Biochemical and genetic responses in cuticular mutants

The cuticle layer is essential for maintaining organ integrity and protecting plants against biotic and abiotic stresses present in their natural environment. Arabidopsis thaliana mutants with a defective cuticle layer may help to precisely determine which genes are actually taking part in the formation of the cuticle. The functions of some cuticular genes seem to be conserved between monocots and dicots as we could complement the Arabidopsis fiddlehead (fdh) mutant with the barley allele of the FDH gene. In addition to a defective cuticle, fdh, bodyguard (bdg), lacerata (lcr) and the novel htm Arabidopsis mutant show ectopic organ fusions and other developmental phenotypes. As the isolation of pure cutin from cuticular mutants is not doable, we analysed the cell wall bound lipid composition from leaves of the fdh and lcr mutants. As a consequence of fdh and lcr mutations, a significant increase in cutin monomer amount takes place. Even though FDH and LCR enzymes have very different functions, similar alterations in cell wall bound lipid composition were observed in their mutants. As it was observed in bdg earlier, we could also show that both fdh and lcr accumulated more wax. Even though each of these three mutants has its own particularities that, mutations in three genes coding for so different enzymes trigger similar phenotypical alterations was quite unexpected. This brought us to study the compensatory response to cuticular damage and to compare the transcriptome of mutants using microarrays. This analysis supports the idea that plants are able to compensate for cuticle defects specifically altering gene expression. We characterised the expression pattern of the recently cloned HTM gene using promoter and protein fusion reporter constructs. We found that the HTM gene is epidermis specific, and is particularly strongly expressed in petals and sepals. This goes well in hand with the phenotype of the htm mutant which is characterised by postgenital organ fusion within floral buds. The bound lipid analysis revealed that whereas htm deposits similar quantity of monomers on its outer cell wall, there is a shift in monomer proportions. The wax composition of this mutant was not found different from that of the wild type but a global decrease of 20% was observed. Thus it is more likely that HTM is indirectly involved in wax and cutin biosynthesis. We also characterised insertion lines in genes putatively involved in cutin biosynthesis, respectively encoding an aldehyde dehydrogenase, an epoxide hydrolase-like enzyme, and a member of the α/β hydrolase-fold super family

Dissection of the Complex Phenotype in Cuticular Mutants of Arabidopsis Reveals a Role of SERRATE as a Mediator

Mutations in LACERATA (LCR), FIDDLEHEAD (FDH), and BODYGUARD (BDG) cause a complex developmental syndrome that is consistent with an important role for these Arabidopsis genes in cuticle biogenesis. The genesis of their pleiotropic phenotypes is, however, poorly understood. We provide evidence that neither distorted depositions of cutin, nor deficiencies in the chemical composition of cuticular lipids, account for these features, instead suggesting that the mutants alleviate the functional disorder of the cuticle by reinforcing their defenses. To better understand how plants adapt to these mutations, we performed a genome-wide gene expression analysis. We found that apparent compensatory transcriptional responses in these mutants involve the induction of wax, cutin, cell wall, and defense genes. To gain greater insight into the mechanism by which cuticular mutations trigger this response in the plants, we performed an overlap meta-analysis, which is termed MASTA (MicroArray overlap Search Tool and Analysis), of differentially expressed genes. This suggested that different cell integrity pathways are recruited in cesA cellulose synthase and cuticular mutants. Using MASTA for an in silico suppressor/enhancer screen, we identified SERRATE (SE), which encodes a protein of RNA–processing multi-protein complexes, as a likely enhancer. In confirmation of this notion, the se lcr and se bdg double mutants eradicate severe leaf deformations as well as the organ fusions that are typical of lcr and bdg and other cuticular mutants. Also, lcr does not confer resistance to Botrytis cinerea in a se mutant background. We propose that there is a role for SERRATE-mediated RNA signaling in the cuticle integrity pathway

The Epidermis-Specific Extracellular BODYGUARD Controls Cuticle Development and Morphogenesis in Arabidopsis

The outermost epidermal cell wall is specialized to withstand pathogens and natural stresses, and lipid-based cuticular polymers are the major barrier against incursions. The Arabidopsis thaliana mutant bodyguard (bdg), which exhibits defects characteristic of the loss of cuticle structure not attributable to a lack of typical cutin monomers, unexpectedly accumulates significantly more cell wall–bound lipids and epicuticular waxes than wild-type plants. Pleiotropic effects of the bdg mutation on growth, viability, and cell differentiation are also observed. BDG encodes a member of the α/β-hydrolase fold protein superfamily and is expressed exclusively in epidermal cells. Using Strep-tag epitope-tagged BDG for mutant complementation and immunolocalization, we show that BDG is a polarly localized protein that accumulates in the outermost cell wall in the epidermis. With regard to the appearance and structure of the cuticle, the phenotype conferred by bdg is reminiscent of that of transgenic Arabidopsis plants that express an extracellular fungal cutinase, suggesting that bdg may be incapable of completing the polymerization of carboxylic esters in the cuticular layer of the cell wall or the cuticle proper. We propose that BDG codes for an extracellular synthase responsible for the formation of cuticle. The alternative hypothesis proposes that BDG controls the proliferation/differentiation status of the epidermis via an unknown mechanism

