Arabidopsis SDG8 Potentiates the Sustainable Transcriptional Induction of the Pathogenesis-Related Genes PR1 and PR2 During Plant Defense Response.

Post-translational covalent modifications of histones play important roles in modulating chromatin structure and are involved in the control of multiple developmental processes in plants. Here we provide insight into the contribution of the histone lysine methyltransferase SET DOMAIN GROUP 8 (SDG8), implicated in histone H3 lysine 36 trimethylation (H3K36me3), in connection with RNA polymerase II (RNAPII) to enhance Arabidopsis immunity. We showed that even if the sdg8-1 loss-of-function mutant, defective in H3K36 methylation, displayed a higher sensitivity to different strains of the bacterial pathogen Pseudomonas syringae, effector-triggered immunity (ETI) still operated, but less efficiently than in the wild-type (WT) plants. In sdg8-1, the level of the plant defense hormone salicylic acid (SA) was abnormally high under resting conditions and was accumulated similarly to WT at the early stage of pathogen infection but quickly dropped down at later stages. Concomitantly, the transcription of several defense-related genes along the SA signaling pathway was inefficiently induced in the mutant. Remarkably, albeit the defense genes PATHOGENESIS-RELATED1 (PR1) and PR2 have retained responsiveness to exogenous SA, their inductions fade more rapidly in sdg8-1 than in WT. At chromatin, while global levels of histone methylations were found to be stable, local increases of H3K4 and H3K36 methylations as well as RNAPII loading were observed at some defense genes following SA-treatments in WT. In sdg8-1, the H3K36me3 increase was largely attenuated and also the increases of H3K4me3 and RNAPII were frequently compromised. Lastly, we demonstrated that SDG8 could physically interact with the RNAPII C-terminal Domain, providing a possible link between RNAPII loading and H3K36me3 deposition. Collectively, our results indicate that SDG8, through its histone methyltransferase activity and its physical coupling with RNAPII, participates in the strong transcriptional induction of some defense-related genes, in particular PR1 and PR2, to potentiate sustainable immunity during plant defense response to bacterial pathogen.

Jasmonic Acid Oxidase 2 Hydroxylates Jasmonic Acid and Represses Basal Defense and Resistance Responses against Botrytis cinerea Infection.

Jasmonates (JAs) orchestrate immune responses upon wound/herbivore injury or infection by necrotrophic pathogens. Elucidation of catabolic routes has revealed new complexity in jasmonate metabolism. Two integrated pathways attenuate signaling by turning over the active hormone jasmonoyl-isoleucine (JA-Ile) through ω-oxidation or deconjugation, and define an indirect route forming the derivative 12OH-JA. Here, we provide evidence for a second 12OH-JA formation pathway by direct jasmonic acid (JA) oxidation. Three jasmonic acid oxidases (JAOs) of the 2-oxoglutarate dioxygenase family catalyze specific oxidation of JA to 12OH-JA, and their genes are induced by wounding or infection by the fungus Botrytis cinerea. JAO2 exhibits the highest basal expression, and its deficiency in jao2 mutants strongly enhanced antifungal resistance. The resistance phenotype resulted from constitutive expression of antimicrobial markers rather than from their higher induction in infected jao2 plants and could be reversed by ectopic expression of any of the three JAOs in jao2. Elevated defense in jao2 was dependent on the activity of JASMONATE RESPONSE 1 (JAR1) and CORONATINE-INSENSITIVE 1 (COI1) but was not correlated with enhanced JA-Ile accumulation. Instead, jao2 mutant lines displayed altered accumulation of several JA species in healthy and challenged plants, suggesting elevated metabolic flux through JA-Ile. Collectively, these data identify the missing enzymes hydroxylating JA and uncover an important metabolic diversion mechanism for repressing basal JA defense responses.

The Rise and Fall of Jasmonate Biological Activities.

