A Study On The Molecular Physiological Mechanism Of Cold Resistance In LSi1-overexpressing Rice

Rice?Oryza sativa?is normally grown in tropical climate zones and it is sensitive to even mild cold stress,particularly,at the early stages of seedling development.Tolerance to cold injury at the Seedling Stage?CTSS?is a complex trait controlled by multiple genes.Silicon?Si?can overcome this complexity as it is considered as a broad spectrum alleviator to combat biotic and abiotic stresses.Rice requires several fold more Si than the essential macronutrients,such as N,P,and K for enhancing stress tolerance.LSil characterization,as a silicon transporter,has opened silicon uptake system in rice and facilitated genetic up regulation of the silicon intake capacity by LSil overexpression.Here,we characterized the enhanced copy number role of the Low silicon 1 gene?LSi1?to enhance cold tolerance using LSi1-overexpression Transgene Dular?TD?as experiment material and its wild Dular?WD?line as control.Study included phonotypical,anatomical,physiological,quantitative and comparative proteomics,qPCR and Western Blot approaches.The results provided here a new insight into the underlying cold tolerance molecular mechanisms of the TD and are summarized below:1-Initially,LSi1 gene overexpression functionality was confirmed through TAIL-PCR which depicted LSil gene was inserted in non-coding region without disturbing any functional gene in rice genome.Further,on cold treatment,phenotypic response of TD was found cold tolerant with green seedlings as compared to WD which turned yellow.LSil gene expression in TD root was detected significantly higher through semi-quantitative PCR as well as the amount of silicon content in the form of monosilicic acid?H4SiO4?enhanced significantly?p<0.05?in TD leaves as compared to WD leaves.Further,TD root Si uptake competency on 3 h,6 h,9 h,12 h and 24 h time interval was detected which continuously enhanced at each time point.At 3 h,TD root silicon uptake was 15 ?g/g DW which reached up to 30 ?g/g DW at 24 h,while WD root uptake at initial stage was detected 9 ?g/g DW which could only reach up to 14 ?g/g.As well as,silicon uptake of TD leaf increased from 8?g/g DW to 17 ?g/g DW,while WD leaf could only uptake from 5 ?g/g DW to 8 ?g/g DW within 24 h of cold stress.As well as,LSil relative expression in TD root was also consistently up regulated with increasing Si nutrition concentration from 0.5 mM to 2 mM which depicted that LSil overexpression significantly enhanced genetic ability of TD rice root to uptake silicon under cold stress.2-Silicon absorbed by root in the form of monosilicic acid?H4Si04?transported from root to canopy through transpiration route.Where,it deposits as silica bodies under cuticle to form silica-cuticle double layer,we confirmed this silicon deposition pattern in leaf canopy by microscopic studies.Under confocal microscopy,significant increase of silicon deposition as silica opal was observed in the motor cells of the TD leaf blades as compared to WD leaves.For in-detailed structural study,under scanning electron microscopy?SEM?,we found significantly higher deposition of conical shaped silica bodies on upper TD leaf epidermis,dumbbell shaped silica bodies on lower TD leaf epidermis and also amorphous silicon aggregation in bulliform cells of TD leaves at transverse view forming a silica-cuticle bilayer.Structurally,this silica-cuticle bilayer protected chloroplast machinery from cold stress,improved light interception being translucent for light to regulate photosynthesis.In consistency,chlorophyll a,b contents were found significantly?p<0.05?higher in TD leaves as compared to WD leaves.3-This silica-cuticle bilayer cell also prevented TD leaf cells from excessive transpiration water loss and assisted to maintain cells relative water content?RWC?