Small-Scale Variability in Bacterial Community Structure in Different Soil Types.

Microbial spatial distribution has mostly been studied at field to global scales (i.e., ecosystem scales). However, the spatial organization at small scales (i.e., centimeter to millimeter scales), which can help improve our understanding of the impacts of spatial communities structure on microbial functioning, has received comparatively little attention. Previous work has shown that small-scale spatial structure exists in soil microbial communities, but these studies have not compared soils from geographically distant locations, nor have they utilized community ecology approaches, such as the core and satellite hypothesis and/or abundance-occupancy relationships, often used in macro-ecology, to improve the description of the spatial organization of communities. In the present work, we focused on bacterial diversity (i.e., 16S rRNA gene sequencing) occurring in micro-samples from a variety of locations with different pedo-climatic histories (i.e., from semi-arid, alpine, and temperate climates) and physicochemical properties. The forms of ecological spatial relationships in bacterial communities (i.e., occupancy-frequency and abundance-occupancy) and taxa distributions (i.e., habitat generalists and specialists) were investigated. The results showed that bacterial composition differed in the four soils at the small scale. Moreover, one soil presented a satellite mode distribution, whereas the three others presented bimodal distributions. Interestingly, numerous core taxa were present in the four soils among which 8 OTUs were common to the four sites. These results confirm that analyses of the small-scale spatial distribution are necessary to understand consequent functional processes taking place in soils, affecting thus ecosystem functioning.

Arabidopsis cell wall composition determines disease resistance specificity and fitness.

Plant cell walls are complex structures subject to dynamic remodeling in response to developmental and environmental cues and play essential functions in disease resistance responses. We tested the specific contribution of plant cell walls to immunity by determining the susceptibility of a set of Arabidopsis cell wall mutants (cwm) to pathogens with different parasitic styles: a vascular bacterium, a necrotrophic fungus, and a biotrophic oomycete. Remarkably, most cwm mutants tested (29/34; 85.3%) showed alterations in their resistance responses to at least one of these pathogens in comparison to wild-type plants, illustrating the relevance of wall composition in determining disease-resistance phenotypes. We found that the enhanced resistance of cwm plants to the necrotrophic and vascular pathogens negatively impacted cwm fitness traits, such as biomass and seed yield. Enhanced resistance of cwm plants is not only mediated by canonical immune pathways, like those modulated by phytohormones or microbe-associated molecular patterns, which are not deregulated in the cwm tested. Pectin-enriched wall fractions isolated from cwm plants triggered immune responses in wild-type plants, suggesting that wall-mediated defensive pathways might contribute to cwm resistance. Cell walls of cwm plants show a high diversity of composition alterations as revealed by glycome profiling that detect specific wall carbohydrate moieties. Mathematical analysis of glycome profiling data identified correlations between the amounts of specific wall carbohydrate moieties and disease resistance phenotypes of cwm plants. These data support the relevant and specific function of plant wall composition in plant immune response modulation and in balancing disease resistance/development trade-offs.

Arabidopsis Response Regulator 6 (ARR6) Modulates Plant Cell-Wall Composition and Disease Resistance.

The cytokinin signaling pathway, which is mediated by Arabidopsis response regulator (ARR) proteins, has been involved in the modulation of some disease-resistance responses. Here, we describe novel functions of ARR6 in the control of plant disease-resistance and cell-wall composition. Plants impaired in ARR6 function (arr6) were more resistant and susceptible, respectively, to the necrotrophic fungus Plectosphaerella cucumerina and to the vascular bacterium Ralstonia solanacearum, whereas Arabidopsis plants that overexpress ARR6 showed the opposite phenotypes, which further support a role of ARR6 in the modulation of disease-resistance responses against these pathogens. Transcriptomics and metabolomics analyses revealed that, in arr6 plants, canonical disease-resistance pathways, like those activated by defensive phytohormones, were not altered, whereas immune responses triggered by microbe-associated molecular patterns were slightly enhanced. Cell-wall composition of arr6 plants was found to be severely altered compared with that of wild-type plants. Remarkably, pectin-enriched cell-wall fractions extracted from arr6 walls triggered more intense immune responses than those activated by similar wall fractions from wild-type plants, suggesting that arr6 pectin fraction is enriched in wall-related damage-associated molecular patterns, which trigger immune responses. This work supports a novel function of ARR6 in the control of cell-wall composition and disease resistance and reinforces the role of the plant cell wall in the modulation of specific immune responses.