Jasmonates (JAs) constitute a major class of plant regulators that coordinate responses to biotic and abiotic threats and important aspects of plant development. The core biosynthetic pathway converts linolenic acid released from plastid membrane lipids to the cyclopentenone cis-oxo-phytodienoic acid (OPDA) that is further reduced and shortened to jasmonic acid (JA) in peroxisomes. Abundant pools of OPDA esterified to plastid lipids also occur upon stress, mainly in the Arabidopsis genus. Long thought to be the bioactive hormone, JA only gains its pleiotropic hormonal properties upon conjugation into jasmonoyl-isoleucine (JA-Ile). The signaling pathway triggered when JA-Ile promotes the assembly of COI1-JAZ (Coronatine Insensitive 1-JAsmonate Zim domain) co-receptor complexes has been the focus of most recent research in the jasmonate field. In parallel, OPDA and several other JA derivatives are recognized for their separate activities and contribute to the diversity of jasmonate action in plant physiology. We summarize in this chapter the properties of different bioactive JAs and review elements known for their perception and signal transduction. Much progress has also been gained on the enzymatic processes governing JA-Ile removal. Two JA-Ile catabolic pathways, operating through ω-oxidation (cytochromes P450) or conjugate cleavage (amido hydrolases) shape signal dynamics to allow optimal control on defense. JA-Ile turnover not only participates in signal attenuation, but also impact the homeostasis of the entire JA metabolic pathway.

Histone H2B monoubiquitination is involved in the regulation of cutin and wax composition in Arabidopsis thaliana.

The plant cuticle is a chemically heterogeneous lipophilic layer composed of a cutin polymer matrix and waxes which covers the aerial parts of plants. This layer plays an essential role in the survival of plants by protecting them from desiccation and (a)biotic stresses. Knowledge on the gene networks and mechanisms regulating the synthesis of cuticle components during organ expansion or stress response remains limited however. Here, using five loss-of-function mutants for histone monoubiquitination, we report on the role of two RING E3 ligases, namely HISTONE MONOUBIQUITINATION 1 and 2 (HUB1 and HUB2), in the selective transcriptional activation of four cuticle biosynthesis genes in Arabidopsis thaliana. Microscopy observations showed that in hub1-6 and hub2-2 mutants irregular epidermal cells and disorganized cuticle layers were present in rosette leaves. Water loss measurements on excised rosettes demonstrated that cuticular permeability was significantly increased in the mutants. Chemical analysis of cuticle components revealed that the wax composition was changed and that cutin 16:0 dicarboxylic acid was significantly reduced in all hub mutants. Analysis of transcript levels of selected genes indicated that LACS2, ATT1 and HOTHEAD involved in cutin biosynthesis and CER1 involved in wax biosynthesis were down-regulated in the hub mutants, while the expression of LACERATA, CER3, CER6 and CER10 remained unchanged. Chromatin immunoprecipitation assays further showed that hub mutants are impaired in dynamic changes of histone H2B monoubiquitination at several loci of down-regulated genes. Taken together, these data establish that the regulation of cuticle composition involves chromatin remodeling by H2B monoubiquitination.

Chromatin modification and remodelling: a regulatory landscape for the control of Arabidopsis defence responses upon pathogen attack.

Due to their sessile lifestyle, plants have to cope with an ever-changing environment and to defend themselves against a multitude of biotic aggressors that compromise their development and reproduction. Responses to various biotic stresses largely depend on the plant's capacity to modulate rapidly and specifically its transcriptome. In a stress type-dependent manner, external signals are translocated into the nucleus to activate transcription factors, resulting in the increased expression of particular sets of defence-related genes. Among mechanisms of transcriptional regulation, chromatin remodelling accomplished through the activity of histone-modifying enzymes and ATP-dependent chromatin-remodelling complexes is emerging as a key process in the orchestration of plant biotic stress responses. In this review, we summarize and discuss roles that chromatin-remodelling mechanisms may play in regulating Arabidopsis defence responses.

Arabidopsis SET DOMAIN GROUP2 is required for H3K4 trimethylation and is crucial for both sporophyte and gametophyte development.

