Characterization of CHARK, an unusual cytokinin receptor of rice.
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ABSTRACT:
SUBMITTER: Halawa M
PROVIDER: S-EPMC7814049 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Cytokinins play important roles in regulating plant development, including shoot and root meristems, leaf longevity, and grain yield. However, the in planta functions of rice cytokinin receptors have not been genetically characterized yet. Here we isolated a rice mutant, Osckt1, with enhanced tolerance to cytokinin treatment. Further analysis showed that Osckt1 was insensitive to aromatic cytokinins but responded normally to isoprenoid and phenylurea-type cytokinins. Map-based cloning revealed that the mutation occurred in a putative cytokinin receptor gene, histidine kinase 6 (OsHK6). OsCKT1 was found to be expressed in various tissues throughout the plant and the protein was located in the endoplasmic reticulum. In addition, whole-genome gene expression profiling analysis showed that OsCKT1 was involved in cytokinin regulation of a number of biological processes, including secondary metabolism, sucrose and starch metabolism, chlorophyll synthesis, and photosynthesis. Our results demonstrate that OsCKT1 plays important roles in cytokinin perception and control of root development in rice.
| S-EPMC5281565 | biostudies-literature
Project description:BACKGROUND:Cytokinins are one of the five major hormones families in plants and are important for their normal growth and environmental adaptability. In plants, cytokinins are mostly present as glycosides in plants, and their glycosylation modifications are catalyzed by family 1 glycosyltransferases. Current research on cytokinin glycosylation has focused on the biochemical identification of enzymes and the analysis of metabolites in Arabidopsis. There are few studies that examine how cytokinin glycosylation affects its synthesis and accumulation in plants. It is particularly important to understand these processes in food crops such as rice (Oryza sativa); however, to date, cytokinin glycosyltransferase genes in rice have not been reported. RESULTS:In this study, we identified eight rice genes that were functionally homologous to an Arabidopsis cytokinin glycosyltransferase gene. These genes were cloned and expressed in a prokaryotic system to obtain their purified proteins. Through enzymatic analysis and liquid chromatography-mass spectrometry, a single rice glycosyltransferase, Os6, was identified that glycosylated cytokinin in vitro. Os6 was overexpressed in Arabidopsis, and the extraction of cytokinin glycosides showed that Os6 is functionally active in planta. CONCLUSIONS:The identification and characterization of the first cytokinin glycosyltransferase from rice is important for future studies on the cytokinin metabolic pathway in rice. An improved understanding of rice cytokinin glycosyltransferases may facilitate genetic improvements in rice quality.
| S-EPMC6439077 | biostudies-literature
Project description:The major components of the cytokinin (CK) signaling pathway have been identified from the receptors to their downstream transcription factors. However, since signaling proteins are encoded by multigene families, characterizing and quantifying the contribution of each component or their combinations to the signaling cascade have been challenging. Here, we describe a transient gene expression system in rice (Oryza sativa) protoplasts suitable to reconstitute CK signaling branches using the CK reporter construct TCSn:fLUC, consisting of a synthetic CK-responsive promoter and the firefly luciferase gene, as a sensitive readout of signaling output. We used this system to systematically test the contributions of CK signaling components, either alone or in various combinations, with or without CK treatment. The type-B response regulators (RRs) OsRR16, OsRR17, OsRR18, and OsRR19 all activated TCSn:fLUC strongly, with OsRR18 and OsRR19 showing the strongest induction by CK. Cotransfecting the reporter with OsHP01, OsHP02, OsHP05, or OsHK03 alone resulted in much weaker effects relative to those of the type-B OsRRs. When we tested combinations of OsHK03, OsHPs, and OsRRs, each combination exhibited distinct CK signaling activities. This system thus allows the rapid and high-throughput exploration of CK signaling in rice.
