Genome-Wide Analysis of the Rab Gene Family in Melilotus albus Reveals Their Role in Salt Tolerance

Melilotus albus is a high-quality forage, due to its high protein content, and aboveground biomass and salt tolerance. Rab (Ras-related protein in the brain) proteins are the largest GTPase family which play a key role in intracellular membrane transport, and many Rab genes have been identified in eukaryotes. The growth and distribution of M. albus are severely hampered by soil salinization. However, little is known about candidate genes for salt tolerance in M. albus. In this study, 27 Rab family genes were identified for the first time from M. albus, and divided into eight groups (Groups A-H). The number of introns in MaRabs ranged from one to seven, with most genes containing one intron. In addition, most MaRab proteins showed similarities in motif composition. Phylogenetic analysis and structural-domain comparison indicated that Rab family genes were highly conserved in M. albus. Members of the MaRab gene family were distributed across all eight chromosomes, with the largest distribution on chromosome 1. Prediction of the protein interaction network showed that 24 Rab proteins exhibited protein–protein interactions. Analysis of the promoter cis-acting elements showed that MaRab-gene family members are extensively involved in abiotic stress responses. RNA-seq data analysis of the MaRab-gene-expression patterns suggested that the Rab gene family possesses differentially expressed members in five organs and under salt stress, drought stress, and ABA (Abscisic Acid) treatment. Differentially expressed genes under drought stress, salt stress and ABA stress were validated by quantitative real-time PCR. Furthermore, heterologous expression in yeast was used to characterize the functions of MaRab1 and MaRab17, which were upregulated in reaction to salt stress. In summary, this study provided valuable information for further research into the molecular mechanism of the response of M. albus to saline stress, as well as the possibility of developing cultivars with high salt-resistance characteristics.     

Sodium Accumulation in Infected Cells and Ion Transporters Mistargeting in Nodules of Medicago truncatula: Two Ugly Items That Hinder Coping with Salt Stress Effects

The maintenance of intracellular nitrogen-fixing bacteria causes changes in proteins’ location and in gene expression that may be detrimental to the host cell fitness. We hypothesized that the nodule’s high vulnerability toward salt stress might be due to alterations in mechanisms involved in the exclusion of Na⁺ from the host cytoplasm. Confocal and electron microscopy immunolocalization analyses of Na⁺/K⁺ exchangers in the root nodule showed the plasma membrane (MtNHX7) and endosome/tonoplast (MtNHX6) signal in non-infected cells; however, in mature infected cells the proteins were depleted from their target membranes and expelled to vacuoles. This mistargeting suggests partial loss of the exchanger’s functionality in these cells. In the mature part of the nodule 7 of the 20 genes encoding ion transporters, channels, and Na⁺/K⁺ exchangers were either not expressed or substantially downregulated. In nodules from plants subjected to salt treatments, low temperature-scanning electron microscopy and X-ray microanalysis revealed the accumulation of 5–6 times more Na⁺ per infected cell versus non-infected one. Hence, the infected cells’ inability to withstand the salt may be the integral result of preexisting defects in the localization of proteins involved in Na⁺ exclusion and the reduced expression of key genes of ion homeostasis, resulting in premature senescence and termination of symbiosis.     

Genome-Wide Analysis of the Soybean Calmodulin-Binding Protein 60 Family and Identification of GmCBP60A-1 Responses to Drought and Salt Stresses

Calmodulin-binding protein 60 (CBP60) members constitute a plant-specific protein family that plays an important role in plant growth and development. In the soybean genome, nineteen CBP60 members were identified and analyzed for their corresponding sequences and structures to explore their functions. Among GmCBP60A-1, which primarily locates in the cytomembrane, was significantly induced by drought and salt stresses. The overexpression of GmCBP60A-1 enhanced drought and salt tolerance in Arabidopsis, which showed better state in the germination of seeds and the root growth of seedlings. In the soybean hairy roots experiment, the overexpression of GmCBP60A-1 increased proline content, lowered water loss rate and malondialdehyde (MDA) content, all of which likely enhanced the drought and salt tolerance of soybean seedlings. Under stress conditions, drought and salt response-related genes showed significant differences in expression in hairy root soybean plants of GmCBP60A-1-overexpressing and hairy root soybean plants of RNAi. The present study identified GmCBP60A-1 as an important gene in response to salt and drought stresses based on the functional analysis of this gene and its potential underlying mechanisms in soybean stress-tolerance.     

