S-type Anion Channels SLAC1 and SLAH3 Function as Essential Negative Regulators of Inward K+ Channels and Stomatal Opening in Arabidopsis.

Drought stress induces stomatal closure and inhibits stomatal opening simultaneously. However, the underlying molecular mechanism is still largely unknown. Here we show that S-type anion channels SLAC1 and SLAH3 mainly inhibit inward K+ (K+in) channel KAT1 by protein-protein interaction, and consequently prevent stomatal opening in Arabidopsis. Voltage-clamp results demonstrated that SLAC1 inhibited KAT1 dramatically, but did not inhibit KAT2. SLAH3 inhibited KAT1 to a weaker degree relative to SLAC1. Both the N terminus and the C terminuses of SLAC1 inhibited KAT1, but the inhibition by the N terminus was stronger. The C terminus was essential for the inhibition of KAT1 by SLAC1. Furthermore, drought stress strongly up-regulated the expression of SLAC1 and SLAH3 in Arabidopsis guard cells, and the over-expression of wild type and truncated SLAC1 dramatically impaired K+in currents of guard cells and light-induced stomatal opening. Additionally, the inhibition of KAT1 by SLAC1 and KC1 only partially overlapped, suggesting that SLAC1 and KC1 inhibited K+in channels using different molecular mechanisms. Taken together, we discovered a novel regulatory mechanism for stomatal movement, in which singling pathways for stomatal closure and opening are directly coupled together by protein-protein interaction between SLAC1/SLAH3 and KAT1 in Arabidopsis.

Cyclic nucleotide-gated channel 18 is an essential Ca2+ channel in pollen tube tips for pollen tube guidance to ovules in Arabidopsis.

In flowering plants, pollen tubes are guided into ovules by multiple attractants from female gametophytes to release paired sperm cells for double fertilization. It has been well-established that Ca(2+) gradients in the pollen tube tips are essential for pollen tube guidance and that plasma membrane Ca(2+) channels in pollen tube tips are core components that regulate Ca(2+) gradients by mediating and regulating external Ca(2+) influx. Therefore, Ca(2+) channels are the core components for pollen tube guidance. However, there is still no genetic evidence for the identification of the putative Ca(2+) channels essential for pollen tube guidance. Here, we report that the point mutations R491Q or R578K in cyclic nucleotide-gated channel 18 (CNGC18) resulted in abnormal Ca(2+) gradients and strong pollen tube guidance defects by impairing the activation of CNGC18 in Arabidopsis. The pollen tube guidance defects of cngc18-17 (R491Q) and of the transfer DNA (T-DNA) insertion mutant cngc18-1 (+/-) were completely rescued by CNGC18. Furthermore, domain-swapping experiments showed that CNGC18's transmembrane domains are indispensable for pollen tube guidance. Additionally, we found that, among eight Ca(2+) channels (including six CNGCs and two glutamate receptor-like channels), CNGC18 was the only one essential for pollen tube guidance. Thus, CNGC18 is the long-sought essential Ca(2+) channel for pollen tube guidance in Arabidopsis.

The S-Type Anion Channel ZmSLAC1 Plays Essential Roles in Stomatal Closure by Mediating Nitrate Efflux in Maize.

Diverse stimuli induce stomatal closure by triggering the efflux of osmotic anions, which is mainly mediated by the main anion channel SLAC1 in plants, and the anion permeability and selectivity of SLAC1 channels from several plant species have been reported to be variable. However, the genetic identity as well as the anion permeability and selectivity of the main S-type anion channel ZmSLAC1 in maize are still unknown. In this study, we identified GRMZM2G106921 as the gene encoding ZmSLAC1 in maize, and the maize mutants zmslac1-1 and zmslac1-2 harboring a mutator (Mu) transposon in ZmSLAC1 exhibited strong insensitive phenotypes of stomatal closure in response to diverse stimuli. We further found that ZmSLAC1 functions as a nitrate-selective anion channel without obvious permeability to chloride, sulfate and malate, clearly different from SLAC1 channels of Arabidopsis thaliana, Brassica rapa ssp. chinensis and Solanum lycopersicum L. Further experimental data show that the expression of ZmSLAC1 successfully rescued the stomatal movement phenotypes of the Arabidopsis double mutant atslac1-3atslah3-2 by mainly restoring nitrate-carried anion channel currents of guard cells. Together, these findings demonstrate that ZmSLAC1 is involved in stomatal closure mainly by mediating the efflux of nitrate in maize.

