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The plant mitogen-activated protein kinase (MAPK) cascade plays an important role in mediating responses to biotic and abiotic stresses and is the main pathway through which extracellular stimuli are transduced intracellularly as signals. Our previous research showed that the GhMKK6-GhMPK4 cascade signaling pathway plays an important role in cotton immunity. To further analyze the role and regulatory mechanism of the GhMKK6-GhMPK4 cascade signaling pathway in cotton resistance to Fusarium wilt, we functionally analyzed GhMPK4. Our results show that silencing GhMPK4 reduces cotton tolerance to Fusarium wilt and reduces the expression of several resistance genes. Further experiments revealed that GhMPK4 is similar to GhMKK6, both of whose overexpression cause unfavorable cotton immune response characteristics. By using a yeast two-hybrid screening library and performing a bioinformatics analysis, we screened and identified a negative regulator of the MAPK kinase-protein phosphatase AP2C1. Through the functional analysis of AP2C1, it was found that, after being silenced, GhAP2C1 increased resistance to Fusarium wilt, but GhAP2C1 overexpression caused sensitivity to Fusarium wilt. These findings show that GhAP2C1 interacts together with GhMPK4 to regulate the immune response of cotton to Fusarium oxysporum , which provides important data for functionally analyzing and studying the feedback regulatory mechanism of the MAPK cascade and helps to clarify the regulatory mechanism through which the MAPK cascade acts in response to pathogens.

Key Message: We report that GhWRKY21, a WRKY transcription factor, plays essential roles in regulating the intensity of the drought-induced ABA signalling pathway by facilitating the expression of GhHAB in cotton (Gossypium hirsutum). Abscisic acid (ABA) is one of the most important plant hormones in response to abiotic stress. However, activation of the ABA signalling pathway often leads to growth inhibition. The mechanisms that regulate the intensity of ABA signals are poorly understood. Here, we isolated and analysed the cotton group IId WRKY transcription factor (TF) gene GhWRKY21. Functional analysis indicated that GhWRKY21 plays a negative role in the drought response of cotton. Silencing of GhWRKY21 in cotton dramatically increased drought tolerance, whereas ectopic GhWRKY21 overexpression in Nicotiana benthamiana decreased drought tolerance. Furthermore, the GhWRKY21-mediated drought tolerance was ABA dependent. To clarify the mechanism underlying the GhWRKY21-mediated regulation of drought tolerance, 17 clade-A-type type 2C protein phosphatase (PP2C) genes, which are negative regulators of ABA signalling, were identified in cotton. Notably, GhWRKY21 interacted specifically with the W-box element within the promoter of GhHAB and regulated its expression. Silencing of GhHAB in cotton yielded a phenotype similar to that of GhWRKY21-silenced cotton. These results suggest that GhWRKY21 regulates the intensity of ABA signals by facilitating the expression of GhHAB. In summary, these findings dramatically improve our understanding of the function of WRKY TFs and provide insights into the mechanism of ABA-mediated drought tolerance., (© 2020. Springer-Verlag GmbH Germany, part of Springer Nature.)

The protein kinase (PK, kinome) family is one of the largest families in plants and regulates almost all aspects of plant processes, including plant development and stress responses. Despite their important functions, comprehensive functional classification, evolutionary analysis and expression patterns of the cotton PK gene family has yet to be performed on PK genes. In this study, we identified the cotton kinomes in the Gossypium raimondii, Gossypium arboretum, Gossypium hirsutum and Gossypium barbadense genomes and classified them into 7 groups and 122-24 subfamilies using software HMMER v3.0 scanning and neighbor-joining (NJ) phylogenetic analysis. Some conserved exon-intron structures were identified not only in cotton species but also in primitive plants, ferns and moss, suggesting the significant function and ancient origination of these PK genes. Collinearity analysis revealed that 16.6 million years ago (Mya) cotton-specific whole genome duplication (WGD) events may have played a partial role in the expansion of the cotton kinomes, whereas tandem duplication (TD) events mainly contributed to the expansion of the cotton RLK group. Synteny analysis revealed that tetraploidization of G. hirsutum and G. barbadense contributed to the expansion of G. hirsutum and G. barbadense PKs. Global expression analysis of cotton PKs revealed stress-specific and fiber development-related expression patterns, suggesting that many cotton PKs might be involved in the regulation of the stress response and fiber development processes. This study provides foundational information for further studies on the evolution and molecular function of cotton PKs.

