Annexin 1 regulates the H2O2‐induced calcium signature in Arabidopsis thaliana roots

Hydrogen peroxide is the most stable of the reactive oxygen species (ROS) and is a regulator of development, immunity and adaptation to stress. It frequently acts by elevating cytosolic free Ca²⁺ ([Ca²⁺]_cyt) as a second messenger, with activation of plasma membrane Ca²⁺‐permeable influx channels as a fundamental part of this process. At the genetic level, to date only the Ca²⁺‐permeable Stelar K⁺ Outward Rectifier (SKOR) channel has been identified as being responsive to hydrogen peroxide. We show here that the ROS‐regulated Ca²⁺ transport protein Annexin 1 in Arabidopsis thaliana (AtANN1) is involved in regulating the root epidermal [Ca²⁺]_cyt response to stress levels of extracellular hydrogen peroxide. Peroxide‐stimulated [Ca²⁺]_cyt elevation (determined using aequorin luminometry) was aberrant in roots and root epidermal protoplasts of the Atann1 knockout mutant. Similarly, peroxide‐stimulated net Ca²⁺ influx and K⁺ efflux were aberrant in Atann1 root mature epidermis, determined using extracellular vibrating ion‐selective microelectrodes. Peroxide induction of GSTU1 (Glutathione‐S‐Transferase1 Tau 1), which is known to be [Ca²⁺]_cyt‐dependent was impaired in mutant roots, consistent with a lesion in signalling. Expression of AtANN1 in roots was suppressed by peroxide, consistent with the need to restrict further Ca²⁺ influx. Differential regulation of annexin expression was evident, with AtANN2 down‐regulation but up‐regulation of AtANN3 and AtANN4. Overall the results point to involvement of AtANN1 in shaping the root peroxide‐induced [Ca²⁺]_cyt signature and downstream signalling.     

Cyclic adenosine 5′‐diphosphoribose (cADPR) cyclic guanosine 3′,5′‐monophosphate positively function in Ca2+ elevation in methyl jasmonate‐induced stomatal closure,cADPR is required for methyl jasmonate‐induced ROS accumulation NO production in guard cells

Methyl jasmonate (MeJA) signalling shares several signal components with abscisic acid (ABA) signalling in guard cells. Cyclic adenosine 5′‐diphosphoribose (cADPR) and cyclic guanosine 3′,5′‐monophosphate (cGMP) are second messengers in ABA‐induced stomatal closure. In order to clarify involvement of cADPR and cGMP in MeJA‐induced stomatal closure in Arabidopsis thaliana (Col‐0), we investigated effects of an inhibitor of cADPR synthesis, nicotinamide (NA), and an inhibitor of cGMP synthesis, LY83583 (LY, 6‐anilino‐5,8‐quinolinedione), on MeJA‐induced stomatal closure. Treatment with NA and LY inhibited MeJA‐induced stomatal closure. NA inhibited MeJA‐induced reactive oxygen species (ROS) accumulation and nitric oxide (NO) production in guard cells. NA and LY suppressed transient elevations elicited by MeJA in cytosolic free Ca²⁺ concentration ([Ca²⁺]_cyt) in guard cells. These results suggest that cADPR and cGMP positively function in [Ca²⁺]_cyt elevation in MeJA‐induced stomatal closure, are signalling components shared with ABA‐induced stomatal closure in Arabidopsis, and that cADPR is required for MeJA‐induced ROS accumulation and NO production in Arabidopsis guard cells.     

OsSUV3 dual helicase functions in salinity stress tolerance by maintaining photosynthesis and antioxidant machinery in rice (Oryza sativa L. cv. IR64)

To overcome the salinity‐induced loss of crop yield, a salinity‐tolerant trait is required. The SUV3 helicase is involved in the regulation of RNA surveillance and turnover in mitochondria, but the helicase activity of plant SUV3 and its role in abiotic stress tolerance have not been reported so far. Here we report that the Oryza sativa (rice) SUV3 protein exhibits DNA and RNA helicase, and ATPase activities. Furthermore, we report that SUV3 is induced in rice seedlings in response to high levels of salt. Its expression, driven by a constitutive cauliflower mosaic virus 35S promoter in IR64 transgenic rice plants, confers salinity tolerance. The T₁ and T₂ sense transgenic lines showed tolerance to high salinity and fully matured without any loss in yields. The T₂ transgenic lines also showed tolerance to drought stress. These results suggest that the introduced trait is functional and stable in transgenic rice plants. The rice SUV3 sense transgenic lines showed lesser lipid peroxidation, electrolyte leakage and H₂O₂ production, along with higher activities of antioxidant enzymes under salinity stress, as compared with wild type, vector control and antisense transgenic lines. These results suggest the existence of an efficient antioxidant defence system to cope with salinity‐induced oxidative damage. Overall, this study reports that plant SUV3 exhibits DNA and RNA helicase and ATPase activities, and provides direct evidence of its function in imparting salinity stress tolerance without yield loss. The possible mechanism could be that OsSUV3 helicase functions in salinity stress tolerance by improving photosynthesis and antioxidant machinery in transgenic rice.     

