Systematic characterization of Gossypium GLN family genes reveals a potential function of GhGLN1.1a regulates nitrogen use efficiency in cotton.

The enzyme glutamine synthetase (GLN) is mainly responsible for the assimilation and reassimilation of nitrogen (N) in higher plants. Although the GLN gene has been identified in various plants, there is little information about the GLN family in cotton (Gossypium spp.). To elucidate the roles of GLN genes in cotton, we systematically investigated and characterized the GLN gene family across four cotton species (G. raimondii, G. arboreum, G. hirsutum, and G. barbadense). Our analysis encompassed analysis of members, gene structure, cis-element, intragenomic duplication, and exploration of collinear relationships. Gene duplication analysis indicated that segmental duplication was the primary driving force for the expansion of the GhGLN gene family. Transcriptomic and quantitative real-time reverse-transcription PCR (qRT-PCR) analyses indicated that the GhGLN1.1a gene is responsive to N induction treatment and several abiotic stresses. The results of virus-induced gene silencing revealed that the accumulation and N use efficiency (NUE) of cotton were affected by the inactivation of GhGLN1.1a. This study comprehensively analyzed the GhGLN genes in Gossypium spp., and provides a new perspective on the functional roles of GhGLN1.1a in regulating NUE in cotton.

Genomic loci associated with leaf abscission contribute to machine picking and environmental adaptability in upland cotton (Gossypium hirsutum L.).

INTRODUCTION: Defoliation by applying defoliants before machine picking is an important agricultural practice that enhances harvesting efficiency and leads to increased raw cotton purity. However, the fundamental characteristics of leaf abscission and the underlying genetic basis in cotton are not clearly understood. OBJECTIVES: In this study, we aimed to (1) reveal the phenotypic variations in cotton leaf abscission, (2) discover the whole-genome differentiation sweeps and genetic loci related to defoliation, (3) identify and verify the functions of key candidate genes associated with defoliation, and (4) explore the relationship between haplotype frequency of loci and environmental adaptability. METHODS: Four defoliation-related traits of 383 re-sequenced Gossypium hirsutum accessions were investigated in four environments. The genome-wide association study (GWAS), linkage disequilibrium (LD) interval genotyping and functional identification were conducted. Finally, the haplotype variation related to environmental adaptability and defoliation traits was revealed. RESULTS: Our findings revealed the fundamental phenotypic variations of defoliation traits in cotton. We showed that defoliant significantly increased the defoliation rate without incurring yield and fiber quality penalties. The strong correlations between defoliation traits and growth period traits were observed. A genome-wide association study of defoliation traits identified 174 significant SNPs. Two loci (RDR7 on A02 and RDR13 on A13) that significantly associated with the relative defoliation rate were described, and key candidate genes GhLRR and GhCYCD3;1, encoding a leucine-rich repeat (LRR) family protein and D3-type cell cyclin 1 protein respectively, were functional verified by expression pattern analysis and gene silencing. We found that combining of two favorable haplotypes (HapRDR7 and HapRDR13) improved sensitivity to defoliant. The favorable haplotype frequency generally increased in high latitudes in China, enabling adaptation to the local environment. CONCLUSION: Our findings lay an important foundation for the potentially broad application of leveraging key genetic loci in breeding machine-pickable cotton.

Phosphorus Availability Affects the Photosynthesis and Antioxidant System of Contrasting Low-P-Tolerant Cotton Genotypes.

Phosphorus (P) is an essential macronutrient, and an important component of plant metabolism. However, little is known about the effects of low P availability on P absorption, the photosynthetic electron transport chain, and the antioxidant system in cotton. This study used cotton genotypes (sensitive FJA and DLNTDH and tolerant BX014 and LuYuan343) with contrasting low-P tolerance in a hydroponic experiment under 15 µM, 50 µM, and 500 µM P concentrations. The results showed that low P availability reduced plant development and leaf area, shoot length, and dry weight in FJA and DLNADH, compared to BX014 and LuYuan343. The low P availability decreased the gas-exchange parameters such as the net photosynthetic rate, transpiration rate, and stomatal conductance, and increased the intercellular CO2 concentration. Chlorophyll a fluorescence demonstrated that the leaves' absorption and trapped-energy flux were largely steady. In contrast, considerable gains in absorption and trapped-energy flux per reaction center resulted from decreases in the electron transport per reaction center under low-P conditions. In addition, low P availability reduced the activities of antioxidant enzymes and increased the content of malondialdehyde in the cotton genotypes, especially in FJA and DLNTDH. Moreover, low P availability reduced the activity of PEPC and generated a decline in the content of ATP and NADPH. Our research can provide a theoretical physiological basis for the growth and tolerance of cotton under low-P conditions.

