Absence Of Cytokinin Research Articles

Cytokinins initiate secondary growth in the Arabidopsis root through a set of LBD genes

SummaryDuring primary growth, plant tissues increase their length, and as these tissues mature, they initiate secondary growth to increase thickness.1 It is not known what activates this transition to secondary growth. Cytokinins are key plant hormones regulating vascular development during both primary and secondary growth. During primary growth of Arabidopsis roots, cytokinins promote procambial cell proliferation2,3 and vascular patterning together with the hormone auxin.4, 5, 6, 7 In the absence of cytokinins, secondary growth fails to initiate.8 Enhanced cytokinin levels, in turn, promote secondary growth.8,9 Despite the importance of cytokinins, little is known about the downstream signaling events in this process. Here, we show that cytokinins and a few downstream LATERAL ORGAN BOUNDARIES DOMAIN (LBD) family of transcription factors are rate-limiting components in activating and further promoting secondary growth in Arabidopsis roots. Cytokinins directly activate transcription of two homologous LBD genes, LBD3 and LBD4. Two other homologous LBDs, LBD1 and LBD11, are induced only after prolonged cytokinin treatment. Our genetic studies revealed a two-stage mechanism downstream of cytokinin signaling: while LBD3 and LBD4 regulate activation of secondary growth, LBD1, LBD3, LBD4, and LBD11 together promote further radial growth and maintenance of cambial stem cells. LBD overexpression promoted rapid cell growth followed by accelerated cell divisions, thus leading to enhanced secondary growth. Finally, we show that LBDs rapidly inhibit cytokinin signaling. Together, our data suggest that the cambium-promoting LBDs negatively feed back into cytokinin signaling to keep root secondary growth in balance.

Cytokinin-regulated targets of Cytokinin Response Factor 6 are involved in potassium transport.

Cytokinin (CK) is a plant hormone crucial to plant development and growth. Cytokinin Response Factor 6 (CRF6) is a CK-induced transcription factor that is part of the CK signaling cascade. While the role of CRF6 has been examined in oxidative stress response, there has been surprisingly little investigation of CRF6 in the context of CK signaling, including identifying CK-regulated targets of CRF6. Here, we conduct a transcriptomic study of Arabidopsis examining the CRF6 mutant (crf6) in the presence and absence of CK, revealing 163 downstream CRF6-dependent CK-regulated differentially expressed genes (DEGs). 15.3% of these DEGS were found as overlapping with larger number of standardly identified CK-regulated DEGs, suggesting that CRF6 is involved in regulating a subset of downstream CK responses through these gene targets. The general transcriptional regulation of CRF6-dependent CK-regulated DEGs indicates that CRF6 may function as a negative regulator of CK response. We investigated one subset of CRF6 CK-dependent targets (SKOR, HAK5, and NRT1. 5) involved in an underexamined functional role of CK response: the uptake and transportation of potassium. To determine how CK and CRF6 are involved in potassium acquisition and distribution, ionomic and physiological experiments were conducted on plants grown in media with sufficient and deficient potassium concentrations and in the presence and absence of CK. In order to investigate how CK alone affects potassium transport, similar experiments were performed on skor, hak5, and nrt1.5 mutant lines of these CRF6-dependent CK-regulated targets. These findings indicate novel connections between CK and potassium transport, which appear to be regulated in a CRF6-dependent manner.

Micropropagación de Prosopis pallida (Humb &Bonpl. Ex Willd.) Kunth a partir de yemas apicales

Prosopis pallida, conocido como algarrobo, es una especie emblemática de los bosques secos del norte del Perú. Es de gran importancia económica por su uso en la producción de leña y carbón, así como en la producción de algarrobina proveniente de sus frutos. Actualmente las actividades humanas han deforestado grandes poblaciones de algarrobo en el bosque seco, por lo que es muy importante una propagación masiva para planes de reforestación a gran escala en esos ecosistemas, con la finalidad de conservar la especie y también las características genéticas de individuos élite. El objetivo de la investigación fue establecer un protocolo para la propagación in vitro de algarrobo. Previo a la siembra in vitro, se evaluaron tres tratamientos pregerminativos para poder acelerar la germinación de las semillas con la finalidad de obtener mayor material de propagación. Luego se evaluó el efecto del medio de plantas leñosas con la adición de cuatro concentraciones (0; 0,5; 1,0 y 1,5 mg/L) de citoquininas (BAP y ZEA) sobre la propagación in vitro de Prosopis pallida, habiendo realizado tres ensayos debido a la poca efectividad de brotación de las yemas apicales, resultando mejor la ausencia de citoquininas en yemas apicales con sus dos cotiledones, usando tapas de algodón en los tubos de ensayo. En esta etapa se evaluó el número de nudos, altura de plántula y número de brotes. Para el enraizamiento se ensayó con tres concentraciones (0; 0,5 y 1,0 mg/L) de auxinas (NAA, IBA y IAA), y se evaluó el porcentaje de enraizamiento, longitud de la raíz y número de raíces; obteniéndose mejores resultados con 0,5 mg/L IBA. En la fase de aclimatación se evaluó el porcentaje de aclimatación en dos tipos de sustratos, obteniéndose mejores resultados con sustrato comercial de turba y vermiculita.

