Exogenous Cytokinin Research Articles

Function of histone H2B monoubiquitination in transcriptional regulation of auxin biosynthesis in Arabidopsis

The auxin IAA is a vital plant hormone in controlling growth and development, but our knowledge about its complicated biosynthetic pathways and molecular regulation are still limited and fragmentary. cytokinin induced root waving 2 (ckrw2) was isolated as one of the auxin-deficient mutants in a large-scale forward genetic screen aiming to find more genes functioning in auxin homeostasis and/or its regulation. Here we show that CKRW2 is identical to Histone Monoubiquitination 1 (HUB1), a gene encoding an E3 ligase required for histone H2B monoubiquitination (H2Bub1) in Arabidopsis. In addition to pleiotropic defects in growth and development, loss of CKRW2/HUB1 function also led to typical auxin-deficient phenotypes in roots, which was associated with significantly lower expression levels of several functional auxin synthetic genes, namely TRP2/TSB1, WEI7/ASB1, YUC7 and AMI1. Corresponding defects in H2Bub1 were detected in the coding regions of these genes by chromatin immunoprecipitation (ChIP) analysis, indicating the involvement of H2Bub1 in regulating auxin biosynthesis. Importantly, application of exogenous cytokinin (CK) could stimulate CKRW2/HUB1 expression, providing an epigenetic avenue for CK to regulate the auxin homeostasis. Our results reveal a previously unknown mechanism for regulating auxin biosynthesis via HUB1/2-mediated H2Bub1 at the chromatin level.

Diversified Regulation of Cytokinin Levels and Signaling During Botrytis cinerea Infection in Arabidopsis.

Cytokinins (CKs) can modulate plant immunity to various pathogens, but how CKs are involved in plant defense responses to the necrotrophic pathogen Botrytis cinerea is still unknown. Here, we found that B. cinerea infection induced transcriptional changes in multiple genes involved in the biosynthesis, degradation, and signaling of CKs, as well as their contents, in pathogen-infected Arabidopsis leaves. Among the CKs, the gene expression of CYTOKININ OXIDASE/DEHYDROGENASE 5 (CKX5) was remarkably induced in the local infected leaves and the distant leaves of the same plant without pathogen inoculation. Cis-zeatin (cZ) and its riboside (cZR) accumulated considerably in infected leaves, suggesting an important role of the cis-zeatin type of CKs in the plant response to B. cinerea. Cytokinin double-receptor mutants were more susceptible to B. cinerea infection, whereas an exogenous CK treatment enhanced the expression levels of defense-related genes and of jasmonic acid (JA) and ethylene (ET), but not salicylic acid (SA), resulting in higher resistance of Arabidopsis to B. cinerea. Investigation of CK responses to B. cinerea infection in the JA biosynthesis mutant, jar1-1, and ET-insensitive mutant, ein2-1, showed that CK signaling and levels of CKs, namely, those of isopentenyladenine (iP), isopentenyladenine riboside (iPR), and trans-zeatin (tZ), were enhanced in jar1-1-infected leaves. By contrast, reductions in iP, iPR, tZ, and tZ riboside (tZR) as well as cZR contents occurred in ein2-1-infected leaves, whose transcript levels of CK signaling genes were likewise differentially regulated. The Arabidopsis Response Regulator 5 (ARR5) gene was upregulated in infected leaves of ein2-1 whereas another type-A response regulator, ARR16, was significantly downregulated, suggesting the existence of a complex regulation of CK signaling via the ET pathway. Accumulation of the cis-zeatin type of CKs in B. cinerea-infected leaves depended on ET but not JA pathways. Collectively, our findings provide evidence that CK responds to B. cinerea infection in a variety of ways that are differently modulated by JA and ET pathways in Arabidopsis.

Polyamine Metabolism Is Involved in the Direct Regeneration of Shoots from Arabidopsis Lateral Root Primordia.