Fusarium wilt is a major biotic stress affecting the productivity of cotton (Gossypium hirsutum). Although mitogen-activated protein kinase (MAPK) cascades play critical roles in plant disease resistance, their intricate regulation under fungal stress remains unclear, especially with regards to microRNA-mediated regulation of MAPK gene expression. In this study, we report that the MAPK kinase gene GhMKK6 and ghr-miR5272a work together in cotton resistance to Fusarium wilt. Silencing GhMKK6 in cotton decreased resistance to F. oxysporum by repressing the expression of known disease-resistance genes. Furthermore, although GhMKK6 played a positive role in disease resistance, excessive GhMKK6 activation caused an excessive hypersensitive response. ghr-miR5272a, a major regulator, prevents this excessive response by regulating GhMKK6 expression. ghr-miR5272a targets the GhMKK6 3'-untranslated region in cotton. Overexpressing miR5272a decreased the expression of GhMKK6 and disease-resistance genes, and increased sensitivity to F. oxysporum, yielding a similar phenotype to GhMKK6-silenced cotton. Overall, these results demonstrate that the ghr-miR5272a-mediated regulation of GhMKK6 expression contributes to the immune response in cotton, and reveal a new feedback loop mechanism in plant disease response., (© The Author 2017. Published by Oxford University Press on behalf of the Society for Experimental Biology.)

Mitogen-activated protein kinase kinase kinases (MAPKKKs) function at the top level of MAPK cascades and play important roles in plant development and stress responses. Although MAPKKKs comprise the largest family in the MAPK cascades, very few Raf-like MAPKKKs have been functionally identified, especially in the economically important crop cotton. In this study, a Raf-like MAPKKK gene, GhRaf19, was characterized for the first time in cotton. Our data show that the expression of GhRaf19 was inhibited by PEG and NaCl and induced by cold (4°C) and H 2 O 2 . Furthermore, when GhRaf19 was silenced in cotton using virus-induced gene silencing (VIGS), tolerance to drought and salt stress were enhanced, the accumulation of reactive oxygen species (ROS) was reduced, and ROS-related gene expression was increased. Consistent with these results, in N. benthamiana, overexpressing-GhRaf19 reduced tolerance to drought and salt. However, GhRaf19-silenced plants showed lowered resistance to cold in cotton, and this effect was correlated with the accumulation of ROS. In contrast, overexpressing GhRaf19 in N. benthamiana increased resistance to cold by inducing higher levels of expression and activity of ROS-related antioxidant genes/enzymes. These results indicate that GhRaf19 negatively regulates tolerance to drought and salt and positively regulates resistance to cold stress by modulating cellular ROS in cotton., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

Mitogen-activated protein kinase (MAPK) cascades play critical roles in signal transduction processes in eukaryotes. The MAPK kinases (MAPKKs) that link MAPKK kinases (MAPKKKs) and MAPKs are key components of MAPK cascades. However, the intricate regulatory mechanisms that control MAPKKs under drought stress conditions are not fully understood, especially in cotton (Gossypium hirsutum) Here, we isolated and characterized the cotton group B MAPKK gene GhMKK3 Overexpressing GhMKK3 in Nicotiana benthamiana enhanced tolerance to drought, and the results of RNA sequencing (RNA-seq) and quantitative real-time PCR (qRT-PCR) assays suggest that GhMKK3 plays an important role in responses to abiotic stresses by regulating stomatal responses and root hair growth. Further evidence demonstrated that overexpressing GhMKK3 promoted root growth and ABA-induced stomatal closure. In contrast, silencing GhMKK3 in cotton using virus-induced gene silencing (VIGS) resulted in the opposite phenotypes. More importantly, we identified an ABA- and drought-induced MAPK cascade that is composed of GhMKK3, GhMPK7 and GhPIP1 that compensates for deficiency in the MAPK cascade pathway in cotton under drought stress conditions. Together, these findings significantly improve our understanding of the mechanism by which GhMKK3 positively regulates drought stress responses., (© The Author 2016. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

WRKY transcription factors constitute a very large family of proteins in plants and participate in modulating plant biological processes, such as growth, development and stress responses. However, the exact roles of WRKY proteins are unclear, particularly in non-model plants. In this study, Gossypium hirsutum WRKY41 (GhWRKY41) was isolated and transformed into Nicotiana benthamiana. Our results showed that overexpression of GhWRKY41 enhanced the drought and salt stress tolerance of transgenic Nicotiana benthamiana. The transgenic plants exhibited lower malondialdehyde content and higher antioxidant enzyme activity, and the expression of antioxidant genes was upregulated in transgenic plants exposed to osmotic stress. A β-glucuronidase (GUS) staining assay showed that GhWRKY41 was highly expressed in the stomata when plants were exposed to osmotic stress, and plants overexpressing GhWRKY41 exhibited enhanced stomatal closure when they were exposed to osmotic stress. Taken together, our findings demonstrate that GhWRKY41 may enhance plant tolerance to stress by functioning as a positive regulator of stoma closure and by regulating reactive oxygen species (ROS) scavenging and the expression of antioxidant genes.

