Diverging cell wall strategies for drought adaptation in two maize inbreds with contrasting lodging resistance.

The plant cell wall is a plastic structure of variable composition that constitutes the first line of defence against environmental challenges. Lodging and drought are two stressful conditions that severely impact maize yield. In a previous work, we characterised the cell walls of two maize inbreds, EA2024 (susceptible) and B73 (resistant) to stalk lodging. Here, we show that drought induces distinct phenotypical, physiological, cell wall, and transcriptional changes in the two inbreds, with B73 exhibiting lower tolerance to this stress than EA2024. In control conditions, EA2024 stalks had higher levels of cellulose, uronic acids and p-coumarate than B73. However, upon drought EA2024 displayed increased levels of arabinose-enriched polymers, such as pectin-arabinans and arabinogalactan proteins, and a decreased lignin content. By contrast, B73 displayed a deeper rearrangement of cell walls upon drought, including modifications in lignin composition (increased S subunits and S/G ratio; decreased H subunits) and an increase of uronic acids. Drought induced more substantial changes in gene expression in B73 compared to EA2024, particularly in cell wall-related genes, that were modulated in an inbred-specific manner. Transcription factor enrichment assays unveiled inbred-specific regulatory networks coordinating cell wall genes expression. Altogether, these findings reveal that B73 and EA2024 inbreds, with opposite stalk-lodging phenotypes, undertake different cell wall modification strategies in response to drought. We propose that the specific cell wall composition conferring lodging resistance to B73, compromises its cell wall plasticity, and renders this inbred more susceptible to drought.

A novel pectin methylesterase inhibitor, PMEI3, in common bean suggests a key role of pectin methylesterification in Pseudomonas resistance.

The mechanisms underlying susceptibility to and defense against Pseudomonas syringae (Pph) of the common bean (Phaseolus vulgaris) have not yet been clarified. To investigate these, 15-day-old plants of the variety Riñón were infected with Pph and the transcriptomic changes at 2 h and 9 h post-infection were analysed. RNA-seq analysis showed an up-regulation of genes involved in defense/signaling at 2 h, most of them being down-regulated at 9 h, suggesting that Pph inhibits the transcriptomic reprogramming of the plant. This trend was also observed in the modulation of 101 cell wall-related genes. Cell wall composition changes at early stages of Pph infection were associated with homogalacturonan methylation and the formation of egg boxes. Among the cell wall genes modulated, a pectin methylesterase inhibitor 3 (PvPMEI3) gene, closely related to AtPMEI3, was detected. PvPMEI3 protein was located in the apoplast and its pectin methylesterase inhibitory activity was demonstrated. PvPMEI3 seems to be a good candidate to play a key role in Pph infection, which was supported by analysis of an Arabidopsis pmei3 mutant, which showed susceptibility to Pph, in contrast to resistant Arabidopsis Col-0 plants. These results indicate a key role of the degree of pectin methylesterification in host resistance to Pph during the first steps of the attack.

Plant Cell Wall Plasticity under Stress Situations.

This Special Issue, entitled "Plant Cell Wall Plasticity under Stress Situations", is a compilation of five articles, whose authors deepen our understanding of the roles of different cell wall components under biotic and abiotic stress [...].

Immune Priming Triggers Cell Wall Remodeling and Increased Resistance to Halo Blight Disease in Common Bean.

The cell wall (CW) is a dynamic structure extensively remodeled during plant growth and under stress conditions, however little is known about its roles during the immune system priming, especially in crops. In order to shed light on such a process, we used the Phaseolus vulgaris-Pseudomonas syringae (Pph) pathosystem and the immune priming capacity of 2,6-dichloroisonicotinic acid (INA). In the first instance we confirmed that INA-pretreated plants were more resistant to Pph, which was in line with the enhanced production of H2O2 of the primed plants after elicitation with the peptide flg22. Thereafter, CWs from plants subjected to the different treatments (non- or Pph-inoculated on non- or INA-pretreated plants) were isolated to study their composition and properties. As a result, the Pph inoculation modified the bean CW to some extent, mostly the pectic component, but the CW was as vulnerable to enzymatic hydrolysis as in the case of non-inoculated plants. By contrast, the INA priming triggered a pronounced CW remodeling, both on the cellulosic and non-cellulosic polysaccharides, and CW proteins, which resulted in a CW that was more resistant to enzymatic hydrolysis. In conclusion, the increased bean resistance against Pph produced by INA priming can be explained, at least partially, by a drastic CW remodeling.

