Molecular characterization and expression pattern of Rubisco activase gene GhRCAß2 in upland cotton (Gossypium hirsutum L.).

BACKGROUND: Rubisco activase (RCA) is a pivotal enzyme that can catalyse the activation of Rubisco in carbon assimilation pathway. Many studies have shown that RCA may be a potential target for genetic manipulation aimed at enhancing photosynthetic efficiency and crop yield. OBJECTIVE: To understand the biological function of the GhRCAß2 gene in upland cotton, we cloned the coding sequence (CDS) of the GhRCAß2 gene and investigated its sequence features, evolutionary relationship, subcellular localization, promoter sequence and expression pattern. METHODS: The bioinformatics tools were used to analyze the sequence features of GhRCAß2 protein. Transient transformation of Arabidopsis mesophyll protoplasts was performed to determine the subcellular localization of the GhRCAß2 protein. The expression pattern of the GhRCAß2 gene was examined by analyzing transcriptome data and using the quantitative real-time PCR (qRT-PCR). RESULTS: The full-length CDS of GhRCAß2 was 1317 bp, and it encoded a protein with a chloroplast transit peptide. The GhRCAß2 had two conserved ATP-binding domains, and did not have the C-terminal extension (CTE) domain that was unique to the RCA α-isoform in plants. Evolutionarily, GhRCAß2 was clustered in Group A, and had a close evolutionary relationship with the soybean RCA. Western blot analysis demonstrated that GhRCAß2 was immunoreactive to the RCA antibody displaying a molecular weight similar to that of the RCA ß-isoform. The GhRCAß2 protein was found in chloroplast, aligning with its role as a vital enzyme in the process of photosynthesis. The GhRCAß2 gene had a leaf tissue-specific expression pattern, and the yellow-green leaf mutant exhibited a decreased expression of GhRCAß2 in comparison to the wild-type cotton plants. The GhRCAß2 promoter contained several cis-acting elements that respond to light, phytohormones and stress, suggesting that the expression of GhRCAß2 may be regulated by these factors. An additional examination of stress response indicated that GhRCAß2 expression was influenced by cold, heat, salt, and drought stress. Notably, diverse expression pattern was observed across different stress conditions. Additionally, low phosphorus and low potassium stress may result in a notable reduction in the expression of GhRCAß2 gene. CONCLUSION: Our findings will establish a basis for further understanding the function of the GhRCAß2 gene, as well as providing valuable genetic knowledge to improve cotton photosynthetic efficiency and yield under challenging environmental circumstances.

Sequence Characteristics and Expression Analysis of the Gene Encoding Sedoheptulose-1,7-Bisphosphatase, an Important Calvin Cycle Enzyme in Upland Cotton (Gossypium hirsutum L.).

Sedoheptulose-1,7-bisphosphatase (SBPase, EC 3.1.3.37) is a key enzyme in the plant Calvin cycle and one of the main rate-limiting enzymes in the plant photosynthesis pathway. Many studies have demonstrated that the SBPase gene plays an important role in plant photosynthetic efficiency, yield, and stress responses; however, few studies have been conducted on the function and expression of the GhSBPase gene in upland cotton. In this study, our results showed that the coding sequence (CDS) of GhSBPase gene was 1182 bp, encoding a protein with 393 amino acids. The GhSBPase protein had adenosine monophosphate (AMP) binding site and a FIG (FBPase/IMPase/glpX) domain, and had six Cys residues and a CGGT(A/Q)C motif that were involved in redox regulation in plants. Evolutionarily, the GhSBPase protein clustered into the dicotyledon subgroup and was most closely related to the tomato SlSBPase protein. Western-blot analysis further indicated that the GhSBPase gene was indeed the gene encoding the SBPase protein in upland cotton. The GhSBPase protein was localized in chloroplast, which was consistent with its function as a key enzyme in photosynthesis. The GhSBPase gene was specifically highly expressed in leaves, and its expression level was significantly lower in a yellow-green leaf mutant than in the wild type. Moreover, the GhSBPase expression was in response to drought, salt, high- and low-temperature stress, and exhibits different expression patterns. The GhSBPase promoter had the cis-acting elements in response to abiotic stress, phytohormone, and light. In addition, the GhSBPase expression was positively correlated with the chlorophyll fluorescence parameters, suggesting that changes in the expression of the GhSBPase had potential applicability in breeding for enhanced cotton photosynthetic efficiency. These results will help to understand the function of the GhSBPase gene in photosynthesis and the adaptability of plants to external stress and provide important gene information for the high-yield breeding of crops in the future.

