Overexpression of PvWAK3 from seashore paspalum increases salt tolerance in transgenic Arabidopsis via maintenance of ion and ROS homeostasis.

Seashore paspalum (Paspalum vaginatum O. Swartz) is an important warm-season turfgrass species with extreme salt tolerance, but investigations on its salt tolerance mechanism are limited. A salt induced PvWAK3 from halophyte seashore paspalum was identified in this study. Overexpression of PvWAK3 in Arabidopsis led to increased salt tolerance. Transgenic plants had higher levels of seed germination rate, root length, number of lateral roots, shoot weight, survival rate, Fv/Fm, ETR, and NPQ compared with the wild type (WT) under salt stress. Na+ content was increased and K+ content was decreased after salinity treatment, with lower levels of Na+ and Na+/K+ ratio but higher level of K+ in transgenic plants than in WT under salt stress. The improved maintenance of Na+ and K+ homeostasis was associated with the higher transcript levels of K + -Uptake Permease 4 (KUP4), Potassium Transport 2/3 (AKT2), Salt Overly Sensitive 1 (SOS1) and High-Affinity K + Transporter 5 (HAK5) in transgenic plants compared with WT. Superoxide dismutase (SOD), catalase (CAT) and ascorbate-peroxidase (APX) activities, proline concentration, and P5CS1 transcript were increased after salinity treatment, with higher levels in transgenic lines compared with WT, which led to reduced accumulation of O2·- and H2O2 under salt stress. It is suggested that PvWAK3 regulates salt tolerance positively, which is associated with promoted Na+ and K+ homeostasis, activated antioxidant enzymes, and proline biosynthesis under salt stress.

Jasmonate biosynthesis enzyme allene oxide cyclase 2 mediates cold tolerance and pathogen resistance.

Allene oxide cyclase (AOC) is a key enzyme in the biosynthesis of jasmonic acid (JA), which is involved in plant growth and development as well as adaptation to environmental stresses. We identified the cold- and pathogen-responsive AOC2 gene from Medicago sativa subsp. falcata (MfAOC2) and its homolog MtAOC2 from Medicago truncatula. Heterologous expression of MfAOC2 in M. truncatula enhanced cold tolerance and resistance to the fungal pathogen Rhizoctonia solani, with greater accumulation of JA and higher transcript levels of JA downstream genes than in wild-type plants. In contrast, mutation of MtAOC2 reduced cold tolerance and pathogen resistance, with less accumulation of JA and lower transcript levels of JA downstream genes in the aoc2 mutant than in wild-type plants. The aoc2 phenotype and low levels of cold-responsive C-repeat-binding factor (CBF) transcripts could be rescued by expressing MfAOC2 in aoc2 plants or exogenous application of methyl jasmonate. Compared with wild-type plants, higher levels of CBF transcripts were observed in lines expressing MfAOC2 but lower levels of CBF transcripts were observed in the aoc2 mutant under cold conditions; superoxide dismutase, catalase, and ascorbate-peroxidase activities as well as proline concentrations were higher in MfAOC2-expressing lines but lower in the aoc2 mutant. These results suggest that expression of MfAOC2 or MtAOC2 promotes biosynthesis of JA, which positively regulates expression of CBF genes and antioxidant defense under cold conditions and expression of JA downstream genes after pathogen infection, leading to greater cold tolerance and pathogen resistance.

Causal interpretation for groundwater exploitation strategy in a coastal aquifer.

Machine learning models (ML), as a collection of nonparametric or semiparametric estimation methods, can successfully encode the distribution of the problems into its trainable parameters based on observation data. However, the distributions of hydrological variables may change suddenly under complex environmental conditions, leading to biased estimates when using ML models. This work is the first attempt to solve this issue using structural causal models (SCM). Specifically, two SCM were constructed based on hydrological conditions and monitoring data. Then the Propensity Score estimator and the Double Machine Learning estimator were employed to estimate the causal effects of four treatments on the mean Cl- concentration (MCL) in the coastal aquifer. The results showed that pumping groundwater from area A1 or increasing the river level directly leads to a decrease in MCL, while pumping area A3 directly leads to an increase in MCL. Moreover, MCL can be effectively controlled by cooperative-treatment strategies. Finally, two practical exploitation strategies are derived. In the planting month, it should increase groundwater pumping from area A1, limit groundwater pumping from A2, and prohibit groundwater pumping from A3. For the normal month, it is proposed to increase the height of the rubber dam to raise the river level and reduce groundwater pumping from A1 and A2.