Plants can be regenerated from various explants/tissues via de novo shoot meristem formation. Most of these regeneration pathways are indirect and involve callus formation. Besides plant hormones, the role of polyamines (PAs) has been implicated in these processes. Interestingly, the lateral root primordia (LRPs) of Arabidopsis can be directly converted to shoot meristems by exogenous cytokinin application. In this system, no callus formation takes place. We report that the level of PAs, especially that of spermidine (Spd), increased during meristem conversion and the application of exogenous Spd improved its efficiency. The high endogenous Spd level could be due to enhanced synthesis as indicated by the augmented relative expression of PA synthesis genes (AtADC1,2, AtSAMDC2,4, AtSPDS1,2) during the process. However, the effect of PAs on shoot meristem formation might also be dependent on their catabolism. The expression of Arabidopsis POLYAMINE OXIDASE 5 (AtPAO5) was shown to be specifically high during the process and its ectopic overexpression increased the LRP-to-shoot conversion efficiency. This was correlated with Spd accumulation in the roots and ROS accumulation in the converting LRPs. The potential ways how PAO5 may influence direct shoot organogenesis from Arabidopsis LRPs are discussed.

OsRR6, a type-A response regulator in rice, mediates cytokinin, light and stress responses when over-expressed in Arabidopsis

Plants have evolved a complex network of components that sense and respond to diverse signals. In the present study, we have characterized OsRR6, a type-A response regulator, which is part of the two-component sensor-regulator machinery in rice. The expression of OsRR6 is induced by exogenous cytokinin and various abiotic stress treatments, including drought, cold and salinity stress. Organ-specific expression analysis revealed that its expression is high in anther and low in shoot apical meristem. The Arabidopsis plants constitutively expressing OsRR6 (OsRR6OX) exhibited reduced cytokinin sensitivity, adventitious root formation and enhanced anthocyanin accumulation in seeds. OsRR6OX plants were more tolerant to drought and salinity conditions when compared to wild-type. The hypocotyl growth in OsRR6OX seedlings was significantly inhibited under red, far-red and blue-light conditions and also a decline in transcript levels of OsRR6 was observed in rice under the above monochromatic as well as white light treatments. Transcriptome profiling revealed that the genes associated with defense responses and anthocyanin metabolism are up-regulated in OsRR6OX seedlings. Comparative transcriptome analysis showed that the genes associated with phenylpropanoid and triterpenoid biosynthesis are enriched among differentially expressed genes in OsRR6OX seedlings of Arabidopsis, which is in conformity with reanalysis of the transcriptome data performed in rice transgenics for OsRR6. Further, genes like DREB1A/CBF3, COR15A, KIN1, ERD10 and RD29A are significantly upregulated in OsRR6OX seedlings when subjected to ABA and abiotic stress treatments. Thus, a negative regulator of cytokinin signaling, OsRR6, plays a positive role in imparting abiotic stress tolerance.

Ethylene-Cytokinin Interaction Determines Early Defense Response of Wheat against Stagonospora nodorum Berk.

Ethylene, salicylic acid (SA), and jasmonic acid are the key phytohormones involved in plant immunity, and other plant hormones have been demonstrated to interact with them. The classic phytohormone cytokinins are important participants of plant defense signaling. Crosstalk between ethylene and cytokinins has not been sufficiently studied as an aspect of plant immunity and is addressed in the present research. We compared expression of the genes responsible for hormonal metabolism and signaling in wheat cultivars differing in resistance to Stagonospora nodorum in response to their infection with fungal isolates, whose virulence depends on the presence of the necrotrophic effector SnTox3. Furthermore, we studied the action of the exogenous cytokinins, ethephon (2-chloroethylphosphonic acid, ethylene-releasing agent) and 1-methylcyclopropene (1-MCP, inhibitor of ethylene action) on infected plants. Wheat susceptibility was shown to develop due to suppression of reactive oxygen species production and decreased content of active cytokinins brought about by SnTox3-mediated activation of the ethylene signaling pathway. SnTox3 decreased cytokinin content most quickly by its activated glucosylation in an ethylene-dependent manner and, furthermore, by oxidative degradation and inhibition of biosynthesis in ethylene-dependent and ethylene-independent manners. Exogenous zeatin application enhanced wheat resistance against S. nodorum through inhibition of the ethylene signaling pathway and upregulation of SA-dependent genes. Thus, ethylene inhibited triggering of SA-dependent resistance mechanism, at least in part, by suppression of the cytokinin signaling pathway.

