Calcium is involved in the abscisic acid-induced ascorbate peroxidase, superoxide dismutase and chilling resistance in Stylosanthes guianensis

The objective of this work was to test whether Ca²⁺, a second messenger in stress response, is involved in ABA-induced antioxidant enzyme activities in Stylosanthes guianensis. Plants were sprayed with abscisic acid (ABA), calcium channel blocker, LaCl₃, calcium chelator, ethylene glycol-bis(β-amino ethyl ether)-N,N,N′, N′-tetraacetid acid (EGTA), and ABA in combination with LaCl₃ or EGTA. Their effects on superoxide dismutase (SOD) and ascorbate peroxidase (APX) activities and chilling resistance were compared. The results showed that ABA decreased electrolyte leakage and lipid peroxidation but increased maximum photochemical efficiency measured as variable to maximum fluorescence ratio (F_v/F_m) under chilling stress. Treatment with LaCl₃ or EGTA alone and in combination with ABA increased electrolyte leakage and lipid peroxidation, decreased Fv/Fm, suggesting that the block in Ca²⁺ signalling decreased chilling resistance of S. guianensis and the ABA-enhanced chilling resistance. ABA-induced SOD and APX activities were suppressed by LaCl₃ or EGTA. The results suggested that Ca²⁺ is involved in the ABA-enhanced chilling resistance and the ABA-induced SOD and APX activities in S. guianensis.     

Effects of Calcium and Calmodulin Antagonists on Chilling Stress-Induced Proline Accumulation in <Emphasis Type="Italic">Jatropha curcas</Emphasis> L.

Regulation of proline accumulation in plants under chilling stress remains unclear. In this paper, we treated Jatropha curcas seedlings under chilling stress with exogenous calcium chloride (CaCl₂), the plasma membrane Ca²⁺-channel blocker lanthanum chloride (LaCl₃), calmodulin antagonists, chlorpromazine (CPZ), and trifluoperazine (TFP) and investigated the effects of calcium and calmodulin (CaM) on proline accumulation and chilling tolerance. The results showed that CaCl₂ treatment significantly enhanced chilling stress-induced proline accumulation. CaCl₂ also induced an almost immediate and rapid increase of Δ¹-pyrroline-5-carboxylate synthetase (P5CS) and glutamate dehydrogenase activities, the key enzymes in the glutamate pathway of proline biosynthesis, and up-regulated P5CS expression, but it decreased the activity of proline dehydrogenase (ProDH), a key enzyme of proline degradation, and inhibited ProDH expression. Treatment with LaCl₃, CPZ, and TFP exhibited the opposite effects to those by CaCl₂ treatment. Moreover, CaCl₂, LaCl₃, CPZ, and TFP had little effect on the activities of ornithine aminotransferase and arginase, the key enzymes in the ornithine pathway of proline biosynthesis. These results indicated that Ca²⁺-CaM might be involved in signal transduction events, leading to proline accumulation in J. curcas seedlings under chilling stress, and that Ca²⁺-induced proline accumulation is a combined result of the activation of the glutamate pathways of proline biosynthesis and the simultaneous inhibition of the proline degradation pathway. In addition, CaCl₂ treatment increased tissue vitality, decreased the content of the lipid peroxidation product malondialdehyde (MDA), and alleviated electrolyte leakage in J. curcas seedlings under chilling stress, indicating that exogenous Ca²⁺ can enhance chilling tolerance, and proline might be a key factor in this increased chilling tolerance.     

Physiological and Biochemical Mechanisms of Exogenous Calcium Chloride on Alleviating Salt Stress in Two Tartary Buckwheat (<i>Fagopyrum tataricum</i>) Varieties Differing in Salinity Tolerance