Down-regulation of multiple CDK inhibitor ICK/KRP genes promotes cell proliferation, callus induction and plant regeneration in Arabidopsis.

The ICK/KRP cyclin-dependent kinase (CDK) inhibitors are important plant cell cycle regulators sharing only limited similarity with the metazoan CIP/KIP family of CDK inhibitors. Information is still limited regarding the specific functions of different ICK/KRP genes in planta. We have shown previously that down-regulation of multiple CDK inhibitor ICK/KRP genes up-regulates the E2F pathway and increases cell proliferation, and organ and seed sizes in Arabidopsis. In this study, we observed that the quintuple ick1/2/5/6/7 mutant had more cells in the cortical layer of the root apical meristem (RAM) than the wild type (Wt) while its RAM length was similar to that of the Wt, suggesting a faster cell cycle rate in the quintuple mutant. We further investigated the effects of down-regulating ICK genes on tissue culture responses. The cotyledon explants of ick1/2/5/6/7 could form callus efficiently in the absence of cytokinin and also required a lower concentration of 2,4-D for callus induction compared to the Wt plants, suggesting increased competence for callus induction in the mutant. In addition, the quintuple ick mutant showed enhanced abilities to regenerate shoots and roots, suggesting that increased competence to enter the cell cycle in the quintuple mutant might make it possible for more cells to become proliferative and be utilized to form shoots or roots. These findings indicate that CDK activity is a major factor underlying callus induction and increased cell proliferation can enhance in vitro organogenesis.

A type-B response regulator drives the expression of the hydroxymethylbutenyl diphosphate synthase gene in periwinkle

In plant cytokinin (CK) signaling, type-B response regulators (RRs) act as major players in orchestrating the transcriptome changes in response to CK. However, their direct targets are poorly known. The identification of putative type-ARR1 motifs located within the promoter of the CK-responsive hydroxyl methyl butenyl diphosphate synthase (HDS) gene from the methyl erythritol phosphate (MEP) pathway prompted us to investigate the ability of a previously isolated periwinkle type-B RR (CrRR5) that presents high homologies with ARR1 to interact with the promoter. Electrophoretic mobility shift assays (EMSAs) demonstrated that the CrRR5 DNA-binding domain binds specifically type-ARR1 motifs within the HDS promoter. We also established through yellow fluorescent protein (YFP) imaging the targeting of CrRR5 into cell nucleus in accordance with its putative function of transcription factor. In transient assays performed on periwinkle cells cultivated with CK, overexpression of the full-length CrRR5 or a truncated CrRR5 engineering a constitutive active form (35S:ΔDDK) did not affect the HDS promoter activity that reached a threshold. By contrast, in absence of CK, overexpression of CrRR5ΔDDK enhanced promoter activity up to the threshold level observed in cells grown with CK. Our results strongly suggest that CrRR5 directly transactivates the HDS promoter. CrRR5 is the first identified transcription factor mediating the CK signaling that targets a gene from the MEP pathway involved in isoprenoid metabolism. Moreover, CrRR5 could play a role in a regulatory mechanism controlling CK homeostasis in periwinkle cells.

In vitro conservation and low cost micropropagation of Cochlospermum regium (Mart. Ex. Scharank)

Cochlospermum regium (Mart. Ex. Scharank), a Cochlospermaceae is a Cerrado medicinal species showing antimicrobial activity on the female reproductive system. The species risks being genetically eroded since it is highly explored, the biome devastated and the plant roots are employed for phytotherapeutic preparations. This study aims to develop a micropropagation protocol for large scale production of plantlets and establish the species conservation in a germplasm bank, in vitro. Nodal segments were inoculated into Murashige and Skoog (MS) medium supplemented with different concentrations of sucrose, BAP, kinetin, zeatin, Phytagel® and activated charcoal. The bud position effect on the multiplication index was evaluated and the auxin IBA utilized in rooting experiments. The best multiplication response was with the apical bud in culture medium in absence of cytokinins. MS inoculated explants with different IBA concentrations did not root in vitro, butex vitro rooting was satisfactory. Survival of explants, 47.62%, was for 4 months in germplasm bank conditions without culture medium transference. Key words: Cochlospermaceae, Cerrado, “algodaozinho do campo”, medicinal plants, plant growth regulators.

