Asparagine is an important nitrogen storage and transport molecule, but its accumulation as a free amino acid in crops has implications for food safety because free asparagine is a precursor for acrylamide formation during cooking and processing. Asparagine synthesis occurs by the amidation of aspartate, catalysed by asparagine synthetase, and this study concerned the expression of asparagine synthetase (TaASN) genes in wheat. The expression of three genes, TaASN1-3, was studied in different tissues and in response to nitrogen and sulphur supply. The expression of TaASN2 in the embryo and endosperm during mid to late grain development was the highest of any of the genes in any tissue. Both TaASN1 and TaASN2 increased in expression through grain development, and in the grain of field-grown plants during mid-development in response to sulphur deprivation. However, only TaASN1 was affected by nitrogen or sulphur supply in pot-based experiments, showing complex tissue-specific and developmentally-changing responses. A putative N-motif or GCN4-like regulatory motif was found in the promoter of TaASN1 genes from several cereal species. As the study was completed, a fourth gene, TaASN4, was identified from recently available genome data. Phylogenetic analysis showed that other cereal species have similar asparagine synthetase gene families to wheat.

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Asparagine synthetase activity in cereals has become an important issue with the discovery that free asparagine concentration determines the potential for formation of acrylamide, a probably carcinogenic processing contaminant, in baked cereal products. Asparagine synthetase catalyses the ATP-dependent transfer of the amino group of glutamine to a molecule of aspartate to generate glutamate and asparagine. Here, asparagine synthetase-encoding polymerase chain reaction (PCR) products were amplified from wheat (Triticum aestivum) cv. Spark cDNA. The encoded proteins were assigned the names TaASN1, TaASN2, and TaASN3 on the basis of comparisons with other wheat and cereal asparagine synthetases. Although very similar to each other they differed slightly in size, with molecular masses of 65.49, 65.06, and 66.24 kDa, respectively. Chromosomal positions and scaffold references were established for TaASN1, TaASN2, and TaASN3, and a fourth, more recently identified gene, TaASN4. TaASN1, TaASN2, and TaASN4 were all found to be single copy genes, located on chromosomes 5, 3, and 4, respectively, of each genome (A, B, and D), although variety Chinese Spring lacked a TaASN2 gene in the B genome. Two copies of TaASN3 were found on chromosome 1 of each genome, and these were given the names TaASN3.1 and TaASN3.2. The TaASN1, TaASN2, and TaASN3 PCR products were heterologously expressed in Escherichia coli (TaASN4 was not investigated in this part of the study). Western blot analysis identified two monoclonal antibodies that recognized the three proteins, but did not distinguish between them, despite being raised to epitopes SKKPRMIEVAAP and GGSNKPGVMNTV in the variable C-terminal regions of the proteins. The heterologously expressed TaASN1 and TaASN2 proteins were found to be active asparagine synthetases, producing asparagine and glutamate from glutamine and aspartate. The asparagine synthetase reaction was modeled using SNOOPY® software and information from the BRENDA database to generate differential equations to describe the reaction stages, based on mass action kinetics. Experimental data from the reactions catalyzed by TaASN1 and TaASN2 were entered into the model using Copasi, enabling values to be determined for kinetic parameters. Both the reaction data and the modeling showed that the enzymes continued to produce glutamate even when the synthesis of asparagine had ceased due to a lack of aspartate.

ABSTRACT. We analyzed the genetic diversity of 115 barley germplasms, including 112 landraces and three new barley cultivars grown in the Shanghai region, using a set of 11 SSR markers. Sixtysix alleles were observed at the 11 SSR loci, ranged from three to ten, with a mean of six alleles per locus. The polymorphism information content ranged from 0.568 to 0.853, with a mean of 0.732, indicating considerable genetic variation in barley in the Shanghai area. Clustering analysis indicated that these barley accessions could be divided into two categories (A and B). Ninety-seven six-rowed barley cultivars were classified in the A category; sixteen two-rowed and two six-rowed barley cultivars were classified in the B category. This demonstrated genetic differences between two-rowed and six-rowed barley varieties. In addition, we found that the three new barley cultivars are closely related.

