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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 phenologic stage of development provided a useful tool to determine the time when axillary buds switch from the vegetative to the reproductive phase. [ABSTRACT FROM AUTHOR]

Organic solutes, in parlicular glycine betaine and proline, have been detected as osmoprotective compounds in many microorganisms and herhaceous species. How- ever, for woody plants, very little information is available on mechanisms of adaptation to salt stress. In the present study, effects of NaCI treatment on hetaine and free amino acid contents in a clone of Populus trichocarpa × deltoides micropropagated vitroplants were analyzed using HPLC and silicate plate chromatography. The application during 12 days of 50 to 200mM NaCI to vitroplants cultured on Murashige and Skoog medium (Murashige and Skoog 1962. Physol. Plant. 15: 473-497) led to a progressive decrease of growth, leaf senescence, abscission, and apical necrosis. Populus tirichocarpa × deltoids was characterized by the absence of glycine betaine and/or proline accumulation. However, trigonelline was found to increase in vitroplants subjected to 100 mM NaCl. In vitroplants, the content of some amino acids was strongly modified by salt stress. A progressive accumulation of alanine and γ-amino butyrate was particularly significant in roots, whereas the relative concentration of glutamine was strongly enhanced in leaves. Leaf content of glutamate and ornithine attained maximum in the presence of 100 and 150mM NaCl concentrations, and then decreased. Arginine and serine pools were not significantly modified by salt treatment. The variations in vitroplants were of small amplitude compared to those observed in mature poplars where winter rest was associated with a very high arginine level and with a disappearance of nearly all other free amino acids. The results are discussed in relation to previous data obtained with herbaceous species and in relation, to some metabolic pathways in poplars where trigonelline could be considered as a sensitive stress indicator. [ABSTRACT FROM AUTHOR]

Expression of the rolC gene from Agrobacterium rhizogenes causes morphological and developmental alterations in transgenic plants. The histological alterations underlying the macroscopic changes and the cellular localization of the site of expression of the rolC gene have shown that: (i) the expression of the rolC gene is developmentally regulated, (ii) in vegetative transgenic plants, the expression of the rolC gene under the control of its own promoter is restricted to companion and protophloem cells, (iii) the site of action of the product(s) of the activity of the rolC enzyme is distinct from its site of expression, (iv) precise localization of the rolC peptide has been achieved by immunocytochemistry but not by the histochemical GUS assay. These results imply that the sites of action and expression of the rolC gene in trangenic plants are physically separated. Thus the product(s) of the activity of the rolC enzyme must be a factor capable of being transported. Current models for rolC gene action are discussed taking into account the reported results. [ABSTRACT FROM AUTHOR]

It is shown that the simultaneous presence of sucrose (30mg · 1) and kinetin (0.2 to 5mg · 1) is inductive of both lipogenesis and plastidal proteogenesis in Datura innoxia leaf expiants grown in vitro on Murashige and Skog's medium. Ultrastructural examinations reveal, since the end of the first day, an accumulation of lipid inclusions at the cytoplasmic level. At the same time, it occurs an increase in ribosome content and a polyribosome formation preceding the appearance of intraplastidal protein structures. Sucrose alone or kinetin alone have no effect on these two phenomena. The possible interactions between sucrose and kinetin upon attraction and mobilization of nutrients are considered as well as the importance of the creation of such pools for the further cell reactivation. [ABSTRACT FROM AUTHOR]

Considerable transient membrane modifications at the ultrastructural level occurred during the first hours of the in vitro culture of Datura innoxia leaf explants. Pinocytosis, loosening, outgrowths and folds of plasmalemma, plastid membrane invaginations and continuities between the outer layer of the plastid membrane and the plasmalemma were induced when sucrose was added to the medium. These changes were directly connected with the strong amylogenesis that took place from the 12th to the 72th hour. Kinetin and, to a lesser degree, auxin, magnified the phenomenon. Without sucrose, neither membrane revising nor amylogenesis occurred. Relations between these structural membrane modifications and possible alterations in the cell permeability before the induction of dedifferentiation were discussed. [ABSTRACT FROM AUTHOR]

