Search Results (14)

Maize is highly sensitive to water deficit but has high transpiration and biomass production, leading to a substantial water demand. Genetic engineering can overcome reproductive isolation and utilize drought-tolerant genes from distant species. Ammopiptanthus nanus is a relic of the Tertiary glaciation that can adapt to harsh environmental conditions. In our previous study, five maize homozygous T₈ lines overexpressing the AnVP1 gene from Ammopiptanthus nanus were generated and showed the enhancement of drought tolerance. However, the recipient inbred line Zh-1 was poor in yield and agronomic performance. In the present study, the AnVP1 gene was backcrossed from donor parent L10 (one of the five T₈ lines) into recurrent parent Chang 7-2 (one of the elite parents of the commercial hybrid Zhengdan 958). In total, 103 InDel markers were developed and used for assisted background selection. After two generations of foreground selection through glufosinate spraying, the detection of CP4 EPSP MAb1 strips, and the PCR amplification of the AnVP1 gene, along with the similarity of agronomic traits to the recurrent parent, and background selection assisted by these InDel markers, the transgenic AnVP1 gene became homozygous in the BC₂ lines. The average recovery rate of the genetic background of the recurrent parent reached 74.80% in the BC₁ population and 91.93% in the BC₂ population, respectively. The results of RT-PCR and RT-qPCR indicated the stable expression of the AnVP1 gene in the two ultimately selected BC₂F₃ lines, BC₂-36-12 and BC₂-5-15. The drought tolerance of these two BC₂F₃ lines were significantly improved compared to the recurrent parent Chang 7-2, as revealed by their wilting phenotype and survival rate of seedlings. This improvement was related to the enhancement of water-retention ability, as indicated by higher RWC and the reduction in damage, as shown by the decrease in REL, MDA, and H₂O₂ under drought stress. The result of field evaluation in two arid and semi-arid environments indicated that the drought tolerance of Chang 7-2 was significantly improved. This study suggests that the improved Chang 7-2 can be crossed with Zheng 58 to develop the transgenic commercial hybrid Zhengdan 958. Full article

Ammopiptanthus nanus, a Tertiary-era endangered plant, is of great scientific value. In this research, we focus on A. nanus population dynamics in an effort to bridge the divide between micro genetic variation and a macroscopic ecological pattern of distribution. The population structure of 129 wild specimens of A. nanus from eight populations was analyzed using EST-SSR molecular markers in this research. The Mantel test and RDA analysis have been used in this research to investigate the factors that influence the genetic diversity of A. nanus. Using 15 pairs of SSR primers, a total of 227 alleles were detected in 129 samples from 8 populations. The mean number of alleles was 17, and the average expected heterozygosity was 0.405. It is shown that wild A. nanus is divided into six individual populations. A. nanus are significantly affected by wind speed in terms of the variation of genetics. It is suggested that a nature conservation area for A. nanus be established as soon as possible, based on our results and the current natural distribution of the species. It is necessary to focus on the issue of pests and diseases while simultaneously preventing the continuation of anthropogenic woodcutting and disaster. Manual seedling collection should be employed in regions where the environment permits. Through making use of manual breeding techniques, this will contribute to the growth of the natural population of A. nanus. Full article

Drought stress is a primary abiotic stress that causes significant losses to forestry and agricultural production. Therefore, exploring drought-responsive genes and their regulatory mechanism is crucial for plant molecular breeding for forestry and agriculture production safety. Small auxin-up RNA (SAUR) proteins are essential in plant growth and development but show functional diversity in stress response. In this study, the transcriptome sequencing data of Ammopiptanthus nanus seedlings revealed that the expression of AnSAUR50 was continuously downregulated under drought stress. Hence, the AnSAUR50 gene was cloned and functionally analyzed in drought response. The results showed that the coding sequence of AnSAUR50 was 315 bp in length and encoded 104 amino acids. The AnSAUR50 protein showed high conservation, possessed a SAUR-specific domain, and localized in the nucleus and cell membrane. The heterologous expression of the AnSAUR50 gene enhanced the drought sensitivity of the transgenic Arabidopsis with a lower survival rate, biomass, and higher malondialdehyde content and relative electrolyte leakage. Moreover, transgenic plants showed shorter root lengths and bigger stomatal apertures, resulting in facilitating water loss under drought stress. The study indicates that AnSAUR50 negatively regulates drought tolerance by inhibiting root growth and stomatal closure, which provides insights into the underlying function and regulatory mechanism of SAURs in plant stress response. Full article