Perception and First Defense Responses Against Pseudomonas syringae pv. phaseolicola in Phaseolus vulgaris: Identification of Wall-Associated Kinase Receptors.

Common bean (Phaseolus vulgaris) is attacked by several pathogens such as the biotrophic gamma-proteobacterium Pseudomonas syringae pv. phaseolicola. To study the P. syringae pv. phaseolicola-bean interaction during the first stages of infection, leaf discs of a susceptible bean cultivar Riñón were infected with pathogenic P. syringae pv. phaseolicola. Using this experimental system, we tested six new putative wall-associated kinase (WAK) receptors, previously identified in silico. These six P. vulgaris WAKs (PvWAKs) showed high protein sequence homology to the well-described Arabidopsis thaliana WAK1 (AtWAK1) receptor and, by phylogenetic analysis, clustered together with AtWAKs. The expression of PvWAK1 increased at very early stages after the P. syringae pv. phaseolicola infection. Time course experiments were performed to evaluate the accumulation of apoplastic H2O2, Ca2+ influx, total H2O2, antioxidant enzymatic activities, lipid peroxidation, and the concentrations of abscisic acid and salicylic acid (SA), as well as the expression of six defense-related genes: MEKK-1, MAPKK, WRKY33, RIN4, PR1, and NPR1. The results showed that overexpression of PR1 occurred 2 h after P. syringae pv. phaseolicola infection without a concomitant increase in SA levels. Although apoplastic H2O2 increased after infection, the oxidative burst was neither intense nor rapid, and an efficient antioxidant response did not occur, suggesting that the observed cellular damage was caused by the initial increase in total H2O2 early after infection. In conclusion, Riñón can perceive the presence of P. syringae pv. phaseolicola, but this recognition results in only a modest and slow activation of host defenses, leading to high susceptibility to P. syringae pv. phaseolicola.

Elucidating compositional factors of maize cell walls contributing to stalk strength and lodging resistance.

Lodging is one of the causes of maize (Zea mays L.) production losses worldwide and, at least, the resistance to stalk lodging has been positively correlated with stalk strength. In order to elucidate the putative relationship between cell wall, stalk strength and lodging resistance, twelve maize inbreds varying in rind penetration strength and lodging resistance were characterized for cell wall composition and structure. Stepwise multiple regression indicates that H lignin subunits confer a greater rind penetration strength. Besides, the predictive model for lodging showed that a high ferulic acid content increases the resistance to lodging, whereas those of diferulates decrease it. These outcomes highlight that the strength and lodging susceptibility of maize stems may be conditioned by structural features of cell wall rather than by the net amount of cellulose, hemicelluloses and lignin. The results presented here provide biotechnological targets in breeding programs aimed at improving lodging in maize.

Production of Encecalin in Cell Cultures and Hairy Roots of Helianthella quinquenervis (Hook.) A. Gray.

Plant cell and organ cultures of Helianthella quinquenervis, a medicinal plant whose roots are used by the Tarahumara Indians of Chihuahua, Mexico, to relieve several ailments, were established to identify and quantify some chromenes with biological activity, such as encecalin, and to evaluate their potential for biotechnological production. Gas chromatography-mass spectrometry (GC-MS) analysis corroborated the presence of quantifiable amounts of encecalin in H. quinquenervis cell cultures (callus and cell suspensions). In addition, hairy roots were obtained through three transformation protocols (prick, 45-s sonication and co-culture), using wild type Agrobacterium rhizogenes A4. After three months, cocultivation achieved the highest percentage of transformation (66%), and a comparable production (FW) of encecalin (110 µg/g) than the sonication assay (120 µg/g), both giving far higher yields than the prick assay (19 µg/g). Stable integration of rolC and aux1 genes in the transformed roots was confirmed by polymerase chain reaction (PCR). Hairy roots from cocultivation (six months-old) accumulated as much as 1086 µg/g (FW) of encecalin, over three times higher than the cell suspension cultures. The production of encecalin varied with growth kinetics, being higher at the stationary phase. This is the first report of encecalin production in hairy roots of H. quinquenervis, demonstrating the potential for a future biotechnological production of chromenes.

