Cellular responses of two Latin-American cultivars of Lotus corniculatus to low pH and Al stress

Toxic effects of acidic root medium and aluminium were evaluated in two forage cultivars of Lotus corniculatus differing in their tolerance to Al stress. The structural response of most of the root cells exposed to low pH without Al³⁺ differed markedly from that induced by the combined stress. Conspicuous alteration of the nucleus was present only at low pH 4.0 and disintegration of the cytoplasmic components was more drastic than in the roots exposed to acidic solution containing Al³⁺. Cells exposed to low pH without Al, did not produce wall thickenings. Severely damaged cytoplasm and localized death in some cortical cells or groups of cells contrasting with almost intact cells exposed to Al³⁺ stress were found. In this respect, a strong correlation between the occurrence of cell wall thickenings and a better preserved structure of the cytoplasm was observed. The frequency of cell damage in the more tolerant cultivar UFRGS was generally lower, significantly more cortical cells capable of maintaining their resting membrane potential were present than in the sensitive INIA Draco. The difference in their tolerance is related rather to the exudation of citrate and oxalate that was higher in UFRGS than to the accumulation of tannins, which increased after Al treatment in both cultivars.     

蔗糖添加对杉木低磷胁迫响应和蔗糖代谢的影响

为了揭示低磷胁迫下蔗糖对杉木低磷胁迫响应和蔗糖代谢的影响,选用两种不同磷效率杉木家系M32和M28进行低磷胁迫下的蔗糖添加试验,分析蔗糖添加对低磷胁迫下杉木形态特征、生理特性和低磷诱导相关基因表达的影响。结果表明：蔗糖添加促进了低磷胁迫下杉木苗高、根长、根表面积、根平均直径、根体积、根叶组织蔗糖含量和根叶组织无机磷含量的增加,但仍明显低于正常供磷处理下添加蔗糖处理的杉木增量。低磷促进杉木叶中花青素的积累,而正常供磷和低磷胁迫下的蔗糖添加处理都显著促进了叶片花青素含量的增加。随着胁迫时间的延长,M28与M32在根、叶组织的蔗糖含量存在显著差异,且M28根叶组织中的蔗糖合成酶活性和蔗糖磷酸合成酶活性都高于M32。蔗糖合成酶ClSuSy在M28和M32根系中受低磷胁迫诱导下调表达,但蔗糖添加处理明显诱导ClSuSy表达量升高,M28在正常供磷并添加蔗糖处理下的ClSuSy表达量显著高于其它处理。蔗糖转运蛋白SUT4、磷转运蛋白ClPht1;4、紫色酸性磷酸酶PAP1和PAP11在M28和M32根系中总体上受低磷胁迫诱导上调表达,且受蔗糖添加处理诱导下调表达。低磷胁迫下,添加或不添加蔗糖处理的M32根系SUT4的表达量均在15d时显著升高,并在45d时回落到正常水平。ClPht1;4和PAP1在低磷胁迫15d的表达量显著高于45d时的表达量,且ClPht1;4在M32根系中的表达量远高于M28。本研究表明,蔗糖对杉木低磷胁迫响应和糖代谢有重要的影响作用,低磷胁迫下添加蔗糖处理能够在一定程度上缓解杉木低磷胁迫响应。     

