The innards of the cell: studies of water dipolar relaxation using the ACDAN fluorescent probe.

This article reviews the use of the 6-acetyl-2-(dimethylamino)naphthalene (ACDAN) fluorophore to study dipolar relaxation in cells, tissues, and biomimetic systems. As the most hydrophilic member of the 6-acyl-2-(dimethylamino)naphthalene series, ACDAN markedly partitions to aqueous environments. In contrast to 6-lauroyl-2-(dimethylamino)naphthalene (LAURDAN), the hydrophobic and best-known member of the series used to explore relaxation phenomena in biological (or biomimetic) membranes, ACDAN allows mapping of spatial and temporal water dipolar relaxation in cytosolic and intra-organelle environments of the cell. This is also true for the 6-propionyl-2-(dimethylamino)naphthalene (PRODAN) derivative which, unlike LAURDAN, partitions to both hydrophobic and aqueous environments. We will (i) summarize the mechanism which underlies the solvatochromic properties of the DAN probes, (ii) expound on the importance of water relaxation to understand the intracellular environment, (iii) discuss technical aspects of the use of ACDAN in eukaryotic cells and some specialized structures, including liquid condensates arising from processes leading to liquid immiscibility and, (iv) present some novel studies in plant cells and tissues which demonstrate the kinds of information that can be uncovered using this approach to study dipolar relaxation in living systems.

Autophagy modulates growth and development in the moss Physcomitrium patens.

Physcomitrium patens apical growing protonemal cells have the singularity that they continue to undergo cell divisions as the plant develops. This feature provides a valuable tool to study autophagy in the context of a multicellular apical growing tissue coupled to development. Herein, we showed that the core autophagy machinery is present in the moss P. patens, and characterized the 2D and 3D growth and development of atg5 and atg7 loss-of-function mutants under optimal and nutrient-deprived conditions. Our results showed that 2D growth of the different morphological and functional protonemata apical growing cells, chloronema and caulonema, is differentially modulated by this process. These differences depend on the protonema cell type and position along the protonemal filament, and growth condition. As a global plant response, the absence of autophagy favors the spread of the colony through protonemata growth at the expense of a reduction of the 3D growth, such as the buds and gametophore development, and thus the adult gametophytic and reproductive phases. Altogether this study provides valuable information suggesting that autophagy has roles during apical growth with differential responses within the cell types of the same tissue and contributes to life cycle progression and thus the growth and development of the 2D and 3D tissues of P. patens.

Leaf structure and ultrastructure changes induced by heat stress and drought during seed filling in field-grown soybean and their relationship with grain yield.

Studies focusing on terminal drought combined with heat impacts on plants of agronomic value remain scarce, and even less under field conditions. The objective of this study was to investigate leaf structural and ultrastructural changes induced by heat stress (HS) and drought stress (DS) during seed filling and their relationship with physiological variables and yield determination. Two soybean cultivars were grown in field conditions. During seed filling four treatments were applied, including a control (without manipulation, at ambient temperature and field capacity), HS (episodes exceeding 32°C for 6 h d-1) during 21-d, DS (20% of field capacity soil water content) during 35-d, and HS×DS. Drought principally reduced leaf area, whereas heat decreased leaf thickness, possible as acclimation strategies, but also irreversible reducing CO2 assimilation sites. Both stresses damaged the outer and inner membranes of chloroplasts, causing swollen chloroplasts and accumulation of plastoglobules, loss of chlorophyll content, and negatively affecting chlorophyll fluorescence. Thus, the performance and integrity of the photosynthetic machinery were reduced. Through a morpho-functional perspective and a holistic multiscale approach, our results provide evidence of photosynthesis impairment and yield drops under stressful conditions which were associated with structural and ultrastructural (particularly at the level of chloroplasts) modifications of leaves.

Leaf photosynthesis and senescence in heated and droughted field-grown soybean with contrasting seed protein concentration.

