CDPK protein in cotton: genomic-wide identification, expression analysis, and conferring resistance to heat stress.

BACKGROUND: Calcium-dependent protein kinase (CDPK) plays a key role in cotton tolerance to abiotic stress. However, its role in cotton heat stress tolerance is not well understood. Here, we characterize the GhCDPK gene family and their expression profiles with the aim of identifying CDPK genes associated with heat stress tolerance. RESULTS: This study revealed 48 GhCDPK members in the cotton genome, distributed on 18 chromosomes. Tree phylogenetic analysis showed three main clustering groups of the GhCDPKs. Cis-elements revealed many abiotic stress and phytohormone pathways conserved promoter regions. Similarly, analysis of the transcription factor binding sites (TFBDS) in the GhCDPK genes showed many stress and hormone related sites. The expression analysis based on qRT-PCR showed that GhCDPK16 was highly responsive to high-temperature stress. Subsequent protein-protein interactions of GhCDPK16 revealed predictable interaction with ROS generating, calcium binding, and ABA signaling proteins. Overexpression of GhCDPK16 in cotton and Arabidopsis improved thermotolerance by lowering ROS compound buildup. Under heat stress, GhCDPK16 transgenic lines upregulated heat-inducible genes GhHSP70, GHSP17.3, and GhGR1, as demonstrated by qRT-PCR analysis. Contrarily, GhCDPK16 knockout lines in cotton exhibited an increase in ROS accumulation. Furthermore, antioxidant enzyme activity was dramatically boosted in the GhCDPK16-ox transgenic lines. CONCLUSIONS: The collective findings demonstrated that GhCDPK16 could be a viable gene to enhance thermotolerance in cotton and, therefore, a potential candidate gene for improving heat tolerance in cotton.

Physiological and Biochemical Properties of Cotton Seedlings in Response to Cu2+ Stress.

Copper(II) (Cu2+) is essential for plant growth and development. However, high concentrations are extremely toxic to plants. We investigated the tolerance mechanism of cotton under Cu2+ stress in a hybrid cotton variety (Zhongmian 63) and two parent lines with different Cu2+ concentrations (0, 0.2, 50, and 100 µM). The stem height, root length, and leaf area of cotton seedlings had decreased growth rates in response to increasing Cu2+ concentrations. Increasing Cu2+ concentration promoted Cu2+ accumulation in all three cotton genotypes' roots, stems, and leaves. However, compared with the parent lines, the roots of Zhongmian 63 were richer in Cu2+ and had the least amount of Cu2+ transported to the shoots. Moreover, excess Cu2+ also induced changes in cellular redox homeostasis, causing accumulation of hydrogen peroxide (H2O2) and malondialdehyde (MDA). Conversely, antioxidant enzyme activity increased, while photosynthetic pigment content decreased. Our findings indicated that the hybrid cotton variety fared well under Cu2+ stress. This creates a theoretical foundation for the further analysis of the molecular mechanism of cotton resistance to copper and suggests the potential of the large-scale planting of Zhongmian 63 in copper-contaminated soils.

Transcriptome and Metabolome Profiling Unveil Pigment Formation Variations in Brown Cotton Lines (Gossypium hirsutum L.).

Naturally brown colored cotton (NBCC) is becoming increasingly popular due to its natural properties of coloration. However, poor fiber quality and color fading are key issues that are hindering the cultivation of naturally colored cotton. In this study, based on transcriptome and metabolome of 18 days post-anthesis (DPA), we compared the variations of pigment formation in two brown cotton fibers (DCF and LCF), with white cotton fiber (WCF) belonging to a near-isogenic line. A transcriptome study revealed a total of 15,785 differentially expressed genes significantly enriched in the flavonoid biosynthesis pathway. Furthermore, for flavonoid biosynthesis-related genes, such as flavonoid 3'5'-hydroxylase (F3'5'H), anthocyanidin synthase (ANS), anthocyanidin reductase (ANR), chalcone synthase (CHS), dihydroflavonol 4-reductase (DFR), and chalcone isomerase (CHI), their expressions significantly increased in LCF compared with DCF and WCF. Moreover, transcription factors MYB and bHLH were significantly expressed in LCF and DCF. Most flavonoid-related metabolites (myricetin naringenin, catechin, epicatechin-epiafzelechin, and epigallocatechin) were found to be more highly up-regulated in LCF and DCF than WCF. These findings reveal the regulatory mechanism controlling different brown pigmentation in cotton fibers and elucidate the need for the proper selection of high-quality brown cotton fiber breeding lines for promising fiber quality and durable brown color pigmentation.