In vitro characterization of root extracellular trap and exudates of three Sahelian woody plant species.

MAIN CONCLUSION: Arabinogalactan protein content in both root extracellular trap and root exudates varies in three Sahelian woody plant species that are differentially tolerant to drought. At the root tip, mature root cap cells, mainly border cells (BCs)/border-like cells (BLCs) and their associated mucilage, form a web-like structure known as the "Root Extracellular Trap" (RET). Although the RET along with the entire suite of root exudates are known to influence rhizosphere function, their features in woody species is poorly documented. Here, RET and root exudates were analyzed from three Sahelian woody species with contrasted sensitivity to drought stress (Balanites aegyptiaca, Acacia raddiana and Tamarindus indica) and that have been selected for reforestation along the African Great Green Wall in northern Senegal. Optical and transmission electron microscopy show that Balanites aegyptiaca, the most drought-tolerant species, produces only BC, whereas Acacia raddiana and Tamarindus indica release both BCs and BLCs. Biochemical analyses reveal that RET and root exudates of Balanites aegyptiaca and Acacia raddiana contain significantly more abundant arabinogalactan proteins (AGPs) compared to Tamarindus indica, the most drought-sensitive species. Root exudates of the three woody species also differentially impact the plant soil beneficial bacteria Azospirillum brasilense growth. These results highlight the importance of root secretions for woody species survival under dry conditions.

Remote sensing monitoring of land restoration interventions in semi-arid environments with a before-after control-impact statistical design.

Restoration interventions to combat land degradation are carried out in arid and semi-arid areas to improve vegetation cover and land productivity. Evaluating the success of an intervention over time is challenging due to various constraints (e.g. difficult-to-access areas, lack of long-term records) and the lack of standardised and affordable methodologies. We propose a semi-automatic methodology that uses remote sensing data to provide a rapid, standardised and objective assessment of the biophysical impact, in terms of vegetation cover, of restoration interventions. The Normalised Difference Vegetation Index (NDVI) is used as a proxy for vegetation cover. Recognising that changes in vegetation cover are naturally due to environmental factors such as seasonality and inter-annual climate variability, conclusions about the success of the intervention cannot be drawn by focussing on the intervention area only. We therefore use a comparative method that analyses the temporal variations (before and after the intervention) of the NDVI of the intervention area with respect to multiple control sites that are automatically and randomly selected from a set of candidates that are similar to the intervention area. Similarity is defined in terms of class composition as derived from an ISODATA classification of the imagery before the intervention. The method provides an estimate of the magnitude and significance of the difference in greenness change between the intervention area and control areas. As a case study, the methodology is applied to 15 restoration interventions carried out in Senegal. The impact of the interventions is analysed using 250-m MODIS and 30-m Landsat data. Results show that a significant improvement in vegetation cover was detectable only in one third of the analysed interventions, which is consistent with independent qualitative assessments based on field observations and visual analysis of high resolution imagery. Rural development agencies may potentially use the proposed method for a first screening of restoration interventions.

High-throughput microanalysis of large lignocellulosic sample sets by pyrolysis-gas chromatography/mass spectrometry.

High-throughput analytical techniques to assess the chemistry of lignocellulosic plant material are crucial to plant cell-wall research. We have established an analytical platform for this purpose and demonstrated its usefulness with two applications. The system is based on analytical pyrolysis, coupled to gas chromatography/mass spectrometry - a technique particularly suited for analysis of lignocellulose. Automated multivariate-based data-processing methods are used to obtain results within a few hours after analysis, with an experimental batch of 500 analyzed samples. The usefulness of multivariate sample discrimination methods and hierarchical clustering of samples is demonstrated. We have analyzed an Arabidopsis mutant collection consisting of 300 samples representing 31 genotypes. The mutant collection is presented through cluster analysis, based on chemotypic difference, with respect to wild type. Further, we have analyzed 500 thin sections from five biological replicate trees to create a spatial highly resolved profile of the proportions of syringyl-, guaiacyl- and p-hydroxyphenyl lignin across phloem, developing and mature wood in aspen. The combination of biologically easy to interpret information, the low demand of sample amount and the flexibility in sample types amenable to analysis makes this technique a valuable extension to the range of established high-throughput biomaterial analytical platforms.

