The cAMP-dependent phosphorylation footprint in response to heat stress.

KEY MESSAGE: cAMP modulates the phosphorylation status of highly conserved phosphosites in RNA-binding proteins crucial for mRNA metabolism and reprogramming in response to heat stress. In plants, 3',5'-cyclic adenosine monophosphate (3',5'-cAMP) is a second messenger that modulates multiple cellular targets, thereby participating in plant developmental and adaptive processes. Although its role in ameliorating heat-related damage has been demonstrated, mechanisms that govern cAMP-dependent responses to heat have remained elusive. Here we analyze the role cAMP-dependent phosphorylation during prolonged heat stress (HS) with a view to gain insight into processes that govern plant responses to HS. To do so, we performed quantitative phosphoproteomic analyses in Nicotiana tabacum Bright Yellow-2 cells grown at 27 °C or 35 °C for 3 days overexpressing a molecular "sponge" that reduces free intracellular cAMP levels. Our phosphorylation data and analyses reveal that the presence of cAMP is an essential factor that governs specific protein phosphorylation events that occur during prolonged HS in BY-2 cells. Notably, cAMP modulates HS-dependent phosphorylation of proteins that functions in mRNA processing, transcriptional control, vesicular trafficking, and cell cycle regulation and this is indicative for a systemic role of the messenger. In particular, changes of cAMP levels affect the phosphorylation status of highly conserved phosphosites in 19 RNA-binding proteins that are crucial during the reprogramming of the mRNA metabolism in response to HS. Furthermore, phosphorylation site motifs and molecular docking suggest that some proteins, including kinases and phosphatases, are conceivably able to directly interact with cAMP thus further supporting a regulatory role of cAMP in plant HS responses.

The Arabidopsis Mitochondrial Nucleoid-Associated Protein WHIRLY2 Is Required for a Proper Response to Salt Stress.

In the last years, plant organelles have emerged as central coordinators of responses to internal and external stimuli, which can induce stress. Mitochondria play a fundamental role as stress sensors being part of a complex communication network between the organelles and the nucleus. Among the different environmental stresses, salt stress poses a significant challenge and requires efficient signaling and protective mechanisms. By using the why2 T-DNA insertion mutant and a novel knock-out mutant prepared by CRISPR/Cas9-mediated genome editing, this study revealed that WHIRLY2 is crucial for protecting mitochondrial DNA (mtDNA) integrity during salt stress. Loss-of-function mutants show an enhanced sensitivity to salt stress. The disruption of WHIRLY2 causes the impairment of mtDNA repair that results in the accumulation of aberrant recombination products, coinciding with severe alterations in nucleoid integrity and overall mitochondria morphology besides a compromised redox-dependent response and misregulation of antioxidant enzymes. The results of this study revealed that WHIRLY2-mediated structural features in mitochondria (nucleoid compactness and cristae) are important for an effective response to salt stress.

The Role of Glutamine Synthetase (GS) and Glutamate Synthase (GOGAT) in the Improvement of Nitrogen Use Efficiency in Cereals.

Cereals are the most broadly produced crops and represent the primary source of food worldwide. Nitrogen (N) is a critical mineral nutrient for plant growth and high yield, and the quality of cereal crops greatly depends on a suitable N supply. In the last decades, a massive use of N fertilizers has been achieved in the desire to have high yields of cereal crops, leading to damaging effects for the environment, ecosystems, and human health. To ensure agricultural sustainability and the required food source, many attempts have been made towards developing cereal crops with a more effective nitrogen use efficiency (NUE). NUE depends on N uptake, utilization, and lastly, combining the capability to assimilate N into carbon skeletons and remobilize the N assimilated. The glutamine synthetase (GS)/glutamate synthase (GOGAT) cycle represents a crucial metabolic step of N assimilation, regulating crop yield. In this review, the physiological and genetic studies on GS and GOGAT of the main cereal crops will be examined, giving emphasis on their implications in NUE.

Quantitative phosphoproteomics reveals novel roles of cAMP in plants.

