Exogenous Cytokinin Treatment Research Articles

Functional analysis of RsWUSb with Agrobacterium-mediated in planta transformation in radish (Raphanus sativus L.)

The WUSCHEL-related homeobox (WOX) family genes are involved in cell division and differentiation throughout various stages of plant growth and development. Till now, the function of WOX genes in radish remains unclear. In this study, a total of 23 RsWOX genes were identified from the radish genome. RT-qPCR analysis showed that the expression level of RsWUSb was significantly increased with exogenous cytokinin treatment. Overexpression of RsWUSb in Arabidopsis resulted in apical shoot clustering. Notably, the RsWUSb-overexpressed radish plants were generated by Agrobacterium-mediated in planta transformation approach. Compared with the wide-type (WT) plants, the RsWUSb-OE plants displayed clustered apical shoots, more leaves, and weak apical dominance. Moreover, yeast one-hybrid (Y1H) and dual-luciferase assay (DLA) showed that the RsRR18-1 (RESPONSE REGULATOR 18-1) bound to the promoter of RsWUSb and upregulated its transcription. Furthermore, the expression of RsRR18-1 was significantly activated by exogenous cytokinin, suggesting that cytokinin signaling might regulate the formation and development of radish shoots via RsRR18-1-RsWUSb module. Collectively, these findings indicated that in planta transformation approach is a simple and effective method for generating transgenic plants and gene function validation in radish.

Evolutionary and expression dynamics of LRR-RLKs and functional establishment of KLAVIER homolog in shoot mediated regulation of AON in chickpea symbiosis

Chickpea shoot exogenously treated with cytokinin showed stunted phenotype of root, shoot and significantly reduced nodule numbers. Genome-wide identification of LRR-RLKs in chickpea and Medicago resulted in 200 and 371 genes respectively. Gene duplication analysis revealed that LRR-RLKs family expanded through segmental duplications in chickpea and tandem duplications in Medicago. Expression profiling of LRR-RLKs revealed their involvement in cytokinin signaling and plant organ development. Overexpression of KLAVIER ortholog of chickpea, Ca_LRR-RLK147, in roots revealed its localization in the membrane but showed no effect on root nodulation despite increased cle peptide levels. Two findings (i) drastic effect on nodule number by exogenous cytokinin treatment to only shoot and restoration to normal nodulation by treatment to both root and shoot tissue and (ii) no effect on nodule number by overexpression of Ca_LRR-RLK147 establishes the fact that despite presence of cle peptides in root, the function of Ca_LRR-RLK147 was shoot mediated during AON.

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.

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.

Identification and expression analysis of cytokinin metabolic genes IPTs, CYP735A and CKXs in the biofuel plant Jatropha curcas.

The seed oil of Jatropha curcas is considered a potential bioenergy source that could replace fossil fuels. However, the seed yield of Jatropha is low and has yet to be improved. We previously reported that exogenous cytokinin treatment increased the seed yield of Jatropha. Cytokinin levels are directly regulated by isopentenyl transferase (IPT), cytochrome P450 monooxygenase, family 735, subfamily A (CYP735A), and cytokinin oxidase/dehydrogenase (CKX). In this study, we cloned six IPT genes, one JcCYP735A gene, and seven JcCKX genes. The expression patterns of these 14 genes in various organs were determined using real-time quantitative PCR. JcIPT1 was primarily expressed in roots and seeds, JcIPT2 was expressed in roots, apical meristems, and mature leaves, JcIPT3 was expressed in stems and mature leaves, JcIPT5 was expressed in roots and mature leaves, JcIPT6 was expressed in seeds at 10 days after pollination, and JcIPT9 was expressed in mature leaves. JcCYP735A was mainly expressed in roots, flower buds, and seeds. The seven JcCKX genes also showed different expression patterns in different organs of Jatropha. In addition, CK levels were detected in flower buds and seeds at different stages of development. The concentration of N6-(Δ2-isopentenyl)-adenine (iP), iP-riboside, and trans-zeatin (tZ) increased with flower development, and the concentration of iP decreased with seed development, while that of tZ increased. We further analyzed the function of JcCYP735A using the CRISPR-Cas9 system, and found that the concentrations of tZ and tZ-riboside decreased significantly in the Jccyp735a mutants, which showed severely retarded growth. These findings will be helpful for further studies of the functions of cytokinin metabolic genes and understanding the roles of cytokinins in Jatropha growth and development.

Analysis of the transcriptional responses in inflorescence buds of Jatropha curcas exposed to cytokinin treatment.

