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Abstract Background Thymus mongolicus (family Lamiaceae) is a Thyme subshrub with strong aroma and remarkable environmental adaptability. Limited genomic information limits the use of this plant. Results Chromosome-level 605.2 Mb genome of T. mongolicus was generated, with 96.28% anchored to 12 pseudochromosomes. The repetitive sequences were dominant, accounting for 70.98%, and 32,593 protein-coding genes were predicted. Synteny analysis revealed that Lamiaceae species generally underwent two rounds of whole genome duplication; moreover, species-specific genome duplication was identified. A recent LTR retrotransposon burst and tandem duplication might play important roles in the formation of the Thymus genome. Using comparative genomic analysis, phylogenetic tree of seven Lamiaceae species was constructed, which revealed that Thyme plants evolved recently in the family. Under the phylogenetic framework, we performed functional enrichment analysis of the genes on nodes that contained the most gene duplication events (> 50% support) and of relevant significant expanded gene families. These genes were highly associated with environmental adaptation and biosynthesis of secondary metabolites. Combined transcriptome and metabolome analyses revealed that Peroxidases, Hydroxycinnamoyl-CoA shikimate/quinate hydroxycinnamoyl transferases, and 4-coumarate-CoA ligases genes were the essential regulators of the phenylpropanoid–flavonoid pathway. Their catalytic products (e.g., apigenin, naringenin chalcone, and several apigenin-related compounds) might be responsible for the environmental tolerance and aromatic properties of T. mongolicus. Conclusion This study enhanced the understanding of the genomic evolution of T. mongolicus, enabling further exploration of its unique traits and applications, and contributed to the understanding of Lamiaceae genomics and evolutionary biology.

Grassland plants that endure livestock grazing exhibit a dwarf phenotype, which can be transmitted to clonal offspring. Yet to date, it remains poorly understood whether such transgenerational dwarf effects alter the plants’ response to shade. Here, we conducted a common garden experiment under sunlight and shade conditions with clonal Leymus chinensis offspring, the parents of which had endured livestock overgrazing (OG) and non-grazing (NG) in the field, respectively. Plant morphological, physiological, and transcriptomic analyses were carried out. The results indicated that NG offspring showed greater shade avoidance than OG offspring. That is, NG offspring exhibited greater plasticity of vegetative height and leaf width, which may be contributed to their greater photosynthetic capacity and gibberellin (GA3) content compared with OG offspring when treated with shade. In addition, RNA-Seq profiling showed that differentially expressed genes in NG offspring were mainly enriched in RNA modification and metabolic processes, which facilitated rapid response to shade. Phytochrome interacting factors (PIFs) promoted downstream shade marker genes in NG offspring by significantly downregulating the expression of PHYC, SPY, and DELLA. Our findings suggest that light conditions should be taken into account to better understand transgenerational dwarf effects induced by livestock grazing on grassland ecosystems. These results provide new insights into the inducible factors of phenotypic variations in grassland plants that experience grazing.

Grazing disturbance can change the structure of plant rhizosphere microbial communities and thereby alter the feedback to promote plant growth or induce plant defenses. However, little is known about how such changes occur and vary under different grazing pressures or the roles of root metabolites in altering the composition of rhizosphere microbial communities. In this study, the effects of different grazing pressures on the composition of microbial communities were investigated, and the mechanisms by which different grazing pressures changed rhizosphere microbiomes were explored with metabolomics. Grazing changed composition, functions, and co-expression networks of microbial communities. Under light grazing (LG), some saprophytic fungi, such as Lentinus sp., Ramichloridium sp., Ascobolus sp. and Hyphoderma sp., were significantly enriched, whereas under heavy grazing (HG), potentially beneficial rhizobacteria, such as Stenotrophomonas sp., Microbacterium sp., and Lysobacter sp., were significantly enriched. The beneficial mycorrhizal fungus Schizothecium sp. was significantly enriched in both LG and HG. Moreover, all enriched beneficial microorganisms were positively correlated with root metabolites, including amino acids (AAs), short-chain organic acids (SCOAs), and alkaloids. This suggests that these significantly enriched rhizosphere microbial changes may be caused by these differential root metabolites. Under LG, it is inferred that root metabolites, especially AAs such as L-Histidine, may regulate specific saprophytic fungi to participate in material transformations and the energy cycle and promote plant growth. Furthermore, to help alleviate the stress of HG and improve plant defenses, it is inferred that the root system actively regulates the synthesis of these root metabolites such as AAs, SCOAs, and alkaloids under grazing interference, and then secretes them to promote the growth of some specific plant growth-promoting rhizobacteria and fungi. To summarize, grasses can regulate beneficial microorganisms by changing root metabolites composition, and the response strategies vary under different grazing pressure in typical grassland ecosystems.

