Antonio Masi

Changes in antioxidant and pigment pool dimensions in UV-B irradiated maize seedlings

Abstract Ultraviolet-B (UV-B) radiation (280–320 nm) is an environmental challenge affecting a number of metabolic functions through the generation of reactive oxygen species (ROS). Plants protect themselves from this harmful radiation by synthesizing flavonoids, which act as a screen inside the epidermal cell layer, and by making adjustments to the antioxidant systems at both cell and whole organism level. This study describes the flavonoid content, the photosynthetic pigment composition and the proline, tocopherol and ascorbate content in UV-B exposed maize plants. Following exposure, the tocopherol content was slightly, but significantly lower, pointing to the membrane environment as a primary target for UV-B radiation. The water-soluble antioxidant content was largely unaffected, but an enhanced turnover in the ascorbate–glutathione cycle might be needed for tocopherol regeneration.

A plant spermine oxidase/dehydrogenase regulated by the proteasome and polyamines

Polyamine oxidases (PAOs) are flavin-dependent enzymes involved in polyamine catabolism. In Arabidopsis five PAO genes (AtPAO1-AtPAO5) have been identified which present some common characteristics, but also important differences in primary structure, substrate specificity, subcellular localization, and tissue-specific expression pattern, differences which may suggest distinct physiological roles. In the present work, AtPAO5, the only so far uncharacterized AtPAO which is specifically expressed in the vascular system, was partially purified from 35S::AtPAO5-6His Arabidopsis transgenic plants and biochemically characterized. Data presented here allow AtPAO5 to be classified as a spermine dehydrogenase. It is also shown that AtPAO5 oxidizes the polyamines spermine, thermospermine, and N(1)-acetylspermine, the latter being the best in vitro substrate of the recombinant enzyme. AtPAO5 also oxidizes these polyamines in vivo, as was evidenced by analysis of polyamine levels in the 35S::AtPAO5-6His Arabidopsis transgenic plants, as well as in a loss-of-function atpao5 mutant. Furthermore, subcellular localization studies indicate that AtPAO5 is a cytosolic protein undergoing proteasomal control. Positive regulation of AtPAO5 expression by polyamines at the transcriptional and post-transcriptional level is also shown. These data provide new insights into the catalytic properties of the PAO gene family and the complex regulatory network controlling polyamine metabolism.

Protein Expression Changes in Maize Roots in Response to Humic Substances

Humic substances are known to affect plant metabolism at different levels. We characterized humic substances extracted from earthworm feces by diffuse reflectance infrared Fourier transform (DRIFT) spectroscopy and used them to treat corn, Zea mays L., seedlings to investigate changes in patterns of root protein expression. After root plasma membrane extraction and purification, proteins were separated by two-dimensional gel electrophoresis, and differential spot intensities were evaluated by image analysis. Finally, 42 differentially expressed proteins were identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The majority of them were downregulated by the treatment with humic substances. The proteins identified included malate dehydrogenase, ATPases, cytoskeleton proteins, and different enzymes belonging to the glycolytic/gluconeogenic pathways and sucrose metabolism. The identification of factors involved in plant responses to humic substances may improve our understanding of plant–soil cross-talk, and enable a better management of soil resources.

Measuring low-molecular-weight thiols by detecting the fluorescence of their SBD-derivatives: application to studies of diurnal and UV-B induced changes in Zea mays L.

Summary The composition of low-molecular-weight thiols in Zea mays L. was investigated by HPLC separation following derivatisation with SBD-F (ammonium 7-fluoro 2,1,3-benzooxadiazole-4-sulfonate) as a fluorescent label. This method was applied to describing variations in the acid-soluble thiol pool in a normal dark/light cycle, and with supplemental UV-B radiation causing moderate photo-oxidative stress. We provide evidence of an increase in leaf η-glu-cys content during the night. This finding suggests that η-glu-cys accumulation in the absence of light is a general phenomenon, though it is not yet clear whether glycine availability from photorespiration is the underlying mechanism in C4 plants too. UV-B radiation caused a significant increase in η-glu-cys content in both shoots and roots. A faster loss of GSH in shoots, in experiments where the glutathione biosynthesis inhibitor buthionine sulfoximine was used, indicates that UV-B radiation enhances glutathione degradation. This is consistent with a higher content of cys-gly (reported to be a η-glutamyl-transpeptidase degradation product of glutathione or its conjugates) in shoots of UV-B exposed seedlings.

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.

Nitrate sensing by the maize root apex transition zone: a merged transcriptomic and proteomic survey

Highlight A combined untargeted approach was adopted to achieve a picture of the transcriptional and proteomic profiles typifying the maize root transition zone in response to nitrate.

Low-molecular-weight thiols in plants: Functional and analytical implications

Low-molecular-weight (LMW) thiols are a class of highly reactive compounds massively involved in the maintenance of cellular redox homeostasis. They are implicated in plant responses to almost all stress factors, as well as in the regulation of cellular metabolism. The most studied LMW thiols are glutathione and its biosynthetically related compounds (cysteine, γ-glutamylcysteine, cysteinylglycine, and phytochelatins). Other LMW thiols are described in the literature, such as thiocysteine, cysteamine, homocysteine, lipoic acid, and many species-specific volatile thiols. Here, we review the known LMW thiols in plants, briefly describing their physico-chemical properties, their relevance in post-translational protein modification, and recently-developed thiol detection methods. Current research points to a huge thiol biodiversity in plants and many species-specific and organ-specific thiols remain to be identified. Recent advances in technology should help researchers in this very challenging task, helping us to decipher the roles of thiols in plant metabolism.

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.