Exogenous Application Of Cytokinin Research Articles

Identification of a conserved set of cytokinin-responsive genes expressed in the fruits of Prunus persica

Ethylene plays an essential role in the ripening of peach, a climacteric stone fruit. However, several studies have shown that other phytohormones may alter fruit organoleptic properties, nutritional value, and overall quality. Although cytokinin levels are highest during fruit set and early stages of fruit development, exogenous cytokinin application on the late stages of peach fruit development (post-lignification stage) alters the peach fruit ripening indicators and delays ripening. To better understand the molecular mechanisms by which cytokinin alters fruit ripening and fruit quality traits, we identified a conserved set of 26 peach cytokinin-regulated orthologs of the Arabidopsis cytokinin-regulated genes. All but two of these genes show expression in ripe peach fruits. The peach cytokinin-regulated genes map throughout the Prunus persica genome, located on all eight chromosome-scale pseudomolecules. However, eight of these orthologs are located on pseudomolecule 1, while five orthologs are on pseudomolecule 2. Gene Ontology enrichment analyses revealed that many of these genes are associated with metabolic processes. The region upstream of the transcription start site contained Cytokinin Response Elements (CKREs) in 77% (20/26) of these putative cytokinin-regulated genes. The cytokinin responsiveness of eight of these genes (PpeBPH1, PpeCKX4, PpeCPK28, PpeCYP18-2, PpeHSP90-7, PpeNRP2, PpeNIP1-1, and PpePCRP1) was confirmed by RT-qPCR analyses of ripe peach fruits treated exogenously with trans-zeatin for one hour. Therefore, by using a comparative genomic analysis between peach and Arabidopsis, we have identified a conserved set of cytokinin-regulated genes expressed in peach fruits that respond to exogenous application of trans-zeatin and, based upon the conserved cis-regulatory regions, are most likely regulated by the cytokinin Type-B Response Regulators.

Exogenous application of phytohormones mitigates the effect of salt stress on Carica papaya plants

ABSTRACT The salinity has complex effects on the physiological, nutritional and hormonal interactions of plants. This study aimed to evaluate the mitigating action of exogenous application of different types and concentrations of phytohormones on Carica papaya under salt stress. The experiment was conducted in greenhouse, using a randomized block design with treatments formed from a 2 x 3 x 5 factorial scheme, relative to two concentrations of salts in the irrigation water (0.6 and 2.4 dS m-1), three types of phytohormones [Auxin = indole-3-acetic acid; Cytokinin = CPPU (N-(2-chloro-pyridyl)-N-phenylurea) and Gibberellin = gibberellic acid(AG3)], and five concentrations of the phytohormone (0, 12.5, 25.0, 37.5 and 50.0 µM L-1), with three repetitions, totaling 90 plots, and the experimental unit consisted of six plants. C. papaya cv. ‘Sunrise Solo’ was grown on polyethylene trays of 162 cells, with capacity for 50 mL. The plants were evaluated for survival percentage and phytomass accumulation until 25 days after sowing. Increase in irrigation water salinity has deleterious effects on C. papaya plants regardless of the application of phytohormones. Exogenous application of gibberellin has a positive effect in mitigating salt stress on C. papaya plants, greater than the effects of auxins and cytokinins. Exogenous application of gibberellin and auxin at concentration of 37.5 µM L-1 mitigates the effects of salinity on C. papaya plants. Exogenous application of cytokinin at concentrations of 12.5 to 50.0 µM L-1 is not viable for C. papaya plants.

