Cytokinin Signal Transduction Research Articles

Dissecting the Roles of the Cytokinin Signaling Network: The Case of De Novo Shoot Apical Meristem Formation.

Numerous biotechnological applications require a fast and efficient clonal propagation of whole plants under controlled laboratory conditions. For most plant species, the de novo regeneration of shoots from the cuttings of various plant organs can be obtained on nutrient media supplemented with plant hormones, auxin and cytokinin. While auxin is needed during the early stages of the process that include the establishment of pluripotent primordia and the subsequent acquisition of organogenic competence, cytokinin-supplemented media are required to induce these primordia to differentiate into developing shoots. The perception of cytokinin through the receptor ARABIDOPSIS HISTIDINE KINASE4 (AHK4) is crucial for the activation of the two main regulators of the establishment and maintenance of shoot apical meristems (SAMs): SHOOTMERISTEMLESS (STM) and the WUSCHEL-CLAVATA3 (WUS-CLV3) regulatory circuit. In this review, we summarize the current knowledge of the roles of the cytokinin signaling cascade in the perception and transduction of signals that are crucial for the de novo establishment of SAMs and lead to the desired biotechnological output-adventitious shoot multiplication. We highlight the functional differences between individual members of the multigene families involved in cytokinin signal transduction, and demonstrate how complex genetic regulation can be achieved through functional specialization of individual gene family members.

Genome-wide identification and expression profiles analysis of the authentic response regulator gene family in licorice.

As one of the traditional Chinese medicinal herbs that were most generally used, licorice attracts lots of interest due to its therapeutic potential. Authentic response regulators (ARRs) are key factors in cytokinin signal transduction and crucial for plant growth and stress response processes. Nevertheless, the characteristics and functions of the licorice ARR genes are still unknown. In present study, a systematic genome-wide identification and expression analysis of the licorice ARR gene family were conducted and 51 ARR members were identified. Collinearity analysis revealed the significant roles of segmental duplications in the expansion of licorice ARR genes. The cis-acting elements associated with development, stress and phytohormone responses were identified, implying their pivotal roles in diverse regulatory processes. RNA-seq and qRT-PCR results suggested that A-type, but not B-type ARRs were induced by zeatin. Additionally, ARRs participated in diverse abiotic stresses and phytohormones responses. Yeast one-hybrid assay demonstrated that GuARR1, GuARR2, GuARR11, GuARR12, GuARR10-1, GuARR10-2 and GuARR14 were able to bind to the promoter of GuARR8-3, and GuARR1, GuARR12 bound to the GuARR8-1 promoter. GuARR1, GuARR2, GuARR11 and GuARR10-2 bound to the GuARR6-2 promoter as well as GuARR12 and GuARR10-2 bound to the GuARR6-1 promoter. Collectively, these findings provide a basis for future ARR genes function investigations, shedding light on the potential medicinal properties and agricultural applications of licorice.

Dual transcriptomic and metabolomic analyses provide novel insights into the role of vitamins A and B metabolism in ectomycorrhizal symbiosis between Pinus yunnanensis and Lactarius deliciosus

AbstractEctomycorrhizal (EM) fungi play important roles in nutrient cycling and plant community establishment in forest ecosystems. Effects of EM formation on global alterations of the transcriptome and metabolome during plant‐fungal interaction and the key metabolites involved in EM development are largely unknown. Here, dual RNA‐Seq and untargeted metabolomic analyses were used to reveal stage‐specific and core responses of Pinus yunnanensis and Lactarius deliciosus during mycorrhizal colonization. We found that L. deliciosus colonization in P. yunnanensis roots induced different transcriptional changes across three interaction stages, with a small core of genes consistently regulated at all stages. Concentrations of retinol (vitamin A) and retinoic acid increased while that of B group vitamins decreased during EM formation, which was coordinately regulated by these two plant‐fungus partners, with L. deliciosus possibly playing a dominant role. Exogenous retinol altered the diameter and mantle thickness of P. yunnanensis ‐ L. deliciosus EM tips and affected host plant growth and phosphorus acquisition. In the absence of L. deliciosus, exogenous retinol increased the root diameter and the number of root tips of P. yunnanensis. Furthermore, the concentration of auxin increased, but that of abscisic acid decreased during EM formation, and the genes involved in plant hormone signal transduction were gradually activated, and auxin and cytokinin signal transduction potentially played a positive role in this EM symbiosis. In conclusion, we propose that the interaction of P. yunnanensis and L. deliciosus alters vitamin metabolism, which may further affect plant hormone biosynthesis and signal transduction, modulating root morphology and EM traits.

