Lionel Hill

Abscisic acid has a key role in modulating diverse plant-pathogen interactions

We isolated an activation-tagged Arabidopsis (Arabidopsis thaliana) line, constitutive disease susceptibility2-1D (cds2-1D), that showed enhanced bacterial growth when challenged with various Pseudomonas syringae strains. Systemic acquired resistance and systemic PATHOGENESIS-RELATED GENE1 induction were also compromised in cds2-1D. The T-DNA insertion adjacent to NINE-CIS-EPOXYCAROTENOID DIOXYGENASE5 (NCED5), one of six genes encoding the abscisic acid (ABA) biosynthetic enzyme NCED, caused a massive increase in transcript level and enhanced ABA levels >2-fold. Overexpression of NCED genes recreated the enhanced disease susceptibility phenotype. NCED2, NCED3, and NCED5 were induced, and ABA accumulated strongly following compatible P. syringae infection. The ABA biosynthetic mutant aba3-1 showed reduced susceptibility to virulent P. syringae, and ABA, whether through exogenous application or endogenous accumulation in response to mild water stress, resulted in increased bacterial growth following challenge with virulent P. syringae, indicating that ABA suppresses resistance to P. syringae. Likewise ABA accumulation also compromised resistance to the biotrophic oomycete Hyaloperonospora arabidopsis, whereas resistance to the fungus Alternaria brassicicola was enhanced in cds2-1D plants and compromised in aba3-1 plants, indicating that ABA promotes resistance to this necrotroph. Comparison of the accumulation of salicylic acid and jasmonic acid in the wild type, cds2-1D, and aba3-1 plants challenged with P. syringae showed that ABA promotes jasmonic acid accumulation and exhibits a complex antagonistic relationship with salicylic acid. Our findings provide genetic evidence that the abiotic stress signal ABA also has profound roles in modulating diverse plant-pathogen interactions mediated at least in part by cross talk with the jasmonic acid and salicylic acid biotic stress signal pathways.

AtMYB12 regulates caffeoyl quinic acid and flavonol synthesis in tomato: expression in fruit results in very high levels of both types of polyphenol

Plant polyphenolics exhibit a broad spectrum of health-promoting effects when consumed as part of the diet, and there is considerable interest in enhancing the levels of these bioactive molecules in plants used as foods. AtMYB12 was originally identified as a flavonol-specific transcriptional activator in Arabidopsis thaliana, and this has been confirmed by ectopic expression in tobacco. AtMYB12 is able to induce the expression of additional target genes in tobacco, leading to the accumulation of very high levels of flavonols. When expressed in a tissue-specific manner in tomato, AtMYB12 activates the caffeoyl quinic acid biosynthetic pathway, in addition to the flavonol biosynthetic pathway, an activity which probably mirrors that of the orthologous MYB12-like protein in tomato. As a result of its broad specificity for transcriptional activation in tomato, AtMYB12 can be used to produce fruit with extremely high levels of multiple polyphenolic anti-oxidants. Our data indicate that transcription factors may have different specificities for target genes in different plants, which is of significance when designing strategies to improve metabolite accumulation and the anti-oxidant capacity of foods.

Disruption of adenosine-5'-phosphosulfate kinase in Arabidopsis reduces levels of sulfated secondary metabolites

Plants can metabolize sulfate by two pathways, which branch at the level of adenosine 5′-phosphosulfate (APS). APS can be reduced to sulfide and incorporated into Cys in the primary sulfate assimilation pathway or phosphorylated by APS kinase to 3′-phosphoadenosine 5′-phosphosulfate, which is the activated sulfate form for sulfation reactions. To assess to what extent APS kinase regulates accumulation of sulfated compounds, we analyzed the corresponding gene family in Arabidopsis thaliana. Analysis of T-DNA insertion knockout lines for each of the four isoforms did not reveal any phenotypical alterations. However, when all six combinations of double mutants were compared, the apk1 apk2 plants were significantly smaller than wild-type plants. The levels of glucosinolates, a major class of sulfated secondary metabolites, and the sulfated 12-hydroxyjasmonate were reduced approximately fivefold in apk1 apk2 plants. Although auxin levels were increased in the apk1 apk2 mutants, as is the case for most plants with compromised glucosinolate synthesis, typical high auxin phenotypes were not observed. The reduction in glucosinolates resulted in increased transcript levels for genes involved in glucosinolate biosynthesis and accumulation of desulfated precursors. It also led to great alterations in sulfur metabolism: the levels of sulfate and thiols increased in the apk1 apk2 plants. The data indicate that the APK1 and APK2 isoforms of APS kinase play a major role in the synthesis of secondary sulfated metabolites and are required for normal growth rates.