Soybean plants under heat and drought generate a multiplicity of responses in photosynthesis and senescence, impairing growth, yield, and seed quality. The goal of this study was to analyze and quantify independent and combined effects of heat and drought during seed filling on photosynthesis and senescence, and its subsequent effects on the filling duration in soybean genotypes contrasting on seed protein. Two field experiments were conducted using high and low seed protein genotypes. During seed filling plants were exposed to four treatments: control (ambient temperature and soil water content near field capacity), heat stress (HS, episodes above 32 °C 6 h d-1) during 15-d, drought stress (DS, soil water content ≤ 25% of field capacity) during the entire seed filling, and HS × DS. We found non-genotypic variation in leaf photosynthesis in both experiments. Irrigated HS, did no alter photosynthesis and senescence. Drought, regardless of heat, reduced photosynthesis, carbohydrate production and affected membranes integrity, leading to premature leaf senescence and shortening the filling duration. The magnitude of responses was similar between drought alone and stresses combined, indicating a dominant role of drought over heat. The seed filling duration was not shorter in high protein compared to low protein genotype, nor was senescence pattern altered across treatments. These results indicated that the higher seed protein content exhibited by some genotypes are not necessarily associated with an earlier onset of senescence and shortening of the filling period as suggested by previous studies analyzing genotypes differing in protein concentration.

Lipid Hydroperoxidation Effect on the Dynamical Evolution of the Conductance Process in Bilayer Lipid Membranes: A Condition Toward Criticality.

Cell membranes are one of the main targets of oxidative processes mediated by reactive oxygen species (ROS). These chemical species interact with unsaturated fatty acids of membrane lipids, triggering an autocatalytic chain reaction, producing lipid hydroperoxides (LOOHs) as the first relatively stable product of the ROS-mediated lipid peroxidation (LPO) process. Numerous biophysical and computational studies have been carried out to elucidate the LPO impact on the structure and organization of lipid membranes. However, although LOOHs are the major primary product of LPO of polyunsaturated fatty acids (PUFAs), to the best of our knowledge, there is no experimental evidence on the effects of the accumulation of these LPO byproducts on the electrical properties and the underlying dynamics of lipid membranes. In this work, bilayer lipid membranes (BLMs) containing 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocoline (POPC) with increasing hydroperoxidized POPC (POPC-OOH) molar proportions (BLMPC/PC-OOH) are used as model membranes to investigate the effect of LOOH-mediated LPO propagation on the electrical behavior of lipid bilayers. Voltage-induced ion current signals are analyzed by applying the fractal method of power spectrum density (PSD) analysis. We experimentally prove that, when certain LOOH concentration and energy threshold are overcome, oxidized membranes evolve toward a critical state characterized by the emergence of non-linear electrical behavior dynamics and the pore-type metastable structures formation. PSD analysis shows that temporal dynamics exhibiting "white" noise (non-time correlations) reflects a linear relationship between the input and output signals, while long-term correlations (ß > 0.5) begin to be observed closely to the transition (critical point) from linear (Ohmic) to nonlinear (non-Ohmic) behavior. The generation of lipid pores appears to arise as an optimized energy dissipation mechanism based on the system's ability to self-organize and generate ordered structures capable of dissipating energy gradients more efficiently under stressful oxidative conditions.

Reactive oxygen species are involved in the signaling of equine sperm chemotaxis.

Sperm chemotaxis may facilitate the finding of the oocyte. Only capacitated spermatozoa can orient their movement by chemotaxis, which as well as capacitation, is regulated in part by the cAMP-PKA pathway. Reactive oxygen species (ROS) are produced during sperm capacitation which is closely related to chemotaxis. Then, the ROS participation in the chemotactic signaling can be expected. Here we studied the role of ROS in the chemotaxis signaling of equine spermatozoa which produce high quantities of ROS because of their energy metabolism. The level of capacitated and chemotactic spermatozoa was increased with 0.1 and 0.2 mM hydrogen peroxide (H2O2), which was involved in the chemotactic signaling. By combining a concentration gradient of H2O2 with inhibitors/chelators of some of the signaling pathway elements, we showed that the activation of NOX (membrane NADPH oxidase) increases the intracellular ROS which activate the chemotaxis AMPc-PKA pathway. Our results provide evidence about the participation of ROS in the chemotactic signaling mediated by progesterone (P).

Interactions between soybean, Bradyrhizobium japonicum and Soybean mosaic virus: the effects depend on the interaction sequence.

