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Cytokinin is considered to be an important driver of seed yield. To increase the yield of cotton while avoiding the negative consequences caused by constitutive overproduction of cytokinin, we carpel-specifically downregulated cytokinin oxidase/dehydrogenase (CKX), a key negative regulator of cytokinin levels, in transgenic cotton. The carpel-speci...
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... plants of tobacco (Nicotiana tabacum) and cotton 'Jimian 14' were generated using the method described before (Luo et al., 2007), and grown in the green house at over 25 °C under a 16:8 h light-dark photoperiod or field condition. Gene specific primers used for plasmid construction are listed in Supplementary Table S1. ...Context 2
... were captured on a microscope (CKX41, Olympus, Japan). Gene specific primers are listed in Supplementary Table S1. ...Context 3
... High Prime DNA Labeling and Detection Starter Kit II (Roche, Germany) were employed to prepare digoxigenin-labeled probes and to detect hybridization signals. Gene specific primers are listed in Supplementary Table S1. ...Context 4
... Dual-Glo Luciferase Assay System (Promega, USA) was employed to measure the LUC activity, which was calculated based on the ratio of LUC/Renilla luciferase (REN). Gene specific primers are listed in Supplementary Table S1. ...Context 5
... a nontransgenic negative control, a transgenic pro35S::GhCKXRNAi line, 35SCR6, which showed a moderate increase of cytokinins with normal growth, and had the best yield performance among the pro35S::GhCKX-RNAi cottons ( Zhao et al., 2015), was used as a positive control. In carpels of ACR1 and ACR5 transgenic cotton, the total cytokinins were 29.53 ± 1.46 ng g −1 and 27.46 ± 1.63 ng g −1 , respectively, significantly higher than in the 35SCR6 line (23.46 ± 1.81 ng g −1 ) and the non-transgenic control (15.05 ± 1.73 ng g −1 ) (Table 1), indicating a carpel-specific increase of cytokinins by proAGIP::GhCKX3b-RNAi. Immunolocalization assays supported that tZ and tZR signals, which made up the majority (85.0%) of the active CKs (Table 1), mainly appeared at the position where the ovule initiated, and the signals from ACR1 and ACR5 were visibly stronger than those in 35SCR6 and the control (Fig. 4). ...Context 6
... carpels of ACR1 and ACR5 transgenic cotton, the total cytokinins were 29.53 ± 1.46 ng g −1 and 27.46 ± 1.63 ng g −1 , respectively, significantly higher than in the 35SCR6 line (23.46 ± 1.81 ng g −1 ) and the non-transgenic control (15.05 ± 1.73 ng g −1 ) (Table 1), indicating a carpel-specific increase of cytokinins by proAGIP::GhCKX3b-RNAi. Immunolocalization assays supported that tZ and tZR signals, which made up the majority (85.0%) of the active CKs (Table 1), mainly appeared at the position where the ovule initiated, and the signals from ACR1 and ACR5 were visibly stronger than those in 35SCR6 and the control (Fig. 4). ...Context 7
... thus designed a GhCKX3b-RNAi sequence, which could interfere with the expression of GhCKX3b, GhCKX3c, GhCKX5a, and GhCKX6b (Figs 3, 5A; Supplementary Fig. S3; Supplementary Table S5). The cytokinin content in the carpels of transgenic proAGIP::GhCKX3b-RNAi cotton lines was significantly higher not only over the wild-type control but also over the positive control pro35S::GhCKX3b-RNAi carpels (Table 1; Fig. 4). Compared with pro35S::GhCKX3b-RNAi cotton, of which some exhibited abnormal phenotypes, all proAGIP::GhCKX3b-RNAi cotton lines grew well without any cytokinin overproduction-related aberrations. ...Citations
... In addition, GasHSP25, GasHSP32, GhesHSP26, and GhesHSP32 from the MTΙ/CP subfamily might play important roles in ovule development. A fertilized ovule can produce a seed in cotton (Zeng et al., 2022). Thus, GasHSP25, GasHSP32, GhesHSP26, and GhesHSP32 might be closely related to seed development and yield. ...