Molecular analysis and biochemical characteristics of degenerated strains of Cordyceps militaris.

Cordyceps militaris has commercially been cultivated, but its degenerated subcultures have gradually resulted in the reduced production. In this study, the biological characteristics and DNA change of degenerated strains of C. militaris were analyzed in detail. The results showed that the degenerated strains exhibited the lower growth rate, and the deficiency in fruit body formation and pigment production. The degradation of strains was not attributable to DNA changes identified by RAPD and SRAP. Compared to normal strains, the biochemical indexes of degradation strains and normal strains showed that the carotenoid content of degradation strains was significantly lower, the activities of cellulase and amylase of degradation strains were slight lower, and the EPS content was lower, but the IPS was higher. All these results suggested that the degradation of C. militaris may be caused by the inhibition or in harmony of metabolite synthesis involved in the metabolic regulation, which should be further verified.

Protein kinase OsSAPK8 functions as an essential activator of S-type anion channel OsSLAC1, which is nitrate-selective in rice.

MAIN CONCLUSION: OsSAPK8 is an essential activator of OsSLAC1 by phosphorylation, and OsSLAC1 is a nitrate-selective anion channel. S-type anion channel AtSLAC1 and protein kinase AtOST1 have been well-characterized as two core components of ABA signaling cascade in Arabidopsis guard cells, and AtOST1 functions as a main upstream activator of AtSLAC1 for drought stress- and ABA-induced stomata closure. However, the identity of the ortholog of AtOST1 in rice, the main activator of OsSLAC1, is still unknown. Here, we report that protein kinase OsSAPK8 interacts with and activates OsSLAC1 mainly by phosphorylating serine 129 (S129) of OsSLAC1, and this phosphorylating site corresponds to the specific phosphorylating site serine 120 (S120) of AtSLAC1 for AtOST1. Additionally, we found that OsSLAC1 is a nitrate-selective anion channel without obvious permeability to chloride, malate, and sulfate, and the expression of OsSLAC1 in Arabidopsis slac1-3 (atslac1-3) mutant successfully rescued the hypersensitive phenotype of this mutant to drought stress. Together, this research suggests that OsSAPK8 is a counterpart of AtOST1 for the activation of OsSLAC1, which is a nitrate-selective anion channel.

The response of Serratia marcescens JG to environmental changes by quorum sensing system.

Many bacterial cells are known to regulate their cooperative behaviors and physiological processes through a molecular mechanism called quorum sensing. Quorum sensing in Serratia marcescens JG is mediated by the synthesis of autoinducer 2 (AI-2) which is a furanosyl borate diester. In this study, the response of quorum sensing in S. marcescens JG to environment changes such as the initial pH, carbon sources and boracic acid was investigated by a bioreporter and real-time PCR analysis. The results show that glucose can affect AI-2 synthesis to the greatest extent, and 2.0 % glucose can stimulate S. marcescens JG to produce more AI-2, with a 3.5-fold increase in activity compared with control culture. Furthermore, the response of quorum sensing to changes in glucose concentration was performed by changing the amount of luxS RNA transcripts. A maximum of luxS transcription appeared during the exponential growth phase when the glucose concentration was 20.0 g/L. AI-2 production was also slightly impacted by the low initial pH. It is significant for us that the addition of boracic acid at microdosage (0.1-0.2 g/L) can also induce AI-2 synthesis, which probably demonstrated the feasible fact that the 4,5-dihydroxy-2, 3-pentanedione cyclizes by the addition of borate and the loss of water, is hydrated and is converted to the final AI-2 in S. marcescens JG.

The dependence of quorum sensing in Serratia marcescens JG on the transcription of luxS gene.

Bacteria communicate with one another using chemical signal molecules. This phenomenon termed quorum sensing enables the bacteria to monitor the environment for other bacteria and to alter behavior on a population-wide scale in response to cell density. Serratia marcescens JG, a quorum sensing bacterium, can secrete a furanosyl borate diester autoinducer (AI-2) in the exponential phase of growth. In this study, to further investigate the regulation of AI-2 production in S. marcescens JG, the pfs and luxS promoter fusions to an operon luxCDABE reporter were constructed in a low-copy-number vector pBR322K, which allows an examination of transcription of the genes in the pathway for signal synthesis. The results show that the luxS expression is constitutive, and the transcription of luxS is tightly correlated with AI-2 production in S. marcescens JG because the peaks of AI-2 production and transcriptional level of luxS appear at the same time point. The close relation of the profiles of luxS transcription and AI-2 production was also confirmed with real-time PCR technology. These results support the hypothesis that the quorum sensing in S. marcescens JG is luxS dependent.

