Cytokinin Treatment Research Articles

The effect of auxins in inducing organogenesis or somatic embryogenesis in mature sunflower zygotic embryo derived apex

Induction of shoots or of somatic embryos is the key step for gaining the morphogenetic potential in sunflower (Helianthus annuus L.), species known as recalcitrant to in vitro regeneration. In the immature zygotic embryo derived tissues or in other juvenile tissues resulted from seedlings, the acquisition of the competence for regeneration can be achieved directly by cytokinin treatment or by preconditioning the explants on cytokinin containing medium. In this paper is presented a new type of explant for sunflower in vitro culture, consisting of the apex with primordial leaves, resulted from ungerminated mature zygotic embryo, in which a specific morphogenetic response was triggered by the exogenously applied auxins. Among the auxins tested, indole-3-acetic acid, indole-3-butyric acid and 1-naphthaleneacetic acid are inducers of an organogenetic response, apical/axillary shoots and adventitious buds being regenerated while 2,4-dichlorophenoxyacetic acid, 3,6-dichloro-2-methoxybenzoic acid and 4-amino-3,5,6-trichloropicolinic acid led to somatic embryo formation. Among the auxins tested only 4-amino-3,5,6-trichloropicolinic acid sustains the embryos development up to mature stage. A high amount of sucrose (120 g L-1) supplied during the auxin treatment promotes the maturation of the embryos directly on the induction medium for all tested auxins with embryogenic effect. These findings show that regardless of the type of morphogenetic response aimed in sunflower meristematic tissues resulted from mature embryos, the presence of auxins is mandatory.

Callus culture as a new approach for the production of high added value compounds in Ilex paraguariensis: genotype influence, medium optimization and compounds identification.

Ilex paraguariensis (yerba mate) is a native species from South America and is a rich source of bioactive compounds. There is a lack of research efforts on the phytochemical investigation of callus culture from this species. In the present study, an effort was made to optimize callus culture conditions and to identify secondary compounds. Calli were induced from 10 genotypes using leaf explants and the best genotype was selected to evaluate the effects of cytokinin types and concentrations on callus induction and biomass accumulation. The best genotype and cytokinin treatment were used to conduct one last experiment with sucrose concentrations in culture media and its effects on calli biomass, antioxidant activity and secondary compounds accumulation. Callus initiation was genotype dependent, and the 6-156-6 line had the best response. Zeatin supplemented medium showed higher callus induction rate (82%) and higher biomass accumulation after 120 days (328.2 mg). Higher biomass and secondary compounds accumulation were observed for calli on 3% sucrose medium. Antioxidant activity was not affected by sucrose concentrations. Yerba mate callus culture allowed the accumulation of chlorogenic acid, cryptochlorogenic acid, neochlorogenic acid, 3,4-dicaffeoylquinic acid, 3,5-dicaffeoylquinic acid, 4,5-dicaffeoylquinic acid, theobromine and caffeine.

Plant Hormones Differentially Control the Sub-Cellular Localization of Plasma Membrane Microdomains during the Early Stage of Soybean Nodulation.

Phytohormones regulate the mutualistic symbiotic interaction between legumes and rhizobia, nitrogen-fixing soil bacteria, notably by controlling the formation of the infection thread in the root hair (RH). At the cellular level, the formation of the infection thread is promoted by the translocation of plasma membrane microdomains at the tip of the RH. We hypothesize that phytohormones regulate the translocation of plasma membrane microdomains to regulate infection thread formation. Accordingly, we treated with hormone and hormone inhibitors transgenic soybean roots expressing fusions between the Green Fluorescent Protein (GFP) and GmFWL1 or GmFLOT2/4, two microdomain-associated proteins translocated at the tip of the soybean RH in response to rhizobia. Auxin and cytokinin treatments are sufficient to trigger or inhibit the translocation of GmFWL1 and GmFLOT2/4 to the RH tip independently of the presence of rhizobia, respectively. Unexpectedly, the application of salicylic acid, a phytohormone regulating the plant defense system, also promotes the translocation of GmFWL1 and GmFLOT2/4 to the RH tip regardless of the presence of rhizobia. These results suggest that phytohormones are playing a central role in controlling the early stages of rhizobia infection by regulating the translocation of plasma membrane microdomains. They also support the concept of crosstalk of phytohormones to control nodulation.

