Genome-Wide Identification and Expression Analysis of the HSF Gene Family in Ammopiptanthus mongolicus.

Ammopiptanthus mongolicus is an ancient remnant species from the Mediterranean displaying characteristics such as high-temperature tolerance, drought resistance, cold resistance, and adaptability to impoverished soil. In the case of high-temperature tolerance, heat shock transcription factors (HSFs) are integral transcriptional regulatory proteins exerting a critical role in cellular processes. Despite extensive research on the HSF family across various species, there has been no analysis specifically focused on A. mongolicus. In this study, we identified 24 members of the AmHSF gene family based on the genome database of A. mongolicus, which were unevenly distributed over 9 chromosomes. Phylogenetic analysis showed that these 24 members can be categorized into 5 primary classes consisting of a total of 13 subgroups. Analysis of the physical and chemical properties revealed significant diversity among these proteins. With the exception of the AmHSFB3 protein, which is localized in the cytoplasm, all other AmHSF proteins were found to be situated in the nucleus. Comparison of amino acid sequences revealed that all AmHSF proteins contain a conserved DNA-binding domains structure, and the DNA-binding domains and oligomerization domains of the AmHSF gene exhibit conservation with counterparts across diverse species; we investigated the collinearity of AmHSF genes in relation to those of three other representative species. Through GO enrichment analysis, evidence emerged that AmHSF genes are involved in heat stress responses and may be involved in multiple transcriptional regulatory pathways that coordinate plant growth and stress responses. Finally, through a comprehensive analysis using transcriptome data, we examined the expression levels of 24 AmHSFs under 45 °C. The results revealed significant differences in the expression profiles of AmHSFs at different time intervals during exposure to high temperatures, highlighting their crucial role in responding to heat stress. In summary, these results provide a better understanding of the role and regulatory mechanisms of HSF in the heat stress response of A. mongolicus, meanwhile also establishing a foundation for further exploration of the biological functions of AmHSF in the adversity response of A. mongolicus.

First report of Neocosmospora pisi causing blight on Ammopiptanthus mongolicus in China.

Ammopiptanthus mongolicus is the only evergreen broad-leaved shrub in the desert region of Northwest China, which is one of the dominant species in the desert vegetation of the region, playing an important role in maintaining the stability of the local desert ecosystem. A. mongolicus is also very hardy and drought resistant and can survive extreme temperatures (Liu et al. 2013; Yang et al. 2022). The large-scale death of A. mongolicus could cause desertification in the region. Two months after the discovery of Fusarium verticillioides causing blight on A. mongolicus in Etuoke county, Inner Mongolia Autonomous Region in September 2023 (Yang et al. 2024), a large number of A. mongolicus plants with symptoms of blights were found in Lingwu city, Ningxia Hui Autonomous Region, China (106.442368°E, 37.734026°N) in November 2023. The incidence of diseased plants in this field was about 30%. The field symptoms in Lingwu city were similar to those observed in Etuoke county. The diseased leaves initially turned yellow, then wilted and dehisced, eventually resulting in plant death (Figure 1). The roots of the diseased plants were cut diagonally and the central cylinder showed a brown color (Figure 2). In order to investigate whether the death of A. mongolicus was caused by the same pathogen as those identified previously, 30 roots were collected from 10 diseased plants. After rinsing and surface sterilization (70% ethanol for 3 min and 2.5% NaClO for 5 min, rinsed 3 times with sterile distilled water), diseased tissues (10×10 mm) were placed on potato dextrose agar (PDA) (3 pieces per plate) and incubated from 3 to 5 days at 25°C. The strain AmP5 was isolated and used for further study. After 3 days on PDA medium, fungal colonies were white to milky, the undersides of the cultures were yellowish to orange-brown (Figure 3). After 7 days on synthetic nutrient-poor agar (SNA), microconidia were ovoidal or with a rounded apex and truncate base, 10.5 ± 1.5 µm × 1.6 ± 0.2 µm (×400). The macroconidia were slightly curved or arcuate, 40.5 ± 3.5 µm × 5 ± 0.5 µm (×400) (Figure 4) (Sisic et al. 2018). The pathogen was confirmed to be Neocosmospora pisi by multigene phylogenetic analysis of TEF, RPB1 and RPB2 genes using primers EF1/EF2, F5/G2R and 5F2/11AR, respectively (O'Donnell et al. 2022). The sequences of PCR products were deposited in GenBank with accession numbers OR944631 (RPB1), OR988086 (TEF) and OR988087 (RPB2), respectively. The results of pairwise alignment in Fusarioid-ID database (Crous et al. 2021) showed 99.84% similarity and 83.96% overlap of the EF1-α sequence to the corresponding sequence LR583636 of ex-epitype CBS 123669 of Neocosmospora pisi (syn. Fusarium solani f. sp. pisi), 99.72% similarity and 85.66% overlap of the RPB1 sequence to the corresponding sequence MW834242 of ex-epitype CBS 123669 of N. pisi, and 99.47% similarity and 78.26% overlap of RPB2 sequence to the corresponding sequence LR583862 of ex-epitype CBS 123669 of N. pisi. Moreover, the result of polyphasic identification in the Fusarioid-ID database also showed EF1-a, RPB1, and RPB2 sequences had 99.15% similarity to the corresponding sequences of CBS 1233669. The pathogenicity of AmP5 was tested on potted 64 days old seedlings A. mongolicus plants. The roots of 3 seedlings were inoculated with conidial suspension (1×106 /ml), and another 3 used as controls were inoculated with sterile water, by gently peeling off the soil around the roots during inoculation, and pouring the conidial suspension around the roots (10 ml/seedling). All plants were placed in a growth chamber at 18-25℃ (10 h light; 14 h dark). After incubation for 3-5 days, the symptoms similar to those observed in the field (Figure 5), including brown rot of steles (Figure 6), developed on plants inoculated with conidial suspension, whereas no symptoms were observed on the control plants. The same pathogen was reisolated from inoculated roots and confirmed as N. pisi based on morphological and molecular analyses (TEF, RPB1 and RPB2). To our knowledge, this is the first report of blight on A. mongolicus caused by N. pisi in China. This study also indicates that blight on A. mongolicus can be caused by different fungal pathogens. Blight caused by different pathogens may have different in terms of control measures and pathogenic mechanisms, so the study of blight caused by different pathogens is of profound value.

