Search Results (23)

Soybean is a dual-purpose crop for food and oil, playing a crucial role in China’s grain production. Seed size and weight are key agronomic traits directly influencing the yield. Cytokinin oxidases/dehydrogenases (CKXs) specifically degrade certain isoforms of endogenous cytokinins (CKs), thereby modulating plant growth and seed development. However, their role in soybeans remains largely uncharacterized. In a previous genome-wide association study of 250 soybean core germplasms, we identified GmCKX3 as a yield-related gene. To elucidate its function, we developed GmCKX3-deficient mutants using CRISPR/Cas9 gene editing in soybean Williams82 and conducted a three-year phenotypic analysis. Loss of GmCKX3 function significantly enhanced the seed size and weight, which was attributed to an increased cell size and fat accumulation in the endosperm. This enhancement was driven by elevated endogenous CK levels resulting from suppressed GmCKX3 expression. Subcellular localization revealed that GmCKX3 resides in the endoplasmic reticulum and predominantly degrades the isopentenyladenine (iP)-type CK. Integrated transcriptomic and metabolomic analyses uncovered key genes and pathways involved in CK regulation, supporting GmCKX3’s central role in seed-trait modulation. These findings advance our understanding of cytokinin-mediated seed development and offer promising targets for molecular breeding aimed at improving the soybean yield. Full article

Cytokinin oxidases/dehydrogenases (CKXs) play a crucial role in modulating plant stress resistance by degrading cytokinins. The ‘duli’ pear (Pyrus betulifolia Bunge), a highly stress-resistant cultivar, is widely used as a rootstock in pear cultivation. This study aims to comprehensively identify and characterize the PbCKX gene family in ‘duli’. A total of 10 PbCKX genes were identified, which are unevenly distributed across five chromosomes and classified into four groups based on sequence similarity and phylogenetic relationships. The PbCKX genes exhibit a high degree of conservation in motifs and structural features, although exon structure variations are observed. Comparative analysis revealed 10 homologous gene pairs between ‘duli’ and Arabidopsis and 14 pairs between ‘duli’ and apple. Additionally, cis-acting elements related to abiotic stress, hormone responses, and light responsiveness were identified in the promoter regions of the PbCKX genes. RNA-seq analysis showed that PbCKX1 and PbCKX2 were predominantly expressed in roots, while PbCKX3 to PbCKX10 had higher expression in leaves. The PbCKX genes responded to both exogenous hormones and salt stress, with salt stress inducing a more pronounced response. Most abiotic stress treatments led to the downregulation of PbCKX4 and PbCKX9, while PbCKX6 and PbCKX8 were upregulated. Notably, treatments with Abscisic acid and NaCl significantly enhanced CKX enzyme activity in ‘duli’ over 20 days, reducing levels of zeatin and isopentenyladenine. Conversely, treatments with gibberellin, cytokinin, and auxin significantly reduced CKX enzyme activity and increased concentrations of zeatin and isopentenyladenine over the same period. These findings provide valuable insights for future studies on the functional role of PbCKX genes in abiotic stress responses in ‘duli’. Full article

Lodging is one of the major problems in rice production. However, few genes that can explain the culm strength within the temperate japonica subspecies have been identified. In this study, we identified OsRLCK191, which encodes receptor-like cytoplasmic kinase and plays critical roles in culm strength. OsRLCK191 mutants were produced by the CRISPR-Cas9 DNA-editing system. Compared with wild types (WTs), the bending moment of the whole plant (WP), the bending moment at breaking (BM), and the section modulus (SM) were decreased in rlck191 significantly. Although there is no significant decrease in the culm length of rlck191 compared with the WT; in the mutant, except the length of the fourth internode being significantly increased, the lengths of other internodes are significantly shortened. In addition, the yield traits of panicle length, thousand-seed weight, and seed setting rate decreased significantly in rlck191. Moreover, RNA-seq experiments were performed at an early stage of rice panicle differentiation in shoot apex. The differentially expressed genes (DEGs) are mainly involved in cell wall biogenesis, cell wall polysaccharide metabolic processes, cellar component biogenesis, and DNA-binding transcription factors. Transcriptome analysis of the cell wall biological process pathways showed that major genes that participated in the cytokinin oxidase/dehydrogenase family, cellulose synthase catalytic subunit genes, and ethylene response factor family transcription factor were related to culm strength. Our research provides an important theoretical basis for analyzing the lodging resistance mechanism and lodging resistance breeding of temperate japonica. Full article

