Map-based Cloning And Functional Analysis Of TWI1 Controlling Vascular Development In Rice

Leaf veins are distributed in the mesophyll(MS)tissues and play important roles in the transport of minerals,water,photosynthetic products and provide physical support for leaves.Rice veins are parallel veins and consist of midrib vein,large veins,and small veins.The density of small veins(The number of small veins per unit leaf width)is particularly important for the transport of photosynthetic products.C4 crops have a higher density of small veins than C3 plants,and thus have a higher rate of photosynthetic products transport.Study of the molecular mechanisms underlying the regulation of rice vein density,it will help solve the basic scientific problems,such as the revolution and development of vascular bundles,and more importantly,can lay a solid theoretical foundation to enhance rice photosynthetic efficiency through modifying the small vein density and engineering C4 like vasculature structures in rice.In this study,a C4-like mutant was obtained through T-DNA mutant library screening,TWI1 gene was cloned through map-based cloning,histological analysis was conducted to investigate the structure that caused the mutant phenotype,and the molecular mechanism underlying the TWI1 regulating small vein differentiation was revealed.Main conclusions are as below:1.To investigate the molecular mechanism underlying cell fate specification in rice leaves,a rice T-DNA insertion mutant library(Oryza sativa L.ssp.japonica variety Nipponbare)was screened for abnormal leaf shape.There are normally nine small veins per 2mm leaf in width in Oryza.Sativa L.japonica.Mutant with 14 small veins in the 2mm leaf in width was obtained,and the mutant also showed twisted leaf,so named as twi1(twisted-leaf1).Histological analysis indicates MS was absent,bundle sheath(BS)cells failed to develop an integrated layer surrounding the vasculature,minor veins failed to form sclerenchyma cells in twi1 leaves,and finally resulted higher photosynthesis efficiency in twi1 mutant.2.TWI1 was cloned through map-based cloning,and sequence analysis showed that TWI1 encodes a 591-amino acid RADICAL-INDUCED CELL DEATH protein with RCD-SRO-TAF4 conserved domain;as to a single base substitution(T to A)in the first exon in the twi1 allele,resulting in a premature termination codon;and TWI1 is a transcription factor binding protein,localized in the nuclear and the cytoplasm.Expression profile analysis showed that TWI1 expressed in the young seedling,mature root,stem,leaf,flower and glume,and higher expression was detected in the young seedling and mature leaves.A complementation analysis was next performed to confirm the genetic role of twi1 in the observed twisted leaf phenotype.The rescued leaf shape was observed in the TWI1 expressing twi1 plants,thus confirmed the genetic role of twi1 in the observed twisted leaf phenotype.3.Yeast-two hybrids analysis showed that TWI1 interacts with OSH15,and the interaction was verified by BiFC and Co-IP,also RST domain of TWI1 and HD domain of OSH15 are necessary and sufficient for OSH15 binding.Utilizing 2A self-cleaving,we showed that OSH15 has the capacity to move cell to cell through plasmodesmata and this capacity is compromised in the presence of TWI1.Meanwhile,we confirmed this by trichome rescue assay,TWI1 and RST could inhibit the cell-to-cell trafficking of both GL1-KN1 and GL1-OSH15,thereby preventing trichome development,whereas in the presence of TW1-?C trichome development could still occur,which supports the hypothesis that OSH15 is a non-cell-autonomous protein whose trafficking ability is controlled by TWI1.4.Immuno-histological analysis of median longitudinal sections of the shoot apex indicated that,in WT tissues,signal was present in the flanks of the SAM and at the boundary of the SAM and leaf primordia.While in the twi1 mutant,signal was present in both the SAM and leaf primordia,with leaf primordium 2 having ectopic OSH15 in almost all cells,which confirmed that the presence of TWI1 is responsible for the mutant phenotype.In contrast to the twi1 mutant,twi1/osh15 double mutant and transgenic twi1/RST and twi1/OSH15 RNAi rice lines all exhibited WT leaf morphology,the presence of TWI1 is necessary to control the cellular distribution of OSH15 in developing rice leaves,and TWI1 play important roles in the lignification and specification of vascular bundle cells,which claimed the function of TWI1 gene in the regulation of small vein initiation,formation,anatomical structure development and density in rice.