First report of Fusarium verticillioides causing blight on Ammopiptanthus mongolicus in China.

Ammopiptanthus mongolicus is the only evergreen broadleaf shrub and constructive species in the northwest desert of China (Hu et al. 2021). It also is listed as one of the national second-class endangered plants. Ammopiptanthus mongolicus has a good effect of water conservation, windbreak and sand fixation because its deep root system (Zhou et al. 2012; Dong et al. 2023). A large number of dead plants of Ammopiptanthus mongolicus were found in Etuoke county, Inner Mongolia Autonomous Region, China (40°4'28″-40°4'34″ N, 106°53'5″-106°53'31″ E). In September 2023, the investigation and research in the region found that the incidence of diseased plants in this field was about 30%, and for individual plant, the incidence of diseased branches was about 60%. The leaves of diseased branches initially became from green to yellow and then wilt and fall. Eventually the plant dies. (Figure 1). The miter cut of the root showed that the root steles of diseased plants had obvious black and brown color (Figure 2). For isolation, the 30 tissue blocks (10×10 mm) of from 10 symptomatic roots diseased were surface sterilized with 70% ethanol for 3 minutes and sodium hypochlorite (2.5% available chlorine) for 5 minutes, and rinsed three times with sterilized distilled water. Then, these tissue blocks were placed on potato dextrose agar (PDA) medium, and incubated from 3 to 5 days at 25°C. After 3 days on PDA, the surface of the colony was rough, the color were white-pink at the beginning, and deep purple pigment were produced in the later stage, making the colony bluish-purple to gray-purple, their undersides were bluish-purple. Mycelia were white. After 7 days on SNA, Microconidia were typical of the clavicular type, 8.5 ± 2.5 µm × 2.3 ± 0.2 µm(×400). Microconidia were usually very long conidial chains, sometimes the spore chain collapses and the conidia clump together to form an approximate pseudocephaly. The macroconidia were slender and long, slightly falcate or straight, 42.8 ± 3.4 µm × 3.8 ± 0.7 µm(×400) (Figure 4). Species identity was confirmed by sequencing the EF1-α gene (EF1 and EF2 primers)(O'Donnell et al. 1998), RPB1 (F5 and G2R primers)(O'Donnell et al. 2022) and RPB2 (5F2 and 11AR)(O'Donnell et al. 2022). The amplified sequences of a representative isolate (AmP10) have deposited in GenBank with accession number OR594338 (EF1-α), OR841329 (RPB1) and OR841331 (RPB2). Thee results of pairwise alignment in Fusarioid-ID datebase(Crous et al. 2021) showed that EF1-α sequence was 99.54% similarity and 89.96% overlap to the corresponding sequence KF499582 of ex-epitype CBS 218.76 of Fusarium verticillioides, Fusarium fujikuroi species complex (FFSC, previously GFSC) (Lecellier et al. 2014), RPB1 sequence was 100% similarity and 100% overlap to the corresponding sequence MW402638 of ex-epitype CBS 218.76 of Fusarium verticillioides, Fusarium fujikuroi species complex (FFSC, previously GFSC) (Yilmaz et al. 2021), RPB2 sequence was 99.94% similarity and 87.83% overlap to the corresponding sequence MW928835 of ex-epitype CBS 218.76 of Fusarium verticillioides, Fusarium fujikuroi species complex (FFSC, previously GFSC) (Crous et al. 2021). Moreover, the result of polyphasic identification in Fusarioid-ID datebase also showed EF1-α, RPB1 and RPB2 sequences were 100% similarity to the corresponding sequences of ex-epitype CBS 218.76 of Fusarium verticillioides, Fusarium fujikuroi species complex (FFSC, previously GFSC). To test the pathogenicity, the healthy green seedlings (64 days old) were planted into plastic pots containing sterilized soil in the greenhouse after the seeds of Ammopiptanthus mongolicus were surface sterilized with 70% ethanol for 3 minutes and 2.5% sodium hypochlorite for 3 minutes. The roots of 3 seedlings were inoculated with 1×106 /ml of the conidial suspension, and another 3 used as controls with inoculated sterile water. Then, all pots were placed in a greenhouse maintained at 18°C to 25°C. After incubation for 3-5 days, the typical symptoms similar to the symptoms in the field (Figure 5), brown root steles (Figure 6), developed on the plants inoculated with conidial suspension, whereas no symptoms were observed on the control plants. The same pathogen was consistently reisolated from the inoculated roots and confirmed as Fusarium verticillioides based on morphological and molecular analyses. To our knowledge, this is the first report of Fusarium verticillioides on Ammopiptanthus mongolicus in China. This study provides a basis for identifying pathogens causing blight on Ammopiptanthus mongolicus and managing the disease.

