Histological and immunohistochemical studies on flower induction in the olive tree (Olea europaea L.).

The aim of this research was to study flower bud differentiation processes in two oil olive cultivars from Tuscan germplasm (Leccino and Puntino). The effect of fruit-set was studied using 'ON' (with fruits) and 'OFF' (without fruits) shoots. Axillary buds were periodically collected at different phenological stages, from endocarp sclerification (July) until budbreak in the following spring. Thin sections were analysed using histology (apex size), histochemistry (RNA, starch and soluble carbohydrates) and cytokinin immunocytochemistry (zeatin localisation). The micromorphological observations and histochemical procedures did not allow us to distinguish axillary buds sampled from 'ON' and 'OFF' shoots. Cytokinin immunocytochemistry revealed early different localisation patterns between 'ON' and 'OFF' samples. Zeatin accumulated only in 'OFF' axillary bud meristems, particularly in July, when endocarp sclerification of fruits from the previous flowering is taking place. At this time, a strong RNA signal was also observed. Both these signals were correlated with floral evocation, and their coincidence with a phenological stage of development provided a useful tool to determine the time when axillary buds switch from the vegetative to the reproductive phase.

Dynamics of cytokinins in apical shoot meristems of a day-neutral tobacco during floral transition and flower formation

This study considered cytokinin distribution in tobacco (Nicotiana tabacum L.) shoot apices in distinct phases of development using immunocytochemistry and quantitative tandem mass spectrometry. In contrast to vegetative apices and flower buds, we detected no free cytokinin bases (zeatin, dihydrozeatin, or isopentenyladenine) in prefloral transition apices. We also observed a 3-fold decrease in the content of cytokinin ribosides (zeatin riboside, dihydrozeatin riboside, and isopentenyladenosine) during this transition phase. The group concluded that organ formation (e.g. leaves and flowers) is characterized by enhanced cytokinin content, in contrast to the very low endogenous cytokinin levels found in prefloral transition apices, which showed no organogenesis. The immunocytochemical analyses revealed a differing intracellular localization of the cytokinin bases. Dihydrozeatin and isopentenyladenine were mainly cytoplasmic and perinuclear, whereas zeatin showed a clear-cut nuclear labeling. To our knowledge, this is the first time that this phenomenon has been reported. Cytokinins do not seem to act as positive effectors in the prefloral transition phase in tobacco shoot apices. Furthermore, the differences in distribution at the cellular level may be indicative of a specific physiological role of zeatin in nuclear processes.

Modified development in transgenic tobacco plants expressing a rolA::GUS translational fusion and subcellular localization of the fusion protein.

The rolA gene is transferred naturally by Agrobacterium rhizogenes to the genome of host plants, where it induces dramatic changes in development of transformed plants, including dwarfism and leaf wrinkling. The predicted translation product of the rolA gene is a small (11.4 kDa), basic (pI = 11.2) protein, which has no clearly significant similarity to sequences in the data bases. We have introduced into the tobacco genome a gene encoding a rolA::GUS fusion protein. Expression of this gene led to synthesis of an RNA and a protein of expected size, and the transformed plants exhibited the dwarfism and leaf wrinkling typical of rolA plants, but to a lesser degree than plants transformed with the wild-type rolA gene. The distribution of beta-glucuronidase (GUS) activity was compared in subcellular fractions of leaf extracts from plants expressing either the rolA::gus gene or a control gus construct. As expected, in the control plants, GUS activity was essentially cytosolic. In contrast, in plants expressing the rolA::gus gene the highest specific activity was associated with the plasmalemma fraction.

Instability of phenotype and gene expression in long-term culture of carrot hairy root clones.

Hairy root clones were established from carrot root discs inoculated with an agropine-type strain of Agrobacterium rhizogenes A4 harbouring the gus reporter gene on the TL-DNA. The clones were periodically examined for their phenotypic characteristics and for their ability to express the gus gene, to produce opines and to grow in the presence of NAM. The presence of the gus gene in the roots was confirmed by Southern blot hybridisation. The clones displayed various morphologies which were generally not correlated with the transformation events, and they were highly unstable throughout the successive subcultures, both for their phenotype and for their ability to express the transgenes. Reversible inactivation of the gus gene expression was associated with a high gus copy number. This could have some consequences for fundamental studies and practical uses of hairy roots.

