Altitude-dependent metabolite biomarkers reveal the mechanism of plateau pika adaptation to high altitudes.

The harsh environment in the Tibetan plateau, the highest place in the world, poses thermoregulatory challenges and hypoxic stress to animals. The impacts of plateau environment on animal physiology and reproduction include external factors such as strong ultraviolet radiation and low temperature, and internal factors such as animal metabolites and gut microbiota. However, it remains unclear how plateau pika adapt to high altitudes through the combination of serum metabolites and gut microbiota. To this end, we captured 24 wild plateau pikas at the altitudes of 3400, 3600, or 3800 m a.s.l. in a Tibetan alpine grassland. Using the machine learning algorithms (random forest), we identified five biomarkers of serum metabolites indicative of the altitudes, that is, dihydrotestosterone, homo-l-arginine, alpha-ketoglutaric-acid, serotonin, and threonine, which were related to body weight, reproduction, and energy metabolism of pika. Those metabolic biomarkers were positively correlated with Lachnospiraceae_ Agathobacter, Ruminococcaceae, or Prevotellaceae_Prevotella, suggesting the close relationship between metabolites and gut microbiota. By identifying the metabolic biomarkers and gut microbiota analysis, we reveal the mechanisms of adaptation to high altitudes in plateau pika.

Transcriptomic Analysis Reveals Key Genes Involved in Oil and Linoleic Acid Biosynthesis during Artemisia sphaerocephala Seed Development.

Artemisia sphaerocephala seeds are rich in polysaccharides and linoleic acid (C18:2), which have been widely used as traditional medicine and to improve food quality. The accumulation patterns and molecular regulatory mechanisms of polysaccharides during A. sphaerocephala seed development have been studied. However, the related research on seed oil and C18:2 remain unclear. For this study, A. sphaerocephala seeds at seven different development stages at 10, 20, 30, 40, 50, 60, and 70 days after flowering (designated as S1~S7), respectively, were employed as experimental samples, the accumulation patterns of oil and fatty acids (FA) and the underlying molecular regulatory mechanisms were analyzed. The results revealed that oil content increased from 10.1% to 20.0% in the early stages of seed development (S1~S2), and up to 32.0% in mature seeds, of which C18:2 accounted for 80.6% of the total FA. FA and triacylglycerol biosynthesis-related genes jointly involved in the rapid accumulation of oil in S1~S2. Weighted gene co-expression network analysis showed that transcription factors FUS3 and bHLH played a critical role in the seed oil biosynthesis. The perfect harmonization of the high expression of FAD2 with the extremely low expression of FAD3 regulated the accumulation of C18:2. This study uncovered the gene involved in oil biosynthesis and molecular regulatory mechanisms of high C18:2 accumulation in A. sphaerocephala seeds; thus, advancing research into unsaturated fatty acid metabolism in plants while generating valuable genetic resources for optimal C18:2 breeding.

Transcriptome and co-expression network analysis reveal molecular mechanisms of mucilage formation during seed development in Artemisia sphaerocephala.

Seed mucilage has significant economic value. However, the identification of key regulatory genes in mucilage formation and their molecular regulatory mechanism remain unknown. Artemisia sphaerocephala seeds are rich in mucilage. In this study, A. sphaerocephala seeds in 10, 20, 30, 40, 50, 60 and 70 days after flowering were used as materials to reveal their molecular regulatory mechanism in mucilage formation by RNA-sequencing and weighted gene co-expression network analysis (WGCNA). 21 key regulatory genes for mucilage formation were identified, including AsKNAT7 and AsTTG1 genes, as well as AsNAM and AsAP2 gene families. From 10-30 days after flowering, both AsNAM and AsAP2 supported mucilage formation. From 40-70 days after flowering, promotion by AsNAM and AsAP2 was weakened and the up-regulation of AsKNAT7 inhibited mucilage formation, leading to no further increases in mucilage content. This in depth elucidation of seed mucilage formation lays the foundation for the application of mucilage.

Cloning and functional analysis of the FAD2 gene family from desert shrub Artemisia sphaerocephala.

BACKGROUND: Linoleic acid is an important polyunsaturated fatty acid, required for all eukaryotes. Microsomal delta-12 (Δ12) oleate desaturase (FAD2) is a key enzyme for linoleic acid biosynthesis. Desert shrub Artemisia sphaerocephala is rich in linoleic acid, it has a large FAD2 gene family with twenty-six members. The aim of this work is to unveil the difference and potentially functionality of AsFAD2 family members. RESULTS: Full-length cDNAs of twenty-one AsFAD2 genes were obtained from A. sphaerocephala. The putative polypeptides encoded by AsFAD2 family genes showed a high level of sequence similarity and were relatively conserved during evolution. The motif composition was also relatively conservative. Quantitative real-time PCR analysis revealed that the AsFAD2-1 gene was strongly expressed in developing seeds, which may be closely associated with the high accumulating ability of linoleic acid in A. sphaerocephala seeds. Although different AsFAD2 family members showed diverse response to salt stress, the overall mRNA levels of the AsFAD2 family genes was stable. Transient expression of AsFAD2 genes in the Nicotiana benthamiana leaves revealed that the encoded proteins were all located in the endoplasmic reticulum. Heterologous expression in Saccharomyces cerevisiae suggested that only three AsFAD2 enzymes, AsFAD2-1, - 10, and - 23, were Δ12 oleate desaturases, which could convert oleic acid to linoleic acid, whereas AsFAD2-1 and AsFAD2-10 could also produce palmitolinoleic acid. CONCLUSIONS: This research reported the cloning, expression studies, subcellular localization and functional identification of the large AsFAD2 gene family. These results should be helpful in understanding fatty acid biosynthesis in A. sphaerocephala, and has the potential to be applied in the study of plant fatty acids traits.

