Chloride, glutathiones, and insect-derived elicitors introduced into the xylem trigger electrical signaling.

Ricca assays allow the direct introduction of compounds extracted from plants or the organisms that attack them into the leaf vasculature. Using chromatographic fractionation of Arabidopsis (Arabidopsis thaliana) leaf extracts, we found glutamate was the most active low mass elicitor of membrane depolarization. However, other known elicitors of membrane depolarization are generated in the wound response. These include unstable aglycones generated by glucosinolate (GSL) breakdown. None of the aglycone-derived GSL-breakdown products, including nitriles and isothiocyanates, that we tested using Ricca assays triggered electrical activity. Instead, we found that glutathione and the GSL-derived compound sulforaphane glutathione triggered membrane depolarizations. These findings identify a potential link between GSL breakdown and glutathione in the generation of membrane depolarizing signals. Noting that the chromatographic fractionation of plant extracts can dilute or exchange ions, we found that Cl- caused glutamate receptor-like3.3-dependent membrane depolarizations. In summary, we show that, in addition to glutamate, glutathione derivatives as well as chloride ions will need to be considered as potential elicitors of wound-response membrane potential change. Finally, by introducing aphid (Brevicoryne brassicae) extracts or the flagellin-derived peptide flg22 into the leaf vasculature we extend the use of Ricca assays for the exploration of insect/plant and bacteria/plant interactions.

Mechanosensation in leaf veins.

Whether the plant vasculature has the capacity to sense touch is unknown. We developed a quantitative assay to investigate touch-response electrical signals in the leaves and veins of Arabidopsis thaliana. Mechanostimulated electrical signaling in leaves displayed strong diel regulation. Signals of full amplitude could be generated by repeated stimulation at the same site after approximately 90 minutes. However, the signals showed intermediate amplitudes when repeatedly stimulated in shorter timeframes. Using intracellular electrodes, we detected touch-response membrane depolarizations in the phloem. On the basis of this, we mutated multiple Arabidopsis H+-ATPase (AHA) genes expressed in companion cells. We found that aha1 aha3 double mutants attenuated touch-responses, and this was coupled to growth rate reduction. Moreover, propagating membrane depolarizations could be triggered by mechanostimulating the exposed primary vasculature of wild-type plants but not of aha1 aha3 mutants. Primary veins have autonomous mechanosensory properties which depend on P-type proton pumps.

Jasmonate Precursor Biosynthetic Enzymes LOX3 and LOX4 Control Wound-Response Growth Restriction.

Wound-response plant growth restriction requires the synthesis of potent mediators called jasmonates (JAs). Four 13-lipoxygenases (13-LOXs) produce JA precursors in Arabidopsis (Arabidopsis thaliana) leaves, but the 13-LOXs responsible for growth restriction have not yet been identified. Through loss-of-function genetic analyses, we identified LOX3 and LOX4 as the principal 13-LOXs responsible for vegetative growth restriction after repetitive wounding. Additional genetic studies were carried out in the gain-of-function fatty acid oxygenation 2 (fou2) mutant that, even when undamaged, shows JA-dependent leaf growth restriction. The fou2 lox3 lox4 triple mutant suppressed the fou2 JA-dependent growth phenotype, confirming that LOX3 and LOX4 function in leaf growth restriction. The fou2 mutation affects the TWO PORE CHANNEL1 (TPC1) ion channel. Additional genetic approaches based on this gene were used to further investigate LOX3 function in relation to leaf growth. To activate LOX3-dependent JA production in unwounded plants, we employed hyperactive TPC1 variants. Expression of the TPC1ΔCa i variant in phloem companion cells caused strongly reduced rosette growth in the absence of wounding. Summarizing, in parallel to their established roles in male reproductive development in Arabidopsis, LOX3 and LOX4 control leaf growth rates after wounding. The process of wound-response growth restriction can be recapitulated in unwounded plants when the LOX3 pathway is activated genetically using a hyperactive vacuolar cation channel.

Comparative Analysis of Phosphoproteome Remodeling After Short Term Water Stress and ABA Treatments versus Longer Term Water Stress Acclimation.

Several studies have used short term dehydration, osmotic stress or Abscisic Acid (ABA) treatments to identify the initial protein phosphorylation-dephosphorylation responses to drought and low water potential or ABA treatments. However, longer term drought acclimation leads to altered expression of many kinases and phosphatases suggesting that it may also produce unique changes in phosphoproteome composition. To get a better overview of the state of drought-related phosphoproteomics and investigate this question of short versus longer term phosphoproteome regulation, we compared three Arabidopsis thaliana studies analyzing short term phosphoproteome changes to recent data from our laboratory analyzing phosphoproteome changes after a longer drought acclimation treatment. There was very little overlap of phosphoproteins with putative stress-induced phosphorylation or dephosphorylation among these studies. While some of this is due to technical limitations and limited coverage of the phosphoproteome achieved by each study, biological differences and the type of stress treatment used also play a role. This comparative analysis emphasized how both short and long term analysis of physiologically relevant stress treatments, as well as validation of phosphoproteomic data, will be needed to move past just scratching the surface of the stress phosphoproteome. In drought acclimation experiments, distinguishing between changes in protein abundance versus phosphorylation stoichiometry is a key challenge. We discuss initial work in using Arabidopsis seedling transient expression combined with Phos-tag gel analysis as a way to validate drought-induced phosphorylation-dephosphorylation of candidate proteins.

Dynamic proline metabolism: importance and regulation in water limited environments.

Drought-induced proline accumulation observed in many plant species has led to the hypothesis that further increases in proline accumulation would promote drought tolerance. Here we discuss both previous and new data showing that proline metabolism and turnover, rather than just proline accumulation, functions to maintain growth during water limitation. Mutants of Δ (1)-Pyrroline-5-Carboxylate Synthetase1 (P5CS1) and Proline Dehydrogenase1 (PDH1), key enzymes in proline synthesis and catabolism respectively, both have similar reductions in growth during controlled soil drying. Such results are consistent with patterns of natural variation in proline accumulation and with evidence that turnover of proline can act to buffer cellular redox status during drought. Proline synthesis and catabolism are regulated by multiple cellular mechanisms, of which we know only a few. An example of this is immunoblot detection of P5CS1 and PDH1 showing that the Highly ABA-induced (HAI) protein phosphatase 2Cs (PP2Cs) have different effects on P5CS1 and PDH1 protein levels despite having similar increases in proline accumulation. Immunoblot data also indicate that both P5CS1 and PDH1 are subjected to unknown post-translational modifications.