Genotypic variation in carbon and nitrogen metabolism in the cotton subtending leaves and seed cotton yield under various nitrogen levels.

BACKGROUND: Nitrogen (N) is the key nutrient required for high cotton production; however, its excessive use can increase the cost of production and environmental problems. Reducing the application of N while sustaining the yield is an important issue to be solved. Therefore, this study was designed to investigate the genotypic variations in subtending leaf physiology and its contribution to seed cotton yield of contrasting N-efficient cotton genotypes under various N levels in pot and field conditions. RESULTS: The results showed that the application of N increased the enzymatic activities related to carbon (C) and N metabolisms. Under the same N level, the C/N metabolisms of the N-efficient genotypes were significantly higher than N-inefficient genotypes, indicating a strong N assimilation and photoassimilation ability in N-efficient genotypes, especially under low N level. Moreover, the antioxidant enzymatic activities were significantly higher, whereas malondialdehyde content was lower in N-efficient cotton genotypes than in N-inefficient ones. Therefore, N-efficient cotton genotypes showed strong resistance, higher C/N metabolisms, and provided sufficient dry matter for boll development. As a result, the yield, N use efficiency, and value cost ratio of the N-efficient cotton genotypes were higher than in the N-inefficient genotypes. CONCLUSION: It was confirmed that the higher C/N metabolisms in the cotton subtending leaves of N-efficient cotton genotypes could support higher seed cotton yield under relatively low N application. © 2022 Society of Chemical Industry.

Genetic variation in morphological traits in cotton and their roles in increasing phosphorus-use-efficiency in response to low phosphorus availability.

Phosphorus (P) is an essential macronutrient required for fundamental processes in plants. Trait plasticity is crucial for plant adaptation to environmental change. Variations in traits underlie diverse phosphorus (P) acquisition strategies among plants. Nevertheless, how the intraspecific plasticity and integration of morphological traits contribute to Phosphorus-Use-Efficiency (PUE) in cotton is unknown. In this study, 25 morphological traits were evaluated in 384 cotton genotypes grown with low P (LP, 10µmol. L-1) and normal nutrition (CK, 500µmol. L-1) to assess the genetic variability of morphological traits and their relationship to phosphorus use efficiency. Results revealed a large genetic variation in mostly morphological traits under low P. Significant enhancement in root traits and phosphorus efficiency-related traits like PUE was observed at LP as compared to CK conditions. In response to low P availability, cotton genotypes showed large plasticity in shoot and total dry biomass, phosphorus, and nitrogen efficiency-related traits (i.e., phosphorus/nitrogen use efficiency, phosphorus/nitrogen uptake efficiency), and most root traits, but a limited response in root dry biomass, taproot length, root surface area, root volume, and SPAD value. In addition, significant correlations were observed between PUtE (phosphorus uptake efficiency), NUE (nitrogen use efficiency), TDB (total dry biomass), and RTD (root tissue density) with PUE under both P supply level and phosphorus stress index, which may be a key indicator for improving PUE under LP conditions. Most root traits are most affected by genotypes than nutrition level. Conserved PUE is more affected by the nutrition level than the genotype effect. Principal component analysis depicted the comprehensive indicators under two P supply conditions were mainly reflected in root-related traits and morphological indicators such as dry matter biomass. These results indicate that interspecific variations exist within these cotton genotypes and traits. Our study provides suggestions for future research to enhance the ability of the earth system model to predict how crops respond to environmental interference and provide target quality for cotton breeding in phosphorus-deficient areas.

Identification and Expression Analysis of the NPF Genes in Cotton.

