Recent advances in the genetics underlying wheat grain protein content and grain protein deviation in hexaploid wheat.

Wheat is one of the most important global crops and selection for better performance has been ongoing since ancient times. As a quantitative trait controlled by the interplay of several genomic loci and under the strong influence of the environment, grain protein content (GPC) is of major interest in breeding programs. Here, we review the most recent contributions to the genetics underlying wheat GPC and grain protein deviation (GPD, representing the relationship between grain protein content and yield), together with the performance of genomic prediction models characterizing these traits. A total of 364 significant loci related to GPC and GPD are positioned on the hexaploid wheat genome, highlighting genomic regions where significant independent QTL overlap, with special focus on two regions located on chromosomes 3A and 5A. Some of the corresponding homoeologous sequences co-locate with significant independent QTL reported on the B and D subgenomes. Overlapping independent QTL from different studies are indicative of genomic regions exhibiting stability across environments and genotypes, with promising candidates for improving grain quality.

Leaf senescence and nutrient remobilisation in barley and wheat.

Extensive studies have been undertaken on senescence processes in barley and wheat and their importance for the nitrogen use efficiency of these crop plants. During the senescence processes, proteins are degraded and nutrients are re-mobilised from senescing leaves to other organs, especially the developing grain. Most of the proteins degraded reside in the chloroplasts, with Rubisco constituting the most dominant protein fraction. Despite intensive studies, the proteases responsible for Rubisco degradation have not yet been identified. Evidence for degradation of stromal proteins outside of chloroplasts is summarised. Rubisco is thought to be released from chloroplasts into vesicles containing stroma material (RCB = Rubisco-containing bodies). These vesicles may then take different routes for their degradation. Transcriptome analyses on barley and wheat senescence have identified genes involved in degradative, metabolic and regulatory processes that could be used in future strategies aimed at modifying the senescence process. The breeding of crops for characters related to senescence processes, e.g. higher yields and better nutrient use efficiency, is complex. Such breeding has to cope with the dilemma that delayed senescence, which could lead to higher yields, is correlated with a decrease in nutrient use efficiency. Pinpointing regulatory genes involved in senescence might lead to tools that could effectively overcome this dilemma.

Up-regulation of the ascorbate-dependent antioxidative system in barley leaves during powdery mildew infection.

Abstract The ascorbate-dependent antioxidative system was studied in leaves of barley infected with the barley powdery mildew fungus Blumeria graminis f.sp. hordei (Bgh). Increased ascorbate peroxidase (APX) activity was detected upon infection, especially in the compatible interaction. APX activity was determined in epidermal and total leaf tissues. A relatively higher increase in APX activity was found in the epidermis compared to total leaf 72 h after inoculation in the compatible interaction, but the increase was not restricted to the epidermis. Activity assays in native gels and Northern blot hybridization indicated that the increase in APX activity was caused by a cytosolic APX isoform. 'Inverse Northern blot' hybridization results with the cDNA of a cytosolic APX supported the relatively higher increase in epidermal APX activity compared to total leaf activity. In the compatible interaction, monodehydroascorbate reductase (MDHAR) activity increased in temporal and spatial patterns similar to that of APX activity. In contrast to this, dehydroascorbate reductase and glutathione reductase activities either decreased or were unaffected by Bgh infection. The increase in APX and MDHAR activities in the compatible interaction continued until severe infection of the leaves. Thus, an up-regulation of the antioxidative system of the host cells could play a role for maintenance of the biotrophic relationship between Bgh and the barley leaf by preventing proliferating oxidative processes, which would otherwise be harmful to the living plant cell on which the biotrophic powdery mildew fungus depends.

A chalcone synthase with an unusual substrate preference is expressed in barley leaves in response to UV light and pathogen attack.