Appropriate grazing pressure and fertilizer application of nitrogen (N) and phosphorus (P) are effective measures to increase grassland productivity. In this study, we report on the interactive effects of grazing intensity and fertilizer application on soil properties, enzyme characteristics, and soil bacterial community compositions. The experiment was set up in a typical grassland in Xilingol, Inner Mongolia, and had 12 treatments (CC, CN, CP, CNP, LC, LN, LP, LNP, HC, HN, HP, and HNP). These consisted of three grazing intensity levels crossed with four fertilizer application treatments: no fertilizer, N fertilizer, P fertilizer addition alone, and both N and P fertilizers addition, subjected to field sampling and laboratory analysis. The results showed that soil alkaline hydrolysis nitrogen was increased by 15 and 13.6% in LN over LC in the 0–10 and 10–20 cm soil depth layers, respectively. Soil available P was 135.6% higher in LP than in LC at 0–10 cm but similar between LP and LC at 10–20 cm. Soil urease activity rose by 46.8 and 39.3% in 0–10 cm soil and was augmented by 63.1 and 60.3% in 10–20 cm soil of LN and LP relative to LC, respectively. Soil catalase (CAT) activity was decreased in response to LNP by 29.4, 23.5, and 26.5% vis-à-vis LC, LN, and LP in the 0–10 cm layer. Soil CAT activity also decreased in 0–20 cm layer for HN and HNP in comparison with HP. The relative abundance of Actinobacteria increased by 38.1 and 45.0% in HC over that in CC and LC, respectively, in 0–10 cm soil; compared with LC, it increased by 35.8 and 21.7% in LN and LNP, respectively. The relative abundance of Proteobacteria was increased in LNP versus LC in 0–10 cm soil. Overall, fertilizer application coupled with a light grazing intensity promoted key soil properties and the relative abundance of a dominant bacterial phylum.

Soil seed banks are the main seed reservoirs globally and are critical for the regeneration of grassland plant populations. Grazing often affects the density and diversity of grassland soil seed banks; however, it is not clear how these effects vary on a global scale with grazing intensity, grazing duration, and climatic conditions. Here, we screened 92 independent published articles and performed a meta-analysis of the extracted data. The effects of grazing on four soil seed bank density and diversity indices were analyzed. Four alpha diversity indices, namely Shannon–Wiener, Margalef, Pielou, and Simpson indices, were selected in this study. The Shannon–Wiener index reflects the species complexity of the community. The Margalef index reflects species richness of the seed bank. The Pielou index reflects whether the number of seeds of all species in the seed bank is evenly distributed, and the Simpson’s index reflects the status of dominant plants in the seed bank. We found that grazing significantly decreased the total density of soil seed banks. Perennial plant seed banks were more affected by grazing than annual plant seed banks. Heavy grazing significantly decreased the Margalef index of seed banks, while moderate grazing significantly decreased the Simpson’s alpha diversity index. Long-term grazing (>10 years) had the greatest impact on Simpson’s alpha diversity index, while medium-term grazing (5–10 years) had a significant negative impact on Shannon–Wiener, Margalef, and Pielou indices. In addition, our results show that grazing has a greater effect on seed banks in arid areas than those in temperate areas. Knowledge of climate-specific effects will contribute to understanding the risk of local extinctions in grassland populations and help decision-makers maintain the health of grassland ecosystems.

