Study On Carbon And Nitrogen Metabolism And Proteomics During Growth And Development Of Flowering Chinese Cabbage

Flowering Chinese cabbage(Brassica campestris L.ssp.Chinensis var.Utilis Tsen et Lee) can grow for all year in south China. Flowering Chinese cabbage can bolt and flower without vernalization, however, low temperature in seed or seedling stage can induce early bolting, and thus result in a severe yield reduction and imbalance of market supply. In this research, we used three cultivars with different maturity, including “Youqing 49”(Early maturity),“Youlvcutai”(Middle maturity) and“Cuilv 80 days”(Late maturity), to explore the relationship of carbon and nitrogen metabolism and flower stalk formation during the growth and development of flowering Chinese cabbage. At the same time, we used late maturity cultivar “ Cuilv 80 days ” as material to study the effect of low temperature treatment of seed on plant growth and development.Three different temperature were set,including 5 ℃, 15℃ and 25 ℃(CK). And i TRAQ technique was adopted to analyse the protein change in the leaves of plants after low temperature treatment during flower bud differentiation stage. The main results were as below.1. The contents of soluble sugar, sucrose, fructose and reducing sugar in three cultivars had little change from early to middle stage of flower bud differentiation, but increased rapidly from middle stage of flower bud differentiation to budding stage, and gone up or down steady from budding to harvest stage. After flower bud differentiation, all kinds of sugar contents in the leaves were lower than those in the stems. The contents of sugars both in leaves and stems in the late maturity cultivar were the highest among three types of cultivars,and had no significant difference in the leaves between early and middle maturity cultivar, but the sugar contents in the stems of the middle maturity cultivar were higher than those in early maturity cultivar. There were significant positively correlations between neutral invertase(NI)and acid invertase(AI) activities in the leaves as well as sucrose synthase(SS) and sucrose phosphate synthase(SPS) activities in the stems and the contents of soluble sugar, sucrose,fructose and reducing sugar; while there were no significant correlations between SS and SPS activities in leaves as well as NI and AI activities in the stems and the contents of soluble sugar, sucrose, fructose and reducing sugar. This illustrated that sugar accumulation was the fastest from middle stage of flower bud differentiation to budding stage. After flower buddifferentiation, the sugars gradually transported from leaves to stems for providing enough energy for flower stalk growing. During the whole growing stage, sugars in the leaves continuously degraded, but those in the stems continuously synthesized and accumulated. The sugar contents and activities of NI and AI in leaves as well as sugar contents and activities of SS and SPS in stem had close relationships. Therefore, higher activities of SS and SPS in stems could promote the accumulation of sugars, and improve flower stalk quality.2. Nitrogen metabolism was different during whole period among different maturity of flowering Chinese cabbage variety. The soluble protein content in the early and middle maturing variety gradually increased from bud differentiation stage to the squaring stage, but it was different in the late maturing variety. At the early stage of flower stalk formation stalk,there was a higher content of soluble protein in late maturing variety, while in the middle and late stage of flower stalk formation, the early maturing variety was higher than other variety.The content of free amino acid in the early and middle maturing variety changed little in the formation of flower stalk, while it was present with an evident single-peak curve in the late maturing variety and appeared value apex at the stage of middle bud differentiation. There was a slight change of the glutamine synthase(GS) activity, but the activity in the early maturing variety is the highest, and then the middle maturing variety, the late maturing variety is the lowest. The activity of glutamate dehydrogenase(GDH) enhanced gradually in the growth of flowering Chinese cabbage, and it was higher in the early maturing variety. The GDH activity was no significant between the middle and the late maturing variety. This showed that the early and the middle maturing variety had greater ability of absorbing and assimilating nitrogen, which helped the accumulation of protein in the formation of flower stalk, while the late maturing variety consumed protein to form its flower stalk.3. Low temperature treatment significantly influenced osmotic substances and reactive oxygen metabolism system of flowering Chinese cabbage. Lower temperature treatment enhanced the contents of soluble sugar and soluble protein in leaves of flowering Chinese cabbage, and the contents of soluble protein in leaves decreased following the order of 5℃,25℃(CK) and 15℃. The content of soluble protein under 5℃ treatment in the leaves was significantly higher than those under 15℃ and 25℃ treatment, but there was no significant difference between 15℃ and 25℃ treatment. With the decrease of temperature, the contents of proline(Pro) and reduced glutathione tablets(GSH) in the leaves were remarkably increased. The 5℃ and 15℃ low temperature treatment significantly improved the contentsof malondialdehyde(MDA) and ascorbic acid(ASA) in the leaves compared with the control treatment, but there was no difference between 5℃ and 15℃. The activities of superoxide dismutase(SOD), aseorbateperoxidase(APX) and glutathione reductase(GR) enhanced significantly under low temperature treatment, but the activity of peroxidase(POD) was significantly decreased. 15℃ low temperature treatment enhanced the activity of SOD in the leaves greater than the 5℃ treatment, but there was no significant in the activities of POD and APX between them. These results showed that when flowering Chinese cabbage suffered from low temperature stress, the content of MDA increased, lipid peroxidation aggravated,but low temperature also strengthened the removal ability of reactive oxygen by increasing the contents of Pro, GSH and ASA and the activities of SOD, APX and GR in flowering Chinese cabbage, which maintained the balance of ROS and reduced the damage for flowering Chinese cabbage.4. Isobaric tags for relative and absolute quantitation(i TRAQ) technique was used to screen and identify differentially expressed proteins in late maturity cultivar treated with 5℃,15℃ and 25℃(CK). We identified 4073 proteins in total, the differentially expressed proteins were 367 in the leaves between 5℃ and 25℃ treatments, 359 between 15℃ and 25℃treatments, 532 between 5 ℃ and 15 ℃. 64 proteins might be attributed to the low temperature induce and plant growth and development. These proteins respectively belonged to: photosynthesis(12), sugar metabolism(6), amino acid metabolism(13), plant hormone(3),stress(23) and energy(7).5. Low temperature treatment repressed the photosynthesis. It also improved sugar metabolism including the up-regulation of β-glucosidase glucose 19 and the down-regulation of 1-glucose-adenosine acyltransferase large subunit protein and fructose kinase 1 in the leaves of flowering Chinese cabbage, which contributed to the accumulation of fructose and glucose, and thus promoted flower bud differentiation. The activities of two nitrate reductase increased, and thus promoted nitrogen absorption and assimilation. The significant up-regulation of 3-Hydroxy-3-methylglutaryl-coenzyme A synthase(HMGS) and sterol24-C-methyltransferase 1(SMT1) and down-regulation of geranylgeranyl diphosphate synthase(GGPS), which were related to plant hormone accelerated the biosynthesis and transformation of sterols and cytokinin, might be closely associated with flower bud differentiation. Changes of some stress-related proteins improved low temperature endurance by restoration of protein folding, desaturation of fatty acid and elimination of reactive oxygenspecies(ROS). In addition, low temperature reduced the activity of V-ATP enzyme and improved the activity of NADH dehydrogenase to promote the synthesis of ATP, which could provide enough energy to protect flowering Chinese cabbage from chilling stress and to promote the flower bud differentiation.