The NPF (NITRATE TRANSPORTER 1/PEPTIDE TRANSPORTER FAMILY) transports various substrates, including nitrogen (N), which is essential for plant growth and development. Although many NPF homologs have been identified in various plants, limited studies on these proteins have been reported in cotton. This study identified 75, 71, and 150 NPF genes in Gossypium arboreum, G. raimondii, and G. hirsutum, respectively, via genome-wide analyses. The phylogenetic tree indicated that cotton NPF genes are subdivided into eight subgroups, closely clustered with Arabidopsis orthologues. The chromosomal location, gene structure, motif compositions, and cis-elements have been displayed. Moreover, the collinearity analysis showed that whole-genome duplication event has played an important role in the expansion and diversification of the NPF gene family in cotton. According to the transcriptome and qRT-PCR analyses, several GhNPFs were induced by the nitrogen deficiency treatment. Additional functional experiments revealed that virus-induced silencing (VIGS) of the GhNPF6.14 gene affects the growth and N absorption and accumulation in cotton. Thus, this study lays the foundation for further functional characterization of NPF genes in cotton.

Ameliorative Effects of Silicon against Salt Stress in Gossypium hirsutum L.

Silicon (Si) could alleviate the adverse effect of salinity in many crops, but the effect in cotton remains unclear. In this study, we evaluated the role of Si in regulating the salt stress tolerance of cotton by analyzing the induced morpho-physiological changes. A hydroponic experiment was conducted by using contrasting salt-tolerant cotton genotypes (sensitive Z0102; tolerant Z9807) and four treatments (CK, control; CKSi, 0.4 mM Si; NaCl, 150 mM NaCl; NaClSi, 150 mM NaCl+0.4 mM Si). The results showed that Si significantly enhanced the net photosynthesis rate and improved the growth of cotton seedling under salt stress in both salt-sensitive and salt-tolerant genotypes. Exogenous Si significantly reduced the accumulation of reactive oxygen species (ROS) and decreased the malondialdehyde (MDA) content in salt-stressed cotton. In addition, the application of Si up-regulated the expression of CAT1, SODCC and POD, and significantly enhanced the antioxidant enzymatic activities, such as catalase (CAT) and peroxidase (POD), of the salt-stressed cotton seedlings. Further, Si addition protected the integrity of the chloroplast ultrastructure, including key enzymes in photosynthesis such as ferredoxin-NADP reeducates (FNR), ATP synthase (Mg2+Ca2+-ATPase) and ribulose-1, 5-bisphosphate carboxylase/oxygenase (RubisCO), and the structure and function of the photosynthetic apparatus PSII from salt stress. Moreover, Si significantly increased the effective stomatal density and stomatal aperture in the salt-stressed cotton seedlings. Taken together, Si could likely ameliorate adverse effects of salt stress on cotton by improving the ROS scavenging ability and photosynthetic capacity.

Evaluation of Cotton (Gossypium hirsutum L.) Leaf Abscission Sensitivity Triggered by Thidiazuron through Membership Function Value.

Chemical defoliation is an essential agricultural practice in cotton production for mechanic harvesting. Thidiazuron (TDZ) is the active ingredient of the chemical defoliant used on cotton. So far, few studies havefocused on the method of identifying the sensitivity of cotton cultivars to TDZ. Therefore, a greenhouse soil culture experiment was performed by using two widely cultivatedupland cotton cultivars CRI 49 and CRI 12 treated with seven different concentrations (0, 100, 200, 300, 400, 500, and 1000 mg L-1) of TDZ at the seedling stage to establish a screening system. Principal component analysis and the membership function value (MFV) method was used to analyze the physiological and phenotypic characters, including abscission rate, amino acids content, net photosynthetic rate (Pn), etc. Finally, we developed a mathematical evaluation model, selected 100 mg L-1 TDZ as the optimal concentration and identified reliable characters net photosynthetic rate (Pn), stomatal conductance (Gs), and transpiration rate (Tr) to evaluate cotton leaf abscission sensitivity. These results also confirmed that CRI 12 was more sensitive to TDZ than CRI 49. This is the first time using a mathematical evaluation method to evaluate the cotton leaf abscission sensitivity triggered by TDZ at the seedling stage and the results were also confirmed in the field experiment. Furthermore, it will be valuable that MFV method is applied to stress sensitivity evaluation in other crop species under stress environment.

Chemical Defoliant Promotes Leaf Abscission by Altering ROS Metabolism and Photosynthetic Efficiency in Gossypium hirsutum.