Ectopic expression of Arabidopsis CYCD2 and CYCD3 in tobacco has distinct effects on the structural organization of the shoot apical meristem.

Transgenic tobacco lines expressing Arath-CYCD2 or Arath-CYCD3 genes under a cauliflower mosaic virus 35S promoter are modified in the timing of their development, but not in the phenotype of their vegetative organs. They display an increased rate of leaf initiation, which is shown to be associated with distinct changes in the structural organization of their shoot apical meristem (SAM). Constitutive expression of Arath-CYCD2 leads to a progressive modification of the SAM structural organization with predominant periclinal divisions in the L3 layer and to the loss of the classical cytophysiological zonation, the central zone being reduced to the central cells of the L1 and L2 layers. These changes reveal a particular sensitivity of the corpus cells (L3) to Arath-CYCD2 over-expression and suggest a role for CYCD2 in controlling the planes of cell division in these cells. The SAM structural modifications in the Arath-CYCD3 over-expressing lines are less drastic; only an increased cell number together with a reduced cell size, particularly in the L1 layer, characterizes the peripheral zones. This could be related to the shortening of the G1-phase duration that renders cell growth incomplete between successive mitoses. Cell proliferation continues beyond the SAM in the developing internodes and confers a delayed senescence to Arath-CYCD3 over-expressing juvenile tissues. In addition, the ploidy levels of mature stem tissues in both types of transgenic lines are unaffected, suggesting that the studied G1 to S cell-cycle genes have no effect on the extent of endoreduplication in tobacco stem tissues.

Effect of the differentiated or dedifferentiated state of tobacco pith tissue on its behaviour after inoculation with Agrobacterium rhizogenes.

The ability of Nicotiana tabacum (cv. Wisconsin 38) pith tissue to give rise to transformed roots after inoculation with Agrobacterium rhizogenes (agropine type strain 1855) has been examined in relation to its state of differentiation. In young plants, all the stem tissues are able to initiate transformed roots while pith of adult plants does not react. Mature pith, composed mostly of polyploid and non-dividing cells, is able to recover susceptibility when placed in vitro on a medium containing auxin and kinetin. The optimal production of transformed roots is obtained after 6-10 days, when proliferating cells redifferentiated "cambial-like" layers and tracheids. The relationships between the inability of fully differentiated pith to give rise to roots and the underlying cell states are discussed.

In situ localization of endogenous cytokinins during shooty tumor development on Eucalyptus globulus Labill.

Our previous results demonstrated that endogenous cytokinins are involved in the shooty potential of tumors initiated on Eucalyptus globulus plantlets inoculated with Agrobacterium tumefaciens strain 82.139 [A. Azmi et al. (1997a) Plant Sci 127: 81-90]. In order to investigate whether or not these hormones are distributed homogeneously in the tumors prior to the onset of bud regeneration, decapitated hypocotyls were inoculated with the strain C58pMP90/T139 GUS-INT harboring the wild transferred DNA (T-DNA) of strain 82.139 tagged with the beta-glucuronidase (gus)-reporter gene. In situ immunolocalization of zeatin, dihydrozeatin and isopentenyladenine was performed in the developing tumors and combined with the histo-enzymological beta-glucuronidase assay. It was found that the expression of the T-DNA was restricted to only some small areas located deeply in the tumors. These sites were also provided with a high cytokinin signal while the untransformed parts of the tumors displayed a weaker signal, except in the early differentiating tracheary elements. The regenerated buds were untransformed and originated from superficial parts of the tumors provided with a moderate signal for cytokinins. The method of colocalization of both cytokinins and gus expression developed here might be helpful for further studies concerning the role of these hormones in controlling gene expression at cell and tissue levels.

The plant oncogene rolC is responsible for the release of cytokinins from glucoside conjugates.