Histone H3 lysine 4 trimethylation (H3K4me3) is abundant in euchromatin and is in general associated with transcriptional activation in eukaryotes. Although some Arabidopsis thaliana SET DOMAIN GROUP (SDG) genes have been previously shown to be involved in H3K4 methylation, they are unlikely to be responsible for global genome-wide deposition of H3K4me3. Most strikingly, sparse knowledge is currently available about the role of histone methylation in gametophyte development. In this study, we show that the previously uncharacterized SDG2 is required for global H3K4me3 deposition and its loss of function causes wide-ranging defects in both sporophyte and gametophyte development. Transcriptome analyses of young flower buds have identified 452 genes downregulated by more than twofold in the sdg2-1 mutant; among them, 11 genes, including SPOROCYTELESS/NOZZLE (SPL/NZZ) and MALE STERILITY1 (MS1), have been previously shown to be essential for male and/or female gametophyte development. We show that both SPL/NZZ and MS1 contain bivalent chromatin domains enriched simultaneously with the transcriptionally active mark H3K4me3 and the transcriptionally repressive mark H3K27me3 and that SDG2 is specifically required for the H3K4me3 deposition. Our data suggest that SDG2-mediated H3K4me3 deposition poises SPL/NZZ and MS1 for transcriptional activation, forming a key regulatory mechanism in the gene networks responsible for gametophyte development.

The E2 ubiquitin-conjugating enzymes, AtUBC1 and AtUBC2, play redundant roles and are involved in activation of FLC expression and repression of flowering in Arabidopsis thaliana.

Post-translational modifications of proteins by addition of ubiquitin can regulate protein degradation and localization, protein-protein interactions and transcriptional activation. In the ubiquitylation system, substrate specificity is primarily determined by the E2 ubiquitin-conjugating enzyme (UBC) and the E3 ubiquitin ligase. The Arabidopsis thaliana genome contains 37 genes encoding UBC homologs. However, the biological functions of these genes remain largely uncharacterized. Here, we report reverse genetic characterization of AtUBC1 and AtUBC2. While the loss-of-function single mutants Atubc1-1 and Atubc2-1 only show weak phenotypes, the double mutant Atubc1-1 Atubc2-1 shows a dramatically reduced number of rosette leaves and an early-flowering phenotype. Consistent with these results, the transcript levels of the floral repressor genes FLOWERING LOCUS C (FLC), MADS ASSOCIATED FLOWERING 4 (MAF4) and MAF5 are reduced in the double mutant. Loss-of-function mutants of HISTONE MONOUBIQUITINATION 1 (HUB1) and HUB2, which were previously reported to encode an E3 involved in histone H2B ubiquitylation, also show an early-flowering phenotype and reduced levels of FLC, MAF4 and MAF5 transcripts. In both Atubc1-1 Atubc2-1 and hub2-2 mutants, H2B mono-ubiquitylation is drastically reduced. Taken together, our results indicate that E2s AtUBC1/AtUBC2 and E3s HUB1/HUB2 together mediate H2B ubiquitylation, which is involved in the activation of floral repressor genes as well as in other processes as indicated by the pleiotropic phenotypes of the mutants.

Novel beta-1,3-, 1,6-oligoglucan elicitor from Alternaria alternata 102 for defense responses in tobacco.

A novel elicitor that induces chitinases in tobacco BY-2 cells was isolated from Alternaria alternata 102. Six other fungi, including A. alternata IFO 6587, could not induce, or weakly induce chitinase activity. The purified elicitor was soluble in 75% methanol and showed the chitinase-inducing activity when applied at concentrations of as low as 25 ng x mL(-1). Structural determination by methylation analysis, reducing-end analysis, MALDI-TOF/MS, and NMR spectroscopy indicated that the elicitor was a mixture of beta-1,3-, 1,6-oligoglucans mostly with a degree of polymerization of between 8 and 17. Periodate oxidation of the elicitor suggested that the 1,6-linked and nonreducing terminal residues are essential for the elicitor activity. Further analysis of the elicitor responses in BY-2 cells indicated that the activity of this beta-1,3-, 1,6-glucan elicitor was about 1000 times more potent than that of laminarin, which is a known elicitor of defense responses in tobacco. Analyzing the expression of defense-related genes indicated that a phenylalanine ammonia-lyase gene and a coumaroyl-CoA O-methyltransferase gene were transiently expressed by this beta-1,3-, 1,6-glucan elicitor. The elicitor induced a weak oxidative burst but did not induce cell death in the BY-2 cells. In the tissue of tobacco plants, this beta-1,3-, 1,6-glucan elicitor induced the expression of basic PR-3 genes, the phenylpropanoid pathway genes, and the sesquiterpenoid pathway genes. In comparison with laminarin and laminarin sulfate, which are reported to be potent elicitors of defense responses in tobacco, the expression pattern of genes induced by the purified beta-1,3-, 1,6-glucan elicitor was more similar to that induced by laminarin than to that induced by laminarin sulfate.