Cytokinins (CKs) are a group of phytohormones that are involved in plant growth, development, and disease resistance. The isopentenyl transferase (IPT) and cytokinin oxidase/dehydrogenase (CKX) families comprise key enzymes controlling CK biosynthesis and degradation. However, an integrated analysis of these two gene families in radish has not yet been explored. In this study, 13 RsIPT and 12 RsCKX genes were identified and characterized, most of which had four copies in Brassica napus and two copies in radish and other diploid Brassica species. Promoter analysis indicated that the genes contained at least one phytohormone or defense and stress responsiveness cis-acting element. RsIPTs and RsCKXs were expanded through segmental duplication. Moreover, strong purifying selection drove the evolution of the two gene families. The expression of the RsIPT and RsCKX genes distinctly showed diversity in different tissues and developmental stages of the root. Expression profiling showed that RsCKX1-1/1-2/1-3 was significantly upregulated in club-resistant materials during primary infection, suggesting their vital function in clubroot resistance. The interaction network of CKX proteins with similar 3D structures also reflected the important role of RsCKX genes in disease resistance. This study provides a foundation for further functional study on the IPT and CKX genes for clubroot resistance improvement in Raphanus. Full article

6-BA, a small molecule compound of cytokinins, has been proven to delay leaf senescence in different species, including Chinese flowering cabbage; however, its specific mechanism remains relatively unknown. In this study, the application of external 6-BA delayed leaf senescence in Chinese flowering cabbage, showing that 6-BA effectively prevented the decrease in the maximum quantum yield (Fv/Fm) and overall chlorophyll content and suppressed the expression of the senescence-associated gene BrSAG12 over a 7-day period of storage. Moreover, treatment with 6-BA decreased the respiratory rate, NAD(H) content, the activities of hexose phosphate isomerase (PHI), succinate dehydrogenase (SDH), cytochrome c oxidase (CCO), and ascorbic acid oxidase (AAO) using enzyme-linked immunosorbent assay, and the transcriptional abundance of related genes by real-time quantitative polymerase chain reaction. Furthermore, 6-BA also increased the activity and expression levels of glucose-6-phosphate dehydrogenase (G6PDH) and 6-phosphate gluconate dehydrogenase (6-PGDH). The group treated with 6-BA retained elevated levels of NADP (H), ATP, total ATPase, and nicotinamide adenine dinucleotide kinase (NADK) activity, as well as the expression of respiratory enzymes. Molecular docking indicated that 6-BA hinders the glycolysis pathway (EMP), tricarboxylic acid cycle (TCA), and cytochrome pathway (CCP), and sustains elevated levels of the pentose phosphate pathway (PPP) through interactions with the PHI, SDH, 6-PGDH, G6PDH, CCO, and AAO proteins, consequently delaying postharvest leaf senescence in Chinese flowering cabbage. Full article

Members of the TaCKX gene family (GFM) encode oxidase/dehydrogenase cytokinin degrading enzymes (CKX), which play an important role in the homeostasis of phytohormones, affecting wheat development and productivity. Therefore, the objective of this investigation was to test how the expression patterns of the yield-related TaCKX genes and TaNAC2-5A (NAC2) measured in 7 days after pollination (DAP) spikes and the seedling roots of parents are inherited to apply this knowledge in the breeding process. The expression patterns of these genes were compared between parents and their F₂ progeny in crosses of one mother with different paterns of awnless cultivars and reciprocal crosses of awned and awnless lines. We showed that most of the genes tested in the 7 DAP spikes and seedling roots of the F₂ progeny showed paternal expression patterns in crosses of awnless cultivars as well as reciprocal crosses of awned and awnless lines. Consequently, the values of grain yield in the F₂ progeny were similar to the pater; however, the values of seedling root mass were similar to the mother or both parents. The correlation analysis of TaCKX GFMs and NAC2 in spikes and spikes per seedling roots reveals that the genes correlate with each other specifically with the pater and the F₂ progeny or the mother and the F₂ progeny, which shape phenotypic traits. The numbers of spikes and semi-empty spikes are mainly correlated with the specific coexpression of the TaCKX and NAC2 genes expressed in spikes or spikes per roots of the pater and F₂ progeny. Variable regression analysis of grain yield and root mass with TaCKX GFMs and NAC2 expressed in the tested tissues of five crosses revealed a significant dependency of these parameters on the mother and F₂ and/or the pater and F₂ progeny. We showed that the inheritance of yield-related traits depends on the specific cooperative expression of some TaCKX GFMs, in some crosses coupled with NAC2, and is strongly dependent on the genotypes used for the crosses. Indications for parental selection in the breeding of high-yielding lines are discussed. Full article