The rolC gene of Agrobacterium rhizogenes, which drastically affects growth and development of transgenic plants, codes for a cytokinin-beta-glucosidase. Indeed, rolC protein expressed in Escherichia coli as a fusion protein hydrolyses cytokinin glucosides, thus liberating free cytokinins. Furthermore, beta-glucosidase activity present in E. coli extracts expressing the rolC protein was inhibited by affinity-purified antibodies specific for the rolC protein. Finally, rolC proteins expressed in transgenic plants were shown to be responsible for cytokinin-beta-glucosidase activity. Morphological and phytohormonal analysis, performed on transgenic plants that are somatic mosaics for the expression of the rolC gene, extend and confirm our interpretation that the developmental, physiological and morphological alterations caused by rolC expression in transgenic plants are primarily due to a modification of the cytokinin balance. These observations shed new light on the control of growth and differentiation in plants by growth factors.

Competency of Nicotiana tabacum L. stem tissues to dedifferentiate is associated with differential levels of cell cycle gene expression and endogenous cytokinins.

The expression of the mitotic cyclin Arath; CYCB1;1 and of the cyclin-dependent kinase Arath; CDC2a was located by beta-glucuronidase histochemical detection and in situ hybridization, and was quantified by 4-methylumbelliferyl beta- D-glucuronide assay in tobacco stem tissues during both in vivo differentiation and in vitro dedifferentiation. The changes in localization of endogenous cytokinins were also determined during both processes using quantitative analysis and in situ immunocytochemistry. The CDC2a promoter was expressed continuously during stem development, with particularly high expression in the shoot apical meristem and in the internal and external primary phloem. CYCB1 expression was not restricted to the dividing cells; its expression in the shoot apical meristem was particularly high in the leaf-forming peripheral cells but the gene was also expressed throughout development in the internal and external phloem in which the rate of cell division was reduced or zero. Following in vitro culture, the internal phloem cells appeared to be particularly competent to re-enter the cell cycle within a short lag phase while the pith tissue reactivated later. In culture, cells that resumed division were found to accumulate cytokinins. The high competency of primary phloem to dedifferentiate was associated with its capacity to express CDC2a and CYCB genes and the presence of high cytokinin levels, providing some insights into the determinants of competency for resuming cell division.

Induction of male sterility in plants by metabolic engineering of the carbohydrate supply.

Extracellular invertase mediates phloem unloading via an apoplastic pathway. The gene encoding isoenzyme Nin88 from tobacco was cloned and shown to be characterized by a specific spatial and temporal expression pattern. Tissue-specific antisense repression of Nin88 under control of the corresponding promoter in tobacco results in a block during early stages of pollen development, thus, causing male sterility. This result demonstrates a critical role of extracellular invertase in pollen development and strongly supports the essential function of extracellular sucrose cleavage for supplying carbohydrates to sink tissues via the apoplast. The specific interference with phloem unloading, the sugar status, and metabolic signaling during pollen formation will be a potentially valuable approach to induce male sterility in various crop species for hybrid seed production.

Tissue-specific expression of the rolA gene mediates morphological changes in transgenic tobacco.

The spatial and temporal activity of the entire and individual promoter domains of the rolA gene of Agrobacterium rhizogenes was investigated and correlated with the distinctive features of the phenotypes of transgenic tobacco plants. The GUS assay was performed in the presence of an oxidative catalyst during the development of transgenic plants expressing chimeric genes containing the beta-glucuronidase coding sequence under the control of the different promoter domains. In situ hybridization was also used on transgenic plants harbouring rolA under the control of the entire or deleted promoter. This paper demonstrates for the first time that the entire rolA promoter, composed of domains, A, B and C, is silent in seeds, then activated at the onset of germination in the cotyledons and in the elongation zone of the radicle and is finally expressed throughout the vegetative and floral phases. Domains B + C, which were sufficient to induce wrinkled leaves and short internodes, were active in all the stem tissues, but only in the companion cells of the phloem strands of the leaves. Domain C, which specified a dwarf phenotype with normal leaves, was weakly expressed in the stem vascular bundles and in the leaf internal phloem. These results indicate that the vascular bundles are the primary targets for the generation of the short internode phenotype. Furthermore, the local expression of rolA in the stem vascular bundles induced a size reduction of the surrounding parenchyma cells, suggesting the existence of some diffusible factor(s) associated with the expression of the rolA gene.