酸枣根系结构可塑性对自然梯度干旱生境的适应机制

根系是植物吸收水分和养分的主要器官,是直接接触土壤最先感受土壤逆境胁迫的部位。在干旱环境中,植物根系的结构特征必定发生改变以维持正常的生物机能而生存。目前,关于根系解剖结构的研究大多集中于根系的某一特定结构对单一逆境因子的响应。以生长在烟台-石家庄-银川-吐鲁番不同地域气候条件形成的自然梯度干旱环境中的酸枣为试验材料,应用植物显微技术研究酸根系结构的可塑性对不同自然梯度干旱环境的适应机制,结果表明:酸枣根的初生结构包括表皮、皮层和维管柱,表皮位于幼根的最外层,由单层体积较小、紧密排列的表皮细胞组成。皮层占根初生结构的大部分比例,由体积较大的多层薄壁细胞组成,薄壁细胞近似圆球形,数目众多,呈环形分布。维管柱位于最内层,细胞小而密集,由中柱鞘、初生木质部、初生韧皮部及薄壁细胞组成。随生境干旱加剧,酸枣根初生结构表皮细胞的厚度和宽度逐渐增加,皮层薄壁细胞的厚度和宽度、皮层薄壁细胞层数和皮层厚度均以宁夏银川样地的最大。酸枣根的次生结构包括周皮(木栓层、木栓形成层、栓内层)和次生维管组织(次生韧皮部、维管形成层和次生木质部)。从烟台至新疆吐鲁番随生境干旱加剧,酸枣植株根系周皮逐渐加厚、致密度提高。次生木质部中,导管的数量增加,管径增大。干旱环境中,酸枣植株根系结构上的变化一方面提高了吸水能力和输水效率,另一方面增强了保水能力,减少水分散失,这可能是其适应干旱逆境的机制之一。     

Accumulation of reactive oxygen species in arbuscular mycorrhizal roots

We investigated the accumulation of reactive oxygen species (ROS) in arbuscular mycorrhizal (AM) roots from Medicago truncatula, Zea mays and Nicotiana tabacum using three independent staining techniques. Colonized root cortical cells and the symbiotic fungal partner were observed to be involved in the production of ROS. Extraradical hyphae and spores from Glomus intraradices accumulated small levels of ROS within their cell wall and produced ROS within the cytoplasm in response to stress. Within AM roots, we observed a certain correlation of arbuscular senescence and H₂O₂ accumulation after staining by diaminobenzidine (DAB) and a more general accumulation of ROS close to fungal structures when using dihydrorhodamine 123 (DHR 123) for staining. According to electron microscopical analysis of AM roots from Z. mays after staining by CeCl₃, intracellular accumulation of H₂O₂ was observed in the plant cytoplasm close to intact and collapsing fungal structures, whereas intercellular H₂O₂ was located on the surface of fungal hyphae. These characteristics of ROS accumulation in AM roots suggest similarities to ROS accumulation during the senescence of legume root nodules.     

Preliminary results from a microscopic examination on the effects of aluminium on the root tips of wheat

Root tips from aluminium (Al) tolerant (Waalt) and Al sensitive (Warigal) wheat (Triticum aestivum (L). Thell.) cultivars exposed to low concentrations of Al (10 M) for 10, 24 and 72 hours were examined under the light and electron microscope. After fixing and embedding, longitudinal and transverse thin and ultrathin sections were cut. There was no evidence of Al damage to the root tips of the Al tolerant cultivar under both the light and electron microscope. For the Al sensitive cultivar, Al had no observable effect on the root tips 10 hours after Al addition when examined under the light microscope. When examined under an electron microscope, electron dense globular deposits were observed between the cell wall and cell membrane of the epidermal cells. There was not obvious damage to the cell cytoplasm. Two or 3 days after Al addition, light microscopy showed that the cells in the root tips had become swollen and extensively vacuolated. The tissues appeared disorganised and degenerate, particularly in the epidermis and outer cortical cells. The electron microscope also revealed a thickening of the cell wall. The cell wall was broken down, particularly in the epidermis in the region 4–6 mm from the root tip. The tissue in the meristematic area was largely intact.     

Effect of Osmotic Stress on Abscisic Acid Efflux and Compartmentation in the Roots of Two Maize Lines Differing in Drought Susceptibility

Roots of two Zea mays L. lines (drought-resistant Polj 17, and drought-susceptible F-2) were exposed to osmotic stress induced by sorbitol (osmotic potential –1.0 MPa). The following parameters were determined in cortex cells: membrane permeability for abscisic acid (ABA), ABA fluxes across membranes, pH values and ABA content in cytoplasm and vacuole. Osmotic stress induced different distribution of ABA within cell compartments in the investigated lines. ABA transport in the F-2 line occurred according to the intracellular pH gradient and the anion trap concept. In Polj 17, however, osmotic stress did not cause any significant effect on pH gradient and compartmental ABA content, but had a stimulating effect on ABA efflux from cytoplasm to apoplast and than via xylem to the leaf. These findings indicate different mechanisms of ABA transport between the investigated lines in response to osmotic stress.     

