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Interactions between members of two homeodomain protein families in Arabidopsis thaliana Pidkowich, Mark Stephen

Abstract

Recessive mutations in the BEL1I (BEL1) gene of Arabidopsis thaliana cause defective ovule development in which the inner integument fails to initiate and the outer integument develops abnormally. Thus, BEL1 is believed to be an important regulator of ovule morphogenesis. Interestingly, BEL1 is also expressed in several tissues which are apparently unaffected by a loss of BEL1 function. One attractive hypothesis is that additional BEL1-tike genes may function redundantly in these tissues such that loss-offunction mutations in BEL1 alone have no deleterious effects. Consistent with this hypothesis, BEL1 belongs to a family of twelve BEL1-Like Homeobox genes (BLH) in Arabidopsis. The predicted protein product of each BLH gene contains several domains which are shared between all members of the family. First, all BLH proteins contain an atypical TALE (Three Amino Acid Loop Extension) homeodomain which suggests that these proteins function as DNA-binding transcription factors. Second, all BLH proteins contain two domains, the SKY and BEL1 domains, which are predicted to mediate interactions with additional proteins. Therefore, all BLH proteins may function as heterodimers with similar protein partners. Third, all BLH proteins share sequences resembling a nuclear export sequence (NES) which suggests that subcellular localization may be a common mechanism of regulating BLH function. Expression analyses indicate that, like BEL1 and ATH1, four more members of the BLH family, BLH1-BLH4, are also expressed in several plant tissues. Thus, there is considerable potential for several BLH genes to be functioning with BEL1 in Arabidopsis. Moreover, in situ hybridization experiments demonstrate that BLH2 is expressed in floral apices in a manner that overlaps with BEL1 The function of an individual transcription factor can depend heavily on the formation of heterodimeric complexes with other proteins. Indeed, the sequence comparison suggests that each BLH protein contains at least two domains that could possibly mediate such protein-protein interactions. Interactions between TALE homeodomain proteins, and their influence in various developmental processes, have been well established in fungi and animals. Even though members of two different TALE subclasses in plants are important developmental regulators, the existence of interactions between plant TALE proteins has been previously unexplored. I have used the yeast two-hybrid system to demonstrate that BLH proteins can selectively heterodimerize with specific KNAT homeodomain proteins. Using a series of BEL1 deletion mutants, I further show that interaction requires BEL1 sequences N-terminal to the homeodomain. Analysis of the KNAT clones isolated in two-hybrid system screens using BEL1 as bait indicates that the MEINOX domain of KNAT proteins is sufficient to mediate interactions with BLH partners. These findings validate a previous hypothesis that the MEINOX domain has been conserved between plants and animals as an interaction domain for developmental regulators. Finally, the overlapping patterns of BLH2 and SHOOT MERISTEMLESS (STM) expression within the apex of the young floral primordium suggest the potential for BLH2/STM heterodimer function in determining the position of lateral organ formation.

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