Analysis On The Dynamics And Regulation Of Cell Lignification In The Stem Of Plant In Zizania Latifolia

The development of swollen stem was induced by the infection of Ustilago esculenta in Zizania latifolia,fillowing with the significant inhibition of lignification in the stem cells which played an important role in the quality formation and postharvest.Based on the synthesizing of lignin and thickening of secondary cell wall,Lignification could significantly affect the quality and taste of fruits and vegetables.Most fruits and vegetables had experienced lignification during their growth and development,accompanied by continuous lignification during their senescence.There had been a lot of studies on the regulation of lignification in higher plants,and also on the lignification related to the postharvest quality of Z.latifolia.However,the dynamics and molecular regulation mechanism of lignification during the formation and development of swollen stem of Z.latifolia had not been studied.In this research,Based on the lignification changes of the swollen stem before and after morphogenesis in normal“Jiaobai”during plant development,cell growth and lignification staining observation,total lignin content and monomer content in stem were used to preliminarily elucidate the lignification dynamics of stem in the plant of Z.latifolia.Combined with the expression pattern analysis of important genes of phenylpropane metabolism pathway involved in lignification regulation in the stem of Z.latifolia.The regulation mode of lignification during the formation and development of swollen stem of Z.latifolia were discussed,and the function of lignin monomer synthesis genes were analyzed,which would provide technical support and theoretical basis for the molecular mechanism of lignin synthesis and regulation related to stem swolling and development of Z.latifolia.The main results obtained in this study were as follows:1.Microscopic observation of cell growth and lignin distribution in the swolling stems of Z.latifoliaBased on the microscopic observation of tissue sections and lignin staining,the comparative analysis of cell growth and lignification in the swelling stem was carried out between the normal Jiaobai and wild Jiaobai.The color and distribution areas of lignification staining in normal Jiaobai stems were significantly lower than those of Z.latifolia,indicating that the degree of cell lignification in the swolling stem of normal Jiaobai was significantly lower than that of Z.latifolia.Statistical observation of stem cell growth showed that there were significant differences in cell growth among different nodes of normal stem.The number of vascular bundles per unit area in the middle node was the highest,the average size of thin-walled cells（measured at the smallest point）was the largest（32μm）,and the average number of parenchyma cells per unit area in the vascular bundle tissues of stem was the least,and the number of vascular bundles in the upper node was the smallest,the number of parenchyma cells was the smallest.The average cell size was the smallest（17μm）,and the average number of parenchyma cells per unit area was the largest.The number of vascular bundles and parenchyma cells in the lower node were between the upper and middle nodes.The swolling stem of Z.latifolia started at the upper node,and the number of parenchyma cells between vascular bundle tissues increased significantly.The swollen stem was realized through the formation of vascular bundle and the extension of parenchyma cells at the middle node.Further electron microscopic observation showed that the secondary wall thickness of the cell wall was significantly inhibited during the development of normal Jiaobai compared with that of wild Jiaobai,and the thickness of the secondary wall of the cell wall was significantly higher in the lower nodes than in the upper and middle nodes.During the development of normal Jiaobai stem expansion,the lignification of stem cells was significantly reduced,mainly in the distribution of lignification and inhibition of secondary wall thickening,which was related to the growth and development period of the stem.2.Dynamic detection of total lignin and monomer content in stem cells of Z. latifoliaBased on the significant differences of lignification during the development stem of normal Jiaobai,the total lignin and monomer content of normal Jiaobai and wild Jiaobai stems were dynamically detected at different developmental periods.The total lignin content of the nodes in wild Jiaobai showed a gradual increase from top to bottom,and the total lignin content decreased slightly in the more mature lignified nodes,but there was no significant difference,which was related to the growth and development period of the nodes in the stem.The