A calmodulin-like protein (CML10) interacts with cytosolic enzymes GSTU8 and FBA6 to regulate cold tolerance.

Calmodulin-like proteins (CMLs) are calcium (Ca2+) sensors involved in plant growth and development as well as adaptation to environmental stresses; however, their roles in plant responses to cold are not well understood. To reveal the role of MsCML10 from alfalfa (Medicago sativa) in regulating cold tolerance, we examined transgenic alfalfa and Medicago truncatula overexpressing MsCML10, MsCML10-RNAi alfalfa, and a M. truncatula cml10-1 mutant and identified MsCML10-interacting proteins. MsCML10 and MtCML10 transcripts were induced by cold treatment. Upregulation or downregulation of MsCML10 resulted in increased or decreased cold tolerance, respectively, while cml10-1 showed decreased cold tolerance that was complemented by expressing MsCML10, suggesting that MsCML10 regulates cold tolerance. MsCML10 interacted with glutathione S-transferase (MsGSTU8) and fructose 1,6-biphosphate aldolase (MsFBA6), and the interaction depended on the presence of Ca2+. The altered activities of Glutathione S-transferase and FBA and levels of ROS and sugars were associated with MsCML10 transcript levels. We propose that MsCML10 decodes the cold-induced Ca2+ signal and regulates cold tolerance through activating MsGSTU8 and MsFBA6, leading to improved maintenance of ROS homeostasis and increased accumulation of sugars for osmoregulation, respectively.

Effectiveness and comparison of physical barriers on seawater intrusion and nitrate accumulation in upstream aquifers.

Physical barrier is a coastal engineering widely used to prevent seawater intrusion. However, previous studies have not evaluated the combined influence of type and structure of the physical barrier on nitrate accumulation in upstream aquifers. Furthermore, the mechanisms of nitrate accumulation caused by the physical barriers are still unclear. In this study, numerical simulations were conducted to investigate the mechanisms and influence of the physical barrier on seawater intrusion and NO3- accumulation. The results show that constructing physical barrier can result in the reduction of nitrate discharge to the sea and accumulation of nitrate in upstream aquifers. The accumulation degree is significant if the barrier height is large; the barrier location is near the sea; the nitrate infiltration rate is large; the infiltration nitrate concentration is large; the inflow is weak, and the inflow DOC concentration is low. A cut-off wall is more likely to cause nitrate accumulation than a subsurface dam. It can result in mean nitrate concentration in groundwater upstream increasing by more than 30 %. Because a nitrate accumulation zone is formed behind the cut-off wall where the flow is slow and dissolved oxygen carbon is difficult to be replenished so that the denitrification is weak. Despite the subsurface dam may not result in a significant increase in nitrate concentration, it cannot be applied to the areas where SI has occurred due to the residual seawater problem. The nitrate accumulation in the upstream aquifer is a long-term process that lasts for more than three years to reach a pseudo-steady state. Seasonal variations of inflow and infiltration lead to fluctuation of mean nitrate concentration, thereby the nitrate accumulation rate increased after April and weakened between July and December.

Genome-Wide Identification and Characterization of Short-Chain Dehydrogenase/Reductase (SDR) Gene Family in Medicago truncatula.

Short-chain dehydrogenase/reductase (SDR) belongs to the NAD(P)(H)-dependent oxidoreductase superfamily. Limited investigations reveal that SDRs participate in diverse metabolisms. A genome-wide identification of the SDR gene family in M. truncatula was conducted. A total of 213 MtSDR genes were identified, and they were distributed on all chromosomes unevenly. MtSDR proteins were categorized into seven subgroups based on phylogenetic analysis and three types including 'classic', 'extended', and 'atypical', depending on the cofactor-binding site and active site. Analysis of the data from M. truncatula Gene Expression Atlas (MtGEA) showed that above half of MtSDRs were expressed in at least one organ, and lots of MtSDRs had a preference in a tissue-specific expression. The cis-acting element responsive to plant hormones (salicylic acid, ABA, auxin, MeJA, and gibberellin) and stresses were found in the promoter of some MtSDRs. Many genes of MtSDR7C,MtSDR65C, MtSDR110C, MtSDR114C, and MtSDR108E families were responsive to drought, salt, and cold. The study provides useful information for further investigation on biological functions of MtSDRs, especially in abiotic stress adaptation, in the future.