BackgroundJatropha curcas L. is a potential biofuel plant. Application of exogenous cytokinin (6-benzyladenine, BA) on its inflorescence buds can significantly increase the number of female flowers, thereby improving seed yield. To investigate which genes and signal pathways are involved in the response to cytokinin in J. curcas inflorescence buds, we monitored transcriptional activity in inflorescences at 0, 3, 12, 24, and 48 h after BA treatment using a microarray.ResultsWe detected 5,555 differentially expressed transcripts over the course of the experiment, which could be grouped into 12 distinct temporal expression patterns. We also identified 31 and 131 transcripts in J. curcas whose homologs in model plants function in flowering and phytohormonal signaling pathways, respectively. According to the transcriptional analysis of genes involved in flower development, we hypothesized that BA treatment delays floral organ formation by inhibiting the transcription of the A, B and E classes of floral organ-identity genes, which would allow more time to generate more floral primordia in inflorescence meristems, thereby enhancing inflorescence branching and significantly increasing flower number per inflorescence. BA treatment might also play an important role in maintaining the flowering signals by activating the transcription of GIGANTEA (GI) and inactivating the transcription of CONSTITUTIVE PHOTOMORPHOGENIC 1 (COP1) and TERMINAL FLOWER 1b (TFL1b). In addition, exogenous cytokinin treatment could regulate the expression of genes involved in the metabolism and signaling of other phytohormones, indicating that cytokinin and other phytohormones jointly regulate flower development in J. curcas inflorescence buds.ConclusionsOur study provides a framework to better understand the molecular mechanisms underlying changes in flowering traits in response to cytokinin treatment in J. curcas inflorescence buds. The results provide valuable information related to the mechanisms of cross-talk among multiple phytohormone signaling pathways in woody plants.Electronic supplementary materialThe online version of this article (doi:10.1186/s12870-014-0318-z) contains supplementary material, which is available to authorized users.

OsARF16 Is Involved in Cytokinin-Mediated Inhibition of Phosphate Transport and Phosphate Signaling in Rice (Oryza sativa L.)

BackgroundPlant responses to phytohormone stimuli are the most important biological features for plants to survive in a complex environment. Cytokinin regulates growth and nutrient homeostasis, such as the phosphate (Pi) starvation response and Pi uptake in plants. However, the mechanisms underlying how cytokinin participates in Pi uptake and Pi signaling are largely unknown. In this study, we found that OsARF16 is required for the cytokinin response and is involved in the negative regulation of Pi uptake and Pi signaling by cytokinin.Principal FindingsThe mutant osarf16 showed an obvious resistance to exogenous cytokinin treatment and the expression level of the OsARF16 gene was considerably up-regulated by cytokinin. Cytokinin (6-BA) application suppressed Pi uptake and the Pi starvation response in wild-type Nipponbare (NIP) and all these responses were compromised in the osarf16 mutant. Our data showed that cytokinin inhibits the transport of Pi from the roots to the shoots and that OsARF16 is involved in this process. The Pi content in the osarf16 mutant was much higher than in NIP under 6-BA treatment. The expressions of PHOSPHATE TRANSPORTER1 (PHT1) genes, phosphate (Pi) starvation-induced (PSI) genes and purple PAPase genes were higher in the osarf16 mutant than in NIP under cytokinin treatment.ConclusionOur results revealed a new biological function for OsARF16 in the cytokinin-mediated inhibition of Pi uptake and Pi signaling in rice.

Exogenous cytokinin treatment maintains cyclin homeostasis in rice seedlings that show changes of cyclin expression when the photoperiod is rapidly changed

Cyclin is a fundamental regulator of the plant cell cycle. Five different types of cyclin genes (the A-, B-, C-, D-, and H-types) have been reported in Oryza sativa. However, except for Os;cycA1;1, Os;cycB2;1, and Os;cycB2;2, the mechanisms of expression of these cyclin genes have not yet been studied. The interactions of cyclins with cytokinin, an important trigger for cell cycle regulation, have also not been well studied. Here we used semi-quantitative RT-PCR in rice seedlings to analyze the effect of cytokinin on photomorphogenesis and the expression of six cyclin genes. Fifteen-day-old seedlings were grown in a 16/8 h light/dark cycle and then transferred to either constant light or constant dark. The expression of all the cyclin genes tested, except the C-type, decreased after 1 hour in the dark, but did not change after transfer to the light or when kinetin was added to the medium. Similarly, seedlings grown in the dark had decreased expression of the cyclin genes, except Os;cycB2;2, after transfer to the light, a decrease that was prevented by kinetin treatment. Thus, exogenous cytokinin plays an important role in maintaining homeostasis of cyclin gene expression following rapid changes of photoperiod.