Photosynthetic attributes and biological yield of wheat genotypes as influenced by exogenous trehalose and cytokinins under field drought stress

Present investigation was aimed at studying the effect of exogenous application of cytokinins like kinetin (Kn) and benzyl adenine (BA) in combination with osmoprotectant like trehalose (Tre) at two stages i.e vegetative stage (VS) and flag leaf stage (FLS) on photosynthetic parameters and biological yield of drought stressed wheat genotypes. Under drought stress, significant decrease in leaf net photosynthetic rate (Pn), stomatal conductance (gs), transpiration rate, sub-stomatal CO2 concentration and decrease in photosynthetic water use efficiency has been observed. Foliar application of both cytokinins and trehalose had ameliorative effect on photosynthetic rate, stomatal conductance, transpiration rate, substomatal conductance of carbon dioxide. Significant increase was recorded in chlorophyll a, b, total chlorophyll content and carotenoid content of wheat genotypes with exogenous application of cytokinins and trehalose. Exogenous application of Tre @ 1.5mM along with Kn @ 40mg/L, was observed more effective in mitigation of adverse effect of drought stress in wheat genotypes.

The Rice G Protein \u03b3 Subunit DEP1/qPE9\u20131 Positively Regulates Grain-Filling Process by Increasing Auxin and Cytokinin Content in Rice Grains

Heterotrimeric G protein-mediated signal transduction is one of the most important and highly conserved signaling pathways in eukaryotes, which involves in the regulation of many important biological processes. As compared with those in mammals and Arabidopsis thaliana, the functions of rice heterotrimeric G protein and their molecular mechanisms are largely unknown. The rice genome contains a single Gα (RGA1) and Gβ (RGB1), and five Gγ (RGG1, RGG2, GS3, DEP1/qPE9–1, and GGC2) subunits. Recent genetic studies have shown that DEP1/qPE9–1, an atypical putative Gγ protein, is responsible for the grain size as well as the dense and erect panicles, but the biochemical and molecular mechanisms underlying the control of grain size are not well understood. Here, we report that rice plants carrying DEP1/qPE9–1 have more endosperm cells per grain than plants contain the dep1/qpe9–1 allele. The DEP1/qPE9–1 line has a higher rate and more prolonged period of starch accumulation than the dep1/qpe9–1 line. Additionally, the expression of several essential genes encoding enzymes catalyzing sucrose metabolism and starch biosynthesis is higher in the DEP1/qPE9–1 line than in the dep1/qpe9–1 line, especially from the mid to late grain-filling stage. Grains of the DEP1/qPE9–1 line also have higher contents of three phytohormones, ABA, auxin and cytokinin. Exogenous application of auxin or cytokinin enhanced the starch accumulation and the expression of genes encoding grain-filling-related enzymes in the grains of dep1/qpe9–1, whereas ABA produced no effects. Based on these results, we conclude that DEP1/qPE9–1 positively regulates starch accumulation primarily through auxin and cytokinin, which enhance the expression of genes encoding starch biosynthesis during the mid to late grain-filling stage, resulting in increased duration of the grain-filling process.

Auxin and cytokinin coordinate the dormancy and outgrowth of axillary bud in strawberry runner

BackgroundAxillary 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.ResultsIn 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.ConclusionHere, 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.

Transcriptome analysis of two inflorescence branching mutants reveals cytokinin is an important regulator in controlling inflorescence architecture in the woody plant Jatropha curcas