Kribbella deserti sp. nov., isolated from rhizosphere soil of Ammopiptanthus mongolicus.

A Gram-stain-positive, aerobic bacterial strain, designated SL15-1T, was isolated from desert soil which was sampled from the rhizosphere of Ammopiptanthus mongolicus, Hangjin Banner, Ordos, Inner Mongolia, northern China. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain SL15-1T was clustered with Kribbella strains, sharing the highest similarity of 16S rRNA gene sequence (96.97 %) with Kribbella sandramycini DSM 15626T. Strain SL15-1T contained diphosphatidylglycerol, phosphatidylglycerol, phosphatidylcholine, an unknown phospholipid, an unknown lipid and two unknown aminophospholipids as the major polar lipids. MK-9(H4) was the predominant menaquinone, while anteiso-C15 : 0, iso-C16 : 0, C17 : 1ω8c and iso-C14 : 0 were the major cellular fatty acids. Its genomic DNA G+C content was 65.3 mol%. The results of physiological and biochemical tests allowed the discrimination of strain SL15-1T from its phylogenetic relatives. Kribbella deserti sp. nov. is therefore proposed with strain SL15-1T (=CGMCC 1.15906T=KCTC 39825T) as the type strain.

Quantitative Phosphoproteomic Analysis Provides Insight into the Response to Short-Term Drought Stress in Ammopiptanthus mongolicus Roots.

Drought is one of the major abiotic stresses that negatively affects plant growth and development. Ammopiptanthus mongolicus is an ecologically important shrub in the mid-Asia desert region and used as a model for abiotic tolerance research in trees. Protein phosphorylation participates in the regulation of various biological processes, however, phosphorylation events associated with drought stress signaling and response in plants is still limited. Here, we conducted a quantitative phosphoproteomic analysis of the response of A. mongolicus roots to short-term drought stress. Data are available via the iProx database with project ID IPX0000971000. In total, 7841 phosphorylation sites were found from the 2019 identified phosphopeptides, corresponding to 1060 phosphoproteins. Drought stress results in significant changes in the abundance of 103 phosphopeptides, corresponding to 90 differentially-phosphorylated phosphoproteins (DPPs). Motif-x analysis identified two motifs, including [pSP] and [RXXpS], from these DPPs. Functional enrichment and protein-protein interaction analysis showed that the DPPs were mainly involved in signal transduction and transcriptional regulation, osmotic adjustment, stress response and defense, RNA splicing and transport, protein synthesis, folding and degradation, and epigenetic regulation. These drought-corresponsive phosphoproteins, and the related signaling and metabolic pathways probably play important roles in drought stress signaling and response in A. mongolicus roots. Our results provide new information for understanding the molecular mechanism of the abiotic stress response in plants at the posttranslational level.

Complex interactions among residues within pore region determine the K+ dependence of a KAT1-type potassium channel AmKAT1.