The role of cell wall phenolics during the early remodelling of cellulose-deficient maize cells.

The habituation of cultured cells to cellulose biosynthesis inhibitors such as dichlobenil (dichlorobenzonitrile, DCB) has proven a valuable tool to elucidate the mechanisms involved in plant cell wall structural plasticity. Our group has demonstrated that maize cells cope with DCB through a modified cell wall in which cellulose is replaced by a more extensive network of highly cross-linked feruloylated arabinoxylans. In order to gain further insight into the contribution of phenolics to the early remodelling of cellulose-deficient cell walls, a comparative HPLC-PAD analysis was carried out of hydroxycinnamates esterified into nascent and cell wall polysaccharides obtained from non-habituated (NH) and habituated to low DCB concentrations (1.5 µM; H) maize suspension-cultured cells. Incipient DCB-habituated cell walls showed significantly higher levels of esterified ferulic acid and p-coumaric acid throughout the culture cycle. In terms of cell wall fortification, ferulic acid is associated to arabinoxylan crosslinking whereas the increase of p-coumaric suggests an early lignification response. As expected, the level of hydroxycinnamates esterified into nascent polysaccharides was also higher in DCB-habituated cells indicating an overexpression of phenylpropanoid pathway. Due to their key role in cell wall strengthening, special attention was paid into the dimerization pattern of ferulic acid. A quantitative comparison of diferulate dehydrodimers (DFAs) between cell lines and cell compartments revealed that an extra dimerization took place in H cells when both nascent and mature cell wall polysaccharides were analysed. In addition, qualitative differences in the ferulic acid coupling pattern were detected in H cells, allowing us to suggest that 8-O-4'-DFA and 8-5'-DFA featured the ferulic acid dimerization when it occurred in the protoplasmic and cell wall fractions respectively. Both qualitative and quantitative differences in the phenolic profile between NH and H cells point to a regioselectivity in the ferulate dehydrodimerization.

Necrotic and Cytolytic Activity on Grapevine Leaves Produced by Nep1-Like Proteins of Diplodia seriata.

Many phytopathogenic fungi produce necrosis and ethylene inducing peptide 1 (Nep1-like proteins or NLP) that trigger leaf necrosis and the activation of defense mechanisms. These proteins have been widely studied in plant pathogens as Moniliophthora perniciosa or Botrytis cinerea between others, but little is known about their biological roles in grapevine trunk pathogens. Advances in the sequencing of genomes of several fungi involved in grapevine trunk diseases have revealed that these proteins are present in several copies in their genomes. The aim of this project was to analyze the presence of genes encoding NLP proteins in the Diplodia seriata genome and to characterize their putative role as virulence factors associated to grapevine trunk diseases. In this study, we characterized four NLPs from Diplodia seriata. All proteins showed highly similar amino acid sequences and contained the characteristic peptide motifs of NLPs. DserNEPs slightly reduced the viability of Vitis vinifera L. cell cultures. The cytolytic activity from DserNEP1 was stronger than that from DserNEP2, even at low concentrations. Purified DserNEPs also produced necrosis in leaves when they were inoculated into micropropagules of V. vinifera L. This is the first record of Nep1-like proteins from a fungus associated with grapevine trunk diseases and also from a member of the Botryosphaeriaceae family.

Effect of ancymidol on cell wall metabolism in growing maize cells.