Phylogeny, gene structures, and expression patterns of the auxin response factor (GhARF2) in upland cotton (Gossypium hirsutum L.).

BACKGROUND: Auxin response factors (ARFs) are a class of transcription factors that regulate the expression of auxin-responsive genes and play important functions in plant growth and development. To understand the biological functions of the auxin response factor GhARF2 gene in upland cotton, the coding sequence (CDS) of GhARF2 gene was cloned, and its protein sequence, evolutionary relationship, subcellular localization and expression pattern were analysed. METHODS: The CDS sequence of GhARF2 gene was cloned from upland cotton variety Baimian No.1, and its protein sequence was analyzed by bioinformatics method. The subcellular localization of GhARF2 protein was detected by tobacco epidermal transient transformation system, and the tissue expression and stress expression pattern of GhARF2 were analyzed by quantitative Real­Time PCR (qRT-PCR). RESULTS: The full-length CDS of GhARF2 gene was 2583 bp, encoded 860 amino acids, and had a molecular weight and an isoelectric point of 95.46 KDa and 6.02, respectively. The GhARF2 protein had multiple phosphorylation sites, no transmembrane domain, and secondary structures dominated by random coils and alpha helix. The GhARF2 protein had 3 conserved typical domains of ARF gene family members, including the B3 DNA binding domain, the Auxin_resp domain, and the Aux/IAA domain. Phylogenetic analysis revealed that ARF2 proteins in different species were clustered in the Group A subgroup, in which GhARF2 was closely related to TcARF2 of Theobroma cacao L. (Malvaceae). The subcellular localization results showed that the GhARF2 protein was localized in the nucleus. Analysis of tissue expression pattern showed that the GhARF2 gene was expressed in all tested tissues, with the highest expression levels in sepal, followed by leaf, and the lowest expression levels in fiber. Further stress expression analysis showed that the GhARF2 gene was induced by drought, high-temperature, low-temperature and salt stress, and had different expression patterns under different stress conditions. CONCLUSION: These results established a foundation for understanding the functions of GhARF2 and breeding varieties with high-stress tolerance in cotton.

Sequence Characteristics and Expression Analysis of GhCIPK23 Gene in Upland Cotton (Gossypium hirsutum L.).

CIPK (calcineurin B-like-interacting protein kinase) is a kind of serine/threonine protein kinase widely existing in plants, and it plays an important role in plant growth and development and stress response. To better understand the biological functions of the GhCIPK23 gene in upland cotton, the coding sequence (CDS) of the GhCIPK23 gene was cloned in upland cotton, and its protein sequence, evolutionary relationship, subcellular localization, expression pattern and cis-acting elements in the promoter region were analyzed. Our results showed that the full-length CDS of GhCIPK23 was 1368 bp, encoding a protein with 455 amino acids. The molecular weight and isoelectric point of this protein were 50.83 KDa and 8.94, respectively. The GhCIPK23 protein contained a conserved N-terminal protein kinase domain and C-terminal regulatory domain of the CIPK gene family member. Phylogenetic tree analysis demonstrated that GhCIPK23 had a close relationship with AtCIPK23, followed by OsCIPK23, and belonged to Group A with AtCIPK23 and OsCIPK23. The subcellular localization experiment indicated that GhCIPK23 was located in the plasma membrane. Tissue expression analysis showed that GhCIPK23 had the highest expression in petals, followed by sepals, and the lowest in fibers. Stress expression analysis showed that the expression of the GhCIPK23 gene was in response to drought, salt, low-temperature and exogenous abscisic acid (ABA) treatment, and had different expression patterns under different stress conditions. Further cis-acting elements analysis showed that the GhCIPK23 promoter region had cis-acting elements in response to abiotic stress, phytohormones and light. These results established a foundation for understanding the function of GhCIPK23 and breeding varieties with high-stress tolerance in cotton.