KAT1-type channels mediate K(+) influx into guard cells that enables stomatal opening. In this study, a KAT1-type channel AmKAT1 was cloned from the xerophyte Ammopiptanthus mongolicus. In contrast to most KAT1-type channels, its activation is strongly dependent on external K(+) concentration, so it can be used as a model to explore the mechanism for the K(+) -dependent gating of KAT1-type channels. Domain swapping between AmKAT1 and KAT1 reveals that the S5-pore-S6 region controls the K(+) dependence of AmKAT1, and residue substitutions show that multiple residues within the S5-Pore linker and Pore are involved in its K(+) -dependent gating. Importantly, complex interactions occur among these residues, and it is these interactions that determine its K(+) dependence. Finally, we analyzed the potential mechanism for the K(+) dependence of AmKAT1, which could originate from the requirement of K(+) occupancy in the selectivity filter to maintain its conductive conformation. These results provide new insights into the molecular basis of the K(+) -dependent gating of KAT1-type channels.

Functional identification of a GORK potassium channel from the ancient desert shrub Ammopiptanthus mongolicus (Maxim.) Cheng f.

KEY MESSAGE: A GORK homologue K(+) channel from the ancient desert shrub Ammopiptanthus mongolicus (Maxim.) Cheng f. shows the functional conservation of the GORK channels among plant species. Guard cell K(+) release through the outward potassium channels eventually enables the closure of stomata which consequently prevents plant water loss from severe transpiration. Early patch-clamp studies with the guard cells have revealed many details of such outward potassium currents. However, genes coding for these potassium-release channels have not been sufficiently characterized from species other than the model plant Arabidopsis thaliana. We report here the functional identification of a GORK (for Gated or Guard cell Outward Rectifying K(+) channels) homologue from the ancient desert shrub Ammopiptanthus mongolicus (Maxim.) Cheng f. AmGORK was primary expressed in shoots, where the transcripts were regulated by stress factors simulated by PEG, NaCl or ABA treatments. Patch-clamp measurements on isolated guard cell protoplasts revealed typical depolarization voltage gated outward K(+) currents sensitive to the extracelluar K(+) concentration and pH, resembling the fundamental properties previously described in other species. Two-electrode voltage-clamp analysis in Xenopus lavies oocytes with AmGORK reconstituted highly similar characteristics as assessed in the guard cells, supporting that the function of AmGORK is consistent with a crucial role in mediating stomatal closure in Ammopiptanthus mongolicus. Furthermore, a single amino acid mutation D297N of AmGORK eventually abolishes both the voltage-gating and its outward rectification and converts the channel into a leak-like channel, indicating strong involvement of this residue in the gating and voltage dependence of AmGORK. Our results obtained from this anciently originated plant support a strong functional conservation of the GORK channels among plant species and maybe also along the progress of revolution.

Chemical and Transcriptomic Analyses of Leaf Cuticular Wax Metabolism in Ammopiptanthus mongolicus under Osmotic Stress.

Plant cuticular wax forms a hydrophobic structure in the cuticle layer covering epidermis as the first barrier between plants and environments. Ammopiptanthus mongolicus, a leguminous desert shrub, exhibits high tolerances to multiple abiotic stress. The physiological, chemical, and transcriptomic analyses of epidermal permeability, cuticular wax metabolism and related gene expression profiles under osmotic stress in A. mongolicus leaves were performed. Physiological analyses revealed decreased leaf epidermal permeability under osmotic stress. Chemical analyses revealed saturated straight-chain alkanes as major components of leaf cuticular wax, and under osmotic stress, the contents of total wax and multiple alkane components significantly increased. Transcriptome analyses revealed the up-regulation of genes involved in biosynthesis of very-long-chain fatty acids and alkanes and wax transportation under osmotic stress. Weighted gene co-expression network analysis identified 17 modules and 6 hub genes related to wax accumulation, including 5 enzyme genes coding KCS, KCR, WAX2, FAR, and LACS, and an ABCG transporter gene. Our findings indicated that the leaf epidermal permeability of A. mongolicus decreased under osmotic stress to inhibit water loss via regulating the expression of wax-related enzyme and transporter genes, further promoting cuticular wax accumulation. This study provided new evidence for understanding the roles of cuticle lipids in abiotic stress tolerance of desert plants.

AmDHN4, a winter accumulated SKn-type dehydrin from Ammopiptanthus mongolicus, and regulated by AmWRKY45, enhances the tolerance of Arabidopsis to low temperature and osmotic stress.

