Cytosolic fumarase acts as a metabolic fail-safe for both high and low temperature acclimation of Arabidopsis thaliana.

Plants acclimate their photosynthetic capacity (Pmax) in response to changing environmental conditions. In Arabidopsis thaliana, photosynthetic acclimation to cold requires the accumulation of the organic acid fumarate, catalysed by a cytosolically localized fumarase, FUM2. However, the role of this accumulation is currently unknown. Here, we use an integrated experimental and modelling approach to examine the role of FUM2 and fumarate across the physiological temperature range. We have studied three genotypes: Col-0; a fum2 mutant in a Col-0 background; and C24, an accession with reduced FUM2 expression. While low temperature causes an increase in Pmax in the Col-0 plants, this parameter decreases following exposure of plants to 30 °C for 7 d. Plants in which fumarate accumulation is partially (C24) or completely (fum2) abolished show a reduced acclimation of Pmax across the physiological temperature range (i.e. Pmax changes less in response to changing temperature). To understand the role of fumarate accumulation, we have adapted a reliability engineering technique, Failure Mode and Effect Analysis (FMEA), to formalize a rigorous approach for ranking metabolites according to the potential risk that they pose to the metabolic system. FMEA identifies fumarate as a low-risk metabolite, while its precursor, malate, is shown to be high risk and liable to cause system instability. We propose that the role of FUM2 is to provide a fail-safe in order to control malate concentration, maintaining system stability in a changing environment. We suggest that FMEA is a technique that is not only useful in understanding plant metabolism but can also be used to study reliability in other systems and synthetic pathways.

The Cytochrome b 6 f Complex Is Not Involved in Cyanobacterial State Transitions.

Photosynthetic organisms must sense and respond to fluctuating environmental conditions in order to perform efficient photosynthesis and to avoid the formation of dangerous reactive oxygen species. The excitation energy arriving at each photosystem permanently changes due to variations in the intensity and spectral properties of the absorbed light. Cyanobacteria, like plants and algae, have developed a mechanism, named "state transitions," that balances photosystem activities. Here, we characterize the role of the cytochrome b 6 f complex and phosphorylation reactions in cyanobacterial state transitions using Synechococcus elongatus PCC 7942 and Synechocystis PCC 6803 as model organisms. First, large photosystem II (PSII) fluorescence quenching was observed in State II, a result that does not appear to be related to energy transfer from PSII to PSI (spillover). This membrane-associated process was inhibited by betaine, Suc, and high concentrations of phosphate. Then, using different chemicals affecting the plastoquinone pool redox state and cytochrome b 6 f activity, we demonstrate that this complex is not involved in state transitions in S. elongatus or Synechocystis PCC6803. Finally, by constructing and characterizing 21 protein kinase and phosphatase mutants and using chemical inhibitors, we demonstrate that phosphorylation reactions are not essential for cyanobacterial state transitions. Thus, signal transduction is completely different in cyanobacterial and plant (green alga) state transitions.

Revisiting cyanobacterial state transitions.

Photosynthetic organisms are exposed to a fluctuating environment in which light intensity and quality change continuously. Specific illumination of either photosystem (PSI or PSII) creates an energy imbalance, leading to the reduction or oxidation of the intersystem electron transport chain. This redox imbalance could trigger the formation of dangerous reactive oxygen species. Cyanobacteria, like plants and algae, have developed a mechanism to re-balance this preferential excitation of either reaction center, called state transitions. State transitions are triggered by changes in the redox state of the membrane-soluble plastoquinone (PQ) pool. In plants and green algae, these changes in redox potential are sensed by Cytochrome b6f, which interacts with a specific kinase that triggers the movement of the main PSII antenna (the light-harvesting complex II). By contrast, although cyanobacterial state transitions have been studied extensively, there is still no agreement about the molecular mechanism, the PQ redox state sensor and the signaling pathways involved. In this review, we aimed to critically evaluate the results published on cyanobacterial state transitions, and discuss the "new" and "old" models in the subject. The phycobilisome and membrane contributions to this physiological process were addressed and the current hypotheses regarding its signaling transduction pathway were discussed.