Misexpression of FATTY ACID ELONGATION1 in the Arabidopsis Epidermis Induces Cell Death and Suggests a Critical Role for Phospholipase A2 in This Process[W]

Very-long-chain fatty acids (VLCFAs) are important functional components of various lipid classes, including cuticular lipids in the higher plant epidermis and lipid-derived second messengers. Here, we report the characterization of transgenic Arabidopsis thaliana plants that epidermally express FATTY ACID ELONGATION1 (FAE1), the seed-specific β-ketoacyl-CoA synthase (KCS) catalyzing the first rate-limiting step in VLCFA biosynthesis. Misexpression of FAE1 changes the VLCFAs in different classes of lipids but surprisingly does not complement the KCS fiddlehead mutant. FAE1 misexpression plants are similar to the wild type but display an essentially glabrous phenotype, owing to the selective death of trichome cells. This cell death is accompanied by membrane damage, generation of reactive oxygen species, and callose deposition. We found that nuclei of arrested trichome cells in FAE1 misexpression plants cell-autonomously accumulate high levels of DNA damage, including double-strand breaks characteristic of lipoapoptosis. A chemical genetic screen revealed that inhibitors of KCS and phospholipase A2 (PLA2), but not inhibitors of de novo ceramide biosynthesis, rescue trichome cells from death. These results support the functional role of acyl chain length of fatty acids and PLA2 as determinants for programmed cell death, likely involving the exchange of VLCFAs between phospholipids and the acyl-CoA pool

Parallel evolution of the <i>POQR</i> prolyl oligo peptidase gene conferring plant quantitative disease resistance

<div><p>Plant pathogens with a broad host range are able to infect plant lineages that diverged over 100 million years ago. They exert similar and recurring constraints on the evolution of unrelated plant populations. Plants generally respond with quantitative disease resistance (QDR), a form of immunity relying on complex genetic determinants. In most cases, the molecular determinants of QDR and how they evolve is unknown. Here we identify in <i>Arabidopsis thaliana</i> a gene mediating QDR against <i>Sclerotinia sclerotiorum</i>, agent of the white mold disease, and provide evidence of its convergent evolution in multiple plant species. Using genome wide association mapping in <i>A</i>. <i>thaliana</i>, we associated the gene encoding the POQR prolyl-oligopeptidase with QDR against <i>S</i>. <i>sclerotiorum</i>. Loss of this gene compromised QDR against <i>S</i>. <i>sclerotiorum</i> but not against a bacterial pathogen. Natural diversity analysis associated <i>POQR</i> sequence with QDR. Remarkably, the same amino acid changes occurred after independent duplications of <i>POQR</i> in ancestors of multiple plant species, including <i>A</i>. <i>thaliana</i> and tomato. Genome-scale expression analyses revealed that parallel divergence in gene expression upon <i>S</i>. <i>sclerotiorum</i> infection is a frequent pattern in genes, such as <i>POQR</i>, that duplicated both in <i>A</i>. <i>thaliana</i> and tomato. Our study identifies a previously uncharacterized gene mediating QDR against <i>S</i>. <i>sclerotiorum</i>. It shows that some QDR determinants are conserved in distantly related plants and have emerged through the repeated use of similar genetic polymorphisms at different evolutionary time scales.</p></div

Natural diversity at <i>POQR</i> locus in <i>A</i>. <i>thaliana</i> associates sequence and expression polymorphism with QDR.

<p>(A) Distribution of disease severity index for accessions harboring either a C or a T at position 7061677 of chromosome 1, corresponding respectively to a proline or serine at POQR amino acid position 5 (*** Student’s t test p-value<0.01). <b>(B)</b> Maximum likelihood phylogenetic tree of POQR protein sequences in 46 <i>A</i>. <i>thaliana</i> accessions. Identical sequences were collapsed; nodes are sized proportionally to the number of accessions with identical POQR isoform, and colored according to the average disease severity index after <i>S</i>. <i>sclerotiorum</i> inoculation, indicated at the center of each node. Nodes are labeled with accessions forming the corresponding group. Portions of the network corresponding to clades A, S and R are highlighted with colored background. Branches carrying POQR isoforms with a proline at position 5 are shown as bold dotted lines. Support corresponding to an SH-like approximate likelihood-ratio test is shown for major branches. <b>(C)</b> Distribution of disease severity index 6 days after <i>S</i>. <i>sclerotiorum</i> inoculation in major POQR clades (*** Student’s t test p-value<0.01).</p

Parallel sequence evolution of POQR homologs in multiple plant lineages.