3',5'-cyclic adenosine monophosphate (cAMP) is finally recognized as an essential signaling molecule in plants where cAMP-dependent processes include responses to hormones and environmental stimuli. To better understand the role of 3',5'-cAMP at the systems level, we have undertaken a phosphoproteomic analysis to elucidate the cAMP-dependent response of tobacco BY-2 cells. These cells overexpress a molecular "sponge" that buffers free intracellular cAMP level. The results show that, firstly, in vivo cAMP dampening profoundly affects the plant kinome and notably mitogen-activated protein kinases, receptor-like kinases, and calcium-dependent protein kinases, thereby modulating the cellular responses at the systems level. Secondly, buffering cAMP levels also affects mRNA processing through the modulation of the phosphorylation status of several RNA-binding proteins with roles in splicing, including many serine and arginine-rich proteins. Thirdly, cAMP-dependent phosphorylation targets appear to be conserved among plant species. Taken together, these findings are consistent with an ancient role of cAMP in mRNA processing and cellular programming and suggest that unperturbed cellular cAMP levels are essential for cellular homeostasis and signaling in plant cells.

Comparative Analysis of Bioactive Compounds in Two Globe Artichoke Ecotypes Sanitized and Non-Sanitized from Viral Infections.

Globe artichoke ecotypes sanitized from plant pathogen infections are characterized by high vegetative vigor, productivity, and quality of capitula. The recent availability on the market of these plants has renewed the interest of farmers and pharmaceutical industries in the crop. Globe artichoke exhibits interesting nutraceutical properties due to the high content of health-promoting bioactive compounds (BACs), such as polyphenols, that could be extracted from waste biomass. The production of BACs depends on several factors including the plant portion considered, the globe artichoke variety/ecotype, and the physiological status of the plants, linked to biotic and abiotic stresses. We investigated the influence of viral infections on polyphenol accumulation in two Apulian late-flowering ecotypes "Locale di Mola tardivo" and "Troianella", comparing sanitized virus-free material (S) vs. naturally virus-infected (non-sanitized, NS) plants. Transcriptome analysis of the two ecotypes highlighted that differentially expressed genes (DEGs), in the two tested conditions, were mainly involved in primary metabolism and processing of genetic/environmental information. The up-regulation of the genes related to the biosynthesis of secondary metabolites and the analysis of peroxidase activity suggested that their modulation is influenced by the phytosanitary status of the plant and is ecotype-dependent. Conversely, the phytochemical analysis showed a remarkable decrease in polyphenols and lignin accumulation in S artichokes compared to NS plants. This unique study analyzes the potential of growing vigorous, sanitized plants, in order to have high amounts of 'soft and clean' biomass, finalized for BAC extraction for nutraceutical purposes. This, in turn, opens new perspectives for a circular economy of sanitized artichokes, in line with the current phytosanitary standards and sustainable development goals.

Redox Signaling in Plant Heat Stress Response.

The increase in environmental temperature due to global warming is a critical threat to plant growth and productivity. Heat stress can cause impairment in several biochemical and physiological processes. Plants sense and respond to this adverse environmental condition by activating a plethora of defense systems. Among them, the heat stress response (HSR) involves an intricate network of heat shock factors (HSFs) and heat shock proteins (HSPs). However, a growing amount of evidence suggests that reactive oxygen species (ROS), besides potentially being responsible for cellular oxidative damage, can act as signal molecules in HSR, leading to adaptative responses. The role of ROS as toxic or signal molecules depends on the fine balance between their production and scavenging. Enzymatic and non-enzymatic antioxidants represent the first line of defense against oxidative damage and their activity is critical to maintaining an optimal redox environment. However, the HS-dependent ROS burst temporarily oxidizes the cellular environment, triggering redox-dependent signaling cascades. This review provides an overview of the redox-activated mechanisms that participate in the HSR.

Enhanced Natural Attenuation of Groundwater Cr(VI) Pollution Using Electron Donors: Yeast Extract vs. Polyhydroxybutyrate.