A Genome-Wide Expression Profile of Salt-Responsive Genes in the Apple Rootstock Malus zumi

In some areas of cultivation, a lack of salt tolerance severely affects plant productivity. Apple, Malus x domestica Borkh., is sensitive to salt, and, as a perennial woody plant the mechanism of salt stress adaption will be different from that of annual herbal model plants, such as Arabidopsis. Malus zumi is a salt tolerant apple rootstock, which survives high salinity (up to 0.6% NaCl). To examine the mechanism underlying this tolerance, a genome-wide expression analysis was performed, using a cDNA library constructed from salt-treated seedlings of Malus zumi. A total of 15,000 cDNA clones were selected for microarray analysis. In total a group of 576 cDNAs, of which expression changed more than four-fold, were sequenced and 18 genes were selected to verify their expression pattern under salt stress by semi-quantitative RT-PCR. Our genome-wide expression analysis resulted in the isolation of 50 novel Malus genes and the elucidation of a new apple-specific mechanism of salt tolerance, including the stabilization of photosynthesis under stress, involvement of phenolic compounds, and sorbitol in ROS scavenging and osmoprotection. The promoter regions of 111 genes were analyzed by PlantCARE, suggesting an intensive cross-talking of abiotic stress in Malus zumi. An interaction network of salt responsive genes was constructed and molecular regulatory pathways of apple were deduced. Our research will contribute to gene function analysis and further the understanding of salt-tolerance mechanisms in fruit trees.     

Overexpression of MsRCI2D and MsRCI2E Enhances Salt Tolerance in Alfalfa (Medicago sativa L.) by Stabilizing Antioxidant Activity and Regulating Ion Homeostasis

Rare cold-inducible 2 (RCI2) genes from alfalfa (Medicago sativa L.) are part of a multigene family whose members respond to a variety of abiotic stresses by regulating ion homeostasis and stabilizing membranes. In this study, salt, alkali, and ABA treatments were used to induce MsRCI2D and MsRCI2E expression in alfalfa, but the response time and the expression intensity of the MsRCI2D,-E genes were different under specific treatments. The expression intensity of the MsRCI2D gene was the highest in salt- and alkali-stressed leaves, while the MsRCI2E gene more rapidly responded to salt and ABA treatment. In addition to differences in gene expression, MsRCI2D and MsRCI2E differ in their subcellular localization. Akin to MtRCI2D from Medicago truncatula, MsRCI2D is also localized in the cell membrane, while MsRCI2E is different from MtRCI2E, localized in the cell membrane and the inner membrane. This difference might be related to an extra 20 amino acids in the C-terminal tail of MsRCI2E. We investigated the function of MsRCI2D and MsRCI2E proteins in alfalfa by generating transgenic alfalfa chimeras. Compared with the MsRCI2E-overexpressing chimera, under high-salinity stress (200 mmol·L⁻¹ NaCl), the MsRCI2D-overexpressing chimera exhibited a better phenotype, manifested as a higher chlorophyll content and a lower MDA content. After salt treatment, the enzyme activities of SOD, POD, CAT, and GR in MsRCI2D- and -E-overexpressing roots were significantly higher than those in the control. In addition, after salt stress, the Na⁺ content in MsRCI2D- and -E-transformed roots was lower than that in the control; K⁺ was higher than that in the control; and the Na⁺/K⁺ ratio was lower than that in the control. Correspondingly, H⁺-ATPase, SOS1, and NHX1 genes were significantly up-regulated, and the HKT gene was significantly down-regulated after 6 h of salt treatment. MsRCI2D was also found to regulate the expression of the MsRCI2B and MsRCI2E genes, and the MsRCI2E gene could alter the expression of the MsRCI2A, MsRCI2B, and MsRCI2D genes. MsRCI2D- and -E-overexpressing alfalfa was found to have higher salt tolerance, manifested as improved activity of antioxidant enzymes, reduced content of reactive oxygen species, and sustained Na⁺ and K⁺ ion balance by regulating the expression of the H⁺-ATPase, SOS1, NHX1, HKT, and MsRCI2 genes.     

