The ethylene response factor gene, ThDRE1A, is involved in abscisic acid- and ethylene-mediated cadmium accumulation in Tamarix hispida.

Tamarix hispida is highly tolerant to salt, drought and heavy metal stress and is a potential material for the remediation of cadmium (Cd)-contaminated soil under harsh conditions. In this study, T. hispida growth and chlorophyll content decreased, whereas flavonoid and carotenoid contents increased under long-term Cd stress (25 d). The aboveground components of T. hispida were collected for RNA-seq to investigate the mechanism of Cd accumulation. GO and KEGG enrichment analyses revealed that the differentially expressed genes (DEGs) were significantly enriched in plant hormone-related pathways. Exogenous hormone treatment and determination of Cd2+ levels showed that ethylene (ETH) and abscisic acid (ABA) antagonists regulate Cd accumulation in T. hispida. Twenty-five transcription factors were identified as upstream regulators of hormone-related pathways. ThDRE1A, which was previously identified as an important regulatory factor, was selected for further analysis. The results indicated that ThABAH2.5 and ThACCO3.1 were direct target genes of ThDRE1A. The determination of Cd2+, ABA, and ETH levels indicated that ThDRE1A plays an important role in Cd accumulation through the antagonistic regulation of ABA and ETH. In conclusion, these results reveal the molecular mechanism underlying Cd accumulation in plants and identify candidate genes for further research.

The transcription factor ThDOF8 binds to a novel cis-element and mediates molecular responses to salt stress in Tamarix hispida.

Salt stress is a common abiotic factor that restricts plant growth and development. As a halophyte, Tamarix hispida is a good model plant for exploring salt-tolerance genes and regulatory mechanisms. DNA-binding with one finger (DOF) is an important transcription factor (TF) that influences and controls various signaling substances involved in diverse biological processes related to plant growth and development, but the regulatory mechanisms of DOF TFs in response to salt stress are largely unknown in T. hispida. In the present study, a newly identified Dof gene, ThDOF8, was cloned from T. hispida, and its expression was found to be induced by salt stress. Transient overexpression of ThDOF8 enhanced T. hispida salt tolerance by enhancing proline levels, and increasing the activities of the antioxidant enzymes superoxide dismutase (SOD) and peroxidase (POD). These results were also verified in stably transformed Arabidopsis. Results from TF-centered yeast one-hybrid (Y1H) assays and EMSAs showed that ThDOF8 binds to a newly identified cis-element (TGCG). Expression profiling by gene chip analysis identified four potential direct targets of ThDOF8, namely the cysteine-rich receptor-like kinases genes, CRK10 and CRK26, and two glutamate decarboxylase genes, GAD41, and GAD42, and these were further verified by ChIP-quantitative-PCR, EMSAs, Y1H assays, and ß-glucuronidase enzyme activity assays. ThDOF8 can bind to the TGCG element in the promoter regions of its target genes, and transient overexpression of ThCRK10 also enhanced T. hispida salt tolerance. On the basis of our results, we propose a new regulatory mechanism model, in which ThDOF8 binds to the TGCG cis-element in the promoter of the target gene CRK10 to regulate its expression and improve salt tolerance in T. hispida. This study provides a basis for furthering our understanding the role of DOF TFs and identifying other downstream candidate genes that have the potential for improving plant salt tolerance via molecular breeding.

Insights into the phylogenetic relationships and species boundaries of the Myricaria squamosa complex (Tamaricaceae) based on the complete chloroplast genome.

Myricaria plants are widely distributed in Eurasia and are helpful for windbreak and embankment protection. Current molecular evidence has led to controversy regarding species boundaries within the Myricaria genus and interspecific phylogenetic relationships between three specific species-M. bracteata, M. paniculata and M. squamosa-which have remained unresolved. This study treated these three unresolved taxa as a species complex, named the M. squamosa complex. The genome skimming approach was used to determine 35 complete plastome sequences and nuclear ribosomal DNA sequences for the said complex and other closely related species, followed by de novo assembly. Comparative analyses were conducted across Myricaria to identify the genome size, gene content, repeat type and number, SSR (simple sequence repeat) abundance, and codon usage bias of chloroplast genomes. Tree-based species delimitation results indicated that M. bracteata, M. paniculata and M. squamosa could not be distinguished and formed two monophyletic lineages (P1 and P2) that were clustered together. Compared to plastome-based species delimitation, the standard nuclear DNA barcode had the lowest species resolution, and the standard chloroplast DNA barcode and group-specific barcodes delimitated a maximum of four out of the five species. Plastid phylogenomics analyses indicated that the monophyletic M. squamosa complex is comprised of two evolutionarily significant units: one in the western Tarim Basin and the other in the eastern Qinghai-Tibet Plateau. This finding contradicts previous species discrimination and promotes the urgent need for taxonomic revision of the threatened genus Myricaria. Dense sampling and plastid genomes will be essential in this effort. The super-barcodes and specific barcode candidates outlined in this study will aid in further studies of evolutionary history.

