Deposition of callose in treatment of cells of the tomato plant (<Emphasis Type="Italic">Lycopersicon esculentum</Emphasis> L.) with biotic elicitors

The dynamics of induced accumulation of callose in tomato cells is studied. Localization of callose in the cells is studied by methods of fluorescence microscopy and the time needed for quantitative determination of callosa is optimized. The quantity of callose in tomato cells treated with different biotic elicitors is established. A nonlinear dependence of the deposition of callose on the concentration of chitin oligomers containing 3–5 fragments of N-acetylglucosamine is established and a proportional increase in the content of callose in the cells with increasing concentration of chitin dimer and chitosan in the culture medium is demonstrated.     

Overexpression of Pto activates defense responses and confers broad resistance.

The tomato disease resistance (R) gene Pto specifies race-specific resistance to the bacterial pathogen Pseudomonas syringae pv tomato carrying the avrPto gene. Pto encodes a serine/threonine protein kinase that is postulated to be activated by a physical interaction with the AvrPto protein. Here, we report that overexpression of Pto in tomato activates defense responses in the absence of the Pto-AvrPto interaction. Leaves of three transgenic tomato lines carrying the cauliflower mosaic virus 35S::Pto transgene exhibited microscopic cell death, salicylic acid accumulation, and increased expression of pathogenesis-related genes. Cell death in these plants was limited to palisade mesophyll cells and required light for induction. Mesophyll cells of 35S::Pto plants showed the accumulation of autofluorescent compounds, callose deposition, and lignification. When inoculated with P. s. tomato without avrPto, all three 35S::Pto lines displayed significant resistance and supported less bacterial growth than did nontransgenic lines. Similarly, the 35S::Pto lines also were more resistant to Xanthomonas campestris pv vesicatoria and Cladosporium fulvum. These results demonstrate that defense responses and general resistance can be activated by the overexpression of an R gene.     

Absence of the endo-beta-1,4-glucanases Cel1 and Cel2 reduces susceptibility to Botrytis cinerea in tomato

Cel1 and Cel2 are members of the tomato (Solanum lycopersicum Mill) endo-beta-1,4-glucanase (EGase) family that may play a role in fruit ripening and organ abscission. This work demonstrates that Cel1 protein is present in other vegetative tissues and accumulates during leaf development. We recently reported the downregulation of both the Cel1 mRNA and protein upon fungal infection, suggesting the involvement of EGases in plant-pathogen interactions. This hypothesis was confirmed by assessing the resistance to Botrytis cinerea infection of transgenic plants expressing both genes in an antisense orientation (Anti-Cel1, Anti-Cel2 and Anti-Cel1-Cel2). The Anti-Cel1-Cel2 plants showed enhanced resistance to this fungal necrotroph. Microscopical analysis of infected leaves revealed that tomato plants accumulated pathogen-inducible callose within the expanding lesion. Anti-Cel1-Cel2 plants presented a faster and enhanced callose accumulation against B. cinerea than wild-type plants. The inhibitor 2-deoxy-d-glucose, a callose synthesis inhibitor, showed a direct relationship between faster callose accumulation and enhanced resistance to B. cinerea. EGase activity appears to negatively modulate callose deposition. The absence of both EGase genes was associated with changes in the expression of the pathogen-related genes PR1 and LoxD. Interestingly, Anti-Cel1-Cel2 plants were more susceptible to Pseudomonas syringae, displaying severe disease symptoms and enhanced bacterial growth relative to wild-type plants. Analysis of the involvement of Cel1 and Cel2 in the susceptibility to B. cinerea in fruits was done with the ripening-impaired mutants Never ripe (Nr) and Ripening inhibitor (rin). The data reported in this work support the idea that enzymes involved in cell wall metabolism play a role in susceptibility to pathogens.     

