RNA-Seq analysis of gene expression for floral development in crested wheatgrass (Agropyron cristatum L.).

Crested wheatgrass [Agropyron cristatum L. (Gaertn.)] is widely used for early spring grazing in western Canada and the development of late maturing cultivars which maintain forage quality for a longer period is desired. However, it is difficult to manipulate the timing of floral transition, as little is known about molecular mechanism of plant maturity in this species. In this study, RNA-Seq and differential gene expression analysis were performed to investigate gene expression for floral initiation and development in crested wheatgrass. Three cDNA libraries were generated and sequenced to represent three successive growth stages by sampling leaves at the stem elongation stage, spikes at boot and anthesis stages. The sequencing generated 25,568,846; 25,144,688 and 25,714,194 qualified Illumina reads for the three successive stages, respectively. De novo assembly of all the reads generated 311,671 transcripts with a mean length of 487 bp, and 152,849 genes with an average sequence length of 669 bp. A total of 48,574 (31.8%) and 105,222 (68.8%) genes were annotated in the Swiss-Prot and NCBI non-redundant (nr) protein databases, respectively. Based on the Kyoto Encyclopedia of Genes and Genome (KEGG) pathway database, 9,723 annotated sequences were mapped onto 298 pathways, including plant circadian clock pathway. Specifically, 113 flowering time-associated genes, 123 MADS-box genes and 22 CONSTANS-LIKE (COL) genes were identified. A COL homolog DN52048-c0-g4 which was clustered with the flowering time genes AtCO and OsHd1 in Arabidopsis (Arabidopsis thaliana L.) and rice (Oryza sativa L.), respectively, showed specific expression in leaves and could be a CONSTANS (CO) candidate gene. Taken together, this study has generated a new set of genomic resources for identifying and characterizing genes and pathways involved in floral transition and development in crested wheatgrass. These findings are significant for further understanding of the molecular basis for late maturity in this grass species.

Uncovering the Genetic Architecture of Seed Weight and Size in Intermediate Wheatgrass through Linkage and Association Mapping.

Intermediate wheatgrass [IWG; (Host) Barkworth & D.R. Dewey subsp. ] is being developed as a new perennial grain crop that has a large allohexaploid genome similar to that of wheat ( L.). Breeding for increased seed weight is one of the primary goals for improving grain yield of IWG. As a new crop, however, the genetic architecture of seed weight and size has not been characterized, and selective breeding of IWG may be more intricate than wheat because of its self-incompatible mating system and perennial growth habit. Here, seed weight, seed area size, seed width, and seed length were evaluated across multiple years, in a heterogeneous breeding population comprised of 1126 genets and two clonally replicated biparental populations comprised of 172 and 265 genets. Among 10,171 DNA markers discovered using genotyping-by-sequencing (GBS) in the breeding population, 4731 markers were present in a consensus genetic map previously constructed using seven full-sib populations. Thirty-three quantitative trait loci (QTL) associated with seed weight and size were identified using association mapping (AM), of which 23 were verified using linkage mapping in the biparental populations. About 37.6% of seed weight variation in the breeding population was explained by 15 QTL, 12 of which also contributed to either seed length or seed width. When performing either phenotypic selection or genomic selection for seed weight, we observed the frequency of favorable QTL alleles were increased to >46%. Thus, by combining AM and genomic selection, we can effectively select the favorable QTL alleles for seed weight and size in IWG breeding populations.

Cloning and expression analysis of cysteine protease gene (MwCP) in Agropyron mongolicum Keng.

In this study, a cysteine protease gene (MwCP) from Agropyron mongolicum Keng was isolated using RACE. Sequence analysis indicated that MwCP was 1473 bp, and it contained a 1134-bp open reading frame, which encoded 377 amino acids with a 24-amino acid N-terminal signal peptide. The results indicated that the MwCP protein was a new member of the papain C1A family, and it was predicted to be an extracellular, secretory stable hydrophilic protein. The secondary structure of MwCP was mainly composed of α-helices and random coils, and the space structure primarily contained α-helices, ß-sheets, and ß-turns. Homology analyses showed the 98% homology between MwCP amino acids and a cysteine protease found in Triticum aestivum (GenBank accession No. AAW21813.1). Analysis of mRNA using semi-quantitative RT-PCR indicated that during a 48-h drought stress period, MwCP was expressed during the 4th hour, and the expression level peaked during the 6th hour before declining to the original level. The results revealed that MwCP was involved in drought-resistant physiological processes of A. mongolicum. Moreover, the MwCP expression levels were highest in leaves, intermediate in roots, and lowest in stems.

Linking ethylene to nitrogen-dependent leaf longevity of grass species in a temperate steppe.

