| Systematic networks of leaf vein vascular strands play important roles in leaf morphology,leaf size,leaf spatial distribution and plant biomass.Available evidences indicate that transcription factors,enzymatic molecules and hormonal signals are involved in leaf vein morphogenesis.Birch trees(Betula)are tall and deciduous.Due to the presence of excellent tree structures and white barks,birch trees which harbor specific ornamental values are widely used for courtyard ornamentation and landscaping.Leaf vein morphogenesis is of great significance to the growth and development of birch trees.To date,knowledge on the molecular mechanisms underlying leaf vein development remains poorly understood.To reveal the key regulators for patterning leaf vein network,plants of Betula pendula 'Dalecarlica' which is an intraspecies variant of B.pendula were employed as our sampling collection objects.Based on the anatomical observation on the leaf vein in B.pendula'Dalecarlica',we performed transcriptome sequencing on the plant materials of the apical buds(DSO)and primary veins from young to mature stages(DS1,DS2,DS3,DS4,DS5 and DS6).Construction of leaf vein genetic regulatory network was on account of Bottom-up graphic Gaussian model(GGM).Further,to characterize the molecular function of the differentially expressed genes from our leaf vein genetic regulatory network,two plant homeodomain genes(BpPHD3 and BpPHD4)were selected for functional characterization using birch transgenic technology.The results were as follows:(1)Analysis of the anatomical observation on the primary vein in B.pendula 'Dalecarlica'showed that the ground tissues,xylem,phloem,cambium and mechanical tissues were major structural components.With the development of primary vein,the thickness of each of the structural components was gradually increased.Of these,the dynamical growth characters of mechanical tissues at the developmental stages from DS1 to DS6 were consistent with the growth of primary vein.Transcriptome profiles in B.pendula 'Dalecarlica' suggested that compared to other developmental stages,there was an increased number of differentially expressed genes in the primary vein at DS1 and DS4 stages.Moreover,analysis of the leaf vein genetic regulatory network revealed that BpPHD3 and BpPHD4 showed weak expression in the primary vein at DSO stage and showed gradually increased expression at the developmental stages from DS1 to DS6.(2)We cloned the BpPHD3 and BpPHD4 from birch tissues.Analysis of the sequences showed that the full-length coding sequence of BpPHD3 was 1389 bp.This gene encoded 462 amino acids and harbored the typical conserved domain of plant homeodomain-like(PHD-L);the full-length coding sequence of BpPHD4 was 1323 bp.This gene encoded 440 amino acids and harbored the typical conserved domain of PHD-L.(3)The Agrobacterium-mediated transformation procedure was retrieved for the generation of BpPHD3 overepression and suppression transgenic birch.Analysis of the phenotypic traits suggested that compared to non-transgenic birch,the seedling height,leaf length,leaf width,leaf area and leaf volume were all declined in transgenic lines.Particularly,these front traits were significantly declined in overexpression transgenic lines.However,the leaf epidermal cells were increased in size and decreased in number in transgenic lines.Moreover,the overexpression transgenic lines displayed changes in leaf thickness and leaf internal structure.Briefly,the leaf thickness of the overexpression transgenic lines was thin,with the decreased thickness in upper epidermis,lower epidermis,palisade tissue and mesophyll tissue.The leaf thickness of suppression transgenic lines was thick,with the increase in thickness of all structural traits except for lower epidermis.With regards to the leaf vein characters,the primary vein length,secondary veins number and the distance between the secondary veins were reduced in transgenic birch,while the major secondary angle to primary vein was raised.Moreover,the primary vein diameter was decreased in overexpression transgenic lines and increased in suppression transgenic lines.The results of endogenous auxin content measurement suggested that IAA(Indoleacetic Acid)content in the leaves of transgenic birch showed decline in levels.ZR(Zeatin Riboside)content in the leaves was significantly increased in overexpression transgenic lines and significantly decreased in suppression transgenic lines.The content of IAA and ZR was both significantly reduced in the primary veins of transgenic birch.The IAA content of the secondary veins in transgenic birch was significantly decreased.The ZR content was decreased in overexpression transgenic lines and increased in suppression transgenic lines.The measurement of reactive oxygen species(ROS)showed that transgenic lines rich in ROS accumulation were found.Of these,there was more ROS accumulation in overexpression transgenic lines than those of suppression transgenic lines.Based on the expression patterns of genes related to leaf vein development,many genes involved in cell cycles,transcription factors and auxin signal transduction were expressed at altered levels in the leaves of transgenic birch.Of these,genes encoding the key enzymatic products of cell cycles showed decrease in expression in overexpression transgenic lines and increase in expression in suppression transgenic lines.(4)The Agrobacterium-mediated transformation procedure was retrieved for the generation of BpPHD4 overexpression and suppression transgenic birch.Analysis of the phenotypic characters,hormone content and ROS accumulation showed that compared to non-transgenic birch,BpPHD4 overexpression lines displayed the decreased values in seedling height,leaf length,leaf width,leaf area,leaf thickness and leaf volume,upper epidermis thickness,lower epidermis thickness,palisade tissue thickness and mesophyll tissue thickness,IAA content in the leaves,ZR content in the leaves,primary vein,secondary vein number,primary vein diameter,IAA content in the primary vein,ZR content in the primary vein,IAA content in the secondary veins,ZR content in the secondary veins.Meanwhile,BpPHD4 overexpression transgenic lines had epidermal cells in small size and number,and increased ROS accumulation.In reference to suppression transgenic lines,these front values were contrary to those of overexpression transgenic lines.In terms of the expression profiles of genes related to leaf vein development,many genes associated with cell cycles,transcription factors and auxin signal transduction were differentially expressed in the leaves of transgenic birch.Of which,cell cycle-related genes were down-regulated in overexpression transgenic lines and up-regulated in supression transgenic lines.(5)Analysis of our transcriptome profiles of BpPHD4 overexpression transgenic birch showed that many genes related to cell wall components,defense mechanisms and auxin synthesis and signaling transduction were weakly expressed in overexpression transgenic lines.Many genes coding for the enzymatic products of cell number,cell cycle and chromatin organization were strongly expressed in overexpression transgenic lines.Combining the transcriptome profiles of BpPHD4 overexpression transgenic birch with the genome data of B.pendula,28 genes involved in defense mechanisms,cell cycles,plant hormone and other developmental processes were predicted as candidate targeted genes for BpPHD4.Taken together,among leaf vein genetic regulatory network in birch,BpPHD3 and BpPHD4 were considered as inportant transcription factors in leaf morphogenesis and vein development.Of these,BpPHD3 could regulate the hormone homeostasis,the expression of genes related to leaf vein development and ROS accumulation,thereby helping alter the processes of leaf size and vein patterns.BpPHD4 probably suppressed the synthesis of endogenous hormone,increased ROS accumulation and altered the expression of genes involved in leaf vein development thereby affecting the leaf size and vein outgrowth.Our study from the molecular biology perspective analyzed the molecular mechanisms underlying leaf vein development The results of our study not only enrich the basic theory of leaf morphogenesis,but also provide the valuable reference for molecular design and genetic improvement of plant leaves. |
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