Proximity to Photosystem II is necessary for activation of Plastid Terminal Oxidase (PTOX) for photoprotection.

The Plastid Terminal Oxidase (PTOX) is a chloroplast localized plastoquinone oxygen oxidoreductase suggested to have the potential to act as a photoprotective safety valve for photosynthesis. However, PTOX overexpression in plants has been unsuccessful at inducing photoprotection, and the factors that control its activity remain elusive. Here, we show that significant PTOX activity is induced in response to high light in the model species Eutrema salsugineum and Arabidopsis thaliana. This activation correlates with structural reorganization of the thylakoid membrane. Over-expression of PTOX in mutants of Arabidopsis thaliana perturbed in thylakoid stacking also results in such activity, in contrast to wild type plants with normal granal structure. Further, PTOX activation protects against photoinhibition of Photosystem II and reduces reactive oxygen production under stress conditions. We conclude that structural re-arrangements of the thylakoid membranes, bringing Photosystem II and PTOX into proximity, are both required and sufficient for PTOX to act as a Photosystem II sink and play a role in photoprotection.

N-acetylcysteine reduces markers of differentiation in 3T3-L1 adipocytes.

Oxidative stress plays a critical role in the pathogenesis of diabetes, hypertension and atherosclerosis. Some authors reported that fat accumulation correlates to systemic oxidative stress in humans and mice, but the relationship of lipid production and oxidative metabolism is still unclear. In our laboratory we used 3T3-L1 preadipocytes, which are able to differentiate into mature adipocytes and accumulate lipids, as obesity model. We showed that intracellular reactive oxygen species (ROS) and antioxidant enzymes superoxide dismutase (SOD) and glutathione peroxidase (GPx) activities increased in parallel with fat accumulation. Meanwhile N-acetylcysteine (NAC), a well known antioxidant and Glutathione (GSH) precursor, inhibited ROS levels as well as fat accumulation in a concentration-dependent manner. NAC also inhibited both adipogenic transcription factors CCAAT/enhancer binding protein beta (C/EBP ß) and peroxisomal proliferator activated receptor gamma (PPAR γ) expression; we suggested that intracellular GSH content could be responsible for these effects.

A Holistic Approach to Study Photosynthetic Acclimation Responses of Plants to Fluctuating Light.

Plants in natural environments receive light through sunflecks, the duration and distribution of these being highly variable across the day. Consequently, plants need to adjust their photosynthetic processes to avoid photoinhibition and maximize yield. Changes in the composition of the photosynthetic apparatus in response to sustained changes in the environment are referred to as photosynthetic acclimation, a process that involves changes in protein content and composition. Considering this definition, acclimation differs from regulation, which involves processes that alter the activity of individual proteins over short-time periods, without changing the abundance of those proteins. The interconnection and overlapping of the short- and long-term photosynthetic responses, which can occur simultaneously or/and sequentially over time, make the study of long-term acclimation to fluctuating light in plants challenging. In this review we identify short-term responses of plants to fluctuating light that could act as sensors and signals for acclimation responses, with the aim of understanding how plants integrate environmental fluctuations over time and tailor their responses accordingly. Mathematical modeling has the potential to integrate physiological processes over different timescales and to help disentangle short-term regulatory responses from long-term acclimation responses. We review existing mathematical modeling techniques for studying photosynthetic responses to fluctuating light and propose new methods for addressing the topic from a holistic point of view.

Different roles for ApcD and ApcF in Synechococcus elongatus and Synechocystis sp. PCC 6803 phycobilisomes.

