Identification Of The Genes Involved In G-protein Signaling In Mulberry And Their Regulation In Response To Abiotic Stresses

Drought,heat,cold,nutritional deficiency,soil salinity and heavy metal stress are most severe abiotic stresses limiting plant growth,development and reproduction.In order to adapt to these abiotic stresses,plants positively cope with stresses by changing initially their physiological and biochemical processes.Plants have evolved a series of physiological pathways to help plant tolerance to stresses,including signal recognition and transduction,transcription regulation,and stress responses.Signal recognition and transduction play key roles in plant abiotic stresses responses,and stress signal sensors,receptor-like protein kinases,second messengers,Ca²⁺signal pathway,ABA signal transduction,SOS signal pathway,MAPK cascades,ROS signal pathway and transcription factors participate in this process.Heterotrimeric guanine-nucleotide-binding proteins?G proteins?,a class of second messengers,are evolutionarily conserved signaling intermediates that regulate signal recognition,signal transduction,hormone perception and immune responses.Plant G proteins are involved in morphology,light signal reception,sugar sensing,responses to biotic and abiotic stresses and other physiological processes.The mechanism of G proteins response to abiotic stresses has been well studied in model plants,but very little is known about their function in woody plants,and several key pathways and regulation modes mediated by G proteins remain unclear or controversial.Mulberry is a deciduous and economically important perennial tree,and its leaves are the main source of food for silkworms.Besides,mulberry has multiple uses in ecology,food uses and pharmaceuticals.Mulberry has better adaptation to drought,salt stress,water logging,metal stress and other important environmental stresses,and thus it can be used as a potential plant to ameliorate ecologically fragile areas,but little is known regarding the molecular mechanisms.Also,there is no report on the functions of the G protein signal pathway in mulberry.It is notable that the expression of mulberry G protein signaling genes showed higher connectivity with many stress responsive genes by weighted gene co-expression network analysis?WGCNA?on the salt transcriptome data.Therefore,it is significant that conducting the functional analysis of mulberry G protein signaling for exploring the mechanism of mulberry abiotic stresses responses,which will also help to supplement and improve the research on G protein signaling in woody plants,especially for the research on several unclear or controversial pathways regulated by G proteins.To identify the key salt-stress responsive genes and analyze the connectivity of G protein signaling genes with other stress responsive genes,the mulberry tree“Cesha No.6 hybrids”and“Cesha No.12 hybrids”from Hetian Sericulutre Institute of Xinjiang Autonomous Region were used for salt stress treatment and transcriptome sequencing in this study.Bioinformatics method,gene cloning and yeast two-hybrid?Y2H?assay were used to identify the genes involved in mulberry G protein signaling.The expression patterns of these genes in different tissues and the seedlings under different G protein effectors,abiotic stresses and signaling molecules treatments were analyzed by quantitative real-time PCR.The obtained genes were transformed into tobacco or Arabidopsis and the tolerance of the transgenic plants to drought and salt stresses was analyzed.In addition,the proteins interacted with G protein signaling proteins were screened and characterized by Y2H,three-binding hybrid?Y3H?and bimolecular fluorescence complementation?BiFC?assays.The main results obtained in this study are as follows:1.Transcriptome sequence analysis in mulberry under salt stress.The seedlings of“Cesha No.6 hybrids”and“Cesha No.12 hybrids”were treated with salt stress and used for transcriptome analysis by RNA-seq technology.A total of 101589 unigenes were obtained from 24 mulberry salinity subtranscriptomes,and 34.72%of the assembled reads showed significant similarity to the sequences in the NCBI,KEGG,SwissProt,Pfam and other bioinformatics databases.The differentially expressed genes?DEGs?in response to salt stress were analyzed by using the DEGseq?2010?R package.It is found that the genes involved in ion transport,ROS scavenging system,ABA signal transduction,Ca²⁺signal pathway and MAPK signal cascades showed response to salt stress,and the genes encoding receptor-like protein kinases,transcription factors,heat shock proteins,aquaporin,late embryogenesis abundant proteins,osmotic proteins and?1-pyrroline-5-carboxylic