R2R3-MYB transcription factors (TFs) participate in the modulation of plant development, secondary metabolism, and responses to environmental stresses. Ammopiptanthus nanus, a leguminous dryland shrub, tolerates a high degree of environmental stress, including drought and low-temperature stress. The systematic identification, structural analysis, evolutionary analysis, and gene profiling of R2R3-MYB TFs under cold and osmotic stress in A. nanus were performed. Up to 137 R2R3-MYB TFs were identified and clustered into nine clades, with most A. nanus R2R3-MYB members belonging to clade VIII. Tandem and segmental duplication events drove the expansion of the A. nanus R2R3-MYB family. Expression profiling revealed that multiple R2R3-MYB genes significantly changed under osmotic and cold stress conditions. MiR858 and miR159 targeted 88 R2R3-MYB genes. AnaMYB87, an miR858-targeted clade VIII R2R3-MYB TF, was up-regulated under both osmotic and cold stress. A transient expression assay in apples showed that the overexpression of AnaMYB87 promoted anthocyanin accumulation. A luciferase reporter assay in tobacco demonstrated that AnaMYB87 positively affected the transactivation of the dihydroflavonol reductase gene, indicating that the miR858-MYB87 module mediates anthocyanin accumulation under osmotic stress by regulating the dihydroflavonol reductase gene in A. nanus. This study provides new data to understand the roles of R2R3-MYB in plant stress responses. Full article

Ammopiptanthus possesses ancestral traits and, as a tertiary relict, is one of the surviving remnants of the ancient Mediterranean retreat and climate drought. It is also the only genus of super xerophytic, evergreen, broad-leaved shrubs. Ammopiptanthus nanus, one of the two species in this genus, is predominantly found in extremely arid and frigid environments, and is increasingly threatened with extinction. Study of the species’ genetic diversity is thus beneficial for its survival and the efficacy of ex situ conservation efforts. Based on transcriptome data, 15 pairs of effective EST-SSR were screened to evaluate A. nanus genetic diversity. In all, 87 samples from three populations were evaluated, the results of which show that ex situ conservation in the Wuqia region needs to be supplemented. Conservation and breeding of individual A. nanus offspring should be strengthened in the future to ensure their progeny continue to exhibit high genetic diversity and variation. Full article

Ammopiptanthus nanus (M. Pop.) Cheng f. is a very important resource plant that integrates soil and water conservation, afforestation of barren mountains, and ornamental, medicinal, and scientific research functions and is also a critically endangered plant in China, remaining in only six small fragmented populations in the wild. These populations have been suffering from severe anthropomorphic disturbances, causing further losses in genetic diversity. However, its genetic diversity level and genetic differentiation degree among the fragmented populations are still unclear. Inthis study, DNA was extracted from fresh leaves from the remnant populations of A. nanus, and the inter-simple-sequence repeat (ISSR) molecular marker system was used to assess its level of genetic diversity and differentiation. The result was that its genetic diversity is low at both species and population levels, with only 51.70% and 26.84% polymorphic loci, respectively. The Akeqi population had the highest genetic diversity, whereas the Ohsalur and Xiaoerbulak populations had the lowest. There was significant genetic differentiation among the populations, and the value of the genetic differentiation coefficient (Gst) was as high as 0.73, while the gene flow value was as low as 0.19 owing to spatial fragmentation and a serious genetic exchange barrier among the populations. It is suggested that a nature reserve and germplasm banks should be established as soon as possible for elimination of the anthropomorphic disturbances, and mutual introductions between the populations and introduced patches of the species, such as with habitat corridors or stepping stones, should be performed simultaneously to improve the genetic diversity of the isolated populations for the conservation of this plant. Full article

Long non-coding RNAs (lncRNAs) have been shown to play critical regulatory roles in plants. Ammopiptanthus nanus can survive under severe low-temperature stress, and lncRNAs may play crucial roles in the gene regulation network underlying the cold stress response in A. nanus. To investigate the roles of lncRNAs in the cold stress response of A. nanus, a combined lncRNA and mRNA expression profiling under cold stress was conducted. Up to 4890 novel lncRNAs were identified in A. nanus and 1322 of them were differentially expressed under cold stress, including 543 up-regulated and 779 down-regulated lncRNAs. A total of 421 lncRNAs were found to participate in the cold stress response by forming lncRNA–mRNA modules and regulating the genes encoding the stress-related transcription factors and enzymes in a cis-acting manner. We found that 31 lncRNAs acting as miRNA precursors and 8 lncRNAs acting as endogenous competitive targets of miRNAs participated in the cold stress response by forming lncRNA–miRNA–mRNA regulatory modules. In particular, a cold stress-responsive lncRNA, TCONS00065739, which was experimentally proven to be an endogenous competitive target of miR530, contributed to the cold stress adaptation by regulating TZP in A. nanus. These results provide new data for understanding the biological roles of lncRNAs in response to cold stress in plants. Full article

