Guillermo Baena

The vesicle trafficking SYNTAXIN OF PLANTS132 (SYP132) drives hormone-regulated endocytic traffic to suppress the density and function of plasma membrane (PM) H⁺-ATPases. In response to bacterial pathogens, it also promotes secretory traffic of antimicrobial pathogenesis-related (PR) proteins. These seemingly opposite actions of SYP132 raise questions about the mechanistic connections between the two, likely independent, membrane trafficking pathways intersecting plant growth and immunity. To study SYP132 and associated trafficking of PM H⁺-ATPase 1 (AHA1) and PATHOGENESIS-RELATED PROTEIN1 (PR1) during pathogenesis, we used the virulent Pseudomonas syringae pv. tomato DC3000 (Pst DC3000) bacteria for infection of Arabidopsis (Arabidopsis thaliana) plants. SYP132 overexpression suppressed bacterial infection in plants through the stomatal route. However, bacterial infection was enhanced when bacteria were infiltrated into leaf tissue to bypass stomatal defenses. Tracking time-dependent changes in native AHA1 and SYP132 abundance, cellular distribution, and function, we discovered that bacterial pathogen infection triggers AHA1 and SYP132 internalization from the plasma membrane. AHA1 bound to SYP132 through its regulatory SNARE Habc domain, and these interactions affected PM H⁺-ATPase traffic. Remarkably, using the Arabidopsis aha1 mutant, we discovered that AHA1 is essential for moderating SYP132 abundance and associated secretion of PR1 at the plasma membrane for pathogen defense. Thus, we show that during pathogenesis SYP132 coordinates AHA1 with opposing effects on the traffic of AHA1 and PR1.

Phosphoenolpyruvate carboxylase (PEPC) is an enzyme with key roles in carbon and nitrogen metabolisms. The mechanisms that control enzyme stability and turnover are not well known. This paper investigates the degradation of PEPC via selective autophagy, including the role of the monoubiquitination of the enzyme in this process. In Arabidopsis, the genetic inhibition of autophagy increases the amount of monoubiquitinated PEPC in the atg2, atg5, and atg18a lines. The same is observed in nbr1, which is deficient in a protein that recruits monoubiquitinated substrates for selective autophagy. In cultured tobacco cells, the chemical inhibition of the degradation of autophagic substrates increases the quantity of PEPC proteins. When the formation of the autophagosome is blocked with 3-methyladenine (3-MA), monoubiquitinated PEPC accumulates as a result. Finally, pull-down experiments with a truncated version of NBR1 demonstrate the recovery of intact and/or fragmented PEPC in Arabidopsis leaves and roots, as well as cultured tobacco cells. Taken together, the results show that a fraction of PEPC is cleaved via selective autophagy and that the monoubiquitination of the enzyme has a role in its recruitment towards this pathway. Although autophagy seems to be a minor pathway, the results presented here increase the knowledge about the role of monoubiquitination and the regulation of PEPC degradation.

Main conclusion Carbonylation inactivates sorghum C ₄ PEPCase while nitrosylation has little impact on its activity but holds back carbonylation. This interplay could be important to preserve photosynthetic C₄ PEPCase activity in salinity. Previous work had shown that nitric acid (NO) increased phosphoenolpyruvate carboxylase kinase (PEPCase-k) activity, promoting the phosphorylation of phosphoenolpyruvate carboxylase (PEPCase) in sorghum leaves (Monreal et al. in Planta 238:859–869, 2013b). The present work investigates the effect of NO on C₄ PEPCase in sorghum leaves and its interplay with carbonylation, an oxidative modification frequently observed under salt stress. The PEPCase of sorghum leaves could be carbonylated in vitro and in vivo, and this post-translational modification (PTM) was accompanied by a loss of its activity. Similarly, PEPCase could be S-nitrosylated in vitro and in vivo, and this PTM had little impact on its activity. The S-nitrosylated PEPCase showed increased resistance towards subsequent carbonylation, both in vitro and in vivo. Under salt shock, carbonylation of PEPCase increased in parallel with decreased S-nitrosylation of the enzyme. Subsequent increase of S-nitrosylation was accompanied by decreased carbonylation. Taken together, the results suggest that S-nitrosylation could contribute to maintain C₄ PEPCase activity in stressed sorghum plants. Thus, salt-induced NO synthesis would be protecting photosynthetic PEPCase activity from oxidative inactivation while promoting its phosphorylation, which will guarantee its optimal functioning in suboptimal conditions.

Phosphoenolpyruvate carboxylase (PEPC; E.C. 4.1.1.31) was characterized in developing and germinating sorghum seeds, focusing on the transcript and polypeptide abundance of multiple plant-type phosphoenolpyruvate carboxylase (PTPC) genes, and the post-translational modification of each isoenzyme by phosphorylation versus monoubiquitination during germination. We observed high levels of SbPPC4 (Sb07g014960) transcripts during early development (stage I), and extensive transcript abundance of SbPPC2 (Sb02g021090) and SbPPC3 (Sb04g008720) throughout the entire life cycle of the seed. Although tandem mass spectrometry (MS) analysis of immunopurified PTPC indicated that four different PTPC isoenzymes were expressed in the developing and germinating seeds, SbPPC3 was the most abundant isozyme of the developing seed, and of the embryo and the aleurone layer of germinating seeds. In vivo phosphorylation of the different PTPC isoenzymes at their conserved N-terminal seryl phosphorylation site during germination was also established by MS/MS analysis. Furthermore, three of the four isoenzymes were partially monoubiquitinated, with MS/MS pinpointing SbPPC2 and SbPPC3 monoubiquitination at the conserved Lys-630 and Lys-624 residues, respectively. Our results demonstrate that monoubiquitination and phosphorylation simultaneously occur in vivo with different PTPC isozymes during seed germination. In addition, we show that PTPC monoubiquitination in germinating sorghum seeds always increases at stage II (emergence of the radicle), is maintained during the aerobic period of rapid cell division and reserve mobilization, and remains relatively constant until stage IV–V when coleoptiles initiate the formation of the photosynthetic tissues.