Reshaping the Primary Cell Wall: Dual Effects on Plant Resistance to Ralstonia solanacearum and Heat Stress Response.

Temperature elevation drastically affects plant defense responses to Ralstonia solanacearum and inhibits the major source of resistance in Arabidopsis thaliana, which is mediated by the receptor pair RRS1-R/RPS4. In this study, we refined a previous genome-wide association (GWA) mapping analysis by using a local score approach and detected the primary cell wall CESA3 gene as a major gene involved in plant response to R. solanacearum at both 27°C and an elevated temperature, 30°C. We functionally validated CESA3 as a susceptibility gene involved in resistance to R. solanacearum at both 27 and 30°C through a reverse genetic approach. We provide evidence that the cesa3mre1 mutant enhances resistance to bacterial disease and that resistance is associated with an alteration of root cell morphology conserved at elevated temperatures. However, even by forcing the entry of the bacterium to bypass the primary cell wall barrier, the cesa3mre1 mutant still showed enhanced resistance to R. solanacearum with delayed onset of bacterial wilt symptoms. We demonstrated that the cesa3mre1 mutant had constitutive expression of the defense-related gene VSP1, which is upregulated at elevated temperatures, and that during infection, its expression level is maintained higher than in the wild-type Col-0. In conclusion, this study reveals that alteration of the primary cell wall by mutating the cellulose synthase subunit CESA3 contributes to enhanced resistance to R. solanacearum, remaining effective under heat stress. We expect that these results will help to identify robust genetic sources of resistance to R. solanacearum in the context of global warming. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Functional investigation of five R2R3-MYB transcription factors associated with wood development in Eucalyptus using DAP-seq-ML.

A multi-tiered transcriptional network regulates xylem differentiation and secondary cell wall (SCW) formation in plants, with evidence of both conserved and lineage-specific SCW network architecture. We aimed to elucidate the roles of selected R2R3-MYB transcription factors (TFs) linked to Eucalyptus wood formation by identifying genome-wide TF binding sites and direct target genes through an improved DAP-seq protocol combined with machine learning for target gene assignment (DAP-seq-ML). We applied this to five TFs including a well-studied SCW master regulator (EgrMYB2; homolog of AtMYB83), a repressor of lignification (EgrMYB1; homolog of AtMYB4), a TF affecting SCW thickness and vessel density (EgrMYB137; homolog of PtrMYB074) and two TFs with unclear roles in SCW regulation (EgrMYB135 and EgrMYB122). Each DAP-seq TF peak set (average 12,613 peaks) was enriched for canonical R2R3-MYB binding motifs. To improve the reliability of target gene assignment to peaks, a random forest classifier was developed from Arabidopsis DAP-seq, RNA-seq, chromatin, and conserved noncoding sequence data which demonstrated significantly higher precision and recall to the baseline method of assigning genes to proximal peaks. EgrMYB1, EgrMYB2 and EgrMYB137 predicted targets showed clear enrichment for SCW-related biological processes. As validation, EgrMYB137 overexpression in transgenic Eucalyptus hairy roots increased xylem lignification, while its dominant repression in transgenic Arabidopsis and Populus reduced xylem lignification, stunted growth, and caused downregulation of SCW genes. EgrMYB137 targets overlapped significantly with those of EgrMYB2, suggesting partial functional redundancy. Our results show that DAP-seq-ML identified biologically relevant R2R3-MYB targets supported by the finding that EgrMYB137 promotes SCW lignification in planta.

Overexpression of EgrIAA20 from Eucalyptus grandis, a Non-Canonical Aux/IAA Gene, Specifically Decouples Lignification of the Different Cell-Types in Arabidopsis Secondary Xylem.