PIRIN2 stabilizes cysteine protease XCP2 and increases susceptibility to the vascular pathogen Ralstonia solanacearum in Arabidopsis.

PIRIN (PRN) is a member of the functionally diverse cupin protein superfamily. There are four members of the Arabidopsis thaliana PRN family, but the roles of these proteins are largely unknown. Here we describe a function of the Arabidopsis PIRIN2 (PRN2) that is related to susceptibility to the bacterial plant pathogen Ralstonia solanacearum. Two prn2 mutant alleles displayed decreased disease development and bacterial growth in response to R.  solanacearum infection. We elucidated the underlying molecular mechanism by analyzing PRN2 interactions with the papain-like cysteine proteases (PLCPs) XCP2, RD21A, and RD21B, all of which bound to PRN2 in yeast two-hybrid assays and in Arabidopsis protoplast co-immunoprecipitation assays. We show that XCP2 is stabilized by PRN2 through inhibition of its autolysis on the basis of PLCP activity profiling assays and enzymatic assays with recombinant protein. The stabilization of XCP2 by PRN2 was also confirmed in planta. Like prn2 mutants, an xcp2 single knockout mutant and xcp2 prn2 double knockout mutant displayed decreased susceptibility to R. solanacearum, suggesting that stabilization of XCP2 by PRN2 underlies susceptibility to R. solanacearum in Arabidopsis.

Arabidopsis WAT1 is a vacuolar auxin transport facilitator required for auxin homoeostasis.

The plant hormone auxin (indole-3-acetic acid, IAA) has a crucial role in plant development. Its spatiotemporal distribution is controlled by a combination of biosynthetic, metabolic and transport mechanisms. Four families of auxin transporters have been identified that mediate transport across the plasma or endoplasmic reticulum membrane. Here we report the discovery and the functional characterization of the first vacuolar auxin transporter. We demonstrate that WALLS ARE THIN1 (WAT1), a plant-specific protein that dictates secondary cell wall thickness of wood fibres, facilitates auxin export from isolated Arabidopsis vacuoles in yeast and in Xenopus oocytes. We unambiguously identify IAA and related metabolites in isolated Arabidopsis vacuoles, suggesting a key role for the vacuole in intracellular auxin homoeostasis. Moreover, local auxin application onto wat1 mutant stems restores fibre cell wall thickness. Our study provides new insight into the complexity of auxin transport in plants and a means to dissect auxin function during fibre differentiation.

[The international man-media Tessékéré (OHMi) Observatory: a research tool to study the complexity of arid ecosystems in Sahel]. / L'Observatoire hommes-milieux international Tessékéré (OHMi) : un outil de recherche pour étudier la complexité des écosystèmes arides du Sahel.

In the Sahelian zone, the drought phenomenon, combined with anthropic factors (monoculture, bush fires, defect or deficit of manure, overgrazing, etc.), has seriously affected ecological great balances, involving a degradation of the natural resources as well as a fall in agricultural productions, pointing to a process of desertification. To face these challenges, in the course of the 8th ordinary session of the conference of the Heads of States of the African Union held in January 2007 in Addis Ababa (Ethiopia), 11 countries adopted the Panafrican project called the Green Great Wall (GGW). The total objective of the GGW is to contribute i) to the fight against the desert's advance, ii) to the development of the Saharan-Sahelian zones toward a durable management of the natural resources, and iii) to the fight against poverty. It deals with the construction of a set of zones of afforestation crossing the whole African continent in the long term (7000km of which are in the west). Even if some decisions in the launching phase the GGW must be taken quickly, one cannot do without investment in interdisciplinary research. In particular, associating fundamental research and applied research will allow us to ensure the success in the medium and long term of such a large-scale reforestation project. Research segmented in compartmentalized knowledge fields needed to get adequate tools, among which OHMi Tessékéré, initiated by INNEE (Centre national de la recherche scientifique [CNRS]), in partnership with UCAD, constitutes an example. This suitable scientific tool, capable of action flexibility, of self-financing capacity, anchored in civil society, ready to implement a pragmatic and local interdisciplinarity founded currently on the concept of socio-ecological system (SES), is the one we chose to conduct our studies on the Ferlo arid ecosystems.

