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The molecular actions of mitogen-activated protein kinases (MAPKs) are ultimately accomplished by the substrate proteins where phosphorylation affects their molecular properties and function(s), but knowledge regarding plant MAPK substrates is currently still fragmentary. Here, we uncovered a previously uncharacterized protein family consisting of three proline/serine-rich proteins (PRPs) that are substrates of stress-related MAPKs. We demonstrated the importance of a MAPK docking domain necessary for protein–protein interaction with MAPKs and consequently also for phosphorylation. The main phosphorylated site was mapped to a residue conserved between all three proteins, which when mutated to a non-phosphorylatable form, differentially affected their protein stability. Together with their distinct gene expression patterns, this differential accumulation of the three proteins upon phosphorylation probably contributes to their distinct function(s). Transgenic over-expression of PRP, the founding member, led to plants with enhanced resistance to Pseudomonas syringae pv. tomato DC3000. Older plants of the over-expressing lines have curly leaves and were generally smaller in stature. This growth phenotype was lost in plants expressing the phosphosite variant, suggesting a phosphorylation-dependent effect. Thus, this novel family of PRPs may be involved in MAPK regulation of plant development and / or pathogen resistance responses. As datamining associates PRP expression profiles with hypoxia or oxidative stress and PRP-overexpressing plants have elevated levels of reactive oxygen species, PRP may connect MAPK and oxidative stress signaling.

Kelch repeat-containing proteins are involved in diverse cellular processes, but only a small subset of plant kelch proteins has been functionally characterized. Thiocyanate-forming protein (TFP) from field-penny cress, Thlaspi arvense (Brassicaceae), is a representative of specifier proteins, a group of kelch proteins involved in plant specialized metabolism. As components of the glucosinolate-myrosinase system of the Brassicaceae, specifier proteins determine the profile of bioactive products formed when plant tissue is disrupted and glucosinolates are hydrolyzed by myrosinases. Here, we describe the crystal structure of TaTFP at a resolution of 1.4 Å. TaTFP crystallized as homodimer. Each monomer forms a six-blade β-propeller with a wide “top” and a narrower “bottom” opening with distinct strand-connecting loops protruding far beyond the lower propeller surface. Molecular modeling and mutational analysis identified residues for glucosinolate aglucone and Fe2+ cofactor binding within these loops. As the first experimentally determined structure of a plant kelch protein, the crystal structure of TaTFP not only enables more detailed mechanistic studies on glucosinolate breakdown product formation, but also provides a new basis for research on the diverse roles and mechanisms of other kelch proteins in plants.

As components of the glucosinolate-myrosinase system, specifier proteins contribute to the diversity of chemical defenses that have evolved in plants of the Brassicales order as a protection against herbivores and pathogens. Glucosinolates are thioglucosides that are stored separately from their hydrolytic enzymes, myrosinases, in plant tissue. Upon tissue disruption, glucosinolates are hydrolyzed by myrosinases yielding instable aglucones that rearrange to form defensive isothiocyanates. In the presence of specifier proteins, other products, namely simple nitriles, epithionitriles and organic thiocyanates, can be formed instead of isothiocyanates depending on the glucosinolate side chain structure and the type of specifier protein. The biochemical role of specifier proteins is largely unresolved. We have used two thiocyanate-forming proteins and one epithiospecifier protein with different substrate/product specificities to develop molecular models that, in conjunction with mutational analyses, allow us to propose an active site and docking arrangements with glucosinolate aglucones that may explain some of the differences in specifier protein specificities. Furthermore, quantum-mechanical calculations support a reaction mechanism for benzylthiocyanate formation including a catalytic role of the TFP involved. These results may serve as a basis for further theoretical and experimental investigations of the mechanisms of glucosinolate breakdown that will also help to better understand the evolution of specifier proteins from ancestral proteins with functions outside glucosinolate metabolism.

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Here we report the production of marker-free transgenic plants expressing phenolic compounds with high pharmacological value. Our strategy consisted in simultaneous delivery of lox-target and cre-containing constructs into the plant genome by cotransformation. In the Cre-vector, the cre recombinase gene was controlled by a seed-specific napin promoter. In the lox-target construct the selectable bar gene was placed between two lox sites in direct orientation, while a napin promoter driven vstI gene was inserted outside of the lox sites. Upon seed-specific cre induction the bar expression cassette was excised from the tobacco genome. Genetic and molecular analysis of T1 progeny plants indicated DNA excision in all 10 transgenic lines tested. RP-HPLC analysis demonstrated that the expression of the vstI gene resulted in accumulation of trans-resveratrol and its glycosylated derivative piceid in seeds of all marker free lines. These findings indicate that the seed-specific marker gene excision did not interfere with the expression of the gene of interest. Our data demonstrated the feasi of a developmentally controlled cre gene to mediate site-specific excision in tobacco very efficiently.