Mitogen-activated protein kinase (MAPK) cascades mediate various responses in plants. As the top component, MAP3Ks deserve more attention; however, little is known about the role of MAP3Ks, especially in cotton, a worldwide economic crop. In this study, a gene encoding a putative Raf-like MAP3K, GhMAP3K40, was isolated. GhMAP3K40 expression was induced by stress and multiple signal molecules. The plants overexpressing GhMAP3K40 had an enhanced tolerance to drought and salt stress at the germination stage. However, at the seedling stage, the transgenic plants suffered more severe damage after drought, exposure to pathogens and oxidative stress. The defence-related genes and the antioxidant system were activated in transgenic palnts, suggesting that GhMAP3K40 positively regulate the defence response. The transgenic plants were less able to prevent pathogenic invasion, which was due to defects in the cell structure of the leaves. The root system of the control plants were stronger compared with the transgenic plants. These results indicated a negative role of GhMAP3K40 in growth and development and GhMAP3K40 possibly caused the defects by down-regulating the lignin biosynthesis. Overall, these results suggest that GhMAP3K40 may positively regulate defence response but cause reduced tolerance to biotic and abiotic stress by negatively regulating growth and development., (Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.)

The WRKY transcription factors modulate numerous physiological processes, including plant growth, development and responses to various environmental stresses. Currently, our understanding of the functions of the majority of the WRKY family members and their possible roles in signalling crosstalk is limited. In particular, very few WRKYs have been identified and characterised from an economically important crop, cotton. In this study, we characterised a novel group IIc WRKY gene, GhWRKY68, which is induced by different abiotic stresses and multiple defence-related signalling molecules. The β-glucuronidase activity driven by the GhWRKY68 promoter was enhanced after exposure to drought, salt, abscisic acid (ABA) and H2O2. The overexpression of GhWRKY68 in Nicotiana benthamiana reduced resistance to drought and salt and affected several physiological indices. GhWRKY68 may mediate salt and drought responses by modulating ABA content and enhancing the transcript levels of ABA-responsive genes. GhWRKY68-overexpressing plants exhibited reduced tolerance to oxidative stress after drought and salt stress treatments, which correlated with the accumulation of reactive oxygen species (ROS), reduced enzyme activities, elevated malondialdehyde (MDA) content and altered ROS-related gene expression. These results indicate that GhWRKY68 is a transcription factor that responds to drought and salt stresses by regulating ABA signalling and modulating cellular ROS.

Drought and high salinity are two major environmental factors that significantly limit the productivity of agricultural crops worldwide. WRKY transcription factors play essential roles in the adaptation of plants to abiotic stresses. However, WRKY genes involved in drought and salt tolerance in cotton (Gossypium hirsutum) are largely unknown. Here, a group IId WRKY gene, GhWRKY17, was isolated and characterized. GhWRKY17 was found to be induced after exposure to drought, salt, H2O2 and ABA. The constitutive expression of GhWRKY17 in Nicotiana benthamiana remarkably reduced plant tolerance to drought and salt stress, as determined through physiological analyses of the germination rate, root growth, survival rate, leaf water loss and Chl content. GhWRKY17 transgenic plants were observed to be more sensitive to ABA-mediated seed germination and root growth. However, overexpressing GhWRKY17 in N. benthamiana impaired ABA-induced stomatal closure. Furthermore, we found that GhWRKY17 modulated the increased sensitivity of plants to drought by reducing the level of ABA, and transcript levels of ABA-inducible genes, including AREB, DREB, NCED, ERD and LEA, were clearly repressed under drought and salt stress conditions. Consistent with the accumulation of reactive oxygen species (ROS), reduced proline contents and enzyme activities, elevated electrolyte leakage and malondialdehyde, and lower expression of ROS-scavenging genes, including APX, CAT and SOD, the GhWRKY17 transgenic plants exhibited reduced tolerance to oxidative stress compared with wild-type plants. These results therefore indicate that GhWRKY17 responds to drought and salt stress through ABA signaling and the regulation of cellular ROS production in plants., (© The Author 2014. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