Due to their extensive growth potential, transgenic root systems arising from inoculation with Agrobacterium rhizogenes became popular inthe last decade as model systems in domains as diverse as productionof secondary metabolites, interactions with pathogens and symbionts,examination of gene importance in control of root development or in regulation of gene expression in roots. Wild-type bacterial strains have also been considered as useful tools to stimulate rooting on recalcitrant cuttings or microcuttings as they cause abundant root initiation at the site of inoculation. Root initiation and the in vitro growth characteristics of transformed roots result from the transfer of genes located on the root-inducing plasmid (Ri) to plant cells and their expression therein. Two sets of pRi genes are involved in tlhe root induction process: the rol (root loci) genes located in the TL region and the aux genes of the TR region. Some of these genes being able to interact, the system appears also as a new tool to study the role of auxin in the process of root initiation. The distinctive phenotype of the transformed roots which are capable of hormone autonomous growth seems to be controlled mainly by the rol genes. These rol genes, i.e. the genetic loci rol A, rol B, rol C and rol D correspond to open reading frames ORFs 10, 11, 12 and 15. In vitro experiments determined the functions of the Rol B and Rol C proteins but the functionsof Rol A and Rol D are still unknown. Altered metabolism of developmental regulators or modified sensitivity to auxin have been suspectedto mediate root induction and morphological abnormalities of transformed roots and plants. The target cells for transformation and the cells which are competent for root initiation will be characterized as well as the subsequent development of transgenic roots provided with various constructs from the whole T-DNA to single rol genes. Results dealing with auxin contents in relation with root growth kinetics, phenoty [ABSTRACT FROM AUTHOR]

Pollination in flowering plants is initiated by germination of pollen grains on stigmas followed by fast growth of pollen tubes representing highly energy-consuming processes. The symplastic isolation of pollen grains and tubes requires import of Suc available in the apoplast. We show that the functional coupling of Suc cleavage by invertases and uptake of the released hexoses by monosaccharide transporters are critical for pollination in tobacco (Nicotiana tabacum). Transcript profiling, in situ hybridization, and immunolocalization of extracellular invertases and two monosaccharide transporters in vitro and in vivo support the functional coupling in supplying carbohydrates for pollen germination and tube growth evidenced by spatiotemporally coordinated expression. Detection of vacuolar invertases in maternal tissues by these approaches revealed metabolic cross talk between male and female tissues and supported the requirement for carbohydrate supply in transmitting tissue during pollination. Tissue-specific expression of an invertase inhibitor and addition of the chemical invertase inhibitor miglitol strongly reduced extracellular invertase activity and impaired pollen germination. Measurements of (competitive) uptake of labeled sugars identified two import pathways for exogenously available Suc into the germinating pollen operating in parallel: direct Suc uptake and via the hexoses after cleavage by extracellular invertase. Reduction of extracellular invertase activity in pollen decreases Suc uptake and severely compromises pollen germination. We further demonstrate that Glc as sole carbon source is sufficient for pollen germination, whereas Suc is supporting tube growth, revealing an important regulatory role of both the invertase substrate and products contributing to a potential metabolic and signaling-based multilayer regulation of pollination by carbohydrates., (© 2017 American Society of Plant Biologists. All Rights Reserved.)

We have established a detailed framework for the process of shoot regeneration from Arabidopsis root and hypocotyl explants grown in vitro. Using transgenic plant lines in which the GUS or GFP genes were fused to promoters of developmental genes (WUS, CLV1, CLV3, STM, CUC1, PLT1, RCH1, QC25), or to promoters of genes encoding indicators of the auxin response (DR5) or transport (PIN1), cytokinin (CK) response (ARR5) or synthesis (IPT5), or mitotic activity (CYCB1), we showed that regenerated shoots originated directly or indirectly from the pericycle cells adjacent to xylem poles. In addition, shoot regeneration appeared to be partly similar to the formation of lateral root meristems (LRMs). During pre-culture on a 2, 4-dichlorophenoxyacetic acid (2, 4-D)-rich callus-inducing medium (CIM), xylem pericycle reactivation established outgrowths that were not true calli but had many characteristics of LRMs. Transfer to a CK-rich shoot-inducing medium (SIM) resulted in early LRM-like primordia changing to shoot meristems. Direct origin of shoots from the xylem pericycle occurred upon direct culture on CK-containing media without prior growth on CIM. Thus, it appeared that the xylem pericycle is more pluripotent than previously thought. This pluripotency was accompanied by the ability of pericycle derivatives to retain diploidy, even after several rounds of cell division. In contrast, the phloem pericycle did not display such developmental plasticity, and responded to CKs with only periclinal divisions. Such observations reinforce the view that the pericycle is an 'extended meristem' that comprises two types of cell populations. They also suggest that the founder cells for LRM initiation are not initially fully specified for this developmental pathway.

