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Journal of Experimental Botany logoLink to Journal of Experimental Botany
. 2022 Jul 6;73(19):6758–6772. doi: 10.1093/jxb/erac303

Carpel-specific down-regulation of GhCKXs in cotton significantly enhances seed and fiber yield

Jianyan Zeng 1, Xingying Yan 2, Wenqin Bai 3, Mi Zhang 4, Yang Chen 5, Xianbi Li 6, Lei Hou 7, Juan Zhao 8, Xiaoyan Ding 9, Ruochen Liu 10, Fanlong Wang 11, Hui Ren 12, Jingyi Zhang 13, Bo Ding 14, Haoru Liu 15, Yuehua Xiao 16, Yan Pei 17,✉
Editor: Zoe Wilson18
PMCID: PMC9629787  PMID: 35792654

Carpel-specific enhancement of cytokinin significantly increases cotton ovule number and cotton yield without detrimental phenotypes.

Keywords: AG subfamily gene, carpel-specific, cotton yield, cytokinin, cytokinin oxidase, ovule initiation

Abstract

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 down-regulated specifically the carpel genes for cytokinin oxidase/dehydrogenase (CKX), a key negative regulator of cytokinin levels, in transgenic cotton. The carpel-specific down-regulation of CKXs significantly enhanced cytokinin levels in the carpels. The elevated cytokinin promoted the expression of carpel- and ovule-development-associated genes, GhSTK2, GhAG1, and GhSHP, boosting ovule formation and thus producing more seeds in the ovary. Field experiments showed that the carpel-specific increase of cytokinin significantly increased both seed yield and fiber yield of cotton, without resulting in detrimental phenotypes. Our study details the regulatory mechanism of cytokinin signaling for seed development, and provides an effective and feasible strategy for yield improvement of seed crops.

Introduction

Cotton is a major source of natural fibers for the global textile industry. Cotton seeds contain about 23% protein and 21% oil, and therefore are also an important source of foodstuff, feed, and edible oil (Zhang et al., 2002; Sunilkumar et al., 2006; Chen et al., 2007; Ma et al., 2016). A cotton boll contains approximately 30 seeds, and approximately 25% of the ovular epidermal cells of each ovule are able to differentiate into the commercially important lint fibers (Kim and Triplett, 2001; Mansoor and Paterson, 2012). Hence, the seed size and seed number of cotton determine the yield of fibers and seeds. However, seed size is negatively correlated with seed number due to the limitation of space and nutrition, and enhancing fiber abundance usually results in smaller seeds (J. Zhang et al., 2005; M. Zhang et al., 2011).

Generally, a seed is derived from a fertilized ovule, and thus, ovule development is critical to seed yield. The number of ovules per ovary depends on the ovule identity and ovule primordia initiation, which are controlled by an array of genes (Franks et al., 2002; Favaro et al., 2003; Sridhar et al., 2006; Azhakanandam et al., 2008; Jiang et al., 2020), hormone signals (Higuchi et al., 2004; Bartrina et al., 2011; Bencivenga et al., 2012; Zu et al., 2022), and environmental factors (Meyer, 1966; Reddy et al., 1991; Ritchie et al., 2007; Sita et al., 2017; Jiang et al., 2019). Ovules originally arise from carpel (ovary) tissue as new meristematic formation, and the cell fate in proliferating ovule primordia is specified by particular ovule identity factors, such as MADS box AG subfamily members Seedstick (STK), Shatterproof (SHP1/2), and Agamous (AG) (Favaro et al., 2003; Pinyopich et al., 2003; Brambilla et al., 2007; Zu et al., 2022). It was reported that ectopic expression of their genes can promote the formation of carpels and ovules (Favaro et al., 2003; Pinyopich et al., 2003; Xu et al., 2004; Guo et al., 2007; Liu et al., 2009, 2010; de Moura et al., 2017; Nardeli et al., 2018).

Cytokinins (CKs) are an important group of phytohormones that regulate the proliferation and differentiation of plant cells (Ioio et al., 2008) and control many developmental and physiological processes in plants, including leaf senescence (Gan and Amasino, 1995; Hönig et al., 2018), organ formation (Lohar et al., 2004; Zhao et al., 2009), nutrient uptake and allocation (Séguéla et al., 2008; Gu et al., 2018), as well as biotic and abiotic stress (Siemens et al., 2006; Rivero et al., 2007; Choi et al., 2010; Peleg et al., 2011; Cortleven et al., 2019). A noteworthy role of cytokinins is to regulate seed yield (Sawan et al., 2000; Ashikari et al., 2005; Bartrina et al., 2011).

The signal transduction pathway of cytokinins has been well investigated in Arabidopsis. The signaling pathway is initiated by binding of cytokinin to Arabidopsis histidine kinase receptors (AHKs), following phosphoryl group transport via Arabidopsis histidine proteins (AHPs) to Arabidopsis response regulators (ARRs). The B-type ARRs that contain conserved GARP DNA binding and activation domains can bind to a short 5ʹ-(A/G)GAT(T/C)-3ʹ core DNA sequence to activate the transcription of downstream genes (Sakai et al., 2000, 2001; Mason et al., 2004, 2005; Argyros et al., 2008; Werner and Schmülling, 2009; Argueso et al., 2010; Zubo and Schaller, 2020). Among ARR-activated genes, members of the AG subfamily, such as STK, AG1, and SHP, have been known to be involved in ovule initiation (Bartrina et al., 2011; Zu et al., 2022). However, details about cytokinin signaling pathway in carpel development need to be investigated.

Cytokinin levels are regulated through biosynthesis, activation, degradation, and conjugation of the bioactive molecules (Sakakibara, 2006; Jameson and Song, 2016). Cytokinin oxidase/dehydrogenase (CKX), which catalyses the irreversible degradation of the cytokinins by oxidative side chain cleavage, is a crucial regulator controlling endogenous cytokinin levels in the plant kingdom (Jones and Schreiber, 1997; Schmülling et al., 2003; Kowalska et al., 2010). Down-regulation of CKX genes could significantly increase cytokinin levels in transgenic plants and contribute to the enhancement of seed (grain) yield (Ashikari et al., 2005; Zalewski et al., 2012; Yeh et al., 2015; Holubová et al., 2018). However, constitutive down-regulation of CKXs in plants usually causes cytokinin overproduction phenotypes, including dwarfism, sterility, and root growth inhibition (Zalewski et al., 2012; Gao et al., 2014; Zhao et al., 2015; Gasparis et al., 2019). Thus, the key to regulating CKXs for seed yield improvement is to express the genes at the right time in the right place (Daskalova et al., 2007; Werner et al., 2010; Gao et al., 2014; Ramireddy et al., 2018).

In this study, we used a chimeric carpel- and stamen-specific promoter (Busch et al., 1999; Deyholos and Sieburth, 2000), proAGIP, to down-regulate a functional cotton CKX gene (proAGIP::GhCKX3b-RNAi) in cotton carpels. We showed that the carpel-specific down-regulation of GhCKXs could enhance CK levels at the position where the ovule initiates in the carpel, without causing abnormal growth phenotypes. Cytokinin-activated GhARR1, GhARR2a, and GhARR11 in turn promoted the expression of GhSTK2, GhAG1, and GhSHP by binding their promoter sequences, thus boosting carpel development and ovule formation. As a result, seed and fiber yield of proAGIP::GhCKX3b-RNAi cotton was significantly increased compared with that of the non-transgenic control. Our results indicate the biotechnological potential of manipulation of CKs in cotton carpels for the concurrent improvement of seed and fiber yield.

Materials and methods

Plasmid construction and plant materials

The construction of GhCKX3b-RNAi (previously named GhCKX-RNAi) and the genotype of pro35S::GhCKX-RNAi transgenic cotton have been described previously (Zeng et al., 2012; Zhao et al., 2015). To construct the vectors used for genetic transformation, the 1653 bp sequence of the 3ʹ end of the second intron of the AtAG gene was amplified from the gDNA of Arabidopsis and fused with a 46 bp minimal CaMV35S promoter to create a functional proAGIP promoter, as previously described (Fang et al., 1989; Deyholos and Sieburth, 2000). For the proAGIP::GhCKX3b-RNAi construct, the proAGIP promoter was linked with the GhCKX3b-RNAi fragment and assembled with HindIII and EcoRI-linearized p5 vector (Luo et al., 2007). proAGIP::GUS was constructed by replacing the CaMV35S promoter of the pBI121 vector with the proAGIP. To construct the vectors used for dual-luciferase and protein subcellular localization assays, the coding regions of GhARR1, GhARR2a, GhARR2b, GhARR11, and GhARR12 were amplified from carpel cDNA of upland cotton ‘Jimian 14’ (with or without stop codon). Similarly, Yellow Fluorescent Protein (YFP) was amplified from plasmid pLGN-pro35S::GhPIN3a::YFP (Zeng et al., 2019) (with or without stop codon). For the pro35S::GhARR1, pro35S::GhARR2a, pro35S::GhARR2b, pro35S::GhARR11, and pro35S::GhARR12 constructs, the coding regions of GhARR1, GhARR2a, GhARR2b, GhARR11, and GhARR12 (with stop codon), respectively, were assembled with SpeI and SalI-linearized pLGN vector (Zeng et al., 2019). For the pro35S::GhARR1::YFP, pro35S::GhARR2a::YFP, and pro35S:GhARR2b::YFP constructs, the coding regions of GhARR1, GhARR2a, and GhARR2b (without stop codon), respectively, were fused to the 5ʹ terminal region of YFP (with stop codon) and assembled with SpeI and EcoRI-linearized pLGN vector. For the pro35S::YFP::GhARR11 and pro35S::YFP::GhARR12 constructs, the coding regions of GhARR11 and GhARR12 (with stop codon), respectively, were fused to the 3ʹ terminal region of YFP (without stop codon) and assembled with SpeI and EcoRI-linearized pLGN vector. For the proGhSTK1::LUC, proGhSTK2::LUC, proGhAG1::LUC, and proGhSHP::LUC constructs, a 2998 bp upstream region from the start codon of GhSTK1 (proGhSTK1, −2998 to −1 bp), a 2493 bp upstream region from the start codon of GhSTK2 (proGhSTK2, −2493 to −1 bp), a 3000 bp upstream region from the start codon of GhAG1 (proGhAG1, −3000 to −1 bp), and a 2998 bp upstream region from the start codon of GhSHP (proGhSHP, −2998 to −1 bp), respectively, were amplified from gDNA of upland cotton ‘Jimin 14’, and assembled with HindIII and BamHI-linearized pGreenII 0800-LUC vector (Hellens et al., 2005). For yeast one-hybrid (Y1H) assays, the partial sequences of proGhSTK2 (−1491 to −476 bp), proGhAG1 (−2425 to −1 bp), and proGhSHP (−2814 to −340 bp), respectively, were inserted into the pAbAi vector (Takara Bio, Japan) between sites KpnI and XhoI, HindIII and XhoI, and KpnI and XhoI to construct the pAbAi bait vectors. The coding regions of GhARR1, GhARR2a, and GhARR11, respectively, were inserted into the pGADT7 vector (Takara Bio, Japan) between sites NdeI and BamHI, BamHI and XhoI, and EcoRI and XhoI to construct the pGADT7 prey vectors. The constructs used for dual-luciferase and protein subcellular localization assays were transformed into Agrobacterium tumefaciens strain GV3101. The constructs used for Y1H assays were introduced into Y1H Gold Yeast (Takara Bio, Japan). The constructs used for genetic transformation were transformed into Agrobacterium tumefaciens strain LBA4404. Transgenic 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.

