Xiaoshuang Li

Overexpression of Tamarix albiflonum TaMnSOD increases drought tolerance in transgenic cotton

Drought is a major environmental stress that limits cotton (Gossypium hirsutum L.) production worldwide. TaMnSOD plays a crucial role as a peroxidation scavenger. In this study, TaMnSOD cDNA of Tamarix albiflonum was overexpressed in the cotton cultivar fy11 by Agrobacterium tumefaciens-mediated transformation. The transformed plants were assessed by gDNA PCR, RT-PCR and DNA gel blot analysis. The physiological and biochemical characters of two independent transgenic lines and control plants were tested and compared, and the morphological traits (biomass, root and lateral root length, leaf number) were also detected after recovery from water-withholding stress. When water was withheld from pot-grown 6-week-old seedlings for 18xa0days (watering to 8xa0% of field capacity), transgenic cotton plants accumulated more proline and soluble sugar than wild-type plants (WT). The activity of antioxidant enzymes such as superoxide dismutase and peroxidase was enhanced in transgenic plants under drought stress. Cell membrane integrity was also considerably improved under water stress, as indicated by reduced malondialdehyde content relative to control plants. Furthermore, net photosynthesis, stomatal conductance and transpiration rate were increased in transgenic plants compared with wild type. Transgenic cotton showed increases in biomass as well as root and leaf systems compared with WT after 2xa0weeks recovery from stress. These results suggest that TaMnSOD transgenic cotton plants acquired improved drought tolerance through enhanced development of the root and leaf system and the regulation of superoxide scavenging.

De novo transcriptome characterization and gene expression profiling of the desiccation tolerant moss Bryum argenteum following rehydration

BackgroundThe desiccation-tolerant moss Bryum argenteum is an important component of the Biological Soil Crusts (BSCs) found in the Gurbantunggut desert. Desiccation tolerance is defined as the ability to revive from the air dried state. To elucidate the molecular mechanisms related to desiccation tolerance, we employed RNA-Seq and digital gene expression (DGE) technologies to study the genome-wide expression profiles of the dehydration and rehydration processes in this important desert plant.ResultsWe applied a two-step approach to investigate the gene expression profile upon rehydration in the moss Bryum argenteum using Illumina HiSeq2000 sequencing platform. First, a total of 57,247 transcript assembly contigs (TACs) were obtained from 54.79 million reads by de novo assembly, with an average length of 863xa0bp and N50 of 1,372xa0bp. Among the reconstructed TACs, 36,916 (64.5xa0%) revealed similarity with existing protein sequences in the public databases. 23,509 and 21,607 TACs were assigned GO and KEGG annotation information, respectively. Second, samples were taken from 3 hydration stages: desiccated (Dry), rehydrated 2xa0h (R2) and rehydrated 24xa0h (R24), and DEG libraries were constructed for Differentially Expressed Genes (DEGs) discovery. 4,081 and 6,709 DEGs were identified in R2 and R24, compared with Dry, respectively. Compared to the desiccated sample, up-regulated genes after two hours of hydration are primarily related to stress responses. GO function enrichment network, EKGG metabolic pathway and MapMan analysis supports the idea of the rapid recovery of photosynthesis after 24xa0h of rehydration. We identified 770 transcription factors (TFs) which were classified into 50 TF families. 142 TF transcripts were up-regulated upon rehydration including 23 members of the ERF family.ConclusionsIn this study, we constructed a pioneering, high-quality reference transcriptome in B. argenteum and generated three DGE libraries to elucidate the changes of gene expression upon rehydration. Expression profiles consistent with the rapid recovery of photosynthesis (at R2) and the re-establishment of a positive carbon balance following rehydration (at R24) were observed. Our study will extend our knowledge of bryophyte transcriptomes and provide further insight into the molecular mechanisms related to rehydration and desiccation-tolerance.