The maintenance service network is always designed as a multi-level service network to provide timely maintenance service for failed machinery, and is rarely studied in agriculture. Thus, this paper focuses on a three-level maintenance service network location–allocation problem in agriculture, which contains several spare part centres, service stations, and service units. This research aims to obtain the optimal location of spare part centres and service stations while determining service vehicle allocation results for service stations, and the problem can be called a multi-level facility location and allocation problem (MLFLAP). Considering contiguity constraints and hierarchical relationships, the proposed MLFLAP is formulated as a mixed-integer linear programming (MILP) model integrating with P-region and set covering location problems to minimize total service costs, including spare part centre construction costs, service vehicle usage costs, and service mileage costs of service stations. The Benders decomposition-based solution method with several improvements is then applied to decompose the original MLFLAP into master problem and subproblems to find the optimal solutions effectively. Finally, a real-world case in China is proposed to evaluate the performance of the model and algorithm in agriculture, and sensitivity analysis is also conducted to demonstrate the impact of several parameters.

Abstract We study the vertical impact of a droplet onto a cubic pillar of comparable size placed on a flat surface, by means of numerical simulations and experiments. Strikingly, during the impact a large volume of air is trapped around the pillar side faces. Impingement upon different positions of the pillar top surface strongly influences the size and the position of the entrapped air. By comparing the droplet morphological changes during the impact from both computations and experiments, we show that the direct numerical simulations, based on the Volume of Fluid method, provide additional and new insight into the droplet dynamics. We elucidate, with the computational results, the three-dimensional air entrapment process as well as the evolution of the entrapped air into bubbles.

Long-term overgrazing (OG) is one of the key drivers of global grassland degradation with severe loss of productivity and ecosystem functions, which may result in stress memory such as smaller stature of grassland plants. However, how the OG-induced stress memory could be regulated by phytohormones is unknown. In this study, we investigated the changes of four phytohormones of cloned offspring of Leymus chinensis that were developed from no-grazing (NG) plants and OG plants with a grazing history of 30 years. The concentrations of auxin (IAA) and gibberellic acid (GA) in OG plant leaves were 45% and 20% lower than control, respectively. Meanwhile, the level of abscisic acid (ABA) in OG leaves nearly doubled compared with that in NG leaves. The situation was quite similar in roots. Unexpectedly, no significant changes in the jasmonic acid (JA) level were observed between OG and NG plants. The changes in gene expression patterns between OG and NG plants were also investigated by transcriptomic analysis. In total, 302 differentially expressed genes (DEGs) were identified between OG and NG plants, which were mainly classified into the functions of synthesis, receptor, and signal transduction processes of phytohormones. The expression of 24 key genes related to the biosynthesis and signal transduction of IAA and GA was downregulated in OG plants. Among them, OASA1 and AO1 (regulating the biosynthesis of IAA and ABA, respectively) were reduced significantly by 88 and 92%, respectively. In addition, the content of secondary metabolites related to plant defense such as flavonoids and phenols was also increased in leaves. Taken together, the decrease of positive plant growth-related hormones (IAA and GA) together with the increase of plant stress-related hormones or factors (ABA, flavonoids, and phenols) induced the growth-defense trade-offs for L. chinensis adaptation to long-term OG stress. The findings reported in this study shed new light on the mechanism of plant–animal interaction in the grassland ecosystem and provide a deeper insight into optimizing grazing management and sustainable utilization of grassland.