RNA extraction and real-time quantitative PCR

Total RNA was extracted using the EASY spin plant RNA extraction kit (Aidlab Biotech, China). Approximately 1 μg RNA was transcribed into first-strand cDNA using the NovoScript Plus All-in-one First Strand cDNA Synthesis SuperMix (gDNA Purge, Novoprotein, China). The real-time quantitative PCR (RT-qPCR) assays were performed on a CFX Connect Real-Time System (Bio-Rad Laboratories) with 2×NovoStart SYBR qPCR SuperMix plus (Novoprotein, China). GhHis3 (Zhang et al., 2011; Wan et al., 2016; Wu et al., 2018) and GhUbiquitin (Walford et al., 2011; Wu et al., 2018) served as internal references. Gene specific primers used for RT-qPCR are listed in Supplementary Table S2. The expression data were calculated with the ΔΔCt method. For the RT-qPCR analysis, three individual biological replicates with two technical replicates for each gene were used. Mean values and standard errors were calculated using the data from the three replicates. The compliance of the RT-qPCR experiments with the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) is shown in a MIQE checklist (Supplementary Table S3).

Sequence retrieval, phylogenetic analysis, and sequence alignment

The amino acid sequences of AtCKXs and B-type AtARRs were obtained from the Arabidopsis genome databases (https://www.Arabidopsis.org/, accessed on 30 December 2019). The GhCKX and B-type GhARR homologs were identified using the BLASTP tool with default parameters in the CottonFGD database (https://cottonfgd.org/, accessed on 30 December 2019) (Zhu et al., 2017) using AtCKX and B-type AtARR sequences, respectively. The cutoff values were 0 for the sequence retrieval of GhCKXs and 1.0−140 for the sequence retrieval of B-type GhARRs. Then, the selected GhCKX sequences were used for further identification of GrCKXs and GaCKXs by searching the databases https://phytozome.jgi.doe.gov/pz/portal.html and https://cottonfgd.org/, respectively. The phylogenetic trees of deduced CKX and B-type ARR amino acids were constructed by the neighbor-joining algorithm with default parameters, with 1000 bootstrap replicates as implemented in MEGA5.0 software. The GhCKX sequences were aligned using Megalign DNAstar software (Burland, 2000) and analysed with the Gendoc software (Nicholas, 1997).

Histochemical staining and quantification of β-glucuronidase activity

Histochemical staining of β-glucuronidase (GUS) was performed as previously described by Jefferson et al. (1987). Briefly, detached or hand-sectioned tissues were immediately immersed in the staining solution (Zeng et al., 2019) and then placed in the dark at 37 °C for 12 h. The stained samples were bleached and fixed in 75% ethanol before photographing. Images were captured on a stereo-microscope imaging system (SteREO Discovery V20, Zeiss, Germany). Fluorometric assays of GUS activity in vegetative and reproductive organs of proTCS::GUS transgenic cotton were performed as described by Hou et al. (2008). Each sample was ground in liquid nitrogen. Protein estimation was performed using the method of Bradford (1976). GUS activity was calculated as pmol 4-methylumbelliferone (4-MU) per minute per microgram protein and each test was represented by three biological replicates.

In situ hybridization

The linearized DNA template of the gene-specific GhCKX3b probe was amplified directly from a vector carrying the coding sequence of GhCKX3b. In situ hybridization of GhCKX3b mRNA was performed following the method described in Zhang et al. (2017). The sections incubated with the sense RNA probe served as the negative control. Images were captured on a microscope (CKX41, Olympus, Japan). Gene specific primers are listed in Supplementary Table S1.

Quantification of endogenous cytokinin

Buds of cotton were harvested at the pinhead square stage (approximately −21 d post-anthesis; DPA), and then carpels (100 mg fresh weight) were separated and ground in liquid nitrogen. Extraction and detection of endogenous cytokinin was performed following the method described in Yoshimoto et al. (2009) and Zeng et al. (2019). Analytical parameters of LC-MS/MS are listed in Supplementary Table S4.

Immunohistochemical localization of cytokinins

Immunolocalization of cytokinins was performed following the method described by Zhang et al. (2017) with some modifications. Briefly, sections (10 μm) of cotton bud at the pinhead square stage were incubated with antibody (against trans-zeatin riboside (tZR) and trans-zeatin (tZ), Agrisera, Sweden) and then the signal was detected by using DyLight 550-labeled secondary antibody (Abcam, UK), and visualized on a laser-scanning confocal microscope (SP8, Leica, Germany). Sections incubated without the primary antibody served as the negative control.

Transient expression

Four-week-old leaves of Nicotiana benthamiana were used for transient expression. Agrobacterium tumefaciens strain GV3101 containing a plant expression vector was cultured overnight at 28 °C to OD600 of 1.0. The pelleted cells were resuspended and diluted with infiltration buffer (Chen et al., 2021) to OD600 of 0.01–0.05. The infiltrated leaves were used for analysis 3 d later.

Microscopic observations

GUS-stained samples were observed using a stereo-microscope imaging system (SteREO Discovery V20, Zeiss, Germany). Fiber initiation was observed on an S-3400N scanning electron microscope (Hitachi, Japan) as described previously by Zeng et al. (2019). The protein subcellular localization and cytokinin immunolocalization was observed on a laser-scanning confocal microscope (SP8, Leica, Germany). The fluorescence signal was detected by an HyD detector under a ×40 oil immersion objective lens. The imaging condition was set up manually: DyLight 550 (excitation: 552 nm, emission: 560–600 nm) and YFP (excitation: 514 nm, emission: 520–560 nm). The intensity of fluorescence signal was quantified using software Leica Application Suite X.

Southern blot

Southern blot was conducted as previously described by Zhang et al. (2011). Briefly, gDNA of proAGIP::GhCKX3b-RNAi transformants and the non-transgenic control cotton was digested overnight (10 µg) with the restriction enzyme EcoRI. An NPTII fragment amplified from the vector p5 served as the probe. DIG 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.

Yeast one-hybrid assay

Y1H assays were performed as previously described by Li et al. (2021). In brief, the pAbAi bait vector was transformed into the yeast strain Y1H Gold by the LiAc-mediated method. The pGADT7 prey vector was transformed into Y1H Gold cells harboring the Bait-pAbAi. Transformants were screened in the synthetic dextrose medium (SDM) containing 100 or 200 ng ml−1 aureobasidin A (AbA) and lacking Ura and Leu (SDM/+AbA/−Leu). The yeast growth was captured after 5 d inoculation at 30 °C.

Dual-luciferase reporter assay

A dual-luciferase reporter assay was performed as previously described by Hellens et al. (2005) and Yan et al. (2018). Briefly, the promoter fragments were amplified and ligated into the pGreenII 0800-LUC vector to produce firefly luciferase (LUC) reporters, and the coding regions of GhARRs were amplified and ligated into the pLGN vector to produce effectors. The reporters and effectors were co-infiltrated into 4-week-old leaves of N. benthamiana as described in ‘Transient expression’. The effector expressing pro35S::YFP served as internal control. After 3 d of growth at 25 °C, 10 μM tZ was infiltrated into the leaves 5 h before sampling. The leaves were infiltrated with the same volume of dimethyl sulfoxide (DMSO) as the negative control (0 μM tZ). The 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.

Statistical analysis

Statistical analysis was performed with Student’s t-test or one-way ANOVA followed by Tukey multiple comparisons test (P<0.05). Each experiment comprised at least three replicates. The intensity of fluorescence signal was calculated using Leica Application Suite X software. Standard errors and standard deviations were calculated using Microsoft Excel (2016) and IBM SPSS Statistics (version 19).

Results

GhCKX3b is preferentially expressed in carpels

By detecting GUS activity, we estimated cytokinin activities in vegetative and reproductive organs of cotton expressing the cytokinin signaling reporter proTCS::GUS (Müller and Sheen, 2008; Zeng et al., 2019). The maximum GUS activity was discernible in the flower bud at the pinhead square stage (approximately −21 DPA) when the ovule formation begins (Fig. 1A, B), suggesting that a high activity of cytokinins is required for ovule morphogenesis.

Fig. 1.

Fig. 1.

