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ATP-binding cassette transporter G (ABCG) has been shown to be engaged in export of broad-spectrum compounds with structural differences, but little is known concerning its role in cutin formation of cotton (Gossypium spp.). In this study, we conduct a genome-wide survey and detected 69, 71, 124 and 131 ABCG genes within G. arboretum, G. raimondii, G. hirsutum and G. barbadense, separately. The above ABCGs could be divided into four groups (Ia, Ib, Ic, II). Some ABCG genes such as GhABCG15, whose homologous gene transports cuticular lipid in Arabidopsis, was preferentially expressed in the development of fiber. A weighted gene co-expression network analysis (WGCNA) demonstrated that GhABCG expression was significantly associated with the amount of 16-Hydroxypalmitate (a main component of cutin precursor) in cotton fibers. Further, silencing of GhABCG15 by virus-induced gene silencing (VIGS) in cotton generated brightened and crinkled leaves as well as reduced thickness of cuticle and increased permeability. Chemical composition analysis showed the cutin content in GhABCG15-silenced leaves had decreased while the wax content had increased. Our results provide an insight for better understanding of the role of the Gossypium ABCG family and revealed the essential role of GhABCGs in cotton cutin formation.

Seed size is a key factor affecting crop yield and a major agronomic trait concerned in peanut (Arachis hypogaea L.) breeding. However, little is known about the regulation mechanism of peanut seed size. In the present study, a peanut small seed mutant1 (ssm1) was identified through irradiating peanut cultivar Luhua11 (LH11) using 60Coγ ray. Since the globular embryo stage, the embryo size of ssm1 was significantly smaller than that of LH11. The dry seed weight of ssm1 was only 39.69% of the wild type LH14. The seeds were wrinkled with darker seed coat. The oil content of ssm1 seeds were also decreased significantly. Seeds of ssm1 and LH11 were sampled 10, 20, and 40 days after pegging (DAP) and were used for RNA-seq. The results revealed that genes involved in plant hormones and several transcription factors related to seed development were differentially expressed at all three stages, especially at DAP10 and DAP20. Genes of fatty acid biosynthesis and late embryogenesis abundant protein were significantly decreased to compare with LH11. Interestingly, the gene profiling data suggested that PKp2 and/or LEC1 could be the key candidate genes leading to the small seed phenotype of the mutant. Our results provide valuable clues for further understanding the mechanisms underlying seed size control in peanut.

Salt cress (

BackgroundPlant height, mainly decided by main stem height, is the major agronomic trait and closely correlated to crop yield. A number of studies had been conducted on model plants and crops to understand the molecular and genetic basis of plant height. However, little is known on the molecular mechanisms of peanut main stem height.ResultsIn this study, a semi-dwarf peanut mutant was identified from60Co γ-ray induced mutant population and designated as semi-dwarf mutant 2 (sdm2). The height ofsdm2was only 59.3% of its wild line Fenghua 1 (FH1) at the mature stage. Thesdm2has less internode number and short internode length to compare with FH1. Gene expression profiles of stem and leaf from bothsdm2and FH1 were analyzed using high throughput RNA sequencing. The differentially expressed genes (DEGs) were involved in hormone biosynthesis and signaling pathways, cell wall synthetic and metabolic pathways. BR, GA and IAA biosynthesis and signal transduction pathways were significantly enriched. The expression of several genes in BR biosynthesis and signaling were found to be significantly down-regulated insdm2as compared to FH1. Many transcription factors encoding genes were identified as DEGs.ConclusionsA large number of genes were found differentially expressed betweensdm2and FH1. These results provide useful information for uncovering the molecular mechanism regulating peanut stem height. It could facilitate identification of causal genes for breeding peanut varieties with semi-dwarf phenotype.

Background Grafting, an ancient agronomic technique, is an artificial mode of asexual reproduction in plants. Recently, grafting research has gradually shifted from modifying agronomic traits to the study of molecular mechanism. Grafting is an excellent tool to study long-range signaling processes in plants. And the grafting between species will help elucidate the molecular mechanisms underlying contrasting differences between different species. Arabidopsis thaliana is a salt-sensitive model glycophyte and Eutrema salsugineum (previously Thellungiella salsuginea, salt cress) is a salt-tolerant model halophyte. Successful grafting of these two model plants will help further study the physiological and molecular mechanisms underlying salt tolerance in plants. The aim of this study was to demonstrate two sterile micro-grafting methods for Arabidopsis and salt cress. Results We developed the methods for sterile grafting between A. thaliana and E. salsugineum; this is the first report on inter-generic grafting between Arabidopsis and Eutrema. The method involves cut-in grafting under sterile conditions. The grafted plant part was placed in half strength Murashige and Skoog medium with 1% agar and 1% sugar, and then cultured vertically with 22 °C/18 °C short-day/night cycles. The plants were then transferred to half strength Hoagland nutrient solution for hydroponics. The reported method is simple and easy to operate. Self-grafted Arabidopsis–Arabidopsis and Eutrema–Eutrema plants were used as controls, which were obtained with an improved hypocotyl-cutting grafting method. Ion contents in grafted plants were detected by inductively coupled plasma optical emission spectroscopy. The results showed that the ion content in salt cress and Arabidopsis changed to different degrees after grafting. Conclusions The inter-species grafting technique described here makes it possible to study hybrid plants between Arabidopsis and Eutrema and will contribute to further understanding of long-distance communications in plants. This technique also provides a reference for improving plant varieties using grafting, such as gardening plants, as well as fruit and vegetable crops.

