Cytokinin Response Regulators Research Articles

Identification of abiotic stress responsive genes: A genome wide analysis of the cytokinin response regulator gene family in rice

Identification of abiotic stress responsive genes: A genome wide analysis of the cytokinin response regulator gene family in rice

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.

Ectopic expression of the apple cytokinin response regulator MdRR9 gene in tomatoes promotes shoot branching

Response regulator 9 (RR9) is a typical member of type-A RRs whose transcription is rapidly increased by exogenous cytokinin (CTK). CTK stimulates the outgrowth of axillary buds in apples (Malus domestica). Previous studies showed that the decreased bud outgrowth activity of type-A rr mutants is paradoxical given that type-A RRs act as negative feedback regulators of CTK signaling in Arabidopsis. However, no systematic study of RR9 has been performed in apples. In this study, the genomic sequences of MdRR9, located on chromosome 14, were obtained. Sequencing identified MdRR9 as having an open reading frame (ORF) of 648 bp. Expression pattern showed MdRR9 has the highest expression level in the stem. MdRR9 was up-regulated earlier in response to 6-benzylaminopurine (6-BA, a synthetic type of CTK) than decapitation treatment. The subcellular localization of MdRR9 was in the nucleus. Ectopic expression of MdRR9 in tomatoes indicates that MdRR9 plays a positive role in branch control. A yeast double-hybrid assay showed that MdRR9 may interact with MADS-box transcription factor 22-like (MdAGL9), which gives us a new perspective to study the regulation of the bud outgrowth mechanism of RR9 in the future.

Mining key genes related to root morphogenesis through genome-wide identification and expression analysis of RR gene family in citrus.

Morphogenesis of root is a vital factor to determine the root system architecture. Cytokinin response regulators (RRs) are the key transcription factors in cytokinin signaling, which play important roles in regulating the root morphogenesis. In this study, 29 RR proteins, including 21 RRs and 8 pseudo RRs, were identified from the genome of citrus, and termed as CcRR1-21 and CcPRR1-8, respectively. Phylogenetic analysis revealed that the 29 CcRRs could be classified into four types according to their representative domains. Analysis of cis-elements of CcRRs indicated that they were possibly involved in the regulation of growth and abiotic stress resistance in citrus. Within the type A and type B CcRRs, CcRR4, CcRR5, CcRR6 and CcRR16 highly expressed in roots and leaves, and dramatically responded to the treatments of hormones and abiotic stresses. CcRR2, CcRR10, CcRR14 and CcRR19 also highly expressed in roots under different treatments. Characteristic analysis revealed that the above 8 CcRRs significantly and differentially expressed in the three zones of root, suggesting their functional differences in regulating root growth and development. Further investigation of the 3 highly and differentially expressed CcRRs, CcRR5, CcRR10 and CcRR14, in 9 citrus rootstocks showed that the expression of CcRR5, CcRR10 and CcRR14 was significantly correlated to the length of primary root, the number of lateral roots, and both primary root and the number of lateral roots, respectively. Results of this study indicated that CcRRs were involved in regulating the growth and development of the root in citrus with different functions among the members.

Genome-Wide Identification and Expression Analysis of Cytokinin Response Regulator (RR) Genes in the Woody Plant Jatropha curcas and Functional Analysis of JcRR12 in Arabidopsis.

