Combined lncRNA and mRNA Expression Profiles Identified the lncRNA-miRNA-mRNA Modules Regulating the Cold Stress Response in Ammopiptanthus nanus.

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.

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.

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.

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.

The Characterization of R2R3-MYB Genes in Ammopiptanthus nanus Uncovers That the miR858-AnaMYB87 Module Mediates the Accumulation of Anthocyanin under Osmotic Stress.

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.

The complete chloroplast genome of Thermopsis lanceolata: genome structure and its phylogenetic relationships within the family Fabaceae.

Thermopsis lanceolata R. Br. belongs to the genus Thermopsis, Fabaceae. The alkaloids of T. lanceolata have anti-cancer, anti-heart rate disorders and other pharmacological effects. To explore the chloroplast genome of T. lanceolata and its phylogenetic relationship, a complete chloroplast genome of T. lanceolata was sequenced and annotated, and a phylogenetic tree was constructed. The complete chloroplast genome of T. lanceolata is a circular molecule of 151,526 bp, consisting of a large single copy (LSC) region of 83,780 bp, a small single copy (SSC) region of 15,566 bp, and a pair of inverted repeats (IRa and IRb) of 26,090 bp. The chloroplast genome of T. lanceolata contained 130 genes, including 85 protein-coding genes, 37 transfer RNA genes, and 8 ribosomal RNA genes. Phylogenetic analysis revealed a close relationship between T. lanceolata and T. turkestanica.

The complete chloroplast genome sequencing analysis revealed an unusual IRs reduction in three species of subfamily Zygophylloideae.

Tetraena mongolica, Zygophyllum xanthoxylon, and Z. fabago are three typical dryland plants with important ecological values in subfamily Zygophylloideae of Zygophyllaceae. Studies on the chloroplast genomes of them are favorable for understanding the diversity and phylogeny of Zygophyllaceae. Here, we sequenced and assembled the whole chloroplast genomes of T. mongolica, Z. xanthoxylon, and Z. fabago, and performed comparative genomic and phylogenetic analysis. The total size, structure, gene content and orders of these three chloroplast genomes were similar, and the three chloroplast genomes exhibited a typical quadripartite structure with a large single-copy region (LSC; 79,696-80,291 bp), a small single-copy region (SSC; 16,462-17,162 bp), and two inverted repeats (IRs; 4,288-4,413 bp). A total of 107 unique genes were identified from the three chloroplast genomes, including 70 protein-coding genes, 33 tRNAs, and 4 rRNAs. Compared with other angiosperms, the three chloroplast genomes were significantly reduced in overall length due to an unusual 16-24 kb shrinkage of IR regions and loss of the 11 genes which encoded subunits of NADH dehydrogenase. Genome-wide comparisons revealed similarities and variations between the three species and others. Phylogenetic analysis based on the three chloroplast genomes supported the opinion that Zygophyllaceae belonged to Zygophyllales in Fabids, and Z. xanthoxylon and Z. fabago belonged to Zygophyllum. The genome-wide comparisons revealed the similarity and variations between the chloroplast genomes of the three Zygophylloideae species and other plant species. This study provides a valuable molecular biology evidence for further studies of phylogenetic status of Zygophyllaceae.