Plant height is a fundamentally crucial agronomic trait to control crop growth and high yield cultivation. Several studies have been conducted on the understanding ofmolecular genetic bases of plant height in model plants and crops. However, the molecular mechanism underlying peanut plant height development is stilluncertain. In the present study, we created a peanut mutant library by fast neutron irradiation using peanut variety SH13 and identified a semi-dwarf mutant 1 (sdm1). At 84 DAP (days after planting), the main stem of sdm1 was only about 62% of SH13. The internode length of sdm1 hydroponic seedlings was found significantly shorter than that of SH13 at 14 DAP. In addition, the foliar spraying of exogenous IAA could partially restore the semi-dwarf phenotype of sdm1. Transcriptome data indicated that the differentially expressed genes (DEGs) between sdm1 and SH13 significantly enriched in diterpenoid biosynthesis, alpha-linolenic acid metabolism, brassinosteroid biosynthesis, tryptophan metabolism and plant hormone signal transduction. The expression trend of most of the genes involved in IAA and JA pathway showed significantly down- and up- regulation, which may be one of the key factors of the sdm1 semi-dwarf phenotype. Moreover, several transcription factorsand cell wall relatedgenes were expressed differentially between sdm1 and SH13. Conclusively, this research work not only provided important clues to unveil the molecular mechanism of peanut plant height regulation, but also presented basic materials for breeding peanut cultivars with ideal plant height.

Quinoa is a cold-resistant and nutrient-rich crop. To decipher the cold stress response of quinoa, the full-length transcriptomes of the cold-resistant quinoa variety CRQ64 and the cold-sensitive quinoa variety CSQ5 were compared. We identified 55,389 novel isoforms and 6432 novel genes in these transcriptomes. Under cold stress, CRQ64 had more differentially expressed genes (DEGs) and differentially alternative splicing events compared to non-stress conditions than CSQ5. DEGs that were specifically present only in CRQ64 were significantly enriched in processes which contribute to osmoregulation and ROS homeostasis in plants, such as sucrose metabolism and phenylpropanoid biosynthesis. More genes with differential alternative splicing under cold stress were enriched in peroxidase functions in CRQ64. In total, 5988 transcription factors and 2956 long non-coding RNAs (LncRNAs) were detected in this dataset. Many of these had altered expression patterns under cold stress compared to non-stress conditions. Our transcriptome results demonstrate that CRQ64 undergoes a wider stress response than CSQ5 under cold stress. Our results improved the annotation of the quinoa genome and provide new insight into the mechanisms of cold resistance in quinoa.

All eukaryotes rely on the ubiquitin-proteasome system (UPS) and autophagy to control the abundance of key regulatory proteins and maintain a healthy intracellular environment. In the UPS, damaged or superfluous proteins are ubiquitinated and degraded in the proteasome, mediated by three types of ubiquitin enzymes: E1s (ubiquitin activating enzymes), E2s (ubiquitin conjugating enzymes), and E3s (ubiquitin protein ligases). Conversely, in autophagy, a vesicular autophagosome is formed that transfers damaged proteins and organelles to the vacuole, mediated by a series of ATGs (autophagy related genes). Despite the use of two completely different componential systems, the UPS and autophagy are closely interconnected and mutually regulated. During autophagy, ATG8 proteins, which are autophagosome markers, decorate the autophagosome membrane similarly to ubiquitination of damaged proteins. Ubiquitin is also involved in many selective autophagy processes and is thus a common factor of the UPS and autophagy. Additionally, the components of the UPS, such as the 26S proteasome, can be degraded via autophagy, and conversely, ATGs can be degraded by the UPS, indicating cross regulation between the two pathways. The UPS and autophagy cooperate and jointly regulate homeostasis of cellular components during plant development and stress response.

