Altering cold-regulated gene expression decouples the salicylic acid-growth trade-off in Arabidopsis.

In Arabidopsis (Arabidopsis thaliana), overproduction of salicylic acid (SA) increases disease resistance and abiotic stress tolerance but penalizes growth. This growth-defense trade-off has hindered the adoption of SA-based disease management strategies in agriculture. However, investigation of how SA inhibits plant growth has been challenging because many SA-hyperaccumulating Arabidopsis mutants have developmental defects due to the pleiotropic effects of the underlying genes. Here, we heterologously expressed a bacterial SA synthase gene in Arabidopsis and observed that elevated SA levels decreased plant growth and reduced the expression of cold-regulated (COR) genes in a dose-dependent manner. Growth suppression was exacerbated at below-ambient temperatures. Severing the SA-responsiveness of individual COR genes was sufficient to overcome the growth inhibition caused by elevated SA at ambient and below-ambient temperatures while preserving disease- and abiotic-stress-related benefits. Our results show the potential of decoupling SA-mediated growth and defense trade-offs for improving crop productivity.

Dirigent isoflavene-forming PsPTS2: 3D structure, stereochemical, and kinetic characterization comparison with pterocarpan-forming PsPTS1 homolog in pea.

Pea phytoalexins (-)-maackiain and (+)-pisatin have opposite C6a/C11a configurations, but biosynthetically how this occurs is unknown. Pea dirigent-protein (DP) PsPTS2 generates 7,2'-dihydroxy-4',5'-methylenedioxyisoflav-3-ene (DMDIF), and stereoselectivity toward four possible 7,2'-dihydroxy-4',5'-methylenedioxyisoflavan-4-ol (DMDI) stereoisomers was investigated. Stereoisomer configurations were determined using NMR spectroscopy, electronic circular dichroism, and molecular orbital analyses. PsPTS2 efficiently converted cis-(3R,4R)-DMDI into DMDIF 20-fold faster than the trans-(3R,4S)-isomer. The 4R-configured substrate's near ß-axial OH orientation significantly enhanced its leaving group abilities in generating A-ring mono-quinone methide (QM), whereas 4S-isomer's α-equatorial-OH was a poorer leaving group. Docking simulations indicated that the 4R-configured ß-axial OH was closest to Asp51, whereas 4S-isomer's α-equatorial OH was further away. Neither cis-(3S,4S)- nor trans-(3S,4R)-DMDIs were substrates, even with the former having C3/C4 stereochemistry as in (+)-pisatin. PsPTS2 used cis-(3R,4R)-7,2'-dihydroxy-4'-methoxyisoflavan-4-ol [cis-(3R,4R)-DMI] and C3/C4 stereoisomers to give 2',7-dihydroxy-4'-methoxyisoflav-3-ene (DMIF). DP homologs may exist in licorice (Glycyrrhiza pallidiflora) and tree legume Bolusanthus speciosus, as DMIF occurs in both species. PsPTS1 utilized cis-(3R,4R)-DMDI to give (-)-maackiain 2200-fold more efficiently than with cis-(3R,4R)-DMI to give (-)-medicarpin. PsPTS1 also slowly converted trans-(3S,4R)-DMDI into (+)-maackiain, reflecting the better 4R configured OH leaving group. PsPTS2 and PsPTS1 provisionally provide the means to enable differing C6a and C11a configurations in (+)-pisatin and (-)-maackiain, via identical DP-engendered mono-QM bound intermediate generation, which PsPTS2 either re-aromatizes to give DMDIF or PsPTS1 intramolecularly cyclizes to afford (-)-maackiain. Substrate docking simulations using PsPTS2 and PsPTS1 indicate cis-(3R,4R)-DMDI binds in the anti-configuration in PsPTS2 to afford DMDIF, and the syn-configuration in PsPTS1 to give maackiain.

Impact of SDH Mutations in Alternaria solani on Recently Developed SDHI Fungicides Adepidyn and Solatenol.