Phenylurea-type Cytokinin ameliorates the performance of young pea plants under salt stress

Cytokinins are known to enhance stress tolerance in plants. The present study aimed to assess the possible protective effect of exogenous phenylurea-type cytokinin (4PU-30) on alleviating salt (NaCl) stress. Young pea (Pisum sativum L.) plants were sprayed with cytokinin 4PU-30 and were subsequently subjected to NaCl treatment. The effect of 4PU-30 on cell membrane stability was assessed based on electrolyte leakage from leaves of control and NaCl stressed plants. A previously established model system employing the kinetics of ion leakage served to evaluate the effect of the 4PU-30 application on plants response to salinity. Salt treatment caused a moderate decrease in leaf water content. Accumulation of proline, malondialdehyde (MDA), and hydrogen peroxide (H2O2) in the leaves of NaCl treated plants indicated the development of oxidative stress, which was significantly alleviated by pretreatment with phenylurea-type cytokinin 4PU-30. Foliar application of 4PU-30 reduced the damaging effect of NaCl, as evidenced by decreased electrolyte leakage. Distinct roles of cell walls and plasmalemma in the processes of ion efflux due to salt stress are discussed.

Application of Exogenous Phytohormones at Silking Stage Improve Grain Quality under Post-Silking Drought Stress in Waxy Maize.

The application of exogenous plant growth regulator can improve plant resistance to drought stress. The effects of application of exogenous cytokinin (CTK), brassinolide (BR), or gibberellic acid (GA) at the silking time on the grain quality of two waxy maize hybrids under drought stress at grain formation stage were studied. Grain weight of both hybrids was unaffected by exogenous phytohormones under control conditions but increased under drought conditions with the application of BR. The grain starch content in response to drought varied with hybrid and phytohormone. Starch granule size and protein content in grains were increased by drought under all conditions, but various phytohormones exerted different forms of influence. The starch λmax in Yunuo7 was unaffected by single or interaction of phytohormones and water deficit, λmax in Jingkenuo2000 with BR was unaffected but with CTK or GA increased by drought. Relative crystallinity was reduced by drought without the application of phytohormone, but with phytohormones in response to drought it was different. Flour peak viscosity was reduced by drought. The value was increased with BR spraying under control and drought conditions. Retrogradation percentage under drought conditions was unaffected by exogenous phytohormones in Jingkenuo2000. In Yunuo7, retrogradation percentage was unaffected by BR but reduced by CTK and GA. In conclusion, spraying phytohormones at the silking stage can affect grain weight and starch quality, grains with a sticky taste can be improved by applying BR, and grains with low retrograde tendency can be produced by applying CTK.

Targeted Transgene Expression in Rice Using a Callus Strong Promoter for Selectable Marker Gene Control.

Precise expression of a transgene in the desired manner is important for plant genetic engineering and gene function deciphering, but it is a challenge to obtain specific transgene expression free from the interference of the constitutive promoters used to express the selectable marker gene, such as the Cauliflower mosaic virus (CaMV) 35S promoter. So, the solutions to avoid these inappropriate regulations are largely demanded. In this study, we report the characterization of a callus strong promoter (CSP1) in rice and its application for accurate transgene expression. Our results indicate that the high expression of the CSP1 promoter in the callus enables efficient selection of hygromycin equivalent to that provided by the CaMV 35S promoter, whereas its expression in other tissues is low. To evaluate possible leaky effects, the expression of a β-glucuronidase reporter driven by six specific promoters involving hormone signaling, pathogen response, cell fate determination, and proliferation was observed in transgenic rice plants generated by CSP1-mediated selection. Distinct β-glucuronidase expression was found consistently in most of the transgenic lines obtained for each promoter. In addition, we applied these specific marker lines to investigate the root cellular responses to exogenous cytokinin and auxin treatment. The results reveal that the root growth inhibition by cytokinin was differently regulated at high and low concentrations. In summary, we have established the feasibility of using callus-specific promoter-dependent selection to mitigate the transgene misexpression in rice. By enabling efficient transformation, rice plants with reliable transgene expression will be easily acquired for broad applications.

