| Salinity and drought are major abiotic stress affecting cotton(Gossypium hirsutum L.)production.Exploration of salt and drought-tolerant genes is essential for the genetic improvement of salt and drought tolerance in cotton.KTI12 protein has been regarded as putative regulator protein of Elongator complex which functions in plant development and abiotic stress tolerance.However,our knowledge in the specific role of GhKTI12 gene are largely unknown.Here,the GhKTI12 gene was isolated in upland cotton(Gossypium hirsutum)and investigated its effect in relation to plant growth and stress response,by inserting this gene into the model plant Tobacco(Nicotiana tabacum)and cotton(Gossypium hirsutum)for stable and temporary gene expression analysis.Firstly,we determined that tissue specific expression of GhKTI12 transcript and RT-PCR analysis showed that GhKTI12 was strongly expressed in reproductive parts such as anther and pistil followed by root and stem in vegetative parts.It is suggested that the highest expression in anther and pistil could provide for seed production whereas root and stem expression could support for abiotic stress response and plant growth rate.Based on putative cis-regulatory elements in the GhKTI12 gene promoter sequence that occupy 48.97 % of stress responsiveness elements,and subsequently,expression of GhKTI12 protein was highly induced by Na Cl and polyethylene glycol(PEG).Therefore,we hypothesized that overexpression of GhKTI12 could improve the seed yield production through regulation of the plant growth and development and abiotic stress tolerance.Through transient gene expression,leaf disc assay showed that leaves of TRV-GhKTI12 silenced cotton plants subjected to severe necrosis under salinity and osmotic stress solution for 2 days as compared to TRV2-00 control plants.In accordance with leaf color deterioration,chlorophyll content was reduced by 46 % and 30 % in silenced plant in salinity and osmotic stress,respectively.Twomonth-old silenced cotton plants irrigated by 200 m M Na Cl and 400 m M mannitol solution for one month revealed the higher sensitivity to stress condition by reduction in leaf size,plant height and early flowering phenotype.To explore the molecular mechanism of the significant phenotypes in TRV-GhKTI12 silenced cotton plants,leaves samples of 60-days old silenced cotton transgenic and TRV-00 seedlings under salinity and drought treatments were subjected to RNA-Seq analysis,which has identified(10112)and(7197)differently expressed genes(DEGs)under salinity and drought stress between silenced TRVGhKTI12 plants and TRV-00 control plants,respectively.GO and KEGG enrichment analysis revealed that large number of genes downregulated were chlorophyll a/b-binding(LHCB)proteins related with photosynthesis in cotton under salinity stress.Among(73)downregulated DEGs in plant hormone stress responsive genes in plant-hormone signaling pathway,30 % of auxin biosynthesis genes which could enhance drought tolerance by modulating root architecture,exhibited the lower expression level in cotton under drought stress.In addition,we also identified three ABA biosynthesis genes such as ABF3,ABI5,ABI2,which can enhance osmotic stress by stimulating stomata closure,were downregulated by drought stress.Under both salinity and drought stresses,large proportion of calcium-binding proteins(CML45,CML11,CML27,CML23,CML19)which involved in mediating plant responses to salt stress and drought stress,were commonly downregulated.A combination of RNA-Seq analysis and gene expression analysis by q PCR analysis confirmed that GhKTI12 is involved in response to salt and drought tolerance in cotton.To further verify our hypothesis,we investigated the influence of GhKTI12 protein on plant growth and its response to salinity and drought stress in GhKTI12 overexpressed tobacco plants.In terms of plant development effect,constitutive overexpression of GhKTI12 severely affected plant architecture by generating very large leaves and preventing reproduction.Notably,the robust increase in leaf size was mainly due to significant changes in leaf length direction increased by 34.31.% rather than leaf width direction increased by only 16.88 %.In addition to generate vigorous leaves,GhKTI12 tobacco plants revealed to increase in plant height that was approximately 37.6-45.3 % taller than that those in wild type plants.GhKTI12 tobacco plant displayed the late-flowering phenotype about 30 days that is also associated with a substantial increase in plant biomass production and seed yield due to significant increase in capsule number.It is surprisingly that constitutive overexpression of GhKTI12 have resulted in increase in both vegetative and reproductive organ growth despite maintaining delay flowering phenotype.The microscopic observation indicated that transgenic tobacco showed the approximately 55.26 % increase in leave epidermal cell number and 54.83 % in stem parenchyma cells number compared with the wild type,suggesting that GhKTI12 is positively regulated the cell proliferation.To understand the molecular mechanism of the significant phenotypes in GhKTI12 transgenic tobacco plants,shoot apical meristem(SAM)of 60-days old transgenic and WT seedlings was subjected to RNA-Seq analysis,which has identified 11168 differently expressed genes(DEGs)between WT and GhKTI12 transgenic plants,respectively.GO and KEGG enrichment analysis of shoot apical meristematic transcriptome revealed that large number of DEGs upregulated were involved in cell proliferation and cell membrane synthesis.Furthermore,overexpression of GhKTI12 suppressed the expression of MADS box genes related with flowering time such as AG-like genes,SEP1,SEP2,AP1 and AP3.A combined analysis of RNA-Seq data and gene expression confirmed by q PCR analysis suggested that GhKTI12 is involved in the regulation organ growth and plant growth through increasing the expression level of mitochondria respiratory proteins that could provide cell energy demand for cell proliferation.In addition to controlling organ growth through cell division,GhKTI12 caused to delay flowering via suppressing the transcription level of flowering enhancer genes.Taken together,protein KTI12 homolog,named GhKTI12 gene,has shown to improve the crop productivity through regulation of both vegetative and reproductive organ growth across prolonged plant growth period by promoting cell proliferation.In addition to provide the plant growth and development,constitutive expression of KTI12 significantly enhanced tolerance to drought and salt stress in Tobacco.In vitro condition,two-weeks old transgenic seedlings had longer taproots than in the wild type after 200 m M Na Cl and 300 m M mannitol treatment for two weeks.The transgenic tobacco accumulates a higher-level of relative water content(RWC)than wild-type plants due to increased stomatal closure and consequently,the survival rate of GhKTI12 overexpressed plants was higher than wild type under both stresses.The accumulation of proline,osmotic adjustment substances,in GhKTI12 transgenic plants was greater than that in WT plants.The greater competency of antioxidative activity in transgenic plants was shown by lower malondialdehyde(MDA)content,relative electrical conductivity,and reactive oxygen species(ROS)accumulation by enhancing by the enzyme activities of superoxide dismutase(SOD),peroxidase(POD),catalase(CAT)under stress conditions.This result indicated overexpression of GhKTI12 increased drought and salt tolerance.Collectively,our results suggest that GhKTI12 may be used as a candidate gene for cotton molecular breeding to improve crop yield and stress-tolerance both. |
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