GhPYL9-5D and GhPYR1-3 A positively regulate Arabidopsis and cotton responses to ABA, drought, high salinity and osmotic stress.

BACKGROUND: Abscisic acid (ABA) receptor pyrabactin resistance 1/PYR1-like/regulatory components of ABA receptor proteins (PYR/PYL/RCARs) have been demonstrated to play pivotal roles in ABA signaling and in response to diverse environmental stimuli including drought, salinity and osmotic stress in Arabidopsis. However, whether and how GhPYL9-5D and GhPYR1-3A, the homologues of Arabidopsis PYL9 and PYR1 in cotton, function in responding to ABA and abiotic stresses are still unclear. RESULTS: GhPYL9-5D and GhPYR1-3A were targeted to the cytoplasm and nucleus. Overexpression of GhPYL9-5D and GhPYR1-3A in Arabidopsis wild type and sextuple mutant pyr1pyl1pyl2pyl4pyl5pyl8 plants resulted in ABA hypersensitivity in terms of seed germination, root growth and stomatal closure, as well as seedling tolerance to water deficit, salt and osmotic stress. Moreover, the VIGS (Virus-induced gene silencing) cotton plants, in which GhPYL9-5D or GhPYR1-3A were knocked down, showed clearly reduced tolerance to polyethylene glycol 6000 (PEG)-induced drought, salinity and osmotic stresses compared with the controls. Additionally, transcriptomic data revealed that GhPYL9-5D was highly expressed in the root, and GhPYR1-3A was strongly expressed in the fiber and stem. GhPYL9-5D, GhPYR1-3A and their homologs in cotton were highly expressed after treatment with PEG or NaCl, and the two genes were co-expressed with redox signaling components, transcription factors and auxin signal components. These results suggest that GhPYL9-5D and GhPYR1-3A may serve important roles through interplaying with hormone and other signaling components in cotton adaptation to salt or osmotic stress. CONCLUSIONS: GhPYL9-5D and GhPYR1-3A positively regulate ABA-mediated seed germination, primary root growth and stomatal closure, as well as tolerance to drought, salt and osmotic stresses likely through affecting the expression of multiple downstream stress-associated genes in Arabidopsis and cotton.

Systematic analysis and expression of Gossypium 2ODD superfamily highlight the roles of GhLDOXs responding to alkali and other abiotic stress in cotton.

BACKGROUND: 2-oxoglutarate-dependent dioxygenase (2ODD) is the second largest family of oxidases involved in various oxygenation/hydroxylation reactions in plants. Many members in the family regulate gene transcription, nucleic acid modification/repair and secondary metabolic synthesis. The 2ODD family genes also function in the formation of abundant flavonoids during anthocyanin synthesis, thereby modulating plant development and response to diverse stresses. RESULTS: Totally, 379, 336, 205, and 204 2ODD genes were identified in G. barbadense (Gb), G. hirsutum (Gh), G. arboreum (Ga), and G. raimondii (Gb), respectively. The 336 2ODDs in G. hirsutum were divided into 15 subfamilies according to their putative functions. The structural features and functions of the 2ODD members in the same subfamily were similar and evolutionarily conserved. Tandem duplications and segmental duplications served essential roles in the large-scale expansion of the cotton 2ODD family. Ka/Ks values for most of the gene pairs were less than 1, indicating that 2ODD genes undergo strong purifying selection during evolution. Gh2ODDs might act in cotton responses to different abiotic stresses. GhLDOX3 and GhLDOX7, two members of the GhLDOX subfamily from Gh2ODDs, were significantly down-regulated in transcription under alkaline stress. Moreover, the expression of GhLDOX3 in leaves was significantly higher than that in other tissues. These results will provide valuable information for further understanding the evolution mechanisms and functions of the cotton 2ODD genes in the future. CONCLUSIONS: Genome-wide identification, structure, and evolution and expression analysis of 2ODD genes in Gossypium were carried out. The 2ODDs were highly conserved during evolutionary. Most Gh2ODDs were involved in the regulation of cotton responses to multiple abiotic stresses including salt, drought, hot, cold and alkali.