total lignin at the upper nodes was higher than that at the other two nodes,which would be related to the high cell division and cell density at the upper nodes.Further detection of lignin monomers revealed that the lignin monomer content in the swolling stems in normal Jiaobai was lower than that of wild Jiaobai stems.The S-lignin monomer content was significantly lower in the lower expansion nodes and the moss tube of the normal Jiaobai stems than in the wild Jiaobai stems;the S-lignin and G-lignin contents of the normal Jiaobai expansion nodes were significantly lower than those of the moss tube,but there was no significant change among the different nodes in the swollen stems of normal Jiaobai,but the S-lignin content of the moss tube in old Jiaobai was significantly higher than that of the normal Jiaobai.The inhibition of lignification during stem swolling development of Z.latifolia was associated with the reduction of total lignin in stem cells and the change of monomer content,and the change of S-lignin monomer content in swollen stem cells would play an important role in the regulation of lignification process.3.Expression pattern analysis of genes related to lignin synthesis pathway during the stem development of Z.latifoliaBased on the genomic and transcriptomic data of Z.latifolia,the important genes of the phenylpropane metabolic pathway related to lignin synthesis were selected and their expression changes were analyzed during the stems development of Z.latifolia.The expression of Zl-4CL1 was significantly down regulated during the stem development of Z.latifolia,The similar phenomenon also occurred in Zl-4CL3 and Zl-4CL5.The expression of three genes were significantly decreased at the 8-leaf stage of swolling stem.The expression of Zl-4CAD gene in the stem of normal Jiaobai was significantly lower than that of wild Jiaobai at the 8-leaf stage and the initial stage of stem expansion.Therefore,the regulation of lignification process during stem swolling could be closely related to the down-regulation of genes related to lignin synthesis.The analysis of the genes Zl-F5H and Zl-COMT related to S-lignin monomer synthesis in the upper node showed that the expression levels of the four Zl-F5H genes were significantly down regulated after the initial swolling and development（8-leaf stage and early expansion stage）of the stem in normal Jiaobai compared with the stem of wild Jiaobai.The expression of Zl-COMT was significantly decreased at the initial stage of swolling stem,but increased at the early start of swollen stem.The inhibition of lignification during swollen stem was closely related to the synthesis of S-lignin monomer,and the regulation period could mainly occur at the initial stage of swolling stem.4.Full-length cloning and subcellular localization of Zl-F5H4 gene in Z.latifoliaBased on the genomic and transcriptomic data,combined with the analysis of gene expression pattern during the stem development of Z.latifolia,The Zl-F5H4gene was selected and cloned.The full-length DNA sequence of Zl-F5H4 was 1326bp,with the c DNA full-length of 1245 bp and one intron,which encoded 415 amino acids.Sequence analysis showed that Zl-F5H4 contained cytochrome P450.The protein had a theoretical molecular weight of 47.05 k Da and an isoelectric point of6.08.The main amino acids were Ala,Arg,Asp,Leu and Glu,of which the total number of negatively charged residues（Asp+Glu）was 60 and the total number of positively charged residues（Arg+Lys）was 53.The molecular formula of Zl-F5H4was C₂₀₈₈H₃₃₁₅N₅₈₇O₆₃₀S₂₄.There were no signal peptide and no transmembrane region structure in Zl-F5H4.The subcellular localization of Zl-F5H4 was predicted to be in the endoplasmic reticulum.Phylogenetic tree analysis revealed that Zl-F5H4could be clustered with homologous genes in Oryza sativa Japonica Group,which may be related to the close kinship between the genus.The over-expression genetic transformation vector of p FGC-Egfp-Zl-F5H4 was constructed and transient expression analysis was performed in tobacco leaves by the method of Agrobacterium-mediated.Microscopic observation of GFP fluorescence signal showed that the Zl-F5H4 gene was localized in the endoplasmic reticulum of sarcomeres in tobacco leaf,which was consistent with related studies reported in other plants.In addition,the expression of Zl-F5H4 in transiently expressed tobacco leaves was analyzed by fluorescence quantitative PCR.The expression of Zl-F5H4 gene in over-expressed tobacco leaves was significantly higher than that of the null and control,which initially verified the correctness of Zl-F5H4 over-expression genetic transformation vector construction,and the genetic transformation analysis of Z.latifolia plants had been carried out by seedling-end transformation method.