Genome-wide identification of the melon (Cucumis melo L.) response regulator gene family and functional analysis of CmRR6 and CmPRR3 in response to cold stress

The response regulator (RR) gene family play crucial roles in cytokinin signal transduction, plant development, and resistance to abiotic stress. However, there are no reports on the identification and functional characterization of RR genes in melon. In this study, a total of 18 CmRRs were identified and classified into type A, type B, and clock PRRs, based on phylogenetic analysis. Most of the CmRRs displayed tissue-specific expression patterns, and some were induced by cold stress according to two RNA-seq datasets. The expression patterns of CmRR2/6/11/15 and CmPRR2/3 under cold treatment were confirmed by qRT-PCR. Subcellular localization assays indicated that CmRR6 and CmPRR3 were primarily localized in the nucleus and chloroplast. Furthermore, when either CmRR6 or CmPRR3 were silenced using tobacco ringspot virus (TRSV), the cold tolerance of the virus-induced gene silencing (VIGS) melon plants were significantly enhanced, as evidenced by measurements of chlorophyll fluorescence, ion leakage, reactive oxygen, proline, and malondialdehyde levels. Additionally, the expression levels of CmCBF1, CmCBF2, and CmCBF3 were significantly increased in CmRR6-silenced and CmPRR3-silenced plants under cold treatment. Our findings suggest that CmRRs contribute to cold stress responses and provide new insights for further pursuing the molecular mechanisms underlying CmRRs-mediated cold tolerance in melon.

Genome-wide identification of CaARR-Bs transcription factor gene family in pepper and their expression patterns under salinity stress

In plants, ARRs-B transcription factors play a crucial role in regulating cytokinin signal transduction, abiotic stress resistance, and plant development. A number of adverse environmental conditions have caused severe losses for the pepper (Capsicum annuum L.)—a significant and economically important vegetable. Among the transcription factors of the type B-ARRs family, multiple members have different functions. In pepper, only a few members of the ARRs-B family have been reported and characterized. The current study aimed to characterize ARRs-B transcription factors in C. annuum, including phylogenetic relationships, gene structures, protein motif arrangement, and RT-qPCR expression analyses and their role in salinity stress. In total, ten genes encode CaARRs-B transcription factors (CaARR1 to CaARR10) from the largest subfamily of type-B ARRs were identified in C. annum. The genome-wide analyses of the CaARRs-B family in C. annuum were performed based on the reported ARRs-B genes in Arabidopsis. An analysis of homologous alignments of candidate genes, including their phylogenetic relationships, gene structures, conserved domains, and qPCR expression profiles, was conducted. In comparison with other plant ARRs-B proteins, CaARRs-B proteins showed gene conservation and potentially specialized functions. In addition, tissue-specific expression profiles showed that CaARRs-B genes were differentially expressed, suggesting functionally divergent. CaARRs-B proteins had a typical conserved domain, including AAR-like (pfam: PF00072) and Myb DNA binding (pfam: PF00249) domains. Ten of the CaARRs-B genes were asymmetrically mapped on seven chromosomes in Pepper. Additionally, the phylogenetic tree of CaARRs-B genes from C. annuum and other plant species revealed that CaARRs-B genes were classified into four clusters, which may have evolved conservatively. Further, using quantitative real-time qRT-PCR, the study assessed the expression patterns of CaARRs-B genes in Capsicum annuum seedlings subjected to salt stress. The study used quantitative real-time qRT-PCR to examine CaARRs-B gene expression in Capsicum annuum seedlings under salt stress. Roots exhibited elevated expression of CaARR2 and CaARR9, while leaves showed decreased expression for CaARR3, CaARR4, CaARR7, and CaARR8. Notably, no amplification was observed for CaARR10. This research sheds light on the roles of CaARRs-B genes in pepper’s response to salinity stress. These findings enrich our comprehension of the functional implications of CaARRs-B genes in pepper, especially in responding to salinity stress, laying a solid groundwork for subsequent in-depth studies and applications in the growth and development of Capsicum annuum.