Salt stress is one of the most serious abiotic stresses limiting plant growth and development. Calcium as an essential nutrient element and important signaling molecule plays an important role in ameliorating the adverse effect of salinity on plants. This study aimed to investigate the impact of exogenous calcium on improving salt tolerance in Tartary buckwheat cultivars, cv. Xinong9920 (salt-tolerant) and cv. Xinong9909 (salt-sensitive). Four-week-old Tartary buckwheat seedlings under 100 mM NaCl stress were treated with and without exogenous calcium chloride (CaCl₂), Ca²⁺ chelator ethylene glycol tetraacetic acid (EGTA) and Ca²⁺-channel blocker lanthanum chloride (LaCl₃) for 10 days. Then, some important physiological and biochemical indexes were determined. The results showed that salt stress significantly reduced seedling growth, decreased photosynthetic pigments, inhibited antioxidants and antioxidant enzyme activities. However, it increased the reactive oxygen species (ROS) levels in the two Tartary buckwheat cultivars. Exogenous 10 mM CaCl₂ application on salt-stressed Tartary buckwheat seedlings obviously mitigated the negative effects of NaCl stress and partially restored seedlings growth. Ca²⁺-treated salt-stressed seedlings diplayed a suppressed accumulation of ROS, increased the contents of total chlorophyll, soluble protein, proline and antioxidants, and elevated the activities of antioxidant enzymes compared with salt stress alone. On the contrary, the addition of 0.5 mM LaCl₃ and 5 mM EGTA on salt-stressed Tartary buckwheat seedlings exhibited the opposite effects to those with CaCl₂ treatment. These results indicate that exogenous Ca²⁺ can enhance salt stress tolerance and Ca²⁺ supplementation may be an effective practice to cultivate Tartary buckwheat in saline soils.     

Differential metabolic responses of perennial grass Cynodon transvaalensis×Cynodon dactylon (C₄) and Poa Pratensis (C₃) to heat stress

Differential metabolic responses to heat stress may be associated with variations in heat tolerance between cool‐season (C₃) and warm‐season (C₄) perennial grass species. The main objective of this study was to identify metabolites associated with differential heat tolerance between C₄ bermudagrass and C₃ Kentucky bluegrass by performing metabolite profile analysis using gas chromatography‐mass spectrometry. Plants of Kentucky bluegrass (Poa Pratensis‘Midnight’) and hybrid bermudagrass (Cynodon transvaalensis×Cynodon dactylon‘Tifdwarf’) were grown under optimum temperature conditions (20/15°C for Kentucky bluegrass and 30/25°C for bermudagrass) or heat stress (35/30°C for Kentucky bluegrass and 45/40°C for bermudagrass). Physiological responses to heat stress were evaluated by visual rating of grass quality, measuring photochemical efficiency (variable fluorescence to maximal fluorescence) and electrolyte leakage. All of these parameters indicated that bermudagrass exhibited better heat tolerance than Kentucky bluegrass. The metabolite analysis of leaf polar extracts revealed 36 heat‐responsive metabolites identified in both grass species, mainly consisting of organic acids, amino acids, sugars and sugar alcohols. Most metabolites showed higher accumulation in bermudagrass compared with Kentucky bluegrass, especially following long‐term (18 days) heat stress. The differentially accumulated metabolites included seven sugars (sucrose, fructose, galactose, floridoside, melibiose, maltose and xylose), a sugar alcohol (inositol), six organic acids (malic acid, citric acid, threonic acid, galacturonic acid, isocitric acid and methyl malonic acid) and nine amino acids (Asn, Ala, Val, Thr, γ‐Aminobutyric acid, IIe, Gly, Lys and Met). The differential accumulation of those metabolites could be associated with the differential heat tolerance between C₃ Kentucky bluegrass and C₄ bermudagrass.     

Responses of antioxidant enzymes to cold and high light are not correlated to freezing tolerance in natural accessions of Arabidopsis thaliana

Low temperatures and high light cause imbalances in primary and secondary reactions of photosynthesis, and thus can result in oxidative stress. Plants employ a range of low‐molecular weight antioxidants and antioxidant enzymes to prevent oxidative damage, and antioxidant defence is considered an important component of stress tolerance. To figure out whether oxidative stress and antioxidant defence are key factors defining the different cold acclimation capacities of natural accessions of the model plant Arabidopsis thaliana, we investigated hydrogen peroxide (H₂O₂) production, antioxidant enzyme activity and lipid peroxidation during a time course of cold treatment and exposure to high light in four differentially cold‐tolerant natural accessions of Arabidopsis (C24, Nd, Rsch, Te) that span the European distribution range of the species. All accessions except Rsch (from Russia) had elevated H₂O₂ in the cold, indicating that production of reactive oxygen species is part of the cold response in Arabidopsis. Glutathione reductase activity increased in all but Rsch, while ascorbate peroxidase and superoxide dismutase were unchanged and catalase decreased in all but Rsch. Under high light, the Scandinavian accession Te had elevated levels of H₂O₂. Te appeared most sensitive to oxidative stress, having higher malondialdehyde (MDA) levels in the cold and under high light, while only high light caused elevated MDA in the other accessions. Although the most freezing‐tolerant, Te had the highest sensitivity to oxidative stress. No correlation was found between freezing tolerance and activity of antioxidant enzymes in the four accessions investigated, arguing against a key role for antioxidant defence in the differential cold acclimation capacities of Arabidopsis accessions.     