Phylogenetic analysis of upland cotton MATE gene family reveals a conserved subfamily involved in transport of proanthocyanidins.

The multidrug and toxic compound extrusion (MATE) protein belongs to a secondary transporter family, which plays a role in transporting different kinds of substrates like phytohormones and secondary metabolites. In plant, MATE transporters related to the endogenous and exogenous mechanisms of detoxification for secondary metabolites such as alkaloids, flavonoids, anthocyanins and other secondary metabolites have been studied. However, a genome-wide analysis of the MATE family is rarely reported in upland cotton (Gossypium hirsutum L.). In the study, a total of 72 GhMATEs were identified from the genome of upland cotton, which were classified into four subfamilies with possible diverse functions such as transport of proanthocyanidins (PAs), accumulation of alkaloids, extrusion of xenobiotic compounds, regulation of disease resistance and response to abiotic stresses. Meanwhile, the gene structure, evolutionary relationship, physical location, conservative motifs, subcellular localization and gene expression pattern of GhMATEs have been further analysed. Three of these MATE genes (GhMATE12, GhMATE16 and GhMATE38) were identified as candidate genes due to their functions in transport of PA similar to GhTT12. These results provide a new perspective on upland cotton MATE gene family for their potential roles in transport of PA and a theoretical basis for further analyzing the function of MATE genes and improving the fiber quality of brown cotton.

Genomic Comparison of the P-ATPase Gene Family in Four Cotton Species and Their Expression Patterns in Gossypium hirsutum.

Plant P-type H⁺-ATPase (P-ATPase) is a membrane protein existing in the plasma membrane that plays an important role in the transmembrane transport of plant cells. To understand the variety and quantity of P-ATPase proteins in different cotton species, we combined four databases from two diploid cotton species (Gossypium raimondii and G. arboreum) and two tetraploid cotton species (G. hirsutum and G. barbadense) to screen the P-ATPase gene family and resolved the evolutionary relationships between the former cotton species. We identified 53, 51, 99 and 98 P-ATPase genes from G. arboretum, G. raimondii, G. barbadense and G. hirsutum, respectively. The structural and phylogenetic analyses revealed that the gene structure was consistent between P-ATPase genes, with a close evolutionary relationship. The expression analysis of P-ATPase genes showed that many P-ATPase genes were highly expressed in various tissues and at different fiber developmental stages in G. hirsutum, suggesting that they have potential functions during growth and fiber development in cotton.

Genome-wide analysis of the GST gene family in Gossypium hirsutum L.

Glutathione-S-transferase (GST) is a ubiquitous multi-functional protein superfamily that plays important roles in plant primary and secondary metabolism, stress and intercellular signal transduction. Concomitantly, it also functions as a ligand in the metabolism of plant hormones and substance transport. In order to understand the GST gene family in upland cotton (Gossypium hirsutum L.), herein we analyzed the species, evolutionary relationship, physical location, gene structure, conserved motifs and expression patterns. We identified 70 GST genes in the whole genome of upland cotton, and divided them into U, F, T, Z, EF1Bγ and TCHQD groups by phylogenetic tree and gene structure analyses. The gene mapping analysis indicated that the GST genes were on every chromosome except chromosome AD/At2, AD/At4, AD/At5, AD/Dt5 and AD/Dt10. Moreover, the GST gene cluster appeared on four chromosomes (AD/At9, AD/Dt7, AD/Dt12 and AD/Dt13). qRT-PCR assays showed that eight genes (GhGSTF2-9) were expressed in the root, stem, leave and fiber of different developmental stages while GhGSTF1 might be a pseudogene. Combining qRT-PCR and bioinformatic analysis, we speculated that GhGSTF8 might be involved in the transport and accumulation of proanthocyanidins/anthocyanins; GhGSTF4, 6 and 9 might play roles in regulating the growth and stress response of upland cotton; the function of GhGSTF2, 3, 5 and 7 remains to be further investigated. Our work provides a theoretical basis for further studies on the molecular evolution and function of the GST gene family in upland cotton.

A putative molybdate transporter LjMOT1 is required for molybdenum transport in Lotus japonicus.