Role of bZIP Transcription Factors in Response to NaCl Stress in Tamarix ramosissima under Exogenous Potassium (K+).

Salt stress is a significant environmental factor affecting plant growth and development, with NaCl stress being one of the most common types of salt stress. The halophyte, Tamarix ramosissima Ledeb (T. ramosissima), is frequently utilized for the afforestation of saline-alkali soils. Indeed, there has been limited research and reports by experts and scholars on the regulatory mechanisms of basic leucine zipper (bZIP) genes in T. ramosissima when treated with exogenous potassium (K+) to alleviate the effects of NaCl stress. This study focused on the bZIP genes in T. ramosissima roots under NaCl stress with additional KCl applied. We identified key candidate genes and metabolic pathways related to bZIP and validated them through quantitative real-time PCR (qRT-PCR). The results revealed that under NaCl stress with additional KCl applied treatments at 0 h, 48 h, and 168 h, based on Pfam protein domain prediction and physicochemical property analysis, we identified 20 related bZIP genes. Notably, four bZIP genes (bZIP_2, bZIP_6, bZIP_16, and bZIP_18) were labeled with the plant hormone signal transduction pathway, showing a predominant up-regulation in expression levels. The results suggest that these genes may mediate multiple physiological pathways under NaCl stress with additional KCl applied at 48 h and 168 h, enhancing signal transduction, reducing the accumulation of ROS, and decreasing oxidative damage, thereby enhancing the tolerance of T. ramosissima to NaCl stress. This study provides gene resources and a theoretical basis for further breeding of salt-tolerant Tamarix species and the involvement of bZIP transcription factors in mitigating NaCl toxicity.

Transcriptome profiling reveals multiple regulatory pathways of Tamarix chinensis in response to salt stress.

KEY MESSAGE: Multiple regulatory pathways of T. chinensis to salt stress were identified through transcriptome data analysis. Tamarix chinensis (Tamarix chinensis Lour.) is a typical halophyte capable of completing its life cycle in soils with medium to high salinity. However, the mechanisms underlying its resistance to high salt stress are still largely unclear. In this study, transcriptome profiling analyses in different organs of T. chinensis plants in response to salt stress were carried out. A total number of 2280, 689, and 489 differentially expressed genes (DEGs) were, respectively, identified in roots, stems, and leaves, with more DEGs detected in roots than in stems and leaves. Gene Ontology (GO) term analysis revealed that they were significantly enriched in "biological processes" and "molecular functions". Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis revealed that "Beta-alanine metabolism" was the most differentially enriched pathway in roots, stems, and leaves. In pair-to-pair comparison of the most differentially enriched pathways, a total of 14 pathways, including 5 pathways in roots and leaves, 6 pathways in roots and stems, and 3 pathways in leaves and stems, were identified. Furthermore, genes encoding transcription factor, such as bHLH, bZIP, HD-Zip, MYB, NAC, WRKY, and genes associated with oxidative stress, starch and sucrose metabolism, and ion homeostasis, were differentially expressed with distinct organ specificity in roots, stems, and leaves. Our findings in this research provide a novel approach for exploring the salt tolerance mechanism of halophytes and identifying new gene targets for the genetic breeding of new plant cultivars with improved resistance to salt stress.

Population structure and genetic diversity of Tamarix chinensis as revealed with microsatellite markers in two estuarine flats.