Ammopiptanthus mongolicus, a rare temperate evergreen broadleaf shrub, exhibits remarkable tolerance to low temperature and drought stress in winter. Late embryogenesis abundant (LEA) proteins, a kind of hydrophilic protein with a protective function, play significant roles in enhancing plant tolerance to abiotic stress. In this present study, we analyzed the evolution and expression of LEA genes in A. mongolicus, and investigated the function and regulatory mechanism of dehydrin under abiotic stresses. Evolutionary analysis revealed that 14 AmLEA genes underwent tandem duplication events, and 36 AmLEA genes underwent segmental duplication events Notably, an expansion in SKn-type dehydrins was observed. Expression analysis showed that AmDHN4, a SKn-type dehydrin, was up-regulated in winter and under low temperature and osmotic stresses. Functional analysis showcased that the heterologous expression of the AmDHN4 enhanced the tolerance of yeast and tobacco to low temperature stress. Additionally, the overexpression of AmDHN4 significantly improved the tolerance of transgenic Arabidopsis to low temperature, drought, and osmotic stress. Further investigations identified AmWRKY45, a downstream transcription factor in the jasmonic acid signaling pathway, binding to the AmDHN4 promoter and positively regulating its expression. In summary, these findings contribute to a deeper understanding of the functional and regulatory mechanisms of dehydrin.

Characterization of NAC Gene Family in Ammopiptanthus mongolicus and Functional Analysis of AmNAC24, an Osmotic and Cold-Stress-Induced NAC Gene.

The NAC family of transcription factors (TFs) is recognized as a significant group within the plant kingdom, contributing crucially to managing growth and development processes in plants, as well as to their response and adaptation to various environmental stressors. Ammopiptanthus mongolicus, a temperate evergreen shrub renowned for its remarkable resilience to low temperatures and drought stress, presents an ideal subject for investigating the potential involvement of NAC TFs in stress response mechanisms. Here, the structure, evolution, and expression profiles of NAC family TFs were analyzed systematically, and a cold and osmotic stress-induced member, AmNAC24, was selected and functionally characterized. A total of 86 NAC genes were identified in A. mongolicus, and these were divided into 15 groups. Up to 48 and 8 NAC genes were generated by segmental duplication and tandem duplication, respectively, indicating that segmental duplication is a predominant mechanism in the expansion of the NAC gene family in A. mongolicus. A considerable amount of NAC genes, including AmNAC24, exhibited upregulation in response to cold and osmotic stress. This observation is in line with the detection of numerous cis-acting elements linked to abiotic stress response in the promoters of A. mongolicus NAC genes. Subcellular localization revealed the nuclear residence of the AmNAC24 protein, coupled with demonstrable transcriptional activation activity. AmNAC24 overexpression enhanced the tolerance of cold and osmotic stresses in Arabidopsis thaliana, possibly by maintaining ROS homeostasis. The present study provided essential data for understanding the biological functions of NAC TFs in plants.

Isolation and functional characterization of cold-induced gene (AmCIP) promoter from Ammopiptanthus mongolicus.

AmCIP is a dehydrin-like protein which involved in abiotic stress tolerance in xerophytes evergreen woody plant A. mongolicus. AmCIP could be induced in the cotyledon and radicle during cold acclimation. To further elucidate the regulation of the upstream region of the gene, we isolated and characterized the promoter of AmCIP. Herein, a 1115 bp 5'-flanking region of AmCIP genomic DNA was isolated and cloned by genome walking from A. mongolicus and the segment sequence was identified as "PrAmCIP" promoter. Analysis of the promoter sequence revealed the presences of some basic cis-acting elements, which were related to various environmental stresses and plant hormones. GUS histochemical staining of transgene tobacco showed that PrAmCIP was induced by 4℃, 55℃, NaCl, mannitol and ABA, whereas it could hardly drive GUS gene expression under normal conditions. Furthermore, we constructed three deletion fragments and genetically transformed them into Arabidopsis thaliana. GUS histochemical staining showed that the MYCATERD1 element of the CP7 fragment (-189 âˆ¼ -1) may be a key element in response to drought. In conclusion, we provide an inducible promoter, PrAmCIP, which can be applied to the development of transgenic plants for abiotic stresse tolerance.

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The stoichiometric characteristics of leaves can reflect environmental adaptation of plants, and thus the study of the relationship between them is helpful for exploring plant adaptation strategies. In this study, taking the national second-level key protection species, Ammopiptanthus mongolicus, as the research object, we set up 26 plots to collect samples, and measured the content of carbon (C), nitrogen (N), phosphorus (P) and water use efficiency (WUE) of leaves. We analyzed the relationship between leaf stoichiometric characteristics and WUE, and quantified the contributions of soil, climate, and water use efficiency to the variations of leaf stoichiometry. The results showed that C, N, and P contents in the leaves were (583.99±27.93), (24.31±2.09), and (1.83±0.06) mg·g-1, respectively. The coefficients of variation were 4.8%, 8.6%, and 3.2%, respectively, all belonging to weak variability, indicating that foliar contents of C, N and P tended to a certain stable value. The average value of N:P was 13.3, indicating that the growth of A. mongolicus was mainly limited by N. WUE was not correlated with leaf C content, but was significantly positively correlated with leaf N and P contents and N:P, and significantly negatively correlated with C:N and C:P, indicating that there was a linear synergistic trend between WUE and leaf nutrient content. The main factors influencing leaf C content and C:P were climatic factors, the leaf N content and N:P were mainly affected by soil factors, and the water use efficiency mainly affected leaf P content and C:N, indicating that the driving factors of different stoichiometric characteristics were different. The results could help eva-luate the habitat adaptation of desert plants, which would provide a theoretical basis for the conservation and management of A. mongolicus.