Comparative Proteomic Analysis of Tolerant and Sensitive Varieties Reveals That Phenylpropanoid Biosynthesis Contributes to Salt Tolerance in Mulberry

Mulberry, an important woody tree, has strong tolerance to environmental stresses, including salinity, drought, and heavy metal stress. However, the current research on mulberry resistance focuses mainly on the selection of resistant resources and the determination of physiological indicators. In order to clarify the molecular mechanism of salt tolerance in mulberry, the physiological changes and proteomic profiles were comprehensively analyzed in salt-tolerant (Jisang3) and salt-sensitive (Guisangyou12) mulberry varieties. After salt treatment, the malondialdehyde (MDA) content and proline content were significantly increased compared to control, and the MDA and proline content in G12 was significantly lower than in Jisang3 under salt stress. The calcium content was significantly reduced in the salt-sensitive mulberry varieties Guisangyou12 (G12), while sodium content was significantly increased in both mulberry varieties. Although the Jisang3 is salt-tolerant, salt stress caused more reductions of photosynthetic rate in Jisang3 than Guisangyou12. Using tandem mass tags (TMT)-based proteomics, the changes of mulberry proteome levels were analyzed in salt-tolerant and salt-sensitive mulberry varieties under salt stress. Combined with GO and KEGG databases, the differentially expressed proteins were significantly enriched in the GO terms of amino acid transport and metabolism and posttranslational modification, protein turnover up-classified in Guisangyou12 while down-classified in Jisang3. Through the comparison of proteomic level, we identified the phenylpropanoid biosynthesis may play an important role in salt tolerance of mulberry. We clarified the molecular mechanism of mulberry salt tolerance, which is of great significance for the selection of excellent candidate genes for saline-alkali soil management and mulberry stress resistance genetic engineering.     

Characterization in Effective Stimulation on the Magnitude,Gating, Frequency Dependence,and Hysteresis of INa Exerted by Picaridin (or Icaridin), a Known Insect Repellent

Picaridin (icaridin), a member of the piperidine chemical family, is a broad-spectrum arthropod repellent. Its actions have been largely thought to be due to its interaction with odorant receptor proteins. However, to our knowledge, to what extent the presence of picaridin can modify the magnitude, gating, and/or the strength of voltage-dependent hysteresis (Hys_(V)) of plasmalemmal ionic currents, such as, voltage-gated Na⁺ current [I_Na], has not been entirely explored. In GH₃ pituitary tumor cells, we demonstrated that with exposure to picaridin the transient (I_Na(T)) and late (I_Na(L)) components of voltage-gated Na⁺ current (I_Na) were differentially stimulated with effective EC₅₀’s of 32.7 and 2.8 μM, respectively. Upon cell exposure to it, the steady-state current versus voltage relationship I_Na(T) was shifted to more hyperpolarized potentials. Moreover, its presence caused a rightward shift in the midpoint for the steady-state inactivate curve of the current. The cumulative inhibition of I_Na(T) induced during repetitive stimuli became retarded during its exposure. The recovery time course from the I_Na block elicited, following the conditioning pulse stimulation, was satisfactorily fitted by two exponential processes. Moreover, the fast and slow time constants of recovery from the I_Na block by the same conditioning protocol were noticeably increased in the presence of picaridin. However, the fraction in fast or slow component of recovery time course was, respectively, increased or decreased with an increase in picaridin concentrations. The Hys_(V)’s strength of persistent I_Na (I_Na(P)), responding to triangular ramp voltage, was also enhanced during cell exposure to picaridin. The magnitude of resurgent I_Na (I_Na(R)) was raised in its presence. Picaritin-induced increases of I_Na(P) or I_Na(R) intrinsically in GH₃ cells could be attenuated by further addition of ranolazine. The predictions of molecular docking also disclosed that there are possible interactions of the picaridin molecule with the hNa_V1.7 channel. Taken literally, the stimulation of I_Na exerted by the exposure to picaridin is expected to exert impacts on the functional activities residing in electrically excitable cells.     