A cDNA clone was isolated by differential hybridization from a library prepared from barley leaves inoculated with the fungus Blumeria graminis f.sp. hordei (Bgh). The open reading frame of the insert (designated HvCHS2) encoded a polypeptide with 72-79% identity to chalcone synthases (CHS) and 65-68% identity to stilbene synthases. Alignments of the amino acid sequence of HvCHS2 with the consensus sequence of naringenin-CHS (EC 2.3.1.74) reveals significant differences between HvCHS2 and naringenin-CHS. HvCHS2 transcripts accumulate strongly in barley leaves in response to inoculation with Bgh, whereas only insignificant accumulation of barley naringenin-CHS (CHS1) transcripts is seen upon the inoculation. The accumulation of HvCHS2 transcripts is also elicited by UV light. To compare the activity of HvCHS2 with the activity of CHS1, the two enzymes were expressed in Escherichia coli. Both HvCHS2 and CHS1 catalyse the formation of chalcones. However, HvCHS2 and CHS1 differ in their substrate requirements. CHS1 uses cinnamoyl-CoA and 4-coumaroyl-CoA at comparable rates whereas feruloyl-CoA is a poor substrate for this enzyme. In contrast, HvCHS2 converts feruloyl-CoA and caffeoyl-CoA at the highest rate whereas cinnamoyl-CoA is a poor substrate. Thus, HvCHS2 is a novel pathogen and UV light induces homoeriodictyol/eriodictyol CHS involved in the direct production of flavonoids possessing multi-substituted B-rings.

Molecular characterization of the oxalate oxidase involved in the response of barley to the powdery mildew fungus.

Previously we reported that oxalate oxidase activity increases in extracts of barley (Hordeum vulgare) leaves in response to the powdery mildew fungus (Blumeria [syn. Erysiphe] graminis f.sp. hordei) and proposed this as a source of H2O2 during plant-pathogen interactions. In this paper we show that the N terminus of the major pathogen-response oxalate oxidase has a high degree of sequence identity to previously characterized germin-like oxalate oxidases. Two cDNAs were isolated, pHvOxOa, which represents this major enzyme, and pHvOxOb', representing a closely related enzyme. Our data suggest the presence of only two oxalate oxidase genes in the barley genome, i.e. a gene encoding HvOxOa, which possibly exists in several copies, and a single-copy gene encoding HvOxOb. The use of 3' end gene-specific probes has allowed us to demonstrate that the HvOxOa transcript accumulates to 6 times the level of the HvOxOb transcript in response to the powdery mildew fungus. The transcripts were detected in both compatible and incompatible interactions with a similar accumulation pattern. The oxalate oxidase is found exclusively in the leaf mesophyll, where it is cell wall located. A model for a signal transduction pathway in which oxalate oxidase plays a central role is proposed for the regulation of the hypersensitive response.

A flavonoid 7-O-methyltransferase is expressed in barley leaves in response to pathogen attack.

We have shown previously that transcripts corresponding to the cDNA clone pBH72-F1, with similarities to O-methyltransferases (OMT), accumulated in barley leaves in response to attack by the pathogenic fungus Blumeria graminis (Plant Mol Biol 26 (1994) 1797). To investigate the accumulation pattern in the defence response and the organ localization of the pBH72-F1-encoded polypeptide (F1-OMT), an antiserum was raised against Escherichia coli expressed F1-OMT. The 43 kDa protein was absent in normal leaves but accumulated strongly in response to pathogen attack. The F1-OMT protein accumulated faster in barley lines inoculated with an avirulent B. graminis isolates compared to a virulent isolate. Additionally, F1-OMT related proteins were detected in developing kernels. F1-OMT was expressed as a functional enzyme in E. coli and the substrate specificity was investigated. The enzyme exhibited OMT activity towards flavonoid aglycones with the highest activity against apigenin (4',5,7-trihydroxyflavone). In contrast, caffeic acid did not serve as substrate for F1-OMT. The product of F1-OMT was analyzed by HPLC and GC-MS and found to be genkwanin (4',5-dihydroxy-7-methoxyflavone). Initial velocity data were best represented by a sequential bi-bi mechanism, and kinetic parameters of KSAM = 10.9 microM, Kapigenin = 4.6 microM and a specific activity of 0.45 mukat/g were obtained. Barley F1-OMT, apigenin 7-O-methyltransferase, is suggested to be involved in the production of a methylated flavonoid phytoalexin.

An epidermis/papilla-specific oxalate oxidase-like protein in the defence response of barley attacked by the powdery mildew fungus.

