The Arabidopsis thaliana Knockout Mutant for Phytochelatin Synthase1 (cad1-3) Is Defective in Callose Deposition, Bacterial Pathogen Defense and Auxin Content, But Shows an Increased Stem Lignification

Abstract

The enzyme phytochelatin synthase (PCS) has long been studied with regard to its role in metal(loid) detoxification in several organisms, i.e., plants, yeasts, and nematodes. It is in fact widely recognized that PCS detoxifies a number of heavy metals by catalyzing the formation of thiol-rich oligomers, namely phytochelatins, from glutathione and related peptides. However, recent investigations have highlighted other possible roles played by the PCS enzyme in the plant cell, e.g., the control of pathogen-triggered callose deposition. In order to examine novel aspects of Arabidopsis thaliana PCS1 (AtPCS1) functions and to elucidate its possible roles in the secondary metabolism, metabolomic data of A. thaliana wild-type and cad1-3 mutant were compared, the latter lacking AtPCS1. HPLC-ESI-MS analysis showed differences in the relative levels of metabolites from the glucosinolate and phenylpropanoid pathways between cad1-3 and wild-type plants. Specifically, in control (Cd-untreated) plants, higher levels of 4-methoxy-indol-3-ylmethylglucosinolate were found in cad1-3 plants vs. wild-type. Moreover, the cad1-3 mutant showed to be impaired in the deposit of callose after Cd exposure, suggesting that AtPCS1 protects the plant against the toxicity of heavy metals not only by synthesizing PCs, but also by contributing to callose deposition. In line with the contribution of callose in counteracting Cd toxicity, we found that another callose-defective mutant, pen2-1, was more sensitive to high concentrations of Cd than wild-type plants. Moreover, cad1-3 plants were more susceptible than wild-type to the hemibiotrophic bacterial pathogen Pseudomonas syringae. The metabolome also revealed differences in the relative levels of hydroxycinnamic acids and flavonols, with consequences on cell wall properties and auxin content, respectively. First, increased lignification in the cad1-3 stems was found, probably aimed at counteracting the entry of Cd into the inner tissues. Second, in cad1-3 shoots, increased relative levels of kaempferol 3,7 dirhamnoside and quercetin hexoside rhamnoside were detected. These flavonols are endogenous inhibitors of auxin transport in planta; auxin levels in both roots and shoots of the cad1-3 mutant were in fact lower than those of the wild-type. Overall, our data highlight novel aspects of AtPCS1 functions in A. thaliana.

Moonlighting Function of Phytochelatin Synthase1 in Extracellular Defense against Fungal Pathogens

“…As indicated by the aberrant accumulation of 4MI3G and IG-metabolism products in pcs1 plants upon flg22 or Cd 21 treatment, or inoculation with P. infestans (Clay et al, 2009;De Benedictis et al, 2018;Matern et al, 2019), PCS1, like PEN2, is required for the pathogen-inducible metabolism of IGs. Similarly, upon inoculation with Bgh, pen2 and pen4 hyperaccumulate 4MI3G (Fig.…”

“…The same enzyme is also needed for heavy metal ion tolerance, as glutathione is the precursor of heavy metal-chelating polypeptides, called phytochelatins (PCs), which in Arabidopsis are predominantly produced by PC synthase1 (PCS1; Grill et al, 1985Grill et al, , 1989Howden et al, 1995;Rea, 2006). Notably, pcs1 mutants display elevated cell death responses in leaves upon inoculation with the oomycete pathogen Phytophthora infestans, enhanced susceptibility to the bacterial phytopathogen Pseudomonas syringae DC3000, and reduced callose deposition upon treatment with the peptide-epitope flg22 derived from the motor protein of P. syringae, indicating that apart from a function in tolerance toward heavy metal ions, PCS1 has also a role in Arabidopsis immunity (Clay et al, 2009;Kühnlenz et al, 2015;De Benedictis et al, 2018). Moreover, given that a PCS from Caenorhabditis elegans was able to restore PC production, but not to revert the cell death phenotype in the pcs1 mutant background, it was suggested that PCS1 function in plant immunity is not dependent on PC accumulation (Kühnlenz et al, 2015).…”

An expanded role of the SNARE-containing regulon as it relates to the defense process that Glycine max has to Heterodera glycines

“…The work presented here experimentally tests components that would be expected to function within the regulon, but lie outside of the genetically defined apparatus (Humphry et al 2010). The selected genes encoding myosin XI and callose synthase (CS) relate to their previously reported defense roles and how the roles relate to vesicle trafficking (Ellinger et al 2013;Ellinger, Glöckner, et al 2014;Ellinger, Sode, et al 2014;Yang et al 2014;Peremyslov et al 2015;Leslie et al 2016;De Benedictis et al 2018;Sassmann et al 2018;Wang et al 2018). Furthermore, an examination of CS has been performed due to our previous observations that have shown an apparent increase in callose labeling at sites of engineered resistance in transgenic lines overexpressing GmSYP121-1 (GmSYP121-1-OE), leading to a successful defense response while in contrast GmSYP121-1-RNAi transgenic lines exhibit an impaired defense response and an apparent decrease in callose at sites of successful parasitism (Sharma et al 2016).…”

Arabidopsis phytochelatin synthase 1, but not phytochelatin synthesis, functions in extracellular defense against multiple fungal pathogens

“…As suggested by the accumulation of 4-methoxyindol-3-ylmethyl glucosinolate (4MI3G) in pcs1 plants upon flg22 or Cd 2+ treatment (Clay et al, 2009;De Benedictis et al, 2018), PCS1, like PEN2, is required for the pathogen-inducible metabolism of IGs. Upon inoculation with a fungal pathogen, pen2 and pen4 not only hyperaccumulate 4MI3G (Figure4B), but additionally are not able to produce indol-3-ylmethylamine (I3A, Figure 4A) and raphanusamic acid (RA, Figure 4D).…”