Background: Tamarix chinensis Lour. is a 3-6-meter-tall small tree with high salt- and alkali- tolerance and aggressive invasiveness, mainly distributed in the eastern part of China in warm-temperate and subtropical climate zones, yet there is little information available regarding genetic diversity and population structure. Methods: A total of 204 individuals of nine T. chinensis populations were investigated for genetic diversity and population structure using a set of 12 highly polymorphic microsatellite markers. Results: The total number of alleles detected was 162, the average number of effective allele was 4.607, the average polymorphism information content (PIC) value of the 12 loci was 0.685, and the mean observed heterozygosity (Ho) and the mean expected heterozygosity (He) was 0.653 and 0.711, respectively. Analysis of molecular variance (AMOVA) showed a 5.32% genetic variation among T. chinensis populations. Despite a low population differentiation, Bayesian clustering analysis, discriminant analysis of principal components (DAPC) and the unweighted pair group method with arithmetic mean (UPGMA) clearly identified three genetic clusters correlated to the populations' geographic origin: the northern populations including those from Yellow River Delta, the Fangshan (FS) population from Beijing, the Changyi (CY) population from Bohai Bay, the Huanjiabu (HHJ) population from Hangzhou Bay, and the remaining two populations from Hangzhou Bay. There was a significant relationship between the genetic distance and geographical distance of the paired populations. Gene flow (Nm) was 4.254 estimated from FST. Conclusion: T. chinensis possessed high genetic diversity comparable to tree species, and although the population differentiation is shallow, our results classified the sampled populations according to sampling localities, suggesting the different origins of the study populations.

Overexpression of ThSCL32 confers salt stress tolerance by enhancing ThPHD3 gene expression in Tamarix hispida.

GRAS transcription factors belong to the plant-specific protein family. They are not only involved in plant growth and development but also in plant responses to a variety of abiotic stresses. However, to date, the SCL32(SCARECROW-like 32) gene conferring the desired resistance to salt stresses has not been reported in plants. Here, ThSCL32, a homologous gene of ArabidopsisthalianaAtSCL32, was identified. ThSCL32 was highly induced by salt stress in Tamarix hispida. ThSCL32 overexpression in T. hispida gave rise to improved salt tolerance. ThSCL32-silenced T. hispida plants were more sensitive to salt stress. RNA-seq analysis of transient transgenic T. hispida overexpressing ThSCL32 revealed significantly enhanced ThPHD3 (prolyl-4-hydroxylase domain 3 protein) gene expression. ChIP-PCR further verified that ThSCL32 probably binds to the novel cis-element SBS (ACGTTG) in the promoter of ThPHD3 to activate its expression. In brief, our results suggest that the ThSCL32 transcription factor is involved in salt tolerance in T. hispida by enhancing ThPHD3 expression.

Proteome Dynamics Analysis Reveals the Potential Mechanisms of Salinity and Drought Response during Seed Germination and Seedling Growth in Tamarix hispida.

Understanding the molecular mechanisms of seed germination and seedling growth is vital for mining functional genes for the improvement of plant drought in a desert. Tamarix hispida is extremely resistant to drought and soil salinity perennial shrubs or trees. This study was the first to investigate the protein abundance profile of the transition process during the processes of T. hispida seed germination and seedling growth using label-free proteomics approaches. Our data suggested that asynchronous regulation of transcriptomics and proteomics occurs upon short-term seed germination and seedling growth of T. hispida. Enrichment analysis revealed that the main differentially abundant proteins had significant enrichment in stimulus response, biosynthesis, and metabolism. Two delta-1-pyrroline-5-carboxylate synthetases (P5CS), one Ycf3-interacting protein (Y3IP), one low-temperature-induced 65 kDa protein-like molecule, and four peroxidases (PRX) were involved in both water deprivation and hyperosmotic salinity responses. Through a comparative analysis of transcriptomics and proteomics, we found that proteomics may be better at studying short-term developmental processes. Our results support the existence of several mechanisms that enhance tolerance to salinity and drought stress during seedling growth in T. hispida.

The R2R3-MYB transcription factor ThRAX2 recognized a new element MYB-T (CTTCCA) to enhance cadmium tolerance in Tamarix hispida.