BackgroundIn higher plants, inflorescence architecture is an important agronomic trait directly determining seed yield. However, little information is available on the regulatory mechanism of inflorescence development in perennial woody plants. Based on two inflorescence branching mutants, we investigated the transcriptome differences in inflorescence buds between two mutants and wild-type (WT) plants by RNA-Seq to identify the genes and regulatory networks controlling inflorescence architecture in Jatropha curcas L., a perennial woody plant belonging to Euphorbiaceae.ResultsTwo inflorescence branching mutants were identified in germplasm collection of Jatropha. The duo xiao hua (dxh) mutant has a seven-order branch inflorescence, and the gynoecy (g) mutant has a three-order branch inflorescence, while WT Jatropha has predominantly four-order branch inflorescence, occasionally the three- or five-order branch inflorescences in fields. Using weighted gene correlation network analysis (WGCNA), we identified several hub genes involved in the cytokinin metabolic pathway from modules highly associated with inflorescence phenotypes. Among them, Jatropha ADENOSINE KINASE 2 (JcADK2), ADENINE PHOSPHORIBOSYL TRANSFERASE 1 (JcAPT1), CYTOKININ OXIDASE 3 (JcCKX3), ISOPENTENYLTRANSFERASE 5 (JcIPT5), LONELY GUY 3 (JcLOG3) and JcLOG5 may participate in cytokinin metabolic pathway in Jatropha. Consistently, exogenous application of cytokinin (6-benzyladenine, 6-BA) on inflorescence buds induced high-branch inflorescence phenotype in both low-branch inflorescence mutant (g) and WT plants. These results suggested that cytokinin is an important regulator in controlling inflorescence branching in Jatropha. In addition, comparative transcriptome analysis showed that Arabidopsis homologous genes Jatropha AGAMOUS-LIKE 6 (JcAGL6), JcAGL24, FRUITFUL (JcFUL), LEAFY (JcLFY), SEPALLATAs (JcSEPs), TERMINAL FLOWER 1 (JcTFL1), and WUSCHEL-RELATED HOMEOBOX 3 (JcWOX3), were differentially expressed in inflorescence buds between dxh and g mutants and WT plants, indicating that they may participate in inflorescence development in Jatropha. The expression of JcTFL1 was downregulated, while the expression of JcLFY and JcAP1 were upregulated in inflorescences in low-branch g mutant.ConclusionsCytokinin is an important regulator in controlling inflorescence branching in Jatropha. The regulation of inflorescence architecture by the genes involved in floral development, including TFL1, LFY and AP1, may be conservative in Jatropha and Arabidopsis. Our results provide helpful information for elucidating the regulatory mechanism of inflorescence architecture in Jatropha.

Influence of Blue-Light Signaling Components on the Regulation of Cytokinin-Dependent Arabidopsis thaliana Seedlings’ Greening

De-etiolation of plant seedlings is controlled by a complex system of light and hormonal signaling. This process is accompanied by chloroplast development and initiation of chlorophyll biosynthesis leading to cotyledon greening. In this study, peculiaritis of regulation of expression of the key genes encoding enzymes of chlorophyll biosynthesis in wild type plants and mutants of perception and transduction of blue light in Arabidopsis thaliana (L.) Heynh. during cytokinin (CK)-dependent greening of seedlings were investigated. Inactivation of blue-light signaling components (??RY1, CRY2 and HY5) led to retarded chlorophyll accumulation and decrease of the expression of the key genes responsible for its biosynthesis. Exogenous application of cytokinin accelerated de-etiolation of wild type and knockout-mutants cry1cry2 and hy5 seedlings that was manifested itself in elevating chlorophyll a fluorescence and higher level of transcripts of a number of genes involved in chlorophyll biosynthesis. Taken together the results may provide evidence for the significant contribution of blue-light signaling to the greening regulation at early steps of de-etiolation of seedlings and involvement of HY5 protein in realization of the positive effect of CK at early steps of photomorphogenesis.

The effects of novel synthetic cytokinin derivatives and endogenous cytokinins on the in vitro growth responses of hemp (Cannabis sativa L.) explants

The biotechnological utilization (genetic transformation, gene editing) of industrial hemp (Cannabis sativa L.) has been hampered by a lack of robust regeneration/in vitro multiplication protocols. In order to break this barrier we propose an approach combining standard application of exogenous growth regulators (auxins, cytokinins) with the knowledge of (1) endogenous cytokinin concentrations in primary explants based on latest analytical techniques and (2) the exogenous application of novel synthetic cytokinins or the regulators of their activity/function. We have tested eight explant types isolated from aseptically germinated seedlings in order to induce shoot regeneration/multiple shoot formation. The main problems appeared were callus formation and strong apical dominance. The latter phenomenon was suppressed using the auxin antagonist α-(2-oxo-2-phenylethyl)-1H-indole-3-acetic acid (PEO-IAA), and multiple shoot cultures were established from isolated apical meristems using media containing the novel synthetic cytokinin derivative 6-benzylamino-9-(tetrahydroxypyranyl)purin (BAP9THP). Endogenous cytokinin concentrations were measured in primary explants and explants cultured under diverse conditions to clarify the interactions between endogenous and exogenously applied cytokinins and their synthetic derivatives. These measurements were subsequently used to optimize the applied concentrations and timing of application of specific cytokinin derivatives to achieve desirable in vitro responses and regeneration including inhibition of callus formation, single shoot formation, and induction of multiple shoots. The well-developed shoots were rooted on media containing auxin (α-naphthaleneacetic acid). The protocol was developed using the variety USO-31 and has been successfully applied to four other industrial hemp varieties: Tygra, Monoica, Bialobrzeskie, Fibrol.