%in between 100-80%up to 9th day of cold stress while in WD leaves RWC%delined up to 50%,resulted in ceasing of the photosynthesis,increment of the respiration and reactive oxygen species?ROS?accumulation.Along with silica-cuticle bilayer,soluble silicon in ionic form interacted with metabolic machinery of TD leaf cell and resulted in significant antioxidants activity up regulation such as superoxide dismutase?SOD?,catalase?CAT?and peroxidase?POD?while,significant decrease in the electrolytes leakage?EL?and malonedialdehyde?MDA?content in TD leaves.Lower level of EL and MDA depicted membrane integrity of TD cells,while,increased SOD,CAT and POD activity depicted a competent defense network to combat cold stress through ROS scavenging at physiological level.4-Further,quantitative and comparative proteomics revealed molecular network triggered by silicon bioactivity in regulation of cold defense.As,silicon?Si?OH?4?has preferential attraction with the hydroxyl unit of amino acids found in proteins,enzymes and hormones which may points its role as proteins bio-activator in regulation of plant defense mechanism.4.1-In TD leaf,based on silicon induced protein expressional cascade,it is hypothesized that for transient and quick cold stress response,guanine nucleotide-binding?GNB?protein?loc_os01g49290.1?as "signaling switch" up regulated to transmit cold signals outside a cell to its interior part through alternative binding and hydrolyzing cycles of GTP?guanosine triphosphate?to GDP?guanosine diphosphate?.On GTP binding,this signaling switch activated G protein-coupled receptors?GPCRs?that spanned the cell membrane.Cold signaling molecules binded to a domain of the GPCR located outside the cell,and an intracellular GPCR domain then in turn activated GTPases enzymes to alter cell function in response to cold stress.GTPases enzymes included GTP-binding nuclear protein Ran2?loc_os05g49890.1?up regulation transduced cold signal into the nucleus to switch on stress responsive gene transcription along with post-transcriptional regulation including mRNA stability,nucleo-cytoplasmic trafficking of mRNA and proteins through Ran dependent karyopherin?importin beta?proteins and nuclear envelope?NE?assembly.Along with controlling signaling and transcriptional events,silicon triggered cytosolic calcium?Ca2+?concentration through calmodulin-related protein?loc_os01g59530.1?up regulation.Resultant high Ca2+ level served as messenger of cold stress and modulated cellular processes via high-affinity,Ca+-binding proteins to regulate diverse systematic functions such as cell homeostasis.Under cold stress,chloroplast is the main site of oxidative burst as a by-product of enhanced oxygen metabolism.Silicon activated superoxide dismutase 1?loc_os03g22810.1?and superoxide dismutase?loc_os08g44770.1?proteins to detoxify the superoxide?O2-?radical into either ordinary molecular oxygen?O2?or hydrogen peroxide?H2O2?located in chloroplast of TD leaf cells.Being ROS molecule,hydrogen peroxide detoxification was further scavanged by strong network of OsAPxl?loc_os03g17690.2?,OsAPx2?loc_os07g49400.2?,OsAPx4?loc_os08g43560.2?,OsAPx6?loc_os12g07820.1?and thioredoxin M-type?loc_os12g08730.1?both from chloroplast and other cell organelles to maintain TD leaf cells redox homeostasis.Hydrogen peroxide?H2O2?also served as a signalling molecule along with G-proteins coupled and calcium signaling to modulate downstream signaling events in TD leaf cells.A cascade of cold stress responsive gene expression including HSPs,auxin-binding protein ABP20 and disease resistance response 206 proteins also up regulated in response to enhanced silicon content.Stromal 70 kDa heat shock-related protein?loc_os05g23740.1?and heat shock protein 82?loc_os04g01740.1?prevented protein dysfunction,assisted proper protein folding,provided thermo tolerance on exposure to low temperature stress through repairment of damaged proteins especially in chloroplast.Disease resistance response protein 206?loc os11g42500.1?up regulation strengthened TD leaf cell wall through the biosynthesis of flavonolignans,lignans,and alkaloids.Auxin-binding protein ABP20?loc_os08g35760.1?played a wide range of regulatory roles through direct interaction with salicylic acid?SA?for cold stress adaptation responses.Silicon also induced up regulation of histone H2B.1 protein?loc_os01g05630.1?,protein splicing factor U2af 38 kDa?loc_os05g48960.3?,elongation factor 1-delta 2?loc_os03g29260.2?,30S ribosomal protein S1?loc_os03g20100.1?and 50S ribosomal protein L12-1?loc_os01g47330.1?involved in translation machinery stabilization of chloroplast to synthesize chloroplastic proteins/enzymes while,up regulation of TOC75?loc_os03g16440.1?and SRP;probable signal recognition particle 43 kDa?loc_os03g03990.1?mediated proteins targeting to the outer envelope membrane and interior of chloroplast.While,up regulation of ATP-dependent Clp protease ATP-binding subunit cIpA?loc_os03g 14280.1?,proteasome subunit alpha type 6?loc_os03g0 8280.2?and APs,aspartic proteinase