MAIN CONCLUSION: Ancymidol inhibits the incorporation of cellulose into cell walls of maize cell cultures in a gibberellin-independent manner, impairing cell growth; the reduction in the cellulose content is compensated with xylans. Ancymidol is a plant growth retardant which impairs gibberellin biosynthesis. It has been reported to inhibit cellulose synthesis by tobacco cells, based on its cell-malforming effects. To ascertain the putative role of ancymidol as a cellulose biosynthesis inhibitor, we conducted a biochemical study of its effect on cell growth and cell wall metabolism in maize cultured cells. Ancymidol concentrations ≤ 500 µM progressively reduced cell growth and induced globular cell shape without affecting cell viability. However, cell growth and viability were strongly reduced by ancymidol concentrations ≥ 1.5 mM. The I50 value for the effect of ancymidol on FW gain was 658 µM. A reversal of the inhibitory effects on cell growth was observed when 500 µM ancymidol-treated cultures were supplemented with 100 µM GA3. Ancymidol impaired the accumulation of cellulose in cell walls, as monitored by FTIR spectroscopy. Cells treated with 500 µM ancymidol showed a ~ 60% reduction in cellulose content, with no further change as the ancymidol concentration increased. Cellulose content was partially restored by 100 µM GA3. Radiolabeling experiments confirmed that ancymidol reduced the incorporation of [14C]glucose into α-cellulose and this reduction was not reverted by the simultaneous application of GA3. RT-PCR analysis indicated that the cellulose biosynthesis inhibition caused by ancymidol is not related to a downregulation of ZmCesA gene expression. Additionally, ancymidol treatment increased the incorporation of [3H]arabinose into a hemicellulose-enriched fraction, and up-regulated ZmIRX9 and ZmIRX10L gene expression, indicating an enhancement in the biosynthesis of arabinoxylans as a compensatory response to cellulose reduction.

Characterization of structural cell wall polysaccharides in cattail (Typha latifolia): Evaluation as potential biofuel feedstock.

Second generation bioethanol produced from lignocellulosic biomass is attracting attention as an alternative energy source. In this study, a detailed knowledge of the composition and structure of common cattail (Typha latifolia L.) cell wall polysaccharides, obtained from stem or leaves, has been conducted using a wide set of techniques to evaluate this species as a potential bioethanol feedstock. Our results showed that common cattail cellulose content was high for plants in the order Poales and was accompanied by a small amount of cross-linked polysaccharides. A high degree of arabinose-substitution in xylans, a high syringyl/guaiacyl ratio in lignin and a low level of cell wall crystallinity could yield a good performance for lignocellulose saccharification. These results identify common cattail as a promising plant for use as potential bioethanol feedstock. To the best of our knowledge, this is the first in-depth analysis to be conducted of lignocellulosic material from common cattail.

Phenolic metabolism and molecular mass distribution of polysaccharides in cellulose-deficient maize cells.

As a consequence of the habituation to low levels of dichlobenil (DCB), cultured maize cells presented an altered hemicellulose cell fate with a lower proportion of strongly wall-bound hemicelluloses and an increase in soluble extracellular polymers released into the culture medium. The aim of this study was to investigate the relative molecular mass distributions of polysaccharides as well as phenolic metabolism in cells habituated to low levels of DCB (1.5 µM). Generally, cell wall bound hemicelluloses and sloughed polymers from habituated cells were more homogeneously sized and had a lower weight-average relative molecular mass. In addition, polysaccharides underwent massive cross-linking after being secreted into the cell wall, but this cross-linking was less pronounced in habituated cells than in non-habituated ones. However, when relativized, ferulic acid and p-coumaric acid contents were higher in this habituated cell line. Feasibly, cells habituated to low levels of DCB synthesized molecules with a lower weight-average relative molecular mass, although cross-linked, as a part of their strategy to compensate for the lack of cellulose.

Quinclorac-habituation of bean (Phaseolus vulgaris) cultured cells is related to an increase in their antioxidant capacity.

The habituation of bean cells to quinclorac did not rely on cell wall modifications, contrary to what it was previously observed for the well-known cellulose biosynthesis inhibitors dichlobenil or isoxaben. The aim of the present study was to investigate whether or not the bean cells habituation to quinclorac is related to an enhancement of antioxidant activities involved in the scavenging capacity of reactive oxygen species. Treating non-habituated bean calluses with 10 µM quinclorac reduced the relative growth rate and induced a two-fold increase in lipid peroxidation. However, the exposition of quinclorac-habituated cells to a concentration of quinclorac up to 30 µM neither affected their growth rate nor increased their lipid peroxidation levels. Quinclorac-habituated calluses had significantly higher constitutive levels of three antioxidant activities (class-III peroxidase, glutathione reductase, and superoxide dismutase) than those observed in non-habituated calluses, and the treatment of habituated calluses with 30 µM quinclorac significantly increased the level of class III-peroxidase and superoxide dismutase. The results reported here indicate that the process of habituation to quinclorac in bean callus-cultured cells is related, at least partially, to the development of a stable antioxidant capacity that enables them to cope with the oxidative stress caused by quinclorac. Class-III peroxidase and superoxide dismutase activities could play a major role in the quinclorac-habituation. Changes in the antioxidant status of bean cells were stable, since the increase in the antioxidant activities were maintained in quinclorac-dehabituated cells.