Wood (secondary xylem) formation is regulated by auxin, which plays a pivotal role as an integrator of developmental and environmental cues. However, our current knowledge of auxin-signaling during wood formation is incomplete. Our previous genome-wide analysis of Aux/IAAs in Eucalyptus grandis showed the presence of the non-canonical paralog member EgrIAA20 that is preferentially expressed in cambium. We analyzed its cellular localization using a GFP fusion protein and its transcriptional activity using transactivation assays, and demonstrated its nuclear localization and strong auxin response repressor activity. In addition, we functionally tested the role of EgrIAA20 by constitutive overexpression in Arabidopsis to investigate for phenotypic changes in secondary xylem formation. Transgenic Arabidopsis plants overexpressing EgrIAA20 were smaller and displayed impaired development of secondary fibers, but not of other wood cell types. The inhibition in fiber development specifically affected their cell wall lignification. We performed yeast-two-hybrid assays to identify EgrIAA20 protein partners during wood formation in Eucalyptus, and identified EgrIAA9A, whose ortholog PtoIAA9 in poplar is also known to be involved in wood formation. Altogether, we showed that EgrIAA20 is an important auxin signaling component specifically involved in controlling the lignification of wood fibers.

Wood Architecture and Composition Are Deeply Remodeled in Frost Sensitive Eucalyptus Overexpressing CBF/DREB1 Transcription Factors.

Eucalypts are the most planted trees worldwide, but most of them are frost sensitive. Overexpressing transcription factors for CRT-repeat binding factors (CBFs) in transgenic Eucalyptus confer cold resistance both in leaves and stems. While wood plays crucial roles in trees and is affected by environmental cues, its potential role in adaptation to cold stress has been neglected. Here, we addressed this question by investigating the changes occurring in wood in response to the overexpression of two CBFs, taking advantage of available transgenic Eucalyptus lines. We performed histological, biochemical, and transcriptomic analyses on xylem samples. CBF ectopic expression led to a reduction of both primary and secondary growth, and triggered changes in xylem architecture with smaller and more frequent vessels and fibers exhibiting reduced lumens. In addition, lignin content and syringyl/guaiacyl (S/G) ratio increased. Consistently, many genes of the phenylpropanoid and lignin branch pathway were upregulated. Most of the features of xylem remodeling induced by CBF overexpression are reminiscent of those observed after long exposure of Eucalyptus trees to chilling temperatures. Altogether, these results suggest that CBF plays a central role in the cross-talk between response to cold and wood formation and that the remodeling of wood is part of the adaptive strategies to face cold stress.

Implementing the CRISPR/Cas9 Technology in Eucalyptus Hairy Roots Using Wood-Related Genes.

Eucalypts are the most planted hardwoods worldwide. The availability of the Eucalyptus grandis genome highlighted many genes awaiting functional characterization, lagging behind because of the lack of efficient genetic transformation protocols. In order to efficiently generate knock-out mutants to study the function of eucalypts genes, we implemented the powerful CRISPR/Cas9 gene editing technology with the hairy roots transformation system. As proofs-of-concept, we targeted two wood-related genes: Cinnamoyl-CoA Reductase1 (CCR1), a key lignin biosynthetic gene and IAA9A an auxin dependent transcription factor of Aux/IAA family. Almost all transgenic hairy roots were edited but the allele-editing rates and spectra varied greatly depending on the gene targeted. Most edition events generated truncated proteins, the prevalent edition types were small deletions but large deletions were also quite frequent. By using a combination of FT-IR spectroscopy and multivariate analysis (partial least square analysis (PLS-DA)), we showed that the CCR1-edited lines, which were clearly separated from the controls. The most discriminant wave-numbers were attributed to lignin. Histochemical analyses further confirmed the decreased lignification and the presence of collapsed vessels in CCR1-edited lines, which are characteristics of CCR1 deficiency. Although the efficiency of editing could be improved, the method described here is already a powerful tool to functionally characterize eucalypts genes for both basic research and industry purposes.

A systems biology view of wood formation in Eucalyptus grandis trees submitted to different potassium and water regimes.