Chemical defoliation is an important part of cotton mechanical harvesting, which can effectively reduce the impurity content. Thidiazuron (TDZ) is the most used chemical defoliant on cotton. To better clarify the mechanism of TDZ promoting cotton leaf abscission, a greenhouse experiment was conducted on two cotton cultivars (CRI 12 and CRI 49) by using 100 mg L-1 TDZ at the eight-true-leaf stage. Results showed that TDZ significantly promoted the formation of leaf abscission zone and leaf abscission. Although the antioxidant enzyme activities were improved, the reactive oxygen species and malondialdehyde (MDA) contents of TDZ increased significantly compared with CK (water). The photosynthesis system was destroyed as net photosynthesis (Pn), transpiration rate (Tr), and stomatal conductance (Gs) decreased dramatically by TDZ. Furthermore, comparative RNA-seq analysis of the leaves showed that all of the photosynthetic related genes were downregulated and the oxidation-reduction process participated in leaf shedding caused by TDZ. Consequently, a hypothesis involving possible cross-talk between ROS metabolism and photosynthesis jointly regulating cotton leaf abscission is proposed. Our findings not only provide important insights into leaf shedding-associated changes induced by TDZ in cotton, but also highlight the possibility that the ROS and photosynthesis may play a critical role in the organ shedding process in other crops.

Nitrogen Enhances Salt Tolerance by Modulating the Antioxidant Defense System and Osmoregulation Substance Content in Gossypium hirsutum.

Increasing soil salinity suppresses both productivity and fiber quality of cotton, thus, an appropriate management approach needs to be developed to lessen the detrimental effect of salinity stress. This study assessed two cotton genotypes with different salt sensitivities to investigate the possible role of nitrogen supplementation at the seedling stage. Salt stress induced by sodium chloride (NaCl, 200 mmol·L-1) decreased the growth traits and dry mass production of both genotypes. Nitrogen supplementation increased the plant water status, photosynthetic pigment synthesis, and gas exchange attributes. Addition of nitrogen to the saline media significantly decreased the generation of lethal oxidative stress biomarkers such as hydrogen peroxide, lipid peroxidation, and electrolyte leakage ratio. The activity of the antioxidant defense system was upregulated in both saline and non-saline growth media as a result of nitrogen application. Furthermore, nitrogen supplementation enhanced the accumulation of osmolytes, such as soluble sugars, soluble proteins, and free amino acids. This established the beneficial role of nitrogen by retaining additional osmolality to uphold the relative water content and protect the photosynthetic apparatus, particularly in the salt-sensitive genotype. In summary, nitrogen application may represent a potential strategy to overcome the salinity-mediated impairment of cotton to some extent.

Transcriptome Analysis Reveals Differences in Key Genes and Pathways Regulating Carbon and Nitrogen Metabolism in Cotton Genotypes under N Starvation and Resupply.

Nitrogen (N) is the most important limiting factor for cotton production worldwide. Genotype-dependent ability to cope with N shortage has been only partially explored in cotton, and in this context, the comparison of molecular responses of cotton genotypes with different nitrogen use efficiency (NUE) is of particular interest to dissect the key molecular mechanisms underlying NUE. In this study, we employed Illumina RNA-Sequencing to determine the genotypic difference in transcriptome profile using two cotton genotypes differing in NUE (CCRI-69, N-efficient, and XLZ-30, N-inefficient) under N starvation and resupply treatments. The results showed that a large genetic variation existed in differentially expressed genes (DEGs) related to amino acid, carbon, and nitrogen metabolism between CCRI-69 and XLZ-30. Further analysis of metabolic changes in cotton genotypes under N resupply showed that nitrogen metabolism and aromatic amino acid metabolism pathways were mainly enriched in CCRI-69 by regulating carbon metabolism pathways such as starch and sucrose metabolism, glycolysis/gluconeogenesis, and pentose phosphate pathway. Additionally, we performed an expression network analysis of genes related to amino acid, carbon, and nitrogen metabolism. In total, 75 and 33 genes were identified as hub genes in shoots and roots of cotton genotypes, respectively. In summary, the identified hub genes may provide new insights into coordinating carbon and nitrogen metabolism and improving NUE in cotton.

