Barley systematics and taxonomy foreseen by seed morphometric variation.

Since its Neolithic domestication in the Fertile Crescent, barley has spread to all continents and represents a major cereal in many modern agrarian systems. Current barley diversity includes thousands of varieties divided into four main categories corresponding to 2-row and 6-row subspecies and naked and hulled types, each of them with winter and spring varieties. This diversity is associated to different uses and allow cultivation in diverse environments. We used a large dataset of 58 varieties of French origin, (1) to assess the taxonomic signal in barley grain measurements comparing 2-row and 6-row subspecies, and naked and hulled types; (2) to test the impact of the sowing period and interannual variation on the grains size and shape; (3) to investigate the existence of morphological differences between winter and spring types; and finally (4) to contrast the relationship between the morphometric and genetic proximity. Size and shape of 1980 modern barley caryopses were quantified through elliptic Fourier Transforms and traditional size measurements. Our results indicate that barley grains record morphological diversity of the ear (89.3% classification accuracy between 2-row/6-row subspecies; 85.2% between hulled and naked type), sowing time of the grains (from 65.6% to 73.3% within barley groups), and environmental conditions during its cultivation and varietal diversity. This study opens perspectives for studying archaeological barley seeds and tracing the barley diversity and evolution since the Neolithic.

Proteomic and morphological analysis of early stages of wheat grain development.

The identification of 249 proteins in the first 2 wks of wheat grain development enabled the chronological description of the early processes of grain formation. Cell division involved expression of the enzymes and proteins of the cytoskeleton and structure, DNA repair and replication enzymes and cellular metabolism enzymes (synthesis of amino acids, cell wall initiation, carbon fixation and energy production, cofactors and vitamins) with a peak expression at 125 degrees C day (degrees day after anthesis). After the first synthesis of amino acids, protein transport mechanisms, translation signals, sugar metabolism (polymerization of protein) and stress/defence proteins were activated with stable expression between 150 and 280 degrees C day. Proteins responsible for folding and degradation, including different subunits of proteasome, were highly expressed at 195 degrees C day. Proteins associated with starch granules (GBSS type 1) were present at the beginning of grain formation and increased regularly up to 280 degrees C day. Heat shock proteins (HSP70, 80, 90) were expressed throughout the early grain development stages.

Extraction and proteome analysis of starch granule-associated proteins in mature wheat kernel (Triticum aestivum L.).

Starch consists of the two glucose polymers, amylose and amylopectin, and is deposited as semicrystalline granules inside plastids. The starch granule proteome is particularly challenging to study due to the amount of interfering compounds (sugars, storage proteins), the very low starch granule-associated protein content and also the dynamic range of abundant proteins. Here we present the protocol for extraction and 2-DE of wheat starch granule-associated proteins whose most important steps are: (i) washing and sonication to remove interfering compounds (storage proteins) from the surface of the granules, (ii) scanning electron microscopy (SEM) observations to monitor purification and granules swelling, (iii) appropriate protein extraction and solubilization to obtain enough proteins for Coomassie blue staining and proteomic analysis. Our objective was to minimize the amount of contamination by storage proteins and to preserve the structure of the starch and of starch-associated proteins and to maximize the number of polypeptides that can be resolved. For quantitative proteomic analysis of proteins associated with wheat starch granules, we developed a two-step protein extraction protocol including TCA/acetone precipitation and phenol extraction. With this protocol, proteins were extracted from wheat starch granules and solubilized and satisfactory blue-stained 2-DE protein maps were obtained. The majority of the spots associated with starch granules were identified by peptide mass fingerprinting and MS/MS and functionally classified into carbohydrate metabolism and stress defense.

A laboratory protocol for determining glucose and maximum ethanol production from wheat grain: application to a complete genetic set of near-isogenic waxy lines.

A laboratory protocol was developed to assess glucose and ethanol yields from wheat. The impact of the analyzed wholemeal flour quantity and the saccharification on the amount of released glucose was estimated. The whole process including the analytical methods (glucose and ethanol) was repeatable and reproducible. This protocol was used to assess the glucose and ethanol yields of six varieties and of a complete set of hexaploid near-isogenic waxy lines of cv. Trémie grown in three locations. As compared to the normal line of Trémie, double null (AnBnD) and triple null (nAnBnD) isogenic lines showed a low Hagberg falling number (218, 65, and 63 s, respectively), a higher grain protein content (10.7, 11.5, and 12.1% DM, respectively), a lower glucose yield (728, 703, and 707 kg/t, respectively), and a lower ethanol yield (463, 453, and 452 L/t, respectively). These values indicate a strong involvement of alleles encoded at Wx-B1 and Wx-D1 loci in grain composition.