oryzasin-1?loc_os05g49200.1?maintained protein quality control through removal of cold induced damaged or misfolded proteins.Low temperature induced enzymes inefficiency and destabilized chloroplast translation machinery in WD.While in TD leaves,due to up regulation of above discussed chloroplastic splicing factors,elongation factors,ribosomal 50S and 30S proteins,TOC75 and SRP,the chloroplast translation machinery stabilized to synthesize structural and enzymatic proteins.This enhanced synthesis of structural and enzymatic chloroplastic proteins resulted in photosynthesis regulation of TD leaf cells.CO2 fixation cycle was regulated through ribulose bisphosphate carboxylase?loc_os05g35330.1?,transketolase?loc_os06g04270.1?,sedoheptulose-1,7-bisphosphatase?loc_os04g16680.1?and ribulose-phosphate 3-epimerase?loc_os03g07300.1?up regulation to utilize ATP and NADPH generated by PSI for carbon fixation.While,oxygen-evolving enhancer protein 1?loc_os01g31690.1?,cytochrome b6-f complex iron-sulfur subunit?loc_os07g37030.1?and ATP synthase beta chain?loc_os05g47980.1?up regulated PSI photosynthetic continuity.chlorophyll a-b binding M9?loc_os11g13890.6?,chlorophyll a-b binding protein 6A?loc_os06g21590.2?and chlorophyll a-b binding?loc_os03g39610.2?proteins up regulated photosynthetic light harvesting process and balanced the excitation energy between the photosystems I and II to avoid photoinhibition in TD seedlings.Silicon also activated protochlorophyllide reductase A?loc_os04g58200.1?expression which also assisted chlorophyll biosynthesis in TD leaf cells through phototransformation of protochlorophyllide?Pchlide?to chlorophyllide?Chlide?and minimized the 1O2 generation and also malonedialdehyde?MDA?production but maximized quantum efficiency.Further,photosynthesis regulation based on silicon bioactivity,up regulated carbohydrate metabolism including glucose-6-phosphate isomerase?loc_os09g29070.1?,glyceraldehyde-3-phosphate dehydrogenase B?loc_os03g03720.2?proteins activation and TCA cycle including NAD-dependent malic enzyme 62kDa isoform?EC 1.1.1.39,loc_os07g31380.1?,and malate dehydrogenase?loc_os05g49880.1?enzymes activation to meet cellular energy demand for cold adaptation of TD leaves.4.2-Based on cold induced expressional profile in TD root,it is hypothesizd that GNB?loc_os0lg49290.1?overexpression acted as molecular switch to activate cysteine-rich receptor kinase protein?NCRK;loc_os04g56430.1?.NCRK?Rop GTPase?,as universal master regulators,transmited cold signals from outside the cell to intracellular signaling cascades.Ran2 GTPase helped to transduce cold signal into the nucleus to switch on stress responsive gene transcription and regulated post-transcriptional events such as pre-mRNA processing,mRNA stability,RNA/protein export/import across nuclear membrane same as in TD leaf cells.Along with G-proteins signaling cascade,silicon induced over-expression of IAA-amino acid hydrolase ILR1-like 3?loc_os01g37960.1?involved in auxin synthesis and lipoxygenase 3?loc_os03g49350.1?involved in jasmonate synthesis.The coordinated arrays of regulatory networks through the induction of these endogenous hormones transduced signals to up regulate expression of stress responsive genes and redox homeostasis in TD root cells.Redox homeostasis included cytosolic ascorbate peroxidase,OsAPxl?loc_os03g17690.2?,cytosolic ascorbate peroxidase,OsAPx2?loc_os07g49400.2?,cytosolic peroxiredoxin Prdx?loc_os07g44440₁?,catalase isozyme A?loc_os02g02400.2?up regulation to detoxify hydrogen peroxide?H2O2?in TD root cells.Overexpression of stress responsive genes such as Hsp 70-kDa heat-shock cognate?loc_osl lg47760.6?maintained TD root cells proteins in their functional conformations,avoided proteins unnecessary aggregation,assisted refolding of non-native proteins,regulated protein import and translocation,and facilitated the proteolytic degradation of unstable proteins through the proteins targeting into lysosomes or proteasomes.Silicon interaction also activated germin-like protein subfamily 8-2?loc_os08g08960.1?and germin-like protein subfamily 2-4?loc_os02g32980.1?to assist metabolic flow of polysaccharides,starch,glycolipids and glycoproteins towards Td root cell wall,while,mannitol dehydrogenase?loc_os09g23530.1,loc_os09g23550.1,loc_os09g23540.1?and chalcone-flavonone isomerase?loc os03g60509.2?activation up regulated phenylpropanoids lignin biosynthesis and flavonoids chalcones biosynthesis