GhNHX3D, a Vacuolar-Localized Na+/H+ Antiporter, Positively Regulates Salt Response in Upland Cotton.

Vacuolar sodium/proton (Na+/H+) antiporters (NHXs) can stabilize ion contents to improve the salt tolerance of plants. Here, GhNHX3D was cloned and characterized from upland cotton (Gossypium hirsutum). Phylogenetic and sequence analyses showed that GhNHX3D belongs to the vacuolar-type NHXs. The GhNHX3D-enhanced green fluorescent protein (eGFP) fusion protein localized on the vacuolar membrane when transiently expressed in Arabidopsis protoplasts. The quantitative real-time PCR (qRT-PCR) analysis showed that GhNHX3D was induced rapidly in response to salt stress in cotton leaves, and its transcript levels increased with the aggravation of salt stress. The introduction of GhNHX3D into the salt-sensitive yeast mutant ATX3 improved its salt tolerance. Furthermore, silencing of GhNHX3D in cotton plants by virus-induced gene silencing (VIGS) increased the Na+ levels in the leaves, stems, and roots and decreased the K+ content in the roots, leading to greater salt sensitivity. Our results indicate that GhNHX3D is a member of the vacuolar NHX family and can confer salt tolerance by adjusting the steady-state balance of cellular Na+ and K+ ions.

Genome-Wide Identification and Comparative Analysis of the 3-Hydroxy-3-methylglutaryl Coenzyme A Reductase (HMGR) Gene Family in Gossypium.

Terpenes are the largest and most diverse class of secondary metabolites in plants and play a very important role in plant adaptation to environment. 3-Hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) is a rate-limiting enzyme in the process of terpene biosynthesis in the cytosol. Previous study found the HMGR genes underwent gene expansion in Gossypium raimondii, but the characteristics and evolution of the HMGR gene family in Gossypium genus are unclear. In this study, genome-wide identification and comparative study of HMGR gene family were carried out in three Gossypium species with genome sequences, i.e., G. raimondii, Gossypium arboreum, and Gossypium hirsutum. In total, nine, nine and 18 HMGR genes were identified in G. raimondii, G. arboreum, and G. hirsutum, respectively. The results indicated that the HMGR genes underwent gene expansion and a unique gene cluster containing four HMGR genes was found in all the three Gossypium species. The phylogenetic analysis suggested that the expansion of HMGR genes had occurred in their common ancestor. There was a pseudogene that had a 10-bp deletion resulting in a frameshift mutation and could not be translated into functional proteins in G. arboreum and the A-subgenome of G. hirsutum. The expression profiles of the two pseudogenes showed that they had tissue-specific expression. Additionally, the expression pattern of the pseudogene in the A-subgenome of G. hirsutum was similar to its paralogous gene in the D-subgenome of G. hirsutum. Our results provide useful information for understanding cytosolic terpene biosynthesis in Gossypium species.

Terpene synthases GhTPS6 and GhTPS47 participate in resistance to Verticillium dahliae in upland cotton.

Terpene synthases (TPSs) are enzymes responsible for catalyzing the production of diverse terpenes, the largest class of secondary metabolites in plants. Here, we identified 107 TPS gene loci encompassing 92 full-length TPS genes in upland cotton (Gossypium hirsutum L.). Phylogenetic analysis showed they were divided into six subfamilies. Segmental duplication and tandem duplication events contributed greatly to the expansion of TPS gene family, particularly the TPS-a and TPS-b subfamilies. Expression profile analysis screened out that GhTPSs may mediate the interaction between cotton and Verticillium dahliae. Three-dimensional structures and subcellular localizations of the two selected GhTPSs, GhTPS6 and GhTPS47, which belong to the TPS-a subfamily, demonstrated similarity in protein structures and nucleus and cytoplasm localization. Virus-induced gene silencing (VIGS) of the two GhTPSs yielded plants characterized by increased wilting and chlorosis, more severe vascular browning, and higher disease index than control plants. Additionally, knockdown of GhTPS6 and GhTPS47 led to the down-regulation of cotton terpene synthesis following V. dahliae infection, indicating that these two genes may positively regulate resistance to V. dahliae through the modulation of disease-resistant terpene biosynthesis. Overall, our study represents a comprehensive analysis of the G. hirsutum TPS gene family, revealing their potential roles in defense responses against Verticillium wilt.

GhCLCc-1, a Chloride Channel Gene from Upland Cotton, Positively Regulates Salt Tolerance by Modulating the Accumulation of Chloride Ions.

The ionic toxicity induced by salinization has adverse effects on the growth and development of crops. However, researches on ionic toxicity and salt tolerance in plants have focused primarily on cations such as sodium ions (Na+), with very limited studies on chloride ions (Cl-). Here, we cloned the homologous genes of Arabidopsis thaliana AtCLCc, GhCLCc-1A/D, from upland cotton (Gossypium hirsutum), which were significantly induced by NaCl or KCl treatments. Subcellular localization showed that GhCLCc-1A/D were both localized to the tonoplast. Complementation of Arabidopsis atclcc mutant with GhCLCc-1 rescued its salt-sensitive phenotype. In addition, the silencing of the GhCLCc-1 gene led to an increased accumulation of Cl- in the roots, stems, and leaves of cotton seedlings under salt treatments, resulting in compromised salt tolerance. And ectopic expression of the GhCLCc-1 gene in Arabidopsis reduced the accumulation of Cl- in transgenic lines under salt treatments, thereby enhancing salt tolerance. These findings elucidate that GhCLCc-1 positively regulates salt tolerance by modulating Cl- accumulation and could be a potential target gene for improving salt tolerance in plants.