Caspase-like proteases and the phytohormone cytokinin as determinants of S-RNAse-based self-incompatibility-induced PCD in Petunia hybrida L.

S-RNAse-based self-incompatibility (SI) in petunia (Petunia hybrida L.) is a self-/non-self-recognition system underlying the pistil rejection of self-pollen. Using different methods, including a TUNEL assay, we have recently shown that programmed cell death (PCD) is a factor of the SI in petunia. Here, we show that the growth of self-incompatible pollen tubes in the style tissues during 4h after pollination is accompanied by five-sixfold increase in a caspase-like protease (CLP) activity. Exogenous cytokinin (CK) inhibits the pollen tube growth and stimulates the CLP activity in compatible pollen tubes. The actin depolymerization with latrunculin B induces a sharp drop in the CLP activity in self-incompatible pollen tubes and its increase in compatible pollen tubes. Altogether, our results suggest that a CLP is involved in the SI-induced PCD and that CK is a putative activator of the CLP. We assume that CK provokes acidification of the cytosol and thus promotes the activation of a CLP. Thus, our results suggest that CK and CLP are involved in the S-RNAse-based SI-induced PCD in petunia. Potential relations between these components in PCD signaling are discussed.

Arabidopsis AZG2 transports cytokinins in vivo and regulates lateral root emergence.

Cytokinin and auxin are key regulators of plant growth and development. During the last decade transport mechanisms have turned out to be the key for the control of local and long-distance hormone distributions. In contrast with auxin, cytokinin transport is poorly understood. Here, we show that Arabidopsis thaliana AZG2, a member of the AZG purine transporter family, acts as cytokinin transporter involved in root system architecture determination. Even though purines are substrates for both AZG1 and AZG2, we found distinct transport mechanisms. The expression of AZG2 is restricted to a small group of cells surrounding the lateral root (LR) primordia and induced by auxins. Compared to the wild-type (WT), mutants carrying loss-of-function alleles of AZG2 have higher LR density, suggesting that AZG2 is part of a regulatory pathway in LR emergence. Moreover, azg2 is partially insensitive to exogenous cytokinin, which is consistent with the observation that the cytokinin reporter TCSnpro :GFP showed lower fluorescence signal in the roots of azg2 compared to the WT. These results indicate a defective cytokinin signalling pathway in the region of LR primordia. The integration of AZG2 subcellular localization and cytokinin transport capacity data allowed us to propose a local cytokinin:auxin signalling model for the regulation of LR emergence.

A GRF-GIF chimeric protein improves the regeneration efficiency of transgenic plants.

The potential of genome editing to improve the agronomic performance of crops is often limited by low plant regeneration efficiencies and few transformable genotypes. Here, we show that expression of a fusion protein combining wheat GROWTH-REGULATING FACTOR 4 (GRF4) and its cofactor GRF-INTERACTING FACTOR 1 (GIF1) substantially increases the efficiency and speed of regeneration in wheat, triticale and rice and increases the number of transformable wheat genotypes. GRF4-GIF1 transgenic plants were fertile and without obvious developmental defects. Moreover, GRF4-GIF1 induces efficient wheat regeneration in the absence of exogenous cytokinins, which facilitates selection of transgenic plants without selectable markers. We also combined GRF4-GIF1 with CRISPR-Cas9 genome editing, generating 30 edited wheat plants with disruptions in the gene Q (AP2L-A5). Finally, we show that a dicot GRF-GIF chimera improves regeneration efficiency in citrus, suggesting that this strategy can be applied to dicot crops.

The pre-mRNA splicing factor RDM16 regulates root stem cell maintenance in Arabidopsis.

