KCl Stress Research Articles

Accumulation of astaxanthin in Microcystis aeruginosa under NaCl and KCl stresses

Astaxanthin is a high-value natural antioxidant, and can be accumulated in Microcystis aeruginosa. To enhance astaxanthin accumulation in the microalgae by using salt stress, the cell growth, photosynthetic abilities, reactive oxygen species (ROS) levels, astaxanthin and its precursor content, and gene expression were investigated under NaCl and KCl stresses. The two salt stresses inhibited the cell growth by lowering photosynthetic abilities and raising ROS levels. During the 6-day treatment, the two salt stresses improved the levels of astaxanthin, precursors (β-carotene and zeaxanthin) and carotenoids, which might be caused by the raised ROS up-regulating expression of 7 related genes. At the same concentration, KCl stress showed stronger inducing effect on astaxanthin and its precursor production than NaCl stress, due to higher expression of related genes. Therefore, NaCl and KCl stresses have obvious ion differences on astaxanthin accumulation, of which KCl stress is more suitable for the high-value antioxidant production from microalgae.

Pps1, phosphatidylserine synthase, regulates the salt stress response in Schizosaccharomyces pombe.

Phosphatidylserine (PS) is important for maintaining growth, cytoskeleton, and various functions in yeast; however, its role in stress responses is poorly understood. In Schizosaccharomyces pombe, the PS synthase deletion (pps1∆) mutant shows defects in growth, morphology, cytokinesis, actin cytoskeleton, and cell wall integrity, and these phenotypes are rescued by ethanolamine supplementation. Here, we evaluated the role of Pps1 in the salt stress response in S. pombe. We found that pps1∆ cells are sensitive to salt stresses such as KCl and CaCl2 even in the presence of ethanolamine. Loss of the functional cAMP-dependent protein kinase (git3∆ or pka1∆) or phospholipase B Plb1 (plb1∆) enhanced the salt stress-sensitive phenotype in pps1∆ cells. Green fluorescent protein (GFP)-Pps1 was localized at the plasma membrane and endoplasmic reticulum regardless of the stress conditions. In pka1∆ cells, GFP-Pps1 was accumulated around the nucleus under the KCl stress. Pka1 was localized in the nucleus and the cytoplasm under normal conditions and transferred from the nucleus to the cytoplasm under salt-stress conditions. Pka1 translocated from the nucleus to the cytoplasm during CaCl2 stress in the wild-type cells, while it remained localized in the nucleus in pps1∆ cells. Expression and phosphorylation of Pka1-GFP were not changed in pps1∆ cells. Our results demonstrate that Pps1 plays an important role in the salt stress response in S. pombe.

Melatonin enhances KCl salinity tolerance by maintaining K+ homeostasis in Malus hupehensis.

Large amounts of potash fertilizer are often applied to apple (Malus domestica) orchards to enhance fruit quality and yields, but this treatment aggravates KCl-based salinity stress. Melatonin (MT) is involved in a variety of abiotic stress responses in plants. However, its role in KCl stress tolerance is still unknown. In the present study, we determined that an appropriate concentration (100 μm) of MT significantly alleviated KCl stress in Malus hupehensis by enhancing K+ efflux out of cells and compartmentalizing K+ in vacuoles. Transcriptome deep-sequencing analysis identified the core transcription factor gene MdWRKY53, whose expression responded to both KCl and MT treatment. Overexpressing MdWRKY53 enhanced KCl tolerance in transgenic apple plants by increasing K+ efflux and K+ compartmentalization. Subsequently, we characterized the transporter genes MdGORK1 and MdNHX2 as downstream targets of MdWRKY53 by ChIP-seq. MdGORK1 localized to the plasma membrane and enhanced K+ efflux to increase KCl tolerance in transgenic apple plants. Moreover, overexpressing MdNHX2 enhanced the KCl tolerance of transgenic apple plants/callus by compartmentalizing K+ into the vacuole. RT-qPCR and LUC activity analyses indicated that MdWRKY53 binds to the promoters of MdGORK1 and MdNHX2 and induces their transcription. Taken together, our findings reveal that the MT-WRKY53-GORK1/NHX2-K+ module regulates K+ homeostasis to enhance KCl stress tolerance in apple. These findings shed light on the molecular mechanism of apple response to KCl-based salinity stress and lay the foundation for the practical application of MT in salt stress.