BACKGROUND AND AIMS: Leaf longevity is an important plant functional trait that often varies with soil nitrogen supply. Ethylene is a classical plant hormone involved in the control of senescence and abscission, but its role in nitrogen-dependent leaf longevity is largely unknown. METHODS: Pot and field experiments were performed to examine the effects of nitrogen addition on leaf longevity and ethylene production in two dominant plant species, Agropyron cristatum and Stipa krylovii, in a temperate steppe in northern China. KEY RESULTS: Nitrogen addition increased leaf ethylene production and nitrogen concentration but shortened leaf longevity; the addition of cobalt chloride, an ethylene biosynthesis inhibitor, reduced leaf nitrogen concentration and increased leaf longevity. Path analysis indicated that nitrogen addition reduced leaf longevity mainly through altering leaf ethylene production. CONCLUSIONS: These findings provide the first experimental evidence in support of the involvement of ethylene in nitrogen-induced decrease in leaf longevity.

Hierarchical reproductive allocation and allometry within a perennial bunchgrass after 11 years of nutrient addition.

Bunchgrasses are one of the most important plant functional groups in grassland ecosystems. Reproductive allocation (RA) for a bunchgrass is a hierarchical process; however, how bunchgrasses adjust their RAs along hierarchical levels in response to nutrient addition has never been addressed. Here, utilizing an 11-year nutrient addition experiment, we examined the patterns and variations in RA of Agropyron cristatum at the individual, tiller and spike levels. We evaluated the reproductive allometric relationship at each level by type II regression analysis to determine size-dependent and size-independent effects on plant RA variations. Our results indicate that the proportion of reproductive individuals in A. cristatum increased significantly after 11 years of nutrient addition. Adjustments in RA in A. cristatum were mainly occurred at the individual and tiller levels but not at the spike level. A size-dependent effect was a dominant mechanism underlying the changes in plant RA at both individual and tiller levels. Likewise, the distribution of plant size was markedly changed with large individuals increasing after nutrient addition. Tiller-level RA may be a limiting factor for the adjustment of RA in A. cristatum. To the best of our knowledge, this study is the first to examine plant responses in terms of reproductive allocation and allometry to nutrient enrichment within a bunchgrass population from a hierarchical view. Our findings have important implications for understanding the mechanisms underlying bunchgrass responses in RA to future eutrophication due to human activities. In addition, we developed a hierarchical analysis method for disentangling the mechanisms that lead to variation in RA for perennial bunchgrasses.

The invasive grass Agropyron cristatum doubles belowground productivity but not soil carbon.

Root dynamics are among the largest knowledge gaps in determining how terrestrial carbon (C) cycles will respond to environmental change. Increases in productivity accompanying plant invasions and introductions could increase ecosystem C storage, but belowground changes are unknown, even though roots may account for 50-90% of production in temperate ecosystems. We examined whether the introduction of a widespread invasive grass with relatively high shoot production also increased belowground productivity and soil C storage, using a multiyear rhizotron study in 50-year-old stands dominated either by the invasive C3 grass Agropyron cristatum or by largely C4 native grasses. Relative to native vegetation, stands dominated by the invader had doubled root productivity. Soil carbon isotope values showed that the invader had made detectable contributions to soil C. Soil C content, however, was not significantly different between invader-dominated stands (0.42 mg C/g soil) and native vegetation (0.45 mg C/g soil). The discrepancy between enhanced production and lack of soil C changes was attributable to differences in root traits between invader-dominated stands and native vegetation. Relative to native vegetation, roots beneath the invader had 59% more young white tissue, with 80% higher mortality and 19% lower C:N ratios (all P < 0.05). Such patterns have previously been reported for aboveground tissues of invaders, and we show that they are also found belowground. If these root traits occur in other invasive species, then the global phenomenon of increased productivity following biological invasion may not increase soil C storage.

Chelator-enhanced lead accumulation in Agropyron elongatum cv Szarvasi-1 in hydroponic culture.

Hydroponic culture was applied to compare the efficiency of K2EDTA and citrate in mobilizing Pb for accumulation in Agropyron elongatum cv. Szarvasi-1 and their effects on some physiological characteristics of the plants. The plants were grown in nutrient solutions containing 0, 10, and 100 microM Pb(NO3)2 combined with chelating agents added to the nutrient solutions after 21 days of growth, in 3 concentrations (0, 100, and 500 microM). The effects were measured after eight days. The energy grass proved to be greatly resistant to the treatments, as was reflected in the slight inhibition of growth, the resistance of the photosynthetic electron transport chain and the chlorophyll composition and the lack of change in the malone-dialdehyde content. Fundamental differences can be identified between the effects of EDTA and citrate. Citrate had only a little effect on the physiological parameters, which may be due to the strongly increasing lead accumulation with increasing concentration of Pb in the nutrient solution. Additionally, citrate ensured a higher biomass yield with higher shoot Pb accumulation compared to EDTA in almost all treatments. Concerning biomass reduction, 10 microM Pb applied together with K2EDTA has the most deleterious effects on energy grass. The effects correlated with the concentration of EDTA.