Genome-Wide Identification of the RR Gene Family and Its Expression Analysis in Response to TDZ Induction in Rhododendron delavayi.

The cytokinin response regulator (RR) gene is essential for cytokinin signal transduction, which plays a crucial role in plant growth and development. Here, we applied bioinformatics to Rhododendron delavayi's genome to identify its RR gene family and systematically analyzed their gene characteristics, phylogenetic evolution, chromosomal localization, collinearity analysis, promoter cis-elements, and expression patterns. Overall, 33 RdRR genes were distinguished and classified into three types. All these genes harbored motif 5 (YEVTTVNSGLEALELLRENKB), the most conserved one, along with the plant-conserved domain (REC domain), and could be mapped to 10 chromosomes with four gene pairs of segmental replication events but no tandem replication events; 13 RdRR genes showed collinearity with Arabidopsis thaliana genes. Promoter analysis revealed multiple hormone-related cis-elements in the RR genes. After a TDZ (thidiazuron) treatment, 13 genes had higher expression levels than the control, whose magnitude of change depended on the developmental stage of leaves' adventitious buds. The expression levels of RdRR14, RdRR17, RdRR20, and RdRR24 agreed with the average number of adventitious buds post-TDZ treatment. We speculate that these four genes could figure prominently in bud regeneration from R. delavayi leaves in vitro. This study provides detailed knowledge of RdRRs for research on cytokinin signaling and RdRR functioning in R. delavayi.

Influence of cultivation practices on the metabolism of cytokinin and its correlation in rice production

AbstractCytokinin (CTK) has an important regulatory effect on plant morphology, physiology, and yield, and is one of the main factors regulating nitrogen absorption, transport, and metabolism. This paper reviews the research progress in nitrogen absorption, transport and metabolism, cytokinin synthesis, transport and signal transduction, etc., focusing on the physiological mechanisms by which CTK and nitrogen collaborate to regulate the root–shoot relationship and its impact on agronomic traits of crops. The synthesis of trans‐zeatin (tZ) and trans‐zeatin riboside (tZR) is generally regulated by root nitrogen, which is transported them to the sprouts, regulates nitrogen allocation and metabolism, and affects photosynthetic characteristics and yield. In young shoots, nitrogen upregulates the synthesis of isopentenyl, adenine (iP), and iP‐nucleoside (iPR) and transports to roots through the phloem, reducing nitrogen uptake and transport, affecting root morphology. Based on this, the role of, CTK in coordinating relationships between source repositories and enhancing injection. We conducted an analysis on how various cultivation methods impact CTK metabolism and its relationship with plant growth. Additionally, we discussed the challenges associated with applying CTK functions to paddy field production and suggested future research directions.

Dynamic transcriptome profiling provides insights into rhizome enlargement in ginger (Zingiber officinale Rosc.).