The SMO1 Family of Sterol 4α-Methyl Oxidases Is Essential for Auxin- and Cytokinin-Regulated Embryogenesis.

In the plant sterol biosynthetic pathway, sterol 4α-methyl oxidase1 (SMO1) and SMO2 enzymes are involved in the removal of the first and second methyl groups at the C-4 position, respectively. SMO2s have been found to be essential for embryonic and postembryonic development, but the roles of SMO1s remain unclear. Here, we found that the three Arabidopsis (Arabidopsis thaliana) SMO1 genes displayed different expression patterns. Single smo1 mutants and smo1-1 smo1-3 double mutants showed no obvious phenotype, but the smo1-1 smo1-2 double mutant was embryo lethal. The smo1-1 smo1-2 embryos exhibited severe defects, including no cotyledon or shoot apical meristem formation, abnormal division of suspensor cells, and twin embryos. These defects were associated with enhanced and ectopic expression of auxin biosynthesis and response reporters. Consistently, the expression pattern and polar localization of PIN FORMED1, PIN FORMED7, and AUXIN RESISTANT1 auxin transporters were dramatically altered in smo1-1 smo1-2 embryos. Moreover, cytokinin biosynthesis and response were reduced in smo1-1 smo1-2 embryos. Tissue culture experiments further demonstrated that homeostasis between auxin and cytokinin was altered in smo1-1 smo1-2 heterozygous mutants. This disturbed balance of auxin and cytokinin in smo1-1 smo1-2 embryos was accompanied by unrestricted expression of the quiescent center marker WUSCHEL-RELATED HOMEOBOX5 Accordingly, exogenous application of either auxin biosynthesis inhibitor or cytokinin partially rescued the embryo lethality of smo1-1 smo1-2 Sterol analyses revealed that 4,4-dimethylsterols dramatically accumulated in smo1-1 smo1-2 heterozygous mutants. Together, these data demonstrate that SMO1s function through maintaining correct sterol composition to balance auxin and cytokinin activities during embryogenesis.

Exogenous application of cytokinin during dark senescence eliminates the acceleration of photosystem II impairment caused by chlorophyll b deficiency in barley

Recent studies have shown that chlorophyll (Chl) b has an important role in the regulation of leaf senescence. However, there is only limited information about senescence of plants lacking Chl b and senescence-induced decrease in photosystem II (PSII) and photosystem I (PSI) function has not even been investigated in such plants. We have studied senescence-induced changes in photosynthetic pigment content and PSII and PSI activities in detached leaves of Chl b-deficient barley mutant, chlorina f2f2 (clo). After 4 days in the dark, the senescence-induced decrease in PSI activity was smaller in clo compared to WT leaves. On the contrary, the senescence-induced impairment in PSII function (estimated from Chl fluorescence parameters) was much more pronounced in clo leaves, even though the relative decrease in Chl content was similar to wild type (WT) leaves (Hordeum vulgare L., cv. Bonus). The stronger impairment of PSII function seems to be related to more pronounced damage of reaction centers of PSII. Interestingly, exogenously applied plant hormone cytokinin 6-benzylaminopurine (BA) was able to maintain PSII function in the dark senescing clo leaves to a similar extent as in WT. Thus, considering the fact that without BA the senescence-induced decrease in PSII photochemistry in clo was more pronounced than in WT, the relative protective effect of BA was higher in Chl b-deficient mutant than in WT.