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Cytokinin activities and GhCKX3b transcription levels in different cotton tissues. (A, B) The endogenous cytokinin activities indicated by GUS activities (A) and GUS staining (B) of the proTCS::GUS reporter system in cotton tissues. GUS activities were measured by methylumbelliferyl glucuronide assay in proTCS::GUS transgenic cotton roots (10 d after sowing; DAS), stems (the third internode from the apex at 110 DAS cotton plants), leaves (the third leaf from the apex), flower buds (approximately −21 DPA), petals, stamens, pistils, and ovules at anthesis. Wild-type flower buds were used as the negative control. Error bars indicate standard deviation (SD) of three biological repeats. Buds at approximately −21 DPA were used for GUS staining. (C, D) The GhCKX3b transcription levels detected by RT-qPCR (C) and mRNA in situ hybridization (D) in wild-type roots (10 DAS), stems (the third internode from the apex at 110 DAS cotton plants), leaves (the third leaf from the apex), carpels (ovaries), bracts, sepals petals, and stamens at approximately −21 DPA. GhHis3 and GhUbiquitin served as internal control. Error bars indicate SD of three replicates. Bud sections of 10 µm at approximately −21 DPA were used for in situ hybridization with gene-specific probe GhCKX3b. The sections incubated with sense RNA probe served as the negative control. The lower panels in (D) show the enlarged image of carpels (ovaries). Scale bars=500 μm. Car, carpel; Op, ovule primordia; Pe, petal; Sep, sepal; Sta, stamen.

To identify cotton CKXs, which are the key negative regulators of cytokinins in plants (Schmülling et al., 2003), we screened the genomic database of Gossypium hirsutum (https://cottonfgd.org/). Twenty-seven GhCKX homologous genes to Arabidopsis AtCKXs (https://www.arabidopsis.org/) were identified (Supplementary Fig. S1). Among them, GhCKX3b, GhCKX3c, GhCKX5a, and GhCKX6b were expressed preferentially in the carpel and stamen (Fig. 1C; Supplementary Fig. S2). Our previous study demonstrated that GhCKX3b is a functional cytokinin oxidase (previously named GhCKX; Zeng et al., 2012; Zhao et al., 2015). In situ mRNA hybridization confirmed a strong signal of GhCKX3b transcript in the carpel at the place where ovules formed (Fig. 1D).

Specific down-regulation of GhCKX3b increases CK levels in carpel

Down-regulation of CKXs could significantly increase cytokinin levels in transgenic plants (Ashikari et al., 2005; Zalewski et al., 2012; Yeh et al., 2015; Holubová et al., 2018). However, constitutional down-regulation of CKXs in cotton usually resulted in cytokinin overproduction phenotypes, including dwarfism, sterility, and shortened root (Fig. 2C; Zhao et al., 2015).

Fig. 2.

Fig. 2.

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Molecular and phenotypic identification of proAGIP::GhCKX3b-RNAi transgenic cotton. (A) GhCKX3b transcription levels in cotton carpels (ovaries) at the pinhead square stage (approximately −21 DPA) of T0proAGIP::GhCKX3b-RNAi transgenic lines and wild type. GhHis3 and GhUbiquitin served as internal control. Error bars indicate SD of three replicates. (B) Southern blot analysis of T1proAGIP::GhCKX3b-RNAi transgenic lines ACR1 and ACR5. Red arrows indicate the positive hybridization signal. (C) Phenotypes of 120 DAS cotton plants grown in the field. Control, non-transgenic segregated line of proAGIP::GhCKX3b-RNAi transgenic cotton; 35SCR13 and 35SCR17, lines #13 and #17 of pro35S::GhCKX3b-RNAi transgenic cotton; scale bar=10 cm. (D) Fiber initiation on 0 DPA ovules; scale bar=50 μm. (E) The phenotype of 30 DPA cotton bolls; scale bar=1 cm.

To avoid the side effects on plant growth from the global overproduction of cytokinins, while increasing the seed number through cytokinin manipulation, we decided to increase the cytokinin content spatiotemporally in carpels. To this end, we used proAGIP, a chimeric carpel- and stamen-specific promoter (Busch et al., 1999; Deyholos and Sieburth, 2000), to direct the transcription of a GhCKX3b-RNAi sequence (Supplementary Fig. S3). The GUS pattern in proAGIP::GUS transgenic tobacco (Nicotiana tabacum) confirmed the carpel-specificity of proAGIP promoter (Supplementary Fig. S4; Deyholos and Sieburth, 2000; Wang et al., 2008).

No discernable phenotypic alteration in plant growth and development was observed in transgenic cotton holding proAGIP::GhCKX3b-RNAi (ACR) (Fig. 2C–E). RT-qPCR showed that the transcriptional level of GhCKX3b in the carpel of transgenic lines was obviously reduced. Two transgenic lines, ACR1 and ACR5, in which GhCKX3b was largely down-regulated, were selected for further study (Figs 2A, B, 3).

Fig. 3.

Fig. 3.

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GhCKXs transcription levels in cotton carpels at the pinhead square stage. Total RNAs were extracted from cotton carpels at the pinhead square stage (approximately −21 DPA). The relative transcription levels were determined by RT-qPCR. GhHis3 and GhUbiquitin served as internal control. Error bars indicate SD of three replicates. Control, non-transgenic segregated line of proAGIP::GhCKX3b-RNAi transgenic cotton. ACR, T2proAGIP::GhCKX3b-RNAi transgenic cotton. 35SCR6, line #6 of T5pro35S::GhCKX3b-RNAi transgenic cotton.

Then, we performed LC-MS/MS to detect the content of six primarily active cytokinins, namely tZ, tZR, N6-isopentenyladenine, N6-isopentenyladenosine, dihydrozeatin, and dihydrozeatin riboside, in carpels of cotton. Besides a non-transgenic negative control, a transgenic pro35S::GhCKX-RNAi 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).

Table 1.

Active CK content in carpels during cotton ovule initiation (ng g−1 FW)

Line tZ tZR iP iPR DZ DZR Total CKs
Control 5.17 ± 0.51 7.63 ± 0.78 0.47 ± 0.16 1.50 ± 0.21 0.19 ± 0.05 0.09 ± 0.01 15.05 ± 1.73 (c)
ACR1 10.04 ± 0.31 15.62 ± 0.84 0.90 ± 0.07 2.51 ± 0.22 0.28 ± 0.00 0.19 ± 0.01 29.53 ± 1.46 (a)
ACR5 10.68 ± 0.28 14.23 ± 0.15 0.56 ± 0.29 1.53 ± 0.80 0.28 ± 0.10 0.17 ± 0.01 27.46 ± 1.63 (a)
35SCR6 8.57 ± 0.43 12.20 ± 0.80 0.76 ± 0.13 1.58 ± 0.37 0.21 ± 0.07 0.14 ± 0.01 23.46 ± 1.81 (b)
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Sampled carpels (ovaries) from buds at the pinhead square stage (approximately −21 DPA). Total CKs, sum of contents of tZ (trans-zeatin), tZR (tZ riboside), iP (N6-isopentenyladenine), iPR (N6-isopentenyladenosine), DZ (dihydrozeatin), and DZR (DZ riboside). Data are presented as means ±SD (n=3). Within each column, means that are followed different letters are significantly different at P<0.05 by one-way ANOVA with a Tukey multiple comparisons test. FW, fresh weight; Control, non-transgenic segregated line of proAGIP::GhCKX3b-RNAi transgenic cotton; ACR, proAGIP::GhCKX3b-RNAi transgenic cotton; 35SCR6, line #6 of pro35S::GhCKX3b-RNAi transgenic cotton.

Fig. 4.

Fig. 4.

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Immunolocalization of active CKs in carpels at the pinhead square stage. (A–O) Carpels at the pinhead square stage (approximately −21 DPA) were used for the immunolocalization of CKs. Bud sections of 10 µm were incubated with the antibody (against tZR and tZ) and then the signal was detected by a secondary antibody conjugated to DyLight 550. Sections incubated without the primary antibody served as the negative control. Arrows indicate where ovules formed. Scale bar=100 μm. (P) Fluorescence intensity along the white dashed arrows (in G–J, respectively). The fluorescence intensity is given in arbitrary units (A.U.). Control, non-transgenic segregated line of proAGIP::GhCKX3b-RNAi transgenic cotton; ACR, T2proAGIP::GhCKX3b-RNAi transgenic cotton; 35SCR6, line #6 of T5pro35S::GhCKX3b-RNAi transgenic cotton.

Increase of cytokinin promotes the expression of ovule initiation-related genes

It has been reported that the expression of genes of the AG subfamily, such as those for Seedstick (STK), Shatterproof (SHP), and Agamous (AG), was able to promote the formation of carpels and ovules (Favaro et al., 2003; Pinyopich et al., 2003; Xu et al., 2004; Guo et al., 2007; Liu et al., 2009, 2010; de Moura et al., 2017; Nardeli et al., 2018). In line with previous reports, AG subfamily genes, including GhSTK1, GhSTK2, GhSHP, and GhAG1 were up-regulated in the carpel of the ACR1 and ACR5 lines (Fig. 5A; Supplementary Table S5). To confirm these results, we treated 0 DPA wild-type ovules with 50 μΜ tZ, an active cytokinin, for 12 h. The exposure resulted in a noticeable increase in the expression of AG subfamily genes (Fig. 5B), confirming the promotive effect of the cytokinin on the expression of these genes.

Fig. 5.

Fig. 5.

