Change in Auxin and Cytokinin Levels Coincides with Altered Expression of Branching Genes during Axillary Bud Outgrowth in Chrysanthemum.
ABSTRACT: In the production and breeding of Chrysanthemum sp., shoot branching is an important quality aspect as the outgrowth of axillary buds determines the final plant shape. Bud outgrowth is mainly controlled by apical dominance and the crosstalk between the plant hormones auxin, cytokinin and strigolactone. In this work the hormonal and genetic regulation of axillary bud outgrowth was studied in two differently branching cut flower Chrysanthemum morifolium (Ramat) genotypes. C17 is a split-type which forms an inflorescence meristem after a certain vegetative period, while C18 remains vegetative under long day conditions. Plant growth of both genotypes was monitored during 5 subsequent weeks starting one week before flower initiation occurred in C17. Axillary bud outgrowth was measured weekly and samples of shoot apex, stem and axillary buds were taken during the first two weeks. We combined auxin and cytokinin measurements by UPLC-MS/MS with RT-qPCR expression analysis of genes involved in shoot branching regulation pathways in chrysanthemum. These included bud development genes (CmBRC1, CmDRM1, CmSTM, CmLsL), auxin pathway genes (CmPIN1, CmTIR3, CmTIR1, CmAXR1, CmAXR6, CmAXR2, CmIAA16, CmIAA12), cytokinin pathway genes (CmIPT3, CmHK3, CmRR1) and strigolactone genes (CmMAX1 and CmMAX2). Genotype C17 showed a release from apical dominance after floral transition coinciding with decreased auxin and increased cytokinin levels in the subapical axillary buds. As opposed to C17, C18 maintained strong apical dominance with vegetative growth throughout the experiment. Here high auxin levels and decreasing cytokinin levels in axillary buds and stem were measured. A differential expression of several branching genes accompanied the different hormonal change and bud outgrowth in C17 and C18. This was clear for the strigolactone biosynthesis gene CmMAX1, the transcription factor CmBRC1 and the dormancy associated gene CmDRM1, that all showed a decreased expression in C17 at floral transition and an increased expression in C18 with continuous vegetative growth. These results offer a case study for Chrysanthemum, showing an altered cytokinin to auxin balance and differential gene expression between vegetative growth with apical dominance and transition to generative growth with loss of apical dominance and axillary bud outgrowth. This suggests a conservation of several aspects of the hormonal and genetical regulation of bud outgrowth in Chrysanthemum. Furthermore, 15 previously uncharacterised genes in chrysanthemum, were described in this study. Of those genes involved in axillary bud outgrowth we identified CmDRM1, CmBRC1 and CmMAX1 as having an altered expression preceding axillary bud outgrowth, which could be useful as markers for bud activity.
Project description:The diverse plasticity of plant architecture is largely determined by shoot branching. Shoot branching is an event regulated by multiple environmental, developmental and hormonal stimuli through triggering lateral bud response. After perceiving these signals, the lateral buds will respond and make a decision on whether to grow out. TCP transcriptional factors, BRC1/TB1/FC1, were previously proven to be involved in local inhibition of shoot branching in Arabidopsis, pea, tomato, maize and rice. To investigate the function of BRC1, we isolated the BRC1 homolog from chrysanthemum. There were two transcripts of DgBRC1 coming from two alleles in one locus, both of which complemented the multiple branches phenotype of Arabidopsis brc1-1, indicating that both are functionally conserved. DgBRC1 was mainly expressed in dormant axillary buds, and down-regulated at the bud activation stage, and up-regulated by higher planting densities. DgBRC1 transcripts could respond to apical auxin supply and polar auxin transport. Moreover, we found that the acropetal cytokinin stream promoted branch outgrowth whether or not apical auxin was present. Basipetal cytokinin promoted outgrowth of branches in the absence of apical auxin, while strengthening the inhibitory effects on lower buds in the presence of apical auxin. The influence of auxin and strigolactons (SLs) on the production of cytokinin was investigated, we found that auxin locally down-regulated biosynthesis of cytokinin in nodes, SLs also down-regulated the biosynthesis of cytokinin, the interactions among these phytohormones need further investigation.