The cytokinin (CK) response regulator (RR) gene family plays a pivotal role in regulating the developmental and environmental responses of plants. Axillary bud outgrowth in the perennial woody plant Jatropha curcas is regulated by the crosstalk between CK and gibberellins (GA). In this study, we first analyzed the effects of gibberellin A3 (GA3), lovastatin (a CK synthesis inhibitor), decapitation, and their interaction, on the outgrowth of axillary buds. The results indicate that lovastatin completely inhibited GA-promoted axillary bud outgrowth and partially weakened the decapitation-promoted axillary bud outgrowth. To further characterize and understand the role of CK signaling in promoting the development of female flowers and branches, we performed bioinformatics and expression analyses to characterize the CK RR gene (JcRR) family in J. curcas. A total of 14 members of the JcRR family were identified; these genes were distributed on 10 chromosomes. Phylogenetic analysis indicated that the corresponding RR proteins are evolutionarily conserved across different plant species, and the Myb-like DNA-binding domain divides the 14 members of the JcRR family into type-A and type-B proteins. Further analysis of cis-acting elements in the promoter regions of JcRRs suggests that JcRRs are expressed in response to phytohormones, light, and abiotic stress factors; thus, JcRRs may be involved in some plant development processes. Genomic sequence comparison revealed that segmental duplication may have played crucial roles in the expansion of the JcRR gene family, and five pairs of duplicated genes were all subjected to purifying selection. By analyzing RNA sequencing (RNA-seq) and quantitative reverse transcription-polymerase chain reaction (qRT–PCR) data, we characterized that the temporospatial expression patterns of JcRRs during the development of various tissues and the response of these genes to phytohormones and abiotic stress. The JcRRs were mainly expressed in the roots, while they also exhibited differential expression patterns in other tissues. The expression levels of all six type-A and one type-B JcRRs increased in response to 6-benzylaminopurine (6-BA), while the four type-B JcRRs levels decreased. The expression levels of two type-B JcRRs increased in response to exogenous GA3 treatment, while those of three type-A and three type-B JcRRs decreased. We found that type-A JcRRs may play a positive role in the continuous growth of axillary buds, while the role of type-B JcRRs might be the opposite. In response to abiotic stress, the expression levels of two type-A and three type-B JcRRs strongly increased. The overexpression of JcRR12 in Arabidopsis thaliana slightly increased the numbers of rosette branches after decapitation, but not under normal conditions. In conclusion, our results provide detailed knowledge of JcRRs for further analysis of CK signaling and JcRR functions in J. curcas.

Repeated turnovers keep sex chromosomes young in willows

BackgroundSalicaceae species have diverse sex determination systems and frequent sex chromosome turnovers. However, compared with poplars, the diversity of sex determination in willows is poorly understood, and little is known about the evolutionary forces driving their turnover. Here, we characterized the sex determination in two Salix species, S. chaenomeloides and S. arbutifolia, which have an XY system on chromosome 7 and 15, respectively.ResultsBased on the assemblies of their sex determination regions, we found that the sex determination mechanism of willows may have underlying similarities with poplars, both involving intact and/or partial homologs of a type A cytokinin response regulator (RR) gene. Comparative analyses suggested that at least two sex turnover events have occurred in Salix, one preserving the ancestral pattern of male heterogamety, and the other changing heterogametic sex from XY to ZW, which could be partly explained by the “deleterious mutation load” and “sexually antagonistic selection” theoretical models. We hypothesize that these repeated turnovers keep sex chromosomes of willow species in a perpetually young state, leading to limited degeneration.ConclusionsOur findings further improve the evolutionary trajectory of sex chromosomes in Salicaceae species, explore the evolutionary forces driving the repeated turnovers of their sex chromosomes, and provide a valuable reference for the study of sex chromosomes in other species.

A chromosome-level Populus qiongdaoensis genome assembly provides insights into tropical adaptation and a cryptic turnover of sex determination.

Populus species have long been used as model organisms to study the adaptability of trees and the evolution of sex chromosomes. As a species belonging to the section Populus and limited to tropical areas, the P. qiongdaoensis genome contains important information for tropical poplar studies and protection. Here, we report a chromosome-level genome assembly and annotation of a female P. qiongdaoensis. Gene family clustering, positive selection detection and historical reconstruction of population dynamics revealed the tropical adaptation of P. qiongdaoensis, and showed convergent evolution with another tropical poplar, P. ilicifolia, at the molecular level, especially on some functional genes (e.g., PIF3 and PIL1). In addition, we also identified a ZW sex determination system on chromosome 19 of P. qiongdaoensis, and inferred that it seems to have a similar sex determination mechanism to other poplars, controlled by a type-A cytokinin response regulator (RR) gene. However, comparison and phylogenetic analysis of the sex determination regions confirmed a cryptic sex turnover event in the section Populus, which may be caused by the translocation and duplication of the RR gene driven by Helitron-like transposable elements. Our study provides new insights into the environmental adaptation and sex chromosome evolution of poplars, and emphasizes the importance of using long read sequencing in ecological and evolutionary inferences of plants.