The rhizome is an economically important part of ginger (Zingiber officinale Rosc.). However, the mechanism of ginger rhizome enlargement remains unclear. In this study, we performed an integrated analysis of the hormone content and transcriptome of ginger at three rhizome enlargement stages: initial enlargement (S1), middle enlargement (S2), and peak enlargement (S3). With rhizome enlargement, the levels of the hormones zeatin (ZT), gibberellic acid (GA), indole acetic acid (IAA), and jasmonic acid (JA) were significantly increased, and this increase was positively correlated with rhizome diameter. Transcriptomic analysis identified a large number of differentially expressed genes (DEGs); the number of DEGs were 2,206 in the transition from S1 to S2, and 1,151 in the transition from S2 to S3. The expression of several genes related to hormone biosynthesis and signalling and cell division or expansion, and transcription factors was significantly altered, which suggests that these genes play essential roles in rhizome enlargement. The results of correlation analysis suggested that the process of ginger rhizome enlargement may be primarily related to the regulation of endogenous cytokinin, GA3, auxin, and JA biosynthesis pathways and signal transduction; GRAS, HB, MYB, MYB122, bZIP60, ARF1, ARF2, E2FB1, and E2FB2, which may regulate the expression of rhizome formation-related genes; and CYC2, CDKB1, CDKB2, EXPA1, and XTH7, which may mediate cell division and expansion. These results provide gene resources and information that will be useful for the molecular breeding in ginger.

Transcriptome Analysis Reveals the Hormone Signalling Coexpression Pathways Involved in Adventitious Root Formation in Populus

Adventitious roots (ARs) occur naturally in many species and are important for plants to absorb nutrients and water. AR formation can also be induced from explants of trees, whose clonal propagation is needed. AR formation is gridlock for many woody plant mass propagations. Plant hormones have been regarded as playing a key role in AR formation, and the molecular regulatory mechanisms need to be elucidated. In this study, RNA-Seq was performed to reveal the molecular mechanisms in the different periods of AR formation from hybrid poplar clone 84K (Populus alba × P. glandulosa) and AUXIN SIGNALING F-BOX (PagFBL1-OE). To understand the importance of differentially expressed genes (DEGs), we found that many genes involved in signal transduction mechanisms were induced at 12, 24 and 48 h in 84K and PagFBL1-OE cells by NOG classification. We also found that many DEGs were enriched in hormone signal transduction only for the first 12 h in 84K and PagFBL1-OE by KEGG pathway enrichment. Notably, more DEGs appeared in indole-3-acetic acid (IAA), abscisic acid (ABA), ethylene (ETH), jasmonic acid (JA), brassinolide (BR), cytokinin (CTK) and gibberellin (GA) signal transduction for the first 12 h in PagFBL1-OE than in 84K. Moreover, ARF (Pop_G01G075686), IAA14 (Pop_A10G047257), SAURs (Pop_A03G019756, Pop_A12G067965, Pop_G03G055849 and Pop_G12G008821), JAR1s (Pop_A14G000375 and Pop_G14G044264), CTR1 (Pop_A17G052594 and Pop_G09G030293), CRE1s (Pop_G07G086605 and Pop_G07G086618), GID1 (Pop_A04G026477), BKI1 (Pop_A02G066155), PYR/PYLs (Pop_A03G050217 and Pop_G01G089222), and TGAs (Pop_A04G059310, Pop_G04G060065 and Pop_G05G008153) were only specifically expressed in PagFBL1-OE and could play an important role in AR formation, especially in the first 12 h under plant hormone signal transduction. These results show that the complex biological process of AR formation is primarily influenced by the hormone signalling pathway in Populus. This study reveals the initial regulation of AR formation in woody plant cuttings and thus contributes to further elucidating the molecular mechanism by which hormones interact.

CcRR5 interacts with CcRR14 and CcSnRK2s to regulate the root development in citrus.