Stress-induced accumulation and tissue-specific localization of dehydrins in Arabidopsis thaliana

Stress-induced accumulation of five (COR47, LTI29, ERD14, LTI30 and RAB18) and tissue localization of four (LTI29, ERD14, LTI30 and RAB18) dehydrins in Arabidopsis were characterized immunologically with protein-specific antibodies. The five dehydrins exhibited clear differences in their accumulation patterns in response to low temperature, ABA and salinity. ERD14 accumulated in unstressed plants, although the protein level was up-regulated by ABA, salinity and low temperature. LTI29 mainly accumulated in response to low temperature, but was also found in ABA- and salt-treated plants. LTI30 and COR47 accumulated primarily in response to low temperature, whereas RAB18 was only found in ABA-treated plants and was the only dehydrin in this study that accumulated in dry seeds.Immunohistochemical localization of LTI29, ERD14 and RAB18 demonstrated tissue and cell type specificity in unstressed plants. ERD14 was present in the vascular tissue and bordering parenchymal cells, LTI29 and ERD14 accumulated in the root tip, and RAB18 was localized to stomatal guard cells. LTI30 was not detected in unstressed plants. The localization of LTI29, ERD14 and RAB18 in stress-treated plants was not restricted to certain tissues or cell types. Instead these proteins accumulated in most cells, although cells within and surrounding the vascular tissue showed more intense staining. LTI30 accumulated primarily in vascular tissue and anthers of cold-treated plants.This study supports a physiological function for dehydrins in certain plant cells during optimal growth conditions and in most cell types during ABA or cold treatment. The differences in stress specificity and spatial distribution of dehydrins in Arabidopsis suggest a functional specialization for the members of this protein family.     

OBPC Symposium: Maize 2004 &; beyond—Intracellular colonization of cereals and other crop plants by nitrogen-fixing bacteria for reduced inputs of synthetic nitrogen fertilizers

Summary The problem of environmental nitrogen enrichment is most likely to be solved by reducing the inputs of synthetic nitrogen fertilizers through the creation of cereals that, like legumes, are able to fix nitrogen. In legumes, rhizobia present intracellularly in vesicles in the cytoplasm of nodule cells fix nitrogen endosymbiotically. Rhizobia within these membrane-bounded compartments are supplied with energy from plant photosynthates and, in return, the bacteria provide the plant with biologically fixed nitrogen. Recently, we have demonstrated, using novel inoculation conditions with very low numbers of bacteria, that cells of the root meristems of maize, rice, wheat, and other major non-legume crops can be colonized intracellularly by the non-rhizobial, non-nodulating, nitrogen-fixing bacterium, Gluconacetobacter diazotrophicus, that occurs naturally in sugarcane. G. diazotrophicus expressing nitrogen-fixing genes is present in membrane-bounded compartments in the cytoplasm of cells of the root meristems of the target cereals and non-legume species, similar to the intracellular colonization of legume root nodule cells by rhizobia. In order to obtain an indication of the likelihood of adequate growth and yield of maize, for example, with reduced inputs of synthetic nitrogen fertilizers, we are determining the extent to which nitrogen fixation is correlated with systemic intracellular colonization by G. diazotrophicus, with minimal or zero inputs of synthetic nitrogen fertilizer.     

