UNDERSTANDING FUNCTIONS OF AP2-DOMAIN CONTAINING TRANSCRIPTION FACTORS IN RICE

Abstract

Plants produce various organs throughout their life cycle, and the process of forming any new organ is known as organogenesis. Plant organogenesis requires cellular reprogramming, associated with dedifferentiation, pluripotency acquisition, cell cycle reactivation, new cell fate acquisition, and re/trans-differentiation processes. The root formation in plants is one of the best suitable systems for studying cellular reprogramming in plants. In higher plants, two types of root systems can be observed: the tap and the fibrous root systems. The dicots, for example, Arabidopsis thaliana, a dicot model plant, contains the tap root system. The tap root system comprises an embryonic-derived primary root (PR) and root-borne post-embryonic lateral roots (LRs). The monocots, including cereals, contain the fibrous root system comprising shoot borne crown roots (CRs), brace roots (BRs), root-borne LRs, and an embryonic-derived PR. It is believed that the PR in cereals is short-lived, and they depend on the shoot-borne roots that make up a major root system along with LRs in lateral stages. The shoot-borne root formation that is highly relevant to cereal crop production is not naturally present in Arabidopsis, thus all of its knowledge cannot be transferable to the other plants. Rice (Oryza sativa) belongs to a grass family called Poaceae and is considered to be a perfect model for cereal crop research because of its relatively small genome size. Also, a well established and highly efficient transformation system, extensive genetic resources, and synteny with the sequences of other cereal crops strengthen rice as a model organism. Rice is a staple food crop and feeds a significant population worldwide. The rice develops a fibrous root system that contains the post-embryonic shoot-borne CRs and root-borne LRs. An embryonic PR provides anchorage, water absorption, and nutrient uptake at the early stages of rice seedlings. As PR is short-lived, rice depends on post-embryonic roots to complete its life cycle. In the rice stem base, the inner ground meristem cells close to the vascular tissues initiate the CR formation in rice. Meanwhile, the LRs initiate from the endodermal and pericycle cells located opposite to the protophloem in rice. The post-embryonic root formation in rice requires cellular reprogramming. During crown root primordium (CRP) initiation, the innermost ground meristem cells (shoot cells) dedifferentiate and enter into cell division to produce CR initials. The initial cells further divide and differentiate into root meristems that emerged in the CRP outgrowth stage. Our previous laser-captured CRP transcriptome profiling study revealed that six PLETHORA (OsPLT) genes are sharply induced during CRP initiation and outgrowth stages in rice. In rice, OsPLT genes encode AP2-domain containing transcription factors. There are ten PLT genes present in rice and categorized into two subclades: clade A (OsPLT1~6) and clade B (OsPLT7~10). Based on the expression pattern and phylogenetic analysis from the literature, I have chosen OsPLT1, OsPLT6, OsPLT7, and OsPLT9 for their functional characterization in rice root development in this study. Using loss-of-function and gain function approaches, we have identified the functional role of these PLTs in rice. OsPLT1 positively regulates both CR and LR formation in rice. While osplt1 mutants produced fewer CRs and LRs, the OsPLT-GR overexpression plants produced a robust root system in rice. Further, the ectopic root formation from leaf tissues and the induced scutellum-derived callus formation were observed in OsPLT1-GR lines. It suggests that overexpression of OsPLT1 alone can induce root cellular reprogramming in rice. Next, the osplt6 mutants produced fewer CRs and LRs than the wild type. However, in OsPLT6-GR plants, we observed that the shoot and root tissues were converted into callus-like tissues. The scutellum-derived callus formation was also promoted in OsPLT6-GR plants. Further, the CR formation was induced in the OsPLT6 GR plants. Therefore, OsPLT6 plays a key role in differentiation and cellular reprogramming, regulating CR root development in a dose-dependent manner in rice. The phytohormone auxin regulates the OsWOX11-ERF3-OsRR2 module, which plays an important role in rice crown root development. Using qRT-PCR analysis, our study found that the expression of WOX11-ERF3-OsRR2 module was affected in osplt1 and osplt6 mutants. In contrast, the expression of these genes was significantly upregulated in overexpression lines consistent with the induced CR and LR formation in rice. Further, we observed that WOX11 and ERF3 expression were upregulated in OsPLT6 overexpression lines. Although the plants have been converted into a callus, the CR formation was promoted in these lines due to induction in the ERF3 gene expression. Therefore, OsPLT1 and OsPLT6 genes regulate the expression of the WOX11-ERF3-OsRR2 module during CR development in rice. Next, through ChIP-qPCR, we identified OsYUC3, an auxin biosynthesis gene, is the direct target of OsPLT1 in rice. We also identified that OsYUC3, OsYUC6, OsYUC7, and OsYUC9 genes were significantly upregulated in OsPLT6-GR plants. Therefore, OsPLT1 and OsPLT6 function upstream of the auxin signaling and regulate auxin-mediated developmental processes, including root development in rice. Further, we observed that the CR and LR development was impaired in OsPLT7 mutants. However, further studies are required to study the functional roles of OsPLT7 and OsPLT9 in rice.