The phycobilisome, the cyanobacterial light harvesting complex, is a huge phycobiliprotein containing extramembrane complex, formed by a core from which rods radiate. The phycobilisome has evolved to efficiently absorb sun energy and transfer it to the photosystems via the last energy acceptors of the phycobilisome, ApcD and ApcE. ApcF also affects energy transfer by interacting with ApcE. In this work we studied the role of ApcD and ApcF in energy transfer and state transitions in Synechococcus elongatus and Synechocystis PCC6803. Our results demonstrate that these proteins have different roles in both processes in the two strains. The lack of ApcD and ApcF inhibits state transitions in Synechocystis but not in S. elongatus. In addition, lack of ApcF decreases energy transfer to both photosystems only in Synechocystis, while the lack of ApcD alters energy transfer to photosystem I only in S. elongatus. Thus, conclusions based on results obtained in one cyanobacterial strain cannot be systematically transferred to other strains and the putative role(s) of phycobilisomes in state transitions need to be reconsidered.

The increase of photosynthetic carbon assimilation as a mechanism of adaptation to low temperature in Lotus japonicus.

Low temperature is one of the most important factors affecting plant growth, it causes an stress that directly alters the photosynthetic process and leads to photoinhibition when severe enough. In order to address the photosynthetic acclimation response of Lotus japonicus to cold stress, two ecotypes with contrasting tolerance (MG-1 and MG-20) were studied. Their chloroplast responses were addressed after 7 days under low temperature through different strategies. Proteomic analysis showed changes in photosynthetic and carbon metabolism proteins due to stress, but differentially between ecotypes. In the sensitive MG-1 ecotype acclimation seems to be related to energy dissipation in photosystems, while an increase in photosynthetic carbon assimilation as an electron sink, seems to be preponderant in the tolerant MG-20 ecotype. Chloroplast ROS generation was higher under low temperature conditions only in the MG-1 ecotype. These data are consistent with alterations in the thylakoid membranes in the sensitive ecotype. However, the accumulation of starch granules observed in the tolerant MG-20 ecotype indicates the maintenance of sugar metabolism under cold conditions. Altogether, our data suggest that different acclimation strategies and contrasting chloroplast redox imbalance could account for the differential cold stress response of both L. japonicus ecotypes.

Polyamines and Legumes: Joint Stories of Stress, Nitrogen Fixation and Environment.

Polyamines (PAs) are natural aliphatic amines involved in many physiological processes in almost all living organisms, including responses to abiotic stresses and microbial interactions. On other hand, the family Leguminosae constitutes an economically and ecologically key botanical group for humans, being also regarded as the most important protein source for livestock. This review presents the profuse evidence that relates changes in PAs levels during responses to biotic and abiotic stresses in model and cultivable species within Leguminosae and examines the unreviewed information regarding their potential roles in the functioning of symbiotic interactions with nitrogen-fixing bacteria and arbuscular mycorrhizae in this family. As linking plant physiological behavior with "big data" available in "omics" is an essential step to improve our understanding of legumes responses to global change, we also examined integrative MultiOmics approaches available to decrypt the interface legumes-PAs-abiotic and biotic stress interactions. These approaches are expected to accelerate the identification of stress tolerant phenotypes and the design of new biotechnological strategies to increase their yield and adaptation to marginal environments, making better use of available plant genetic resources.

Rice tolerance to suboptimal low temperatures relies on the maintenance of the photosynthetic capacity.

The purpose of this research was to identify differences between two contrasting rice cultivars in their response to suboptimal low temperatures stress. A transcriptomic analysis of the seedlings was performed and results were complemented with biochemical and physiological analyses. The microarray analysis showed downregulation of many genes related with PSII and particularly with the oxygen evolving complex in the sensitive cultivar IR50. Complementary studies indicated that the PSII performance, the degree of oxygen evolving complex coupling with the PSII core and net photosynthetic rate diminished in this cultivar in response to the stress. However, the tolerant cultivar Koshihikari was able to maintain its energy equilibrium by sustaining the photosynthetic capacity. The increase of oleic acid in Koshihikari could be related with membrane remodelling of the chloroplasts and hence contribute to tolerance. Overall, these results work as a ground for future analyses that look forward to characterize possible mechanisms to tolerate this stress.

