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Grain hardness is an important quality trait of cereal crops. In wheat, it is mainly determined by the Hardness locus that harbors genes encoding puroindoline A (PINA) and puroindoline B (PINB). Any deletion or mutation of these genes leading to the absence of PINA or to single amino acid changes in PINB leads to hard endosperms. Although it is generally acknowledged that hardness is controlled by adhesion strength between the protein matrix and starch granules, the physicochemical mechanisms connecting puroindolines and the starch-protein interactions are unknown as of this time. To explore these mechanisms, we focused on PINA. The overexpression in a hard wheat cultivar (cv. Courtot with the Pina-D1a and Pinb-D1d alleles) decreased grain hardness in a dose-related effect, suggesting an interactive process. When PINA was added to gliadins in solution, large aggregates of up to 13 μm in diameter were formed. Turbidimetry measurements showed that the PINA-gliadin interaction displayed a high cooperativity that increased with a decrease in pH from neutral to acid (pH 4) media, mimicking the pH change during endosperm development. No turbidity was observed in the presence of isolated α- and γ-gliadins, but non-cooperative interactions of PINA with these proteins could be confirmed by surface plasmon resonance. A significant higher interaction of PINA with γ-gliadins than with α-gliadins was observed. Similar binding behavior was observed with a recombinant repeated polypeptide that mimics the repeat domain of gliadins, i.e., (Pro-Gln-Gln-Pro-Tyr)8. Taken together, these results suggest that the interaction of PINA with a monomeric gliadin creates a nucleation point leading to the aggregation of other gliadins, a phenomenon that could prevent further interaction of the storage prolamins with starch granules. Consequently, the role of puroindoline-prolamin interactions on grain hardness should be addressed on the basis of previous observations that highlight the similar subcellular routing of storage prolamins and puroindolines., Competing Interests: The authors have declared that no competing interests exist.

Key Message: A large, 53-kbp, intact DNA fragment was inserted into the wheat ( Triticum aestivum L.) genome. FISH analyses of individual transgenic events revealed multiple insertions of intact fragments. Transferring large intact DNA fragments containing clusters of resistance genes or complete metabolic pathways into the wheat genome remains a challenge. In a previous work, we showed that the use of dephosphorylated cassettes for wheat transformation enabled the production of simple integration patterns. Here, we used the same technology to produce a cassette containing a 44-kb Arabidopsis thaliana BAC, flanked by one selection gene and one reporter gene. This 53-kb linear cassette was integrated in the bread wheat (Triticum aestivum L.) genome by biolistic transformation. Our results showed that transgenic plants harboring the entire cassette were generated. The inheritability of the cassette was demonstrated in the T1 and T2 generation. Surprisingly, FISH analysis performed on T1 progeny of independent events identified double genomic insertions of intact fragments in non-homoeologous positions. Inheritability of these double insertions was demonstrated by FISH analysis of the T1 generation. Relative conclusions that can be drawn from molecular or FISH analysis are discussed along with future prospects of the engineering of large fragments for wheat transformation or genome editing.

The wheat genome encodes some 100,000 genes. To understand how the expression of these genes is regulated it will be necessary to carry out many genetic transformation experiments. Robust protocols that allow scientists to transform a wide range of wheat genotypes are therefore required. In this chapter, we describe a protocol for biolistic transformation of wheat that uses immature embryos and small quantities of DNA cassettes. An original method for DNA cassette purification is also described. This protocol can be used to transform a wide range of wheat genotypes and other related species.

For important food crops such as wheat and rice, grain yield depends on grain number and size. In rice (Oryza sativa), GW2 was isolated from a major quantitative trait locus for yield and encodes an E3 RING ligase that negatively regulates grain size. Wheat (Triticum aestivum) has TaGW2 homologues in the A, B, and D genomes, and polymorphisms in TaGW2-A were associated with grain width. Here, to investigate TaGW2 function, RNA interference (RNAi) was used to down-regulate TaGW2 transcript levels. Transgenic wheat lines showed significantly decreased grain size-related dimensions compared with controls. Furthermore, TaGW2 knockdown also caused a significant reduction in endosperm cell number. These results indicate that TaGW2 regulates grain size in wheat, possibly by controlling endosperm cell number. Wheat and rice GW2 genes thus seem to have divergent functions, with rice GW2 negatively regulating grain size and TaGW2 positively regulating grain size. Analysis of transcription of TaGW2 homoeologues in developing grains suggested that TaGW2-A and -D act in both the division and late grain-filling phases. Furthermore, biochemical and molecular analyses revealed that TaGW2-A is a functional E3 RING ubiquitin ligase with nucleocytoplasmic subcellular partitioning. A functional nuclear export sequence responsible for TaGW2-A export from the nucleus to the cytosol and retention in the nucleolus was identified. Therefore, these results show that TaGW2 acts in the regulation of grain size and may provide an important tool for enhancement of grain yield.