WRKY transcription factors form one of the largest transcription factor families and function as important components in the complex signaling processes that occur during plant stress responses. However, relative to the research progress in model plants, far less information is available on the function of WRKY proteins in cotton. In the present study, we identified the GhWRKY40 gene in cotton (Gossypium hirsutum) and determined that the GhWRKY40 protein is targeted to the nucleus and is a stress-inducible transcription factor. The GhWRKY40 transcript level was increased upon wounding and infection with the bacterial pathogen Ralstonia solanacearum. The overexpression of GhWRKY40 down-regulated most of the defense-related genes, enhanced the wounding tolerance and increased the susceptibility to R. solanacearum. Consistent with a role in multiple stress responses, we found that the GhWRKY40 transcript level was increased by the stress hormones salicylic acid (SA), methyl jasmonate (MeJA) and ethylene (ET). Moreover, GhWRKY40 interacted with the MAPK kinase GhMPK20, as shown using yeast two-hybrid and bimolecular fluorescence complementation systems. Collectively, these results suggest that GhWRKY40 is regulated by SA, MeJA and ET signaling and coordinates responses to wounding and R. solanacearum attack. These findings highlight the importance of WRKYs in regulating wounding- and pathogen-induced responses.

Key Message: Our results indicate that overexpression of the GhWRKY39 - 1 gene enhances resistance to pathogen infection and tolerance to high salt and oxidative stress in transgenic Nicotiana benthamiana., Abstract: WRKY transcription factor genes play significant roles in the response to biotic and abiotic stresses. Cotton (Gossypium hirsutum) is an important fiber and oil crop worldwide. We isolated and characterized GhWRKY39-1, which is a group IId WRKY gene that is present as a single copy in the cotton genome. Quantitative PCR analyses indicated that GhWRKY39-1 was induced by pathogen infection, defense-related signaling molecules, and abiotic stresses, such as NaCl and methyl viologen. An analysis of the subcellular localization of the GhWRKY39-1 protein indicated that it localized to the nucleus. Furthermore, constitutive overexpression of GhWRKY39-1 in Nicotiana benthamiana conferred a greater resistance to infection by both the bacterial pathogen Ralstonia solanacearum and the fungal pathogen Rhizoctonia solani. The transgenic plants also exhibited elevated mRNA levels of several pathogen-related (PR) genes, including PR1c, PR2 and PR4. Moreover, transgenic plants displayed an enhanced tolerance to salt and oxidative stress and elevated expression of several oxidation-related genes, including APX, CAT, GST and SOD. Overall, these results indicate that GhWRKY39-1 functions as a positive regulator of plant defense against pathogen infection and responses to salt stress and reactive oxygen species.

Mitogen-activated protein kinase (MAPK) cascades are involved in plant development, stress responses and hormonal signal transduction. MAPK kinases (MAPKKs), as the key nodes in these cascades, link MAPKs and MAPKK kinases (MAPKKKs). In this study, GhMKK4, a novel group C MAPKK gene from cotton (Gossypium hirsutum), was isolated and identified. Its expression can be induced by various stresses and signalling molecules. The overexpression of GhMKK4 in Nicotiana benthamiana enhanced its susceptibility to bacterial and fungal pathogens, but had no significant effects on salt or drought tolerance. Notably, the overexpressing plants showed increased sensitivity to abscisic acid (ABA) and gibberellin A3 (GA3), and ABA and gibberellin (GA) signalling were affected on infection with Ralstonia solanacearum bacteria. Furthermore, the overexpressing plants showed more reactive oxygen species (ROS) accumulation and stronger inhibition of catalase (CAT), a ROS-scavenging enzyme, than control plants after salicylic acid (SA) treatment. Interestingly, two genes encoding ornithine decarboxylase (ODC) and S-adenosylmethionine decarboxylase (SAMDC), the key enzymes in polyamine synthesis, exhibited reduced R. solanacearum-induced expression in overexpressing plants. These findings broaden our knowledge about the functions of MAPKKs in diverse signalling pathways and the negative regulation of disease resistance in the cotton crop., (© 2013 BSPP AND JOHN WILEY & SONS LTD.)

Mitogen-activated protein kinase (MAPK) cascades play important roles in the perception of external signals and the generation of suitable responses. Cotton (Gossypium hirsutum) is an important fibre-producing and oil-producing crop worldwide. However, few MAPKs and their interaction partners have been functionally characterized in cotton. In the present study, the group A MAPK G. hirsutum (Gh)MPK6a was identified and characterized. GhMPK6a expression can be induced through multiple defence-related signal molecules and abiotic and biotic stresses. The ectopic expression of GhMPK6a in Nicotiana benthamiana reduced drought and salt tolerance, with elevated malondialdehyde content, higher reactive oxygen species content and lower abscisic acid content than in wild-type plants. Moreover, plants overexpressing GhMPK6a were sensitive to the bacterial pathogen Ralstonia solanacearum. Histochemical analysis of β-glucuronidase activity revealed that GhMPK6a showed tissue-specific expression during postgermination development, mixed bud differentiation, and pollination. Most importantly, GhMPK6a interacts with the upstream MAPK kinase GhMKK4, as shown by the use of yeast two-hybrid and bimolecular fluorescence complementation systems, compensating for a deficiency of MAPK interaction partners in cotton crops. Taken together, these results suggest that GhMPK6a negatively regulates osmotic stress and bacterial infection, and plays an important role in developmental processes. These results provide useful information for elucidating the roles of MAPK cascades in cotton crops., Structured Digital Abstract: GhMPK6a physically interacts with GhMPK4 by two hybrid (View interaction) GhMPK6a and GhMPK4 physically interact by bimolecular fluorescence complementation (View interaction)., (© 2013 FEBS.)