Genome-Wide Characterization of Cytokinin Response Regulator in Grape and Expression Analyses during Berry Set Process

Genome-Wide Characterization of Cytokinin Response Regulator in Grape and Expression Analyses during Berry Set Process

Gain-of-function of the cytokinin response activator ARR1 increases heat shock tolerance in Arabidopsis thaliana

ABSTRACT In addition to its well-established role in plant development, the hormone cytokinin regulates plant responses to biotic and abiotic stresses. It was previously shown that cytokinin signaling acts negatively upon drought and osmotic stress tolerance and that gain-of-function of the cytokinin response regulator ARR1 causes osmotic stress hypersensitivity. Here we show that increased ARR1 action increases tolerance to heat shock and that this is correlated with increased accumulation of the heat shock proteins Hsp17.6 and Hsp70. These results show that the heat shock tolerance of plants can be elevated by increasing the expression of a cytokinin response activator.

Time-Resolved Analysis of Candidate Gene Expression and Ambient Temperature During Bud Dormancy in Apple.

Winter dormancy – a period of low metabolic activity and no visible growth – appears as an adaptation to harsh winter conditions and can be divided into different phases. It is tightly controlled by environmental cues, with ambient temperature playing a major role. During endodormancy, a cultivar-specific amount of cold needs to be perceived, and during ecodormancy, heat hours accumulate before bud burst and anthesis in spring. Expression analysis, performed in several key fruit tree species, proved to be very useful in elucidating the molecular control of onset and release of dormancy. However, the time resolution of these experiments has been limited. Therefore, in this study, dense time-series expression analysis was conducted for 40 candidate genes involved in dormancy control, under the cool-temperate climate conditions in Dresden. Samples were taken from the cultivars ‘Pinova’ and ‘Gala,’ which differ in flowering time. The set of candidate genes included well-established dormancy genes such as DAM genes, MdFLC-like, MdICE1, MdPRE 1, and MdPIF4. Furthermore, we tested genes from dormancy-associated pathways including the brassinosteroid, gibberellic acid, abscisic acid (ABA), cytokinin response, and respiratory stress pathways. The expression patterns of well-established dormancy genes were confirmed and could be associated with specific dormancy phases. In addition, less well-known transcription factors and genes of the ABA signaling pathway showed associations with dormancy progression. The three ABA signaling genes HAB1_chr15, HAI3, and ABF2 showed a local minimum of gene expression in proximity of the endodormancy to ecodormancy transition. The number of sampling points allowed us to correlate expression values with temperature data, which revealed significant correlations of ambient temperature with the expression of the Malus domestica genes MdICE1, MdPIF4, MdFLC-like, HAB1chr15, and the type-B cytokinin response regulator BRR9. Interestingly, the slope of the linear correlation of temperature with the expression of MdPIF4 differed between cultivars. Whether the strength of inducibility of MdPIF4 expression by low temperature differs between the ‘Pinova’ and ‘Gala’ alleles needs to be tested further.

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.

Shy Girl, a kiwifruit suppressor of feminization, restricts gynoecium development via regulation of cytokinin metabolism and signalling.

Kiwifruit (Actinidia chinensis) is a dioecious, long-living woody perennial vine. Reduced generation time and induction of hermaphroditism can accelerate crop improvement and facilitate alternative farming for better food security in the face of climate change. Previous studies identified that CENTRORADIALIS genes CEN and CEN4 act to repress flowering, whilst the male-specific Shy Girl (SyGl) gene with homology to type-C cytokinin response regulators could repress gynoecium development in model plants. Here we use CRISPR/Cas9 to mutagenize CEN, CEN4 and SyGl in the male kiwifruit A. chinensis 'Bruce'. Biallelic mutations of CEN and CEN4 generated rapid-flowering male plants, and simultaneous targeting of CEN4 and SyGl gave rise to rapid-flowering hermaphrodites with restored gynoecial function and viable pollen, providing functional evidence for the role of SyGl in suppression of feminization. Analysis of ovary tissues identified genes that contribute to carpel development and revealed that SyGl affected both cytokinin profiles and the expression of genes involved in cytokinin metabolism and signalling. The plant lines generated by CEN4/SyGl knockout could self-pollinate and produce fast-flowering offspring. These results establish that SyGI acts as the suppressor of feminization in kiwifruit and demonstrate the potential for accelerated breeding in an outcrossing horticultural woody perennial.