LcFIN2, a novel chloroplast protein gene from sheepgrass,enhances tolerance to low temperature in Arabidopsis and rice

Adverse environmental stresses affect plant growth and crop yields. Sheepgrass (Leymus chinensis (Trin.) Tzvel), an important forage grass that is widely distributed in the east of Eurasia steppe, has high tolerance to extreme low temperature. Many genes that respond to cold stress were identified in sheepgrass by RNA‐sequencing, but more detailed studies are needed to dissect the function of those genes. Here, we found that LcFIN2, a sheepgrass freezing‐induced protein 2, encoded a chloroplast‐targeted protein. Expression of LcFIN2 was upregulated by freezing, chilling, NaCl and abscisic acid (ABA) treatments. Overexpression of LcFIN2 enhanced the survival rate of transgenic Arabidopsis after freezing stress. Importantly, heterologous expression of LcFIN2 in rice exhibited not only higher survival rate but also accumulated various soluble substances and reduced membrane damage in rice under chilling stress. Furthermore, the chlorophyll content, the quantum photochemistry efficiency of photosystem II (ΦPSII), the non‐photochemical quenching (NPQ), the net photosynthesis rate (Pn) and the expression of some chloroplast ribosomal‐related and photosynthesis‐related genes were higher in the transgenic rice under chilling stress. These findings suggested that the LcFIN2 gene could potentially be used to improve low‐temperature tolerance in crops.     

Phosphorus-induced micronutrient disorders in hybrid poplar

The effect of calcium on the nodulation of lucerne was studied using EGTA, a specific calcium-chelator. First, the effects of the chelator were tested on hydroponically grown plants at pH 7.0. Optimal numbers of nodules were obtained in nutrient solution containing 0.2 mM CaCl₂. When 0.4 mM EGTA was given additionally, nodulation was completely inhibited. Nodulation was restored specifically with CaCl₂, but not with MgCl₂. For studies in an acid soil (pH-H₂O 5.2), lucerne seedlings were grown in rhizotrons. 67% of the seedlings became nodulated when the soil around the seed was neutralized locally with 1.0 μmol of K₂CO₃ in drops of 12 μL volume. When native calcium was removed with 2 μmol of EGTA, nodulation was reduced to 12%. However, addition of EGTA to soil resulted in a drop of pH from 6.1 to 5.2. A phosphate buffer could also not keep soil-pH sufficiently stable. Such pH-decreases could be avoided by placing agar blocks containing 6 μmol of EGTA for three hours on freshly developed roots. This treatment reduced nodulation from 87% to 32%, with soil-pH lowering only from 6.2 to 6.0. Nodulation could be restored by adding 2 μmol of CaCl₂. The depletion of soil-calcium could depress nodule formation only during the first day after inoculation.     

Physiological and metabolomic responses of bermudagrass (Cynodon dactylon) to alkali stress

Bermudagrass (Cynodon dactylon) is a widely used warm‐season turfgrass species with superior stress tolerance except for cold. In this study, a comparative analysis of the responses to alkali stress in bermudagrass at the physiological and metabolomic levels were performed. Mild alkali with relatively low pH slightly inhibited growth of bermudagrass as evidenced by lower electrolyte leakage, more rapid growth and higher survival rate when compared to moderate and severe alkali treatments. Moreover, the amount of 37 metabolites including amino acids, organic acids, sugars and sugar alcohols were modulated by the alkali treatments. Among them, 15 metabolites were involved in carbon and amino acid metabolic pathways. Under mild alkali stress, bermudagrass possibly slowed down metabolisms to maintain basic growth. However, moderate and severe alkali‐stressed plants accumulated significantly higher amount of carbohydrates which might result in carbon starvation. Taken together, alkali stress had severely inhibitory effect partially due to combined ionic stress and high pH stress. These results suggested that bermudagrass employed different strategies in response to alkali stresses with different pH and ionic values.     