Hydrogen peroxide (H2 O2 ) is generated in many metabolic processes. As a signaling molecule, H2 O2 plays important roles in plant growth and development, as well as environmental stress response. In Arabidopsis, there are three catalase genes, CAT1, CAT2, and CAT3. The encoded catalases are predominately peroxisomal proteins and are critical for scavenging H2 O2 . Since CAT1 and CAT3 are linked on chromosome 1, it has been almost impossible to generate cat1/3 and cat1/2/3 mutants by traditional genetic tools. In this study, we constructed cat1/3 double mutants and cat1/2/3 triple mutants by CRISPR/Cas9 to investigate the role of catalases. The cat1/2/3 triple mutants displayed severe redox disturbance and growth defects under physiological conditions compared with wild-type and the cat2/3 double mutants. Transcriptome analysis showed a more profound transcriptional response in the cat1/2/3 triple mutants compared to the cat2/3 mutants. These differentially expressed genes are involved in plant growth regulation as well as abiotic and biotic stress responses. In addition, expression of OXI1 (OXIDATIVE SIGNAL INDUCIBLE 1) and several MAPK cascade genes were changed dramatically in the catalase triple mutant, suggesting that H2 O2 produced in peroxisomes could serve as a peroxisomal retrograde signal.

A VIGS method by agroinoculation of cotton seeds was developed for gene silencing in young seedlings and roots, and applied in functional analysis of GhBI-1 in response to salt stress. Virus-induced gene silencing (VIGS) has been widely used to investigate the functions of genes expressed in mature leaves, but not yet in young seedlings or roots of cotton (Gossypium hirsutum L.). Here, we developed a simple and effective VIGS method for silencing genes in young cotton seedlings and roots by soaking naked seeds in Agrobacterium cultures carrying tobacco rattle virus (TRV)-VIGS vectors. When the naked seeds were soaked in Agrobacterium cultures with an OD600 of 1.5 for 90 min, it was optimal for silencing genes effectively in young seedlings as clear photo-bleaching phenotype in the newly emerging leaves of pTRV:GhCLA1 seedlings were observed at 12-14 days post inoculation. Silencing of GhPGF (cotton pigment gland formation) by this method resulted in a 90% decrease in transcript abundances of the gene in roots at the early development stage. We further used the tool to investigate function of GhBI-1 (cotton Bax inhibitor-1) gene in response to salt stress and demonstrated that GhBI-1 might play a protective role under salt stress by suppressing stress-induced cell death in cotton. Our results showed that the newly established VIGS method is a powerful tool for elucidating functions of genes in cotton, especially the genes expressed in young seedlings and roots.

BackgroundPlant height is the major agronomic trait and closely correlated to crop yield. Plant height development is an important area of plant developmental biology and numerous studies about its molecular genetic basis had been conducted on model plants and crops. However, little is known on the molecular mechanisms of peanut plant height.ResultsIn this study, a semi-dwarf peanut mutant was identified from Co60 gama ray induced mutant population and designated as semi-dwarf mutant 2 (sdm2). The height of sdm2 was only 59.3% of its wild line Fenghua 1 (FH1) at the mature stage. Both the internode number and internode length of sdm2 were less than those of FH1. Gene expression profiles of stem and leaf from both sdm2 and FH1 were analyzed using high throughput RNA sequencing. The identified differentially expressed genes (DEGs) were involved in hormone biosynthesis and signaling pathways, cell wall synthetic and metabolic pathways. BR, GA and IAA biosynthesis and signal transduction pathways were significantly enriched. The expression of several genes in both BR biosynthesis and signaling were found to be significantly down-regulated in stem and leaf of sdm2 as compared to FH1. Many transcription factor genes were identified as DEGs.ConclusionsA large number of genes were found to be differentially expressed between sdm2 and FH1. These results provide useful information for uncovering the molecular mechanism regulating peanut stem height. It could facilitate identification of causal genes for breeding advanced peanut varieties with semi-dwarf phenotype.

The opening and closing of stomata are crucial for plant photosynthesis and transpiration. Actin filaments undergo dynamic reorganization during stomatal closure, but the underlying mechanism for this cytoskeletal reorganization remains largely unclear. In this study, we identified and characterized Arabidopsis thaliana casein kinase 1-like protein 2 (CKL2), which responds to abscisic acid (ABA) treatment and participates in ABA- and drought-induced stomatal closure. Although CKL2 does not bind to actin filaments directly and has no effect on actin assembly in vitro, it colocalizes with and stabilizes actin filaments in guard cells. Further investigation revealed that CKL2 physically interacts with and phosphorylates actin depolymerizing factor 4 (ADF4) and inhibits its activity in actin filament disassembly. During ABA-induced stomatal closure, deletion of CKL2 in Arabidopsis alters actin reorganization in stomata and renders stomatal closure less sensitive to ABA, whereas deletion of ADF4 impairs the disassembly of actin filaments and causes stomatal closure to be more sensitive to ABA. Deletion of ADF4 in the ckl2 mutant partially recues its ABA-insensitive stomatal closure phenotype. Moreover, Arabidopsis ADFs from subclass I are targets of CKL2 in vitro. Thus, our results suggest that CKL2 regulates actin filament reorganization and stomatal closure mainly through phosphorylation of ADF.