Abstract Utilizing methods such as scanning electron microscopy observation and mercury intrusion porosimetry, this paper investigates the basic microstructure and pore structure properties of polymer-cement composite joint sealants for pavements, and analyzes the effects and rules of various material types, ratio parameters and processing conditions. Further, the fractal characteristics and variation rules of pore size distribution are investigated for the joint sealants by introducing the fractal theory. The results show that changes in material type, ratio parameter and processing condition produce insignificant effects on the basic microstructure properties and configuration of joint sealants, with effects reflected primarily in the change of sealant pore structure. Measures like increasing the powder-liquid ratio and cement ratio, blending with sulphoaluminate cement or mica powder, adding latex powder or coupling agent, cold drawing and hot pressing, as well as ultraviolet irradiation treatment are all capable of reducing the total pore volume of joint sealants and refining their pore structure. In contrast, opposite effects are yielded when low-grade cement is used, styrene-acrylic emulsion is blended, or plasticizer is added. Additionally, after blending with talc powder or adding carbon fiber additive, the total pore volume of joint sealants remains basically unchanged or reduced, despite the coarsened pore structure. The total pore volume of joint sealants increases after wet–dry cycling treatment, while no obvious change in the pore size distribution is observed. Pore size distribution of the studied joint sealants presents distinct fractal characteristics, and the corresponding fractal dimension of pore surface area ranges between 2.6 and 2.8.

Abstract Background Overgrazing is a primary contributor to severe reduction in forage quality and production in Inner Mongolia, leading to extensive ecosystem degradation, sheep health impairment and growth performance reduction. Further studies to identify serum biomarkers that reflect changes in sheep health and nutritional status following overgrazing would be beneficial. We hereby hypothesize that reduced sheep growth performance under overgrazing conditions would be associated with metabolic and immune response alterations. This study used an untargeted metabolomics analysis by conducting ultra-high-performance liquid chromatography combined with quadrupole time-of-flight mass spectrometry (UHPLC-Q-TOF/MS) of sheep serum under overgrazing and light grazing conditions to identify metabolic disruptions in response to overgrazing. Results The sheep body weight gains as well as serum biochemical variables associated with immune responses and nutritional metabolism (immunoglobulin G, albumin, glucose, and nonesterified fatty acids) were significantly decreased with overgrazing compared with light grazing condition. In contrast, other serum parameters such as alanine and aspartate aminotransferase, alkaline phosphatase, total bilirubin, blood urea nitrogen, and interleukin-8 were markedly higher in the overgrazing group. Principal component analysis discriminated the metabolomes of the light grazing from the overgrazing group. Multivariate and univariate analyses revealed changes in the serum concentrations of 15 metabolites (9 metabolites exhibited a marked increase, whereas 6 metabolites showed a significant decrease) in the overgrazing group. Major changes of fatty acid oxidation, bile acid biosynthesis, and purine and protein metabolism were observed. Conclusions These findings offer metabolic evidence for putative biomarkers for overgrazing-induced changes in serum metabolism. Target-identification of these particular metabolites may potentially increase our knowledge of the molecular mechanisms of altered immune responses, nutritional metabolism, and reduced sheep growth performance under overgrazing conditions.

Abstract Background Overgrazing is a major factor that causes steppe degradation in Inner Mongolian, resulting in extensive ecosystem damage. Scarcity of grass means sheep are smaller and therefore mutton and cashmere production is greatly reduced, which has resulted in massive annual economic losses. Liver is the primary metabolic organ in mammals. It is also the key source of energy supply and detoxification of metabolites in animals, has a close relationship with animal growth. However, investigations on the responses of sheep induced by consequence of overgrazing, particularly those relating to liver-related molecular mechanisms and related metabolic pathways, remain elusive. Results The body weight daily gain of sheep, immune organ indices (liver and spleen), and serum parameters related to immune response, protein synthesis and energy supply (IgG, albumin, glucose and non-esterified fatty acid) were significantly lower in the overgrazing group. Other serum parameters including alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, total bilirubin, blood urea nitrogen and interleukin-6 were significantly higher in the overgrazing group. For the RNA-Seq results, we identified approximately 50 differentially expressed genes, of which half of were up-regulated and the other half were down-regulated (overgrazing group versus light grazing group). Bioinformatics analysis identified two enriched KEGG pathways including peroxisome proliferator-activated receptor (PPAR) signaling pathway (related to lipolysis) and ECM-receptor interaction (related to liver injury and apoptosis). Additionally, several of the down-regulated genes were related to detoxification and immune response. Conclusions Overall, based on the high-throughput RNA sequencing profile integrated with the results of serum biochemical analyses, consequences of lower forage availability and quality under overgrazing condition induced altered expression levels of genes participating in energy metabolism (particularly lipid metabolism) and detoxification and immune responses, causing lipolysis and impaired health status, which might be key reasons for the reduced growth performance of sheep. This investigation provides a novel foundation for the development of sheep hepatic gene interactive networks that are a response to the degraded forage availability under overgrazing condition.