Big Grain3, encoding a purine permease, regulates grain size via modulating cytokinin transport in rice.

Grain size is an important agronomic trait affecting grain yield, but the underlying molecular mechanisms remain to be elucidated. Here, we isolated a dominant mutant, big grain3 (bg3-D), which exhibits a remarkable increase of grain size caused by activation of the PURINE PERMEASE gene, OsPUP4. BG3/OsPUP4 is predominantly expressed in vascular tissues and is specifically suppressed by exogenous cytokinin application. Hormone profiling revealed that the distribution of different cytokinin forms, in roots and shoots of the bg3-D mutant, is altered. Quantitative reverse transcription-PCR (qRT-PCR) analysis indicated that expression of rice cytokinin type-A RESPONSE REGULATOR (OsRR) genes is enhanced in the roots of the bg3-D mutant. These results suggest that OsPUP4 might contribute to the long-distance transport of cytokinin, by reinforcing cytokinin loading into vascular bundle cells. Furthermore, plants overexpressing OsPUP7, the closest homolog of OsPUP4, also exhibited a similar phenotype to the bg3-D mutant. Interestingly, subcellular localization demonstrated that OsPUP4 was localized on the plasma membrane, whereas OsPUP7 was localized to the endoplasmic reticulum. Based on these findings, we propose that OsPUP4 and OsPUP7 function in a linear pathway to direct cytokinin cell-to-cell transport, affecting both its long-distance movement and local allocation.

Local and Systemic Effect of Cytokinins on Soybean Nodulation and Regulation of Their Isopentenyl Transferase (IPT) Biosynthesis Genes Following Rhizobia Inoculation.

Cytokinins are important regulators of cell proliferation and differentiation in plant development. Here, a role for this phytohormone group in soybean nodulation is shown through the exogenous application of cytokinins (6-benzylaminopurine, N6-(Δ2-isopentenyl)-adenine and trans-zeatin) via either root drenching or a petiole feeding technique. Overall, nodule numbers were reduced by treatment with high cytokinin concentrations, but increased with lower concentrations. This was especially evident when feeding the solutions directly into the vasculature via petiole feeding. These findings highlight the importance of cytokinin in nodule development. To further investigate the role of cytokinin in controlling nodule numbers, the IPT gene family involved in cytokinin biosynthesis was characterized in soybean. Bioinformatic analyses identified 17 IPT genes in the soybean genome and homeologous duplicate gene partners were subsequently identified including GmIPT5 and GmIPT6, the orthologs of LjIPT3. Expression of GmIPT5 was upregulated in the shoot in response to nodulation, but this was independent of a functional copy of the autoregulation of nodulation (AON) receptor, GmNARK, which suggests it is unlikely to have a role in the negative feedback system called AON. Legumes also control nodule numbers in the presence of soil nitrogen through nitrate-dependent regulation of nodulation, a locally acting pathway in soybean. Upon nitrate treatment to the root, the tandem duplicates GmIPT3 and GmIPT15 were upregulated in expression indicating a role for these genes in the plant’s response to soil nitrogen, potentially including the nitrate-dependent regulation of legume nodulation pathway. Additional roles for cytokinin and their IPT biosynthetic genes in nodulation and the control of nodule numbers are discussed.

Biochemical and molecular characterisation of exogenous cytokinin application on grain filling in rice

