Santiago Signorelli

Water stress induces a differential and spatially distributed nitro-oxidative stress response in roots and leaves of Lotus japonicus

Water stress is one of the most severe problems for plant growth and productivity. Using the legume Lotus japonicus exposed to water stress, a comparative analysis of key components in metabolism of reactive nitrogen and oxygen species (RNS and ROS, respectively) were made. After water stress treatment plants accumulated proline 23 and 10-fold in roots and leaves respectively, compared with well-watered plants. Significant changes in metabolism of RNS and ROS were observed, with an increase in both protein tyrosine nitration and lipid peroxidation, which indicate that water stress induces a nitro-oxidative stress. In roots, ·NO content was increased and S-nitrosoglutathione reductase activity was reduced by 23%, wherein a specific protein nitration pattern was observed. As part of this response, activity of NADPH-generating dehydrogenases was also affected in roots resulting in an increase of the NADPH/NADP(+) ratio. Our results suggest that in comparison with leaves, roots are significantly affected by water stress inducing an increase in proline and NO content which could highlight multiple functions for these metabolites in water stress adaptation, recovery and signaling. Thus, it is proposed that water stress generates a spatial distribution of nitro-oxidative stress with the oxidative stress component being higher in leaves whereas the nitrosative stress component is higher in roots.

Proline does not quench singlet oxygen: evidence to reconsider its protective role in plants.

Plants are commonly subjected to several environmental stresses that lead to an overproduction of reactive oxygen species (ROS). As plants accumulate proline in response to stress conditions, some authors have proposed that proline could act as a non-enzymatic antioxidant against ROS. One type of ROS aimed to be quenched by proline is singlet oxygen ((1)O(2))-molecular oxygen in its lowest energy electronically excited state-constitutively generated in oxygenic, photosynthetic organisms. In this study we clearly prove that proline cannot quench (1)O(2) in aqueous buffer, giving rise to a rethinking about the antioxidant role of proline against (1)O(2).

Connecting Proline and γ-Aminobutyric Acid in Stressed Plants through Non-Enzymatic Reactions

The accumulation of proline (Pro) in plants exposed to biotic/abiotic stress is a well-documented and conserved response in most vegetal species. Stress conditions induce the overproduction of reactive oxygen species which can lead to cellular damage. In vitro assays have shown that enzyme inactivation by hydroxyl radicals (·OH) can be avoided in presence of Pro, suggesting that this amino acid could act as an ·OH scavenger. We applied Density Functional Theory coupled with a polarizable continuum model to elucidate how Pro reacts with ·OH. In this work we suggest that Pro reacts favourably with ·OH by H–abstraction on the amine group. This reaction produces the spontaneous decarboxylation of Pro leading to the formation of pyrrolidin-1-yl. In turn, pyrrolidin-1-yl can easily be converted to Δ1-pyrroline, the substrate of the enzyme Δ1-pyrroline dehydrogenase, which produces γ-aminobutyric acid (GABA). GABA and Pro are frequently accumulated in stressed plants and several protective roles have been assigned to these molecules. Thereby we present an alternative non-enzymatic way to synthetize GABA under oxidative stress. Finally this work sheds light on a new beneficial role of Pro accumulation in the maintenance of photosynthetic activity.

Antioxidant and photosystem II responses contribute to explain the drought–heat contrasting tolerance of two forage legumes

Identification of metabolic targets of environmental stress factors is critical to improve the stress tolerance of plants. Studying the biochemical and physiological responses of plants with different capacities to deal with stress is a valid approach to reach this objective. Lotus corniculatus (lotus) and Trifolium pratense (clover) are legumes with contrasting summer stress tolerances. In stress conditions, which are defined as drought, heat or a combination of both, we found that differential biochemical responses of leaves explain these behaviours. Lotus and clover showed differences in water loss control, proline accumulation and antioxidant enzymatic capacity. Drought and/or heat stress induced a large accumulation of proline in the tolerant species (lotus), whereas heat stress did not cause proline accumulation in the sensitive species (clover). In lotus, Mn-SOD and Fe-SOD were induced by drought, but in clover, the SOD-isoform profile was not affected by stress. Moreover, lotus has more SOD-isoforms and a higher total SOD activity than clover. The functionality and electrophoretic profile of photosystem II (PSII) proteins under stress also exhibited differences between the two species. In lotus, PSII activity was drastically affected by combined stress and, interestingly, was correlated with D2 protein degradation. Possible implications of this event as an adaption mechanism in tolerant species are discussed. We conclude that the stress-tolerant capability of lotus is related to its ability to respond to oxidative damage and adaption of the photosynthetic machinery. This reveals that these two aspects should be included in the evaluation of the tolerance of species to stress conditions.

Learning To Breathe: Developmental Phase Transitions in Oxygen Status

Plants are developmentally disposed to significant changes in oxygen availability, but our understanding of the importance of hypoxia is almost entirely limited to stress biology. Differential patterns of the abundance of oxygen, nitric oxide (•NO), and reactive oxygen species (ROS), as well as of redox potential, occur in organs and meristems, and examples are emerging in the literature of mechanistic relationships of these to development. We describe here the convergence of these cues in meristematic and reproductive tissues, and discuss the evidence for regulated hypoxic niches within which oxygen-, ROS-, •NO-, and redox-dependent signalling curate developmental transitions in plants.

