Lentil adaptation to drought stress: response, tolerance, and breeding approaches.

Lentil (Lens culinaris Medik.) is a cool season legume crop that plays vital roles in food and nutritional security, mostly in the least developed countries. Lentil is often cultivated in dry and semi-dry regions, where the primary abiotic factor is drought, which negatively impacts lentil growth and development, resulting in a reduction of yield. To withstand drought-induced multiple negative effects, lentil plants evolved a variety of adaptation strategies that can be classified within three broad categories of drought tolerance mechanisms (i.e., escape, avoidance, and tolerance). Lentil adapts to drought by the modulation of various traits in the root system, leaf architecture, canopy structure, branching, anatomical features, and flowering process. Furthermore, the activation of certain defensive biochemical pathways as well as the regulation of gene functions contributes to lentil drought tolerance. Plant breeders typically employ conventional and mutational breeding approaches to develop lentil varieties that can withstand drought effects; however, little progress has been made in developing drought-tolerant lentil varieties using genomics-assisted technologies. This review highlights the current understanding of morpho-physiological, biochemical, and molecular mechanisms of lentil adaptation to drought stress. We also discuss the potential application of omics-assisted breeding approaches to develop lentil varieties with superior drought tolerance traits.

GUARD CELL HYDROGEN PEROXIDE-RESISTANT1 functions upstream of reactive carbonyl species production in Arabidopsis guard-cell abscisic acid signaling.

GUARD CELL HYDROGEN PEROXIDE-RESISTANT1 (GHR1), a leucine-rich repeat receptor-like kinase, is involved in abscisic acid (ABA)-induced stomatal closure. We investigated the role of GHR1 in reactive oxygen species (ROS) signaling for ABA-induced stomatal closure. Abscisic acid induced ROS production in wild type (WT) and the ghr1 of Arabidopsis thaliana. Hydrogen peroxide induced stomatal closure, accompanying the generation of acrolein in guard cells. The reactive carbonyl species (RCS) scavengers inhibited the ABA- and H2O2-induced stomatal closure in WT. In the ghr1, H2O2 failed to induce acrolein production and stomatal closure while RCS induced stomatal closure. Thus, GHR1 functions downstream of ROS and is required for the generation of RCS in guard-cell ABA signaling. In the ghr1, Ca2+ induced stomatal closure but RCS did not activate ICa channels. The GHR1 may be also involved in a Ca2+-independent pathway for ABA-induced stomatal closure.

Outward-rectifying potassium channels GORK and SKOR function in regulation of root growth under salt stress in Arabidopsis thaliana.

Plants often face high salinity as a significant environmental challenge with roots being the first responders to this stress. Maintaining K+/Na+ ratio within plant cells is crucial for survival, as the intracellular K+ level decreases and the intracellular Na+ level increases under saline conditions. However, knowledge about the molecular regulatory mechanisms of K+ loss in response to salt stress through outward-rectifying K+ channels in plants is largely unknown. In this study, we found that the Arabidopsis double mutant gorkskor, in which the GORK and SKOR genes are disrupted, showed an improved primary root growth under salt stress compared to wild-type (WT) and the gork and skor single-mutant plants. No significant differences in the sensitivity to mannitol stress between the WT and gorkskor mutant were observed. Accumulation of ROS induced by salt stress was reduced in the gorkskor roots. The gorkskor mutant seedlings had significantly higher K+ content, lower Na+ content, and a greater resultant K+/Na+ ratio than the WT under salt stress. Moreover, salt-stress-induced elevation of cytosolic free Ca2+ concentration was reduced in the gorkskor roots. Taken together, these results suggest that Arabidopsis Shaker-type outward-rectifying K+ channels GORK and SKOR may redundantly function in regulation of primary root growth under salt stress and are involved in not only the late-stage response (e.g. K+ leakage) but also the early response including ROS production and [Ca2+]cyt elevation.

Quercetin Attenuates Acetaldehyde-Induced Cytotoxicity via the Heme Oxygenase-1-Dependent Antioxidant Mechanism in Hepatocytes.