Settlement of the diazotrophic,phytoeffective bacterial strain Pantoea agglomerans on and within winter wheat: An investigation using ELISA and transmission electron microscopy

The aim of this study was to investigate the ability of Pantoea agglomerans, a plant growth-promoting bacterium, to colonize various regions and tissues of the wheat plant (Triticum aestivum L.) by using different inoculation methods and inoculum concentrations. In addition, the enzyme-linked immunosorbent assay (ELISA) and transmission electron microscopy (TEM) were used to determine: (a) the ability of the bacterial cells to grow and survive both on the surface and within internal tissue of the plant and (b) the response of the plant to bacterial infection. After inoculation, cells of the diazotrophic bacterial strain P. agglomerans were found to be located in roots, stems and leaves. Colony development of bacterial cells was only detected within intercellular spaces of the root and on the root surface. However, single bacterial cells were observed in leaves and stems on the surface of the epidermis, in the vicinity to stomatal cells, within intercellular spaces of the mesophyll and within xylem vessels. Inoculated bacterial cells were found to be able to enter host tissues, to multiply in the plant and to maintain a delicate relationship between endophyte and host. The density of bacterial settlement in the plant in all experiments was about 10⁶ to 10⁷ cells per mL root or shoot sap. Establishment was confirmed by a low coefficient of variation of ELISA means at these concentrations.     

盐胁迫对竹柳幼苗生理响应及结构解剖的研究

为了解盐胁迫对竹柳(Salix spp.)幼苗生长的影响,采用土培方法,对NaCl胁迫下半年生竹柳扦插苗的成活率、生理响应和根叶部结构进行研究。结果表明,在0.25%Na Cl胁迫(轻度盐胁迫)下竹柳能正常生长,而在0.5%NaCl(中度、重度盐胁迫)下生长受到抑制,推断竹柳的耐盐阈值是0.5%。随着NaCl浓度的增大,叶片相对含水量、叶绿素a含量、叶绿素总含量和叶绿素a/b均呈下降趋势;但叶绿素b含量、脯氨酸含量和MDA含量呈升高趋势。在轻度盐胁迫下,叶片SOD活性和可溶性蛋白含量均升高,在中、重度盐胁迫下显著下降。从根叶解剖结构来看,叶片、角质层、栅栏组织厚度和根部周皮和直径在轻度盐胁迫时最大,但在中度盐胁迫时叶片栅栏组织细胞长度减小且排列越来越疏松,根部输导组织细胞不正常。这表明竹柳在轻度胁迫时具有一定的耐盐性,但在中高度盐胁迫下生长不良。     

Secondary root growth in Rhynchosia edulis Griseb. (Leguminosae): Origin of cambia and their products

This study focuses on the development of a secondary root structure in Rhynchosia edulis Griseb. (Leguminosae). Its principal objectives are (i) to study the origin of cambia and the nature of their products; (ii) to correlate root structure to habitat; and (iii) to compare this anatomy with that of other Leguminosae species growing in the same environment. Serial transverse cuts of the main root show that the secondary root structure in this species results from several phenomena, namely (1) a cambium arising from procambial and pericycle cells; (2) a lateral meristem producing cell layers from the periphery towards the inner part of the root and from which vascular bundles, whose cambia fuse forming a continuous ring, originate; and (3) the formation of “elliptical cambia” in the mid portion of the root giving rise to vascular bundles in reverse orientation. The comparison of secondary root growth in R. edulis with other root structures in Leguminosae species growing in hilly areas shows different structural patterns. Nonetheless, these different patterns have the same objective: to enlarge storage parenchyma tissue enabling survival within an environment having limited water availability.     

Asexual reproduction of <Emphasis Type="Italic">Hygrophorus olivaceoalbus</Emphasis> by intracellular microsclerotia in root cells of <Emphasis Type="Italic">Picea abies</Emphasis> – A winner of ozone stress?