GhCLCg-1, a Vacuolar Chloride Channel, Contributes to Salt Tolerance by Regulating Ion Accumulation in Upland Cotton.

Soil and freshwater salinization is increasingly becoming a problem worldwide and has adversely affected plant growth. However, most of the related studies have focused on sodium ion (Na+) stress, with relatively little research on chloride ion (Cl-) stress. Here, we found that upland cotton (Gossypium hirsutum) plants accumulated Cl- and exhibited strong growth inhibition under NaCl or KCl treatment. Then, a chloride channel gene (GhCLCg-1) was cloned from upland cotton. Phylogenetic and sequence analyses indicated that GhCLCg-1 was highly homologous to AtCLCg and also have conserved voltage_CLC and CBS domains. The subcellular localization assay showed that GhCLCg-1 was localized on the vacuolar membrane. Gene expression analyses revealed that the expression of GhCLCg-1 increased rapidly in cotton in response to chloride stress (NaCl or KCl), and the transcript levels increased as the chloride stress intensified. The overexpression of GhCLCg-1 in Arabidopsis thaliana changed the uptake of ions with a decrease of the Na+/K+ ratios in the roots, stems, and leaves, and enhanced salt tolerance. In contrast, silencing GhCLCg-1 in cotton plants increased the Cl- contents in the roots, stems, and leaves and the Na+/K+ ratios in the stems and leaves, resulting in compromised salt tolerance. These results provide important insights into the toxicity of chloride to plants and also indicate that GhCLCg-1 can positively regulates salt tolerance by adjusting ion accumulation in upland cotton.

Transcriptional characterization and response to defense elicitors of mevalonate pathway genes in cotton (Gossypium arboreum L.).

The mevalonate (MVA) pathway is responsible for the biosynthesis of cytosolic terpenes including gossypol and its derivatives, which play an important role in the cotton plant's defense against pathogens and herbivores. In this study, we identified and cloned 17 potentially functional genes encoding enzymes that catalyze the six steps of the MVA pathway in Gossypium arboreum. Expression pattern analysis by qRT-PCR demonstrated that these genes had tissue-specific expression profiles and were most prevalently expressed in roots. Moreover, these genes were up-regulated in response to several elicitors, including methyl jasmonate and salicylic acid, as well as Verticillium dahliae infection and Helicoverpa armigera infestation. This indicates that the MVA pathway genes are involved in the signaling pathway regulated by exogenous hormones and the resistance of cotton plants to pathogens and herbivores. Our results improve the understanding of cytosolic terpene biosynthesis in Gossypium species and lay the foundation for further research on gossypol biosynthesis.

Evolutionary Conservation and Divergence of Genes Encoding 3-Hydroxy-3-methylglutaryl Coenzyme A Synthase in the Allotetraploid Cotton Species Gossypium hirsutum.

Polyploidization is important for the speciation and subsequent evolution of many plant species. Analyses of the duplicated genes produced via polyploidization events may clarify the origin and evolution of gene families. During terpene biosynthesis, 3-hydroxy-3-methylglutaryl coenzyme A synthase (HMGS) functions as a key enzyme in the mevalonate pathway. In this study, we first identified a total of 53 HMGS genes in 23 land plant species, while no HMGS genes were detected in three green algae species. The phylogenetic analysis suggested that plant HMGS genes may have originated from a common ancestral gene before clustering in different branches during the divergence of plant lineages. Then, we detected six HMGS genes in the allotetraploid cotton species (Gossypium hirsutum), which was twice that of the two diploid cotton species (Gossypium raimondii and Gossypium arboreum). The comparison of gene structures and phylogenetic analysis of HMGS genes revealed conserved evolution during polyploidization in Gossypium. Moreover, the expression patterns indicated that six GhHMGS genes were expressed in all tested tissues, with most genes considerably expressed in the roots, and they were responsive to various phytohormone treatments and abiotic stresses. The sequence and expression divergence of duplicated genes in G. hirsutum implied the sub-functionalization of GhHMGS1A and GhHMGS1D as well as GhHMGS3A and GhHMGS3D, whereas it implied the pseudogenization of GhHMGS2A and GhHMGS2D. Collectively, our study unraveled the evolutionary history of HMGS genes in green plants and from diploid to allotetraploid in cotton and illustrated the different evolutionary fates of duplicated HMGS genes resulting from polyploidization.

[Ecophysiological characteristics and productivity of wheat-cotton community].

The eco-physiology and productivity of 6-2-2 wheat-early maturing cotton-middle maturing cotton were studied in comparison with traditional 6-2 wheat-middel maturing cotton. The results showed that after wheat harvested, the LAI of cotton increased fast, the leaf area duration(LAD) and CGR were raised obviously, the dry matter rose rapidly, and the accumulated amount was larger than CK. Light use efficiency was 1.13%, 21% more than CK. The effective blossom period was extended 18 d, and the lint yield in both 1998 and 1999 was over 2250 kg.hm-2, 21-22% more than CK.