Molybdenum (Mo) is an essential micronutrient that is required for plant growth and development, and it affects the formation of root nodules and nitrogen fixation in legumes. In this study, Lotus japonicus was grown on MS solid media containing 0 nmol l-1 (-Mo), 103 nmol l-1 (+Mo) and 1030 nmol l-1 (10 × Mo) of Mo. The phenotypes of plants growing on the three different media showed no obvious differences after 15 days, but the plants growing on -Mo for 45 days presented typical symptoms of Mo depletion, such as a short taproot, few lateral roots and yellowing leaves. A Mo transporter gene, LjMOT1, was isolated from L. japonicus. It encoded 468 amino acids, including two conserved motifs, and was predicted to locate to chromosome 3 of the L. japonicus genome. A homology comparison indicated that LjMOT1 had high similarities to other MOT1 proteins and was closely related to GmMOT1. Subcellular localization indicated that LjMOT1 is localized to the plasma membrane. qRT-PCR analyses showed that increasing Mo concentrations regulated the relative expression level of LjMOT1. Moreover, the Mo concentration in shoots was positively correlated to the expression of LjMOT1, but there was no such evident correlation in the roots. In addition, changes in the nitrate reductase activity were coincident with changes in the Mo concentration. These results suggest that LjMOT1 may be involved in the transport of Mo and provide a theoretical basis for further understanding of the mechanism of Mo transport in higher plants.

[Cloning and prokaryotic expression of casein kinase II subunit beta gene fragment of Dirofilaria immitis].

OBJECTIVE: To clone and express the partial fragment of Csnk2b gene of Dirofilaria immitis in prokaryotic cells, and analyze the immunoreactivity. METHODS: The partial fragment of Csnk2b gene was amplified by PCR with a pair of specific primers. The PCR product was cloned into pMD18-T, and then sub-cloned to pGEX-4T-1 expression vector. The constructed plasmid pGEX-4T-1-Csnk2b was transformed into E. coli Rosetta (DE3) and followed by expression of the protein induced by IPTG. The recombinant protein was analyzed by SDS-PAGE and identified by Western blotting. RESULTS: The PCR product was about 700 bp. Enzyme digestion and DNA sequencing confirmed that the recombinant plasmid pGEX-4T-1-Csnk2b was constructed. SDS-PAGE results showed that the relative molecular weight (M(r)) of the fusion protein (GST-Csnk2b) was about 45 000. GST-Csnk2b reacted positively with mouse anti-D. immitis serum. CONCLUSION: The partial Csnk2b gene has been expressed in prokaryotic expression system and shows immunoreactivity.

Transcriptomic and metabolomic analyses reveal the potential mechanism of waterlogging resistance in cotton (Gossypium hirsutum L.).

Introduction: Cotton (Gossypium hirsutum L.) is susceptible to long-term waterlogging stress; however, genomic information of cotton response mechanisms toward long days of waterlogging is quite elusive. Methods: Here, we combined the transcriptome and metabolome expression level changes in cotton roots after 10 and 20 days of waterlogging stress treatment pertaining to potential resistance mechanisms in two cotton genotypes. Results and discussion: Numerous adventitious roots and hypertrophic lenticels were induced in CJ1831056 and CJ1831072. Transcriptome analysis revealed 101,599 differentially expressed genes in cotton roots with higher gene expression after 20 days of stress. Reactive oxygen species (ROS) generating genes, antioxidant enzyme genes, and transcription factor genes (AP2, MYB, WRKY, and bZIP) were highly responsive to waterlogging stress among the two genotypes. Metabolomics results showed higher expressions of stress-resistant metabolites sinapyl alcohol, L-glutamic acid, galactaric acid, glucose 1-phosphate, L-valine, L-asparagine, and melibiose in CJ1831056 than CJ1831072. Differentially expressed metabolites (adenosine, galactaric acid, sinapyl alcohol, L-valine, L-asparagine, and melibiose) significantly correlated with the differentially expressed PRX52, PER1, PER64, and BGLU11 transcripts. This investigation reveals genes for targeted genetic engineering to improve waterlogging stress resistance to enhance abiotic stress regulatory mechanisms in cotton at the transcript and metabolic levels of study.

Genome-wide identification of ABCC gene family and their expression analysis in pigment deposition of fiber in brown cotton (Gossypium hirsutum).