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Increase of cytokinin promoted the expression of ovule initiation-related genes. (A) Comparison of the relative expression levels of AG subfamily genes in T2 ACR1 and ACR5 transgenic lines with those of the T5 35SCR6 and the non-transgenic control. Total RNAs were extracted from cotton carpels at the pinhead square stage (approximately −21 DPA). The relative transcription levels were determined by RT-qPCR. GhHis3 and GhUbiquitin served as internal control. Error bars indicate SD of three replicates. Control, non-transgenic segregated line of proAGIP::GhCKX3b-RNAi transgenic cotton; ACR, proAGIP::GhCKX3b-RNAi transgenic cotton; 35SCR6, line #6 of pro35S::GhCKX3b-RNAi transgenic cotton. (B) The relative transcription levels of AG subfamily genes in tZ-treated ovules and the control. Total RNA was extracted from 0 DPA ovules treated with 50 μM tZ or the same volume of DMSO for the negative control, for 12 h. (C) Schematic representation of the constructs used in the dual-luciferase assay. (D) The promoters of AG subfamily genes involved in ovule initiation were activated under the expression of GhARRs and treatment with 10 μM tZ via a dual-luciferase assay. The effector expressing pro35S::YFP served as internal control. The leaves were infiltrated with 10 μM tZ or the same volume of DMSO for the negative control (0 μM tZ). The transactivation activity of AG subfamily gene promoters by GhARRs was calculated based on the ratio LUC/REN. Error bars indicate SD of three biological replicates. Asterisks represent significant difference (versus internal control, or 0 μM tZ) as determined by Student’s t-test (*P<0.05; **P<0.01). (E) Yeast one-hybrid assay of GhARRs binding to the promoters of AG subfamily genes. Promoter fragments were inserted into the pAbAi vector to construct pAbAi bait vectors, and the full-length coding sequences of GhARRs were inserted into the pGADT7 vector to construct pGADT7 prey vectors. The pGADT7 prey vector was transformed into Y1H Gold cells harboring the Bait-pAbAi. The Bait-pAbAi and 53-pGADT7 (53-AD) vectors served as the positive control. Possible interactions were screened in synthetic dextrose medium (SDM) containing 100 or 200 ng ml−1 aureobasidin A (AbA) and lacking Ura and Leu (SDM/+AbA/−Leu).

Cytokinins regulate the expression of downstream signal transduction genes by activating B-type ARRs (Hwang and Sheen, 2001; Meng et al., 2017). To identify B-type ARR genes that are involved in cotton carpel development, we conducted a comparative phylogenetic analysis using data from Arabidopsis (https://www.arabidopsis.org/) and Gossypium hirsutum (https://cottonfgd.org/) (D’Agostino et al., 2000; Sakai et al., 2000; Mason et al., 2004, 2005; Zubo and Schaller, 2020). Five carpel expressive GhARRs, i.e. GhARR1, GhARR2a, GhARR2b, GhARR11, and GhARR12, which predominantly localize to the nuclei of N. benthamiana pavement cells, were identified (Supplementary Fig. S5). Dual-luciferase reporter and Y1H assays indicated that GhARR1 could bind directly to the promoter sequences of the AG subfamily genes GhSTK2 and GhAG1, GhARR2a to the promoter of GhAG1, and GhARR11 to the promoter of GhSHP (Fig. 5E; Supplementary Fig. S6). This binding could activate the transcription of these AG subfamily genes (Fig. 5C–D), thereby promoting the expression of AG downstream genes.

Carpel-specific down-regulation of GhCKXs significantly increases seed and fiber yield

By counting, we found that the number of ovules per locule (0 DPA) of ACR1 and ACR5 was significantly higher than that of the non-transgenic control as well as the constitutively down-regulated 35SCR6 line (Fig. 6; Supplementary Fig. S7). The average number of ovules per locule of the ACR1 and ACR5 lines was 9.6 ± 0.5 and 9.3 ± 0.4, respectively, increasing to 21.5% and 17.7%, respectively, over that of the non-transgenic control (7.9 ± 0.6), and 11.6% and 8.1%, respectively, over that of the 35SCR6 line (8.6 ± 0.4). For ACR1 locules, the majority (59.2%) contained 10 or 11 ovules in each locule. In contrast, for the non-transgenic cotton, the majority (77.5%) had seven or eight ovules, and only a small part (1.1%) had 10 ovules. For the 35SCR6 cotton, 4.3% of locules had 10 ovules, and none had 11 ovules. The increase of ovules per locule of ACR cotton was also constant during the flower stage of cotton development (Supplementary Fig. S8).

Fig. 6.

Fig. 6.

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Comparison of ovule number in ACR1 and ACR5 transgenic lines with those of the 35SCR6 and the non-transgenic control. (A) Phenotypes of 0 DPA bolls peeled off shells. Bolls of T2 ACR1 and ACR5 and T5 35SCR6, as well as the non-transgenic control at 0 DPA, were harvested and the shells were peeled off. (B) Locule percentage. The ratio represents the percentage of locules containing a different number of ovules. (C, D) Ovule number per locule or boll. Error bars indicate SD of 45 bolls in each experiment. Different letters in (C, D) represent significant differences at P<0.05 by one-way ANOVA followed by Tukey multiple comparisons test. Control, non-transgenic segregated line of proAGIP::GhCKX3b-RNAi transgenic cotton; ACR, proAGIP::GhCKX3b-RNAi transgenic cotton; 35SCR6, line #6 of pro35S::GhCKX3b-RNAi transgenic cotton.

More ovules per locule usually means more seeds per boll. To assess the agronomic performance of the proAGIP::GhCKX3b-RNAi transgenic cotton, we conducted field trials at the experimental farm located in Chongqing, China (29°49ʹ11″N, 106°24ʹ43″E) in 2018 and 2019. The consecutive two-year field experiments showed a significant increase of seeds per boll in ACR lines (Table 2). The seed number per boll of ACR1 and ACR5 was 31.1 ± 0.7 and 29.8 ± 0.6, respectively, significantly higher than that of 35SCR6 (26.1 ± 0.9) and the control (24.8 ± 0.2). Consequently, the seed yield of ACR1 and ACR5 increased 22.4% and 26.5%, respectively, as compared with that of the control. The increased seeds resulted in a significant increase of lint yield. The lint yield of ACR1 and ACR5 was increased by 32.3% and 25.8%, respectively, compared with the control. The seed yield of the ACR1 line was lower (but not statistically significant) than that of 35SCR6 cotton, due to a slight decrease in seed size (indicated by seed index, the weight in grams of 100 seeds). Nevertheless, the lint yield of the ACR1 line was still significantly higher than that of the 35SCR6 line (Table 2), with the advantage of more seeds per boll. The fiber quality, fiber length, and fiber strength of ACR1 were lower than those of the control; but the values of ACR5 were not significantly different from those of the control (Supplementary Table S6; Supplementary Fig. S9).

Table 2.

Comparison of yield-related traits among transgenic cottons and non-transgenic segregated lines in the field trial

Year Line Boll
number per plant		 Seed number per boll Seed index (g) Lint index (g) Seed cotton weight per boll (g) Seed yield
(kg/plot)		 Lint yield (kg/plot)
2018 Control 32.0 ± 0.2 (c) 24.8 ± 0.1 (b) 10.1 ± 0.1 (b) 6.3 ± 0.1 (a) 4.1 ± 0.1 (c) 4.8 ± 0.1 (b) 3.0 ± 0.0 (c)
ACR1 33.3 ± 0.1 (b) 30.5 ± 0.4 (a) 9.5 ± 0.0 (c) 6.4 ± 0.1 (a) 4.9 ± 0.1 (a) 5.8 ± 0.1 (a) 3.9 ± 0.1 (a)
ACR5 33.7 ± 0.2 (b) 29.7 ± 0.6 (a) 10.1 ± 0.2 (b) 6.4 ± 0.1 (a) 4.9 ± 0.1 (a) 6.1 ± 0.2 (a) 3.8 ± 0.1 (a)
35SCR6 34.5 ± 0.1 (a) 25.3 ± 0.1 (b) 11.5 ± 0.2 (a) 6.7 ± 0.2 (a) 4.6 ± 0.1 (b) 6.0 ± 0.1 (a) 3.5 ± 0.1 (b)
2019 Control 32.7 ± 0.2 (b) 25.0 ± 0.3 (c) 10.3 ± 0.2 (b), (c) 6.4 ± 0.2 (a) 4.1 ± 0.1 (d) 5.0 ± 0.0 (b) 3.1 ± 0.1 (d)
ACR1 33.9 ± 0.4 (b) 31.7 ± 0.1 (a) 9.6 ± 0.3 (c) 6.7 ± 0.1 (a) 5.2 ± 0.1 (a) 6.2 ± 0.2 (a) 4.3 ± 0.0 (a)
ACR5 33.7 ± 0.1 (b) 29.9 ± 0.7 (a) 10.3 ± 0.3 (b) 6.5 ± 0.3 (a) 5.0 ± 0.1 (b) 6.2 ± 0.2 (a) 3.9 ± 0.0 (b)
35SCR6 34.6 ± 0.3 (a) 26.8 ± 0.5 (b) 10.7 ± 0.2 (a) 6.6 ± 0.1 (a) 4.7 ± 0.1 (c) 6.0 ± 0.0 (a) 3.7 ± 0.0 (c)
Average over 2 years Control 32.3 ± 0.4 (c) 24.8 ± 0.2 (b) 10.2 ± 0.2 (b) 6.4 ± 0.1 (a) 4.1 ± 0.1 (c) 4.9 ± 0.1 (b) 3.1 ± 0.0 (d)
ACR1 33.6 ± 0.4 (b) 31.1 ± 0.7 (a) 9.6 ± 0.2 (c) 6.6 ± 0.2 (a) 5.0 ± 0.2 (a) 6.0 ± 0.2 (a) 4.1 ± 0.2 (a)
ACR5 33.7 ± 0.1 (b) 29.8 ± 0.6 (a) 10.2 ± 0.2 (b) 6.5 ± 0.2 (a) 5.0 ± 0.1 (a) 6.2 ± 0.2 (a) 3.9 ± 0.1 (b)
35SCR6 34.5 ± 0.1 (a) 26.1 ± 0.9 (b) 11.1 ± 0.4 (a) 6.6 ± 0.2 (a) 4.6 ± 0.1 (b) 5.9 ± 0.1 (a) 3.6 ± 0.1 (c)
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Cotton plants were grown at the experimental farm located in Chongqing, China (29°49ʹ11″N, 106°24ʹ43″E). The experiment was designed as a randomized comparative trial with three replicates. Plots were arranged randomly with an area of 18 m2 (4 m×4.5 m). Results are presented as means ±SD (n=3). Within each column, means that are followed by different letters are significantly different at P<0.05 by one-way ANOVA with a Tukey multiple comparisons test. Control, non-transgenic segregated line of proAGIP::GhCKX3b-RNAi transgenic cotton; ACR, proAGIP::GhCKX3b-RNAi transgenic cotton; 35SCR6, line #6 of pro35S::GhCKX3b-RNAi transgenic cotton.

