cis-element in the promoter of an ocamylase gene and may contribute to gibberellin and sugar regulation of oc-amylase gene expression (Lu et al., 2002). M Y B 1 R proteins also include telomeric D N A binding proteins. For example, RTBP1 identified in rice binds to the conserved tandem repeat sequence in plant telomeric D N A (Yu et al., 2000). The single M Y B DNA-binding domain in RTBP1 is well conserved in the telomeric D N A binding proteins identified from different species, such as yeast and mammals (Broccoli et al., 1997; Spink et a l , 2000; Vassetzky et al., 1999). Interestingly, unlike canonical M Y B transcription factors, the single M Y B repeats in the telomeric D N A binding proteins are located at the carboxy-ternimi. The binding of proteins to telomeric D N A is proposed to maintain chromosome integrity and protect telomers from degradation and end fusion (Greider, 1996).  1.4.2 Plant MYB Factors with Three Repeats  Through genome sequencing, genes encoding three M Y B repeats (MYB3R) have been identified from Arabidopsis (Braun and Grotewold, 1999). Such genes have been also detected  in all the major plant lineages, including mosses, ferns and monocots. These genes constitute a small gene family in plant lineages, which is similar in size to the families in vertebrates and Drosophila (Kranz et al., 2000). Five M Y B 3 R genes are annotated in the Arabidopsis genome (Stracke et al., 2001. Table 1-2). The plant M Y B 3 R proteins and the vertebrate c-Myb share a high degree of amino acid identity in the M Y B DNA-binding domain (over 60%, compared to 40%-60% between the R2R3-type proteins and the vertebrate c-Myb) (Braun and Grotewold, 1999). The intron-exon structures are also conserved in M Y B 3 R genes from vertebrates and plants (Braun and Grotewold, 1999; Kranz et al., 2000). In vertebrates, M Y B transcription factors play an important role in the control of cell proliferation, differentiation and cell death through the cell cycle regulation (Thompson and Ramsay, 1995; Weston, 1998). The closer relationship between the plant M Y B 3 R proteins and vertebrate M Y B proteins in sequence suggests that the plant M Y B 3 R proteins may have similar functions. In support of this speculation, three M Y B 3 R proteins identified in tobacco have been found to bind to the conserved cis-element M S A in the promoters of plant B-type cyclin genes and modulate the promoter activity (Ito et al., 2001). However, the exact function of plant M Y B 3 R proteins is not known yet.  1.4.3 R2R3-Type Plant MYB Factors  R2R3-type M Y B genes constitute a large gene family and are only present in the plant lineage (Riechmann et al., 2000). However functions of these genes are poorly understood. From known cases, they are involved in many aspects of plant growth and development, and appear to regulate plant-specific processes. (1) Regulation of phenylpropanoid metabolism. Extensive genetic and molecular studies have shown that the accumulation of specific flavonoid end products, such as anthocyanins and flavonols in plants, is under the control of M Y B transcription factors. It was first revealed in maize that anthocyanin biosynthesis requires the interaction of the M Y B factor  C l / P l with the basic helix-loop-helix (bHLH, Myc) member R l / B l (Cone et al., 1986; Paz-Ares et al., 1987; Ludwig et al., 1989). Each family contains several alleles and multiple paralogs to control pigmentation in different tissues and organs. For instance, C I is required for anthocyanin accumulation in the aleurone layers, whereas PI controls pigmentation in other mature tissues (Dooner and Robbin, 1991). In contrast to C l / P l , the M Y B factor P acts alone to regulate a subset of the structural genes in the flavonoid pathway, leading to the biosynthesis of phlobaphene, another flavonoid product that accumulates in the maize kernel pericarp and floral organs (Grotewold et al., 1994). The biosynthesis of flavonoids in dicots is also under the control of M Y B factors. In petunia, expression of the late flavonoid genes in flowers is controlled by at least three regulatory genes, AN1 (encoding a b H L H factor), AN2 (encoding a M Y B factor), and AN11 (encoding a WD40 repeat protein) (Quattrocchio et al., 1993, 1999; de Vetten et al., 1997; Spelt et al., 2000). Expression of AN2 is essentially restricted to the corolla limb. The M Y B gene AN4 in petunia might be a paralog of AN2 in the control of pigmentation in anthers (Spelt et al., 2000). In Arabidopsis, the control of flavonoid synthesis follows a similar scenario. At least three regulator genes, 7TG7, TT8 and 7T2 are required for flavonoid synthesis in seeds. Similar to petunia, they encode a WD40 protein, a b H L H factor and a M Y B factor, respectively (Nesi et al., 2000, 2001). Seeds of tt2 (atmybl23) mutants are yellow or buff in color as a result of the lack of proanthocyanidin in the seed coat. Expression of late flavonoid biosynthesis genes, such as DFR and BAN, are dramatically down-regulated in the mutant. 7T2 expression is restricted to the seed during early embryogenesis, which is consistent with expression of the structural gene BAN and proanthocyanidin deposition. Overexpression of 7T2 is able to induce the ectopic expression of DFR and BAN, but is not sufficient for the ectopic accumulation of the proanthocyanidin product (Nesi et al., 2001). These data suggest that 7T2 plays a key role in the control of the spatial and temporal expression of flavonoid biosynthesis genes and thus proanthocyanidin accumulation.  Interestingly, overexpression of PAP1 (AtMYB75) by activation tagging, a member closely related to 7T2 in Arabidopsis, results in intense purple pigmentation in the whole plant. Many other phenylpropanoid products are also increased. Expression of the general phenylpropanoid pathway genes, such as PAL, and genes specific for anthocyanin synthesis and transport, such as chalcone synthase (CHS), are also massively enhanced. Ectopic expression of PAP1 and the most closely related member PAP2 (AtMYB90) in tobacco causes the same phenotype. These results suggest that these two MYB genes are involved in the regulation of the phenylpropanoid gene expression. PAP1 and PAP2 may be functionally redundant (Borevitz et al., 2000). A MYB gene that controls the specific structural gene expression in anthocyanin biosynthesis has been also demonstrated in grape (Kobayashi et al., 2002). In addition to the flavonoid pathway, other phenylpropanoid branch pathways are also under the control of MYB genes. Ectopic expression of Antirrhirum MYB308 in tobacco results in significant reduction of all major soluble phenolic compounds and lignin. Expression of the structural genes in the phenylpropanoid pathway, C4H, 4CL, CAD (encoding cinnamyl alcohol dehydrogenase, a key enzyme in lignin synthesis), and CHS (encoding a key enzyme in flavonoid synthesis) are all down-regulated in the transgenic tobacco plant. In addition, the plant is developmentally retarded (Tamagnone et al., 1998). These results suggest that an AmMYB308 ortholog in tobacco may play a role in negatively regulating the phenylpropanoid pathway. The function of AtMYB4, an AmMYB308 ortholog in Arabidopsis, has been characterized by a knockout mutation. The level of sinapoyl malate, the major soluble phenolic compound in Arabidopsis leaves, is significantly increased in the atmyb4 insertion-knockout. The increase of the sinapoyl ester causes the mutant plant to be more resistant to U V - B irradiation. A t M Y B 4 appears to be a transcriptional repressor, functioning principally to down-regulate C4H expression. This is the first direct evidence to show that an AtMYB gene regulates expression of one of the structural genes in the general phenylpropanoid pathway. However, expression of the other two structural genes in this pathway, PAL and 4CL, is not changed in the atmyb4 knockout mutant (Jin et al., 2000).  Besides the phenylpropanoid pathway, M Y B transcription factors are also involved in regulating other metabolic pathways. AtMYB61 controls the accumulation of polysaccharides in the seed coat. Seeds of atmybôl insertion-knockout mutants are deficient in seed mucilage extrusion. Accordingly, AtMYBôl is preferentially expressed in siliques, and the expression is localized in the seed coat (Penfield et al., 2001). ATR1 (AtMYB34) is involved in the regulation of the tryptophan pathway by up-regulating ASA1 expression, a gene encoding the catalytic subunit of the first enzyme in the tryptophan pathway (Bender and Fink, 1998). In plants, tryptophan provided by the tryptophan pathway serves as a precursor for protein synthesis and the biosynthesis of an array of metabolites, such as the plant hormone indole-3-acetic acid (Radwanski and Last, 1995). This demonstrates that transcriptional control by M Y B factors is not limited to genes in secondary metabolic pathways. (2) Control of the development and determination of cell fate and identity. M Y B factors GL1 (AtMYBO) and W E R (AtMYB66) have been well characterized for their roles in control of trichome and root atrichoblast specification. In Arabidopsis, GL1 specifies trichome formation on the surface of leaves and stems. Expression of GL1 is restricted to the epidermal cells in developing shoot tissues, gll mutations generate a non-trichome leaf phenotype (Koornneef et al., 1982; Oppenheimer et al., 1991; Larkin et al., 1993). W E R is required for nonhair cell (atrichoblast) identity in the root epidermis. Accordingly, expression of WER is limited to a subset of developing epidermal cells in the root and hypocotyl. wer mutations generate extra-hairy roots (Lee and Schiefelbein, 1999). GL1 and W E R share a high degree of amino acid identity. Either gene promoter driving the other gene could functionally complement for each other (Lee and Schiefeldein, 2001). The M Y B factor M I X T A from Antirrhinum affects epidermal cell shape in the petal. In the mixta mutant, the epidermal cells of the petal fail to form the normal conical shape (Noda et al., 1994). The genetic locus ASYMMETRICAL LEAVES1 (AS1) in Arabidopsis has been found to encode a M Y B factor (AtMYB91) (Byrne et al., 2000). AS1 negatively interacts with KNOX genes to identify stem cells and lateral organ founder cells within the shoot meristem in  Arabidopsis. In stem cells, the KNOX gene STM negatively regulates AS1. In founder cells, the reduced STM expression allows AS1 is expressed. AS1 in turn down-regulates another two KNOX genes, KNAT1 and KNAT2 (Byrne et al., 2000). AS1 is an ortholog of Antirrhinum PHAN and maize RS2. Their products share a high degree of amino acid identity throughout the entire sequences (Timmermans et al., 1999). (3) Participation in plant response to environmental stimuli and in mediating plant hormone actions. A gibberellin (GA) responsive MYB gene GAMYB has been identified from barley aleurone layers. GAMYB expression is an early event in GA-regulated gene expression during seed germination and precedes a-amylase gene expression. G A M Y B is able to bind to the oc-amylase gene promoter and activates the promoter activity (Gubler et al., 1995). Recently, three rice MYB cDNAs encoding single M Y B repeat proteins were proposed to have the similar function of regulating a-amylase gene expression in response to G A and sugar (Lu et al., 2002). In Arabidopsis, expression of AtMYB2 is induced by abscisic acid (ABA), dehydration and salinity treatments (Urao et al., 1993). A t M Y B 2 along with the b H L H factor A t M Y C 2 binds to the promoter of the Arabidopsis dehydration-responsive gene rd22 to activate the promoter activity (Ade et al., 1997). A t M Y B 2 also binds to the promoter of the alcohol dehydrogenase gene AtADHl to activate this promoter activity (Hoeren et al., 1998). Transgenic Arabidopsis plants overexpressing both AtMYB2 and AtMYC2 are more sensitive to exogenous A B A . Accordingly, ABA-induced expression of rd22 is at a higher magnitude. Expression of AtADHl is also strongly increased in the double-overexpression line (Abe et al., 2003). AtMYB30 is proposed to be a positive regulator during hypersensitive response (HR) signaling. AtMYB30 expression is rapidly induced during hypersensitive cell death upon bacterial attack (Daniel et al., 1999). Overexpression of AtMYB30 in Arabidopsis and tobacco accelerates the plant HR and increases resistance to bacterial and fungal pathogens. Suppression of AtMYB30 expression by antisense R N A in transgenic Arabidopsis strongly decreases hypersensitive cell death (Vailleau et al., 2002). Elicitor and wounding responsive MYB genes have also been described in tobacco and rice (Sugimoto et al., 2000; Lee et al. 2001). NtMYB2  from tobacco has been shown to activate the PAL promoter activity, and overexpression of NtMYB2 in transgenic tobacco plants induces PAL expression (Sugimoto et al., 2000). These results suggest that NtMYB2 is involved in defense response by regulating gene expression in the general phenylpropanoid pathway. It is interesting to see that functionally related MYB genes cluster together in phylogenetic trees (Stracke et al., 2001). This point can be illustrated in several phylogenetic subgroups. In subgroup 5, both members TT2 (AtMYB123) and ZmMYBCl control pigmentation in Arabidopsis seeds and maize kernels. When expanded to the closely related subgroup 6, it can be seen that three members in this subgroup, PhMYBAN2, PAP1 (AtMYB75) and PAP2 (AtMYB90) are also involved in regulating plant pigmentation in petunia and Arabidopsis. In subgroup 15, WER (AtMYB66) and GL1 (AtMYBO) both control epidermal cell differentiation in different organs of Arabidopsis and are functionally equivalent. Their different biological functions are solely caused by their different spatial expression patterns, which are conferred by specific regulatory sequences (Lee and Schiefelbein, 2001). Another example is AS1 (AtMYB91), AmMYBPHAN and the closely related gene ZmMYBRS2. These genes negatively interact with KNOX genes to distinguish the stem cell and founder cell identity in shoot meristems in different plant species. The above data suggest a way to approach plant MYB gene functional studies. For example, although the function of AtMYB113 in the subgroup 6 and AiMYB23 in the subgroup 15 are unknown, it may be related to those of other subgroup members. Indeed, it has been shown that overexpression of AtMYB23 can partially rescue the gll mutant phenotype. This result implies that AtMYB23 may have partial functional overlap with GL1 (Kirik et al., 2001).  1.5 Combinatorial Control: Protein-Protein Interactions  Transcriptional control of a given gene usually requires the combined action of several transcription factors in eukaryotes (Riechmann and Ratcliffe, 2000). This combined action involves interactions between multiple proteins to give the specificity of regulatory effects, or  modulate transcription activities with a limited number of transcription factors (Singh, 1998). The yeast-based two-hybrid system provides a useful tool to study protein-protein interactions (Field and Song, 1989). Several examples of physical interactions between M Y B and other transcription factors have been described in plants. Extensive studies have shown that in maize, the M Y B factor C l / P l requires the b H L H factor R/B in the regulation of anthocyanin biosynthetic genes. Using the yeast two-hybrid system, a direct interaction between C I and B factors in the regulation of the target promoter has been demonstrated. The M Y B DNA-binding domain of C I and the amino-terminal region of B are required for the interaction (Goff et al., 1992). The maize M Y B protein P controls another branch of flavonoid metabolism. P and C I activate expression of some common genes in the flavonoid biosynthesis pathway, such as Al, by binding to an identical cis-element with different affinities. However, C I in association with the R/B factor can activate additional genes specific for anthocyanin synthesis, such as Bzl, which P can not, although P and C I share nearly 80% identity in the M Y B DNA-binding domain (Grotewold et al., 1994). These data indicate that a M Y B transcription factor can obtain a new D N A binding specificity by interacting with another transcription factor. The amino acid residues in the first helix of the M Y B R3 repeat are critical for the interaction of C I and R. Substitution of these residues in P is sufficient for conferring the regulatory specificity of CI to the P factor (Grotewold et al, 2000). This further demonstrates that the M Y B DNA-binding domain is important for the protein-protein interaction. In Arabidopsis, several developmental processes are regulated by M Y B proteins in cooperation with other regulatory factors. For example, the M Y B protein GL1 with T T G l , GL3 in the control of trichome cell fate (Oppenheimer et al., 1991), and AtMYB61 with T T G l and CL2 in the regulation of seed mucilage extrusion (Penfield et al., 2001). It has been demonstrated that the b H L H protein GL3 physically interacts with the M Y B protein GL1, similar to the interaction between B and C I in maize, in the regulation of trichome cell development (Payne et al., 2000). The promoter of the dehydration responsive gene rd22 has  both the M Y B and b H L H binding sites. Overexpression of both AtMYB2 and AtMYC2 enhances the ABA-induced expression of rd.22 (Abe et al., 1997 and 2003). These results also suggesting that these two proteins might have physical interaction.  1.6 The Way Forward in Plant MYB Gene Functional Studies  Currently, sequence analysis of genes is more advanced than their functional characterization. A variety of reverse genetic approaches provide useful tools for functional studies of genes identified by sequencing of the plant genome. The most common method involves large populations of mutants created by either T-DNA, or transposon insertions (Maes et al., 1999; Walbot, 2000; Krysan et al., 1999). Originally, these populations were used to screen for insertion mutations in a particular gene using a PCR-based strategy with a gene specific primer in combination with T-DNA (or transposon) border primers. It is calculated that Arabidopsis T - D N A insertion lines generated by seed transformation have an average of 1.5 TD N A inserts per line (Krysan et al., 1996). Thus, by searching a large enough insertion population, one should be able to find an insertion in any gene of interest. In one of the first uses of reverse genetic approaches, T - D N A insertions within any member of the Arabidopsis actin gene family were identified in a single screen using gene degenerate primers. By searching 5,300 Arabidopsis T-DNA insertion lines, insertions in two actin genes were found out of a total of 10 (McKinney et al., 1995). Using gene-specific primers to screen 9,100 Arabidopsis T - D N A lines, insertions in seventeen genes were found in a total of 63 genes examined (Krysan et al., 1996). A systematic screen for insertion mutations in a large gene family has been carried out in the Arabidopsis P450 gene family (Winkler et al., 1998). Recently, T - D N A collections have been generated in which T - D N A insertion junctions are sequenced and compiled in databases. These databases can be easily searched on-line for potential T-DNA insertions in a gene of interest.  