A General Model to Explain Repeated Turnovers of Sex Determination in the Salicaceae.

Dioecy, the presence of separate sexes on distinct individuals, has evolved repeatedly in multiple plant lineages. However, the specific mechanisms by which sex systems evolve and their commonalities among plant species remain poorly understood. With both XY and ZW sex systems, the family Salicaceae provides a system to uncover the evolutionary forces driving sex chromosome turnovers. In this study, we performed a genome-wide association study to characterize sex determination in two Populus species, P. euphratica and P. alba. Our results reveal an XY system of sex determination on chromosome 14 of P. euphratica, and a ZW system on chromosome 19 of P. alba. We further assembled the corresponding sex-determination regions, and found that their sex chromosome turnovers may be driven by the repeated translocations of a Helitron-like transposon. During the translocation, this factor may have captured partial or intact sequences that are orthologous to a type-A cytokinin response regulator gene. Based on results from this and other recently published studies, we hypothesize that this gene may act as a master regulator of sex determination for the entire family. We propose a general model to explain how the XY and ZW sex systems in this family can be determined by the same RR gene. Our study provides new insights into the diversification of incipient sex chromosomes in flowering plants by showing how transposition and rearrangement of a single gene can control sex in both XY and ZW systems.

Type-B cytokinin response regulators link hormonal stimuli and molecular responses during the transition from endo- to ecodormancy in apple buds.

Cytokinin together with MdoBRR1, MdoBRR8 and MdoBRR10 genes participate in the downregulation of MdoDAM1, contributing to the transition from endo- to ecodormancy in apple buds. The final step of cytokinin (CK) signaling pathway culminates in the activation of type-B response regulators (BRRs), important transcriptional factors in the modulation of CK-responsive genes. In this study, we performed a genome-wide analysis aiming to identify apple BRR family members and understand their involvement in bud dormancy control. The investigation identified ten MdoBRR protein-coding genes. A higher expression of three MdoBRR (MdoBRR1, MdoBRR9 and MdoBRR10) was observed in dormant buds in comparison to other developmental stages. Interestingly, in ecodormant buds these three MdoBRR genes were upregulated in a CK-dependent manner. Transcription profiles, determined during dormancy cycle under field and artificially controlled conditions, revealed that MdoBRR1 and MdoBRR8 played important roles in the transition from endo- to ecodormancy, probably mediated by endogenous CK stimuli. The expression of MdoBRR7, MdoBRR9, and MdoBRR10 was induced in ecodormant buds exposed to warm temperatures, indicating a putative role in growth resumption after chilling requirement fulfillment. Contrasting expression patternsin vivo between MdoBRRs and MdoDAM1, an essential dormancy establishment regulator, were observed during dormancy cycle and in CK-treated buds. Thereafter, in vivo transactivation assays showed that CK stimuli combined with transient overexpression of MdoBRR1, MdoBRR8, and MdoBRR10 resulted in downregulation of the reporter gene gusA driven by the MdoDAM1 promoter. These pieces of evidences point to the integration of CK-triggered responses through MdoBRRs that are able to downregulate MdoDAM1, contributing to dormancy release in apple.

Identification of Genes Related to Mesocarp Development in Cucumber

Cucumber (Cucumis sativus L.) is an agriculturally and economically important vegetable crop worldwide. The thickness of mesocarp is an important quality pertaining to edibility. The development of mesocarp, however, has received little attention. In this study, we selected one thick mesocarp line D8 to analyze the gene expression profiles of mesocarp at 0, 3, 6, and 9 days after pollination (DAP) by RNA sequencing. Genes mainly related to ribosomes, carbon metabolism, biosynthesis of amino acids, and oxidative phosphorylation were up-regulated at 3 DAP. From 3 to 6 DAP, the genes related to oxidative phosphorylation, photosynthesis, and phagosome were highly induced. From 6 to 9 DAP, the genes related to plant hormone signal transduction were highly induced. Differentially expressed genes linked to cyclin, hormone biosynthesis and signaling, carbon metabolism, and transcription factors MYB and WRKY were common up-regulated at 3, 6, 9 DAP in comparison with 0 DAP. The auxin-responsive and cytokinin-responsive genes were significantly up-regulated in mesocarp development. The higher expression levels of the auxin-responsive gene indole-3-acetic acid-amido synthetase 3.6 and the cytokinin response regulator 3/4b in the thick mesocarp cultivar D8 than in the thin mesocarp cultivar XUE1 might have explained the different growth rates of the mesocarp.