The Rice Cation/H+ Exchanger Family Involved in Cd Tolerance and Transport

Cadmium (Cd), a heavy metal toxic to humans, easily accumulates in rice grains. Rice with unacceptable Cd content has become a serious food safety problem in many rice production regions due to contaminations by industrialization and inappropriate waste management. The development of rice varieties with low grain Cd content is seen as an economic and long-term solution of this problem. The cation/H⁺ exchanger (CAX) family has been shown to play important roles in Cd uptake, transport and accumulation in plants. Here, we report the characterization of the rice CAX family. The six rice CAX genes all have homologous genes in Arabidopsis thaliana. Phylogenetic analysis identified two subfamilies with three rice and three Arabidopsis thaliana genes in both of them. All rice CAX genes have trans-member structures. OsCAX1a and OsCAX1c were localized in the vacuolar while OsCAX4 were localized in the plasma membrane in rice cell. The consequences of qRT-PCR analysis showed that all the six genes strongly expressed in the leaves under the different Cd treatments. Their expression in roots increased in a Cd dose-dependent manner. GUS staining assay showed that all the six rice CAX genes strongly expressed in roots, whereas OsCAX1c and OsCAX4 also strongly expressed in rice leaves. The yeast (Saccharomyces cerevisiae) cells expressing OsCAX1a, OsCAX1c and OsCAX4 grew better than those expressing the vector control on SD-Gal medium containing CdCl₂. OsCAX1a and OsCAX1c enhanced while OsCAX4 reduced Cd accumulation in yeast. No auto-inhibition was found for all the rice CAX genes. Therefore, OsCAX1a, OsCAX1c and OsCAX4 are likely to involve in Cd uptake and translocation in rice, which need to be further validated.     

Epigenomic Features and Potential Functions of K+ and Na+ Favorable DNA G-Quadruplexes in Rice

DNA G-quadruplexes (G4s) are non-canonical four-stranded DNA structures involved in various biological processes in eukaryotes. Molecularly crowded solutions and monovalent cations have been reported to stabilize in vitro and in vivo G4 formation. However, how K⁺ and Na⁺ affect G4 formation genome-wide is still unclear in plants. Here, we conducted BG4-DNA-IP-seq, DNA immunoprecipitation with anti-BG4 antibody coupled with sequencing, under K⁺ and Na⁺ + PEG conditions in vitro. We found that K⁺-specific IP-G4s had a longer peak size, more GC and PQS content, and distinct AT and GC skews compared to Na⁺-specific IP-G4s. Moreover, K⁺- and Na⁺-specific IP-G4s exhibited differential subgenomic enrichment and distinct putative functional motifs for the binding of certain trans-factors. More importantly, we found that K⁺-specific IP-G4s were more associated with active marks, such as active histone marks, and low DNA methylation levels, as compared to Na⁺-specific IP-G4s; thus, K⁺-specific IP-G4s in combination with active chromatin features facilitate the expression of overlapping genes. In addition, K⁺- and Na⁺-specific IP-G4 overlapping genes exhibited differential GO (gene ontology) terms, suggesting they may have distinct biological relevance in rice. Thus, our study, for the first time, explores the effects of K⁺ and Na⁺ on global G4 formation in vitro, thereby providing valuable resources for functional G4 studies in rice. It will provide certain G4 loci for the biotechnological engineering of rice in the future.     