SES1 is vital for seedling establishment and post-germination growth under high-potassium stress conditions in Arabidopsis thaliana.

The potassium ion (K+) plays an important role in maintaining plant growth and development, while excess potassium in the soil can cause stress to plants. The understanding of the molecular mechanism of plant's response to high KCl stress is still limited. At the seed stage, wild type (WT) and SENSITIVE TO SALT1 (SES1) mutants were exposed to different concentrations of potassium treatments. Tolerance was assayed as we compared their performances under stress using seedling establishment rate and root length. Na+content, K+content, and K+/Na+ ratio were determined using a flame atomic absorption spectrometer. In addition, the expressions of KCl-responding genes and ER stress-related genes were also detected and analyzed using qRT-PCR. SES1 mutants exhibited seedling establishment defects under high potassium concentration conditions and exogenous calcium partially restored the hypersensitivity phenotype of ses1 mutants. The expression of some K+ transporter/channel genes were higher in ses1-2, and the ratio of potassium to sodium (K+/Na+) in ses1-2 roots decreased after KCl treatment compared with WT. Further analysis showed that the ER stress marker genes were dramatically induced by high K+ treatment and much higher expression levels were detected in ses1-2, indicating ses1-2 suffers a more serious ER stress than WT, and ER stress may influence the seedling establishment of ses1-2 under high KCl conditions. These results strongly indicate that SES1 is a potassium tolerance relevant molecule that may be related to maintaining the seedling K+/Na+ balance under high potassium conditions during seedling establishment and post-germination growth. Our results will provide a basis for further studies on the biological roles of SES1 in modulating potassium uptake, transport, and adaptation to stress conditions.

Tuz Stresinin (Potasyum Klorür) Aspir (Carthamus tinctorius L.) Çeşitlerinin Ekolojik ve Fizyolojik Özelliklerine Etkisi

Salinity, which is a significant abiotic stress factor, is among the most important factors that limit product quality and yield. In this study investigated phenological and physiological changes that occurred in three different safflower varieties that were exposed to KCl stress at concentrations of 0, 50, 100 and 150 mM. It was found that, based on increasing salt concentrations, the most significant decrease was in all phenological parameters at the concentrations of 100 and 150 mM KCl. The SOD and CAT activities showed a significant increase at high salt concentrations in the Balcı and Dinçer varieties. The APX and GR activities showed a parallelism in all safflower varieties, and they showed a significant increase based on increased concentration at the applications of 100 and 150 mM KCl. Among the three safflower varieties, the most resistant variety to salt application was Dinçer, while the most sensitive one was Remzibey. The Balcı variety was closer to the Dinçer variety in terms of its tolerance against salt stress.

Resveratrol Alleviates the KCl Salinity Stress of Malus hupehensis Rhed.

Applying large amounts of potash fertilizer in apple orchards for high apple quality and yield aggravates KCl stress. As a phytoalexin, resveratrol (Res) participates in plant resistance to biotic stress. However, its role in relation to KCl stress has never been reported. Herein we investigated the role of Res in KCl stress response of Malus hupehensis Rehd., a widely used apple rootstock in China which is sensitive to KCl stress. KCl-stressed apple seedlings showed significant wilting phenotype and decline in photosynthetic rate, and the application of 100 μmol Res alleviated KCl stress and maintained photosynthetic capacity. Exogenous Res can strengthen the activities of peroxidase and catalase, thus eliminating reactive oxygen species production induced by KCl stress. Moreover, exogenous Res can decrease the electrolyte leakage by accumulating proline for osmotic balance under KCl stress. Furthermore, exogenous Res application can affect K+/Na+ homeostasis in cytoplasm by enhancing K+ efflux outside the cells, inhibiting Na+ efflux and K+ absorption, and compartmentalizing K+ into vacuoles through regulating the expression of K+ and Na+ transporter genes. These findings provide a theoretical basis for the application of exogenous Res to relieve the KCl stress of apples.