Early blight, caused by Alternaria solani, is observed annually in all midwestern potato production areas. The use of foliar fungicides remains a primary management strategy. However, A. solani has developed reduced sensitivity or resistance to many single-site fungicides such as quinone outside inhibitor (QoI, FRAC group 11), succinate dehydrogenase inhibitor (SDHI, FRAC group 7), demethylation inhibitor (DMI, FRAC group 3), and anilinopyrimidine (AP, FRAC group 9) fungicides. Boscalid, fluopyram, solatenol, and adepidyn are EPA-registered SDHI fungicides used commercially on a variety of crops, including potato. Five SDH mutations have been characterized previously in A. solani that affect the efficacy of boscalid while only one of these mutations has been demonstrated to negatively affect fluopyram efficacy. Conidial germination assays were used to determine if a shift in sensitivity has occurred in these SDHI fungicides. A. solani isolates collected prior to the commercial application of SDHI fungicides (baseline) were compared with recently collected isolates (nonbaseline). Greenhouse evaluations were conducted also to evaluate the efficacy of boscalid, fluopyram, solatenol, and adepidyn on A. solani isolates possessing individual SDH mutations. Additionally, field trials were conducted to determine the effects of application of these SDHI fungicides on the frequency of SDH mutations. Fluopyram, solatenol, and adepidyn had high intrinsic activity against A. solani when compared with boscalid, based on in vitro assays. The application of adepidyn and solatenol resulted in greater early blight control than the application of boscalid and fluopyram in greenhouse experiments. Molecular characterization of A. solani isolates collected from the field trials determined that the frequency of the H134R-mutation can increase in response to more recently developed SDHI fungicides. In contrast, the H278R/Y- and H133R-mutations decreased to the point of being nearly absent in these field experiments.

Macroevolution of leaf defenses and secondary metabolites across the genus Helianthus.

Leaf defenses are widely recognized as key adaptations and drivers of plant evolution. Across environmentally diverse habitats, the macroevolution of leaf defenses can be predicted by the univariate trade-off model, which predicts that defenses are functionally redundant and thus trade off, and the resource availability hypothesis, which predicts that defense investment is determined by inherent growth rate and that higher defense will evolve in lower resource environments. Here, we examined the evolution of leaf physical and chemical defenses and secondary metabolites in relation to environmental characteristics and leaf economic strategy across 28 species of Helianthus (the sunflowers). Using a phylogenetic comparative approach, we found few evolutionary trade-offs among defenses and no evidence for defense syndromes. We also found that leaf defenses are strongly related to leaf economic strategy, with higher defense in more resource-conservative species, although there is little support for the evolution of higher defense in low-resource habitats. A wide variety of physical and chemical defenses predict resistance to different insect herbivores, fungal pathogens, and a parasitic plant, suggesting that most sunflower defenses are not redundant in function and that wild Helianthus represents a rich source of variation for the improvement of crop sunflower.

Dirigent protein subfamily function and structure in terrestrial plant phenol metabolism.

Aquatic plant transition to land, and subsequent terrestrial plant species diversification, was accompanied by the emergence and massive elaboration of plant phenol chemo-diversity. Concomitantly, dirigent protein (DP) and dirigent-like protein subfamilies, derived from large multigene families, emerged and became extensively diversified. DP biochemical functions as gateway entry points into new and diverse plant phenol skeletal types then markedly expanded. DPs have at least eight non-uniformly distributed subfamilies, with different DP subfamily members of known biochemical/physiological function now implicated as gateway entries to lignan, lignin, aromatic diterpenoid, pterocarpan and isoflavene pathways. While some other DP subfamily members have jacalin domains, both these and indeed the majority of DPs throughout the plant kingdom await discovery of their biochemical roles. Methods and approaches were developed to discover DP biochemical function as gateway entry points to distinct plant phenol skeletal types in land plants. Various DP 3D X-ray structural determinations enabled structure-based comparative sequence analysis and modeling to understand similarities and differences among the different DP subfamilies. We consider that the core DP ß-barrel fold and associated characteristics are likely common to all DPs, with several residues conserved and nearly invariant. There is also considerable variation in residue composition and topography of the putative substrate binding pockets, as well as substantial differences in several loops, such as the ß1-ß2 loop. All DPs likely bind and stabilize quinone methide intermediates, while guiding distinctive regio- and/or stereo-chemical entry into Nature's chemo-diverse land plant phenol metabolic classes.

