Physiological And Molecular Mechanism Of Strigolactones-Regulated Chilling Stress Tolerance In Pepper

As a typical thermophilous vegetable,the growth and yield of peppers are easily limited by chilling conditions.Strigolactones（SLs）,a carotenoid-derived plant hormone,play an important role in plant growth and development and adaptation to biotic and abiotic stresses.In this study,we used the‘Hangjiao No.4’pepper seedlings,to examine the effect of exogenous rac-GR24（Synthetic analogue of strigolactone,SL）and Tis108（Strigolactones biosynthesis inhibitor,Tis）on the growth,photosynthesis,antioxidant defense,and endogenous plant hormone levels of pepper seedlings under chilling stress.In addition,we used RNA-seq to carry out transcriptome analysis of pepper leaves to reveal the role of Strigolactones in response to chilling.The main result are as follows.（1）Chilling stress suppressed the growth of pepper seedlings,including plant fresh,dry weight and root architecture,destroyed leaf tissue and biological membrane system,increased the content of soluble sugar,soluble protein and proline.The inhibition of growth and the damage of leaf tissue and membrane system in plant with SL treatment are less than that in plant without SL/Tis treatment,and are less than that in plant with Tis treatment.During chilling stress,the accumulation of the above-mentioned osmotic adjustment substances is further increased by SL and decreased by Tis.（2）Under chilling stress,SL alleviates the decline of photosynthetic pigment content and net photosynthetic rate（Pn）in pepper leaves.Meanwhile,SL application increased the closure degree of stomata in the early stage of chilling stress（24 h）,resulting in significantly lower stomatal conductance（Gs）than plants without SL/Tis treatment.With the prolong of stress（72h）,the closure degree of stomata in plants with SL pretreatment was reduced,resulting in higher stomatal conductance.The effect of Tis pretreatment was opposite.（3）Under chilling stress,SL application alleviates the inhibition of photosynthetic electron transfer from O_A to Q_B（V_J,φEo）,protects the terminal electron acceptor on the PSI acceptor side（φRo）,optimizes the energy distribution in unit cross section and single active reaction center,reduces the loss of ATP,and maintains the Calvin cycle,thereby increasing the metabolic consumption of the excitation energy[（1-q P）/NPQ],lead to alleviating the degree of photoinhibition during initial chilling stress in the pepper seedlings（Fv/Fm）,improving the actual photochemical efficiency（φPSII）.The effect of Tis pretreatment was opposite.（4）Under long-term chilling stress（120 h）,SL pretreatment increases the accumulation of lutein,zeaxanthin,antheraxanthin,and violaxanthin in pepper leaves,improves the activity of violaxanthin deepoxidase（VDE）,and promotes degree of de-epoxidation of the xanthophyll cycle[（Z+A）/（Z+A+V）],increasing the energy dissipation（NPQ）,reducing the excessive excitation energy of the PSII reaction center[（1-q P）/NPQ],finally alleviated the photoinhibition degree（Fv/Fm）induced by chilling in pepper seedlings.（5）Under chilling stress,the Fv/Fm of the leaves of plants treated with streptomycin sulfate（D1 protein synthesis inhibitor,SM）were all lower than those of plants without SM treatment.However,the Fv/Fm of plants with SL and Tis was still significantly higher and lower than that of plants without SL and Tis in the presence of SM,indicating that the protection of strigolactone on PSII does not depend on the turnover of D1 protein.（6）Under chilling stress,SL application activated the expression of Ca Cu/Zn-SOD,Ca Fe-SOD,Ca CAT,Ca APX,Ca DHAR,Ca MDHAR and Ca GR,increasing the activity of corresponding antioxidant enzymes,and increasing the ratio of As A/DHA and GSH/GSSH,to reduce the accumulation of O₂.^-and H₂O₂.It also reduced the level of auxin（IAA）,and further increased the level of cytokinin（ZT）,salicylic acid（SA）and jasmonic acid（JA）,and dynamically changed the level of abscisic acid（ABA）.（7）Twelve c DNA libraries were constructed from four different treatments of normal control（NT）,chilling stress（LT）alone,chilling stress of SL pretreated plants（SL＿LT）,and chilling stress of Tis pretreated plants（Tis＿LT）.A total of 77.19 Gb of clean reads were obtained by transcriptome sequencing,and 8776 differentially expressed genes were screened among the three chilling treatment groups,including 4473（51.0%）upregulated genes and 4303（49.0%）downregulated genes.GO functional characterization found that,based on the gene expression level of NT treatment,the term that Tis＿LT is significantly enriched to cell components,and mostly related to photosynthesis（photosystem,photosystem II,photosystem I,photosynthetic membrane,thylakoid,thylakoid Membrane）,and are all down-regulated expression.Based on the gene expression level of LT treatment,SL＿LT and Tis＿LT were also significantly enriched in the cell component term,and related to photosynthesis,but they were up-regulated and expressed respectively.KEGG pathway annotation found that,Tis＿LT vs NT was significantly annotated to the photosynthesis-antenna protein pathway,SL＿LT vs LT is mainly annotated to the signal transduction pathway of plant hormones,Tis＿LT vs LT is significantly annotated to the photosynthesis-antenna protein pathway,SL＿LT vs Tis＿LT significantly annotated the KEGG pathways of photosynthesis-antenna protein,photosynthesis,carbon fixation of photosynthetic organisms,glyoxylic acid and dicarboxylic acid metabolism,porphyrin and chlorophyll metabolism,carbon metabolism,and nitrogen metabolism.The porphyrin and chlorophyll metabolism,photosynthesis-antenna protein,photosynthesis,carbon fixation of photosynthetic organisms and carotenoid synthesis pathways were further analyzed.The results were annotated to 18,21,29,31 and 15 differentially expressed genes respectively.In basically,those genes were highly expressed under SL＿LT treatment,moderately expressed under LT treatment,and low expressed under Ti S＿LT treatment.