Defense and resistance-inducing activities in tobacco of the sulfated beta-1,3 glucan PS3 and its synergistic activities with the unsulfated molecule.

Laminarin, a beta-1,3 glucan with single beta-glucose branches at position 6, was chemically sulfated to produce PS3 with a degree of sulfation of 2.4. PS3 has previously been shown to activate the salicylic acid (SA) signaling pathway in infiltrated tobacco and Arabidopsis thaliana leaf tissues. Here, we investigated whether PS3 induces systemic defense and resistance responses in tobacco. Using a radiolabeled compound, it was first demonstrated that PS3 remains strictly localized to the infiltrated tissues. PS3 is also resistant to beta-glucanase degradation. In transgenic PR1-beta-glucuronidase (GUS) tobacco plants, PS3 causes a strong increase in GUS activity in treated tissues but none in untreated leaves. PS3-infiltrated tissues challenged with tobacco mosaic virus (TMV) 8 d after elicitor application show a decrease in both the lesion number and the lesion size, whereas treatment with laminarin, the unsulfated native glucan, affected only the lesion number. PS3 does not induce systemic acquired resistance to TMV. PS3 and laminarin show synergistic effects in promoting the oxidative burst in tobacco cell suspensions and in increasing the expression of genes encoding O-methyltransferases of the phenylpropanoid pathway in tobacco plants. No synergistic effect was observed on the expression of either the SA-dependent acidic PR1 gene or the ethylene-dependent basic PR5 gene in tobacco plants.

Beta-1,3 glucan sulfate, but not beta-1,3 glucan, induces the salicylic acid signaling pathway in tobacco and Arabidopsis.

Sulfate substituents naturally occurring in biomolecules, such as oligosaccharides and polysaccharides, can play a critical role in major physiological functions in plants and animals. We show that laminarin, a beta-1,3 glucan with elicitor activity in tobacco (Nicotiana tabacum), becomes, after chemical sulfation, an inducer of the salicylic acid (SA) signaling pathway in tobacco and Arabidopsis thaliana. In tobacco cell suspensions, the oxidative burst induced by the laminarin sulfate PS3 was Ca2+ dependent but partially kinase independent, whereas laminarin triggered a strickly kinase-dependent oxidative burst. Cells treated with PS3 or laminarin remained fully responsive to a second application of laminarin or PS3, respectively, suggesting two distinct perception systems. In tobacco leaves, PS3, but not laminarin, caused electrolyte leakage and triggered scopoletin and SA accumulation. Expression of different families of Pathogenesis-Related (PR) proteins was analyzed in wild-type and mutant tobacco as well as in Arabidopsis. Laminarin induced expression of ethylene-dependent PR proteins, whereas PS3 triggered expression of ethylene- and SA-dependent PR proteins. In Arabidopsis, PS3-induced PR1 expression was also NPR1 (for nonexpressor of PR genes1) dependent. Structure-activity analysis revealed that (1) a minimum chain length is essential for biological activity of unsulfated as well as sulfated laminarin, (2) the sulfate residues are essential and cannot be replaced by other anionic groups, and (3) moderately sulfated beta-1,3 glucans are active. In tobacco, PS3 and curdlan sulfate induced immunity against Tobacco mosaic virus infection, whereas laminarin induced only a weak resistance. The results open new routes to work out new molecules suitable for crop protection.