Project description:Chrysanthemum (Dendranthema grandiflorum cv. Jinba) shoot branching is determined by bud outgrowth during the vegetative growth stage. The degree of axillary bud outgrowth is highly influenced by environmental conditions, such as nutrient availability. Here, we demonstrated that phosphorus (Pi) starvation significantly reduces axillary bud outgrowth in chrysanthemum. A strigolactone (SL) biosynthesis gene, DgCCD7, was isolated and characterized as an ortholog of MAX3/DAD3/RMS5/D17. By using ultra-performance liquid chromatography coupled with mass spectrometry (UPLC-MS), three putative SLs were identified and levels of all three SLs showed strong increase under Pi starvation conditions. Determinations of the distribution of SLs and regulation of DgCCD7/8 in response to Pi changes in root indicate that SL acts systemically. However, temporal expression patterns of biosynthesis and signaling genes in nodes revealed that Pi starvation causes a local response of SL pathway. Treatment of node segments with or without auxin and Pi revealed that in the absence of exogenous auxin, Pi delayed axillary buds outgrowth and up-regulated local SL pathway genes. These data indicated that an auxin-SL regulatory loop responded to Pi starvation for delaying bud outgrowth locally, root biosynthesized SLs were transported acropetally and functioned in shoot branching inhibition under Pi starvation. We proposed that SLs contributed to chrysanthemum shoot branching control in response to Pi-limiting conditions in a systemic way.
Project description:For almost a century the plant hormone auxin has been central to theories on apical dominance, whereby the growing shoot tip suppresses the growth of the axillary buds below. According to the classic model, the auxin indole-3-acetic acid is produced in the shoot tip and transported down the stem, where it inhibits bud growth. We report here that the initiation of bud growth after shoot tip loss cannot be dependent on apical auxin supply because we observe bud release up to 24 h before changes in auxin content in the adjacent stem. After the loss of the shoot tip, sugars are rapidly redistributed over large distances and accumulate in axillary buds within a timeframe that correlates with bud release. Moreover, artificially increasing sucrose levels in plants represses the expression of BRANCHED1 (BRC1), the key transcriptional regulator responsible for maintaining bud dormancy, and results in rapid bud release. An enhancement in sugar supply is both necessary and sufficient for suppressed buds to be released from apical dominance. Our data support a theory of apical dominance whereby the shoot tip's strong demand for sugars inhibits axillary bud outgrowth by limiting the amount of sugar translocated to those buds.
Project description:BACKGROUND:Axillary buds allow the production of either vegetative or reproductive shoots, which display a plastic developmental potential of the plant to suit the prevailing environmental changes. Strawberry represents one of many plant species which displays horizontal above-ground growth of shoot development for asexual reproduction. Two distinct runner growth patterns exist in different strawberry species: one is called sympodial type such as Fragaria vesca, and the other one is called monopodial type such as Fragaria pentaphylla. Despite the runner growth morphology of these strawberry species have been well known, the mechanisms that determine the distinct patterns have rarely been reported. RESULTS:In this study, we used Fragaria vesca Hawaii-4 and Fragaria pentaphylla as model species, and captured the initiated dormant bud and non-dormant bud as materials to compare their transcriptome profiles and phytohormone content. Comparisons revealed that relatively higher auxin activity is present in the dormant bud and relatively higher cytokinin activity is in the non-dormant bud. Decapitation and pharmacological experiments on dormant buds showed that the reduction of auxin accumulation triggers the regeneration of vegetative shoots in dormant buds, and exogenous cytokinin application triggers cell fate turnover and generation of reproductive shoots. CONCLUSION:Here, we uncover a mechanism by which auxin and cytokinin coordinate the dormancy and outgrowth of axillary bud in strawberry runner. Our results suggest a contrasting behavior of auxin and cytokinin in control of axillary bud development, facilitating a preliminary understanding of shoot architecture formation in strawberry.