Expression profiling of miRNAs indicates crosstalk between phytohormonal response and rhizobial infection in chickpea

Legumes develop root nodules in which bacteria fix nitrogen for plants. The phytohormones auxin and cytokinin regulate nodule organogenesis by recruiting various genes to effect symbiosis. Moreover, these genes are regulated by the action of microRNAs also. To understand the complex regulatory network involving miRNAs in response to phytohormones and rhizobial interactions in chickpea roots, a miRNA expression profiling was performed. Indole acetic acid and 6-benzylaminopurine at concentrations of 0.1, 1 and 10 µM were used for auxin and cytokinin exogenous treatment and Mesorhizobium ciceri to study rhizobial interaction with chickpea root. Expression profiling of a set of 11 miRNAs was performed. Further, the targets of the candidate miRNAs were identified, followed by functional annotation. This analysis revealed that cat-miR160, cat-miR164, cat-miR396 and cat-miR398 were responsive to auxin and cytokinin. cat-miR319 was found to be only auxin responsive and is known to regulate auxin signalling by targeting TEOSINTE BRANCHED/CYCLOIDEA/PCF (TCP) which interacts with auxin inducible genes. Further, cytokinin elicited a response at very low concentration of 0.1 µM, and most of the miRNAs investigated were responsive to cytokinin. Interactome analysis revealed that cat-miR164 and cat-miR168 work in conjunction to regulate auxin signalling. Interestingly, cat-miR169 and cat-miR482 were low expressing during auxin treatment and M. ciceri infection but their expression spiked during cytokinin treatment, indicating a cytokinin mediated mode of action. The miRNA expression profiling in response to phytohormones and rhizobia and the reported function of their target genes suggested a crosstalk among the phytohormonal responses during chickpea nodulation.

Timely removal of exogenous cytokinin and the prevention of auxin transport from the shoot to the root affect the regeneration potential of Arabidopsis roots

In vitro regeneration of Arabidopsis from roots is generally achieved via indirect organogenesis. First, transdifferentiation of lateral root primordia to calli is achieved by a balanced auxin-to-cytokinin ratio that is followed by the induction of shoot meristem formation using a high cytokinin level. Here we demonstrate that if the root explants were transferred onto a hormone-free medium after a transient (4-days) cytokinin treatment, embryogenic marker genes (LEC1, LEC2, FUS3) started to be expressed. App. 50% of the regeneration foci developed into plantlets with trichome-less cotyledon-like leaves. Moreover, the somatic embryogenesis defective lec1 mutant could regenerate only shoots with trichome-bearing leaves under this condition. Based on these observations, the mixed accomplishment of shoot organogenesis and somatic embryogenesis is hypothesized in the Arabidopsis root explants cultured under hormone-free conditions following cytokinin induction. Using whole seedlings instead of root explants in the same experimental set up, no regenerates were formed on the roots. Applying the auxin transport inhibitor TIBA to the root-to-shoot junction of the seedlings, the regeneration ability of the root could be restored. The observations indicate that shoot-derived endogenous auxin blocks the cytokinin-induced regeneration process in the roots of whole seedlings. The expression of the wound-induced transcription factor WIND1 could be detected in the roots of unwounded seedlings if the shoot-to-root auxin transport was inhibited. Manipulating the exogenous cytokinin level together with the endogenous shoot-to-root auxin transport therefore could mimic the effect of wounding (removal of shoot) on plant regeneration from roots.

Mutations in tetrapyrrole biosynthesis pathway uncouple nuclear WUSCHEL expression from de novo shoot development in Arabidopsis

Plant de novo organogenesis in tissue culture systems has long been exploited to study the plasticity of pluripotency. External application of high cytokinin-to-auxin ratio in cultured medium stimulates greening of tissue and promotes nascent shoot apical meristem (SAM) formation. The stem cell niche in SAM is maintained by a negative feedback loop between CLAVATA and WUSCHEL (WUS) signaling. The fact that cytokinin is able to induce expression of SAM master regulator WUS suggests that the capacity of de novo shoot development is largely dependent on WUS activity. However, the molecular mechanism of WUS expression remains obscure. Here we found that WUS expression during de novo SAM formation is affected by the altered tetrapyrrole metabolism catalyzed in the plastid. Loss-of-function mutations in HEME OXYGENASE/LONG HYPOCOTYL 1 (hy1), Mg-CHELATASE H (chlh), Mg-CHELATASE I1 (chli1), and the regulator of Mg-CHELATASE, GENOME-UNCOUPLED 4 (gun4), result in elevated WUS expression but shoot regeneration efficiency is decreased whereas loss-of-function mutation in PROTOPORPHYRIN IX FERROCHELATASE 2 (fc2) exhibits compromised WUS expression with a reduced number of shoots when mutant root explants are cultured on shoot induction medium. Our genetic results show that nuclear WUS expression is affected by tetrapyrrole intermediate(s) under the varying plastid status stimulated by external cytokinin treatment during de novo organogenesis. We propose novel regulatory mechanism of nuclear WUS expression that is likely modulated by tetrapyrrole intermediate(s) under the varying plastid status stimulated by external cytokinin treatment during de novo organogenesis.

