Effects Of Two Rain-Shelter Modes On The Physiological Characteristics And Gene Expression Of Sweet Cherry In Guizhou Province,China

Sweet cherry（Prunus avium L.）has been widely grown in many countries and has become an important cultivated tree due to its excellent fruit quality and high economic value.In recent years,the sweet cherry industry has rapidly extended to South China,including Southwest China.However,excessive rain in Southwest China has severely inhibited flower bud differentiation,pollination and fruit setting;therefore,fruit yield and quality are highly restricted in this region.Rain-shelter cultivation can effectively reduce the adverse effects of rain on sweet cherry.To date,however,there have been few studies concerning the influence of rain-shelter modes on the physiological characteristics and gene expression of sweet cherry.Therefore,the leaf morphology and structure,photosynthesis and chlorophyll fluorescence characteristics,as well as the fruit yield and quality of sweet cherries on the 35th,45th and 55th days after the full flowering period（DAF35,DAF45 and DAF55）,were investigated after exposure to three cultivation modes i.e.,arched-cover,umbrella-cover and open-field in 2020 and 2021.Additionally,transcriptomic and metabonomic analysis technology were used to identify the differentially expressed genes,metabolites and metabolic pathways involved in environmental adaptation when trees were subjected to rain shelters.The main results were as follows:1)The effect of rain shelters on the leaf structure and antioxidants of sweet cherryCompared with open-field,arched-cover and umbrella-cover modes reduced the light intensity by 19.59%and 14.74%,respectively.The leaf morphology and cell structure of sweet cherries cultivated under the two kinds of rain shelters changed significantly.Compared with the open-field,the length,width and area of the leaves were significantly increased,and the thickness of the leaf palisade tissue and sponge tissue,the thickness of the epidermis,the thickness of the leaf and the value of palisade tissue/sponge tissue were reduced to varying degrees.At the same time,rain shelters also increased the superoxide dismutase（SOD）,peroxidase（POD）and catalase（CAT）activities of leaves and reduced the content of malondialdehyde（MDA）during DAF45 and DAF55.There were no obvious differences in leaf morphology,cell structure or antioxidant enzyme activity between the two rain shelter cultivation modes.2)The effect of rain shelters on the photosynthetic characteristics of sweet cherryThe two rain-shelter modes significantly increased the leaf chlorophyll a（Chla）,chlorophyll b（Chlb）and carotenoid（Car）contents and reduced the Chla/Chlb value.The photosynthetic"midday break"phenomenon existed in leaves under the three cultivation modes,but the accumulation of sunlight on arched-cover and umbrella-cover leaves was higher than that from the open-field,and there was a significant difference compared to the open-field at both DAF45 and DAF55.Compared with open-field,rain shelters significantly increased the apparent quantum efficiency（AQY）of the leaf,reduced the light compensation point（LSP）and light saturation point（LCP）,and enhanced the utilization efficiency of weak light of the leaf;at the same time,these shelters improved the apparent carboxylation efficiency（ACE）,reduced the CO₂compensation point（CCP）and the CO₂saturation point（CSP）,and enhanced the utilization efficiency of CO₂of the leaf.The two kinds of rain shelters increased the actual photochemical efficiency（ΦPSII）,PSII electron transfer rate（ETR）,photochemical quenching coefficient（q P）and PSII maximum light energy conversion efficiency（Fv/Fm）values of sweet cherry leaves but reduced the nonphotochemical quenching coefficient（NPQ）,whereΦPSII and q P were significantly different from those of the open-field.The two rain shelters also increased the soluble sugar（SS）and soluble protein（SP）contents of leaves to varying degrees.However,the difference in the photosynthetic pigment content,photosynthetic characteristics,chlorophyll fluorescence characteristic parameters and accumulation of organic matter between the two rain-shelter modes was not obvious.Among them,arched-cover had a more obvious influence on leaf morphology and physiological characteristics than umbrella cover.3)The effect of rain shelters on the yield and quality of sweet cherry fruitDuring the fruit development period,the fruit under arched-cover had the highest growth rate,which was characterized by the largest polar and suture diameters and single fruit weight,followed by fruit under umbrella-cover and open-field;at DAF55in 2021,arched-cover significantly increased the polar and suture diameters and single fruit weight.The coloring effect of the fruits under the two rain-shelter modes was better than that of the open-field,and the arched-cover had the best coloring effect.Arched-cover significantly improved the springiness and cohesiveness of the fruit and improved the texture of the fruit;both rain shelters increased the total soluble solids（TSS）,soluble protein（SP）,soluble sugar（SS）,ascorbic acid（As A）and anthocyanin（Anth）contents of the fruit and reduced the titratable acid（TA）content.Among them,arched-cover had a more significant effect on TSS,SS and SP,while umbrella-cover had a more significant effect on As A.The comprehensive evaluation results of the principal component analysis of fruit quality showed that the fruit quality under arched-cover was the best,followed by that under umbrella-cover and open-field.Arched-cover and umbrella-cover both