Monitoring genome-wide changes in gene expression in response to endogenous cytokinin reveals targets in Arabidopsis thaliana

Cytokinins have been implicated in developmental and growth processes in plants including cell division, chloroplast biogenesis, shoot meristem initiation and senescence. The regulation of these processes requires changes in cytokinin-responsive gene expression. Here, we induced the expression of a bacterial isopentenyl transferase gene, IPT, in transgenic Arabidopsis thaliana seedlings to study the regulation of genome-wide gene expression in response to endogenous cytokinin. Using MPSS (massively parallel signature sequencing) we identified 823 and 917 genes that were up- and downregulated, respectively, following 24 h of IPT induction. When comparing the response to cytokinin after 6 and 24 h, we identified different clusters of genes showing a similar course of regulation. Our study provides researchers with the opportunity to rapidly assess whether genes of interest are regulated by cytokinins.     

Effect of human immunodeficiency virus type 1 protein R (vpr) gene expression on basic cellular function of fission yeast Schizosaccharomyces pombe.

The human immunodeficiency virus type 1 (HIV-1) Vpr protein affects cell morphology and prevents proliferation of human cells by induction of cell cycle G2 arrest. In this study, we used the fission yeast Schizosaccharomyces pombe as a model system to investigate the cellular effects of HIV-1 vpr gene expression. The vpr gene was cloned into an inducible fission yeast gene expression vector and expressed in wild-type S. pombe cells, and using these cells, we were able to demonstrate the specific Vpr-induced effects by induction and suppression of vpr gene expression. Induction of HIV-1 vpr gene expression affected S. pombe at the colonial, cellular, and molecular levels. Specifically, Vpr induced small-colony formation, polymorphic cells, growth delay, and cell cycle G2 arrest. Additionally, Vpr-induced G2 arrest appeared to be independent of cell size and morphological changes. The cell cycle G2 arrest correlated with increased phosphorylation of p34cdc2, suggesting negative regulation of mitosis by HIV-1 Vpr. Treatment of Vpr-induced cell with a protein phosphatase inhibitor, okadaic acid, transiently suppressed cell cycle arrest and morphological changes. This observation implicates possible involvement of protein phosphatase(s) in the effects of Vpr. Together, these data showed that the HIV-1 Vpr-induced cellular changes in S. pombe are similar to those observed in human cells. Therefore, the S. pombe system is suited for further investigation of the HIV-1 vpr gene functions.     

Design and synthesis of photolabile caged cytokinin

Cytokinins are phytohormones that regulate diverse developmental processes throughout the life of a plant. trans-Zeatin, kinetin, benzyladenine and dihydrozeatin are adenine-type cytokinins that are perceived by the AHK cytokinin receptors. Endogenous cytokinin levels are critical for regulating plant development. To manipulate intracellular cytokinin levels, caged cytokinins were designed on the basis of the crystal structure of the AHK4 cytokinin receptor. The caged cytokinin was photolyzed to release the cytokinin molecule inside the cells and induce cytokinin-responsive gene expression. The uncaging of intracellular caged cytokinins demonstrated that cytokinin-induced root growth inhibition can be manipulated with photo-irradiation. This caged cytokinin system could be a powerful tool for cytokinin biology.     

Cytokinin and auxin inhibit abscisic acid-induced stomatal closure by enhancing ethylene production in Arabidopsis

Cytokinins and auxins are major phytohormones involved in various aspects of plant growth and development. These phytohormones are also known to antagonize the effects of abscisic acid (ABA) on stomatal movement, and to affect ethylene biosynthesis. As ethylene has an antagonistic effect on ABA-induced stomatal closure, the possibility that the antagonistic effects of these phytohormones on ABA were mediated through ethylene biosynthesis was investigated. Both the cytokinin, 6-benzyladenine (BA), and the auxin, 1-naphthaleneacetic acid (NAA), antagonized ABA-induced stomatal closure in a manner similar to that following application of the ethylene precursor, 1-aminocyclopropane-1-carboxylic acid (ACC). However, these effects were negated when ethylene signalling, perception, or biosynthesis were blocked. As stomatal aperture is regulated by changes in guard cell volume, ABA application was found to reduce the volume of the guard cell protoplasts (GCP). It was found that BA, NAA, or ACC application compensated perfectly for the reduction in GCP volume by ABA application in WT plants. The above observations suggest that cytokinins and auxins inhibit ABA-induced stomatal closure through the modulation of ethylene biosynthesis, and that ethylene inhibits the ABA-induced reduction of osmotic pressure in the guard cells.     

