Overexpression of KNAT1 in lettuce shifts leaf determinate growth to a shoot-like indeterminate growth associated with an accumulation of isopentenyl-type cytokinins.
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Leaves are specialized organs characterized by defined developmental destiny and determinate growth. The overexpression of Knotted1-like homeobox genes in different species has been shown to alter leaf shape and development, but a definite role for this class of genes remains to be established. Transgenics that overexpress Knotted1-like genes present some traits that are characteristic of altered cytokinin physiology. Here we show that lettuce (Lactuca sativa) leaves that overexpress KNAT1, an Arabidopsis kn1-like gene, acquire characteristics of indeterminate growth typical of the shoot and that this cell fate change is associated with the accumulation of specific types of cytokinins. The possibility that the phenotypic effects of KNAT1 overexpression may arise primarily from the modulation of local ratios of different cytokinins is discussed. (+info)
Regulation of plant growth by cytokinin.
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Cytokinins are a class of plant-specific hormones that play a central role during the cell cycle and influence numerous developmental programs. Because of the lack of biosynthetic and signaling mutants, the regulatory roles of cytokinins are not well understood. We genetically engineered cytokinin oxidase expression in transgenic tobacco plants to reduce their endogenous cytokinin content. Cytokinin-deficient plants developed stunted shoots with smaller apical meristems. The plastochrone was prolonged, and leaf cell production was only 3-4% that of wild type, indicating an absolute requirement of cytokinins for leaf growth. In contrast, root meristems of transgenic plants were enlarged and gave rise to faster growing and more branched roots. These results suggest that cytokinins are an important regulatory factor of plant meristem activity and morphogenesis, with opposing roles in shoots and roots. (+info)
4-Coumarate:coenzyme A ligase and isoperoxidase expression in Zinnia mesophyll cells induced to differentiate into tracheary elements.
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When cultured in inductive medium containing adequate auxin and cytokinin, isolated mesophyll cells of Zinnia elegans L. cv Envy differentiate into tracheary elements with lignified secondary wall thickenings. Differentiation does not occur when cells are cultured in control medium, which has reduced levels of auxin and/or cytokinin. The activities of two enzymes involved in lignin synthesis, 4-coumarate:coenzyme A ligase and peroxidase, were examined. An induction-specific cationic isoperoxidase, visualized by low pH polyacrylamide gel electrophoresis, is detectable in soluble and wall fractions of cultured Zinnia cells long before tracheary elements visibly differentiate and is thus an early marker of differentiation. Compounds (such as antiauxins, anticytokinins, and tunicamycin) that inhibit or delay differentiation alter the expression of this isoperoxidase. 4-Coumarate:coenzyme A ligase activity increases dramatically only as cells differentiate. Together, these results suggest that the onset of lignification in differentiating Zinnia cells might be controlled by the availability of precursors synthesized by way of 4-coumarate:coenzyme A ligase. These precursors would then be polymerized into lignin in the cell wall by the induction-specific isoperoxidase. (+info)
Kinetics of determination in the differentiation of isolated mesophyll cells of Zinnia elegans to tracheary elements.
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Mechanically isolated mesophyll cells of Zinnia elegans L. cv Envy differentiate to tracheary elements when cultured in inductive medium containing 0.5 micromolar alpha-naphthaleneacetic acid and 0.5 micromolar benzyladenine. The cells do not differentiate when cultured in medium in which the concentration of auxin and/or cytokinin has been reduced to 0.005 micromolar. Cells require an initial 24-hour exposure to inductive cytokinin and 56-hour exposure to inductive auxin for differentiation at 72 hours of culture. Freshly isolated Zinnia cells can be maintained in medium having low concentrations of both auxin and cytokinin for only 1 day without significant loss of potential to differentiate upon transfer to inductive medium. Initial culture for up to 2 days in medium having high auxin and low cytokinin, or low auxin and high cytokinin, allows full differentiation on the third day after transfer to inductive medium and potentiates the early differentiation of some cells. (+info)
The Arabidopsis AMP1 gene encodes a putative glutamate carboxypeptidase.