Response regulator (RR) is an important component of the cytokinin (CK) signal transduction system associated with root development and stress resistance in model plants. However, the function of RR gene and the molecular mechanism on regulating the root development in woody plants such as citrus remain unclear. Here, we demonstrate that CcRR5, a member of the type A RR, regulates the morphogenesis of root through interacting with CcRR14 and CcSnRK2s in citrus. CcRR5 is mainly expressed in root tips and young leaves. The activity of CcRR5 promoter triggered by CcRR14 was proved with transient expression assay. Seven SnRK2 family members with highly conserved domains were identified in citrus. Among them, CcSnRK2.3, CcSnRK2.6, CcSnRK2.7, and CcSnRK2.8 can interact with CcRR5 and CcRR14. Phenotypic analysis of CcRR5 overexpressed transgenic citrus plants indicated that the transcription level of CcRR5 was associated with root length and lateral root numbers. This was also correlated to the expression of root-related genes and thus confirmed that CcRR5 is involved in the root development. Taken together, the results of this study indicate that CcRR5 is a positive regulator of root growth and CcRR14 directly regulates the expression of CcRR5. Both CcRR5 and CcRR14 can interact with CcSnRK2s.

Cytokinin Response Factor 9 Represses Cytokinin Responses in Flower Development.

A multi-step phosphorelay system is the main conduit of cytokinin signal transduction. However, several groups of additional factors that also play a role in this signaling pathway have been found-among them the Cytokinin Response Factors (CRFs). In a genetic screen, CRF9 was identified as a regulator of the transcriptional cytokinin response. It is mainly expressed in flowers. Mutational analysis indicates that CRF9 plays a role in the transition from vegetative to reproductive growth and silique development. The CRF9 protein is localized in the nucleus and functions as a transcriptional repressor of Arabidopsis Response Regulator 6 (ARR6)-a primary response gene for cytokinin signaling. The experimental data suggest that CRF9 functions as a repressor of cytokinin during reproductive development.

Insights on the stem elongation of spur-type bud sport mutant of ‘Red Delicious’ apple

The decreased capacity of auxin-, CTK-, and BR-mediated cell division and cell enlargement pathways, combined with the enhanced capacity of GA and ETH-, JA-, ABA-, SA-mediated stress-resistant pathways were presumed to be the crucial reasons for the formation of spur-type 'Red Delicious' mutants. Vallee Spur', which exhibit short internodes and compact tree shape, is the fourth generation of the spur-type bud sport mutant of 'Red Delicious'. However, the underlying molecular mechanism of these properties remains unclear. Here, comparative phenotypic, full-length transcriptome and phytohormone analyses were performed between 'Red Delicious' (NSP) and 'Vallee Spur' (SP). The new shoot internode length of NSP was ˃ 1.53-fold higher than that of the SP mutant. Cytological analysis showed that the stem cells of the SP mutant were smaller and more tightly arranged relative to the NSP. By Iso-Seq, a total of 1426 differentially expressed genes (DEGs) were detected, including 808 upregulated and 618 downregulated genes in new shoot apex with 2 leaves of the SP mutant. Gene expressions involved in auxin, cytokinin (CTK), and brassinosteroid (BR) signal transduction were mostly downregulated in the SP mutant, whereas those involved in gibberellin (GA), ethylene (ETH), jasmonate (JA), ABA, and salicylic acid (SA) signal transduction were mostly upregulated. The overall thermogram analysis of hormone levels in the shoot apex carrying two leaves detected by LC-MS/MS absolute quantification showed that the levels of IAA-Asp, IAA, iP7G, OPDA, and 6-deoxyCS were significantly upregulated in the SP mutant, while the remaining 28 hormones were significantly downregulated. It is speculated that the decreased capacity of auxin, CTK, and BR-mediated cell division and cell enlargement pathways is crucial for the formation of the SP mutant. GA and stress-resistant pathways of ETH, JA, ABA, and SA also play vital roles in stem elongation. These results highlight the involvement of phytohormones in the formation of stem elongation occurring in 'Red Delicious' spur-type bud sport mutants and provide information for exploring its biological mechanism.

