Transcriptomic And Metabolomic Analysis Of The Regulation Of Stalk Development In Flowering Chinese Cabbage

Unlike Arabidopsis thaliana,stalk is the main attribute of stalk vegetables,and the stalk in addition to elongation and thickening is also accompanied by the accumulation of nutrients;these two processes determine the yield and quality of the vegetable.Therefore,it is of great economic and scientific value to study the regulation mechanism of stalk formation.Flowering Chinese cabbage(Brassica campestris L.ssp.Chinesis(L.)Makino var.Utilis Tsen et Lee)is a subspecies of Chinese cabbage originally found in Southern China.Flowering Chinese cabbage is an important stalk vegetable,and is a well-known specialty vegetable of China.It occupies the largest cultivation area and has the highest yield in Southern China,and is now planted throughout the country.Bud differentiation of flowering Chinese cabbage is not sensitive to light and temperature;suitable varieties exhibit flowering in all four seasons of the year.However,when the plant encounters an adversity(such as low temperature),flowering Chinese cabbage showed early bolting and flowering,which in turn affects the yield and quality.However,there is limited research on the regulation of stalk development in flowering Chinese cabbage,and the regulation mechanism of stalk formation and nutrient accumulation is still not clear.In the present study,transcriptomics and metabolomics were used to examine the gene expression characteristics and the change in metabolites at three key developmental stages of the flowering Chinese cabbage,and investigate the effect of low temperature on stalk development by transcriptomics and study the regulation mechanism of gibberellin(GA)in stalk formation.The main results were as follows:1.The genes of cell cycle and flowering time involved in the regulation of stalk formation.Total of 35327 genes were obtained by RNA sequencing at three key stages(S1,S3,and S5)of stalk development in shoot apices of flowering Chinese cabbage;among these,11514 were differentially expressed genes(DEGs),identified by pairwise comparisons at three stages—i.e.,7015,7083,and 4361 DEGs from S1 vs S3,S1 vs S5,and S3 vs S5,respectively.Kyoto Encyclopedia of Genes and Genomes(KEGG)and Gene ontology(GO)enrichment analysis revealed that these DEGs were significantly enriched in ‘starch and sucrose metabolism’ and ‘plant hormone signal transduction’ pathways and were involved in plant morphogenesis and flowering related GO terms,including ‘flower development,’ ‘floral organ morphogenesis,’ ‘pollen development,’ ‘cell differentiation,’ and ‘cell division’.According to morphological and structural characteristics of shoot apex cells,gene regulation of plant hormone signal transduction,cell cycle regulation,and flowering time process were analyzed.The results showed that cell cycle was differentially expressed,including CYCD3,CYCA2,CYCB1,CDKB,CDKC,BR,E2 Fa.The flowering time regulation genes were differentially expressed,including RGA1,RGA2-like,FVE,SPL3,SPL5,SPL9,AP1,LFY,SOC1,SVP.2.Metabonomics results revealed the nutrient dynamic change process during stalk formation.Four hundred and ninety-three types of metabolites(31 classes)were identified in the stalk of flowering Chinese cabbage using metabonomic analysis.There were 14 carbohydrates,28 amino acids,56 organic acids,16 fatty acids,and 13 vitamins in the primary metabolites.Additionally,active secondary metabolites,such as flavonoids,anthocyanins,phenolic acids,etc.were identified.The different metabolite analyses found that carbohydrate content increased during the course of the stalk development and reached the maximum during the commercial harvest period(S5).At the same time,9 amino acids and 3 vitamins also accumulated during stalk development,but most of the phenolics were decreased.3.Intergrative metabolomic and transcriptomic analyses unveil the regulation mechanism of sucrose phloem unloading and lignin biosynthesis.Coalition analysis with metabonomics and transcriptomics showed an up-regulated expression of sucrose transporter gene SUCs,SWEETs,sugar transporter protein gene STPs,and cell wall invertase gene CWINVs during stalk formation,and accumulation of sucrose and glucose in the stalk.Based on this result,we established the regulation of symplast and apoplastic way on sucrose phloem unloading and transport to the pith cell storage of.At the same time,metabolomics and transcriptomics analysis showed that the content of coniferyl alcohol(lignin monomer)and its precursors(ferulic acid and coniferylaldehyde)were significantly up-regulated,and the expression of key genes in lignin biosynthesis was also significantly up-regulated.4.Transcriptome unveil the mechanism of low temperature regulation of bolting,flowering and sucrose transportation of flowering Chinese cabbage.Low temperature treatment can improve the content of endogenous GA and indole-3-acetic acid(IAA),improve the pith cell length,promote bolting,and early flowering time of the flowering Chinese cabbage.Transcriptome analysis showed that there were 2503 DEGs under low temperature treatment,of which 2211 up-regulated genes were mainly enriched in cell wall biosynthesis,cell wall components,cell morphogenesis,and cell elongation GO terms and carbohydrate transmembrane transport activity GO terms.Further analysis showed that the cell wall relaxation and remodeling key genes EXPAs,XTHs,and PPMEs were up-regulated under low temperature,and low temperature also up-regulated the expression of sucrose accumulation related genes SWEETs,SUCs,STPs,and CWINVs.In addition,low temperature induced an up-regulation in the expression of a number of MADS-box transcription factors and advance the flowering time.5.GA,uniconazole(GA synthesis inhibitor)and low-temperature treatment were used to explore the role of GA in bolting and flowering regulation in flowering Chinese cabbage.The results showed that GA and low temperature can promote bolting and flowering time whereas the effect of uniconazole was converse,these results indicated that GA plays a decisive role in stalk elongation.Gene expression analysis showed that GA can up-regulate gibberellin signal response gene GASA6,cell wall relaxation and remodeling key genes EXPA11,XTH3,and flowering regulation genes SOC1 and AP1,and promote bolting and flowering time of flowering Chinese cabbage.Low temperature can induce up-regulation of SOC1 and AP1 expression not through GA pathway,and thereby promote the development of flowering Chinese cabbage.The above research results indicate that the genes of cell cycle and flowering time involved in the regulation of stalk formation.During the development of flowering Chinese cabbage,sucrose was unloading to the phloem by the symplast and apoplastic way,and transported to the pith cell storage in the form of sucrose and glucose.GA plays a decisive role in stalk elongation,low temperature can regulate the stalk elongation,accumulation of nutrition and flowering by increasing the content of GA.In addition,low temperature can also advance the flowering time of flowering Chinese cabbage by other regulation ways.