Effect of Zinc Sulfate and Boric Acid on the Hormonal Status of Potato Plants in Relation to Tuberization

Potato (Solanum tuberosum L.) plants were grown in a greenhouse using zinc- and boron-deficient soil. The effects of seed-tuber treatment with 3 mM zinc sulfate and 8 mM boric acid on the content and ratio of phytohormones in the leaves and mature tubers, the indices of photosynthetic activity, the rate and NaF-sensitivity of respiration, and the tuber growth were studied. Zinc-sulfate treatment shifted the hormonal balance toward a substantial increase in the cytokinin content and the cytokinin/ABA ratio, as well as a decrease in the IAA/cytokinin ratio. Boric-acid treatment resulted in an increase in the IAA content and IAA/cytokinin ratio. Zinc-sulfate treatment abolished the apical dominance and increased the tuber weight due to their increased number and the number of phellem (cork) cell layers. Boric-acid treatment increased cell diameter in the tuber perimedullary zone; an increase in tuber weight per plant was related to tuber growth. A relationship between changes in the plant hormonal status induced by zinc-sulfate and boric-acid treatments and the activity of physiological processes is discussed.     

Membrane lipid alteration during phosphate starvation is regulated by phosphate signaling and auxin/cytokinin cross-talk

During phosphate (Pi) starvation in plants, membrane phospholipid content decreases concomitantly with an increase in non-phosphorus glycolipids. Although several studies have indicated the involvement of phytohormones in various physiological changes upon Pi starvation, the regulation of Pi-starvation induced membrane lipid alteration remains unknown. Previously, we reported the response of type B monogalactosyl diacylglycerol synthase genes (atMGD2 and atMGD3) to Pi starvation, and suggested a role for these genes in galactolipid accumulation during Pi starvation. We now report our investigation of the regulatory mechanism for the response of atMGD2/3 and changes in membrane lipid composition to Pi starvation. Exogenous auxin activated atMGD2/3 expression during Pi starvation, whereas their expression was repressed by cytokinin treatment in the root. Moreover, auxin inhibitors and the axr4 aux1 double mutation in auxin signaling impaired the increase of atMGD2/3 expression during Pi starvation, showing that auxin is required for atMGD2/3 activation. The fact that hormonal effects during Pi starvation were also observed with regard to changes in membrane lipid composition demonstrates that both auxin and cytokinin are indeed involved in the dynamic changes in membrane lipids during Pi starvation. Phosphite is not metabolically available in plants; however, when we supplied phosphite to Pi-starved plants, the Pi-starvation response disappeared with respect to both atMGD2/3 expression and changes in membrane lipids. These results indicate that the observed global change in plant membranes during Pi starvation is not caused by Pi-starvation induced damage in plant cells but rather is strictly regulated by Pi signaling and auxin/cytokinin cross-talk.     

Temporal and spatial order of events during the induction of cortical cell divisions in white clover by Rhizobium leguminosarum bv. trifolii inoculation or localized cytokinin addition

We examined the timing and location of several early root responses to Rhizobium leguminosarum bv. trifolii infection, compared with a localized addition of cytokinin in white clover, to study the role of cytokinin in early signaling during nodule initiation. Induction of ENOD40 expression by either rhizobia or cytokinin was similar in timing and location and occurred in nodule progenitor cells in the inner cortex. Inoculation of rhizobia in the mature root failed to induce ENOD40 expression and cortical cell divisions (ccd). Nitrate addition at levels repressing nodule formation inhibited ENOD40 induction by rhizobia but not by cytokinin. ENOD40 expression was not induced by auxin, an auxin transport inhibitor, or an ethylene precursor. In contrast to rhizobia, cytokinin addition was not sufficient to induce a modulation of the auxin flow, the induction of specific chalcone synthase genes, and the accumulation of fluorescent compounds associated with nodule initiation. However, cytokinin addition was sufficient for the localized induction of auxin-induced GH3 gene expression and the initiation of ccd. Our results suggest that rhizobia induce cytokinin-mediated events in parallel to changes in auxin-related responses during nodule initiation and support a role of ENOD40 in regulating ccd. We propose a model for the interactions of cytokinin with auxin, ENOD40, flavonoids, and nitrate during nodulation.     

A rice isoflavone reductase-like gene, OsIRL, is induced by rice blast fungal elicitor

We have isolated and characterized a rice isoflavone reductase-like gene, OsIRL, whose expression is induced by a fungal elicitor. The OsIRL cDNA contains 1203 bp with an open reading frame of 942 nucleotides encoding 314 amino acids. The deduced amino acid sequence of OsIRL has a putative pyridine nucleotide binding domain and is 68% homologous with the maize isoflavone reductase-like gene. Southern blot analysis revealed that OsIRL belongs to a small multigene family. Expression of OsIRL was induced by treatment with a fungal elicitor and jasmonic acid as well as by inoculation with rice blast fungus. Cycloheximide (1 microM), strongly inhibited the induction of OsIRL by the fungal elicitor, indicating that new protein synthesis is required. The protein kinase inhibitor, staurosporine (1 microM), had little effect, but the phosphatase inhibitor, calyculin A (1 microM), strongly inhibited induction. Treatment with salicylic acid (SA, 5 mM) strongly inhibited expression of OsIRL in response to fungal elicitor and JA, while abscisic acid (ABA, 200 microM) also strongly antagonized OsIRL induction by JA, but had only a weak effect on induction by the fungal elicitor. These results suggest that the expression of OsIRL is positively regulated by phytohormones such as JA, and negatively by phytohormones such as SA, ABA.     