acid synthetases were also regulated by salt stress.In this study,the mulberry seeds were treated by salt stress during germination period and the seedlings generated from the seeds with better germination ability showed stronger salt stress tolerance,which indicates that pre-expose to salt stress during germination period can produce high salt-tolerant plants and endowed the plant with stress memory that improving resistance to future stresses.The numbers of DEGs in low salt-tolerant genotypes are more than that in high salt-tolerant genotypes under salt stress,which indicates low salt-tolerant genotypes are more sensitive to salt stress than high salt-tolerant genotypes.It is showed that the number of DEGs in root are more than that in leaf and stem by comparing the changes in transcript levels between high-and low salt-tolerant genotypes under normal conditions,which indicates the differences between high-and low salt-tolerant genotypes are mainly reflect on root.Besides,WGCNA analysis indicated that the genes from cyan?darkgrey and violet modules were enriched in“spliceosome”,“RNA transport”,“ribosome biogenesis in eukaryotes”,“ubiquitin mediated proteolysis”,“basal transcription factors”,“mRNA surveillance pathway”,“protein processing in endoplasmic reticulum”and“endocytosis”.Above results indicated that pre-expose to salt stress during germination period may change the resistance of plant to future stresses and endowed the plant with stress memory at RNA and protein levels.Expression analysis of mulberry G protein signaling genes showed that G?and G?2 were divided in brown module,G?and RGS were divided in black module,G?1 and RACK1 were divided in green module.The genes encoding RLKs,WRKY,E3 ligase,CDPK,cytochrome P450,PPR,ERF and calmodulin were found to be co-expressed with G protein signaling genes.41ribosomal protein encoding genes were found to be co-expressed with RACK1,which suggests RACK1 protein may regulate the activities of ribosome in plants.Above data indicates G protein may be one of the key factors involved in mulberry stress responses.2.Study on the molecular mechanism of mulberry G protein signaling genes in response to abiotic stressIn this study,we identified and cloned six G protein signaling genes from mulberry using bioinformatics methods,including one G?,one G?,two G?,one RGS and one RACK1.The exon-intron organization of the mulberry G proteins was relatively similar to G protein genes identified in Arabidopsis,Brassica rapa and Glycine max.Multiple sequence alignment analysis showed that mulberry G?,G?,G?1,G?2,RGS and RACK1 shared high similarity with those in other land plants,the highest similarity is more than 90%.The interaction between MaG?and MaG?,between MaG?and MaG?and between MaG?and MaRGS were confirmed in yeast by Y2H assay.Expression of mulberry G protein signaling genes was detected in this study.The results showed that mulberry G protein signaling genes were ubiquitously but differentially expressed in various tissues.The expression levels of all these genes were affected by abiotic stresses and signal molecules.Besides,G protein effectors?e.g.cholera toxin,pertussis toxin,GTP?S,GDP?and suramin?were firstly used in plant,which can regulate the expression of mulberry G protein signal genes.Mulberry G protein signaling genes were transformed into tobacco and the transgenic plants were used for resistance identification experiment.The results showed that overexpression of MaG?and MaRGS reduce plant tolerance to drought and salt stresses,while overexpression of MaG?,MaG?1 and MaG?2 improve plant tolerance to drought and salt stresses.Further analysis indicated overexpression of MaG?and MaRGS reduced the detoxification of ROS but overexpression of MaG?,MaG?1 and MaG?2 elevated the detoxification of ROS.The above work is the first time to fully analyze the function of G protein signaling genes in regulating plant drought and salt stresses responses and several controversial regulation modes mediated by G proteins in Arabidopsis were elucidated.It is found that the soluble sugar content of mulberry and tobacco were significantly enhanced by NaCl and PEG treatments.The expression levels of WNK and ATG genes of mulberry and tobacco showed responses to NaCl,mannitol and glucose treatments.Besides,expression levels of WNK and ATG genes showed more sensitive to glucose in transgenic plants than wild type tobacco,which suggests glucose and autophagy may participate in plant G protein responses to abiotic stresses.Based on above analysis and previous study,we hypothesize a proposed mechanism of G protein signaling in response to drought and salt stresses.When plants are exposed to drought and salt stresses,the glucose content and the accumulation of ATG were induced,and WNK proteins were recruited.And then,RGS was internalized and G protein?subunit was uncoupled from RGS,G proteins were subsequently dissociated into G?