| In this paper, Various concentrations of sulfuretted hydrogen donor sodium bisulfide were added to birch suspension cultured cells in order to find the optimal treatment concentration and time for Cell growth and secondary metabolism(the content of triterpene, flavone and polyphenol).At the same time, biosynthetic enzymes or gene of IAA, ABA, JA and PAs were analyzed.Then,both hormone and NaSH were added to to birch suspension cultured cells, we analyzed the relationship between hormone and the secondary metabolites. The aim was to have a preliminary study that the effect of H2S on the endogenous hormones and secondary metabolites accumulation in birch suspension cultured cells,at the same time, lay the foundation for Secondary metabolites accumulation mechanism The main findings are as follows:1. Effect of H2S on physiological state in birch suspension cells0.1mmol/L,0.5mmol/L,1mmol/L and 5mmol/L NaHS were added into 8d birch suspension cultured cells for 3-96h. Research found 0.1mmol/L NaHS groups strengthen cells vitality and cells growth.However, lmmol/L NaHS groups reduce cells vitality and cells growth.Among them,0.1mmol/L NaHS treatment for 96h induce the highest cells vitality, the increasing rate was 37.66%.1mmol/L NaHS treatment for 96h induce the lowesat cells vitality, only 24.09% of contral groups.Research on pH,electrical conductivity and malondiadehyde content found 0.1mmol/L NaHS treatment had a little influence on on pH,electrical conductivity and malondiadehyde content. However, 1mmol/L NaHS treatment reduced pH,at the same time improved the electrical conductivity and malondiadehyde content. Among them,after 5mmol/L NaHS treatment for 96h,the pH reduce 0.73, electrical conductivity increased 89.15% and the content of malondiadehyde increased 44.62%.Research on enzyme activities (SOD, POD, CAT and PAL)found all enzyme activities were increased except 0.1mmol/L NaHS treatment. Among them,The maximum SOD activities were induced by 1mmol/L H2S for 96h, the increasing rate was 41.04%, The maximum POD activities were induced by lmmol/L H2S for 24h, the increasing rate was 94.55%, The maximum CAT activities were induced by lmmol/L H2S for 48h, the increasing rate was 150% The maximum PAL activities were induced by 0.5 mmol/L H2S for 24h, the increasing rate was 71.43%.2. Effect of H2S on the secondary metabolism in birch suspension cellsTreament same as above.The NaHS treatment improved the secondary metabolites accumulation except the 5mmol/L NaHS groups. Among them,The most secondary metabolites accumulation were induced by 1mmol/L H2S for 24h,the content of triterpene, flavone and polyphenol increase 36.67%,28.57% and 36.89% respectively.It can be seen, low concentration H2S is conducive to cell growth and high concentration H2S is conducive to secondary metabolites accumulation.The effection of H2S polyphenol> triterpene> flavone.3. Effect of H2S on endogenous hormone in birch suspension cellsTreament same as above.The NaHS treatment decreased the content of IAA and increase the content of ABA, JA and PAs. Correlation analysis showed that the content of IAA was significantly and negatively correlated with the secondary metabolites accumulation,but the content of ABA, JA and PAs were significantly and positively correlated with the secondary metabolites accumulation (1) The content of IAA gradually decreased after more than 0.5mmol/L NaHS treatment.The content of IAA was only 46.78% of control groups after lmmol/L NaHS treating for 24h, at the same time the expression of GH3.5A、GH3.5B、 GH3.9A were increased 158.12%,307.47% and 509.73%. (2) The content of ABA increased in initial stage and then declined. The highest content of ABA was induced by lmmol/L NaHS for 12h, the increasing rate was 196.46%, The highest expression of NCED4 was induced by 1 mmol/L H2S for 6h, the increasing rate was 431.85%. (3) The content of PAs increased after the NaHS treatment. The highest activity of ADC was induced by lmmol/L H2S for 12h. the increasing rate was 15.91%. The highest content of Put was induced by 1mmol/L NaHS for 96h, the increasing rate was 20.34%.The highest activity of SAMDC was induced by 0.5mmol/L NaHS for 48h, the increasing rate was 74.15%.At the same time the content of Spm increased 8.41%. (4) The content of JA increased in initial stage and then declined. The highest activity of LOX was induced by 1mmol/L NaHS for 12h, the increasing rate was 22.39%, The highest content of JA was induced by lmmol/L NaHS for 24h, the increasing rate was 67.17%.It can be seen,the expression of geen and enzyme changed,then the content of hormone changed.The next section we analyze the relation between hormone and secondary metabolism.4. H2S work with hormone on secondary metabolism accumulate in birch suspension cellsDifferent cpntent of IAA, ABA, Put. Spd and Spm were added into 8d birch suspension cultured cells ensure that final concentration were 0. 1mmol/L、0.5mmol/L、1mmol/L and 5mmol/L,at the same time those hormone work with lmmol/L NaHS were added into birch suspension cultured cells for 24h. Research found IAA decrease the content of second metabolism,however ABA and PAs increase the content of second metabolism. (1) The lowest content of second matabolism was induced by 5mmol/L IAA,the content of triterpene, flavone and polyphenol were only 88.28%、86.44% and 78.91% of contral groups.5mmol/L IAA work with 1mmol/L NaHS increase the second metabolism accumulation, the content of triterpene, flavone and polyphenol were 111.16%、107.39% and 93.82% of contral groups. (2) The highest content of second matabolism was induced by lmmol/L ABA,the content of triterpene, flavone and polyphenol increased 17.85%,23.48% and 16.73% respectively.1mmol/L ABA work with lmmol/L NaHS increased the second metabolism accumulation, the content of triterpene, flavone and polyphenol increased 58.19%, 46.02% and 57.30% respectively. (3) The highest content of triterpene was induced by 5mmol/L Put,the content of triterpene increased 22.91%,work with lmmol/L NaHS the content of triterpene increased 68.00%. The highest content of flavone was induced by lmmol/L Spm,the content of flavone increased 31.09%,work with 1mmol/L NaHS the content of flavone increased 48.62%. The highest content of polyphenol was induced by 0.5mmol/L Spd,the content of polyphenol increased 37.17%,work with lmmol/L NaHS the content of polyphenol increased 75.91%. Correlation analysis showed that IAA, ABA, PUT and SPD were significantly and positively correlated with the secondary metabolites accumulation.Work with NaHS, the relativity between IAA, ABA, Put and secondary metabolites further increased.So,IAA, ABA, JA and PAs work with H2S induce the secondary metabolites accumulation. |
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