Enhancing stimulation of cyaniding, GhLDOX3 activates reactive oxygen species to regulate tolerance of alkalinity negatively in cotton.

Anthocyanins belong to flavonoid secondary metabolites that act as plant pigments to give flowers and fruits different colors and as "scavengers" of reactive oxygen species (ROS) to protect plants from abiotic and biotic stresses. Few studies linked anthocyanins to alkaline resistance so far. In this study, anthocyanin synthesis-related gene leucoanthocyanidin dioxygenase (LDOX) was screened as a candidate gene to explore its relationship with alkali stress. The results found that pYL156: GhLDOX3 lines treated with 50 mM Na2CO3 (pH 11.11) for 24 h showed a significant increase in peroxidase (POD) activity, a decrease in total anthocyanin content and an increase in cyanidin content and a decrease in ROS accumulation compared to pYL156. The overexpressed (OE) lines, ldox mutant and wild-type (WT) lines in Arabidopsis were treated with 50 mM Na2CO3, 100 mM Na2CO3 and 150 mM Na2CO3 for 8 d, respectively. The wilted degree of the OE lines was more severe than WT lines, and less severe in the mutant lines in the 150 mM Na2CO3 treatment. After treatment, the expression levels of AtCAT and AtGSH genes related to antioxidant system in OE lines were significantly lower than in WT, and the expression levels of AtCAT and AtGSH in mutant lines were significantly higher than in WT. In conclusion, the above results suggest GhLDOX3 played a negative regulatory role in the mechanism of resisting Na2CO3 stress. Therefore, it can be considered in cotton breeding to improve the alkali tolerance of cotton by regulating the expression of related genes.

GhAP1-D3 positively regulates flowering time and early maturity with no yield and fiber quality penalties in upland cotton.

Flowering time (FTi) is a major factor determining how quickly cotton plants reach maturity. Early maturity greatly affects lint yield and fiber quality and is crucial for mechanical harvesting of cotton in northwestern China. Yet, few quantitative trait loci (QTLs) or genes regulating early maturity have been reported in cotton, and the underlying regulatory mechanisms are largely unknown. In this study, we characterized 152, 68, and 101 loci that were significantly associated with the three key early maturity traits-FTi, flower and boll period (FBP) and whole growth period (WGP), respectively, via four genome-wide association study methods in upland cotton (Gossypium hirsutum). We focused on one major early maturity-related genomic region containing three single nucleotide polymorphisms on chromosome D03, and determined that GhAP1-D3, a gene homologous to Arabidopsis thaliana APETALA1 (AP1), is the causal locus in this region. Transgenic plants overexpressing GhAP1-D3 showed significantly early flowering and early maturity without penalties for yield and fiber quality compared to wild-type (WT) plants. By contrast, the mutant lines of GhAP1-D3 generated by genome editing displayed markedly later flowering than the WT. GhAP1-D3 interacted with GhSOC1 (SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1), a pivotal regulator of FTi, both in vitro and in vivo. Changes in GhAP1-D3 transcript levels clearly affected the expression of multiple key flowering regulatory genes. Additionally, DNA hypomethylation and high levels of H3K9ac affected strong expression of GhAP1-D3 in early-maturing cotton cultivars. We propose that epigenetic modifications modulate GhAP1-D3 expression to positively regulate FTi in cotton through interaction of the encoded GhAP1 with GhSOC1 and affecting the transcription of multiple flowering-related genes. These findings may also lay a foundation for breeding early-maturing cotton varieties in the future.

Wheat TaTIP4;1 Confers Enhanced Tolerance to Drought, Salt and Osmotic Stress in Arabidopsis and Rice.