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Arabidopsis amp1 mutants show pleiotropic phenotypes, including altered shoot apical meristems, increased cell proliferation, polycotyly, constitutive photomorphogenesis, early flowering time, increased levels of endogenous cytokinin, and increased cyclin cycD3 expression. We have isolated the AMP1 gene by map-based cloning. The AMP1 cDNA encodes a 706;-amino acid polypeptide with significant similarity to glutamate carboxypeptidases. The AMP1 mRNA was expressed in all tissues examined, with higher expression in roots, stems, inflorescences, and siliques. Microarray analysis identified four mRNA species with altered expression in two alleles of amp1, including upregulation of CYP78A5, which has been shown to mark the shoot apical meristem boundary. The similarity of the AMP1 protein to glutamate carboxypeptidases, and in particular to N-acetyl alpha-linked acidic dipeptidases, suggests that the AMP1 gene product modulates the level of a small signaling molecule that acts to regulate a number of aspects of plant development, in particular the size of the apical meristem. (+info)
Srchi24, a chitinase homolog lacking an essential glutamic acid residue for hydrolytic activity, is induced during nodule development on Sesbania rostrata.
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The interaction between the tropical legume Sesbania rostrata and the bacterium Azorhizobium caulinodans results in the formation of nodules on both stem and roots. Stem nodulation was used as a model system to isolate early markers by differential display. One of them, Srchi24 is a novel early nodulin whose transcript level increased already 4 h after inoculation. This enhancement depended on Nod factor-producing bacteria. Srchi24 transcript levels were induced also by exogenous cytokinins. In situ hybridization and immunolocalization experiments showed that Srchi24 transcripts and proteins were present in the outermost cortical cell layers of the developing nodules. Sequence analyses revealed that Srchi24 is similar to class III chitinases, but lacks an important catalytic glutamate residue. A fusion between a maltose-binding protein and Srchi24 had no detectable hydrolytic activity. A function in nodulation is proposed for the Srchi24 protein. (+info)
Effects of P(SAG12)-IPT gene expression on development and senescence in transgenic lettuce.
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An ipt gene under control of the senescence-specific SAG12 promoter from Arabidopsis (P(SAG12)-IPT) significantly delayed developmental and postharvest leaf senescence in mature heads of transgenic lettuce (Lactuca sativa L. cv Evola) homozygous for the transgene. Apart from retardation of leaf senescence, mature, 60-d-old plants exhibited normal morphology with no significant differences in head diameter or fresh weight of leaves and roots. Induction of senescence by nitrogen starvation rapidly reduced total nitrogen, nitrate, and growth of transgenic and azygous (control) plants, but chlorophyll was retained in the lower (outer) leaves of transgenic plants. Harvested P(SAG12)-IPT heads also retained chlorophyll in their lower leaves. During later development (bolting and preflowering) of transgenic plants, the decrease in chlorophyll, total protein, and Rubisco content in leaves was abolished, resulting in a uniform distribution of these components throughout the plants. Homozygous P(SAG12)-IPT lettuce plants showed a slight delay in bolting (4-6 d), a severe delay in flowering (4-8 weeks), and premature senescence of their upper leaves. These changes correlated with significantly elevated concentrations of cytokinin and hexoses in the upper leaves of transgenic plants during later stages of development, implicating a relationship between cytokinin and hexose concentrations in senescence. (+info)
On the "activation" of cytokinins.
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A number of cytokinin analogs containing modifications in the heterocyclic moiety were prepared. These compounds were tested for activity as cytokinins and anticytokinins in the tabacco bioassay and the results were used to determine whether any position(s) of the heterocyclic nucleus of cytokinins may require derivatization as part of an over-all "activation" process. 3-substituted 4-alkylaminopyrazolo [3,4-d]pyrimidines and 4-alkylaminopyrrolo[2,3-d]pyrimidines, for example, have (substituted) carbon rather than nitrogen atoms at positions 3 and 5, respectively (analogous to position 7 in purines) and would be predicted to be metabolically stable at these positions. The finding that these compounds had cytokinin activity suggested that modification at the metabolically stable positions. The finding that these compounds had cytokinin activity suggested that modification at the metabolically stable position, and by extension at position 7 in cytokinin analogues which are purines, is not a prerequisite for the expression of cytokinin activity. Similar consideration of other heterocyclic analogs which have cytokinin activity suggests that the active form of a cytokinin can be the exogenous compound itself. Certain structural analogs of cytokinins were found to inhibit the growth of tobacco callus promoted by 6-(3-methyl-2-butenylamino)purine. These compounds were studied as potential cytokinin antagonists, i.e. having activity analogous to the 7-alkylamino-3-methylpyrazolo[4,3-d]pyrimidines (Hecht, S. M., 2068-2610; Skoog, F., Schmitz, R.Y., Hecht, S.M., and Bock, R. M. (1973) Phytochemistry 12, 25-37). The activity of these compounds is discussed and criteria are proposed to distinguish between those species which are specific anticytokinins and those which otherwise inhibit growth. (+info)