Comparative transcriptome analysis of NaCl and KCl stress response in Malus hupehensis Rehd. Provide insight into the regulation involved in Na+ and K+ homeostasis

BackgroundApple is among the most widely cultivated perennial fruit crops worldwide. It is sensitive to salt stress, which seriously affects the growth and productivity of apple trees by destroying the homeostasis of Na+ and K+. Previous studies focused on the molecular mechanism underlying NaCl stress. However, signaling transduction under KCl stress has not been thoroughly studied. ResultsWe comprehensively analyzed the salt tolerance of Malus hupehensis Rehd., which is a widely used rootstock in apple orchards, by using RNA-Seq. Roots and leaves were treated with NaCl and KCl. Based on mapping analyses, a total of 762 differentially expressed genes (DEGs) related to NaCl and KCl stress in the roots and leaves were identified. Furthermore, we identified seven hub genes by WGCNA Analysis. The Gene Ontology (GO) terms were enriched in ion transmembrane transporter and oxidoreductase activity under NaCl and KCl stress. The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways focused on the plant hormone signal transduction and mitogen-activated protein kinase signaling pathway. We also screened out 28 candidate genes from 762 DEGs and verified their expression by quantitative reverse transcription polymerase chain reaction (qRT-PCR). All of these enriched genes were closely related to NaCl and KCl stress and take part in mediating the Na+ and K+ homeostasis in M. hupehensis. ConclusionsThis transcriptome analysis provides a valuable resource for elucidating the signaling pathway of NaCl and KCl stress and is a substantial genetic resource for discovering genes related to the NaCl and KCl stress response.

Exogenous Strigolactones alleviate KCl stress by regulating photosynthesis, ROS migration and ion transport in Malus hupehensis Rehd

AimsIn recent years, the application of large amounts of potash fertilizer in apple orchards leads to worsening KCl stress. Strigolactone (SL), as a novel phytohormone, reportedly participates in plant tolerance to NaCl and drought stresses. However, the underlying mechanism and the effects of exogenous SL on the KCl stress of apple seedlings remain unclear. MethodsWe sprayed different concentrations of exogenous SL on Malus hupehensis Rehd. under KCl stress and measured the physiological indexes like, photosynthetic parameter, content of ROS, osmolytes and mineral element. In addition, the expressions of KCl-responding genes and SL-signaling genes were also detected and analyzed. ResultsApplication of exogenous SL protected the chlorophyll and maintained the photosynthetic rate of apple seedlings under KCl stress. Exogenous SL strengthened the enzyme activities of peroxidase and catalase, thereby eliminating reactive oxygen species production induced by KCl stress, promoting the accumulation of proline, and maintaining osmotic balance. Exogenous SL expelled K+ outside of the cytoplasm and compartmentalized K+ into the vacuole, increased the contents of Na+, Mg2+, Fe2+, and Mn2+ in the cytoplasm to maintain the ion homeostasis under KCl stress. ConclusionsExogenous SL can regulate photosynthesis, ROS migration and ion transport in apple seedlings to alleviate KCl stress.

IAA production and maize crop growth promoting potential of endophyte Aspergillus niger (AO11) under salt stress

Maize is cultivated under a broad range of soil conditions and environments. Maize is slightly vulnerable to salt stress and therefore it is seriously affected by soil salinity all over the world. Recognizing the responses of maize to salt stress and making a good strategy to overcome this problem could aid to develop solutions in saline areas to improve maize productivity. We investigated in this research the impacts, tolerance and salt stress management in corn. Many endophytic fungi can produce the Indole-3-acetic acid (IAA) is known for their role in plant growth and development both with and without salt stress conditions. The current study was focused on the production of IAA by endophytic fungi (Aspergillus niger) and maize seeds germination and promotion of seedling growth and vigor. In order to evaluate the defense response of maize plant, in relation to A. niger, an experiment was designed with three replications of treatments (control, salt stressed, salt stressed inoculated with A. niger, and only A. niger inoculated plants. It was determined that A. niger has the ability to produce the IAA in NaCl and KCl stress peaking 53 μg/ml and was not significantly by alternating the nitrogen and carbon sources in the nutrient broth but increasing the tryptophan concentration raised its production level. High concentration stress of sodium chloride and potassium chloride decrease maize plant seeds germination percentage, shoot and root length also affected the fresh and dry weight of maize. A. niger improves salt resistance in maize and also increased the germination percentage up to 30%, also improved the chlorophyll level and it was proved an effective approach for improving maize germination and growth under salt stress.

Changes in the Survivability of Marine Vibrio sp. under Hyper KCl Stress in the Presence of Betaine as Well as with Exposure to 37ºC.