Discussion

The promotive effect of cytokinins on seed yield was first observed by exogenous application of cytokinins in various crops (Dyer et al., 1987; Atkins and Pigeaire, 1993; Sawan et al., 2000; Zuñiga-Mayo et al., 2018), followed by genetic expression of cytokinin biosynthetic genes, such as the gene for isopentenyltransferase, to endogenously increase the cytokinin levels in transgenic plants (Ma et al., 2008; Atkins et al., 2011; Peleg et al., 2011). CKX, which inactivates cytokinin irreversibly in plant cells, has been identified as a key negative regulator of cytokinin content in monocots and dicots (Jones and Schreiber, 1997; Schmülling et al., 2003; Kowalska et al., 2010; Zeng et al., 2012; Zhao et al., 2015; Ogonowska et al., 2019). Accumulating evidence has demonstrated that CKX is a key regulator for seed number, and down-regulation of CKXs is an effective strategy for yield improvement of seed crops (Zalewski et al., 2010; Bartrina et al., 2011; Li et al., 2013; Yeh et al., 2015; Schwarz et al., 2020). In our previous study, we generated cytokinin-enhanced transgenic cotton in which a CKX gene was constitutively down-regulated. We found that some transgenic cottons displayed cytokinin overproduction-related aberrations, including dwarf growth, shortened root, and sterility. Only a moderate increase of cytokinin could show a positive effect on cotton yield (Zhao et al., 2015). In this study, we revealed that GhCKX3b, GhCKX3c, GhCKX5a, and GhCKX6b were expressed preferentially in the carpel and stamen (Fig. 1C; Supplementary Fig. S2), suggesting a possible function of these genes in carpel development. We 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. More importantly, the fiber yield of the two transgenic proAGIP::GhCKX3b-RNAi cotton lines was significantly higher than that of both wild-type control and the pro35S::GhCKX3b-RNAi line (Table 2), indicating an advantage of the tissue-specific regulation strategy over the constitutive one.

Cytokinins regulate growth and development of plants through the cytokinin–ARR signaling pathway (Hwang and Sheen, 2001; Meng et al., 2017). Among ARR-activated genes, AG subfamily genes STK2, AG1, and SHP are positive regulators of ovule identity and initiation (Favaro et al., 2003; Pinyopich et al., 2003; Xu et al., 2004; Guo et al., 2007; Liu et al., 2009, 2010; de Moura et al., 2017; Nardeli et al., 2018). Our transcriptome and RT-qPCR data showed that the expression of AG subfamily genes was obviously increased in the ACR transgenic cotton carpels at the place where ovules formed (Fig. 5A; Supplementary Table S5). Our Y1H and dual-luciferase assays further revealed that GhARR1, GhARR2a, and GhARR11 could directly bind to the upstream sequences of GhAG1 and GhSTK2, GhAG1, and GhSHP, respectively, and in turn activate the transcription of these AG subfamily genes (Fig. 5C–E; Supplementary Fig. S6). The enhanced expression of these AG subfamily genes could in turn promote carpel development and ovule formation, thus increasing the yield of seeds. The mechanism for increasing cotton yield by the carpel-specific enhancement of cytokinins is summarized in Fig. 7.

Fig. 7.

Fig. 7.

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A model showing the promotive effect of carpel-specific enhancement of cytokinins (CKs) on cotton yield. The carpel-specific down-regulation of GhCKXs results in an increase of CK levels at the place where ovules initiate. The increased cytokinin content stimulates the activation of B-type ARR (Hwang and Sheen, 2001; Meng et al., 2017), which in turn promotes the transcription of AG subfamily genes (e.g. GhSTK2, GhAG1, and GhSHP) by binding the upstream region of the genes. The up-regulation of these AG subfamily genes boosts carpel differentiation and ovule formation (Favaro et al., 2003; Pinyopich et al., 2003; Xu et al., 2004; Guo et al., 2007; Liu et al., 2009; Liu et al., 2010; de Moura et al., 2017; Nardeli et al., 2018). This promotion of ovule formation results in production of more seeds in a boll, thus increasing the yield of both seeds and fibers of cotton.

Fiber length, strength, and fineness are the three major traits determining the quality and economic value of cotton (Han et al., 2013; Long et al., 2018). It has been reported that a high concentration of kinetin (>5 µM), a type of cytokinin, inhibits fiber elongation, whereas a low concentration (<0.5 µM) stimulates fiber elongation (Beasley and Ting, 1974; Yu et al., 2000b). In addition, fiber elongation was inhibited in transgenic cotton expressing cytokinin biosynthesis isopentenyltransferase gene, ipt, under the control of seed-specific promoter Ph/P (Yu et al., 2000a). Our previous study demonstrated that constitutive overexpression of GhCKX-RNAi had little negative effect on fiber quality, such as length, strength, and fineness (Zhao et al., 2015). In this current study, we found that the fiber length and fiber strength of the ACR1 transgenic line was significantly decreased, whereas there was no significant alteration in ACR5. We noticed that the seed number per boll of ACR1 (31.1 ± 0.7) was higher than that of ACR5 (29.8 ± 0.6), while its seed size (indicated by the seed index) declined (Table 2). One explanation for the deterioration in quality of transgenic ACR1 fibers is that more seeds per boll means a lower nutritional share, which may reduce the quality of the fibers. The impact of manipulation of cytokinins on fiber quality awaits further investigation.

Taken together, our data indicate that without any hindrance of plant development, carpel-specific up-regulation of endogenous cytokinins by down-regulation of CKX is a feasible and effective strategy for seed yield improvement, not only for cotton but perhaps also for other dicotyledons, such as canola and soybean.

Supplementary data

The following supplementary data are available at JXB online.

Fig. S1. Phylogenetic analysis of CKX proteins.

Fig. S2. Transcription levels of GhCKXs in different wild-type upland cotton tissues.

Fig. S3. Sequence alignment of partial GhCKXs which were preferentially expressed in the carpel.

Fig. S4. The activity pattern of proAGIP in Nicotiana tabacum.

Fig. S5. Expression patterns and nuclear localization of GhARRs.

Fig. S6. Diagram of ARR binding elements in the AG subfamily gene promoters.

Fig. S7. Comparison of ovule number per 0 DPA locule or boll between T0proAGIP::GhCKX3b-RNAi transgenic cottons and wild type.

Fig. S8. Comparison of ovule number per 0 DPA locule or boll between T2proAGIP::GhCKX3b-RNAi transgenic cotton and the non-transgenic segregated line at different growth temperatures.

Fig. S9. Phenotypes of proAGIP::GhCKX3b-RNAi transgenic cotton bolls and seeds.

Table S1. Primer and fragment information for plasmid construction.

Table S2. Primers pairs used for RT-qPCR assay.

Table S3. MIQE checklist.

Table S4. Analysis parameters for CKs using LC-MS/MS.

Table S5. Transcriptomic analysis of transcripts that were significantly changed between proAGIP::GhCKX3b-RNAi transgenic cotton and the non-transgenic segregated line.

Table S6. Comparison of mature fiber qualities of proAGIP::GhCKX3b-RNAi transgenic cottons and the non-transgenic segregated line in field trial.

erac303_suppl_Supplementary_Figures_and_Tables
Click here for additional data file. (2MB, pdf)

Glossary

Abbreviations

AG

Agamous

AHK

Arabidopsis histidine kinase receptor

AHP

Arabidopsis histidine protein

ARR

Arabidopsis response regulator

CK

cytokinin

CKX

cytokinin oxidase/dehydrogenase

DPA

days post-anthesis

RT-qPCR

real-time quantitative PCR

SHP

Shatterproof

STK

Seedstick

tZ

trans-zeatin

tZR

trans-zeatin riboside

Contributor Information

Jianyan Zeng, Biotechnology Research Center, Southwest University, Beibei, Chongqing, P. R. China.

Xingying Yan, Biotechnology Research Center, Southwest University, Beibei, Chongqing, P. R. China.

Wenqin Bai, Biotechnology Research Center, Southwest University, Beibei, Chongqing, P. R. China.

Mi Zhang, Biotechnology Research Center, Southwest University, Beibei, Chongqing, P. R. China.

Yang Chen, Biotechnology Research Center, Southwest University, Beibei, Chongqing, P. R. China.

Xianbi Li, Biotechnology Research Center, Southwest University, Beibei, Chongqing, P. R. China.

Lei Hou, Biotechnology Research Center, Southwest University, Beibei, Chongqing, P. R. China.

Juan Zhao, Biotechnology Research Center, Southwest University, Beibei, Chongqing, P. R. China.

Xiaoyan Ding, Biotechnology Research Center, Southwest University, Beibei, Chongqing, P. R. China.

Ruochen Liu, Biotechnology Research Center, Southwest University, Beibei, Chongqing, P. R. China.

Fanlong Wang, Biotechnology Research Center, Southwest University, Beibei, Chongqing, P. R. China.

Hui Ren, Biotechnology Research Center, Southwest University, Beibei, Chongqing, P. R. China.

Jingyi Zhang, Biotechnology Research Center, Southwest University, Beibei, Chongqing, P. R. China.

Bo Ding, Biotechnology Research Center, Southwest University, Beibei, Chongqing, P. R. China.

Haoru Liu, Biotechnology Research Center, Southwest University, Beibei, Chongqing, P. R. China.

Yuehua Xiao, Biotechnology Research Center, Southwest University, Beibei, Chongqing, P. R. China.

Yan Pei, Biotechnology Research Center, Southwest University, Beibei, Chongqing, P. R. China.

