| Research and development of the energy plant Jat ropha curcas L.is a strategical demand for Yunnan province and our country.Drought is a key limiting factor for development and production of J.curcas.The results from our laboratory showed that drought-hardening could significantly enhance drought resistance of J.curcas seedlings.Based on our previous work and Illumina Hiseq 2000 high-throughput sequencing platform,using three-week old J.curcas seedlings as experimental materials,which were pretreated with 10% PEG 6000 for drought hardening for 2 days,then followed by air drought stress 3 days,the lea RNA was extracted at different time points and high-throughput sequencing was carried out by RNA-seq,then the differentially expressed genes of non-structural carbohydrate metabolisms were significantly enriched and the metabolic pathways of non-structural carbohydrate were constructed in J.curcas seedlings under the drought-hardening and the following drought stress.The content of non-structural carbohydrate was determined and the spatiotemporal expression patterns of key enzyme genes were analyzed in leaves and stems of J.curcas seedlings,to fomulate the involvement of non-structural carbohydrate metabolism pathways in response and adaptation mechanism of J.curcas seedlings to drought stress.The main findings are as follows:1.During the drought hardening and drought stress,the starch synthase(SS)and starch branching enzyme(SBE)genes of starch synthetic in J.curcas leaves were continuously up-regulated.The SSS gene expression was up to 33-fold,and the SBE gene expression was up to 55-fold.The starch content of leaves in J.curcas increased rapidly during the early stage of drought hardening,indicated that the accumulation of starch responded rapidly to drought stress.With the prolonged drought time,the starch content of leaves decreased significantly,and the drought-hardening group was always higher than the control,which resulted from amylolysis to soluble sugars to relieve drought stress.These results suggested that the seedlings with drought-hardening could decomposed the starch into soluble sugars to relieve the following severe drought stress.2.Sucrose synthase(SSS),sucrose phosphates synthase(SPS),invertase(InV)were co-regulated the sucrose content in J.curcas seedlings under the drought response and adaptation.The SPS of sucrose synthesis in leaves reached 10 fold at DS72,SS gene increased 35 fold at DS48,and InV gene achieves 12 fold at DS24,which indicated the contents of glucose and fructose decreased gradually and sucrose contents increased during drought hardening.The content of sucrose was significantly reduced,while the glucose and fructose rose rapidly in leaves under drought stress.The contents of sucrose,glucose and fructose in the stems showed opposite trend,which showed the contents of sucrose,glucose and fructose did not change significantly.During the drought stress,the content of sucrose accumulated obviously,and the content of glucose and fructose decreased significantly,which may be related to characteristics of sucrose transport in vivo.3.The expression of galaetinol synthase(GS)and raffinose synthase(RS)genes in leaves were significantly up-regulated in the process of raffinose biosynthesis under drought stress.The expression of GS genes were up-regulated up to 80-fold,and the RS genes increased up to 2000-fold.The accumulation of raffinose oligosaccharides was promoted,indicated that drought hardening was conducive to the production of osmotic adjustment substances such as raffinose oligosaccharides which could relieve injury of drought stress.The GS and RS genes of the raffinose oligosaccharides biosynthesis in stems were up-regulated,but were not pronounced in leaves.This suggested that the GS and RS genes in the leaves of J.curcas seedlings were more sensitive than those in stems.4.The genes of trehalose phosphate synthase(TPS)and trehalose phosphate phosphatase(TPP)were up-regulated,and the genes of trehalase(THase)were down-regulated which were beneficial to the accumulation of trehalose that reached a peak of 98.2 mg/g DW at DS24 in response and adapatation of J.curcas seedlings.The genes of TPS,TPP,and THase in the stems of J.curcas seedlings were up-regulated during the early drought hardening stage,indicated that the stems were more sensitive to drought stress during trehalose metabolism.With the prolongation of drought stress,the content of trehalose was gradually decreased in leaves and stems,but the content of experimental group was always higher than the control,which were beneficial to the accumulation of trehalose in J.curcas seedlings and enhancement of their drought tolerance.In general,based on the determination of the content of non-structural carbohydrate metabolisms in leaves and stems of J.curcas seedlings and the analysis of spatiotemporal expression patterns of these related genes in their metabolisms,our results showed the metabolic pathways of starch,sucrose,raffinose,trehalose were improved soluble sugar content corresponding to stabilize the osmotic pressure,then alleviated damage of drought stress,and maintained the basic process of growth in J.curcas.These results will lay the theoretical foundation to some extent for explaining the molecular mechanisms of drought resistance and molecular breed ing in J.curcas plants. |
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