IDENTIFICATION AND FUNCTIONAL ANALYSIS OF KEY REGULATORS DURING RICE CROWN ROOT DEVELOPMENT

Abstract

The plant root system is composed of the primary embryonic roots and various post-embryonic roots for its essential functions of water and nutrients absorption, anchorage, food storage, and also provides an interface for plant-microbe interaction. In rice (Oryza sativa), the mature root system has a short pole-borne primary/seminal root and shoot-borne adventitious roots (also called crown roots) and root-borne lateral roots. Crown roots from the major part of the mature rice root architecture. The origin of post-embryonic roots is highly diverse in plant species. Lateral roots in Arabidopsis originate from the xylem pole pericycle cells of the primary root. In contrast, lateral root in rice originates from endodermal and pericycle cells located opposite to protophloem. Similarly, the adventitious roots in Arabidopsis naturally originate from the pericycle cells at the xylem pole. In contrast, in rice, crown roots arise from the innermost ground meristem cells peripheral to the vascular cylinder at the stem base. Various events of crown root development have been divided into seven stages, starting from an initial cell establishment for crown root primordia until its emergence. To dissect out the molecular determinants and genetic networks during rice crown root development, we have categorized these developmental events into two broader stages, early stage and late stage. During the early stage, crown root primordia are established by the specification of crown root primordia initial cells, which eventually undergo the process of anticlinal and periclinal cell divisions to produce initials for epidermis-endodermis, root cap, and central stele. These initials then differentiate into the epidermis, endodermis, root cap, and stele tissues. The endodermal cells also produce a cortex layer via periclinal cell division. During late-stage, further tissue differentiation leads to the establishment of the fundamental organization of root primordia. Columella cells are formed at the root cap, and vascular tissues are established in the central region. Once crown root primordia are about to emerge out, cells at the basal region of the primordia initiate cell elongation and vacuolation. We have used laser capture microdissection (LCM) to collect cells from early and late-stage crown root primordia. Tissues from several early and late-stage primordia were collected. Since the innermost ground tissues peripheral to the vascular cylinder have the competency to initiate a crown root-specific developmental program when proper cues are perceived, we collected these tissues as background control tissues. Next, to uncover the modulations in dynamics of gene expression pattern and gene regulatory network instrumental for crown root primordia specification/differentiation and tissue organization, we performed RNA-seq of total RNA extracted from crown root primordia vi collected by LCM (LCM-seq). Genome-wide analysis of early and late developmental stages of crown root primordia revealed that genes with their putative functions in epigenetic regulation, transcriptional gene expression, and cell cycle were specifically induced in early crown root primordia. In contrast, genes of protein synthesis, protein metabolism, and cell division/differentiation were induced in later stage crown root primordia. We further confirmed the spatial-temporal regulation of a subset of genes identified in LCM-seq during crown root primordia development by RNA in-situ hybridization. Plant hormones like auxin and cytokinin play an important role during rice crown root development. We have studied the effects of auxin and cytokinin during root development and observed that auxin and cytokinin have antagonistic effects on CR initiation but similar effects on their growth. In a collaborative project with Prof. Mukesh Jain, JNU, New Delhi, we have globally identified genes whose expression is regulated by auxin and cytokinin in crown tissues. This transcriptome analysis revealed that significantly large numbers of transcription factors were de-regulated upon hormonal treatment. We studied tempo-spatial expression patterns of selected genes by RNA-RNA in situ hybridization in developing crown root primordia. Our analysis has identified some transcription factors which are specifically expressed in developing crown root primordia. Moreover, we have also analysed auxin responsiveness of the genes identified from our LCM-seq studies and validated their regulation by auxin for some by qRT-PCR. Finally, we have shown the role of an auxin-inducible crown-root expressed transcription factor of the WOX gene family by down-regulating its endogenous expression in the transgenic rice lines. Thus, our overall LCM-seq analysis together with tempo-spatial expression pattern studies not only reveals the genetic networks operating during the early and late stage of crown root primordia but also demonstrate hormonal regulation of crown root primordia abundant genes during rice crown root development.