Zoe Wilson, University of Nottingham, UK.

Author contributions

YP and JZ conceived the experiments and wrote the manuscript; JZ, XY, WB, YC, JZ, XD, RL, FW, HR, JZ, BD, HL, MZ, XL, LH, and YX performed the experiments and analysed the data; YP, JZ, YX, XY, and MZ contributed to the interpretation of the results. All authors reviewed and approved the manuscript.

Conflict of interest

The authors declare no conflicts of interest.

Funding

This work was supported by the National Natural Sciences Foundation of China (NSFC, Grant No. 31130039 to YP and U2003209 to YX), the Chongqing Postdoctoral Science Foundation (Grant No. cstc2020jcyj-bshX0019 to JZ), the China Postdoctoral Science Special Foundation (Grant No. 2021T140569 to JZ), and the China Postdoctoral Science Foundation (Grant No. 2020M673104 to JZ).

Data availability

The original contributions presented in the study are included in the article and its supplementary data, and further enquiries can be directed to the corresponding author.

References

  1. Argueso CT, Raines T, Kieber JJ.. 2010. Cytokinin signaling and transcriptional networks. Current Opinion in Plant Biology 13, 533–539. [DOI] [PubMed] [Google Scholar]
  2. Argyros RD, Mathews DE, Chiang Y-H, Palmer CM, Thibault DM, Etheridge N, Argyros DA, Mason MG, Kieber JJ, Schaller GE.. 2008. Type B response regulators of Arabidopsis play key roles in cytokinin signaling and plant development. The Plant Cell 20, 2102–2116. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Ashikari M, Sakakibara H, Lin S, Yamamoto T, Takashi T, Nishimura A, Angeles ER, Qian Q, Kitano H, Matsuoka M.. 2005. Cytokinin oxidase regulates rice grain production. Science 309, 741–745. [DOI] [PubMed] [Google Scholar]
  4. Atkins CA, Emery RN, Smith PM.. 2011. Consequences of transforming narrow leafed lupin (Lupinus angustifolius [L.]) with an ipt gene under control of a flower-specific promoter. Transgenic Research 20, 1321–1332. [DOI] [PubMed] [Google Scholar]
  5. Atkins C, Pigeaire A.. 1993. Application of cytokinins to flowers to increase pod set in Lupinus angustifolius L. Australian Journal of Agricultural Research 44, 1799–1819. [Google Scholar]
  6. Azhakanandam S, Nole-Wilson S, Bao F, Franks RG.. 2008. SEUSS and AINTEGUMENTA mediate patterning and ovule initiation during gynoecium medial domain development. Plant Physiology 146, 1165–1181. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Bartrina I, Otto E, Strnad M, Werner T, Schmülling T.. 2011. Cytokinin regulates the activity of reproductive meristems, flower organ size, ovule formation, and thus seed yield in Arabidopsis thaliana. The Plant Cell 23, 69–80. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Beasley C, Ting IP.. 1974. Effects of plant growth substances on in vitro fiber development from unfertilized cotton ovules. American Journal of Botany 61, 188–194. [Google Scholar]
  9. Bencivenga S, Simonini S, Benková E, Colombo L.. 2012. The transcription factors BEL1 and SPL are required for cytokinin and auxin signaling during ovule development in Arabidopsis. The Plant Cell 24, 2886–2897. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Bradford MM. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry 72, 248–254. [DOI] [PubMed] [Google Scholar]
  11. Brambilla V, Battaglia R, Colombo M, Masiero S, Bencivenga S, Kater MM, Colombo L.. 2007. Genetic and molecular interactions between BELL1 and MADS box factors support ovule development in Arabidopsis. The Plant Cell 19, 2544–2556. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Burland TG. 2000. DNASTAR’s Lasergene sequence analysis software. In: Misener S, Krawetz SA, eds. Bioinformatics methods and protocols. Methods in Molecular Biology, vol 132. Totowa: Humana Press, 71–91. [DOI] [PubMed] [Google Scholar]
  13. Busch MA, Bomblies K, Weigel D.. 1999. Activation of a floral homeotic gene in Arabidopsis. Science 285, 585–587. [DOI] [PubMed] [Google Scholar]
  14. Chen Y, Zhang M, Wang L, et al. 2021. GhKWL1 upregulates GhERF105 but its function is impaired by binding with VdISC1, a pathogenic effector of Verticillium dahliae. International Journal of Molecular Sciences 22, 7328. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Chen ZJ, Scheffler BE, Dennis E, et al. 2007. Toward sequencing cotton (Gossypium) Genomes. Plant Physiology 145, 1303–1310. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Choi J, Huh SU, Kojima M, Sakakibara H, Paek K-H, Hwang I.. 2010. The cytokinin-activated transcription factor ARR2 promotes plant immunity via TGA3/NPR1-dependent salicylic acid signaling in Arabidopsis. Developmental Cell 19, 284–295. [DOI] [PubMed] [Google Scholar]
  17. Cortleven A, Leuendorf JE, Frank M, Pezzetta D, Bolt S, Schmülling T.. 2019. Cytokinin action in response to abiotic and biotic stresses in plants. Plant, Cell & Environment 42, 998–1018. [DOI] [PubMed] [Google Scholar]
  18. D’Agostino IB, Deruere J, Kieber JJ.. 2000. Characterization of the response of the Arabidopsis response regulator gene family to cytokinin. Plant Physiology 124, 1706–1717. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Daskalova S, McCormac A, Scott N, Van Onckelen H, Elliott M.. 2007. Effect of seed-specific expression of the ipt gene on Nicotiana tabacum L. seed composition. Plant Growth Regulation 51, 217–229. [Google Scholar]
  20. de Moura SM, Artico S, Lima C, Nardeli SM, Berbel A, Oliveira-Neto OB, Grossi-de-Sá MF, Ferrándiz C, Madueño F, Alves-Ferreira M.. 2017. Functional characterization of AGAMOUS-subfamily members from cotton during reproductive development and in response to plant hormones. Plant Reproduction 30, 19–39. [DOI] [PubMed] [Google Scholar]
  21. Deyholos MK, Sieburth LE.. 2000. Separable whorl-specific expression and negative regulation by enhancer elements within the AGAMOUS second intron. The Plant Cell 12, 1799–1810. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Dyer DJ, Carlson DR, Cotterman CD, Sikorski JA, Ditson SL.. 1987. Soybean pod set enhancement with synthetic cytokinin analogs. Plant Physiology 84, 240–243. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Fang R-X, Nagy F, Sivasubramaniam S, Chua N-H.. 1989. Multiple cis regulatory elements for maximal expression of the cauliflower mosaic virus 35S promoter in transgenic plants. The Plant Cell 1, 141–150. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Favaro R, Pinyopich A, Battaglia R, Kooiker M, Borghi L, Ditta G, Yanofsky MF, Kater MM, Colombo L.. 2003. MADS-box protein complexes control carpel and ovule development in Arabidopsis. The Plant Cell 15, 2603–2611. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Franks RG, Wang C, Levin JZ, Liu Z.. 2002. SEUSS, a member of a novel family of plant regulatory proteins, represses floral homeotic gene expression with LEUNIG. Development 129, 253–263. [DOI] [PubMed] [Google Scholar]
  26. Gan S, Amasino RM.. 1995. Inhibition of leaf senescence by autoregulated production of cytokinin. Science 270, 1986–1988. [DOI] [PubMed] [Google Scholar]
  27. Gao S, Fang J, Xu F, Wang W, Sun X, Chu J, Cai B, Feng Y, Chu C.. 2014. Cytokinin oxidase/dehydrogenase4 integrates cytokinin and auxin signaling to control rice crown root formation. Plant Physiology 165, 1035–1046. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Gasparis S, Przyborowski M, Kała M, Nadolska-Orczyk A.. 2019. Knockout of the HvCKX1 or HvCKX3 gene in barley (Hordeum vulgare L.) by RNA-guided Cas9 nuclease affects the regulation of cytokinin metabolism and root morphology. Cells 8, 782. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Gu J, Li Z, Mao Y, Struik PC, Zhang H, Liu L, Wang Z, Yang J.. 2018. Roles of nitrogen and cytokinin signals in root and shoot communications in maximizing of plant productivity and their agronomic applications. Plant Science 274, 320–331. [DOI] [PubMed] [Google Scholar]
  30. Guo Y, Zhu Q, Zheng S, Li M.. 2007. Cloning of a MADS box gene (GhMADS3) from cotton and analysis of its homeotic role in transgenic tobacco. Journal of Genetics and Genomics 34, 527–535. [DOI] [PubMed] [Google Scholar]