Changes in cytokinin activities before, during and after floral initiation in Polianthes tuberosa

The contents of endogenous cytokinin in tuberose corms ( Polianthes tuberosa) at vegetative, early floral initiation, and flower development stages were investigated. We also determined the influence of exogenous cytokinin treatment on the corm apex at three different growth stages in relation to floral initiation and development in tuberose. The exogenous cytokinin effectively induced floral initiation and development, especially at the early floral initiation and flower development stages. Endogenous cytokinins were higher in early floral initiation and development stages in comparison to the vegetative stage. During floral initiation stage, the zeatin and dihydrozeatin increased significantly, while the cytokinins, zeatin riboside, dihydrozeatin riboside, 6N- ( δ 2 -isopentenyl) adenine, and 6N -( δ 2 -isopentenyl) adenine riboside at consistently low levels. The increase of cytokinin levels in tuberose corms during floral induction suggests a role for cytokinins in tuberose apex evocation. Moreover, these results indicate that cytokinins seem to promote the development of flower buds rather than inducing flowering in tuberose.

Stomatal response to exogenous cytokinin treatment of the hemiparasite Melampyrum arvense L. before and after attachment to the host

The effect of cytokinins (CKs) and K+ on stomatal behaviour in darkness were studied in the root hemiparasite Melampyrum arvense before (the preparasitic stage) and after attachment to the host (Capsella bursa pastoris L. Med.). The solutes were applied with xylem stream. The stomatal apparatus of the attached hemiparasite was insensitive to externally supplied CKs and K+. Contrary to this finding, the stomatal aperture of hemiparasite in the preparasitic stage increased to about 25, 40 and 69% of the value obtained in light, respectively, after treatment with 200 mM KCl and 10-5 M zeatin riboside ([9R]Z), applied separately or together. CKs influenced K+ transport. The treatment with KCl and [9R]Z,separately or together, increased the content of K+ in guard cell pairs to about 32, 46 and 79 % of the value obtained in light, respectively. Other CKs had a smaller effect (45 - 16 %) in comparison with that of [9R]Z; isopentyladenine was nearly inactive.

Effect of cytokinin on alkaloid accumulation in periwinkle callus cultures transformed with a light-inducible ipt gene

The effect of cytokinins on accumulation of indole alkaloids in periwinkle callus cultures was investigated. Firstly, it was found that exogenously-applied cytokinin increased the ajmalicine and serpentine content of untransformed callus culture obtained from cotyledons. Secondly, periwinkle cotyledons were transformed with the isopentenyl transferase ( ipt) gene under the control of a light-inducible promoter and two transformed callus lines were used in order to investigate whether endogenously-produced cytokinin could also increase the alkaloid production. We found that the ipt-transgenic tissues accumulated higher levels of isopentenyl transferase transcripts as well as zeatin riboside, even under non-inductive condition, but lower concentration of alkaloids compared to that of untransformed tissues. A 28 kDa polypeptide whose accumulation was previously found to be associated with alkaloid production in a periwinkle cell suspension was also present in the non-transformed tissue and its level was increased in parallel to the CK-enhanced alkaloid production. Neither light induction condition, nor exogenous cytokinin treatment led to the increase of the 28 kDa polypeptide accumulation in the transformed tissues. All these data show that endogenously-produced cytokinin does not mimic the effect of exogenously-applied cytokinin on the alkaloid production in periwinkle calli.

Role of cytokinin in enhanced productivity of maize supplied with NH4 + and NO3 −

Supplying both N forms (NH4 ++NO3 −) to the maize (Zea mays L.) plant can optimize productivity by enhancing reproductive development. However, the physiological factors responsible for this enhancement have not been elucidated, and may include the supply of cytokinin, a growth-regulating substance. Therefore, field and gravel hydroponic studies were conducted to examine the effect of N form (NH4 ++NO3 − versus predominantly NO3 −) and exogenous cytokinin treatment (six foliar applications of 22 μM 6-benzylaminopurine (BAP) during vegetative growth versus untreated) on productivity and yield of maize. For untreated plants, NH4 ++NO3 − nutrition increased grain yield by 11% and whole shoot N content by 6% compared with predominantly NO3 −. Cytokinin application to NO3 −-grown field plants increased grain yield to that of NH4 ++NO3 −-grown plants, which was the result of enhanced dry matter partitioning to the grain and decreased kernel abortion. Likewise, hydroponically grown maize supplied with NH4 ++NO3 − doubled anthesis earshoot weight, and enhanced the partitioning of dry matter to the shoot. NH4 ++NO3 − nutrition also increased earshoot N content by 200%, and whole shoot N accumulation by 25%. During vegetative growth, NH4 ++NO3 − plants had higher concentrations of endogenous cytokinins zeatin and zeatin riboside in root tips than NO3 −-grown plants. Based on these data, we suggest that the enhanced earshoot and grain production of plants supplied with NH4 ++NO3 − may be partly associated with an increased endogenous cytokinin supply.