Mitogen-activated protein kinase kinases (MAPKK) mediate a variety of stress responses in plants. So far little is known on the functional role of MAPKKs in cotton. In the present study, Gossypium hirsutum MKK1 (GhMKK1) function was investigated. GhMKK1 protein may activate its specific targets in both the nucleus and cytoplasm. Treatments with salt, drought, and H2O2 induced the expression of GhMKK1 and increased the activity of GhMKK1, while overexpression of GhMKK1 in Nicotiana benthamiana enhanced its tolerance to salt and drought stresses as determined by many physiological data. Additionally, GhMKK1 activity was found to up-regulate pathogen-associated biotic stress, and overexpression of GhMKK1 increased the susceptibility of the transgenic plants to the pathogen Ralstonia solanacearum by reducing the expression of PR genes. Moreover, GhMKK1-overexpressing plants also exhibited an enhanced reactive oxygen species scavenging capability and markedly elevated activities of several antioxidant enzymes. These results indicate that GhMKK1 is involved in plants defence responses and provide new data to further analyze the function of plant MAPK pathways.

Background: As a large family of regulatory proteins, WRKY transcription factors play essential roles in the processes of adaptation to diverse environmental stresses and plant growth and development. Although several studies have investigated the role of WRKY transcription factors during these processes, the mechanisms underlying the function of WRKY members need to be further explored, and research focusing on the WRKY family in cotton crops is extremely limited., Results: In the present study, a gene encoding a putative WRKY family member, GhWRKY15, was isolated from cotton. GhWRKY15 is present as a single copy gene, and a transient expression analysis indicated that GhWRKY15 was localised to the nucleus. Additionally, a group of cis-acting elements associated with the response to environmental stress and plant growth and development were detected in the promoter. Consistently, northern blot analysis showed that GhWRKY15 expression was significantly induced in cotton seedlings following fungal infection or treatment with salicylic acid, methyl jasmonate or methyl viologen. Furthermore, GhWRKY15-overexpressing tobacco exhibited more resistance to viral and fungal infections compared with wild-type tobacco. The GhWRKY15-overexpressing tobacco also exhibited increased RNA expression of several pathogen-related genes, NONEXPRESSOR OF PR1, and two genes that encode enzymes involved in ET biosynthesis. Importantly, increased activity of the antioxidant enzymes POD and APX during infection and enhanced expression of NtAPX1 and NtGPX in transgenic tobacco following methyl viologen treatment were observed. Moreover, GhWRKY15 transcription was greater in the roots and stems compared with the expression in the cotyledon of cotton, and the stems of transgenic plants displayed faster elongation at the earlier shooting stages compared with wide type tobacco. Additionally, exposure to abiotic stresses, including cold, wounding and drought, resulted in the accumulation of GhWRKY15 transcripts., Conclusion: Overall, our data suggest that overexpression of GhWRKY15 may contribute to the alteration of defence resistance to both viral and fungal infections, probably through regulating the ROS system via multiple signalling pathways in tobacco. It is intriguing that GhWRKY15 overexpression in tobacco affects plant growth and development, especially stem elongation. This finding suggests that the role of the WRKY proteins in disease resistance may be closely related to their function in regulating plant growth and development.

Mitogen-activated protein kinase (MAPK) cascades are involved in various processes from plant growth and development to biotic and abiotic stress responses. MAPK kinases (MAPKKs), which link MAPKs and MAPKK kinases (MAPKKKs), play crucial roles in MAPK cascades to mediate a variety of stress responses in plants. However, few MAPKKs have been functionally characterized in cotton (Gossypium hirsutum). In this study, a novel gene, GhMKK5, from cotton belonging to the group C MAPKKs was isolated and characterized. The expression of GhMKK5 can be induced by pathogen infection, abiotic stresses, and multiple defence-related signal molecules. The overexpression of GhMKK5 in Nicotiana benthamiana enhanced the plants' resistance to the bacterial pathogen Ralstonia solanacearum by elevating the expression of pathogen resistance (PR) genes, including PR1a, PR2, PR4, PR5, and NPR1, but increased the plants' sensitivity to the oomycete pathogen Phytophthora parasitica var. nicotianae Tucker. Importantly, GhMKK5-overexpressing plants displayed markedly elevated expression of reactive oxygen species-related and cell death marker genes, such as NtRbohA and NtCDM, and resulted in hypersensitive response (HR)-like cell death characterized by the accumulation of H(2)O(2). Furthermore, it was demonstrated that GhMKK5 overexpression in plants reduced their tolerance to salt and drought stresses, as determined by statistical analysis of seed germination, root length, leaf water loss, and survival rate. Drought obviously accelerated the cell death phenomenon in GhMKK5-overexpressing plants. These results suggest that GhMKK5 may play an important role in pathogen infection and the regulation of the salt and drought stress responses in plants.