Genome-Wide Identification and Expression Analysis of AMT and NRT Gene Family in Pecan (Carya illinoinensis) Seedlings Revealed a Preference for NH4+-N

Nitrogen (N) is a major limiting factor for plant growth and crop production. The use of N fertilizer in forestry production is increasing each year, but the loss is substantial. Mastering the regulatory mechanisms of N uptake and transport is a key way to improve plant nitrogen use efficiency (NUE). However, this has rarely been studied in pecans. In this study, 10 AMT and 69 NRT gene family members were identified and systematically analyzed from the whole pecan genome using a bioinformatics approach, and the expression patterns of AMT and NRT genes and the uptake characteristics of NH₄⁺ and NO₃⁻ in pecan were analyzed by aeroponic cultivation at varying NH₄⁺/NO₃⁻ ratios (0/0, 0/100,25/75, 50/50, 75/25,100/0 as CK, T1, T2, T3, T4, and T5). The results showed that gene duplication was the main reason for the amplification of the AMT and NRT gene families in pecan, both of which experienced purifying selection. Based on qRT-PCR results, CiAMTs were primarily expressed in roots, and CiNRTs were majorly expressed in leaves, which were consistent with the distribution of pecan NH₄⁺ and NO₃⁻ concentrations in the organs. The expression levels of CiAMTs and CiNRTs were mainly significantly upregulated under N deficiency and T4 treatment. Meanwhile, T4 treatment significantly increased the NH₄⁺, NO₃⁻, and NO₂⁻ concentrations as well as the Vmax and Km values of NH₄⁺ and NO₃⁻ in pecans, and Vmax/Km indicated that pecan seedlings preferred to absorb NH₄⁺. In summary, considering the single N source of T5, we suggested that the NH₄⁺/NO₃⁻ ratio of 75:25 was more beneficial to improve the NUE of pecan, thus increasing pecan yield, which provides a theoretical basis for promoting the scale development of pecan and provides a basis for further identification of the functions of AMT and NRT genes in the N uptake and transport process of pecan.     

HKT1;1 and HKT1;2 Na+ Transporters from Solanum galapagense Play Different Roles in the Plant Na+ Distribution under Salinity

Salt tolerance is a target trait in plant science and tomato breeding programs. Wild tomato accessions have been often explored for this purpose. Since shoot Na⁺/K⁺ is a key component of salt tolerance, RNAi-mediated knockdown isogenic lines obtained for Solanum galapagense alleles encoding both class I Na⁺ transporters HKT1;1 and HKT1;2 were used to investigate the silencing effects on the Na and K contents of the xylem sap, and source and sink organs of the scion, and their contribution to salt tolerance in all 16 rootstock/scion combinations of non-silenced and silenced lines, under two salinity treatments. The results show that SgHKT1;1 is operating differently from SgHKT1;2 regarding Na circulation in the tomato vascular system under salinity. A model was built to show that using silenced SgHKT1;1 line as rootstock would improve salt tolerance and fruit quality of varieties carrying the wild type SgHKT1;2 allele. Moreover, this increasing effect on both yield and fruit soluble solids content of silencing SgHKT1;1 could explain that a low expressing HKT1;1 variant was fixed in S. lycopersicum during domestication, and the paradox of increasing agronomic salt tolerance through silencing the HKT1;1 allele from S. galapagense, a salt adapted species.     