It is still unclear whether or how quercetin influences the toxic events induced by acetaldehyde in hepatocytes, though quercetin has been reported to mitigate alcohol-induced mouse liver injury. In this study, we evaluated the modulating effect of quercetin on the cytotoxicity induced by acetaldehyde in mouse hepatoma Hepa1c1c7 cells, the frequently used cellular hepatocyte model. The pretreatment with quercetin significantly inhibited the cytotoxicity induced by acetaldehyde. The treatment with quercetin itself had an ability to enhance the total ALDH activity, as well as the ALDH1A1 and ALDH3A1 gene expressions. The acetaldehyde treatment significantly enhanced the intracellular reactive oxygen species (ROS) level, whereas the quercetin pretreatment dose-dependently inhibited it. Accordingly, the treatment with quercetin itself significantly up-regulated the representative intracellular antioxidant-related gene expressions, including heme oxygenase-1 (HO-1), glutamate-cysteine ligase, catalytic subunit (GCLC), and cystine/glutamate exchanger (xCT), that coincided with the enhancement of the total intracellular glutathione (GSH) level. Tin protoporphyrin IX (SNPP), a typical HO-1 inhibitor, restored the quercetin-induced reduction in the intracellular ROS level, whereas buthionine sulphoximine, a representative GSH biosynthesis inhibitor, did not. SNPP also cancelled the quercetin-induced cytoprotection against acetaldehyde. These results suggest that the low-molecular-weight antioxidants produced by the HO-1 enzymatic reaction are mainly attributable to quercetin-induced cytoprotection.

Evaluation of quercetin as a potential cytoprotector against acetaldehyde using the cultured hepatocyte model with aldehyde dehydrogenase isozyme deficiency.

Protective effect of quercetin against acetaldehyde was evaluated using the cultured hepatocyte models with aldehyde dehydrogenase (ALDH) isozyme deficiency (aldh2-kd and aldh1a1-kd). The quercetin-induced cytoprotection against acetaldehyde in the ALDH1A1-deficient mutant (aldh1a1-kd) was weaker than that in the wild type. Furthermore, quercetin did not enhance the ALDH activity in aldh1a1-kd cells, suggesting that ALDH1A1 is involved in quercetin-induced cytoprotection.

Negative regulation of chitosan-induced stomatal closure by glutathione in Arabidopsis thaliana.

Chitosan (CHT) is a deacylated derivative of chitin and improves growth and yield performance, activates defensive genes, and also induces stomatal closure in plants. Glutathione (GSH) has significant functions in the growth, development, defense systems, signaling, and gene expression. GSH negatively regulates abscisic acid-, methyl jasmonate-, and salicylic acid-induced stomatal closure. However, the negative regulation by GSH of CHT-induced stomatal closure is still unknown. Regulation of CHT-induced stomatal closure by GSH in guard cells was investigated using two GSH-deficient mutants, cad2-1 and chlorina 1-1 (ch1-1), and a GSH-decreasing chemical, 1-chloro-2,4-dinitrobenzene (CDNB). The cad2-1 and ch1-1 mutations and CDNB treatment enhanced CHT-induced stomatal closure. Treatment with glutathione monoethyl ester restored the GSH level in the guard cells of cad2-1 and ch1-1 and complemented the stomatal phenotype of the mutants. These results indicate that GSH negatively regulates CHT-induced stomatal closure in Arabidopsis thaliana.

Enhancing effect of the coexisting alpha-tocopherol on quercetin absorption and metabolism.

The aim of this study is to investigate the modulating effect of coexisting food components on the absorption and metabolism of quercetin and blood plasma antioxidant potentials. The combination of quercetin with α-tocopherol (αT), cellulose, or a commercially available vegetable beverage containing αT and dietary fiber was orally administered to mice. Compared to the single administration of quercetin aglycone, the coadministration of αT with quercetin significantly increased the plasma quercetin concentration at 0.5 h, whereas the combination of quercetin and cellulose decreased it. Interestingly, the administration of quercetin mixed with the vegetable beverage showed no significant change in the quercetin concentration in the mice plasma. The treatment of the cells with the blood plasma after the coadministration of αT with quercetin significantly upregulated the gene expression of the antioxidant enzyme (heme oxygenase-1), whereas the quercetin and cellulose combination did not. In the plasma of the quercetin-administered mice, eight types of quercetin metabolites were detected, and their quantities were affected by the combination with αT. The potentials of the heme oxygenase-1 gene expression by these metabolites were very limited, although several metabolites showed radical scavenging activities comparable to aglycone in the in vitro assays. These results suggested that the combination of αT potentiates the quercetin absorption and metabolism and thus the plasma antioxidant potentials, at least in part, by the quantitative changes in the quercetin metabolites.