The uplift of the Qinghai Tibet Plateau has led to a drastic change in the climate in Central Asia, from warm and rainy, to dry and less rainfall. Ammopiptanthus nanus, a rare evergreen broad-leaved shrub distributed in the temperate desert region of Central Asia, has survived the drastic climate change in Central Asia caused by the uplift of the Qinghai-Tibet Plateau. Ascorbate oxidase (AO) regulates the redox status of the apoplast by catalyzing the oxidation of ascorbate acid to dehydroascorbic acid, and plays a key role in the adaptation of plants to environmental changes. Analyzing the evolution, environmental response, and biological functions of the AO family of A. nanus is helpful for understanding how plant genome evolution responds to climate change in Central Asia. A total of 16 AOs were identified in A. nanus, all of which contained the ascorbate oxidase domain, most of which contained transmembrane domain, and many were predicted to be localized in the apoplast. Segmental duplication and tandem duplication are the main factors driving the gene amplification of the AO gene family in A. nanus. Gene expression analysis based on transcriptome data and fluorescence quantitative PCR, as well as enzyme activity measurements, showed that the expression levels of AO genes and total enzyme activity decreased under short-term osmotic stress and low-temperature stress, but the expression of some AO genes (AnAO5, AnAO13, and AnAO16) and total enzyme activity increased under 7 days of cold stress. AnAO5 and AnAO11 are targeted by miR4415. Further functional studies on AnAO5 showed that AnAO5 protein was localized in the apoplast. The expression of AnAO5 in yeast cells and the transient expression in tobacco enhanced the tolerance of yeast and tobacco to low-temperature stress, and the overexpression of AnAO5 enhanced the tolerance of Arabidopsis seedlings to cold stress. Our research provides important data for understanding the role of AOs in plant adaptation to environmental change. Full article

Thaumatin-like proteins (TLPs), a family of proteins with high sequence similarity to thaumatin, are shown to be involved in plant defense, and are thus classified into the pathogenesis related protein family 5. Ammopiptanthus nanus is a rare evergreen broad-leaved shrub distributed in the temperate zone of Central Asia, which has a high tolerance to low-temperature stress. To characterize A. nanus TLPs and understand their roles in low-temperature response in A. nanus, a comprehensive analysis of the structure, evolution, and expression of TLP family proteins was performed. A total of 31 TLP genes were detected in the A. nanus genome, and they were divided into four groups based on their phylogenetic positions. The majority of the AnTLPs contained the conserved cysteine residues and were predicted to have the typical three-dimensional structure of plant TLPs. The primary modes of gene duplication of the AnTLP family genes were segmental duplication. The promoter regions of most AnTLP genes contain multiple cis-acting elements related to environmental stress response. Gene expression analysis based on transcriptome data and fluorescence quantitative PCR analysis revealed that several AnTLP genes were involved in cold-stress response. We further showed that a cold-induced AnTLP gene, AnTLP13, was localized in apoplast, and heterologous expression of the AnTLP13 in Escherichia coli and yeast cells and tobacco leaves enhanced low-temperature stress tolerance when compared with the control cells or seedlings. Our study provided important data for understanding the roles of TLPs in plant response to abiotic stress. Full article

Sucrose non-fermenting-1 (SNF1)-related protein kinase 2’s (SnRK2s) are plant-specific serine/threonine protein kinases and play crucial roles in the abscisic acid signaling pathway and abiotic stress response. Ammopiptanthus nanus is a relict xerophyte shrub and extremely tolerant of abiotic stresses. Therefore, we performed genome-wide identification of the AnSnRK2 genes and analyzed their expression profiles under osmotic stresses including drought and salinity. A total of 11 AnSnRK2 genes (AnSnRK2.1-AnSnRK2.11) were identified in the A. nanus genome and were divided into three groups according to the phylogenetic tree. The AnSnRK2.6 has seven introns and others have eight introns. All of the AnSnRK2 proteins are highly conserved at the N-terminus and contain similar motif composition. The result of cis-acting element analysis showed that there were abundant hormone- and stress-related cis-elements in the promoter regions of AnSnRK2s. Moreover, the results of quantitative real-time PCR exhibited that the expression of most AnSnRK2s was induced by NaCl and PEG-6000 treatments, but the expression of AnSnRK2.3 and AnSnRK2.6 was inhibited, suggesting that the AnSnRK2s might play key roles in stress tolerance. The study provides insights into understanding the function of AnSnRK2s. Full article

