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Nuclear factor YA (NF-YA) genes play important roles in many biological processes, such as leaf growth, nitrogen nutrition, drought resistance, and salt stress. The functions of NF-YA genes in cotton have not been elucidated. The current study identified a total of 16, 16, 31, and 29 genes from Gossypium raimondii, G. arboretum, G. barbadense, and G. hirsutum, respectively. The NF-YA genes in cotton were phylogenetically classified into 4 groups. Analysis of gene structure, conserved motifs and multiple sequence alignments supported the evolutionary conservation of NF-YA family genes in cotton. Analysis of the expression patterns of GhNF-YAs in cotton suggested that GhNF-YAs play important roles in plant growth, development, and stress responses. The quantitative real-time PCR (qRT-PCR) validation of selected genes suggested that GhNF-YA genes are induced in response to salt, drought, ABA, and MeJA treatments. GhNF-YA genes may regulate salt and drought stress via the ABA or MeJA pathway. Silencing of GhNF-YA10 and GhNF-YA23 significantly reduced the salt tolerance of cotton seedlings, indicating that these genes participate in the regulation of the response of cotton to salt stress. These results establish a foundation for subsequent functional studies of the NF-YA gene family in cotton.

Hybrid breakdown (HB) functions as a common reproductive barrier and reduces hybrid fitness in many species, including cotton. However, the related genes and the underlying genetic mechanisms of HB in cotton remain unknown. Here, we found that the photosensitive genetic male sterile line CCRI9106 was a hybrid progeny of Gossypium hirsutum and Gossypium barbadense and probably a product of HB. Fine mapping with F2 s (CCRI9106 × G. hirsutum/G. barbadense lines) identified a pair of male sterility genes GoFLA19s (encoding fasciclin-like arabinogalactan family protein) located on chromosomes A12 and D12. Crucial variations occurring in the fasciclin-like domain and the arabinogalactan protein domain were predicted to cause the non-functionalization of GbFLA19-D and GhFLA19-A. CRISPR/Cas9-mediated knockout assay confirmed the effects of GhFLA19s on male sterility. Sequence alignment analyses showed that variations in GbFLA19-D and GhFLA19-A likely occurred after the formation of allotetraploid cotton species. GoFLA19s are specifically expressed in anthers and contribute to tapetal development, exine assembly, intine formation, and pollen grain maturation. RNA-sequencing and quantitative reverse transcriptase-polymerase chain reaction analyses illustrated that genes related to these biological processes were significantly downregulated in the mutant. Our research on male sterility genes, GoFLA19s, improves the understanding of the molecular characteristics and evolutionary significance of HB in interspecific hybrid breeding.

Background B-BOX (BBX) proteins are zinc-finger transcription factors with one or two BBX domains and sometimes a CCT domain. These proteins play an essential role in regulating plant growth and development, as well as in resisting abiotic stress. So far, the BBX gene family has been widely studied in other crops. However, no one has systematically studied the BBX gene in cotton. Results In the present study, 17, 18, 37 and 33 BBX genes were detected in Gossypium arboreum, G. raimondii, G. hirsutum and G. barbadense, respectively, via genome-wide identification. Phylogenetic analysis showed that all BBX genes were divided into 5 main categories. The protein motifs and exon/intron structures showed that each group of BBX genes was highly conserved. Collinearity analysis revealed that the amplification of BBX gene family in Gossypium spp. was mainly through segmental replication. Nonsynonymous (Ka)/ synonymous (Ks) substitution ratios indicated that the BBX gene family had undergone purification selection throughout the long-term natural selection process. Moreover, transcriptomic data showed that some GhBBX genes were highly expressed in floral organs. The qRT-PCR results showed that there were significant differences in GhBBX genes in leaves and shoot apexes between early-maturing materials and late-maturing materials at most periods. Yeast two-hybrid results showed that GhBBX5/GhBBX23 and GhBBX8/GhBBX26 might interact with GhFT. Transcriptome data analysis and qRT-PCR verification showed that different GhBBX genes had different biological functions in abiotic stress and phytohormone response. Conclusions Our comprehensive analysis of BBX in G. hirsutum provided a basis for further study on the molecular role of GhBBXs in regulating flowering and cotton resistance to abiotic stress.

Lint percentage is a major yield component in cotton breeding programmes. To identify quantitative trait loci (QTLs) and candidate genes related to lint percentage, we used an intraspecific recombinant inbred line population of 137 lines derived from Gossypium hirsutum cv. CCRI36 and G. hirsutum acc. G2005 for QTL mapping of lint percentage. Based on a high-density genetic map and phenotype data collected in four growing environments, we identified a total of 28 QTLs for lint percentage. Three stable QTLs (qLP-At5-2, qLP-Dt7-1 and qLP-Dt7-2) were detected in at least two environments. Two genes (Gh_A05G1584 and Gh_A05G1689) containing nonsynonymous single nucleotide polymorphisms (SNPs) were identified by association analysis using published data. The quantitative real-time PCR results showed that the expression levels of Gh_A05G1584 were higher in cv. CCRI36 than in acc. G2005 during all fibre development stages; Gh_A05G1689 was mainly expressed in 15 and 25 days post-anthesis fibres and its expression level was higher in cv. CCRI36. These results suggest candidate genes for lint percentage and provide molecular information for use in cotton breeding programmes aimed at improving yield.