Effects of Heat Stress on Carbon Transport from Tomato Leaves

Export of radioactive carbon from two cultivars of tomato, (Lycopersiconesculentum Mill.) leaves was inhibited in response to heat stress.Increasing temperatures resulted in a marked decrease in leafstarch levels. The depletion of starch concentration in theleaves was primarily due to hydrolysis and an inhibition ofstarch formation. At high temperatures, starch hydrolysis wasinhibited in Roma VF, a heat sensitive cultivar, while Saladette,a heat tolerant cultivar was not similarly affected. Calloseformation was found on phloem sieve tube plates of leaf petiolesexposed to 72 h of high temperatures. More sieve tube plateswere covered with a thicker callose layer in Roma VF than inSaladette. Lycopersicon esculentum (Mill.), tomato, carbon translocation, starch hydrolysis, callose, heat stress     

Small RNA Derived from the Virulence Modulating Region of the Potato spindle tuber viroid Silences callose synthase Genes of Tomato Plants

The tomato (Solanum lycopersicum) callose synthase genes CalS11-like and CalS12-like encode proteins that are essential for the formation of callose, a major component of pollen mother cell walls; these enzymes also function in callose formation during pathogen infection. This article describes the targeting of these callose synthase mRNAs by a small RNA derived from the virulence modulating region of two Potato spindle tuber viroid variants. More specifically, viroid infection of tomato plants resulted in the suppression of the target mRNAs up to 1.5-fold, depending on the viroid variant used and the gene targeted. The targeting of these mRNAs by RNA silencing was validated by artificial microRNA experiments in a transient expression system and by RNA ligase-mediated rapid amplification of cDNA ends. Viroid mutants incapable of targeting callose synthase mRNAs failed to induce typical infection phenotypes, whereas a chimeric viroid obtained by swapping the virulence modulating regions of a mild and a severe variant of Potato spindle tuber viroid greatly affected the accumulation of viroids and the severity of disease symptoms. These data provide evidence of the silencing of multiple genes by a single small RNA derived from a viroid.     

Regulation of Callose Metabolism in Higher Plant Cells in vitro

Temporary accumulation of callose in suspension-cultured wheat (Triticum timopheevii Zhuk.) cells at the exponential growth phase was correlated with the mitotic index due to the formation of the cell plates in dividing cells. Callose disappeared in expanding cells owing to enhanced activities of endo- and exoglucanases. The exogluconase activity was reduced when the cells were treated with cycloheximide, an inhibitor of protein synthesis. A similar pattern was observed when elicitors experimentally enhanced callose synthesis. Apparently, in such cases, callose behaves as a temporary component repairing the cell wall. We presume that plant cells comprise a universal mechanism for regulating callose synthesis.     

Investigation on the Ontogeny of Pollen in Pinus thunbergii Parl

The microsporogenesis in Pinus thunbergii occurs in early to middle March, and the development of pollen in late March to early April. A mature pollen grain consists of four cells. The starch grains aggregate in the equators during meiosis Ⅰ to Ⅱ. The callose materials appear first in inner side of the wall of meiocyte and then in the equator. Sometimes the 3-or 4-sac- cate pollen grains have been found. In the ontogeny of pollen in Pinus thunbergii following events are noteworthy. (1) During meiosis the equatorial aggregation of starch grains in me iocyte becomes conspicuous. (2) During formation of tetrad the callose wall appears in regular activity. It is noteworthy that there is itself feature for the accumulation of callose in Pinus thunbergii. After formation of prothallial cells the callose appears mainly in the area between body and saccus. So the prominent symmetry of distribution of callose occurs in the pollen grains. But almost no callose accumulation takes place i,n tenuity in distal face of pollen grain. On the contrary, there are certain callose accumulation around the prothallial cells in the proximal face, forming the polar distribution of callose in pollen grains.     

Caffeine inhibits callose deposition in the cell plate and the depolymerization of microtubules in the central region of the phragmoplast

Treatment of tobacco BY-2 cells with 10 mM caffeine that was started after the cells had entered the mitotic phase did not completely inhibit the deposition of callose in the cell plate and allowed the centrifugal redistribution of phragmoplast microtubules. On the other hand, when treatment with caffeine was started before the cells entered the mitotic phase, the deposition of callose was completely inhibited and the redistribution of phragmoplast microtubules was also inhibited. As the inhibition of redistribution of phragmoplast microtubules seems to be caused by the inhibition of depolymerization of microtubules at the central region of the phragmoplast, these results strongly suggest that the deposition of callose in the cell plate is tightly linked with the depolymerization of phragmoplast microtubules. Callose deposition was observed in phragmoplasts isolated from caffeine-treated cells as well as in those isolated from non-caffeine-treated cells, and caffeine did not inhibit callose synthesis in isolated phragmoplast, indicating that caffeine neither inhibits the accumulation of callose synthase at the equatorial regions of the phragmoplast nor arrests callose synthase itself.     