BLADE-ON-PETIOLE genes are not involved in the transition from protonema to gametophore in the moss Physcomitrella patens.

The timing of the transition between developmental phases is a critical determinant of plant form. In the moss Physcomitrella patens, the transition from protonema to gametophore is a particularly important step as it results in a change from two-dimensional to three-dimensional growth of the plant body. It is well known that this transition is promoted by cytokinin (CK), however, the underlying mechanisms are poorly understood. Previously, it was reported that P. patens orthologs of BLADE-ON-PETIOLE (BOP) genes (PpBOPs) work downstream of CK to promote the transition to gametophore. To further understand the role of PpBOPs in the control of this transition, we performed functional analyses of PpBOP genes. We simultaneously disrupted the function of all three PpBOP genes in P. patens using CRISPR technology, however, no abnormal phenotypes were observed in the triple mutant during either the gametophytic or the sporophytic growth stages. CK treatment did not alter the phase change in the triple mutant. We conclude that PpBOP genes are unnecessary in the control of P. patens development under normal conditions. We propose that BOP genes are not involved in the control of developmental processes in bryophytes and other basal land plants, but may function in physiological processes such as in the defense response.

The cytokinin trans-zeatine riboside increased resistance to heavy metals in the halophyte plant species Kosteletzkya pentacarpos in the absence but not in the presence of NaCl

Heavy metals such as cadmium and zinc constitute major pollutants in coastal areas and frequently accumulate in salt marshes. The wetland halophyte plant species Kosteletzkya pentacarpos is a promising species for phytostabilization of contaminated areas. In order to assess the role of the antisenescing phytohormone cytokinin in heavy metal resistance in this species, seedlings were exposed for two weeks to Cd (10 μM), Zn (100 μM) or Cd + Zn (10 μM + 100 μM) in the presence or absence of 50 mM NaCl and half of the plants were sprayed every two days with the cytokinin trans-zeatine riboside (10 μM). Zinc reduced the endogenous cytokinin concentration. Exogenous cytokinin increased plant growth, stomatal conductance, net photosynthesis and total ascorbate and reduced oxidative stress estimated by malondialdehyde in Zn-treated plants maintained in the absence of NaCl. Heavy metal induced an increase in the senescing hormone ethylene which was reduced by cytokinin treatment. Plants exposed to the mixed treatment (Cd + Zn) exhibited a specific hormonal status in relation to accumulation of abscisic acid and depletion of salicylic acid. Non-protein thiols (glutathione and phytochelatins) accumulated in response to Cd and Cd + Zn. It is concluded that toxic doses of Cd and Zn have different impacts on the plant behavior and that the simultaneous presence of the two elements induces a specific physiological constraint at the plant level. Salinity helps the plant to cope with heavy metal toxicities and the plant hormone cytokinin assumes key function in Zn resistance but its efficiency is lower in the presence of NaCl.

Cytokinin Signaling Is Essential for Organ Formation in Marchantia polymorpha.

Cytokinins are known to regulate various physiological events in plants. Cytokinin signaling is mediated by the phosphorelay system, one of the most ancient mechanisms controlling hormonal pathways in plants. The liverwort Marchantia polymorpha possesses all components necessary for cytokinin signaling; however, whether they respond to cytokinins and how the signaling is fine-tuned remain largely unknown. Here, we report cytokinin function in Marchantia development and organ formation. Our measurement of cytokinin species revealed that cis-zeatin is the most abundant cytokinin in Marchantia. We reduced the endogenous cytokinin level by overexpressing the gene for cytokinin oxidase, MpCKX, which inactivates cytokinins, and generated overexpression and knockout lines for type-A (MpRRA) and type-B (MpRRB) response regulators to manipulate the signaling. The overexpression lines of MpCKX and MpRRA, and the knockout lines of MpRRB, shared phenotypes such as inhibition of gemma cup formation, enhanced rhizoid formation and hyponastic thallus growth. Conversely, the knockout lines of MpRRA produced more gemma cups and exhibited epinastic thallus growth. MpRRA expression was elevated by cytokinin treatment and reduced by knocking out MpRRB, suggesting that MpRRA is upregulated by the MpRRB-mediated cytokinin signaling, which is antagonized by MpRRA. Our findings indicate that when plants moved onto land they already deployed the negative feedback loop of cytokinin signaling, which has an indispensable role in organogenesis.