Functional analysis of a novel Cys2/His2-type zinc finger protein involved in salt tolerance in rice.

The Cys2/His2-type zinc finger proteins have been implicated in different cellular processes involved in plant development and stress responses. Through microarray analysis, a salt-responsive zinc finger protein gene ZFP179 was identified and subsequently cloned from rice seedlings. ZFP179 encodes a 17.95 kDa protein with two C2H2-type zinc finger motifs having transcriptional activation activity. The real-time RT-PCR analysis showed that ZFP179 was highly expressed in immature spikes, and markedly induced in the seedlings by NaCl, PEG 6000, and ABA treatments. Overexpression of ZFP179 in rice increased salt tolerance and the transgenic seedlings showed hypersensitivity to exogenous ABA. The increased levels of free proline and soluble sugars were observed in transgenic plants compared to wild-type plants under salt stress. The ZFP179 transgenic rice exhibited significantly increased tolerance to oxidative stress, the reactive oxygen species (ROS)-scavenging ability, and expression levels of a number of stress-related genes, including OsDREB2A, OsP5CS OsProT, and OsLea3 under salt stress. Our studies suggest that ZFP179 plays a crucial role in the plant response to salt stress, and is useful in developing transgenic crops with enhanced tolerance to salt stress.

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Some fungi engage symbiosis with bacteria, which can effectively promote the metabolism and growth of fungi. The diversity and community structure of bacteria can reflect the growth and substrate utilization of fungi. In this study, we analyzed the effects of different sawdust dosages on the community structure and diversity of bacteria associated with the hyphae of Pleurotus eryngii using high-throughput sequencing technology (HST) based on PCR-amplified 16S rRNA V3-V4 fragments. The results showed that the high-quality sequences from five groups of mycelia samples were clustered into 25 phyla, 52 classes, 114 orders, 199 families, and 406 genera. Proteobacteria (35.0%-85.9%) and Firmicutes (6.5%-38.4%) were the most abundant bacterial phyla, while Acinetobacter (14.8%-71.6%) and Pseudomonas (1.7%-22.3%) were the dominant symbiotic genera. Compared with the mycelia grown on the complete culture medium, sawdust addition could increase the diversity of bacteria coexisting with P. eryngii mycelia, and change the community structure of 10 dominant phyla and 9 dominant genera. P. eryngii cultivated in substrate containing 5 g sawdust had the fastest mycelium growth rate, thick mycelia, and neatly edges. Furthermore, judging from the abundance and diversity, Pseudomonas and Lactobacillus became the dominant genera, which were positively correlated with the mycelia growth vigour. Sawdust, as an important carbon source, could affect the growth and development of P. eryngii and the community structure and diversity of bacteria coexisting with mycelia. This study would lay a theoretical foundation for exploring the molecular mechanism of sawdust and mycelium symbiosis affecting the growth and development of P. eryngii.

Cyclic Dipeptides Mediating Quorum Sensing and Their Biological Effects in Hypsizygus Marmoreus.

A novel quorum sensing (QS) system was discovered in Serratia odorifera, the symbiotic bacterium of Hypsizygus marmoreus. This system uses cyclo(Pro-Phe), cyclo(Pro-Tyr), cyclo(Pro-Val), cyclo(Pro-Leu), cyclo(Tyr-Leu), and cyclo(Tyr-Ile) as autoinducers. This discovery is the first attempt to characterize cyclic dipeptides as QS signaling molecules in S. odorifera and improves the classical QS theory. Significantly, except for cyclo(Tyr-Leu), these QS autoinducers can increase the transcription level of lignin-degrading enzyme genes of H.marmoreus. The cyclo(Pro-Phe) can increase the activity of extracellular laccase (1.32-fold) and manganese peroxidase (20%), which may explain why QS potentially regulates the hyphal growth, primordium formation, and fruit body development of H. marmoreus. Furthermore, it was demonstrated that the cyclo(Tyr-Ile) biosynthesis in S. odorifera was catalyzed by the nonribosomal peptide synthetase (NRPS). This study supports exploring the growth and development of H.marmoreus promoted by its symbiotic bacteria at QS signal transduction level.

Three CNGC Family Members, CNGC5, CNGC6, and CNGC9, Are Required for Constitutive Growth of Arabidopsis Root Hairs as Ca2+-Permeable Channels.