Engineered silver nanoparticles are sensed at the plasma membrane and dramatically modify the physiology of Arabidopsis thaliana plants

Silver nanoparticles (Ag NPs) are the world's most important nanomaterial and nanotoxicant. The aim of this study was to determine the early stages of interactions between Ag NPs and plant cells, and to investigate their physiological roles. We have shown that the addition of Ag NPs to cultivation medium, at levels above 300 mg L^?1, inhibited Arabidopsis thaliana root elongation and leaf expansion. This also resulted in decreased photosynthetic efficiency and the extreme accumulation of Ag in tissues. Acute application of Ag NPs induced a transient elevation of [Ca²⁺]_cyt and the accumulation of reactive oxygen species (ROS; partially generated by NADPH oxidase). Whole‐cell patch‐clamp measurements on root cell protoplasts demonstrated that Ag NPs slightly inhibited plasma membrane K⁺ efflux and Ca²⁺ influx currents, or caused membrane breakdown; however, in excised outside‐out patches, Ag NPs activated Gd³⁺‐sensitive Ca²⁺ influx channels with unitary conductance of approximately 56 pS. Bulk particles did not modify the plasma membrane currents. Tests with electron paramagnetic resonance spectroscopy showed that Ag NPs were not able to catalyse hydroxyl radical generation, but that they directly oxidized the major plant antioxidant, l ‐ascorbic acid. Overall, the data presented shed light on mechanisms of the impact of nanosilver on plant cells, and show that these include the induction of classical stress signalling reactions (mediated by [Ca²⁺]_cyt and ROS) and a specific effect on the plasma membrane conductance and the reduced ascorbate.     

Phosphatidylinositol‐hydrolyzing phospholipase C4 modulates rice response to salt and drought

Phosphatidylinositol‐specific phospholipase C (PI‐PLC) is involved in stress signalling but its signalling function remains largely unknown in crop plants. Here, we report that the PI‐PLC4 from rice (Oryza sativa cv), OsPLC4, plays a positive role in osmotic stress response. Two independent knockout mutants, plc4‐1 and plc4‐2, exhibited decreased seedling growth and survival rate whereas overexpression of OsPLC4 improved survival rate under high salinity and water deficiency, compared with wild type (WT). OsPLC4 hydrolyses PI, phosphatidylinositol 4‐phosphate (PI4P), and phosphatidylinositol‐4,5‐bisphosphate (PIP₂) to generate diacylglycerol (DAG) in vitro. Knockout of OsPLC4 attenuated salt‐induced increase of phosphatidic acid (PA) whereas overexpression of OsPLC4 decreased the level of PI4P and PIP₂ under salt treatment. Applications of DAG or PA restored the growth defect of plc4‐1 to WT but DAG kinase inhibitor 1 blocked the complementary effect of DAG in plc4‐1 under salt stress. In addition, the loss of OsPLC4 compromised the increase of inositol triphosphate and free cytoplasmic Ca²⁺ ([Ca²⁺]_cyt) and inhibited the induction of genes involved in Ca²⁺ sensor and osmotic stress response to salt stress. The results indicate that OsPLC4 modulates the activity of two signalling pathways, PA and Ca²⁺, to affect rice seedling response to osmotic stress.     

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.     