Project description:Cremastra appendiculata has become endangered due to reproductive difficulties. Specifically, vegetative reproduction is almost its only way to reproduce, and, under natural conditions, it cannot grow branches, resulting in an extremely low reproductive coefficient (reproductive percentage). Here, we performed RNA-Seq and a differentially expressed gene (DEG) analysis of the three stages of lateral bud development in C. appendiculata after decapitation-dormancy (D2), transition (TD2), and emergence (TG2)-and the annual axillary bud natural break (G1) to gain insight into the molecular regulatory network of shoot branching in this plant. Additionally, we applied the auxin transport inhibitors N-1-naphthylphthalamic acid (NPA) and 2,3,5-triiodibenzoic acid (TIBA) to a treated pseudobulb string of C. appendiculata to verify the conclusions obtained by the transcriptome. RNA-Seq provided a wealth of valuable information. Successive pairwise comparative transcriptome analyses revealed 5988 genes as DEGs. GO (Gene Ontology) and KEGG (Kyoto encyclopedia of genes and genomes) analyses of DEGs showed significant enrichments in phytohormone biosynthesis and metabolism, regulation of hormone levels, and a hormone-mediated signaling pathway. qRT-PCR validation showed a highly significant correlation (p < 0.01) with the RNA-Seq generated data. High-performance liquid chromatography (HPLC) and qRT-PCR results showed that, after decapitation, the NPA- and TIBA-induced lateral buds germinated due to rapidly decreasing auxin levels, caused by upregulation of the dioxygenase for auxin oxidation gene (DAO). Decreased auxin levels promoted the expression of isopentenyl transferase (IPT) and cytochrome P450 monooxygenase, family 735, subfamily A (CYP735A) genes and inhibited two carotenoid cleavage dioxygenases (CCD7 and CCD8). Zeatin levels significantly increased after the treatments. The increased cytokinin levels promoted the expression of WUSCHEL (WUS) and inhibited expression of BRANCHED1 (BRC1) in the cytokinin signal transduction pathway and initiated lateral bud outgrowth. Our data suggest that our theories concerning the regulation of shoot branching and apical dominance is really similar to those observed in annual plants. Auxin inhibits bud outgrowth and tends to inhibit cytokinin levels. The pseudobulb in the plant behaves in a similar manner to that of a shoot above the ground.
Project description:Tulip vegetative reproduction. Tulips reproduce asexually by the outgrowth of their axillary meristems located in the axil of each bulb scale. The number of axillary meristems in one bulb is low, and not all of them grow out during the yearly growth cycle of the bulb. Since the degree of axillary bud outgrowth in tulip determines the success of their vegetative propagation, this study aimed at understanding the mechanism controlling the differential axillary bud activity. We used a combined physiological and "bottom-up" molecular approach to shed light on this process and found that first two inner located buds do not seem to experience dormancy during the growth cycle, while mid-located buds enter dormancy by the end of the growing season. Dormancy was assessed by weight increase and TgTB1 expression levels, a conserved TCP transcription factor and well-known master integrator of environmental and endogenous signals influencing axillary meristem outgrowth in plants. We showed that TgTB1 expression in tulip bulbs can be modulated by sucrose, cytokinin and strigolactone, just as it has been reported for other species. However, the limited growth of mid-located buds, even when their TgTB1 expression is downregulated, points at other factors, probably physical, inhibiting their growth. We conclude that the time of axillary bud initiation determines the degree of dormancy and the sink strength of the bud. Thus, development, apical dominance, sink strength, hormonal cross-talk, expression of TgTB1 and other possibly physical but unidentified players, all converge to determine the growth capacity of tulip axillary buds.
Project description:BACKGROUND: In saffron (Crocus sativus), new corms develop at the base of every shoot developed from the maternal corm, a globular underground storage stem. Since the degree of bud sprouts influences the number and size of new corms, and strigolactones (SLs) suppress growth of pre-formed axillary bud, it was considered appropriate to investigate SL involvement in physiology and molecular biology in saffron. We focused on two of the genes within the SL pathway, CCD7 and CCD8, encoding carotenoid cleavage enzymes required for the production of SLs. RESULTS: The CsCCD7 and CsCCD8 genes are the first ones isolated and characterized from a non-grass monocotyledonous plant. CsCCD7 and CsCCD8 expression showed some overlapping, although they were not identical. CsCCD8 was highly expressed in quiescent axillary buds and decapitation dramatically reduced its expression levels, suggesting its involvement in the suppression of axillary bud outgrowth. Furthermore, in vitro experiments showed also the involvement of auxin, cytokinin and jasmonic acid on the sprouting of axillary buds from corms in which the apical bud was removed. In addition, CsCCD8 expression, but not CsCCD7, was higher in the newly developed vascular tissue of axillary buds compared to the vascular tissue of the apical bud. CONCLUSIONS: We showed that production and transport of auxin in saffron corms could act synergistically with SLs to arrest the outgrowth of the axillary buds, similar to the control of above-ground shoot branching. In addition, jasmonic acid seems to play a prominent role in bud dormancy in saffron. While cytokinins from roots promote bud outgrowth. In addition the expression results of CsCCD8 suggest that SLs could positively regulate procambial activity and the development of new vascular tissues connecting leaves with the mother corm.