Remediation interventions based on the native bacteria's capability to reduce Cr(VI) represent a valid strategy in terms of economic and environmental sustainability. In this study, a bioremediation test was carried out using viable microcosms set with groundwater and deep soil (4:1), collected from the saturated zone of an industrial site in Southern Italy that was polluted by ~130 µg L-1 of Cr(VI). Conditions simulating the potential natural attenuation were compared to the enhanced natural attenuation induced by supplying yeast extract or polyhydroxybutyrate. Sterile controls were set up to study the possible Cr(VI) abiotic reduction. No pollution attenuation was detected in the unamended viable reactors, whereas yeast extract provided the complete Cr(VI) removal in 7 days, and polyhydroxybutyrate allowed ~70% pollutant removal after 21 days. The incomplete abiotic removal of Cr(VI) was observed in sterile reactors amended with yeast extract, thus suggesting the essential role of native bacteria in Cr(VI) remediation. This was in accordance with the results of Pearson's coefficient test, which revealed that Cr(VI) removal was positively correlated with microbial proliferation (n = 0.724), and also negatively correlated with pH (n = -0.646), dissolved oxygen (n = -0.828) and nitrate (n = -0.940). The relationships between the Cr(VI) removal and other monitored parameters were investigated by principal component analysis, which explained 76.71% of the total variance.

GUN1 involvement in the redox changes occurring during biogenic retrograde signaling.

Chloroplast biogenesis requires a tight communication between nucleus and plastids. By retrograde signals, plastids transmit information about their functional and developmental state to adjust nuclear gene expression, accordingly. GENOMES UNCOUPLED 1 (GUN1), a chloroplast-localized protein integrating several developmental and stress-related signals, is one of the main players of retrograde signaling. Here, we focused on the interplay between GUN1 and redox regulation during biogenic retrograde signaling, by investigating redox parameters in Arabidopsis wild type and gun1 seedlings. Our data highlight that during biogenic retrograde signaling superoxide anion (O2-) and hydrogen peroxide (H2O2) play a different role in response to GUN1. Under physiological conditions, even in the absence of a visible phenotype, gun1 mutants show low activity of superoxide dismutase (SOD) and ascorbate peroxidase (APX), with an increase in O2- accumulation and lipid peroxidation, suggesting that GUN1 indirectly protects chloroplasts from oxidative damage. In wild type seedlings, perturbation of chloroplast development with lincomycin causes H2O2 accumulation, in parallel with the decrease of ROS-removal metabolites and enzymes. These redox changes do not take place in gun1 mutants which, in contrast, enhance SOD, APX and catalase activities. Our results indicate that in response to lincomycin, GUN1 is necessary for the H2O2-dependent oxidation of cellular environment, which might contribute to the redox-dependent plastid-to nucleus communication.

Chloroplast-localized GUN1 contributes to the acquisition of basal thermotolerance in Arabidopsis thaliana.

Heat stress (HS) severely affects different cellular compartments operating in metabolic processes and represents a critical threat to plant growth and yield. Chloroplasts are crucial for heat stress response (HSR), signaling to the nucleus the environmental challenge and adjusting metabolic and biosynthetic functions accordingly. GENOMES UNCOUPLED 1 (GUN1), a chloroplast-localized protein, has been recognized as one of the main players of chloroplast retrograde signaling. Here, we investigate HSR in Arabidopsis wild-type and gun1 plantlets subjected to 2 hours of HS at 45°C. In wild-type plants, Reactive Oxygen Species (ROS) accumulate promptly after HS, contributing to transiently oxidize the cellular environment and acting as signaling molecules. After 3 hours of physiological recovery at growth temperature (22°C), the induction of enzymatic and non-enzymatic antioxidants prevents oxidative damage. On the other hand, gun1 mutants fail to induce the oxidative burst immediately after HS and accumulate ROS and oxidative damage after 3 hours of recovery at 22°C, thus resulting in enhanced sensitivity to HS. These data suggest that GUN1 is required to oxidize the cellular environment, participating in the acquisition of basal thermotolerance through the redox-dependent plastid-to-nucleus communication.

Cyclic AMP mediates heat stress response by the control of redox homeostasis and ubiquitin-proteasome system.