Genetically modified (GM) crops possess some superior characteristics, such as high yield and insect resistance, but their biosafety has aroused broad public concern. Some genetic engineering technologies have recently been proposed to remove exogenous genes from GM crops. Few approaches have been applied to maintain advantageous traits, but excising exogenous genes in seeds or fruits from these hybrid crops has led to the generation of harvested food without exogenous genes. In a previous study, split-Cre mediated by split intein could recombine its structure and restore recombination activity in hybrid plants. In the current study, the recombination efficiency of split-Cre under the control of ovule-specific or pollen-specific promoters was validated by hybridization of transgenic Arabidopsis containing the improved expression vectors. In these vectors, all exogenous genes were flanked by two loxP sites, including promoters, resistance genes, reporter genes, and split-Cre genes linked to the reporter genes via LP4/2A. A gene deletion system was designed in which NCre was driven by proDD45, and CCre was driven by proACA9 and proDLL. Transgenic lines containing NCre were used as paternal lines to hybridize with transgenic lines containing CCre. Because this hybridization method results in no co-expression of the NCre and CCre genes controlled by reproduction-specific promoters in the F1 progeny, the desirable characteristics could be retained. After self-crossing in F1 progeny, the expression level and protein activity of reporter genes were detected, and confirmed that recombination of split-Cre had occurred and the exogenous genes were partially deleted. The gene deletion efficiency represented by the quantitative measurements of GUS enzyme activity was over 59%, with the highest efficiency of 73% among variable hybrid combinations. Thus, in the present study a novel dual reproductive cell-specific promoter-mediated gene deletion system was developed that has the potential to take advantage of the merits of GM crops while alleviating biosafety concerns.

Background: B-BOX (BBX) proteins are zinc-finger transcription factors with one or two BBX domains and sometimes a CCT domain. These proteins play an essential role in regulating plant growth and development, as well as in resisting abiotic stress. So far, the BBX gene family has been widely studied in other crops. However, no one has systematically studied the BBX gene in cotton. Results: In the present study, 17, 18, 37 and 33 BBX genes were detected in Gossypium arboreum, G. raimondii, G. hirsutum and G. barbadense, respectively, via genome-wide identification. Phylogenetic analysis showed that all BBX genes were divided into 5 main categories. The protein motifs and exon/intron structures indicated that each group of BBX genes was highly conserved. Collinearity analysis revealed that the amplification of BBX gene family in Gossypium spp. was mainly through segmental replication. Nonsynonymous (Ka)/ synonymous (Ks) substitution ratios indicated that the BBX gene family had undergone purification selection throughout the long-term natural selection process. Moreover, transcriptomic data showed that some GhBBX genes were highly expressed in floral organs. Transcriptome data analysis and qRT-PCR verification showed that different GhBBX genes had different biological functions in flower bud differentiation, abiotic stress and stress response. Conclusions: Our comprehensive analysis of BBX in G. hirsutum provides a basis for further study on the molecular role of GhBBXs in regulating flowering and cotton resistance to abiotic stress.

Background Histone deacetylases (HDACs) catalyze histone deacetylation and suppress gene transcription during various cellular processes. Within the superfamily of HDACs, RPD3/HDA1-type HDACs are the most studied, and it is reported that RPD3 genes play crucial roles in plant growth and physiological processes. However, there is a lack of systematic research on the RPD3/HDA1 gene family in cotton. Results In this study, genome-wide analysis identified 9, 9, 18, and 18 RPD3 genes in Gossypium raimondii, G. arboreum, G. hirsutum, and G. barbadense, respectively. This gene family was divided into 4 subfamilies through phylogenetic analysis. The exon-intron structure and conserved motif analysis revealed high conservation in each branch of the cotton RPD3 genes. Collinearity analysis indicated that segmental duplication was the primary driving force during the expansion of the RPD3 gene family in cotton. There was at least one presumed cis-element related to plant hormones in the promoter regions of all GhRPD3 genes, especially MeJA- and ABA-responsive elements, which have more members than other hormone-relevant elements. The expression patterns showed that most GhRPD3 genes had relatively high expression levels in floral organs and performed higher expression in early-maturity cotton compared with late-maturity cotton during flower bud differentiation. In addition, the expression of GhRPD3 genes could be significantly induced by one or more abiotic stresses as well as exogenous application of MeJA or ABA. Conclusions Our findings reveal that GhRPD3 genes may be involved in flower bud differentiation and resistance to abiotic stresses, which provides a basis for further functional verification of GhRPD3 genes in cotton development and a foundation for breeding better early-maturity cotton cultivars in the future.