Post mortem function of AtMC9 in xylem vessel elements.

Cell death of xylem elements is manifested by rupture of the tonoplast and subsequent autolysis of the cellular contents. Metacaspases have been implicated in various forms of plant cell death but regulation and execution of xylem cell death by metacaspases remains unknown. Analysis of the type II metacaspase gene family in Arabidopsis thaliana supported the function of METACASPASE 9 (AtMC9) in xylem cell death. Progression of xylem cell death was analysed in protoxylem vessel elements of 3-d-old atmc9 mutant roots using reporter gene analysis and electron microscopy. Protoxylem cell death was normally initiated in atmc9 mutant lines, but detailed electron microscopic analyses revealed a role for AtMC9 in clearance of the cell contents post mortem, that is after tonoplast rupture. Subcellular localization of fluorescent AtMC9 reporter fusions supported a post mortem role for AtMC9. Further, probe-based activity profiling suggested a function of AtMC9 on activities of papain-like cysteine proteases. Our data demonstrate that the function of AtMC9 in xylem cell death is to degrade vessel cell contents after vacuolar rupture. We further provide evidence on a proteolytic cascade in post mortem autolysis of xylem vessel elements and suggest that AtMC9 is part of this cascade.

Identification of novel transcription factors regulating secondary cell wall formation in Arabidopsis.

The presence of lignin in secondary cell walls (SCW) is a major factor preventing hydrolytic enzymes from gaining access to cellulose, thereby limiting the saccharification potential of plant biomass. To understand how lignification is regulated is a prerequisite for selecting plant biomass better adapted to bioethanol production. Because transcriptional regulation is a major mechanism controlling the expression of genes involved in lignin biosynthesis, our aim was to identify novel transcription factors (TFs) dictating lignin profiles in the model plant Arabidopsis. To this end, we have developed a post-genomic approach by combining four independent in-house SCW-related transcriptome datasets obtained from (1) the fiber cell wall-deficient wat1 Arabidopsis mutant, (2) Arabidopsis lines over-expressing either the master regulatory activator EgMYB2 or (3) the repressor EgMYB1 and finally (4) Arabidopsis orthologs of Eucalyptus xylem-expressed genes. This allowed us to identify 502 up- or down-regulated TFs. We preferentially selected those present in more than one dataset and further analyzed their in silico expression patterns as an additional selection criteria. This selection process led to 80 candidates. Notably, 16 of them were already proven to regulate SCW formation, thereby validating the overall strategy. Then, we phenotyped 43 corresponding mutant lines focusing on histological observations of xylem and interfascicular fibers. This phenotypic screen revealed six mutant lines exhibiting altered lignification patterns. Two of them [Bel-like HomeoBox6 (blh6) and a zinc finger TF] presented hypolignified SCW. Three others (myb52, myb-like TF, hb5) showed hyperlignified SCW whereas the last one (hb15) showed ectopic lignification. In addition, our meta-analyses highlighted a reservoir of new potential regulators adding to the gene network regulating SCW but also opening new avenues to ultimately improve SCW composition for biofuel production.

Disease resistance or growth: the role of plant hormones in balancing immune responses and fitness costs.