Role of Cytokinin, Strigolactone, and Auxin Export on Outgrowth of Axillary Buds in Apple.

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.

The 26S Proteasome Is Required for the Maintenance of Root Apical Meristem by Modulating Auxin and Cytokinin Responses Under High-Boron Stress.

Boron (B), an essential micronutrient, causes adverse effects on the growth and development of plants when highly accumulated. By the analysis of Arabidopsis mutants hypersensitive to high-boron (high-B) stress, we have shown that 26S proteasome (26SP) is required to maintain the morphology of the root apical meristem (RAM) under high-B stress. To further understand the molecular function of 26SP in tolerance to high-B stress in the RAM, in this study we investigated the pathways regulated by 26SP using a 26SP subunit mutant, rpt5a, which is hypersensitive to high-B stress. Expression of RPT5a was induced by high-B stress in the entire RAM accompanied by its strong expression in the stele, including the stem cells. Analysis of stele organization in the rpt5a mutant revealed that 26SP is especially important for maintenance of the stele under high-B stress condition (3 mM B treatment). Expression analyses of an auxin-response reporter revealed that auxin responses were enhanced in the stele and the stem cell niche by high-B stress, especially in the rpt5a mutant. In contrast, the expression of TCS::GFP representing cytokinin signaling in the stem cell niche was unchanged in the wild type and extremely weak in the rpt5a mutant, irrespective of B condition. The drastically aberrant auxin and cytokinin responses in the rpt5a mutant under high-B stress were supported by transcriptome analysis using root tips. These results suggest that the collapse of hormonal crosstalk in the stele including the stem cells occurred in the rpt5a mutant, especially under high-B stress. Treatment with the auxin signaling inhibitor α-(phenyl ethyl-2-one)-indole-3-acetic acid (PEO-IAA) reduced sensitivity to high-B stress in the wild type and restored the RAM morphology in the rpt5a mutant under the high-B stress condition. In addition, cytokinin treatment conferred the rpt5a mutant with tolerance to high-B stress in RAM morphology. It is concluded that 26SP containing RPT5a is required for maintenance of auxin/cytokinin balance in the stele, which is crucial for preventing defects in RAM morphology under high-B stress.

Many rice genes are differentially spliced between roots and shoots but cytokinin has minimal effect on splicing.

Alternatively spliced genes produce multiple spliced isoforms, called transcript variants. In differential alternative splicing, transcript variant abundance differs across sample types. Differential alternative splicing is common in animal systems and influences cellular development in many processes, but its extent and significance is not as well known in plants. To investigate differential alternative splicing in plants, we examined RNA‐Seq data from rice seedlings. The data included three biological replicates per sample type, approximately 30 million sequence alignments per replicate, and four sample types: roots and shoots treated with exogenous cytokinin delivered hydroponically or a mock treatment. Cytokinin treatment triggered expression changes in thousands of genes but had negligible effect on splicing patterns. However, many genes were differentially spliced between mock‐treated roots and shoots, indicating that our methods were sufficiently sensitive to detect differential splicing between data sets. Quantitative fragment analysis of reverse transcriptase‐PCR products made from newly prepared rice samples confirmed 9 of 10 differential splicing events between rice roots and shoots. Differential alternative splicing typically changed the relative abundance of splice variants that co‐occurred in a data set. Analysis of a similar (but less deeply sequenced) RNA‐Seq data set from Arabidopsis showed the same pattern. In both the Arabidopsis and rice RNA‐Seq data sets, most genes annotated as alternatively spliced had small minor variant frequencies. Of splicing choices with abundant support for minor forms, most alternative splicing events were located within the protein‐coding sequence and maintained the annotated reading frame. A tool for visualizing protein annotations in the context of genomic sequence (ProtAnnot) together with a genome browser (Integrated Genome Browser) were used to visualize and assess effects of differential splicing on gene function. In general, differentially spliced regions coincided with conserved protein domains, indicating that differential alternative splicing is likely to affect protein function between root and shoot tissue in rice.