Survivability at hyper KCl stress was examined at 30ºC and 37ºC in the presence and absence of an osmoprotectant by using resting cells prepared from marine Vibrio sp. grown at early stationary phase. Survivability was decided by counting colonies. The number of initial cells, 107.1 ± 0.2 (CFU·mL-1), was reduced to 105.1 ± 0.5 and < 101.0 (CFU·mL-1) at 30ºC and 37ºC, respectively, by the exposure of resting cells, that were prepared from cells grown for 8 h at 0.5 M NaCl at 30ºC, to 1.2 M KCl and 50 mM NaCl for 3 h. Betaine externally existed as a final concentration of 50 mM mitigated hyper KCl stress to the resting cells at 37ºC. The number of surviving cells was maintained 104.9 ± 0.3 (CFU·mL-1) when resting cells, 106.5 ± 0.1 (CFU·mL-1), that were prepared from pre-adapted cells to relatively high concentration of KCl in the growth for 10 h at 0.8 M KCl and 50 mM NaCl at 37ºC, were exposed to 1.2 M KCl, 50 mM NaCl, and 50 mM betaine at 37ºC for 3 h. The results indicate that osmoadaptation system(s) in resting cells is temperature sensitive and betaine functions to mitigate hyper KCl stress to the resting cells at 37ºC.

Mitigation of Hyper KCl Stress at 42ºC with Externally Existing Sodium Glutamate to a Halotolerant &lt;i&gt;Brevibacterium&lt;/i&gt; sp. JCM 6894

Halotolerant Brevibacterium sp. JCM 6894 grew at 37ºC in the presence of 2.3 M KCl, while the growth was repressed with the same concentration of NaCl. When resting cells, 107.4 ± 0.1 (CFU·mL-1), prepared from cells grown in the absence of salts at 30ºC, were exposed to 3.3 M NaCl for 36 h at 42ºC, reduction of the number of resting cells was maintained within a 1-log cycle in the presence of proline, betaine, or ectoine (50 mM). In the presence of 3.3 M KCl, the most functional osmoprotectant was sodium glutamate (50 mM), and the value was 107.2 ± 0.1 (CFU·mL-1) when exposed for 72 h at 42ºC. In the absence of osmoprotectants, the value was reduced to four orders of magnitude in each experimental condition. The number of resting cells, 106.8 ± 0.1 (CFU·mL-1), prepared from grown cells pre-adapted to 2.3 M KCl at 37ºC, was hardly reduced when exposed to 3.3 M KCl in the presence of sodium glutamate more than 50 mM for 72 h at 42ºC. Those results indicate that the isolate can sense the difference in hyper KCl stress as opposed to hyper NaCl stress, and different kinds of osmoadaptation systems can function to cope with each hyper salt stress.

Enhanced Lipid Production and Molecular Dynamics under Salinity Stress in Green Microalga Chlamydomonas reinhardtii (137C).

Microalgal lipids are a source of valuable nutritional ingredients in biotechnological industries, and are precursors to biodiesel production. Here, the effects of salt-induced stresses, including NaCl, KCl, and LiCl stresses, on the production of lipid in green microalga Chlamydomonas reinhardtii (137c) were investigated. NaCl stress dramatically increased saturated fatty acids (SFAs), which accounted for 70.2% of the fatty acid methyl ester (FAMEs) under stress. In contrary, KCl stress led to a slight increase in SFAs (47.05%) with the remaining being polyunsaturated fatty acids (PUFAs) (45.77%). RT-PCR analysis revealed that the genes involved in FA biosynthesis, such as PDH2, ACCase, MAT and KAS2, were up-regulated by NaCl-induced stress. Conversely, the genes responsible for the Kennedy pathway were suppressed. The alteration of FA homeostasis was further assessed by overexpressing MAT, the enzyme responsible for the production of malonyl-ACP, a key building block for FA biosynthesis, in the cyanobacterium Synechococcus elongatus PCC 7942. Intracellular FA composition was affected, with a predominant synthesis of SFAs in transformed cells. Owing to the diversity and relative abundance of SFAs, monounsaturated fatty acid (MUFAs) and PUFAs enable the feasibility of using microorganisms as a source of microalgal lipids or valuable nutritional ingredients; salt-induced stress and expression of MAT are useful in providing precursors for enhanced lipid production.