Wood production in fast-growing Eucalyptus grandis trees is highly dependent on both potassium (K) fertilization and water availability but the molecular processes underlying wood formation in response to the combined effects of these two limiting factors remain unknown. E. grandis trees were submitted to four combinations of K-fertilization and water supply. Weighted gene co-expression network analysis and MixOmics-based co-regulation networks were used to integrate xylem transcriptome, metabolome and complex wood traits. Functional characterization of a candidate gene was performed in transgenic E. grandis hairy roots. This integrated network-based approach enabled us to identify meaningful biological processes and regulators impacted by K-fertilization and/or water limitation. It revealed that modules of co-regulated genes and metabolites strongly correlated to wood complex traits are in the heart of a complex trade-off between biomass production and stress responses. Nested in these modules, potential new cell-wall regulators were identified, as further confirmed by the functional characterization of EgMYB137. These findings provide new insights into the regulatory mechanisms of wood formation under stressful conditions, pointing out both known and new regulators co-opted by K-fertilization and/or water limitation that may potentially promote adaptive wood traits.

Special trends in CBF and DREB2 groups in Eucalyptus gunnii vs Eucalyptus grandis suggest that CBF are master players in the trade-off between growth and stress resistance.

Annotation of the Eucalyptus grandis genome showed a large amplification of the dehydration-responsive element binding 1/C-repeat binding factor (DREB1/CBF) group without recent DREB2 gene duplication compared with other plant species. The present annotation of the CBF and DREB2 genes from a draft of the Eucalyptus gunnii genome sequence reveals at least one additional CBF copy in the E. gunnii genome compared with E. grandis, suggesting that this group is still evolving, unlike the DREB2 group. This study aims to investigate the redundancy/neo- or sub-functionalization of the duplicates and the relative involvement of the two groups in abiotic stress responses in both E. grandis and E. gunnii (lower growth but higher cold resistance). A comprehensive transcriptional analysis using high-throughput quantitative real-time polymerase chain reaction (qRT-PCR) was performed on leaves, stems and roots from the two Eucalyptus species after cold, heat or drought treatment. A large CBF cluster accounted for most of the cold response in all the organs, whereas heat and drought responses mainly involved a small CBF cluster and the DREB2 genes. In addition, CBF putative target genes, known to be involved in plant tolerance and development, were found to be cold-regulated. The higher transcript amounts of both the CBF and target genes in the cold tolerant E. gunnii contrasted with the higher CBF induction rates in the fast growing E. grandis. Altogether, the present results, in agreement with previous data about Eucalyptus transgenic lines over-expressing CBF, suggest that these factors, which promote both stress protection and growth limitation, participate in the trade-off between growth and resistance in this woody species.

Tomato GDSL1 is required for cutin deposition in the fruit cuticle.

The plant cuticle consists of cutin, a polyester of glycerol, hydroxyl, and epoxy fatty acids, covered and filled by waxes. While the biosynthesis of cutin building blocks is well documented, the mechanisms underlining their extracellular deposition remain unknown. Among the proteins extracted from dewaxed tomato (Solanum lycopersicum) peels, we identified GDSL1, a member of the GDSL esterase/acylhydrolase family of plant proteins. GDSL1 is strongly expressed in the epidermis of growing fruit. In GDSL1-silenced tomato lines, we observed a significant reduction in fruit cuticle thickness and a decrease in cutin monomer content proportional to the level of GDSL1 silencing. A significant decrease of wax load was observed only for cuticles of the severely silenced transgenic line. Fourier transform infrared (FTIR) analysis of isolated cutins revealed a reduction in cutin density in silenced lines. Indeed, FTIR-attenuated total reflectance spectroscopy and atomic force microscopy imaging showed that drastic GDSL1 silencing leads to a reduction in ester bond cross-links and to the appearance of nanopores in tomato cutins. Furthermore, immunolabeling experiments attested that GDSL1 is essentially entrapped in the cuticle proper and cuticle layer. These results suggest that GDSL1 is specifically involved in the extracellular deposition of the cutin polyester in the tomato fruit cuticle.

Cell-type-specific control of secondary cell wall formation by Musashi-type translational regulators in Arabidopsis.

