Characterization of Arabidopsis pyrabactin-like ABA receptor (py14 and py17) and transcription factor (rav and abi5) activities in transiently transformed Nicotiana benthamiana and stable transgenic lines of cotton (Gossypium hirsutum)



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Abscisic acid (ABA) is a plant hormone that regulates numerous developmental processes and adaptive stress responses in plants. Although many ABA signaling components have been identified, understanding in the functional and structural conservation of ABA signaling networks across species remains to be elucidated. The family of homologous proteins encoding pyrabactin resistance (PYR), pyrabactin resistance 1-like (PYL), and regulatory component of ABA receptor (RCAR) have been discovered by forward genetic screens in Arabidopsis and shown to function as ABA receptors.

The main objective of this study is to test the functions of select PYL ABA receptors and downstream transcription factors in heterologous systems (cotton, tobacco) to gain insight into conserved ABA signaling modules in plants. The transient expression of Arabidopsis effectors of ABA signaling in .Nicotiana benthamiana and upland cotton, Gossypium hirsutum, was undertaken to gain deeper insights into the utility of ABA receptors and transcription factors for genetic engineering of agronomic traits such as stress adaptation or stress avoidance to improve yields and seed value. The potential significance is a deeper understanding of efficacy and molecular mechanisms of deeply conserved ABA gene effectors. I found via RNA blot analysis that AtPYL7 overexpression results in agonist (inductive) ABA signaling activity in N. benthamiana. AtRAV1, a known ABA transcription factor (TF) and positive effector of ABA sensitivity also showed similar agonist activity on endogenous ABA-inducible gene expression of Late Embryogenesis Abundant (LEA) in N. benthamiana. The results were strengthened by comparative data analysis across three endogenous LEA target genes with significant inductive correlations across genes that were of similar magnitude of agonist activity as exogenous ABA treatment of N. benthamiana leaves. I further correlated protein expression for these effectors to functional effects via immunoblots. These data together at the transcriptional and translational levels support the claim that Nicotiana benthamiana transient assays are a robust and facile system to functionally characterize the various effectors of ABA signaling pathways and study their molecular interactions.

In previous studies from the Rock lab, transgenic cotton (Gossypium hirsutum L.) that over-express cDNAs encoding the Arabidopsis transcription factors Related to ABA-Insensitive3/Vivivparous1 (AtRAV1/2), encoding a B3 domain class TF,and ABA-Insensitive5 (AtABI5), a bZIP class TF, showed improved photosynthesis in the field and greater root and leaf biomass under deficit irrigation. We investigated a subset of these transgenic lines to further characterize RAV1 and ABI5 expression at the protein level in seeds and to address mechanisms of drought tolerance and adaptation by morphological and physiological assays of cotyledonary leaves and greenhouse-grown plants subjected to drought stress. I hypothesized that the transgenic lines may have enhanced responses to abscisic acid (ABA), resulting in greater water use efficiency under drought stress. I measured stomatal density whereas Dr. Fiene and collaborators measured absolute and relative sizes (i.e. pore area/guard cell area) of guard cell apertures. I characterized transgene expression in seeds by immunoblot. AtRAV1 cotyledons had significantly higher stomatal densities, and 26% smaller guard cell apertures than control line Coker312 under drought stress, raising the possibility that smaller guard cell pores and greater stomatal densities may contribute to water use efficiencies measured in AtRAV1 plants in the greenhouse and field. These results are consistent with the hypothesis that over-expression of AtRAV1 resulted in an ABA-hypersensitive phenotype manifest as lower levels of endogenous ABA in cotyledons associated with greater reductions in pore apertures during stress and increased stomatal density. Our results further substantiate the potential for engineering drought tolerance in agricultural crops such as cotton by over-expression of AtABI5 and AtRAVs.



Abscisic acid (ABA), Pyrabactin resistance, Pyrabactin resistance 1-like, Late embryogenesis abundant, Type 2C protein phosphatases, Related to ABA-Insensitive3/Vivivparous1