The WRKY family of transcription factors plays important roles in plant growth and responses to biotic and abiotic stresses. Ammopiptanthus nanus, the only evergreen broadleaf shrub endemic to the desert and semi-desert regions of northwestern China, is highly tolerant to various stresses. However, a systematic study of WRKY proteins in A. nanus has not been reported. In the present study, we identified 63 WRKY genes in the A. nanus genome. Based on the conserved WRKY domains, zinc finger structures, and phylogenetic relationships in their encoded proteins, we classified these genes into four groups (group I–IV) and several subgroups (subgroup IIa–IIe). Conserved motif analysis showed that all motifs except those within the WRKY domains had a subfamily-specific distribution. Expression analysis revealed that the AnWRKY genes had distinct expression patterns, with some being more responsive to herbivory and drought stresses than others. Based on the results of our current study, we speculate that AnWRKY40 and AnWRKY48 are positive regulators of the plant’s response to drought and herbivory stresses, respectively. Our results indicate that AnWRKY genes contribute to the ability of A. nanus plants to withstand harsh, dry conditions. Full article

APETALA2/ethylene-responsive factor (AP2/ERF) is one of the largest transcription factor (TF) families in plants, which play important roles in regulating plant growth, development, and response to environmental stresses. Ammopiptanthus nanus, an unusual evergreen broad-leaved shrub in the arid region in the northern temperate zone, demonstrates a strong tolerance to low temperature and drought stresses, and AP2/ERF transcription factors may contribute to the stress tolerance of A. nanus. In the current study, 174 AP2/ERF family members were identified from the A. nanus genome, and they were divided into five subfamilies, including 92 ERF members, 55 dehydration-responsive element binding (DREB) members, 24 AP2 members, 2 RAV members, and 1 Soloist member. Compared with the other leguminous plants, A. nanus has more members of the DREB subfamily and the B1 group of the ERF subfamily, and gene expansion in the AP2/ERF family is primarily driven by tandem and segmental duplications. Promoter analysis showed that many stress-related cis-acting elements existed in promoter regions of the DREB genes, implying that MYB, ICE1, and WRKY transcription factors regulate the expression of DREB genes in A. nanus. Expression profiling revealed that the majority of DREB members were responsive to osmotic and cold stresses, and several DREB genes such as EVM0023336.1 and EVM0013392.1 were highly induced by cold stress, which may play important roles in cold response in A. nanus. This study provided important data for understanding the evolution and functions of AP2/ERF and DREB transcription factors in A. nanus. Full article

Expansins, cell-wall loosening proteins, play an important role in plant growth and development and abiotic stress tolerance. Ammopiptanthus nanus (A. nanus) is an important plant to study to understand stress resistance in forestry. In our previous study, two α-type expansins from A. nanus were cloned and named AnEXPA1 and AnEXPA2. In this study, we found that they responded to different abiotic stress and hormone signals. It suggests that they may play different roles in response to abiotic stress. Their promoters show some of the same element responses to abiotic stress and hormones, but some special elements were identified between the expansins that could be essential for their expression. In order to further testify the reliability of the above results, we conducted an analysis of β-glucuronidase (GUS) dyeing. The analysis showed that AnEXPA1 was only induced by cold stress, whereas AnEXPA2 responded to hormone induction. AnEXPA1 and AnEXPA2 transgenic Arabidopsis plants showed better tolerance to cold and drought stresses. Moreover, the ability to scavenge reactive oxygen species (ROS) was significantly improved in the transgenic plants, and expansin activity was enhanced. These results suggested that AnEXPA1 and AnEXPA2 play an important role in the response to abiotic stress. Our research contributes to a better understanding of the regulatory network of expansins and may benefit agricultural production. Full article

Chitinase is a kind of hydrolase with chitin as a substrate and is proposed to play an essential role in plant defense system by functioning against fungal pathogens through degrading chitin. Recent studies indicated chitinase is also involved in abiotic stress response in plants, helping plants to survive in stressful environments. A. nanus, a rare evergreen broad-leaved shrub distrusted in deserts in Central Asia, exhibits a high level of tolerance to drought and low temperature stresses. To identify the chitinase gene involved in drought and low temperature responses in A. nanus, we performed genome-wide identification, classification, sequence alignment, and spatio-temporal gene expression analysis of the chitinases in A. nanus under osmotic and low temperature stress. A total of 32 chitinase genes belonging to glycosyl hydrolase 18 (GH18) and GH19 families were identified from A. nanus. Class III chitinases appear to be amplified quantitatively in A. nanus, and their genes carry less introns, indicating their involvement in stress response in A. nanus. The expression level of the majority of chitinases varied in leaves, stems, and roots, and regulated under environmental stress. Some chitinases, such as EVM0022783, EVM0020238, and EVM0003645, are strongly induced by low temperature and osmotic stress, and the MYC/ICE1 (inducer of CBF expression 1) binding sites in promoter regions may mediate the induction of these chitinases under stress. These chitinases might play key roles in the tolerance to these abiotic stress in A. nanus and have potential for biotechnological applications. This study provided important data for understanding the biological functions of chitinases in A. nanus. Full article