The microRNA miR393 re‐directs secondary metabolite biosynthesis away from camalexin and towards glucosinolates

flg22 treatment increases levels of miR393, a microRNA that targets auxin receptors. Over-expression of miR393 renders plants more resistant to biotroph pathogens and more susceptible to necrotroph pathogens. In contrast, over-expression of AFB1, an auxin receptor whose mRNA is partially resistant to miR393 degradation, renders the plant more susceptible to biotroph pathogens. Here we investigate the mechanism by which auxin signalling and miR393 influence plant defence. We show that auxin signalling represses SA levels and signalling. We also show that miR393 represses auxin signalling, preventing it from antagonizing SA signalling. In addition, over-expression of miR393 increases glucosinolate levels and decreases the levels of camalexin. Further studies on pathogen interactions in auxin signalling mutants revealed that ARF1 and ARF9 negatively regulate glucosinolate accumulation, and that ARF9 positively regulates camalexin accumulation. We propose that the action of miR393 on auxin signalling triggers two complementary responses. First, it prevents suppression of SA levels by auxin. Second, it stabilizes ARF1 and ARF9 in inactive complexes. As a result, the plant is able to mount a full SA response and to re-direct metabolic flow toward the most effective anti-microbial compounds for biotroph resistance. We propose that miR393 levels can fine-tune plant defences and prioritize resources.

Evidence that glucose 6-phosphate is imported as the substrate for starch synthesis by the plastids of developing pea embryos.

The aim of this work was to determine in what form carbon destined for starch synthesis crosses the membranes of plastids in developing pea (Pisum sativum L.) embryos. Plastids were isolated mechanically and incubated in the presence of ATP with the following 14C-labelled substrates: glucose, fructose, glucose 6-phosphate, glucose 1-phosphate, fructose 6-phosphate, fructose 1,6-bisphosphate, dihydroxyacetone phosphate. Glucose 6-phosphate was the only substrate that supported physiologically relevant rates of starch synthesis. Incorporation of label from glucose 6-phosphate into starch was dependent upon the integrity of the plastids and the presence of ATP. The rate of incorporation approached saturation at a glucose 6-phosphate concentration of less than 1 mM. It is argued that glucose 6-phosphate is likely to enter the plastid as the source of carbon for starch synthesis in vivo.

Pseudomonas sax Genes Overcome Aliphatic Isothiocyanate–Mediated Non-Host Resistance in Arabidopsis

Natural-product effectors of disease resistance in Arabidopsis reveal complementary disabling mechanisms in the pathogen. Most plant-microbe interactions do not result in disease; natural products restrict non-host pathogens. We found that sulforaphane (4-methylsulfinylbutyl isothiocyanate), a natural product derived from aliphatic glucosinolates, inhibits growth in Arabidopsis of non-host Pseudomonas bacteria in planta. Multiple sax genes (saxCAB/F/D/G) were identified in Pseudomonas species virulent on Arabidopsis. These sax genes are required to overwhelm isothiocyanate-based defenses and facilitate a disease outcome, especially in the young leaves critical for plant survival. Introduction of saxCAB genes into non-host strains enabled them to overcome these Arabidopsis defenses. Our study shows that aliphatic isothiocyanates, previously shown to limit damage by herbivores, are also crucial, robust, and developmentally regulated defenses that underpin non-host resistance in the Arabidopsis-Pseudomonas pathosystem.