Two guard cell‐preferential MAPKs,MPK9 and MPK12, regulate YEL signalling in Arabidopsis guard cells

We report that two mitogen‐activated protein kinases (MAPKs), MPK9 and MPK12, positively regulate abscisic acid (ABA)‐induced stomatal closure in Arabidopsis thaliana. Yeast elicitor (YEL) induced stomatal closure accompanied by intracellular reactive oxygen species (ROS) accumulation and cytosolic free calcium concentration ([Ca²⁺]_cyt) oscillation. In this study, we examined whether these two MAP kinases are involved in YEL‐induced stomatal closure using MAPKK inhibitors, PD98059 and U0126, and MAPK mutants, mpk9, mpk12 and mpk9 mpk12. Both PD98059 and U0126 inhibited YEL‐induced stomatal closure. YEL induced stomatal closure in the mpk9 and mpk12 mutants but not in the mpk9 mpk12 mutant, suggesting that a MAPK cascade involving MPK9 and MPK12 functions in guard cell YEL signalling. However, YEL induced extracellular ROS production, intracellular ROS accumulation and cytosolic alkalisation in the mpk9, mpk12 and mpk9 mpk12 mutants. YEL induced [Ca²⁺]_cyt oscillations in both wild type and mpk9 mpk12 mutant. These results suggest that MPK9 and MPK12 function redundantly downstream of extracellular ROS production, intracellular ROS accumulation, cytosolic alkalisation and [Ca²⁺]_cyt oscillation in YEL‐induced stomatal closure in Arabidopsis guard cells and are shared with ABA signalling.     

CAX1 suppresses Cd‐induced generation of reactive oxygen species in Arabidopsis halleri

The molecular analysis of metal hyperaccumulation in species such as Arabidopsis halleri offers the chance to gain insights into metal homeostasis and into the evolution of adaptation to extreme habitats. A prerequisite of metal hyperaccumulation is metal hypertolerance. Genetic analysis of a backcross population derived from Arabidopsis lyrata × A. halleri crosses revealed three quantitative trait loci for Cd hypertolerance. A candidate gene for Cdtol2 is AhCAX1, encoding a vacuolar Ca²⁺/H⁺ antiporter. We developed a method for the transformation of vegetatively propagated A. halleri plants and generated AhCAX1‐silenced lines. Upon Cd²⁺ exposure, several‐fold higher accumulation of reactive oxygen species (ROS) was detectable in roots of AhCAX1‐silenced plants. In accordance with the dependence of Cdtol2 on external Ca²⁺ concentration, this phenotype was exclusively observed in low Ca²⁺ conditions. The effects of external Ca²⁺ on Cd accumulation cannot explain the phenotype as they were not influenced by the genotype. Our data strongly support the hypothesis that higher expression of CAX1 in A. halleri relative to other Arabidopsis species represents a Cd hypertolerance factor. We propose a function of AhCAX1 in preventing a positive feedback loop of Cd‐elicited ROS production triggering further Ca²⁺‐dependent ROS accumulation.     

Ectopic expression of phloem motor protein pea forisome PsSEO-F1 enhances salinity stress tolerance in tobacco

Key message

PsSEOF-1 binds to calcium and its expression is upregulated by salinity treatment. PsSEOF - 1 -overexpressing transgenic tobacco showed enhanced salinity stress tolerance by maintaining cellular ion homeostasis and modulating ROS-scavenging pathway.

Abstract

Calcium (Ca²⁺) plays important role in growth, development and stress tolerance in plants. Cellular Ca²⁺ homeostasis is achieved by the collective action of channels, pumps, antiporters and by Ca²⁺ chelators present in the cell like calcium-binding proteins. Forisomes are ATP-independent mechanically active motor proteins known to function in wound sealing of injured sieve elements of phloem tissue. The Ca²⁺-binding activity of forisome and its role in abiotic stress signaling were largely unknown. Here we report the Ca²⁺-binding activity of pea forisome (PsSEO-F1) and its novel function in promoting salinity tolerance in transgenic tobacco. Native PsSEO-F1 promoter positively responded in salinity stress as confirmed using GUS reporter. Overexpression of PsSEO-F1 tobacco plants confers salinity tolerance by alleviating ionic toxicity and increased ROS scavenging activity which probably results in reduced membrane damage and improved yield under salinity stress. Evaluation of several physiological indices shows an increase in relative water content, electrolyte leakage, proline accumulation and chlorophyll content in transgenic lines as compared with null-segregant control. Expression of several genes involved in cellular homeostasis is perturbed by PsSEO-F1 overexpression. These findings suggest that PsSEO-F1 provides salinity tolerance through cellular Ca²⁺ homeostasis which in turn modulates ROS machinery providing indirect link between Ca²⁺ and ROS signaling under salinity-induced perturbation. PsSEO-F1 most likely functions in salinity stress tolerance by improving antioxidant machinery and mitigating ion toxicity in transgenic lines. This finding should make an important contribution in our better understanding of the significance of calcium signaling in phloem tissue leading to salinity stress tolerance.