| S-EPMC8037374 | biostudies-literature
Project description:Cytokinins (CKs) are a class of plant hormones that regulate many aspects of growth and development, including cell division, apical dominance, leaf senescence, nutrient signaling, and shoot differentiation. In the past decade, substantial progress has been made in understanding CK biosynthesis, metabolism and signal transduction. Much of this knowledge is based on research in Arabidopsis, a dicotyledonous model plant. Although cytokinin plays an important role for growth and development in the Gramineae, our knowledge of cytokinin responsive genes in monocotyledonous species is very limited compared to Arabidopsis. The search for genes whose expression is modified by CK has yielded a number of valuable tools that have been used to understand CK signaling and the complex developmental processes under control of this hormone. We tried to identify rice genes regulated by CK using an Affymetrix rice genome array. Keywords: time_course; hormone_response; organ_specific_response
2007-02-15 | GSE6719 | GEO
Project description:BackgroundThe response regulators represent the elements of bacterial two-component system and have been characterized from dicot plants like Arabidopsis but little information is available on the monocots, including the cereal crops. The aim of this study was to characterize type-A response regulator genes from rice, and to investigate their expression in various organs as well as in response to different hormones, including cytokinin, and environmental stimuli.ResultsBy analysis of the whole genome sequence of rice, we have identified ten genes encoding type-A response regulators based upon their high sequence identity within the receiver domain. The exon-intron organization, intron-phasing as well as chromosomal location of all the RT-PCR amplified rice (Oryza sativa) response regulator (OsRR) genes have been analyzed. The transcripts of OsRR genes could be detected by real-time PCR in all organs of the light- and dark-grown rice seedlings/plants, although there were quantitative differences. The steady-state transcript levels of most of the OsRR genes increased rapidly (within 15 min) on exogenous cytokinin application even in the presence of cycloheximide. Moreover, the expression of the OsRR6 gene was enhanced in rice seedlings exposed to salinity, dehydration and low temperature stress.ConclusionTen type-A response regulator genes identified in rice, the model monocot plant, show overlapping/differential expression patterns in various organs and in response to light. The induction of OsRR genes by cytokinin even in the absence of de novo protein synthesis qualifies them to be primary cytokinin response genes. The induction of OsRR6 in response to different environmental stimuli indicates its role in cross-talk between abiotic stress and cytokinin signaling. These results provide a foundation for further investigations on specific as well as overlapping cellular functions of type-A response regulators in rice.
| S-EPMC1382228 | biostudies-literature
Project description:Strigolactones (SLs), a group of terpenoid lactones derived from carotenoids, are plant hormones that control numerous aspects of plant development. Although the framework of SL signaling that the repressor DWARF 53 (D53) could be SL-dependently degraded via the SL receptor D14 and F-box protein D3 has been established, the downstream response genes to SLs remain to be elucidated. Here we show that the cytokinin (CK) content is dramatically increased in shoot bases of the rice SL signaling mutant d53 By examining transcript levels of all the CK metabolism-related genes after treatment with SL analog GR24, we identified CYTOKININ OXIDASE/DEHYDROGENASE 9 (OsCKX9) as a primary response gene significantly up-regulated within 1 h of treatment in the wild type but not in d53 We also found that OsCKX9 functions as a cytosolic and nuclear dual-localized CK catabolic enzyme, and that the overexpression of OsCKX9 suppresses the browning of d53 calli. Both the CRISPR/Cas9-generated OsCKX9 mutants and OsCKX9-overexpressing transgenic plants showed significant increases in tiller number and decreases in plant height and panicle size, suggesting that the homeostasis of OsCKX9 plays a critical role in regulating rice shoot architecture. Moreover, we identified the CK-inducible rice type-A response regulator OsRR5 as the secondary SL-responsive gene, whose expression is significantly repressed after 4 h of GR24 treatment in the wild type but not in osckx9 These findings reveal a comprehensive plant hormone cross-talk in which SL can induce the expression of OsCKX9 to down-regulate CK content, which in turn triggers the response of downstream genes.
| S-EPMC6628823 | biostudies-literature
Project description:Angustmycin A has anti-mycobacterial and cytokinin activities, and contains an intriguing structure in which an unusual sugar with C5′-C6′ dehydration is linked to adenine via an N-glycosidic bond. However, the logic underlying the biosynthesis of this molecule has long remained obscure. Here, we address angustmycin A biosynthesis by the full deciphering of its pathway. We demonstrate that AgmD, C, A, E, and B function as d-allulose 6-phosphate 3-epimerase, d-allulose 6-phosphate pyrophosphokinase, adenine phosphoallulosyltransferase, phosphoribohydrolase, and phosphatase, respectively, and that these collaboratively catalyze the relay reactions to biosynthesize angustmycin C. Additionally, we provide evidence that AgmF is a noncanonical dehydratase for the final step to angustmycin A via a self-sufficient strategy for cofactor recycling. Finally, we have reconstituted the entire six-enzyme pathway in vitro and in E. coli leading to angustmycin A production. These results expand the enzymatic repertoire regarding natural product biosynthesis, and also open the way for rational and rapid discovery of other angustmycin related antibiotics. Angustmycin A is a nucleoside antibiotic having anti-mycobacterial and cytokinin activities. Here, the authors report the whole pathway leading to angustmycin A biosynthesis in Streptomyces and achieve the heterologous production of angustmycin A in E. coli.
