Cytokinin activation of Arabidopsis cell division through a D-type cyclin.
Cytokinins are plant hormones that regulate plant cell division. The D-type cyclin CycD3 was found to be elevated in a mutant of Arabidopsis with a high level of cytokinin and to be rapidly induced by cytokinin application in both cell cultures and whole plants. Constitutive expression of CycD3 in transgenic plants allowed induction and maintenance of cell division in the absence of exogenous cytokinin. Results suggest that cytokinin activates Arabidopsis cell division through induction of CycD3 at the G1-S cell cycle phase transition. (+info)
Detection of membrane-bound cytokinin-binding proteins in Arabidopsis thaliana cells.
In order to isolate cytokinin-binding proteins (CBPs), we have developed new affinity probes constituted of a cytokinin such as zeatin riboside ([9R]Z) conjugated to a carrier protein. These probes were used for detecting CBPs in an ELISA procedure. The efficiency of the cytokinin conjugate in detecting CBPs was controlled with protein model: proteins having an affinity for cytokinin such as the monoclonal anti-[9R]Z antibodies did bind the cytokinin conjugate whereas proteins unable to bind cytokinin such as bovine serum albumin did not. Using these new affinity probes, we showed that CBPs are present in the membrane fraction of in vitro cultured Arabidopsis thaliana cells. The nature of the protein at the detected binding sites was demonstrated by submitting the microsomal proteins to a proteolytic treatment, which was found to eradicate the binding. Free biologically active cytokinins or monoclonal anti-[9R]Z antibodies inhibited the binding, thus showing the specificity of the interaction. The detected CBPs were partially solubilized from the membranes with potassium chloride, indicating their peripheral membrane location. The separation by anion exchange chromatography of solubilized microsomal proteins revealed the existence of two different CBPs. They were present at higher levels in cells during the exponential growth phase. (+info)
A gene encoding the cytokinin enzyme zeatin O-xylosyltransferase of Phaseolus vulgaris.
Zeatin is the most active and ubiquitous form of the naturally occurring cytokinins. Glycosyl conjugates of zeatin are found in many plant tissues and are considered important for storage and protection against degradative enzymes. Two enzymes catalyzing the formation of O-glycosyl derivatives of zeatin have been characterized, O-glucosyltransferase and O-xylosyltransferase, occurring in seeds of lima bean (Phaseolus lunatus) and bean (Phaseolus vulgaris), respectively. Recently, the ZOG1 gene (zeatin O-glucosyltansferase) was isolated from P. lunatis (). Based on the ZOG1 sequence, the ZOX1 gene (zeatin O-xylosyltransferase) was cloned from P. vulgaris. ZOX1 contains an open reading frame of 1362 bp that codes for a 454-amino acid peptide of 51 kD. The recombinant protein has properties identical to the native enzyme: it catalyzes O-xylosylzeatin formation with UDP-Xyl as a glycosyl donor but does not recognize UDP-Glucose as a substrate. The ZOX1 and ZOG1 genes exhibit 93% identity at the nucleotide level and 90% similarity at the amino acid level. Neither gene contains introns. These zeatin-specific genes and their promoters will be useful for studies of the regulation of active versus storage forms of cytokinins. Comparison of sequences encoding similar enzymes with distinct substrate specificity may lead to identification of epitopes specific to cytokinin and glycosyl donor molecules. (+info)
Leaf senescence is delayed in tobacco plants expressing the maize homeobox gene knotted1 under the control of a senescence-activated promoter.
Leaf senescence is an active process involving remobilization of nutrients from senescing leaves to other parts of the plant. Whereas senescence is accompanied by a decline in leaf cytokinin content, supplemental cytokinin delays senescence. Plants that overexpress isopentenyl transferase (ipt), a cytokinin-producing gene, or knotted1 (kn1), a homeobox gene, have many phenotypes in common. Many of these phenotypes are characteristic of altered cytokinin physiology. The effect of kn1 on leaf senescence was tested by driving its expression using the promoter of the senescence-associated gene SAG12. SAG:kn1 tobacco plants showed a marked delay in leaf senescence but otherwise developed normally. The delay in senescence was revealed by an increase in chlorophyll content in SAG:kn1 leaves relative to leaves of the control plants and by a decrease in the number of dead leaves. Senescence was also delayed in detached leaves of SAG:kn1 plants. Delayed senescence was accompanied by increased leaf cytokinin content in older leaves expressing kn1. These experiments extend the current understanding of kn1 function and suggest that in addition to mediating meristem maintenance, kn1 is capable of regulating the onset of senescence in leaves. (+info)
Multiubiquitin chain binding subunit MCB1 (RPN10) of the 26S proteasome is essential for developmental progression in Physcomitrella patens.