The small heat shock proteins (sHSPs) are important components in plant growth and development, and stress response. However, a systematical understanding of the sHSP family is yet to be reported in five diploid Gossypium species. In this study, 34 GlsHSPs, 36 GrsHSPs, 37 GtsHSPs, 37 GasHSPs, and 38 GhesHSPs were identified in Gossypium longicalyx, Gossypium raimondii, Gossypium turneri, Gossypium arboreum, and Gossypium herbaceum, respectively. These sHSP members can be clustered into 10 subfamilies. Different subfamilies had different member numbers, motif distributions, gene structures, gene duplication events, gene loss numbers, and cis‐regulatory elements. Besides, the paleohexaploidization event in cotton ancestor led to expanding the sHSP members and it was also inherited by five diploid Gossypium species. After the cotton ancestor divergence, the sHSP members had the relatively conserved evolution in five diploid Gossypium species. The comprehensive evolutionary history of the sHSP family was revealed in five diploid Gossypium species. Furthermore, several GasHSPs and GhesHSPs were important candidates in plant growth and development, and stress response. These current findings can provide valuable information for the molecular evolution and further functional research of the sHSP family in cotton.
... AtCKX overexpression in Arabidopsis induces CK deficiency which enhances salt tolerance and drought tolerance [23]. Moderate increase in CK levels by down-regulating GhCKXs expression resulted in higher fiber and seed yield in cotton [24,25]. A CKX gene was isolated from Medicago sativa, MsCKX, and its expression was found to increase under salt stress and abscisic acid (ABA) treatment. ...
... The genes common to both screens were set as the final CKX members. CKXs were renamed according to their homology with Arabidopsis thaliana [25]. ...
... gov/) for Arabidopsis thaliana. The software MEGA5 was used for sequence alignment, the results were analyzed to construct intraspecific (Neighbor Joining (NJ) method) [25]. ...
Cytokinin oxidase/dehydrogenase (CKX) is a key regulatory enzyme for the irreversible degradation of the plant hormone cytokinin (CK), which is important in growth and development and response to abiotic stresses in cotton. In this study, 27, 28, 14 and 14 CKXs were screened by FAD structural domain and cytokinin binding structural domain in Gossypium hirsutum, Gossypium barbadense, Gossypium arboreum and Gossypium raimondii, respectively. Their phylogenetic relationships and expression patterns were analyzed, and most GhCKXs were found to be tissue-specific and responsive to various abiotic stresses such as cold, heat, salt and PEG. GhCKX6b-Dt was selected for gene silencing in evolutionary branch II for salt stress, because its expression increased after salt stress in cotton plants. An increase in PRO and MDA content and a decrease in SOD activity due to this gene were found after inducing salt stress, contributing to oxidative damage and decreased salt tolerance. In this study, CKXs were analyzed to reveal the possible role of GhCKXs against abiotic stresses in cotton, which provides a basis for further understanding of the biological functions of CK in plants such as growth and development and stress resistance.
... And apart from that, the high expression of GhCKX2/3/16/17 during ovule and fiber development was in agreement with previous studies in upland cotton [51]. Additionally, downregulation of GhCKX3b significantly increased cytokinin contents to enhanc seed yield and fiber yield of cotton found in a recent study [52]. ...
Background:
Cytokinin oxidase/dehydrogenase (CKX) plays a vital role in response to abiotic stress through modulating the antioxidant enzyme activities. Nevertheless, the biological function of the CKX gene family has yet to be reported in cotton.
Result:
In this study, a total of 27 GhCKXs were identified by the genome-wide investigation and distributed across 18 chromosomes. Phylogenetic tree analysis revealed that CKX genes were clustered into four clades, and most gene expansions originated from segmental duplications. The CKXs gene structure and motif analysis displayed remarkably well conserved among the four groups. Moreover, the cis-acting elements related to the abiotic stress, hormones, and light response were identified within the promoter regions of GhCKXs. Transcriptome data and RT-qPCR showed that GhCKX genes demonstrated higher expression levels in various tissues and were involved in cotton's abiotic stress and phytohormone response. The protein-protein interaction network indicates that the CKX family probably participated in redox regulation, including oxidoreduction or ATP levels, to mediate plant growth and development. Functionally identified via virus-induced gene silencing (VIGS) found that the GhCKX14 gene improved drought resistance by modulating the antioxidant-related activitie.