RDR6 (RNA-dependent RNA polymerase 6) is not only involved in virus resistance but also plays an important role in natural plant development. In the present study, a novel RDR gene, named GhRDR6 (Gossypium hirsutum RDR6), was isolated from cotton (G. hirsutum L.). Alignment and evolutionary relationship analyses showed that GhRDR6 was more closely related to RDR6 than to other RDRs. Expression analysis indicated that this single-copy gene is constitutively expressed in the roots, stems and leaves. Semi-quantitative RT-PCR (reverse transcription-PCR) showed that GhRDR6 was up-regulated by the application of various phytohormones, including MeJA [methyl JA (jasmonate)], ABA (abscisic acid), JA, α-naphthylacetic acid, gibberellins and ET (ethylene). In addition, GhRDR6 expression increased in response to wounding, cold (4°C) and NaCl treatments, but not by drought. Furthermore, overexpression of GhRDR6 in transgenic Nicotiana benthamiana plants resulted in root lengths longer than the wide-type during the seeding stage. Interestingly, the GhRDR6-overexpressing plants displayed reduced tolerance to oxidative damage, resulting in reduced ABA-sensitivity, but they tolerated freezing. Moreover, resistance to potato virus Y was enhanced in transgenic N. benthamiana plants. These results suggest that GhRDR6 may play an important role in plant defence responses and a pivotal role in plant development.

Mitogen-activated protein kinase (MAPK) cascades play important roles in mediating biotic and abiotic stress responses. In plants, MAPKs are classified into four major groups (A-D) according to their sequence homology and conserved phosphorylation motifs. Compared with well-studied MAPKs in groups A and B, little is known about group C. In this study, we functionally characterised a stress-responsive group C MAPK gene (GhMPK2) from cotton (Gossypium hirsutum). Northern blot analysis indicated that GhMPK2 was induced by abscisic acid (ABA) and abiotic stresses, such as NaCl, PEG, and dehydration. Subcellular localization analysis suggested that GhMPK2 may activate its specific targets in the nucleus. Constitutive overexpression of GhMPK2 in tobacco (Nicotiana tabacum) conferred reduced sensitivity to ABA during both seed germination and vegetative growth. Interestingly, transgenic plants had a decreased rate of water loss and exhibited enhanced drought and salt tolerance. Additionally, transgenic plants showed improved osmotic adjustment capacity, elevated proline accumulation and up-regulated expression of several stress-related genes, including DIN1, Osmotin and NtLEA5. β-glucuronidase (GUS) expression driven by the GhMPK2 promoter was clearly enhanced by treatment with NaCl, PEG, and ABA. These results strongly suggest that GhMPK2 positively regulates salt and drought tolerance in transgenic plants.

Background: Mitogen-activated protein kinase (MAPK) cascades play pivotal roles in mediating biotic and abiotic stress responses. In plants, MAPKs are classified into four major groups (A-D) according to their sequence homology and conserved phosphorylation motifs. Members of group A and B have been extensively characterized, but little information on the group D MAPKs has been reported., Results: In this study, we isolated and characterised GhMPK16, the first group D MAPK gene found in cotton. Southern blot analysis suggests GhMPK16 is single copy in the cotton genome, and RNA blot analysis indicates that GhMPK16 transcripts accumulate following pathogen infection and treatment with multiple defense-related signal molecules. The analysis of the promoter region of GhMPK16 revealed a group of putative cis-acting elements related to stress responses. Subcellular localization analysis suggests that GhMPK16 acts in the nucleus. Transgenic Arabidopsis overexpressing GhMPK16 displayed significant resistance to fungi (Colletotrichum nicotianae and Alternaria alternata) and bacteria (Pseudomonas solanacearum) pathogen, and the transcripts of pathogen-related (PR) genes were more rapidly and strongly induced in the transgenic plants. Furthermore, transgenic Arabidopsis showed reduced drought tolerance and rapid H2O2 accumulation., Conclusion: These results suggest that GhMPK16 might be involved in multiple signal transduction pathways, including biotic and abiotic stress signaling pathways.