Genome-Wide Identification,Classification and Expression Analysis of m6A Gene Family in Solanum lycopersicum

Advanced knowledge of messenger RNA (mRNA) N⁶-methyladenosine (m⁶A) and DNA N⁶-methyldeoxyadenosine (6 mA) redefine our understanding of these epigenetic modifications. Both m⁶A and 6mA carry important information for gene regulation, and the corresponding catalytic enzymes sometimes belong to the same gene family and need to be distinguished. However, a comprehensive analysis of the m⁶A gene family in tomato remains obscure. Here, 24 putative m⁶A genes and their family genes in tomato were identified and renamed according to BLASTP and phylogenetic analysis. Chromosomal location, synteny, phylogenetic, and structural analyses were performed, unravelling distinct evolutionary relationships between the MT-A70, ALKBH, and YTH protein families, respectively. Most of the 24 genes had extensive tissue expression, and 9 genes could be clustered in a similar expression trend. Besides, SlYTH1 and SlYTH3A showed a different expression pattern in leaf and fruit development. Additionally, qPCR data revealed the expression variation under multiple abiotic stresses, and LC-MS/MS determination exhibited that the cold stress decreased the level of N⁶ 2′-O dimethyladenosine (m⁶Am). Notably, the orthologs of newly identified single-strand DNA (ssDNA) 6mA writer–eraser–reader also existed in the tomato genome. Our study provides comprehensive information on m⁶A components and their family proteins in tomato and will facilitate further functional analysis of the tomato N⁶-methyladenosine modification genes.     

Deficiency of Thyroid Hormone Reduces Voltage-Gated Na+ Currents as Well as Expression of Na+/K+-ATPase in the Mouse Hippocampus

Mice lacking functional thyroid follicular cells, Pax8^−/− mice, die early postnatally, making them suitable models for extreme hypothyroidism. We have previously obtained evidence in postnatal rat neurons, that a down-regulation of Na⁺-current density could explain the reduced excitability of the nervous system in hypothyroidism. If such a mechanism underlies the development of coma and death in severe hypothyroidism, Pax8^−/− mice should show deficits in the expression of Na⁺ currents and potentially also in the expression of Na⁺/K⁺-ATPases, which are necessary to maintain low intracellular Na⁺ levels. We thus compared Na⁺ current densities in postnatal mice using the patch-clamp technique in the whole-cell configuration as well as the expression of three alpha and two beta-subunits of the Na⁺/K⁺-ATPase in wild type versus Pax8^−/− mice. Whereas the Na⁺ current density in hippocampal neurons from wild type mice was upregulated within the first postnatal week, the Na⁺ current density remained at a very low level in hippocampal neurons from Pax8^−/− mice. Pax8^−/− mice also showed significantly decreased protein expression levels of the catalytic α1 and α3 subunits of the Na⁺/K⁺-ATPase as well as decreased levels of the β2 isoform, with no changes in the α2 and β1 subunits.     

A Comprehensive Analysis of the DUF4228 Gene Family in Gossypium Reveals the Role of GhDUF4228-67 in Salt Tolerance

Soil salinization conditions seriously restrict cotton yield and quality. Related studies have shown that the DUF4228 proteins are pivotal in plant resistance to abiotic stress. However, there has been no systematic identification and analysis of the DUF4228 gene family in cotton and their role in abiotic stress. In this study, a total of 308 DUF4228 genes were identified in four Gossypium species, which were divided into five subfamilies. Gene structure and protein motifs analysis showed that the GhDUF4228 proteins were conserved in each subfamily. In addition, whole genome duplication (WGD) events and allopolyploidization might play an essential role in the expansion of the DUF4228 genes. Besides, many stress-responsive (MYB, MYC) and hormone-responsive (ABA, MeJA) related cis-elements were detected in the promoters of the DUF4228 genes. The qRT-PCR results showed that GhDUF4228 genes might be involved in the response to abiotic stress. VIGS assays and the measurement of relative water content (RWC), Proline content, POD activity, and malondialdehyde (MDA) content indicated that GhDUF4228-67 might be a positive regulator of cotton response to salt stress. The results in this study systematically characterized the DUF4228s in Gossypium species and will provide helpful information to further research the role of DUF4228s in salt tolerance.