무 뿌리에서 이차 성장 및 기관 재생에 관여하는 형성층 조절자의 규명

학위논문 (석사)-- 서울대학교 대학원 : 자연과학대학 생명과학부, 2018. 8. 이지영.Raphanus sativus or radish is one of the most socioeconomically important root crops in northeast Asia. Radish root develops an edible thick storage root whose growth has been proven to be caused by cell division in the cambium. Although the radish genome has been studied and compared to other members of the Brassicaceae, its broad comprehension is yet to be completed. In this research, we looked at the radish root gene expression during its key development periods, 5, 7 and 9 weeks post seed planting, in two phenotypically different radish lines. We identified conserved cambium regulators as well as new cambium enriched genes that might play a novel function in the vascular cambium. Additionally, we discovered the ability of the radish cambium to reprogram itself to produce adventitious roots (AR). To further understand this process, we investigated how auxin induces AR formation in radish. We found that the auxin-induced WOX11/12 molecular pathway that produces AR in Arabidopsis, also plays an important role in radish AR formation, thus confirming the evolutionary conservation of the pathway. We also analyzed how cambium secondary growth regulators change their expression during AR organogenesis. Interestingly, we found that RsWOX4and RsPXY, key regulators of cambium cell division, change their expression pattern seemingly to aid the vascularization of the AR primordium. This thesis provides an overview of the molecular dynamics in radish taproot cambium, alternative functions of the cambium.Table of Contents Abstract ii List of Figures vii 1 Introduction 1 1.1 A brief introduction to the vascular cambium. 1 1.2 Raphanus sativus as a study model for secondary growth in roots. 5 1.3 Transcription regulators in the cambium and procambium. 8 1.4 Root organogenesis and its regulators. 13 1.5 Previous Research: Root Yield depend on the radial growth of radish 16 2 Materials and Methods 21 2.1 Plant materials and growth conditions. 21 2.2 RNA in situ hybridization 21 2.2.1 cDNA library preparation 21 2.2.2 Gene cloning 22 2.2.3 RNA probe preparation. 23 2.2.4 Tissue preparation 24 2.2.5 Slide pretreatment 25 2.2.6 Prehybridization and hybridization 25 2.2.7 Post hybridization washes 26 2.2.8 Signal detection 26 2.3 Toluidine blue staining 27 2.4 Imaging 27 2.5 Real-time quantitative PCR. 28 2.6 Scanning electron microscopy (SEM) 29 2.7 Primers list 30 2.7.1 Primers used for cloning 30 2.7.2 Primers used for probes 31 2.7.3 Primers used for qRT-PCR 34 3 Results 36 3.1 Cross-species comparison identifies cambium-enriched transcription factors in radish. 36 3.2 Gene selection for RNAseq validation. 38 3.3 Study of cambium enriched genes in Raphanus sativus. 40 3.4 Cambium gene expression dynamics. 44 3.5 Cross-line comparison provides thickening related gene candidates. 47 3.6 Wounded radish can be reprogrammed to produce adventitious roots. 50 3.7 Gene expression change during AR formation. 53 3.8 Enrichment of auxin pathway in the cambium. 56 3.9 Auxin induces adventitious roots formation in radish. 58 3.10 Auxin induces transcription factors related to AR formation. 60 3.11 In situ Hybridization suggest a reprogramming of cambium regulators during organogenesis 63 4 Discussion 68 4.1 Evolutionary conservation of cambium regulators 68 4.2 The vascular cambium of the Raphanus sativus showed capability to generate adventitious roots. 71 4.3 Auxin induces 2 transcriptional pathways during the transcriptional reprogramming of the vascular cambium for adventitious root formation in Raphanus sativus. 72 Bibliography 77 국문 초록 85Maste

Local auxin competition explains fragmented differentiation patterns

Trajectories of cellular ontogeny are tightly controlled and often involve feedback-regulated molecular antagonism. For example, sieve element differentiation along developing protophloem cell files of Arabidopsis roots requires two antagonistic regulators of auxin efflux. Paradoxically, loss-of-function in either regulator triggers similar, seemingly stochastic differentiation failures of individual sieve element precursors. Here we show that these patterning defects are distinct and non-random. They can be explained by auxin-dependent bistability that emerges from competition for auxin between neighboring cells. This bistability depends on the presence of an auxin influx facilitator, and can be triggered by either flux enhancement or repression. Our results uncover a hitherto overlooked aspect of auxin uptake, and highlight the contributions of local auxin influx, efflux and biosynthesis to protophloem formation. Moreover, the combined experimental-modeling approach suggests that without auxin efflux homeostasis, auxin influx interferes with coordinated differentiation

Local auxin competition explains fragmented differentiation patterns

Trajectories of cellular ontogeny are tightly controlled and often involve feedback-regulated molecular antagonism. For example, sieve element differentiation along developing protophloem cell files of Arabidopsis roots requires two antagonistic regulators of auxin efflux. Paradoxically, loss-of-function in either regulator triggers similar, seemingly stochastic differentiation failures of individual sieve element precursors. Here we show that these patterning defects are distinct and non-random. They can be explained by auxin-dependent bistability that emerges from competition for auxin between neighboring cells. This bistability depends on the presence of an auxin influx facilitator, and can be triggered by either flux enhancement or repression. Our results uncover a hitherto overlooked aspect of auxin uptake, and highlight the contributions of local auxin influx, efflux and biosynthesis to protophloem formation. Moreover, the combined experimental-modeling approach suggests that without auxin efflux homeostasis, auxin influx interferes with coordinated differentiation