Cold induction of EARLI1, a putative Arabidopsis lipid transfer protein, is light and calcium dependent

As sessile organisms, plants must adapt to their environment. One approach toward understanding this adaptation is to investigate environmental regulation of gene expression. Our focus is on the environmental regulation of EARLI1, which is activated by cold and long‐day photoperiods. Cold activation of EARLI1 in short‐day photoperiods is slow, requiring several hours at 4 °C to detect an increase in mRNA abundance. EARLI1 is not efficiently cold‐activated in etiolated seedlings, suggesting that photomorphogenesis is necessary for its cold activation. Cold activation of EARLI1 is inhibited in the presence of the calcium channel blocker lanthanum chloride or the calcium chelator EGTA. Addition of the calcium ionophore Bay K8644 results in cold‐independent activation of EARLI1. These data suggest that EARLI1 is not an immediate target of the cold response, and that calcium flux affects its expression. EARLI1 is a putative secreted protein and has motifs found in lipid transfer proteins. Over‐expression of EARLI1 in transgenic plants results in reduced electrolyte leakage during freezing damage, suggesting that EARLI1 may affect membrane or cell wall stability in response to low temperature stress.     

Electrical resistance changes during exposure to low temperature measure chilling and freezing tolerance in olive tree (Olea europaea L.) plants

Electrical resistance changes in different organs of four olive tree (Olea europaea L.) varieties, characterized by different tolerance to chilling and freezing, were examined, during exposure to low temperature. Apparent critical temperatures (CT) and freezing temperatures (T_fr) were identified on the basis of the electrical resistance changes. Both temperatures were lower for the more chilling‐tolerant genotypes. From the apparent critical temperatures, the absolute critical temperature (CT_abs) and the time delay of the chilling signal transduction process were calculated. In shoots, CT_abs varied from 8·8 °C for Ascolana (chilling‐tolerant variety) to 13·6 °C for Coratina (chilling‐sensitive variety). The magnitude of the transduction time was very similar (about 2 min) for the three genotypes that are more sensitive to chilling, whereas it was significantly higher (about 3 min) for the most tolerant genotype. Different freezing temperatures were observed for different organs. It would appear from this experiment that the order of sensitivity is roots > leaves > shoots > vegetative buds. Accord was found between the absolute critical temperature of electrical resistance and the critical temperature of membrane potential. The occurrence of electrical resistance changes in the tissues of the olive trees exposed to low temperature suggests the use of this experimental procedure as a quick, easy and non‐destructive tool to screen plant tissues for chilling tolerance. The strong dependence of the electrical resistance on low temperature, and the critical temperature of around 10 °C, can yield interesting information about the lowest thermal limits for the continuation of normal physiological processes and therefore about the adaptability of plants to particular environments.     

Chilling‐induced Ca2+ overload enhances production of active oxygen species in maize (Zea mays L.) cultured cells: the effect of abscisic acid treatment

Chilling (4 °C) induced a prolonged high level of intracellular Ca²⁺ (Ca²⁺ overload) and lipid peroxidation in maize (Zea mays L. cv Black Mexican Sweet) cultured cells. However, such Ca²⁺ overload and enhanced lipid peroxidation were not seen in abscisic acid (ABA)‐treated cells, which had an improved chilling tolerance. A Ca²⁺ ionophore, A23187, caused Ca²⁺ overload in both ABA‐treated maize cells and the untreated control, whereas an enhanced lipid peroxidation was detected only in the control. The high level of active oxygen species (AOS) in the control during chilling at 4 °C could be reduced by the presence of lanthanum (La³⁺), a Ca²⁺ channel blocker, in the medium. Moreover, both the A23187‐induced lipid peroxidation and AOS production in the control could be reduced by extracellular EGTA, a Ca²⁺ chelator. Laser‐scanning confocal microscopy revealed that mitochondria were one of the major AOS sources under chilling and during A23187 treatment. In vitro assays showed that superoxide production in isolated maize mitochondria was enhanced by the presence of Ca²⁺. Findings suggest that chilling‐induced Ca²⁺ influx in the control triggers a marked generation of AOS, which in turn results in the enhanced lipid peroxidation. The ability of ABA‐treated cells to avoid the chilling‐induced Ca²⁺ influx may serve as a mechanism that prevents the chilling‐induced oxidative stress and thus results in less chilling injury.     