The symbiotic interaction between soybean and nitrogen-fixing rhizobia can lead to plant growth promotion and induced systemic responses. Symbiotic interactions may increase tolerance/resistance to abiotic/biotic stress conditions, but are also sensitive to environmental conditions. Soybean mosaic virus (SMV), which is transmitted by seed and aphids, severely affects crop yields in many areas of the world, consequently virus infection may precede rhizobium infection or vice versa in the field. With the hypothesis that sequence of interaction is a key determinant of the resulting responses; growth, primary metabolism and defence responses were evaluated in different interaction sequences. Results showed that vegetative growth was promoted by Bradyrhizobium japonicum (Bj) inoculation and drastically impaired by SMV infection. The negative effect of SMV single infection on soybean growth parameters was correlated with photosynthesis decrease, sugar accumulation, oxidative damage, and increases in salicylic acid levels. Bj inoculation partially reversed virus-induced symptoms, mainly at Bj-SMV sequence. However, this symptom attenuation did not correlate with less virus accumulation. Nodulation was negatively affected by SMV, particularly when virus infection was previous to Bj inoculation (SMV-Bj). Defence related hormones (salicylic acid (SA)/jasmonic acid (JA)) and the expression of defence-related genes were dependent on the sequence of tripartite interaction. The present study showed that the sequence of the tripartite interaction among soybean, Bj and SMV determinates the tolerance/susceptibility to SMV infection, through changes in the defence mechanism and metabolic alteration.

Redox Systemic Signaling and Induced Tolerance Responses During Soybean-Bradyrhizobium japonicum Interaction: Involvement of Nod Factor Receptor and Autoregulation of Nodulation.

The symbiotic relationship between legumes and nitrogen-fixing rhizobia induces local and systemic responses, which ultimately lead to nodule formation. The autoregulation of nodulation (AON) is a systemic mechanism related to innate immunity that controls nodule development and involves different components ranging from hormones, peptides, receptors to small RNAs. Here, we characterized a rapid systemic redox changes induced during soybean-Bradyrhizobium japonicum symbiotic interaction. A transient peak of reactive oxygen species (ROS) generation was found in soybean leaves after 30 min of root inoculation with B. japonicum. The ROS response was accompanied by changes in the redox state of glutathione and by activation of antioxidant enzymes. Moreover, the ROS peak and antioxidant enzyme activation were abolished in leaves by the addition, in either root or leaf, of DPI, an NADPH oxidase inhibitor. Likewise, these systemic redox changes primed the plant increasing its tolerance to photooxidative stress. With the use of non-nodulating nfr5-mutant and hyper-nodulating nark-mutant soybean plants, we subsequently studied the systemic redox changes. The nfr5-mutant lacked the systemic redox changes after inoculation, whereas the nark-mutant showed a similar redox systemic signaling than the wild type plants. However, neither nfr5- nor nark-mutant exhibited tolerance to photooxidative stress condition. Altogether, these results demonstrated that (i) the early redox systemic signaling during symbiotic interaction depends on a Nod factor receptor, and that (ii) the induced tolerance response depends on the AON mechanisms.

Phosphatidylinositol 3-kinase function at very early symbiont perception: a local nodulation control under stress conditions?

Root hair curling is an early and essential morphological change required for the success of the symbiotic interaction between legumes and rhizobia. At this stage rhizobia grow as an infection thread within root hairs and are internalized into the plant cells by endocytosis, where the PI3K enzyme plays important roles. Previous observations show that stress conditions affect early stages of the symbiotic interaction, from 2 to 30 min post-inoculation, which we term as very early host responses, and affect symbiosis establishment. Herein, we demonstrated the relevance of the very early host responses for the symbiotic interaction. PI3K and the NADPH oxidase complex are found to have key roles in the microsymbiont recognition response, modulating the apoplastic and intracellular/endosomal ROS induction in root hairs. Interestingly, compared with soybean mutant plants that do not perceive the symbiont, we demonstrated that the very early symbiont perception under sublethal saline stress conditions induced root hair death. Together, these results highlight not only the importance of the very early host-responses on later stages of the symbiont interaction, but also suggest that they act as a mechanism for local control of nodulation capacity, prior to the abortion of the infection thread, preventing the allocation of resources/energy for nodule formation under unfavorable environmental conditions.

An Autophagy-Related Kinase Is Essential for the Symbiotic Relationship between Phaseolus vulgaris and Both Rhizobia and Arbuscular Mycorrhizal Fungi.