Molecular cloning, expression analysis, and heterologous characterization of a novel sodium/hydrogen exchanger from a mangrove plant, Rhizophora apiculata

Mangroves are natural halophytes confined to the tropical and subtropical coastal region of the world. Rhizophora apiculata is a small mangrove tree with a natural reservoir of many unknown salt tolerance mechanisms which is completely unexplored. In the present study, we cloned the full-length coding sequence of a putative vacuolar Na+/H+ exchanger from R. apiculata (RaNHX1). In-silico analysis predicted highly conserved signature domain, motifs such as cation/proton exchanger 1 (CPA1), Na+/H+ exchanger domain, amiloride-binding motif, and 12 transmembrane helices, which confirmed they are a member of plant NHX family. Transcript analysis of RaNHX1 revealed differential transcript regulation pattern in various tissues and under salt stress libraries. Total Na+ and K+ analysis in leaves, stem, and root tissues under salt stress revealed that leaves are major Na+ storage tissues in R. apiculata. The heterologous expression of pYES2.1-RaNHX1 construct in AXT3 yeast mutant strain showed partial complementation of sensitive phenotype under NaCl and KCl stress indicating their involvement in sequestering Na+ and K+. In slight alkaline condition, AXT3-RaNHX1 partially complemented the mutant phenotype under Na+ and K+ supplements, while at acidic pH phenotype was less conclusive. Total Na+ and K+ analysis of AXT3-RaNHX1 showed higher accumulation in yeast cells compared to AXT3 mutant in NaCl stress conditions. Taken together, this is a first attempt to functionally characterize RaNHX1 member from R. apiculata which may contribute to better understanding of salt tolerance mechanism in R. apiculata.

Endothelial-Dependent and Independent Vascular Relaxation Effect of Tetrahydropalmatine on Rat Aorta.

Tetrahydropalmatine (THP) is an active natural alkaloid isolated from Corydalis yanhusuo W.T. Wang which has been widely used for treating pain and cardiovascular disease in traditional Chinese medicine. Previous studies suggested THP have various pharmacological effects in neural and cardio tissue while the vascular reactivity of THP was not fully established. The present study found that THP relaxed rat aorta which contracted by phenylephrine (Phe), KCl, and U46619. The vascular relaxation effect of THP was partially attenuated by PI3K inhibitor wortmannin, Akt inhibitor IV, endothelial nitric oxide synthetase (eNOS) inhibitor L-NAME, guanylate cyclase inhibitors and the mechanical removal of endothelium. Also, the eNOS substrate L-arginine reversed the inhibition effect of L-NAME on THP-induced vascular relaxation. THP also induced intracellular NO production in human umbilical vein endothelial cells. However, Pre-incubation with β-adrenergic receptor blocker propranolol, angiotensin II receptor 1 (AT1) inhibitor losartan, angiotensin II receptor 2 (AT1) inhibitor PD123319 or angiotensin converting enzyme inhibitor enalapril enhanced the vascular relaxation effect of THP. THP did not affect the angiotensin II induced vascular contraction. Cyclooxygenase-2 (COX2) inhibitor indomethacin did not affect the vascular relaxation effect of THP. Furthermore, pre-treatment THP attenuated KCl and Phe induced rat aorta contraction in standard Krebs solution. In Ca2+ free Krebs solution, THP inhibited the Ca2+ induced vascular contraction under KCl or Phe stress and reduced KCl stressed Ca2+ influx in rat vascular smooth muscle cells. THP also inhibited intracellular Ca2+ release induced vascular contraction by blocking Ryr or IP3 receptors. In addition, the voltage-dependent K+ channel (Kv) blocker 4-aminopyridine, ATP-sensitive K+ channel (KATP) blocker glibenclamide and inward rectifying K+ channel blocker BaCl2 attenuated THP induced vascular relaxation regardless of the Ca2+-activated K+ channel (KCa) blocker tetraethylammonium. Thus, we could conclude that THP relaxed rat aorta in an endothelium-dependent and independent manner. The underlying mechanism of THP relaxing rat aorta involved PI3K/Akt/eNOS/NO/cGMP signaling path-way, Ca2+ channels and K+ channels rather than COX2, β-adrenergic receptor and renin-angiotensin system (RAS). These findings indicated that THP might be a potent treatment of diseases with vascular dysfunction like hypertension.