Characterization of reference genes for RT-qPCR in the desert moss Syntrichia caninervis in response to abiotic stress and desiccation/rehydration

Syntrichia caninervis is the dominant bryophyte of the biological soil crusts found in the Gurbantunggut desert. The extreme desert environment is characterized by prolonged drought, temperature extremes, high radiation and frequent cycles of hydration and dehydration. S. caninervis is an ideal organism for the identification and characterization of genes related to abiotic stress tolerance. Reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) expression analysis is a powerful analytical technique that requires the use of stable reference genes. Using available S. caninervis transcriptome data, we selected 15 candidate reference genes and analyzed their relative expression stabilities in S. caninervis gametophores exposed to a range of abiotic stresses or a hydration-desiccation-rehydration cycle. The programs geNorm, NormFinder, and RefFinder were used to assess and rank the expression stability of the 15 candidate genes. The stability ranking results of reference genes under each specific experimental condition showed high consistency using different algorithms. For abiotic stress treatments, the combination of two genes (α-TUB2 and CDPK) were sufficient for accurate normalization. For the hydration-desiccation-rehydration process, the combination of two genes (α-TUB1 and CDPK) were sufficient for accurate normalization. 18S was among the least stable genes in all of the experimental sets and was unsuitable as reference gene in S. caninervis. This is the first systematic investigation and comparison of reference gene selection for RT-qPCR work in S. caninervis. This research will facilitate gene expression studies in S. caninervis, related moss species from the Syntrichia complex and other mosses.

EFFECTS OF DROUGHT STRESS ON THE SEED GERMINATION AND EARLY SEEDLING GROWTH OF THE ENDEMIC DESERT PLANT EREMOSPARTON SONGORICUM (FABACEAE)

Eremosparton songoricum (Litv.) Vass. is an endemic and extremely drought-resistant desert plant with populations that are gradually declining due to the failure of sexual recruitment. The effects of drought stress on the seed germination and physiological characteristics of seeds and seedlings were investigated. The results showed that the germination percentage decreased with an increase of polyethylene glycol 6000 (PEG) concentration: -0.3 MPa (5 % PEG) had a promoting effect on seed germination, -0.9 MPa (15 % PEG) dramatically reduced germination, and -1.8 MPa (30 % PEG) was the threshold for E. songoricum germination. However, the contents of proline and soluble sugars and the activity of CAT increased with increasing PEG concentrations. At the young seedling stage, the proline content and CAT, SOD and POD activities all increased at 2 h and then decreased; except for a decrease at 2 h, the MDA content also increased compared to the control (0 h). These results indicated that 2 h may be a key response time point for E. songoricum to resist drought stress. The above results demonstrate that drought stress can suppress and delay the germination of E. songoricum and that the seeds accumulate osmolytes and augment the activity of antioxidative enzymes to cope with drought injury. E. songoricum seedlings are sensitive to water stress and can quickly respond to drought but cannot tolerate drought for an extended period. Although such physiological and biochemical changes are important strategies for E. songoricum to adapt to a drought-prone environment, they may be, at least partially, responsible for the failure of sexual reproduction under natural conditions.

Reference Gene Selection in the Desert Plant Eremosparton songoricum

Eremosparton songoricum (Litv.) Vass. (E. songoricum) is a rare and extremely drought-tolerant desert plant that holds promise as a model organism for the identification of genes associated with water deficit stress. Here, we cloned and evaluated the expression of eight candidate reference genes using quantitative real-time reverse transcriptase polymerase chain reactions. The expression of these candidate reference genes was analyzed in a diverse set of 20 samples including various E. songoricum plant tissues exposed to multiple environmental stresses. GeNorm analysis indicated that expression stability varied between the reference genes in the different experimental conditions, but the two most stable reference genes were sufficient for normalization in most conditions. EsEF and Esα-TUB were sufficient for various stress conditions, EsEF and EsACT were suitable for samples of differing germination stages, and EsGAPDHand EsUBQ were most stable across multiple adult tissue samples. The Es18S gene was unsuitable as a reference gene in our analysis. In addition, the expression level of the drought-stress related transcription factor EsDREB2 verified the utility of E. songoricum reference genes and indicated that no single gene was adequate for normalization on its own. This is the first systematic report on the selection of reference genes in E. songoricum, and these data will facilitate future work on gene expression in this species.