Functional divergence of GhAP1.1 and GhFUL2 associated with flowering regulation in upland cotton (Gossypium hirsutum L.).

The AP1/FUL transcription factors are important for floral development, but the underlying molecular mechanisms remain unclear. In this study, we cloned and identified two AP1/FUL-like genes, GhAP1.1 and GhFUL2, in upland cotton, which is a commonly cultivated economically valuable crop. Sequence alignment and phylogenetic analysis indicated that GhAP1.1 and GhFUL2, which are encoded by genes in the AP1/FUL clade, have conserved N-terminal regions but diverse C-terminal domains. Quantitative real-time PCR analysis revealed that GhAP1.1 and GhFUL2 were expressed in the flower and root, and showed opposite expression patterns during shoot apical meristem development. The upregulated expression of GhAP1.1 in Arabidopsis did not result in significant changes to the flowering time or floral organ development, and the transcript levels of the florigen FT increased and those of LFY decreased. Overexpression of GhFUL2 in Arabidopsis delayed flowering and promoted bolting by decreasing FT and LFY transcript levels. Silencing GhFUL2 in cotton dramatically increased the expression of GhFT and GhAP1.3 and promoted flowering. Yeast two-hybrid and bimolecular fluorescence complementation assays indicated that GhAP1.1 could interact with the SVP homolog GhSVP2.2, whereas GhFUL2 formed heterodimers with GhSEP3/GhSEP4 homologs and GhSVP2.2. The present results demonstrated that the functional divergence of GhAP1.1 and GhFUL2, which involved changes in sequences and expression patterns, influenced the regulation of cotton flower development.

Evolutionary Relationships and Divergence of KNOTTED1-Like Family Genes Involved in Salt Tolerance and Development in Cotton (Gossypium hirsutum L.).

The KNOX (KNOTTED1-like homeobox) transcription factors play an important role in leaf, shoot apical meristem and seed development and respond to biotic and abiotic stresses. In this study, we analyzed the diversity and evolutionary history of the KNOX gene family in the genome of tetraploid cotton (Gossypium hirsutum). Forty-four putative KNOX genes were identified. All KNOX genes from seven higher plant species were classified into KNOXI, KNOXII, and KNATM clades based on a phylogenetic analysis. Chromosomal localization and collinearity analysis suggested that whole-genome duplication and a polyploidization event contributed to the expansion of the cotton KNOX gene family. Analyses of expression profiles revealed that the GhKNOX genes likely responded to diverse stresses and were involved in cotton growth developmental processes. Silencing of GhKNOX2 enhanced the salt tolerance of cotton seedlings, whereas silencing of GhKNOX10 and GhKNOX14 reduced seedling tolerance to salt stress. Silencing of GhSTM3 influenced the cotton flowering time and plant development. These findings clarify the evolution of the cotton KNOX gene family and provide a foundation for future functional studies of KNOX proteins in cotton growth and development and response to abiotic stresses.

Cloning and expression of the chloroplast copper/zinc-superoxide dismutase gene in upland cotton (Gossypium hirsutum L.).

A full-length 1,043-base-pair cDNA clone encoding a chloroplast copper/zinc superoxide dismutase (Cu/Zn-SOD) of upland cotton was first isolated by rapid amplification of cDNA ends (RACE) from the leaves of the cotton (Gossypium hirsutum L.) variety 'CRI36'. Nucleotide sequence analysis of the clone revealed that it contained the complete coding sequence of the mature SOD isozyme subunit, along with a 60-amino acid transit peptide at N-terminal. The amino acid sequence predicted from the full-length clone showed 66%-74% homology with the amino acid sequences of Cu/Zn-SOD from several other plants. This gene was found to be expressed in the leaves and stems, but not in roots, flowers, and hypocotyls, indicating that the gene was expressed only in green tissues. Also, its expression was found to be most active at seedling stage and declined gradually in later development stages. Expression of this cotton Cu/Zn-SOD gene by using the pET-21a (+) expression vector in E. coli BL21 (DE3) led to the production of a novel 29 kD polypeptide with SOD enzyme activity, confirming that the cloned cotton Cu/Zn-SOD cDNA was indeed encoding a functioning SOD enzyme.