Molecular Regulation Of Lignin Biosynthesis In Fruit Development Of Oil-Camellia

The genus Camellia includes more than 280 species,of which have oil value can broadly referred as oil-tea or oil-Camellia.Oil-Camellia is a unique superior woody oil tree species in southern hillside area of China,which have important economic,social and ecological value.In these oil-Camellia species,the fruit characteristics,such as morphology,kernel oil content and oil quality,are different.There are only a few studies focusing on these differences.In the previous studies,we observed that the main reason for the difference in seed rate of oil-Camellia fruit was the difference of pericarp.The lignification of pericarp,which is the result of lignin accumulation and regulated by lignin biosynthesis genes and transcription factors,might have an important effect on the development and rate of seed of oil-Camellia fruit.In this work,six oil-Camellia species are used for analysis.The mature fruit phenotypic traits were compared.The fruit growth and development dynamics and the accumulation of lignin in the fruit were characterized in detail.The genes associated with lignin accumulation of oil-Camellia fruit were analyzed and screened through transcriptome and small RNA sequencing.The key regulatory genes of lignin biosynthesis of oil-Camellia fruit were cloned,and their expression patterns and function were analyzed.The main results were as follows.1.Comparison of oil-Camellia mature fruit phenotypic traits and pericarp component.The seed yield of fresh fruit in six different oil-Camellia from large to small,in turn,were Camellia yuhsienensis(70.32 %),C.meiocarpa(60.40 %),C.oleifera ‘Changlin 53#'(52.65 %),C.oleifera ‘Changlin 166#'(52.65 %),C.chekiangoleosa(16.34 %)and C.octopetala(7.81 %),and were negatively correlated with the thickness of the pericarp.The largest difference coefficient of fruit weight indexes was the pericarp weight(3.59 %).The G-lignin and S-lignin content in pericarp of C.chekiangoleosa and C.octopetala which had thicker pericarp was significantly lower than that of others oil-Camellia.2.Effect of lignin accumulation on the growth and development of oil-Camellia fruit.The fruit development of oil-Camellia species had a similar S-type three stages.The fruit development of oil-Camellia composed of pericarp development and seed development.The pericarp development was characterized as an Expansion-Extension process which was identified by the lignification of pericarp.Seed development was divided into three periods: predevelopment(until seed-filled seed coat space),seed formation(until seed kernel molding)and seed expansion(until seed coat lignification completely).The development of the pericarp thickness was completed in early stage of fruit development,the thickness of pericarp was no longer increased after lignification.The pattern of lignification in pericarp of oil-Camellia fruit could be divided into two types: outside-to-in and inside-to-out.In the oil-Camellia which had“outside-to-in” pattern of lignification in pericarp,we found that the earlier the lignification of the pericarp,the thinner the pericarp.3.Identification of lignin regulatory genes in oil-Camellia fruits based on omics.Through the difference expression genes analysis of transcriptome data of floral bud and different fruit tissues of C.chekiangoleosa,1083 genes might be related to lignin accumulation of fruit of C.chekiangoleosa had been identified,of which 586 genes had a high expression in the endocarp and seed coat which had high lignification.Through homologous sequence alignment,32 lignin biosynthesis and transcriptional regulation genes were filtrated,of which 21 genes had a high expression in the seed coat.Through the analysis of small RNA sequencing of C.chekiangoleosa,103 mi RNAs were identified,of which 77 mi RNAs were conservative in other plants and 26 were unique to C.chekiangoleosa.According to the expression of 103 mi RNAs of C.chekiangoleosa in floral bud and different fruit tissues,it could be divided into five categories.The precursor sequence of Cch-mi R156 a was cloned,the cut site of its target gene was analyzed,and the in situ hybridization analysis showed that Cch-mi R156 a was specifically enriched in stamen and embryo.Through the difference expression genes analysis of transcriptome data of pericarp and seed of C.oleifera at different developmental stages,we had identified 1770 genes might be related to lignin accumulation of C.oleifera fruit,of which 701 genes had a high expression in the early stages pericarp which the lignin accumulation was rapid.Through homologous sequence alignment,43 lignin biosynthesis and transcriptional regulation genes were filtrated,of which 27 genes had a high expression in the pericarp at early stages.The transcriptome analysis of C.chekiangoleosa and C.oleifera showed that the expression of lignin biosynthesis and transcriptional regulation genes was related to the lignin accumulation process.4.Cloning and functional analysis of lignin regulation genes in oil-Camellia.Combined with the results of transcriptome analysis and pertinent literature,the NACs and BELs key transcription factors of lignin biosynthesis were analyzed.The DN47942(Cch NST1)and DN40632(Cch BLH6)were in the same branch with NST1 and BLH6/7 respectively,which were the key transcription factors of Arabidopsis.The Cch NST1 and Cch BLH6 were cloned.Cch NST1 encodes 396 amino acids,N-terminal has a NAM domain,is a typical NAC transcription factor.Cch BLH6 encodes 632 amino acids,has a POX domain and a HOX domain,is a typical BEL transcription factor.In the fruit of C.chekiangoleosa,Cch NST1 and Cch BLH6 had high expression levels in both endocarp and seed coat.The variation of the expression of Cch NST1 in the pericarp and seed was basically consistent with the observed variation of fruit pericarp lignin accumulation.The lignification such as root lignification,cell layer and width of the stem xylem,showed an increase in overexpress Cch NST1 and Cch BLH6 transgenic Arabidopsis,compared with the wild type.The leaf of Cch NST1 transgenic Arabidopsis showed an upward-curling phenotype and may be related to a flattening lignification area of leaf midrib.Overexpression Cch NST1 increased the expression of the downstream genes of At NST1,including MYB46,MYB83 and BLH6 in Arabidopsis.The lignification of the stem of Cch NST1 transgenic Populus,was significantly higher than that of the wild type Populus.The phloem fiber cells displayed a great degree of thickening in transgenic Populus.In the overexpression Cch NST1 transgenic Populus,the expression of Pd SND1 which was the homologous gene of NST1 in Populus,and its downstream genes Pd MYB21 and Pd MYB74,were increased significantly when compared with wildtype Populus.Subcellular localization analysis showed that Cch NST1 and Cch BLH6 were localized in the nucleus.