Novel DREB A-5 subgroup transcription factors from desert moss (Syntrichia caninervis) confers multiple abiotic stress tolerance to yeast ☆

Syntrichia caninervis Mitt. is a typical desiccation tolerant moss from a temperate desert which has been a good resource for stress tolerant gene isolation. Dehydration responsive element binding proteins (DREBs) was proven to play an important role in responding to abiotic stress, which has been identified in many plants, and were rarely reported in moss. In this study, we cloned ten DREB genes from S. caninervis, and investigated their abiotic stress response and stress tolerance. The results showed that ten ScDREB proteins belonged to the A-5 sub-group of the DREB sub-family. Six genes, ScDREB1, ScDREB2, ScDREB4, ScDREB6, ScDREB7, and ScDREB8 were involved in the ABA-dependent signal pathway and the desiccation, salt, and cold stress response. ScDREB3 also responded to desiccation, salt, and cold stresses, but was insensitive to ABA treatment. Another gene, ScDREB5, was involved in an ABA-independent cold stress-responsive signal pathway. Two genes, ScDREB9 and ScDREB10, responded slightly or had no response to neither stress factor or ABA treatment. We transformed four typical genes into yeast cells and the stress tolerance ability of transgenic yeast was evaluated. The results showed that ScDREB3 and ScDREB5 enhanced the yeasts cold and salt tolerance. ScDREB8 and ScDREB10 conferred the osmotic, salt, cold, and high temperature stresses tolerance, especially for osmotic and salt stresses. Our results indicated that A-5 sub-group DREB genes in S. caninervis played important roles in abiotic stresses response and enhanced stress tolerance to transgenic yeast. To our knowledge, this is the first report on DREB genes characterization from desiccation tolerant moss, and this study will not only provide insight into the molecular mechanisms of S. caninervis adaptation to environmental stresses, but also provides valuable gene candidates for plant molecular breeding.

Transcriptome-Wide Identification, Classification, and Characterization of AP2/ERF Family Genes in the Desert Moss Syntrichia caninervis

APETALA2/Ethylene Responsive Factor (AP2/ERF) is a large family of plant transcription factors which play important roles in the control of plant metabolism and development as well as responses to various biotic and abiotic stresses. The desert moss Syntrichia caninervis, due to its robust and comprehensive stress tolerance, is a promising organism for the identification of stress-related genes. Using S. caninervis transcriptome data, 80 AP2/ERF unigenes were identified by HMM modeling and BLASTP searching. Based on the number of AP2 domains, multiple sequence alignment, motif analysis, and gene tree construction, ScAP2/ERF genes were classified into three main subfamilies (including 5 AP2 gene members, 72 ERF gene members, and 1 RAV member) and two Soloist members. We found that the ratio for each subfamily was constant between S. caninervis and the model moss Physcomitrella patens, however, as compared to the angiosperm Arabidopsis, the percentage of ERF subfamily members in both moss species were greatly expanded, while the members of the AP2 and RAV subfamilies were reduced accordingly. The amino acid composition of the AP2 domain of ScAP2/ERFs was conserved as compared with Arabidopsis. Interestingly, most of the identified DREB genes in S. caninervis belonged to the A-5 group which play important roles in stress responses and are rarely reported in the literature. Expression profile analysis of ScDREB genes showed different gene expression patterns under dehydration and rehydration; the majority of ScDREB genes demonstrated a stronger response to dehydration relative to rehydration indicating that ScDREB may play an important role in dehydrated moss tissues. To our knowledge, this is the first study to detail the identification and characterization of the AP2/ERF gene family in a desert moss. Further, this study will lay the foundation for further functional analysis of these genes, provide greater insight to the stress tolerance mechanisms in S. caninervis and provide a reference for AP2/ERF gene family classification in other moss species.