Resistance (R) genes protect plants very effectively from disease, but many of them are rapidly overcome when present in widely grown cultivars. To overcome this lack of durability, strategies that increase host resistance diversity have been proposed. Among them is the use of multilines composed of near-isogenic lines (NILs) containing different disease resistance genes. In contrast to classical R-gene introgression by recurrent backcrossing, a transgenic approach allows the development of lines with identical genetic background, differing only in a single R gene. We have used alleles of the resistance locus Pm3 in wheat, conferring race-specific resistance to wheat powdery mildew (Blumeria graminis f. sp. tritici), to develop transgenic wheat lines overexpressing Pm3a, Pm3c, Pm3d, Pm3f or Pm3g. In field experiments, all tested transgenic lines were significantly more resistant than their respective nontransformed sister lines. The resistance level of the transgenic Pm3 lines was determined mainly by the frequency of virulence to the particular Pm3 allele in the powdery mildew population, Pm3 expression levels and most likely also allele-specific properties. We created six two-way multilines by mixing seeds of the parental line Bobwhite and transgenic Pm3a, Pm3b and Pm3d lines. The Pm3 multilines were more resistant than their components when tested in the field. This demonstrates that the difference in a single R gene is sufficient to cause host-diversity effects and that multilines of transgenic Pm3 wheat lines represent a promising strategy for an effective and sustainable use of Pm3 alleles., (© 2011 The Authors. Plant Biotechnology Journal © 2011 Society for Experimental Biology, Association of Applied Biologists and Blackwell Publishing Ltd.)

In the present work, an endopin-like elastase inhibitor was purified for the first time from bovine muscle. A three-step chromatography procedure was developed including successively SP-Sepharose, Q-Sepharose and EMD-DEAE 650. This procedure provides about 300 microg of highly pure inhibitor from 500 g of bovine diaphragm muscle. The N-terminal sequence of the muscle elastase inhibitor, together with the sequence of a trypsin-generated peptide, showed 100% similarity with the cDNA deduced sequence of chromaffin cell endopin 1. Hence, the muscle inhibitor was designated muscle endopin 1 (mEndopin 1). mEndopin 1 had a molecular mass of 70 kDa, as assessed by both gel filtration and SDS/PAGE. According to the association rates determined, mEndopin 1 is a potent inhibitor of elastase (kass=2.41x10(7) M(-1).s(-1)) and trypsin (kass=3.92x10(6) M(-1).s(-1)), whereas plasmin (kass=1.78x10(3) M(-1).s(-1)) and chymotrypsin (kass=1.0x10(2) M(-1).s(-1)) were only moderately inhibited. By contrast, no inhibition was detected against several other selected serine proteinases, as well as against cysteine proteinases of the papain family. The cellular location of mEndopin in muscle tissue and its tissue distribution were investigated using a highly specific rabbit antiserum. The results obtained demonstrate an intracellular location and a wide distribution in bovine tissues.

The aim of this study was to determine whether exogenous glucose metabolism influences the pH in superfused EDL muscle from growing rats fed or starved for 48 h (body weight 55 and 45 g, respectively). Energy state and intracellular pH of muscle were repeatedly monitored by 31P-nuclear magnetic resonance spectroscopy (31P-NMRS); glycogen and other energy metabolites were assayed enzymatically in muscle extracts at the end of the experiment. In EDL muscles from starved rats superfused with glucose for 4 h, intracellular pH was elevated (7-7.3), lactate concentration low, glycogen repletion very intense and citrate synthase activity high. We conclude that glucose was routed mainly toward both oxidative phosphorylation and glycogen synthesis in EDL muscles after food deprivation of rats. In contrast, the major pathway in muscles from fed rats may be glycolysis because the glycogen pool remained constant throughout the experiment. The additional and minor pH component (in the range of 6.5 to 6.8) seen in muscles from fed rats, even in the presence of exogenous glucose, might be due to impaired glucose utilization because this component appears also in muscles from starved rats superfused without glucose or with a nonmetabolizable analog of glucose. Consequently, direct pH measurement by 31P-NMR may be considered to be a precise criterion for evaluation of differences in metabolic potentialities of muscle studied ex vivo in relation to the nutritional state of rats.