A novel Medicago truncatula calmodulin-like protein (MtCML42) regulates cold tolerance and flowering time.

Calmodulin-like proteins (CMLs) are one of the Ca2+ sensors in plants, but the functions of most CMLs remain unknown. The regulation of cold tolerance and flowering time by MtCML42 in Medicago truncatula and the underlying mechanisms were investigated using MtCML42-overexpressing plants and cml42 Medicago mutants with a Tnt1 retrotransposon insertion. Compared with the wild type (WT), MtCML42-overexpressing lines had increased cold tolerance, whereas cml42 mutants showed decreased cold tolerance. The impaired cold tolerance in cml42 could b complemented by MtCML42 expression. The transcript levels of MtCBF1, MtCBF4, MtCOR413, MtCAS15, MtLTI6A, MtGolS1 and MtGolS2 and the concentrations of raffinose and sucrose were increased in response to cold treatment, whereas higher levels were observed in MtCML42-overexpressing lines and lower levels were observed in cml42 mutants. In addition, early flowering with upregulated MtFTa1 and downregulated MtABI5 transcripts was observed in MtCML42-overexpressing lines, whereas delayed flowering with downregulated MtFTa1 and upregulated MtABI5 was observed in cml42. MtABI5 expression could complement the flowering phenotype in the Arabidopsis mutant abi5. Our results suggest that MtCML42 positively regulates MtCBF1 and MtCBF4 expression, which in turn upregulates the expression of some COR genes, MtGolS1 and MtGolS2, which leads to raffinose accumulation and increased cold tolerance. MtCML42 regulates flowering time through sequentially downregulating MtABI5 and upregulating MtFTa1 expression.

Calmodulin-Like (CML) Gene Family in Medicago truncatula: Genome-Wide Identification, Characterization and Expression Analysis.

Calcium is an important second messenger in mediating adaptation responses of plants to abiotic and biotic stresses. Calmodulin-like (CML) protein is an important calcium-signaling protein that can sense and decode Ca2+ signal in plants. Medicago truncatula is a model legume plant; however, investigations of MtCML proteins are limited. Using genome analysis and BLAST database searches, fifty MtCML proteins that possess EF-hand motifs were identified. Phylogenetic analysis showed that CML homologs between M. truncatula, Arabidopsis thaliana and Oryza sativa shared close relationships. Gene structure analysis revealed that these MtCML genes contained one to four conserved EF-hand motifs. All MtCMLs are localized to eight chromosomes and underwent gene duplication. In addition, MtCML genes were differentially expressed in different tissues of M. truncatula. Cis-acting elements in promoter region and expression analysis revealed the potential response of MtCML protein to abiotic stress and hormones. The results provide a basis of further functional research on the MtCML gene family and facilitate their potential use for applications in the genetic improvement on M. truncatula in drought, cold and salt stress environments.

Effects of carbon availability in a woody carbon source on its nitrate removal behavior in solid-phase denitrification.

Woody biomass is the most common natural carbon source applied in solid-phase denitrification (SPD). However, its denitrification ability is low in the SPD process due to its poor carbon availability. In this study, sawdust samples were pretreated to various degrees, and then filled into SPD bioreactors to reveal the relationship between carbon availability and denitrification behaviors. The behaviors include the denitrification process, internal effects of major factors (carbon availability, pH and temperature), and the presence of bacterial communities. Results shown that the long-term denitrification rate of pretreated sawdust was increased by 4.5-4.8 times over that of untreated sawdust (29.3 mg N L-1 sawdust d-1). However, despite improving the pretreatment degree of the sawdust in the bioreactor, the long-term denitrification rate shown no further increase. The denitrification rate was most influenced by the temperature, followed by the pH, and then the sawdust pretreatment degree. The denitrification rate increased with decreasing pH and rising temperature of the pretreated sawdust. The removed nitrate was rarely converted into nitrite or nitrous oxide, but ammonium was produced at high pH and temperature for the pretreated sawdust. The adverse effects of ammonium and dissolved organic carbon (DOC) reduced when the pH of the pretreated sawdust was lowered to 6.5. Hydrolytic and denitrifying bacteria formed the main SPD bioreactor bacteria, whose abundances increased with increasing sawdust pretreatment degree. The results were beneficial to reduce the hydrolytic retention time and adverse products for the SPD system using woody carbon source.