Reactivation of Meristem Activity and Sprout Growth in Potato Tubers Require Both Cytokinin and Gibberellin

Reactivation of dormant meristems is of central importance for plant fitness and survival. Due to their large meristem size, potato (Solanum tuberosum) tubers serve as a model system to study the underlying molecular processes. The phytohormones cytokinins (CK) and gibberellins (GA) play important roles in releasing potato tuber dormancy and promoting sprouting, but their mode of action in these processes is still obscure. Here, we established an in vitro assay using excised tuber buds to study the dormancy-releasing capacity of GA and CK and show that application of gibberellic acid (GA(3)) is sufficient to induce sprouting. In contrast, treatment with 6-benzylaminopurine induced bud break but did not support further sprout growth unless GA(3) was administered additionally. Transgenic potato plants expressing Arabidopsis (Arabidopsis thaliana) GA 20-oxidase or GA 2-oxidase to modify endogenous GA levels showed the expected phenotypical changes as well as slight effects on tuber sprouting. The isopentenyltransferase (IPT) from Agrobacterium tumefaciens and the Arabidopsis cytokinin oxidase/dehydrogenase1 (CKX) were exploited to modify the amounts of CK in transgenic potato plants. IPT expression promoted earlier sprouting in vitro. Strikingly, CKX-expressing tubers exhibited a prolonged dormancy period and did not respond to GA(3). This supports an essential role of CK in terminating tuber dormancy and indicates that GA is not sufficient to break dormancy in the absence of CK. GA(3)-treated wild-type and CKX-expressing tuber buds were subjected to a transcriptome analysis that revealed transcriptional changes in several functional groups, including cell wall metabolism, cell cycle, and auxin and ethylene signaling, denoting events associated with the reactivation of dormant meristems.

Nutrient responses differ between node and organogenic cultures of Corymbia torelliana×C. citriodora (Myrtaceae)

Hybrids between Corymbia torelliana (F.Muell.) K.D.Hill & L.A.S.Johnson and C. citriodora subsp. variegata (F.Muell.) A.R.Bean & M.W.McDonald are used extensively for plantation forestry in subtropical eastern Australia; however, plantation establishment has been hampered by inadequate seed supply and variable amenability to propagation as rooted cuttings. The present study investigated node-culture and organogenic-culture methods for in vitro propagation of two families of juvenile Corymbia hybrids. The effects of nutrient and auxin concentrations on root formation, shoot elongation and shoot proliferation, and subsequent conversion to plantlets in an in vitro soil-less system, were assessed. The response to the nutrient concentration differed between organogenic and node cultures. Half-strength, compared with full-strength, medium with auxin increased shoot proliferation by 58% for one family in the node culture, in which many shoots form roots and elongate rapidly in the absence of cytokinin. However, full-strength medium increased shoot proliferation by 213% over that in the half-strength medium for the other family in the organogenic culture, in which shoots do not form roots in the presence of cytokinin, and nutrient uptake occurs via callus and stem tissue rather than roots. The auxin, naphthalene acetic acid, sometimes stimulated root formation and shoot elongation in the node culture, although it had no effect on the final number of shoots. Most shoots (57–100%) subsequently formed roots in the in vitro soil-less medium. The high rates of proliferation and plantlet conversion in the present study provide the means, not only for simultaneous laboratory storage and field-testing of Corymbia clones before selection of desired genotypes, but also for en masse plantlet production of selected clones or families for plantation establishment.