| S-EPMC8599513 | biostudies-literature
Project description:The expression of a KNOX class 1 gene OSH1 is induced by cytokinin during regeneration of shoots from callus in Oryza sativa L. (rice). This cytokinin-induced expression was enhanced by overexpression of homologues of cytokinin-signalling phosphorelay genes such as a histidine kinase gene OHK3, a phosphotransmitter gene OHP2 and a response regulator gene ORR1 in cultured cells. Regionally overlapped expression of these genes and OSH1 was observed in shoot apex. These results suggest that these cytokinin-signalling genes are positive regulators of the expression of OSH1, and mediate the OSH expression upon shoot regeneration from callus in rice.
| S-EPMC6879368 | biostudies-literature
Project description:BackgroundCytokinins play many essential roles in plant growth and development, mainly through signal transduction pathways. Although the cytokinin signaling pathway in rice has been clarified, no synthetic reporter for cytokinin signaling output has been reported for rice. The sensitive synthetic reporter two-component signaling sensor (TCSn) is used in the model plant Arabidopsis; however, whether the reporter reflects the cytokinin signaling output pattern in rice remains unclear.ResultsEarly-cytokinin-responsive type-A OsRR-binding element (A/G)GAT(C/T) was more clustered in the 15 type-A OsRRs than in the 13 control genes. Quantitative polymerase chain reaction analysis showed that the relative expression of seven type-A OsRRs in roots and shoots was significantly induced by exogenous cytokinin application, and that of seven OsRRs, mainly in roots, was inhibited by exogenous auxin application. We constructed a transgenic rice plant harboring a beta-glucuronidase (GUS) driven by the synthetic promoter TCSn. TCSn::GUS was expressed in the meristem of germinated rice seed and rice seedlings. Furthermore, TCSn::GUS expression in rice seedlings was induced specifically by exogenous cytokinin application and decreased by exogenous auxin application. Moreover, no obvious reduction in GUS levels was observed after three generations of selfing of transgenic plants, indicating that TCSn::GUS is not subject to transgene silencing.ConclusionsWe report here a robust and sensitive synthetic sensor for monitoring the transcriptional output of the cytokinin signaling network in rice.
| S-EPMC5658958 | biostudies-literature
Project description:The rice root system is important for growth. The crosstalk between auxin and cytokinin mediates root initiation and elongation. However, it remains unclear how the transcriptional network upstream of the auxin and cytokinin signalling pathways determines root development. Here, we observed that the knockdown of OsNAC2, which encodes a NAC transcription factor, increased the primary root length and the number of crown roots. OsNAC2 predominantly expressed in primary root tips, crown roots and lateral root primordia, implying it influences root development. Molecular analyses revealed that the expressions of auxin- and cytokinin-responsive genes were affected in OsNAC2-overexpressing (OsNAC2-OX; ON7 and ON11), RNA interference (OsNAC2-RNAi; RNAi25 and RNAi31) and CRISPR/Cas9 plants. Additionally, OsNAC2 can directly bind to the promoters of IAA inactivation-related genes (GH3.6 and GH3.8), an IAA signalling-related gene (OsARF25), and a cytokinin oxidase gene (OsCKX4). Furthermore, genetic analysis of ON11/osgh3.6 and RNAi31/osckx4 homozygote confirmed that OsCKX4 and OsGH3.6 functioned downstream of OsNAC2. The mRNA levels of CROWN ROOTLESS (CRL) genes and cyclin-dependent protein kinase (CDK) genes increased in OsNAC2-RNAi and OsNAC2-cas9 lines while reduced in OsNAC2-OX lines. Thus, we describe that OsNAC2 functions as an upstream integrator of auxin and cytokinin signals that affect CRL and CDK production to regulate cell division during root development. This novel auxin-OsNAC2-cytokinin model should provide a new insight into the understanding of NAC TFs and crosstalk of auxin and cytokinin pathway, and can be potentially applied in agriculture to enhance rice yields by genetic approaches.
| S-EPMC6953191 | biostudies-literature