Heat stress (HS), causing impairment in several physiological processes, is one of the most damaging environmental cues for plants. To counteract the harmful effects of high temperatures, plants activate complex signalling networks, indicated as HS response (HSR). Expression of heat shock proteins (HSPs) and adjustment of redox homeostasis are crucial events of HSR, required for thermotolerance. By pharmacological approaches, the involvement of cAMP in triggering plant HSR has been recently proposed. In this study, to investigate the role of cAMP in HSR signalling, tobacco BY-2 cells overexpressing the 'cAMP-sponge', a genetic tool that reduces intracellular cAMP levels, have been used. in vivo cAMP dampening increased HS susceptibility in a HSPs-independent way. The failure in cAMP elevation during HS caused a high accumulation of reactive oxygen species, due to increased levels of respiratory burst oxidase homolog D, decreased activities of catalase and ascorbate peroxidase, as well as down-accumulation of proteins involved in the control of redox homeostasis. In addition, cAMP deficiency impaired proteasome activity and prevented the accumulation of many proteins of ubiquitin-proteasome system (UPS). By a large-scale proteomic approach together with in silico analyses, these UPS proteins were identified in a specific cAMP-dependent network of HSR.

Cyclic AMP: A Polyhedral Signalling Molecule in Plants.

The cyclic nucleotide cAMP (3',5'-cyclic adenosine monophosphate) is nowadays recognised as an important signalling molecule in plants, involved in many molecular processes, including sensing and response to biotic and abiotic environmental stresses. The validation of a functional cAMP-dependent signalling system in higher plants has spurred a great scientific interest on the polyhedral role of cAMP, as it actively participates in plant adaptation to external stimuli, in addition to the regulation of physiological processes. The complex architecture of cAMP-dependent pathways is far from being fully understood, because the actors of these pathways and their downstream target proteins remain largely unidentified. Recently, a genetic strategy was effectively used to lower cAMP cytosolic levels and hence shed light on the consequences of cAMP deficiency in plant cells. This review aims to provide an integrated overview of the current state of knowledge on cAMP's role in plant growth and response to environmental stress. Current knowledge of the molecular components and the mechanisms of cAMP signalling events is summarised.

Vitamin C in Plants: From Functions to Biofortification.

Vitamin C (l-ascorbic acid) is an excellent free radical scavenger, not only for its capability to donate reducing equivalents but also for the relative stability of the derived monodehydroascorbate radical. However, vitamin C is not only an antioxidant, since it is also a cofactor for numerous enzymes involved in plant and human metabolism. In humans, vitamin C takes part in various physiological processes, such as iron absorption, collagen synthesis, immune stimulation, and epigenetic regulation. Due to the functional loss of the gene coding for l-gulonolactone oxidase, humans cannot synthesize vitamin C; thus, they principally utilize plant-based foods for their needs. For this reason, increasing the vitamin C content of crops could have helpful effects on human health. To achieve this objective, exhaustive knowledge of the metabolism and functions of vitamin C in plants is needed. In this review, the multiple roles of vitamin C in plant physiology as well as the regulation of its content, through biosynthetic or recycling pathways, are analyzed. Finally, attention is paid to the strategies that have been used to increase the content of vitamin C in crops, emphasizing not only the improvement of nutritional value of the crops but also the acquisition of plant stress resistance.

Plant Cell Cultures as Model Systems to Study Programmed Cell Death.

The study of programmed cell death (PCD) activated in a certain group of cells is complex when analyzed in the whole plant. Plant cell suspension cultures are useful when investigating PCD triggered by environmental and developmental stimuli. Due to their homogeneity and the possibility to synchronize their responses induced by external stimuli, these cultures are used for studying the signaling pathways leading to PCD. The first problem in the analysis of PCD in cell cultures is the quantification of cell viability/death over time. Cultured cells from different plant species may have specific mitotic patterns leading to calli or cell chains mixed to single cell suspensions. For this reason, not all cell cultures allow morphological parameters to be investigated using microscopy analysis, and adapted or ad hoc methods are needed to test cell viability.Here we report on some accurate methods to establish and propagate cell cultures from different plant species, including crops, as well as to determine cell viability and PCD morphological and genetic markers. In particular, we describe a protocol for extracting nucleic acids required for real-time PCR analysis which has been optimized for those cell cultures that do not allow the use of commercial kits.