To dissect Arabidopsis R2R3-type MYB gene functions, a systematic screen for MYB gene insertion mutations has been performed with several T - D N A or transposon tagged populations. This resulted in the identification of 47 insertions in 36 genes by searching a total of 73 MYB genes (Meissner et al., 1999). So far insertion knockouts in only one gene, AtMYB61, exhibited a mutant phenotype (Penfield et al., 2001). The lack of phenotypic changes is probably due to functional redundancy in the family members. The AtMYB4 function has also been elucidated by an insertion knockout (Jin et al., 2000). Another common method in plants for producing loss-of-function mutants for a specific gene is the use of antisense R N A or R N A interference (RNAi) to suppress gene expression (Schuch, 1991; de Lange et al., 1995; Hamilton et a l , 1995; Finnegan et al., 1996). This method is based on interfering with gene expression. Using this approach, combined with overexpression, AtMYB30 has been functionally characterized (Vaiieau et al., 2002). The efficiency of antisense R N A suppression is usually low. A new approach to suppress gene expression is R N A i . In this method, double-strand R N A is used to block gene expression (Bosher and Labouesse, 2000). Gene repression using R N A i in a sequence-specific manner has been reported in Arabidopsis. R N A i transformants exhibited mutant phenotypes at a much higher frequency than the plants transformed with either the antisense or the sense gene alone (Chuang and Meyerowitz, 2000). However its efficacy is not yet clear. Epigenetic phenotypes could be a problem in this method (Que and Jorgensen, 1998). However, with the use of loss-offunction approaches, a gene with redundant functions can not be easily characterized. A gain-of-function in which a gene is constitutively expressed is another approach for a functional study. One of the methods is using T-DNA carrying the 35S enhancer as an activation tag (Weigel et al., 2000). Using this approach, PAP1 (AtMYB75) and PAP2 (AtMYB90) have been successfully identified (Borevitz et al., 2000). However, a phenotype revealed in this way may not truly reflect gene function, since it could be caused by interference with other genes. The study of AtMYB4 by its knockout mutation has shown that AtMYB4 primarily negatively regulates C4H expression. However, when overexpressing AtMYB4 under the control of the 35S  promoter, not only C4H, but also 4CL and CHS are down-regulated (Jin et al., 2000). This suggests that the specificity of a transcription factor might be reduced under conditions of overexpression. Thus, gene function revealed by a gain-of-function allele should be interpreted with caution.  1.7 Research Objectives and Approaches  In this thesis, based on evidence revealed at the time (reviewed in Section 3.1), I set out to identify MYB genes involved in the regulation of transcription initiation of genes encoding enzymes in the lignin biosynthetic pathway in Arabidopsis, using At4CLl as a marker. Taking advantage of the ongoing Arabidopsis Expressed Sequence Tag (EST) project that was revealing new MYB genes at the time I started my thesis, my objective was to search for MYB genes that had expression patterns similar to that of At4CLl. Alternatively, I would clone MYB cDNAs from Arabidopsis bolting stems, where At4CLl was highly expressed, and then characterize these genes. This approach appeared to be reasonable, since in some cases, such as the examples reviewed in this chapter, expression of regulatory genes occurred coordinately with expression of target structural genes. In a second project, a MYB gene with a T-DNA insertion in its promoter region was identified by Dr. Aviva Pri-Hadash in the Douglas laboratory, which was a potential gene knockout. My further study showed that this gene had an interesting expression pattern. I set out to continue characterization of this gene by its expression profiling, and to characterize its function using reverse genetic approaches.  Chapter 2 Materials and Methods  2.1 Plant Material Preparation  2.1.1 Plant Growth Conditions  For growing seedlings on defined media, Arabidopsis thaliana seeds were surfacesterilized with 20% bleach plus 0.02% Triton-XlOO, and then planted on A T (ABRC) or MS salt (Murashige and Skoog, 1962) plus 1% sucrose media. In the experiments where the harvesting or observation of roots was necessary, seedlings were grown vertically in square plates. All plates were put in a growth chamber at 20°C under continuous light. For hydroponic cultures, seedlings were grown in A T liquid media plus 0.5% sucrose in a growth chamber at 20°C with 16 h light / 8 h dark cycle. Plants were grown to maturity in soil in a growth chamber at 20°C with continuous light or a 16 h light / 8 h dark cycle.  2.1.2 Plant Treatments  Expression of AtMYB68 in response to external stimuli was examined as follows. In the following treatments, A T liquid media plus 0.5% sucrose and 23 m M MES served as basic media. Seedlings were treated under the light period, except as indicated otherwise. For the NOy treatment, Arabidopsis seedlings were grown in A T liquid media in the absence of N 0 " for 4 days. K N 0 was added to the media to a final concentration of 2 mM. 3  3  Whole seedlings were harvested after a 6-hour treatment. Seedlings maintained in the medium without K N 0 served as a control. 3  For the plant hormone treatments, seven- or eight-day old Arabidopsis seedlings grown in A T media were transferred to new media supplemented with I A A , B A P , G A , A B A or A C C 3  respectively. Each plant hormone was made to a final concentration of 10 p M in the medium,  except I A A at 100 u M . Whole seedlings were harvested after a 6-hour treatment respectively. Seedlings in the medium without any plant hormones served as a control. For the cold treatment, seven-day old Arabidopsis seedlings grown in A T liquid media without MES were transferred to a 4°C dairy fridge for 24 hours. For the desiccation treatment, seedlings from the same batch were removed from the medium, put on Whatman paper in a Petri dish and left room temperature 30 minutes (Kranz et al., 1998). For the salinity treatment, seedlings from the same batch were transferred to the new medium supplemented with NaCl in a concentration of 200 m M for 6 hours. Whole seedlings were harvested after each treatment. Seedlings grown in A T media under the normal conditions served as a control. AtMYB68 overexpression and knockout lines were examined for phenotypes as follows. In the following treatments, M S media supplemented with 1% sucrose served as basic media. Seedlings were grown under continuous light. Primary root length was measured using a ruler. Lateral root numbers were visualized under a dissecting microscope. A B A treatment: four-day old seedlings grown on MS plates were transferred to plates supplemented with 1.0 (iM A B A . Seedlings were grown vertically on the A B A plates. Measurements were taken after 6 or 7 days. I A A treatment: seven-day old seedlings grown on M S plates were transferred to plates supplemented with 1.0 u,M I A A . Seedlings were grown vertically on the I A A plates. Measurements were taken after 5 days. Salinity stress: four-day old seedlings grown on M S plates were transferred to plates supplemented with a series of concentrations (50, 100 and 150 mM) of NaCl. Seedlings were grown vertically with roots upside down on the NaCl plates (Wu et al., 1996), for observing root-bending. Drought stress: 20-day old soil-grown plants were not watered for 2 weeks. To minimize variation, the same number of plants was grown in each pot. The root obstacle-touching response was examined as described by Rutherford and Masson (1996).  2.1.3 Plant Transformation  Arabidopsis Columbia (Col) plants were transformed according to the protocol described by Clough and Bent (1998). Briefly, Agrobacterium GV3303 strains carrying binary vectors with inserts were grown in L B media plus kanamycin (100 mg/L), gentamycin (25 mg/L) and rifampicin (25 mg/L) to O D  600  -0.8. The Agrobacterium cells were collected by centrifugation  then resuspended in 5% sucrose. Silwet L-77 was added to a concentration of 0.05%. Aerial parts of plants with many immature flower clusters and few siliques were dipped in the Agrobacterium solution. The plants were then covered by Saran wrap and kept under a lab bench for a day. The transformation procedure was repeated once after a 3-day interval for the antisense AtMYB68 transformation. Seeds of transformed plants ( T l generation) were harvested and plated on kanamycin-containing (50 mg/L) A T media to identify transformants.  2.2 General Molecular Methods  2.2.1 Plasmid DNA Preparation and DNA Sequencing  Plasmid D N A was prepared by the alkaline-lysis/PEG precipitation procedure (the NAPS laboratory of the University of British Columbia, 1995), or using the QIAprep Spin Miniprep Kit (QIAGEN), following the manufacture's instructions. D N A sequencing was done using the BigDye™ Terminators Sequencing Kit and carried out in the NAPS laboratory.  2.2.2 Plant Genomic DNA Isolation  Plant genomic D N A was isolated according to the 'Short D N A Quick-Prep' protocol from the University of Wisconsin Biotechnology Center (http://www.biotech.wisc.edu).  2.2.3 Total RNA and Poly(A) RNA Isolation +  Total R N A was extracted from Arabidopsis seedlings and mature organs using the RNeasy™ Plant Mini Kit (QIAGEN), following the manufacture's instructions. Poly(A) R N A +  was prepared using the Dynabeads™ Oligo (dT) ( D Y N A L ) , following the manufacture's 25  instruction.  2.2.4 Northern and Southern Blot Analysis  Total R N A or poly(A) R N A was separated on 1.2% agarose gels containing 0.22 M +  formaldehyde in I X MOPS buffer. After separation, the gel was washed in water for 1 hour, and then blotted on Hybond™-N nylon membranes (Amersham) in 10X SSC. R N A samples were fixed by baking blots at 80°C for 2 hours. P-radiolabeled probes were hybridized to blots in 32  0.25 M sodium phosphate buffer (pH 7.2) containing 7% SDS (Church and Gilbert, 1984), at 65°C in a hybridization oven overnight. The blots were washed at low stringency (2X SSC, 0.1% SDS at 65 °C) and high stringency (0.2X SSC, 0.1% SDS at 65 °C). Signals were detected by exposure to an x-ray film (Kodak). For Southern blot analyses, D N A was separated on 1% agarose gels, and then blotted on Hybond™-N+ nylon membranes (Amersham) in 0.4 M NaOH. Blots were hybridized and washed at the same conditions as those in northern blot analyses. Signals were detected as above. Hybridization probes used in northern and Southern blot analyses were generated from PCR fragments, cDNAs, or genomic D N A , as indicated in each experiment. P-radiolabeled 32  probes were generated using the Random Primers D N A Labeling System (Gibco BRL) according to the manufacturer's instructions.  Blots were sometimes stripped for re-hybridization. For R N A blots, the blots were stripped by incubating blots in 5 m M Tris (pH 8.0) buffer containing 2 m M E D T A and 0.1 X Denhardt at 65 °C for 2 hours. For D N A blots, blots were stripped by pouring boiling 0.1% SDS on the blots, and then allowing the solution to cool to room temperature. This procedure was sometimes repeated. Successful stripping was ensured by exposure to an x-ray film before the blots were used for the next hybridization.  2.2.5 Quantitative RT-PCR Analysis  Total R N A (100 ng) was used in reverse transcription (RT) followed by PCR amplification using the QIAGEN™ OneStep RT-PCR Kit (QIAGEN), except for AtMYB84 where 2-step RT-PCR was used. One gene-specific primer (forward) was designed to span an intron to avoid amplification from any contaminating D N A . The QuantumRNA™ 18S Internal Standards (Ambion) were used as an internal control. The products were separated on 3.5% mini polyacrylamide gels, and then post-stained by Syber green I (Molecular Probes) for 30 minutes. Fluorescent signals were detected and quantified using a Storm Phosphorimager and the ImageQuant software (Amersham-Pharmacia). First, the PCR linear amplification range for each target gene was determined, in which PCR products were increased exponentially with the cycle number. This was done by performing identical RT-PCR reactions for different numbers of PCR cycles. The results were quantified and plotted. An example of such an experiment is shown in Figure 2-1. A cycle number close to the middle of the linear range was used in the experiment. Second, the optimal ratio of 18S primers to Competimers™ in the kit (Ambion) was determined. This was done by performing several RT-PCR reactions with different ratios of 18S primers to Competimers at the number of PCR cycles determined above. The results were assessed on a gel. The ratio that gave a level of 18S product similar to that of the gene specific product was chosen. Primers for each gene used in the quantitative RT-PCR analyses are listed in Table 2-1.  Table 2-1. Primers Used in Quantitative RT-PCR Analyses Gene  Primer  AtMYB68  Forward 5'-GCCTCAGAAAATTGGTTTAAGG-3'  Product Size (bp) 1008  Reverse 5 ' -T AC AC ATG ATTTGGCGC ATTG A- 3 ' AtMYB84  Forward 5' -TTGGTAGCAGGTGGTCTATAATC-3 '  750  Reverse 5 ' -TCC AT AT AT ACGTT ACGT ACCCCC- 3 ' AtMYB36  Foreard  5'-TGCCTCAGAAAATTGGGCTGAAG-3'  930  Reverse 5 ' -TTATCCATCCCTATAGTTACGCA-3'  Cycle Number 26  28  30  V_>  32  LJ  34  ^  AIMYB68  10000000 -,  24  26  28  30  32  34  36  Cycle Number  Figure 2-1. Determination of the Linear Range for RT-PCR. Total R N A from 4-day old Col seedlings was used in RT-PCR to determine the linear range for AtMYB68 amplification. RT-PCR aliquots were removed from the thermocycler at the indicated cycle numbers and resolved on a polyacrylamide gel. Cycle numbers are plotted against the log scales of signal intensity.  2.3 Cloning  2.3.1 Cloning of MYB cDNAs  Poly(A) R N A (1 u.g) extracted from Arabidopsis Col bolting stems was used to generate +  MYB cDNAs, using the Marathon™ cDNA Amplification Kit (CLONTECH) following the manufacture's instructions. The 5' regions of MYB cDNAs were amplified using 5'-RACE. A MYB degenerate primer corresponding to the region in the M Y B R3 repeat, 5'TTGAC(T)T(CA)T(G)CA(G)TTG(A)G(T)CG(AT)GTC(T)CT(G)TCC-3', was used in 5'R A C E . The amplified products were separated on a gel and tested by a Southern blot analysis. A mixture of c D N A fragments corresponding to the M Y B DNA-binding domain was used as a probe. These c D N A fragments were derived from M Y B EST clones 95L7T7, 106H10T7 and 1B4T7P by first digesting with Hindlll then with Sali (see Figure 2-2-A). The SaWHindlll fragments were recovered from a gel. The 5 ' - R A C E products of the predicted size were cloned into the T A vector (Invitrogen) and sequenced. The 3' region of the STM31 cDNA was amplified using 3 ' - R A C E , with the Expand™ High Fidelity PCR System (Boehringer Mannhein). A gene-specific primer, 5'T A G A G C A C C G T G C T T T T T C G C A G - 3 ' , was used in 3 ' - R A C E . The amplified fragment was also cloned into the T A vector and sequenced. The full-length STM31 c D N A was generated by joining the 5'-cDNA and the 3'-cDNA at the Sad site in the overlapping region.  2.3.2 Generation of the AtMYB68::GUS Construct  PCR was used to generate a 1.9 kb AtMYB68 promoter fragment with Hindlll and BamHl sites on the 5' and 3' ends respectively from Arabidopsis Col genomic D N A , using the Expand™  High Fidelity PCR System (Boehringer Mannhein). Primers, 5'ctcaa^Ç^CTTTrCATCTCrrTTGG-3', and Hindlll  5 ' -tgcggaTCCCATT/ITCTTGATTCTTG-3 ', were used for PCR. The BamHl  PCR fragment was cloned into the pBluescript KS vector, and sequenced to ensure no mutations were introduced during amplification. A 1.9 kb fragment was released by BamHl digestion and Hindlll partial digestion. This fragment was inserted 5' to the GUS gene in the pBI121 binary vector (CLONTECH), replacing the 35S promoter (Figure 2-2-E). The AtMYB68 promoter was translationally fused to the GL75 in the vector. The plasmid was transformed into the Agrobacterium GV3303 strain.  2.3.3 Cloning of the AtMYB68 cDNA into E. coli Expression Vectors  The 3' c D N A of AtMYB68 was derived from the EST clone 110F12T7 by Hindlll digestion. A fragment of -880 bp was recovered from a gel and cloned into the Hindlll site of the expression vector pQE-30 (QIAGEN) (Figure 2-2-D). The sense orientation clone was selected by Xbal digestion. The clone was also sequenced to confirm that the cDNA was in frame with the A T G from the vector. This plasmid was transformed in the E. coli M15[pREP4] strain using a method recommended by the manufacture. The empty vector pQE-30 in M15[pREP4] was used as a negative control.  2.3.4 Generation of Constructions to Sense and Antisense AtMYB68  A sense construct for overexpression of AtMYB68 was generated as follows. The fulllength AtMYB68 c D N A was released from EST clone 110F12T7 by SaWNoil digestion. Two overhangs were removed by Klenow, and then the cDNA was inserted into the Smal site behind the 35S promoter of the pRTlOl vector (Topfer et a l , 1987) (Figure 2-2-B). The sense  orientation clone was selected by HindIII digestion. A 35S-ArMyBf55-poly-A cassette was released by Hindlll partial digestion. A fragment of ~2 kb was selected and inserted into the Hindlll site of the pBinl9 binary vector. An antisense AtMYB68 construct was generated as follows. The 3' c D N A of AtMYB68 was released from EST clone 110F12T7 by Hindlll digestion. Two overhangs were removed by Klenow, and then the c D N A was inserted into the Smal site behind the 35S promoter of the pRTlOl vector (Figure 2-2-C). The antisense orientation clone was selected by Hindi digestion. A 35S-AtMYB68 (-)-poly-A cassette was released by Hindlll digestion and then inserted into the same site of the pBinl9 binary vector. Both sense and antisense constructs in pBinl9 were transformed into the Agrobacterium GV3303 strain.  2.4 Protein Analysis  2.4.1 Protein Extraction  The recombinant AtMYB68 protein was prepared and purified according to the QIAexpressionist™ instructions (QIAGEN). Briefly, an E. coli culture of 500 ml (OD =0.7600  0.9) containing the expression plasmids was induced by 1 m M IPTG for 2 hours. The bacterial pellet was collected by centrifugation. The recombinant protein was extracted and purified under denaturing conditions as follows. The pellet was resuspended and lysed in 6 M guanidine-HCl buffer. The recombinant protein was purified with Ni-NTA resin (QIAGEN) columns, and finally eluted with 8 M urea buffer (pH 4.5). Plant proteins were extracted by grinding tissues in a buffer containing 25 m M Tris (pH 8.0), 10 m M NaCl, 10 m M MgC12, 5 m M E D T A , 10 m M (3-mercaptoethanol, 1 m M PMSF (Kwok et al., 1998), plus 1 tablet Complete™ (Roche) per 10 ml buffer to prevent protein degradation. The homogenate was centrifuged at 4°C to remove cellular debris.  R2  R3  AtMYB68  3 E S T 110F12T7  I  \s L  (AtMYB68)  Lamda-pRL2  AtMYB68  pRTlOl  D  AtMYB68  pQE-30  pBI121  Figure 2-2. Plasmids Generated for Various AtMYB68 Constructs. (A) The predicted A t M Y B 6 8 protein from the cDNA is shown on the top. Black and grey boxes represent the M Y B R2 and R3 repeats. (B) AtMYB68 sense D N A construct. (C) AtMYB68 antisense D N A construct. (D) AtMYB68 expression construct. The grey box represents 6X histidine-tag on the vector. The translation start and stop codons are shown on the figure. M C S , multi-cloning site. (E) AtMYB68::GUS construct. A 1.9 kb AtMYB68 promoter with the A T G codon was put in frame to replace the 35S promoter. Pro, promoter. Ter, terminator.  Protein concentrations were quantified by the Bradford (1976) method using the Bio-Rad Protein Assay Solution™ (Bio-Rad) with B S A as a standard. 2.4.2 Generation of Anti-Serum against the AtMYB68 Recombinant Protein  Polyclonal antibodies were raised in New Zealand White rabbits, cared for at the Animal Care Center in the University of British Columbia. Two female rabbits were pre-bled and the pre-immune sera were tested for cross-reaction to E. coli and plant proteins. The two rabbits were injected with -100 pg purified recombinant AtMYB68 (in the urea buffer) mixed with Complete Freund's Adjuvant in the total volume of 1 ml. They then received two subsequent booster injections of -100 pg recombinant AtMYB68 mixed with Incomplete Freund's Adjuvant at about 3-week intervals. The rabbits were sacrificed and their blood was recovered. The blood was placed in Falcon tubes with toothpicks at the edge at 37 °C for 1 hour, then transferred to 4 °C overnight to induce clotting. The clot was removed and the serum were centrifuged to remove blood cell debris. A few crystals of sodium azide were put in the serum to prevent contamination. The serum was stored at -80 °C.  2.4.3 AtMYB68 Antiserum Purification  The antiserum from one of the rabbits was purified using a CNBr-activated Sepharose™ 4B (Pharmacia Biotech) affinity column, according to a protocol described by Fuller et al. (1997), combined with the CNBr-activated Sepharose™ 4B manufacture's instructions. The purified recombinant AtMYB68 protein in the urea buffer was concentrated and changed to the coupling buffer (0.1 M N a H C 0 , pH 8.3, 0.5 M NaCl) before it was bound to the Sepharose. 