Research into molecular mechanisms of self-incompatibility (SI) in plants can be observed in representatives of various families, including Solanaceae. Earlier studies of the mechanisms of S-RNase-based SI in petunia (Petunia hybrida E. Vilm.) demonstrate that programmed cell death (PCD) is an SI factor. These studies suggest that the phytohormon cytokinin (CK) is putative activator of caspase-like proteases (CLPs). In this work, data confirming this hypothesis were obtained in two model objects—petunia and tomato (six Solanaceae representatives). The exogenous zeatin treatment of tomato and petunia stigmas before a compatible pollination activates CLPs in the pollen tubes in vivo, as shown via the intravital imaging of CLP activities. CK at any concentration slows down the germination and growth of petunia and tomato male gametophytes both in vitro and in vivo; shifts the pH of the cytoplasm (PHc) to the acid region, thereby creating the optimal conditions for CLP to function and inhibiting the F-actin formation and/or destructing the cytoskeleton in pollen tubes to point foci during SI-induced PCD; and accumulates in style tissues during SI response. The activity of the ISOPENTENYLTRANSFERASE 5 (IPT5) gene at this moment exceeds its activity in a cross-compatible pollination, and the levels of expression of the CKX1 and CKX2 genes (CK OXIDASE/DEHYDROGENASE) are significantly lower in self-incompatible pollination. All this suggests that CK plays a decisive role in the mechanism underlying SI-induced PCD. Full article

Liriodendron chinense is a tree species of the Magnoliaceae family, an ancient relict plant mainly used for landscaping and timber production due to its excellent material properties and ornamental value. The cytokinin oxidase/dehydrogenase (CKX) enzyme regulates cytokinin levels and plays an important role in plant growth, development, and resistance. However, too-high or too-low temperatures or soil drought can limit the growth of L. chinense, representing a key issue for research. Here, we identified the CKX gene family in the L. chinense genome and examined its transcriptional responses to cold, drought, and heat stresses. A total of five LcCKX genes, distributed on four chromosomes and divided into three phylogenetic groups, were identified across the whole L. chinense genome. Further analysis showed that multiple hormone- and stress-responsive cis-acting elements are located in the promoter regions of LcCKXs, indicating a potential role of these LcCKXs in plant growth, development, and response to environmental stresses. Based on existing transcriptome data, LcCKXs, especially LcCKX5, were found to transcriptionally respond to cold, heat, and drought stresses. Furthermore, quantitative reverse-transcription PCR (qRT-PCR) showed that LcCKX5 responds to drought stress in an ABA-dependent manner in stems and leaves and in an ABA-independent manner in roots. These results lay a foundation for functional research on LcCKX genes in the resistance breeding of the rare and endangered tree species of L. chinense. Full article

Cytokinin oxidase/dehydrogenase (CKX) catalyzes the irreversible breakdown of active cytokinins, which are a class of plant hormones that regulate cell division. According to conserved sequences of CKX genes from monocotyledons, PCR primers were designed to synthesize a probe for screening a bamboo genomic library. Cloned results of three genes encoding cytokinin oxidase were named as follows: BoCKX1, BoCKX2, and BoCKX3. In comparing the exon-intron structures among the above three genes, there are three exons and two introns in BoCKX1 and BoCKX3 genes, whereas BoCKX2 contains four exons and three introns. The amino acid sequence of BoCKX2 protein shares 78% and 79% identity with BoCKX1 and BoCKX3 proteins, respectively. BoCKX1 and BoCKX3 genes are particularly closely related given that the amino acid and nucleotide sequence identities are more than 90%. These three BoCKX proteins carried putative signal peptide sequences typical of secretion pathway, and a GHS-motif was found at N-terminal flavin adenine dinucleotide (FAD) binding domain, suggesting that BoCKX proteins might covalently conjugate with an FAD cofactor through a predicted histidine residue. Full article