BackgroundPoor filling of grains in the basal spikelets of large size panicles bearing numerous spikelets has been a major limitation in attempts to increase the rice production to feed the world’s increasing population. Considering that biotechnological intervention could play important role in overcoming this limitation, the role of cytokinin in grain filling was investigated based on the information on cell proliferating potential of the hormone and reports of its high accumulation in immature seeds.ResultsA comparative study considering two rice varieties differing in panicle compactness, lax-panicle Upahar and compact-panicle OR-1918, revealed significant difference in grain filling, cytokinin oxidase (CKX) activity and expression, and expression of cell cycle regulators and cytokinin signaling components between the basal and apical spikelets of OR-1918, but not of Upahar. Exogenous application of cytokinin (6-Benzylaminopurine, BAP) to OR-1918 improved grain filling significantly, and this was accompanied by a significant decrease in expression and activity of CKX, particularly in the basal spikelets where the activity of CKX was significantly higher than that in the apical spikelets. Cytokinin application also resulted in significant increase in expression of cell cycle regulators like cyclin dependent kinases and cyclins in the basal spikelets that might be facilitating cell division in the endosperm cells by promoting G1/S phase and G2/M phase transition leading to improvement in grain filling. Expression studies of type-A response regulator (RR) component of cytokinin signaling indicated possible role of OsRR3, OsRR4 and OsRR6 as repressors of CKX expression, much needed for an increased accumulation of CK in cells. Furthermore, the observed effect of BAP might not be solely because of it, but also because of induced synthesis of trans-zeatin (tZ) and N6-(Δ2-isopentenyl)adenine (iP), as reflected from accumulation of tZR (tZ riboside) and iPR (iP riboside), and significantly enhanced expression of an isopentenyl transferase (IPT) isoform.ConclusionThe results suggested that seed-specific overexpression of OsRR4 and OsRR6, and more importantly of IPT9 could be an effective biotechnological intervention towards improving the CK level of the developing caryopses leading to enhanced grain filling in rice cultivars bearing large panicles with numerous spikelets, and thereby increasing their yield potential.

Fluorescent reporter lines for auxin and cytokinin signalling in barley (Hordeum vulgare).

The phytohormones auxin and cytokinin control development and maintenance of plant meristems and stem cell systems. Fluorescent protein reporter lines that monitor phytohormone controlled gene expression programmes have been widely used to study development and differentiation in the model species Arabidopsis, but equivalent tools are still missing for the majority of crop species. Barley (Hordeum vulgare) is the fourth most abundant cereal crop plant, but knowledge on these important phytohormones in regard to the barley root and shoot stem cell niches is still negligible. We have now analysed the role of auxin and cytokinin in barley root meristem development, and present fluorescent protein reporter lines that allow to dissect auxin and cytokinin signalling outputs in vivo. We found that application of either auxin or cytokinin to barley seedlings negatively impacts root meristem growth. We further established a barley cytokinin reporter, TCSnew, which revealed significant cytokinin signalling in the stele cells proximal to the QC, and in the differentiated root cap cells. Application of exogenous cytokinin activated signalling in the root stem cell niche. Commonly employed auxin reporters DR5 or DR5v2 failed to respond to auxin in barley. However, analysis of putative auxin signalling targets barley PLETHORA1 (HvPLT1) is expressed in a similar pattern as its orthologue AtPLT1 from Arabidopsis, i.e. in the QC and the surrounding cells. Furthermore, the PINFORMED1 (HvPIN1) auxin efflux carrier was found to be expressed in root and shoot meristems, where it polarly localized to the plasma membrane. HvPIN1 expression is negatively regulated by cytokinin and its intracellular localisation is sensitive to brefeldinA (BFA). With this study, we provide the first fluorescent reporter lines as a tool to study auxin and cytokinin signalling and response pathways in barley.

Effect of decapitation and exogenous application of gibberellic acid (GA3) and cytokinin (CK) on some physiological characteristics of stevia.

Stevia (Stevia rebaudiana B.) has auxiliary buds that often remain dormant for a long time and sometimes remain dormant until the plants change at the reproductive stage. This study was designed out to investigate whether decapitation and exogenous application of plant growth regulators enhance the productivity of stevia through breaking the apical dominance and increasing physiological characteristics. Experiment was carried out as a factorial in randomized complete block design with three replications. Factors were consisted two agricultural practices (Decapitation and No-decapitation) and eight foliar spray including without spray as control, water spray, GA3 (300, 600 and 900 µm) and CK (100, 200 and 400 µm). The results of the present investigation indicated a positive response on number of branches and leaves, leaves and stem fresh weight and total dry weight, in both harvests not only from the decapitation of apical buds but also from foliar application of CK (400 µM). Thus, it can be concluded that the decapitation practices in conjunction with foliar application of CK (400 µM) could be used to increase the dry-leaf yield of stevia. However, further studies are required to standardize the dose of CK (400 µM) to improve the yield and quality of stevia.