Role of calcium signaling in cadmium stress mitigation by indol-3-acetic acid and gibberellin in chickpea seedlings.

Given the adverse impacts of heavy metals on plant development and physiological processes, the present research investigated the protective role of indole-3-acetic acid (IAA) and gibberellic acid (GA3) against cadmium (Cd)-induced injury in chickpea seedlings. Therefore, seeds germinated for 6 days in a medium containing 200 µM Cd alone or combined with 10 µM GA3 or 10 µM IAA. Both GA3 and IAA mitigated Cd-imposed growth delays in roots and shoots (80% and 50% increase in root and shoot length, respectively). This beneficial effect was accompanied by a significant reduction in Cd2+ accumulation in both roots (74% for IAA and 38% for GA3) and shoots (68% and 35%, respectively). Furthermore, these phytohormones restored the cellular redox state by reducing the activity of NADPH oxidase and downregulating the transcription level of RbohF and RbohD genes. Likewise, hydrogen peroxide contents were reduced by GA3 and IAA supply. Additionally, GA3 and IAA countered the Cd-induced reduction in total phenols, flavonoids, and reducing sugars in both roots and shoots. The exogenous effectors enhanced the activities of catalase, ascorbate peroxidase, and thioredoxin, as well as the corresponding gene expressions. Interestingly, adding GA3 and IAA to the Cd-contaminated germination media corrected the level of calcium (Ca2+) ion within seedling tissues. This effect coincided with the upregulation of key genes associated with stress sensing and signal transduction, including auxin-binding protein (ABP19a), mitogen-activated protein kinase (MAPK2), calcium-dependent protein kinase (CDPK1), and calmodulin (CaM). Overall, the current results suggest that GA3 and IAA sustain the Ca2+ signaling pathway, resulting in metal phytotoxicity relief. Amendment of agricultural soils contaminated with heavy metals with GA3 or IAA could represent an effective practice to improve crop yield.

An outward-rectifying plant K+ channel SPORK2 exhibits temperature-sensitive ion-transport activity.

Temperature sensing is critical for the survival of living organisms.1,2 Thermosensitive transient receptor-potential (TRP) cation channels function as thermosensors in mammals.2,3,4,5,6 In contrast to animals, land plants lack TRP genes.7,8,9 Previous patch-clamp studies in plant cells suggested the presence of ion channels whose activities are related to temperature, implying the presence of TRP-like channels.10,11,12,13,14 However, the molecular entities of such temperature-sensitive ion channels were still unknown in land plants. In this study, we observed that the unique rainfall-induced leaf-folding movement of the legume tree Samanea saman15 was temperature-sensitive by using a rainfall-mimicking assay. Chilling-induced leaf folding in S. saman was shown to be related to the swelling of the motor cells16,17 at the base of the leaflet. This swelling suggested involvement of temperature-sensitive inactivation of K+ currents, independent of fluctuations in ion channel gene expression in motor cells. These findings led us to examine the temperature sensitivity of an outward-rectifying K+ channel, SPORK2, which was reported as an ion channel responsible for the nyctinastic (circadian-rhythmic) leaf movement of S. saman.18 We also discovered that SPORK2 exhibits temperature-sensitive K+ transport activity in the Xenopus oocyte expression system. Using chimeric channels, we showed that two domains of SPORK2 regulated the temperature sensitivity. Furthermore, heterologously expressed SPORK2 in Arabidopsis guard cells induced temperature-dependent stomatal closure. Therefore, SPORK2 is an ion channel in land plants with temperature-sensitive ion-transport activity that functions similarly to mammalian TRP channels. Our current findings advance the molecular understanding of temperature-sensing mechanisms in plants.

The effect of exogenous dihydroxyacetone and methylglyoxal on growth, anthocyanin accumulation, and the glyoxalase system in Arabidopsis.

Dihydroxyacetone (DHA) occurs in wide-ranging organisms, including plants, and can undergo spontaneous conversion to methylglyoxal (MG). While the toxicity of MG to plants is well-known, the toxicity of DHA to plants remains to be elucidated. We investigated the effects of DHA and MG on Arabidopsis. Exogenous DHA at up to 10 mm did not affect the radicle emergence, the expansion of green cotyledons, the seedling growth, or the activity of glyoxalase II, while DHA at 10 mm inhibited the root elongation and increased the activity of glyoxalase I. Exogenous MG at 1.0 mm inhibited these physiological responses and increased both activities. Dihydroxyacetone at 10 mm increased the MG content in the roots. These results indicate that DHA is not so toxic as MG in Arabidopsis seeds and seedlings and suggest that the toxic effect of DHA at high concentrations is attributed to MG accumulation by the conversion to MG.