Nitrogen and water addition reduce leaf longevity of steppe species.

BACKGROUND AND AIMS: Changes in supplies of resources will modify plant functional traits. However, few experimental studies have addressed the effects of nitrogen and water variations, either singly or in combination, on functional traits. METHODS: A 2-year field experiment was conducted to test the effects of nitrogen and water addition on leaf longevity and other functional traits of the two dominant (Agropyron cristatum and Stipa krylovii) and three most common species (Cleistogenes squarrosa, Melilotoides ruthenica and Potentilla tanacetifolia) in a temperate steppe in northern China. KEY RESULTS: Additional nitrogen and water increased leaf nitrogen content and net photosynthetic rate, and changed other measured functional traits. Leaf longevity decreased significantly with both nitrogen addition (-6 days in 2007 and -5·4 days in 2008; both P < 0·001) and watering (-13 days in 2007 and -9·9 days in 2008; both P < 0·001), and significant differences in leaf longevity were also found among species. Nitrogen and water interacted to affect leaf longevity and other functional traits. Soil water content explained approx. 70 % of the shifts in leaf longevity. Biomass at both species and community level increased under water and nitrogen addition because of the increase in leaf biomass production per individual plant. CONCLUSIONS: The results suggest that additional nitrogen and water supplies reduce plant leaf longevity. Soil water availability might play a fundamental role in determining leaf longevity and other leaf functional traits, and its effects can be modified by soil nitrogen availability in semi-arid areas. The different responses of species to resource alterations may cause different global change ramifications under future climate change scenarios.

Species-specific response of photosynthesis to burning and nitrogen fertilization.

The present study was conducted to examine photosynthetic characteristics of three dominant grass species (Agropyron cristatum, Leymus chinensis, and Cleistogenes squarrosa) and their responses to burning and nitrogen fertilization in a semiarid grassland in northern China. Photosynthetic rate (P(n)), stomatal conductance (g(s)), and water use efficiency (WUE) showed strong temporal variability over the growing season. C. squarrosa showed a significantly higher P(n) and WUE than A. cristatum and L. chinensis. Burning stimulated P(n) of A. cristatum and L. chinensis by 24-59% (P < 0.05) in the early growing season, but not during other time periods. Light-saturated photosynthetic rate (P(max)) in A. cristatum and the maximum apparent quantum yield (Phi(max)) in A. cristatum and L. chinensis were significantly enhanced by burning (16-67%) in both the fertilized and unfertilized plots. The main effect of burning on P(n), P(max) and Phi(max) was not significant in C. squarrosa. The burning-induced changes in soil moisture could explain 51% (P = 0.01) of the burning-induced changes in P(n) of the three species. All three species showed positive responses to N fertilization in terms of P(n), P(max), and Phi(max). The stimulation of P(n) under N fertilization was mainly observed in the early growing season when the soil extractable N content was significantly higher in the fertilized plots. The N fertilization-induced changes in soil extractable N content could explain 66% (P = 0.001) of the changes in P(n) under N fertilization. The photosynthetic responses of the three species indicate that burning and N fertilization will potentially change the community structure and ecosystem productivity in the semiarid grasslands of northern China.

A new species of Epichloë symbiotic with Chinese grasses.

Epichloë species are fungal symbionts (endophytes) of grasses, six European and four North American biological species in genus Epichloë have been described in previous researches. In this study we describe a new Epichloë species, Epichloë yangzii Li et Wang, found in natural symbioses with Roegneria kamoji native to China. We investigated the host specificity, morphology, interfertility tests and molecular phylogenetic evidences of this new species. The results indicated that E. yangzii is host specific and seedborne. Most morphological characteristics of this new species are typical in the genus. However differences are evident in several features including size of perithecia, asci and ascospores. In mating tests E. yangzii was not interfertile with E. elymi isolates from related hosts in genera Elymus. Phylogenetic relationships based on sequences of beta-tubulin gene (tub2) introns and translation elongation factor 1-alpha gene (tef1) introns showed that members of the new species grouped into exclusive clades with high bootstrap value.

Asymmetric somatic hybridization between wheat (Triticum aestivum L.) and Agropyron elongatum (Host) Nevishi.

Suspension-derived protoplasts of Agropyron elongatum irradiated by ultra-violet light (UV) were fused with the suspension-derived protoplasts of Triticum astivum using PEG. Fertile intergeneric somatic hybrid plants were produced and various hybrid lines have been selected and propagated in successive generations. Their hybrid nature was confirmed by analysis of profiles of isozymes, RAPDs, and 5S rDNA spacer sequences, and via GISH analysis. By the procedure described, the phenotype and chromosome number of wheat could be maintained besides transfer of a few chromosomes and chromosomal fragments from the donor A. elongatum. The results above indicated that highly asymmetric fertile hybrid plants and hybrid progenies of wheat were produced via somatic hybridization.