respectively.This increased level of phenolics including lignin,flavonoids,polysaccharides,starch,glycolipids and glycoproteins and their subsequent incorporation into the TD root cell wall enhanced structural strength and repairment ability after cold injury.Along with cell wall strength enhancement,silicon regulated TD root cells growth through overexpression of beta-expansin la?loc_os10g40720.1?,an endogenous protein,involved in cell wall extension and tubulin alpha-7 chain,tubulin alpha-3 chain,tubulin beta-4 chain,actin-2?loc_os03g56810.1,loc_osllg14220.1,loc_os01g59150.1?involved in root cell morphogenesis and growth regulation.Silicon based on up regulation of elongation factor 1-delta 1?loc_os07g42300.1?,S-adenosylmethionine synthetase 1?loc_os05g04510.1?,cysteine synthase?loc_os01g74650.3?,ethylmalonate-semialdehyde dehydrogenase?loc_os07g09060.1?and isovaleryl-CoA dehydrogenase?loc_os05g03480.5?induced mitochondrial translation machinery of TD root cells.Mitochondrial translation machinery synthesized dihydrolipoyl dehydrogenase?loc_os01g22520.1?,succinyl-CoA ligase beta-chain?loc_os02g40830.2?,malate dehydrogenase?loc_os10g33800.1?and NADP-dependent malic enzyme?loc_os01g52500.4?involved in mitochondria tricarboxylic acid?TCA?energy cycle to generate energy-rich ATP?adenosine triphosphate?molecules.Carbon and nitrogen metabolism as the second source of energy for root cells were also up regulated in response to LSi1 overexpression in regulation of cold tolerance.Nitrogen metabolism related proteins such as S-adenosylmethionine synthetase 1?loc_os05g04510.1?,glutamine synthetase root isozyme 5?loc_os03g12290.1?and glutamine synthetase root isozyme 3?loc_os02g50240.1?up regulation reflected the enhancement of the root cells nitrogen metabolic activity to convert nitrogen sources into energy currency.In addition,proteins involved in carbohydrate metabolism like fructokinase-2?loc_os08g02120.1?,triosephosphate isomerase?loc_os01g62420.4?,glucose-1-phosphate adenylyltransferase?loc_os08g25734.1?,1,4-alpha-glucan branching enzyme?loc_os06g51084.1?and alpha-amylase precursor?loc_os02g52710.1?up regulated to trigger carbon sources for energy synthesis.This up regulated synthesis of energy-rich ATP?adenosine triphosphate?molecules provided energy for root cells metabolic regulation including water,nutrients and silicon uptake from soil.As,low temperature induces hydraulic imbalance in plant due to water lost through arial transpiration and reduced water intake capacity by root.In TD seedling,silicon deposition reduced cuticular transpirational water loss by deposition of Si beneath the cuticle and promoted root water intake through up regulation of major intrinsic protein,aquaporin PIP1.2?loc_os04g47220.1?.Cysteine-rich receptor kinase protein?loc_os04g56430.1?overexpression also contributed at molecular level in the transport of water and mineral salts being involved in developing tracheary cells,responsible for hydraulic characteristics in vascular plants.As well as aquaporin NIP2.1?loc_os02g51110.1?protein specific to silicon transporter also up regulated for efficient silicon uptake.5-The timing and the amount of gene expression at mRNA level can have a profound effect on the gene functions of cell.Up regulation of signaling molecules mRNA response within 24 h of cold stress such as GTPase related OsRAN2?loc_os05g49890.1?,GNB?loc_os01g49290.1?and calcium signaling related calmodulin?loc_os01g59530.1?molecules in leaf,while,NCRK?loc_os04g56430.1?,OsRAN2?loc_os05g49890.1?,GNB?loc_os01849290.1?GTPases and hormonal signaling Lipoxygenase 3?loc_os03g49350.1?genes in root triggered a biochemical chain of events as primary cold response inside the leaf and root cells.mRNA relative expression of G-protein signaling genes cascade stabled upto 7th day of cold stress while hormonal and calcium signaling significantly declined on 3rd and 7th day.Along with G-protein signaling genes,mRNA response of antioxidant genes such as SODCC1?loc_os03g22810.1?,CATA?loc_os02g02400.2?and APX1?loc_os03g17690.2?also up regulated within 24 h of cold stress and competency of these enzymes at mRNA level were stabled upto 7th day of cold stress,in consistency with G-proteins signaling genes expression pattern.While,mRNA response of HSP and GLP defense genes were up regulated after 24 h cold stress,which might serve as second cold stress responsive cascade after signaling and redox up