Project description:Single-flower cut Chrysanthemum (Dendranthema grandiflorum 'Jinba') holds a unique status in global floriculture industry. However, the extensive axillary bud outgrowth presents a major drawback. Shade is an environment cue that inhibits shoot branching. Present study was aimed at investigating the effect of ratio of red to far-red light (R:FR) in regulating the lateral bud outgrowth of Chrysanthemum and the detailed mechanism. Results showed that the fate of axillary buds at specific positions in stem exhibited difference in response to R:FR. Decreasing R:FR resulted in elevation of abscisic acid (ABA) accumulation in axillary buds. Expression of ABA, indole-3-acetic acid (IAA) and strigolactones (SL) -related metabolism and signal transduction genes was significantly changed in response to low R:FR. In addition, low R:FR caused the re-distribution of sucrose across the whole plant, driving more sucrose towards bottom buds. Our results indicate that low R:FR not always inhibits bud outgrowth, rather its influence depends on the bud position in the stem. ABA, SL and auxin pathways were involved in the process. Interestingly, sucrose also appears to be involved in the process which is necessary to pay attention in the further studies. The present study also lays the foundation for developing methods to regulate axillary bud outgrowth in Chrysanthemum.
Project description:We show that MAX1, a specific repressor of vegetative axillary bud outgrowth in Arabidopsis, acts a positive regulator of the flavonoid pathway, including 11 structural genes and the transcription factor An2. Repression of bud outgrowth requires MAX1-dependent flavonoid gene expression. As the flavonoidless state leads to lateral outgrowth in Arabidopsis, our data suggest that a flavonoid-based mechanism regulates axillary bud outgrowth and that this mechanism is under the control of MAX1. Flavonoid gene expression results in the diminished expression of auxin transporters in the bud and stem, and this, in turn, decreases the rate of polar auxin transport. We speculate that MAX1 could repress axillary bud outgrowth via regulating flavonoid-dependent auxin retention in the bud and underlying stem. Because MAX1 is implicated in synthesis of the carotenoid-derived branch regulator(s) from the root, it likely links long-distance signaling with local control of bud outgrowth.
Project description:Shoot branching is regulated by phytohormones, including cytokinin (CK), strigolactone (SL), and auxin in axillary buds. The correlative importance of these phytohormones in the outgrowth of apple axillary buds remains unclear. In this study, the outgrowth dynamics of axillary buds of a more-branching mutant (MB) and its wild-type (WT) of Malus spectabilis were assessed using exogenous chemical treatments, transcriptome analysis, paraffin section, and reverse transcription-quantitative PCR analysis (RT-qPCR). High contents of CK and abscisic acid coincided in MB axillary buds. Exogenous CK promoted axillary bud outgrowth in the WT but not in MB, whereas exogenous gibberellic had no significant effect on bud outgrowth in the WT. Functional analysis of transcriptome data and RT-qPCR analysis of gene transcripts revealed that MB branching were associated with CK signaling, auxin transport, and SL signaling. Transcription of the SL-related genes MsMAX1, MsD14, and MsMAX2 in the axillary buds of MB was generally upregulated during bud outgrowth, whereas MsBRC1/2 were generally downregulated both in WT and MB. Exogenous SL inhibited outgrowth of axillary buds in the WT and the apple varieties T337, M26, and Nagafu 2, whereas axillary buds of the MB were insensitive to SL treatment. Treatment with N-1-naphthylphalamic acid (NPA; an auxin transport inhibitor) inhibited bud outgrowth in plants of the WT and MB. The transcript abundance of MsPIN1 was generally decreased in response to NPA and SL treatments, and increased in CK and decapitation treatments, whereas no consistent pattern was observed for MsD14 and MsMAX2. Collectively, the present results suggest that in apple auxin transport from the axillary bud to the stem may be essential for the outgrowth of axillary buds, and at least, is involved in the process of bud outgrowth.