The genetic identities of Ca2+ channels in root hair (RH) tips essential for constitutive RH growth have remained elusive for decades. Here, we report the identification and characterization of three cyclic nucleotide-gated channel (CNGC) family members, CNGC5, CNGC6, and CNGC9, as Ca2+ channels essential for constitutive RH growth in Arabidopsis. We found that the cngc5-1cngc6-2cngc9-1 triple mutant (designated shrh1) showed significantly shorter and branching RH phenotypes as compared with the wild type. The defective RH growth phenotype of shrh1 could be rescued by either the expression of CNGC5, CNGC6, or CNGC9 single gene or by the supply of high external Ca2+, but could not be rescued by external K+ supply. Cytosolic Ca2+ imaging and patch-clamp data in HEK293T cells showed that these three CNGCs all function as Ca2+-permeable channels. Cytosolic Ca2+ imaging in growing RHs further showed that the Ca2+ gradients and their oscillation in RH tips were dramatically attenuated in shrh1 compared with those in the wild type. Phenotypic analysis revealed that these three CNGCs are Ca2+ channels essential for constitutive RH growth, with different roles in RHs from the conditional player CNGC14. Moreover, we found that these three CNGCs are involved in auxin signaling in RHs. Taken together, our study identified CNGC5, CNGC6, and CNGC9 as three key Ca2+ channels essential for constitutive RH growth and auxin signaling in Arabidopsis.

Increased tolerance of rice to cold, drought and oxidative stresses mediated by the overexpression of a gene that encodes the zinc finger protein ZFP245.

ZFP245 is a cold- and drought-responsive gene that encodes a zinc finger protein in rice. The ZFP245 protein localizes in the nucleus and exhibits trans-activation activity. Transgenic rice plants overexpressing ZFP245 were generated and found to display high tolerance to cold and drought stresses. The transgenic plants did not exhibit growth retardation, but showed growth sensitivity against exogenous abscisic acid, increased free proline levels and elevated expression of rice pyrroline-5-carboxylatesynthetase and proline transporter genes under stress conditions. Overproduction of ZFP245 enhanced the activities of reactive oxygen species-scavenging enzymes under stress conditions and increased the tolerance of rice seedlings to oxidative stress. Our data suggest that ZFP245 may contribute to the tolerance of rice plants to cold and drought stresses by regulating proline levels and reactive oxygen species-scavenging activities, and therefore may be useful for developing transgenic crops with enhanced tolerance to abiotic stress.

Molecular spectrum of lanthanide complexes with 2,3-dichlorobenzoic acid and 2,2-bipyridine.

With 2,3-dichlorobenzoic acid as the first ligands and 2,2'-bipyridine as the second ligands, the lanthanide complexes [Ln(2,3-DClBA)3bipy]2 [Ln=Nd(a), Sm(b), Eu(c), Tb(d), Dy(e), Ho(f)] have been synthesized. By using Infrared (IR) and Raman (R) spectra, the characteristics of the groups can be identified. The bands of lanthanide complexes have been analyzed and attributed, and clearly demonstrated with the use of the complementarity of IR and R. The experiment reveals that the bands of complexes are affected by lanthanide elements (Ln). The frequency of stretching vibration and breathing vibration of ring, together with the stretching vibration of the carbonyl group (νCO), tends to be rising as the atomic number of lanthanide increasing. Meanwhile, crystallography data demonstrate that the six carbonyl groups have different bond length and bond angle, which can lead to different vibration frequency. The second derivatives of IR show that there are multiple vibration frequencies existing in the symmetrical stretching vibration of the carbonyl group (νsCO). Therefore the second derivative of IR spectrum is a characteristic band of different coordination modes of carbonyl group.

Overexpression of the Qc-SNARE gene OsSYP71 enhances tolerance to oxidative stress and resistance to rice blast in rice (Oryza sativa L.).

OsSYP71 is an oxidative stress and rice blast response gene that encodes a Qc-SNARE protein in rice. Qc-SNARE proteins belong to the superfamily of SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors), which function as important components of the vesicle trafficking machinery in eukaryotic cells. In this paper, 12 Qc-SNARE genes were isolated from rice, and expression patterns of 9 genes were detected in various tissues and in seedlings challenged with oxidative stresses and inoculated with rice blast. The expression of OsSYP71 was clearly up-regulated under these stresses. Overexpression of OsSYP71 in rice showed more tolerance to oxidative stress and resistance to rice blast than wild-type plants. These results indicate that Qc-SNAREs play an important role in rice response to environmental stresses, and OsSYP71 is useful in engineering crop plants with enhanced tolerance to oxidative stress and resistance to rice blast.

