Influence of formic acid on fungal flora of barley and on aflatoxin production inAspergillus flavus link

In recent yearsAspergillus flavus and aflatoxin production have been noted on several occasions in grain preserved with formic acid. Samples of mouldy barley treated with formic acid and stored in an open bin were investigated for the presence of fungi. In the lower part of the bin there was a clear dominance ofFusarium sporotrichioides, and deoxynivalenol and neosolaniol were detected.A. flavus andA. fumigatus were also present.Paecilomyces variotii occurred, almost as a pure culture, in the upper part of the bin, but no patulin was found. Cultivation of four fungal isolates from these genera on laboratory substrates containing formic acid showedP. variotii to be the most tolerant to formic acid, withstanding 150 mM, but still without patulin production.F. sporotrichioides andA. fumigatus tolerated only 6 mM formic acid. The growth ofA. flavus was reduced and atypical at 60 mM formic acid. Pretreatment ofA. flavus spores with formic acid increased aflatoxin production about 800 times.     

Components of partial resistance of barley to Rhynchosporium secalis: use of seedling tests to predict field resistance

Components of partial resistance [disease incidence (DI), infection frequency (IF), latent period (LP), spores per lesion (SPL)] were assessed on glasshouse-grown barley seedlings following inoculation with spore suspensions of Rhynchosporium secalis at growth stage 12 (Zadoks, Chang & Konzak, 1974). Four experiments were carried out at different times during 1988. Three spring barley cultivars [two from Cyprus (cvs Kantara and Athenais) and one from the UK (cv. Triumph)] were used in the first three experiments. In the fourth experiment eight additional UK cultivars with NIAB resistance ratings ranging from 3 to 9 were used. Two races of R. secalis were used in the first three experiments and three in the fourth. The three cultivars, Kantara, Athenais and Triumph, were examined in all four experiments and significant differences detected for virtually all components of partial resistance in each. Differences, however, were often small and ranking of cultivars varied in different experiments. The greater susceptibility of cv. Kantara compared to cv. Athenais, observed under field conditions in Cyprus, would not be anticipated from the small differences in components of partial resistance observed in these experiments, but, for these cultivars, the possibility of a marked genotype x environment interaction cannot be discounted. Mean values for the components of partial resistance differed in the four experiments. LP was correlated with mean glasshouse temperature from inoculation to the onset of sporulation but differences in IF and SPL were not correlated with temperature. For these components, light quality and/or duration appeared to be more important. Overall, there were no differences between races but significant race X cultivar interactions were observed in two experiments. In the fourth experiment, examining 11 cultivars, there were significant differences between cultivars for all components of partial resistance. IF and LP were correlated but neither of these components was correlated with SPL indicating independent control of this latter component. Both IF and LP were correlated with field performance (NIAB ratings) but there was no correlation with SPL. However, combining IF with mean values of SPL restricted to the 5 days following the end of the LP, produced the best correlation (r= 0.92) with NIAB ratings. Problems of assessing components of partial resistance and possible means of improving assessments are discussed.     

Protease-stable integration of Lhcb1 into thylakoid membranes is dependent on chlorophyll b in allelic chlorina-f 2 mutants of barley (Hordeum vulgare L.)