ABC (ATP-binding cassette) transporters are a class of superfamily transmembrane proteins that are commonly observed in natural organisms. The ABCC (ATP-binding cassette C subfamily) protein belongs to a subfamily of the ABC protein family and is a multidrug resistance-associated transporter that localizes to the tonoplast and plays a significant role in pathogenic microbial responses, heavy metal regulation, secondary metabolite transport, and plant growth. Recent studies have shown that the ABCC protein is also involved in the transport of anthocyanins/proanthocyanidins (PAs). To clarify the types and numbers of ABCC genes involved in PA transport in Gossypium hirsutum, the phylogenetic evolution, physical location, and structure of ABCC genes were classified by bioinformatic methods in the upland cotton genome, and the expression levels of these genes were analyzed at different developmental stages of the cotton fiber. The results showed that 42 ABCC genes were initially identified in the whole genome of upland cotton; they were designated GhABCC1-42. The gene structure and phylogenetic analysis showed that the closely related ABCC genes were structurally identical. The analysis of chromosomal localization demonstrated that there were no ABCC genes on the chromosomes of AD/At2, AD/At5, AD/At6, AD/At10, AD/At12, AD/At13, AD/Dt2, AD/Dt6, AD/Dt10, and AD/Dt13. Outside the genes, there were ABCC genes on other chromosomes, and gene clusters appeared on the two chromosomes AD/At11 and AD/Dt8. Phylogenetic tree analysis showed that some ABCC proteins in G. hirsutum were clustered with those of Arabidopsis thaliana, Vitis vinifera and Zea mays, which are known to function in anthocyanin/PA transport. The protein structure prediction indicated that the GhABCC protein structure is similar to the AtABCC protein in A. thaliana, and most of these proteins have a transmembrane domain. At the same time, a quantitative RT-PCR analysis of 42 ABCC genes at different developmental stages of brown cotton fiber showed that the relative expression levels of GhABCC24, GhABCC27, GhABCC28, GhABCC29 and GhABCC33 were consistent with the trend of PA accumulation, which may play a role in PA transport. These results provide a theoretical basis for further analysis of the function of the cotton ABCC genes and their role in the transport of PA.

Molecular cloning, expression analysis and subcellular localization of a Transparent Testa 12 ortholog in brown cotton (Gossypium hirsutum L.).

Transparent Testa 12 (TT12) is a kind of transmembrane transporter of proanthocyanidins (PAs), which belongs to a membrane-localized multidrug and toxin efflux (MATE) family, but the molecular basis of PAs transport is still poorly understood. Here, we cloned a full-length TT12 cDNA from the fiber of brown cotton (Gossypium hirsutum), named GhTT12 (GenBank accession No. KF240564), which comprised 1733 bp with an open reading frame (ORF) of 1503 bp and encoded a putative protein containing 500 amino acid residues with a typical MATE conserved domain. The GhTT12 gene had 96.8% similarity to AA genome in Gossypium arboretum. Quantitative RT-PCR analysis denoted that the relative expression of GhTT12 in brown cotton was 1-5 folds higher than that in white cotton. The mRNA level was the highest at 5 days post anthesis (DPA) and reduced gradually during the fiber development. Expressing GhTT12-fused green fluorescent protein (GFP) in Nicotiana tabacum showed that GhTT12-GFP was localized in the vacuole membrane. The content of PAs increased firstly and decreased afterwards, and reached the maximum at 15 DPA in brown cotton. But for white cotton, the content of PAs remained at a low level during the fiber development. We speculate that GhTT12 may participate in the transportation of PAs from the cytoplasmic matrix to the vacuole. Taken together, our data revealed that GhTT12 was functional as a PAs transmembrane transporter.

Isolation and molecular characterization of an ethylene response factor NtERF1-1 in Nicotiana tabacum cv. Xanthi.

Apetala2/Ethylene Response Factors (AP2/ERF) play important roles in regulating gene expression under abiotic and biotic stress in the plant kingdom. Here, we isolated a member of the AP2/ERF transcription factors, NtERF1-1, from Nicotiana tabcum cv. Xanthi NN carrying the N gene, which is resistant to Tobacco mosaic virus (TMV). NtERF1-1 encoded a putative protein of 229 amino acids with a predicted molecular mass of 24.58 kDa. Nucleotide sequence analysis showed that NtERF1-1 contained a conserved DNA-binding domain at the N-terminal. Comparison of amino acid sequences revealed that NtERF1-1 possessed high similarities to ERFs from diverse plants. Semi-quantitative and real-time quantitative RT-PCR analyses indicated that NtERF1-1 was up-regulated following TMV infection. In addition, we speculated that NtERF1-1 might participate in the signal transduction pathway of defence response inducted by the interaction between the N gene and TMV.