Combined Effect of Salicylic Acid and Proline Mitigates Drought Stress in Rice (Oryza sativa L.) through the Modulation of Physiological Attributes and Antioxidant Enzymes.

Salicylic acid (SA) and proline exhibit protective effects against a wide range of stresses. However, the combined impact of SA and proline on rice under drought stress is still unknown. Therefore, we investigated the protective roles of SA and/or proline in conferring drought tolerance in rice. There were eight treatments comprising the control (T1; 95-100% FC), 1.5 mM SA (T2), 2 mM proline (T3), 0.75 mM SA + 1 mM proline (T4), 45-50% FC (T5, drought stress), T5 + 1.5 mM SA (T6), T5 + 2 mM proline (T7), and T5 + 0.75 mM SA + 1 mM proline (T8), and two rice varieties: BRRI dhan66 and BRRI dhan75. Drought stress significantly decreased the plant growth, biomass, yield attributes, photosynthetic rate (Pn), stomatal conductance (gs), transpiration rate (Tr), photosynthetic pigments (chlorophyll and carotenoids content), relative water content (RWC), membrane stability index (MSI), soluble sugar and starch content, and uptake of N, P and K+ in roots and shoots. Drought-induced oxidative stress in the form of increased hydrogen peroxide (H2O2) production and lipid peroxidation (MDA) was observed. The combined application of SA (0.75 mM) + proline (1 mM) was found to be more effective than the single application of either for drought stress mitigation in rice. A combined dose of SA + proline alleviated oxidative stress through boosting antioxidant enzymatic activity in contrast to their separate application. The application of SA + proline also enhanced proline, soluble sugar and starch content, which resulted in the amelioration of osmotic stress. Consequently, the combined application of SA and proline significantly increased the gas exchange characteristics, photosynthetic pigments, RWC, MSI, nutrient uptake, plant growth, biomass and yield of rice. Therefore, the combined application of SA and proline alleviated the detrimental impacts of drought stress more pronouncedly than their separate application did by increasing osmoprotectants, improving nutrient transport, up-regulating antioxidant enzyme activity and inhibiting oxidative stress.

Seed pretreatment with melatonin confers cadmium tolerance to chickpea seedlings through cellular redox homeostasis and antioxidant gene expression improvement.

Several phytoremediation strategies have been undertaken to alleviate cadmium (Cd)-mediated injury to crop yield resulting from agricultural land pollution. In the present study, the potentially beneficial effect of melatonin (Me) was appraised. Therefore, chickpea (Cicer arietinum L.) seeds were imbibed for 12 H in distilled water or Me (10 µM) solution. Then, the seeds germinated in the presence or the absence of 200 µM CdCl2 for 6 days. Seedlings obtained from Me-pretreated seeds exhibited enhanced growth traits, reflected by fresh biomass and length increase. This beneficial effect was associated with a decreased Cd accumulation in seedling tissues (by 46 and 89% in roots and shoots, respectively). Besides, Me efficiently protected the cell membrane integrity of Cd-subjected seedlings. This protective effect was manifested by the decreased lipoxygenase activity and the subsequently reduced accumulation of 4-hydroxy-2-nonenal. Melatonin counteracted the Cd-mediated stimulation of the pro-oxidant NADPH-oxidase (90 and 45% decrease compared to non-pretreated Cd-stressed roots and shoots, respectively) and NADH-oxidase activities (almost 40% decrease compared to non-pretreated roots and shoots), preventing, thus, hydrogen peroxide overaccumulation (50 and 35% lesser than non-pretreated roots and shoots, respectively). Furthermore, Me enhanced the cellular content of pyridine nicotinamide reduced forms [NAD(P)H] and their redox state. This effect was associated with the Me-mediated stimulation of the glucose-6-phosphate dehydrogenase (G6PDH) and malate dehydrogenase activities, concomitantly with the inhibition of NAD(P)H-consuming activities. These effects were accompanied by the up-regulation of G6PDH gene expression (45% increase in roots) and the down-regulation of the respiratory burst oxidase homolog protein F (RBOHF) gene expression (53% decrease in roots and shoots). Likewise, Me induced an increased activity and gene transcription of the Asada-Halliwell cycle, namely ascorbate peroxidase, monodehydroascorbate reductase, dehydroascorbate reductase, and glutathione reductase, concomitantly with a reduction of the glutathione peroxidase activity. This modulating effect led to the restoration of the redox homeostasis of the ascorbate and the glutathione pools. Overall, current results attest that seed pretreatment with Me is effective in Cd stress relief and can be a beneficial crop-protective approach.