The excess use of nitrogen fertilizers causes many problems, including higher costs of crop production, lower nitrogen use efficiency, and environmental damage. Crop breeding for low-nitrogen tolerance, especially molecular breeding, has become the major route to solving these issues. Therefore, in crops such as barley (Hordeum vulgare L.), it is crucial to understand the mechanisms of low-nitrogen tolerance at the molecule level. In the present study, two barley cultivars, BI-04 (tolerant to low nitrogen) and BI-45 (sensitive to low nitrogen), were used for gene expression analysis under low-nitrogen stress, including 10 genes related to primary nitrogen metabolism. The results showed that the expressions of HvNIA2 (nitrite reductase), HvGS2 (chloroplastic glutamine synthetase), and HvGLU2 (ferredoxin-dependent glutamate synthase) were only induced in shoots of BI-04 under low-nitrogen stress, HvGLU2 was also only induced in roots of BI-04, and HvGS2 showed a rapid response to low-nitrogen stress in the roots of BI-04. The expression of HvASN1 (asparagine synthetase) was reduced in both cultivars, but it showed a lower reduction in the shoots of BI-04. In addition, gene expression and regulation differences in the shoots and roots were also compared between the barley cultivars. Taken together, the results indicated that the four above-mentioned genes might play important roles in low-nitrogen tolerance in barley.

Salinity is one of the major abiotic stresses that affect crop productivity. Identification of the potential novel genes responsible for salt tolerance in barley will contribute to understanding the molecular mechanism of barley responses to salt stress. We compared changes in transcriptome between Hua 11 (a salt-tolerant genotype) and Hua 30 (a salt sensitive genotype) in response to salt stress at the seedling stage using barley cDNA microarrays. In total, 557 and 247 salt-responsive genes were expressed exclusively in the shoot and root tissue of the salt-tolerant genotype, respectively. Among these genes, a number of signal-related genes, transcription factors and compatible solutes were identified and some of these genes were carefully discussed. Notably, a LysM RLK was firstly found involved in salt stress response. Moreover, key enzymes in the pathways of jasmonic acid biosynthesis, lipid metabolism and indole-3-acetic acid homeostasis were specifically affected by salt stress in salt tolerance genotype. These salt-responsive genes and biochemical pathways identified in this study could provide further information for understanding the mechanisms of salt tolerance in barley.

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In vitro mutagenesis via isolated microspore culture provides an efficient way to produce numerous double haploid (DH) lines with mutation introduction and homozygosity stabilization, which can be used for screening directly. In this study, 356 DH lines were produced from the malt barley (Hordeum vulgare L.) cultivar Hua-30 via microspore mutagenic treatment with ethyl methane sulfonate or pingyangmycin during in vitro culture. The lines were subjected to field screening under high nitrogen (HN) and low nitrogen (LN) conditions, and the number of productive tillers was used as the main screening index. Five mutant lines (A1-28, A1-84, A1-226, A16-11, and A9-29) with high numbers of productive tillers were obtained over three consecutive years of screening. In the fifth year, components related to N-use efficiency (NUE), including N accumulation, utilization, and translocation, were characterized for these lines based on N uptake efficiency (NUpE), N utilization efficiency (NUtE), and N translocation efficiency (NTE). The results show that the NUpE of four mutant lines (A1-84, A1-226, A9-29, and A16-11) improved significantly under HN, whereas that of two lines (A1-84 and A9-29) improved under LN. As a result, their NUE improved greatly. No improvement in NUtE was observed in any of the five mutant lines. A1-84 and A9-29 were selected as an enhanced genotype in N uptake, and A1-28 showed improved NTE at the grain-filling stage. Our results imply that high-NUpE mutants can be produced through microspore mutagenesis and field screening, and that improvement of NUE in barley depends on enhancement of N uptake.