AnDREB5.1, a A5 group DREB gene from desert shrub Ammopiptanthus nanus, confers osmotic and cold stress tolerances in transgenic tobacco.

The Dehydration-Responsive Element Binding (DREB) subfamily of transcription factors plays crucial roles in plant abiotic stress response. Ammopiptanthus nanus (A. nanus) is an eremophyte exhibiting remarkable tolerance to environmental stress and DREB proteins may contribute to its tolerance to water deficit and low-temperature stress. In the present study, an A. nanus DREB A5 group transcription factor gene, AnDREB5.1, was isolated and characterized in terms of structure and function in abiotic stress tolerance. AnDREB5.1 protein is distributed in the nucleus, possesses transactivation capacity, and is capable of binding to DRE core cis-acting element. The transcription of AnDREB5.1 was induced under osmotic and cold stress. Tobacco seedlings overexpressing AnDREB5.1 displayed higher tolerance to cold stress, osmotic stress, and oxidative stress compared to wild-type tobacco (WT). Under osmotic and cold stress, overexpression of AnDREB5.1 increased antioxidant enzyme activity in tobacco leaves, inhibiting excessive elevation of ROS levels. Transcriptome sequencing analysis showed that overexpression of AnDREB5.1 raised the tolerance of transgenic tobacco seedlings to abiotic stress by regulating multiple genes, including antioxidant enzymes, transcription factors, and stress-tolerant related functional genes like NtCOR413 and NtLEA14. This study provides new evidence for understanding the potential roles of the DREB A5 subgroup members in plants.

Genome-wide identification and functional analysis of the cellulose synthase-like gene superfamily in common oat (Avena sativa L.).

Hemicelluloses constitute approximately one-third of the plant cell wall and can be used as a dietary fiber and food additive, and as raw materials for biofuels. Although genes involved in hemicelluloses synthesis have been investigated in some model plants, no comprehensive analysis has been conducted in common oat at present. In this study, we identified and systematically analyzed the cellulose synthase-like gene (Csl) family members in common oat and investigated them using various bioinformatics tools. The results showed that there are 76 members of the oat Csl gene family distributed on 17 chromosomes, and phylogenetic analysis indicated that the 76 Csl genes belong to the CslA, CslC, CslD, CslE, CslF, CslH, and CslJ subfamilies. A total of 14 classes of cis-acting elements were identified in the promoter regions, including hormone response, light response, cell development, and defense stress elements. The collinearity analysis identified 28 pairs of segmentally duplicated genes, most of which were found on chromosomes 2D and 6A. Expression pattern analysis showed that oat Csl genes display strong tissue-specific expression; of the 76 Csl genes, 33 were significantly up-regulated in stems and 30 were up-regulated in immature seeds. The expression of most members of the AsCsl gene family is repressed by abiotic stress, while the expression of some members is up-regulated by light. Immunoelectron microscopy shows that the product of AsCsl61, a member of CslF subfamily, mediates (1,3; 1,4)-ß-D-glucan synthesis in transgenic Arabidopsis. These findings provide a fundamental understanding of the structural, functional, and evolutionary features of the oat Csl genes and may contribute to our general understanding of hemicellulose biosynthesis. Moreover, this information will be helpful in designing experiments for genetic manipulation of mixed-linkage glucan (MLG) synthesis with the goal of quality improvement in oat.

Responses of Manila Grass (Zoysia matrella) to chilling stress: From transcriptomics to physiology.

Manila grass (Zoysia matrella), a warm-season turfgrass, usually wilts and browns by late autumn because of low temperature. To elucidate the molecular mechanisms regarding Manila grass responses to cold stress, we performed transcriptome sequencing of leaves exposed to 4°C for 0 (CK), 2h (2h_CT) and 72h (72h_CT) by Illumina technology. Approximately 250 million paired-end reads were obtained and de novo assembled into 82,605 unigenes. A total of 34,879 unigenes were annotated by comparing their sequence to public protein databases. At the 2h- and 72h-cold time points, 324 and 5,851 differentially expressed genes (DEGs) were identified, respectively. Gene ontology (GO) and metabolism pathway (KEGG) enrichment analyses of DEGs indicated that auxin, gibberellins, ethylene and calcium took part in the cold signal transduction in the early period. And in the late cold period, electron transport activities, photosynthetic machinery and activity, carbohydrate and nitrogen metabolism, redox equilibrium and hormone metabolism were disturbed. Low temperature stress triggered high light, drought and oxidative stress. At the physiological level, cold stress induced a decrease in water content, an increase in levels of total soluble sugar, free proline and MDA, and changes in bioactive gibberellins levels, which supported the changes in gene expression. The results provided a large set of sequence data of Manila grass as well as molecular mechanisms of the grass in response to cold stress. This information will be helpful for future study of molecular breeding and turf management.

A DREB1 gene from zoysiagrass enhances Arabidopsis tolerance to temperature stresses without growth inhibition.

