Genome-Wide Exploration of the Grape GLR Gene Family and Differential Responses of VvGLR3.1 and VvGLR3.2 to Low Temperature and Salt Stress

Grapes,one of the oldest tree species globally,are rich in vitamins.However,environmental conditions such as low temperature and soil salinization significantly affect grape yield and quality.The glutamate receptor(GLR)family,comprising highly conserved ligand-gated ion channels,regulates plant growth and development in response to stress.In this study,11 members of the VvGLR gene family in grapes were identified using whole-genome sequence analysis.Bioinformatic methods were employed to analyze the basic physical and chemical properties,phylogenetic trees,conserved domains,motifs,expression patterns,and evolutionary relationships.Phylogenetic and collinear analyses revealed that the VvGLRs were divided into three subgroups,showing the high conservation of the grape GLR family.These members exhibited 2 glutamate receptor binding regions(GABAb and GluR)and 3-4 transmembrane regions(M1,M2,M3,and M4).Real-time quantitative PCR analysis demonstrated the sensitivity of all VvGLRs to low temperature and salt stress.Subsequent localization studies in Nicotiana tabacum verified that VvGLR3.1 and VvGLR3.2 proteins were located on the cell membrane and cell nucleus.Additionally,yeast transformation experiments confirmed the functionality of VvGLR3.1 and VvGLR3.2 in response to low temperature and salt stress.Thesefindings highlight the significant role of the GLR family,a highly conserved group of ion channels,in enhancing grape stress resistance.This study offers new insights into the grape GLR gene family,providing fundamental knowledge for further functional analysis and breeding of stress-resistant grapevines.

Genome Wide Characterization of CBL-CIPK Family Genes and Their Responsive Expression in Rosa chinensis

Calcium(Ca^(2+))plays a pivotal role in various signal transduction pathways.Calcineurin B-like proteins(CBLs)are a unique group of Ca^(2+)sensors that decode Ca^(2+)signals by activating the plant specific protein kinase known as the CBL-interacting protein kinase(CIPK).In plants,the CBL-CIPK signaling network regulates multiple signals in response to different extracellular cues including abiotic stress.However,the genome wide annotation and expression patterns of CBLs and CIPKs in woody cutting flower plants are still unclear.In this study,a total number of 7 CBLs(RcCBLs)and 17 CIPKs(RcCIPKs)genes,divided into four and five subfamilies,respectively,were identified from the rose genome.All RcCBLs possess a classic elongation factor-hand(EF-hand)domain,while all RcCIPKs possess both the classic kinase and NAF domains.Most RcCBLs were predicted to be plasma membrane localized,whereas most RcCIPKs were predicted to be cytoplasmic localized.Synteny analysis showed that one RcCBL gene pair and five RcCIPK gene pairs have gone through whole genome duplication events.Promoter cis-element prediction assays indicated that RcCBLs and RcCIPKs could function in different abiotic stress responses in rose plants.Further quantitative real-time PCR analysis demonstrated that RcCBLs and RcCIPKs were expressed in different organs with overlapped but distinct patterns in response to various abiotic stresses.The findings in this work will provide fundamental information and gene resources for further functional research on RcCBLs and RcCIPKs.

Development of a stable attenuated double-mutant of tobacco mosaic virus for cross-protection

Tobacco(Nicotiana tabacum)and tomato(Solanum lycopersicum)are two major economic crops in China.Tobacco mosaic virus(TMV;genus Tobamovirus)is the most prevalent virus infecting both crops.Currently,some widely cultivated tobacco and tomato cultivars are susceptible to TMV and there is no effective strategy to control this virus.Cross-protection can be a safe and environmentally friendly strategy to prevent viral diseases.However,stable attenuated TMV mutants are scarce.In this study,we found that the substitutions in the replicase p126,arginine at position 196(R^(196))with aspartic acid(D),glutamic acid at position 614(E^(614))with glycine(G),serine at position 643(S^(643))with phenylalanine(F),or D at position 730(D^(730))with S,significantly reduced the virulence and replication of TMV.However,only the mutation of S^(643) to F reduced the RNA silencing suppression activity of TMV p126.A double-mutant TMV-E614G-S643F induced no visible symptom and was genetically stable through six successive passages in tobacco plants.Furthermore,our results showed that TMV-E614G-S643F double-mutant could provide effective protection against the wild-type TMV infection in tobacco and tomato plants.This study reports a promising mild mutant for cross-protection to control TMV in tobacco and tomato plants.

Genome-Wide Identification and Expression Profiling of the Shaker K+ Channel and HAK/KUP/KT Transporter Gene Families in Grape (Vitis vinifera L.)

Potassium(K+)is an essential macronutrient for plants to maintain normal growth and development.Shaker-like K+channels and HAK/KUP/KT transporters are critical components in the K+acquisition and translocation.In this study,we identified 9 Shaker-like K+channel(VvK)and 18 HAK/KUP/KT transporter(VvKUP)genes in grape,which were renamed according to their distributions in the genome and relative linear orders among the distinct chromosomes.Similar structure organizations were found within each group according to the exon/intron structure and protein motif analysis.Chromosomal distribution analysis showed that 9 VvK genes and 18 VvKUP genes were unevenly distributed on 7 or 10 putative grape chromosomes.Three pairs of tandem duplicated genes and one pair of segmental duplicated genes were observed in the expansion of the grape VvKUP genes.Gene expression omnibus(GEO)data analysis showed that VvK and VvKUP genes were expressed differentially in distinct tissues.Various cis-acting regulatory elements pertinent to phytohormone responses and abiotic stresses,including K+deficiency response and drought stress,were detected in the promoter region of VvK and VvKUP genes.This study provides valuable information for further functional studies of VvK and VvKUP genes,and lays a foundation to explore K+uptake and utilization in fruit trees.