Early habituation of maize (Zea mays) suspension-cultured cells to 2,6-dichlorobenzonitrile is associated with the enhancement of antioxidant status.

The cellulose biosynthesis inhibitor 2,6-dichlorobenzonitrile (DCB) has been widely used to gain insights into cell wall composition and architecture. Studies of changes during early habituation to DCB can provide information on mechanisms that allow tolerance/habituation to DCB. In this context, maize-cultured cells with a reduced amount of cellulose (∼20%) were obtained by stepwise habituation to low DCB concentrations. The results reported here attempt to elucidate the putative role of an antioxidant strategy during incipient habituation. The short-term exposure to DCB of non-habituated maize-cultured cells induced a substantial increase in oxidative damage. Concomitantly, short-term treated cells presented an increase in class III peroxidase and glutathione S-transferase activities and total glutathione content. Maize cells habituated to 0.3-1 µM DCB (incipient habituation) were characterized by a reduction in the relative cell growth rate, an enhancement of ascorbate peroxidase and class III peroxidase activities, and a net increment in total glutathione content. Moreover, these cell lines showed increased levels of glutathione S-transferase activity. Changes in antioxidant/conjugation status enabled 0.3 and 0.5 µM DCB-habituated cells to control lipid peroxidation levels, but this was not the case of maize cells habituated to 1 µM DCB, which despite showing an increased antioxidant capacity were not capable of reducing the oxidative damage to control levels. The results reported here confirm that exposure and incipient habituation of maize cells to DCB are associated with an enhancement in antioxidant/conjugation activities which could play a role in incipient DCB habituation of maize-cultured cells.

Ectopic lignification in primary cellulose-deficient cell walls of maize cell suspension cultures.

Maize (Zea mays L.) suspension-cultured cells with up to 70% less cellulose were obtained by stepwise habituation to dichlobenil (DCB), a cellulose biosynthesis inhibitor. Cellulose deficiency was accompanied by marked changes in cell wall matrix polysaccharides and phenolics as revealed by Fourier transform infrared (FTIR) spectroscopy. Cell wall compositional analysis indicated that the cellulose-deficient cell walls showed an enhancement of highly branched and cross-linked arabinoxylans, as well as an increased content in ferulic acid, diferulates and p-coumaric acid, and the presence of a polymer that stained positive for phloroglucinol. In accordance with this, cellulose-deficient cell walls showed a fivefold increase in Klason-type lignin. Thioacidolysis/GC-MS analysis of cellulose-deficient cell walls indicated the presence of a lignin-like polymer with a Syringyl/Guaiacyl ratio of 1.45, which differed from the sensu stricto stress-related lignin that arose in response to short-term DCB-treatments. Gene expression analysis of these cells indicated an overexpression of genes specific for the biosynthesis of monolignol units of lignin. A study of stress signaling pathways revealed an overexpression of some of the jasmonate signaling pathway genes, which might trigger ectopic lignification in response to cell wall integrity disruptions. In summary, the structural plasticity of primary cell walls is proven, since a lignification process is possible in response to cellulose impoverishment.

The biosynthesis and wall-binding of hemicelluloses in cellulose-deficient maize cells: an example of metabolic plasticity.

