Foliar manganese spray induces the resistance of cucumber to Colletotrichum lagenarium.

Micronutrients provide a potentially interesting alternative to fungicides for the protection of crops against fungal pathogens. Here we studied the effect of foliar-applied manganese (Mn) in the form of MnSO4 on severity of anthracnose disease, caused by Colletotrichum lagenarium in cucumber (Cucumis sativus L.) plant. The study was done aimed to characterize the optimum dose and application time of Mn fertilizer on disease suppression as well as to identify the defense mechanisms by which Mn-treated plants resist to fungal disease. In preliminary tests, Mn was applied at different concentrations (1.8, 4.5 and 7.2 mM) and various time points (three days before or two hours before inoculation, or three days after inoculation). Results showed that application of Mn either before or after inoculation suppressed the fungal infection in leaves and cotyledons, with a higher efficiency when applied three days prior to inoculation. However, all applied concentrations of Mn equally reduced the disease severity. Mn treatment in the absence of the pathogen promoted lignification and reactive oxygen species (ROS) accumulation. Also, pre-inoculation Mn treatment enhanced pathogen-induced lignification, callose or ROS production and reduced pathogen-induced cell death. The increase of lignin, callose and ROS induction by Mn application were 34, 30 and 31 % compared to control, respectively. Together, the results suggested the effectiveness of Mn treatments on anthracnose alleviation in cucumber plants. The findings here have a practical importance in plant physiology studies to identify the resistance-relevant mechanisms to pathogens and in sustainable agriculture to control the fungal diseases by a safe method.

Regulated trafficking of cellulose synthases.

New findings reveal that proteins involved in cellulose biosynthesis undergo regulated trafficking between intracellular compartments and the plasma membrane. The coordinated secretion and internalization of these proteins involve both the actin and cortical microtubule cytoskeletons. This regulated trafficking allows the dynamic remodeling of cellulose synthase complex (CSC) secretion during cell expansion and differentiation. Several new actors of the cellulose synthesis machinery have been recently identified.

Approaches to understanding the functional architecture of the plant cell wall.

Cell wall polysaccharides are some of the most complex biopolymers known, and yet their functions remain largely mysterious. Advances in imaging methods permit direct visualisation of the molecular architecture of cell walls and the modifications that occur to polymers during growth and development. To address the structural and functional relationships of individual cell wall components, we need to better characterise a broad range of structural and architectural alterations in cell walls, appearing as a consequence of developmental regulation, environmental adaptation or genetic modification. We have developed a rapid method to screen large numbers of plants for a broad range of cell wall phenotypes using Fourier transform infrared microspectroscopy and Principal Component Analysis. We are using model systems to uncover the genes that encode some of the cell-wall-related biosynthetic and hydrolytic enzymes, and structural proteins.

Temperature-sensitive alleles of RSW2 link the KORRIGAN endo-1,4-beta-glucanase to cellulose synthesis and cytokinesis in Arabidopsis.

An 8.5-kb cosmid containing the KORRIGAN gene complements the cellulose-deficient rsw2-1 mutant of Arabidopsis. Three temperature-sensitive alleles of rsw2 show single amino acid mutations in the putative endo-1,4-beta-glucanase encoded by KOR. The F1 from crosses between kor-1 and rsw2 alleles shows a weak, temperature-sensitive root phenotype. The shoots of rsw2-1 seedlings produce less cellulose and accumulate a short chain, readily extractable glucan resembling that reported for rsw1 (which is defective in a putative glycosyltransferase required for cellulose synthesis). The double mutant (rsw2-1 rsw1) shows further reductions in cellulose production relative to both single mutants, constitutively slow root growth, and enhanced temperature-sensitive responses that are typically more severe than in either single mutant. Abnormal cytokinesis and severely reduced birefringent retardation in elongating root cell walls of rsw2 link the enzyme to cellulose production for primary cell walls and probably cell plates. The Rsw2(-) phenotype generally resembles the Kor(-) and cellulose-deficient Rsw1(-) phenotypes, but anther dehiscence is impaired in Rsw2-1(-). The findings link a second putative enzyme activity to cellulose synthesis in primary cell walls of Arabidopsis and further increases the parallels to cellulose synthesis in Agrobacterium tumefaciens where the celA and celC genes are required and encode a putative glycosyltransferase and an endo-1,4-beta-glucanase related to RSW1 and KOR, respectively.