Expression of female sterility in alfalfa (<Emphasis Type="Italic">Medicago sativa</Emphasis> L.)

Female sterility associated with the presence of callose in the nucellus at anthesis was studied in an F₁ progeny of two alfalfa plants displaying 5 and 81% ovule sterility. Transgressive segregation was observed and 100% sterile plants were obtained. Two of the sterile plants were used for cytological analyses on sectioned and stain-cleared whole ovules, in comparison to a 100% fertile full sib plant. The first sign of sterility was callose deposition in the nucellus cell walls surrounding the sporogenous cells of the young ovules. At the same stage, no trace of callose was present in ovule primordia of the fertile plant. Megaspore mother cells differentiated in both fertile and sterile ovules and meiosis was initiated, as indicated by chromatin patterning typical of a zygotene stage. However, meiosis was never completed in the sterile plants. In the control, callose was deposited around the meiocyte and as sects between the cells of the dyads and tetrads during meiosis, and disappeared after the completion of meiosis; an embryo sac developed and female fertility was normal. In the sterile ovules, some nucellus cells enlarged and callose accumulation continued forming thick deposits. At anthesis, the sterile ovules lacked an embryo sac and showed massive callose accumulation in the nucellus. Male fertility was normal in female-sterile plants, thus a female-specific arrest of sporogenesis appears to be the cause of sterility. Pistil development was aberrant in some sterile genotypes, even with arrested pistil growth in early flower buds.     

Cellular change and callose accumulation in zygotic embryos of Eleutherococcus senticosus caused by plasmolyzing pretreatment result in high frequency of single-cell-derived somatic embryogenesis

Summary. Eleutherococcus senticosus zygotic embryos were pretreated with 1.0 M mannitol or sucrose for 3–24 h. This pretreatment resulted in a high frequency of somatic-embryo formation on hormone-free medium. All the somatic embryos developed directly and independently from single epidermal cells on the surface of zygotic embryos after plasmolyzing pretreatment. Scanning electron microscopic observation revealed that the epidermal cells of hypocotyls rapidly became irregular and showed a random orientation before somatic-embryo development commenced. At the same time, the epidermal cells in the untreated control remained regular. Callose concentration determined by fluorometric analysis increased sharply in E. senticosus zygotic embryos after plasmolyzing pretreatment but remained low in the untreated control. Aniline blue fluorescent staining of callose showed that the plasmolyzing pretreatment of zygotic embryos resulted in heavy accumulation of callose between the plasma membrane and cell walls. On the basis of these results, we suggest that plasmolyzing pretreatment of zygotic embryos induces the accumulation of callose, and the interruption of cell-to-cell communication imposed by this might stimulate the reprogramming of epidermal cells into embryogenically competent cells and finally induce somatic-embryo development from single cells. Correspondence and reprints: Division of Forest Resources, College of Forest Sciences, Kangwon National University, Chunchon 200-701, Republic of Korea.     

A glycine-rich protein that facilitates exine formation during tomato pollen development

Formation of the unique and highly diverse outer cell wall, or exine, of pollen is essential for normal pollen function and survival. However, little is known about the many contributing proteins and processes involved in the formation of this wall. The tomato gene LeGRP92 encodes for a glycine-rich protein produced specifically in the tapetum. LeGRP92 is found as four major forms that accumulate differentially in protein extracts from stamens at different developmental stages. The three largest molecular weight forms accumulated during early microspore development, while the smallest molecular weight form of LeGRP92 was present in protein extracts from stamens from early microsporogenesis through anther dehiscence, and was the only form present in dehisced pollen. Light microscopy immunolocalization experiments detected LeGRP92 at only two stages, late tetrad and early free microspore. However, we observed accumulation of the LeGRP92 at the early tetrad stage of development by removing the callose wall from tetrads, which allowed LeGRP92 detection. Transmission electron microscopy confirmed the LeGRP92 accumulation from microspore mother cells, tetrads through anther dehiscence. It was observed in the callose surrounding the microspore mother cells and tetrads, the exine of microspores and mature pollen, and orbicules. Plants expressing antisense RNA had reduced levels of LeGRP92 mRNA and protein, which correlated to pollen with altered exine formation and reduced pollen viability and germination. These data suggest that the LeGRP92 has a role in facilitating sporopollenin deposition and uniform exine formation and pollen viability.     