Mitogen-activated protein kinase (MAPK) cascades play important roles in mediating pathogen responses and reactive oxygen species signaling. In plants, MAPKs are classified into four major groups (A-D). Previous studies have mainly focused on groups A and B, but little is known about group C. In this study, we functionally characterized a stress-responsive group C MAPK gene (GhMPK2) from cotton. Northern blot analysis indicated that GhMPK2 was induced not only by signaling molecules, such as ethylene and methyl jasmonate, but also by methyl viologen-mediated oxidative stress. Transgenic tobacco (Nicotiana tabacum) plants that overexpress GhMPK2 displayed enhanced resistance to fungal and viral pathogens, and the expression of the pathogenesis-related (PR) genes, including PR1, PR2, PR4, and PR5, was significantly increased. Interestingly, the transcription of 1-aminocyclopropane-1-carboxylic acid synthase (ACS) and 1-aminocyclopropane-1-carboxylic acid oxidase (ACO) was significantly upregulated in transgenic plants, suggesting that GhMPK2 positively regulates ethylene synthesis. Moreover, overexpression of GhMPK2 elevated the expression of several antioxidant enzymes, conferring on transgenic plants enhanced reactive oxygen species scavenging capability and oxidative stress tolerance. These results increased our understanding of the role of the group C GhMPK2 gene in multiple defense-signaling pathways, including those that are involved in responses to pathogen infection and oxidative stress., (© 2011 The Authors Journal compilation © 2011 FEBS.)

WRKY proteins play important roles in plant defense responses. In the present study, a novel WRKY gene, nominated as GhWRKY3, was isolated from cotton (Gossypium hirsutum L.). The full-length cDNA of GhWRKY3 is 1,705 bp in length and encodes a protein with 507 amino acids containing two typical WRKY domains and two zinc finger motifs. Amino acid sequence alignment revealed that GhWRKY3 shares a high degree of identity with other higher plant WRKY proteins. The subcellular localization assay indicated that GhWRKY3 is localized to the nucleus. Analysis of 5'-flanking region of GhWRKY3 revealed a group of putative cis-acting elements. The results of expression analysis indicated that GhWRKY3 is constitutively expressed in roots, stems and leaves. Semi-quantitative RT-PCR showed that GhWRKY3 is up-regulated by application of various phytohormones including salicylic acid (SA), methyl jasmonate (MeJA), abscisic acid (ABA), gibberellins (GAs) and ethylene (ET). Furthermore, the transcripts of GhWRKY3 are enhanced after infection with Rhizoctonia solani, Colletotrichum gossypii and Fusarium oxysporum f. sp. vasinfectum, respectively. Also, GhWRKY3 can be induced by wounding treatment, but not by cytokinin (6-benzylaminopurine, 6-BA), auxin analogue, drought, NaCl, and cold (4°C). These data suggested that GhWRKY3 might play an important role in plant defense responses and fulfill a pivotal role in plant development.

The RNA-dependent RNA polymerase (RdRP) cDNA, designated as Gossypium hirsutum RdRP (GhRdRP) was cloned from cotton by rapid amplification of cDNA ends-polymerase chain reaction (RACE-PCR). The full-length cDNA was 3,672 bp in size and encoded an open reading frame (ORF) of 1,110 amino acids which contained the RdRP conserved functional domain and the signature motif DbDGD. Amino acid sequence alignment indicated that GhRdRP shared the highest identity (66.37%) with AtRdRP1 and had homology with other plant, fungal, yeast and nematode RdRPs. The corresponding genomic DNA containing five exons and four introns, was isolated and analyzed. Also a 5'-flanking region was cloned, and a group of putative cis-acting elements were identified. Southern blot analysis revealed a single copy of the GhRdRP gene in cotton genome. The expression analysis by semi-quantitative RT-PCR showed that GhRdRP was induced by salicylic acid (SA), 5-chloroSA (5-CSA) and fungal infection of Rhizoctonia solani Kuhn. The cloning and characterization of the GhRdRP gene will be useful for further studies of biological roles of GhRdRP in plants.

