AUXIN-CYTOKININ RESPONSES AND FUNCTIONAL SIGNIFICANCE OF AUXIN DURING RICE CROWN ROOT DEVELOPMENT

Abstract

Rice root system is a fibrous root system, composed of primary/seminal, adventitious/crown and lateral roots. Proper establishment of rice root system is a key determinant of productivity. Plant hormones such as auxin and cytokinin have been shown to play critical role in during crown root (CR) development but the dynamic responses and interactions between these hormones have not been extensively studied during crown root differentiation. The broad aim of this thesis was to study responses of auxin and cytokinin signaling and explore deeper insight on the functional significance of auxin during rice crown root development. The study first focused on identifying auxin and cytokinin responsive domains during various stages of CR development. Auxin and cytokinin responsive synthetic promoter::reporter constructs, DR5::erYFP and TCSn::erGFP, were generated for monitoring auxin and cytokinin responses, respectively. Stable transgenic rice lines generated raised with these constructs were used to monitor hormonal responses in rice stem base containing developing CRs. Using RNA-RNA in situ hybridization and fluorescence analysis, we observed show that in the early crown root primordia, auxin response is abundant in the early root cap cells of the root tips whereas TCSn::erGFP signals were relatively broader in the root tips. Importantly, TCSn::erGFP signals were significantly less in the auxin responsive domains in the root tip. Auxin-cytokinin cross talk is well established during root development in plants. During CR development, we also identified some narrow domains with overlapping auxin and cytokinin responses that might be a domain where auxin and cytokinin signaling directly interact with each other. Next, in order to understand the functional importance of auxin in the auxin-cytokinin interaction domain, we genetically altered active pool of endogenous auxin in the cytokinin responsive domain by expressing auxin inactivating gene, OsMGH3 using TCSn promoter. We observed that reducing active auxin resulted pleiotropic abnormalities such as stunted plant growth, loss-of apical dominance and root architecture in the transgenic plants, indicating key role of auxin and cytokinin interaction during CR development. Further, to dissect out mechanism of auxin-regulated crown root formation, we selected two plant-specific AP2-domain containing specialized transcription factors (OsAP2/ERF-40 and OsAP2/PLT3) for functional studies using reverse genetics-based approaches. RNA in situ hybridization and RT-PCR analyses showed that both genes are specifically expressed in the developing crown root primordia. Interestingly, and the expression of OsAP2/ERF-40 was induced upon exogenous auxin treatment induced whereas OsAP2/PLT3 was not affected in the rice stem base upon auxin treatment. We show x that down-regulation and ectopic over-expression of OsAP2/ERF-40 in the transgenic rice display defects in the crown root development in the transgenic rice. These, together with the regulatory gene expression analysis revealed that OsAP2/ERF-40 is sufficient to induce adventitious root and to trigger the root developmental program in a dose dependent manner. OsAP2/PLT3 is a member of PLETHORA gene family, key regulators for stem cell specification and maintenance in the root apical meristem. High genetic redundancy in the PLT family compelled us to use mis-expression approach to decipher the function of OsAP2/PLT3. We observed that ectopic over-expression of OsAP2/PLT3 causes defects in root architecture, radial growth, leaf angle and plant fertility. Overall, our studies not only reveal specific and overlapping auxin and cytokinin response domains but also provide evidence for two novel transcription factors regulating adventitious/crown root developmental program, a key agronomically important quantitative trait.