3  This was done by ultrafiltration using the Ultrafree™-4 Centrifugal Filter Unit (MWCO 10,000, Millipore). The concentrated recombinant protein was finally dialyzed in the coupling buffer overnight. CNBr-activated Sepharose 4B powder (1.5 g) was swollen and washed by 1 m M HC1 followed by the coupling buffer, and then mixed with recombinant AtMYB68 at a concentration  of -2.4 mg/ml in 10 ml coupling buffer overnight at 4°C. The gel was washed with the coupling buffer, and then incubated with 15 ml of 1 M ethanolamine (pH 8.0) for 5 hours at 4°C to block excess remaining active groups. The gel was washed alternatively with the coupling buffer (pH 8.3) and the wash buffer (0.1 M sodium acetate, 0.5 M NaCl, pH 4.0) for three cycles to remove excess of uncoupled antigen. The gel slurry was prepared in PBS buffer at the ratio 3:1, and then packed in a 20 cm-long glass column and equilibrated with PBS buffer. The antiserum (8 ml) was diluted in 3X volume of PBS buffer and filtered through a 0.2 p,m filter, and then loaded to the column. The flow rate was controlled at -200 ml/hr. The column was washed by PBS buffer until an A  2 8 0  < 0.005 was present in the wash. The bound antibody was eluted by glycine-HCl  buffer (pH 2.3) and neutralized in 1/4 volume of 0.5 M sodium phosphate buffer (pH 7.7). The purified antibody was finally concentrated to -0.1 (Xg/u.1 using the Ultrafree™-15 Centrifugal Filter Device (MWCO 10,000, Millipore), and examined on SDS-PAGE.  2.4.4 Western Blot Analysis  Bacterial or plant proteins were separated on 10% SDS-polyacrylamide separating gels (Laemmli, 1970), and then blotted on P V D F membranes (Amersham-Pharmacia) at 30 V overnight. Duplicated gels were stained by 0.1% Coomassie Brilliant Blue as loading controls. Signals were detected by using E C L Plus™ detection reagents (Amersham-Pharmacia) according to the manufacturer's instruction. The blots were reacted with a 1:1000 dilution of the rabbit antiserum raised against the recombinant AtMYB68, or a 1:5000 dilution of the purified antibody. Horseradish peroxidase (HRP) conjugated anti-rabbit IgG (Amersham-Pharmacia) was used as the secondary antibody at a 1:20000 dilution. Chemiluminescent signals were detected by exposure to x-ray films. E C L protein molecular weight markers (Amersham-Pharmacia) were also loaded on the blots.  2.5 Histology and Histochemistry  2.5.1 Histochemical GUS Staining  Seedlings were incubated in a GUS reaction solution, containing 0.5 m M X-Gluc substrate in 0.1 M sodium phosphate buffer (pH 7.0), and 0.5 m M each potassium ferri- and ferrocyanide as an oxidation catalyst, plus 10 m M E D T A and 0.1% Triton X-100, at 37 °C overnight (Stomp, 1992). Stained seedlings were then cleared as described by Malamy and Benfey (1997) and mounted in 50% glycerol on glass microscope slides. Samples were observed under a dissecting microscope. Pictures were taken with Ektachrome Tungsten films (Kodak).  2.5.2 Root Section Preparation  Two-three days old Col seedlings harboring the AtMYB68::GUS fusion, or 7-day old wol seedlings harboring the same fusion were pre-stained for GUS activity as described above for 9 hours, and then fixed in 4% formaldehyde in PBS buffer overnight at 4 °C. The seedlings were dehydrated via an ethanol series at room temperature, 30%, 50%, 70%, 85%, 95%, 100%, 30 minute in each, 100% ethanol for 1 hour. Ethanol exchanged for Spurr resin by passing through a series of ethanokresin mixtures, 3:1, 1:1, 1:3, 100% resin (X2), 1 hour in each, and then embedded in 100% resin and baked at 65 °C overnight. The Spurr resin was prepared following a recipe in the E M facility of the University of British Columbia. The seedling root was sectioned to ~3 pm on the Ultracut microtome E (Reichert-Jung), and mounted on glass slides. Samples were observed under a Leitz microscope with the DIC field.  2.6 Primers Used in Screening T-DNA Tagged Populations  AtMYB68 primers used for screening for AtMYB68 knockout mutations in various TD N A tagged populations are listed in Table 2-2. T-DNA border primers used in the screens were specified by the generator of each collection.  Table 2-2. AtMYB68 Primers Used for Screening T-DNA Tagged Populations Name  Primer  myt>13-3  Reverse 5 ' -TTCT ACC ACTCCCT A A AG AC AC AG- 3 '  m13295  Forward 5 ' -CCATCCTCCTCATTCTCTTCATCATCAAC-3 ' Wisconsin (U of  ml3293III  Reverse 5'-AACTTTTCT ACC ACTCCCT AAAGAC AC AG-3' Wisconsin)  ml3293  T-DNA Population Screened Jack (ABRC)  Nested 5 ' - AGA A A ACCCCTCTTT ATGT ATGT A AGT AC-3 '  ml3293III( to confirm)  Garlic (TMRI)  Chapter 3 Searching for Arabidopsis MYB Genes Involved in Regulation of the Phenylpropanoid Pathway  3.1 Introduction  M Y B transcription factors appear to play an important role in the regulation of phenylpropanoid metabolism. A random-binding site selection experiment has shown a core binding site of the maize M Y B protein P (Grotewold et al., 1994). This core binding sequence conforms to the conserved cis-elements P- and L-box present in PAL, 4CL and other genes encoding enzymes in phenylpropanoid metabolism (Table 3-1, Douglas, 1996). A tobacco M Y B related protein has been also shown to bind to a similar sequence (Sablowski et al., 1994). Ectopic expression of the Antirrhinum MYB gene AmMYB305 can activate the bean PAL2 promoter activity in tobacco mesophyll cells (Sablowski et al., 1995).  Table 3-1. Conserved Cis-Elements in Promoters of Phenylpropanoid Genes and M Y B P Binding Site (According to Douglas, 1996) Sequence  Cis-Element  Gene  Plant  ACCAACCC  Core binding site  P (MYB)  Maize  4CL1  Parsley  ACCAACCC  FP4 FP8  4CL1  Parsley  ACAAACCC  PBox  PALI  Parsley  ACCTACCA  LBox  PALI  Parsley  ACCTACCA  AC-I  PALI  Bean  ACCAACCC  AC-II  PALI  Bean  ACCTAACT  AC-III  PAL]  Bean  AACAACCC  AC  CAD  Eucalyptus gunnii  (T)  (A)  ACCAACCC  A few years ago, based on the analysis of cis-elements in the promoters of the genes encoding enzymes in phenylpropanoid metabolism, we hypothesized that M Y B transcription factors were involved in the regulation of tissue/cell type-specific expression of the genes  encoding enzymes in phenylpropanoid metabolism. Recent research on Arabidopsis MYB genes has revealed several AtMYB genes, such as AtMYB4 and TT2, are involved in the regulation of different branch pathways in phenylpropanoid metabolism (reviewed in Chapter 1). However, another important branch pathway, that of lignin biosynthesis, has not been reported to be under the control of M Y B proteins. Lignin biosynthesis requires the coordinate action of both the enzymes in the general phenylpropanoid pathway (PAL, C 4 H and 4CL) and the enzymes specific for lignin biosynthesis, such as C A D (Hahlbrock and Scheel, 1989). The M Y B binding site has been found in the promoters of the genes encoding these enzymes (Douglas, 1996; Table 3-1). Therefore we expect to see that coordinate control of gene transcription initiation, which brings about coordinate enzyme action in this pathway, is under the control of M Y B transcription factors. The Arabidopsis bolting stem is a major site for lignin biosynthesis, and At4CLl is a good marker for lignin biosynthesis since its expression is temporally and spatially coordinated with lignin deposition (Lee et al., 1995). In this chapter, I describe the initial work to identify AtMYB genes involved in the regulation of expression of the genes encoding enzymes required by lignin biosynthesis. I searched for representative AtMYB genes in the publicly available EST database at the time that had expression patterns similar to that of the marker gene At4CLl. I also tried to identify such AtMYB genes through cloning MYB cDNAs from the bolting stem, where At4CLl was strongly expressed. Before doing these experiments, I first characterized differential expression patterns of Arabidopsis 4CL genes.  3.2 Expression of Arabidopsis 4CL and 4CL-Like Genes  Since At4CLl and At4CL2 cDNAs shared a high degree of identity, I first needed to know how similar they were in expression patterns. The expression pattern of At4CLl was determined before (Lee et al., 1995). The expression pattern of At4CL2 was examined in seedlings and mature organs by a northern blot analysis. A -1.5 kb genomic D N A fragment of  At4CL2 containing the 1 exon and a -400 bp upstream region was used as a probe. This st  fragment did not hybridize to the At4CLl cDNA as tested on a Southern blot (data not shown). For comparison, the At4CLl c D N A was hybridized to the same blot after the At4CL2 probe was removed. Figure 3-1-A shows that the level of the At4CL2 transcript was generally lower than that of At4CLl (probes were labeled to have similar specific activities). Both had similar expression patterns in seedlings. Accumulation of the transcripts was increased as the seedling grew from 2day old to 3-day old. It was noted that some seedlings at this age accumulated anthocyanin pigments in the cotyledons and hypocotyls under the light conditions used. Among organs, the At4CLl transcript accumulated highly in bolting stems. In contrast, the level of the At4CL2 transcript was low in this organ. Both had moderate expression in the root, but had low expression in rosette leaves, flowers and siliques. At the time, by searching the Arabidopsis EST database, we found that EST clone 139B10T7 had some similarity to At4CLl (now, with completion of the Arabidopsis genome project, this gene is known to be one of many related to 4CL). The predicted protein has 41.5% identity to A t 4 C L l . It contains a partially conserved sequence 'GEICIRG' (motif II), common to all 4CL enzymes (Becker-Andre et al., 1991), and the motif I, which was postulated to be part of the AMP-binding domain that is common to ATP-dependent enzymes (Schroder, 1989) (Figure 3-2). Thus, the expression pattern of this 4CL-\ikt gene was also examined. A northern blot analysis showed that the transcript of this gene accumulated ubiquitously at a low level in all the tissues/organs tested. The transcript level was slightly higher in 3-day old seedlings. This could be related to light induced anthocyanin synthesis in these seedlings (Figure 3-1, B).  3.3 Expression Patterns of Arabidopsis MYB Genes  When this project was started in 1995, a search of the Arabidopsis EST database revealed 25 M Y B related EST clones (Table 3-2). These clones were ordered from the A B R C Stock  fp <P &  ^  RT ST LF FL SL  m  mt  «*  At4CL2  H  Figure 3-1. Northern Blot Analysis of Expression Patterns of Arabidopsis 4CL Genes. (A) Expression patterns of At4CLl and At4CL2. ThcAt4CLl cDNA was hybridized to the same blot after the At4CL2 probe was removed. (B) Expression Pattern of 4CL-like EST 139B10T7. Total R N A was extracted from Col seedlings and mature organs. 10 pg total R N A was loaded on each lane. 2D-SD, 2-day old seedlings. 3D-SD, 3-day old seedlings. RT, 10-day old seedling roots. ST, bolting stems. L F , rosette leaves. FL, flowers. SL, siliques. A ribosomal R N A probe was hybridized to the same blot as a loading control after a blot was striped. Blots were washed at high stringency.  At4CLl 139B10T7  MAPQEQAVSQ  VMEKQSNNNN  SDVIFRSKLP  D I Y I P N . . H L  SLHDYIFQNI  50  MEKSGYGR.  .DGIYRSLRP  TLVLPKDPNT  SLVSFLFRNS  50  5 5  SEFATKPCLI  NGPTGHVYTY  SDVHVISRQI  AANFHKLGVN  QNDWMLLLP  100  SSYPSKLAIA  DSDTGDSLTF  SQLKSAVARL  AHGFHRLGIR  KNDWLIFAP  100  At4CLl 10 139B10T7 10  NCPEFVLSFL  AASFRGATAT  AANPFFTPAE  IAKQAKASNT  KLIITEARYV  150  NSYQFPLCFL  AVTAIGGVFT  TANPLYTVNE  VSKQIKDSNP  KIIISVNQLF  150  At4CLl 139B10T7  15  DKIKPLQNDD  G W I V C I D D N  ESVPIPEGCL  RFTELTQSTT  E A S E V I D S . .  200  139B10T7 15  D K I K G F . . D L  P W L L G S K D .  .TVEIPPGSN  SKILSFDNVM  ELSEPVSEYP  200  At4CLl 20 139B10T7 20  . V E I S P D D W  ALPYSSGTTG  LPKGVMLTHK  GLVTSVAQQV  DGENPNLYFH  250  FVEIKQSDTA  ALLYSSGTTG  TSKGVELTHG  NFIAASLMVT  . . MDQDLMGE  250  At4CLl  motif I At4CLl 25 139B10T7 25  SDDVILCVLP  MFHIYALNSI  MLCGLRVGAA  ILIMPKFEIN  LLLELIQRCK  300  YHGVFLCFLP  MFHVFGLAVI  TYSQLQRGNA  LVSMARFELE  LVLKNIEKFR  300  At4CLl 30 139B10T7 30  VTVAPMVPPI  VLAIAKSSET  EKYDLSSIRV  VKSGAAPLGK  ELEDAVNAKF  350  VTHLWWPPV  FLALSKQSIV  KKFDLSSLKY  IGSGAAPLGK  DLMEECGRNI  350  At4CLl 35 139B10T7 35  PNAKLGQGYG  MTEAGPVLAM  SLGFAKEPFP  V K S G A C G T W  RNAEMKIVDP  400  PNVLLMQGYG  MTETCGIVSV  E D P R L G K . . .  RNSGSAGMLA  PGVEAQIVSV  400  At4CLl 40 139B10T7 40  DTGDSLSRNQ  PGEICIRGHQ  IMKGYLNNPA  ATAETIDKDG  WLHTGDIGLI  450  ETGKSQPPNQ  QGEIWVRGPN  MMKGYLNNPQ  ATKETIDKKS  WVHTGDLGYF  450  motif II At4CLl  45  DDDDELFIVD  RLKELIKYKG  FQVAPAELEA  LLIGHPDITD  VAWAMKEEA  500  139B10T7 45  NEDGNLYWD  RIKELIKYKG  FQVAPAELEG  LLVSHPDILD  A W I P F P D E E  500  At4CLl 50 139B10T7 50  A G E V P V A F W  KSKDSELSED  DVKQFVSKQV  VFYKRINKVF  FTESIPKAPS  550  A G E V P I A F W  RSPNSSITEQ  DIQKFIAKQV  APYKRLRRVS  FISLVPKSAA  550  At4CLl  55  GKILRKDLRA  KLANGL  139B10T7 55  GKILRRELVQ  QVRSKM  566 566  Figure 3-2. Alignment of A t 4 C L l and the 4CL-Like Protein Encoded by EST 139B10T7. The shaded area indicates conserved amino acids in both proteins. Motif I and II are conserved in all 4CLs.  Center, and checked by sequencing to ensure that the right clones had been received. They represented 21 unique genes since redundancy existed in three pairs of ESTs (clones 4 and 7; 6 and 21; and 16 and 23), and one EST clone (clone 20) can no longer be assigned as a MYBrelated gene, as most of Arabidopsis genes have been annotated today. Three (clones 3, 18 and 25) were not R2R3-type MYB genes.  Table 3-2. Examined Arabidopsis MYB EST Clones Douglas EST Clone  GenBank Accession  Lab No.  for EST Clone  Corresponding Gene*  Gene Code  AtMYB47  Atlgl8710  AtMYB45  At3g48920  1 2  165L19T7 170K1T7  R30551 R65084  4  111E18T7  T42232  AtMYB94 (AtMYBCP70)  At3g47600  7  111F18T7  T42245  AtMYB94  At3g47600  5  178M4T7  H36793  AtMYB93  Atlg34670  6  95L7T7  T21333  AtMYB44 (AtMYBrl)  At5g67300  21 8  93E13T7 99M15T7  T21228  AtMYB44  At5g67300  T22593  AtMYB51  Atlgl8570  9 10  99P9T7 106H10T7  T22223 T22697  AtMYB75 (PAP1) AtMYB63  Atlg56650 Atlg79180  11  157G9T7  T88197  AtMYB92 (AtMYB64)  At5gl0280  12  122N17T7  T43719  AtMYB6  At4g09460  13  110F12T7  T42065  AtMYB68  At5g65790  14  1B4T7P  T04118  AtMYB3  Atlg22640  15  186019T7  R89962  AtMYBlô (AtMIXTA)  At5gl5310  16  114F24T7  T42666  AtMYB59  At5g59780  23  206N11T7  N37592  AtMYB59  At5g59780  17  15604T7  T88476  AtMYB36  At5g57620  19  220A8T7  N38154  AtMYB4  At4g38620  22  193M15T7  H76020  AtMYB90 (PAP2)  Atlg66390  24  E11A6T7  AtMYB96 (mybcovl)  At5g62470  3*  109M16T7  AA041019 T41952  Encoding MYB-like protein  At4gl7780  18* 25*  135E3T7 130A10T7  T46559  Encoding putative M Y B protein  Atlg71030  T44683  Encoding Myb-related  At5g47390  N65339  transcription activator-like protein ?  20*  226M4T7  #AtMYB gene systematic name in Stracke et al. (2001). *EST clones not included in the Stracke et al. (2001) AtMYB gene list.  The non-conserved 3' region of each clone was PCR amplified using a degenerate primer corresponding to the end of the conserved M Y B DNA-binding domain and a vector primer. No product was amplified from clone 25. The PCR fragments were gel-purified and used as probes for northern analyses. For clone 25, the whole cDNA fragment was used as a probe. The results of northern blot analyses are summarized in Table 3-3, and accumulation of the transcripts detected by northern blot analyses is shown in Figure 3-3. Total R N A from 2- and  Table 3-3. Expression Patterns of Examined Arabidopsis MYB Genes EST Clone  Tissue Detected  (Gene)  2D-Seedlings 3-4D-Seedlings Roots Bolting Stems Rosette Leaves Flowers Siliques  165L19T7 (AtMYB47)  —  —  N/A  —  —  —  N/A  170K1T7 (AtMYB45)  —  —  N/A  —  —  —  N/A  111E18T7 (AtMYB94) 11 IF\871 (AtMYB94)  Not Done —  —  —  —  —  —  N/A  178M4T7 (AtMYB93)  —  —  N/A  —  —  —  N/A  95L7T7 (AtMYB44)  +  ++  +++  ++  +++  +  +  93E13T7 (AtMYB44)  +  ++  N/A  +++  +++  +  99M15T7 (AtMYBSl)  —  —  N/A  —  +  —  99P9T7 (AtMYB75)  +  —  N/A  —  —  —  N/A  106H10T7 (AtMYB63)  —  —  N/A  —  —  —  N/A  157G9T7 (AtMYB92)  —  —  N/A  —  —  —  N/A  122N17T7 (AtMYB6)  —  —  N/A  —  —  —  N/A  UQVUT1  —  +  ++  —  —  —  —  N/A  —  —  —  —  —  —  N/A*  +  N/A  +  +  N/A  N/A  (AtMYB68)  1B4T7P (AtMYB3)  .N/A N/A  186019T7 (ATMYB16)  ++  ++  N/A  —  —  +  N/A  114F24T7(AfMra59)  —  —  N/A  —  —  —  N/A  206N11T7 (AtMYB59)  —  —  N/A  —  —  —  N/A  15604T7 (AtMYB36)  N/A  +  ++  —  —  —  —  220A8T7 (AtMYB4)  +  ++  ++  +  +  +  ++  193M15T7 (AtMYB90)  —  —  N/A  —  —  —  N/A  E11A6T7 (AtMYB96)  —  —  N/A  —  —  —  N/A  —  —  N/A  109M16T7  —  —  N/A  —  135E3T7  N/A  —  —  —  N/ \ -  +  N/A  +(-)  +  N/A  N/A  130A10T7  +  ++  +  +  ++  +  +  +++, moderate; ++, weak; +, very weak; +(-), just detectable; -, not detectable; N/A, not done. * Signal detected on poly(A) R N A blot. +  3-day old seedlings, bolting stems, rosette leaves and flowers was commonly examined. Total R N A from roots and siliques was only examined with 3 clones (expression of AtMYB68 is shown in Chapter 4). The transcript levels of these MYB-homolog genes were generally low, except that the level of the AtMYB44 transcript was relatively high. Of all 21 genes examined, expression of 13 genes was not detectable in the tissues/organs tested on the total R N A northern blots. Expression of 8 genes was detectable by a northern analysis with various expression patterns (Figure 3-3). In particular, expression of 99M15T7 (AtMYB51) was only detected in rosette leaves (Figure 3-3, C), and 99P9T7 (AtMYB75) expression was only detected in 2-day old seedlings (Figure 3-3, D). Expression of 15604T7 (AtMYB36) was mainly detected in roots, and detectable in 3 day-old seedlings (Figure 3-3, F). This pattern is the same as that of 110F12T7 (AtMYB68) (Chapter 4). Expression of 186019T7 (AtMYB 16) was relatively high in seedlings, and just detectable in flowers (Figure 3-3, E). 95L7T7 (and 93E13T7 for AtMYB44), 220A8T7 (AtMYB4) and 130A10T7 had more or less constitutive expression patterns (Figure 3-3, A , B and G). In these, expression of AtMYB44 was higher in roots and rosette leaves, and expression of AtMYB4 was higher in roots, as compared to other organs. Expression of 135E3T7 and 1B4T7P (AtMYB3) was tested using both total R N A and poly(A) R N A on a northern blot. Neither was detectable using total R N A . Using poly(A) R N A , +  +  both had constitutive expression patterns in the tissues/organs tested, including 4-day old seedlings, bolting stems and rosette leaves (Figure 3-3, H). Among all the above AtMYB genes tested, none had an expression pattern that coordinated with that of At4CLl, which was most highly expressed in bolting stems among organs. Thus, none of these MYB genes were obvious candidates in my search for a regulator of the expression of At4CLl, or a set of genes encoding the enzymes in the lignin biosynthesis pathway.  4> 4>  F L L F ST  "P  ^  RT ST L F F L SL  UibOWll (AtMYBlb) 95L7T7 (A1MYB44)  rRNA rRNA  B  •• ^  RT ST L F F L SL  F L ST L F  15604T7(.4/A/}'«i6)  93E13T7(/Ort/}«-«)  1 1  r/JA.4 RT  ST  LF  FL  SL 220A8T7 (AtMYB4)  -p'  ' L F ST F L 130A10T7  99M15T7 (AlMYBSl)  crrn J  rRNA  r«A4  II -V ' ^ 5  L F ST F L  Total RNA &  RT  ST L F F L SL &  mRNA ST L F  99P9T7 (AtMYB75) IB4T7P (AlMYBS) rRNA  I35E3T7 ROC1  3.4 Cloning MYB cDNAs from Arabidopsis Bolting Stems  Using the EST clones available at the time, I did not find any AtMYB genes that were coordinately expressed with At4CLl. Since a characteristic of At4CLl was high expression in bolting stems, likely associated with lignin biosynthesis, I tried to clone new AtMYB cDNAs from bolting stems where they might be specifically or preferentially expressed, and potentially involved in regulating expression of At4CLl and other genes in the lignin biosynthetic pathway. Poly(A) R N A isolated from Arabidopsis bolting stems was used for reverse transcription +  to generate cDNAs. The 5' regions of cDNAs encoding the conserved M Y B DNA-binding domains were amplified by 5 ' - R A C E using a MYB degenerate primer. The predicted size for an amplified fragment corresponding to the R2R3-type M Y B DNA-domain was -400 bp. Several distinct PCR fragments ranging in size from 300 bp to 450 bp were observed by gel electrophoresis (data not shown). These fragments were tested on a Southern blot using a MYB cDNA probe. The Southern blot analysis showed hybridization signals from the fragments (data not shown), which were then cloned into T A vectors (Invitrogen). The cloned inserts were also tested on Southern blots. Nine out of 54 clones tested had inserts that hybridized to the MYB probe (data not shown). These 9 positive clones were sequenced. Seven of them had identical sequences corresponding to the conserved M Y B DNA-binding domain, and the longest sequence  Figure 3-3. Northern Blot Analysis of Expression Patterns of Arabidopsis MYB Genes. Total R N A was extracted from Col (except R N A on blot H from WS ecotype) seedlings and mature organs. 