Hydrogen peroxide (H

Peanut (Arachis hypogaea L.), as a source of oil and protein, is the second-most important grain legume cultivated in the world. Peanut is a relatively drought-tolerant crop; however, the molecular biology research of peanut is far behind other crops; the molecular mechanisms of its stress resistance are poorly understood. DELLA proteins, negative regulators of gibberellin signaling pathway in plants, promote survival of plants in adverse environments. In this study, four DELLA homologue genes were isolated using peanut transcriptome sequences. Molecular phylogenetic analysis revealed that these four AhDELLAs fall into three distinct groups: AhDELLA1 and AhDELLA2 clustered into two distinct groups, while AhDELLA3 and AhDELLA4 were in one group, which was separated from other DELLAs. qRT-PCR results showed that these four DELLA genes were expressed differentially in various peanut tissues. The AhDELLA1 and AhDELLA2 genes were expressed ubiquitously in different tissues. AhDELLA3 and AhDELLA4 showed much higher expression level in flowers and seeds as compared with other organs. The expression of four AhDELLA genes was temporally induced by PEG 6000 treatment. The AhDELLA3 and AhDELLA4 transcripts were significantly induced by NaCl treatment, while the expression of AhDELLA1 and AhDELLA2 did not change much under salt stress. The possible role of DELLA proteins in peanut development and responses to abiotic stresses is discussed.

Carbonic anhydrase (CA; EC 4.2.1.1) catalyzes the interconversion of CO2 and HCO3- and plays an important role in photosynthetic carbon assimilation. We report that the effect of manipulating AtβCA6 expression on the growth and biomass accumulation of Arabidopsis thaliana shows that AtβCA6 was expressed in mitochondria. Overexpression of AtβCA6 increased the fresh weight, dry weight and rosette leaf area and it was also linked to a slight decrease in the rate of respiration. By contrast, when the respiration rate in the AtβCA6 knock-out mutant SALK_065611 increased, the fresh weight, dry weight and rosette leaf area decreased. The expression level of AtβCA6 mainly affected the expression of the genes that were related to metabolism, photosynthesis and respiration. We discuss these data with respect to a potential role of AtβCA6 in refixation of CO2 released from respiration and its potential as an option to increase biomass production.

Nonspecific lipid transfer proteins (nsLTPs), a group of small, basic proteins that are ubiquitously distributed throughout the plant kingdom, are thought to participate in cutin formation as well as in defense reactions against abiotic and biotic stresses. However, whether nsLTPs are involved in cuticular wax deposition remains unknown. We identified a salt-induced gene, TsnsLTP4, encoding an nsLTP from Thellungiella salsuginea. TsnsLTP4 expression was significantly induced by salt, polyethylene glycol (PEG), abscisic acid (ABA), and high (37 °C) and low (4 °C) temperatures. Transgenic onion epidermal cells transiently expressing a TsnsLTP4-DsRed fusion protein demonstrated that TsnsLTP4 was targeted to the cell wall. Overexpression of TsnsLTP4 in transgenic Arabidopsis plants resulted in increased epicuticular wax deposition, particularly of wax components with a carbon chain length of more than C27. Moreover, the amount of wax deposited was strongly reduced in RNA interference (RNAi)-knockdown TsnsLTP4 transgenic T. salsuginea lines. Cuticle permeability was inversely related to the expression level of TsnsLTP4. Further analyses indicated that overexpression of TsnsLTP4 resulted in significantly enhanced drought and salt tolerance in transgenic Arabidopsis. In summary, our studies suggested that TsnsLTP4 may play a role in wax deposition and in plant tolerance against abiotic stresses.

Galactinol synthase (GOLS, EC 2.4.1.123), a key enzyme in the synthesis of raffinose family oligosaccharides (RFOs), catalyzes the condensation of UDP-galactose with myo-inositol to produce galactinol as the sole donor for the synthesis of RFOs. RFOs have been implicated in mitigating effects of environmental stresses on plants. TsGOLS2, was cloned from Thellungiella salsuginea with high homology to AtGOLS2. TsGOLS2 was up-regulated by several abiotic stresses. We overexpressed TsGOLS2 in Arabidopsis thaliana. The contents of galactinol, raffinose, and α-ketoglutaric acid were significantly increased in transgenic plants. Compared to wild type plants, salt-stressed transgenic A. thaliana exhibited higher germination rate, photosynthesis ability, and seedling growth. After being treated with osmotic stress by high concentration of sorbitol, transgenic plants retained high germination rates and grew well during early development. These results indicated that overexpression of TsGOLS2 in A. thaliana improved the tolerance of transgenic plants to high salinity and osmotic stress.