(+)-Pisatin biosynthesis: from (-) enantiomeric intermediates via an achiral 7,2'-dihydroxy-4',5'-methylenedioxyisoflav-3-ene.

(+)-Pisatin, produced by peas (Pisum sativum L.), is an isoflavonoid derivative belonging to the pterocarpan family. It was the first chemically identified phytoalexin, and subsequent research has demonstrated that most legumes produce pterocarpans with the opposite stereochemistry. Studies on the biosynthesis of (+)-pisatin have shown that (-) enantiomeric compounds are intermediates in (+)-pisatin synthesis. However, the steps from the (-)-7,2'-dihydroxy-4',5'-methylenedioxyisoflavanone [(-)-sophorol] intermediate to (+)-6a-hydroxymaackiain intermediate are undetermined. Chemical reduction of (-)-sophorol using sodium borohydride (NaBH4) produced two isomers of (-)-7,2'-dihydroxy-4',5'-methylenedioxyisoflavanol [(-)-DMDI] with optimal UV absorbance at 299.3 and 300.5 nm, respectively. In contrast, enzymatic reduction of (-)-sophorol by the pea enzyme sophorol reductase (SOR) produced only the 299.3 nm (-)-DMDI isomer. Proton nuclear magnetic resonance ((1)H NMR) analysis of the 299.3 nm (-)-DMDI isomer demonstrated that this isomer had the same NMR spectrum as previously reported for cis-isoflavanol isomers, indicating that cis-(-)-DMDI is an intermediate in (+)-pisatin biosynthesis. Enzyme assays using protein extracts from pea tissue treated with CuCl2 as an elicitor converted the cis-(-)-DMDI isomer into an achiral isoflavene, 7,2'-dihydroxy-4',5'-methylenedioxyisoflav-3-ene (DMDIF), and the trans-(-)-DMDI isomer was not metabolized by the same protein preparation. A comparison of the enzyme activities on cis-(-)-DMDI with protein preparations from elicited tissue versus non-elicited tissue showed a threefold increase in the amount of activity in the proteins from the elicited tissue. Proteins from the elicited tissues of alfalfa, bean, and chickpea converted cis-(-)-DMDI into either (-)-maackiain and/or (-)-sophorol, while proteins from the elicited tissues of broccoli and pepper produced no detectable product. These results are consistent with the involvement of cis-(-)-DMDI and the achiral DMDIF as intermediates in (+)-pisatin biosynthesis.

Inducible expression of Pisum sativum xyloglucan fucosyltransferase in the pea root cap meristem, and effects of antisense mRNA expression on root cap cell wall structural integrity.

Mitosis and cell wall synthesis in the legume root cap meristem can be induced and synchronized by the nondestructive removal of border cells from the cap periphery. Newly synthesized cells can be examined microscopically as they differentiate progressively during cap development, and ultimately detach as a new population of border cells. This system was used to demonstrate that Pisum sativum L. fucosyl transferase (PsFut1) mRNA expression is strongly expressed in root meristematic tissues, and is induced >2-fold during a 5-h period when mitosis in the root cap meristem is increased. Expression of PsFut1 antisense mRNA in pea hairy roots under the control of the CaMV35S promoter, which exhibits meristem localized expression in pea root caps, resulted in a 50-60% reduction in meristem localized endogenous PsFut1 mRNA expression measured using whole mount in situ hybridization. Changes in gross levels of cell wall fucosylated xyloglucan were not detected, but altered surface localization patterns were detected using whole mount immunolocalization with CCRC-M1, an antibody that recognizes fucosylated xyloglucan. Emerging hairy roots expressing antisense PsFut1 mRNA appeared normal macroscopically but scanning electron microscopy of tissues with altered CCRC-M1 localization patterns revealed wrinkled, collapsed cell surfaces. As individual border cells separated from the cap periphery, cell death occurred in correlation with extrusion of cellular contents through breaks in the wall.