In vivo and in vitro approaches demonstrate proline is not directly involved in the protection against superoxide, nitric oxide, nitrogen dioxide and peroxynitrite

Plants accumulate proline under diverse types of stresses, and it has been suggested that this α-amino acid has the capacity to protect against oxidative stress. However, it is still controversial whether its protection is due to the direct scavenging of reactive oxygen species (ROS). To solve this issue and considering that nitrosative stress is directly related with an oxidative stress condition, we evaluated whether proline can protect against nitrosative damage. Using proteins of Lotus japonicus (Regel) K.Larsen leaves exposed to a peroxynitrite (ONOO-/ONOOH) generator in presence and absence of 100mM proline, the potential of proline to protect was analysed by the protein nitration profile and NADP-dependent isocitrate dehydrogenase activity, which is inhibited by nitration. In both cases, the presence of proline did not diminish the peroxynitrite effects. Additionally, proline biosynthesis Arabidopsis knockout (KO) mutant plants of Δ(1)-pyrroline-5-carboxylate synthetase1 (P5CS1) gene, designated as Atp5cs1-1 and Atp5cs1-4, showed similar protein nitration levels as wild-type plants under salinity-induced oxidative stress, despite mutants having higher levels of lipid oxidation, H2O2 and superoxide (O2·-). Finally, by a fluorometric assay using specific fluorescent probes, it was determined that the presence of 100mM proline did not affect the time-course content of peroxynitrite or nitric oxide generation in vitro. Our results reveal the relevance of proline accumulation in vivo under stress, but unequivocally demonstrate that proline is not a direct scavenger of peroxynitrite, superoxide, ·NO and nitrogen dioxide (·NO2).

The Fermentation Analogy: A Point of View for Understanding the Intriguing Role of Proline Accumulation in Stressed Plants

The accumulation of proline under environmental stress is a conserved response of plants. Five decades have passed since the first report of proline accumulation in plants (Barnnet and Naylor, 1966). Many hypotheses have been put forward regarding assignment of a function to proline. These proposed roles include antioxidant capacity, osmoprotection, signaling, developmental function, and contributing in redox and cellular homeostasis (Smirnoff and Cumbes, 1989; Mattioli et al., 2008; Szabados and Savoure, 2010; Kishor et al., 2015). Among these, the osmotic adjustment, osmoprotectant, and antioxidant role have probably been the earliest and most widely accepted functions of proline. It is known that compatible osmolytes contribute in the retention of water, but also some of them force proteins to adopt a compactly folded structure, preventing the unfolding and reducing the exposed surface of the protein to damaging compounds (Attri et al., 2010). In case of proline this was demonstrated both in vitro and in single cell organisms. For example, it was observed that proline overproducing E. coli mutants had greater osmotolerance (Csonka et al., 1988) and also proline can inhibit protein aggregation in vivo (Ignatova and Gierasch, 2006). However, neither osmotic adjustment nor osmoprotection has been clearly confirmed in plants (Maggio et al., 2002; Kavi Kishor and Sreenivasulu, 2014). This is probably more complex in plants because plants have different osmoregulatory mechanisms. Controversy is present regarding its proposed antioxidant role because recent evidence demonstrates that proline cannot scavenge singlet oxygen, superoxide, nitric oxide, peroxynitrite nor nitrogen dioxide (Signorelli et al., 2013, 2016). In view of the lack of activity in previously suggested roles it has recently been proposed that proline acts exclusively as a hydroxyl radical scavenger (Signorelli et al., 2014, 2015), but more evidence for the physiological importance of this role is needed.

Developmental control of hypoxia during bud burst in grapevine

Dormant or quiescent buds of woody perennials are often dense and in the case of grapevine (Vitis vinifera L.) have a low tissue oxygen status. The precise timing of the decision to resume growth is difficult to predict, but once committed, the increase in tissue oxygen status is rapid and developmentally regulated. Here, we show that more than a third of the grapevine homologues of widely conserved hypoxia-responsive genes and nearly a fifth of all grapevine genes possessing a plant hypoxia-responsive promoter element were differentially regulated during bud burst, in apparent harmony with resumption of meristem identity and cell-cycle gene regulation. We then investigated the molecular and biochemical properties of the grapevine ERF-VII homologues, which in other species are oxygen labile and function in transcriptional regulation of hypoxia-responsive genes. Each of the 3 VvERF-VIIs were substrates for oxygen-dependent proteolysis in vitro, as a function of the N-terminal cysteine. Collectively, these data support an important developmental function of oxygen-dependent signalling in determining the timing and effective coordination bud burst in grapevine. In addition, novel regulators, including GASA-, TCP-, MYB3R-, PLT-, and WUS-like transcription factors, were identified as hallmarks of the orderly and functional resumption of growth following quiescence in buds.

Roles for Light, Energy, and Oxygen in the Fate of Quiescent Axillary Buds

The decision of a quiescent axillary bud to commit to regrowth is governed by both metabolic and signaling functions, driven by light, energy, and oxygen availability.

Identification of Δ1-pyrroline 5-carboxylate synthase (P5CS) genes involved in the synthesis of proline in Lotus japonicus

ABSTRACT Proline accumulation is a common response of plants to different biotic and abiotic stresses. In the model legume Lotus japonicus, osmotic stress-induced proline accumulation is one of the first responses of the plant, converting proline in a reliable stress marker. The main biosynthetic pathway of proline is from glutamate and the reaction catalyzed by the enzyme Δ1-pyrroline 5-carboxylate synthase (P5CS) is the rate limiting step. L. japonicus has been suggested to have three different P5CS genes. Here the predicted P5CS genes of L. japonicus were analyzed in silico and their expression under osmotic stress was determined. Contrary to previous suggestions this study demonstrated that L. japonicus has two different P5CS genes, as most dicotyledonous plants do. The gene that is inducible by osmotic stress and is located on chromosome 1, was called LjP5CS1, and the one located on chromosome 2 and not inducible by osmotic stress was called LjP5CS2.