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Studies on Arabidopsis MYB transcription factor genes : potential regulators of the phenylpropanoid pathway and analysis of the root preferentially expressed gene AtMYB68 Wang, Qing

Abstract

Genes encoding MYB 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-DNA 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 IAA ~2-fold, and by ABA ~3-fold. In order to determine AtMYB68 function, transgenic lines overexpressing and underexpressing AtMYB68 were generated in Arabidopsis. In addition, by searching several T-DNA 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.

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