Floral zygomorphy is proved to have evolved many times independently from actinomorphy, exhibiting two kinds of asymmetry, dorsoventral (DV) and organ internal (IN) asymmetry, in flowering plants. Multiple underlying regulators are involved in establishing the complex flowering mechanism during plant development. To understand how genes control the floral dorsoventral asymmetry in soybean, we isolated and analyzed the role of two different copies of CYC-like genes in Glycine max. It was demonstrated that CYClike genes, GmTCP1 and GmTCP2, encoded the TCP transcription factors that were specifically expressed in the dorsal region of floral meristem. However, the two copies were proved to be functionally divergent during petal development, and by altering the expression of CYC-like genes in transgenic soybean, similar to CYC and DICH, it was found that CYC-like genes had dorsalizing activity. The expression pattern of transgenic plants indicated that the GmTCP2 gene could specifically regulate downst...

To obtain strong inducible promoters to drive abiotic stress-inducible transgene expression with minimal negative effects, we constructed three artificial synthetic promoters (EKCM, EKCRM, and ECCRM) comprising multiple cis-acting stress-response elements. Each promoter was fused independently to the β-glucuronidase (GUS) reporter gene, and GUS expression was analyzed in stable expression systems in Arabidopsis thaliana. T2 transgenic progenies showed integration of the promoter-GUS construct in their genome. RT-PCR assays and histochemical staining analysis showed that GUS expression driven by each promoter increased under desiccation, cold, and high salt conditions. The activity of synthetic promoters, assessed by fluorometric quantitative analysis of GUS enzyme activity, was significantly higher than that of the rd29A promoter under various stress treatments. The most powerful promoter, EKCM, allowed about 1.29-fold in GUS activity relative to the rd29A promoter, on average, under dehydration conditions. All three synthetic promoters could drive stress-inducible GUS expression in different organs of transgenic Arabidopsis. These synthetic promoters represent valuable tools for improving the stress tolerance of crops.

The contribution of SOS1 (for Salt Overly Sensitive 1), encoding a sodium/proton antiporter, to plant salinity tolerance was analyzed in wild-type and RNA interference (RNAi) lines of the halophytic Arabidopsis (Arabidopsis thaliana)-relative Thellungiella salsuginea. Under all conditions, SOS1 mRNA abundance was higher in Thellungiella than in Arabidopsis. Ectopic expression of the Thellungiella homolog ThSOS1 suppressed the salt-sensitive phenotype of a Saccharomyces cerevisiae strain lacking sodium ion (Na+) efflux transporters and increased salt tolerance of wild-type Arabidopsis. thsos1-RNAi lines of Thellungiella were highly salt sensitive. A representative line, thsos1-4, showed faster Na+ accumulation, more severe water loss in shoots under salt stress, and slower removal of Na+ from the root after removal of stress compared with the wild type. thsos1-4 showed drastically higher sodium-specific fluorescence visualized by CoroNa-Green, a sodium-specific fluorophore, than the wild type, inhibition of endocytosis in root tip cells, and cell death in the adjacent elongation zone. After prolonged stress, Na+ accumulated inside the pericycle in thsos1-4, while sodium was confined in vacuoles of epidermis and cortex cells in the wild type. RNAi-based interference of SOS1 caused cell death in the root elongation zone, accompanied by fragmentation of vacuoles, inhibition of endocytosis, and apoplastic sodium influx into the stele and hence the shoot. Reduction in SOS1 expression changed Thellungiella that normally can grow in seawater-strength sodium chloride solutions into a plant as sensitive to Na+ as Arabidopsis.

Soil salinity is a major abiotic stress that lowers agricultural production around the world. Mainly caused by elevated levels of NaCl, it induces a wide range of responses in plants. In addition to ion toxicity, high salt levels can induce oxidative stress with the formation and accumulation of reactive oxygen species (ROS). We introduced the transcription factor YAP1, originally from yeast (Saccharomyces cerevisiae), into Arabidopsis thaliana (ecotype Columbia). When treated with various NaCl concentrations, transgenic plants showed increased activities of antioxidant enzymes catalase, superoxide dismutase, ascorbate peroxidase, peroxidase, glutathione S-transferase, and glutathione reductase compared with the wild-type Arabidopsis. This demonstrated that an active oxygen scavenging system was enhanced to protect plants from salt stress by equilibrating ROS metabolism. Transgenic Arabidopsis maintained higher photosynthesis levels and lower amounts of H2O2, suggesting that ROS production was reduced. Physiological analysis implied that transgenic Arabidopsis might employ multiple mechanisms to improve its salt tolerance.