Gigaspora margarita with and without its endobacterium shows adaptive responses to oxidative stress.

Arbuscular mycorrhizal (AM) fungi experience oxidative stress during the plant-fungal interaction, due to endogenous reactive oxygen species (ROS) produced by fungal metabolism and exogenous ROS produced by plant cells. Here, we examine the responses to H2O2 in Gigaspora margarita, an AM fungus containing the endobacterial symbiont Candidatus Glomeribacter gigasporarum (CaGg). Previous studies revealed that G. margarita with its endobacterium produces more ATP and has higher respiratory activity than a cured line that lacks the endobacterium. This higher bioenergetic potential leads to higher production of ROS and to a higher ROS-detoxifying capacity, suggesting a direct or indirect role of the endobacterium in modulating fungal antioxidant responses. To test the hypothesis that the fungal-endobacterial symbiosis may enhance the fitness of the AM fungus in the presence of oxidative stress, we treated the fungus with a sublethal concentration of H2O2 and performed RNA-seq analysis. Our results demonstrate that (i) irrespective of the endobacterium presence, G. margarita faces oxidative stress by activating multiple metabolic processes (methionine oxidation, sulfur uptake, the pentose phosphate pathway, activation of ROS-scavenger genes); (ii) in the presence of its endobacterium, G. margarita upregulates some metabolic pathways, like chromatin status modifications and iron metabolism; and (iii) contrary to our hypothesis, the cured line responds to H2O2 by activating the transcription of specific ROS scavengers. We confirmed the RNA-seq findings by measuring the glutathione and ascorbate concentration, which was the same in both lines after H2O2 treatment. We conclude that both fungal lines may face oxidative stress, but they activate alternative strategies.

Constitutive cyclic GMP accumulation in Arabidopsis thaliana compromises systemic acquired resistance induced by an avirulent pathogen by modulating local signals.

The infection of Arabidopsis thaliana plants with avirulent pathogens causes the accumulation of cGMP with a biphasic profile downstream of nitric oxide signalling. However, plant enzymes that modulate cGMP levels have yet to be identified, so we generated transgenic A. thaliana plants expressing the rat soluble guanylate cyclase (GC) to increase genetically the level of cGMP and to study the function of cGMP in plant defence responses. Once confirmed that cGMP levels were higher in the GC transgenic lines than in wild-type controls, the GC transgenic plants were then challenged with bacterial pathogens and their defence responses were characterized. Although local resistance was similar in the GC transgenic and wild-type lines, differences in the redox state suggested potential cross-talk between cGMP and the glutathione redox system. Furthermore, large-scale transcriptomic and proteomic analysis highlighted the significant modulation of both gene expression and protein abundance at the infection site, inhibiting the establishment of systemic acquired resistance. Our data indicate that cGMP plays a key role in local responses controlling the induction of systemic acquired resistance in plants challenged with avirulent pathogens.

An interdomain network: the endobacterium of a mycorrhizal fungus promotes antioxidative responses in both fungal and plant hosts.

Arbuscular mycorrhizal fungi (AMF) are obligate plant biotrophs that may contain endobacteria in their cytoplasm. Genome sequencing of Candidatus Glomeribacter gigasporarum revealed a reduced genome and dependence on the fungal host. RNA-seq analysis of the AMF Gigaspora margarita in the presence and absence of the endobacterium indicated that endobacteria have an important role in the fungal pre-symbiotic phase by enhancing fungal bioenergetic capacity. To improve the understanding of fungal-endobacterial interactions, iTRAQ (isobaric tags for relative and absolute quantification) quantitative proteomics was used to identify differentially expressed proteins in G. margarita germinating spores with endobacteria (B+), without endobacteria in the cured line (B-) and after application of the synthetic strigolactone GR24. Proteomic, transcriptomic and biochemical data identified several fungal and bacterial proteins involved in interspecies interactions. Endobacteria influenced fungal growth, calcium signalling and metabolism. The greatest effects were on fungal primary metabolism and respiration, which was 50% higher in B+ than in B-. A shift towards pentose phosphate metabolism was detected in B-. Quantification of carbonylated proteins indicated that the B- line had higher oxidative stress levels, which were also observed in two host plants. This study shows that endobacteria generate a complex interdomain network that affects AMF and fungal-plant interactions.

