The Role Of GhHHO2 Gene In Response To Low Nitrogen Stress In Cotton

Nitrogen is one of the important macronutrients to promote plant growth and crop productivity.Plants have evolved various mechanisms to adapt to nitrogen imbalance.In the agricultural system,in order to meet the increasing production demands,nitrogen fertilizer have been applied in large quantities throughout the world to maximize crop yields.In 2007,the global nitrogen fertilizer application was 110 million tons.By 2050,it is expected to increase to 125 to 236 million tons.However,a large part of the nitrogen deposited in the soil cannot be absorbed by plants and lost to the environment,resulting in serious environmental pollution and ecological imbalance.Therefore,it is a huge challenge to meet the needs of the growing world population by developing high-yield agriculture while ensuring cotton quality and environmental quality,and cultivating new crop varieties that efficiently use nitrogen fertilizer under reduced nitrogen fertilizer applications is the key to solving this dilemma.Transcription factors have the ability to coordinately regulate the expression of a group of genes.Therefore,it is a more economical and effective method to improve nitrogen utilization efficiency by regulating the expression of this gene with little or no nitrogen fertilizer.The preliminary transcriptome data of the research group showed that the transcription level of GhHHO2 gene was significantly up-regulated in upland cotton roots under low nitrogen level.In order to clarify whether the transcription factor is involved in the response process of cotton to low nitrogen stress,relevant researches were carried out and the following results were obtained:1.Bioinformatics analysis of GhHHO2 gene in upland cottonWe first cloned GhHHO2 gene（Gh＿A12G1550）from upland cotton（Gossypium hirsutum）.Bioinformatics analysis revealed that the gene was located on chromosome12,contained an open reading frame of 1140 bp,encoding 379 amino acid residues and its protein molecular weight was 41 k Da.A total of 16 upland cotton homologous genes were obtained by searching the cotton database Cotton FGD for HHO related genes.Previous studies have shown that there are six HHO homologous genes in Arabidopsis.Phylogenetic tree analysis of upland cotton and Arabidopsis HHO homologous genes shows that GhHHO2 gene is closest to GhHHO3 gene in upland cotton,and it has the highest homology with At HHO2 in Arabidopsis.In order to understand the expression pattern of HHO homologous genes in upland cotton,a search was performed using the upland cotton gene digital expression profiling library of our laboratory,and a heat map analysis was performed using Heml software.The results showed that the expression of GhHHO2 gene was relatively high in roots and 25-day-old fiber,and its expression pattern was similar to GhHHO5 gene（Gh＿A12G0642）.2.Analysis of GhHHO2 gene expression characteristicsReal-time PCR technology was used to investigate the expression characteristics of GhHHO2 gene during cotton growth.The results showed that the expression level of GhHHO2 gene was highest in roots,higher in sepals and cotyledons,and lowest in 10DPA ovules.Cotton seedlings were treated with different concentrations of nitrate nutrient solution.The results showed that low concentration of nitrate could induce the expression of GhHHO2 gene,and the induction effect in roots was more obvious.When the cotton seedlings were treated with different nitrogen sources,it was found that under high-concentration ammonium salt culture conditions,the roots obviously turned yellow and suffered ammonium poisoning.The expression level of GhHHO2 gene was significantly up-regulated relative to nitrate treatment.It is speculated that GhHHO2gene may also play a certain role in removal of ammonium poisoning.Using the Plant Care website to predict the cis-acting elements of the GhHHO2gene promoter sequence,it was found that the P_GhHHO2sequence is rich in six types of elements including hormone response,light response and stress response,indicating that GhHHO2 gene is inseparable from these plant growth and development processes.In order to confirm the expression pattern of GhHHO2 gene in Arabidopsis,the 2911 bp promoter sequence was fused with GUS reporter gene,and the p BI121-P_GhHHO2::GUS transgenic Arabidopsis plants were subjected to histochemical staining.The results showed that there were very obvious GUS staining signals in