A reference-grade genome of the xerophyte Ammopiptanthus mongolicus sheds light on its evolution history in legumes and drought-tolerance mechanisms.

Plants that grow in extreme environments represent unique sources of stress-resistance genes and mechanisms. Ammopiptanthus mongolicus (Leguminosae) is a xerophytic evergreen broadleaf shrub native to semi-arid and desert regions; however, its drought-tolerance mechanisms remain poorly understood. Here, we report the assembly of a reference-grade genome for A. mongolicus, describe its evolutionary history within the legume family, and examine its drought-tolerance mechanisms. The assembled genome is 843.07 Mb in length, with 98.7% of the sequences successfully anchored to the nine chromosomes of A. mongolicus. The genome is predicted to contain 47 611 protein-coding genes, and 70.71% of the genome is composed of repetitive sequences; these are dominated by transposable elements, particularly long-terminal-repeat retrotransposons. Evolutionary analyses revealed two whole-genome duplication (WGD) events at 130 and 58 million years ago (mya) that are shared by the genus Ammopiptanthus and other legumes, but no species-specific WGDs were found within this genus. Ancestral genome reconstruction revealed that the A. mongolicus genome has undergone fewer rearrangements than other genomes in the legume family, confirming its status as a "relict plant". Transcriptomic analyses demonstrated that genes involved in cuticular wax biosynthesis and transport are highly expressed, both under normal conditions and in response to polyethylene glycol-induced dehydration. Significant induction of genes related to ethylene biosynthesis and signaling was also observed in leaves under dehydration stress, suggesting that enhanced ethylene response and formation of thick waxy cuticles are two major mechanisms of drought tolerance in A. mongolicus. Ectopic expression of AmERF2, an ethylene response factor unique to A. mongolicus, can markedly increase the drought tolerance of transgenic Arabidopsis thaliana plants, demonstrating the potential for application of A. mongolicus genes in crop improvement.

Difference of water source of two Ammopiptanthus mongolicus communities in Ulan Buh Desert, China.

To understand the adaptation of water use strategy of plant community to habitat heterogeneity, we measured the δD and δ18O values of xylem water of shrubs and potential water sources (soil water in different layers or groundwater) of Ammopiptanthus mongolicus communities on sand dune and Gobi from April to September in 2021 in the Ulan Buh Desert. Employing the MixSIAR model, we examined the seasonal dynamics of water source of each shrub by quantifying the contribution of different potential water sources. The results showed that A. mongolicus and Artemisia xerophytica on sand dune mainly used soil water of 10-25 cm in April and May after heavy rain in early spring, whereas Artemisia ordosica mainly used soil water of 10-200 cm. During the drought event within summer from June to August, A. mongolicus increasingly used soil water of 100-200 cm and groundwater, but A. xerophytica and A. ordosica increased the usage of 50-200 cm soil water. After the moderate rain in September, A. mongolicus evenly used soil water in all layers and groundwater, whereas two Artemisia shrubs preferred soil water of 10-50 cm. On Gobi, A. mongolicus and Nitraria sphaerocarpa evenly used soil water in all layers in April and May, mainly used 50-150 cm soil water from June to August and used 10-50 cm soil water in September. Convolvulus tragacanthoides mainly used soil water of 10-50 cm (from April to May), 25-150 cm (from June to August), and 10-25 cm (in September), separately. There were seasonal differences in water use of three shrubs on sand dune and Gobi A. mongolicus communities. During drought, A. mongolicus on sand dune could use deep soil water and groundwater, and that on Gobi relied only on deep soil water, which was more sensitive to rainfall.

The alteration of proteins and metabolites in leaf apoplast and the related gene expression associated with the adaptation of Ammopiptanthus mongolicus to winter freezing stress.