Cytokinin Modulates Responses to Phytomelatonin in Arabidopsis thaliana under High Light Stress

Fine-tuned interactions between melatonin (MT) and hormones affected by environmental inputs are crucial for plant growth. Under high light (HL) conditions, melatonin reduced photodamage in Arabidopsis thaliana and contributed to the restoration of the expression of the cytokinin (CK) synthesis genes IPT3, IPT5 and LOG7 and genes for CK signal transduction AHK2,3 and ARR 1, 4, 5 and 12 which were downregulated by stress. However, CK signaling mutants displayed no significant changes in the expression of CK genes following HL + MT treatment, implying that a fully functional cytokinin signaling pathway is a prerequisite for MT-CK interactions. In turn, cytokinin treatment increased the expression of the key melatonin synthesis gene ASMT under both moderate and HL in wild-type plants. This upregulation was further accentuated in the ipt3,5,7 mutant which is highly sensitive to CK. In this mutant, in addition to ASMT, the melatonin synthesis genes SNAT and COMT, as well as the putative signaling genes CAND2 and GPA1, displayed elevated transcript levels. The results of the study suggest that melatonin acts synergistically with CK to cope with HL stress through melatonin-associated activation or repression of the respective hormonal genes.

Histidine kinase inhibitors impair shoot regeneration in Arabidopsis thaliana via cytokinin signaling and SAM patterning determinants.

Reversible protein phosphorylation is a post-translational modification involved in virtually all plant processes, as it mediates protein activity and signal transduction. Here, we probe dynamic protein phosphorylation during de novo shoot organogenesis in Arabidopsis thaliana. We find that application of three kinase inhibitors in various time intervals has different effects on root explants. Short exposures to the putative histidine (His) kinase inhibitor TCSA during the initial days on shoot induction medium (SIM) are detrimental for regeneration in seven natural accessions. Investigation of cytokinin signaling mutants, as well as reporter lines for hormone responses and shoot markers, suggests that TCSA impedes cytokinin signal transduction via AHK3, AHK4, AHP3, and AHP5. A mass spectrometry-based phosphoproteome analysis further reveals profound deregulation of Ser/Thr/Tyr phosphoproteins regulating protein modification, transcription, vesicle trafficking, organ morphogenesis, and cation transport. Among TCSA-responsive factors are prior candidates with a role in shoot apical meristem patterning, such as AGO1, BAM1, PLL5, FIP37, TOP1ALPHA, and RBR1, as well as proteins involved in polar auxin transport (e.g., PIN1) and brassinosteroid signaling (e.g., BIN2). Putative novel regeneration determinants regulated by TCSA include RD2, AT1G52780, PVA11, and AVT1C, while NAIP2, OPS, ARR1, QKY, and aquaporins exhibit differential phospholevels on control SIM. LC-MS/MS data are available via ProteomeXchange with identifier PXD030754.

Genome-Wide Identification and Analysis of the Growth-Regulating Factor Family in Zanthoxylum armatum DC and Functional Analysis of ZaGRF6 in Leaf Size and Longevity Regulation.

Growth-regulating factors (GRFs) are plant-specific transcription factors that play an important role in plant growth and development. In this study, fifteen GRF gene members containing QLQ and WRC domains were identified in Zanthoxylum armatum. Phylogenetic and collinearity analysis showed that ZaGRFs were closely related to CsGRFs and AtGRFs, and distantly related to OsGRFs. There are a large number of cis-acting elements related to hormone response and stress induction in the GRF gene promoter region of Z. armatum. Tissue-specific expression analysis showed that except for ZaGRF7, all the ZaGRFs were highly expressed in young parts with active growth and development, including terminal buds, seeds, and young flowers, suggesting their key roles in Z. armatum growth and development. Eight ZaGRFs were selected to investigate the transcriptional response to auxin, gibberellin and drought treatments. A total of six ZaGRFs in the NAA treatment, four ZaGRFs in the GA3 treatment, and six ZaGRFs in the PEG treatment were induced and significantly up-regulated. Overexpression of ZaGRF6 increased branching and chlorophyll content and delayed senescence of transgenic Nicotiana benthamiana. ZaGRF6 increased the expression of CRF2 and suppressed the expression of ARR4 and CKX1, indicating that ZaGRF6 is involved in cytokinin metabolism and signal transduction. These research results lay a foundation for further analysis of the GRF gene function of Z. armatum and provide candidate genes for growth, development, and stress resistance breeding of Z. armatum.