AHK3-Mediated Cytokinin Signaling Is Required for the Delayed Leaf Senescence Induced by SSPP.

Leaf senescence is a highly-programmed developmental process regulated by an array of multiple signaling pathways. Our group previously reported that overexpression of the protein phosphatase-encoding gene SSPP led to delayed leaf senescence and significantly enhanced cytokinin responses. However, it is still unclear how the delayed leaf senescence phenotype is associated with the enhanced cytokinin responses. In this study, we introduced a cytokinin receptor AHK3 knockout into the 35S:SSPP background. The phenotypic analysis of double mutant revealed that AHK3 loss-of-function reversed the delayed leaf senescence induced by SSPP. Moreover, we found the hypersensitivity of 35S:SSPP to exogenous cytokinin treatment disappeared due to the introduction of AHK3 knockout. Collectively, our results demonstrated that AHK3-mediated cytokinin signaling is required for the delayed leaf senescence caused by SSPP overexpression and the detailed mechanism remains to be further elucidated.

Multifaceted activity of cytokinin in leaf development shapes its size and structure in Arabidopsis.

The phytohormone cytokinin has been shown to affect many aspects of plant development ranging from the regulation of the shoot apical meristem to leaf senescence. However, some studies have reported contradictory effects of cytokinin on leaf physiology. Therefore cytokinin treatments cause both chlorosis and increased greening and both lead to decrease or increase in cell size. To elucidate this multifaceted role of cytokinin in leaf development, we have employed a system of temporal controls over the cytokinin pool and investigated the consequences of modulated cytokinin levels in the third leaf of Arabidopsis. We show that, at the cell proliferation phase, cytokinin is needed to maintain cell proliferation by blocking the transition to cell expansion and the onset of photosynthesis. Transcriptome profiling revealed regulation by cytokinin of a gene suite previously shown to affect cell proliferation and expansion and thereby a molecular mechanism by which cytokinin modulates a molecular network underlying the cellular responses. During the cell expansion phase, cytokinin stimulates cell expansion and differentiation. Consequently, a cytokinin excess at the cell expansion phase results in an increased leaf and rosette size fueled by higher cell expansion rate, yielding higher shoot biomass. Proteome profiling revealed the stimulation of primary metabolism by cytokinin, in line with an increased sugar content that is expected to increase turgor pressure, representing the driving force of cell expansion. Therefore, the developmental timing of cytokinin content fluctuations, together with a tight control of primary metabolism, is a key factor mediating transitions from cell proliferation to cell expansion in leaves.

Cytokinin treatment modifies litchi fruit pericarp anatomy leading to reduced susceptibility to post-harvest pericarp browning

Litchi (Litchi chinensis Sonn.) is a subtropical fruit known for its attractive red pericarp color, semi-translucent white aril and unique flavor and aroma. Rapid post-harvest pericarp browning strictly limits litchi fruit marketing. In the current research, we hypothesized that modification of litchi fruit pericarp anatomy by hormone application may reduce fruit susceptibility to post-harvest pericarp browning. In this context, we hypothesized that cytokinin treatment, known to induce cell division, may yield fruit with thicker pericarp and reduced susceptibility for fruit surface micro-crack formation, water loss and post-harvest pericarp browning. Exogenous cytokinin treatment was applied at different stages along the course of litchi fruit development and the effect on fruit pericarp anatomy, fruit maturation and postharvest pericarp browning was investigated. Interestingly, cytokinin treatment, applied 4 weeks after full female bloom (WFB), during the phase of pericarp cell division, led to mature fruit with thicker pericarp, reduced rate of post-harvest water loss and reduced susceptibility to post-harvest pericarp browning, as compared to non-treated control fruit. Histological sections ascribe the difference in pericarp anatomy to increased cell proliferation in the parenchymatic tissue and the highly-lignified brachysclereid cell layer. In contrast, exogenous cytokinin treatment applied 7 WFB, following the phase of pericarp cell division, significantly increased epidermal-cell proliferation but had no significant effect on overall fruit pericarp thickness and only minor affect on post-harvest water loss or pericarp browning. Interestingly, the late cytokinin treatment also significantly postponed fruit maturation-associated anthocyanin accumulation and chlorophyll degradation, as previously reported, but had no effect on other parameters of fruit maturation, like total soluble sugars and total titratable acids typically modified during aril maturation. In conclusion, exogenous cytokinin treatment at different stages in fruit development differentially modifies litchi fruit pericarp anatomy by induction of cell-type specific cell proliferation. Early cytokinin treatment during the phase of pericarp cell division may prolong litchi fruit storage by reducing fruit susceptibility to post-harvest water loss and pericarp browning.

