FUNCTIONAL GENOMICS OF HORMONAL REGULATION OF CROWN ROOT DEVELOPMENT IN RICE

Abstract

The Oryza sativa (rice) root system is primarily composed of shoot-borne adventitious/crown roots (ARs/CRs) that develop from the coleoptile base, and therefore, it is an excellent model system for studying the shoot-to-root trans-differentiation process. The root system of monocot cereal plants is composed of seminal embryonic roots from the radicle and post-embryonic adventitious roots (ARs) from the coleoptile nodes. In rice, ARs arise constitutively from the basal coleoptile/stem nodes in a circular pattern, also known as nodal or crown roots (CRs). These post-embryonic shoot-borne CRs dominate the mature root system of the rice plant. Various phytohormones, transcription factors, and their interaction are a major determinants of various stages of CR development. Auxin is a key regulator of root organogenesis in plants, and an auxin maximum is a prerequisite for CRP initiation. Besides, it also regulates later stages of CRP outgrowth and CR emergence. Auxin signaling is activated by post-translational degradation of Aux/IAA repressor proteins that otherwise inhibit the activity of auxin response factors. In our previous work, we performed genome-wide identification and characterization of genes regulated by auxin and cytokinin in rice crown tissues. The study provided a list of global genes and transcription factors (TFs), which are commonly and specifically regulated by auxin and cytokinin. The data provide a rich source of fore-mining novel gene functions. To validate the list of differentially regulated genes upon auxin and cytokinin induction, we performed a qRTPCR analysis. We selected a list of a few genes belonging to important plant transcription factors family, hormonal biosynthesis and signaling, cell signaling, and transport genes, which are commonly regulated by auxin and cytokinin induction during CR development. These genes mainly have predicted functions as TFs or hormonal/cell signaling pathways. Next, using our RNA-Seq data, we prepared a list of genes specifically and solely regulated by auxin and cytokinin individually. The genes regulated by auxin belong to important plant transcription factors such as AP2/ERF, bHLH, ZIP, DOF, NAC, and HSF gene family, which have been shown to have a crucial role in the development, physiology, abiotic stress, and cellular signaling.However, there is no linear correlation between transcript and protein abundance. We reveal global changes in protein and protein phosphorylation in response to an auxin stimulus during CR development. Using a label-free, quantitative (phospho)proteomics-based approach, we have identified proteins whose abundance is altered and phosphorylated upon auxin treatment. The analyses of developing crown root primordia (CRP) and emerged CRs identified 334 proteins and 12 amino acids, respectively, that were differentially regulated upon auxin treatment. Gene ontology enrichment analysis of global proteome data uncovered the biological processes associated with chromatin conformational change, gene expression, and cell cycle that were regulated by auxin signaling. Spatial gene expression pattern analysis of differentially abundant proteins disclosed their stage-specific dynamic expression pattern during CRP development. Further, our tempo-spatial gene expression and functional analyses revealed that auxin creates a regulatory module during CRP development and activates ethylene biosynthesis exclusively during CRP initiation. Further, the phosphoproteome analysis identified 8,220 phosphosites, which could be mapped to 1,594 phosphoproteins and of which 66 phosphosites were differentially phosphorylated upon auxin treatment. Importantly, we observed differential phosphorylation of the cyclin-dependent kinase G-2 (OsCDKG;2) and cell wall proteins, in response to auxin signaling, suggesting that auxindependent phosphorylation may be required for cell cycle activation and cell wall synthesis during root organogenesis. Thus, our study provides evidence for the translational and posttranslational regulation during CR development downstream of the auxin signaling pathway. CR meristem provides an excellent system to study the factors and mechanisms operating to regulate pluripotency, cellular memory, and cell-fate transition in plants. One gene family that has been shown to have a role in meristem establishment and tissue differentiation is the PLETHORA (PLT) gene family in plants. To study the role of OsPLT3 in CR development, we used the miss expression approach and raised pUbi::OsPLT3-GR transgenic rice plants. Upon treatment with 1μM dexamethasone, we could induce the downstream targets of OsPLT3 resulting in a plethora of pleiotropic effects suggesting its role in a gain of pluripotency. Further, we used pUbi::dsRNAiOsPLT3 transgenic lines to study the effect of OsPLT3 on rice root architecture, pluripotency, and regeneration efficiency. The next gene that we studied for its functional characterization is OsAP2/ERF-40, its overexpression induces ectopic CR roots and elongated stem internodes, suggesting that it is sufficient to induce the CR developmental signaling in rice plants. Further, to investigate its role during CR meristem establishment and development, we employed the RNA-RNA interference technique. The pUbi::dsRNAiOsAP2/ERF-40 transgenic lines showed a reduction in CR number and we could observe the reduced number of transcripts in Line #1 in three biological replicates. Further, to identify downstream targets of OsAP2/ERF-40, we used the inducible construct pUbi::OsAP2/ERF-40-GR.