Background: Salinity is one of the most significant environmental factors limiting the productivity of cotton. However, the key genetic components responsible for the reduction in cotton yield in saline-alkali soils are still unclear. Results: Here, we evaluated three main components of lint yield, single boll weight (SBW), lint percentage (LP) and boll number per plant (BNPP), across 316 G. hirsutum accessions under four salt conditions over two years. Phenotypic analysis indicated that LP was unchanged under different salt conditions, however BNPP decreased significantly and SBW increased slightly under high salt conditions. Based on 57,413 high-quality single nucleotide polymorphisms (SNPs) and genome-wide association study (GWAS) analysis, a total of 42, 91 and 25 stable quantitative trait loci (QTLs) were identified for SBW, LP and BNPP, respectively. Phenotypic and QTL analysis suggested that there was little correlation among the three traits. For LP, 8 stable QTLs were detected simultaneously in four different salt conditions, while fewer repeated QTLs for SBW or BNPP were identified. Gene Ontology (GO) analysis indicated that their regulatory mechanisms were also quite different. Via transcriptome profile data, we detected that 10 genes from the 8 stable LP QTLs were predominantly expressed during fiber development. Further, haplotype analyses found that a MYB gene (GhMYB103), with the two SNP variations in cis-regulatory and coding regions, was significantly correlated with lint percentage, implying a crucial role in lint yield. We also identified that 40 candidate genes from BNPP QTLs were salt-inducible. Genes related to carbohydrate metabolism and cell structure maintenance were rich in plants grown in high salt conditions, while genes related to ion transport were active in plants grown in low salt conditions, implying different regulatory mechanisms for BNPP at high and low salt conditions. Conclusions: This study provides a foundation for elucidating cotton salt tolerance mechanisms and contributes gene resources for developing upland cotton varieties with high yields and salt stress tolerance.

Aquaporins (AQPs) are integral membrane proteins from a larger family of major intrinsic proteins (MIPs) and function in a huge variety of processes such as water transport, plant growth and stress response. The availability of the whole-genome data of different cotton species allows us to study systematic evolution and function of cotton AQPs on a genome-wide level. Here, a total of 53, 58, 113 and 111 AQP genes were identified in G. arboreum, G. raimondii, G. hirsutum and G. barbadense, respectively. A comprehensive analysis of cotton AQPs, involved in exon/intron structure, functional domains, phylogenetic relationships and gene duplications, divided these AQPs into five subfamilies (PIP, NIP, SIP, TIP and XIP). Comparative genome analysis among 30 species from algae to angiosperm as well as common tandem duplication events in 24 well-studied plants further revealed the evolutionary conservation of AQP family in the organism kingdom. Combining transcriptome analysis and Quantitative Real-time PCR (qRT-PCR) verification, most AQPs exhibited tissue-specific expression patterns both in G. raimondii and G. hirsutum. Meanwhile, a bias of time to peak expression of several AQPs was also detected after treating G. davidsonii and G. hirsutum with 200 mM NaCl. It is interesting that both PIP1;4 h/i/j and PIP2;2a/e showed the highly conserved tandem structure, but differentially contributed to tissue development and stress response in different cotton species. These results demonstrated that cotton AQPs were structural conservation while experienced the functional differentiation during the process of evolution and domestication. This study will further broaden our insights into the evolution and functional elucidation of AQP gene family in cotton.

Ubiquitination plays important roles in modulating the abiotic stress tolerance of plants. Drought seriously restricts agricultural production, but how ubiquitination participates in regulating drought tolerance remains largely unknown. Here, we identified a drought-inducible gene, MYB30-INTERACTING E3 LIGASE 1 (GhMIEL1), which encodes a RING E3 ubiquitin ligase in cotton (Gossypium hirsutum). GhMIEL1 was strongly induced by polyethylene glycol (PEG-6000) and the phytohormone abscisic acid (ABA). Overexpression and knockdown of GhMIEL1 in cotton substantially enhanced and reduced drought tolerance, respectively. GhMIEL1 interacted with the MYB transcription factor GhMYB66 and could ubiquitinate and degrade it in vitro. GhMYB66 directly bound to the LONELY GUY 5 (GhLOG5) promoter, a gene encoding cytokinin riboside 5'-monophosphate phosphoribohydrolase, to repress its transcription. Overexpression of GhMIEL1 and silencing of GhMYB66 altered the homeostasis of cytokinin of plant roots, increased total root length and number of root tips, and enhanced plant drought tolerance. Conversely, silencing GhLOG5 decreased total root length and number of root tips and reduced plant drought tolerance. Our studies reveal that the GhMIEL1-GhMYB66-GhLOG5 module positively regulates drought tolerance in cotton, which deepens our understanding of plant ubiquitination-mediated drought tolerance and provides insights for improving drought tolerance.