Altered pectin composition in primary cell walls of korrigan, a dwarf mutant of Arabidopsis deficient in a membrane-bound endo-1,4-beta-glucanase.

Korrigan (kor) is a dwarf mutant of Arabidopsis thaliana (L.) Heynh. that is deficient in a membrane-bound endo-1,4-beta-glucanase. The effect of the mutation on the pectin network has been studied in kor by microscopical techniques associated with various probes specific for different classes of pectic polysaccharides. The localisation of native crystalline cellulose was also examined using the cellobiohydrolase I-gold probe. The investigations were focused on the external cell walls of the epidermis, a cell layer that, in a number of plant species, has been shown to be growth limiting. Anionic sites associated with pectic polymers were quantified using the cationic gold probe. Homogalacturonans were quantified using polyclonal anti-polygalacturonic acid/rhamnogalacturonan I antibodies recognising polygalacturonic acid, and monoclonal JIM7 and JIM5 antibodies recognising homogalacturonans with a high or low degree of methyl-esterification, respectively. Rhamnogalacturonans were quantified with two monoclonal antibodies, LM5, recognising beta-1,4 galactan side chains of rhamnogalacturonan I, and CCRCM2. Our results show a marked increase in homogalacturonan epitopes and a decrease in rhamnogalacturonan epitopes in kor compared to the wild type. A substantial decrease in cellobiohydrolase I-gold labelling was also observed in the mutant cell walls. These findings demonstrate that a deficiency in an endo-1,4-beta-glucanase, which is in principle not directly implicated in pectin metabolism, can induce important changes in pectin composition in the primary cell wall. The changes indicate the existence of feedback mechanisms controlling the synthesis and/or deposition of pectic polysaccharides in primary cell walls.

BOTERO1 is required for normal orientation of cortical microtubules and anisotropic cell expansion in Arabidopsis.

Mutants at the BOTERO1 locus are affected in anisotropic growth in all non-tip-growing cell types examined. Mutant cells are shorter and broader than those of the wild type. Mutant inflorescence stems show a dramatically reduced bending modulus and maximum stress at yield. Our observations of root epidermis cells show that the cell expansion defect in bot1 is correlated with a defect in the orientation of the cortical microtubules. We found that in cells within the apical portion of the root, which roughly corresponds to the meristem, microtubules were loosely organized and became much more highly aligned in transverse arrays with increasing distance from the tip. Such a transition was not observed in bot1. No defect in microtubule organization was observed in kor-1, another mutant with a radial cell expansion defect. We also found that in wild-type root epidermal cells, cessation of radial expansion precedes the increased alignment of cortical microtubules into transverse arrays. Bot1 roots still show a gravitropic response, which indicates that ordered cortical microtubules are not required for differential growth during gravitropism. Interestingly, the fact that in the mutant, these major changes in microtubule organization cause relatively subtle changes in cell morphology, suggest that other levels of control of growth anisotropy remain to be discovered. Together, these observations suggest that BOT1 is required for organizing cortical microtubules into transverse arrays in interphase cells, and that this organization is required for consolidating, rather than initiating, changes in the direction of cell expansion.

PROCUSTE1 encodes a cellulose synthase required for normal cell elongation specifically in roots and dark-grown hypocotyls of Arabidopsis.

Mutants at the PROCUSTE1 (PRC1) locus show decreased cell elongation, specifically in roots and dark-grown hypocotyls. Cell elongation defects are correlated with a cellulose deficiency and the presence of gapped walls. Map-based cloning of PRC1 reveals that it encodes a member (CesA6) of the cellulose synthase catalytic subunit family, of which at least nine other members exist in Arabidopsis. Mutations in another family member, RSW1 (CesA1), cause similar cell wall defects in all cell types, including those in hypocotyls and roots, suggesting that cellulose synthesis in these organs requires the coordinated expression of at least two distinct cellulose synthase isoforms.

Gibberellin and ethylene control endoreduplication levels in the Arabidopsis thaliana hypocotyl.