The sodium/hydrogen antiporter (NHX) gene family with the Na+/H+ exchange protein domain is a transporter of sodium and hydrogen ions and plays an important role in the response of plants to salt stress. Studying the response of cotton to salt stress through comprehensive identification and analysis of NHX genes in several species and their roles in salt tolerance mechanisms is of great significance. In this study, 23, 24, 12, and 12 NHX genes were identified from Gossypium hirsutum (Gh), G. barbadense, G. arboreum and G. raimondii, respectively. Phylogenetic analysis showed that these genes were mainly divided into three clades with significant subcellular localization, namely, endosome (Endo-class), plasma membrane (PM-class) and vacuole (Vac-class). By analyzing the structure of NHX genes and proteins, each branch of the NHX gene family was found to be structurally conserved, and collinearity analysis showed that NHX genes were mainly expressed through whole genome and segmental duplication. The non-synonymous (Ka)/synonymous (Ks) values showed that the NHX gene family experienced strong purifying selection during long-term evolution. Cis-acting element analysis showed that the NHX gene family may be related to the regulation of abscisic acid (ABA) and methyl jasmonate (MeJA) hormones. Additionally, transcriptomic data analysis and qRT-PCR showed that GhNHXs exhibited different expression patterns in each tissue and under different salinities. These results provide an important reference for us to further understand and analyze the molecular regulation mechanism of cotton NHX genes.

Salt-induced developmental plasticity in a plant root system strongly depends on auxin signaling. However, the molecular events underlying this process are poorly understood. MicroRNA390 (miR390), trans-actin small interfering RNAs (tasiRNAs), and AUXIN RESPONSE FACTORs (ARFs) form a regulatory module involved in controlling lateral root (LR) growth. Here, we found that miR390 expression was strongly induced by exposure to salt during LR formation in poplar (Populus spp.) plants. miR390 overexpression stimulated LR development and increased salt tolerance, whereas miR390 knockdown caused by a short tandem target mimic repressed LR growth and compromised salt resistance. ARF3.1, ARF3.2, and ARF4 expression was inhibited significantly by the presence of salt, and transcript abundance was decreased dramatically in the miR390-overexpressing line but increased in the miR390-knockdown line. Constitutive expression of ARF4m harboring mutated trans-acting small interfering ARF-binding sites removed the salt resistance of the miR390 overexpressors. miR390 positively regulated auxin signaling in LRs subjected to salt, but ARF4 inhibited auxin signaling. Salinity stabilized the poplar Aux/IAA repressor INDOLE-3-ACETIC ACID17.1, and overexpression of an auxin/salt-resistant form of this repressor suppressed LR growth in miR390-overexpressing and ARF4-RNA interfering lines in the presence of salt. Thus, the miR390/TAS3/ARFs module is a key regulator, via modulating the auxin pathway, of LR growth in poplar subjected to salt stress.

ATBS1 INTERACTING FACTORs (AIFs) are atypical basic helix-loop-helix (bHLH) proteins play important roles in the growth and development of plants. There are no comprehensive reports on the genomic identification and functional analysis of AIFs in cotton. In this study, 8, 11, 16, and 15 AIFs were identified in Gossypium arboreum L., Gossypium raimondii L., Gossypium hirsutum L., and Gossypium barbadense L., respectively. AIFs were also screened in other species: 3 in Theobroma cacao L., 4 in Populus trichocarpa C. and 5 in Zea mays L. Based on a neighbour-joining phylogenetic tree, 66 AIF proteins identified in these species were divided into 3 groups. Multiple sequence alignment analysis showed that the AIF protein sequences were relatively conserved. Analysis of gene chromosome position and gene replication showed that AIFs amplification in cotton might be due to fragment and tandem replication. And cold stress response and plant hormone cis-elements were identified in the promoter regions of GhAIFs. Published transcriptional data showed that GhAIFs were differentially expressed in various tissues, especially in floral organs and fibers. qRT-PCR results showed that most of GhAIFs were highly expressed at the fiber initiation stage. Analysis of the expression of selected GhAIFs under abiotic stress (GA, IAA, SA, ABA, and 4 °C treatment) suggested that GhAIFs might be involved in the regulation of abiotic stress. The comprehensive analysis of the AIF gene family in upland cotton provides a foundation for further studies on the function of atypical bHLH-GhAIFs family members and screening of fiber development, and stress-resistant candidate genes in cotton.

Pentatricopeptide repeat (PPR) proteins, which are characterized by tandem 30–40 amino acid sequence motifs, constitute of a large gene family in plants. Some PPR proteins have been identified to play important roles in organellar RNA metabolism and organ development in Arabidopsis and rice. However, functions of PPR genes in woody species remain largely unknown. Here, we identified and characterized a total of 626 PPR genes containing PPR motifs in the Populus trichocarpa genome. A comprehensive genome-wide analysis of the poplar PPR gene family was performed, including chromosomal location, phylogenetic relationships and gene duplication. Genome-wide transcriptomic analysis showed that 154 of the PtrPPR genes were induced by biotic and abiotic treatments, including Marssonina brunnea, salicylic acid (SA), methyl jasmonate (MeJA), mechanical wounding, cold and salinity stress. Quantitative RT-PCR analysis further investigated the expression profiles of 11 PtrPPR genes under different stresses. Our results contribute to a comprehensive understanding the roles of PPR proteins and provided an insight for improving the stress tolerance in poplar.