Quinoa (Chenopodium quinoa) is a well-known climate-resilient crop and has been introduced into multiple marginal lands across the world, including China, to improve food security and/or balanced nutrient supplies. Conventional breeding has been widely applied in the selection and breeding of quinoa varieties in China since 1980s; however, few studies have been implemented on the genetic variances among different varieties developed by diversity breeding objectives. In this study, the phenotypic and genetic differences between two varieties (Longli-4 and CA3-1) from China were systematically analyzed. A total of 407,651 and 2,731,411 single nucleotide polymorphisms (SNPs) and 212,724 and 587,935 small insertion and deletion (INDELs) were detected for Longli-4 and CA3-1, respectively, when compared with the reference genome of PI614886. The SNPs/INDELs were unevenly distributed across each chromosome for both varieties. There were 143,996 SNPs and 83,410 INDELs shared between Longli-4 and CA3-1, accounting for 4% of the total variances. The variation was then screened based on the SNP effects. There were 818 and 73 genes with the variety-specific non-synonymous and stop-gain variation in Longli-4, whereas there were 13,701 and 733 genes in CA3-1. Specifically, 3501 genes with the non-synonymous variation and 74 genes with the stop-gain variation were found in both Longli-4 and CA3-1. These results suggest that convergent selection occurred during the different breeding processes. A set of candidate genes related to agronomic traits and domestication were further selected to detect the genetic divergence in detail in the two varieties. Only one domestication gene was identified having Longli-4-specific stop-gain variation. Twelve candidate genes related to betalain (1), flowering (4), seed size (2), domestication (1), and saponin (4) were identified having CA3-1-specific stop-gain variation. Interestingly, one seed size gene homologous of CKX1 (cytokinin oxidase/dehydrogenase 1) had the stop-gain variation in both varieties. This research will therefore provide guidance for the molecular-assisted breeding in quinoa. Full article

Cytokinin oxidase/dehydrogenase (CKX) is a specific enzyme affecting plant growth, development, and yield by mediating the metabolism of endogenous cytokinins in a dual catalytic mode. This study aims to reveal the distribution and associated properties of CKX gene family members in the whole genome of Brassica juncea, lay a theoretical basis for further exploration of the biological function of BjuCKX genes, and provide genetic resources to improve the breeding quality of B. juncea. We screened BjuCKX genes with typical FAD-binding and CK binding domains and identified them using bioinformatics methods. In addition, we analyzed physicochemical properties, phylogenetic relationship, gene structure, conservative motif, cis-acting element of promoter, and expression pattern of BjuCKX gene family members. Endogenous hormone levels (GA3, ZR, IAA, ABA, BR and MeJA) were also determined in different developmental periods using an indirect enzyme-linked immunosorbent assay (ELISA). A total of 23 BjuCKX genes were identified, and they were renamed BjuCKX01~BjuCKX23. Further analysis revealed that the amino acid number of 23 BjuCKX proteins ranged from 333 to 1337 aa, the molecular weight ranged 36.58 to 148.49 kDa, whereas the theoretical isoelectric point ranged from 4.94 to 9.10. The phylogenetic tree clustering analysis can group family members into four subgroups. Collinearity analysis revealed that genes were not evenly distributed on the chromosome, with a pair of tandem repeats. Meanwhile, BjuCKX genes located on each chromosome revealed cross collinearity caused by fragment replication. The genes were more conserved in structure. In the upstream region of promoter, there were several cis-acting elements, including plant growth and development, hormone response, and biological and abiotic stress. Combined with transcriptome data, BjuCKX gene expression has been demonstrated to be different at varying developmental stages of the stem. RT-qPCR further confirmed that BjuCKX genes were involved in stem development and affects growth by regulating endogenous hormone levels. Full article