Tonoplast aquaporins (intrinsic proteins, TIPs) have been indicated to play important roles in plant tolerance to water deficit and salinity. However, the functions of wheat TIPs in response to the stresses are largely unknown. In this study, we observed that transgenic plants overexpressing wheat TaTIP4;1 in Arabidopsis and rice displayed clearly enhanced seed germination and seedling growth under drought, salt and osmotic stress. Compared with wild type plants, Arabidopsis and rice overexpression lines had heightened water contents, reduced leaf water loss, lowered levels of Na+, Na+/K+, H2O2 and malondialdehyde, and improved activities of catalase and/or superoxide dismutase, and increased accumulation of proline under drought, salinity and/or osmotic stresses. Moreover, the expression levels of multiple drought responsive genes clearly elevated upon water dehydration, and the transcription of some salt responsive genes was markedly induced by NaCl treatment in the overexpression lines. Also, the yeast cells containing TaTIP4;1 showed increased tolerance to NaCl and mannitol, and mutation in one of three serines of TaTIP4;1 caused decreased tolerance to the two stresses. These results suggest that TaTIP4;1 serves as an essential positive regulator of seed germination and seedling growth under drought, salt and/or osmotic stress through impacting water relations, ROS balance, the accumulation of Na+ and proline, and stimulating the expression of dozens of stress responsive genes in Arabidopsis and rice. Phosphorylation may modulate the activity of TaTIP4;1.

Knockdown of Gh_A05G1554 (GhDHN_03) and Gh_D05G1729 (GhDHN_04) Dehydrin genes, Reveals their potential role in enhancing osmotic and salt tolerance in cotton.

In this investigation, whole-genome identification and functional characterization of the cotton dehydrin genes was carried out. A total of 16, 7, and 7 dehydrin proteins were identified in G. hirsutum, G. arboreum and G. raimondii, respectively. Through RNA sequence data and RT-qPCR validation, Gh_A05G1554 (GhDHN_03) and Gh_D05G1729 (GhDHN_04) were highly upregulated, and knockdown of the two genes, significantly reduced the ability of the plants to tolerate the effects of osmotic and salt stress. The VIGS-plants recorded significantly higher concentration levels of oxidants, hydrogen peroxide (H2O2) and malondialdehyde (MDA), furthermore, the four stress responsive genes GhLEA2, Gh_D12G2017 (CDKF4), Gh_A07G0747 (GPCR) and a transcription factor, trihelix, Gh_A05G2067, were significantly downregulated in VIGS-plants, but upregulated in wild types under osmotic and salt stress condition. The result indicated that dehydrin proteins are vital for plants and can be exploited in developing a more osmotic and salt stress-resilient germplasm to boost and improve cotton production.

Versatile Roles of Aquaporins in Plant Growth and Development.

Aquaporins (AQPs) are universal membrane integrated water channel proteins that selectively and reversibly facilitate the movement of water, gases, metalloids, and other small neutral solutes across cellular membranes in living organisms. Compared with other organisms, plants have the largest number of AQP members with diverse characteristics, subcellular localizations and substrate permeabilities. AQPs play important roles in plant water relations, cell turgor pressure maintenance, the hydraulic regulation of roots and leaves, and in leaf transpiration, root water uptake, and plant responses to multiple biotic and abiotic stresses. They are also required for plant growth and development. In this review, we comprehensively summarize the expression and roles of diverse AQPs in the growth and development of various vegetative and reproductive organs in plants. The functions of AQPs in the intracellular translocation of hydrogen peroxide are also discussed.

Redox Components: Key Regulators of Epigenetic Modifications in Plants.

