The Physio-ecological Effects And Molecular Mechanisms Of Enhanced UV-B Radiation On Peanut (Arachis Hypogaea L.)

A large number of chlorofluorocarbons (CFCs) discharged into the air due to human industrial activity lead to thinning of the ozone layer. As a result, ultraviolet-B (UV-B,280-320nm) in the solar radiation reach to the ground is enhanced. Many researches have pointed out that elevated UV-B radiation had significant impact on individuals, populations of terrestrial plants and total ecosystems. Peanut (Arachis hypogaea L.) is an annual legume crop, which is rich in fat and protein. At present, the effects of enhanced UV-B radiation on physio-ecological of peanut is seldom reported, as well as the molecular mechanisms. In this study, cultivar "Xiaojingsheng" peanuts was chosen as test materials, the effects of enhanced UV-B radiation on factors including seedling morphology, gas exchange parameters, changes in chloroplast ultrastructure, ROS levels, antioxidant enzyme activity, antioxidant content, and proteome expression profiling have been analyzed. Fructose-1,6-bisphosphate aldolase (FBA) gene AhFBA which was down-regulated by enhanced UV-B radiation, were cloned and analyzed the transcription level of gene AhFBA treaed under enhanced UV-B by real-time quantitative reverse transcription polymerase chain reaction. AhFBA gene in E. coli BL21(DE3) were also expressed and observed the growth curve of the host cell contained recombinant vector pET28a-AhFBA treated with UV-B. The main purpose of this paper is to answer the following questions:(1) What are the effects of enhanced UV-B radiation on peanut in physio-ecology level?(2) How does peanut proteome respond to enhanced UV-B radiation?(3) Whether FBA proteins play a role in resisting enhanced UV-B radiation? The results are as follows:The impact of UV-B radiation on photosynthetic related parameters and chloroplast ultrastructure of A. hypogaea were studied, after an enhanced UV-B irradiation performed8h per day for3days with ultraviolet light (280-320nm) that maintained about54uW/cm2radiations. Contents of chlorophyll a and chlorophyll (a+b) declined significantly after radiation for3days (P<0.05), concomitantly, gas exchange parameters were significantly affected (P<0.05), showing a35.42%reduction in net photosynthesis (Pn),28.12%in stomatal conductance (Gs),26.37%in transpiration rate (Tr) and20.25%in stomata limitation(Ls), however, intercellular CO2concentration (Ci) was increased by16.22%, showing significant difference with that in control (P<0.05), at the end of3days UV-B treatment. Similarly, the fluorescence parameters of Fv/Fm and Fv/Fo were also significantly reduced (P<0.05). However, the differences of above indexes were not significant compareed with the control treated with UV-B after1and2days (P>0.05). Transmission electron microscope (TEM) images revealed that the peanut treated for3d whose chloroplast membranes were swelled and disintegrated, and stromal thylakoids were relaxed and parallel to each other, which might be responsible for worse performance of the photosynthesis. As a result, the size and number of starch grains decreased.The contents of superoxide anion (O2-), flavonoids, and soluble proteins, the activities of catalase (CAT) and peroxidase (POD) in peanut leaves were increased significantly treated with supplementary ultraviolet-B (UV-B), as well as relative electrical conductivity (P<0.05). The activity of superoxide dismutase (SOD) increased at first and then decreased. Nevertheless, there were no significant changes in the contents of hydrogen peroxide (H2O2), malondialdehyde (MDA), ascorbic acid (AsA) and Glutathione (GSH), the activities of ascorbate peroxidase (APX) in leaves (P>0.05). These results indicated that elevated UV-B could increase the content of reactive oxygen species (ROS) in peanut leaves, and O2-was the principal factor for oxidative stress. Peanut seedlings resisted the stress of ROS mainly through increasing the content flavonoid and the activities of SOD, CAT and POD. However, As A-GSH circulatory system had no unobvious effect on clearing ROS.A total of39protein spots were differentially expressed by at least2.5fold compared with the controls (22proteins were down-regulated and17were up-regulated) after treatment with supplementary UV-B radiation. Of those protein spots,27were successfully identified by MALDI TOF/TOF MS after a database search. Those27proteins could be classified into eight categories according to their functions:class1, photosynthesis (plastocyanin, ribulose-1,5-bisphosphate carboxylase small subunit, oxygen-evolving enhancer protein1, PsbP domain-containing protein6); class11, carbohydrate metabolism (malate dehydrogenase, and fructose-bisphosphate aldolase); class Ⅲ, energy synthesis (ATP synthase); class IV, amino acid biosynthesis (cysteine synthase); class V, protein biosynthesis (ribosome