GENETICALLY STABLE SOMATIC EMBRYOS OF F1 CUCUMBER (CUCUMIS SATIVUS) HYBRIDS

Explants were taken from hypocotyls, cotyledons and leaves of five cucumber cultivars. The maximum callus weight in the induction medium was 1215.4 mg with the cv. Natica using hypocotyl explants and the minimum was 487.6 mg with cv Mascot using leaf explants. When auxin was available in the induction phase somatic embryos were obtained; the absence of cytokinin caused a reduction in the number o: embryos. The number of somatic embryos increased with sucrose concentration to maximum of 9% sucrose. Three weeks on induction medium was needed for produc tion of somatic embryos; prolonged incubation (6 weeks) gave poor somatic embryo numbers. As auxin concentration decreased in maturation medium somatic embryo yield increased; the highest number was obtained in the absence of auxin. Generally the culture of callus on maturation medium in darkness was superior to 16 h light foi somatic embryogenesis. Leaf explants of the cultivar Profito gave 37.3 embryos pei replicate which was the highest number recorded. In addition, this cultivar yieldec embryos in the shortest time period (6 weeks). Satisfactory somatic embryo numbers were obtained after three weeks on induction medium, plus three weeks on maturation medium, or four weeks on induction medium, plus two weeks on maturation medium, or five weeks on induction medium, plus one week on maturation medium Comparison of characteristics of cucumber plants derived from somatic embryogenesis with those obtained from seed showed no significant difference in phenotype ol plants, or fruits, or genotype (using RAPD DNA tests).

Effect of ammonium ions and cytokinins on hyperhydricity and multiplication rate of in vitro regenerated shoots of Aloe polyphylla

In search for the optimal culture conditions resulting in a high production of healthy plants and low occurrence of hyperhydricity in tissue cultured regenerants of Aloe polyphylla, we investigated the relationship between ammonium ions in the medium, applied cytokinins (CKs) and CK concentrations in the induction of hyperhydricity. Shoots were grown on media with different NH4+ concentrations (10.3, 20.6 and 61.8 mM) and supplemented with N6-benzyladenine (BA), zeatin or thidiazuron (TDZ) at 0, 5 or 15 μM. Elevating the levels of NH4+, in the absence of CKs, could not induce hyperhydricity. Similarly, very low hyperhydricity was observed when CKs were added to media containing low NH4+ (10.3 mM). However, in the presence of higher NH4+ concentrations, CKs increased hyperhydricity in a concentration-dependant manner, suggesting that they were capable of inducing this syndrome only when other factors in the culture system were not optimised. High numbers of healthy looking shoots were produced on media with low NH4+ and low BA or zeatin (5 μM). The use of TDZ resulted in the formation of buds, which did not develop into shoots. Identifying the factors responsible for hyperhydricity is an important step in the successful use of the micropropagation technique for the conservation of this species.

Cytokinins Regulate a Bidirectional Phosphorelay Network in Arabidopsis

The cytokinin class of plant hormones plays key roles in regulating diverse developmental and physiological processes. Arabidopsis perceives cytokinins with three related and partially redundant receptor histidine kinases (HKs): CRE1 (the same protein as WOL and AHK4), AHK2, and AHK3 (CRE-family receptors). It is suggested that binding of cytokinins induces autophosphorylation of these HKs and subsequent transfer of the phosphoryl group to a histidine phosphotransfer protein (HPt) and then to a response regulator (RR), ultimately regulating downstream signaling events. Here we demonstrate that, in vitro and in a yeast system, CRE1 is not only a kinase that phosphorylates HPts in the presence of cytokinin but is also a phosphatase that dephosphorylates HPts in the absence of cytokinin. To explore the roles of these activities in planta, we replaced CRE1 with mutant versions of the gene or with AHK2. Replacing CRE1 with CRE1(T278I), which lacks cytokinin binding activity and is locked in the phosphatase form, decreased cytokinin sensitivity. Conversely, replacing CRE1 with AHK2, which favors kinase activity, increased cytokinin sensitivity. These results indicate that in the presence of cytokinins, cytokinin receptors feed phosphate to phosphorelay-integrating HPt proteins. In the absence of cytokinins, CRE1 removes phosphate from HPt proteins, decreasing the system phosphoload.

Activation of the Oryza sativa non-symbiotic haemoglobin-2 promoter by the cytokinin-regulated transcription factor, ARR1.