TaARR1, a cytokinin response regulator gene in Triticum aestivum, is essential in plant N starvation tolerance via regulating the N acquisition and N assimilation

Plant N starvation response is closely associated with the N signaling components that involve transduction of the low-N cues. In this study, we functionally characterized TaARR1, a cytokinin (CK) response regulator gene in Triticum aestivum, in mediating the N starvation adaptation in plants. TaARR1 harbors two conserved domains specified by plant ARR family members; subcellular localization analysis indicated its target onto nucleus after endoplasmic reticulum assortment. TaARR1 displayed modified expression upon the N starvation stressor, showing upregulated expression in roots and leaves over a 27-h N starvation treatment and whose induced transcripts were gradually recovered along with progression of the N recovery treatment. The tobacco lines overexpressing TaARR1 displayed improved low-N stress tolerance, displaying enlarged phenotype, increased biomass and N accumulation, and enhanced glutamine synthetase (GS) activities compared with wild type (WT) following the N starvation treatment. Expression analysis on genes encoding the nitrate transporter (NRT) and GS proteins in Nicotiana tabacum revealed that NtNRT2.2 and NtGS3 are upregulated in expression in the N-deprived transgenic lines, whose expression patterns were contrasted to other above family genes that were unaltered on transcripts between the transgenic lines and WT. Transgene analysis validated the function of NtNRT2.2 and NtGS3 in regulating N accumulation, GS activity, growth traits, and N use efficiency in plants. These results suggested the internal connection between the TaARR1-mediated N starvation tolerance and the modified transcription of distinct N acquisition- and assimilation-associated genes. Our investigation together indicates that TaARR1 is essential in plant N starvation adaptation due to the gene function in transcriptionally regulating distinct NRT and GS genes that affect plant N uptake and assimilation under the N starvation condition.

Involvement of cytokinin response regulator RhRR1 in the control of flowering

Flowering at a suitable time is critical for ensuring reproductive success in the plant life cycle. The transition from vegetative growth to reproduction development is finely tuned by environmental and endogenous signals. To date, control of flowering involves five genetically defined pathways. However, the role of type-A response regulator genes in regulation of this process remains largely unclear. In the present study, we cloned and characterized a type-A response regulator gene (RhRR1) in rose. The expression of RhRR1 significantly increased in axillary bud during the transition from the vegetative growth to the start of floral differentiation, and in rose flowers in response to exogenous cytokinin or 1-methylcyclopropene (1-MCP) treatments, while that expression was markedly repressed by ethylene treatment. RhRR1 has the highest degree of sequence homology to AtARR8 and AtARR9, and is localized in the nucleus. Ectopic expression RhRR1 in Arabidopsis promoted early flowering, accompanied with the less rosette leaf number at bolting, and shorter bolting time after transferring the plants into pots. In addition, the expression of flowering regulatory genes in RhRR1 transgenic Arabidopsis, including FLOWERING LOCUS D, GA REQUIRING 1, LUMINIDEPENDENS, LEAFY, and TWIN SISTER OF FT clearly increased. These results allow us to infer that RhRR1 plays a key role in the control of flowering.