Hygrophorus olivaceoalbus has long been known as an ectomycorrhizal fungus, formerly designated with the artificial binomen Piceirhiza gelatinosa. Recently, it has been found to be abundant and very frequent under double ambient ozone free air fumigation of mature Norway spruce trees. As it has already been reported that this fungus can form intercellular hyphae within the root meristem, a more detailed study was performed to clarify its type of root colonization. The present study not only revealed intercellular hyphae within the meristematic zone but also intracellular hyphae within root cortex cells which grow towards and into large tannin droplets—phenolic compounds usually deposited as defensive aids—to finally fill them completely. The hyphal assemblages become globular which at first sit as separated hyphal balls within cells still containing cytoplasm. Later on, they are apparently released from the root cells, presumably as microsclerotia for dispersal of the species. Old ectomycorrhizae (ECM) show an apical pore, and later a large orifice of a tube-like cylinder formed by the thick, persistent hyphal mantle. The root tissue is progressively degrading towards proximal parts. Disintegrating root cells apparently liberate the microsclerotia through the orifice. Further studies have to find out the mechanism by which the microsclerotia are liberated and whether they operate as asexual propagules and lastly how and by whom they are propagated. As these ECM are found under ozone stress, and with identical features at higher altitudes, stress impact on trees might be the causative agent for the high frequency and abundance of Hygrophorus olivaceoalbus ECM.     

The use of excised roots from in vitro culture for the determination of superoxide production in plants

The production of reactive oxygen species (ROS) in plants is a common event in metabolic and physiological processes as well as in the response to biotic and abiotic stress. In this paper we will report that root tissue from axenically grown tomato cultivars and Lycopersicon wild species can be used for the determination of superoxide production. Superoxide generation was evaluated following the treatment of root tissues with two general elicitors of the defence response: laminarin and calcium ionophore A23187. Results demonstrated that elicitor reactivity in terms of superoxide generation of the tomato cultivars and the wild species used was different. This suggested varying levels of competence for non-specific active defence. The proposed technique merges the advantages of in vitro cultures and of whole tissues and also demonstrates that root tissue is a suitable material for evaluating free radical release.     

Soil acidity factors and nodulation ofTrifolium repens

Summary Effects of factors associated with soil acidity (low pH, low calcium, high aluminium and high manganese) on theTrifolium repens-Rhizobium trifolii symbiosis were investigted under laboratory conditions using an axenic solution-culture technique. 200 μM manganese increased root elongation in the range pH 4.3–5.5, but had no effect on root hair formation, the number of Rhizobium in the rhizosphere, or nodule formation. Root elongation and root hair formation were unaffected at pH 4.3 when 500 or 1000μM calcium was supplied, whereas multiplication of Rhizobium in the rhizosphere and nodulation were inhibited at pH 4.3 and 4.7.50–1000μM calcium had no effect either on the multiplication of Rhizobium in the range pH 4.3–5.5, or on nodule formation in the absence of aluminium. 50 μM aluminium inhibited, root elongation and root hair formation at pH 4.3 and 4.7; the effect on root elongation was reduced by increasing the calcium concentration from 50 to 1000μM. 50μM aluminium also inhibited Rhizobium multiplication in the rhizosphere and reduced nodule formation at pH 5.5 (at which aluminium precipitated out of solution), but root elongation and root hair formation were unaffected. These, effects of aluminium at pH 5.5 may explain the poor response to inoculation by white clover in acid mineral soils after liming.     

The effects of salinity on ion concentrations within the root cells of Zea mays L.

Zea mays is a salt-sensitive crop species which in saline (100 mol m^-3 NaCl) conditions suffers considerable growth reduction correlated with elevated Na⁺ and Cl^- concentration within the leaves. To increase understanding of the regulation of ion uptake and transport by the roots in saline conditions, ion concentrations within individual root cortical cells were determined by X-ray microanalysis. There was variation in Na⁺, K⁺ and Cl^- distributions among individual cells, which could not be correlated with their spatial position in the roots. Generally, however, in response to saline growth conditions (100 mol m³ NaCl) Na⁺ and Cl^- were mostly localized in the vacuoles, although their concentrations were also sometimes increased in the cytoplasm and cell walls. The concentration of K⁺ in the cytoplasm was usually maintained at a level (mean 79 mol m^-3) compatible with the biochemical functions ascribed to this ion.Abbreviation (T)AEM (Transmission) analytical electron microscopy     