Cyclic AMP deficiency negatively affects cell growth and enhances stress-related responses in tobacco Bright Yellow-2 cells.

Cyclic adenosine 3',5'-monophosphate (cAMP) is a recognized second messenger; however, knowledge of cAMP involvement in plant physiological processes originates primarily from pharmacological studies. To obtain direct evidence for cAMP function in plants, tobacco Bright Yellow-2 (BY-2) cells were transformed with the cAMP sponge, which is a genetically encoded tool that reduces cAMP availability. BY-2 cells expressing the cAMP sponge (cAS cells), showed low levels of free cAMP and exhibited growth inhibition that was not proportional to the cAMP sponge transcript level. Growth inhibition in cAS cells was closely related to the precocious inhibition of mitosis due to a delay in cell cycle progression. The cAMP deficiency also enhanced antioxidant systems. Remarkable changes occurred in the cAS proteomic profile compared with that of wild-type (WT) cells. Proteins involved in translation, cytoskeletal organization, and cell proliferation were down-regulated, whereas stress-related proteins were up-regulated in cAS cells. These results support the hypothesis that BY-2 cells sense cAMP deficiency as a stress condition. Finally, many proteasome subunits were differentially expressed in cAS cells compared with WT cells, indicating that cAMP signaling broadly affects protein degradation via the ubiquitin/proteasome pathway.

Role of redox homeostasis in thermo-tolerance under a climate change scenario.

BACKGROUND: Climate change predictions indicate a progressive increase in average temperatures and an increase in the frequency of heatwaves, which will have a negative impact on crop productivity. Over the last decade, a number of studies have addressed the question of how model plants or specific crops modify their metabolism when exposed to heat stress. SCOPE: This review provides an overview of the redox pathways that contribute to how plants cope with heat stress. The focus is on the role of reactive oxygen species (ROS), redox metabolites and enzymes in the signalling pathways leading to the activation of defence responses. Additional attention is paid to the regulating mechanisms that lead to an increase in specific ROS-scavenging systems during heat stress, which have been studied in different model systems. Finally, increasing thermo-tolerance in model and crop plants by exposing them to heat acclimation or to exogenous treatments is discussed. CONCLUSIONS: Although there is clear evidence that several strategies are specifically activated according to the intensity and the duration of heat stress, as well as the capacity of the different species or genotypes to overcome stress, an alteration in redox homeostasis seems to be a common event. Different mechanisms that act to enhance redox systems enable crops to overcome heat stress more effectively. Knowledge of thermo-tolerance within agronomic biodiversity is thus of key importance to enable researchers to identify new strategies for overcoming the impacts of climate change, and for decision-makers in planning for an uncertain future with new choices and options open to them.

Involvement of DNA methylation in the control of cell growth during heat stress in tobacco BY-2 cells.

The alteration of growth patterns, through the adjustment of cell division and expansion, is a characteristic response of plants to environmental stress. In order to study this response in more depth, the effect of heat stress on growth was investigated in tobacco BY-2 cells. The results indicate that heat stress inhibited cell division, by slowing cell cycle progression. Cells were stopped in the pre-mitotic phases, as shown by the increased expression of CycD3-1 and by the decrease in the NtCycA13, NtCyc29 and CDKB1-1 transcripts. The decrease in cell length and the reduced expression of Nt-EXPA5 indicated that cell expansion was also inhibited. Since DNA methylation plays a key role in controlling gene expression, the possibility that the altered expression of genes involved in the control of cell growth, observed during heat stress, could be due to changes in the methylation state of their promoters was investigated. The results show that the altered expression of CycD3-1 and Nt-EXPA5 was consistent with changes in the methylation state of the upstream region of these genes. These results suggest that DNA methylation, controlling the expression of genes involved in plant development, contributes to growth alteration occurring in response to environmental changes.