Physiological, Transcriptomic and Metabolomic Analyses of Overwintering Cryptomeria fortunei Needles

Low temperatures affect plant growth, development, and geographical distribution. Cryptomeriafortunei (Chinese cedar) is a major industrial tree species used for timber manufacturing in southern China. However, its popularization and application in northern China are limited due to its poor low-temperature resistance (approximately −6 °C), and its overwintering mechanism remains unclear. Here, we performed physiological, metabolomic, and transcriptomic analyses of overwintering C.fortunei needles at three stages: before winter, during winter, and in early spring. Physiological analyses showed that electrolyte leakage, H2O2, malondialdehyde (MDA), soluble sugar, and protein contents increased, while the maximum quantum yield of photosystem II (PSII) (Fv/Fm), effective quantum yield of PSII (YII), and chlorophyll content decreased as overwintering progressed. Metabolomic and transcriptomic analyses revealed that downregulated gibberellin (GA), salicylic acid (SA), cytokinin (CTK), and auxin signal transduction and upregulated abscisic acid (ABA), ethylene, brassinosteroids (BR), and jasmonic acid (JA) signal transduction pathways promoted the winter acclimation of C.fortunei, while the opposite expression pattern promoted the transition from dormancy to growth. In addition, upregulated genes/metabolites involved in phenylpropanoid and flavonoid biosynthesis, starch and sucrose metabolism, cold-related protein and fatty acid desaturases, and downregulated photosynthesis-related pathways promoted winter acclimation, while five (WRKY, AP2/ERF, NAC, MYB, and bHLH) and three (AP2/ERF, MYB, and bHLH) transcription factors were associated with winter acclimation and early spring transition from dormancy to growth, respectively. In summary, we report the first transcriptome of overwintering C.fortunei, providing a foundation for the cultivation of and research on overwintering varieties.

Distinct Hormone Signalling-Modulation Activities Characterize Two Maize Endosperm-Specific Type-A Response Regulators.

ZmTCRR1 and 2 are type-A response regulators expressed in the maize endosperm transfer cells (TC). While type-B response regulators transcriptionally control canonical type-A response regulators, as part of the cytokinin signal transduction mechanism, the ZmTCRRs are regulated by ZmMRP1, a master regulator of TC identity. In addition, the corresponding proteins are not detected in the TC, accumulating in the inner endosperm cells instead. These features suggest these molecules are not involved in classical, cell-autonomous, cytokinin signalling pathways. Using transgenic Arabidopsis plants ectopically expressing these genes, we have shown that ZmTCRR1 and 2 can modulate auxin and cytokinin signalling, respectively. In Arabidopsis, the ectopic expression of ZmTCRR2 blocked, almost completely, cytokinin perception. Given the conservation of these signalling pathways at the molecular level, our results suggest that the ZmTCRRs modulate cytokinin and auxin perception in the inner endosperm cells.

Identification of BcARR Genes and CTK Effects on Stalk Development of Flowering Chinese Cabbage.

