Functional analyses of EMP2 and HSBP2, paralogous HEAT SHOCK FACTOR BINDING proteins in maize.

Plants exhibit an evolutionarily conserved series of molecular/biochemical responses to heat stress, which is essential to the survival of these sessile organisms. This heat shock response (HSR) entails the upregulation of numerous genes encoding heat shock proteins (HSPs); alleviation of heat stress correlates with rapid attenuation of this transcriptional activation. Heat stress related crop losses to US agriculture average more than $12 billion annually. Despite this impact on crop production, the mechanisms of HSR regulation are inadequately understood. Previous analyses in animal systems suggested that the coiled-coil domain protein HEAT SHOCK BINDING PROTEIN1 (HSBP1) negatively-regulates the expression of hsps during attenuation of the heat shock response via its interaction with the HEAT SHOCK transcription FACTOR1 (HSF1). The absence of hsbp1 mutants in animal systems, however, has hindered genetic analyses of HSBP1 function. Maize contains two HSBP1 paralogues, EMPTY PERICARP2 (EMP2) and HEAT SHOCK BINDING PROTEIN2 HSBP2), which show differential accumulation during maize development and heat stress. Mutations in emp2 are embryo lethal, and lethality of emp2 mutant embryos correlates with an unregulated HSR and overexpression of maize hsps. Clonal sectors of emp2 mutant tissue reveal that EMP2 is not required for regulation of hsp transcription in post-embryonic tissues, whereas the HSBP2 protein is upregulated in heat-stressed seedlings. EMP2 and HSBP2 exhibit gene-specific, non-overlapping interactions with distinct maize HSFs in yeast. We hypothesize that EMP2 and HSBP2 may perform non-redundant, tissue-specific functions during negative regulation of the maize HSR. Molecular genetic and biochemical dissection of non-redundant HSBP function in maize are testing this model, and will provide novel insight into the regulation of the HSR in an important crop plant.

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Recessive mutations in the maize discolored1 (dsc1) locus prevent normal kernel development.

Embryo growth in homozygous dsc1- kernels is delayed compared to wild type kernels, at maturity most of the embryonic tissue is degraded. The development of endosperm tissues is also abnormal in dsc1 mutant kernels. A portion of the dsc1 gene was isolated by transposon tagging and reveals sequence homology to the VAN3 gene of Arabidopsis, which encodes an AdP Ribosylation Factor GTPase Activating Protein (ARF-GAP). ARF-GAP proteins are evolutionarily conserved proteins involved in the endocytic pathway and vesicle trafficking. Currently, we are working to identify the entire dsc1 locus and its function during maize kernel development. We are especially interested in understanding the molecular basis for the disparate phenotypes of dsc1 and van3 in maize and Arabidopsis.

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SEMAPHORE1 is required for auxin transport and knox gene regulation.

Recessive mutations in semaphore1 result in the ectopic expression of knox genes in leaf and endosperm tissue, as well as reductions in polar auxin transport. Genetic analyses suggest that SEMAPHORE1 may regulate knox gene expression in a different developmental pathway than ROUGH SHEATH2, the first-identified regulator of knox gene expression in maize. Mutations at semaphore1 are pleiotropic, disrupting specific domains of the shoot. However, unlike previously described mutations that cause ectopic knox gene expression, semaphore1 mutations affect development of the embryo, endosperm, lateral roots, and pollen as well as leaves and vasculature. Moreover, polar transport of the phytohormone auxin is significantly reduced in semaphore1 mutant shoots. The data suggest that many of the pleiotropic semaphore1 phenotypes result from defective polar auxin transport (PAT) in sem1 mutant shoots, and support models correlating down-regulated knox gene expression and PAT in maize shoots. In collaboration with Frantisek Baluska (U Bonn), cell biological investigation have shown that auxin (IAA) distrubution and F-actin localization are disturbed in semaphore mutant root cells, and accumulation of maize PIN (a putative auxin efflux protein) proteins is decreased. Efforts are directed now toward map-based cloning the semaphore gene, and to generate additional mutant alleles

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