Deciphering the mechanism of secondary cell wall/SCW formation in plants is key to understanding their development and the molecular basis of biomass recalcitrance. Although transcriptional regulation is essential for SCW formation, little is known about the implication of post-transcriptional mechanisms in this process. Here we report that two bonafide RNA-binding proteins homologous to the animal translational regulator Musashi, MSIL2 and MSIL4, function redundantly to control SCW formation in Arabidopsis. MSIL2/4 interactomes are similar and enriched in proteins involved in mRNA binding and translational regulation. MSIL2/4 mutations alter SCW formation in the fibers, leading to a reduction in lignin deposition, and an increase of 4-O-glucuronoxylan methylation. In accordance, quantitative proteomics of stems reveal an overaccumulation of glucuronoxylan biosynthetic machinery, including GXM3, in the msil2/4 mutant stem. We showed that MSIL4 immunoprecipitates GXM mRNAs, suggesting a novel aspect of SCW regulation, linking post-transcriptional control to the regulation of SCW biosynthesis genes.

Down-regulation of a single auxin efflux transport protein in tomato induces precocious fruit development.

The PIN-FORMED (PIN) auxin efflux transport protein family has been well characterized in the model plant Arabidopsis thaliana, where these proteins are crucial for auxin regulation of various aspects of plant development. Recent evidence indicates that PIN proteins may play a role in fruit set and early fruit development in tomato (Solanum lycopersicum), but functional analyses of PIN-silenced plants failed to corroborate this hypothesis. Here it is demonstrated that silencing specifically the tomato SlPIN4 gene, which is predominantly expressed in tomato flower bud and young developing fruit, leads to parthenocarpic fruits due to precocious fruit development before fertilization. This phenotype was associated with only slight modifications of auxin homeostasis at early stages of flower bud development and with minor alterations of ARF and Aux/IAA gene expression. However, microarray transcriptome analysis and large-scale quantitative RT-PCR profiling of transcription factors in developing flower bud and fruit highlighted differentially expressed regulatory genes, which are potential targets for auxin control of fruit set and development in tomato. In conclusion, this work provides clear evidence that the tomato PIN protein SlPIN4 plays a major role in auxin regulation of tomato fruit set, possibly by preventing precocious fruit development in the absence of pollination, and further gives new insights into the target genes involved in fruit set.

NMR-Based Tissular and Developmental Metabolomics of Tomato Fruit.

Fruit is a complex organ containing seeds and several interconnected tissues with dedicated roles. However, most biochemical or molecular studies about fleshy fruit development concern the entire fruit, the fruit without seeds, or pericarp only. We studied tomato (Solanum lycopersicum) fruit at four stages of development (12, 20, 35, and 45 days post-anthesis). We separated the seeds and the other tissues, exocarp, mesocarp, columella with placenta and locular tissue, and analyzed them individually using proton NMR metabolomic profiling for the quantification of major polar metabolites, enzymatic analysis of starch, and LC-DAD analysis of isoprenoids. Pericarp tissue represented about half of the entire fruit mass only. The composition of each fruit tissue changed during fruit development. An ANOVA-PCA highlighted common, and specific metabolite trends between tissues e.g., higher contents of chlorogenate in locular tissue and of starch in columella. Euclidian distances based on compositional data showed proximities within and between tissues. Several metabolic regulations differed between tissues as revealed by the comparison of metabolite networks based on correlations between compounds. This work stressed the role of specific tissues less studied than pericarp but that impact fruit organoleptic quality including its shape and taste, and fruit processing quality.

Distinct leaf transcriptomic response of water deficient Eucalyptus grandis submitted to potassium and sodium fertilization.

While potassium fertilization increases growth yield in Brazilian eucalyptus plantations, it could also increase water requirements, making trees more vulnerable to drought. Sodium fertilization, which has been shown to promote eucalyptus growth compared to K-deficient trees, could partially mitigate this adverse effect of potassium. However, little is known about the influence of K and Na fertilization on the tree metabolic response to water deficit. The aim of the present study was thus to analyze the transcriptome of leaves sampled from Eucalyptus grandis trees subjected to 37% rainfall reduction, and fertilized with potassium (K), sodium (Na), compared to control trees (C). The multifactorial experiment was set up in a field with a throughfall exclusion system. Transcriptomic analysis was performed on leaves from two-year-old trees, and data analyzed using multifactorial statistical analysis and weighted gene co-expression network analysis (WGCNA). Significant sets of genes were seen to respond to rainfall reduction, in interaction with K or Na fertilization, or to fertilization only (regardless of the water supply regime). The genes were involved in stress signaling, primary and secondary metabolism, secondary cell wall formation and photosynthetic activity. Our focus on key genes related to cation transporters and aquaporins highlighted specific regulation of ion homeostasis, and plant adjustment to water deficit. While water availability significantly affects the transcriptomic response of eucalyptus species, this study points out that the transcriptomic response is highly dependent on the fertilization regime. Our study is based on the first large-scale field trial in a tropical region, specifically designed to study the interaction between water availability and nutrition in eucalyptus. To our knowledge, this is the first global transcriptomic analysis to compare the influence of K and Na fertilization on tree adaptive traits in water deficit conditions.