10 pg total R N A (except 20 pg on blot B) was loaded on each lane. Poly(A)  +  R N A was extracted from WS seedlings and selected mature organs. ~2 ug poly(A) R N A (blot +  H) was loaded on each mRNA lane. 2D-SD, 2-day old seedlings. 3D-SD, 3-day old seedlings. RT, 10-16 days old seedling roots. ST, bolting stems. L F , rosette leaves. F L , flowers. SL, siliques. For blots A and G, a ribosomal R N A probe was hybridized to the same blot as a loading control after the blot was striped. For blots B, C, D and E , ribosomal R N A bands were stained by ethidium bromide as a loading control. For blot H , ROC1 c D N A (encoding Arabidopsis cytosolic cyclophilin) was hybridized to the same blot as a loading control after the blot was striped.  was 414 bp in length (data not shown). This MYB sequence was not identical to any known MYB EST or gene at the time. The 3' region of the c D N A was amplified by 3 ' - R A C E using a gene-specific primer. An amplified fragment was cloned into the T A vector. Three clones were sequenced and had identical sequences. The full-length cDNA was generated by joining the 5'-clone and 3'-clone together. This gene was designated as STM31. The ORF encoded 320 amino acids (Figure 3-4). Later, this MYB gene was also identified by another group (Romero, et al., 1998). In that study this gene was systematically named as AtMYB73 (At4g37260). The other two positive clones identified on the Southern blot had sequences identical to the known EST clone 1B4T7P in the M Y B DNA-binding domain (data not shown). Thus 3' cloning for this gene was not carried out. The expression pattern of STM31 was examined by a northern blot analysis. The analysis showed that STM31 had a constitutive expression pattern in various tissues/organs tested, including 2-day and 3-day old seedlings, seedling roots, bolting stems, rosette leaves, flowers and siliques (Figure 3-5). Expression of the EST clone 1B4T7P, identical to the second clone identified from the 5'-RACE, is shown in Figure 3-3-H. Thus, these two MYB genes cloned as cDNAs from Arabidopsis bolting stems did not have expression patterns similar to At4CLl, making it unclear whether they could be involved in regulating At4CLl expression.  A TTTCTTCCCA  ACACACACAA  AAAAAAAAAC  TGTAACGACC  AAAAAAAAAG  AGGACGCGTT  AGAAGAAAAA  AAAAGAACAA  TCCGATAAAA  AGATCCGGCG  ISHTCAAACC  CGACCCGTAA  GAATATGGAG  AGGATTAAAG  GTCCATGGAG  TCCAGAAGAA  GATGATCTGT  TGCAGAGGCT  TGTTCAGAAA  CATGGTCCGA  GGAACTGGTC  TTTGATTAGC  AAATCAATCC  CTGGACGTTC  CGGCAAATCT  TGTCGTCTCC  GGTGGTGTAA  CCAGCTATCT  CCGGAGGTAG  AGCACCGTGC  TTTTTCGCAG  GAAGAAGACG  AGACGATTAT  TCGAGCTCAC  GCTCGGTTTG  GTAACAAGTG  GGCTACGATC  TCTCGTCTTC  TCAATGGACG  AACCGATAAC  GCTATCAAGA  ATCATTGGAA  CTCGACGCTG  AAGCGAAAAT  GCAGCGTCGA  AGGGCAAAGT  TGTGATTTTG  GTGGTAATGG  AGGGTATGAT  GGTAATTTAG  GAGAAGAGCA  ACCGTTGAAA  CGTACGGCGA  GTGGTGGTGG  TGGTGTCTCG  ACTGGCTTGT  ATATGAGTCC  CGGAAGTCCA  TCGGGATCTG  ACGTCAGCGA  GCAATCTAGT  GGTGGTGCAC  ACGTGTTTAA  ACCAACGGTT  AGATCTGAGG  TTACAGCGTC  ATCGTCTGGT  GAAGATCCTC  CAACTTATCT  TAGTTTGTCT  CTTCCTTGGA  CTGACGAGAC  GGTTCGAGTC  AACGAGCCGG  TTCAACTTAA  CCAGAATACG  GTTATGGACG  GTGGTTATAC  GGCGGAGCTG  TTTCCGGTTA  GAAAGGAAGA  GCAAGTGGAA  GTAGAAGAAG  AAGAAGCGAA  GGGGATATCT  GGTGGATTCG  GTGGTGAGTT  CATGACGGTG  GTTCAGGAGA  TGATAAGGAC  GGAGGTGAGG  AGTTACATGG  CGGATTTACA  GCGAGGAAAC  GTCGGTGGTA  GTAGTTCTGG  CGGCGGAGGT  GGCGGTTCGT  GTATGCCACA  AAGTGTAAAC  AGCCGTCGTG  TTGGGTTTAG  AGAGTTTATA  GTGAACCAAA  TCGGAATTGG  GAAGATGGAG  HSBAGTGATA AGAACAAGTT  TTCCCTCTGT  TTCTCGGGAA  AATAAAAGTT  TCAGGCTTTT  CATGTATAGA  GCCAGTAGTA  GGGACGACGA  AAGGAGGAAG  CGAAATCAAA  AAATGTTTTG  TTATCATCAT  CATATGATCT  TCTTTCGTGA  TTCAATTAAA  AGCAAAGCAA  GATCACTTAT  TAAAAAAAAA  AAAAAAAA  12  58  B RIKGPWSPEE DDLLQRLVQK HGPRNWSLIS KSIPGRSGKS PEVEHRAFSQ EEDETIXRAii ARFGNKWATI SRLLHGRTDN AÏKNHWNSTJÙ  MSNPTRKNME  CRLRWCNQLS KRKCSVEGQS  CDFGGNGGYD  GNLGEEQPLK  RTASGGGGVS. TGLYMSPGSP  SGSDVSEQSS  RSEVTASSSG  EDPPTYLSLS  LPWTDETVRV  NEPVQLNQNT  VMDGGYTAEL  GGAHVFKPTV FPVRKEEQVE  VEEEEAKGIS  GGFGGEFMTV  VQEMIRTEVR  SYMADLQRGN  VGGSSSGGGG  GGSCMPQSVN  SRRVGFREFI  VNQIGIGKME  *  Figure 3-4. Nucleotide and Deduced Amino Acid Sequences of Arabidopsis MYB STM31 (AtMYB73). (A) The D N A sequence. Shaded nucleotides indicate the putative translation start and stop sites. (B) The deduced amino acid sequence. Grey amino acids, the dark grey (R2) and the light grey (R3) indicate the conserved M Y B binding domain.  V>  T  RT  ST  LE  FL  SL  STM31  rRNA  Figure 3-5. Northern Blot Analysis of  ./Expression.  Total R N A was extracted from Col seedlings and mature organs. 10 fig total R N A was loaded on each lane. 2D-SD, 2-day old seedlings. 3D-SD, 3-day old seedlings. RT, 10-day old seedling roots. ST, bolting stems. L F , rosette leaves. FL, flowers. SL, siliques. A ribosomal R N A probe was hybridized to the same blot as a loading control after the blot was striped.  3.5 Discussion  Strikingly, among various organs, At4CLl is strongly expressed in the bolting stems but not At4CL2. This data plus enzyme activity analyses (Ehlting, et al., 1999), suggest that although At4CLl and At4CL2 share a high degree of similarity, At4CLl is the only member in the Arabidopsis 4CL gene family that plays a primary role in phenylpropanoid metabolism in bolting stems, a major site for lignin synthesis. Both genes also have moderate expression in seedling roots. This result suggests that At4CLl and At4CL2 might have overlapping functions in the root, which may reflect an evolutionary adaptation of roots for response to stress. Various phenolic compounds derived from the phenylpropanoid pathway appear to play an important role in the  plant defense response (Dixon and Paiva, 1995). Both At4CLl and At4CL2 expression are induced by pathogen infection, wounding and U V irradiation (Lee, et al., 1995; Ehlting, et al., 1999). The protein encoded by the 4CL-like EST clone 139B10T7 is only partially conserved in the two motifs present in 4 C L enzymes. Thus it was not surprising to see that this 4CL-like protein did not have 4 C L enzyme activity (Samaeian and Douglas, unpublished data). What biochemical function this 4CL-like protein has needs to be tested further, but is not apparent from its expression pattern. Of thel25 Arabidopsis R2R3-MYB genes, I examined expression of 19 genes by northern blot analyses (plus one by RT-PCR, Chapter 5). These represent 16% of the members of the Arabidopsis R2R3-MF5 gene family. These genes are scattered in different phylogenetic subgroups. For 13 out of 21 MYB genes examined here, expression was not detectable using total R N A on northern blots. This result indicates that AtMYB transcripts generally accumulate at a low level. It also should be noted that for most of the genes, the expression was not examined in roots and siliques. The reason for this was that in my screen for AtMYB genes involved in the regulation of the phenylpropanoid pathway, the bolting stem was our primary target. Thus, it can not be excluded that some of these AtMYB genes are specifically or preferentially expressed in roots or siliques, such as AtMYB68 and AtMYB36. Another possibility for not seeing the expression is that the transcription of some of the genes may be induced under specific conditions. These conditions were not included in my assays. Three years after I started this project, expression patterns of Arabidopsis MYB genes were systematically tested by 'reverse northern' analyses (Kranz, et al., 1998). Most of my gene expression data matches their results with minor exceptions. Currently, examination of gene expression profiles on a large scale can be much more easily achieved by using the microarray technique. However, either reverse northern or RNA-array data should be interpreted with caution, since in these techniques, sizes of transcripts are not included and therefore the problem of cross-hybridization to other genes can be involved (Kranz, et al., 1998). Recently, expression  patterns of over 95% of Arabidopsis genes have been examined by using the MPSS (Massively Parallel Signature Sequencing) technique, a more precise and sensitive way to quantify gene expression (Brenner et al., 2000a and 2000b; http://mpss.ucdavis.edu). M y expression data also matches MPSS data. For some of the MYB genes, expression was not detectable in my study, it was also not detectable or detected at a very low level by MPSS. Some of the MYB genes, examined for the expression pattern in this study, have also been analyzed in other groups later. In particular, AtMYB4 has been found to negatively regulate the biosynthesis of sinapate ester, a U V protector in Arabidopsis leaves (Jin et al., 2000). M y data and MPSS data revealed that this gene has a more or less constitutive expression pattern. Expression of both AtMYB90 and AtMYB75 are not detectable by MPSS. In my experiment, the AtMYB75 transcript was only detected at a very low level in 2-day old seedlings. It is interesting to see that overexpressing these two genes with such a low expression level, results in dramatic increase of a set of phenylpropanoid products (Borevitz et al., 2000). Expression of AtMYB44 has also been found to increase at late stages of embryogenesis, and A t M Y B 4 4 maybe related to embryogenesis (Kirik, et al., 1998). Here I described one approach to identify a regulatory gene, by searching for coordinate expression with a target gene. In some cases, this approach could work because gene function can be reflected from its expression pattern. AtMYB genes TT2 and AtMYBôl are such examples. Both are involved in different aspects of seed development. Accordingly, the expression is seed specific or preferential (Nesi et al., 2001; Penfield et al., 2001). However, this is not always necessary true. Studies of animal and plant M Y B transcription factors, such as the maize C I , revealed that the activity of some M Y B factors requires interactions with partner proteins (Weston, 1998; Goff et al., 1992). In this case, expression of one of the regulatory genes may not be similar to that of a target gene, since the combination of transcription factors leads to gene regulation. This could be the case in regulation of gene expression in the phenylpropanoid pathway. Thus other ways to identify a MYB gene involved in the regulation of the phenylpropanoid pathway should be considered.  Another approach used in this study was to clone MYB cDNAs preferentially expressed in the bolting stem, where At4CLl was most highly expressed. However, neither cDNA identified was preferentially expressed in this organ. Thus, like the other AtMYB genes analyzed in this study, there were no obvious candidates for genes regulating gene expression in the lignin biosynthetic pathway. In addition, interesting results were obtained with another AtMYB gene (described in Chapter 4 and 5), therefore I did not study these genes further. Now we know there are over 130 MYB genes in Arabidopsis (Stracke, et al., 2001). However, using the R A C E method, I only obtained two clones from the bolting stem, obviously missing some. The problem could be caused by the MYB degenerate primer used in the 5'R A C E . Although the primer was designed corresponding to the highly conserved amino acid stretch in the M Y B R3 repeat, it may not have been able to amplify all the members of such a big gene family. On the other hand, this result also reflected low expression of AtMYB genes in general. Another study in which Arabidopsis MYB genes were systematically identified by a PCR-based strategy revealed the same thing. More than twice as many genes were amplified from a genomic library than from cDNA libraries despite different combinations of several degenerate primers used (Romero et al., 1998).  Chapter 4 AtMYB68: a Root Preferentially Expressed MYB Gene — Expression Profile Studies  4.1 Introduction  Plant roots are adapted for growing through soil and absorbing water and mineral nutrients. Roots grow and develop from their distal ends. The external morphology of the root tip can be divided into four distinct but overlapping zones: the root cap, the meristematic zone, the elongation zone and the maturation zone (Figure 4-1, A). The meristematic zone is characterized by small cells overlaid by the root cap. The elongation zone is the site of rapid and extensive cell elongation. Both the meristematic zone and the elongation zone are about a quarter of a millimeter long in the Arabidopsis root. The maturation zone is the region in which the elongated cells acquire their differentiated characteristics. Differentiation may begin earlier, but the cells do not achieve the mature state until they reach this zone (Dolan et al., 1993; Taiz and Zeiger, 2002) Transverse sections reveal that the Arabidopsis root has a simple and radial organization. A typical section from the mature region of the root is composed of the central vascular core surrounded by four radial cell layers (Figure 4-1, B). From the outside, they are the epidermis, cortex, endodermis and pericycle. Each cell type is present in a single layer. In Arabidopsis, there are usually 8 and 12 cells in the endodermis and pericycle respectively. The central vascular core is composed of the phloem and xylem. The xylem axis consists of two types of cells, the protoxylem cells at two ends and the central metaxylem cells. Two poles of the protoxylem cells are adjacent to pericycle cells (Dolan et al., 1993; Cano-Delgado et al., 2000). Longitudinally, each cell type forms a vertical file of cells that can be traced to meristematic initials in the root apical meristem. There are four sets of meristematic initials in Arabidopsis roots. Respectively, these give rise to the epidermis and the lateral root-cap, the columella root cap, the cortex and endodermis, and the stele tissues including the pericycle and  vascular tissues (Dolan et al., 1993). These initials appear to be established during embryogenesis (Scheres et al., 1994).  Figure 4-1. Schematic View of Root Anatomy. (A) Stylized typical root longitudinal section (from Plant Physiology (Taiz and Zeiger), copyright © 2002 Sinauer). (B) Arabidopsis root cross-section. Ep, Epidermis. C, Cortex. En, Endodermis. Pe, Pericycle. X , Xylem. Ph, Phloem (from D i Laurenzio et al., copyright © 1996 Cell).  The elegant simplicity of the Arabidopsis root anatomy and its clear cell lineages make it a useful tool for developmental study with the aid of mutant screening. One class of root mutations in Arabidopsis causes radial pattern defects. An example of such a mutation is wooden leg (wol), which has highly reduced root growth as compared to the wild type. The root of the wooden leg mutant has a reduced cell number in the vascular tissue and all these vascular cells differentiate into protoxylem, resulting in the vascular cylinder lacking phloem and consisting of  xylem only (Scheres et al., 1995). The WOL gene encodes a putative two component histidine kinase. The wol-1 allele contains a single point mutation converting T  278  to I  278  in the W O L  protein, resulting in a non-conservative amino acid change in the conserved putative extracellular receptor domain. It is postulated that the WOL gene product is a receptor molecule that controls the asymmetric cell divisions of the vascular initials through a certain signal transduction pathway (Mahonen et al., 2000). Lateral root development provides an interesting example of postembryonic organogenesis in higher plants. In angiosperms, lateral roots are derived from the differentiated pericycle layer of a parent root (McCully, 1975). Their formation involves several key stages (Malamy and Benfey, 1997). First, the mature pericycle cells are stimulated and dedifferentiated. Interestingly, not all pericycle cells can become founder cells for lateral roots. Lateral roots arise only from files of pericycle cells adjacent to the two xylem poles (Steeves and Sussex, 1989; Charlton, 1991). It is not known how this bipolar pattern is established. The initiation of lateral roots occurs some distance away from the root apical meristem. There is a consistent relationship between the position of the first founder cell division and the root apex (Casimiro et al., 2001). Second, ordered cell divisions and cell differentiation occur to generate a highly organized lateral root primordium. This process is initiated from a pair of adjacent xylem pole pericycle cells in a single file that undergo several transverse divisions, followed by a periclinal division to give rise to two cell layers. This step represents the critical event during the onset of lateral root formation. Third, the lateral root primordium grows through the overlying cell layers and eventually emerges from the parent root. This process largely depends on cell expansion. In Arabidopsis, emergence of lateral roots is observed -20 mm from the primary root apex (Casimiro et al., 2001). Fourth, an active lateral root meristem allows continued lateral root growth. The plant hormone auxin is a key regulator of lateral root development. It is known that exogenous auxin induces lateral root primordium formation, but inhibits root elongation (Evans et al., 1994). Several auxin-related lateral root mutants have been isolated in Arabidopsis. In  these mutants, lateral root development was arrested at different stages. This suggests that indole-3-acetic acid (IAA) is required at several stages of lateral root development (Celenza et al., 1995). It is also proposed that I A A is initially required to establish a population of rapidly dividing pericycle cells and that the subsequent derivatives form a hormone-autonomous meristem (Laskowski et al., 1995). Auxin polar transport plays an important role in lateral root formation. It has been demonstrated that blocking auxin basipetal transport (from the shoot apex toward the base) results in a reduced lateral root number in Arabidopsis seedlings (Reed et al., 1998). Another experiment shows that treating the Arabidopsis root with the auxin polar transport inhibitor arrests lateral root development at the initiation stage. Endogenous I A A is found to accumulate in the root apex while reducing in the upper zone where lateral roots initiate. This result suggests that the initiation of the xylem pole pericycle cells for lateral root formation requires a threshold concentration of auxin (Casimiro et al., 2001). Root development including root branching is also largely depending on nutrient supply. One striking example is that many plant species respond to an uneven distribution of nutrients (e.g. N0 ", N H 3  + 4  or inorganic phosphate) by developing their lateral roots preferentially within  nutrient-rich zones (Robinson, 1994; Leyser and Fitter, 1998). This is believed to be important for a plant to compete for limiting resources (Hutchings and de Kroon, 1994). However, the mechanisms are poorly understood. Studies on N 0 " have shown that N 0 " supply modulates 3  3  lateral root growth with two opposite effects. Low concentrations of N 0 " locally stimulate 3  lateral root growth, but at high concentrations it has an inhibitory effect on lateral root growth (Ericsson, 1995; Zhang et al., 1999). Two distinct signal pathways have been proposed. The stimulatory effect is localized to the lateral roots directly exposed to N0 ". It acts through an 3  N0 " specific pathway. The Arabidopsis ANR1 gene, encoding a MADS-box transcription factor, 3  was identified in this pathway (Zhang and Forde, 1998). In contrast, the inhibitory effect is systematic. It more likely depends on the plant N status. N sources other than N0 " produce a 3  response, and this pathway might be mediated by the N/C ratio (Zhang et al., 1999).  