Isolation and Characterization of a Cytokinin Up-Regulated Gene from Tobacco Mesophyll Protoplasts

We have isolated a cytokinin up-regulated cDNA clone, H13, froman early stage of cultured tobacco mesophyll protoplasts bya differential display method. The expression of this gene wasspecifically induced by natural and synthetic cytokinins includingN-(2-chloro-4-pyridyl)-N'-phenylurea (4PU30), a diphenylurea-typecytokinin, although the simultaneous presence of auxin was alsorequired. It seems that the preceding treatment of the tobaccomesophyll protoplasts by auxin is necessary for the gene torespond to cytokinin. The addition of a cytokinin antagonist,compound 182, which suppressed the induction of cell divisionin tobacco mesophyll protoplasts, completely abolished the expressionof this gene. Though the predicted gene product of H13 did notsuggest us any sequences of defined functions, two domains ofthe predicted sequence had significant homology to several reportedsequences in the data base. The gene product of H13 is proposedto have a role in regenerating cell wall in cultured protoplasts,since a cDNA clone E6, from cotton fiber cells, which has themost closely related structure to H13, has been isolated fromcells which showed active cellulose synthesis. This suppositionis supported by the evidence that in the absence of cytokinin,cell wall regeneration was significantly suppressed, resultingin failure of the induction of cell division. Thus, the geneproduct of H13 is supposed to have a role in regenerating cellwalls and facilitating the progression of the cell cycle, resultingin the sustained cell division of tobacco mesophyll protoplasts. ¹These authors are equally contributed to this work.     

Characterization of genes involved in cytokinin signaling and metabolism from rice

Two-component signaling elements play important roles in plants, including a central role in cytokinin signaling. We characterized two-component elements from the monocot rice (Oryza sativa) using several complementary approaches. Phylogenetic analysis reveals relatively simple orthologous relationships among the histidine kinases in rice and Arabidopsis (Arabidopsis thaliana). In contrast, the histidine-containing phosphotransfer proteins (OsHPs) and response regulators (OsRRs) display a higher degree of lineage-specific expansion. The intracellular localizations of several OsHPs and OsRRs were examined in rice and generally found to correspond to the localizations of their dicot counterparts. The functionality of rice type-B OsRRs was tested in Arabidopsis; one from a clade composed of both monocot and dicot type-B OsRRs complemented an Arabidopsis type-B response regulator mutant, but a type-B OsRR from a monocot-specific subfamily generally did not. The expression of genes encoding two-component elements and proteins involved in cytokinin biosynthesis and degradation was analyzed in rice roots and shoots and in response to phytohormones. Nearly all type-A OsRRs and OsHK4 were up-regulated in response to cytokinin, but other cytokinin signaling elements were not appreciably affected. Furthermore, multiple cytokinin oxidase (OsCKX) genes were up-regulated by cytokinin. Abscisic acid treatment decreased the expression of several genes involved in cytokinin biosynthesis and degradation. Auxin affected the expression of a few genes; brassinosteroid and gibberellin had only modest effects. Our results support a shared role for two-component elements in mediating cytokinin signaling in monocots and dicots and reveal how phytohormones can impact cytokinin function through modulating gene expression.     

AUXIN UP-REGULATED F-BOX PROTEIN1 regulates the cross talk between auxin transport and cytokinin signaling during plant root growth

Plant root development is mediated by the concerted action of the auxin and cytokinin phytohormones, with cytokinin serving as an antagonist of auxin transport. Here, we identify the AUXIN UP-REGULATED F-BOX PROTEIN1 (AUF1) and its potential paralog AUF2 as important positive modifiers of root elongation that tether auxin movements to cytokinin signaling in Arabidopsis (Arabidopsis thaliana). The AUF1 mRNA level in roots is strongly up-regulated by auxin but not by other phytohormones. Whereas the auf1 single and auf1 auf2 double mutant roots grow normally without exogenous auxin and respond similarly to the wild type upon auxin application, their growth is hypersensitive to auxin transport inhibitors, with the mutant roots also having reduced basipetal and acropetal auxin transport. The effects of auf1 on auxin movements may be mediated in part by the misexpression of several PIN-FORMED (PIN) auxin efflux proteins, which for PIN2 reduces its abundance on the plasma membrane of root cells. auf1 roots are also hypersensitive to cytokinin and have increased expression of several components of cytokinin signaling. Kinematic analyses of root growth and localization of the cyclin B mitotic marker showed that AUF1 does not affect root cell division but promotes cytokinin-mediated cell expansion in the elongation/differentiation zone. Epistasis analyses implicate the cytokinin regulator ARR1 or its effector(s) as the target of the SKP1-Cullin1-F Box (SCF) ubiquitin ligases assembled with AUF1/2. Given the wide distribution of AUF1/2-type proteins among land plants, we propose that SCF(AUF1/2) provides additional cross talk between auxin and cytokinin, which modifies auxin distribution and ultimately root elongation.     