Eukaryotes contain three types of lipid kinases that belong to the phosphatidylinositol 3-kinase (PI3K) family. In plants and Saccharomyces cerevisiae, only PI3K class III family members have been identified. These enzymes regulate the innate immune response, intracellular trafficking, autophagy, and senescence. Here, we report that RNAi-mediated downregulation of common bean (Phaseolus vulgaris) PI3K severely impaired symbiosis in composite P. vulgaris plants with endosymbionts such as Rhizobium tropici and Rhizophagus irregularis Downregulation of Pv-PI3K was associated with a marked decrease in root hair growth and curling. Additionally, infection thread growth, root-nodule number, and symbiosome formation in root nodule cells were severely affected. Interestingly, root colonization by AM fungi and the formation of arbuscules were also abolished in PI3K loss-of-function plants. Furthermore, the transcript accumulation of genes encoding proteins known to interact with PI3K to form protein complexes involved in autophagy was drastically reduced in these transgenic roots. RNAi-mediated downregulation of one of these genes, Beclin1/Atg6, resulted in a similar phenotype as observed for transgenic roots in which Pv-PI3K had been downregulated. Our findings show that an autophagy-related process is crucial for the mutualistic interactions of P. vulgaris with beneficial microorganisms.

Expression of animal anti-apoptotic gene Ced-9 enhances tolerance during Glycine max L.-Bradyrhizobium japonicum interaction under saline stress but reduces nodule formation.

The mechanisms by which the expression of animal cell death suppressors in economically important plants conferred enhanced stress tolerance are not fully understood. In the present work, the effect of expression of animal antiapoptotic gene Ced-9 in soybean hairy roots was evaluated under root hairs and hairy roots death-inducing stress conditions given by i) Bradyrhizobium japonicum inoculation in presence of 50 mM NaCl, and ii) severe salt stress (150 mM NaCl), for 30 min and 3 h, respectively. We have determined that root hairs death induced by inoculation in presence of 50 mM NaCl showed characteristics of ordered process, with increased ROS generation, MDA and ATP levels, whereas the cell death induced by 150 mM NaCl treatment showed non-ordered or necrotic-like characteristics. The expression of Ced-9 inhibited or at least delayed root hairs death under these treatments. Hairy roots expressing Ced-9 had better homeostasis maintenance, preventing potassium release; increasing the ATP levels and controlling the oxidative damage avoiding the increase of reactive oxygen species production. Even when our results demonstrate a positive effect of animal cell death suppressors in plant cell ionic and redox homeostasis under cell death-inducing conditions, its expression, contrary to expectations, drastically inhibited nodule formation even under control conditions.

Redox-related metabolites and gene expression modulated by sugar in sunflower leaves: similarities with Sunflower chlorotic mottle virus-induced symptom.

Sugars are part of an integrated redox system, since they are key regulators of respiration and photosynthesis, and therefore of the levels of reducing power, ATP and ROS. These elements are major determinants of the cellular redox state, which is involved in the perception and regulation of many endogenous and environmental stimuli. Our previous findings suggested that early sugar increase produced during compatible Sunflower chlorotic mottle virus (SuCMoV) infection might modulate chlorotic symptom development through redox state alteration in sunflower. The purpose of this work was to characterize redox-related metabolites and gene expression changes associated with high sugar availability and symptom development induced by SuCMoV. The results show that sugar caused an increase in glutathione, ascorbate, pyridine nucleotides, and ATP. In addition, higher sugar availability reduced hydrogen peroxide and ΦPSII. This finding suggests that high sugar availability would be associated with cellular redox alteration and photoinhibitory process. The expression of the genes analyzed was also strongly affected by sugar, such as the down-regulation of psbA and up-regulation of psbO and cp29. The expression level of cytoplasmic (apx-1 and gr)- and chloroplastic (Fe-sod)-targeted genes was also significantly enhanced in sugar-treated leaves. Therefore, all these responses suggest that sugars induce chloroplastic redox state alteration with photoinhibition process that could be contributing to chlorotic symptom development during SuCMoV infection.

Negative short-term salt effects on the soybean-Bradyrhizobium japonicum interaction and partial reversion by calcium addition.

The short-term (2h) effects of salt stress (50 and 150mM NaCl) on early events of soybean- Bradyrhizobium japonicum (rhizobia) interaction were analysed, determining the following parameters in root hair with or without calcium addition: deformation, apoplastic superoxide radical production (O2⚫-), root hair death and sodium/potassium ion content. We also analysed whether this short-term salt stress influenced later formation of crown and noncrown nodules, determining the number and weight of nodules. The negative effect of salt stress on these characters was attenuated by the addition of 5mM CaCl2. We also analysed the expression of pathogenesis-related proteins (PRP) genes PR-1, PR-2, PR-3, and four isoforms of PR-5. The expression of PR-2 increased under saline conditions and decreased in osmotic treatment and saline treatment supplemented with calcium in the presence of the symbiont. The changes in PR-2 expression levels, together with the death of root hairs provide a possible mechanism for the inhibition of infection by the symbiont under salinity, and suggests a possible overlap with responses to plant pathogens.