Plant growth and response to environmental cues are largely governed by phytohormones. The plant hormones ethylene, jasmonic acid, and salicylic acid (SA) play a central role in the regulation of plant immune responses. In addition, other plant hormones, such as auxins, abscisic acid (ABA), cytokinins, gibberellins, and brassinosteroids, that have been thoroughly described to regulate plant development and growth, have recently emerged as key regulators of plant immunity. Plant hormones interact in complex networks to balance the response to developmental and environmental cues and thus limiting defense-associated fitness costs. The molecular mechanisms that govern these hormonal networks are largely unknown. Moreover, hormone signaling pathways are targeted by pathogens to disturb and evade plant defense responses. In this review, we address novel insights on the regulatory roles of the ABA, SA, and auxin in plant resistance to pathogens and we describe the complex interactions among their signal transduction pathways. The strategies developed by pathogens to evade hormone-mediated defensive responses are also described. Based on these data we discuss how hormone signaling could be manipulated to improve the resistance of crops to pathogens.

Non-cell-autonomous postmortem lignification of tracheary elements in Zinnia elegans.

Postmortem lignification of xylem tracheary elements (TEs) has been debated for decades. Here, we provide evidence in Zinnia elegans TE cell cultures, using pharmacological inhibitors and in intact Z. elegans plants using Fourier transform infrared microspectroscopy, that TE lignification occurs postmortem (i.e., after TE programmed cell death). In situ RT-PCR verified expression of the lignin monomer biosynthetic cinnamoyl CoA reductase and cinnamyl alcohol dehydrogenase in not only the lignifying TEs but also in the unlignified non-TE cells of Z. elegans TE cell cultures and in living, parenchymatic xylem cells that surround TEs in stems. These cells were also shown to have the capacity to synthesize and transport lignin monomers and reactive oxygen species to the cell walls of dead TEs. Differential gene expression analysis in Z. elegans TE cell cultures and concomitant functional analysis in Arabidopsis thaliana resulted in identification of several genes that were expressed in the non-TE cells and that affected lignin chemistry on the basis of pyrolysis-gas chromatography/mass spectrometry analysis. These data suggest that living, parenchymatic xylem cells contribute to TE lignification in a non-cell-autonomous manner, thus enabling the postmortem lignification of TEs.

Galactoglucomannan oligosaccharides are assumed to affect tracheary element formation via interaction with auxin in Zinnia xylogenic cell culture.

KEY MESSAGE: Galactoglucomannan oligosaccharides seem to interact with auxin in xylogenic cell culture, thus influencing mainly metaxylem-like tracheary element differentiation depending on timing with hormones and the process kinetics. Complex mapping of Zinnia mesophyll cell transdifferentiation into tracheary elements with or without prior cell division was documented after palisade and spongy parenchyma cell immobilization during the first 4 days of culture. Here, we report a positive effect of galactoglucomannan oligosaccharides on cell viability and density and higher metaxylem-like tracheary element formation in xylogenic cell culture. The maximal positive effect was achieved by the simultaneous addition of the oligosaccharides and growth hormones (auxin, cytokinin) to the cell culture medium. Moreover, a large number of metaxylem-like tracheary elements were observed in a low-auxin medium supplemented with oligosaccharides, but not in a low-cytokinin medium, suggesting a close relationship between auxin and the oligosaccharides during tracheary element formation.

Arabidopsis wat1 (walls are thin1)-mediated resistance to the bacterial vascular pathogen, Ralstonia solanacearum, is accompanied by cross-regulation of salicylic acid and tryptophan metabolism.

Inactivation of Arabidopsis WAT1 (Walls Are Thin1), a gene required for secondary cell-wall deposition, conferred broad-spectrum resistance to vascular pathogens, including the bacteria Ralstonia solanacearum and Xanthomonas campestris pv. campestris, and the fungi Verticillium dahliae and Verticillium albo-atrum. Introduction of NahG, the bacterial salicylic acid (SA)-degrading salicylate hydroxylase gene, into the wat1 mutant restored full susceptibility to both R. solanacearum and X. campestris pv. campestris. Moreover, SA content was constitutively higher in wat1 roots, further supporting a role for SA in wat1-mediated resistance to vascular pathogens. By combining transcriptomic and metabolomic data, we demonstrated a general repression of indole metabolism in wat1-1 roots as shown by constitutive down-regulation of several genes encoding proteins of the indole glucosinolate biosynthetic pathway and reduced amounts of tryptophan (Trp), indole-3-acetic acid and neoglucobrassicin, the major form of indole glucosinolate in roots. Furthermore, the susceptibility of the wat1 mutant to R. solanacearum was partially restored when crossed with either the trp5 mutant, an over-accumulator of Trp, or Pro35S:AFB1-myc, in which indole-3-acetic acid signaling is constitutively activated. Our original hypothesis placed cell-wall modifications at the heart of the wat1 resistance phenotype. However, the results presented here suggest a mechanism involving root-localized metabolic channeling away from indole metabolites to SA as a central feature of wat1 resistance to R. solanacearum.