Desiccation tolerance in bryophytes: The dehydration and rehydration transcriptomes in the desiccation-tolerant bryophyte Bryum argenteum

The desiccation tolerant bryophyte Bryum argenteum is an important component of desert biological soil crusts (BSCs) and is emerging as a model system for studying vegetative desiccation tolerance. Here we present and analyze the hydration-dehydration-rehydration transcriptomes in B. argenteum to establish a desiccation-tolerance transcriptomic atlas. B. argenteum gametophores representing five different hydration stages (hydrated (H0), dehydrated for 2u2009h (D2), 24u2009h (D24), then rehydrated for 2u2009h (R2) and 48u2009h (R48)), were sampled for transcriptome analyses. Illumina high throughput RNA-Seq technology was employed and generated more than 488.46 million reads. An in-house de novo transcriptome assembly optimization pipeline based on Trinity assembler was developed to obtain a reference Hydration-Dehydration-Rehydration (H-D-R) transcriptome comprising of 76,206 transcripts, with an N50 of 2,016u2009bp and average length of 1,222u2009bp. Comprehensive transcription factor (TF) annotation discovered 978 TFs in 62 families, among which 404 TFs within 40 families were differentially expressed upon dehydration-rehydration. Pfam term enrichment analysis revealed 172 protein families/domains were significantly associated with the H-D-R cycle and confirmed early rehydration (i.e. the R2 stage) as exhibiting the maximum stress-induced changes in gene expression.

Overexpression of ScALDH21 gene in cotton improves drought tolerance and growth in greenhouse and field conditions

Aldehyde dehydrogenase (ALDH) is essential for scavenging redundant aldehydes when plants are exposed to stress. The aim of the present study was to validate the ectopic expression of the ScALDH21 gene, which is isolated from Syntrichia caninervis, an extremely drought-tolerant moss, to improve drought tolerance in cotton (Gossypium hirsutum L.). In our study, the ScALDH21-transformed cotton was identified via PCR, RT-PCR, and DNA gel blotting, and the growth and physiological characteristics related to drought tolerance were compared between the transgenic cotton (TC) and non-transgenic cotton (NT) grown in a greenhouse and in field conditions. The results indicated that TC accumulated approximately 11.8–304xa0% more proline than did NT under drought stress, and produced a lower concentration of lipid peroxidation-derived reactive aldehydes and had a higher peroxidase activity under oxidative stress. Moreover, TC showed reduced loss of the net photosynthetic rate compared with NT. Under field conditions, TC showed greater plant height, larger bolls, and greater cotton fiber yield than NT, but no significant difference in fiber quality between TC and NT following different water-withholding treatments. These results suggest that overexpression of ScALDH21 can greatly improve the drought tolerance of cotton without reduction in yield and fiber quality.

ScDREB8, a novel A-5 type of DREB gene in the desert moss Syntrichia caninervis, confers salt tolerance to Arabidopsis

Salinity is a major limitation factor for crop productivity worldwide. DREB transcription factors broadly participate in plant stress response and have been extensively identified in a wide variety of plants. In this study, we characterized and analyzed the function of a novel A-5 type DREB gene ScDREB8 from the desiccation tolerant moss Syntrichia caninervis. Yeast one-hybrid experiment showed that ScDREB8 had no transactivation activity. Transient expression assay in onion epidermal cells revealed that ScDREB8 is distributed throughout the cell with no apparent specificity. Overexpression of ScDREB8 significantly increased the germination rate of Arabidopsis under salt stress and improved the salt tolerance of Arabidopsis at the seedling stage by up-regulating the expression of downstream stress-related genes and improving ROS scavenging ability. ScDREB8 is a promising candidate gene for improving crop salt stress and will provide greater insight to the molecular mechanism of stress tolerance of A-5 type DREB proteins.