Plants have evolved a sophisticated defense system that employs various hormone pathways to defend against attacks by insect pests. Cytokinin (CK) plays an important role in plant growth and stress tolerance, but the role of CKs in plant–insect interaction remains largely unclear. Here, we report that CKs act as a positive regulator in rice resistance against brown planthopper (BPH), a devastating insect pest of rice. We found that BPH feeding promotes CK biosynthesis and signaling in rice. Exogenous application of CKs significantly increased the rice resistance to BPH. Increasing endogenous CKs by knocking out cytokinin oxidase/dehydrogenase (OsCKXs) led to enhanced resistance to BPH. Moreover, the levels of the plant hormone jasmonic acid (JA) and the expression of JA-responsive genes were elevated by CK treatment and in OsCKXs knockout plants. Furthermore, JA-deficient mutant og1 was more susceptible to BPH, and CK-induced BPH resistance was suppressed in og1. These results indicate that CK-mediated BPH resistance is JA-dependent. Our findings provide the direct evidence for the novel role of CK in promoting insect resistance, and demonstrate that CK-induced insect resistance is JA-dependent. These results provide important guidance for effective pest management strategies in the future. Full article

Barley is an important cereal crop worldwide. Its genetic transformation is now limited to very few cultivars because of the high genotype dependence of embryogenic callus. To reveal the key genes or factors controlling the callus induction and plantlet regeneration in barley, we compared the transcriptomic profiles of immature embryos of Golden Promise and ZU9, which differed dramatically in the efficiency of the genetic transformation. The samples were taken at 0, 5, 10 and 20 days of the culture, respectively. In total, 5386 up-regulated and 6257 down-regulated genes were identified in Golden Promise. Several genes, identified exclusively in GP callus, were selected for further investigation. These genes were mainly involved in protein metabolism, energy metabolism, stress response, detoxification and ubiquitin–proteasome. Four YUCCA flavin monooxygenases, one PIN-FORMED, one tryptophan aminotransferase related, three small auxin up RNA, three indole-3-acetic acid and one adenylate isopentenyl transferase, seven cytokinin oxidase/dehydrogenase, three Arabidopsis histidine kinase, three Arabidopsis histidine phosphotransfer protein, and one Arabidopsis response regulator were differentially expressed in the calli of the two barley genotypes, suggesting that biosynthesis, response and transport of auxin and cytokinin might be associated with cell reprogramming during callus induction. The current results provide insights into molecular mechanisms of callus induction at an early developmental stage and are helpful for optimizing the tissue culture system in barley. Full article

Cytokinin oxidase/dehydrogenases (CKXs) are the key enzymes in cytokinin degradation and have been widely studied in model plants. Little is known about apple’s (Malus×domestica) CKX genes. Here, using genome-wide analysis, we identified 10 MdCKX genes in apple. The phylogenetics, chromosome locations, and genome structures were then tested. Expression analysis showed that MdCKX genes had different expression profiles in apple, pointing to the different roles. Meanwhile, relative expression analysis showed that these genes have different expression patterns in response to several exogenous cytokinin factors, including trans-zeatin (ZT), thidiazuron (TDZ), and N6-furfuryladenine (KT). Finally, we introduced the MdCKX5.2 gene into Arabidopsis to evaluate its functions, and the results suggested the transgenic Arabidopsis displayed phenotypes related to promoting primary root and lateral root development, response to exogenous ZT, and conferring to drought and salt tolerant. Taken together, our results provide insights on the possible application of the MdCKX5.2 gene for molecular breeding in apples. Full article

In chromatin remodeling, the post-translational modification of histone proteins is mediated by multimeric protein complexes. VERNALIZATION INSENSITIVE3 (VIN3) forms a complex with Polycomb Repressive Complex 2 (PRC2), which mediates the trimethylation of H3K27 to repress target gene expression. In rice, four genes (OsVIL1-OsVIL4) encoding the VIN3-like proteins are expressed ubiquitously in various tissues. Null mutants of osvil2 display pleiotropic phenotypes such as altered flowering time, floral organ defects, and reduced tiller size. In contrast, osvil1 mutants did not show significant phenotypes except in fertilization compared with the wild type. However, transgenic plants overexpressing OsVIL1 showed phenotypes of increased biomass and grain yield. Cross-sections of the basal region of elongating stems revealed that the increased biomass was mediated by inducing cell proliferation in the meristem. Chromatin immunoprecipitation assay indicated that OsVIL1 repressed expression of cytokinin oxidase/dehydrogenase gene (OsCKX2) by binding to the promoter and genic regions of OsCKX2. We also observed that OsVIL1 modified the levels of H3K27me3 in the OsCKX2 chromatin. Because OsCKX2 encodes an enzyme that degrades active cytokinin, we conclude that OsVIL1 functions in the regulation of endogenous active cytokinin levels, thereby increasing plant height and productivity. Full article