Epigenetic modifications including DNA methylation, histone modifications, and chromatin remodeling are crucial regulators of chromatin architecture and gene expression in plants. Their dynamics are significantly influenced by oxidants, such as reactive oxygen species (ROS) and nitric oxide (NO), and antioxidants, like pyridine nucleotides and glutathione in plants. These redox intermediates regulate the activities and expression of many enzymes involved in DNA methylation, histone methylation and acetylation, and chromatin remodeling, consequently controlling plant growth and development, and responses to diverse environmental stresses. In recent years, much progress has been made in understanding the functional mechanisms of epigenetic modifications and the roles of redox mediators in controlling gene expression in plants. However, the integrated view of the mechanisms for redox regulation of the epigenetic marks is limited. In this review, we summarize recent advances on the roles and mechanisms of redox components in regulating multiple epigenetic modifications, with a focus of the functions of ROS, NO, and multiple antioxidants in plants.

Histone Deacetylase Inhibitor SAHA Improves High Salinity Tolerance Associated with Hyperacetylation-Enhancing Expression of Ion Homeostasis-Related Genes in Cotton.

Histone acetylation plays an important role in regulation of chromatin structure and gene expression in terms of responding to abiotic stresses. Histone acetylation is modulated by histone deacetylases (HDACs) and histone acetyltransferases. Recently, the effectiveness of HDAC inhibitors (HDACis) for conferring plant salt tolerance has been reported. However, the role of HDACis in cotton has not been elucidated. In the present study, we assessed the effects of the HDACi suberoylanilide hydroxamic acid (SAHA) during high salinity stress in cotton. We demonstrated that 10 µM SAHA pretreatment could rescue of cotton from 250 mM NaCl stress, accompanied with reduced Na+ accumulation and a strong expression of the ion homeostasis-related genes. Western blotting and immunostaining results revealed that SAHA pretreatment could induce global hyperacetylation of histone H3 at lysine 9 (H3K9) and histone H4 at lysine 5 (H4K5) under 250 mM NaCl stress, indicating that SAHA could act as the HDACi in cotton. Chromatin immunoprecipitation and chromatin accessibility coupled with real time quantitative PCR analyses showed that the upregulation of the ion homeostasis-related genes was associated with the elevated acetylation levels of H3K9 and H4K5 and increased chromatin accessibility on the promoter regions of these genes. Our results could provide a theoretical basis for analyzing the mechanism of HDACi application on salt tolerance in plants.

Transcriptome-wide analysis of pseudouridylation of mRNA and non-coding RNAs in Arabidopsis.

Pseudouridine (Ψ) is widely distributed in mRNA and various non-coding RNAs in yeast and mammals, and the specificity of its distribution has been determined. However, knowledge about Ψs in the RNAs of plants, particularly in mRNA, is lacking. In this study, we performed genome-wide pseudouridine-sequencing in Arabidopsis and for the first time identified hundreds of Ψ sites in mRNA and multiple Ψ sites in non-coding RNAs. Many predicted and novel Ψ sites in rRNA and tRNA were detected. mRNA was extensively pseudouridylated, but with Ψs being under-represented in 3'-untranslated regions and enriched at position 1 of triple codons. The phenylalanine codon UUC was the most frequently pseudouridylated site. Some Ψs present in chloroplast 23S, 16S, and 4.5S rRNAs in wild-type Col-0 were absent in plants with a mutation of SVR1 (Suppressor of variegation 1), a chloroplast pseudouridine synthase gene. Many plastid ribosomal proteins and photosynthesis-related proteins were significantly reduced in svr1 relative to the wild-type, indicating the roles of SVR1 in chloroplast protein biosynthesis in Arabidopsis. Our results provide new insights into the occurrence of pseudouridine in Arabidopsis RNAs and the biological functions of SVR1, and will pave the way for further exploiting the mechanisms underlying Ψ modifications in controlling gene expression and protein biosynthesis in plants.

A novel VIGS method by agroinoculation of cotton seeds and application for elucidating functions of GhBI-1 in salt-stress response.