Flowering Chinese cabbage (Brassica campestris L. ssp. Chinensis var. utilis Tsen et Lee) is an important and extensively cultivated vegetable in south China, whose major food product is the stalk. In the process of stalk formation, its initiation and development are regulated by a series of hormonal signals, such as cytokinin and gibberellin. In this study, we analyzed the effects of zeatin (ZT) and gibberellin A3 (GA3), and their interaction, on the bolting of flowering Chinese cabbage. The results indicated that the three-true-leaf spraying of ZT and GA synthesis inhibitor (PAC) inhibited plant height but increased stem diameter. Cytokinin (CTK) synthesis inhibitor (YZJ) and GA3 treatment increased plant height and decreased stem diameter. In addition, ZT and GA3 co-treated plants displayed antagonistic effect. Further, 19 type-B authentic response regulators (ARR-Bs), the positive regulators of cytokinin signal transduction were identified from flowering Chinese cabbage. Comprehensive analysis of phylogeny showed BcARR-Bs clustered into three subfamilies with 10 conserved motifs. Analysis of their expression patterns in different tissues and at various growth stage, and their response to hormone treatment suggest that ARR1-b localized in the nucleus displayed unique highest expression patterns in stem tips, are responsive both to ZT and GA, suggesting a significant role in mediating the crosstalk of ZT and GA in the bolting of flowering Chinese cabbage.

Genome-wide mining of B-type cytokinin response regulators in wheat reveals the involvement of

Context Cytokinin response regulators (RRs) are important components of the two component signal systems that are involved in the regulation of plant growth and development, and in the response to abiotic stresses. Plant cytokinin response regulators (RR) were divided into type A and type B. A-type RR proteins act as negative feedback to regulate cytokinin signals, while B-type RRs could regulate A-type RR gene expression, and B-type RR genes have proved to play important roles in regulating cytokinin signal transduction in various biological processes. Aims We aimed to explore and analyse B-type RR genes in wheat in a preliminary fashion. Methods Using bioinformatics methods, wheat type B RR genes were identified, and type B Triticum aestivum RR (TaRR) genes were analysed using quantitative real-time polymerase chain reaction. In order to further analyse the function of TaRR, staining experiments were performed. Key results Twenty-nine B-type TaRR genes were identified in the wheat genome, divided into three groups according to their phylogenetic relationships. Chromosome mapping showed that 29 TaRRs were evenly distributed on 12 chromosomes, while there were no genes located on the other nine chromosomes, which may have experienced gene loss during evolution. The polymerase chain reaction results showed that TaRRs were significantly up-regulated under polyethylene glycol treatments. Under sodium chloride stress, TaRRs were up-regulated to varying degrees, reaching the maximum at 24 h. The study also found that the expression pattern of TaRRs was different in the root and leaf under different abiotic stresses. In addition, staining experiments also showed that TaRR5.1-6A could induce the self-defence function of leaves. Conclusions These results form the basis for further exploring the role of B-type TaRR genes in plant response to drought stress and salt stress. Implications This study lays the molecular biology foundation for the functional study of the B-type TaRR genes.

The adjuvant activity of two urea derivatives on cytokinins: an example of serendipitous dual effect

The aim of this study was to investigate the action spectrum of two urea derivatives, the 1,3-di(benzo[d]oxazol-5-yl)urea (5-BDPU) and the 1,3-di(benzo[d]oxazol-6-yl)urea (6-BDPU). In order to evaluate a possible adjuvant activity on cytokinins the compounds alone or in the simultaneous presence of different cytokinins were assayed either on in vitro typical cytokinin-related bioassays, or on in planta interaction with cytokinin signal transduction pathway. The compounds ability to activate the cytokinin receptor CRE1/AHK4 was studied either by a heterologous bacterial assay or by a competitive binding assay and docking simulations were performed with the crystal structure of the same receptor. Then, owing to their chemical structure which resembles that of urea-type cytokinins, the ability of 5- and 6-BDPU to inhibit the activity of cytokinin oxidase/dehydrogenase of Zea mays (ZmCKX1) was investigated and docking simulations were performed as well. Accordingly to the experimental results, we speculate that BDPUs could show a dual activity: the blocking of the conformational re-adaption of CRE1/AHK4 receptor maintaining the cytokinin inside its binding pocket, thus possibly enhancing its kinase action; the inhibition of cytokinin oxidase/dehydrogenase activity thus possibly preventing its cleavage of natural cytokinins with isoprenoid side chain.Graphic abstract