Function Of GABA In Cotton Response To Low Nitrogen Stress

Nitrogen is a major component of proteins,phospholipids,nucleic acids,phytohormones,vitamins,and some coenzymes and prosthetic groups in plants,which has a significant effect on the plant growth and development.While it is also an important component of plant chlorophyll and thus can affect the photosynthetic rate of plants.Not only that,nitrogen levels can change the balance of carbon and nitrogen in plants and contribute about 45%to the final crop yield.Moreover,low nitrogen conditions can also affect the expression levels of auxin biosynthesis genes,plant hormone transport related genes,and other plant endogenous hormone levels and signal transduction,which will promote the development of plant lateral roots and increase the absorption efficiency of mineral nutrients.Cotton is a sensitive plant to nitrogen fertilizers.The yield increase depends on the large amount of nitrogen fertilizer applied.However,the utilization efficiency of nitrogen fertilizer by cotton is low,especially in China.Large amounts of nitrogen can not only be absorbed by plants,but also cause soil compaction,further reduce the absorption efficiency of nitrogen fertilizer,leading to water and soil eutrophication,affecting the ecological balance.Gamma-aminobutyric acid?GABA?is a four-carbon,non-protein amino acid that responds to various stress conditions in plants,such as acid-alkali persecution,low temperature,thermal stimulation,high salt,and mechanical damage.It can act either as a nitrogen source by itself or regulate the nitrogen and carbon balance through the GABA shunt.This thesis aimed at exploring the role of GABA in low nitrogen stress in cotton.The results are as follows:1.Effect of low nitrogen stress on GABA level in cottonAfter low nitrogen treatment of Gossypium barbadense cotton for 8 days,GABA content in roots was detected.In the normal treatment group,the content of GABA in'XH12'line was significantly increased from 4.21?g/g?FW?to 5.43?g/g?FW?.The‘XH21'line was also significantly increased from 4.67?g/g?FW?to 5.60?g/g?FW?.Whereas,after 14 days of exposure to low nitrogen stress,the content of GABA in the root of J14 Cotton increased from 2.47?g/g?FW?to 6.11?g/g?FW?.Furthermore,GABA content increased from 2.87?g/g?FW?to 4.47?g/g?FW?after 29 days of low nitrogen treatment.In addition,the GABA content in the low-nitrogen treatment of the T₄ generation GhPOP2 transgenic cotton for 14 days revealed that both the up-regulation and down-regulation of the GhPOP2 transcription level,the GABA content was significantly increased under low-nitrogen conditions.The above results indicated that when cotton suffer from low nitrogen stress,it induce a significant increase in endogenous GABA content.2.Transcriptome analysis of cotton with low nitrogen stressLow-nitrogen treatment was performed on Gossypium barbadense and Gossypium hirsutum with different nitrogen use efficiency?NUE?cotton cultivars.Transcriptome sequencing showed that 3598 and 1801 genes were found to be significantly different in the‘XLZ'and‘XH'cotton cultivars.Under low-nitrogen treatment,there were 433genes with significant differences in low-NUE cotton cultivar compared to high-NUE cotton cultivar,of which 271 genes were significantly up-regulated and 162 genes were significantly down-regulated.Under the normal nitrogen treatment,there were 15 genes with significantly differences in low-NUE cotton cultivar compared to high-NUE cotton cultivar,of which 8 genes were significantly up-regulated and 7 genes were significantlydown-regulated.FurtherKEGGenrichmentanalysisof low-nitrogen-treated Gossypium barbadense and Gossypium hirsutum revealed that these differential genes are mainly in glutamate metabolism,hormone signal transduction,and carotenoid biosynthesis.GABA is closely related to glutamate metabolism,and?-Aminobutyrate transaminase?GABA-T?encoded by GhPOP2 gene is the primary rate-limiting enzyme for GABA degradation metabolism.The transcript level of GhPOP2 gene in the transcriptome was searched.It was found that under low nitrogen stress,the GhPOP2 gene expression levels in Gossypium barbadense and Gossypium hirsutum were increased,while the expression level of glutamate decarboxylase?GAD?was upregulated in Gossypium hirsutum while downregulated in Gossypium barbadense.This may be due to the different demand for glutamate under different nitrogen stress in different cotton cultivars.3.Effects of low nitrogen stress on GhPOP2 transcription in cottonThe promoter of GhPOP2 gene is expressed in various parts of cotton,but it is mainly expressed in reproductive organs such as petals,stamens,stigma,and ovules.It was found that the wild-type cotton after low-nitrogen treatment,root and cotyledon GhPOP2 expression levels were significantly higher compared to the control.Wild-type of J14 was treated with low-nitrogen at different times,detected GhPOP2 expression by RT-PCR,found that the expression level in the roots first increased,and then the nitrogen deficiency signal was transmitted to the hypocotyls and cotyledons in turn.Additionally,the transgenic cotton transferred to P_GhPOP2::GUS was treated with low nitrogen.GUS histochemical staining of roots showed that the plants with low nitrogen treatment for 7 days showed significantly more positive results than the signal in the control,and the guard cells in the leaves were also increased.GUS positive,which also suggests that GABA and plants are resistant to adversity stress.After treatment with Arabidopsis thaliana transformed into P_GhPOP2::GUS,the positive GUS was also found in the root tip.It was initially shown that low nitrogen stress can induce the expression of GhPOP2 gene.This induction is more obvious in the root than others.TobetterunderstandthefunctionoftheGhPOP2gene,a pLGN-35S-GhPOP2::GFP expression vector was constructed and transiently expressed in tobacco leaves.After co-localization with mitochondrial marker,the GhPOP2 gene was located in the mitochondria.4.Overexpression of GhPOP2 gene increases cotton tolerance to low nitrogen stressIn order to change the content of endogenous GABA,an overexpression,antisense,and RNAi plant expression vector of GhPOP2 were constructed.Genetic transformation of cotton was performed and the corresponding transgenic plants were screened.Detection of GABA content in T₄ plants showed that overexpression of GhPOP2reduced GABA content,while antisense and RNAi plant GABA content increased.Phenotypic observations of low-nitrogen-treated GhPOP2 transgenic plants revealed that the tolerance to nitrogen stress in overexpression-plants was significantly higher compared to wild-type,and antisense plants.The leaves in wild-type and antisense plants were significantly yellowed and shedding.Whereas,overexpression of the plant is still a normal leaf phenotype.The reason resulted in the overexpression of GhPOP2increasing the level of GABA degradation and thus providing plants with the required nitrogen source.This increased the plant chlorophyll content and thus promoted the increase of its photosynthetic rate,while down-regulating the expression of GhPOP2led to the opposite result.