Deciphering the route of Ralstonia solanacearum colonization in Arabidopsis thaliana roots during a compatible interaction: focus at the plant cell wall.

The compatible interaction between the model plant, Arabidopsis thaliana, and the GMI1000 strain of the phytopathogenic bacterium, Ralstonia solanacearum, was investigated in an in vitro pathosystem. We describe the progression of the bacteria in the root from penetration at the root surface to the xylem vessels and the cell type-specific, cell wall-associated modifications that accompanies bacterial colonization. Within 6 days post inoculation, R. solanacearum provoked a rapid plasmolysis of the epidermal, cortical, and endodermal cells, including those not directly in contact with the bacteria. Plasmolysis was accompanied by a global degradation of pectic homogalacturonanes as shown by the loss of JIM7 and JIM5 antibody signal in the cell wall of these cell types. As indicated by immunolabeling with Rsol-I antibodies that specifically recognize R. solanacearum, the bacteria progresses through the root in a highly directed, centripetal manner to the xylem poles, without extensive multiplication in the intercellular spaces along its path. Entry into the vascular cylinder was facilitated by cell collapse of the two pericycle cells located at the xylem poles. Once the bacteria reached the xylem vessels, they multiplied abundantly and moved from vessel to vessel by digesting the pit membrane between adjacent vessels. The degradation of the secondary walls of xylem vessels was not a prerequisite for vessel colonization as LM10 antibodies strongly labeled xylem cell walls, even at very late stages in disease development. Finally, the capacity of R. solanacearum to specifically degrade certain cell wall components and not others could be correlated with the arsenal of cell wall hydrolytic enzymes identified in the bacterial genome.

Characterization of a cinnamoyl-CoA reductase 1 (CCR1) mutant in maize: effects on lignification, fibre development, and global gene expression.

Cinnamoyl-CoA reductase (CCR), which catalyses the first committed step of the lignin-specific branch of monolignol biosynthesis, has been extensively characterized in dicot species, but few data are available in monocots. By screening a Mu insertional mutant collection in maize, a mutant in the CCR1 gene was isolated named Zmccr1(-). In this mutant, CCR1 gene expression is reduced to 31% of the residual wild-type level. Zmccr1(-) exhibited enhanced digestibility without compromising plant growth and development. Lignin analysis revealed a slight decrease in lignin content and significant changes in lignin structure. p-Hydroxyphenyl units were strongly decreased and the syringyl/guaiacyl ratio was slightly increased. At the cellular level, alterations in lignin deposition were mainly observed in the walls of the sclerenchymatic fibre cells surrounding the vascular bundles. These cell walls showed little to no staining with phloroglucinol. These histochemical changes were accompanied by an increase in sclerenchyma surface area and an alteration in cell shape. In keeping with this cell type-specific phenotype, transcriptomics performed at an early stage of plant development revealed the down-regulation of genes specifically associated with fibre wall formation. To the present authors' knowledge, this is the first functional characterization of CCR1 in a grass species.

Light-regulated compensation of wat1 (walls are thin1) growth and secondary cell wall phenotypes is auxin-independent.

We previously reported the characterization of walls are thin1 (wat1), an Arabidopsis mutant that exhibits two developmental phenotypes in stems: (1) a severe decrease in fiber secondary cell wall thickness and (2) a reduction in stem height. Auxin concentration and transport were also significantly reduced in the stem base of wat1 plants. In the original study, these characteristics were observed in plants grown under short day conditions (9 h light /15 h dark). Herein, we provide evidence for partial phenotypic complementation of both wat1 developmental phenotypes when grown under a continuous light regime. Interestingly, when auxin concentration and basipetal transport were measured in these plants, neither was restored to wild type levels. These results suggest that free auxin concentration is not responsible for the partial light-regulated complementation of wat1-mediated phenotypes.