Using a reverse genetic approach, a systematic search for knockout mutations in Arabidopsis MYB genes was carried out seven years ago (Pri-Hadash and Douglas, unpublished data). Screens of DuPont and Feldmann T-DNA collections for a total of 14,100 T-DNA lines were performed by PCR using a MYB degenerate primer corresponding to the conserved M Y B binding domain. One T - D N A line with the insertion 536 bp upstream of the putative A T G translation start codon of AtMYB68 (At5g65790) was identified from the Feldmann collection (Figure 4-2). This line was backcrossed two times to the wild type WS ecotype. Since this line had the insertion fairly close to the A T G codon in the 5' promoter region, there was high possibility that this insertion might alter the gene transcription. As well, AtMYB68 had not been functionally studied except that the EST clone had been identified (ABRC). Based on these reasons, it was chosen for further studies. While the subsequent analysis of this AtMYB68 TD N A line (T-14) showed that AtMYB68 expression was not affected (Chapter 5), my preliminary data revealed a root-dominant expression pattern of this gene. The simplicity of Arabidopsis root cell organization provides a good system to study gene expression and function during development. This chapter describes studies on expression of AtMYB68, as the first step towards the goal of determining gene functions.  T-DNA LB Intron 1  TTGTTAX  536 bp  \  R21  Intron 2  \ i '  Figure 4-2. Schematic of a T-DNA Insertion within AtMYB68 in Line T-14. The sequence with a question mark indicates new nucleotides found at the T-DNA-AfMrTifjo  5  junction. The sequence underlined indicates re-arrangement in AtMYB68 at the junction. The dark and light grey boxes indicate the coding region for the two conserved M Y B repeats R2, R3. The hatched box indicated the conserved 5' region. The open box indicates the non-conserved region.  4.2 Organ and Cell-Type Specific Expression of AtMYB68  The organ-specific expression of AtMYB68 was examined by a northern blot analysis. The analysis of total R N A isolated from several Arabidopsis organs and seedlings showed that the AtMYB68 transcript accumulated at a low level in roots, and was also detectable in 4-day old seedlings. It was not detectable in the other organs examined, including bolting stems, rosette leaves, flowers and siliques (Figure 4-3, left panel). Poly(A) R N A isolated from selected +  samples was also prepared on the same blot. The analysis of poly(A) R N A showed that again, +  the AtMYB68 transcript was readily detectable from the 4-day old seedlings, but was still not detectable from either bolting stems or rosette leaves (Figure 4-3, right panel). These northern results were later confirmed by a RT-PCR analysis (Chapter 5). The lower expression of AtMYB68 in the whole seedlings (containing both shoots and roots) could be due to AtMYB68 expression solely contributed in the seedling root, or due to the expression in the shoot as well. This was further tested using quantitative RT-PCR, a more sensitive method than a northern blot. Fourteen-day old Arabidopsis seedlings were separated as shoots (aerial part including hypocotyls) and roots. Total R N A isolated from these two separated parts was subjected to reverse transcription followed by PCR amplification. The quantitative RTPCR analysis revealed that AtMYB68 was expressed at a very low level in the shoot, a level about 20-fold lower than that in the root (Figure 4-4). To determine the spatial and temporal, as well as cell-type specific expression pattern of AtMYB68, the 3-glucuronidase (GUS) reporter gene was fused behind the AtMYB68 promoter. GUS expression would represent where the AtMYB68 promoter is active. A 1.9 kb AtMYB68 promoter fragment including the A T G translation start codon was obtained by PCR amplification from Arabidopsis Col genomic D N A . This PCR product was sequenced and no PCR error was found in the fragment when compared to the Col genome sequence (data not shown). The AtMYB68::GUS translational fusion was generated by inserting the AtMYB68 promoter fragment  Total R N A  m RNA  AtMYBÔX  ROCI  Figure 4-3. Northern Blot Analysis of AîMYB68 Expression in Arabidopsis. Total R N A and poly(A) were extracted from seedlings and mature organs of Arabidopsis WS +  ecotype, except that roots were from 16-day old hydroponically grown seedlings. Leaves contained rosette leaves only. The left panel of the blot was loaded with 10 ug total R N A per lane. The right panel was loaded with ~2 ug poly(A) R N A per lane. The ROCI +  cDNA  (encoding Arabidopsis cytosolic cyclophilin) was hybridized to the same blot as a loading control after the blot was stripped.  SH R T  i  J  AMYB68  I US rRNA  Figure 4-4. RT-PCR Analysis of AtMYB68 Expression in Arabidopsis Seedlings. Reverse transcription followed by PCR amplification was performed against total R N A . Total R N A was extracted from shoots and roots of 14-day old hydroponically grown seedlings. 18S ribosome R N A was used as an internal control. SH, shoot. RT, root.  into the pBI121 binary vector (CLONTECH) in place of the 35S promoter upstream of the GUS gene (Figure 2-2-E). The NPTII gene in the pBI121 vector provided kanamycin resistance as a selectable marker. After transformation into Agrobacterium, the AtMYB68::GUS construct was transformed into Arabidopsis Col plants by the Agrobacterium-mediated floral dip method. Tl-generation transformants were selected on kanamycin plates. The transformation rate was -0.4% (11 out of 3,000 seeds with kanamycin resistance). The kanamycin-resistant T l plants were allowed to set seeds to generate the T2-generation. GUS activity was examined in the T2 generation. Among 4 independent transgenic lines examined, three lines had the same GUS expression pattern, in which the root was a major site of GUS expression (the other line had strong GUS expression in the hypocotyl). One of the lines had strong GUS expression and contained a single locus with the AtMYB68::GUS cassette, indicated by -3:1 segregation ratio of Kan :Kan in the T2-generation R  s  (data not shown). This line was selected to generate the homozygous line, designated 12-2G, for further tests. GUS expression driven by the AtMYB68 promoter was first tested during seedling development. After seeds germinated on plates for 1-2 days, the radicals emerged from seed coats. At this stage, weak GUS staining was visible at and below the shoot-root junction area, apparently in the median stele tissue (Figure 4-5, A). At 3-4 days old, seedlings had two opened green cotyledons. In these seedlings, strong GUS staining was observed through the entire root except the root tip, within the stele tissue. It extended slightly into the hypocotyl (Figure 4-5, B). Two strong staining files adjacent to the xylem vessels were often observed in the upper part of the root (Figure 4-5, E). Close examination of the shoot revealed that GUS staining was also consistently present in the stomata guard cells of the cotyledons (Figure 4-5, C and D). Stained Col wild-type seedlings at the same age were also examined as a control, but no GUS staining was found anywhere in these seedlings (Figure 4-5, F and G). When the seedling was 7-8 days old, lateral roots were emerging from the primary root. At this stage, GUS staining was seen to decline in the old part of the primary root but still  retained in the young part (as shown in the 2-week old seedling, Figure 4-5, H). As lateral roots were developing, the same GUS staining pattern as in the primary root was repeated in the lateral root (Figure 4-5, H). After emergence of true leaves, GUS staining was observed in the guard cells of young leaves but was absent in the cotyledons and old leaves (data not shown). Strong staining was also seen at the petiole bases of expanding leaves when the seedling reached about 2-week old (Figure 4-5,1). GUS activity was also tested in the mature plants. No staining was observed in any mature organs including rosette leaves, bolting stems, flowers and siliques, except in roots (data not shown). To further determine the cellular specificity of AtMYB68 expression, 2-3 days old GUS stained seedlings of Iinel2-2G were embedded in Spurr resin and subjected to sectioning using a microtome. The serial root cross-sections revealed that in general GUS staining was present in the vascular, pericycle and some endodermal cells with a specific expression pattern (Figure 46). The staining was light in the section closest to the root tip (Figure 4-6, A). As the sectioning continued toward the older part of the root, it became obvious that the staining was higher in the cells near the two xylem poles (Figure 4-6, B and C). In the section from the upper part of the root, it was clearly shown that the staining became more restricted to the pericycle cells adjacent to the xylem poles (Figure 4-6, C). The endodermal cells adjacent to xylem side pericycle cells had some spotty staining. This was probably due to the weak staining in these large cells. The staining in the vascular parenchyma cells was generally light. At the root-hypocotyl transition area, the staining was mostly restricted to the two pairs of pericycle cells adjacent to the xylem poles (Figure 4-6, D).  Figure 4-5. Whole-Mount Histochemical Staining of GUS Driven by the AtMYB68 Promoter during Seedling Development in Line 12-2G. GUS staining is shown as a blue color (dark area on the pictures). (A) 1-2 days old seedling. (B) 3-4 days old seedling. (C) Magnification of one of the cotyledons in (B). (D) Higher magnification of (C). (E) Upper part of the root from the 2-3 days old seedling. (F) GUS stained 3-4 days old Col wild type seedling. (G) Magnification of one of the cotyledons in (F). (H) 14-day old seedling. (I) Magnification of the petiole base region in (H).  A  Figure 4-6. Root Cross-Sections of the AtMYB68::GUS Line 12-2G. GUS staining is shown as a blue color (dark area on the pictures). The root was from the 2-3 days old seedling. Sections A to D represent the young part of the root to the root-hypocotyl junction. Ep, Epidermis. C, Cortex. En, Endodermis. Pe, Pericycle. X , Xylem. Ph, Phloem.  4.3 Expression of AtMYB68 in the woodenleg (wol) Mutant Background  The histochemical analysis of root cross-sections revealed that expression of AtMYB68::GUS was higher in the pericycle cells adjacent to the xylem poles in the old part of the root. This cell type-specific pattern led to these questions: What determines this cell typespecific pattern? Do signals from the xylem specify the expression in the xylem pole pericycle cells? The Arabidopsis root mutant woodenleg (wol) provides a useful tool to test this hypothesis. In the wol mutant, the vascular cylinder in the primary root contains only xylem, lacking phloem (Scheres et al., 1995). By expressing AtMYB68::GUS in this mutant background, we would be able to see if the cell type-specific expression of AtMYB68::GUS is altered by the presence of the vascular tissue containing only xylem, without the symmetry of xylem poles. The AtMYB68::GUS construct was introduced into the wol mutant by crossing the AtMYB68::GUS homozygous line 12-2G to the wol-1 homozygous line. The heterozygous F l generation plants set seeds to give rise to the F2-generation. In the F2 generation, about one quarter of seedlings (42 out of 190 examined) had the wol phenotype, characterized by dramatically reduced root growth. In each mutant and wild type sibling population, about 3 quarters of seedlings (31 out of 41 in the mutants and 11 out of 17 in wild-type siblings) had GUS activity, indicating that the GUS transgene and WOL segregate independently. When seedlings were 7-day old, it was easily seen that the wol mutants had much shorter primary roots than wild type siblings (Figure 4-7, A and B). In the mutant seedling, GUS staining was generally in the same pattern as in the wild-type sibling used as a control (Figure 47, compare A and B). The staining was seen in the stele tissue through the entire root except the root tip and slightly extended to the hypocotyl. In the wol background, the staining appeared stronger in the region below the root-hypocotyl junction and the region just basal to the root tip (Figure 4-7, A). In the 15-day old wol seedling, adventitious roots had formed (Figure 4-7, C). The primary root of the wol mutant is not able to produce normal lateral roots, and such adventitious roots come from the hypocotyl. In contrast to the wol primary root, the secondary and later roots have normal vascular tissues (M.-T. Hauser, personal communication). In the wol 61  seedling, strong GUS staining was also present in the secondary roots similarly as in the primary  root. As previously observed, the staining decreased in the old part of the primary root as it grew longer (Figure 4-7, C).  Figure 4-7. Whole-Mount Histochemical Staining of GUS Driven by the AtMYB68 Promoter in the woodenleg Mutant Background. GUS staining is shown as a blue color (dark area on the pictures). (A) 7-day old woodenleg seedling containing the AtMYB68::GUS fusion. (B) 7-day old wild type sibling. (C) 15-day old woodenleg seedling containing the AtMYB68::GUS fusion. PR, primary root. SR, secondary root.  To test if the cell type-specific expression of AtMYB68::GUS was altered in the wol mutant background, it was necessary to look at the expression at the cellular level. Seven-day old wol seedlings harboring the AtMYB68::GUS fusion were stained, and then embedded in Spunresin. These seedlings were subjected to sectioning using a microtome. The serial sections of the wol primary root revealed that the expression pattern of AtMYB68::GUS was much different from that in the wild type root. In the region just basal to the root tip, the staining was stronger than that in the wild type. Strong GUS staining was observed in all pericycle cells, and eventually extended into the whole endodermal layer as well (Figure 4-8, A and B). It was interesting to see that in the youngest section, GUS staining was also clearly present in the nonlignified xylem parenchyma cells within the vascular tissue (Figure 4-8, A). In the central region of the root, the staining was initially present in both the pericycle and endodermis, but later disappeared from the endodermis and became specific to the pericycle, where it was expressed in all the cells (Figure 4-8, C). At the root-hypocotyl transition region, strong GUS staining in the entire layer of the pericycle was maintained, and was evident again in the endodermal layer (Figure 4-8, D). Taken together, these results show that in the wol mutant background, the expression of AtMYB68::GUS was strengthened in the entire layer of pericycle cells and expanded to the endodermal layer, and was no longer restricted to the pericycle cells near the xylem poles. This novel expression pattern correlates with the over production of xylem and loss of symmetry (xylem poles) in the wol vascular tissue. The result suggests that a positive signal from the xylem (or a negative signal from the phloem) may be responsible for the cell typespecific expression of AtMYB68::GUS.  I)  A  Figure 4-8. Root Cross-Sections of the woodenleg Seedling Containing the AtMYB68::GUS fusion. GUS staining was shown as blue color (dark area on the pictures). The root was from 7 day-old seedling. Sections A to D represent the young part of the root to the root-hypocotyl junction. Ep, Epidermis. C, Cortex. En, Endodermis. Pe, Pericycle. X , Xylem. Ph, Phloem.  4.4 Expression of AtMYB68 in Response to External Stimuli  The pericycle cells, where GUS activity driven by the AtMYB68 promoter was higher, have a specific function — they serve as founder cells for lateral root formation (Steeves and Sussex, 1989; Charlton, 1991). This led to the question: Is AtMYB68 related to lateral root formation? To address this question, expression of AtMYB68 in response to N0 " and auxin, 3  two key regulators of lateral root formation, was tested. Meanwhile, AtMYB68 in response to other major plant hormones was also tested, since plant hormones are important signals in many aspects of plant growth and development. As a first step in functional studies of AtMYB68, these tests might give clues to its functions. The quantitative RT-PCR method was used in examination of AtMYB68 expression under various external stimuli. Total R N A extracted from whole seedlings was used for reverse transcription followed by PCR amplification with AtMYB68 specific primers. 18S rRNA primers were also added in the same RT-PCR reaction as an internal control (details described in Chapter 2). The ratio of product intensity of AtMYB68 to 18S rRNA represented the gene relative expression level. As a preliminary experiment, only one typical concentration and treating time were used for each treatment based on those in the literature. For the N 0 " treatment, four-day old Col seedlings grown without N0 " were treated with 3  3  2 m M K N 0 for 6 hours. The RT-PCR analysis showed that the level of the AtMYB68 transcript 3  remained stable when seedlings were treated with N0 " (Figure 4-9, A). 3  For the plant hormone treatments, eight-day old Col seedlings were treated with I A A (a natural auxin), B A P (a synthetic cytokinin), G A (gibberellic acid), A B A (abscisic acid) or A C C 3  (a precursor of ethylene) for 6 hours. Except IAA, used at 10 p M , the other hormones were used at 100 p M in the media. RT-PCR analyses showed that accumulation of the AtMYB68 transcript was down-regulated 2-fold by the I A A treatment, and up-regulated 3-fold by the A B A treatment, as compared to the untreated control respectively. With the other hormone treatments (BAP, G A  3  or ACC), accumulation of the AtMYB68 transcript remained at a stable level (Figure 4-9, B). These results were repeated in two independent experiments.  -N  +N  AtMYB68  LJ  18S rRNA  C/3  '  ta^ 0 8  ^  0.689  0.634  I  °  -N  I Treatment  +N  B con IAA BAP G A3 ABA ACC  AtMYB68  ij ^tmmiJ  ^Km*mi ^SéjlJ'  W*»-^  ^ËHm**^  18S rRNA  5.57  52  I  s  3  1.74  2  _iir  1  9  1  1.39  0.781 con  IAA  BAP  G A3  Treatment  ABA  ACC  con NaCl cold desiccation  V«J  U-J  LJ  i  AtMYB68  18S rRNA  1.80 on 00  1  8  1.6  1  1.56  s o.:i ^ ^  0.6 0.4  o.z  con  NaCl  cold  desiccation  Treatment  Figure 4-9. RT-PCR Analysis of AtMYB68 Expression in Response to External Stimuli. Reverse transcription followed by PCR amplification was performed against total R N A . Total R N A was extracted from whole seedlings after treatments. 18S ribosome R N A was used as an internal control. The ratio of the intensity of AtMYB68 to 18S rRNA represented the relative AtMYB68 mRNA level. The ratios were visualized by bar graphs in the lower panel and values are shown above each bar. RT-PCR reactions were performed at least twice for each treatment to assess reproducibility in quantification. The results were consistent. One of them is shown here. (A) 4-day old seedlings grown hydroponically in absence of NO," were treated with 2mM KNO-, for 6 hours (+N). - N , untreated sample. (B) 8-day old seedlings grown hydroponically were treated with various plant hormones for 6 hours. The concentration of hormones was 100 p M except I A A in 10 p M . The control was in the medium without adding hormones (con). (C) 7-day old seedlings grown hydroponically were treated with abiotic stresses. The seedlings were treated with 200 m M NaCl for 6 hours (NaCl), put in 4°C for 24 hours (cold), or left on Whatman paper on the lab bench for 30 minutes (desiccation), respectively. The control was grown under normal conditions (con).  The inducible expression of AtMYB68 by A B A suggested that AtMYB68 might be related to stress responses, since A B A plays primary regulatory roles in plant response to stress. To test this speculation, expression of AtMYB68 in response to abiotic stresses, in particular, desiccation, cold, and salinity conditions was examined further. For the cold treatment, seven-day old Col seedlings were transferred to 4°C for 24 hours. For the desiccation treatment, the seedlings from the same batch were put on Whatman paper for 30 minutes to induce dehydration. For the salinity treatment, the seedlings from same batch were treated with 200 m M NaCl for 6 hours. RT-PCR analyses showed that the level of the AtMYB68 transcript was down-regulated 2-3 fold by the cold and the desiccation treatments respectively, as compared to the untreated control. The salinity treatment did not significantly change the level of the AtMYB68 transcript (Figure 49, C).  