Effects of Potassium Chloride-Induced Stress on the Carotenoids Canthaxanthin, Astaxanthin, and Lipid Accumulations in the Green Chlorococcal Microalga Strain TISTR 9500.

Microalgae are a diverse group of photosynthetic eukaryotic organisms that are widely distributed globally. They are prolific sources of highly valuable compounds with fascinating chemical structures. Due to their balanced nutritional compositions and health benefits, they are increasingly being used as functional food ingredients. Carotenoid-based pigments and polyunsaturated fatty acids (PUFAs) are examples of high-value nutrients that can be accumulated abundantly in microalgae. Here, the effects of potassium chloride-induced stress on the productions of lipids and carotenoids in the green microalga of the Chlorococcaceae family were investigated. Under normal BG11 medium, this green microalga strain TISTR 9500 accumulated high levels of PUFA and primary carotenoid lutein. Stress tests revealed that KCl enhanced and modulated lipid and carotenoid accumulation levels. The liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis revealed that secondary carotenoids astaxanthin and canthaxanthin were robustly produced under KCl stress with the similar content of lutein. Further, this stress led to a significant increase in the total FA amount with the higher proportion of unsaturated FA than saturated FA. Thus, this green microalga could be an attractive and alternative natural biosource for canthaxanthin and astaxanthin, as well as for functional lipids.

Sodium is the critical factor leading to the positive halotropism of the halophyte Limonium bicolor

Previous studies indicate that the roots of nonhalophytes showed negative halotropism to salt stress to avoid salt damage. However, halotropism of euhalophytes and their possible reasons are little known. Limonium bicolor, a typical recretohalophyte with multicellular salt glands, was used to study halotropism compared with Arabidopsis thaliana under NaCl, KCl and Na2SO4 stress. The elongation of the roots in L. bicolor was significantly promoted by the appropriate concentrations of NaCl, KCl and Na2SO4, but those of A. thaliana was markedly inhibited. However, isosmotic mannitol with 200 mM NaCl did not affect the root growth of both L. bicolor and A. thaliana. The root activity of both L. bicolor and A. thaliana was enhanced by salts. Compared with K+, Cl–, and SO42−, Na+ played a critical role in halotropism of L. bicolor. Furthermore, the gravitropic setpoint angle of L. bicolor increased under NaCl, KCl and Na2SO4 treatments compared with controls, and the phenomenon was most apparent under NaCl treatments. The endogenous IAA content of the NaCl-treated L. bicolor seedlings was significantly higher than that of the controls. These results suggest that the recretohalophyte L. bicolor has positive halotropism and Na+ plays a pivotal role in L. bicolor’s positive root halotropism by regulating IAA.

Proline Formation (Produce) in the Bacteria: under Kcl Stress

Salinity is one of the important abiotic stress affecting microorganism growth and productivity. To survive these stresses most organisms have to stress-adaptation mechanisms. It's one of these things proline. We did not find any similar studies with P. aeruginosa and E. faecalis in our studies. The highest value for proline production at 30 °C 100 rpm was in E. faecalis 11,074 U/ml and in E. coli 6,833 U/ml. The highest proline production in LB medium containing 37 °C 100 rpm KCl was found to be in E. faecalis 14,604 U/ml and in E. coli 6,557 U/ml. However, there are studies with E. coli. This experiment revealed that the extracellular proline concentration is proportionally linked to the KCl stress. We should first mention that studies similar to those we were less common in the literature. We did not find any similar studies with P. aeruginosa and E. faecalis in our studies.

Molecular characterization and functional analysis of a cation-chloride cotransporter gene from trifoliate orange (Poncirus trifoliata L.)

A cation-chloride cotransporter gene PtrCCC from trifoliate orange was identified, its ectopic expression confers salt tolerance in transgenic tobacco. Salinity is one of the most serious environmental stresses that limit crop growth. Cation-chloride cotransporters (CCCs) are membrane proteins that are involved in plant development and salt tolerance. Here we provide an insight into the molecular characterization and functional analysis of CCC-encoding gene (PtrCCC) from trifoliate orange (Poncirus trifoliata L.). The open-reading frame of PtrCCC is 2943 bp in length, which encodes a protein of 980 amino acids. Phylogenetic analysis revealed that PtrCCC is closely related to CCCs from its allied genera as well as woody perennials. Quantitative real-time PCR (qRT-PCR) analysis demonstrated that PtrCCC was expressed in all tested tissues, with higher expression level in root tip and leaf tip. Its expression was up-regulated in the leaves of trifoliate orange under the presence of KCl conditions. Ectopic expression of PtrCCC gene in tobacco exhibited an improvement in salt tolerance. The transgenic plants had higher dry biomass in the roots and shoots than that in wild type plants. Moreover, under KCl and NaCl + KCl stress conditions, the accumulation of shoot Cl− was remarkably decreased in the transgenic lines as compared with the wild type plants. Collectively, these results suggest that PtrCCC plays an important role in salt tolerance, which is partially attributed to its role in ions homeostasis.

