Signal Transduction Characteristics of SA and H2O2in Interactions of Populus Spp. and Botryosphaeria Dothidea
|School||Chinese Academy of Forestry|
|Keywords||Populus spp Botryosphaeria dothidea salicylic acid methyl salicylate hydrogenperoxide|
Differences of salicylic acid （SA）, methyl malicylate （MeSA） and hydrogen peroxide（H2O2） were analyzed in different resistance poplars （Populus tomentosa and Populus×beijingensis） inoculated with Botryosphaeria dothidea. The main results were as follows:1. Difference of endogenous SA content and its related indicators were analyzed in poplarinoculated with B. dothidea, and the role of signaling molecules SA in poplar canker was clear.The results showed that the SA content of the resistant P. tomentosa had increased significantlyby6h after inoculation with B. dothidea and continued to accumulate to191.42ng/g freshweight （FW） by72h, after which it gradually declined. Levels of SA in control plantsremained low （9.98to14.12ng/g FW） throughout the sampling period. When the susceptible P.×beijingensis was infected with B. dothidea, SA levels were significantly lower than in P.tomentosa. Compared with the combination of SA, the free SA content is always relativelysensitive and significant, indicate that the free SA is involved in the defense response of poplarresistance to disease, indicating that the poplar canker resistance and poplar body of free SAaccumulation is closely related.2. Study on the SA biosynthetic pathway in the system of poplar inoculated with B.dothidea, The results showed that the levels of benzoic acid was a downward trend at6-12hafter P.tomentosa inoculation, lower than the non-vaccinated control the content, while theactivity of benzeneformic acid2-hydroxylase （BA2H） was significantly elevated, indicatingthat BA change into SA, activity of phenylalanine ammonia lyase （PAL） was significantlyincreased, while the content of BA increased, this may BA2H activity is inhibited which led tothe increase in BA content; compared with P. tomentosa, P.×beijingensis after inoculation, theBA content of the performance of slow upward trend in the entire vaccination time. The resultsalso showed that P. tomentosa inoculated with B. dothidea after48-72h, the change of free SAand conjugated SA, the change was almost the opposite trend, suggesting the existence of bound SA into free SA of poplar inoculated with B. dothidea, the accumulation of the free stateof SA content may come from different channels, such as vaccination of pre-plant biosynthesisand the latter combined with the participation of the SA, BA and poplar anti-ulcer diseaseareclosely related.3. We analyzed the content of MeSA in resistant P. tomentosa and susceptible P.×beijingensis after inoculation with B. dothidea. The P. tomentosa control plants containedbetween28.65and29.13ng/g FW MeSA, while MeSA levels in infected stems had increasedby6h after inoculation with B. dothidea and remained relatively steady from6to24hpost-inoculation. Then, MeSA content increased significantly in inoculated samples to65.93ng/gFW by48h. No significant induction of MeSA content occurred in the susceptible P.×beijingensis after B. dothidea inoculation. In summary, these quantitative analyses showed thatSA and MeSA production significantly increased in resistant P. tomentosa inoculated with B.dothidea. Moreover, in the susceptible P.×beijingensis, the contents of SA and MeSA were notelevated. Both the basal and induced levels of SA in P. tomentosa plants were increasedcompared with P.×beijingensis. By contrast, the basal level of MeSA in the treated tissue of P.tomentosa plants was similar compared to that in P.×beijingensis. Mock inoculation of thecontrols did not induce SA or MeSA accumulation in treated tissue, indicating that elevatedlevels of these compounds were induced by B. dothidea infection. Here, we identified SAMTand SABP2from P. tomentosa and P.×beijingensis and demonstrated that they shared highsequence similarity to N. tabacum SAMT and P. trichocarpa SABP2, respectively. We alsomeasured the expressions of SABP2and SAMT. We found that SABP2and SAMT were inducedsignificantly and more rapidly in P. tomentosa than in P.