Walls are thin 1 (WAT1), an Arabidopsis homolog of Medicago truncatula NODULIN21, is a tonoplast-localized protein required for secondary wall formation in fibers.

By combining Zinnia elegans in vitro tracheary element genomics with reverse genetics in Arabidopsis, we have identified a new upstream component of secondary wall formation in xylary and interfascicular fibers. Walls are thin 1 (WAT1), an Arabidopsis thaliana homolog of Medicago truncatula NODULIN 21 (MtN21), encodes a plant-specific, predicted integral membrane protein, and is a member of the plant drug/metabolite exporter (P-DME) family (transporter classification number: TC 2.A.7.3). Although WAT1 is ubiquitously expressed throughout the plant, its expression is preferentially associated with vascular tissues, including developing xylem vessels and fibers. WAT1:GFP fusion protein analysis demonstrated that WAT1 is localized to the tonoplast. Analysis of wat1 mutants revealed two cell wall-related phenotypes in stems: a defect in cell elongation, resulting in a dwarfed habit and little to no secondary cell walls in fibers. Secondary walls of vessel elements were unaffected by the mutation. The secondary wall phenotype was supported by comparative transcriptomic and metabolomic analyses of wat1 and wild-type stems, as many transcripts and metabolites involved in secondary wall formation were reduced in abundance. Unexpectedly, these experiments also revealed a modification in tryptophan (Trp) and auxin metabolism that might contribute to the wat1 phenotype. Together, our data demonstrate an essential role for the WAT1 tonoplast protein in the control of secondary cell wall formation in fibers.

Expression of cell wall related genes in basal and ear internodes of silking brown-midrib-3, caffeic acid O-methyltransferase (COMT) down-regulated, and normal maize plants.

BACKGROUND: Silage maize is a major forage and energy resource for cattle feeding, and several studies have shown that lignin content and structure are the determining factors in forage maize feeding value. In maize, four natural brown-midrib mutants have modified lignin content, lignin structure and cell wall digestibility. The greatest lignin reduction and the highest cell wall digestibility were observed in the brown-midrib-3 (bm3) mutant, which is disrupted in the caffeic acid O-methyltransferase (COMT) gene. RESULTS: Expression of cell wall related genes was investigated in basal and ear internodes of normal, COMT antisens (AS225), and bm3 maize plants of the INRA F2 line. A cell wall macro-array was developed with 651 gene specific tags of genes specifically involved in cell wall biogenesis. When comparing basal (older lignifying) and ear (younger lignifying) internodes of the normal line, all genes known to be involved in constitutive monolignol biosynthesis had a higher expression in younger ear internodes. The expression of the COMT gene was heavily reduced, especially in the younger lignifying tissues of the ear internode. Despite the fact that AS225 transgene expression was driven only in sclerenchyma tissues, COMT expression was also heavily reduced in AS225 ear and basal internodes. COMT disruption or down-regulation led to differential expressions of a few lignin pathway genes, which were all over-expressed, except for a phenylalanine ammonia-lyase gene. More unexpectedly, several transcription factor genes, cell signaling genes, transport and detoxification genes, genes involved in cell wall carbohydrate metabolism and genes encoding cell wall proteins, were differentially expressed, and mostly over-expressed, in COMT-deficient plants. CONCLUSION: Differential gene expressions in COMT-deficient plants highlighted a probable disturbance in cell wall assembly. In addition, the gene expressions suggested modified chronology of the different events leading to cell expansion and lignification with consequences far beyond the phenylpropanoid metabolism. The reduced availability of monolignols and S units in bm3 or AS225 plants led to plants also differing in cell wall carbohydrate, and probably protein, composition. Thus, the deficiency in a key-enzyme of the lignin pathway had correlative effects on the whole cell wall metabolism. Furthermore, the observed differential expression between bm3 and normal plants indicated the possible involvement in the maize lignin pathway of genes which up until now have not been considered to play this role.