-GTP and a G??dimer,which leads to activation of downstream stress responsive proteins,elevation of ROS detoxification and plant stresses responses.More studies are needed to support it.3.Study on the molecular mechanism of mulberry MaRACK1 in response to abiotic stressIn this study,overexpression of MaRACK1 in Arabidopsis reduces seed germination rates under drought and salt stresses,and the development of roots were also affected by stresses.Further analysis indicated overexpression of MaRACK1 improves MDA content of transgenic plant but reduces proline production under stresses.Above analysis indicates that MaRACK1 negatively regulates plant drought and salt tolerance.It was confirmed that mulberry and Arabidopsis RACK1 proteins cannot interact with G proteins and RGS by Y2H,Y3H and BiFC assays,which indicates the regulation mode of RACK1in plant drought and salt stresses responses is independent on G proteins.Further analysis confirmed that MaRACK1 cannot interact with the proteins involved in MAPK cascades,but MaMAPK3,MaMAPK5,MaMKKK1 and MaMKKKA can interact with G protein??dimers,which suggests that G proteins are directly connect MAPK cascades proteins and does not require RACK1 scaffold protein and RACK1 have no direct relationship with G protein signaling.The above work is opposite to the point made by Cheng et al^[91]that RACK1 connects heterotrimeric G protein with a MAPK cascade to transduce stress signal.Besides,the stress signal transduction pathways from G protein signaling to MAPK cascades were elucidated and improved.Arabidopsis gene expression was analyzed in ePlant database?http://bar.utoronto.ca/eplant/?in this study.The results showed that the expression of AtRACK1A,AtRACK1B and AtRACK1C were inhibited by drought and salt stresses,AtWNK8 expression was significantly inhibited after 3 h of drought and salt stresses treatments,and the expression of AtSUMO1 were down-regulated by drought and salt stresses.Above data indicates that the declines in the expression of RACK1 gene and the degradation of RACK1 protein may help plant tolerance to stresses.RNA-seq analysis showed that several genes encoding ERF,NAC,WAKY and bHLH were regulated by the overexpression of MaRACK1,and they may also be feedback regulated by RACK1 and participate in the downregulation of RACK1 at the transcriptional level under stress conditions.RNA-seq analysis showed that RACK1 may regulate plant abiotic stresses responses by affecting the accumulation of peroxidase,LEA,cytochrome P450,glutathione S-transferase and HSF proteins.Of these MaRACK1-regulated genes,RAP2-6?AT1G43160?from ERF family has been previously confirmed to be interacted with AtRACK1B,which alters the affinity of RAP2.6for CE1 and GCC cis-acting regulatory elements.AtLTP4?AT5G59310?,AtLTP?AT2G10940?and GLP3?AT5G20630?were found to be bound to RACK1,but their functions in plant abiotic stresses responses are needed to be studied.It is also found that the expression of several genes involved in jasmonic acid synthesis and signal pathway were regulated by MaRACK1 and MaTIFY 10A?MaJAZ1?can interact with MaRACK1,which indicates RACK1 may regulate plant tolerance via jasmonic acid pathway.Mulberry chlorophyll a/b-binding protein MaCP24 and fructose-bisphosphate aldolase MaFBA can interact with MaRACK1 and their encoding genes expression are sensitive to stresses.Besides,the homologous proteins of MaCP24 and MaFBA in Arabidopsis such as AtCAB1?AT1G29930?,AtCAB3?AT1G29910?and AtFBA3?AT2G01140?,can also interact with Arabidopsis RACK1,which suggests these proteins may play important roles in plant stress responses.Based on above analysis,we disapprove the previous view that RACK1 connects heterotrimeric G protein with a MAPK cascade to transduce stress signal.We hypothesize a novel mechanism that drought and salt stresses induced the declines in the expression of RACK1 gene and the degradation of RACK1 protein may help plant tolerance to stresses.Under normal conditions,RACK1 negatively regulates plant drought and salt stresses by affecting stress responsive proteins expression,jasmonic acid signal transduction and photosynthesis.Under drought and salt stresses,ERF,NAC,WAKY and bHLH may attribute to the declines of RACK1 expression and the changes in AtWNK8 and AtSUMO1 accumulation may induce the degradation of RACK1 protein,the negative effect of RACK1 in plant stresses tolerance was eventually eliminated.More studies are needed to elucidate it.