Selection and validation of reference genes for quantitative real-time PCR in Artemisia sphaerocephala based on transcriptome sequence data.

Artemisia sphaerocephala, a dicotyledonous perennial semi-shrub belonging to the Artemisia genus of the Compositae family, is widely distributed in northwestern China. This shrub is one of the most important pioneer plants which is capable of protecting rangelands from wind erosion. It therefore plays a vital role in maintaining desert ecosystem stability. In addition, to its use as a forage grass, it has excellent prospective applications as a source of plant oil and as a plant-based fuel. The use of internal genes is the basis for accurately assessing Real time quantitative PCR. In this study, based on transcriptome data of A. sphaerocephala, we analyzed 21 candidate internal genes to determine the optimal internal genes in this shrub. The stabilities of candidate genes were evaluated in 16 samples of A. sphaerocephala. Finally, UBC9 and TIP41-like were determined as the optimal reference genes in A. sphaerocephala by Delta Ct and three various programs. There were GeNorm, NormFinder and BestKeeper.

Effect of salt stress on fatty acid and α-tocopherol metabolism in two desert shrub species.

MAIN CONCLUSIONS: Compared to Artemisia ordosiea Kraschen, a higher content of α-tocopherol in Artemisia sphaerocephala Kraschen under salt stress inhibits the conversion of linoleic acid (C18:2) into linolenic acid (C18:3), maintains cell membrane stability and contributes to higher salt resistance. Artemisia sphaerocephala Kraschen and Artemisia ordosiea Kraschen are widely distributed in the arid and semiarid desert regions of the northwest of China. Under salt stress, it has been known that α-tocopherol (α-T) improves membrane permeability and maintains Na+/K+ homeostasis; however, the function of α-T in regulating membrane components of fatty acids is unknown. In this study, 100-day-old plants of A. ordosiea and A. sphaerocephala are subjected to various NaCl treatments for 7, 14, and 21 days. Compared to A. ordosiea, A. sphaerocephala has a higher Na+ concentration, higher chlorophyll content and dry weight in all NaCl treatments, but lower relative electric conductivity. The stable unsaturated levels of the lipids in A. sphaerocephala may be attributed to higher level of C18:2. Under 200 mM NaCl treatment, α-T and C18:2 contents in A. sphaerocephala increase significantly, while the Na+, C18:1, C18:3 and jasmonic acid (JA) contents decrease. Moreover, α-T is positively correlated with C18:2, but negatively correlated with C18:3.

Genome-Scale Transcriptome Analysis of the Desert Shrub Artemisia sphaerocephala.

BACKGROUND: Artemisia sphaerocephala, a semi-shrub belonging to the Artemisia genus of the Compositae family, is an important pioneer plant that inhabits moving and semi-stable sand dunes in the deserts and steppes of northwest and north-central China. It is very resilient in extreme environments. Additionally, its seeds have excellent nutritional value, and the abundant lipids and polysaccharides in the seeds make this plant a potential valuable source of bio-energy. However, partly due to the scarcity of genetic information, the genetic mechanisms controlling the traits and environmental adaptation capacity of A. sphaerocephala are unknown. RESULTS: Here, we present the first in-depth transcriptomic analysis of A. sphaerocephala. To maximize the representation of conditional transcripts, mRNA was obtained from 17 samples, including living tissues of desert-growing A. sphaerocephala, seeds germinated in the laboratory, and calli subjected to no stress (control) and high and low temperature, high and low osmotic, and salt stresses. De novo transcriptome assembly performed using an Illumina HiSeq 2500 platform resulted in the generation of 68,373 unigenes. We analyzed the key genes involved in the unsaturated fatty acid synthesis pathway and identified 26 A. sphaerocephala fad2 genes, which is the largest fad2 gene family reported to date. Furthermore, a set of genes responsible for resistance to extreme temperatures, salt, drought and a combination of stresses was identified. CONCLUSION: The present work provides abundant genomic information for functional dissection of the important traits of A. sphaerocephala and contributes to the current understanding of molecular adaptive mechanisms of A. sphaerocephala in the desert environment. Identification of the key genes in the unsaturated fatty acid synthesis pathway could increase understanding of the biological regulatory mechanisms of fatty acid composition traits in plants and facilitate genetic manipulation of the fatty acid composition of oil crops.

Identification and functional analysis of a Δ6-desaturase gene and the effects of temperature and wounding stresses on its expression in Microula sikkimensis leaves.

A Δ6-desaturase gene was isolated from Microula sikkimensis. Sequence analysis indicated that the gene, designated MsD6DES, had an open reading frame of 1,357 bp and encoded 448 amino acids. Heterologous expression in tobacco indicated that MsD6DES can use endogenous substrates to synthesize γ-linolenic acid (GLA, 18:3(Δ 6,9,12)) and octadecatetraenoic acid (OTA, 18:4(Δ 6,9,12,15)). MsD6DES transcripts were distributed in all tested tissues, with high expression levels in seeds and young leaves. The effects of temperature and wounding stresses on MsD6DES expression were analyzed. The results indicated that temperature regulates MsD6DES at the transcriptional level. MsD6DES expression increased first, reaching a maximum 4 h after low-temperature treatment. A slight increase in MsD6DES transcript levels was also observed under high temperature. We found that the response of MsD6DES to temperature stress was different from those of fungi and algae. In addition, MsD6DES was found to be wound-inducible.