KEY MESSAGE: A VIGS method by agroinoculation of cotton seeds was developed for gene silencing in young seedlings and roots, and applied in functional analysis of GhBI-1 in response to salt stress. Virus-induced gene silencing (VIGS) has been widely used to investigate the functions of genes expressed in mature leaves, but not yet in young seedlings or roots of cotton (Gossypium hirsutum L.). Here, we developed a simple and effective VIGS method for silencing genes in young cotton seedlings and roots by soaking naked seeds in Agrobacterium cultures carrying tobacco rattle virus (TRV)-VIGS vectors. When the naked seeds were soaked in Agrobacterium cultures with an OD600 of 1.5 for 90 min, it was optimal for silencing genes effectively in young seedlings as clear photo-bleaching phenotype in the newly emerging leaves of pTRV:GhCLA1 seedlings were observed at 12-14 days post inoculation. Silencing of GhPGF (cotton pigment gland formation) by this method resulted in a 90% decrease in transcript abundances of the gene in roots at the early development stage. We further used the tool to investigate function of GhBI-1 (cotton Bax inhibitor-1) gene in response to salt stress and demonstrated that GhBI-1 might play a protective role under salt stress by suppressing stress-induced cell death in cotton. Our results showed that the newly established VIGS method is a powerful tool for elucidating functions of genes in cotton, especially the genes expressed in young seedlings and roots.

Both AtrbohD and AtrbohF are essential for mediating responses to oxygen deficiency in Arabidopsis.

KEY MESSAGE: Both AtrbohD and AtrbohF promote the increases in activities of ADH, PDC, LDH, and Ca2+ levels, and induce the expression of multiple hypoxia response genes, thus improving Arabidopsis adaptation to oxygen deficiency. NADPH oxidase AtrbohD and AtrbohF cooperatively play key roles in regulation of growth and stress signaling in Arabidopsis. However, reports on AtrbohD and AtrbohF functioning together in hypoxia signaling are scarce, and the underlying mechanisms remain elusive. Here, we show that the double null mutant atrbohD/F is more sensitive to oxygen deprivation compared with wild type (WT) and the single mutant atrbohD and atrbohF. Under oxygen deficiency, enhancements of the transcripts of alcohol dehydrogenase 1 (ADH1) and pyruvate decarboxylase 1 (PDC1) and the activities of ADH, PDC and lactate dehydrogenase in WT are clearly reduced in the single mutants, and more strongly reduced in the double mutant. Moreover, increases in the production of ATP, H2O2 and Ca2+ in WT are significantly arrested in atrbohD, atrbohF, and especially in atrbohD/F. Hypoxia-promoted rise in the expression of some hypoxic responsive genes is also inhibited in atrbohD/F relative to WT, atrbohD and atrbohF. These genes include ethylene response factor 73, lactate dehydrogenase, MYB transcription factor 2, sucrose synthase 1 (SUS1), SUS4, heat stress transcription factor A2 and heat-shock protein 18.2. These results suggest that both AtrbohD and AtrbohF are essential for mediating hypoxia signaling. H2O2 derived from AtrbohD and AtrbohF triggers the Ca2+ increase and induces the expression of multiple hypoxia response genes, thus improving Arabidopsis tolerance to low-oxygen stress. These findings provide new insights into the mechanisms of AtrbohF in regulating the responses to oxygen deprivation in Arabidopsis.

AtrbohD and AtrbohF positively regulate abscisic acid-inhibited primary root growth by affecting Ca2+ signalling and auxin response of roots in Arabidopsis.