R2R3-MYB transcription factors play an important role in plant development and response to various environmental stresses. In this study, a new R2R3-MYB gene, named ThRAX2, was isolated from T. hispida. ThRAX2 has an open reading frame (ORF) of 1191 bp and encodes a protein of 396 amino acids. ThRAX2 was localized in the nucleus. The overexpression of ThRAX2 in Arabidopsis and T. hispida significantly increased Cadmium (Cd) tolerance. Moreover, the accumulation of cadmium in roots and leaves was significantly reduced. The TF-centred Y1H and Y1H results showed that ThRAX2 was able to specifically bind a new cis-element (MYB-T, CTTCCA). The promoters of some Cd-responsive genes, such as ThSOS1, ThCKX3, ThCAX3A, ThMYB78, ThMIP2, ThTPS4, and ThSOD2, all contained 1-3 MYB-T sequences. Furthermore, chromatin immunoprecipitation-polymerase chain reaction (ChIP-PCR) and ChIPquantitative (q)PCR showed that the ThRAX2 gene can bind to ThSOS1, ThCKX3, ThCAX3A and ThMYB78 promoter fragments, including the MYB-T motif. Meanwhile, the qRTPCR results also showed that the expression trends of ThSOS1, ThCKX3, ThCAX3A and ThMYB78 were similar to that of ThRAX2. This finding suggests that Cd tolerance of the ThRAX2 gene may regulate the expression of some downstream genes through specific recognition of the MYB-T motif and participate in regulating intracellular ion homeostasis, transport, and protein activity or enhance antioxidant enzyme activity. This study found a novel cis-acting element that binds ThRAX2 to regulate Cd tolerance, which lays the foundation for the ThRAX2 regulatory mechanism of Cd stress. This study provides a genetic and theoretical basis for the bioremediation of Cd-contaminated land by cultivating transgenic plants in the future.

The multilayered hierarchical gene regulatory network reveals interaction of transcription factors in response to cadmium in Tamarix hispida roots.

Cadmium (Cd) is a toxic metal that affects the normal growth and development of plants. Roots may directly contact Cd and thus serve as the first barrier in the defense responses of plants. In this study, Tamarix hispida (T. hispida) roots treated with 150 µM CdCl2 were collected for RNA-seq. A total of 2004 differentially expressed genes (DEGs) were identified at different time points. Kyoto Encyclopedia of Genes and Genomes enrichment revealed that the DEGs were significantly enriched in phenylpropanoid biosynthesis, flavonoid biosynthesis and other metabolic pathways. To explore the regulatory role of transcription factors (TFs) involved in the Cd stress response, a multilayer hierarchical gene regulatory network (ML-hGRN) was constructed, including 53 TFs and 54 structural genes in ML-hGRN, with 341 predicted regulatory relationships. Binding of DRE1A, MYC1, FEZ, ERF4 and ERF17 to predicted target genes was detected by ChIP-PCR, and DRE1A, MYC1 and FEZ were transiently overexpressed in T. hispida. The results suggest that these TFs play a key role in the Cd stress response by scavenging reactive oxygen species. In conclusion, this study predicts some Cd-responsive TFs that may have an important function under Cd stress and provides useful information for molecular breeding.

ThASR3 confers salt and osmotic stress tolerances in transgenic Tamarix and Arabidopsis.

BACKGROUND: ASR (abscisic acid-, stress-, and ripening-induced) gene family plays a crucial role in responding to abiotic stresses in plants. However, the roles of ASR genes protecting plants against high salt and drought stresses remain unknown in Tamarix hispida. RESULTS: In this study, a salt and drought-induced ASR gene, ThASR3, was isolated from Tamarix hispida. Transgenic Arabidopsis overexpressing ThASR3 exhibited stimulating root growth and increasing fresh weight compared with wild-type (WT) plants under both salt and water deficit stresses. To further analyze the gain- and loss-of-function of ThASR3, the transgenic T. hispida plants overexpressing or RNA interference (RNAi)-silencing ThASR3 were generated using transient transformation. The overexpression of ThASR3 in Tamarix and Arabidopsis plants displayed enhanced reactive oxygen species (ROS) scavenging capability under high salt and osmotic stress conditions, including increasing the activities of antioxidant enzymes and the contents of proline and betaine, and reducing malondialdehyde (MDA) content and electrolyte leakage rates. CONCLUSION: Our results indicate that ThASR3 functions as a positive regulator in Tamarix responses to salt and osmotic stresses and confers multiple abiotic stress tolerances in transgenic plants, which may have an important application value in the genetic improvement of forest tree resistance.

The high pH value of alkaline salt destroys the root membrane permeability of Reaumuria trigyna and leads to its serious physiological decline.