The chlorotic symptom induced by Sunflower chlorotic mottle virus is associated with changes in redox-related gene expression and metabolites.

Systemic infections are commonly associated with changes in host metabolism and gene expression. Sunflower chlorotic mottle virus (SuCMoV) causes systemic infection with sugar increase, photoinhibition and increase in antioxidant enzyme activities before chlorotic symptom appearance in sunflower leaves. The aim of this study was to determine if chlorotic symptom development induced by SuCMoV infection is accompanied by changes in different redox-related metabolites and transcripts. Symptom development was analyzed in the second pair of leaves (systemic infection) at different post-inoculation times: before symptom appearance (BS, 4 dpi), and at an early (ES, 7 dpi) and later stage (LS, 12 dpi) of symptom expression. The results showed that the virus reaches the second pair of leaves at 4 dpi. A positive correlation between chlorotic symptom and number of viral copies was also observed. Changes in hydrogen peroxide, glutathione, pyridine nucleotides and ATP content were observed since symptom appearance (ES, 7 dpi). The expression of some of the genes analyzed was also strongly affected by SuCMoV infection. Specifically, down-regulation of both chloroplast-encoded genes and chloroplast-targeted genes: psbA, rbcS, Cu/Zn sod, Fe sod, phosphoglycolate phosphatase, psbO, psaH and fnr was present, whereas the expression of cytoplasmic-targeted genes, apx1, and Cu/Zn sod was up-regulated. Mitochondrial Mn sod decreased at BS stage and aox decreased only at ES stage. Peroxisomal catalase (cat-2) was lower at BS and LS stages. All these results suggest that SuCMoV infection induces progressive changes in determinants of redox homeostasis associated with chlorotic symptom development.

Are Sunflower chlorotic mottle virus infection symptoms modulated by early increases in leaf sugar concentration?

Symptom development in a susceptible sunflower line inoculated with Sunflower chlorotic mottle virus (SuCMoV) was followed in the second pair of leaves at different post-inoculation times: before symptom expression (BS), at early (ES) and late (LS) symptom expression. Sugar and starch increases and photoinhibition were observed as early effects BS, and were maintained or enhanced later on, however, chlorophyll loss was detected only at LS. Photoinhibition correlated with a drastic decrease in D1 protein level. The progress of infection was accompanied by decreasing levels of apoplastic reactive oxygen species (ROS). In infected leaves, higher antioxidant enzyme activities (superoxide dismutase, SOD; ascorbate peroxidase, APX; glutathione reductase, GR) were observed from BS. The purpose of this work was to evaluate whether the early increases in carbohydrate accumulation may participate in SuCMoV symptom expression. Similar effects on photoinhibition, apoplastic ROS generation and antioxidant activity were generated when healthy leaves were treated with sugars. These results suggest that photoinhibitory processes and lower apoplastic superoxide levels induced by SuCMoV infection may be modulated by sugar increases.

Reductions in maize root-tip elongation by salt and osmotic stress do not correlate with apoplastic O2*- levels.

BACKGROUND AND AIMS: Experimental evidence in the literature suggests that O(2)(*-) produced in the elongation zone of roots and leaves by plasma membrane NADPH oxidase activity is required for growth. This study explores whether growth changes along the root tip induced by hyperosmotic treatments in Zea mays are associated with the distribution of apoplastic O(2)(*-). METHODS: Stress treatments were imposed using 150 mm NaCl or 300 mM sorbitol. Root elongation rates and the spatial distribution of growth rates in the root tip were measured. Apoplastic O(2)(*-) was determined using nitro blue tetrazolium, and H(2)O(2) was determined using 2', 7'-dichlorofluorescin. KEY RESULTS: In non-stressed plants, the distribution of accelerating growth and highest O(2)(*-) levels coincided along the root tip. Salt and osmotic stress of the same intensity had similar inhibitory effects on root elongation, but O(2)(*-) levels increased in sorbitol-treated roots and decreased in NaCl-treated roots. CONCLUSIONS: The lack of association between apoplastic O(2)(*-) levels and root growth inhibition under hyper-osmotic stress leads us to hypothesize that under those conditions the role of apoplastic O(2)(*-) may be to participate in signalling processes, that convey information on the nature of the substrate that the growing root is exploring.