Transcriptomic analysis reveals the regulatory module of apple (Malus \xd7 domestica) floral transition in response to 6-BA

BackgroundInsufficient production of flower buds is an intractable problem in ‘Fuji’ apple orchards. Although cytokinin (CK) promotes flower bud formation in apple trees, little is known about the mechanisms regulating this phenomenon.ResultsIn the present study, high-throughput RNA sequencing (RNA-Seq) of ‘Nagafu No. 2’ buds was conducted to characterize the transcriptional response to 6-BA treatment during key period of floral transition. A weighted gene co-expression network analysis (WGCNA) of the differentially expressed genes identified hormone signal transduction pathways, totaling 84 genes were highly correlated with the expression pattern of flowering-time genes. The up-regulation of CK signal components and a gibberellin (GA) signal repressor were found to contribute to the promotion of floral transition. In relative comparison to non-treated buds, a series of sugar metabolism- and signal- related genes were associated with relatively high levels of sucrose, fructose, and glucose during floral induction in the 6-BA treated buds. Several transcription factors (i.e. SPLs, SOC1, FD, and COL) that are involved in GA, aging, and photoperiod-regulated flowering pathways were also upregulated by the 6-BA treatment. In addition, potential transcription factors integrating CK signaling to trigger floral induction in apple were also assessed; including PHYTO-CHROME-INTERACTING FACTOR (PIF1,3), WUSCHEL-related homeobox (WOX3,13), and CK response regulators (ARR2).ConclusionsThe present study provides insight into the response of flowering and development-related pathways and transcription factors to 6-BA during the period of floral transition in apple. It extends our knowledge of the fundamental mechanisms associated with CK-regulated floral transition in apple trees.

Expression profiles of organogenesis-related genes over the time course of one-step de novo shoot organogenesis from intact seedlings of kohlrabi

Kohlrabi (Brassica oleracea var. gongylodes) is an important vegetable crop that is able to undergo shoot regeneration in culture from intact seedlings in a single-step regeneration process, using cytokinin as the only plant growth regulator. In this work, we present the expression profiles of seven organogenesis-related genes over the time course of shoot regeneration from intact seedlings of kohlrabi cv. Vienna Purple on shoot regeneration media containing trans-zeatin, cis-zeatin, benzyl adenine or thidiazuron. Two auxin transporter genes – PIN3 and PIN4, a cytokinin response regulator – ARR5, two shoot apical meristem-related transcription factors – CUC1 and RGD3, and two cell cycle-related genes – CDKB2;1 and CYCB2;4 – displayed bimodal expression patterns on most cytokinin-containing media when their expression levels were normalized against control plants grown on hormone-free media. The first expression peak corresponded to direct upregulation by cytokinin from the growth media, and the second one reflected transcriptional events related to callus formation and/or acquisition of organogenic competence, corresponding to the shoot regeneration phases that have already been characterized in Arabidopsis thaliana. We demonstrate that the genes involved in the two-step shoot regeneration of Arabidopsis display their expected expression profiles during the single-step shoot regeneration of its close phylogenetic relative kohlrabi confirming the universality of their roles in the distinct phases of the regeneration process in Brassicaceae. The results presented here represent a first step towards genetic characterization of the morphogenetic processes in this important crop species.

RhERF113 Functions in Ethylene-Induced Petal Senescence by Modulating Cytokinin Content in Rose.

In rose (Rosa hybrida), flower senescence is accelerated by ethylene and delayed by cytokinins (CTKs). However, the effectors that regulate these processes are not currently understood. In this study, we identified an APETALA2/ethylene-responsive factor (AP2/ERF) gene, RhERF113, which was induced by ethylene and up-regulated during flower senescence in most floral organs, including sepal, petal, stamen and pistil. The virus-induced gene silencing (VIGS) of RhERF113 expression accelerated rose flower senescence, which was accompanied by a lower CTK content in the flowers. This accelerated senescence could be restored by exogenous CTK treatment. Moreover, the expression levels of genes related to CTK biosynthesis and signaling, including ISOPENTENYL TRANSFERASE 5 (RhIPT5), RhIPT8, HISTIDINE KINASE 2 (RhHK2), RhHK3, CYTOKININ RESPONSE REGULATOR 3 (RhCRR3), RhCRR5, RhCRR8, HOMEOBOX PROTEIN 6 (RhHB6) and PATHOGENESIS-RELATED 10.1 (RhPR10.1), were decreased in the RhERF113-silenced rose flowers. Taken together, our results demonstrate that RhERF113 delays ethylene-induced flower senescence by increasing the CTK content of the floral tissues.