Ultrastructure of infection-thread development during the infection of soybean by Rhizobium japonicum

The location and topography of infection sites in soybean (Glycine max (L.) Merr.) root hairs spot-inoculated with Rhizobium japonicum have been studied at the ultrastructural level. Infections commonly developed at sites created when the induced deformation of an emerging root hair caused a portion of the root-hair cell wall to press against an adjacent epidermal cell, entrapping rhizobia within the pocket between the two host cells. Infections were initiated by bacteria which became embedded in the mucigel in the enclosed groove. Infection-thread formation in soybean appears to involve degradation of mucigel material and localized disruption of the outer layer of the folded hair cell wall by one or more entrapped rhizobia. Rhizobia at the site of penetration are separated from the host cytoplasm by the host plasmalemma and by a layer of wall material that appears similar or identical to the normal inner layer of the hair cell wall. Proliferation of the bacteria results in an irregular, wall-bound sac near the site of penetration. Tubular infection threads, bounded by wall material of the same appearance as that surrounding the sac, emerge from the sac to carry rhizobia roughly single-file into the hair cell. Growing regions of the infection sac or thread are surrounded by host cytoplasm with high concentrations of organelles associated with synthesis and deposition of membrane and cell-wall material. The threads follow a highly irregular path toward the base of the hair cell. Threads commonly run along the base of the hair cell for some distance, and may branch and penetrate into subjacent cortical cells at several points in a manner analagous to the initial penetration of the root hair.     

Early development ofRhizobium-induced root nodules ofParasponia rigida. I. Infection and early nodule initiation

Summary The first of two major steps in the infection process in roots ofParasponia rigida (Ulmaceae) following inoculation byRhizobium strain RP501 involves the invasion ofRhizobium into the intercellular space system of the root cortex. The earliest sign of root nodule initiation is the presence of clumps of multicellular root hairs (MCRH), a response apparently unique amongRhizobium-root associations. At the same time or shortly after MCRH are first visible, cell divisions are initiated in the outer root cortex of the host plant, always subjacent to the MCRH. No infection threads were observed in root hairs or cortical cells in early stages. Rhizobial entry through the epidermis and into the root cortex was shown to occur via intercellular invasion at the bases of MCRH. The second major step in the infection process is the actual infectionper se of host cells by the rhizobia and formation of typical intracellular infection threads with host cell accommodation. This infection step is probably the beginning of the truly symbiotic relationship in these nodules. Rhizobial invasion and infection are accompanied by host cortical cell divisions which result in a callus-like mass of cortical cells. In addition to infection thread formation in some of these host cortical cells, another type of rhizobial proliferation was observed in which large accumulations of rhizobia in intercellular spaces are associated with host cell wall distortion, deposition of electron-dense material in the walls, and occasional deleterious effects on host cell cytoplasm.     

Accumulation of zeatin O-glycosyltransferase in Phaseolus vulgaris and Zea mays following cold stress

Zeatin O-glycosides have been reported as inactive and stable storage forms of cytokinins whose concentrations increase in cold stressed plants. Zeatin O-glycosides accumulation in developing bean seeds has been correlated with an increase of zeatin O-glycosyltransferase , which is specific to trans-zeatin, and catalyzes the conjugation of zeatin O-glycosides. When Phaseolus vulgaris and Zea mays seedlings were grown for 3 days at 25 and then incubated at 4 or 10 for 6 days no further growth was observed in roots. Hypertrophy was observed in the root tips of both species. In shoot-hypocotyl complexes, in contrast, growth occurred when seedlings were incubated at 10 . Western analysis, with Mabs specific to zeatin O-glycosyltransferase, detected antigenically related proteins in roots, shoot tips and cotyledons after seedlings were cold stressed for 1–6 days at 4 or 10 . Immunolocalization, of both maize and bean root sections grown at 25 revealed antigenically related proteins that were detected at low levels in cortical cells. The signal intensified upon cold stress. The localization of zeatin O-glycosyltransferase in Z. mays root tips was directly comparable to the distribution of the zeatin O-glycosides. The enzyme was detected in the nucleus, cytoplasm, and closely associated with the plasma membrane and in the cell wall of Z. mays root cells. Southern analysis suggested that more than one gene in Z. mays that were homologous to zeatin O-glycosyltransferase in P. vulgaris. Zeatin O-glycosyltransferase may be involved in modulation of cytokinins under cold stress.     