Cytokinin deficiency causes pleiotropic developmental changes such as reduced shoot and increased root growth. It was investigated whether cytokinin-deficient tobacco plants, which overproduce different cytokinin oxidase/dehydrogenase enzymes, show changes in different sink and source parameters, which could be causally related to the establishment of the cytokinin deficiency syndrome. Ultrastructural analysis revealed distinct changes in differentiating shoot tissues, including an increased vacuolation and an earlier differentiation of plastids, which showed partially disorganized thylakoid structures later in development. A comparison of the ploidy levels revealed an increased population of cells with a 4C DNA content during early stages of leaf development, indicating an inhibited progression from G2 to mitosis. To compare physiological characteristics of sink leaves, source leaves and roots of wild-type and cytokinin-deficient plants, several photosynthetic parameters, content of soluble sugars, starch and adenylates, as well as activities of enzymes of carbon assimilation and dissimilation were determined. Leaves of cytokinin-deficient plants contained less chlorophyll and non-photochemical quenching of young leaves was increased. However, absorption rate, photosynthetic capacity (F(v)/F(m) and J(CO2 max)) and efficiency (Phi CO(2 app)), as well as the content of soluble sugars, were not strongly altered in source leaves, indicating that chlorophyll is not limiting for photoassimilation and suggesting that source strength did not restrict shoot growth. By contrast, shoot sink tissues showed drastically reduced contents of soluble sugars, decreased activities of vacuolar invertases, and a reduced ATP content. These results strongly support a function of cytokinin in regulating shoot sink strength and its reduction may be a cause of the altered shoot phenotype. Roots of cytokinin-deficient plants contained less sugar compared with wild-type. However, this did not negatively affect glycolysis, ATP content, or root development. It is suggested that cytokinin-mediated regulation of the sink strength differs between roots and shoots.

The outer tissues of dicotyledonous plant roots (i.e. epidermis, cortex, and endodermis) are clearly organized in distinct concentric layers in contrast to the diarch to polyarch vascular tissues of the central stele. Up to now, the outermost layer of the stele, the pericycle, has always been regarded, in accordance with the outer tissue layers, as one uniform concentric layer. However, considering its lateral root-forming competence, the pericycle is composed of two different cell types, with one subset of cells being associated with the xylem, showing strong competence to initiate cell division, whereas another group of cells, associated with the phloem, appears to remain quiescent. Here, we established, using detailed microscopy and specific Arabidopsis thaliana reporter lines, the existence of two distinct pericycle cell types. Analysis of two enhancer trap reporter lines further suggests that the specification between these two subsets takes place early during development, in relation with the determination of the vascular tissues. A genetic screen resulted in the isolation of mutants perturbed in pericycle differentiation. Detailed phenotypical analyses of two of these mutants, combined with observations made in known vascular mutants, revealed an intimate correlation between vascular organization, pericycle fate, and lateral root initiation potency, and illustrated the independence of pericycle differentiation and lateral root initiation from protoxylem differentiation. Taken together, our data show that the pericycle is a heterogeneous cell layer with two groups of cells set up in the root meristem by the same genetic pathway controlling the diarch organization of the vasculature.

Growth and genetic stability of Nicotiana tabacum L. callus were strongly improved by replacing the inorganic nitrogen and phosphorus of the Murashige and Skoog's medium by a soybean peptone and phytate, respectively. Cell proliferation after subcultivation on the modified medium was highly stimulated as evidenced by a strong biomass increase; this improvement was mainly due to the organic N source. In addition, while calluses grown under standard conditions displayed various cell sizes and DNA contents, subcultivation on the modified medium led to homogeneous cell size distribution and stable 4C-8C DNA contents through several subcultures. This improved genetic stability was due to replacement of inorganic P by phytate, provided the presence of peptone. Such new media composition could be useful for slow-growing cell suspensions or calluses.

At the onset of lateral root initiation in Arabidopsis thaliana, the phytohormone auxin activates xylem pole pericycle cells for asymmetric cell division. However, the molecular events leading from auxin to lateral root initiation are poorly understood, in part because the few responsive cells in the process are embedded in the root and are thus difficult to access. A lateral root induction system, in which most xylem pole pericycle cells were synchronously activated by auxin transport inhibition followed by auxin application, was used for microarray transcript profiling. Of 4,600 genes analyzed, 906 significantly differentially regulated genes were identified that could be grouped into six major clusters. Basically, three major patterns were discerned representing induced, repressed, and transiently expressed genes. Analysis of the coregulated genes, which were specific for each time point, provided new insight into the molecular regulation and signal transduction preceding lateral root initiation in Arabidopsis. The reproducible expression profiles during a time course allowed us to define four stages that precede the cell division in the pericycle. These early stages were characterized by G1 cell cycle block, auxin perception, and signal transduction, followed by progression over G1/S transition and G2/M transition. All these processes took place within 6 h after transfer from N-1-naphthylphthalamic acid to 1-naphthalene acetic acid. These results indicate that this lateral root induction system represents a unique synchronized system that allows the systematic study of the developmental program upstream of the cell cycle activation during lateral root initiation.