Allelic chlorina-f2 mutants of barley (Hordeum vulgare L.) growing under different light and temperature conditions demonstrated that the chlorophyll b-free chlorina-f 2^{f  2} and chlorina-f 2¹⁰¹ express a stable phenotype. Only 3 out of 10 light-harvesting chlorophyll a/b-binding proteins, Lhca4 (photosystem I), and Lhcb1 and Lhcb6 (photosystem II), required chlorophyll b for accumulation. The other light-harvesting proteins were found in all chlorina-f2 alleles, indicating that the integration pathway of these proteins into mutant thylakoid membranes was not affected. Chlorina-f2 alleles with a thylakoid membrane capable of fullfilling photosynthesis and transport demands, but with various amounts of chlorophyll b: chlorina-f 2¹⁰¹ (chlorophyll b-free), chlorina-f2¹²³ (27% of chlorophyll b compared with the wild type) and chlorina-f 2¹²² (70% chlorophyll b), were chosen to investigate whether chlorophyll b is necessary for the protease-stable insertion of Lhcb l into mutant thylakoid membranes. The Lhcb1 was affected in almost all alleles and was most sensitive to chlorophyll b deficiency. The Lhcb1 antibody confirmed the heterogeneity of the polypeptides of the light-harvesting chlorophyll a/b-binding protein II (LHCII) and detected in wild-type membranes, two protease-resistant, mature forms of Lhcb1 with apparent molecular masses of 28 and 29 kDa. Only one band reacting with the Lhcb1 antibody could be detected in chlorophyll b-free chlorina-f 2  ^{f  2}. It co-migrated with the 29-kDa band, but was completely digested after treatment of the isolated mutant membranes with exogenous protease. This showed that in chlorina-f 2  f  ² the Lhcb1 precursor was processed at one cleavage site only. The resulting 29-kDa Lhcb1 was not provided with chlorophyll b, and, consequently, not properly folded and inserted into the membrane. It remained susceptible to protease and was inconvertable to a 28-kDa form. Received: 23 March 1998 / Accepted: 9 September 1998     

Chitinase-catalyzed hydrolysis of 4-nitrophenyl penta-N-acetyl-β-chitopentaoside as determined by real-time ESIMS: the 4-nitrophenyl moiety of the substrate interacts with the enzyme binding site

4-Nitrophenyl penta-N-acetyl-β-chitopentaoside [(GlcNAc)₅-pNP] was hydrolyzed by a family GH-19 class II barley chitinase, and the enzymatic reaction was monitored by real-time ESIMS. The wild-type enzyme hydrolyzed (GlcNAc)₅-pNP producing predominantly (GlcNAc)₃-pNP and a lesser amount of (GlcNAc)₂-pNP, indicating that the (GlcNAc)₅ portion of the substrate binds predominantly to subsites −2 ∼ +3 and less frequently to −3 ∼ +2. However, (GlcNAc)₂-pNP was mainly produced from (GlcNAc)₅-pNP by mutated enzymes, in which Trp72 and Trp82 located at +3/+4 were substituted with alanine (W72A and W72A/W82A), indicating that the (GlcNAc)₅ portion of the substrate binds predominantly to subsites −3 ∼ +2 of the mutants. The mutations of the tryptophan residues resulted in a significant shift of the substrate-binding mode to the glycon side, supporting the idea that the indole side chain of Trp72 interacts with the 4-nitrophenyl moiety of the substrate at subsite +4.     

Role of TaALMT1 malate-GABA transporter in alkaline pH tolerance of wheat

Malate exudation through wheat (Triticum aestivum L) aluminium-activated malate transporter 1 (TaALMT1) confers Al³⁺ tolerance at low pH, but is also activated by alkaline pH, and is regulated by and facilitates significant transport of gamma-aminobutyric acid (GABA, a zwitterionic buffer). Therefore, TaALMT1 may facilitate acidification of an alkaline rhizosphere by promoting exudation of both malate and GABA. Here, the performance of wheat near isogenic lines ET8 (Al⁺³-tolerant, high TaALMT1 expression) and ES8 (Al⁺³-sensitive, low TaALMT1 expression) are compared. Root growth (at 5 weeks) was higher for ET8 than ES8 at pH 9. ET8 roots exuded more malate and GABA at high pH and acidified the rhizosphere more rapidly. GABA and malate exudation was enhanced at high pH by the addition of aluminate in both ET8 and transgenic barley expressing TaALMT1. Xenopus laevis oocytes expressing TaALMT1 acidified an alkaline media more rapidly than controls corresponding to higher GABA efflux. TaALMT1 expression did not change under alkaline conditions but key genes involved in GABA turnover changed in accordance with a high rate of GABA synthesis. We propose that TaALMT1 plays a role in alkaline tolerance by exuding malate and GABA, possibly coupled to proton efflux.     