Ammopiptanthus mongolicus, an evergreen broad-leaved plant, can tolerate severe freezing stress (temperatures as low as -20 °C in winter). The apoplast is the space outside the plasma membrane that plays an important role in plant responses to environmental stress. Here, we investigated, using a multi-omics approach, the dynamic alterations in the levels of proteins and metabolites in the apoplast and related gene expression changes involved in the adaptation of A. mongolicus to winter freezing stress. Of the 962 proteins identified in the apoplast, the abundance of several PR proteins, including PR3 and PR5, increased significantly in winter, which may contribute to winter freezing-stress tolerance by functioning as antifreeze proteins. The increased abundance of the cell-wall polysaccharides and cell wall-modifying proteins, including PMEI, XTH32, and EXLA1, may enhance the mechanical properties of the cell wall in A. mongolicus. Accumulation of flavonoids and free amino acids in the apoplast may be beneficial for ROS scavenging and the maintenance of osmotic homeostasis. Integrated analyses revealed gene expression changes associated with alterations in the levels of apoplast proteins and metabolites. Our study improved the current understanding of the roles of apoplast proteins and metabolites in plant adaptation to winter freezing stress.

Leaf-Root-Soil Stoichiometric Characteristics in Different Shrub Ages of Ammopiptanthus mongolicus.

The ecological indicators for the growth and restoration of A. mongolicus populations are important for grasping the regulatory mechanisms of the biogeochemistry cycle, and for providing basic data for the prediction and evaluation of the evolution characteristics of natural A. mongolicus populations. We conducted studies on the eco-stoichiometric characteristics of natural A. mongolicus in different shrub ages, in order to understand the nutrient limitations for the growth and development of A. mongolicus and the synergy between the soil, leaves and roots, and to explore the C, N and P stoichiometric characteristics on A. mongolicus. The results showed the following: (1) The response of C, N and P stoichiometric characteristics in the leaves, roots and soil to changes in shrub age was not completely consistent. The leaf C content was young shrub> mature shrub> middle age shrub. The C content in the root system and C and N content in the soil showed an upward trend with increasing shrub age. The N and P contents of the root system and the P content of the soil showed a downward trend with increasing shrub age. The stoichiometric ratios C:N, C:P and N:P in the leaves, roots and soil showed an upward trend, and the N:P ratios in the leaves and roots were similar. (2) Among the stoichiometric characteristics of the leaves, C, N and P, leaves P and C:P are the most sensitive to shrub age changes, and have ecological implications for the growth and population dynamics of A. mongolicus. The average N:P ratios of young A. mongolicus leaves in young, middle-aged and mature shrubs were 15.32, 18.23 and 21.76, respectively. It can be seen that with an increase in shrub age, the growth of A. mongolicus gradually shifted from being jointly restricted by N and P to being more restricted by P. (3) The N content and the C∶N and N∶P ratios of A. mongolicus are classified as "strictly homoeostasis ", which shows strong plant homoeostasis for environmental adaptability. The N supplemented by symbiotic nitrogen fixation makes A. mongolicus have strong N internal homoeostasis. Therefore, in a desert grassland with low N content, the growth process of A. mongolicus may be easily restricted by P due to the additional N absorbed by it. (4) The C, N and P contents of the leaves, roots and soils of the three shrubs were shown as leaf > root > soil, and the difference was significant (p < 0.05). The correlation analysis showed that the C, N and P contents of the soil, roots and leaves and their stoichiometric ratio characteristics of the three shrubs showed a certain correlation. Among them, the P content of the soil was significantly related to the N:P ratio of the leaves and roots. Therefore, P is likely to become a limiting factor in the plant growth and repair process of the plant ecosystem in the A. mongolicus population. In summary, during the growth of A. mongolicus, special attention should be paid to the balance of nutrients. In order to improve its productivity, it is recommended to reasonably apply P fertilizers in the process of tending management to enhance the soil nutrient status and improve plant nutrient utilization efficiency and homoeostasis.

Characterization of the complete chloroplast genome of Ammopiptanthus mongolicus (Leguminosae).

Ammopiptanthus mongolicus is the only evergreen broadleaf shrub in the northwest desert of China, which can survive in long-term aridity and extremely cold environments. In this study, the complete chloroplast genome sequence of A. mongolicus was reported based on the Illumina NovaSeq Platform (Illumina, San Diego, CA). The chloroplast genome is 156,077 bp in length, containing a pair of inverted repeated (IR) regions (14,698 bp) that are separated by a large single copy (LSC) region of 88,025 bp, and a small single copy (SSC) region of 36,606 bp. Moreover, a total of 115 functional genes were annotated, including 81 mRNA, 30 tRNA genes, and 4 rRNA genes. Phylogenetic analysis based on 16 chloroplast genomics indicates that A. mongolicus is closely related to A. nanus.

The Modulation of Water, Nitrogen, and Phosphorous Supply for Growth Optimization of the Evergreen Shrubs Ammopiptanthus mongolicus for Revegetation Purpose.