We have previously shown that endoreduplication levels in hypocotyls of Arabidopsis thaliana (L.) Heynh. are under negative control of phytochromes. In this study, the hormonal regulation of this process was analysed using a collection of A. thaliana mutants. The results show that two hormones in particular, gibberellin (GA) and ethylene, play distinct roles. Hypocotyl cells of the GA-deficient mutant ga1-11 grown in the dark did not elongate and showed a greatly reduced endoreduplication. Normal endoreduplication could be restored by supplying 10(-9) M of the gibberellin GA(4+7), whereas the restoration of normal cell growth required 100-fold higher concentrations. The GA-insensitive mutant gai showed reduced cell elongation but normal ploidy levels. We conclude that (i) GA(4+7) has a global positive effect on endoreduplication and (ii) that endoreduplication is more sensitive to GA(4+7) than cell elongation. Ethylene had a completely different effect. It induced an extra round of endoreduplication both in light- and dark-grown seedlings and acted mainly on discrete steps rather than having a global effect on endoreduplication. The genes EIN2 and CTR1, components of the ethylene signal transduction pathway were both involved in this process.

A plasma membrane-bound putative endo-1,4-beta-D-glucanase is required for normal wall assembly and cell elongation in Arabidopsis.

Endo-1,4-beta-D-glucanases (EGases) form a large family of hydrolytic enzymes in prokaryotes and eukaryotes. In higher plants, potential substrates in vivo are xyloglucan and non-crystalline cellulose in the cell wall. Gene expression patterns suggest a role for EGases in various developmental processes such as leaf abscission, fruit ripening and cell expansion. Using Arabidopsis thaliana genetics, we demonstrate the requirement of a specialized member of the EGase family for the correct assembly of the walls of elongating cells. KORRIGAN (KOR) is identified by an extreme dwarf mutant with pronounced architectural alterations in the primary cell wall. The KOR gene was isolated and encodes a membrane-anchored member of the EGase family, which is highly conserved between mono- and dicotyledonous plants. KOR is located primarily in the plasma membrane and presumably acts at the plasma membrane-cell wall interface. KOR mRNA was found in all organs examined, and in the developing dark-grown hypocotyl, mRNA levels were correlated with rapid cell elongation. Among plant growth factors involved in the control of hypocotyl elongation (auxin, gibberellins and ethylene) none significantly influenced KOR-mRNA levels. However, reduced KOR-mRNA levels were observed in det2, a mutant deficient for brassinosteroids. Although the in vivo substrate remains to be determined, the mutant phenotype is consistent with a central role for KOR in the assembly of the cellulose-hemicellulose network in the expanding cell wall.

Differential gene expression in Arabidopsis monitored using cDNA arrays.

Large numbers of genes are being discovered on a daily basis for a variety of organisms including Arabidopsis thaliana. To obtain more functional information on these genes, efficient expression monitoring methods need to be developed. In this report we studied the steady-state mRNA levels of over 800 Arabidopsis genes in parallel using high-density arrays of partially sequenced cDNA. The technology is simple and robust and reliably permits the detection of down to twofold variation in mRNA levels. The detection limit lies below 0.01% of the total mRNA population. The comparison of the profiles obtained for light-grown and dark-grown seedlings revealed significant variations in mRNA levels for about 16% of the cDNA investigated. This technology not only provides new functional information on anonymous genes, and thus may guide reverse-genetics approaches, but also constitutes a powerful tool for global gene expression studies, with many potential applications in plant biology.

Phytochrome controls the number of endoreduplication cycles in the Arabidopsis thaliana hypocotyl.

A majority of the cells in the Arabidopsis hypocotyl undergo endoreduplication. The number of endocycles in this organ is partially controlled by light. Up to two cycles occur in light-grown hypocotyls, whereas in the dark about 30% of the cells go through a third cycle. Is the inhibition of the third endocycle in the light an indirect result of the reduced cell size in the light-grown hypocotyl, or is it under independent light control? To address this question, the authors examined the temporal and spacial patterns of endoreduplication in light- or dark-grown plants and report here on the following observations: (i) during germination two endocycles take place prior to any significant cell expansion; (ii) in the dark the third cycle is completed very early during cell growth; and (iii) a mutation that dramatically reduces cell size does not interfere with the third endocycle. The authors then used mutants to study the way light controls the third endocycle and found that the third endocycle is completely suppressed in far red light through the action of phytochrome A and, to a lesser extent, in red light by phytochrome B. Furthermore, no 16C nuclei were observed in dark-grown constitutive photomorphogenic 1 seedlings. And, finally the hypocotyl of the cryptochrome mutant, hy4, grown in blue light was about three times longer than that of the wild-type without a significant difference in ploidy levels. Together, the results support the view that the inhibition of the third endocycle in light-grown hypocotyls is not the consequence of a simple feed-back mechanism coupling the number of cycles to the cell volume, but an integral part of the phytochrome-controlled photomorphogenic program.