The DREB (dehydration-responsive element binding) protein family comprises transcription factors that can increase the survivability of a plant under abiotic stresses by regulating expression of multiple genes and altering downstream metabolism at the cost of growth retardation and developmental delay. In this study, a gene for the DREB1-type transcription factor, designated ZjDREB1.4, was isolated from zoysiagrass (Zoysia japonica Steud.), a popular warm-season turfgrass. This gene contains a conserved AP2/ERF DNA-binding domain flanking the signature sequence of DREB1 and belongs to a DREB1 branch in the grass family that expands in the warm-season species. The expression of ZjDREB1.4 was significantly induced by chilling stress (4-15 °C), moderately induced by salt stress, and only slightly induced by drought stress. The product of ZjDREB1.4 was targeted to the nucleus and showed strong transactivation activity but weak binding to the DRE with ACCGAC as the core sequence. The ZjDREB1.4 protein bound to GCCGAC more preferentially than to ACCGAC. Overexpression of ZjDREB1.4 in Arabidopsis induced the expression of multiple genes including a part of the CBF-regulon, and moderately increased the levels of proline and soluble sugars under normal growth conditions. The transgenic Arabidopsis plants showed an increase in tolerance to high and freezing temperature stresses without obvious growth inhibition and with only a few days delay in bolting. ZjDREB1.4 is potentially useful for producing transgenic plants that are tolerant to high temperature and/or cold stresses with few negative effects.

Long-read sequencing and de novo genome assembly of Ammopiptanthus nanus, a desert shrub.

Background: Ammopiptanthus nanus is a rare broad-leaved shrub that is found in the desert and arid regions of Central Asia. This plant species exhibits extremely high tolerance to drought and freezing and has been used in abiotic tolerance research in plants. As a relic of the tertiary period, A. nanus is of great significance to plant biogeographic research in the ancient Mediterranean region. Here, we report a draft genome assembly using the Pacific Biosciences (PacBio) platform and gene annotation for A. nanus. Findings: A total of 64.72 Gb of raw PacBio sequel reads were generated from four 20-kb libraries. After filtering, 64.53 Gb of clean reads were obtained, giving 72.59× coverage depth. Assembly using Canu gave an assembly length of 823.74 Mb, with a contig N50 of 2.76 Mb. The final size of the assembled A. nanus genome was close to the 889 Mb estimated by k-mer analysis. The gene annotation completeness was evaluated using Benchmarking Universal Single-Copy Orthologs; 1,327 of the 1,440 conserved genes (92.15%) could be found in the A. nanus assembly. Genome annotation revealed that 74.08% of the A. nanus genome is composed of repetitive elements and 53.44% is composed of long terminal repeat elements. We predicted 37,188 protein-coding genes, of which 96.53% were functionally annotated. Conclusions: The genomic sequences of A. nanus could be a valuable source for comparative genomic analysis in the legume family and will be useful for understanding the phylogenetic relationships of the Thermopsideae and the evolutionary response of plant species to the Qinghai Tibetan Plateau uplift.

Global Transcriptome Profiles of 'Meyer' Zoysiagrass in Response to Cold Stress.

A long green period is essential for a turfgrass species with high ornamental value and a wide area of use. Zoysiagrasses (Zoysia spp. Willd.) are perennial turfgrass species popular in tropical, subtropical and temperate zones, possessing many properties necessary to be economically useful turfgrass. They do not have a long green period because of cold sensitivity. A main focus in zoysiagrass research is to develop cold tolerant cultivars. Understanding the cold response in zoysiagrass is a fundamental area of research. In the present study, 'Meyer' zoysiagrass (Zoysia japonica), a widely cultivated variety in the genus, is used. We employed RNA-Seq to investigate genome-wide gene expression profiles in leaves under cold stress (4°C). Using the Illumina sequencing platform, we obtained approximately 206 million high-quality paired-end reads from three libraries (0 h, 2 h, and 72 h cold treatment at 4°C). After de novo assembly and quantitative assessment, 46,412 unigenes were generated with an average length of 998 bp and an N50 of 1,522 bp. A total of 25,644 (55.2%) unigenes were annotated by alignment with public protein databases including NR, SwissProt, KEGG and KOG. Differentially expressed genes (DEGs) were investigated using the RPKM method. A total of 756 DEGs were identified between 0 h and 2 h-cold treatment, with 522 up-regulated and 234 down-regulated; and 5327 DEGs were identified between 0 h and 72 h-cold treatment, with 2453 up-regulated and 2874 down-regulated. The expression profile of 15 DEGs selected randomly was confirmed with qRT-PCR. The results suggest that cold stress can induce desiccation and oxidative stress, inhibit photosynthesis and substance transport. In response to the stress, genes involved in proline synthesis, in starch hydrolysis, in methionine and ascorbic acid metabolism, in SOD activity, and in DREBs response pathway were up-regulated. GA metabolism, ABA and JA stimulus response were affected under cold exposure. This is the first transcriptome sequencing of Z. japonica, providing a large set of sequence data as well as gene expression profiles under cold stress. It will improve our current understanding of the cold response of zoysiagrass and be beneficial in breeding research.