Pre-mRNA (messenger RNA) splicing participates in the regulation of numerous biological processes in plants. For example, alternative splicing shapes transcriptomic responses to abiotic and biotic stress, and controls developmental programs. However, no study has revealed a role for splicing in maintaining the root stem cell niche. Here, a screen for defects in root growth in Arabidopsis thaliana identified an ethyl methane sulfonate mutant defective in pre-mRNA splicing (rdm16-4). The rdm16-4 mutant displays a short-root phenotype resulting from fewer cells in the root apical meristem. The PLETHORA1 (PLT1) and PLT2 transcription factor genes are important for root development and were alternatively spliced in rdm16-4 mutants, resulting in a disordered root stem cell niche and retarded root growth. The root cap of rdm16-4 contained reduced levels of cytokinins, which promote differentiation in the developing root. This reduction was associated with the alternative splicing of genes encoding cytokinin signaling factors, such as ARABIDOPSIS HISTIDINE PHOSPHOTRANSFER PROTEIN5 and ARABIDOPSIS RESPONSE REGULATORS (ARR1, ARR2, and ARR11). Furthermore, expression of the full-length coding sequence of ARR1 or exogenous cytokinin application partially rescued the short-root phenotype of rdm16-4. This reveals that the RDM16-mediated alternative splicing of cytokinin signaling components contributes to root growth.

MERISTEM ACTIVITYLESS (MAL) is involved in root development through maintenance of meristem size in rice.

Rice MERISTEM ACTIVITYLESS (MAL), a RING-H2 finger domain (RFD)-containing gene, regulates meristem cell viability after the initiation of root primordia mediated by cytokinin signaling. Genes in the RING-H2 finger domain (RFD) family play various roles during plant development and in biotic/abiotic stress responses. Rice gene MERISTEM ACTIVITYLESS (MAL), being contained in the RING-H2 finger domain (RFD), is characterized by a transmembrane domain at the N-terminal and a C3H2C3 zinc finger domain at the C-terminal. To elucidate the physiological and molecular functions of MAL, we generated MAL knockdown transgenic plants by RNA interference. MAL RNA-interfered (MRi) transgenic plants exhibited a phenotype with shorter crown root length and lower crown root number, accompanied by a lower cell division rate. The low division rate was observed in the root meristem exactly where MAL was expressed. Furthermore, transcriptome data revealed that cell wall macromolecule metabolism-related genes and redox-related genes were enriched in MAL RNAi lines. Most of these differentially expressed genes (DEGs) were induced by exogenous cytokinin. Hence, we conclude that MAL, as a novel regulatory factor, plays a major role in maintaining cell viability in the meristem after the initiation of root primordial formation, mediated by cytokinin signaling and reactive oxygen species (ROS).

Cytokinin response induces immunity and fungal pathogen resistance, and modulates trafficking of the PRR LeEIX2 in tomato.

Plant immunity is often defined by the immunity hormones: salicylic acid (SA), jasmonic acid (JA), and ethylene (ET). These hormones are well known for differentially regulating defence responses against pathogens. In recent years, the involvement of other plant growth hormones such as auxin, gibberellic acid, abscisic acid, and cytokinins (CKs) in biotic stresses has been recognized. Previous reports have indicated that endogenous and exogenous CK treatment can result in pathogen resistance. We show here that CK induces systemic immunity in tomato (Solanum lycopersicum), modulating cellular trafficking of the pattern recognition receptor (PRR) LeEIX2, which mediates immune responses to Xyn11 family xylanases, and promoting resistance to Botrytis cinerea and Oidium neolycopersici in an SA‐ and ET‐dependent mechanism. CK perception within the host underlies its protective effect. Our results support the notion that CK promotes pathogen resistance by inducing immunity in the host.