Reactive oxygen species (ROS) originating from the NADPH oxidases AtrbohD and AtrbohF play an important role in abscisic acid (ABA)-inhibited primary root growth in Arabidopsis. However, the mechanisms underlying this process remain elusive. In this study, the double mutant atrbohD1/F1 and atrbohD2/F2, in which both AtrbohD and AtrbohF were disrupted, were less sensitive to ABA suppression of root cell elongation than wild-type (WT) plants. Furthermore, the double mutants showed impaired ABA responses in roots, including ROS generation, cytosolic Ca(2+) increases, and activation of plasma membrane Ca(2+)-permeable channels compared with WT. Exogenous H2O2 can activate the Ca(2+) currents in roots of atrbohD1/F1. In addition, exogenous application of the auxin transport inhibitor naphthylphthalamic acid effectively promoted ABA inhibition of root growth of the mutants relative to that of WT. The ABA-induced decreases in auxin sensitivity of the root tips were more pronounced in WT than in atrbohD1/F1. These findings suggest that both AtrbohD and AtrbohF are essential for ABA-promoted ROS production in roots. ROS activate Ca(2+) signalling and reduce auxin sensitivity of roots, thus positively regulating ABA-inhibited primary root growth in Arabidopsis.

NADPH oxidase AtrbohD and AtrbohF function in ROS-dependent regulation of Na⁺/K⁺homeostasis in Arabidopsis under salt stress.

Maintaining cellular Na(+)/K(+) homeostasis is pivotal for plant survival in saline environments. However, knowledge about the molecular regulatory mechanisms of Na(+)/K(+) homeostasis in plants under salt stress is largely lacking. In this report, the Arabidopsis double mutants atrbohD1/F1 and atrbohD2/F2, in which the AtrbohD and AtrbohF genes are disrupted and generation of reactive oxygen species (ROS) is pronouncedly inhibited, were found to be much more sensitive to NaCl treatments than wild-type (WT) and the single null mutant atrbohD1 and atrbohF1 plants. Furthermore, the two double mutant seedlings had significantly higher Na(+) contents, lower K(+) contents, and resultant greater Na(+)/K(+) ratios than the WT, atrbohD1, and atrbohF1 under salt stress. Exogenous H(2)O(2) can partially reverse the increased effects of NaCl on Na(+)/K(+) ratios in the double mutant plants. Pre-treatments with diphenylene iodonium chloride, a widely used inhibitor of NADPH oxidase, clearly enhanced the Na(+)/K(+) ratios in WT seedlings under salt stress. Moreover, NaCl-inhibited inward K(+) currents were arrested, and NaCl-promoted increases in cytosolic Ca(2+) and plasma membrane Ca(2+) influx currents were markedly attenuated in atrbohD1/F1 plants. No significant differences in the sensitivity to osmotic or oxidative stress among the WT, atrbohD1, atrbohF1, atrbohD1/F1, and atrbohD2/F2 were observed. Taken together, these results strongly suggest that ROS produced by both AtrbohD and AtrbohF function as signal molecules to regulate Na(+)/K(+) homeostasis, thus improving the salt tolerance of Arabidopsis.

Nia1 and Nia2 are involved in exogenous salicylic acid-induced nitric oxide generation and stomatal closure in Arabidopsis.

Phytohormone salicylic acid (SA) plays important roles in plant responses to environmental stress. However, knowledge about the molecular mechanisms for SA affecting the stomatal movements is limited. In this paper, we demonstrated that exogenous SA significantly induced stomatal closure and nitric oxide (NO) generation in Arabidopsis guard cells based on genetic and physiological data. These effects were significantly inhibited by the NO scavenger c-PTIO, NO synthase (NOS) inhibitor L-NAME or nitrate reductase suppressor tungstate respectively, implying that NOS and nitrate reductase (NR) participate in SA-evoked stomatal closing. Furthermore, the effects of SA promotion of stomatal closure and NO synthesis are significantly suppressed in NR single mutants of nia1, nia2 or double mutant nia1/nia2, compared with the wild type plants. This suggests that both Nia1 and Nia2 are involved in SA-stimulated stomatal closure. In addition, pharmacological experiments showed that protein kinases, cGMP and cADPR are involved in SA-mediated NO accumulation and stomatal closure induced by SA in Arabidopsis.

Genome-wide identification and expression analysis of NADPH oxidase genes in response to ABA and abiotic stresses, and in fibre formation in Gossypium.