Cell-suspension cultures (Zea mays L., Black Mexican sweet corn) habituated to 2,6-dichlorobenzonitrile (DCB) survive with reduced cellulose owing to hemicellulose network modification. We aimed to define the hemicellulose metabolism modifications in DCB-habituated maize cells showing a mild reduction in cellulose at different stages in the culture cycle. Using pulse-chase radiolabeling, we fed habituated and non-habituated cultures with [(3)H]arabinose, and traced the distribution of (3)H-pentose residues between xylans, xyloglucans and other polymers in several cellular compartments for 5 h. Habituated cells were slower taking up exogenous [(3)H]arabinose. Tritium was incorporated into polysaccharide-bound arabinose and xylose residues, but habituated cells diverted a higher proportion of their new [(3)H]xylose residues into (hetero) xylans at the expense of xyloglucan synthesis. During logarithmic growth, habituated cells showed slower vesicular trafficking of polymers, especially xylans. Moreover, habituated cells showed a decrease in the strong wall-binding of all pentose-containing polysaccharides studied; correspondingly, especially in log-phase cultures, habituation increased the proportion of (3)H-hemicelluloses ([(3)H]xylans and [(3)H]xyloglucan) sloughed into the medium. These findings could be related to the cell walls' cellulose-deficiency, and consequent reduction in binding sites for hemicelluloses; the data could also reflect the habituated cells' reduced capacity to integrate arabinoxylans by extra-protoplasmic phenolic cross-linking, as well as xyloglucans, during wall assembly.

Early cell-wall modifications of maize cell cultures during habituation to dichlobenil.

Studies involving the habituation of plant cell cultures to cellulose biosynthesis inhibitors have achieved significant progress as regards understanding the structural plasticity of cell walls. However, since habituation studies have typically used high concentrations of inhibitors and long-term habituation periods, information on initial changes associated with habituation has usually been lost. This study focuses on monitoring and characterizing the short-term habituation process of maize (Zea mays) cell suspensions to dichlobenil (DCB). Cellulose quantification and FTIR spectroscopy of cell walls from 20 cell lines obtained during an incipient DCB-habituation process showed a reduction in cellulose levels which tended to revert depending on the inhibitor concentration and the length of time that cells were in contact with it. Variations in the cellulose content were concomitant with changes in the expression of several ZmCesA genes, mainly involving overexpression of ZmCesA7 and ZmCesA8. In order to explore these changes in more depth, a cell line habituated to 1.5µM DCB was identified as representative of incipient DCB habituation and selected for further analysis. The cells of this habituated cell line grew more slowly and formed larger clusters. Their cell walls were modified, showing a 33% reduction in cellulose content, that was mainly counteracted by an increase in arabinoxylans, which presented increased extractability. This result was confirmed by immunodot assays graphically plotted by heatmaps, since habituated cell walls had a more extensive presence of epitopes for arabinoxylans and xylans, but also for homogalacturonan with a low degree of esterification and for galactan side chains of rhamnogalacturonan I. Furthermore, a partial shift of xyloglucan epitopes toward more easily extractable fractions was found. However, other epitopes, such as these specific for arabinan side chains of rhamnogalacturonan I or homogalacturonan with a high degree of esterification, seemed to be not affected. In conclusion, the early modifications occurring in maize cell walls as a consequence of DCB-habituation involved quantitative and qualitative changes of arabinoxylans, but also other polysaccharides. Thereby some of the changes that took place in the cell walls in order to compensate for the lack of cellulose differed according to the DCB-habituation level, and illustrate the ability of plant cells to adopt appropriate coping strategies depending on the herbicide concentration and length of exposure time.

Purification and characterization of a soluble ß-1,4-glucan from bean (Phaseolus vulgaris L.)-cultured cells dehabituated to dichlobenil.

Bean cells habituated to grow in the presence of dichlobenil exhibited reduced cellulose and hemicellulose content and an increase in pectic polysaccharides. Furthermore, following the extraction of pectins and hemicelluloses, a large amount of neutral sugars was released. These sugars were found to be part of a soluble ß-1,4-glucan in a preliminary characterization, as reported by Encina et al. (Physiol Plant 114:182-191, 2002). When habituated cells were subcultured in the absence of the herbicide (dehabituated cells), the release of neutral sugars after the extraction of pectins and hemicelluloses was maintained. In this study, we have isolated a soluble ß-1,4-glucan from dehabituated cells by sonication of the wall residue (cellulose fraction) remaining after fractionation. Gel filtration chromatography revealed that its average molecular size was 14 kDa. Digestion of the sample with endocellulase revealed the presence of cellobiose, cellotriose, and cellotetraose. Methylation analysis showed that 4-linked glucose was the most abundant sugar residue, but 4,6-linked glucose, terminal arabinose and 4-linked galactose for xyloglucan, and arabinogalactan were also identified. NMR analysis showed that this 1,4-glucan may be composed of various kinds of substitutions along the glucan backbone together with acetyl groups linked to the OH group of sugar residues. Thus, despite its relatively high molecular mass, the ß-glucan remains soluble because of its unique configuration. This is the first time that a glucan with such characteristics has been isolated and described. The discovery of new molecules, as this ß-glucan with unique features, may help understand the composition and arrangement of the polymers within plant cell walls, contributing to a better understanding of this complex structure.