Exogenous Application of Calcium Ameliorates Salinity Stress Tolerance of Tomato (Solanum lycopersicum L.) and Enhances Fruit Quality.

Tomato is affected by various biotic and abiotic stresses, especially salinity, which drastically hinders the growth and yield of tomato. Calcium (Ca) is a vital macronutrient which plays physiological and biochemical roles in plants. Hence, we studied the protective roles of Ca against salinity stress in tomato. There were eight treatments comprising control (nutrient solution), 5 mM Ca, 10 mM Ca, 15 mM Ca, 12 dS m-1 NaCl, 12 dS m-1 NaCl + 5 mM Ca, 12 dS m-1 NaCl + 10 mM Ca and 12 dS m-1 NaCl + 15 mM Ca, and two tomato varieties: BARI tomato-2 and Binatomato-5. Salinity significantly decreased the plant-growth and yield attributes, relative water content (RWC), photosynthetic pigments (SPAD value) and the uptake of K, Ca and Mg in leaves and roots. Salinity-induced oxidative stress was present in the form of increased Na+ ion concentration, hydrogen peroxide (H2O2) content and lipid peroxidation (MDA). Ca application reduced oxidative stress through the boosting of antioxidant enzymatic activity. Exogenous Ca application enhanced proline and glycine betaine content and reduced Na+ uptake, which resulted in the inhibition of ionic toxicity and osmotic stress, respectively. Hence, Ca application significantly increased the growth and yield attributes, RWC, SPAD value, and uptake of K, Ca and Mg. Calcium application also had a significant effect on the fruit quality of tomato and the highest total soluble solid, total sugar, reducing sugar, ß-carotene, vitamin C and juice pH were found for the combined application of NaCl and Ca. Therefore, application of Ca reversed the salt-induced changes through increasing osmoprotectants, activation of antioxidants enzymes, and by optimizing mineral nutrient status.

Cycloartenyl Ferulate Is the Predominant Compound in Brown Rice Conferring Cytoprotective Potential against Oxidative Stress-Induced Cytotoxicity.

Since brown rice extract is a rich source of biologically active compounds, the present study is aimed to quantify the major compounds in brown rice and to compare their cytoprotective potential against oxidative stress. The content of the main hydrophobic compounds in brown rice followed the order of cycloartenyl ferulate (CAF) (89.00 ± 8.07 nmol/g) >> α-tocopherol (αT) (19.73 ± 2.28 nmol/g) > γ-tocotrienol (γT3) (18.24 ± 1.41 nmol/g) > α-tocotrienol (αT3) (16.02 ± 1.29 nmol/g) > γ-tocopherol (γT) (3.81 ± 0.40 nmol/g). However, the percent contribution of CAF to the radical scavenging activity of one gram of whole brown rice was similar to those of αT, αT3, and γT3 because of its weaker antioxidant activity. The CAF pretreatment displayed a significant cytoprotective effect on the hydrogen peroxide-induced cytotoxicity from 10 µM, which is lower than the minimal concentrations of αT and γT required for a significant protection. CAF also enhanced the nuclear factor erythroid 2-related factor 2 (Nrf2) nuclear translocation coincided with the enhancement of the heme oxygenase-1 (HO-1) mRNA level. An HO-1 inhibitor, tin protoporphyrin IX (SnPP), significantly impaired the cytoprotection of CAF. The cytoprotective potential of CAF is attributable to its cycloartenyl moiety besides the ferulyl moiety. These results suggested that CAF is the predominant cytoprotector in brown rice against hydrogen peroxide-induced cytotoxicity.

The microbiota catabolites of quercetin glycosides concertedly enhance the resistance against acetaldehyde-induced oxidative stress.