Comparative transcriptomics reveals a reduction in carbon capture and flux between source and sink in cytokinin-treated inflorescences of Jatropha curcas L.

The low seed yield of Jatropha curcas has been a stumbling block in realizing its full potential as an ideal bioenergy crop. Low female to male flower ratio is considered as a major limiting factor responsible for low seed yield in Jatropha. An exogenous cytokinin application was performed on floral meristems to increase the seed yield. This resulted in an increase of total flowers count with a higher female to male flower ratio. However, the seed biomass did not increase in the same proportion. The possible reason for this was hypothesized to be the lack of increased photosynthesis efficiency at source tissues which could fulfil the increased demand of photosynthates and primary metabolites in maturing seeds. After cytokinin application, possible molecular mechanisms underlying carbon capture and flux affected between the source and sink in developing flowers, fruits and seeds were investigated. Comparative transcriptome analysis was performed on inflorescence meristems (treated with cytokinin) and control (untreated inflorescence meristems) at time intervals of 15 and 30days, respectively. KEGG-based functional annotation identified various metabolic pathways associated with carbon capture and flux. Pathways such as photosynthesis, carbon fixation, carbohydrate metabolism and nitrogen metabolism were upregulated after 15days of cytokinin treatment; however, those were downregulated after 30days. Five genes FBP, SBP, GS, GDH and AGPase showed significant increase in transcript abundance after 15days of treatment but showed a significant decrease after 30days. These genes, after functional validation, can be suitable targets in designing a suitable genetic intervention strategy to increase overall seed yield in Jatropha.

A sensitive synthetic reporter for visualizing cytokinin signaling output in rice

BackgroundCytokinins play many essential roles in plant growth and development, mainly through signal transduction pathways. Although the cytokinin signaling pathway in rice has been clarified, no synthetic reporter for cytokinin signaling output has been reported for rice. The sensitive synthetic reporter two-component signaling sensor (TCSn) is used in the model plant Arabidopsis; however, whether the reporter reflects the cytokinin signaling output pattern in rice remains unclear.ResultsEarly-cytokinin-responsive type-A OsRR-binding element (A/G)GAT(C/T) was more clustered in the 15 type-A OsRRs than in the 13 control genes. Quantitative polymerase chain reaction analysis showed that the relative expression of seven type-A OsRRs in roots and shoots was significantly induced by exogenous cytokinin application, and that of seven OsRRs, mainly in roots, was inhibited by exogenous auxin application. We constructed a transgenic rice plant harboring a beta-glucuronidase (GUS) driven by the synthetic promoter TCSn. TCSn::GUS was expressed in the meristem of germinated rice seed and rice seedlings. Furthermore, TCSn::GUS expression in rice seedlings was induced specifically by exogenous cytokinin application and decreased by exogenous auxin application. Moreover, no obvious reduction in GUS levels was observed after three generations of selfing of transgenic plants, indicating that TCSn::GUS is not subject to transgene silencing.ConclusionsWe report here a robust and sensitive synthetic sensor for monitoring the transcriptional output of the cytokinin signaling network in rice.

Exogenous cytokinin application to Actinidia chinensis var. deliciosa \u2018Hayward\u2019 fruit promotes fruit expansion through water uptake