CLAVATA3 (CLV3)/endosperm surrounding region (CLE) peptides have been reportedly involved in plant growth and development, as well as responses to abiotic stresses. However, the stress resilience of most CLE genes in cotton remains largely unknown. Here, induced expression pattern analysis showed that GhCLE25 was obviously responsive to osmotic and salt treatments, indicating that GhCLE25 was involved in abiotic stress tolerance. Furthermore, silencing GhCLE25 or the exogenous application of CLE25p effectively led to reduced and enhanced drought tolerance, respectively, as indicated by the activities of the plants’ POD, SOD, CAT, and MDA contents, as well as their height and fresh weight. We found that the knockdown of GhCLE25 promoted seedling growth and development, with a higher plant height and fresh weight in GhCLE25-silenced plants in comparison to control plants. In addition, a comparative transcriptome analysis of TRV:00 versus TRV:GhCLE25 and Mock versus CLE25p revealed that the CLE25-mediated signaling pathway is mainly involved in defense response and phytohormone signaling. Collectively, these findings indicate diverse roles of CLE25 in regulating plant growth and response to environmental stimuli and highlight the potential utilization of CLE25 to improve drought stress in modern agriculture via CLE25p spraying.

The root system architecture plays an essential role in water and nutrient acquisition in plants and is significantly involved in plant adaptation to various environmental stresses. In this study, a panel of 242 cotton accessions was collected to investigate six root morphological traits at the seedling stage, including main root length (MRL), root fresh weight (RFW), total root length (TRL), root surface area (RSA), root volume (RV), and root average diameter (AvgD). The correlation analysis between six root morphological traits revealed a strong positive correlation of TRL with RSA, RV with RSA and AvgD, whereas a significant negative correlation between TRL and AvgD. Subsequently, a genome-wide association study (GWAS) was performed using the root phenotypic data and genotypic data reported previously for 242 accessions using 56,010 single nucleotide polymorphisms (SNPs) from the CottonSNP80K array. A total of 41 quantitative trait loci (QTLs) were identified with 9 for MRL, 6 for RFW, 9 for TRL, 12 for RSA, 12 for RV and 2 for AvgD respectively. Among them, 8 QTLs were repeatedly detected in two or more traits. Integrated with transcriptome analysis, we identified 17 candidate genes with high transcripts of transcripts per million (TPM)≥30 in roots. Furthermore, we verified functionally a candidate gene GH_D05G2106 encoding a WPP domain protein 2 in root development. Virus-induced gene silencing (VIGS) assay showed that knocking down GH_D05G2106 significantly inhibited root development in cotton, indicating its positive role in root system architecture formation. Together, these results provide a theoretical basis and candidate genes for cotton root developmental biology and root-related cotton breeding.

Salt tolerance is an important trait in soybean cultivation and breeding. Plant responses to salt stress include physiological and biochemical changes that affect the movement of water across the plasma membrane. Plasma membrane intrinsic proteins (PIPs) localize to the plasma membrane and regulate the water and solutes flow. In this study, quantitative real-time PCR and yeast two-hybridization were engaged to analyze the early gene expression profiles and interactions of a set of soybean PIPs (GmPIPs) in response to salt stress. A total of 20 GmPIPs-encoding genes had varied expression profiles after salt stress. Among them, 13 genes exhibited a downregulated expression pattern, including GmPIP1;6, the constitutive overexpression of which could improve soybean salt tolerance, and its close homologs GmPIP1;7 and 1;5. Three genes showed upregulated patterns, including the GmPIP1;6 close homolog GmPIP1;4, when four genes with earlier increased and then decreased expression patterns. GmPIP1;5 and GmPIP1;6 could both physically interact strongly with GmPIP2;2, GmPIP2;4, GmPIP2;6, GmPIP2;8, GmPIP2;9, GmPIP2;11, and GmPIP2;13. Definite interactions between GmPIP1;6 and GmPIP1;7 were detected and GmPIP2;9 performed homo-interaction. The interactions of GmPIP1;5 with GmPIP2;11 and 2;13, GmPIP1;6 with GmPIP2;9, 2;11 and GmPIP2;13, and GmPIP2;9 with itself were strengthened upon salt stress rather than osmotic stress. Taken together, we inferred that GmPIP1 type and GmPIP2 type could associate with each other to synergistically function in the plant cell; a salt-stress environment could promote part of their interactions. This result provided new clues to further understand the soybean PIP–isoform interactions, which lead to potentially functional homo- and heterotetramers for salt tolerance.