  31. Han LB, Li YB, Wang HY, et al. 2013. The dual functions of WLIM1a in cell elongation and secondary wall formation in developing cotton fibers. The Plant Cell 25, 4421–4438. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Hellens RP, Allan AC, Friel EN, Bolitho K, Grafton K, Templeton MD, Karunairetnam S, Gleave AP, Laing WA.. 2005. Transient expression vectors for functional genomics, quantification of promoter activity and RNA silencing in plants. Plant Methods 1, 13. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Higuchi M, Pischke MS, Mähönen AP, Miyawaki K, Hashimoto Y, Seki M, Kobayashi M, Shinozaki K, Kato T, Tabata S.. 2004. In planta functions of the Arabidopsis cytokinin receptor family. Proceedings of the National Academy of Sciences, USA 101, 8821–8826. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Holubová K, Hensel G, Vojta P, Tarkowski P, Bergougnoux V, Galuszka P.. 2018. Modification of barley plant productivity through regulation of cytokinin content by reverse-genetics approaches. Frontiers in Plant Science 9, 1676. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Hönig M, Plíhalová L, Husičková A, Nisler J, Doležal K.. 2018. Role of cytokinins in senescence, antioxidant defence and photosynthesis. International Journal of Molecular Sciences 19, 4045. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Hou L, Liu H, Li J, Yang X, Xiao Y, Luo M, Song S, Yang G, Pei Y.. 2008. SCFP, a novel fiber-specific promoter in cotton. Science Bulletin 53, 2639–2645. [Google Scholar]
  37. Hwang I, Sheen J.. 2001. Two-component circuitry in Arabidopsis cytokinin signal transduction. Nature 413, 383–389. [DOI] [PubMed] [Google Scholar]
  38. Ioio RD, Nakamura K, Moubayidin L, Perilli S, Taniguchi M, Morita MT, Aoyama T, Costantino P, Sabatini S.. 2008. A genetic framework for the control of cell division and differentiation in the root meristem. Science 322, 1380–1384. [DOI] [PubMed] [Google Scholar]
  39. Jameson PE, Song J.. 2016. Cytokinin: a key driver of seed yield. Journal of Experimental Botany 67, 593–606. [DOI] [PubMed] [Google Scholar]
  40. Jefferson RA, Kavanagh TA, Bevan MW.. 1987. GUS fusions: beta-glucuronidase as a sensitive and versatile gene fusion marker in higher plants. EMBO Journal 6, 3901–3907. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Jiang H, Hong J, Jiang Y, Yu S, Zhang Y, Shi J, Lin W.. 2020. Genome-wide association analysis identifies candidate genes regulating seed number per silique in Arabidopsis thaliana. Plants 9, 585. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Jiang Y, Lahlali R, Karunakaran C, Warkentin TD, Davis AR, Bueckert RA.. 2019. Pollen, ovules, and pollination in pea: Success, failure, and resilience in heat. Plant, Cell & Environment 42, 354–372. [DOI] [PubMed] [Google Scholar]
  43. Jones RJ, Schreiber BM.. 1997. Role and function of cytokinin oxidase in plants. Plant Growth Regulation 23, 123–134. [Google Scholar]
  44. Kim HJ, Triplett BA.. 2001. Cotton fiber growth in planta and in vitro. Models for plant cell elongation and cell wall biogenesis. Plant Physiology 127, 1361–1366. [PMC free article] [PubMed] [Google Scholar]
  45. Kowalska M, Galuszka P, Frébortová J, Šebela M, Béres T, Hluska T, Šmehilová M, Bilyeu KD, Frébort I.. 2010. Vacuolar and cytosolic cytokinin dehydrogenases of Arabidopsis thaliana: heterologous expression, purification and properties. Phytochemistry 71, 1970–1978. [DOI] [PubMed] [Google Scholar]
  46. Li C, Pei J, Yan X, Cui X, Tsuruta M, Liu Y, Lian C.. 2021. A poplar B-box protein PtrBBX23 modulates the accumulation of anthocyanins and proanthocyanidins in response to high light. Plant, Cell & Environment 44, 3015–3033. [DOI] [PubMed] [Google Scholar]
  47. Li S, Zhao B, Yuan D, et al. 2013. Rice zinc finger protein DST enhances grain production through controlling Gn1a/OsCKX2 expression. Proceedings of the National Academy of Sciences, USA 110, 3167–3172. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Liu X, Zuo K, Xu J, et al. 2010. Functional analysis of GbAGL1, a D-lineage gene from cotton (Gossypium barbadense). Journal of Experimental Botany 61, 1193–1203. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Liu X, Zuo K, Zhang F, Li Y, Xu J, Zhang L, Sun X, Tang K.. 2009. Identification and expression profile of GbAGL2, a C-class gene from Gossypium barbadense. Journal of Biosciences 34, 941–951. [DOI] [PubMed] [Google Scholar]
  50. Lohar DP, Schaff JE, Laskey JG, Kieber JJ, Bilyeu KD, Bird DM.. 2004. Cytokinins play opposite roles in lateral root formation, and nematode and rhizobial symbioses. The Plant Journal 38, 203–214. [DOI] [PubMed] [Google Scholar]
  51. Long Q, Yue F, Liu R, Song S, Li X, Ding B, Yan X, Pei Y.. 2018. The phosphatidylinositol synthase gene (GhPIS) contributes to longer, stronger, and finer fibers in cotton. Molecular Genetics and Genomics 293, 1139–1149. [DOI] [PubMed] [Google Scholar]
  52. Luo M, Xiao Y, Li X, Lu X, Deng W, Li D, Hou L, Hu M, Li Y, Pei Y.. 2007. GhDET2, a steroid 5α-reductase, plays an important role in cotton fiber cell initiation and elongation. The Plant Journal 51, 419–430. [DOI] [PubMed] [Google Scholar]
  53. Ma D, Hu Y, Yang C, et al. 2016. Genetic basis for glandular trichome formation in cotton. Nature Communications 7, 10456. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Ma Q, Wang X, Wang Z.. 2008. Expression of isopentenyl transferase gene controlled by seed-specific lectin promoter in transgenic tobacco influences seed development. Journal of Plant Growth Regulation 27, 68–76. [Google Scholar]
  55. Mansoor S, Paterson AH.. 2012. Genomes for jeans: cotton genomics for engineering superior fiber. Trends in Biotechnology 30, 521–527. [DOI] [PubMed] [Google Scholar]
  56. Mason MG, Li J, Mathews DE, Kieber JJ, Schaller GE.. 2004. Type-B response regulators display overlapping expression patterns in Arabidopsis. Plant Physiology 135, 927–937. [DOI] [PMC free article] [PubMed] [Google Scholar]
  57. Mason MG, Mathews DE, Argyros DA, Maxwell BB, Kieber JJ, Alonso JM, Ecker JR, Schaller GE.. 2005. Multiple type-B response regulators mediate cytokinin signal transduction in Arabidopsis. The Plant Cell 17, 3007–3018. [DOI] [PMC free article] [PubMed] [Google Scholar]
  58. Meng WJ, Cheng ZJ, Sang YL, Zhang MM, Rong XF, Wang ZW, Tang YY, Zhang XS.. 2017. Type-B ARABIDOPSIS RESPONSE REGULATORs specify the shoot stem cell niche by dual regulation of WUSCHEL. The Plant Cell 29, 1357–1372. [DOI] [PMC free article] [PubMed] [Google Scholar]
  59. Meyer VG. 1966. Environmental effects on the differentiation of abnormal cotton flowers. American Journal of Botany 53, 976–980. [Google Scholar]
  60. Müller B, Sheen J.. 2008. Cytokinin and auxin interaction in root stem-cell specification during early embryogenesis. Nature 453, 1094–1097. [DOI] [PMC free article] [PubMed] [Google Scholar]
  61. Nardeli SM, Artico S, Aoyagi GM, de Moura SM, da Franca Silva T, Grossi-de-Sa MF, Romanel E, Alves-Ferreira M.. 2018. Genome-wide analysis of the MADS-box gene family in polyploid cotton (Gossypium hirsutum) and in its diploid parental species (Gossypium arboreum and Gossypium raimondii). Plant Physiology and Biochemistry 127, 169–184. [DOI] [PubMed] [Google Scholar]
  62. Nicholas KB. 1997. GeneDoc: analysis and visualization of genetic variation. EMBNEW News 4, 14. [Google Scholar]
  63. Ogonowska H, Barchacka K, Gasparis S, Jablonski B, Orczyk W, Dmochowska-Boguta M, Nadolska-Orczyk A.. 2019. Specificity of expression of TaCKX family genes in developing plants of wheat and their co-operation within and among organs. PLoS One 14, e0214239. [DOI] [PMC free article] [PubMed] [Google Scholar]
  64. Peleg Z, Reguera M, Tumimbang E, Walia H, Blumwald E.. 2011. Cytokinin-mediated source/sink modifications improve drought tolerance and increase grain yield in rice under water-stress. Plant Biotechnology Journal 9, 747–758. [DOI] [PubMed] [Google Scholar]
  65. Pinyopich A, Ditta GS, Savidge B, Liljegren SJ, Baumann E, Wisman E, Yanofsky MF.. 2003. Assessing the redundancy of MADS-box genes during carpel and ovule development. Nature 424, 85–88. [DOI] [PubMed] [Google Scholar]
  66. Ramireddy E, Hosseini SA, Eggert K, Gillandt S, Gnad H, von Wirén N, Schmülling T.. 2018. Root engineering in barley: increasing cytokinin degradation produces a larger root system, mineral enrichment in the shoot and improved drought tolerance. Plant Physiology 177, 1078–1095. [DOI] [PMC free article] [PubMed] [Google Scholar]
  67. Reddy V, Reddy K, Baker D.. 1991. Temperature effect on growth and development of cotton during the fruiting period. Agronomy Journal 83, 211–217. [Google Scholar]
  68. Ritchie GL, Bednarz CW, Jost PH, Brown SM.. 2007. Cotton growth and development. Bulletin 1252. Athens: University of Georgia. [Google Scholar]
  69. Rivero RM, Kojima M, Gepstein A, Sakakibara H, Mittler R, Gepstein S, Blumwald E.. 2007. Delayed leaf senescence induces extreme drought tolerance in a flowering plant. Proceedings of the National Academy of Sciences, USA 104, 19631–19636. [DOI] [PMC free article] [PubMed] [Google Scholar]
  70. Sakai H, Aoyama T, Oka A.. 2000. Arabidopsis ARR1 and ARR2 response regulators operate as transcriptional activators. The Plant Journal 24, 703–711. [DOI] [PubMed] [Google Scholar]