Variable climatic conditions frequently have harmful effects on plants. Reaumuria trigyna, a salt-secreting xerophytic shrub, occurs in Inner Mongolia, which has a poor environment for plant growth. To explore the physiological and molecular mechanisms of R. trigyna in response to environmental stress, this study investigated the abiotic resistance of R. trigyna in terms of growth regulation, antioxidant defense, osmotic regulation, ion transport, and ion homeostasis-related genes. R. trigyna seedlings were treated with 400 mM NaCl, 400 mM neutral salts (NaCl:Na2SO4 = 9:1), 50 mM alkaline salts (NaHCO3:Na2CO3 = 9:1), 10% polyethylene glycol (PEG), and UV-B. Seedlings under 400 mM NaCl and 400 mM neutral salt stress showed less damage. While alkaline salt, PEG, and UV stress caused more damage, specifically in oxidative damage, proline levels, electrolyte leakage, and activation of antioxidant defenses. Furthermore, under the abiotic stress treatments, the accumulation of Na+ increased while the accumulation of K+ decreased. Further analysis showed that the flow rate of Na+ and K+ under alkaline salt stress was higher than under neutral salt stress. Neutral salt induced high expression of RtNHX1 and RtSOS1, while alkaline salt induced high expression of RtHKT1, and alkaline salt stress significantly reduced the activity of root cells. These results indicated that R. trigyna seedlings were more tolerant to neutral than alkaline salts; this might be because root activity decreased at high pH levels, which impaired membrane permeability and the ion transfer system, leading to an imbalance between Na+ and K+, and in turn to excessive accumulation of reactive oxygen species (ROS) and decreased plant stress resistance.

Effects of Exogenous (K+) Potassium Application on Plant Hormones in the Roots of Tamarix ramosissima under NaCl Stress.

Abiotic stresses such as salt stress seriously affect the growth and yield of plants. Tamarix ramosissima Lcdcb (T. ramosissima) is a widely cultivated halophyte in saline-alkali areas of the world. As an essential element for plant growth and development, K+ plays an irreplaceable role in improving the tolerance of plants to salt stress. However, there are few reports on the mechanism of K+ in promoting plant hormones to reduce the damage of NaCl stress to T. ramosissima. In this study, we sequenced the transcriptome of the roots of T. ramosissima which were treated with exogenous potassium (K+) for 0 h, 48 h and 168 h under NaCl stress, according to the changes in the expression levels of differentially expressed genes (DEGs) in T. ramosissima roots. Key candidate genes and metabolic pathways related to plant hormones were mined for analysis and further verified by quantitative real-time PCR (qRT-PCR). The results showed that under NaCl stress for 48 h and 168 h, there were a large number of DEGs in the roots of T. ramosissima, and the expression levels changed over time. In particular, we found that 56 plant hormone-related genes were annotated to the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway, and with the increase of time, their expression levels were mainly up-regulated and involved in the related metabolic pathways to resist NaCl stress. It is worth noting that 7 DEGs related to abscisic acid (ABA), 28 DEGs related to auxin, 1 DEG related to ethylene (ET), and 1 DEG related to cytokinin (CK) were added within 168 h of exogenous potassium, and they were involved in alleviating the root damage of T. ramosissima under NaCl stress and played an important role. In addition, we found the plant hormone signal transduction pathway, which plays an important role in resistance to NaCl stress. As a result of this study, the molecular mechanism of plant hormones involved in applying exogenous potassium under NaCl stress is further understood, resulting in a better understanding of how exogenous potassium can alleviate the damage caused by NaCl under stress in T. ramosissima.

Effects of Exogenous Potassium (K+) Application on the Antioxidant Enzymes Activities in Leaves of Tamarix ramosissima under NaCl Stress.