This paper is about a study on the mechanical properties of a new polymer-cement composite (PCC) in constant elongation, tension, and shear. The study explored the effects of powder-liquid ratio and cement ratio on the mechanical properties of PCC through detecting the strength, deformation, and energy consumption of specimens under different powder-liquid ratios and cement ratios. In addition, scanning electron microscope and mercury injection apparatus were used for an in-depth analysis on the micromorphology and pore structure features of PCC under different powder-liquid ratios and cement ratios to explore the influence of powder-liquid ratio and cement ratio of the micromechanical properties of PCC. The results showed that, with the increase of powder-liquid ratio and cement ratio, the constant elongation adhesion strength of PCC decreased, and, at a high powder-liquid ratio (0.55) or a high cement ratio (0.5), the constant elongation adhesion strength of PCC completely disappeared. Meanwhile, with the increase of powder-liquid ratio and cement ratio, the tensile shear strength of PCC increased, while the deformation capacity of PCC decreased. The optimal ranges of powder-liquid ratio and cement ratio for PCC were 0.35–0.4 and 0.3-0.4, respectively. Furthermore, the increased powder-liquid ratio and cement ratio made the total pore volume decreased and pore structure refined, which improved the compactness of PCC, thus influencing the performance of PCC macroscopically. An achievement for the study is a flexible composite material, which was formulated with the polymer film as continuous base phase, as well as the inorganic composition and cement hydrates as dispersion phase. The material can effectively improve the economy and practicability of cementation of fissures for airfield pavement.

Grazing, one of the primary utilization modes of grassland, is the main cause of grassland degradation. Historical overgrazing results in dwarf phenotype and decreased photosynthesis of perennial plants. However, it remains unknown what the mechanism underlying of this legacy effect is, and the role of stomata in the resulting decreased photosynthesis also remains unclear. To address these questions, differences in stomatal density, length and width on both adaxial and abaxial epidermis were compared between overgrazing and ungrazed Leymus chinensis offspring by using rhizome buds cultivated in a greenhouse, and the correlation between photosynthetic capacity and stomatal behavior was also investigated. Our results showed that historical grazing significantly impacted phenotype, photosynthesis and stomatal traits of L. chinensis. The offspring plants taken from overgrazed parents were dwarfed compared to those taken from ungrazed parents, and the photosynthesis and stomatal conductance of plants with a grazing history decreased by 28.6% and 21.3%, respectively. In addition, stomatal density and length on adaxial and abaxial leaf surfaces were significantly increased; however, stomatal width on abaxial leaf surfaces of overgrazed L. chinensis was significantly decreased compared with ungrazed individuals. Moreover, the expression patterns of eight genes related to stomatal regulation were tested: seven were down-regulated (2–18 times) and one was up-regulated (three times). Genes, involved in ABC transporter and receptor-like serine/threonine protein kinase were down-regulated. These results suggest that legacy effects of historical grazing affect the stomatal conductance by decreasing the stomatal width in progeny plants, which thus results in lower photosynthesis. Furthermore, changes of stomatal traits and function were regulated by the inhibition of ABC transporter and serine/threonine protein kinase. These findings are helpful for future exploration of the possible mechanisms underlying the response of grassland plants to long-term overgrazing.