The 26S proteasome, a multisubunit complex, is the primary protease of the ubiquitin-mediated proteolytic system in eukaryotes. We have recently characterized MCB1 (RPN10), a subunit of the 26S complex that has affinity for multiubiquitin chains in vitro and as a result may function as a receptor for ubiquitinated substrates. To define the role of MCB1 further, we analyzed its function in Physcomitrella patens by generating MCB1 gene disruptions using homologous recombination. PpMCB1, which is 50 to 75% similar to orthologs from other eukaryotes, is present in the 26S proteasome complex and has a similar affinity for multiubiquitin chains, using a conserved hydrophobic domain within the C-terminal half of the polypeptide. Unlike yeast Deltamcb1 strains, which grow normally, P. patens Deltamcb1 strains are viable but are under developmental arrest, generating abnormal caulonema that are unable to form buds and gametophores. Treatment with auxin and cytokinin restored bud formation and subsequent partial development of gametophores. Complementation of a Deltamcb1 strain with mutated versions of PpMCB1 revealed that the multiubiquitin chain binding site is not essential for the wild-type phenotype. These results show that MCB1 has an important function in the 26S proteasome of higher order eukaryotes in addition to its ability to bind multiubiquitin chains, and they provide further support for a role of the ubiquitin/26S proteasome proteolytic pathway in plant developmental processes triggered by hormones. (+info)
Cytokinins in tobacco and wheat chloroplasts. Occurrence and changes due to light/dark treatment.
Although cytokinins (CKs) affect a number of processes connected with chloroplasts, it has never been rigorously proven that chloroplasts contain CKs. We isolated intact chloroplasts from tobacco (Nicotiana tabacum L. cv SR1) and wheat (Triticum aestivum L. cv Ritmo) leaves and determined their CKs by liquid chromatography/tandem mass spectroscopy. Chloroplasts from both species contained a whole spectrum of CKs, including free bases (zeatin and isopentenyladenine), ribosides (zeatin riboside, and isopentenyladenosine), ribotides (isopentenyladenosine-5'-monophosphate, zeatin riboside-5'-monophosphate, and dihydrozeatin riboside-5'-monophosphate), and N-glucosides (zeatin-N(9)-glucoside, dihydrozeatin-N(9)-glucoside, zeatin-N(7)-glucoside, and isopentenyladenine-N-glucosides). In chloroplasts there was a moderately higher relative amount of bases, ribosides, and ribotides than in leaves, and a significantly increased level of N(9)-glucosides of zeatin and dihydrozeatin. Tobacco and wheat chloroplasts were prepared from leaves at the end of either a dark or light period. After a dark period, chloroplasts accumulated more CKs than after a light period. The differences were moderate for free bases and ribosides, but highly significant for glucosides. Tobacco chloroplasts from dark-treated leaves contained zeatin riboside-O-glucoside and dihydrozeatin riboside-O-glucoside, as well as a relatively high CK oxidase activity. These data show that chloroplasts contain a whole spectrum of CKs and the enzymatic activity necessary for their metabolism. (+info)
Auxin and cytokinin have opposite effects on amyloplast development and the expression of starch synthesis genes in cultured bright yellow-2 tobacco cells.
In cultured Bright Yellow-2 (BY-2) tobacco (Nicotiana tabacum) cells, the depletion of auxin (2,4-dichlorophenoxyacetic acid) in the culture medium induces the accumulation of starch. This is accelerated by the addition of cytokinin (benzyladenine). Light and electron microscopic observations revealed that this amyloplast formation involves drastic changes in plastid morphology. The effects of auxin and cytokinin on amyloplast development were investigated by adding auxin or cytokinin to cells grown in a hormone-free culture. Auxin repressed amyloplast development, whereas cytokinin accelerated starch accumulation regardless of the timing of hormone addition. RNA gel-blot analysis revealed that the accumulation of the ADP-glucose pyrophosphorylase small subunit gene (AgpS), granule-bound starch synthase, and starch branching enzyme transcripts were also affected by hormonal conditions. High levels of AgpS, granule-bound starch synthase, and starch branching enzyme transcripts accumulated in amyloplast-developing cells grown in auxin-depleted conditions. Furthermore, the addition of auxin to the cells cultured in hormone-free medium reduced the level of AgpS transcripts, whereas the addition of cytokinin increased it, irrespective of the timing of hormone addition. These results suggest that auxin and cytokinin exert opposite effects on amyloplast development by regulating the expression of the genes required for starch biosynthesis. (+info)
Cell-division factors from Vinca rosca L. crown gall tumor tissue.
A cell-division factor has been precipitated from extracts of cultured Vinca rosea L. crown gall tumor tissue by using the mercuric acetate procedure previously employed by Wood and colleagues to obtain their "cytokinesin I." On the basis of its mass spectrum, ultraviolet light absorbancy spectra, solubilities, chromatographic migration values, and growth activity, the factor is ribosyl-trans-zeatin, that is, 6-(4-hydroxy-3-methyl-trans-2-butenylamino)-9-beta-D-ribofuranosylpurine. Ribosylzeatin has now been isolated from tumor tissue by four experimental techniques; any possibility that it is an artifact seems to have been eliminated. Contrary to the report by Wood and colleagues, synthetic ribosylzeatin is precipitated from an aqueous solution by mercuric acetate, provided the complete precipitation procedure is utilized. These facts and others discussed strongly support our suggestion that ribosylzeatin was present in the preparation ("cytokinesin I") examined by Wood and colleagues in several biological assays. The reasons advanced by Wood and others for rejecting this suggestion have been found either not to be pertinent to the question or to have insufficient experimental bases. (+info)