leaves,lateral roots,and root hairs of transgenic Arabidopsis,as well as GUS gene was expressed in axillary buds,flower buds and rosette leaves.The transgenic Arabidopsis seedlings were hydroponically treated with different nitrogen supply levels and different nitrogen sources.The staining results were basically consistent with the expression pattern of GhHHO2 gene detected by Real-time PCR.In order to better understand the function of GhHHO2 gene,p LGN-35S-GhHHO2::e GFP and p LGN-35S-e GFP::GhHHO2 expression vectors were constructed and transiently expressed in tobacco leaves.After co-localization with the nuclear dye DAPI,it was found that GhHHO2 gene is located on the nucleus.VIGS silencing GhHHO2 gene of cotton showed a more pronounced nitrogen deficiency phenotype than the control group under low nitrogen stress,and its chlorophyll a and chlorophyll b content was significantly lower than those of the control group,indicating that chlorophyll synthesis in leaves has been affected to some extent after VIGS silencing GhHHO2 gene.3.Heterologous expression of GhHHO2 gene increases Arabidopsis tolerance to low nitrogen stressGhHHO2 gene was heterologously expressed in Arabidopsis,and three transgenic Arabidopsis lines with high expression levels and wild-type Arabidopsis were treated under different nitrate concentrations in the hydroponic culture to observe and compare the phenotypic differences.It was found that under nitrate-deficient（1 m M,3 m M）culture conditions,the three transgenic lines grew more vigorously than the wild-type.As nitrate concentration increased,the differences in growth potential between the transgenic lines and the wild-type gradually narrowed.Root trait statistics show that the GhHHO2 transgenic Arabidopsis plants can significantly increase the length of primary roots under nitrate deficiency condition,especially under the condition of lower nitrate concentration;while under the condition of sufficient nitrogen（10 m M）,there were no significant differences in the length of primary roots from the wild-type.The above data indicates that heterologous overexpression of GhHHO2 gene under low nitrogen stress can increase the length of primary roots of the transgenic Arabidopsis plants,which is conducive to maintaining nitrogen uptake by root system and increasing nitrogen content in the plants,thereby improving tolerance to low nitrogen stress.4.Effects of overexpressing and gene-editing GhHHO2 gene on growth and development of cottonIn order to further study the effects of expressing and gene-editing GhHHO2 gene on nitrogen utilization in cotton,the plant expression vectors were constructed and transformed into cottons.The transgenic cotton plants were obtained through screening.We first determined the content of chlorophyll a,chlorophyll b and carotenoid in the wild-type and two transgenic cotton plants.The results showed that compared with the wild-type cotton plants,the content of chlorophyll a,chlorophyll b and carotenoid in the overexpressing GhHHO2 transgenic cotton plants were almost all up-regulated;the content of chlorophyll a and chlorophyll b in the gene-editing GhHHO2 transgenic cotton plants all were down-regulated,and there was no consistent change in carotenoid content.It was shown that the expression level of GhHHO2 gene had a certain effect on chlorophyll synthesis of cotton.The activity of several key enzymes in nitrogen metabolism of the wild-type and transgenic cottons were also measured.The results showed that under normal nitrogen supply,overexpression of GhHHO2 gene could promote the activity of Ni R and Fd-GOGAT to a certain extent.In order to investigate whether overexpressing and gene-editing GhHHO2 gene have an affect on accumulation of nitrate or ammonium nitrogen in the plants,the content of nitrate and ammonium nitrogen in leaves of the wild-type and two transgenic cottons were determined.It was found that the content of nitrate nitrogen and ammonium nitrogen of the two transgenic cottons were basically the same,which were lower than those of the wild-type.It is speculated that the reason for this situation may be due to enhanced nitrogen metabolism in the overexpression cottons,which assimilated more nitrate and ammonium nitrogen;in the gene-editing cottons,only GhHHO2 gene was edited,which may exist other functionally complementary homologous genes were induced to increase the expression level,resulting in consistent results with the overexpression cottons.