Using in silico methods, several putative phytohormone-responsive cis-elements in the Oryza sativa non-symbiotic haemoglobin (NSHB) 1-4 and Arabidopsis thaliana NSHB1-2 promoters have been identified. An OsNSHB2 promoter::GUS reporter gene fusion shows tissue-specific expression in A. thaliana. GUS expression was observed in roots, the vasculature of young leaves, in flowers, and in the pedicel/stem junction. In transient assays, activity of the OsNSHB2 promoter was significantly up-regulated in the presence of the cytokinin, 6-benzylaminopurine (BA). Deletion analyses indicated that the full-length promoter was required for maximal trans-activation in the presence of cytokinin. Mutation of the single cytokinin-regulated ARR1-binding element abolished promoter activation in response to cytokinin. Constitutive expression of ARR1 under the control of the 35S cauliflower mosaic virus promoter enhanced wild-type OsNSHB2 promoter activity, but had no effect on the activity of the mutated promoter in the absence of cytokinin. However, overexpression of ARR1 in the presence of cytokinin resulted in super-activation of the wild-type promoter. The mutated promoter was only moderately activated in the presence of cytokinin and ARR1, indicating that the OsNSHB2 promoter can be regulated by the ARR1 protein, but requires other cytokinin-induced factors for optimal activation. This is the first report that identifies a trans-acting factor involved in the activation of a NSHB gene.

Efficient regeneration from cotyledon protoplasts in Arabidopsis thaliana

An efficient and fast regeneration system from cotyledon protoplasts was established for Arabidopsis thaliana accessions C24, Columbia, and Wassilewskija. Culture conditions and media compositions were optimised for the development of protoplasts embedded in thin alginate layers. Unexpectedly, the absence of cytokinins had a positive effect on cell development. Moreover, combined adjustment of alpha-naphthylacetic acid and dicamba concentrations resulted in high plating efficiencies of up to 30%, followed by shoot regeneration within only 19 days after protoplast isolation. The protocol is reproducible, efficient, extremely fast, and regenerated plants are fertile. Thus, this cotyledon-based system could prove useful for studying plant cell and molecular biology in A. thaliana.

Control of in vitro organogenesis by cyclin-dependent kinase activities in plants.

Little is known concerning the mechanisms by which auxin and cytokinin exert their effects on proliferation and differentiation. Cyclin-dependent kinases (CDKs) are major regulators of the eukaryotic cell cycle, thus they are assumed to control cell differentiation as well as proliferation in response to phytohormone signals. Here, we overexpressed rice R2 cDNA, which encodes a CDK-activating kinase, in tobacco leaf explants by using the glucocorticoid-mediated transcriptional induction system. Transient expression of R2 during the first 7 days of culture triggered callus formation in the absence of cytokinin. This phenotype was enhanced by higher expression of R2 or coexpression of cyclin H, and suppressed by treatment with roscovitine, a CDK inhibitor. R2 expression at a later stage did not prevent cells from differentiation into roots, suggesting a restricted period for sensing CDK activities that control differentiation fate of cells during organogenesis.

Pasticcino2 is a protein tyrosine phosphatase-like involved in cell proliferation and differentiation in Arabidopsis.

The pasticcino2 (pas2) mutant shows impaired embryo and seedling development associated with cell de-differentiation and proliferation. This process is specifically enhanced in presence of cytokinins leading to callus-like structure of the apical part of the seedling. Cell proliferation concerns localized and stochastic nodules of dividing cells. In absence of cytokinins, cell proliferation leads to small calli on stems but, most often, cell proliferation is associated with post-genital organ fusion. The PAS2 gene was identified by positional cloning. PAS2 expression was found in every plant organ and was not regulated by PAS1 and PAS3 genes. PAS2 encodes the Arabidopsis member of the protein tyrosine phosphatase-like (Ptpl) family, a new PTP family originally described in mice and humans and characterized by a mutated PTP active site. This family of proteins has a yeast homolog that is essential for cell viability. The absence of yeast PAS2 homolog can be functionally replaced by the Arabidopsis PAS2 protein, demonstrating that PAS2 function is conserved between higher and lower eukaryotes.

Isolation and characterization of a cytokinin up-regulated gene from tobacco mesophyll protoplasts.

We have isolated a cytokinin up-regulated cDNA clone, H13, from an early stage of cultured tobacco mesophyll protoplasts by a differential display method. The expression of this gene was specifically induced by natural and synthetic cytokinins including N-(2-chloro-4-pyridyl)-N'-phenylurea (4PU30), a diphenylurea-type cytokinin, although the simultaneous presence of auxin was also required. It seems that the preceding treatment of the tobacco mesophyll protoplasts by auxin is necessary for the gene to respond to cytokinin. The addition of a cytokinin antagonist, compound 182, which suppressed the induction of cell division in tobacco mesophyll protoplasts, completely abolished the expression of this gene. Though the predicted gene product of H13 did not suggest us any sequences of defined functions, two domains of the predicted sequence had significant homology to several reported sequences in the data base. The gene product of H13 is proposed to have a role in regenerating cell wall in cultured protoplasts, since a cDNA clone E6, from cotton fiber cells, which has the most closely related structure to H13, has been isolated from cells which showed active cellulose synthesis. This supposition is supported by the evidence that in the absence of cytokinin, cell wall regeneration was significantly suppressed, resulting in failure of the induction of cell division. Thus, the gene product of H13 is supposed to have a role in regenerating cell walls and facilitating the progression of the cell cycle, resulting in the sustained cell division of tobacco mesophyll protoplasts.