Saline soil is a worldwide distributed resource that seriously harms plants' growth and development. NaCl is the most widely distributed salt in saline soil. As a typical representative of halophytes, Tamarix ramosissima Lcdcb (T. ramosissima) is commonly grown in salinized soil, and halophytes have different abilities to retain more K+ under salt stress conditions. Halophytes can adapt to different salt environments by improving the scavenging activity of reactive oxygen species (ROS) by absorbing and transporting potassium (K+). In this study, electron microscope observation, hydrogen peroxide (H2O2) and malondialdehyde (MDA) contents determination, primary antioxidant enzyme activity determination and transcriptome sequencing analysis were carried out on the leaves of T. ramosissima under NaCl stress at 0 h, 48 h and 168 h. The results showed that H2O2 and MDA contents increased in the 200 mM NaCl + 10 mM KCl and 200 mM NaCl groups, but the content increased the most in the 200 mM NaCl group at 168 h. In addition, the leaves of T. ramosissima in the 200 mM NaCl + 10 mM KCl group had the most salt secretion, and its superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT) activities were all higher than those of the 200 mM NaCl group and significantly higher than those of the control group. According to the results of transcriptome sequencing, it was found that the expression of 39 genes related to antioxidant enzyme activity changed significantly at the transcriptional level. Among them, 15 genes related to antioxidant enzyme activities were upregulated, and 24 genes related to antioxidant enzyme activities were downregulated in the leaves of T. ramosissima when exogenous potassium (K+) was applied under NaCl stress for 48 h; when exogenous potassium (K+) was used for 168 h under NaCl stress, 21 antioxidant enzyme activity-related genes were upregulated, and 18 antioxidant enzyme activity-related genes were downregulated in T. ramosissima leaves. Based on the changes of expression levels at different treatment times, 10 key candidates differentially expressed genes (DEGs) (Unigene0050462, Unigene0014843, Unigene0046159, Unigene0046160, Unigene0008032, Unigene0048033, Unigene0004890, Unigene0015109, Unigene0020552 and Unigene0048538) for antioxidant enzyme activities were further screened. They played an important role in applying exogenous potassium (K+) for 48 h and 168 h to the leaves of T. ramosissima in response to NaCl stress. Their expression levels were dominated by upregulation, which enhanced the activity of antioxidant enzymes, and helped T. ramosissima mitigate NaCl poison and resist NaCl stress. Particularly, Unigene0048538 in glutathione S-transferase (GST) activity had the largest log2 fold-change in the comparison groups of 200 mM NaCl-48 h vs. 200 mM NaCl + 10 mM KCl-48 h and 200 mM NaCl-168 h vs. 200 mM NaCl + 10 mM KCl-168 h. Its expression level was upregulated and played an important role in NaCl toxicity. At the same time, the results of the phylogenetic tree analysis showed that Unigene0048538 had the closest genetic distance to Prunus persica in the evolutionary relationship. In summary, with the increase of exogenous potassium (K+) application time under NaCl stress, T. ramosissima can resist high NaCl stress by enhancing antioxidant enzymes' activity and maintaining the growth of T. ramosissima. Still, it is not enough to completely eliminate NaCl poison. This study provides a theoretical basis for the molecular mechanism of salt tolerance and K+ mitigation of NaCl poison by the representative halophyte T. ramosissima in response to NaCl stress.

Transcriptome and Metabonomic Analysis of Tamarix ramosissima Potassium (K+) Channels and Transporters in Response to NaCl Stress.

Potassium ion (K+) channels and transporters are key components of plant K+ absorption and transportation and play an important role in plant growth and development. This study revealed that K+ channels and transporters are involved in the salt tolerance molecular mechanism and metabolites of the halophyte representative plant Tamarix ramosissima (T. ramosissima) in response to NaCl stress, providing a theoretical basis for the mitigation of salt stress using halophytes. Through transcriptome sequencing and metabolite detection analysis of 0 h, 48 h and 168 h by applying exogenous K+ to the roots of T. ramosissima under NaCl stress, 15 high-quality Clean Data bases were obtained, Q20 reached more than 97%, Q30 reached more than 92%, and GC content reached 44.5%, which is in line with further bioinformatics analysis. Based on the Liquid chromatography−mass spectrometry (LC-MS) analysis, the roots of T. ramosissima were exposed to exogenous potassium for 48 h and 168 h under NaCl stress, and 1510 and 1124 metabolites were identified in positive and negative ion mode, respectively. Through orthogonal projections to latent structures discriminant analysis (OPLS-DA) model analysis, its metabolomic data have excellent predictability and stability. The results of this study showed that there were 37 differentially expressed genes (DEGs) annotated as Class 2 K+ channels (Shaker-like K+ channel and TPK channel) and Class 3 K+ transporters (HAK/KUP/KT, HKT and CPAs transporter families). Among them, 29 DEGs were annotated to the gene ontology (GO) database, and the most genes were involved in the GO Biological Process. In addition, the expression levels of Unigene0014342 in the HAK/KUP/KT transporter and Unigene0088276 and Unigene0103067 in the CPAs transporter both first decreased and then increased when treated with 200 mM NaCl for 48 h and 168 h. However, when treated with 200 mM NaCl + 10 mM KCl for 48 h and 168 h, a continuous upward trend was shown. Notably, the expression level of Unigene0016813 in CPAS transporter continued to increase when treated with 200 mM NaCl and 200 mM NaCl + 10 mM KCl for 48 h and 168 h. 3 DEGs, Unigene0088276, Unigene0016813 and Unigene0103067, were dominated by the positive regulation of their related metabolites, and this correlation was significant. The results showed that these DEGs increased the absorption of K+ and the ratio of K+/Na+ under NaCl stress at 48 h and 168 h after adding exogenous potassium and enhanced the salt tolerance of T. ramosissima. Notably, the expression level of Unigene0103067 in the CPAs transporter was consistently upregulated when 200 mM NaCl + 10 mM KCl was treated for 48 h and 168 h. The positive regulatory metabolites were always dominant, which better helped T. ramosissima resist salt stress. Unigene0103067 plays an important role in enhancing the salt tolerance of T. ramosissima and reducing the toxicity of NaCl in roots. Additionally, phylogenetic tree analysis showed that Unigene0103067 and Reaumuria trigyna had the closest genetic distance in the evolutionary relationship. Finally, 9 DEGs were randomly selected for quantitative real-time PCR (qRT-PCR) verification. Their expression trends were completely consistent with the transcriptome sequencing analysis results, proving that this study's data are accurate and reliable. This study provides resources for revealing the molecular mechanism of NaCl stress tolerance in T. ramosissima and lays a theoretical foundation for cultivating new salt-tolerant varieties.