Initial Bud Outgrowth Occurs Independent of Auxin Flow from Out of Buds.

Apical dominance is the process whereby the shoot tip inhibits the growth of axillary buds along the stem. It has been proposed that the shoot tip, which is the predominant source of the plant hormone auxin, prevents bud outgrowth by suppressing auxin canalization and export from axillary buds into the main stem. In this theory, auxin flow out of axillary buds is a prerequisite for bud outgrowth, and buds are triggered to grow by an enhanced proportional flow of auxin from the buds. A major challenge of directly testing this model is in being able to create a bud- or stem-specific change in auxin transport. Here we evaluate the relationship between specific changes in auxin efflux from axillary buds and bud outgrowth after shoot tip removal (decapitation) in the pea (Pisum sativum). The auxin transport inhibitor 1-N-naphthylphthalamic acid (NPA) and to a lesser extent, the auxin perception inhibitor p-chlorophenoxyisobutyric acid (PCIB), effectively blocked auxin efflux from axillary buds of intact and decapitated plants without affecting auxin flow in the main stem. Gene expression analyses indicate that NPA and PCIB regulate auxin-inducible, and biosynthesis and transport genes, in axillary buds within 3 h after application. These inhibitors had no effect on initial bud outgrowth after decapitation or cytokinin (benzyladenine; BA) treatment. Inhibitory effects of PCIB and NPA on axillary bud outgrowth only became apparent from 48 h after treatment. These findings demonstrate that the initiation of decapitation- and cytokinin-induced axillary bud outgrowth is independent of auxin canalization and export from the bud.

MdMYBL2 helps regulate cytokinin-induced anthocyanin biosynthesis in red-fleshed apple (Malus sieversii f. niedzwetzkyana) callus.

Anthocyanin biosynthesis is induced by cytokinins, and is regulated by MYB transcription factors. However, the underlying molecular mechanisms have not been fully characterised. In the present study, red-fleshed apple callus were induced from the leaves of an R6/R6 homozygous line, which was the hybrid offspring of Malus sieversii f. niedzwetzkyana and 'Fuji'. We analysed the callus anthocyanin contents in response to different cytokinin concentrations. We observed that cytokinin treatments upregulated the expression of anthocyanin structural genes MdDFR and MdUFGT and transcription factor genes MdMYB10 and MdbHLH3. Additionally, the expression of MdMYBL2, which encodes the bHLH and EAR motifs, was inhibited by cytokinin treatments. The MdMYBL2-overexpressing callus had lower anthocyanin contents than the wild-type controls. We noted that the expression levels of anthocyanin biosynthesis structural genes MdDFR and MdUFGT and transcription factor genes MdMYB10 and MdbHLH3 were strongly suppressed in the transgenic callus. Subsequent yeast two-hybrid, bimolecular fluorescence complementation, and pull-down assays indicated that MdMYBL2 interacts with MdbHLH3, which may influence the expression of anthocyanin biosynthesis-related genes. Our findings may provide new insights into how MYB transcription factors influence the cytokinin-regulated anthocyanin biosynthesis in red-fleshed apples.

Effect of constitutive expression of Arabidopsis CLAVATA3 on cell growth and possible role of cytokinins in leaf size control in transgenic tobacco plants

