FUNCTIONAL STUDIES OF AP2/ERF GENES DURING ADVENTITIOUS ROOT DEVELOPMENT IN RICE

Abstract

The Root system architecture (RSA) in plants comprises mainly of two types of root systems- Tap Root system and Fibrous Root system. The dicot plants have tap root system comprising mainly of primary and lateral root. Most Dicots have a tap root system, while monocots have a fibrous root system also known as an adventitious root system or crown root (CR). A fibrous root system which comprises mainly of CR forms a dense network of roots adjacent to the soil surface. The rice being monocot, root system mainly consists of primary root, crown and lateral roots, we are focusing our study on the development of stem-borne adventitious root or crown roots which are dedifferentiated stem tissue in the due process of attaining new function of root. AP2 domain containing transcription factors (TF) are known to regulate growth, development and metabolism in plants. In CR development also, OsAP2/ERF TFs are involve in its development phases. In first part of my study, I have functionally characterized a single AP2-domain transcription factor, OsAP2/ERF-27 using molecular genetics based approaches. By performing RNA in situ hybridization, I investigated dynamic spatial expression pattern of OsAP2/ERF-27 in developing crown root primordia (CRP). Further, by generating loss-of-function (knock-out) and inducible over-expression transgenic rice lines for OsAP2/ERF-27, I uncovered developmental stage-specific function of OsAP2/ERF-27 in establishing root system architecture. My study on function of OsAP2/ERF-27 indicate that it acts downstream of auxin signalling pathway and is specifically expressed in developing CR primordia outgrowth, associating its crucial role during development of CR. Rice lines with CRISPR knock out OsAP2/ERF-27, showed late emergence of adventitious root phenotype with a lesser number of crown roots and increased primary root length at the early stage after germination. Our studies on exogenous effect of IAA, show that dynamic auxin level modulate the expression of OsAP2/ERF-27. All together our studies on these behavioural changes of expression pattern provide convincing evidence for multi-layered regulatory interactions between transcription factor OsAP2/ERF-27 and auxin signalling, instrumental in CR development at early and later stages in rice. In our study, we showed that OsAP2/ERF-27 is necessary for crown root emergence and elongation in rice. As plant-specific transcription factor, many AP2/ERFs contribute in numerous biological and physiological developments and participate in various regulatory roles, such as vegetative, reproductive growth, and also determines crop yield and adaptation in stress condition. In our study, we summarize the functions and prospective applications of OsAP2/ERF-27 in rice. Metabolite profiling are considered as one of the most utilitarian approaches to detect all metabolites and low-molecular-weight molecules in a biological sample. In our study, the seed metabolic profile of rice OsAP2/ERF-27 mutant, and wild type were analysed via widely targeted metabolomics. Several key metabolites were identified and analysed metabolites via PCA and PLS methodology; these metabolites found in mutants responded significantly to promoted seed quality. In the second part of work, I have investigated functions and genetic interaction of couple of two AP2-domain containing transcription factors OsPLT2, as a unique and redundant function in crown root development. As OsPLT2 and OsPLT5 are the closest homolog of Arabidopsis AtPLT5, which functions redundantly with AtPLT3 and AtPLT7 in LR formation. We have functionally characterized OsPLT2 and OsPLT5 in LR, CR formation in rice and their functional redundancy. Osplt2 mutant showed less LR phenotype while double mutant Osplt2Osplt5 exhibited short root (both PR and CR) with less LR as additive phenotype. Overexpression of OsPLT2 lead to high density lengthy LR formation from both PR and CRs. Importantly, the CR number was not significantly affected. This observation indicates that PLT2 has conserved function in regulating root-borne LRs but not in shoot-borne CR formation. This result suggests that PLT-like genes have acquired a species specific expression domain and the function of proteins is conserved, i.e. to promote root primordia irrespective of their developmental origin. This observation indicates that PLT2 has conserved function in regulating root-borne LRs. To conclude, I have uncovered functions of OsAP2/ERF-27, OsPLT2 and OsPLT5 in regulating root architecture in economically important crop plant.