  71. Sakai H, Honma T, Aoyama T, Sato S, Kato T, Tabata S, Oka A.. 2001. ARR1, a transcription factor for genes immediately responsive to cytokinins. Science 294, 1519–1521. [DOI] [PubMed] [Google Scholar]
  72. Sakakibara H. 2006. Cytokinins: activity, biosynthesis, and translocation. Annual Review of Plant Biology 57, 431–449. [DOI] [PubMed] [Google Scholar]
  73. Sawan Z, Mohamed A, Sakr R, Tarrad A.. 2000. Effect of kinetin concentration and methods of application on seed germination, yield components, yield and fiber properties of the Egyptian cotton (Gossypium barbadense). Environmental and Experimental Botany 44, 59–68. [DOI] [PubMed] [Google Scholar]
  74. Schmülling T, Werner T, Riefler M, Krupková E, y Manns IB.. 2003. Structure and function of cytokinin oxidase/dehydrogenase genes of maize, rice, Arabidopsis and other species. Journal of Plant Research 116, 241–252. [DOI] [PubMed] [Google Scholar]
  75. Schwarz I, Scheirlinck M-T, Otto E, Bartrina I, Schmidt R-C, Schmülling T.. 2020. Cytokinin regulates the activity of the inflorescence meristem and components of seed yield in oilseed rape. Journal of Experimental Botany 71, 7146–7159. [DOI] [PubMed] [Google Scholar]
  76. Séguéla M, Briat JF, Vert G, Curie C.. 2008. Cytokinins negatively regulate the root iron uptake machinery in Arabidopsis through a growth-dependent pathway. The Plant Journal 55, 289–300. [DOI] [PubMed] [Google Scholar]
  77. Siemens J, Keller I, Sarx J, Kunz S, Schuller A, Nagel W, Schmülling T, Parniske M, Ludwig-Müller J.. 2006. Transcriptome analysis of Arabidopsis clubroots indicate a key role for cytokinins in disease development. Molecular Plant-Microbe Interactions 19, 480–494. [DOI] [PubMed] [Google Scholar]
  78. Sita K, Sehgal A, Kumar J, Kumar S, Singh S, Siddique KH, Nayyar H.. 2017. Identification of high-temperature tolerant lentil (Lens culinaris Medik.) genotypes through leaf and pollen traits. Frontiers in Plant Science 8, 744. [DOI] [PMC free article] [PubMed] [Google Scholar]
  79. Sridhar VV, Surendrarao A, Liu Z.. 2006. APETALA1 and SEPALLATA3 interact with SEUSS to mediate transcription repression during flower development. Development 133, 3159–3166. [DOI] [PubMed] [Google Scholar]
  80. Sunilkumar G, Campbell LM, Puckhaber L, Stipanovic RD, Rathore KS.. 2006. Engineering cottonseed for use in human nutrition by tissue-specific reduction of toxic gossypol. Proceedings of the National Academy of Sciences, USA 103, 18054–18059. [DOI] [PMC free article] [PubMed] [Google Scholar]
  81. Walford SA, Wu Y, Llewellyn DJ, Dennis ES.. 2011. GhMYB25-like: a key factor in early cotton fibre development. The Plant Journal 65, 785–797. [DOI] [PubMed] [Google Scholar]
  82. Wan Q, Guan X, Yang N, et al. 2016. Small interfering RNA s from bidirectional transcripts of GhMML3_A12 regulate cotton fiber development. New Phytologist 210, 1298–1310. [DOI] [PubMed] [Google Scholar]
  83. Wang H, Hu B, Chen G, Shi N, Zhao Y, Yin Q, Liu J.. 2008. Application of Arabidopsis AGAMOUS second intron for the engineered ablation of flower development in transgenic tobacco. Plant Cell Reports 27, 251–259. [DOI] [PubMed] [Google Scholar]
  84. Werner T, Nehnevajova E, Köllmer I, Novák O, Strnad M, Krämer U, Schmülling T.. 2010. Root-specific reduction of cytokinin causes enhanced root growth, drought tolerance, and leaf mineral enrichment in Arabidopsis and tobacco. The Plant Cell 22, 3905–3920. [DOI] [PMC free article] [PubMed] [Google Scholar]
  85. Werner T, Schmülling T.. 2009. Cytokinin action in plant development. Current Opinion in Plant Biology 12, 527–538. [DOI] [PubMed] [Google Scholar]
  86. Wu H, Tian Y, Wan Q, et al. 2018. Genetics and evolution of MIXTA genes regulating cotton lint fiber development. New Phytologist 217, 883–895. [DOI] [PubMed] [Google Scholar]
  87. Xu H, Li X, Li Q, Bai S, Lu W, Zhang X.. 2004. Characterization of HoMADS 1 and its induction by plant hormones during in vitro ovule development in Hyacinthus orientalis L. Plant Molecular Biology 55, 209–220. [DOI] [PubMed] [Google Scholar]
  88. Yan Q, Wang Y, Li Q, et al. 2018. Up-regulation of GhTT2-3A in cotton fibres during secondary wall thickening results in brown fibres with improved quality. Plant Biotechnology Journal 16, 1735–1747. [DOI] [PMC free article] [PubMed] [Google Scholar]
  89. Yeh S, Chen H, Ng C, Lin C, Tseng T, Li W, Ku MS.. 2015. Down-regulation of cytokinin oxidase 2 expression increases tiller number and improves rice yield. Rice 8, 36. [DOI] [PMC free article] [PubMed] [Google Scholar]
  90. Yoshimoto K, Jikumaru Y, Kamiya Y, Kusano M, Consonni C, Panstruga R, Ohsumi Y, Shirasu K.. 2009. Autophagy negatively regulates cell death by controlling NPR1-dependent salicylic acid signaling during senescence and the innate immune response in Arabidopsis. The Plant Cell 21, 2914–2927. [DOI] [PMC free article] [PubMed] [Google Scholar]
  91. Yu X, Zhu Y, Chen X, Xu Z, Zhou B, Chen S, Shen X.. 2000a. Alterations of root and fiber in transgenic cotton plants with chimeric ph/p-ipt gene expression. Acta Botanica Sinica 42, 59–63. [Google Scholar]
  92. Yu X, Zhu Y, Lu S, Zhang T, Chen X, Xu Z.. 2000b. A comparative analysis of a fuzzless-lintless mutant of Gossypium hirsutum L. cv. Xu-142. Science China Life Sciences 43, 623. [DOI] [PubMed] [Google Scholar]
  93. Zalewski W, Galuszka P, Gasparis S, Orczyk W, Nadolska-Orczyk A.. 2010. Silencing of the HvCKX1 gene decreases the cytokinin oxidase/dehydrogenase level in barley and leads to higher plant productivity. Journal of Experimental Botany 61, 1839–1851. [DOI] [PubMed] [Google Scholar]
  94. Zalewski W, Orczyk W, Gasparis S, Nadolska-Orczyk A.. 2012. HvCKX2 gene silencing by biolistic or Agrobacterium-mediated transformation in barley leads to different phenotypes. BMC Plant Biology 12, 1–12. [DOI] [PMC free article] [PubMed] [Google Scholar]
  95. Zeng Q, Qin S, Song S, Zhang M, Xiao Y, Luo M, Hou L, Pei Y.. 2012. Molecular cloning and characterization of a cytokinin dehydrogenase gene from upland cotton (Gossypium hirsutum L.). Plant Molecular Biology Reporter 30, 1–9. [Google Scholar]
  96. Zeng J, Zhang M, Hou L, Bai W, Yan X, Hou N, Wang H, Huang J, Zhao J, Pei Y.. 2019. Cytokinin inhibits cotton fiber initiation by disrupting PIN3a-mediated asymmetric accumulation of auxin in the ovule epidermis. Journal of Experimental Botany 70, 3139–3151. [DOI] [PMC free article] [PubMed] [Google Scholar]
  97. Zhang J, Guo W, Zhang T.. 2002. Molecular linkage map of allotetraploid cotton (Gossypium hirsutum L. × Gossypium barbadense L.) with a haploid population. Theoretical and Applied Genetics 105, 1166–1174. [DOI] [PubMed] [Google Scholar]
  98. Zhang J, Lu Y, Cantrell R, Hughs E.. 2005. Molecular marker diversity and field performance in commercial cotton cultivars evaluated in the southwestern USA. Crop Science 45, 1483–1490. [Google Scholar]
  99. Zhang M, Zeng J, Long H, Xiao Y, Yan X, Pei Y.. 2017. Auxin regulates cotton fiber initiation via GhPIN-mediated auxin transport. Plant and Cell Physiology 58, 385–397. [DOI] [PubMed] [Google Scholar]
  100. Zhang M, Zheng XL, Song SQ, et al. 2011. Spatiotemporal manipulation of auxin biosynthesis in cotton ovule epidermal cells enhances fiber yield and quality. Nature Biotechnology 29, 453–458. [DOI] [PubMed] [Google Scholar]
  101. Zhao J, Bai W, Zeng Q, et al. 2015. Moderately enhancing cytokinin level by down-regulation of GhCKX expression in cotton concurrently increases fiber and seed yield. Molecular Breeding 35, 60. [DOI] [PMC free article] [PubMed] [Google Scholar]
  102. Zhao Y, Hu Y, Dai M, Huang L, Zhou DX.. 2009. The WUSCHEL-related homeobox gene WOX11 is required to activate shoot-borne crown root development in rice. The Plant Cell 21, 736–748. [DOI] [PMC free article] [PubMed] [Google Scholar]
  103. Zhu T, Liang C, Meng Z, Sun G, Meng Z, Guo S, Zhang R.. 2017. CottonFGD: an integrated functional genomics database for cotton. BMC Plant Biology 17, 101. [DOI] [PMC free article] [PubMed] [Google Scholar]
  104. Zu S, Jiang Y, Chang J, Zhang Y, Xue H, Lin W.. 2022. Interaction of brassinosteroid and cytokinin promotes ovule initiation and increases seed number per silique in Arabidopsis. Journal of Integrative Plant Biology 64, 702–716. [DOI] [PubMed] [Google Scholar]
  105. Zubo YO, Schaller GE.. 2020. Role of the cytokinin-activated type-B response regulators in hormone crosstalk. Plants 9, 166. [DOI] [PMC free article] [PubMed] [Google Scholar]
  106. Zuñiga-Mayo VM, Baños-Bayardo CR, Díaz-Ramírez D, Marsch-Martínez N, de Folter S.. 2018. Conserved and novel responses to cytokinin treatments during flower and fruit development in Brassica napus and Arabidopsis thaliana. Scientific Reports 8, 6836. [DOI] [PMC free article] [PubMed] [Google Scholar]

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