Ethnobotanical study on medicinal plants used by Maonan people in China.

BACKGROUND: This paper is based on an ethnobotanical investigation that focused on the traditional medicinal plants used by local Maonan people to treat human diseases in Maonan concentration regions. The Maonan people have relied on traditional medicine since ancient times, especially medicinal plants. The aim of this study is to document medicinal plants used by the Maonans and to report the status of medicinal plants and associated traditional knowledge. METHODS: Ethnobotanical data were collected from June 2012 to September 2014 in Huanjiang Maonan Autonomous County, northern Guangxi, southwest China. In total, 118 knowledgeable informants were interviewed. Following statistically sampling method, eighteen villages from 5 townships were selected to conduct field investigations. Information was collected through the approache of participatory observation, semi-structured interviews, ranking exercises, key informant interviews, focus group discussions, and participatory rural appraisals. RESULTS: A total of 368 medicinal plant species were investigated and documented together with their medicinal uses by the Maonans, most of which were obtained from the wild ecosystems. The plants were used to treat 95 human diseases. Grinding was a widely used method to prepare traditional herbal medicines. There were significant relationships between gender and age, and between gender and informants' knowledge of medicinal plant use. Deforestation for agricultural purposes was identified as the most destructive factor of medicinal plants, followed by drought and over-harvest. CONCLUSIONS: The species diversity of medicinal plants used by the Maonans in the study area was very rich. Medicinal plants played a significant role in healing various human disorders in the Maonan communities. However, the conflicts between traditional inheriting system and recent socio-economic changes (and other factors) resulted in the reduction or loss of both medicinal plants and associated indigenous knowledge. Thus, conservation efforts and policies, and innovation of inheriting system are necessary for protecting the medicinal plants and associated indigenous knowledge. Awareness is also needed to be raised among local Maonans focusing on sustainable utilization and management of both medicinal plants and traditional knowledge.

Transcriptomic Analysis of Drought Stress Responses in Ammopiptanthus mongolicus Leaves Using the RNA-Seq Technique.

Ammopiptanthus mongolicus (Maxim. Ex Kom.) Cheng f., a relic tree of the Tertiary period, plays a critical role in maintaining desert ecosystems in the Mid-Asia region. Genome-scale gene expression profiling studies will provide deep insight into the molecular mechanism underlying the drought tolerance of A. mongolicus. In the present study, we investigated the transcriptional changes induced by drought treatment in A. mongolicus leaves by establishing a comprehensive transcriptome database and then performing a Digital Gene Expression (DGE) analysis using Solexa sequencing technology. A comprehensive transcriptome database was obtained by assembling the Illumina unigenes with expressed sequence tags (EST) available publicly, and other high throughput sequencing data. To analyze the dynamic and complicated gene regulation network during PEG6000-induced drought treatment in leaves of A. mongolicus, a time-course gene expression analysis was performed using tag-based DGE technology, which identified 437, 1,247 and 802 differentially expressed transcripts in 1, 24 and 72 h drought stress libraries, respectively. GO and KEGG analyses revealed hormone signal transduction and phenylpropanoid biosynthesis were enriched during drought treatment. A batch of drought-regulated transcription factor transcripts were identified, including the subsets of HD-ZIP, bZIP, WRKY, AP2/ERF and bHLH family members, which may play roles in drought response in A. mongolicus. The sequence collection assembled in the present study represents one of the most comprehensive transcriptome databases for A. mongolicus currently. The differentially expressed transcripts identified in our study provide a good start for identifying the key genes in stress response and performing functional analysis to reveal their roles in stress adaptation in planta.

Molecular cloning and expression of a Cu/Zn-Containing superoxide dismutase from Thellungiella halophila.

Superoxide dismutases (SODs) constitute the first line of cellular defense against oxidative stress in plants. SODs generally occur in three different forms with Cu/Zn, Fe, or Mn as prosthetic metals. We cloned the full-length cDNA of the Thellungiella halophila Cu/Zn-SOD gene ThCSD using degenerate RT-PCR and rapid amplification of cDNA ends (RACE). Sequence analysis indicated that the ThCSD gene (GenBank accession number EF405867) had an open reading frame of 456 bp. The deduced 152-amino acid polypeptide had a predicted molecular weight of 15.1 kDa, an estimated pI of 5.4, and a putative Cu/Zn-binding site. Recombinant ThCSD protein was expressed in Escherichia coli and assayed for SOD enzymatic activity in a native polyacrylamide gel. The SOD activity of ThCSD was inactivated by potassium cyanide and hydrogen peroxide but not by sodium azide, confirming that ThCSD is a Cu/Zn-SOD. Northern blotting demonstrated that ThCSD is expressed in roots, stems, and leaves. ThCSD mRNA levels increased by about 30-fold when plants were treated with sodium chloride (NaCl), abscisic acid (ABA), and indole-acetic acid (IAA) and by about 50-fold when treated with UVB light. These results indicate that ThCSD is involved in physiological pathways activated by a variety of environmental conditions.