Dicer-Like Genes Are Required for H2O2 and KCl Stress Responses, Pathogenicity and Small RNA Generation in Valsa mali.

Valsa mali (V. mali) is the causative agent of apple tree Valsa canker, which heavily damages the production of apples in China. However, the biological roles of the RNA interfering (RNAi) pathway in the pathogenicity of V. mali remain unknown. Dicer-like proteins (DCLs) are important components that control the initiation of the RNAi pathway. In this study, VmDCL1 and VmDCL2 were isolated and functionally characterized in V. mali. VmDCL1 and VmDCL2 are orthologous in evolution to the DCLs in Cryphonectria parasitica. The deletion of VmDCL1 and VmDCL2 did not affect vegetative growth when the mutants (ΔVmDCL1, ΔVmDCL2 and ΔVmDCL1DCL2) and wild type strain 03–8 were grown on a PDA medium at 25°C in the dark. However, the colony of ΔVmDCL1 increased by 37.1% compared to the 03–8 colony in a medium containing 0.05% H2O2 3 days after inoculation, and the growth of ΔVmDCL1 was significantly inhibited in a medium containing 0.5 M KCl at a ratio of 25.7%. Meanwhile, in the presence of 0.05% H2O2, the growth of ΔVmDCL2 decreased by 34.5% compared with the growth of 03–8, but ΔVmDCL2 grew normally in the presence of 0.5 M KCl. More importantly, the expression of VmDCL2 was up-regulated 125-fold during the pathogen infection. In the infection assays using apple twigs, the pathogenicity of ΔVmDCL2 and ΔVmDCL1DCL2 was significantly reduced compared with that of 03–8 at a ratio of 24.7 and 41.3%, respectively. All defective phenotypes could be nearly rescued by re-introducing the wild type VmDCL1 and VmDCL2 alleles. Furthermore, the number and length distribution of unique small RNAs (unisRNAs) in the mutants and 03–8 were analyzed using deep sequencing. The number of unisRNAs was obviously lower in ΔVmDCL1, ΔVmDCL2 and ΔVmDCL1DCL2 than that in 03–8, and the length distribution of the sRNAs also markedly changed after the VmDCLs were deleted. These results indicated that VmDCLs function in the H2O2 and KCl stress response, pathogenicity and generation of sRNAs.

KCl induces a caspase-independent programmed cell death in the unicellular green chlorophyte Chlamydomonas reinhardtii (Chlorophyceae)

:Programmed cell death (PCD) plays an important role in mediating adaptation responses under adverse conditions such as high salinity. To understand the molecular mechanism of adaptation of algal cells to salt, the freshwater alga Chlamydomonas reinhardtii was challenged with 200 mM KCl. In the present study, vegetative cells of C. reinhardtii undergo cell death when exposed to 200 mM KCl, this death being dose-dependent, with 100–800 mM KCl causing 16–64% cell death. Within half an hour of KCl exposure, a ~1.9-fold rise in the intracellular H2O2 content followed an increase in antioxidant enzyme (superoxide dismutats, catalase, and ascorbate peroxidase) activities and their transcript levels. Furthermore, apoptotic hallmarks such as disruption of mitochondrial membrane potential, DNA nicks, apoptosis-inducing factor (AIF) release into the cytoplasm, and genomic DNA fragmentation were observed. Interestingly, KCl stress did not stimulate caspase-3-like protease activity. Additionally, cells undergoing PCD showed characteristic shrinkage with an accumulation of lipids and vacuoles, along with degraded chloroplast. These results illustrate that KCl induces reactive oxygen species production that leads to AIF release from mitochondria, causing a caspase-independent cell death in C. reinhardtii.