Anna Rita Trentin

Biochemical and quantitative proteomics investigations in Arabidopsis ggt1 mutant leaves reveal a role for the gamma‐glutamyl cycle in plant's adaptation to environment

The existence of a gamma‐glutamyl cycle consisting of intracellular GSH synthesis, extrusion to the apoplastic space and recovery by gamma‐glutamyl transferase (GGT)‐assisted degradation into its constituent amino acids, has been demonstrated in plants. To address the significance of this cycle in plant cells, we performed integrated biochemical, immunocytochemical, and quantitative proteomics analyses in the Arabidopsis thaliana ggt1 knockout mutant (lacking apoplastic GGT1 isoform) and its corresponding wild‐type (WT). The ggt1 knockout leaves exhibited an increased ascorbate and GSH content, increased apoplastic GSH content, and enhanced protein carbonylations in the low‐molecular weight range compared to WT. The combined iTRAQ and LC‐MS/MS‐based quantitative proteomics approach identified 70 proteins (out of 1013 identified proteins) whose abundance was significantly different in leaves of ggt1 mutant compared to WT, with a fold change ≥1.5. Mining of the proteome data for GSH‐associated genes showed that disruption of gamma‐glutamyl cycle in ggt1 knockout‐leaves was associated with the induction of genes encoding four GSTs in the phi class (GSTF2, GSTF6, GSTF9, and GSTF10), a GSH peroxidase (GPX1), and glyoxylase II. Proteins with a lower abundance compared to the WT are involved in chloroplast functions, carbohydrate/maltose metabolism, and vegetative storage protein synthesis. Present findings suggest that GGT1 plays a role in redox signaling. The disruption of the gamma‐glutamyl cycle in the ggt1 mutant results in pleiotropic effects related to biotic and abiotic stress response, antioxidant metabolism, senescence, carbohydrate metabolism, and photosynthesis, with strong implications for plant adaptation to the environment.

Compensatory expression and substrate inducibility of γ-glutamyl transferase GGT2 isoform in Arabidopsis thaliana

γ-Glutamyl transferases (GGT; EC 2.3.2.2) are glutathione-degrading enzymes that are represented in Arabidopsis thaliana by a small gene family of four members. Two isoforms, GGT1 and GGT2, are apoplastic, sharing broad similarities in their amino acid sequences, but they are differently expressed in the tissues: GGT1 is expressed in roots, leaves, and siliques, while GGT2 was thought to be expressed only in siliques. It is demonstrated here that GGT2 is also expressed in wild-type roots, albeit in very small amounts. GGT2 expression is enhanced in ggt1 knockout mutants, suggesting a compensatory effect to restore GGT activity in the root apoplast. Supplementation with 100 μM glutathione (GSH) resulted in the up-regulation of GGT2 gene expression in wild-type and ggt1 knockout roots, and of GGT1 gene expression in wild-type roots. Glutathione recovery was hampered by the GGT inhibitor serine/borate, suggesting a major role for apoplastic GGTs in this process. These findings can explain the ability of ggt1 knockout mutants to retrieve exogenously added glutathione from the growth medium.

Proteomic analysis of a compatible interaction between sugarcane and Sporisorium scitamineum

Smut caused by Sporisorium scitamineum is one of the important diseases of sugarcane with global significance. Despite the intriguing nature of sugarcane, S. scitamineum interaction, several pertinent aspects remain unexplored. This study investigates the proteome level alterations occurring in the meristem of a S. scitamineum infected susceptible sugarcane cultivar at whip emergence stage. Differentially abundant proteins were identified by 2DE coupled with MALDI‐TOF/TOF‐MS. Comprehensively, 53 sugarcane proteins identified were related to defence, stress, metabolism, protein folding, energy, and cell division; in addition, a putative effector of S. scitamineum, chorismate mutase, was identified. Transcript expression vis‐à‐vis the activity of phenylalanine ammonia lyase was relatively higher in the infected meristem. Abundance of seven candidate proteins in 2D gel profiles was in correlation with its corresponding transcript expression levels as validated by qRT‐PCR. Furthermore, this study has opened up new perspectives on the interaction between sugarcane and S. scitamineum.