×beijingensis after B. dothideainfection, although induction of SABP2occurred in both poplar cultivars after inoculation withthe canker fungus. These results suggested a potential role for SABP2and SAMT in poplardisease resistance responses. Importantly, in P. tomentosa, the expression of SAMT peaked at12h, while SABP2expression peaked at48h, echoing the patterns seen with SA and MeSA. Insummary, SA levels increased in resistant P. tomentosa after inoculation with B. dothidea and peaked at72h after inoculation, SAMT expression was up-regulated, MeSA content increasedby48h after inoculation accompanied by increased expression of SABP2. Although the SAcontent rose in susceptible P.×beijingensis after infection, the increase was not significant andwas not accompanied by decreased MeSA levels and expressions of SAMT and SABP2as in P.tomentosa. Taken together, these data strongly supported the hypothesis that SA and MeSAwere essential parts of the defense response of poplar against pathogen attack. Furthermore, thedata indicated that SA and MeSA in poplar may be converted from one to the other by SAMTand SABP2.4. To investigate the relation between activity differences of hydrogen peroxide （H2O2）and related enzymes in different resistance poplars inoculated with B. dothidea and theresistance of poplar trees，the content of H2O2,the activity dynamic changes of CAT and APXand expression of SABP gene relative were studied. The results showed that the content ofH2O2of Populus tomentosa was higher than P.×beijingensis at the early stage of inoculation,after72hours of inoculation, H2O2content reaches its maximum （737.52mol/g）; afterinoculation with B. dothidea, the activity amplitude of CAT and APX of P.tomentosa weresignificantly higher than P.×beijingensis, the activity of CAT and APX were positivelycorrelated with poplar resistance; and associated with the reduction of CAT activity andcontent increased, Although the SA content arise in Populus×beijingensis, it not significant（7.83μg/g FW）, and not accompanied by decreased CAT activity, the content of H2O2compared with P. tomentosa peak time early and low peak （respectively737.52n mol/g FWand306.99nmol/g FW）, Infect the progress of SA give e-to CAT need the SA content attain akey content, P. tomentosa have SA-H2O2signal pathway; and detection of H2O2in poplarplant phloem by cytochemical method with CeCl3staining, which suggested there were a largenumber of H2O2-CeCl3precipitate particles on both sides of cell wall of P.tomentosa phloem at72h after inoculation;5. SA treatment experiments show that outside of P. tomentosa, H2O2content in SA,dealing with SA treatment plants inoculated with a similar trend and were significantly higher than the control, exogenous SA poplar body H2O2content increased rapidly,48h a peak, SA,after inoculation of poplar body H2O2content trends and SA separate treatment is consistent,but the concentration levels than the SA processed separately to higher SA inoculated plantsinoculated pre-H2O2content significantlyhigher than that of inoculated plants, so that duringthe interactions between Populus tomentosa canker exogenous SA can significantly promotethe H2O2accumulation of poplar body; determination showed that the CAT and APX activity,and exogenous SA can inhibit the APX activity did not change significantly under the CATactivity, exogenous SA to be seen in the interaction system of poplar canker in SA mayprimarily through the regulation of CAT, the accumulation of H2O2disease resistance;Meanwhile, we isolated and identified full-length coding sequence of PtSABP and PbSABPfrom P. tomentosa and P.×beijingensis stems cDNAs, NCBI blast and phylogenetic treeanalysis shows that, proteins of PtSABP and PbSABP belonging to the Catalaselikesuperfamily, their sequences are90%identical to that of tobacco SABP at the amino acid level,RT-PCR analysis showed that gene expression of SABP was inhibited in6-24h after inoculatedwith B. dothidea., suggesting that SA binding with SABP at this time, inhibition of CATactivity, thus contributing increased significantly of the H2O2content at24h after inoculation..The experiments show that the difference of content of SA and H2O2in poplar is due toinoculated with B. dothidea, SABP involved in the disease resistance of poplar and suggestedthat the resistance of poplar to B. dothidea relevant to the accumulation of SA and H2O2.