A novel alternative oxidase (AOX) gene derived from cotton (Gossypium hirsutum), designated as GhAOX1, was cloned with RACE-PCR. The full-length cDNA of GhAOX1was 1,298 bp in size, containing a 996 bp open reading frame (ORF) which corresponds to a precursor protein of 332 amino acid residues with a calculated molecular mass of 37.5 kDa. The predicted amino acid sequence exhibited 68.4%, 68.1%, 59.4%, and 69.8% homology to the alternative oxidases of Arabidopsis thaliana, Nicotiana tabacum, Solanum tuberosum and Glycine max, respectively. Interestingly, striking similarity in several coding regions, such as metal binding and hydrophobic alpha-helix regions was seen between GhAOX1 and other AOX1 proteins. Analysis of the exon/intron structure of the GhAOX1 gene showed that GhAOX1 consisted of four exons and three introns. Southern analysis indicated that the GhAOX1 was a single copy gene belonging to a multi-gene family. Expression analysis by Northern blot revealed that the GhAOX1 exhibited preferential expression in tissues, with the higher expression being found in cotyledons and petals. GhAOX1 was also found to be induced by a variety of stresses stimulation including cold, citrate, SA, KCN and antimycin A in cotton.

A novel gene, designated as GhNPR1 (Gossypium hirsutum non-expressor of pathogenesis-related genes 1), was isolated from G. hirsutum (cotton) by RT-PCR (reverse transcription-PCR) and RACE (rapid amplification of cDNA ends). The full-length cDNA was 2108 bp long and had an ORF (open reading frame) that putatively encoded a polypeptide of 592 amino acids, with a predicted molecular mass of 66 kDa. Comparison of this protein sequence with that of Arabidopsis thaliana, Brassica juncea and Nicotiana tabacum showed that the amino-acid homology was 52.98, 52.32 and 54.98% respectively. Analysis of the exon-intron structure of the GhNPR1 gene showed that GhNPR1 consisted of four exons and three introns. Southern-blot analysis revealed that the GhNPR1 was a single-copy gene in cotton. Northern-blot analysis indicated that GhNPR1 was constitutively expressed in all tested tissues, including roots, stems and leaves, with the high expression in stems and leaves. In addition, GhNPR1 was also found to be induced by signalling molecules for plant defence responses, such as methyl jasmonate, salicylic acid and ethylene, as well as attack by pathogens, such as Fusarium oxysporum and Xanthomonas campestris. These results suggest that GhNPR1 may play an important role in the response to pathogen infections in cotton plants.

Ethylene performs an important function in plant growth and development. 1-aminocyclopropane-1-carboxylate (ACC) synthase (ACS), the key enzyme involved in ethylene biosynthesis, has been the focus of most ethylene studies. Here, a cotton ACS gene referred to as Gossypium hirsutum ACS1 (GhACS1), was isolated. The full-length cDNA of GhACS1 encodes for a 476-amino acid protein which harbors seven conserved regions, 11 invariant amino acid residues, and the PLP binding active site, all of which characterize ACC synthases. Alignment analysis showed that GhACS1 shared a high degree of identity with other known ACC synthases from different species. Two introns were detected in the genomic DNA sequence, and the results of Southern blot analysis suggested that there might be a multi-gene family encoding for ACC synthase in cotton. From the phylogenetic tree constructed with 24 different kinds of ACC synthases, we determined that GhACS1 falls into group II, and was closely associated with the wound-inducible ACS of citrus. The analysis of the 5' flanking region of GhACS1 revealed a group of putative cis-acting elements. The results of expression analysis showed that GhACS1 displayed its transient expression nature after wounding, abscisic acid (ABA), and CuCl(2) treatments. These results indicate that GhACS1, which was transiently expressed in response to certain stimuli, may be involved in the production of ethylene for the transmission of stress signals.

The mitogen-activated protein kinase (MAPK) cascade is one of the major and evolutionally conserved signaling pathways and plays pivotal role in the regulation of stress and developmental signals in plants. Here, a novel gene, termed Gossypium hirsutum MAPK (GhMAPK), was isolated from cotton. The full-length cDNA of GhMAPK encodes for a 372 amino acid protein that contains all 11 of the MAPK conserved subdomains and the phosphorylation-activation motif, TEY. Amino acid sequence alignment revealed that GhMAPK shared high identity with group-C MAPK in plants and showed 83 approximately 89% similarities with MAPKs from Arabidopsis, apricot, pea, petunia, and tobacco. Southern blot analysis indicated that the GhMAPK belonged to a multygene family in cotton. Two introns were found within the region of genomic sequence. Northern blot analysis revealed that the transcripts of GhMAPK accumulated markedly when the cotton seedlings were subjected to various abiotic stimuli such as wounding, cold (4 degrees C), or salinity stress; Furthermore, GhMAPK was upregulated by the exogenous signaling molecules, such as salicylic acid (SA) and hydrogen peroxide (H(2)O(2)), as well as pathogen attacks. These results indicate that the GhMAPK, which has a high degree of identity with group-C plant MAPKs, may also play an important role in response to environmental stresses.