Na+/H+ exchanger catalyzes the countertransport of Na+ and H+ across membranes. Using the rapid amplification of cDNA ends method, a Na+/H+ antiporter gene (ThNHX1) was isolated from a halophytic plant, salt cress (Thellungiella halophila). The deduced amino acid sequence contained 545 amino acid residues with a conserved amiloride-binding domain (87LFFIYLLPPI96) and shared more than 94% identity with that of AtNHX1 from Arabidopsis thaliana. The ThNHX1 mRNA level was upregulated by salt and other stresses (abscisic acid, polyethylene glycol, and high temperature). This gene partially complemented the Na+/Li+-sensitive phenotype of a yeast mutant that was deficient in the endosomal–vacuolar Na+/H+ antiporter ScNHX1. Overexpression of ThNHX1 in Arabidopsis increased salt tolerance of transgenic plants compared with the wild-type plants. In addition, the silencing of ThNHX1 gene in T. halophila caused the transgenic plants to be more salt and osmotic sensitive than wild-type plant. Together, these results suggest that ThNHX1 may function as a tonoplast Na+/H+ antiporter and play an important role in salt tolerance of T. halophila.

In yeast, the plasma membrane Na+/H+ antiporter and Na+-ATPase are key enzymes for salt tolerance.Saccharomyces cerevisiae Na+-ATPase (Enalp ATPase) is encoded by theENA1/PMR2A gene; expression ofENA1 is tightly regulated by Na+ and depends on ambient pH. Although Enalp is active mainly at alkaline pH values inS. cerevisiae, no Na+-ATPase has been found in flowering plants. To test whether this yeast enzyme would improve salt tolerance in plants, we introducedENA1 intoArabidopsis (cv. Columbia) under the control of the cauliflower mosaic virus 35S promoter. Transformants were selected for their ability to grow on a medium containing kanamyin. Southern blot analyses confirmed thatENA1 was transferred into theArabidopsis genome and northern blot analyses showed thatENA1 was expressed in the transformants. Several transgenic homozygous lines and wild-type (WT) plants were evaluated for salt tolerance. No obvious morphological or developmental differences existed between the transgenic and WT plants in the absence of stress. However, overexpression ofENA1 inArabidopsis improved seed germination rates and salt tolerance in seedlings. Under saline conditions, transgenic plants accumulated a lower amount of Na+ than did the wild type, and fresh and dry weights of the former were higher. Other experiments revealed that expression ofENA1 promoted salt tolerance in transgenicArabidopsis under both acidic and alkaline conditions.

A modified Agrobacterium-mediated transformation protocol has been successfully used for transient expression of the intrinsically fluorescent proteins and their fusion proteins in onion epidermis. The mean of the transformed cells rate per peel is about 10.5±0.9%, while that of the particle bombardment method is at the range 2.0±0.4%. To compare with the prevailing method of micro-projectile bombardment, the modified Agrobacterium-mediated transformation may provide with higher efficiency and even more simplified manipulability on a lower budget.

Soybean [Glycine max (L.) Merrill] is an important crop worldwide. In this study, a Chinese local soybean cultivar, Hedou 12, was resequenced by next generation sequencing technology to develop INsertion/DELetion (INDEL) markers for genetic mapping. 49,276 INDEL polymorphisms and 242,059 single nucleotide polymorphisms were detected between Hedou 12 and the Williams 82 reference sequence. Of these, 243 candidate INDEL markers ranging from 5–50 bp in length were chosen for validation, and 165 (68%) of them revealed polymorphisms between Hedou 12 and Williams 82. The validated INDEL markers were also tested in 12 other soybean cultivars. The number of polymorphisms in the pairwise comparisons of 14 soybean cultivars varied from 27 to 165. To test the utility of these INDEL markers, they were used to perform genetic mapping of a crinkly leaf mutant, and the CRINKLY LEAF locus was successfully mapped to a 360 kb region on chromosome 7. This research shows that high-throughput sequencing technologies can facilitate the development of genome-wide molecular markers for genetic mapping in soybean.