Variations in Nitrogen Metabolism are Closely Linked with Nitrogen Uptake and Utilization Efficiency in Cotton Genotypes under Various Nitrogen Supplies.

Cotton production is highly sensitive to nitrogen (N) fertilization, whose excessive use is responsible for human and environmental problems. Lowering N supply together with the selection of N-efficient genotypes, more able to uptake, utilize, and remobilize the available N, could be a challenge to maintain high cotton production sustainably. The current study aimed to explore the intraspecific variation among four cotton genotypes in response to various N supplies, in order to identify the most distinct N-efficient genotypes and their nitrogen use efficiency (NUE)-related traits in hydroponic culture. On the basis of shoot dry matter, CCRI-69 and XLZ-30 were identified as N-efficient and N-inefficient genotypes, respectively, and these results were confirmed by their contrasting N metabolism, uptake (NUpE), and utilization efficiency (NUtE). Overall, our results indicated the key role of shoot glutamine synthetase (GS) and root total soluble protein in NUtE. Conversely, tissue N concentration and N-metabolizing enzymes were considered as the key traits in conferring high NUpE. The remobilization of N from the shoot to roots by high shoot GS activity may be a strategy to enhance root total soluble protein, which improves root growth for N uptake and NUE. In future, multi-omics studies will be employed to focus on the key genes and pathways involved in N metabolism and their role in improving NUE.

Nitrogen preference and genetic variation of cotton genotypes for nitrogen use efficiency.

BACKGROUND: Although nitrogen (N) availability is a major determinant of cotton production, little is known about the importance of plants' preference for ammonium versus nitrate for better growth and nitrogen use efficiency (NUE). We aimed to assess the growth, physiology, and NUE of contrasting N-efficient cotton genotypes (Z-1017, N-efficient and GD-89, N-inefficient) supplied with low and high concentrations of ammonium- and nitrate-N. RESULTS: The results revealed that ammonium fed plants showed poor root growth, lower dry biomass, N content, leaf chlorophyll and gas exchange than those under nitrate irrespective of the concentration. However, the highest N uptake and utilization efficiency were obtained with nitrate fed plants, which also resulted in the highest dry biomass, N content, leaf chlorophyll and gas exchange as well as root growth. The results further confirmed that N-efficient (Z-1017) genotype performed better under both N sources, showing more flexibility to contrasting N condition by increasing growth and NUE in either source of N. Moreover, multivariate analysis showed a strong relationship of root morphological traits with N utilization efficiency, suggesting the physiological importance of roots over shoots in response to low nitrate concentration. CONCLUSION: Thus, it was confirmed that nitrate-N is superior to ammonium-N and the use of nitrate and N-efficient genotype is the best option for optimum cotton growth and NUE. Further, field evaluation is required to confirm the hypothesis that nitrate is a preferred N source for better cotton production and NUE. © 2020 Society of Chemical Industry.

High Nitrogen Enhance Drought Tolerance in Cotton through Antioxidant Enzymatic Activities, Nitrogen Metabolism and Osmotic Adjustment.

Drought is one of the most important abiotic stresses and hampers many plant physiological processes under suboptimal nitrogen (N) concentration. Seedling tolerance to drought stress is very important for optimum growth and development, however, the enhancement of plant stress tolerance through N application in cotton is not fully understood. Therefore, this study investigates the role of high N concentration in enhancing drought stress tolerance in cotton. A hydroponic experiment supplying low (0.25 mM) and high (5 mM) N concentrations, followed by 150 g L-1 polyethylene glycol (PEG)-induced stress was conducted in a growth chamber. PEG-induced drought stress inhibited seedling growth, led to oxidative stress from excessive malondialdehyde (MDA) generation, and reduced N metabolism. High N concentrations alleviated oxidative damage and stomatal limitation by increasing antioxidant enzymatic activities, leaf relative water content, and photosynthesis in cotton seedlings under drought stress. The results revealed that the ameliorative effects of high N concentration may be ascribed to the enhancement of N metabolizing enzymes and an increase in the amounts of osmoprotectants like free amino acids and total soluble protein. The present data suggest that relatively high N concentrations may contribute to drought stress tolerance in cotton through N metabolism, antioxidant capacity, and osmotic adjustment.