We generated transgenic tobacco plants (Nicotiana tabacum L.) with overexpression of the Arabidopsis thaliana CLAVATA3 (CLV3) gene which is known to be a negative regulator of cell division. Surprisingly, most of the 35S::CLV3 transgenic plants showed no phenotypic differences with the wild type plants. However, there were considerable changes in the morphological parameters between 35S::CLV3 overexpressors and wild type plants. As expected, the number of meristematic cells in the shoot apical meristem was reduced in 35S::CLV3 plants as compared to the wild type plants. Moreover, overexpression of CLV3 exerted morphological changes not only to shoot apical meristem but also to leaves and flowers. Thus, transgenic plants were characterized by reduced number of epidermal and mesophyll cells as well as stomatal pores in mature leaves. However, there was a compensatory increase in leaf cell size of 35S::CLV3 plants that contributed to maintenance of organ size within the normal range. We observed that expression of cell expansion-promoted genes, expansin NtEXPA4 and endo-xyloglucan transferase NtEXGT, were elevated in mature leaves. In contrast, there was a decrease in the transcript level of the cell division-related AINTEGUMENTA-like (NtANTL) gene in 35S::CLV3 transgenic plants. In addition, we detected an increase in cytokinin level without any changes in the contents of IAA and ABA in 35S::CLV3 overexpressors. Interestingly, cytokinin treatment was shown to stimulate the expression of NtEXPA4 and NtEXGT genes in 35S::CLV3 transgenic plants. We propose that observed compensatory cell expansion in leaves of 35S::CLV3 transgenic plants may be due, at least in part, to a possible link between cytokinin signalling and cell expansion-related genes.

CUP-SHAPED COTYLEDON1 (CUC1) and CUC2 regulate cytokinin homeostasis to determine ovule number in Arabidopsis.

Seeds derive from ovules upon fertilization and therefore the total number of ovules determines the final seed yield, a fundamental trait in crop plants. Among the factors that co-ordinate the process of ovule formation, the transcription factors CUP-SHAPED COTYLEDON 1 (CUC1) and CUC2 and the hormone cytokinin (CK) have a particularly prominent role. Indeed, the absence of both CUC1 and CUC2 causes a severe reduction in ovule number, a phenotype that can be rescued by CK treatment. In this study, we combined CK quantification with an integrative genome-wide target identification approach to select Arabidopsis genes regulated by CUCs that are also involved in CK metabolism. We focused our attention on the functional characterization of UDP-GLUCOSYL TRANSFERASE 85A3 (UGT85A3) and UGT73C1, which are up-regulated in the absence of CUC1 and CUC2 and encode enzymes able to catalyse CK inactivation by O-glucosylation. Our results demonstrate a role for these UGTs as a link between CUCs and CK homeostasis, and highlight the importance of CUCs and CKs in the determination of seed yield.

Comparative transcriptome analysis reveals the regulatory networks of cytokinin in promoting the floral feminization in the oil plant Sapium sebiferum

BackgroundSapium sebiferum, whose seeds contain high level of fatty acids, has been considered as one of the most important oil plants. However, the high male to female flower ratio limited the seed yield improvement and its industrial potentials. Thus, the study of the sex determination in S. sebiferum is of significant importance in increasing the seed yield.ResultsIn this study, we demonstrated that in S. sebiferum, cytokinin (CK) had strong feminization effects on the floral development. Exogenous application with 6-benzylaminopurine (6-BA) or thidiazuron (TDZ) significantly induced the development of female flowers and increased the fruit number. Interestingly, the feminization effects of cytokinin were also detected on the androecious genotype of S. sebiferum which only produce male flowers. To further investigate the mechanism underlying the role of cytokinin in the flower development and sex differentiation, we performed the comparative transcriptome analysis of the floral buds of the androecious plants subjected to 6-BA. The results showed that there were separately 129, 352 and 642 genes differentially expressed at 6 h, 12 h and 24 h after 6-BA treatment. Functional analysis of the differentially expressed genes (DEGs) showed that many genes are related to the hormonal biosynthesis and signaling, nutrients translocation and cell cycle. Moreover, there were twenty one flowering-related genes identified to be differentially regulated by 6-BA treatment. Specifically, the gynoecium development-related genes SPATULA (SPT), KANADI 2 (KAN2), JAGGED (JAG) and Cytochrome P450 78A9 (CYP79A9) were significantly up-regulated, whereas the expression of PISTILLATA (PI), TATA Box Associated Factor II 59 (TAFII59) and MYB Domain Protein 108 (MYB108) that were important for male organ development was down-regulated in response to 6-BA treatment, demonstrating that cytokinin could directly target the floral organ identity genes to regulate the flower sex.ConclusionsOur work demonstrated that cytokinin is a potential regulator in female flower development in S. sebiferum. The transcriptome analysis of the floral sex transition from androecious to monoecious in response to cytokinin treatment on the androecious S. sebiferum provided valuable information related to the mechanism of sex determination in the perennial woody plants.