The physiologic function of proteasome remains unclear. Evidence suggests a role in degradation of ubiquitin-protein conjugates, MHC antigen presentation, and some specificity of substrate within certain cell types. To explore further the properties of proteasome we have examined its effect on a well defined structure, the myofibril. We find that despite its large size (20S) proteasome is able to degrade myofibrils and intact, permeabilized muscle fibrils. The proteins degraded showed some specificity because actin, myosin and desmin were degraded faster than alpha-actinin, troponin T and tropomyosin. Changes in ultrastructure were slow and included a general loss of structure with Z and I bands effected before the M band and costameres.

Three low molecular mass cysteine proteinase inhibitors were purified from a bovine skeletal muscle crude extract using a three-step procedure. The crude extract was first subjected to gel filtration on a Sephadex G100 column which separated five active fractions (F-I to F-V). Three papain inhibitors, P1, P2 and P3, were fractionated from the F-V fraction by chromatofocalisation on a poly buffer exchanger column. Purification was completed by chromatography on a Mono Q column. After SDS-PAGE, the three inhibitors showed only one band with an M(r) of 14,300. P1, P2 and P3 appeared to be highly resistant to temperature (40-90 degrees C), pH (3-10), reducing agents (5-50 mM) and to be specific for cysteine proteinases since no activity was detected against either serine or aspartyl proteinases. Although to a varying extent, P1, P2 and P3 inhibited papain, cathepsin B and cathepsin L. Analysis of the peptide mixtures of these inhibitors by RP-HPLC after hydrolysis with CNBr or aspartly endoproteinase N together with their amino acid composition revealed that P1, P2 and P3 cysteine proteinase inhibitors are isoforms of the same protein. As their N-terminal ends were blocked, partial sequence of some of these peptides was determined. Computer search in protein identification resources did not reveal any homology of these sequences with proteinase inhibitors of known primary structure. In contrast, they matched well with different parts of the total sequence of a fatty acid binding protein isolated from bovine heart. This homology was supported by the ability of these inhibitors to bind long chain fatty acids.(ABSTRACT TRUNCATED AT 250 WORDS)

Of a total of three Friesian cows, two of which had been treated with adrenalin before slaughter, Mm longissimus (LO), supraspinatus (SS), triceps brachii (TB) and rectus abdominis (RA) were sampled at different times post mortem (pm). pH, calpain/calpastatin activities and degradation of myofibrillar proteins, as evidenced by SDS-PAGE, were assessed. Contraction characteristics were measured by determining myofibrillar ATPase activities. Adrenalin treatment resulted in a high ultimate pH (6.48 +/- 0.40) and a faster decline pm of calpain I activity. The effect was similar in all four investigated muscles (72.4 +/- 5.4% decline at 24 h pm). The decline in calpain I activity in the control muscles was muscle-dependent and ranged from 22.8-74.3% at 24 h pm. Differences in ultimate pH did not lead to distinct rates of breakdown of proteins with molecular weights lower than that of myosin heavy chain. Calpastatin levels were muscle-dependent and correlated with myofibrillar ATPase activity (r = -0.99). In a second experiment Mm rectus abdominis (RA) and psoas major (PM) of adrenalin-treated (n = 6) and control (n = 6) Friesian-Holstein calves were sampled at 1 and 29 h pm for assessment of calpain activities. At seven days pm the M longissimus (LO) was sampled for tenderness evaluation. pH values were measured at 30 min, 4 h and 29 h pm. Adrenalin treatment resulted in a higher ultimate pH in the three muscles. Higher ultimate pH resulted in lower calpain activities in the RA at 29 h pm (P less than or equal to 0.025).(ABSTRACT TRUNCATED AT 250 WORDS)