Plasma membrane NADPH oxidases, also named respiratory burst oxidase homologues (Rbohs), play pivotal roles in many aspects of growth and development, as well as in responses to hormone signalings and various biotic and abiotic stresses. Although Rbohs family members have been identified in several plants, little is known about Rbohs in Gossypium. In this report, we characterized 13, 13, 26 and 19 Rbohs in G. arboretum, G. raimondii, G. hirsutum and G. barbadense, respectively. These Rbohs were conservative in physical properties, structures of genes and motifs. The expansion and evolution of the Rbohs dominantly depended on segmental duplication, and were under the purifying selection. Transcription analyses showed that GhRbohs were expressed in various tissues, and most GhRbohs were highly expressed in flowers. Moreover, different GhRbohs had very diverse expression patterns in response to ABA, high salinity, osmotic stress and heat stress. Some GhRbohs were preferentially and specifically expressed during ovule growth and fiber formation. These results suggest that GhRbohs may serve highly differential roles in mediating ABA signaling, in acclimation to environmental stimuli, and in fiber growth and development. Our findings are valuable for further elucidating the functions and regulation mechanisms of the Rbohs in adaptation to diverse stresses, and in growth and development in Gossypium.

Genome-wide characterization and expression analysis of PP2CA family members in response to ABA and osmotic stress in Gossypium.

Clade A type 2C protein phosphatases (PP2CAs), as central regulators of abscisic acid (ABA) signaling, negative control growth, development and responses to multiple stresses in plants. PP2CA gene families have been characterized at genome-wide levels in several diploid plants like Arabidopsis and rice. However, the information about genome organization, phylogenesis and putative functions of PP2CAs in Gossypium is lacking. Here, PP2CA family members were comprehensively analyzed in four Gossypium species including the diploid progenitor Gossypium arboreum, G. raimondii and the tetraploid G. hirsutum and G. barbadense, and 14, 13, 27, and 23 PP2CA genes were identified in the genomic sequences of these plants, respectively. Analysis results showed that most Gossypium PP2CAs were highly conserved in chromosomal locations, structures, and phylogeny among the four cotton species. Segmental duplication might play important roles in the formation of the PP2CAs, and most PP2CAs may be under purifying selection in Gossypium during evolution. The majority of the PP2CAs were expressed specifically in diverse tissues, and highly expressed in flowers in G. hirsutum. The GhPP2CAs displayed diverse expression patterns in responding to ABA and osmotic stress. Yeast-two hybrid assays revealed that many GhPP2CAs were capable of interaction with the cotton ABA receptors pyrabactin resistance1/PYR1-like/regulatory components of ABA receptors (PYR1/PYL/RCAR) GhPYL2-2D (Gh_D08G2587), GhPYL6-2A (Gh_A06G1418), and GhPYL9-2A (Gh_A11G0870) in the presence and/or absence of ABA. These results gave a comprehensive view of the Gossypium PP2CAs and are valuable for further studying the functions of PP2CAs in Gossypium.

Genetic Detection of Lint Percentage Applying Single-Locus and Multi-Locus Genome-Wide Association Studies in Chinese Early-Maturity Upland Cotton.