Gene flow in a pioneer plant metapopulation (Myricaria germanica) at the catchment scale in a fragmented alpine river system.

River alterations for natural hazard mitigation and land reclamation result in habitat decline and fragmentation for riparian plant species. Extreme events such as floods are responsible for additional local species loss or population decline. Tributaries might provide refugia and subsequent source populations for the colonization of downstream sites in connected riverine networks with metapopulations of plant species. In this study, we analyzed the metapopulation structure of the endangered riparian shrub species Myricaria germanica along the river Isel, Austria, which is part of the Natura 2000 network, and its tributaries. The use of 22 microsatellite markers allowed us to assess the role of tributaries and single populations as well as gene flow up- and downstream. The analysis of 1307 individuals from 45 sites shows the influence of tributaries to the genetic diversity at Isel and no overall isolation by distance pattern. Ongoing bidirectional gene flow is revealed by the detection of first-generation migrants in populations of all tributaries as well as the river Isel, supporting upstream dispersal by wind (seeds) or animals (seeds and pollen). However, some populations display significant population declines and high inbreeding, and recent migration rates are non-significant or low. The genetic pattern at the mouth of river Schwarzach into Isel and shortly thereafter river Kalserbach supports the finding that geographically close populations remain connected and that tributaries can form important refugia for M. germanica in the dynamic riverine network. Conservation and mitigation measures should therefore focus on providing sufficient habitat along tributaries of various size allowing pioneer plants to cope with extreme events in the main channel, especially as they are expected to be more frequent under changing climate.

Transcriptome analysis of Tamarix ramosissima leaves in response to NaCl stress.

Halophyte Tamarix ramosissima. Lcdcb (T. ramosissima) are known as the representative of Tamarix plants that are widely planted in salinized soil. However, molecular mechanisms towards salt tolerance and adaptation are largely rare. In this study, we carried out RNA-sequence and transcriptome analysis of T. ramosissima in response to NaCl stress, screened differentially expressed genes (DEGs) and further verified by qRT-PCR. Results showed that 105702 unigenes were spliced from the raw data of transcriptome sequencing, where 54238 unigenes were retrieved from KEGG, KOG, NR, and SwissProt. After 48 hours of NaCl treatment, the expression levels of 6374 genes were increased, and 5380 genes were decreased in leaves. After 168 hours, the expression levels of 3837 genes were up-regulated and 7808 genes were down-regulated. In particular, 8 transcription factors annotated to the KEGG Pathway were obtained, involving the WRKY and bZIP transcription family. In addition, KEGG pathway annotation showed that expression of 39 genes involved in ROS scavenging mechanisms were significantly changed, in which 21 genes were up-regulated and 18 genes were down-regulated after 48 hours as well as 15 genes were up-regulated and 24 genes were down-regulated after 168h. Simultaneously, the enzyme activities of SOD and POD were significantly enhanced under NaCl treatment, but the enzyme activity of CAT was not significantly enhanced. Moreover, WRKY, MYB and bZIP may participate in the process of salt resistance in T. ramosissima. This study provides gene resources and a theoretical basis for further molecular mechanisms of salt tolerance in T. ramosissima.