Abstract Background This study was designed to reveal potential molecular mechanisms of long-term overgrazing-induced dwarfism in sheepgrass (Leymus chinensis). Methods An electrospray ionisation mass spectrometry system was used to generate proteomic data of dwarf sheepgrass from a long-term overgrazed rangeland and normal sheepgrass from a long-term enclosed rangeland. Differentially expressed proteins (DEPs) between dwarf and normal sheepgrass were identified, after which their potential functions and interactions with each other were predicted. The expression of key DEPs was confirmed by high-performance liquid chromatography mass spectrometry (HPLC–MS) using a multiple reaction monitoring method. Results Compared with normal sheepgrass, a total of 51 upregulated and 53 downregulated proteins were identified in dwarf sheepgrass. The amino acids biosynthesis pathway was differentially enriched between the two conditions presenting DEPs, such as SAT5_ARATH and DAPA_MAIZE. The protein–protein interaction (PPI) network revealed a possible interaction between RPOB2_LEPTE, A0A023H9M8_9STRA, ATPB_DIOEL, RBL_AMOTI and DNAK_GRATL. Four modules were also extracted from the PPI network. The HPLC–MS analysis confirmed the upregulation and downregulation of ATPB_DIOEL and DNAK_GRATL, respectively in dwarf samples compared with in the controls. Conclusions The upregulated ATPB_DIOEL and downregulated DNAK_GRATL as well as proteins that interact with them, such as RPOB2_LEPTE, A0A023H9M8_9STRA and RBL_AMOTI, may be associated with the long-term overgrazing-induced dwarfism in sheepgrass.

Sheepgrass [Leymus chinensis (Trin.) Tzvel] is a valuable forage plant highly significant to the grassland productivity of Euro-Asia steppes. Growth of above-ground tissues of L. chinensis is the major component contributing to the grass yield. Although it is generally known that this species is sensitive to ecosystem disturbance and adverse environments, detailed information of how L. chinensis coping with various nutrient deficiency especially phosphate deprivation (-Pi) is still limited. Here, we investigated impact of Pi-deprivation on shoot growth and biomass accumulation as well as photosynthetic properties of L. chinensis. Growth inhibition of Pi-deprived seedlings was most obvious and reduction of biomass accumulation and net photosynthetic rate (Pn) was 55.3 and 63.3%, respectively, compared to the control plants grown under Pi-repleted condition. Also, we compared these characters with seedlings subjected to low-Pi stress condition. Pi-deprivation caused 18.5 and 12.3% more reduction of biomass and Pn relative to low-Pi-stressed seedlings, respectively. Further analysis of in vivo chlorophyll fluorescence and thylakoid membrane protein complexes using 2D-BN/SDS-PAGE combined with immunoblot detection demonstrated that among the measured photosynthetic parameters, decrease of ATP synthase activity was most pronounced in Pi-deprived plants. Together with less extent of lipid peroxidation of the thylakoid membranes and increased ROS scavenger enzyme activities in the leaves of Pi-deprived seedlings, we suggest that the decreased activity of ATP synthase in their thylakoids is the major cause of the greater reduction of photosynthetic efficiency than that of low-Pi stressed plants, leading to the least shoot growth and biomass production in L. chinensis.

Details on the fall speeds of raindrops are essential in both applications and natural events, such as rain-rate retrieval and soil erosion. Here, we examine the influence of turbulence on the terminal velocity of two water drops of different sizes. For the first time, computations of droplets in turbulent surroundings are conducted with a direct numerical simulation code based on a volume of fluid method. Both the drop surface deformation and internal circulation are captured. The turbulence intensity at the inflow area, as well as the turbulence length scale are varied. In turbulent flow, we find a decline in the terminal velocities for both drops. Based on the study of the wake flow characteristics and drop surface deformation, the decrease in the terminal velocity is found to be directly linked to a shortening of the wake recirculation region resulting from an earlier and more drastic increase in the turbulence kinetic energy in the shear layer. The turbulent surroundings trigger substantial rises in the drop axis ratio amplitude and a slight increase in the drop oscillation frequency, but barely influence the time-averaged drop axis length.

Joint sealant is affected by various environmental factors in service, such as different temperatures, water soaking, ultraviolet and so on. In this paper, the VAE emulsion–cement compositejoint sealant was pretreated under multiple simulation environments. Thereafter, the degradation rules of fixed elongation and tensile properties of joint sealants at different mix proportions were systemically investigated under the action of external environments (temperature, water soaking and ultraviolet), and the influence mechanisms of diverse environmental factors were analyzed. The research results suggested that, under the action of external environments, the VAE emulsion–cement composite joint sealants exhibited degradation effects to varying degrees. After the addition of plasticizer, the joint sealants had reduced cohesion strength in low temperature environment and enhanced flexible deformability. The addition of water repellent improved the water resistance of joint sealants. Meanwhile, adding ultraviolet shield agent partially improved the ultraviolet radiation aging resistance. A greater powder–liquid ratio led to the lower flexibility of joint sealants, but superior water resistance and ultraviolet radiation aging resistance.