Abiotic stress, such as salt, drought and extreme temperature, can result in enhanced production of reactive oxygen species (ROS). Plants have developed both enzymatic ROS-scavenging and non-enzymatic ROS-scavenging systems. The major ROS-scavenging enzymes of plants include superoxide dismutase (SOD), ascorbate peroxidase (APX), catalase (CAT), glutathione peroxidase (GPX) and peroxiredoxins (Prxs). In the present work, we identified a gene encoding chloroplast-located peroxiredoxin Q, SsPrxQ, from Suaeda salsa L. located at chloroplast. Overexpression of SsPrxQ in Arabidopsis leads to an increase in salt and low-temperature tolerance. (Managing editor: Li-Hui Zhao)

Transgenic rice plants co-expressing the Suaeda salsa SsNHX1 (vacuolar membrane Na+/H+ antiporter) and Arabidopsis AVP1 (vacuolar H+-PPase) showed enhanced salt tolerance during 3 d of 300 mM NaCl treatment under outdoor growth conditions. These transgenic rice seedlings also grew better on MS medium containing 150 mM NaCl compared to SsNHX1-transformed lines and non-transformed controls. Measurements on isolated vacuolar membrane vesicles derived from the salt stressed SsNHX1+AVP1-transgenic plants demonstrated that the vesicles had increased V-PPase hydrolytic activity in comparison with the Ss-transgenics and non-transgenics. Moreover the V-PPase activity was closely related to the development period of the SA-transgenic seedlings and markedly higher in 3-week-old seedlings than in 5-week-old seedlings. Statistic analysis indicated that the SA-transgenic rice plants contained relatively more ions with higher K+/Na+ ratio in their shoots compared to the SsNHX1-transformed lines upon salt treatment. Furthermore, these SA-transformants also exhibited relatively higher level of photosynthesis and root proton exportation capacity whereas reduced H2O2 generation in the same plants. In general, these results supported the hypothesis that simultaneous expression of the SsNHX1 and AVP1 conferred greater performance to the transgenic plants than that of the single SsNHX1.

Agricultural productivity is severely affected by soil salinity. One possible mechanism by which plants could survive salt stress is to remove sodium ions from the cytosol via plasma membrane Na + /H + antiporters. In the present study, we demonstrated that expressing the plasma membrane Na + /H + antiporter SOD2 from yeast ( Schizosaccharomyces pombe ) in transgenic rice increased salt tolerance. These transgenic plants accumulated more K + , Ca 2+ , Mg 2+ and less Na + in their shoots compared with those of non-transformed controls. Moreover, measurements on isolated plasma membrane vesicles derived from the SOD2 transgenic rice plant roots showed that the vesicles had enhanced P-ATPase hydrolytic activity. Furthermore, the transformed rice plants maintained higher levels of photosynthesis and root proton exportation capacity, whereas reduced ROS generation. Physiological analysis suggested that transgenic rice plants might employ multiple mechanisms to improve their salt tolerance under salt stress conditions.

Salt stress is one of the most serious factors limiting the productivity of agricultural crops. Increasing evidence has demonstrated that vacuolar Na+/H+ antiporters play a crucial role in plant salt tolerance. In the present study, we expressed the Suaeda salsa vacuolar Na+/H+ antiporter SsNHX1 in transgenic rice to investigate whether this can increase the salt tolerance of rice, and to study how overexpression of this gene affected other salt-tolerant mechanisms. It was found that transgenic rice plants showed markedly enhanced tolerance to salt stress and to water deprivation compared with non-transgenic controls upon salt stress imposition under outdoor conditions. Measurements of ion levels indicated that K+, Ca2+ and Mg2+ contents were all higher in transgenic plants than in non-transformed controls. Furthermore, shoot V-ATPase hydrolytic activity was dramatically increased in transgenics compared to that of non-transformed controls under salt stress conditions. Physiological analysis also showed that the photosynthetic activity of the transformed plants was higher whereas the same plants had reduced reactive oxygen species generation. In addition, the soluble sugar content increased in the transgenics compared with that in non-transgenics. These results imply that up-regulation of a vacuolar Na+/H+ antiporter gene in transgenic rice might cause pleiotropic up-regulation of other salt-resistance-related mechanisms to improve salt tolerance.

The chenopodiaceae Suaeda salsa L. is a leaf succulent euhalophyte. Shoots of the S. salsa are larger and more succulent when grown in highly saline environments. This increased growth and water uptake has been correlated with a large and specific cellular accumulation of sodium. S. salsa does not have salt glands or salt bladders on its leaves. Thus, this plant must compartmentalize the toxic Na(+) in the vacuoles. The ability to compartmentalize sodium may result from a stimulation of the proton pumps that provide the driving force for increased sodium transport into the vacuole. In this work, we isolated the cDNA of the vacuolar membrane proton-translocating inorganic pyrophosphatase (H(+) -PPase) from S. salsa. The SsVP cDNA contains an uninterrupted open reading frame of 2292 bp, coding for a polypeptide of 764 amino acids. Northern blotting analysis showed that SsVP was induced in salinity treated leaves. The activities of both the V-ATPase and the V-PPase in Arabidopsis overexpressing SsVP-2 is higher markedly than in wild-type plant under 200 mM NaCl and drought stresses. The Overexpression of SsVP can increase salt and drought tolerance of transgenic Arabidopsis.

