The International Barley Sequencing Consortium—At the Threshold of Efficient Access to the Barley Genome

Arabidopsis (Arabidopsis thaliana) tryptophan-proline-proline (WPP)-domain proteins, WPP1 and WPP2, are plant-unique, nuclear envelope-associated proteins of unknown function. They have sequence similarity to the nuclear envelope-targeting domain of plant RanGAP1, the GTPase activating protein of the small GTPase Ran. WPP domain-interacting tail-anchored protein 1 (WIT1) and WIT2 are two Arabidopsis proteins containing a coiled-coil domain and a C-terminal predicted transmembrane domain. They are required for RanGAP1 association with the nuclear envelope in root tips. Here, we show that WIT1 also binds WPP1 and WPP2 in planta, we identify the chaperone heat shock cognate protein 70-1 (HSC70-1) as in vivo interaction partner of WPP1 and WPP2, and we show that HSC70-1 interacts in planta with WIT1. WIT1 and green fluorescent protein (GFP)-WIT1 are targeted to the nuclear envelope in Arabidopsis. In contrast, GFP-WIT1 forms large cytoplasmic aggregates when overexpressed transiently in Nicotiana benthamiana leaf epidermis cells. Coexpression of HSC70-1 significantly reduces GFP-WIT1 aggregation and permits association of most GFP-WIT1 with the nuclear envelope. Significantly, WPP1 and WPP2 show the same activity. A WPP1 mutant with reduced affinity for GFP-WIT1 fails to decrease its aggregation. While the WPP-domain proteins act on a region of WIT1 containing the coiled-coil domain, HSC70-1 additionally acts on the C-terminal transmembrane domain. Taken together, our data suggest that both HSC70-1 and the WPP-domain proteins play a role in facilitating WIT1 nuclear envelope targeting, which is, to our knowledge, the first described in planta activity for the WPP-domain proteins.The cytoplasmic Ran GTPase activating protein RanGAP is critical to establishing a functional RanGTP/RanGDP gradient across the nuclear envelope (NE) and is associated with the outer surface of the NE in metazoan and higher plant cells (Matunis et al., 1996; Rose and Meier, 2001). Plant RanGAP1 association with the NE requires a plant-specific targeting domain, named the Trp-Pro-Pro (WPP) domain (Rose and Meier, 2001). Arabidopsis (Arabidopsis thaliana) WPP1 and WPP2 are small (155- and 180-amino-acid residues, respectively) plant-unique proteins of unknown function, which are similar to the WPP domain of RanGAP proteins. WPP1 and WPP2 are located in the cytoplasm, with a concentration at the NE (Patel et al., 2004). They are characterized by a 104-amino-acid-long WPP domain, predicted to consist of a β-strand and three α-helices and shown to be sufficient for NE targeting (Patel et al., 2004). They are also associated with cytoplasmic speckles most likely representing Golgi (Patel et al., 2005). Reduced expression of the WPP protein family causes decreased mitotic activity in roots of Arabidopsis, resulting in shortening of primary roots and decreased number of lateral roots (Patel et al., 2004). RanGAP1 association with the NE in the Arabidopsis root tip requires two families of NE-localized, plant-specific, WPP domain-interacting proteins (WPP domain-interacting protein [WIP] and WPP domain-interacting tail-anchored protein [WIT] families) that are characterized by the presence of a coiled-coil domain and a C-terminal predicted transmembrane domain (TMD; Xu et al., 2007; Zhao et al., 2008). Based on sequence analysis, both the WIP and WIT protein family were classified as putative tail-anchored (TA) proteins, proteins that associate with membranes posttranslationally (Borgese et al., 2003).The heat shock protein 70 family (HSP70) contains both heat-inducible and constitutively expressed members, called heat shock cognate proteins (HSC70). HSC70 chaperones assist in folding newly synthesized proteins (Bukau and Horwich, 1998), are involved in posttranslational translocation of secretory proteins across endoplasmic reticulum (ER) and mitochondrial membranes (Chirico et al., 1988; Deshaies et al., 1988), prevent irreversible aggregation of their substrates (Ngosuwan et al., 2003), and facilitate degradation of misfolded proteins (Meacham et al., 2001). Recently, mammalian HSC70 has also been implied in assisting the membrane insertion of a subset of TA proteins (Abell et al., 2007).The Arabidopsis genome encodes five different cytosolic HSP70s, three of which are expressed constitutively (HSC70-1, HSC70-2, and HSC70-3). While expressed in all organs, Hsc70-1 and Hsc70-2 expression levels are highest in leaves and Hsc70-3 in leaves and roots. All three genes can be further induced by heat shock and cold stress (Sung et al., 2001). Constitutive overexpression of Arabidopsis Hsc70-1 in transgenic plants leads to changes in growth and development, increases thermotolerance (Sung and Guy, 2003), and decreases the plant''s ability to respond to pathogen attack (Noel et al., 2007). Recently, specific interactions of HSC70-1 with SGT1 (for Suppressor of G2 allele of skp1; Noel et al., 2007) and HSC70-3 with turnip mosaic virus RNA-dependent RNA polymerase (Dufresne et al., 2008) were identified, suggesting a role of HSC70 in viral replication and pathogenesis. Both HSC70-1 and HSC70-3 can be detected in the nuclei and the cytoplasm of Nicotiana benthamiana epidermal cells (Noel et al., 2007; Dufresne et al., 2008).Here, we identified Arabidopsis HSC70-1 as an in vivo interaction partner of WPP1 and WPP2 and demonstrated that HSC70-1 associates with WIT1. Using transient expression in N. benthamiana, we show that when expressed at a high level, WIT1 accumulates in large fluorescent bodies in the cytoplasm that may represent aggregates. Upon coexpression in the same system, WPP1, WPP2, and HSC70-1 are all able to prevent the aggregation of overexpressed WIT1 and enable WIT1 association with the NE. While WPP-domain proteins act on a region of WIT1 containing the coiled-coil domain, HSC70-1 additionally acts on the C-terminal TMD. We propose that WPP1 and WPP2 play a chaperone-like role reflected in preventing the aggregation of the coiled-coil region of WIT1 and possibly other coiled-coil TA-type proteins, either in conjunction or independently of HSC70-type chaperones.     