4.5 Expressing A t M Y B 6 8 Recombinant Protein and Raising A t M Y B 6 8 Antiserum  To verify that the cell type-specific expression of AtMYB68::GUS represents native AtMYB68 expression, the in situ hybridization technique was used. Unfortunately, with paraffin sectioning, sections with intact root morphology beyond the root tip region could not be obtained. Other sectioning methodologies that could preserve mature root anatomy were then considered. L R Gold™ plastic sectioning has been successfully used in protein immunolocalization (Samuels et al., 2002). This encouraged me to use this embedding technique combined with immunolocalization to detect localization of native gene expression. In order to do protein immunolocalization for AtMYB68, an AtMYB68 antibody was needed. This antibody would also be useful to detect protein expression levels in gene over-expression lines in functional studies. To raise an AtMYB68-specific antibody, a truncated AtMYB68 recombinant protein was produced in E. coli. To avoid producing antibodies that might cross-react with the conserved M Y B domain, the 3' region of AtMYB68 cDNA, derived from the full-length cDNA clone, was  fused in-frame to the pQE-30 expression vector (QIAexpressionist™). This placed an additional 24 amino acids, including a 6xhistidine-tag, prior to the amino-terminus of the truncated AtMYB68 (Figure 2-2-D). The 6xhistidine-tag would be useful for the recombinant protein purification. This plasmid was transformed into E. coli, and a high level of the recombinant protein was produced upon IPTG induction. The recombinant protein was purified through N i N T A resin. The predicted size of the AtMYB68 recombinant protein was 31.4 kDa. Figure 4-10 shows expression of the AtMYB68 recombinant protein in E. coli and the purification process. The cell lysate from the E. coli strain containing the empty vector was used as a control (lanes 1 and 2). Upon 1 m M IPTG induction, in the total lysate from the E. coli strain containing the AtMYB68 c D N A insert, a product of about the predicted size was clearly seen (indicated by arrow in lane 4 (protein extracted at a small-scale), and lane 5 (protein extracted at a large-scale)). The E. coli cell lysate was passed through a N i - N T A column for purification. The recombinant protein bound to the column and absent in the flow-through fraction (lane 6), and in the wash fraction (lane 7). With a low pH elution (pH 5.9), the recombinant protein was eluted (lane 8), and it was more abundant in a lower pH elute fraction (pH 4.5). Some carboxyl-terminal truncated recombinant proteins were also produced and eluted in this fraction (shown as smaller sizes, lane 9). The average yield of the purified recombinant protein from one liter E. coli culture was about 9 mg calculated by the Bradford method.  Figure 4-10. SDS-PAGE Analysis of AtMYB68 Recombinant Protein Expressed in E. coli and Recombinant Protein Purification. The AtMYB68 c D N A encoding the non-conserved region was put into the pQE-30 expression vector. Proteins in lane 1 to lane 4 were prepared at a small-scale. Proteins in lane 5 were prepared at a large-scale. Lane 1, cell lysate from the E. coli strain containing the empty vector (IPTG absent). Lane 2, cell lysate from the E. coli strain containing the empty vector (IPTG present). Lane 3, cell lysate from the E. coli strain containing the AtMYB68 c D N A insert (IPTG absent). Lane 4 and 5, cell lysate from the E. coli strain containing the AtMYB68 c D N A insert (IPTG present). Lane 6, fraction of flow-through N i - N T A column. Lane 7, wash fraction. Lane 8, elution fraction at pH 5.9. Lane 9, elution fraction at pH 4.5. The arrow indicates the AtMYB68 recombinant protein. M , marker lane with sizes shown at left.  The purified A t M Y B 6 8 recombinant protein (pH 4.5 fraction) was used to immunize two rabbits. The anti-sera from both showed similar reactions to the purified recombinant AtMYB68 and E. coli crude extracts. Characterization of the AtMYB68 antibody by western blot analyses from one is shown here. First, the pre-immune serum was tested for cross-reactions on the western blot shown in Figure 4-11-A. This blot shows that the pre-immune serum did not react with the recombinant AtMYB68 from either the purified fraction (lane 1), or the E. coli total lysate (lane 3). It very slightly reacted with a few other proteins from E. coli total lysates (lane 2 from the strain containing the empty vector, and lane 3 from the strain containing the AtMYB68  insert). It also reacted with a small number of proteins from Arabidopsis shoot and root extracts (lanes 4 and 5). The AtMYB68 antiserum from immunized rabbits was then tested on a western blot shown in Figure 4-11-B. This blot showed that the AtMYB68 antiserum reacted strongly with proteins of about the predicted size (close to 29 kDa, indicated by arrow) from both the purified fraction (lane 1), and the E. coli total lysates (lane 2 (IPTG absent), and lane 3 (IPTG present)). However it also cross-reacted with many other proteins in these preparations. The AtMYB68 antiserum also detected a number of proteins from Arabidopsis shoot and root extracts (lanes 4 and 5). Cross-reactions of the AtMYB68 antiserum with many other proteins from both E. coli and Arabidopsis made it impossible to use in immunodetection. To purify an AtMYB68 antibody from the antiserum, antigen-Sepharose affinity methodology was used as described in Chapter 2. The purified AtMYB68 antibody was tested by the western blot shown in Figure 411-C. In general, the purified antibody detected much less background. It detected a single major band of the purified recombinant AtMYB68 (lane 1), and the major band of the recombinant AtMYB68 from the total lysate of the E. coli strain containing the AtMYB68 c D N A insert (lane 3). The purified antibody did not react with any proteins in the E. coli strain containing the empty vector (lane 2). In the Arabidopsis shoot extract, one major band of -43 kDa in size was detected by the AtMYB68 antibody (lane 4). This size was very close to the predicted size of 42 kDa for the AtMYB68 native protein. Surprisingly, however no bands were clearly detected from the Arabidopsis root extract (lane 5). This result was repeatedly seen with the AtMYB68 antibody using independent protein extracts and blots. This was an unexpected result due to its inconsistency with either northern or RT-PCR analyses, which showed that the AtMYB68 transcript was mainly accumulated in the root but not the shoot (Figure 4-3 and 4-4). There were several possibilities for this anomalous result. The most likely was the AtMYB68 antibody recognized another protein from the shoot. Later, an atmyb68 knockout line, Garlic 94B-18, was identified. In this knockout line, the AtMYB68 transcript was not detectable  (Chapter 5). Thus, the AtMYB68 protein was not expected to be detectable either. This knockout line provided a simple way to test if the anti-AtMYB68 signal from the shoot was an artifact. Protein extracts from shoots and roots of both Col and the knockout homozygous line (94B-18) were prepared. A western blot analysis of these extracts clearly showed that the signals detected by the AtMYB68 antibody were present in both wild type and the knockout shoot extracts. There was no difference in proteins detected in both root extracts either (Figure 4-12). From this, I conclude that the AtMYB68 antibody detected a non-AtMYB68 protein in the Arabidopsis shoot, and failed to detect AtMYB68 in the root. The antibody thus was not able to be used for the protein immunolocalization experiment.  Figure 4-11. Western Blot Analysis of Protein Extracts from E. coli and Arabidopsis Seedlings. Protein preparations were separated on SDS-PAGE then blotted on P V D F membranes. Signals were detected by the E C L Plus system then visualized by exposure to an x-ray film. (A) The pre-immune serum was used as a probe. Lane 1, purified AtMYB68 recombinant protein (-0.05 pg). Lane 2, cell lysate from the E. coli strain containing the empty pQE-30 vector. Lane 3, cell lysate from the E. coli strain containing the AtMYB68 c D N A insert. Lane 4, crude protein extract from 10-day old Arabidopsis seedling shoots (10 pg). Lane 5, crude protein extract from roots of the same seedlings (10 pg). (B) The AtMYB68 Antiserum was used as a probe. Lane 1, purified AtMYB68 recombinant protein (-0.1 pg). Lane 2, cell lysate from the E. coli strain containing the AtMYB68 cDNA insert (IPTG absent). Lane 3, cell lysate from the E. coli strain containing the AtMYB68 cDNA insert (IPTG present). Lane 4, crude protein extract from 10-day old Arabidopsis seedling shoots (10 pg). Lane 5, crude protein extract from roots of the same seedlings (10 pg). The arrow indicates the AtMYB68 recombinant protein position. (C) The purified AtMYB68 antibody was used as a probe. Lane 1, purified AtMYB68 recombinant protein (-0.1 pg). Lane 2, cell lysate from the E. coli strain containing the empty pQE-30 vector. Lane 3, cell lysate from the E. coli strain containing the AtMYB68 cDNA insert. Lane 4, crude protein extract from 10-day old Arabidopsis seedling shoots (10 pg). Lane 5, crude protein extract from roots of the same seedlings (10 pg). Protein marker sizes are shown at left.  94B-I8  Col  SH  RT  SH  RT  kDa 4531-  20.1-  Figure 4-12. Western Blot Analysis of Protein Extracts from an atmyb68 Knockout Line and Wild Type Seedlings. Protein crude extracts were from shoots (SH, 15 pg) and roots (RT, 20 pg) of 8-day old hydroponically grown Col and the atmyb68 knockout line (94B-18). The purified AtMYB68 antibody was used as a probe. Signals were detected by E C L Plus system then visualized by exposure to an x-ray film. Protein marker sizes are shown at left.  4.6 Discussion  As the first step toward understanding the function of AtMYB68 in Arabidopsis growth and development, expression profiles of AtMYB68 were generated. At the organ level, both northern and RT-PCR analyses showed that the AtMYB68 transcript predominantly accumulated in the Arabidopsis root. This result is partially consistent with a previous study (Kranz et al, 1998). In that study, the AtMYB68 transcript was detected in the root but also in the mature leaf.  However, the reverse northern method used in that study can have cross-hybridization problems. MPSS (defined in Section 3.5) data also shows that the level of the AtMYB68 transcript was the highest in the root. However, it did not show expression in shoots of the same age as I used in the RT-PCR analysis, other than expression was detected in immature flowers. Since MPSS is a quite new technique, I do not know how to explain this discrepancy. So far no other functionally characterized plant MYB gene has been reported to have the same organ-specific expression pattern as AtMYB68. At the tissue/cellular level, GUS activity driven by the AtMYB68 promoter was stronger in a unique and specialized cell type in the root, the pericycle cells adjacent to the xylem poles. In this study, the GUS reporter gene fused to the AtMYB68 promoter was used to show the AtMYB68 transcript at the tissue and cell level. It is worth noting that this project started before the Arabidopsis genome project was completed. At the time I followed general procedures and ©  took a 1.9 kb promoter for a fusion to GUS gene. Now we know that the distance from the 5' end of the AtMYB68 ORF to the stop codon of the previous gene is 4450 bp, so that additional information specifying AtMYB68 expression could be located in this region. However, the GUS activity data is consistent with the northern and RT-PCR data on accumulation of the AtMYB68 transcript. All these lines of data showed that the root was the major site of AtMYB68 expression, and that AtMYB68 was expressed much less in small groups of cells in the shoot. These results suggest that the AtMYB68::GUS fusion reflects the site of AtMYB68 expression. It certainly would be good to examine endogenous AtMYB68 expression at the cell level to confirm the GUS data. Therefore I intended to do in situ hybridization. However, for paraplast sectioning, sections with intact root morphology beyond the root tip could not be obtained, since the big cells in the outer layers of the root were too fragile for the xylene treatment. The other sectioning method for in situ hybridization, cryosectioning, would be another choice, but the equipment for this technique was not available until recently in the department. As an alternative to in situ hybridization, I also tried to raise an antibody against the recombinant AtMYB68 to do protein immunolocalization. However the antibody raised  apparently strongly cross-reacted to another Arabidopsis protein rather than recognizing native AtMYB68. In the entire Arabidopsis genome, the closest member to AtMYB68 is AtMYB84. However it did not appear that the antibody cross-reacted to AtMYB84, since the transcript of AtMYB84 accumulated universally at a very low level in all tissues/organs tested (Chapter 5). The signal detected by the AtMYB68 antibody was quite strong and shoot-specific. It is not known what the AtMYB68 antibody reacted to. One would easily suspect that the cDNA cloned into the pQE-30 expression vector was not AtMYB68. However this can be excluded, since the 3' cDNA fragment was released directly from the AtMYB68 full length c D N A clone by restriction enzyme digestion, and the ArMF7J<58-pQE30 clone was confirmed by sequencing. In response to various external stimuli, the level of the AtMYB68 transcript was upregulated by A B A , and down-regulated by IAA, cold and desiccation treatments, but the magnitude of changes was not great. In these initial experiments, I only tested one stimulus concentration at one time point for each treatment. It is possible that these do not represent the optimal conditions to show maximal changes. Thus, further experiments with different concentrations and with time courses are required. Another point is that total R N A from the whole seedling is used for the RT-PCR analysis. However the AtMYB68 transcript mainly accumulates in the root. It would be worth while to examine changes in AtMYB68 expression in the shoot and root separately. Many ABA-responsive genes have a highly conserved sequence, PyACGTG(T/G)C, named the ABA-responsive element (ABRE) in the promoter regions (Giraudat et al., 1994; •Shinozaki and Yamaguchi- Shinozaki, 2000). Another type of A B R E , known as coupling element, hex3 or motif III containing the core sequence C G C G T G has also been identified (Busk and Pages, 1998). However, neither type of ABREs was found in the 1.9 kb promoter region of AtMYB68 or further upstream. Another Arabidopsis MYB gene AtMYB2 is also up-regulated by exogenous A B A , and by drought and high salinity treatments. No A B R E s are found in the promoter of AtMYB2 either (Urao et al., 1993).  M y expression data gave some insights on possible functions of AtMYB68 and a mechanism that regulates AtMYB68 expression. (1) One possible role of A t M Y B 6 8 . The cell-preferential GUS activity driven by the AtMYB68 promoter leads to the speculation that AtMYB68 might be related to lateral root formation, since in root development, the pericycle cells adjacent to the xylem poles serve as progenitors for lateral root formation (Steeves and Sussex, 1989; Charlton, 1991). Several cyclin and cyclin-dependent kinase gene promoters driving GUS activity are also detected in pericycle cells, or the cells adjacent to the xylem poles. For example, the Arabidopsis cyclin gene CycA2;l, and cdc2a, a gene encoding the catalytic subunit p34  cdc2  of the cyclin-dependent kinase  (Burssens et al., 2000; Hemerly et al., 1993). The expression of these cell cycle related genes in this cell type is thought to mark competency for cell division. It is interesting to see that these cell cycle related genes, such as CycA2;l, cycla (CycBl;l) and cdc2a are also expressed in the stomata guard cells of young leaves, which is similar to that of AtMYB68 (Burssens et al., 2000; Serna and Fenoll, 1997). The expression of these cell cycle related genes in the stomata guard cells is also thought to mark them for cell totipotency (Serna and Fenoll, 1997). The similarity of AtMYB68 cell-type expression with that of cell cycle related genes suggests that AtMYB68 might be related to cell competence for division or cell cycle status. Since GUS activity driven by the AtMYB68 promoter declined in the upper part of the root, where lateral roots were producing, and was only retained in the lower part of the root, one possibility would be that AtMYB68 is a factor that blocks the cells from re-entering the cell cycle before conditions are ready. When a signal, such as auxin is present at a proper concentration, AtMYB68 could be down-regulated allowing the cells to undergo cell division. However, no clear separation between the site of lateral root primordium emergence and the region of expression of AtMYB68::GUS was observed. Given that GUS is a quite stable protein, the GUS pattern may not reflect the real turnover time of AtMYB68. Using GFP tagged AtMYB68 may be better to show the dynamic accumulation of AtMYB68. Although the M Y B binding site has been found in the promoter of the Arabidopsis CycBl;! gene (Ito et al., 2001;  Planchais et al., 2002), so far there is no clear evidence to show that any R2R3-type M Y B proteins are involved in regulating the cell cycle. Among two key regulators of lateral root formation examined, the level of the AtMYB68 transcript was only down-regulated in response to exogenous I A A but not responsive to N 0  3  treatment. This result could be explained by the fact that I A A and N 0 " affect different stages of 3  lateral root development. Auxin is thought to represent an important factor for lateral root initiation. A threshold concentration of auxin is required for the xylem pole pericycle cells to enter the cell cycle and initiate lateral root development (Casimiro et al., 2001). In contrast, the effect of N 0 " on lateral root formation affects elongation of emergent lateral roots (Zhang et al., 3  1999). The down-regulation of AtMYB68 expression by I A A suggests that AtMYB68 may be related to initiation of lateral root formation. The induction of AtMYB68 expression by A B A could be a part of the mechanism to prevent those competent cells from dividing before conditions are appropriate. A B A inhibition of cell division has been reported (Newton, 1977; Robertson et al., 1990). This inhibition effect might be accomplished by A B A induced expression of the cyclin-dependent kinase inhibitor ICK1 gene (Wang et al., 1998). (2) Does the xylem send a signal to determine the cell specificity? Only the pericycle cells adjacent to the xylem poles appear to receive a signal to initiate lateral root formation. However, it is not clear what determines these cells' specialization. Since positional cues produce important signaling in root development (Scheres, 1997), I wished to see if the xylem plays a role in xylem pole pericycle cell differentiation, using expression of AtMYB68::GUS as a marker. I demonstrated that cell type-preferential expression of AtMYB68::GUS was indeed changed with the altered xylem pattern in the wol background. With the xylem produced throughout the root vascular tissue in the wol mutant, the expression of AtMYB68::GUS was expanded to the entire layer of pericycle cells, where it appeared stronger than that in the wildtype background, and also expanded to the endodermal layer. This result provides evidence that gene expression in these xylem pole pericycle cells is responsive to a positive signal from the  xylem (or alternatively a negative signal from the phloem). However, the link is between AtMYB68 expression and these cells' role as lateral root founder cells is not known yet. It is also not clear what signal is coming from the root vascular tissue. (3) Other possible roles for A t M Y B 6 8 . Apparently, the xylem pole pericycle cells have multiple functions since genes known with different functions are expressed in these cells. One example is the SOS1 gene. The SOS1 promoter driving GUS expression is more active in the pericycle cells near the xylem poles and the parenchyma cells surrounding the xylem vessels. SOS1 encodes a N a 7 H antiporter located on the plasma membrane. The role of SOS1 in the +  cells surrounding the xylem is proposed to load and unload extra N a to the xylem for long+  distance transport (Shi et al., 2002). At the organ level, AtMYB68 had a root-preferential expression pattern. Expression profiles of 402 potentially stress-related transcription factors, including 121 MYB genes from Arabidopsis have revealed that -15% of these transcription factors are preferentially or specifically expressed in the root, compared with only 6% that are leaf specific. Fifty percent of these root-specific/preferential transcription factors are induced after different types of biotic and abiotic stress treatments (Chen et al., 2002). This data suggests a strong correlation between root-specific/preferential transcription factors and stress responses. The level of the AtMYB68 transcript was mostly up-regulated by A B A among the plant hormone treatments. A B A is known to play a primary regulatory role in the plant response to stress. The endogenous A B A level is usually increased to trigger protective or adaptive responses under adverse environment conditions (Zeevaart and Creelman, 1988; Leung and Giraudat, 1998). Many stress-inducible genes are also responsive to A B A (Bray, 1997; Shinozaki and Yamaguchi-Shinozaki, 2000). Expression profiles of 7,000 Arabidopsis genes treated by A B A , drought, cold and high-salinity have shown that there is a distinct set of induced genes upon each treatment, but there are also overlaps between genes induced by different treatments (Seki et al., 2002). The induction of AtMYB68 expression by A B A also suggests that AtMYB68 might be related to stress responses.  