Effects of spraying exogenous cytokinin or spermine on the starch physicochemical properties of waxy maize exposed to post-silking high temperature

Plant growth regulators (PGR) can be used to alleviate heat injury on the starch quality of crops. In this study, two waxy maize hybrids with exogenous cytokinin or spermine at silking stage were exposed to post-silking ambient temperature (AT; 28/20 °C) and high temperature (HT; 35/27 °C) during grain filling. Results indicated that spraying exogenous cytokinin or spermine increased the grain weight and starch content at both temperature regimes. The starch granule size, amylopectin chain length, and relative crystallinity differed between two hybrids and were affected by PGRs and temperatures. Peak viscosity decreased at HT, unaffected by cytokinin, and increased by spermine at both temperature regimes. The retrogradation percentage at HT with PGR was lower than that without PGR in both hybrids. In conclusion, spraying exogenous cytokinin or spermine at silking stage can increase peak viscosity and reduce retrogradation percentage at HT, thereby alleviating the detrimental effects of post-silking heat injury.

From Survival to Productivity Mode: Cytokinins Allow Avoiding the Avoidance Strategy Under Stress Conditions.

Growth retardation and stress-induced premature plant senescence are accompanied by a severe yield reduction and raise a major agro-economic concern. To improve biomass and yield in agricultural crops under mild stress conditions, the survival must be changed to productivity mode. Our previous successful attempts to delay premature senescence and growth inhibition under abiotic stress conditions by autoregulation of cytokinins (CKs) levels constitute a generic technology toward the development of highly productive plants. Since this technology is based on the induction of CKs synthesis during the age-dependent senescence phase by a senescence-specific promoter (SARK), which is not necessarily regulated by abiotic stress conditions, we developed autoregulating transgenic plants expressing the IPT gene specifically under abiotic stress conditions. The Arabidopsis promoter of the stress-induced metallothionein gene (AtMT) was isolated, fused to the IPT gene and transformed into tobacco plants. The MT:IPT transgenic tobacco plants displayed comparable elevated biomass productivity and maintained growth under drought conditions. To decipher the role and the molecular mechanisms of CKs in reverting the survival transcriptional program to a sustainable plant growth program, we performed gene expression analysis of candidate stress-related genes and found unexpectedly clear downregulation in the CK-overproducing plants. We also investigated kinase activity after applying exogenous CKs to tobacco cell suspensions that were grown in salinity stress. In-gel kinase activity analysis demonstrated CK-dependent deactivation of several stress-related kinases including two of the MAPK components, SIPK and WIPK and the NtOSAK, a member of SnRK2 kinase family, a key component of the ABA signaling cascade. A comprehensive phosphoproteomics analysis of tobacco cells, treated with exogenous CKs under salinity-stress conditions indicated that >50% of the identified phosphoproteins involved in stress responses were dephosphorylated by CKs. We hypothesize that upregulation of CK levels under stress conditions desensitize stress signaling cues through deactivation of kinases that are normally activated under stress conditions. CK-dependent desensitization of environmental stimuli is suggested to attenuate various pathways of the avoidance syndrome including the characteristic growth arrest and the premature senescence while allowing normal growth and metabolic maintenance.

Acid invertase activities of dahlia ‘Kokucho’ petals during flower opening and following cutting and treatment with 6-benzylaminopurine

Extending the vase life of cut dahlia ‘Kokucho’ flowers through enhanced activities of acid invertases (cell wall, CWIN and vacuolar, VIN) on flower opening and senescence after harvest was investigated. The activities of CWIN and VIN were very low in the outer petals at the tight bud stage (stage 1), increased markedly at the half-open stage (stage 2), and decreased at the fully open stage (stage 3) to levels lower than at stage 2. Glucose and fructose concentrations were very low at stage 1, markedly increased at stage 2, and further increased at stage 3. These results suggest that acid invertase activities play a very important role in increasing soluble sugars of petals during flower opening. Following harvest at stage 3, florets or cut flowers were treated with 50 μmol L−1 6-benzylaminopurine (BA) by dipping or spraying, respectively. These treatments extended vase life longer than in the controls. In the florets experiment, CWIN and VIN activities were significantly higher with a BA dip than in the control for petals at 3 and 6 d, but there were no significant differences in sugar concentrations between the control and BA dip groups. In cut flowers at 3 d after cutting, VIN activity and glucose concentration were higher in the outer petals treated with BA spray than in control petals. From these results, application of exogenous cytokinin has some effect of maintaining high acid invertase activity, and high invertase activity itself may be important for delaying senescence in cut dahlia flowers. BA application is useful for extending petal longevity and is expected to be a popular post-harvest treatment in cut dahlia flowers.

