Structural organization of organs in multicellular organisms occurs through intricate patterning mechanisms that often involve complex interactions between transcription factors in regulatory networks. For example, INDEHISCENT (IND), a basic helix-loop-helix (bHLH) transcription factor, specifies formation of the narrow stripes of valve margin tissue, where Arabidopsis thaliana fruits open on maturity. Another bHLH transcription factor, SPATULA (SPT), is required for reproductive tissue development from carpel margins in the Arabidopsis gynoecium before fertilization. Previous studies have therefore assigned the function of SPT to early gynoecium stages and IND to later fruit stages of reproductive development. Here we report that these two transcription factors interact genetically and via protein-protein contact to mediate both gynoecium development and fruit opening. We show that IND directly and positively regulates the expression of SPT, and that spt mutants have partial defects in valve margin formation. Careful analysis of ind mutant gynoecia revealed slight defects in apical tissue formation, and combining mutations in IND and SPT dramatically enhanced both single-mutant phenotypes. Our data show that SPT and IND at least partially mediate their joint functions in gynoecium and fruit development by controlling auxin distribution and suggest that this occurs through cooperative binding to regulatory sequences in downstream target genes.

SUMMARYThe Arabidopsis gynoecium is a complex organ that facilitates fertilization, later developing into a dehiscent silique that protects seeds until their dispersal. Identifying genes important for development is often hampered by functional redundancy. We report unequal redundancy between two closely related genes, SPATULA (SPT) and ALCATRAZ (ALC), revealing previously unknown developmental roles for each. SPT is known to support septum, style and stigma development in the flower, whereas ALC is involved in dehiscence zone development in the fruit. ALC diverged from a SPT-like ancestor following gene duplication coinciding with the At-b polyploidy event. Here we show that ALC is also involved in early gynoecium development, and SPT in later valve margin generation in the silique. Evidence includes the increased severity of early gynoecium disruption, and of later valve margin defects, in spt-alc double mutants. In addition, a repressive version of SPT (35S:SPT-SRDX) disrupts both structures. Consistent with redundancy, ALC and SPT expression patterns overlap in these tissues, and the ALC promoter carries two atypical E-box elements identical to one in SPT required for valve margin expression. Further, SPT can heterodimerize with ALC, and 35S:SPT can fully complement dehiscence defects in alc mutants, although 35S:ALC can only partly complement spt gynoecium disruptions, perhaps associated with its sequence simplification. Interactions with FRUITFULL and SHATTER-PROOF genes differ somewhat between SPT and ALC, reflecting their different specializations. These two genes are apparently undergoing subfunctionalization, with SPT essential for earlier carpel margin tissues, and ALC specializing in later dehiscence zone development.

SummaryThe SPATULA (SPT) gene is involved in generating the septum, style and stigma: specialized tissues that arise from carpel margins. By matching sequences within the extended bHLH region of AtSPT across species databases, twelve orthologues were identified in eudicots, rice and a gymnosperm. Two conserved structural domains were revealed in addition to the bHLH region: an amphipathic helix and an acidic domain. These are conserved in the tomato orthologue, which can restore carpel function to spt mutants of Arabidopsis. The acidic domain is essential for SPT carpel function, and the amphipathic helix supports it. A bipartite sequence overlapping the bHLH domain is required for nuclear localization, and a mutation in the conserved beta strand adjacent to the bHLH C terminus results in the loss of SPT function. SPT apparently acts as a transcriptional activator, as the addition of the SRDX repression domain phenocopies the spt mutant phenotype. Expression of an artificially activating 35S:SPT-VP16 construct can induce carpelloid properties in sepals, and new defects in the gynoecium. These disruptions are associated with ectopic expression of the STYLISH2 gene, although STYLISH2 expression does not require SPT function. Ectopic expression of unmodified SPT does not induce such changes, implying that SPT acts in association with essential coactivators present only in regions where SPT is normally active. Because the VP16 activation domain can compensate to some extent for the loss of the amphipathic helix and acidic domain, these domains may normally interact with such co-activators.