regulation.Hsp70?loc_os05g23740.1?and Hsp82?loc_os04g01740.1?maximally?5-fold?up regulated at 3rd day of cold stress in leaf cells while,Hsc70?loc_os11g47760.6?and germin-like protein subfamily 8-2?loc_os08g08960.1?genes up regulated in root cells with independent relative expression pattern.Maximum Hsc70 expression was found at 3rd day while GL8-2 mRNA maximum expression was found at 7th day of cold stress.GL8-2 maximum expression at 7th day of cold stress might depict its role in long term cold adaptation when short term defense cascade down regulated.qPCR analysis of chloroplast proteins synthesizers i.e.50S ribosomal protein L12-1?loc_os01g47330.1?and 30S ribosomal protein SI?loc_os03g20100.1?genes showed 2-fold relative expression within 24 h of cold stress,and it continued to increase up to 48 h within 4-fold increase.Its expression correlated with the Hsp pattern,which might depict that these ribosomal genes and Hsps coordinately enhanced chloroplast protein synthesis and assisted these chloroplastic proteins regulation.Sequential relative expression of signaling,antioxidant enzymes,defense genes along with stability of chloroplast proteins triggered regulation of energy related processes of photosynthesis,carbohydrate metabolism and TCA cycle such as chlorophyll a-b binding?loc_os03g39610.2?,ribulose bisphosphate carboxylase?Ioc_os05g35330.1?and NADP-dependent malic enzyme?loc_os01852500.4?genes up regulation?5-fold?up to 7th day of cold stress.In consistency,relative expression of PIP 1.2 gene?loc_os04g47220.1?involved in water uptake and NIP2.1 gene?loc_os02g51110.1?involved in silicon uptake also significantly up regulated.This mRNA relative expression genes profile signatured on already discussed LSil induced proteome profile in relation to cold tolerance.6-Western Blot confirmed significant over expression of OsRAN2 GTP-binding nuclear protein in TD leaf and TD root.This OsRAN2 activation might tune TD cell nuclear envelope,signaling transduction up to nucleoplasm for stress responsive genes transcription,mRNA export to cytoplasm for translation and proteins translocation across nuclear membrane.While,in wild Dular,low silicon level resulted in OsRAN2 expression inhibition,nuclear envelope instability and ultimately failure to activate quick cold responsive protein cascade to induce cold tolerance.7-Conclusively,LSil overexpression played important role to enhance silicon uptake under cold stress.Silicon,as insoluble form?silica gel?induced silica-cuticle bilayer and functioned as protective sheet to stabilize chlorophyll contents as well as relative water content of TD leaf cells.While,silicon,as soluble form?ionic form?functioned as bioactive element to trigger physiological responses including antioxidants activity,membrane stability and at molecular level it interacted with a wide range of proteins?structural,enzymatic,hormonal?to regulate cold defense pathway.Silicon,in TD leaf cells,activated G-proteins and Ca+ signaling,redox homeostasis and stress specific proteins expression as primary cold response cascade?PCRC?.Further,silicon regulated transcription,post-transcription,post-translation protein targeting as well as protease events and induced photosynthetic,carbohydrate,mitochondrial proteins network to stabilize leaf cell energy level as secondary cold response cascade?SCRC?in regulation of cold tolerance.While,in TD root cells,silicon activated G-proteins based and hormonal signaling molecules along with activation of antioxidative enzymes as PCRC.Further as SCRC,silicon upregulated flavonoids,polysaccharides,glycoproteins,starch and glycolipids production to enhance cell wall structural strength to resist cold stress.As well as,silicon activated 70-kDa HSP to avoid thermo-stress protein dysfunction,stabilized mitochondrial proteins synthesis machinery,acti-vated TCA,carbohydrate and nitrogen metabolism to regulate Adenosine triphosphate?ATP?synthesis.This ATP production served as energy source for water,nutrients and silicon uptake from soil in regulation of cold tolerance.mRNA studies also depicted coincidence with proteomics expressional cascade.Finally,OsRAN2 was detected as an important cold responsive protein triggered by LSi1 overexpression in both TD leaf and TD root,which assisted nucleo-cytoplasmic transport of macromolecules?signaling,mRNA,proteins?and enabled fine-tuned regulation of biological processes in regulation of cold tolerance in TD.