Upland cotton (Gossypium hirsutum L.) is the most important source of natural fiber in the world. Early-maturity upland cotton varieties are commonly planted in China. Nevertheless, lint yield of early-maturity upland cotton varieties is strikingly lower than that of middle- and late-maturity ones. How to effectively improve lint yield of early maturing cotton, becomes a focus of cotton research. Here, based on 72,792 high-quality single nucleotide polymorphisms of 160 early-maturing upland cotton accessions, we performed genome-wide association studies (GWASs) for lint percentage (LP), one of the most lint-yield component traits, applying one single-locus method and six multi-locus methods. A total of 4 and 45 significant quantitative trait nucleotides (QTNs) were respectively identified to be associated with LP. Interestingly, in two of four planting environments, two of these QTNs (A02_74713290 and A02_75551547) were simultaneously detected via both one single-locus and three or more multi-locus GWAS methods. Among the 42 genes within a genomic region (A02: 74.31-75.95 Mbp) containing the above two peak QTNs, Gh_A02G1269, Gh_A02G1280, and Gh_A02G1295 had the highest expression levels in ovules during seed development from 20 to 25 days post anthesis, whereas Gh_A02G1278 was preferentially expressed in the fibers rather than other organs. These results imply that the four potential candidate genes might be closely related to cotton LP by regulating the proportion of seed weight and fiber yield. The QTNs and potential candidate genes for LP, identified in this study, provide valuable resource for cultivating novel cotton varieties with earliness and high lint yield in the future.

AtrbohD functions downstream of ROP2 and positively regulates waterlogging response in Arabidopsis.

NADPH oxidase AtrbohD plays very important roles in modulating many cellular processes through production of signal molecules reactive oxygen species in Arabidopsis. However, whether it regulates the response to waterlogging stress is unclear. In this report, we showed that expression of AtrbohD was markedly induced by waterlogging stress, and mutation in AtrbohD led to clear sensitivity of Arabidopsis plants to waterlogging stress. Moreover, waterlogging-promoted increases in alcohol dehydrogenase (ADH) activity, ADH1 expression and H2O2 accumulation were significantly attenuated in two mutant lines of AtrbohD. These results indicate that AtrbohD is required for Arabidopsis tolerance to waterlogging stress. Besides, GUS staining experiments revealed that disruption of small G protein ROP2 encoding gene evidently suppressed the increase of AtrbohD expression while defect of AtrbohD did not prominently affect the abundance enhancements of ROP2 transcripts under waterlogged conditions. Together, these data suggest that AtrbohD functions downstream of ROP2 to positively regulate the response to waterlogging stress in Arabidopsis.

Multi-Locus Genome-Wide Association Studies of Fiber-Quality Related Traits in Chinese Early-Maturity Upland Cotton.

Early-maturity varieties of upland cotton are becoming increasingly important for farmers to improve their economic benefits through double cropping practices and mechanical harvesting production in China. However, fiber qualities of early-maturing varieties are relatively poor compared with those of middle- and late- maturing ones. Therefore, it is crucial for researchers to elucidate the genetic bases controlling fiber-quality related traits in early-maturity cultivars, and to improve synergistically cotton earliness and fiber quality. Here, multi-locus genome-wide association studies (ML-GWAS) were conducted in a panel consisting of 160 early-maturing cotton accessions. Each accession was genotyped by 72,792 high-quality single nucleotide polymorphisms (SNPs) using specific-locus amplified fragment sequencing (SLAF-seq) approach, and fiber quality-related traits under four environmental conditions were measured. Applying at least three ML-GWAS methods, a total of 70 significant quantitative trait nucleotides (QTNs) were identified to be associated with five objective traits, including fiber length (FL), fiber strength (FS), fiber micronaire (FM), fiber uniformity (FU) and fiber elongation (FE). Among these QTNs, D11_21619830, A05_28352019 and D03_34920546 were found to be significantly associated with FL, FS, and FM, respectively, across at least two environments. Among 96 genes located in the three target genomic regions (A05: 27.95 28.75, D03: 34.52 35.32, and D11: 21.22 22.02 Mbp), six genes (Gh_A05G2325, Gh_A05G2329, Gh_A05G2334, Gh_D11G1853, Gh_D11G1876, and Gh_D11G1879) were detected to be highly expressed in fibers relative to other eight tissues by transcriptome sequencing method in 12 cotton tissues. Together, multiple favorable QTN alleles and six candidate key genes were characterized to regulate fiber development in early-maturity cotton. This will lay a solid foundation for breeding novel cotton varieties with earliness and excellent fiber-quality in the future.