A cDNA clone of a defence response transcript was isolated from a library prepared from barley leaves expressing papilla resistance towards the powdery mildew fungus, Blumeria (syn. Erysiphe) graminis f.sp. hordei (Bgh). The 904 bp sequence encodes a 229 amino acid polypeptide with a putative signal peptide of 23 amino acids. After cleavage, the protein has a mass of 22.3 kDa and exhibits up to 60% amino acid identity to certain dicot proteins, and 46% amino acid identity to barley oxalate oxidase; therefore we designated it HvOxOLP (for Hordeum vulgare oxalate oxidase-like protein). Single-base substitutions among several cDNA and RACE clones demonstrate a gene of many copies. Both the transcript and protein accumulate from 3 h after inoculation with Bgh. The transcript level peaks at 18-24 h and subsequently decreases, whereas the protein level is stable from 24 h after inoculation. The accumulation patterns are independent of the outcome of the barley/powdery mildew interaction, unlike that of PR proteins, for example. The transcript accumulates specifically in the inoculated epidermal tissue. This temporal and spatial expression pattern suggests a very close relationship to papilla formation. Immunoblot analyses have facilitated a demonstration that HvOxOLP, like oxalate oxidase, is a water-soluble 100 kDa oligomeric protein. The oligomer is heat-stable and SDS-tolerant, and it can be denatured into a 25 kDa monomer. Attempts to demonstrate oxalate oxidase activity for this protein have failed. However, the relationships to oxalate oxidase suggests that HvOxOLP may be involved in H2O2 generation necessary for, for example, cross-linking of cell wall components during formation of papillae.

Purification, characterization, and molecular cloning of basic PR-1-type pathogenesis-related proteins from barley.

Partial amino acid sequences of two proteins, purified from barley leaves reacting hypersensitively to the powdery mildew fungus, showed a high degree of amino acid identity to the PR-1 proteins originally described in tobacco. The proteins, subsequently designated HvPR-1a and HvPR-1b, show apparent pI values of approximately 10.5 and 11, respectively and apparent M(r) 15,000. Independently, differential screening of a cDNA library prepared from barley leaves, exhibiting a compatible interaction with the powdery mildew fungus, resulted in isolation of cDNA species representing two PR-1 homologs. With the exception of one amino acid, the partial amino acid sequences of HvPR-1a and HvPR-1b are identical to internal sequences of the polypeptides derived from the two cDNA species. These derived polypeptides are each 164 amino acids long and both have putative N-terminal leader sequences of 24 amino acids. That these proposed leader sequences are functional is indicated by the observed occurrence of both proteins in the intercellular fluid. The proposed mature proteins (calculated M(r) 14,490 and 15,204) share 91% identical amino acids with each other and 56 to 74% with other PR-1 proteins. Northern blot hybridization and immunoblotting, respectively, show that both transcripts and both proteins accumulate following inoculation of susceptible and hypersensitivity resistant barley leaves with the powdery mildew fungus.

A pathogen-induced gene of barley encodes a protein showing high similarity to a protein kinase regulator.

A full length cDNA of a barley leaf messenger, found to increase in amount during infection attempts by the powdery mildew fungus (Erysiphe graminis), is characterized. The messenger encodes a polypeptide of 261 amino acid residues with a calculated mass of 29.2 kDa and a pI of 4.6. Sequence comparisons as well as serological studies demonstrate that the encoded protein is closely related to a family of mammalian proteins believed to have functions associated with the multifunctional Ca(2+)-dependent protein kinases.

The effect of indomethacin 1% ophthalmic suspension on the pupil during extracapsular cataract surgery.

Fifty-six patients who underwent extracapsular cataract surgery were randomized in two groups with 28 patients in each group. Group 1 was treated with standard preoperative dilation regime and group 2 received in addition indomethacin 1% ophthalmic solution. Horizontal and vertical pupil diameter measurements were taken before capsulotomy, before exprimation of the lens nucleus and before lens implantation. The size of the pupil at the time of capsulotomy and at the time just before lens exprimation was greatest in the non-indomethacin treated group, but at the time of lens implantation the pupil of the indomethacin treated group was greatest. It is concluded that even though indomethacin seems to be able to inhibit surgically induced miosis, probably through its inhibitory effect on prostaglandin synthesis, the effect is only marginal and from a clinical point of view it cannot be recommended.

[Treatment of corneal abrasion]. / Behandlingen af abrasio corneae.

Forty patients with corneal epithelial defects were randomized into two groups. Group 1 (twenty patients) were treated with chloramphenicol ointment and an eye patch for six hours followed by chloramphenicol eyedrops six time a day for six days. Group 2 (twenty patients) were treated with chloramphenicol ointment and double eye patches for twenty-four hours as the only treatment. The patients were examined the next day, and again after a week. Two patients defected in group 1 and two patients from group 1 were excluded from the experiment because they failed to improve after three and four days, respectively. Five patients from group 2 defected. Corneal epithelial wound healing was found to be significantly faster in the group treated with double eye patches and the patients' subjective problems in this group were significantly less. (Significant with a probability less than 1%).