Conclusions:
In this study, the CKX gene family members were analyzed by bioinformatics, and validates the response of GhCKX gene to various phytohormone treatment and abiotic stresses. Our findings established the foundation of GhCKXs in responding to abiotic stress and GhCKX14 in regulating drought resistance in cotton.
Cytokinin oxidase/dehydrogenase (CKX), responsible for irreversible cytokinin degradation, also controls plant growth and development and response to abiotic stress. While the CKX gene has been studied in other plants extensively, its function in cotton is still unknown. Therefore, a genome-wide study to identify the CKX gene family in the four cotton species was conducted using transcriptomics, quantitative real-time PCR (qRT-PCR) and bioinformatics. As a result, in G. hirsutum and G. barbadense (the tetraploid cotton species), 87 and 96 CKX genes respectively and 62 genes each in G. arboreum and G. raimondii, were identified. Based on the evolutionary studies, the cotton CKX gene family has been divided into five distinct subfamilies. It was observed that CKX genes in cotton have conserved sequence logos and gene family expansion was due to segmental duplication or whole genome duplication (WGD). Collinearity and multiple synteny studies showed an expansion of gene families during evolution and purifying selection pressure has been exerted. G. hirsutum CKX genes displayed multiple exons/introns, uneven chromosomal distribution, conserved protein motifs, and cis-elements related to growth and stress in their promoter regions. Cis-elements related to resistance, physiological metabolism and hormonal regulation were identified within the promoter regions of the CKX genes. Expression analysis under different stress conditions (cold, heat, drought and salt) revealed different expression patterns in the different tissues. Through virus-induced gene silencing (VIGS), the GhCKX34A gene was found to improve cold resistance by modulating antioxidant-related activity. Since GhCKX29A is highly expressed during fibre development, we hypothesize that the increased expression of GhCKX29A in fibres has significant effects on fibre elongation. Consequently, these results contribute to our understanding of the involvement of GhCKXs in both fibre development and response to abiotic stress.
Cotton fiber is a raw material for the global textile industry and fiber quality is essential to its industrial application. Carotenoids are plant secondary metabolites that may serve as dietary components, regulate light harvesting, and scavenge reactive oxygen species. Although carotenoids accumulate predominantly in rapidly elongating cotton fibers, their roles in cotton fiber development remain poorly understood. In this study, a fiber-specific promoter proSCFP was applied to drive the expression of GhOR1Del, a positive regulator of carotenoid accumulation, to upregulate the carotenoid level in cotton fiber in planta. Fiber length, strength, and fineness were increased in proSCFP:GhOR1Del transgenic cotton and abscisic acid (ABA) and ethylene contents were increased in elongating fibers. The ABA downstream regulator GhbZIP27a stimulated the expression of the ethylene synthase gene GhACO3 by binding to its promoter, suggesting that ABA promoted fiber elongation by increasing ethylene production. These findings suggest the involvement of carotenoids and ABA signaling in promoting cotton fiber elongation and provide a strategy for improving cotton fiber quality.
Due to the growing human population, the increase in crop yield is an important challenge for modern agriculture. As abiotic and biotic stresses cause severe losses in agriculture, it is also crucial to obtain varieties that are more tolerant to these factors. In the past, traditional breeding methods were used to obtain new varieties displaying demanded traits. Nowadays, genetic engineering is another available tool. An important direction of the research on genetically modified plants concerns the modification of phytohormone metabolism. This review summarizes the state-of-the-art research concerning the modulation of phytohormone content aimed at the stimulation of plant growth and the improvement of stress tolerance. It aims to provide a useful basis for developing new strategies for crop yield improvement by genetic engineering of phytohormone metabolism.