Previous studies of transgenerational plasticity have demonstrated that long-term overgrazing experienced by Leymus chinensis, an ecologically dominant, rhizomatous grass species in eastern Eurasian temperate grassland, significantly affects its clonal growth in subsequent generations. However, there is a dearth of information on the reasons underlying this overgrazing-induced memory effect in plant morphological plasticity. We characterized the relationship between a dwarf phenotype and photosynthesis function decline of L. chinensis from the perspective of leaf photosynthesis by using both field measurement and rhizome buds culture cultivated in a greenhouse. Leaf photosynthetic functions (net photosynthetic rate, stomatal conductance, intercellular carbon dioxide concentration, and transpiration rate) were significantly decreased in smaller L. chinensis individuals that were induced to have a dwarf phenotype by being heavily grazed in the field. This decreased photosynthetic function was maintained a generation after greenhouse tests in which grazing was excluded. Both the response of L. chinensis morphological traits and photosynthetic functions in greenhouse were deceased relative to those in the field experiment. Further, there were significant decreases in leaf chlorophyll content and Rubisco enzyme activities of leaves between bud-cultured dwarf and non-dwarf L. chinensis in the greenhouse. Moreover, gene expression patterns showed that the bud-cultured dwarf L. chinensis significantly down-regulated (by 1.86- to 5.33-fold) a series of key genes that regulate photosynthetic efficiency, stomata opening, and chloroplast development compared with the non-dwarf L. chinensis. This is among the first studies revealing a linkage between long-term overgrazing affecting the transgenerational morphological plasticity of clonal plants and physiologically adaptive photosynthesis function. Overall, clonal transgenerational effects in L. chinensis phenotypic traits heavily involve photosynthetic plasticity.

Stipa grandis P. Smirn. is a dominant plant species in the typical steppe of the Xilingole Plateau of Inner Mongolia. Selection of suitable reference genes for the quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) is important for gene expression analysis and research into the molecular mechanisms underlying the stress responses of S. grandis. In the present study, 15 candidate reference genes (EF1 beta, ACT, GAPDH, SamDC, CUL4, CAP, SNF2, SKIP1, SKIP5, SKIP11, UBC2, UBC15, UBC17, UCH, and HERC2) were evaluated for their stability as potential reference genes for qRT-PCR under different stresses. Four algorithms were used: GeNorm, NormFinder, BestKeeper, and RefFinder. The results showed that the most stable reference genes were different under different stress conditions: EF1beta and UBC15 during drought and salt stresses; ACT and GAPDH under heat stress; SKIP5 and UBC17 under cold stress; UBC15 and HERC2 under high pH stress; UBC2 and UBC15 under wounding stress; EF1beta and UBC17 under jasmonic acid treatment; UBC15 and CUL4 under abscisic acid treatment; and HERC2 and UBC17 under salicylic acid treatment. EF1beta and HERC2 were the most suitable genes for the global analysis of all samples. Furthermore, six target genes, SgPOD, SgPAL, SgLEA, SgLOX, SgHSP90 and SgPR1, were selected to validate the most and least stable reference genes under different treatments. Our results provide guidelines for reference gene selection for more accurate qRT-PCR quantification and will promote studies of gene expression in S. grandis subjected to environmental stress.