Mineral stress affects the cell wall composition of grapevine (Vitis vinifera L.) callus.

Grapevine (Vitis vinifera L.) is one of the most economically important fruit crops in the world. Deficit in nitrogen, phosphorus and sulfur nutrition impairs essential metabolic pathways. The influence of mineral stress in the composition of the plant cell wall (CW) has received residual attention. Using grapevine callus as a model system, 6 weeks deficiency of those elements caused a significant decrease in growth. Callus CWs were analyzed by Fourier transform infrared spectroscopy (FT-IR), by quantification of CW components and by immunolocalization of CW epitopes with monoclonal antibodies. PCA analysis of FT-IR data suggested changes in the main components of the CW in response to individual mineral stress. Decreased cellulose, modifications in pectin methyl esterification and increase of structural proteins were among the events disclosed by FT-IR analysis. Chemical analyses supported some of the assumptions and further disclosed an increase in lignin content under nitrogen deficiency, suggesting a compensation of cellulose by lignin. Moreover, polysaccharides of callus under mineral deficiency showed to be more tightly bonded to the CW, probably due to a more extensive cross-linking of the cellulose-hemicellulose network. Our work showed that mineral stress impacts the CW at different extents according to the withdrawn mineral element, and that the modifications in a given CW component are compensated by the synthesis and/or alternative linking between polymers. The overall results here described for the first time pinpoint the CW of Vitis callus different strategies to overcome mineral stress, depending on how essential they are to cell growth and plant development.

Cellulose biosynthesis inhibitors: comparative effect on bean cell cultures.

The variety of bioassays developed to evaluate different inhibition responses for cellulose biosynthesis inhibitors makes it difficult to compare the results obtained. This work aims (i) to test a single inhibitory assay for comparing active concentrations of a set of putative cellulose biosynthesis inhibitors and (ii) to characterize their effect on cell wall polysaccharides biosynthesis following a short-term exposure. For the first aim, dose-response curves for inhibition of dry-weight increase following a 30 days exposure of bean callus-cultured cells to these inhibitors were obtained. The compound concentration capable of inhibiting dry weight increase by 50% compared to control (I(50)) ranged from subnanomolar (CGA 325'615) to nanomolar (AE F150944, flupoxam, triazofenamide and oxaziclomefone) and micromolar (dichlobenil, quinclorac and compound 1) concentrations. In order to gain a better understanding of the effect of the putative inhibitors on cell wall polysaccharides biosynthesis, the [(14)C]glucose incorporation into cell wall fractions was determined after a 20 h exposure of cell suspensions to each inhibitor at their I(50) value. All the inhibitors tested decreased glucose incorporation into cellulose with the exception of quinclorac, which increased it. In some herbicide treatments, reduction in the incorporation into cellulose was accompanied by an increase in the incorporation into other fractions. In order to appreciate the effect of the inhibitors on cell wall partitioning, a cluster and Principal Component Analysis (PCA) based on the relative contribution of [(14)C]glucose incorporation into the different cell wall fractions were performed, and three groups of compounds were identified. The first group included quinclorac, which increased glucose incorporation into cellulose; the second group consisted of compound 1, CGA 325'615, oxaziclomefone and AE F150944, which decreased the relative glucose incorporation into cellulose but increased it into tightly-bound cellulose fractions; and the third group, comprising flupoxam, triazofenamide and dichlobenil, decreased the relative glucose incorporation into cellulose and increased it into a pectin rich fraction.