Modulation of plant and bacterial polyamine metabolism during the compatible interaction between tomato and Pseudomonas syringae.

The polyamines putrescine, spermidine and spermine participate in a variety of cellular processes in all organisms. Many studies have shown that these polycations are important for plant immunity, as well as for the virulence of diverse fungal phytopathogens. However, the polyamines' roles in the pathogenesis of phytopathogenic bacteria have not been thoroughly elucidated to date. To obtain more information on this topic, we assessed the changes in polyamine homeostasis during the infection of tomato plants by Pseudomonas syringae. Our results showed that polyamine biosynthesis and catabolism are activated in both tomato and bacteria during the pathogenic interaction. This activation results in the accumulation of putrescine in whole leaf tissues, as well as in the apoplastic fluids, which is explained by the induction of its synthesis in plant cells and also on the basis of its excretion by bacteria. We showed that the excretion of this polyamine by P. syringae is stimulated under virulence-inducing conditions, suggesting that it plays a role in plant colonization. However, no activation of bacterial virulence traits or induction of plant invasion was observed after the exogenous addition of putrescine. In addition, no connection was found between this polyamine and plant defence responses. Although further research is warranted to unravel the biological functions of these molecules during plant-bacterial interactions, this study contributes to a better understanding of the changes associated with the homeostasis of polyamines during plant pathogenesis.

Transcriptome Response Mediated by Cold Stress in Lotus japonicus.

Members of the Lotus genus are important as agricultural forage sources under marginal environmental conditions given their high nutritional value and tolerance of various abiotic stresses. However, their dry matter production is drastically reduced in cooler seasons, while their response to such conditions is not well studied. This paper analyzes cold acclimation of the genus by studying Lotus japonicus over a stress period of 24 h. High-throughput RNA sequencing was used to identify and classify 1077 differentially expressed genes, of which 713 were up-regulated and 364 were down-regulated. Up-regulated genes were principally related to lipid, cell wall, phenylpropanoid, sugar, and proline regulation, while down-regulated genes affected the photosynthetic process and chloroplast development. Together, a total of 41 cold-inducible transcription factors were identified, including members of the AP2/ERF, NAC, MYB, and WRKY families; two of them were described as putative novel transcription factors. Finally, DREB1/CBFs were described with respect to their cold stress expression profiles. This is the first transcriptome profiling of the model legume L. japonicus under cold stress. Data obtained may be useful in identifying candidate genes for breeding modified species of forage legumes that more readily acclimate to low temperatures.

The alkaline tolerance in Lotus japonicus is associated with mechanisms of iron acquisition and modification of the architectural pattern of the root.

The response of fifty-four Lotus japonicus ecotypes, and of six selected ecotypes was investigated under alkaline conditions. Sensitive, but not tolerant ecotypes, showed interveinal chlorosis under all alkalinity conditions and high mortality under extreme alkalinity. Interveinal chlorosis was associated with Fe deficiency, as a reduced Fe2+ shoot content was observed in all sensitive ecotypes. In addition, some showed a decline in photosynthesis rate and PSII performance compared to the control. In contrast, some tolerant ecotypes did not change these parameters between treatments. Alkaline tolerance could be explained by a mechanism of Fe acquisition and a root structural modification. This conclusion was based on the fact that all tolerant, but not the sensitive ecotypes, presented high ferric reductase oxidase activity under alkaline stress compared to the control, and a Herringbone root pattern modification. On this basis, the analysis of these mechanisms of alkaline tolerance could be used in screening programs for the selection of new tolerant genotypes in the Lotus genus.

Photosynthetic responses mediate the adaptation of two Lotus japonicus ecotypes to low temperature.