Proteome readjustments in the apoplastic space of Arabidopsis thaliana ggt1 mutant leaves exposed to UV-B radiation

Ultraviolet-B radiation acts as an environmental stimulus, but in high doses it has detrimental effects on plant metabolism. Plasma membranes represent a major target for Reactive Oxygen Species (ROS) generated by this harmful radiation. Oxidative reactions occurring in the apoplastic space are counteracted by antioxidative systems mainly involving ascorbate and, to some extent, glutathione. The occurrence of the latter and its exact role in the extracellular space are not well documented, however. In Arabidopsis thaliana, the gamma-glutamyl transferase isoform (GGT1) bound to the cell wall takes part in the so-called gamma-glutamyl cycle for extracellular glutathione degradation and recovery, and may be implicated in redox sensing and balance. In this work, oxidative conditions were imposed with Ultraviolet-B radiation (UV-B) and studied in redox altered ggt1 mutants. The response of ggt1 knockout Arabidopsis leaves to UV-B radiation was assessed by investigating changes in extracellular glutathione and ascorbate content and their redox state, and in apoplastic protein composition. Our results show that, on UV-B exposure, soluble antioxidants respond to the oxidative conditions in both genotypes. Rearrangements occur in their apoplastic protein composition, suggesting an involvement of Hydrogen Peroxide (H2O2), which may ultimately act as a signal. Other important changes relating to hormonal effects, cell wall remodeling, and redox activities are discussed. We argue that oxidative stress conditions imposed by UV-B and disruption of the gamma-glutamyl cycle result in similar stress-induced responses, to some degree at least. Data are available via ProteomeXchange with identifier PXD001807.

Gamma-glutamyl cycle in plants: a bridge connecting the environment to the plant cell?

1 Dipartimento di Agronomia Animali Alimenti Risorse Naturali e Ambiente (DAFNAE), University of Padova, Legnaro, Italy, 2 Research Laboratory for Biotechnology and Biochemistry, Kathmandu, Nepal, 3 GRADE (Global Research Arch for Developing Education) Academy Private Limited, Birgunj, Nepal, 4 Organization for Educational Initiatives, University of Tsukuba, Tsukuba, Japan, 5 Department of Anatomy I, Showa University School of Medicine, Shinagawa, Japan

Apoplastic gamma-glutamyl transferase activity encoded by GGT1 and GGT2 is important for vegetative and generative development

Gamma-glutamyl transferase (GGT; EC 2.3.2.2) is the only enzyme capable of degrading glutathione (GSH) in extra-cytosolic spaces. In plant cells, the GGT1 and GGT2 isoforms are located in the apoplast, bound respectively to the cell wall and the plasma membrane. GGT1 is expressed throughout plants, mainly in the leaves and vascular system, while GGT2 is more specifically expressed in seeds and trichomes, and weakly in roots. Their role in plant physiology remains to be clarified, however. Obtaining the ggt1/ggt2 double mutant can offer more clues than the corresponding single mutants, and to prevent any compensatory expression between the two isoforms. In this work, ggt1/ggt2 RNAi (RNA interference) lines were generated and characterized in the tissues where both isoforms are expressed. The seed yield was lower in the ggt1/ggt2 RNAi plants due to the siliques being fewer in number and shorter in length, with no changes in thiols and sulfur compounds. Proline accumulation and delayed seed germination were seen in one line. There were also fewer trichomes (which contain high levels of GSH) in the RNAi lines than in the wild type, and the root elongation rate was slower. In conclusion, apoplastic GGT silencing induces a decrease in the number of organs with a high GSH demand (seeds and trichomes) as a result of resource reallocation to preserve integrity and composition.

Nitrate affects transcriptional regulation of UPBEAT1 and ROS localisation in roots of Zea mays L.

Nitrogen (N) is an indispensable nutrient for crops but its availability in agricultural soils is subject to considerable fluctuation. Plants have developed plastic responses to external N fluctuations in order to optimise their development. The coordinated action of nitric oxide and auxin seems to allow the cells of the root apex transition zone (TZ) of N-deprived maize to rapidly sense nitrate (NO3 - ). Preliminary results support the hypothesis that reactive oxygen species (ROS) signalling might also have a role in this pathway, probably through a putative maize orthologue of UPBEAT1 (UPB1). To expand on this hypothesis and better understand the different roles played by different root portions, we investigated the dynamics of ROS production, and the molecular and biochemical regulation of the main components of ROS production and scavenging in tissues of the meristem, transition zone, elongation zone and maturation zone of maize roots. The results suggest that the inverse regulation of ZmUPB1 and ZmPRX112 transcription observed in cells of the TZ in response to nitrogen depletion or NO3 - supply affects the balance between superoxide (O2 •- ) and hydrogen peroxide (H2 O2 ) in the root apex and consequently triggers differential root growth. This explanation is supported by additional results on the overall metabolic and transcriptional regulation of ROS homeostasis.