A Serine Carboxypeptidase-Like Acyltransferase Is Required for Synthesis of Antimicrobial Compounds and Disease Resistance in Oats

Serine carboxypeptidase-like (SCPL) proteins have recently emerged as a new group of plant acyltransferases. These enzymes share homology with peptidases but lack protease activity and instead are able to acylate natural products. Several SCPL acyltransferases have been characterized to date from dicots, including an enzyme required for the synthesis of glucose polyesters that may contribute to insect resistance in wild tomato (Solanum pennellii) and enzymes required for the synthesis of sinapate esters associated with UV protection in Arabidopsis thaliana. In our earlier genetic analysis, we identified the Saponin-deficient 7 (Sad7) locus as being required for the synthesis of antimicrobial triterpene glycosides (avenacins) and for broad-spectrum disease resistance in diploid oat (Avena strigosa). Here, we report on the cloning of Sad7 and show that this gene encodes a functional SCPL acyltransferase, SCPL1, that is able to catalyze the synthesis of both N-methyl anthraniloyl- and benzoyl-derivatized forms of avenacin. Sad7 forms part of an operon-like gene cluster for avenacin synthesis. Oat SCPL1 (SAD7) is the founder member of a subfamily of monocot-specific SCPL proteins that includes predicted proteins from rice (Oryza sativa) and other grasses with potential roles in secondary metabolism and plant defense.

Anthocyanins Double the Shelf Life of Tomatoes by Delaying Overripening and Reducing Susceptibility to Gray Mold

Summary Shelf life is an important quality trait for many fruit, including tomatoes. We report that enrichment of anthocyanin, a natural pigment, in tomatoes can significantly extend shelf life. Processes late in ripening are suppressed by anthocyanin accumulation, and susceptibility to Botrytis cinerea, one of the most important postharvest pathogens, is reduced in purple tomato fruit. We show that reduced susceptibility to B. cinerea is dependent specifically on the accumulation of anthocyanins, which alter the spreading of the ROS burst during infection. The increased antioxidant capacity of purple fruit likely slows the processes of overripening. Enhancing the levels of natural antioxidants in tomato provides a novel strategy for extending shelf life by genetic engineering or conventional breeding.

The Role of the Novel Fem Protein VanK in Vancomycin Resistance in Streptomyces coelicolor

The non-pathogenic, non-glycopeptide-producing actinomycete Streptomyces coelicolor carries a cluster of seven genes (vanSRJKHAX) that confers inducible, high level resistance to vancomycin. The vanK gene has no counterpart in previously characterized vancomycin resistance clusters, yet vanK is required for vancomycin resistance in S. coelicolor. VanK belongs to the Fem family of enzymes, which add the branch amino acid(s) to the stem pentapeptide of peptidoglycan precursors. Upon exposure to vancomycin, the VanRS two-component system switches on expression of all seven van genes, and the VanHAX enzymes reprogram the cell wall such that precursors terminate d-Ala-d-lactate (Lac) rather than d-Ala-d-Ala, thus conferring resistance to vancomycin, which only binds d-Ala-d-Ala-containing precursors. Here we provide biochemical and genetic evidence that VanK is required for vancomycin resistance because the constitutively expressed FemX enzyme, encoded elsewhere on the chromosome, cannot recognize d-Lac-containing precursors as a substrate, whereas VanK can. Consistent with this view, d-Lac-containing precursors carrying the Gly branch are present in the wild type transiently exposed to vancomycin but are undetectable in a vanK mutant treated in the same way. Further, femX null mutants are viable in the presence of vancomycin but die in its absence. Because only VanK can recognize d-Lac-containing precursors, vancomycin-induced expression of VanHAX in a vanK mutant is lethal, and so vanK is required for vancomycin resistance.

A Novel Polyamine Acyltransferase Responsible for the Accumulation of Spermidine Conjugates in Arabidopsis Seed

Hydroxycinnamic acid amides are a class of secondary metabolites distributed widely in plants. We have identified two sinapoyl spermidine derivatives, N-((4′-O-glycosyl)-sinapoyl),N′-sinapoylspermidine and N,N′-disinapoylspermidine, which comprise the two major polyamine conjugates that accumulate in Arabidopsis thaliana seed. Using metabolic profiling of knockout mutants to elucidate the functions of members of the BAHD acyltransferase family in Arabidopsis, we have also identified two genes encoding spermidine disinapoyl transferase (SDT) and spermidine dicoumaroyl transferase (SCT) activities. At2g23510, which is expressed mainly in seeds, encodes a spermidine sinapoyl CoA acyltransferase (SDT) that is required for the production of disinapoyl spermidine and its glucoside in Arabidopsis seed. The structurally related BAHD enzyme encoded by At2g25150 is expressed specifically in roots and has spermidine coumaroyl CoA acyltransferase (SCT) activity both in vitro and in vivo.