significantly increased the fruit setting rate and yield per plant and significantly reduced the fruit cracking rate.However,arched-cover had a more significant impact on the fruit setting rate,fruit cracking rate and yield.Therefore,the arched-cover mode had the effect of significantly increasing fruit setting and yield,reducing the fruit cracking rate,and improving fruit quality.4)The effect of rain shelters on the transcriptional and metabolomic levels of sweet cherry leavesIn the three developmental stages,the numbers of differentially expressed genes（DEGs）in response to arched-cover and umbrella-cover were 3285 and 670,respectively,and the numbers of differentially expressed metabolites（DEMs）in response to arched-cover and umbrella cover were 176 and 77 species,respectively.Therefore,arched-cover had a more significant effect on the transcription and metabolism of sweet cherry leaves than umbrella-cover.Compared with open-field mode,genes involved in the regulation of leaf photosynthesis and antioxidant capacity and other metabolic pathways were also differentially expressed under arched-cover and umbrella-cover modes.Among them,under arched-cover modes,the genes encoding photosynthetic antenna proteins Lhca and Lhcb,the photosynthetic electron transporter Psb D,and the carbon fixation-related gene rbc S were all significantly upregulated.The hem H and Chl H genes involved in chlorophyll synthesis were significantly upregulated,while the chlorophyll decomposition-related genes PAO and Chlase were significantly downregulated,and the carotene synthesis-related genes crt B,ZEP and LUT5 were also significantly upregulated;the genes encoding SOD,POD and glutathione S-transferase GST also showed significant upregulated expression.Under umbrella-cover mode,the transcription levels of Lhca,Psb D,Chl H,hem F,crt B,ZEP and GST were also significantly increased,while the expression of the chlorophyll decomposition-related genes PAO and Chlase was significantly downregulated.L-glutamate,the starting substance involved in amino acid metabolism,chlorophyll synthesis and glutathione metabolism,was significantly upregulated under the arched-cover and umbrella-cover modes,and most of the metabolites that undergo significant changes in the amino acid metabolism pathway were upregulated.Porphobilinogen,a key intermediate metabolite in chlorophyll synthesis,was significantly upregulated under the umbrella-cover mode,and L-cysteine andγ-glutamine,which function in the metabolic synthesis of glutathione,were significantly upregulated under the arched-cover mode.The upregulation of synthetic metabolites in several pathways combined with the upregulated expression of genes regulated by catalytic synthetase promoted the synthesis of final products,which is beneficial to the synthesis and accumulation of amino acids,chlorophyll and glutathione in leaves.5)The effect of rain shelters on the transcriptional and metabolomic levels of sweet cherry fruitsThe transcriptome and metabolome analysis of fruits under arched-cover and open-field modes across three developmental stages revealed a total of 5209differentially expressed genes（DEGs）and 318 differentially expressed metabolites（DEMs）.The metabolic pathway annotations involving sugar metabolism,such as starch and sucrose,galactose and fructose,and mannose metabolism,were found for70 DEGs,among which the important genes SUS,SPS,INV and SDH,which promote the synthesis and accumulation of sucrose,glucose and fructose,were significantly upregulated,while these genes,such as HK and FK,were downregulated.Phenylpropanoid biosynthesis-,flavonoid biosynthesis-,and anthocyanin biosynthesis-related pathways for anthocyanin synthesis were found for a total of 126DEGs and 27 DEMs,including PAL,C4H,F3’H,4CL,DFR,CHS,UFGT,CHI and ANS,which were significantly upregulated;at the same time,phenylalanine,coumaric acid,naringenin,dihydrokaempferol,dihydroquercetin,pelargonidin 3-glucoside,cyanidin 3-glucoside,peonidin 3-glucoside,cyanidin 3-rutinoside,delphinidin3-rutinoside,and cyanidin 3-sophoroside were significantly upregulated,which together promoted the synthesis and accumulation of fruit anthocyanins.The pathways related to polyamine synthesis,such as arginine and proline,cysteine and methionine,and the arginine anabolic pathway,were found for 35 DEGs and 11 DEMs,among which genes such as ADC,SPDS,arg D,SAMS,ODC and TAT were significantly upregulated,while genes such as PAO1,ALDH,MGL and ACC oxidase were significantly downregulated;at the same time,arginine,herring spermine,aspartic acid,homoserine,glutamine,N-acetyl-L-glutamic acid,methionine and other important metabolites were significantly upregulated,which is conducive to the synthesis and accumulation of the final metabolite polyamine.In summary,rain shelters could affect the expression of related genes and metabolites involved in the light energy absorption,CO₂fixation and antioxidant systems of sweet cherry leaves,as well as fruit sugar metabolism,anthocyanin synthesis and polyamine synthesis metabolism.Furthermore,the leaf morphology,light capture ability,carbon fixation ability and organic matter accumulation,as well as the fruit setting of the plant,morphology,color and internal substances of fruit were affected,which resulted in the adaptation of leaves and fruits to the changing microclimate environment under the rain shelter,thereby increasing fruit yield and improving fruit quality.Therefore,rain-shelter cultivation technology for sweet cherry can be implemented in the rainy areas of Guizhou,and the arched-cover mode has a better effect.