3,4-Dihydroxyphenylacetic acid (DOPAC) and 3-hydroxyphenylacetic acid (OPAC) are the predominant catabolites of quercetin glycosides, such as quercetin 4'-O-ß-glucoside from the onion, produced by intestinal microbiota. Although each catabolite has been reported to protect the cells from acetaldehyde-induced cytotoxicity, the effect of their combination remains to be clarified. The purpose of this study was to determine whether the combination of DOPAC and OPAC enhances the resistance against the acetaldehyde-induced oxidative stress in the cultured hepatocytes. The pretreatment of the combination of DOPAC (5 µM) and OPAC (5 µM) showed significant protection against the acetaldehyde- and hydrogen peroxide-induced cytotoxicity, even though each compound at the same concentration did not. This combination also significantly inhibited the intracellular dichlorofluorescin diacetate-detectable reactive oxygen species (ROS) level, whereas the solo treatment did slightly, suggesting that reducing mechanisms of ROS or compounds that enhance ROS production are involved in the cytoprotective effect. The combinatory treatment significantly enhanced the gene expression of not only the aldehyde dehydrogenases (ALDHs), but also glutamate-cysteine ligase, catalytic subunit, the first rate-limiting enzyme of glutathione (GSH) synthesis. Accordingly, both the intracellular GSH level and the total ALDH activity were enhanced by DOPAC plus OPAC. Involvement of GSH in the cytoprotection as well as ALDH up-regulation by the combination was confirmed by the experiments using a GSH biosynthesis inhibitor, buthionine sulfoximine. Taken together, the present results suggested that the quercetin microbiota catabolites concertedly protect the cells from acetaldehyde through a pre-enhanced resistance against oxidative stress by the GSH-dependent up-regulation of ALDHs.

Exogenous nitric oxide promotes salinity tolerance in plants: A meta-analysis.

Nitric oxide (NO) has received much attention since it can boost plant defense mechanisms, and plenty of studies have shown that exogenous NO improves salinity tolerance in plants. However, because of the wide range of experimental settings, it is difficult to assess the administration of optimal dosages, frequency, timing, and method of application and the overall favorable effects of NO on growth and yield improvements. Therefore, we conducted a meta-analysis to reveal the exact physiological and biochemical mechanisms and to understand the influence of plant-related or method-related factors on NO-mediated salt tolerance. Exogenous application of NO significantly influenced biomass accumulation, growth, and yield irrespective of salinity stress. According to this analysis, seed priming and foliar pre-treatment were the most effective methods of NO application to plants. Moreover, one-time and regular intervals of NO treatment were more beneficial for plant growth. The optimum concentration of NO ranges from 0.1 to 0.2 mM, and it alleviates salinity stress up to 150 mM NaCl. Furthermore, the beneficial effect of NO treatment was more pronounced as salinity stress was prolonged (>21 days). This meta-analysis showed that NO supplementation was significantly applicable at germination and seedling stages. Interestingly, exogenous NO treatment boosted plant growth most efficiently in dicots. This meta-analysis showed that exogenous NO alleviates salt-induced oxidative damage and improves plant growth and yield potential by regulating osmotic balance, mineral homeostasis, photosynthetic machinery, the metabolism of reactive oxygen species, and the antioxidant defense mechanism. Our analysis pointed out several research gaps, such as lipid metabolism regulation, reproductive stage performance, C4 plant responses, field-level yield impact, and economic profitability of farmers in response to exogenous NO, which need to be evaluated in the subsequent investigation.

Benzyl isothiocyanate and its metabolites inhibit cell proliferation through protein modification in mouse preosteoclast RAW264.7 cells.

Benzyl isothiocyanate (BITC), derived from cruciferous vegetables, is an organosulfur compound exerting antiproliferative effects in several human cancer cells. In this study, we assessed BITC as a potential osteoclastogenesis inhibitor and investigated its underlying mechanism. BITC at 5 µM significantly decreased the viability of the osteoclast-like differentiating RAW264.7 cells, coinciding with the downregulation of the primary biomarkers for osteoclast differentiation, such as the tartrate-resistant acid phosphatase activity and nuclear factor of activated T-cells gene expression. Not only BITC but also its metabolites, inhibited cell proliferation in the normal RAW264.7 cells, suggesting that BITC shows an anti-osteoclastogenesis effect in vivo after its ingestion and metabolism, possibly through an antiproliferative action. Both BITC and its metabolites also enhanced the DNA fragmentation and the caspase-3 activity, whereas their higher concentrations tended to suppress these effects. BITC was intracellularly accumulated when the cells were treated with its metabolites via their degradation into the free form. A quantitative experiment using the proteolysis/high performance liquid chromatography technique showed that the amount of BITC-lysine thiourea in the cells was also increased in a time-dependent manner, suggesting that lysine modification of the cellular proteins actually took place in the cells treated by BITC. Among the cellular proteins, the cleaved caspase-3 was identified as a potential target for lysine modification by BITC. Taken together, BITC dissociated from its metabolites as well as its free form might modulate osteoclastogenesis, possibly through inhibition of cell proliferation by protein modification.