Isolation of GPD promoter from Tremella fuciformis and driving expression of EGFP gene.

Glyceraldehyde-3-phosphate dehydrogenase (GPD) cDNA was cloned by RT-PCR using total RNA from Tremella fuciformis as template with a pair of degenerate primers. Then, a 500-bp 5'-upstream promoter region of the gene encoding GPD from T. fuciformis genomic DNA was isolated by thermal asymmetric interlaced PCR. The cloned promoter was fused to 5'-upstream of enhanced green fluorescent protein gene to construct T. fuciformis expression vector pCB-TEGFP with hygromycin gene as a selectable marker. Electroporation was performed to transfer plasmid DNA of pCB-TEGFP into yeast-like conidia from T. fuciformis. Molecular evidence, including PCR analysis, fluorescence detection, fluorescence spectra assay, and SDS-PAGE, indicated that the EGFP gene had been integrated into the genome of transgenic T. fuciformis strains and was expressed successfully. The results also showed that this promoter could be used to carry out regulated expression of heterologous gene products in T. fuciformis.

PCR detection of Serratia spp. using primers targeting pfs and luxS genes involved in AI-2-dependent quorum sensing.

Quorum sensing is the ability of bacteria to communicate and coordinate behavior emitting signaling molecules. A series of primers for PCR detection of Serratia spp. has been designed using as targets the pfs and luxS genes involved in AI-2-dependent quorum sensing. The identities of the PCR products (193 and 102 bp) were confirmed by commercial sequencing. Twenty-seven Serratia strains (representing 10 different species) tested positive for the presence of the pfs and luxS genes, while a total of 7 different species of non-Serratia (25 strains) were tested and gave negative results. The sensitivity and specificity of the pfs- and luxS-based PCR assay were also checked in artificially contaminated bacterial samples. In this study we established a novel method to detect Serratia using quorum-sensing genes as diagnostic markers.

Analysis of genetic diversity in Ganoderma population with a novel molecular marker SRAP.

Genetic marker technology designed to detect naturally occurring polymorphisms at the DNA level had become an invaluable and revolutionizing tool for both applied and basic studies of fungi. To eliminate the confusion on the taxonomy of Ganoderma strains, in this study, a collection of 31 accessions representative of morphotypes and some unclassified types was used for analyzing molecular diversity using a novel molecular marker sequence-related amplified polymorphism (SRAP). This collection included commercial cultivars and wild varieties that represented the great diversification of types from different countries and regions. The experimental results showed that 50 out of 95 combinations of primers turned out to be polymorphic, and 85 polymorphism bands were obtained using six combinations. Based on the appearances of markers, the genetic similarity coefficients were calculated, and genetic variations were observed (0 approximately 1) among the 31 different Ganoderma strains. The group of Ganoderma lucidum showed significant differences from the group of Ganoderma sinense. Moreover, G. lucidum in China was also different from G. lucidum in Yugoslavia. At the same time, cluster analysis successfully categorized these 31 Ganoderma strains into five groups. These results revealed the genetic diversity of Ganoderma strains and their correlation with geographic environments. It also suggested SRAP marker could be used in the taxonomic analysis of fungi. To our knowledge, this is the first application of SRAP marker on the systematics of Ganoderma strains within basidiomycetes.

Chromosomal integration of the Vitreoscilla hemoglobin gene and its physiological actions in Tremella fuciformis.

The Vitreoscilla hemoglobin (VHb) gene was expressed in yeast-like conidia (YLCs) of Tremella fuciformis (T. fuciformis) to increase cell density in submerged fermentation by enhancing oxygen uptake. With the intention of doing this, an integrated expression vector containing the VHb gene and the hygromycin B phosphotransferase (hph) gene derived from Escherichia coli (E. coli) as the selectable marker was constructed, and then transformed into protoplasts of YLCs from T. fuciformis with restriction enzyme-mediated DNA integration (REMI). Hygromycin-resistant transformants had been generated during the transformation. Molecular evidences including PCR assay, Southern blotting, and Western blot analysis indicated the VHb gene had been integrated into the genome of transgenic T. fuciformis strains and was expressed successfully. Shake-flask fermentation and bioreactor cultivation results showed that the expression of VHb in this fungus could enhance growth of YLCs. The final cell density was higher in the culture of VHb-expressing strain than that of the wild-type strain. Moreover, these results also suggested that CaMV35S promoter was capable of driving the expression of heterologous genes in T. fuciformis.