Role of calcium and calmodulin antagonist in photosynthesis and salinity tolerance inChlorella vulgaris

To cast light upon the role of Ca¹⁺ and calmodulin on photosynthetic rate (P_n), dark respiration (R_D) and amino acid and protein contents in salinity stressed and non-stressedChlorella cultures, the Ca²⁺ chelator EGTA [ethylene glycol-bis-(2-aminoethyl ether)-N,N- tetraacetate] and the calmodulin antagonist TFP (trifluperazine) were used. TFP markedly inhibited P_N while EGTA exerted a slight, if any, effect on P_N. NaCl tolerance, on the other side, was markedly abolished by TFP that inhibited P_N and lowered rate of proline accumulation. Calmodulin might be involved in osmoregulation and salt tolerance ofChlorella. R_D, however, was markedly enhanced by EGTA and Ca²⁺-free medium and hence the Ca²⁺ deprivation increased stress severity exerted by NaCl. Combinations of Na⁺ and Ca²⁺ enhanced P_N, decreased R_D and proline content in comparison with an osmotically equivalent reference culture containing only NaCl. Addition of Ca²⁺ to TFP treated cultures failed to reactivate calmodulin for proline synthesis. However, when Ca²⁺ was added to EGTA-treated cultures, only relatively reduced proline contents were recorded.     

外源CaCl2对NaCl胁迫下酸枣幼苗氮代谢的影响

为了研究CaCl₂对NaCl胁迫下酸枣幼苗根、茎、叶的氮代谢影响,探索钙缓解幼苗NaCl胁迫的作用途径。该研究以酸枣幼苗为试验材料,检测不同浓度CaCl₂(0、5、10、20 mmol/L)对NaCl(150 mmol/L)胁迫下幼苗叶片H₂O₂、O^-·₂含量,根、茎、叶中硝酸还原酶(NR)、谷氨酰胺合成酶(GS)、谷氨酸合酶(GOGAT)活性及游离氨基酸、可溶性蛋白、硝态氮含量的影响,并采用主成分分析法筛选出评价CaCl₂缓解NaCl胁迫效应的生理指标。结果表明：与NaCl胁迫相比,盐胁迫幼苗叶片的H2O₂、O^-·₂积累量在5、10 mmol/L CaCl₂处理下显著减少;GOGAT活性在5、10 mmol/L CaCl₂处理下的植株根和茎内以及各浓度 CaCl₂处理的叶内均显著升高, GS、NR活性在10、20 mmol/L CaCl₂处理的根内和10 mmol/L CaCl₂处理的茎内以及5、10、20 mmol/L CaCl₂处理的叶内均显著升高;可溶性蛋白含量在5、10、20 mmol/L CaCl₂处理的根、茎、叶内均显著升高,游离氨基酸含量在10、20 mmol/L CaCl₂处理的根和茎内以及10 mmol/L CaCl₂处理的叶内均显著升高,硝态氮含量在10 mmol/L CaCl₂处理的根和茎内以及5、10、20 mmol/L CaCl₂处理的叶内均显著升高。研究发现,150 mmol/L NaCl胁迫对酸枣幼苗造成明显过氧化伤害,抑制了体内氮代谢;外源CaCl₂可通过促进幼苗根和茎内GS/GOGAT循环对NH₄⁺的同化作用,提高叶片NR活性,加快硝态氮的转化速率,从而增强幼苗对NaCl胁迫的适应性,并以10 mmol/L CaCl₂处理缓解效果最佳;游离氨基酸、GOGAT、NR可以作为CaCl₂缓解幼苗NaCl胁迫伤害的评价指标。     

New insights into the genetic basis of natural chilling and cold shock tolerance in rice by genome‐wide association analysis

In order to understand cold adaptability and explore additional genetic resources for the cold tolerance improvement of rice, we investigated the genetic variation of 529 rice accessions under natural chilling and cold shock stress conditions at the seedling stage using genome‐wide association studies; a total of 132 loci were identified. Among them, 12 loci were common for both chilling and cold shock tolerance, suggesting that rice has a distinct and overlapping genetic response and adaptation to the two stresses. Haplotype analysis of a known gene OsMYB2, which is involved in cold tolerance, revealed indica–japonica differentiation and latitude tendency for the haplotypes of this gene. By checking the subpopulation and geographical distribution of accessions with tolerance or sensitivity under these two stress conditions, we found that the chilling tolerance group, which mainly consisted of japonica accessions, has a wider latitudinal distribution than the chilling sensitivity group. We conclude that the genetic basis of natural chilling stress tolerance in rice is distinct from that of cold shock stress frequently used for low‐temperature treatment in the laboratory and the cold adaptability of rice is associated with the subpopulation and latitudinal distribution.