Genome-Wide Identification of Candidate Genes Associated with <i>β</i>-glucan Traits in a Hulled and Hulless Barley (<i>Hordeum vulgare</i> L.) Population

Barley grain is a valuable source of β-glucan, which is an important component of dietary fiber with significant human health benefits. Although the genetic basis of β-glucan biosynthesis has been widely studied, a genome-wide association study (GWAS) is still required for a scan of the candidate genes related to the complex quantitative trait based on the high-quality barley reference genome. In this study, a GWAS was conducted using a population composed of 87 barley landraces (39 hulled and 48 hulless, β-glucan from 2.07% to 6.56%) with 191,098 nucleotide polymorphisms (SNPs) markers to cover the chromosomes with the highest density. The population was divided into four sub-populations (POP1~POP4), and the β-glucan content in POP2 was significantly higher than that in other groups, in which most of the hulless barley landraces are from Qinghai-Tibet Plateau in China. Among seven SNP markers identified by GWAS, two (SNP2 and SNP3) of them showed positive correlation to β-glucan trait and the remaining five (SNP1, SNP4, SNP5, SNP6 and SNP7) showed the negative relationship. Two candidate genes linked to SNP7, HORVU7Hr1G000320 and HORVU7Hr1G000040, belong to the nucleotide triphosphate hydrolase superfamily which is probable to affect the activities of β-glucan synthase. Another candidate gene associated with SNP1, HORVU1Hr1G000010, is possibly involved in sugar response. In conclusion, our results provide new insights into the genetic basis of β-glucan accumulation in barley grains, and the discovery of new SNP markers distributed in each chromosome and the associated candidate genes will be valuable for the breeding of functional barley varieties with high β-glucan.     

Segmental duplications are hot spots of copy number variants affecting barley gene content

Copy number variants (CNVs) are pervasive in several animal and plant genomes and contribute to shaping genetic diversity. In barley, there is evidence that changes in gene copy number underlie important agronomic traits. The recently released reference sequence of barley represents a valuable genomic resource for unveiling the incidence of CNVs that affect gene content and for identifying sequence features associated with CNV formation. Using exome sequencing and read count data, we detected 16 605 deletions and duplications that affect barley gene content by surveying a diverse panel of 172 cultivars, 171 landraces, 22 wild relatives and other 32 uncategorized domesticated accessions. The quest for segmental duplications (SDs) in the reference sequence revealed many low‐copy repeats, most of which overlap predicted coding sequences. Statistical analyses revealed that the incidence of CNVs increases significantly in SD‐rich regions, indicating that these sequence elements act as hot spots for the formation of CNVs. The present study delivers a comprehensive genome‐wide study of CNVs affecting barley gene content and implicates SDs in the molecular mechanisms that lead to the formation of this class of CNVs.     

Predicting polymorphic EST‐SSRs in silico

The public availability of large quantities of gene sequence data provides a valuable resource of the mining of Simple Sequence Repeat (SSR) molecular genetic markers for genetic analysis. These markers are inexpensive, require minimal labour to produce and can frequently be associated with functionally annotated genes. This study presents the characterization of barley EST‐SSRs and the identification of putative polymorphic SSRs from EST data. Polymorphic SSRs are distinguished from monomorphic SSRs by the representation of varying motif lengths within an alignment of sequence reads. Two measures of confidence are calculated, redundancy of a polymorphism and co‐segregation with accessions. The utility of this method is demonstrated through the discovery of 597 candidate polymorphic SSRs, from a total of 452 642 consensus expressed sequences. PCR amplification primers were designed for the identified SSRs. Ten primer pairs were validated for polymorphism in barley and for transferability across species. Analysis of the polymorphisms in relation to SSR motif, length, position and annotation is discussed.