Long cold exposure induces transcriptional and biochemical remodelling of xylem secondary cell wall in Eucalyptus.

Although eucalypts are the most planted hardwood trees worldwide, the majority of them are frost sensitive. The recent creation of frost-tolerant hybrids such as Eucalyptus gundal plants (E. gunnii × E. dalrympleana hybrids), now enables the development of industrial plantations in northern countries. Our objective was to evaluate the impact of cold on the wood structure and composition of these hybrids, and on the biosynthetic and regulatory processes controlling their secondary cell-wall (SCW) formation. We used an integrated approach combining histology, biochemical characterization and transcriptomic profiling as well as gene co-expression analyses to investigate xylem tissues from Eucalyptus hybrids exposed to cold conditions. Chilling temperatures triggered the deposition of thicker and more lignified xylem cell walls as well as regulation at the transcriptional level of SCW genes. Most genes involved in lignin biosynthesis, except those specifically dedicated to syringyl unit biosynthesis, were up-regulated. The construction of a co-expression network enabled the identification of both known and potential new SCW transcription factors, induced by cold stress. These regulators at the crossroads between cold signalling and SCW formation are promising candidates for functional studies since they may contribute to the tolerance of E. gunnii × E. dalrympleana hybrids to cold.

The Woody-Preferential Gene EgMYB88 Regulates the Biosynthesis of Phenylpropanoid-Derived Compounds in Wood.

Comparative phylogenetic analyses of the R2R3-MYB transcription factor family revealed that five subgroups were preferentially found in woody species and were totally absent from Brassicaceae and monocots (Soler et al., 2015). Here, we analyzed one of these subgroups (WPS-I) for which no gene had been yet characterized. Most Eucalyptus members of WPS-I are preferentially expressed in the vascular cambium, the secondary meristem responsible for tree radial growth. We focused on EgMYB88, which is the most specifically and highly expressed in vascular tissues, and showed that it behaves as a transcriptional activator in yeast. Then, we functionally characterized EgMYB88 in both transgenic Arabidopsis and poplar plants overexpressing either the native or the dominant repression form (fused to the Ethylene-responsive element binding factor-associated Amphiphilic Repression motif, EAR). The transgenic Arabidopsis lines had no phenotype whereas the poplar lines overexpressing EgMYB88 exhibited a substantial increase in the levels of the flavonoid catechin and of some salicinoid phenolic glycosides (salicortin, salireposide, and tremulacin), in agreement with the increase of the transcript levels of landmark biosynthetic genes. A change in the lignin structure (increase in the syringyl vs. guaiacyl, S/G ratio) was also observed. Poplar lines overexpressing the EgMYB88 dominant repression form did not show a strict opposite phenotype. The level of catechin was reduced, but the levels of the salicinoid phenolic glycosides and the S/G ratio remained unchanged. In addition, they showed a reduction in soluble oligolignols containing sinapyl p-hydroxybenzoate accompanied by a mild reduction of the insoluble lignin content. Altogether, these results suggest that EgMYB88, and more largely members of the WPS-I group, could control in cambium and in the first layers of differentiating xylem the biosynthesis of some phenylpropanoid-derived secondary metabolites including lignin.

Gene and metabolite regulatory network analysis of early developing fruit tissues highlights new candidate genes for the control of tomato fruit composition and development.