Plug Cell Volume, Main Temperature and the Synthetic Cytokinin 6, Benzyl Aminopurine (BAP) Spray on Lettuce Biomass Accumulation during Nursery at Ambient Temperatures

Although nursery lettuce information is plentiful, the use of exogenous cytokinin (6, benzyl amino purine, BAP) as a plant growth regulator, which would increase vegetable nursery yield has only recent suggested. On the other hand, biomass relationships between plug cell volumes, BAP uses and main temperature during nursery is quite scarce. Our results showed that the growth response of lettuce seedlings during nursery is firstly related to mean air temperature, independently the environmental characteristics of the cultivar tested. From the view of a nursery grower, yield can be optimize when higher plug cell number per tray was used or a BAP spray was applied to limited plug trays (128- or 288-cells). One of the novelty result of this work reside in the objective protocol proposed (the marginal root dry weight accumulation rate) to decide the time to transplant. Finally, our results showed that plug purchaser would be change their visual criteria when they buy BAP-treated lettuce for increased field yield.

Differential growth response of green and variegated Ficus benjamina to exogenous cytokinin and shade

Abstract Benjamin fig (Ficus benjamina) is an important foliage and landscape crop species comprising green and variegated genotypes. The latter develop leaves with yellow and white leaf areas which may impose lower photosynthetic activity, thus resulting in slower growth than green genotypes. In many species, the exogenous supply of cytokinin to pot-grown plants promotes growth, mainly due to enhanced carbon fixation. In this work, we analyze the effect of spraying the cytokinin 6-benzylaminopurine (BAP) on growth and development of green and variegated Ficus benjamina genotypes. Two experiments were carried out in a greenhouse, in which either different number of BAP applications (Experiment 1) or different BAP concentrations (Experiment 2) were tested. In Experiment 2, plants were grown under three different light intensities. BAP sprays promoted rate of leaf appearance, leaf expansion and whole-plant growth, and the effect was stronger in variegated than in green plants. The relative growth rate promotion by BAP was associated with increased net assimilation rate rather than with variation in the leaf area ratio. On the other hand, shading had a more negative impact on growth and development of variegated plants than in green ones. Variegated plants, unlike green ones, developed leaves with high specific leaf area under the lowest light intensity. This led to high leaf area ratio values, which helped to maintain relative growth rates close to those of plants under moderate shading.

The Type-B Cytokinin Response Regulator ARR1 Inhibits Shoot Regeneration in an ARR12-Dependent Manner in Arabidopsis.

Exogenous cytokinin is critical for in vitro shoot regeneration. Proteins involved in the cytokinin signal transduction pathway, including type-B ARABIDOPSIS RESPONSE REGULATORs (ARRs), participate in shoot regeneration in Arabidopsis (Arabidopsis thaliana). Some type-B ARRs (e.g., ARR1 and ARR12) promote shoot regeneration by directly activating WUSCHEL (WUS) expression; however, it is unclear how type-B ARRs inhibit shoot regeneration. Here, we show that ARR12 is a central enhancer of callus formation and shoot regeneration, whereas ARR1 is a strong inhibitor of this process that counteracts the positive effect of ARR12. ARR1 indirectly represses CLAVATA3 (CLV3) expression in an ARR12-dependent manner via competing with ARR12 for binding to the CLV3 promoter, which contributes to its ARR12-dependent inhibitory effect on callus formation and shoot regeneration. In parallel, ARR1 inhibits shoot regeneration through transcriptional activation of INDOLE-3-ACETIC ACID INDUCIBLE17, an auxin response repressor gene, and the consequent indirect repression of WUS expression. Thus, type-B ARRs have diverse effects on callus formation and shoot regeneration. Our study reveals novel molecular pathways linking cytokinin signaling, the CLV3 regulator, and auxin signaling, and sheds light on the mechanism underlying cytokinin-regulated shoot regeneration.