Molecular analysis of cellulose biosynthesis in Arabidopsis.

Cellulose, an abundant, crystalline polysaccharide, is central to plant morphogenesis and to many industries. Chemical and ultrastructural analyses together with map-based cloning indicate that the RSW1 locus of Arabidopsis encodes the catalytic subunit of cellulose synthase. The cloned gene complements the rsw1 mutant whose temperature-sensitive allele is changed in one amino acid. The mutant allele causes a specific reduction in cellulose synthesis, accumulation of noncrystalline beta-1,4-glucan, disassembly of cellulose synthase, and widespread morphological abnormalities. Microfibril crystallization may require proper assembly of the RSW1 gene product into synthase complexes whereas glucan biosynthesis per se does not.

Plant cell expansion: scaling the wall.

The regulation of plant cell size and shape is poorly understood at the molecular level. Recently, two loci required for normal cell expansion in Arabidopsis were cloned. They both encode enzymes involved in the construction of the cell wall. These studies are the first promising examples of the use of Arabidopsis molecular genetics for the study of wall synthesis and assembly during plant cell elongation.

Endoreduplication and development: rule without dividing?

Endoreduplication, a strategy to amplify nuclear DNA without cell division, is very common but poorly understood in plants. Recent findings in Drosophila provide a first picture of the molecular mechanism, which appears to be conserved between plants and animals. In Arabidopsis, the study of trichomes, leaf epidermis and hypocotyl cells sheds new light on the developmental regulation of this process, and its relation to cell expansion.

Cellular basis of hypocotyl growth in Arabidopsis thaliana.

The Arabidopsis thaliana hypocotyl is widely used to study the effects of light and plant growth factors on cell elongation. To provide a framework for the molecular-genetic analysis of cell elongation in this organ, here we describe, at the cellular level, its morphology and growth and identify a number of characteristic, developmental differences between light-grown and dark-grown hypocotyls. First, in the light epidermal cells show a characteristic differentiation that is not observed in the dark. Second, elongation growth of this organ does not involve significant cortical or epidermal cell divisions. However, endoreduplication occurs, as revealed by the presence of 4C and 8C nuclei. In addition, 16C nuclei were found specifically in dark-grown seedlings. Third, in the dark epidermal cells elongate along a steep, acropetal spatial and temporal gradient along the hypocotyl. In contrast, in the light all epidermal cells elongated continuously during the entire growth period. These morphological and physiological differences, in combination with previously reported genetic data (T. Desnos, V. Orbovic, C. Bellini, J. Kronenberger, M. Caboche, J. Traas, H. Höfte [1996] Development 122: 683-693), illustrate that light does not simply inhibit hypocotyl growth in a cell-autonomous fashion, but that the observed growth response to light is a part of an integrated developmental change throughout the elongating organ.

Transgenic Arabidopsis tester lines with dominant marker genes.

The map positions of a set of eight T-DNA insertions in the Arabidopsis genome have been determined by using closely linked visible markers. The insertions are dispersed over four of the five chromosomes. Each T-DNA insert contains one or more of the chimeric marker genes neomycin phosphotransferase (neo), hygromycin phosphotransferase (hpt), phosphinothricin acetyltransferase (bar), beta-glucuronidase (gusA) and indole-3-acetamide hydrolase (iaaH). The neo, hpt and bar marker genes are dominant in a selective germination assay or when used as DNA markers in a polymerase chain reaction. These dominant markers will allow recombinants to be discerned in a germinating F2 population, one generation earlier than with a conventional recessive marker. The transgenic marker lines will speed up and simplify the isolation of recombinants in small genetic intervals, a rate-limiting step in positional cloning strategies. The transgenic lines containing the hpt marker will also be of interest for the isolation of deletion mutants at the T-DNA integration sites.