Surface mining is a critical anthropogenic activity that significantly alters the ecosystem. Revegetation practices are largely utilized to compensate for these detrimental impacts of surface mining. In this study, we investigated the effects of five water (W) regimes [W40: 40%, W48: 48%, W60: 60%, W72: 72%, and W80: 80% of field capacity (FC)], five nitrogen (N) (N0: 0, N24: 24, N60: 60, N96: 96, and N120: 120 mg kg-1 soil), and five phosphorus (P) fertilizer doses (P0: 0, P36: 36, P90: 90, P144: 144, and P180: 180 mg kg-1 soil) on morpho-physiological and biochemical parameters of Ammopiptanthus mongolicus plants to assess the capability of this species to be used for restoration purposes. The results showed that under low W-N resources, A. mongolicus exhibited poor growth performance (i.e., reduced plant height, stem diameter, and dry biomass) in coal-degraded spoils, indicating that A. mongolicus exhibited successful adaptive mechanisms by reducing its biomass production to survive long in environmental stress conditions. Compared with control, moderate to high W and N-P application rates greatly enhanced the net photosynthesis rates, transpiration rates, water-use efficiency, chlorophyll (Chl) a, Chl b, total Chl, and carotenoid contents. Under low-W content, the N-P fertilization enhanced the contents of proline and soluble sugar, as well as the activities of superoxide dismutase, catalase, and peroxidase in leaf tissues, reducing the oxidative stress. Changes in plant growth and metabolism in W-shortage conditions supplied with N-P fertilization may be an adaptive strategy that is essential for its conservation and restoration in the desert ecosystem. The best growth performance was observed in plants under W supplements corresponding to 70% of FC and N and P doses of 33 and 36 mg kg-1 soil, respectively. Our results provide useful information for revegetation and ecological restoration in coal-degraded and arid-degraded lands in the world using endangered species A. mongolicus.

[Ectopic expression of the AmDREB1F gene from Ammopiptanthus mongolicus enhances stress tolerance of transgenic Arabidopsis].

Dehydration-responsive element binding proteins (DREBs) are an important class of transcription factors related to plant stress tolerance. Ammopiptanthus mongolicus is an evergreen broadleaf shrub endemic to desert areas of northwest China, and it has a very high tolerance to harsh environments. In order to reveal the functions and mechanisms of the AmDREB1F gene from this species in enduring abiotic stresses, we performed subcellular localization test, expression pattern analysis, and stress tolerance evaluation of transgenic Arabidopsis harboring this gene. The protein encoded by AmDREB1F was localized in the nucleus. In laboratory-cultured A. mongolicus seedlings, the expression of AmDREB1F was induced significantly by cold and drought but very slightly by salt and heat stresses, and undetectable upon ABA treatment. In leaves of naturally growing shrubs in the wild, the expression levels of the AmDREB1F gene were much higher during the late autumn, winter and early spring than in other seasons. Moreover, the expression was abundant in roots and immature pods rather than other organs of the shrubs. Constitutive expression of AmDREB1F in Arabidopsis induced the expression of several DREB-regulated stress-responsive genes and improved the tolerance of transgenic lines to drought, high salinity and low temperature as well as oxidative stress. The constitutive expression also caused growth retardation of the transgenics, which could be eliminated by the application of gibberellin 3. Stress-inducible expression of AmDREB1F also enhanced the tolerance of transgenic Arabidopsis to all of the four stresses mentioned above, without affecting its growth and development. These results suggest that AmDREB1F gene may play positive regulatory roles in response to abiotic stresses through the ABA-independent signaling pathways.

Heterologous Expression of Three Ammopiptanthus mongolicus Dehydrin Genes Confers Abiotic Stress Tolerance in Arabidopsis thaliana.

Ammopiptanthus mongolicus, a xerophyte plant that belongs to the family Leguminosae, adapts to extremely arid, hot, and cold environments, making it an excellent woody plant to study the molecular mechanisms underlying abiotic stress tolerance. Three dehydrin genes, AmDHN132, AmDHN154, and AmDHN200 were cloned from abiotic stress treated A. mongolicus seedlings. Cytomembrane-located AmDHN200, nucleus-located AmDHN154, and cytoplasm and nucleus-located AmDHN132 were characterized by constitutive overexpression of their genes in Arabidopsis thaliana. Overexpression of AmDHN132, AmDHN154, and AmDHN200 in transgenic Arabidopsis improved salt, osmotic, and cold tolerances, with AmDHN132 having the largest effect, whereas the growth of transformed plants is not negatively affected. These results indicate that AmDHNs contribute to the abiotic stress tolerance of A. mongolicus and that AmDHN genes function differently in response to abiotic stresses. Furthermore, they have the potential to be used in the genetic engineering of stress tolerance in higher plants.