Priming for JA-dependent defenses using hexanoic acid is an effective mechanism to protect Arabidopsis against B. cinerea

Soil drench treatments with hexanoic acid can effectively protect Arabidopsis plants against Botrytis cinerea through a mechanism based on a stronger and faster accumulation of JA-dependent defenses.Plants impaired in ethylene, salicylic acid, abscisic acid or glutathion pathways showed intact protection by hexanoic acid upon B. cinerea infection. Accordingly, no significant changes in the SA marker gene PR-1 in either the SA or ABA hormone balance were observed in the infected and treated plants. In contrast, the JA signaling pathway showed dramatic changes after hexanoic acid treatment, mainly when the pathogen was present. The impaired JA mutants, jin1-2 and jar1, were unable to display hexanoic acid priming against the necrotroph. In addition, hexanoic acid-treated plants infected with B. cinerea showed priming in the expression of the PDF1.2, PR-4 and VSP1 genes implicated in the JA pathways. Moreover, JA and OPDA levels were primed at early stages by hexanoic acid. Treatments also stimulated increased callose accumulation in response to the pathogen. Although callose accumulation has proved an effective IR mechanism against B. cinerea, it is apparently not essential to express hexanoic acid-induced resistance (HxAc-IR) because the mutant pmr4.1 (callose synthesis defective mutant) is protected by treatment.We recently described how hexanoic acid treatments can protect tomato plants against B. cinerea by stimulating ABA-dependent callose deposition and by priming OPDA and JA-Ile production. We clearly demonstrate here that Hx-IR is a dependent plant species, since this acid protects Arabidopsis plants against the same necrotroph by priming JA-dependent defenses without enhancing callose accumulation.     

Isolation of aluminum-tolerant cell lines of tobacco in a simple calcium medium and their responses to aluminum

Aluminum (Al)-tolerant cell lines (ALT301 and ALT401) of tobacco were isolated in a simple calcium (Ca) solution from ethyl methane sulfonate (EMS)-treated suspension cultured tobacco cells ( Nicotiana tabacum L. cv. Samsun, a cell line SL) at the logarithmic phase of growth. A highly tolerant cell line ALT301 exhibited the accumulation of Al and the deposition of callose to the same extent as the parental SL cells during the exposure to Al. However, the Al-treated ALT301 cells grew much better than the Al-treated SL cells after transfer to Al-free growth medium. Compared to SL cells, ALT301 cells were more tolerant to toxicity of copper and iron, but not to that of lanthanum. These results suggest that ALT301 cells have an internal tolerance mechanism, which makes cells grow normally in spite of Al accumulation and Al-induced lesion represented by the deposition of callose. This tolerance mechanism seems also to be effective against copper and iron toxicity. A slightly tolerant cell line ALT401 also accumulated Al to the same degree as SL cells, but deposited significantly less callose than did SL cells (43% of SL). The growth of ALT401 cells after Al treatment was only slightly better than that of SL cells. Thus, it seems that ALT401 cells have a mechanism to protect cells only from the Al-induced deposition of callose, which is not enough to overcome the Al-induced inhibition of growth. The different phenotypes exhibited by these Al-tolerant cell lines suggest that the deposition of callose is not directly related to the inhibition of growth in Al-treated cells.     

Callose Synthase in Pinus sylvestris Response during Infection by Species of Heterobasidion annosum sensu lato with Varied Host Preferences

Strengthening of plant cell walls at the site of fungal entry is one of the earliest plant responses to fungal pathogens. The aim of our study was to characterize the pattern of callose synthase localization and callose deposition in roots of Pinus sylvestris after infection by species of the Heterobasidion annosum s.l. complex with different host specificity: H. annosum s.s., H. parviporum and H. abietinum. To address this, sense‐labelled probes and ribonuclease‐treated samples were used to determine in situ hybridizations of callose synthase by FISH method. Furthermore, determination of callose accumulation within P. sylvestris cells was carried out using aniline blue. The different species of H. annosum s.l. had distinct impacts on the callose synthase staining within plant tissues. Moreover, while inoculation with strains of H. abietinum resulted in callose synthase accumulation at the point of hyphae contact with the host cell, this was not observed with the other species. A significant difference in callose synthesis localization was observed after inoculation with varied species of H. annosum s.l. as a result of the specific interactions with the host.     