Variations in early fruit development and composition may have major impacts on the taste and the overall quality of ripe tomato (Solanum lycopersicum) fruit. To get insights into the networks involved in these coordinated processes and to identify key regulatory genes, we explored the transcriptional and metabolic changes in expanding tomato fruit tissues using multivariate analysis and gene-metabolite correlation networks. To this end, we demonstrated and took advantage of the existence of clear structural and compositional differences between expanding mesocarp and locular tissue during fruit development (12-35 d postanthesis). Transcriptome and metabolome analyses were carried out with tomato microarrays and analytical methods including proton nuclear magnetic resonance and liquid chromatography-mass spectrometry, respectively. Pairwise comparisons of metabolite contents and gene expression profiles detected up to 37 direct gene-metabolite correlations involving regulatory genes (e.g. the correlations between glutamine, bZIP, and MYB transcription factors). Correlation network analyses revealed the existence of major hub genes correlated with 10 or more regulatory transcripts and embedded in a large regulatory network. This approach proved to be a valuable strategy for identifying specific subsets of genes implicated in key processes of fruit development and metabolism, which are therefore potential targets for genetic improvement of tomato fruit quality.

Silencing of the mitochondrial ascorbate synthesizing enzyme L-galactono-1,4-lactone dehydrogenase affects plant and fruit development in tomato.

L-Galactono-1,4-lactone dehydrogenase (EC 1.3.2.3) catalyzes the last step in the main pathway of vitamin C (L-ascorbic acid) biosynthesis in higher plants. In this study, we first characterized the spatial and temporal expression of SlGalLDH in several organs of tomato (Solanum lycopersicum) plants in parallel with the ascorbate content. P(35S):Slgalldh(RNAi) silenced transgenic tomato lines were then generated using an RNAi strategy to evaluate the effect of any resulting modification of the ascorbate pool on plant and fruit development. In all P(35S):Slgalldh(RNAi) plants with reduced SlGalLDH transcript and activity, plant growth rate was decreased. Plants displaying the most severe effects (dwarf plants with no fruit) were excluded from further analysis. The most affected lines studied exhibited up to an 80% reduction in SlGalLDH activity and showed a strong reduction in leaf and fruit size, mainly as a consequence of reduced cell expansion. This was accompanied by significant changes in mitochondrial function and altered ascorbate redox state despite the fact that the total ascorbate content remained unchanged. By using a combination of transcriptomic and metabolomic approaches, we further demonstrated that several primary, like the tricarboxylic acid cycle, as well as secondary metabolic pathways related to stress response were modified in leaves and fruit of P(35S):Slgalldh(RNAi) plants. When taken together, this work confirms the complexity of ascorbate regulation and its link with plant metabolism. Moreover, it strongly suggests that, in addition to ascorbate synthesis, GalLDH could play an important role in the regulation of cell growth-related processes in plants.

The expression of cell proliferation-related genes in early developing flowers is affected by a fruit load reduction in tomato plants.

Changes in photoassimilate partitioning between source and sink organs significantly affect fruit development and size. In this study, a comparison was made of tomato plants (Solanum lycopersicum L.) grown under a low fruit load (one fruit per truss, L1 plants) and under a standard fruit load (five fruits per truss, L5 plants), at morphological, biochemical, and molecular levels. Fruit load reduction resulted in increased photoassimilate availability in the plant and in increased growth rates in all plant organs analysed (root, stem, leaf, flower, and fruit). Larger flower and fruit size in L1 plants were correlated with higher cell number in the pre-anthesis ovary. This was probably due to the acceleration of the flower growth rate since other flower developmental parameters (schedule and time-course) remained otherwise unaffected. Using RT-PCR, it was shown that the transcript levels of CYCB2;1 (cyclin) and CDKB2;1 (cyclin-dependent kinase), two mitosis-specific genes, strongly increased early in developing flower buds. Remarkably, the transcript abundance of CYCD3;1, a D-type cyclin potentially involved in cell cycle regulation in response to mitogenic signals, also increased by more than 5-fold at very early stages of L1 flower development. By contrast, transcripts from fw2.2, a putative negative regulator of cell division in tomato fruit, strongly decreased in developing flower bud, as confirmed by in situ hybridization studies. Taken together, these results suggest that changes in carbohydrate partitioning could control fruit size through the regulation of cell proliferation-related genes at very early stages of flower development.