Influence of Mechanical Stress on Auxin-Stimulated Growth of Excised Pea Stem Sections

The auxin to cytokinin ratios are described for promoting growth in the in vitro cultures of soybean (Glycine max (L.) Merr. cv. Bragg) and perennial clover (Trifolium repens L. cv. Regal Ladinc). Callus growth was induced on somatic tissue with 50:1 auxin to cytokinin (w/w) ratio. A 5:1 ratio served for initiation of cell suspensions from callus and for subsequent growth of callus from cells in suspension. A 1:2 ratio served for regeneration of buds and plantlets from the callus grown from cells. Although (2,4-dichlorophenoxy) acetic acid was the auxin for suspension and regenerative cultures, (2,4,5-trichlorophenoxy)acetic acid was the more effective auxin for initiation of callus on somatic tissue. All cultures were grown with 6-furfurylaminopurine as the cytokinin. The phytohormones strongly influenced the rates of culture growth, but determination of culture type was augmented by dl-alpha tocopherol acetate and iron. Tocopherol and a relatively high complement of iron promoted growth of juvenile cultures, whereas low level of iron and absence of tocopherol favored growth to comparatively more differentiated cultures. Without tocopherol, no callus formed on somatic tissue during the allotted period of incubation. Tocopherol plus a complement of low iron enabled growth of callus on rapidly growing somatic tissue. A high level of iron enabled comparatively more callus growth but suppressed growth of somatic tissue. In suspension cultures tocopherol and a high iron level enhanced dispersion of cells. A low iron complement in the absence of tocopherol induced growth of callus from cells and subsequent regeneration of buds and plantlets from the callus.     

表皮生长因子对neu基因表达的诱导作用(简报)

Shih等首次通过NIH/3T3细胞转染试验在乙基亚硝脲(ENU)诱导的大鼠神经胶质纤维瘤中分离鉴定出一种转化基因,称之为neu基因,其表达可导致培养的NIH/3T3细     

表皮生长因子对neu基因表达的诱导作用（简报）

The erb B2/neu oncogene encodes a protein which sequence is closely similar to the epidermal growth factor receptor (EGFR). We have previously found that EGF can induce the expression of erb B1/EGFR gene in normal and 3H-TdR transformed C3H/10T1/2CL8 mouse embryo fibroblast cells i.e. NC3H10 and TC 3H10 respectively, but we do not know whether the neu oncogene expression can be induced by EGF. In this study, the effect of EGF on NC3H10 and TC3H10 has been observed by Northern blot analysis. The result indicated that EGF had a obvious induction effect on neu oncogene expression in these cells. Thus, the expression of both erbB 1/EGFR gene and erbB 2/neu oncogene can be induced by EGF. This result may provide a novel clue to the molecular mechanism of EGF action in cell nucleus.     

The early nodulin gene SrEnod2 from Sesbania rostrata is inducible by cytokinin

The structure and expression of the early nodulin gene Enod2 from the stem-nodulated tropical legume Sesbania rostrata (SrEnod2) was examined. Genomic clones carrying the single SrEnod2locus were isolated and the DNA sequence of the gene was determined. The SrEnod2 gene was found to lack introns and to encode a protein consisting primarily of a 55-fold repeat of short proline-rich oligopeptides. A putative signal sequence, which may be responsible for targeting of the Enod2 protein to the cell wall, was found to precede this repeat. The temporal expression of the SrEnod2 gene was found to be different in S. rostrata stem versus root nodules induced by Azorhizobium caulinodans ORS571. SrEnod2 gene expression was shown to be induced specifically and rapidly by physiologically significant concentrations of exogenously supplied cytokinins. The SrEnod2 gene was also found to be highly expressed in S. rostrata crown gall tumors induced by wild-type Agrobacterium tumefaciens strains, but not in tumors induced by an A. tumefaciens strain carrying a mutation in the cytokinin biosynthesis gene 4. Implications of these observations with regard to cytokinin-induced plant gene expression and a possible role for cytokinin as a symbiotic signal are discussed.