Some walnut trees (Juglans regia L.) originating from central Asia display an early flowering phenotype. These "Early Mature" (EM) trees may produce flowers within months of germination. Secondary flowering waves are also observed within a growing season. Inflorescences may carry male, female and hermaphrodite flowers. Progeny obtained from selected EM trees were cultured in vitro to initiate clonal propagation of these genotypes. Embryogenic lines were established through the culture of immature zygotic embryos. Microshoot lines were obtained from germinated somatic or zygotic embryos. Plants showing EM phenotypes were recovered through direct conversion of somatic embryos or adventitious rooting of microcuttings. During the in vitro propagation phase, flower buds were observed on microshoots after three to six subcultures. Histological analysis showed that most of these flowers were hermaphrodite. In vitro apical buds were used to clone the walnut orthologous cDNAs of the AGAMOUS and APETALA 3 MADS-box genes. Northern blots revealed a preferential expression of both of these homeotic genes in flowers. The results highlight the usefulness of EM lines to study the genetic cues controlling flowering and sexual maturity in woody perennials.

Twenty seven different yew trees belonging to various genotypes and hybrids have been screened for their capacity to produce significant amounts of taxoids provided with biological activity in the tubulin test. From the three best genotypes selected, Taxus x media "Sargentii" proved to be able to produce viable calluses from excised roots placed in vitro. Taxoid composition at various times of the in vitro culture was determined and the carcinostatic efficiency of the extracts was established using the KB cell cytotoxicity test. In leaves and calluses, respectively, 0.069 and 0.032% paclitaxel (taxol) contents were found. These contents were significantly higher than those previously reported for other genotypes.

The developmental characteristics of a transgenic tobacco line (BIK62) expressing the ipt cytokinin-biosynthetic gene under the control of a tagged promoter were analysed. In situ hybridization and cytokinin immunocytochemistry revealed that the ipt gene was mainly expressed in the axillary buds after the floral transition. The ipt-expressing axillary buds presented morphological alterations such as short and narrow scale-leaflets, and swollen internodes filled with starch grains, giving rise to short and tuberized lateral branches. In addition, the modification of the endogenous cytokinin balance in the axillary meristems resulted in a fast rate of leaf initiation and cytokinins accumulated mostly in the lateral zones of the reactivated axillary meristems, suggesting a role in leaf organogenesis. Cell cycle analysis revealed that the reactivated axillary meristems were characterized by predominant S+G2 nuclei. Terminal internodes displayed low levels of hexose and sucrose concomitant with starch accumulation. Extracellular invertases (EC 3.1.26) were also present in higher amounts in the tuberizing internodes compared to the axillary buds of wild-type tobacco. These results underline the role of cytokinins in cell cycle regulation and in the creation of a sink--source effect. They also provide new information about cytokinin involvement in the process of tuberization and their overproduction in axillary buds giving rise to tuberized lateral branches in a naturally non-tuberizing species.

This report describes the identification of novel plant genes that are required to ensure co-ordinated post-embryonic development. After germination the tumorous shoot development mutants of Arabidopsis thaliana develop disorganized tumorous tissue instead of organized leaves and stems. This results in green callus-like structures, which are capable of unlimited growth in vitro on hormone-free medium. The tsd mutants are recessive and belong to three complementation groups (tsd1, tsd2, tsd3). The genes were mapped to the bottom of chromosomes 5 and 1, and the top of chromosome 3, respectively. Histological analyses showed that the tsd mutants have different developmental defects. The shoot apical meristem of tsd1 formed only rudimentary leaves and was characterized by a degenerating L1 cell layer. tsd2 mutants had reduced cell adhesion and altered cell division planes in the L2 and L3 cell layers. The tumorous tissue of tsd3 mutants originated from the base of the leaf. Cytokinin levels that are inhibitory to the growth of wild-type seedlings bring about an enhanced growth response in all the tsd mutants. The steady state transcript levels of the histidine kinase CKI1 gene and the KNAT1 and STM homeobox genes were increased in tsd mutants, while mRNA levels of cell cycle genes were not altered. We hypothesize that the TSD gene products negatively regulate cytokinin-dependent meristematic activity during vegetative development of Arabidopsis.

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.

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.

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.

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.

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.