Study of priming for callose accumulation in <Emphasis Type="Italic">Allium cepa</Emphasis> during treatment with biotic inducers

The effect of preliminary treatment with inducers of resistance (salicylic and hexanoic acids) at the level of callose synthesis in cells of three onion cultivars have been studied. The ability of these inducers to increase the resistance of onion to pathogenic infections at low concentrations was shown. Using the method of spectrofluorimetry, we demonstrated that the treatment of onion scales with salicylic and hexanoic acids led to the priming of the reaction of callose accumulation.     

Studies on callose and cutin during the expression of competence and determination for organogenic nodule formation from internodes of Humulus lupulus var. Nugget

Callose and cutin deposition were followed by staining with Aniline Blue and Nile Red and by immunolocalization using antibodies raised against callose. Along with morphogenesis induction from internodes of Humulus lupulus var. Nugget, a temporal and spatial differential deposition of callose and cutin was observed. A cutin layer showing bright yellow autofluorescence appears, surrounding cells or groups of cells committed to express morphogenic competence. This cutin layer that evolves to a randomly organized network appeared underneath a callose layer and may create a specific cellular environment with altered permeability and altered receptors providing conditions for entering the cell cycle. The incipient callose accumulation in control explants cultured on basal medium suggests the involvement of callose in the initiation of the morphogenic programme leading to nodule formation. A scanning electron microscopic study during the organogenic process showed that before shoot bud regeneration, the cutin layer increases in thickness and acquires a smooth texture. This cutin layer is specific to nodular organogenic regions and disappeared with plantlet regeneration. This layer may control permeability to water and solute transfer throughout plantlet regeneration.     

Early events responsible for aluminum toxicity symptoms in suspension-cultured tobacco cells

We investigated the aluminum (Al)-induced alterations in zeta potential, plasma membrane (PM) potential and intracellular calcium levels to elucidate their interaction with callose production induced by Al toxicity. A noninvasive confocal laser microscopy has been used to analyse the live tobacco (Nicotiana tabacum) cell events by means of fluorescent probes Fluo-3 acetoxymethyl ester (intracellular calcium) and DiBAC4 (PM potential) as well as to monitor callose accumulation. Log-phase cells showed no detectable changes in the PM potential during the first 30 min of Al treatment, but sustained large depolarization from 60 min onwards. Measurement of zeta potential confirmed the depolarization effect of Al, but the kinetics were different. The Al-treated cells showed a moderate increase in intracellular Ca2+ levels and callose production in 1 h, which coincided with the time course of PM depolarization. Compared with the Al treatment, cyclopiazonic acid, an inhibitor of endoplasmic reticulum Ca(2+)-ATPase, facilitated a higher increase in intracellular Ca2+ levels, but resulted in accumulation of only moderate levels of callose. Calcium channel modulators and Al induced similar levels of callose in the initial 1 h of treatment. Callose production induced by Al toxicity is dependent on both depolarization of the PM and an increase in intracellular Ca2+ levels.     

Callose deposition in the phloem plasmodesmata and inhibition of phloem transport in citrus leaves infected with “Candidatus Liberibacter asiaticus”

Huanglongbing (HLB) is a destructive disease of citrus trees caused by phloem-limited bacteria, Candidatus Liberibacter spp. One of the early microscopic manifestations of HLB is excessive starch accumulation in leaf chloroplasts. We hypothesize that the causative bacteria in the phloem may intervene photoassimilate export, causing the starch to over-accumulate. We examined citrus leaf phloem cells by microscopy methods to characterize plant responses to Liberibacter infection and the contribution of these responses to the pathogenicity of HLB. Plasmodesmata pore units (PPUs) connecting companion cells and sieve elements were stained with a callose-specific dye in the Liberibacter-infected leaf phloem cells; callose accumulated around PPUs before starch began to accumulate in the chloroplasts. When examined by transmission electron microscopy, PPUs with abnormally large callose deposits were more abundant in the Liberibacter-infected samples than in the uninfected samples. We demonstrated an impairment of symplastic dye movement into the vascular tissue and delayed photoassimilate export in the Liberibacter-infected leaves. Liberibacter infection was also linked to callose deposition in the sieve plates, which effectively reduced the sizes of sieve pores. Our results indicate that Liberibacter infection is accompanied by callose deposition in PPUs and sieve pores of the sieve tubes and suggest that the phloem plugging by callose inhibits phloem transport, contributing to the development of HLB symptoms.