Exogenous application of a cytokinin-like compound forchlorfenuron (CPPU) can promote fruit growth, although often at the expense of dry matter (DM), an important indicator of fruit quality. Actinidia chinensis var. deliciosa ‘Hayward’ fruit are very responsive to CPPU treatments, but the mechanism underlying the significant fruit weight increase and associated decrease in DM is unclear. In this study, we hypothesised that CPPU-enhanced growth increases fruit carbohydrate demand, but limited carbohydrate supply resulted in decreased fruit DM. During fruit development, CPPU effects on physical parameters, metabolites, osmotic pressure and transcriptional changes were assessed under conditions of both standard and a high carbohydrate supply. We showed that CPPU increased fruit fresh weight but the dramatic DM decrease was not carbohydrate limited. Enhanced glucose and fructose concentrations contributed to an increase in soluble carbohydrate osmotic pressure, which was correlated with increased water accumulation in CPPU-treated fruit and up-regulation of water channel aquaporin gene PIP2.4 at 49 days after anthesis. Transcipt analysis suggested that the molecular mechanism contributing to increased glucose and fructose concentrations was altered by carbohydrate supply. At standard carbohydrate supply, the early glucose increase in CPPU fruit was associated with reduced starch synthesis and increased starch degradation. When carbohydrate supply was high, the early glucose increase in CPPU fruit was associated with a general decrease in starch synthesis but up-regulation of vacuolar invertase and fructokinase genes. We conclude that CPPU affected fruit expansion by increasing the osmotically-driven water uptake and its effect was not carbohydrate supply-limited.

Comparative Transcriptomic Analysis of Vernalization- and Cytokinin-Induced Floral Transition in Dendrobium nobile

Vernalization is required for floral initiation in Dendrobium. Interestingly, those beneficial effects can also be achieved by exogenous cytokinin application in greenhouses. Thus, an as yet unknown crosstalk/interaction may exist between vernalization and cytokinin signaling pathways. In this study, we showed, by de novo transcriptome assembly using RNA-seq data from both vegetative and reproductive tissue samples, that some floral transition-related genes—DnVRN1, FT, SOC1, LFY and AP1—were differentially expressed in low-temperature-challenged (LT) or thidiazuron (TDZ)-treated plants, compared to those mock-treated (CK). Both LT and TDZ upregulated SOC1, LFY and AP1, while the upregulation of DnVRN1 and FT was only LT-induced. We further found that LT promoted the upregulation of some key cytokinin signaling regulators, including several cytokinin biosynthesis-related genes and type-B response regulator (RR)-encoding genes, and that both LT and TDZ triggered the significant upregulation of some marker genes in the gibberellin (GA) signaling pathway, indicating an important low temperature-cytokinin-GA axis in flowering. Our data thus have revealed a cytokinin-GA signal network underlying vernalization, providing a novel insight into further investigation of the molecular mechanism of floral initiation in Dendrobium.

Evaluating the potential of cytokinins for biomass and lipid enhancement in microalga Acutodesmus obliquus under nitrogen stress

Despite the significant reduction in biomass production, nitrogen stress is the most feasible strategy used for lipid enhancement in microalgae. The present study evaluates the potential for application of exogenous cytokinins to enhance the lipid accumulation and overcome the biomass production constraint of microalgae under nitrogen stressed conditions. This study was undertaken to investigate the effect of exogenously supplied cytokinins viz. kinetin and zeatin at various stages of growth on biomass and lipid productivities of Acutodesmus obliquus under nitrogen stress (ON). The effect of cytokinins on photosynthetic performance and biochemical composition of biomass was also evaluated. Enhanced biomass and lipid productivities were observed upon supplementation of the microalgae with selected cytokinins. Supplementation with kinetin (1mgL−1) and zeatin (0.1mgL−1) resulted in increased biomass productivity up to 50% and 60.7% respectively as compared to ON control. The highest biomass productivity of 176.79mgL−1 d−1 was obtained with the addition of 0.1mgL−1 zeatin (ON-Zi) at the initial log phase. Cytokinin supplementation resulted in improvement in photosynthetic performance and exhibited higher rETR (relative electron transport rate) values as compared to ON control. The addition of kinetin (ON-Km) at mid log phase and Zeatin (ON-Zi) at initial log phase resulted in 64.95% and 63.06% increase in lipid productivity respectively, compared to ON control. Carbohydrate productivity was also found to be increased with zeatin (ON-Zi) supplementation. The supplementation of cytokinins is an easy and scalable strategy for biomass and lipid enhancement in Acutodesmus obliquus under nitrogen stress.