Therefore I further tested expression of AtMYB68 in response to some abiotic stresses. The down-regulation of AtMYB68 expression by both desiccation and cold, and the stable level of AtMYB68 expression upon high salinity, were in contrast to the up-regulation by exogenous A B A . This result suggests that the role of A B A in regulating AtMYB68 expression is distinct from that of these stresses. The down-regulation of AtMYB68 expression by desiccation and cold could be a secondary response, as the plant generally slows metabolism under these adverse conditions. Certainly, it is too early to deduce AtMYB68 function only from its expression profiles. More evidence is needed from other approaches, which is the purpose of experiments reported in the next chapter.  Chapter 5 AtMYB68: a Root Preferentially Expressed MYB Gene — Functional Studies  5.1 Introduction  As reviewed in Chapter 1, when gene sequence information is available, reverse genetic approaches, based on T - D N A or transposon insertion mutagenesis, or T I L L I N G (Targeting Induced Local Lesions in Genomes), are possible for a functional analysis. These have been demonstrated to be very useful for Arabidopsis MYB gene functional studies, although functions of several MYB gene were characterized by the traditional forward genetic approach (Meissner et al., 1999; Penfield et al., 2001; Jin et al., 2000). Another common method for producing loss-offunction mutations in plants, using antisense R N A or R N A i to suppress gene expression, has been also used in the MYB gene functional analysis (Vailleau et al., 2002). In the case of genes with redundant functions, producing gain-of-function mutations is another way to reveal gene function. This has also been used successfully in Arabidopsis MYB gene functional characterization (Borevitz et al., 2000). In this chapter, I describe attempts to reveal AtMYB68 function by disrupting its expression, expressing AtMYB68 under the control of the 35S promoter, repressing the expression by antisense R N A and searching for insertion mutations in T - D N A tagged populations.  5.2 Overexpression of AtMYB68 under the Control of the CaMV 35S Promoter  To create AtMYB68 overexpression lines, the full-length AtMYB68 c D N A of -1.3 kb in size was inserted behind the 35S promoter in the sense orientation in the pRTlOl vector (Figure 2-2-B). The 35S::AtMYB68 cassette was then cloned into the BIN19 binary vector. After transformation into Agrobacterium, the 35S::AtMYB68 construct was transformed into Arabidopsis Col plants by the Agrobacterium-mediated floral dip method. The NPTII gene in  the BIN19 vector provided kanamycin-resistance as a selectable marker. Tl-generation transformants were selected on kanamycin plates. The transformation rate was -0.2% (15 out of 8,000 with kanamycin-resistance). Kanamycin-resistant T l plants were allowed to set seeds to generate the T2-generation. The levels of the AtMYB68 transcript in 35S::AtMYB68 transgenic lines was detected by a northern blot analysis. The first round of detection was performed in the T2 generation. Total R N A was extracted from T2 kanamycin-resistant seedlings. Figure 5-1-A shows that 35S::AtMYB68 was expressed in all the 12 lines tested. Two transcripts with different sizes were detected. They were apparently caused by two transcription terminators in the transgene. The short transcript was likely due to termination at the internal terminator within the AtMYB68 cDNA. The long one was likely due to termination at the external 35S polyA terminator within the pRTlOl vector. The native AtMYB68 transcript (observed in Col) was similar in size to the short one from the 35S::AtMYB68 transgene. AtMYB68 co-suppression lines were not found in these 12 lines based on the northern blot analysis. Homozygous lines with the 35S::AtMYB68 construct were selected from 3 transgenic lines. A northern analysis of the transcript levels of AtMYB68 in the homozygous lines is shown in Figure 5-1-B. Total R N A was extracted from whole seedlings for this analysis. The blot showed that 35S::AtMYB68 was expressed at different levels in these 3 homozygous lines. Line 9-3 had the highest expression level followed by lines 6-4 and 15-1. Line 9-3 was selected for phenotypic examination as presented later.  A 2  3  4  5  6  7  9  Col  8  10  11 16 17  •I B  Col 6-4 9-3 15-1  ?  Figure 5-1. Northern Blot Analysis of AtMYB68 Expression in 35S::AtMYB68 Transgenic Lines. (A) AtMYB68 expression in T2-generation plants. Total R N A was extracted from 18-day old kanamycin-resistant seedlings. (B) AtMYB68 expression in T3 homozygous lines. Total R N A was extracted from 15-day old seedlings. The ethidium bromide stained gel is shown as a loading control. 10 pg total R N A was loaded in each lane. The numbers above the blots indicate the transgenic lines. Col plants were used for transformation. The two transcripts in transgenic lines were caused by the present of two transcription terminators in the transgene. The short transcript was terminated at the internal AtMYB68 terminator. The long transcript was terminated at the external 35S polyA terminator introduced by the vector p R T l O l .  5.3 Repression of AtMYB68  Expression by Antisense RNA  In order to repress AtMYB68 expression, transgenic lines in which AtMYB68 antisense RNA was expressed under the control of the 35S promoter were generated. The 3' end of the AtMYB68 cDNA encoding the non-conserved region was subcloned behind the 35S promoter in the antisense orientation in the pRTlOl vector (Figure 2-2-C). Then the 35S::AtMYB68-3(-) construct was cloned into the BIN19 binary vector. After transformation into Agrobacterium, the 35S::AtMYB68-3 (-) construct was transformed into Arabidopsis Col plants by the Agrobacterium-medwled floral dip method. The NPTII gene in the BIN19 vector provided kanamycin-resistance as a selectable marker. Tl-generation transformants were selected on kanamycin plates. The transformation rate was 0.85% (34 out of 4,000 with kanamycinresistance). Kanamycin-resistant T l plants were allowed to set seeds to generate the T2generation. Homozygous lines with the 35S::AtMYB68-3 (-) construct were selected from 6 transgenic lines. The levels of the native AtMYB68 transcript in the homozygous lines was tested by quantitative RT-PCR (Figure 5-2). Total R N A was extracted from 14-day old seedling roots and subjected to reverse transcription followed by PCR amplification with AtMYB68 specific primers. 18S rRNA was used as an internal control. The ratio of product intensity of AtMYB68 to 18S rRNA represents the gene relative expression level. The RT-PCR analysis showed that in 6 homozygous lines tested, lines 1-2 and 11-1 had the AtMYB68 transcript levels about 2-fold lower than that in the wild type. The other 4 lines generally had the same transcript level as that in the wild type. Since later an atmyb68 knockout line was identified, these 2 repressed lines were not investigated further.  Col  1-2  7-1  C—J »—l  v—J  9ÊÊÊ&ÊÈ$  .2 °'  8  0.772  4MMM0  8-5 11-1 15-2 16-3  i—i i—i  fcn**"*!)^  %^HfeiN^  0.766  AtMYB68  u—»  ^* •  '  2 8 S  t*Ri^J/\,  0.678  0.719  15-2  16-3  M  Pi  0  6  0.2  Transgenic Line  Figure 5-2. RT-PCR Analysis of AtMYB68 Expression in Homozygous Transgenic Lines Harboring the 35S::AtMYB68 Antisense Transgene. Total R N A was extracted from 14-day old hydroponically grown seedling roots and subjected to reverse transcription followed by PCR amplification. 18S ribosome R N A was used as an internal control. The ratio of the intensity of AtMYB68 to 18S rRNA represents the relative AtMYB68 expression level. The ratios are visualized by bar graphs in the lower panel and values are shown above each bar. The numbers above the gel indicate the transgenic lines. Col plants were used for transformation.  5.4 Screen for AtMYB68 Knockout Mutations in T-DNA Tagged Populations  As stated in Section 4.1, one T-DNA line with the insertion 536 bp upstream of the A T G codon of AtMYB68 was identified from the Feldmann T - D N A collection (Pri-Hadash and  Douglas, unpublished data). The level of the AtMYB68 transcript in the homozygous insertion line, T-14, was tested by a northern blot analysis (Figure 5-3). Total R N A was extracted from 12-day old seedling shoots and roots respectively. The analysis showed that abundance of the AtMYB68 transcript in the root of line T-14 was the same as that in the wild-type. The AtMYB68 transcript was not detectable in either shoot RNA. Therefore transcription of AtMYB68 was not affected in this T - D N A insertion line. No mutant phenotypes were observed in line T-14 under normal growth conditions either (data not shown).  WS SH  T-14  RT SH RT  AtMYBàH  ROC1  Figure 5-3. Northern Blot Analysis of AtMYB68 Expression in the T - D N A Insertion Line T-14. Total R N A was extracted from shoots and roots of 12-day old seedlings respectively. 10 u,g total R N A was loaded on each lane. The ROC1 cDNA (encoding Arabidopsis cytosolic cyclophilin) was hybridized to the same blot as a loading control after the blot was stripped. SH, shoot. RT, root. WS ecotype is the wild-type background.  While generating AtMYB68 overexpression and antisense lines, I continued searching for AtMYB68 knockout mutations in publicly available T - D N A tagged populations. After Dr. PriHadash finished screening the DuPont and Feldmann collections, I searched the Jack and Wisconsin T - D N A collections by PCR-based strategies (Table 5-1). AtMYB68 has two small introns, resulting in a total gene size of -1.6 kb, including potential 5' and 3' regulatory sequences. This -1.6 kb region was the target sequence in my screening, since a hit in this region would most likely disrupt gene expression.  Table 5-1. Summary of Screens for a T-DNA Insertion in AtMYB68 T - D N A Collection  No. of T - D N A line  Position of Insertion  DuPont*  8,100  None  Feldmann* (ABRC)  6,000  540 bp upstream of A T G  Jack (ABRC)  6,000  None  Wisconsin  60,480  200 bp upstream of A T G  R N A level  (Sue Gibson lab) Normal +  (U of Wisconsin and A B R C ) Garlic* (TMRI)  100,000  l  s l  intron  Not detectable  * Screened by Dr. Aviva Pri-Hadash + The signal disappeared in 9-line pools. # Directly searched on computer.  For screening the Jack collection, which was composed of 6,000 independent T-DNA lines, an AtMYB68 specific primer slightly outside the 3' end of the coding region combined with T-DNA left and right border primers were used in PCR. The first round of PCR was performed against 6 D N A 1,000-line pools. The PCR reactions were tested by a Southern analysis using the AtMYB68 cDNA as a probe. No positive signals were detected (data not shown). Three years ago, the Wisconsin Biotechnology Center provided a screening service for their T-DNA collection, which was composed of 60,480 independent T - D N A lines. We used this service to screen for an AtMYB68 knockout. AtMYB68 specific primers targeted slightly outside the 5' and 3' ends of the coding region were sent to them for PCR screening. The first round of PCR searched 30 D N A 2,025-line super pools using the combination of the AtMYB68 specific primers and a T - D N A left border primer. The PCR reactions were sent to us, and then they were tested on Southern blots using AtMYB68 genomic D N A as a probe (Figure 5-4). One signal of -1.7 kb in size that was amplified by the AtMYB68 reverse primer and the T - D N A left border primer from super pool 7 was detected. There were also other signals detected on the blots, but the sizes were too big and beyond the target region. The signal of -1.7 kb in size was further reamplified by the nested primers. The re-amplified signal was gel-purified and sequenced.  LB+AtMYB68  M 1  (F)  Super pool  C  M 23  o  n  ->31  ->22 M  LB+AtMYB68 (R) — > 1  >78 9  M  1.7 kb 1.5 kb  M 10  Con >31 M  Sequencing showed that the T - D N A insert was 202 bp upstream of the A T G codon of AtMYB68, and there was a 19 bp unknown sequence at the T-DNA-AtMYB68 junction. This was a promising insertion position, and then we requested a second round of PCR that searched subpools of super pool 7 (9 pools of 225-line). Again, the PCR reactions were separated on a gel after they were received. A distinct band of -1.7 kb in size from sub-super pool 61 was observed (Figure 5-5, indicated by arrow). Sub-super pool 61 was re-amplified along with super pool 7 (X7) as a positive control (Figure 5-6). The band of -1.7 kb in size was re-amplified from sub-super pool 61 (lane 2) using the AtMYB68 specific and T - D N A left border primers, the same as in super pool 7 (lane 1). A band slightly lower in size was re-amplified using the nested primers (lane 3). No band of the same size was re-amplified using the T - D N A left border primer only (lane 4). These results confirmed that the hit was in sub-super pool 61.  Figure 5-4. Southern Blot Analysis of PCR Amplification for a T - D N A Insertion within AtMYB68 Using the Wisconsin Collection. PCR was performed using superpool D N A as templates (superpools 1 to 30) and AtMYB68 specific primers combined with the T - D N A left border primer (LB) (PCR done by the University of Wisconsin). Each superpool contained 2,025 T - D N A transformed lines. The PCR reactions were separated on a gel and blotted. AtMYB68 genomic D N A was used to probe blots. (A) + (B) Left Part, PCR using L B and AtMYB68 forward primers. (B) Right part + (C), PCR using L B and AtMYB68 reverse primers. Lane 31, the PCR control using AtMYB68 forward and reverse primers and wild-type WS genomic D N A as a template. M , marker lane with sizes shown at right.  Sub-super pool M  55 56 57 58 59 60 61 62 63 X7 M  — 3.0 kb — 2.0 kb — 1.6 kb — 1.0 kb  Figure 5-5. Second Round of PCR Amplification for a T - D N A Insertion within AtMYB68 Using the Wisconsin Collection. PCR was performed using sub-super pool D N A of the superpool 7 as templates (sub-super pools 55-63) and the AtMYB68 reverse and the T-DNA left border primers (PCR done by the University of Wisconsin). Each sub-super pool contained 225 T - D N A transformed lines. X7, superpool 7. The arrow indicates the size of the predicted band. M , marker lane with sizes shown at right.  Lastly, the seeds for sub-super pool 61 were ordered, which were composed of 25 pools of 9-line. However one seed pool, J1510, was not sent due to seed shortage. Following the service's instructions, genomic D N A was extracted from 5-day old seedlings of the 24 pools and subjected to PCR amplification using the same conditions as the first two rounds of PCR. The PCR reactions were tested by a Southern analysis as for the first round of PCR. Surprisingly, no signals were detected on the blots except a signal from the control (Figure 5-7). Thus, the signal of the T-DNA insertion in AtMYB68 that had been detected in the first two rounds of PCR was lost for reasons out of my control. The first two rounds of D N A preps and PCR amplification were performed in Wisconsin, and were beyond my ability to troubleshoot, and I was still  missing one 9-line pool of seeds. Due to these uncertainties and my inability to resolve them, this screening was abandoned.  2.0 kb 1.6 kb 1.0 kb 500 bp  Figure 5-6. Re-Amplification of an AtMYB68 -T-DNA Allele from Sub-Super Pool 61 of the Wisconsin Collection. PCR was performed using the second round PCR reaction from sub-super pool 61 as a template. Superpool 7 D N A was used as a positive control. Lane 1, PCR of superpool 7 D N A using the AtMYB68 reverse and T - D N A left border primers (LB). Lane 2, PCR of sub-super pool 61 D N A using L B and the AtMYB68 reverse primers. Lane 3, PCR of sub-super pool 61 D N A using L B and the AtMYB68 nested primers. Lane 4, PCR of sub-super pool 61 D N A using L B primer only. M , marker lane with sizes shown at left.  M N*  ^  WS  Figure 5-7. Southern Blot Analysis of PCR Amplification for an AtMYB68-T-DNA Allele in 9Line Pools of the Wisconsin Collection. PCR was performed using 9-line pool D N A as templates (pools 1486-1509) and the AtMYB68 reverse and T-DNA left border primers. The PCR reactions were separated on a gel and blotted. AtMYB68 genomic D N A was used to probe blots. WS genomic D N A and the AtMYB68 specific primers was used in P C R as a control (WS). M , marker lane with sizes shown at right. (A) 9-line pools 1486 to 1497. (B) 9-line pools 1498 to 1509.  More recently, by searching on-line databases of sequenced T - D N A junctions, we found that a T-DNA line, Garlic 94B, had the insertion in the AtMYB68  coding region in the Torrey  Mesa Research Institute (TMRI) T-DNA collection. This collection was composed of 100,000 independent T-DNA lines (Table 5-1). The seeds from the line Garlic-94B were ordered. To confirm the insert location, rosette leaves from Garlic 94B plants were pooled. Genomic D N A was extracted from the pooled leaves and subjected to PCR amplification with an  AtMYB68  specific primer and the recommended T-DNA left border primer. The predicted size of the PCR product was -1.3 kb based on TMRI data. Figure 5-8 shows that only one band of the right size was amplified. This band was gel-purified and sequenced. Sequencing confirmed that the TD N A insertion was in the predicted location, the first intron of A1MYB68  M  (Figure 5-9).  94B  1.6 kb 1.0  kb  Figure 5-8. PCR Amplification of a T-DNA Insertion within AtMYB68  in the Garlic 94B Line  Using the TMRI Collection. PCR was performed using genomic D N A extracted from Garlic 94B plants as a template and the AtMYB68  specific and T - D N A left border primers. M , marker lane with sizes shown at left.  5'  Intron 1 Intron 2  y  Figure 5-9. Schematic of the T-DNA Insertion within AtMYB68 from the Garlic 94B Line. The dark and light grey boxes indicate the coding region for the two conserved repeats R2, R3 in the M Y B binding domain. The hatched box indicated the conserved 5' region. The open box indicates the non-conserved region.  