     

Calcium acetate‐induced reduction of cadmium accumulation in Oryza sativa: Expression of auto‐inhibited calcium‐ATPase and cadmium transporters

• Calcium (Ca) signalling has an essential role in regulating plant responses to various abiotic stresses.
• This study applied Ca in various forms (Ca acetate and CaCl₂) and concentrations to reduce cadmium (Cd) concentration in rice and propose a possible mechanism through which Ca acts to control the Cd concentration in rice.
• The results showed that supplementation of Cd‐contaminated soil with Ca acetate reduced the Cd concentration in rice after exposure for 7 days in both hydroponic and soil conditions. The possible involvement of the auto‐inhibited Ca²⁺‐ATPase gene (ACA) might act to control the primary signal of the Cd stress response. The messages from ACA3 and ACA13 tended to up‐regulate the low‐affinity cation transporter (OsLCT1) and down‐regulate Cd uptake and the Cd translocation transporter, including the genes, natural resistance‐associated macrophage protein 5 (Nramp5) and Zn/Cd‐transporting ATPase 2 (HMA2), which resulted in a reduction in the Cd concentration in rice. After cultivation for 120 days, the application of Ca acetate into Cd‐contaminated soil inhibited Cd uptake of rice.
• Increasing the Ca acetate concentration in the soil lowered the Cd concentration in rice shoots and grains. Moreover, Ca acetate maintained rice productivity and quality whereas both aspects decreased under Cd stress.

     

Co‐expression of tonoplast Cation/H+ antiporter and H+‐pyrophosphatase from xerophyte Zygophyllum xanthoxylum improves alfalfa plant growth under salinity,drought and field conditions

Salinity and drought are major environmental factors limiting the growth and productivity of alfalfa worldwide as this economically important legume forage is sensitive to these kinds of abiotic stress. In this study, transgenic alfalfa lines expressing both tonoplast NXH and H⁺‐PPase genes, ZxNHX and ZxVP1‐1 from the xerophyte Zygophyllum xanthoxylum L., were produced via Agrobacterium tumefaciens‐mediated transformation. Compared with wild‐type (WT) plants, transgenic alfalfa plants co‐expressing ZxNHX and ZxVP1‐1 grew better with greater plant height and dry mass under normal or stress conditions (NaCl or water‐deficit) in the greenhouse. The growth performance of transgenic alfalfa plants was associated with more Na⁺, K⁺ and Ca²⁺ accumulation in leaves and roots, as a result of co‐expression of ZxNHX and ZxVP1‐1. Cation accumulation contributed to maintaining intracellular ions homoeostasis and osmoregulation of plants and thus conferred higher leaf relative water content and greater photosynthesis capacity in transgenic plants compared to WT when subjected to NaCl or water‐deficit stress. Furthermore, the transgenic alfalfa co‐expressing ZxNHX and ZxVP1‐1 also grew faster than WT plants under field conditions, and most importantly, exhibited enhanced photosynthesis capacity by maintaining higher net photosynthetic rate, stomatal conductance, and water‐use efficiency than WT plants. Our results indicate that co‐expression of tonoplast NHX and H⁺‐PPase genes from a xerophyte significantly improved the growth of alfalfa, and enhanced its tolerance to high salinity and drought. This study laid a solid basis for reclaiming and restoring saline and arid marginal lands as well as improving forage yield in northern China.     

Overexpression of S‐adenosyl‐l‐methionine synthetase increased tomato tolerance to alkali stress through polyamine metabolism

S‐adenosyl‐l ‐methionine (SAM) synthetase is the key enzyme involved in the biosynthesis of SAM, which serves as a common precursor for polyamines (PAs) and ethylene. A SAM synthetase cDNA (SlSAMS1) was introduced into the tomato genome using the Agrobacterium tumefaciens transformation method. Transgenic plants overexpressing SlSAMS1 exhibited a significant increase in tolerance to alkali stress and maintained nutrient balance, higher photosynthetic capacity and lower oxidative stress compared with WT lines. Both in vivo and in vitro experiments indicated that the function of SlSAMS1 mainly depended on the accumulation of Spd and Spm in the transgenic lines. A grafting experiment showed that rootstocks from SlSAMS1‐overexpressing plants provided a stronger root system, increased PAs accumulation, essential elements absorption, and decreased Na⁺ absorption in the scions under alkali stress. As a result, fruit set and yield were significantly enhanced. To our knowledge, this is the first report to provide evidence that SlSAMS1 positively regulates tomato tolerance to alkali stress and plays a major role in modulating polyamine metabolism, resulting in maintainability of nutrient and ROS balance.     

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.