Combined transcriptomic and metabolomic analysis reveals the potential mechanism of seed germination and young seedling growth in Tamarix hispida.

BACKGROUND: Seed germination is a series of ordered physiological and morphogenetic processes and a critical stage in plant life cycle. Tamarix hispida is one of the most salt-tolerant plant species; however, its seed germination has not been analysed using combined transcriptomics and metabolomics. RESULTS: Transcriptomic sequencing and widely targeted metabolomics were used to detect the transcriptional metabolic profiles of T. hispida at different stages of seed germination and young seedling growth. Transcriptomics showed that 46,538 genes were significantly altered throughout the studied development period. Enrichment study revealed that plant hormones, such as auxin, ABA, JA and SA played differential roles at varying stages of seed germination and post-germination. Metabolomics detected 1022 metabolites, with flavonoids accounting for the highest proportion of differential metabolites. Combined analysis indicated that flavonoid biosynthesis in young seedling growth, such as rhoifolin and quercetin, may improve the plant's adaptative ability to extreme desert environments. CONCLUSIONS: The differential regulation of plant hormones and the accumulation of flavonoids may be important for the seed germination survival of T. hispida in response to salt or arid deserts. This study enhanced the understanding of the overall mechanism in seed germination and post-germination. The results provide guidance for the ecological value and young seedling growth of T. hispida.

Genomic analyses provide insights into the evolution and salinity adaptation of halophyte Tamarix chinensis.

BACKGROUND: The woody halophyte Tamarix chinensis is a pioneer tree species in the coastal wetland ecosystem of northern China, exhibiting high resistance to salt stress. However, the genetic information underlying salt tolerance in T. chinensis remains to be seen. Here we present a genomic investigation of T. chinensis to elucidate the underlying mechanism of its high resistance to salinity. RESULTS: Using a combination of PacBio and high-throughput chromosome conformation capture data, a chromosome-level T. chinensis genome was assembled with a size of 1.32 Gb and scaffold N50 of 110.03 Mb. Genome evolution analyses revealed that T. chinensis significantly expanded families of HAT and LIMYB genes. Whole-genome and tandem duplications contributed to the expansion of genes associated with the salinity adaptation of T. chinensis. Transcriptome analyses were performed on root and shoot tissues during salt stress and recovery, and several hub genes responding to salt stress were identified. WRKY33/40, MPK3/4, and XBAT31 were critical in responding to salt stress during early exposure, while WRKY40, ZAT10, AHK4, IRX9, and CESA4/8 were involved in responding to salt stress during late stress and recovery. In addition, PER7/27/57/73 encoding class III peroxidase and MCM3/4/5/7 encoding DNA replication licensing factor maintained up/downregulation during salt stress and recovery stages. CONCLUSIONS: The results presented here reveal the genetic mechanisms underlying salt adaptation in T. chinensis, thus providing important genomic resources for evolutionary studies on tamarisk and plant salt tolerance genetic improvement.

ThHSFA1 Confers Salt Stress Tolerance through Modulation of Reactive Oxygen Species Scavenging by Directly Regulating ThWRKY4.

Heat shock transcription factors (HSFs) play critical roles in several types of environmental stresses. However, the detailed regulatory mechanisms in response to salt stress are still largely unknown. In this study, we examined the salt-induced transcriptional responses of ThHSFA1-ThWRKY4 in Tamarix hispida and their functions and regulatory mechanisms in salt tolerance. ThHSFA1 protein acts as an upstream regulator that can directly activate ThWRKY4 expression by binding to the heat shock element (HSE) of the ThWRKY4 promoter using yeast one-hybrid (Y1H), chromatin immunoprecipitation (ChIP), and dual-luciferase reporter assays. ThHSFA1 and ThWRKY4 expression was significantly induced by salt stress and abscisic acid (ABA) treatment in the roots and leaves of T. hispida. ThHSFA1 is a nuclear-localized protein with transactivation activity at the C-terminus. Compared to nontransgenic plants, transgenic plants overexpressing ThHSFA1 displayed enhanced salt tolerance and exhibited reduced reactive oxygen species (ROS) levels and increased antioxidant enzyme activity levels under salt stress. Therefore, we further concluded that ThHSFA1 mediated the regulation of ThWRKY4 in response to salt stress in T. hispida.