Site-directed mutagenesis identified the key active site residues of alcohol acyltransferase PpAAT1 responsible for aroma biosynthesis in peach fruits

The aroma of peach fruit is predominantly determined by the accumulation of γ-decalactone and ester compounds.A previous study showed that the biosynthesis of these aroma compounds in peach fruit is catalyzed by PpAAT1,an alcohol acyltransferase.In this work,we investigated the key active site residues responsible for γ-decalactone and ester biosynthesis.A total of 14 candidate amino acid residues possibly involved in internal esterification and 9 candidate amino acid residues possibly involved in esterification of PpAAT1 were assessed via site-directed mutagenesis.Analyses of the in vitro enzyme activities of PpAAT1 and its site-directed mutant proteins(PpAAT1-SMs)with different amino acid residue mutations as well as the contents of γ-decalactone in transgenic tobacco leaves and peach fruits transiently expressing PpAAT1 and PpAAT1-SMs revealed that site-directed mutation of H165 in the conserved HxxxD motif led to lost enzymatic activity of PpAAT1 in both internal esterification and its reactions,whereas mutation of the key amino acid residue D376 led to the total loss ofγ-decalactone biosynthesis activity of PpAAT1.Mutations of 9 and 7 other amino acid residues also dramatically affected the enzymatic activity of PpAAT1 in the internal esterification and esterification reactions,respectively.Our findings provide a biochemical foundation for the mechanical biosynthesis ofγ-decalactone and ester compounds catalyzed by PpAAT1 in peach fruits,which could be used to guide the molecular breeding of new peach species with more favorable aromas for consumers.

Overexpression of IbSINA5 Increases Cold Tolerance through a CBF SINA-COR Mediated Module in Sweet Potato

Seven in absentia(SINA)family proteins play a central role in plant growth,development and resistance to abiotic stress.However,their biological function in plant response to cold stress is still largely unknown.In this work,a seven in absentia gene IbSINA5 was isolated from sweet potato.Quantitative real-time polymerase chain reaction(qRT-PCR)analyses demonstrated that IbSINA5 was ubiquitously expressed in various tissues and organs of sweet potato,with a predominant expression in fibrous roots,and was remarkably induced by cold,drought and salt stresses.Subcellular localization assays revealed that IbSINA5-GFP fusion protein was mainly localized in cytoplasm and nucleus.Overexpression of IbSINA5 in sweet potato led to dramatically improved resistance to cold stress in transgenic plants,which was associated with the up-regulated expression of IbCOR(cold-regulated)genes,increased proline production,and decreased malondialdehyde(MDA)and H2O2 accumulation in the leaves of transgenic plants.Furthermore,transient expression of IbCBF3,a C-repeat binding factor(CBF)gene,in the leaf protoplasts of wild type sweet potato plants up-regulated the expression of both IbSINA5 and IbCOR genes.Our results suggest that IbSINA5 could function as a positive regulator in the cold signaling pathway through a CBF-SINA-COR mediated module in sweet potato,and have a great potential to be used as a candidate gene for the future breeding of new plant species with improved cold resistance.

De Novo Transcriptome Analysis in Hevea brasiliensis to Unveil Genes Involved in Low Temperature Stress Response

Low temperature is one of the adversities threatening the growth and development and reduces the yield of rubber trees.However,molecular mechanisms toward rubber trees in response to low temperature are largely unclear.In this study,7,159 and 7,600 differentially expressed genes(DEGs)were identified in‘Reyan 73397’rubber trees.Through GO analysis,the catalytic activity was the representative of the GO term in the only DEGs at the two studied temperatures(room temperature and 4°C,respectively),while KEGG analysis showed that carbon metabolism was the most important grouping under the comparison of these two temperatures.In addition,expression of 9 members of transcription factor MYB family genes were further verified by qRT-PCR,and MYB family genes may play important roles in the regulation of rubber trees under low temperature stress.This study provided a theoretical foundation for(1)revealing the molecular mechanisms of rubber trees in response to low temperature and(2)breeding of tolerant varieties of rubber trees.

De novo Transcriptome Analysis in Leymus mollis to Unveil Genes Involved in Salt Stress Response

Leymus mollis,a wild relative of wheat,is very tolerant to salt stress,and has been considered as a valuable genetic resource for wheat breeding.However,the genetic basis for salt tolerance of this species is still largely unknown.In this study,de novo sequencing,assembly and analysis of L.mollis transcriptome in response to salt stress was performed.A total of 110,323 and 112,846 unigenes were generated for the NaCl-free(CK)and 180 mM NaCl-treated(CT)library,respectively.For the two libraries,73,414 unigenes were successfully annotated in five common protein databases,and 7521 differentially expressed genes(DEGs)between CK and CT libraries were identified.GO enrichment analysis of the DEGs showed that the significantly enriched GO terms were predominantly involved in environmental adaptation(including“response to abiotic stimulus”,“response to water deprivation”),regulation of signaling pathway(such as“regulation of abscisic acid mediated signaling pathway”,“regulation of cell communication”),and photosynthesis(including“response to light stimulus”,“photosynthesis,light harvesting”and“chlorophyll metabolic process”).KEGG pathway enrichment analysis showed that“mRNA surveillance pathway”,“RNA transport”and“plant hormone signal transduction”were predominantly enriched pathways,followed by several secondary metabolic pathways,photosynthesis,carbohydrate metabolism and lipid metabolism.In addition,DEGs related to osmotic stress,ion homeostasis and oxidative stress,including four dehydrins,five aquaporins,an LmNHX2 and several antioxidant enzymes or proteins genes,were found to be up-regulated in response to salt stress.These results will be helpful for further studies on the molecular mechanisms of salt responses in L.mollis.