| Cucumber(Cucumis sativus L.)always suffers from low temperature stress during winter season production.Invesgating its low temperature stress response should be beneficial for developing new methods of cucumber cultivation and breeding new varieties for low temperature conditions.The accumulation of raffinose family oligosaccharides(RFOs)is one of effective ways for plants to resist low temperature stress.Previous studies reported that cucumber,a RFOs-transporting plant,also accumulates large amount of RFOs in the leaves under low temperature,and reduces the RFOs content to normal level after low temperature stress is removed.However,until now the detailed process of RFOs synthesis and catabolism is unclear.Stachyose synthase(STS,EC.2.4.1.67)and a-galactosidase(EC 3.2.1.22)are key enzymes catalyzing stachyose synthesis and decomposition,respectively.There are three different STS transcripts and six different a-galactosidase transcripts in cucumber leaves.In detail,three STS transcripts are from one gene and produced by alternative splicing,while sixα-galactosidase transcripts are encoded by six different genes.The role of these transcripts in RFOs synthesis or decomposition is unclear now.In this research,we first developed a method which can identify the three STS transcripts specially.And then,the stability state and the expression pattern regulated by intracellular sugar levels of these three transcripts were investigated.Furthermore,the expression level,enzyme activity and relative sugars content of these three STS and six α-galactosidase transcripts were measured in the cucumber during low temperature stress and stress removal.In addition,previous studies showed that RFOs accumulates in vacuole,cytoplasm and chloroplasts after low temperature stress,and the level of RFOs in each subcellular space gradually decreased to normal level after low temperature removal.In order to explore the detail of RFOs decomposition in subcellular space,we also studied the subcellular localization of various a-galactosidase.The results are as follows:1."Three-base-anchored-primer" method developed here can be used to distinguish the three transcripts of STS in the real-time qPCR analysis,which provided an effective experimental tool for the following study.2.The expression level of STS transcripts in calli was significantly lower than that in leaves of cucumber.After calli was transferred to sugar free MS medium,the expression level of all three STS transcripts increased.When calli were transferred from the sugar free MS medium to the MS medium with sucrose,raffinose,stachyose or galactose as carbon source,the expression level of all three transcripts decreased.These results indicated that the expression of three STS transcripts were regulated by the level of intracellular sugar.Actinomycin D inhibition test showed that the stability of STS Ⅱ transcript was significantly lower than that of the other two transcripts.3.The expression level of STS Ⅰ and STS Ⅱ in cucumber leaves was significantly higher than that of STS Ⅲ,indicating these two transcripts may play key roles when cucumber plants were suffered from low temperaturestress.After 4℃ treamtment,the soluble sugar content,the expression level of three STS transcripts and the STS activity increased in cucumber leaves.Combined with the results of callus research,it was found that the increases of expression level and activity of STS were induced by low temperature rather than the high sugar level.After cold treatment removal,the content of soluble sugar decreased and the expression of acid a-galactosidase 3 gene(GAL3),alkaline α-galactosidase 2 and 3 gene(AGA2,AGA3),and the activity of GAL and AGA increased.These reuslts indicate that these three genes played an important role in the degradation of RFOs after temperature recovery.4.The results of subcellular localization study showed that GAL1 and GAL2 were located in the cell wall;GAL3 was mainly distributed in the vacuole;AGA1 and AGA2 were mainly distributed in the cytoplasm,whileAGA3 was located in the chloroplast.Combined with previous studies,it suggested that GAL3,AGA2 and AGA3 played roles in degradation of RFOs in vacuole,cytoplasm and chloroplast after low temperature remoral,respectively. |