Tubulin conformation in microtubule-free cells ofVigna sinensis

Summary The primary leaf, epicotyl, and root cells ofVigna sinensis seedlings grown continuously in a 0.08% colchicine solution, become microtubule-free and polyploid. In meristematic root cells a tubulin transformation is detected 1–3 h after the treatment had begun. Tubulin strands are organized at the positions of the pre-existing microtubules. Frequently, the strands converge on or are organized in the cortical cytoplasmic zone where in normal cells the preprophase microtubule band (PMB) is assembled. In meristematic root cells subjected to a 6–12 h colchicine treatment, the tubulin strands become perinuclear, entering the cortical cytoplasm at regions close to the nucleus. One day after the onset of the treatment, tubulin generally forms a continuous reticulum of interconnected strands in all the organs examined. In most cells this reticulum surrounds the nucleus partly or totally or lies close to it, exhibiting variable configurations in different cells. After prolonged treatments, the organization of the tubulin reticulum changes further. Now this consists of crystal-like structures interconnected by thin strands.On thin sections of fixed tissue the tubulin strands consist of paracrystalline material. The distribution of this material in the affected cells coincides with that of tubulin reticulum visualized by immunofluorescence. In transverse planes each strand exhibits circular subunits arranged close to one another in a hexagonal pattern but in longitudinal ones variable images were observed. The paracrystalline material persists in root cells subjected to an 8-day continuous colchicine treatment. The immunolabeled strands seem to be composed of tubulin-colchicine complexes and not pure tubulin.     

Characterization of competent cells and early events of Agrobacterium-mediated genetic transformation in Arabidopsis thaliana

The insertion of foreign DNA in plants occurs through a complex interaction between Agrobacteria and host plant cells. The marker gene β-glucuronidase of Escherichia coli and cytological methods were used to characterize competent cells for Agrobacterium-mediated transformation, to study early cellular events of transformation, and to identify the potential host-cell barriers that limit transformation in Arabidopsis thaliana L. Heynh. In cotyledon and leaf explants, competent cells were mesophyll cells that were dedifferentiating, a process induced by wounding and-or phytohormones. The cells were located either at the cut surface or within the explant after phytohormone pretreatment. In root explants, competent cells were present in dedifferentiating pericycle, and were produced only after phytohormone pretreatment. Irrespective of their origin, the competent cells were small, isodiametric with thin primary cell walls, small and multiple vacuoles, prominent nuclei and dense cytoplasm. In both cotyledon and root explants, histological enumeration and β-glucuronidase assays showed that the number of putatively competent cells was increased by preculture treatment, indicating that cell activation and cell division following wounding were insufficient for transformation without phytohormone treatment. Exposure of explants for 48 h to A. tumefaciens produced no characteristic stress response nor any gradual loss of viability nor cell death. However, in the competent cell, association between the polysaccharide of the host cell wall and that of the bacterial filament was frequently observed, indicating that transformation required polysaccharide-to-polysaccharide contact. Flow cytofluorometry and histological analysis showed that abundant transformation required not only cell activation (an early state exhibiting an increase in nuclear protein) but also cell proliferation (which in cotyledon tissue occurred at many ploidy levels). Noncompetent cells could be made competent with the appropriate phytohormone treatments before bacterial infection: this should aid analysis of critical steps in transformation procedures and should facilitate developing new strategies to transform recalcitrant plants.