Genes involved in cytokinin signal transduction

Although cytokinin plays a central role in plant development, our knowledge about the signal transduction pathway initiated by this plant hormone is fragmentary. By randomly introducing enhancer elements into theArabidopsis genome throughAgrobacterium-mediated transformation, 5 cytokinin independent mutant calli (cki1-1, −2, −3, −4 andcki2) were obtained. These mutants exhibit typical cytokinin responses, including rapid proliferation, chloroplast differentiation, shoot induction and inhibition of root formation, in the absence of cytokinin. TheCKl1 gene encodes a product similar to the sensor histidine kinases of two-component systems, and its overexpression in plants induces typical cytokinin responses (Kakimoto 1996). Here I report that overexpression of this gene did not alter the auxin reqirement ofArabidopsis. Another mutant,many shoots, which was also identified on the same screening, produced many adventitious shoots on cotyledons, petioles and true leaves.

The PASTICCINO genes of Arabidopsis thaliana are involved in the control of cell division and differentiation

The control of cell division by growth regulators is critical to proper plant development. The isolation of single-gene mutants altered in the response to plant hormones should permit the identification of essential genes controlling the growth and development of plants. We have isolated mutants pasticcino belonging to 3 complementation groups (pas1, pas2, pas3) in the progeny of independent ethyl methane sulfonate and T-DNA mutagenized Arabidopsis thaliana plants. The screen was performed in the presence or absence of cytokinin. The mutants isolated were those that showed a significant hypertrophy of their apical parts when grown on cytokinin-containing medium. The pas mutants have altered embryo, leaf and root development. They display uncoordinated cell divisions which are enhanced by cytokinin. Physiological and biochemical analyses show that cytokinins are probably involved in pas phenotypes. The PAS genes have been mapped respectively to chromosomes 3, 5 and 1 and represent new plant genes involved in the control of cell division and plant development.

In vitro regeneration of Pacific silver fir (Abies amabilis) plantlets and histological analysis of shoot formation.

A protocol is described for the in vitro propagation of Abies amabilis (Dougl.) Forbes. Over 60% of the cotyledonary explants from 5-day-old cotyledons formed shoots when cultured for 7 days on Schenk and Hildebrandt's (SH) medium containing 10 micro M N(6)-benzyladenine (BA) followed by another 7 days on SH medium containing 10 micro M each of BA and zeatin. Shoot multiplication was unsuccessful. Seventeen percent rooting was obtained after pulsing for 4 h in 1 mM indole-3-butyric acid and planting pulsed shoots in 1/1 (v/v) perlite/vermiculite. Cell clusters (promeristemoids) of five to seven cells were observed on the cotyledonary explants after 7 days of culture in the presence of cytokinin. These cells developed further into meristematic domes and apical meristems. In the absence of cytokinin, stomata and resin canals reached maturity, whereas cells within the cortex became vacuolated and developed into palisade and spongy mesophyll.

Cytokinin regulation of a soybean pollen allergen gene

Cytokinin treatment of suspension-cultured soybean cells stimulated the accumulation of an mRNA, called cim 1, by a factor of ca. 20 within 4 h. Induction of cim 1 mRNA accumulation occurred at benzyladenine concentrations as low as 10(-8) M. Furthermore, cim 1 mRNA accumulation was stimulated in the absence of cytokinin by staurosporine (an inhibitor of protein kinases) and inhibited in the presence of cytokinin by okadaic acid (an inhibitor of protein phosphatases 1 and 2a), suggesting that cim 1 accumulation in response to cytokinin is dependent on cytokinin-induced dephosphorylation of one or more cellular proteins. The deduced amino acid sequence of the cim 1 protein product, derived from the complete nucleotide sequence of a cim 1 cDNA, was 40% identical to that of a perennial rye grass pollen allergen cDNA (Lol Pl). This sequence also indicated that the cim 1 protein product contains a putative signal peptide followed by predominantly hydrophilic residues, consistent with the hypothesis that it is exported to the apoplast.