A full-length cDNA of SsCCD1 gene encoding carotenoid cleavage dioxygenase was isolated from a halophyte Suaeda salsa. Sequence analysis revealed that SsCCD1 shares a high homology with AtCCD1 from Arabidopsis thaliana. The 1972 bp full-length SsCCD1 has an open reading frame of 1677 bp and encodes a 62.5 KDa protein, with 5′- and 3′-untranslate region of 72 bp and 229 bp, respectively. Northern blotting analysis indicated that the SsCCD1 mRNA levels were significantly up-regulated by NaCl and drought stresses in both root and aerial part tissues. High temperature and ABA also increased the transcriptional level of SsCCD1. Transgenic arabidopsis with overexpression of SsCCD1 gene did not show an increased endogenous ABA concentration, but decreased the carotenoid concentration, indicating that CCD enzyme may be involved in the carotenoid catabolism.

Peroxiredoxin Q, as a member of peroxiredoxin gene family, was most recently cloned from plants. However, its molecular characteristics remain unclear. In this paper, we cloned a peroxiredoxin Q gene from a 400 mM NaCl-treated cDNA library of Suaeda salsa . The SsPrx Q gene (1 003 bp) contains an open reading frame of 214 amino acids with a molecular mass of 23.6 kDa. SsPrx Q showed high homology to the Prx Q of Sedum lineare , with 76% identities in deduced amino acid sequence. Southern blot analysis indicated that SsPrx Q gene has a single copy in S. salsa genome. Northern blot analysis showed that SsPrx Q gene expression was up-regulated by NaCl, mannitol, low temperature, H 2 O 2 , methyl viologen, and ABA. Studies of the recombinant protein produced in E. coli cells exhibited that it actually had a thioredoxin-dependent peroxidase activity, the characteristic of the Prx family. These results indicated that SsPrx Q may play an important role in protecting S. salsa from environmental stresses.

Wheat productivity is severely affected by soil salinity mainly due to Na + toxicity to plant cells. To improve the yield performance of wheat in saline soils, we have generated transgenic wheat expressing a vacuolar Na + /H + antiporter gene AtNHX1 from Arabidopsis thaliana, to enhance the plant capacity of reducing cytosolic Na + by sequestering Na + in the vacuole. The AtNHX1 transgenic wheat lines exhibited improved biomass production at the vegetative growth stage and germination rates in severe saline conditions. A field trial revealed that the transgenic wheat lines produced higher grain yields and heavier and larger grains in the field of saline soils with the electrical conductivity values of soil saturation extracts (EC e ) of 10.6 and 13.7 dS m −1 . The transgenic lines accumulated a lower level of Na + and a higher level of K + in the leaves than non-transgenic plants under saline conditions (100 and 150 mM NaCl). These results indicate that the salt tolerance of wheat and grain yield in saline soils can be improved by enhancing the level of the vacuolar Na + /H + antiporter.

Glycine betaine plays an important role in some plants, including maize, in conditions of abiotic stress, but different maize varieties vary in their capacity to accumulate glycine betaine. An elite maize inbred line DH4866 was transformed with the betA gene from Escherichia coli encoding choline dehydrogenase (EC 1.1.99.1), a key enzyme in the biosynthesis of glycine betaine from choline. The transgenic maize plants accumulated higher levels of glycine betaine and were more tolerant to drought stress than wild-type plants (non-transgenic) at germination and the young seedling stage. Most importantly, the grain yield of transgenic plants was significantly higher than that of wild-type plants after drought treatment. The enhanced glycine betaine accumulation in transgenic maize provides greater protection of the integrity of the cell membrane and greater activity of enzymes compared with wild-type plants in conditions of drought stress.

A Na+/H+ antiporter catalyzes the transport of Na+ and H+ across the tonoplast membrane. We isolated a vacuolar Na+/H+ antiporter cDNA (SsNHX1) clone from a euhalophyte, Suaeda salsa. The nuclear sequence contains 2262 bp with an open reading frame of 1665 bp. The deduced amino acid sequence is similar to that of AtNHX1 and OsNHX1 in rice, with the highest similarities within the predicted transmembrane segments and an amiloride-binding domain. Northern blot analysis shows that the expression of the S. salsa gene was increased by salt stress. The results suggest that the SsNHX1 product is likely a Na+/H+ antiporter and may play important roles in the salt tolerance of S. salsa.