Allele-Dependent Barley Grain β-Amylase Activity

The wild ancestor of cultivated barley, Hordeum vulgare subsp. spontaneum (K. Koch) A. & Gr. (H. spontaneum), is a source of wide genetic diversity, including traits that are important for malting quality. A high β-amylase trait was previously identified in H. spontaneum strains from Israel, and transferred into the backcross progeny of a cross with the domesticated barley cv Adorra. We have used Southern-blot analysis and β-amy1 gene characterization to demonstrate that the high β-amylase trait in the backcross line is co-inherited with the β-amy1 gene from the H. spontaneum parent. We have analyzed the β-amy1 gene organization in various domesticated and wild-type barley strains and identified three distinct β-amy1 alleles. Two of these β-amy1 alleles were present in modern barley, one of which was specifically found in good malting barley cultivars. The third allele, linked with high grain β-amylase activity, was found only in a H. spontaneum strain from the Judean foothills in Israel. The sequences of three isolated β-amy1 alleles are compared. The involvement of specific intron III sequences, in particular a 126-bp palindromic insertion, in the allele-dependent expression of β-amylase activity in barley grain is proposed.     

Adsorptive Effect of Highland Barleyβ-Glucan on Cholesterol

探讨了青稞β-葡聚糖的质量及粒度、胆固醇浓度、吸附温度及时间对其吸附胆固醇作用的影响.结果显示,青稞β-葡聚糖对胆固醇具有较好的吸附作用.该吸附作用随胆固醇浓度和吸附时间的增加而增大,随β-葡聚糖的质量及粒度和温度增大而减小.青稞β-葡聚糖吸附胆固醇的适宜条件为:青稞β-葡聚糖终浓度为2.75～3.00 mg/mL的胆固醇溶液中于30℃时振荡吸附90 min.青稞β-葡聚糖对胆固醇的吸附规律符合Freundlich方程,其吸附方式包括物理吸附和化学吸附.     

Isolation of a Raw Starch-Binding Fragment from Barley α-Amylase

Barley α-amylase was purified by ammonium sulfate fraction, ion-exchange, ultrafiltration, and gel filtration to homogeneity. The purified enzyme was partially digested with trypsin, and the reaction mixture was applied to a cyclohepta-amylose epoxy Sepharose 6B column. Bound fragments were eluted by free cyclohepta-amylose, lyophilized, and separated on Tricine gels. Four fragments were shown to interact with β-cyclodextrin. The fragment that could be identified on the gel with the lowest molecular weight (11 kDa) was electroblotted onto PVDF membrane for sequencing. The N-terminal sequence of this fragment was determined with the N-terminal amino acid corresponding to Ala283 in the whole protein. The trypsin cleavage was at Lys282/Ala283 and the C-terminal cleavage occurred at Lys354/Ile355 to give a fragment size of 11 kDa as estimated by SDS-PAGE. The fragment would be located at the C-terminal region, forming a majority of the antiparallel β-sheets in domain C and the α₇-and α₈-helices of the (α/β)₈ domain.     

Barley proteinase inhibitors: A possible role in grasshopper control?

Young growing barley seedlings contain trypsin and chymotrypsin proteinase inhibitors in their leaf juices. The amount of chymotrypsin inhibitor varies greatly while trypsin inhibitor content is nearly the same in all varieties tested. Compana (CI5438) barley has much more total inhibitor than does Trebi (CI 936), Titan (CI 7055), Horn (CI 926), Hiproly (CI 3947) and Hiproly Normal (CI 4362). The distribution of inhibitors in different barley varieties correlates with the severity of grasshopper damage observed by other workers. Barley leaves could not be induced to accumulate proteinase inhibitors after excision and incubation, wounding, or absorption of crude “proteinase inhibitor inducing factor” preparations. Grasshopper damage experiments as related to proteinase inhibitor should be done in the field using yield as the correlative factor.     