Lotus species are important forage legumes due to their high nutritional value and adaptability to marginal conditions. However, the dry matter production and regrowth rate of cultivable Lotus spp. is drastically reduced during colder seasons. In this work, we evaluated the chilling response of Lotus japonicus ecotypes MG-1 and MG-20. No significant increases were observed in reactive oxygen species and nitric oxide production or in lipid peroxidation, although a chilling-induced redox imbalance was suggested through NADPH/NADP(+) ratio alterations. Antioxidant enzyme catalase, ascorbate peroxidase, and superoxide dismutase activities were also measured. Superoxide dismutase, in particular the chloroplastic isoform, showed different activity for different ecotypes and treatments. Stress-induced photoinhibition also differentially influenced both ecotypes, with MG-1 more affected than MG-20. Data showed that the D2 PSII subunit was more affected than D1 after 1 d of low temperature exposure, although its protein levels recovered over the course of the experiment. Interestingly, D2 recovery was accompanied by improvements in photosynthetic parameters (Asat and Fv/Fm) and the NADPH/NADP(+) ratio. Our results suggest that the D2 protein is involved in the acclimation response of L. japonicus to low temperature. This may provide a deeper insight into the chilling tolerance mechanisms of the Lotus genus.

Transcriptional regulation of 9-cis-epoxycarotenoid dioxygenase (NCED) gene by putrescine accumulation positively modulates ABA synthesis and drought tolerance in Lotus tenuis plants.

The accumulation of putrescine (Put) and increased arginine decarboxylase (ADC, EC 4.1.1.19) activity levels in response to osmotic stress has been reported; however, the biological meaning of this increase remains unclear. To obtain new insights into these questions, we studied the drought response of a transgenic Lotus tenuis line that expresses the oat ADC gene, which is driven by the stress-inducible pRD29A promoter. This line contains high levels of Put with no changes in spermidine and spermine contents, even under water deficits. Our results indicate that the biochemical and morphological responses to dehydration correlate with the Put level and provide evidence that Put controls the ABA content in response to drought by modulating ABA biosynthesis at the transcriptional level.

Response to long-term NaHCO3-derived alkalinity in model Lotus japonicus Ecotypes Gifu B-129 and Miyakojima MG-20: transcriptomic profiling and physiological characterization.

The current knowledge regarding transcriptomic changes induced by alkalinity on plants is scarce and limited to studies where plants were subjected to the alkaline salt for periods not longer than 48 h, so there is no information available regarding the regulation of genes involved in the generation of a new homeostatic cellular condition after long-term alkaline stress. Lotus japonicus is a model legume broadly used to study many important physiological processes including biotic interactions and biotic and abiotic stresses. In the present study, we characterized phenotipically the response to alkaline stress of the most widely used L. japonicus ecotypes, Gifu B-129 and MG-20, and analyzed global transcriptome of plants subjected to 10 mM NaHCO3 during 21 days, by using the Affymetrix Lotus japonicus GeneChip®. Plant growth assessment, gas exchange parameters, chlorophyll a fluorescence transient (OJIP) analysis and metal accumulation supported the notion that MG-20 plants displayed a higher tolerance level to alkaline stress than Gifu B-129. Overall, 407 and 459 probe sets were regulated in MG-20 and Gifu B-129, respectively. The number of probe sets differentially expressed in roots was higher than that of shoots, regardless the ecotype. Gifu B-129 and MG-20 also differed in their regulation of genes that could play important roles in the generation of a new Fe/Zn homeostatic cellular condition, synthesis of plant compounds involved in stress response, protein-degradation, damage repair and root senescence, as well as in glycolysis, gluconeogenesis and TCA. In addition, there were differences between both ecotypes in the expression patterns of putative transcription factors that could determine distinct arrangements of flavonoid and isoflavonoid compounds. Our results provided a set of selected, differentially expressed genes deserving further investigation and suggested that the L. japonicus ecotypes could constitute a useful model to search for common and distinct tolerance mechanisms to long-term alkaline stress response in plants.