A homozygous line with the insert was selected from the same generation using a PCR strategy. The first round of PCR selected plants with the insert within the population. This was done by using the AtMYB68 specific and the T-DNA left border primers in PCR. Figure 5-10-A shows that in a total of 17 plants examined (derived from the Garlic 94B line), a fragment of -1.3 kb in size was amplified from 13 plants, but not from the other 4 (plants 12, 14, 15 and 17). So these 4 plants did not have the T-DNA insert in AtMYB68 and they were the wild type siblings. A second round of PCR selected homozygous plants among those with the insert. This was done by using AtMYB68 specific primers spanning the insertion area for PCR amplification. Since the heterozygous plants would have one copy of a wild-type AtMYB68 allele, it would be amplified to give a fragment of -1.5 kb in size. However the AtMYB68 allele with the insert would be too big to amplify due to the T-DNA insertion, so that no product would be expected in the homozygous plants using these primers. Figure 5-10-B shows that 8 out of 13 plants had a fragment of the predicted size from the wild type copy (plant 4 had the very weak PCR product), suggesting that they were heterozygous. The other 5 plants (plants 5,8, 11, 18 and 19) did not yield the amplification products. Thus they were presumed to be homozygous for the T - D N A insertion allele. Plant 18 line (referred to 94B-18) was selected for further testing.  13 14 15 16 17 18 19 9 4 B M  Figure 5-10. PCR Screening for Homozygous Insertion Lines Derived from Garlic 94B. PCR was performed using genomic D N A from the individual plants derived from Garlic 94B as templates. The numbers above the gels indicate the individual plants. M , marker lane with sizes shown at left. (A) PCR using the AtMYB68 specific and T-DNA left border primers. The Garlic 94B D N A pool was used as a positive control. (B) PCR using the AtMYB68 specific primers. Col D N A was used as a positive control.  The selectable marker on the binary vector used in the Garlic line transformation was the BAR gene, conferring B A S T A resistance. Thus the plants in the Garlic line containing a T-DNA insert should also be BASTA-resistant if T-DNA was intact. Therefore BASTA-resistance was also tested in the Garlic-94B line. The result showed that plants from the homozygous line 94B18 were all BASTA-resistant as expected, but plants from the wild-type sibling line 94B-17 were  also BASTA-resistant. This result indicated that at least one more T - D N A insertion existed in the Garlic-94B line. The level of the AtMYB68 transcript in the homozygous line 94B-18 was tested by RTPCR. Total R N A was extracted from 3-4 days old seedlings. R N A from Col was used as a positive control, and 18S rRNA was used as an internal control for RT-PCR. Figure 5-11-A shows that the level of the AtMYB68 transcript was not detectable in line 94B-18 on an ethidium bromide stained agarose gel, while the product of the AtMYB68 transcript from Col was very abundant. When the same reactions were detected on a polyacrylamide gel stained with Syber Green (sensitivity was increased over 100 times), a trace amount of the AtMYB68 transcript product was seen from line 94B-18. However the abundance was extremely low as compared to Col (Figure 5-11, B), at least 65-fold lower than that in the wild-type. Thus, I referred to the 94B-18 line as an AtMYB68 'knockout' line.  Figure 5-11. RT-PCR Analysis of AtMYB68 Expression in the T - D N A Insertion Line 94B-18. Total R N A extracted from 3-4 days old seedlings was subjected to reverse transcription followed by PCR amplification. 18S rRNA was used as an internal control for RT-PCR. Col R N A was used as a control. (A) RT-PCR reactions separated on an ethidium bromide stained agarose gel. (B) RT-PCR reactions separated on a polyarylamide gel and then stained by Sybre Green.  5.5 Phenotypic Analysis of AtMYB68 Mutant Lines  Morphological mutant phenotypes were examined in both the AtMYB68 overexpression line 9-3, and the knockout line 94B-18. As described in the last section, at least one more TD N A locus other than the insertion in AtMYB68 exists in the Garlic-94B line. Backcrossing the homozygous line 94B-18 to Col to remove the background T - D N A has been initiated. However due to time limitations, the result of this backcrossing is not included in this thesis. To overcome this problem, a sibling of line 94B-18, line 94B-17, lacking the T - D N A insertion in AtMYB68, was used as a control in phenotypic inspection of the knockout line. The genetic background of 94B-17 is identical to the sibling 94B-18. Therefore, any difference on phenotype in line 94B-18 should be solely caused by the disrupted AtMYB68. I first examined phenotypes under normal growth conditions on plates and in soil. No obvious morphological changes were observed in either the overexpression line or the knockout line, except that plants from the overexpression line 9-3 appeared generally shorter than wildtype Col (Figure 5-12, D). Measurements of shoot fresh weight, primary root length and lateral root number were taken from 11-day old seedlings grown on M S plates. These measurements were taken in several batches of seedlings. One set of this data is shown in Table 5-2. No clear statistical differences were shown in different sets of measurements (Table 5-2). The Garlic-94B population generally produced shorter primary roots than Col, but there was no difference in the knockout line 94B-18 and the sibling line 94B-17 (Figure 5-12, A and Table 5-2).  Figure 5-12. Morphology of AtMYB68 Mutant Plants. (A) 10-day old seedlings of the atmyb68 knockout line 94B-18 and controls grown on a plate. (B) Mature plants of the atmyb68 knockout line 94B-18 and the control grown in soil. (C) 11-day old seedlings of the AtMYB68 overexpression line 9-3 and Col grown on plates. (D) Mature plants of the AtMYB68 overexpression line 9-3 and Col grown in soil. 94B-17, a wild-type sibling in the Garlic-94B line. Col, Columbia ecotype.  Table 5-2. Growth of AtMYB68 Mutant Seedlings on Plates* Line  PRL (mm)  L R No./L (cm)"  9-3  64.7±5.6  1.2±0.2  3.211.0  Col  60.0±3.2  1.6±0.3  4.910.7  94B-18  59.0±2.7  1.6±0.4  4.7+1.1  94B-17  62.1±3.5  1.010.2  4.310.8  A  1  SHW (mg)  *Measurements were taken on 11-day old seedlings grown on MS plate plus 3% sucrose. A  The number indicates SD (n=7).  PRL, primary root length. L R No./L, lateral root number per primary root. SHW, shoot fresh weight.  In Section 4.4, the level of the AtMYB68 transcript was shown to be up-regulated by A B A , and down-regulated by I A A , cold and desiccation treatments. These treatments served as guidelines for preliminary phenotypic examinations under specific conditions. Phenotypes were examined in response to two plant hormones, A B A and I A A . For the A B A treatment, 1.0 U.M A B A was added to medium. Shoot fresh weight, primary root growth and lateral root number were measured after 7 days grown on A B A plates. No significant difference was shown with the measurements in either mutant line (Table 5-3). For the I A A treatment, 1.0 jwM I A A was added to medium. The lateral root number was measured after 5 days grown on I A A plates. Again, there was no significant difference in lateral root number increase in response to IAA between the control and AtMYB68 mutant lines (Table 5-4).  Table 5-3. Seedling growth under A B A treatment* Line  A B A (uM)  Root growth (%)  L R No  9-3  0  100  7.7±2.9  100  1.0  80.0±12.0  7.614.1  86.017.31  Col  A  Shoot growth (%)  0  100  10.9±4.02  100  1.0  95.1±4.80  13.012.24  100116.5  100  9.711.7  100  80.8±5.8  5.9+2.5  61.9+6.6  too  8.4+2.3  100  89.4±1.8  7.011.5  67.815.1  94B-18 0 1.0 94B-17 0 1.0  *Measurement were taken after 6 days (for lines 94B-18 and 94B-17), or 7 days (for lines 9-3 and Col) of A B A treatments (1.0 uM). The number indicates SD (n=7).  A  Root and shoot growth are expressed as the relative growth to the control (0 A B A ) . L R No, Lateral root  Table 5-4. Seedling lateral root number under I A A treatment (No./L (cm)' )* 1  IAA (uM)  Overexpression 1ine  Knockout line  9-3  94B-18  94B-17  Col  0  1.9+0.51  2.310.49  2.4+0.37  2.6+0.27  1.0  7.310.97  7.2+0.41  7.810.64  6.910.71  A  *The lateral root number was counted after 5 days of IAA treatments (1.0 |uM). No./L, lateral root number per primary root. A  The number indicates SD (n=7).  Phenotypes were also examined under salinity and drought stresses. For the salinity treatment, inhibition of seedling root elongation on 50, 100 and 150 m M NaCl supplemented media was shown by a root-bending assay. For the drought treatment, soil-grown plants were not watered for 2 weeks. Drought tolerance was observed. Plant roots elongate in a wavy pattern when grown on a hard agar surface at an inclined position. This obstacle-touching response is thought to involve auxin signaling (Okada and Shimura, 1992). Thus, this response was also  tested. Under these conditions, no differences between the control and the overexpression or knockout line were observed (data not shown). Thus, as summarized in Table 5-5, no phenotypic differences were observed between the control and AtMYB68 mutant lines with respect to normal growth or any of the conditions tested (Table 5-5).  Table 5-5. Summary of Phenotypic Examinations of AtMYB68 Mutant Lines Condition  Mutant Phenotype Overexpression line 9-3  Knockout line Garlic 94B-18  Normal growth: MS+3% sucrose  No*  No  Soil  No  No No  Treatments: Abscisic acid  No  Indoleacetic acid  No  Salinity  No  Drought  No  No  Root obstacle-touching  No  No  No J  No  * Mutant phenotype was not observed. Some data are shown in separate tables.  5.6 Expression Patterns of the Members in AtMYB Phylogenetic Subgroup 14  As stated in Chapter 1, the Arabidopsis R2R3-MYB proteins were categorized into 22 phylogenetic subgroups according to similarity outside the conserved M Y B DNA-binding domain (Kranz et al., 1998; Stracke et al., 2001). AtMYB68 was placed in subgroup 14, along with another 5 members, AtMYB84, AtMYB36, AtMYB87, A t M Y B 3 7 and AtMYB38 (Figure 5-13). AtMYB84 is the most closely related to AtMYB68 according to this analysis. To gain some insight on how functionally related these family members might be, I wished to compare their expression patterns, and chose AtMYB84 and AtMYB36, another member of this subgroup less closely related to A t M Y B 6 8 , for the analysis.  —AtMYB36 -AtMYB87 AtMYB84 AtMYB68 AtMYB37 -AtMYB38  Figure 5-13. Re-Drawing of A t M Y B Phylogenetic Subgroup 14 (According to Stracke et al., 2001).  When aligned, it can be seen that the predicted amino acid sequences of these three members were very similar to each other in the conserved amino-terminal M Y B DNA-binding domains. Outside the M Y B DNA-binding domain, AtMYB68 and A t M Y B 8 4 shared two conserved motifs, but they were not well conserved in A t M Y B 3 6 (Figure 5-14). Overall, at the amino acid level, AtMYB68 has 57.3% identity to AtMYB84, and 40.5% identity to AtMYB36. The level of the AtMYB84 transcript was too low to be detected by a northern blot analysis. Therefore, RT-PCR was used to examine expression patterns of these three genes. Total R N A extracted from Arabidopsis seedlings and mature organs was subjected to the quantitative RT-PCR, using 18S rRNA as an internal control. Figure 5-15 shows that AtMYB36 had the expression pattern very similar to that of AtMYB68. Both were mainly detected in roots and also detectable in 3-day old seedlings, but not detectable in the other organs, including bolting stems, rosette leaves, flowers and siliques. The member most closely related to AtMYB68, AtMYB84, had a different expression pattern from the other two. The AtMYB84 transcript was detected at low levels in all the tissues/organs tested. Northern blot analyses of expression of AtMYB68 and AtMYB36 revealed the same result as these RT-PCR data (Figure 4-3 and Figure 3-3-F). These data suggest that AtMYB36 may be functionally related to AtMYB68.  AtMYB68  1 MGRAPCCDKA 1 MGRAPCCDKA 1 MGRAPCCDKA  NVKKGPWSPE NVKKGPWSPE NVKKGPWSPE  EDAKLKDYIE EDAKLKSYIE EDVKLKDYID  NSGTGGNWIA NSGTGGNWIA KYGTGGNWIA  LPQKIGLRRC LPQKIGLKRC LPQKIGLKRC  50 50 50  YLRPNIKHGG  FSEEEDNIIC  100  YLRPNIKHGG  FSEEEENIIC  AtMYB36  51 GKSCRLRWLN  YLRPNIKHGG  FSEEEDRIIL  NLYVTIGSRW SLYLTIGSRW SLYISIGSRW  S11AAQLPGR  AtMYB84  51 GKSCRLRWLN 51 GKSCRLRWLN  TRLKKKLLNK  MKQE- -MVMVI  AtMYB84 AtMYB36  AtMYB68  ^R2  S 11AAQLPGR  100  SI IAAQLPGR  100  MEQQEMVIVMV1  KRQQQG KRQHQ  150 150  TDSTENNLSN  NNNNKSPQNL  150  AIMYB68 i o i  TDNDIKNYWN  AtMYB84 101  TDNDI KNYWN  TRLKKKLI NK  AtMYB36 101  TDNDI KNYWN  TKLKKKLLGR  QRKE-FQEAR QRKE-LQEAC QKQMNRQDSI  AtMYB68 151  QGQGQSNGST  DLYLNNMFGS  SP  WPLLPQLPPP  H H - - QI P - L G  200  AtMYB84 151  QQQI QT  SFMMRQDQTM  FT  WPLH  HHNVQVPALF  200  AtMYB36 151  SNSALEKLQL  HMQLQNLQSP  FSSFYNNPIL  WPKLHPLLQS  TTTNQNPKLA  200  AtMYB68 201  MV1EPTSCNYY  QTTPSCNLEQ  KPLITLKNMV  Kl EEEQERTN  PDHHHQDSVT  250  AtMYB84 201  RIKPTRFATK  KMLSQCSSRT  WSRSKIKNWR  KQTSSSSRFN  AtMYB36 201  SQESFHPLGV  NVDHQHNNTK  LAQINNGASS  AIMYB68 251  NPFD--FSFS  QLLLDPNYYL  AIMYB84 251  NAFDH- LSFS  QLLLDPNHNH  AIMYB36 251  HEFQPNFGFS  AtMYB68 301  SQQQEILQWF  AtMYB84 301  QWF  AtMYB36 301  UWS  AIMYB68 351  AGAAVRAALA  D  250  LYSENVEQSQ  NPA  250  GSGGGGEGDF  AIMSSSTNSP  LPNTSSDQHP  300  LGSGEGFSMN  SILSANTNSP  LLNTSNDN- -  300  QDLRLDNHNM  DFMNRGVS-K  ELFQVGNEFE  LTNGSS  300  GSSNFQTEAI  NDMFINNNNN  NNIVNLETIE  NTKVYGDASV  G- - NFQAETV  N  350  EEVELERKTT  SS  350  GGTTSTSADQ  STISWEDITS  LVNSEDASYF  NAPNHV  350  396  AtMYB84 351  LFS  GASTSTSADQ  STISWEDISS  LVYSDSKQFF  396  AtMYB36 351  S  SWGSASVLDQ  TTEGVTVMLQD  YAQMSYHSV-  396  Figure 5-14. Alignment of AtMYB68 with the other Two Members of Phylogenetic Sub-Group 14. The shaded areas indicate the conserved amino acids. R2 and R3 are the two conserved repeats in the M Y B DNA-binding domain. The underlined sequences indicate conserved motifs outside the M Y B DNA-binding domain.  specific gene  A1MYB68  JSSrRNA  AIMYB84  At MYB 3 6  imé  UJ  L*#  V«#  Figure 5-15. RT-PCR Analysis of Expression Patterns of Three Members in the A t M Y B Phylogenetic Sub-Group 14. Total R N A extracted from Col seedlings and mature organs was subjected to reverse transcription followed by PCR amplification. The upper band is the gene specific band. The lower band is 18S rRNA used as an internal control for RT-PCR. Latter, marker lane.  5.7 Discussion  In this chapter, I described the uses of reverse genetic approaches (knockout of gene expression), and overexpression to determine the function of AtMYB68. However, neither approach revealed any obvious alterations in morphological phenotype under either normal growth conditions or experimental conditions tested. This result is actually consistent with major trends in functional studies of Arabidopsis MYB genes using reverse genetic approaches. In the study done by Meissner et al. (1999), of 47 insertion lines for 36 AtMYB genes identified, none exhibited obvious morphological changes, even in the lines in which gene transcription was reduced or abolished. The lack of phenotypic alterations in the mutants is probably due to functional redundancy among AtMYB genes. Functional redundancy has not been documented in plant MYB genes, but it has been illustrated in the MADS-box transcription factor gene family. MADS-box transcription factors are also encoded by a large gene family in plants. They control many aspects of plant development, most notably flower development, but also fruit and root development (Theissen et al, 2000). The MADS-box SHATTERPROOF genes, SHP1 and SHP2 are both required for fruit dehiscence in Arabidopsis. SHP1 and SHP2 share 87% identity in their amino acid sequences, and have similar expression patterns. Single mutations in either of the two genes did not cause phenotypic changes, but the shplshp2 double mutant resulted in fruits failed to dehisce (Liljegren et al., 2000). Another remarkable example of functional redundancy within the MADS-box gene family is that of the SEPALLATA genes, which are required for the function of some organidentity genes. SEP1, SEP2 and SEP3 are three members closely related to each other. Single insertion mutations or even double mutants caused only subtle changes in phenotype. However, the triple mutant displayed a novel feature: all four flower whorls converted to sepals (Pelaz et al., 2000). From sequence analysis of the Arabidopsis MADS-box genes, it is estimated that more than 40% of the genes might have redundant (or partially redundant) functions (Riechmann and 105  Ratcliffe, 2000). Therefore, the examples in the MADS-box gene family should not be regarded as exceptions and a similar scenario might exist in the AtMYB gene family as well. Studies of MADS-box genes revealed that the genes with redundant functions are usually similar in sequence and related to each other in phylogenetic trees (Riechmann and Ratcliffe, 2000). As reviewed in Chapter 1, the same trend has been shown in functional studies of plant MYB genes as well, such as TT2 (AtMYB123) and ZmMYBCl in the phylogenetic subgroup 5, and PhMYBAN2, PAP1 (AtMYB75) and PAP2 (AtMYB90) in the subgroup 6. Therefore, in order to determine if genes in the same phylogenic subgroup as AtMYB68 could be functionally similar, I examined expression patterns of genes in this subgroup. M y results showed that at least one more member of this subgroup, AtMYB36, had an expression pattern similar to AtMYB68. The member most closely related to AtMYB68, AtMYB84, had a different but overlapping expression pattern. MPSS data shows that AtMYB36 expression is root-specific in the organs examined, and it reveals another root-preferential gene in this subgroup, AtMYB38. These data support our speculation that the members in this subgroup may not only have related sequences, but also related functions. So far, functional characterizations of members in this subgroup have not been reported. We are not able to infer the AtMYB68 function based on known examples. Another possibility for absence of observed phenotypic alterations in the AtMYB68 mutant lines might be due to the limited number of assays that were performed. In the systematic screen for AtMYB mutations by reverse genetics (Meissner et al., 1999), 26 transposon or TD N A insertion lines, in which many lines had reduced or abolished mRNA levels, were examined under different growth conditions based on plate and greenhouse assays. However, only a minority of these lines revealed discernable changes in response to various stimuli. Since there is no evidence that any of the 125 R2R3-type AtMYB genes represents a pseudogene, there must be a function associated to each gene. To find conditions under which gene mutants display phenotypes is a continuing challenge. It is worth noting that in screening for a T-DNA insertion in AtMYB68, we found a total of 3 insertion lines (including the one lost in the Wisconsin collection). Only one of them had an  insert in the ORF, while the other two insertions were in the promoter region >200 bp upstream of the A T G codon. The study by Meissner et al. (1999) had similar results. Only 22 of 47 insertions (including 40 transposon insertion lines and 7 T - D N A insertion lines) were within ORFs. None of 7 T - D N A insertions was located in ORFs, and most of them were within promoter regions >500 bp upstream of translation start codons. These data reveal that efficiency to obtain the knockout insertions with this technique may be low. In this study, although the insertion in the first intron of AtMYB68 reduced the gene expression to an extremely low level, AtMYB68 expression was not totally abolished. This was also observed by Meissner et al. (1999), who found that many insertions, even those in transcribed regions, only reduced but did not abolish gene expression. Although to date the function of AtMYB68 is still unknown, the work presented here definitely provides a good base for future studies. As more and more insertion collections from Arabidopsis are obtained, there is an increasing chance to find a knockout mutation for any particular gene. Besides insertional mutagenesis, other reverse genetics strategies, such as TILLING, are also being used in Arabidopsis (McCallum et al., 2000). Therefore, one strategy is to keep searching for knockouts in members of the same subgroup as AtMYB68, and then to cross the knockouts, such as atmyb36 and atmyb38, to create double or even triple mutants to reveal the function of AtMYB68 and related gene family members. Recently, microarray technology has been developed, which allows the parallel monitoring of expression of thousands of genes, and some Arabidopsis gene chips are commercially available. Using such a chip, it should be feasible to detect global differences in gene expression in the atmyb68 knockout line 94B-18 relative to that in wild-type. 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