recycling factor); class VI, protein processing (heat shock proteins); class VII, defense responses (chitinase, peroxidase, Cu-Zn SOD, caffeic acid3-O-methyltransferase, and germin-like protein); class VIII, unknown proteins. In conclusion, we hypothesized that the enhanced UV-B radiation caused a decrease in the photosynthesis rate of peanut leaves mainly via three mechanisms. First, enhanced UV-B may down-regulate the expression of ribosome recycling factor, which caused a decrease in the expression of subunit PsbP in photosystem Ⅱ, thus destroying the thylakoid membrane structure. Second, the reduced plastocyanin expression may have induced a decrease in photosynthetic electron transport efficiency. Third, the down-regulation of ribulose-1,5-bisphosphate carboxylase and fructose-1,6-bisphosphate aldolase resulted in a decrease in carbon assimilation. At the same time, peanut may also enhance its resistance to UV-B stress by increasing the expressions of antioxidant enzymes and non-enzymatic antioxidants, germin-like proteins, pathogenesis-related proteins, and heat shock proteins.Using soybean(Glycine max) fructose-1,6-bisphosphate aldolase gene (GmFBA, GenBank accession number:AY492006.1) as queries, BLAST searches in peanut EST database on the NCBI website were performanced, peanut EST sequences with high homology to soybean FBA gene were selected, a contig named AhFBA was obtained by in silico cloning technology from the selected ESTs. AhFBA was confirmed by RT-PCR, molecular cloning and sequencing, the complete conding sequence (CDS) of AhFBA was1077bp in length, and the sequnence was submitted to NCBI database with an accession number KF470788.AhFBA gene encoding a protein AhFBA composed of358amino acids with the formula C1706H2725N469O524S6, in which Ala was the most abundant amino acid, the number of acidic amino acids (Asp and Glu) and basic amino acids (Lys and Arg) were all40, the relative molecular mass was38.38kD, isoelectric point was6.73, instability index was30.03, and it had no signal peptide sequence. Bio informatics forecast that AhFBA protein was in most cases located in the cytoplasm with the score of0.65, scores that located in the mitochondrial matrix and chloroplast thylakoid membrane were all0.1and localized in the endoplasmic reticulum was0. AhFBA protein has2amide sites (36and285), a cAMP-dependent protein kinase phosphorylation sites (41),3protein kinase II phosphorylation sites (114,270and309),7protein kinase C phosphorylation sites (50,201,235,247,304,309and336),7N-myristoylation sites (25,97,127,137,232,335and346), an aldolase activity center (217-227bit sequence VLLEGTLLKPN), AhFBA belongs to type I cytoplasmic fructose-bisphosphate aldolase gene.Real-time PCR results showed that:when the A. hypogaea was grew in natural lighting conditions, AhFBA gene was constitutively expressed in roots, leaves, flowers and young fruit, and there is no significant differences in expression levels among them (P>0.05). However, AhFBA gene’s expression up-regulated in leaves more than10times when the plant treated with enhanced UV-B for4h, and then down-regulated after treated8h,16h and24h, reduced the expression of0.75,0.86and0.29times. It indicated AhFBA belongs to UV-B inducible gene, which participated in the UV-B stress response.Primers (forward and reverse primer was introduced BamH I and Hind III restriction enzyme site, respectively) were designed according to AhFBA gene, and the sequence was amplified by PCR. The fragment of vector pET28a and AhFBA was connected after digested with BamH1and Hind III enzymes. PCR and DN A sequencing results verified that the recombinant expression vector pET28a-AhFBA has been successfully constructed containing the target gene AhFBA. Heat shock method was employed to transforme pET28a-AhFBA into E. coli expression strain BL21(DE3), isopropyl thiogalactoside (1PTG) induced the expression of gene AhFBA and about40kD protein band was detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).The host bacteria were irradiated by UV-B for certain minutes after induced by IPTG, then, they were inoculated into new LB medium that contains Kanamycin, OD600value was measured every one hour. The results showed that:there are no significant differences in the growth anmong bacterial BL21(DE3) contain pET28a-AhFBA and pET28a with control (host strain BL21(DE3) contains pET28a unirradiated UV-B) after receiving UV-B irradiation for3min. Accepted9min UV-B irradiation, host strain BL21(DE3) contain pET28a-AhFBA and pET28a growth rates were lower than control, but the former is higher than the latter. The growth rates of BL21(DE3) contain pET28a and pET28a-AhFBA were both lower than control after12min UV-B irradiation, and there is no difference between them, indicating that exogenous recombinant protein AhFBA may help the host bacteria mitigate the adverse effects of UV-B radiation to some extent.