Myo-insositol (MI) is a crucial substance in the growth and developmental processes in plants. It is commonly added to the culture medium to promote adventitious shoot development. In our previous work, MI was found in influencing Agrobacterium-mediated transformation. In this report, a high-throughput RNA sequencing technique (RNA-Seq) was used to investigate differently expressed genes in one-month-old Arabidopsis seedling grown on MI free or MI supplemented culture medium. The results showed that 21,288 and 21,299 genes were detected with and without MI treatment, respectively. The detected genes included 184 new genes that were not annotated in the Arabidopsis thaliana reference genome. Additionally, 183 differentially expressed genes were identified (DEGs, FDR ≤0.05, log2 FC≥1), including 93 up-regulated genes and 90 down-regulated genes. The DEGs were involved in multiple pathways, such as cell wall biosynthesis, biotic and abiotic stress response, chromosome modification, and substrate transportation. Some significantly differently expressed genes provided us with valuable information for exploring the functions of exogenous MI. RNA-Seq results showed that exogenous MI could alter gene expression and signaling transduction in plant cells. These results provided a systematic understanding of the functions of exogenous MI in detail and provided a foundation for future studies.

BACKGROUND:Stipa grandis (Poaceae) is one of the dominant species in a typical steppe of the Inner Mongolian Plateau. However, primarily due to heavy grazing, the grasslands have become seriously degraded, and S. grandis has developed a special growth-inhibition phenotype against the stressful habitat. Because of the lack of transcriptomic and genomic information, the understanding of the molecular mechanisms underlying the grazing response of S. grandis has been prohibited. RESULTS:Using the Illumina HiSeq 2000 platform, two libraries prepared from non-grazing (FS) and overgrazing samples (OS) were sequenced. De novo assembly produced 94,674 unigenes, of which 65,047 unigenes had BLAST hits in the National Center for Biotechnology Information (NCBI) non-redundant (nr) database (E-value < 10-5). In total, 47,747, 26,156 and 40,842 unigenes were assigned to the Gene Ontology (GO), Clusters of Orthologous Group (COG), and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases, respectively. A total of 13,221 unigenes showed significant differences in expression under the overgrazing condition, with a threshold false discovery rate ≤ 0.001 and an absolute value of log2Ratio ≥ 1. These differentially expressed genes (DEGs) were assigned to 43,257 GO terms and were significantly enriched in 32 KEGG pathways (q-value ≤ 0.05). The alterations in the wound-, drought- and defense-related genes indicate that stressors have an additive effect on the growth inhibition of this species. CONCLUSIONS:This first large-scale transcriptome study will provide important information for further gene expression and functional genomics studies, and it facilitated our investigation of the molecular mechanisms of the S. grandis grazing response and the associated morphological and physiological characteristics.

Understanding the mechanism of plant morphological plasticity in response to grazing and clipping of semiarid grassland can provide insight into the process of disturbance-induced decline in grassland productivity. In recent studies there has been controversy regarding two hypotheses: 1) grazing avoidance; and 2) growth limiting mechanisms of morphological plasticity in response to defoliation. However, the experimental evidence presented for the memory response to grazing and clipping of plants has been poorly reported. This paper reports on two experiments that tested these hypotheses in field and in a controlled environment, respectively. We examined the effects of long-term clipping and grazing on the functional traits and their plasticity for Leymus chinensis (Trin.) Tzvelev (the dominate species) in the typical-steppe grassland of Inner Mongolia, China. There were four main findings from these experiments. (i) The majority of phenotypic traits of L. chinensis tended to significantly miniaturize in response to long-term field clipping and grazing. (ii) The significant response of morphological plasticity with and without grazing was maintained in a hydroponic experiment designed to remove environmental variability, but there was no significant difference in L. chinensis individual size traits for the clipping comparison. (iii) Plasticity indexes of L. chinensis traits in a controlled environment were significantly lower than under field conditions indicating that plants had partial and slight memory effect to long-term grazing. (iv) The allometry of various phenotypic traits, indicated significant trade-offs between leaf and stem allocation with variations in plant size induced by defoliation, which were maintained only under grazing in the hydroponic controlled environment experiment. Taken together, our findings suggest that the morphological plasticity of L. chinensis induced by artificial clipping was different with that by livestock grazing. The miniaturization of plant size in long-term grazed grassland may reflect retained characteristics of dwarf memory for adaptation to long-term grazing by large herbivores.