Effects Of FaSnRK1α On Fruit Sucrose Metabolism,Gray Mold And Waterlogging Resistance Of Strawberry

Strawberry fruits are important fresh and processed fruits because of their unique flavor and high nutrient contents.Improving fruit quality and enhancing resistance are the key topics of fruit tree research.Plant sucrose non-fermentable protein kinase-1（SnRK1）plays an important switching role in regulating carbohydrate metabolism and responding to biological and abiotic stresses.It is of great significance to study the effect of SnRK1 on sucrose metabolism of fruit and stress resistance for improving fruit quality and yield.Therefore,the effects of SnRK1 on sucrose metabolism,gray mold resistance and anaerobic respiration metabolism under waterlogging stress were studied using ’Miaoxiang 7’ strawberry as the material.The main results are as follows:1.In this study,a SnRK1 α-subunit gene was cloned from the leaves of strawberry（‘Miaoxiang 7’）using reverse transcription PCR,and was designated as FaSnRK1α.We transiently expressed FaSnRK1α in strawberry fruits and found that overexpression（OE）of the FaSnRK1α gene significantly increased the sucrose content,whereas repression of FaSnRK1α by RNA interference（RNAi）decreased the sucrose content.Further analysis revealed that FaSnRK1α increased the expression of Fa SUS1 and Fa SUS3 as well as the activity of sucrose synthase（SUS）and that Fa SPS1 expression and sucrose phosphate synthase（SPS）activity were strongly downregulated,which decreased the accumulation of sucrose.However,the expression of Fa SPS3,which is reported to contribute to sucrose accumulation,was induced by FaSnRK1α,and Fa NI expression and invertase（INV）activity were upregulated by FaSnRK1α.In addition,FaSnRK1α positively upregulated the expression of the sucrose transporter（SUT）genes Fa SUT1 and Fa SUT5 and interacted with Fa SUS1,Fa SPS1 and Fa SPS3 proteins but not with Fa SUS3,Fa NI,Fa SUT1 or Fa SUT5 proteins.Overall,FaSnRK1α systematically regulates the expression of the genes and activity of key enzymes involved in the sucrose metabolic pathway and promotes the long-distance transport of sucrose,thereby increasing sucrose accumulation.2.In the present study,the FaSnRK1α-OE fruits matured approximately 4 days sooner than did the control fruits,and compared with the control fruits,the FaSnRK1α-RNAi fruits matured approximately 3.5 days later.Compared with those in the control fruits,the ABA and anthocyanin contents in the FaSnRK1α-OE fruits and FaSnRK1α-RNAi fruits were greater and lower,respectively.Manipulation of the FaSnRK1α gene also strongly altered the expression of some genes involved in fruit color and softening,which is consistent with the effects of this gene on the fruit phenotype.Notably,FaSnRK1α-OE increased the expression of Fa PG,Fa PL,Fa EXP1,Fa CHS,Fa CHI and Fa F3 H,while FaSnRK1α-RNAi repressed the expression of these genes.Conversely,FaSnRK1α-OE and FaSnRK1α-RNAi dramatically affected the expression of Fa DFR.Collectively,these results strongly suggest that FaSnRK1αplays a critical role in the regulation of strawberry fruit ripening.3.In order to study the effect of FaSnRK1α on gray mold,the FaSnRK1α-OE and RNAi fruits were inoculated with Botryis cinerea.The result reveals a positive role of FaSnRK1α in the regulation of resistance to gray mold.Further analysis found that FaSnRK1α affects SA content by regulating Fa PAL1 and Fa PAL2 expressions.The genes related to the SA signaling pathway Fa TGA1 and Fa TGA2.1 were significantly increased or decreased in FaSnRK1α-OE or FaSnRK1α-RNAi fruit,respectively.It was also found that FaSnRK1α interacted with the Fa WRKY33.2 protein and negatively regulated Fa WRKY33.2 expression,which acts as a repressor of disease resistance to B.cinerea.Finally,FaSnRK1α regulates six PR genes and the antioxidant enzymes to boost defense response after B.cinerea inoculation.Our findings showed that FaSnRK1α increases the resistance of strawberry fruit to B.cinerea via SA signaling pathway and interaction with the Fa WRKY33.2 transcription factor.4.FaSnRK1α-OE and FaSnRK1α-RNAi strawberry roots were obtained using Agrobacterium rhizogenes-mediated root transformation protocol and then were treated with waterlogging stress.The FaSnRK1α-OE roots had higher root activity and respiratory rate than the FaSnRK1α-RNAi roots,and FaSnRK1α up-regulates the activity of anaerobic respiration related enzymes,including hexokinase（HK）,phosphofructokinase（PFK）,pyruvate kinase（PK）,pyruvate decarboxylase（PDC）,alcohol dehydrogenase（ADH）and lactate dehydrogenase（LDH）.And FaSnRK1α enhanced reactive oxygen species（ROS）metabolism by increasing the antioxidant enzyme activities.This study further sequenced the transcriptomes of the WT and FaSnRK1α-RNAi roots and the DEGs were clearly enriched in defense response,response to biotic stimulus,and cellular carbohydrate metabolic process,and 42 genes in glycolysis,and 30 genes in pyruvate metabolism were significantly regulated in FaSnRK1α-RNAi roots.Furthermore,we analyzed the transcriptional levels of two anaerobic genes and three ERFVIIs,and Fa ADH1,Fa PDC1,Fa HRE2 and Fa RAP2.12 were up-regulated by FaSnRK1α,signifying that FaSnRK1α may be involved in the ethylene signaling pathway to improve submergence tolerance.In conclusion,FaSnRK1α activates the expression of anaerobic response genes and ERFVIIs,thereby enhancing anaerobic respiration metabolism in response to the low oxygen conditions under submergence.