<p><b>(A)</b> Maximum likelihood phylogenetic tree including the 75 best <i>POQR</i> homologs from 40 plant species. Terminal nodes are colored and labeled according to plant species, following the legend shown around the tree. Species belonging to a genus for which <i>S</i>. <i>sclerotiorum</i> infection has not been reported are shown with dotted nodes and labeled in grey (data from <a href="https://nt.ars-grin.gov/fungaldatabases/" target="_blank">https://nt.ars-grin.gov/fungaldatabases/</a>). Support corresponding to an SH-like approximate likelihood-ratio test is shown for major branches. Branches are colored according to amino acid in position 5 of POQR sequences. POQR homologs from <i>Sphagnum fallax</i>, <i>Arabidopsis thaliana</i>, <i>Solanum lycopersicum</i>, <i>Salix purpurea</i> and <i>Oryza sativa</i> are labeled with corresponding identifiers from the Phytozome database. <b>(B)</b> Multiple sequence alignment of POQR homologs from <i>Sphagnum fallax</i>, <i>Arabidopsis thaliana</i>, <i>Solanum lycopersicum</i>, <i>Salix purpurea</i> and <i>Oryza sativa</i> showing diversity at position 5 (triangle) and conservation of the catalytic triad (stars). <b>(C)</b> Measure of leaf area colonized by <i>S</i>. <i>sclerotiorum</i> 24 hours after inoculation in wild type tomato plants, plants silenced for POQR by virus induced gene silencing (VIGS) and plants carrying the empty viral vector (e.v.). Values are shown for n = 16 individual plants per genotype from two independent biological experiments. Significance of the difference from wild type was assessed by a Student’s t test with Benjamini-Hochberg correction for multiple testing (p-values indicated above boxes).</p

Genome wide association mapping in a European <i>A</i>. <i>thaliana</i> population associates <i>At1g20380</i> with quantitative disease resistance against <i>S</i>. <i>sclerotiorum</i>.

<p><b>(A)</b> Distribution of disease severity index (DSI) at 6 days after inoculation by <i>S</i>. <i>sclerotiorum</i> in 84 <i>A</i>. <i>thaliana</i> European accessions. Values shown are averages for 6 to 16 plants per genotype, with error bars showing standard deviation. Points are colored from blue to red according to average DSI. <b>(B)</b> Geographic origin of the accessions used in this work, in relation with their average DSI (same color code as in A). Points are sized according to DSI standard deviation. <b>(C)</b> Manhattan plot of GWAS results for DSI after S. <i>sclerotiorum</i> inoculation. Dotted green lines show false discovery rate thresholds, the red line show the position of the <i>POQR</i> locus. Chr., chromosome. <b>(D)</b> Close-up of the major association peak centered on <i>POQR</i> locus. Only SNPs with association score greater than 1.25 are shown.</p

A model for convergent evolution of the <i>POQR</i> gene in <i>A</i>. <i>thaliana</i> and <i>S</i>. <i>lycopersicum</i>.

<p>Our analyses suggest that a single <i>POQR</i> ancestral gene was inherited by <i>A</i>. <i>thaliana</i> and <i>S</i>. <i>lycopersicum</i> ancestors when lineages diverged, about 120 million years ago (Mya). The ancestral POQR gene then duplicated in parallel in <i>A</i>. <i>thaliana</i> lineage between 88 and 44 Mya (At-β and At-α events) and <i>S</i>. <i>lycopersicum</i> lineage about 64 Mya (Sl-T event). After duplication, <i>POQR</i> ancestral genes underwent parallel amino acid substitutions, notably leading to the emergence of proline 5 (P5) and tyrosine 613 (Y613), and parallel gain or gene induction upon <i>S</i>. <i>sclerotiorum</i> infection (red arrow) in <i>A</i>. <i>thalina</i> and <i>S</i>. <i>lycopersicum</i> lineages.</p

POQR confers enhanced quantitative disease resistance against <i>S</i>. <i>sclerotiorum</i>.

<p><b>(A)</b> Representative pictures of area colonized by <i>S</i>. <i>sclerotiorum</i> expressing GFP, 24 hours after inoculation. Bar = 2.5 mm. <b>(B)</b> Measure of leaf area colonized by <i>S</i>. <i>sclerotiorum</i> 24 hours after inoculation. Values are shown for n = 7 to 17 individual plants per genotype from two independent biological experiments. Significance of the difference from Col-0 was assessed by a Student’s t test with Benjamini-Hochberg correction for multiple testing (p-values indicated above boxes). <b>(C)</b> Pictures of representative symptoms on <i>A</i>. <i>thaliana</i> leaves 10 days after inoculation by <i>X</i>. <i>campestris</i> pv. <i>campestris</i>. <b>(D)</b> Proportion of plants presenting a disease severity index of 1, 2, 3 or 4 at 10 days after inoculation (dpi) by <i>X</i>. <i>campestris</i> pv. <i>campestris</i>. Counts from n = 32 to 48 individual plants per genotype from three independent biological experiments.</p