Malate induces stomatal closure via a receptor-like kinase GHR1- and reactive oxygen species-dependent pathway in Arabidopsis thaliana.

A primary metabolite malate is secreted from guard cells in response to the phytohormone abscisic acid (ABA) and elevated CO2. The secreted malate subsequently facilitates stomatal closure in plants. Here, we investigated the molecular mechanism of malate-induced stomatal closure using inhibitors and ABA signaling component mutants of Arabidopsis thaliana. Malate-induced stomatal closure was impaired by a protein kinase inhibitor, K252a, and also by the disruption of a receptor-like kinase GHR1, which mediates activation of calcium ion (Ca2+) channel by reactive oxygen species (ROS) in guard cells. Malate induced ROS production in guard cells while the malate-induced stomatal closure was impaired by a peroxidase inhibitor, salicylhydroxamic acid, but not by the disruption of Nicotinamide adenine dinucleotide (phosphate) (NAD(P)H) oxidases, RBOHD and RBOHF. The malate-induced stomatal closure was impaired by Ca2+ channel blockers, verapamil, and niflumic acid. These results demonstrate that the malate signaling is mediated by GHR1 and ROS in Arabidopsis guard cells.

Negative regulation of salicylic acid-induced stomatal closure by glutathione in Arabidopsis thaliana.

Salicylic acid (SA) is a ubiquitous phenolic phytohormone that induces stomatal closure. Glutathione (GSH) negatively regulates stomatal closure induced by other plant hormones such as abscisic acid (ABA) and methyl jasmonate (MeJA). However, the involvement of GSH in SA-induced stomatal closure is still unknown. We investigated the regulation of SA signaling by GSH in guard cells using an Arabidopsis thaliana mutant, cad2-1, which is deficient in the first GSH biosynthesis enzyme, γ-glutamylcysteine synthetase. Application of SA decreased stomatal apertures with decreasing intracellular GSH level in guard cells. Decreasing GSH by the cad2-1 mutation and by a GSH-decreasing chemical, 1-chloro-2,4-dinitrobenzene, enhanced the SA-induced stomatal closure. Treatment with glutathione monoethyl ester restored the GSH level in the cad2-1 guard cells and complemented the stomatal phenotype of the mutant. These results indicate that GSH negatively modulates SA-induced stomatal closure in A. thaliana.

Extracellular malate induces stomatal closure via direct activation of guard-cell anion channel SLAC1 and stimulation of Ca2+ signalling.

Plants secrete malate from guard cells to apoplast under stress conditions and exogenous malate induces stomatal closure. Malate is considered an extracellular chemical signal of stomatal closure. However, the molecular mechanism of malate-induced stomatal closure is not fully elucidated. We investigated responses of stomatal aperture, ion channels, and cytosolic Ca2+ to malate. A treatment with malate induced stomatal closure in Arabidopsis thaliana wild-type plants, but not in the mutants deficient in the slow (S-type) anion channel gene SLOW ANION CHANNEL-ASSOCIATED 1 (SLAC1). The treatment with malate increased S-type anion currents in guard-cell protoplasts of wild-type plants but not in the slac1 mutant. In addition, extracellular rather than intracellular application of malate increased the S-type currents of constitutively active mutants of SLAC1, which have kinase-independent activities, in a heterologous expression system using Xenopus oocytes. The treatment with malate transiently increased cytosolic Ca2+ concentration in the wild-type Arabidopsis guard cells and the malate-induced stomatal closure was inhibited by the Ca2+ channel blocker and the Ca2+ chelator. These results indicate that extracellular malate directly activates SLAC1 and simultaneously stimulates Ca2+ signalling in guard cells, resulting in steady and solid activation of SLAC1 for stomatal closure.