Blue Light Induction of Barley Leaf Unfolding. A Phytochrome Reaction?

Low-energy blue light (450, 475 nm) has been found to induced unfolding of etiolated barley leaves (Hordeum rulgare cv. Ingrid). This induction can be reversed by far-red light. Barley leaf unfolding is normally stimulated by red light, reversed by far-red light, and can be considered to be a typical phytochrome controlled response. It is possible to explain the effects by red and blue light as mediated by the same photoreceptor. The phototransformation of this pigment results in two forms, P₂ and P₄, to which physiological activity can be ascribed. The red and blue light affect different steps in a cyclical photoconversion. Calculated theoretical dose response curves are presented for such a model in agreement with the experimental data.     

Intron III-Specific Markers for Screening of β-amylase Alleles in Barley Cultivars

In modern malting barley breeding it is important to increase the level of -amylase activity level in barley. The aim of this study was to investigate if a PCR method for screening -amy1 alleles can be used as an indicator for -amylase activity level in barley. Activity was assayed from 24 cultivars, 7 lines, and a Hordeum spontaneum PI 296897 strain grown in the same field. The -amy1 alleles were identified by amplifying the intron III-specific region of the gene using PCR. No new alleles were detected in addition to the three alleles found earlier: cv Adorra-like, cv Haruna Nijo-like and PI 296897-like -amy1 allele. Samples were grouped according to the nature of their -amy1 locus and enzyme activities were compared between the groups. Cultivars carrying a cv Haruna Nijo-like -amy1 allele had 1.3 times and lines carrying a PI 296897-like -amy1 allele had 2.1 times higher -amylase activity than cultivars carrying a cv Adorra-like -amy1 allele. The mean activities are significantly different in the allele groups (Kruskal–Wallis: for protein H= 11.54, P< 0.01; for meal H= 12.74, P< 0.01). PCR fragments can be used as allele specific markers to predict the level of -amylase activity in breeding when such variation of the intron III is concerned.     

A Functional Raw Starch-Binding Domain of Barley α-Amylase Expressed in Escherichia coli

The mature form of barley seed low-pI α-amylase (BAA1) possesses a raw starch-binding site in addition to the catalytic site. A truncated cDNA encoding the C-terminal region (aa 281–414) and containing the proposed raw starch-binding domain (SBD) but lacking Trp278/Trp279, a previously proposed starch granule-binding site, was synthesized via PCR and expressed in Escherichia coli as an N-terminal His-Tag fusion protein. SBD was produced in the form of insoluble inclusion bodies that were extracted with urea and successfully refolded into a soluble form via dialysis. To determine binding, SBD was purified by affinity chromatography with cycloheptaamylose as ligand cross-linked to Sepharose. This work demonstrates that a SBD is located in the C-terminal region and retains sufficient function in the absence of the N-terminal, catalytic, and Trp278/279 regions.     

Barley β-Galactosidase: Structure, Function, Heterogeneity, and Gene Origin

Barley (Hordeum vulgare) β-galactosidase is composed of a large (45 kDa) and a small (33 kDa) polypeptide. N-terminal sequencing of the polypeptides and antibody reactivity data place the barley enzyme and heterodimeric plant β-galactosidases from jack bean, maize, and wheat in family 35 of the glycosyl hydrolases. Sequence analysis indicates the existence of a subfamily of genes coding for polypeptide precursors that are cleaved to produce the two subunits in heterodimeric β-galactosidases. The heterogeneity of the barley holoenzyme is related, but not restricted, to the N-glycosylation of the small polypeptide. Both polypeptides are essential for the catalytic activity of the enzyme.     

Characterization of Active Barley α-Amylase 1 Expressed and Secreted by Saccharomyces cerevisiae

Recombinant barley α-amylase 1 isozyme was constitutively secreted by Saccharomyces cerevisiae. The enzyme was purified to homogeneity by ultrafiltration and affinity chromatography. The protein had a correct N-terminal sequence of His-Gln-Val-Leu-Phe-Gln-Gly-Phe-Asn-Trp, indicating that the signal peptide was efficiently processed. The purified α-amylase had an enzyme activity of 1.9 mmol maltose/mg protein/min, equivalent to that observed for the native seed enzyme. The k_cat/K_m was 2.7 × 10² mM^?1.s^?1, consistent with those of α-amylases from plants and other sources.     

Barley—Botany,production, harvesting,processing, utilization and economics

Barley, probably the oldest cultivated cereal, is widely grown in cooler areas of the world. The annual world production of nearly two and a half billion bushels exceeds that of rye but is less than that of rice, wheat, corn and oats, respectively. Most of the annual 300 million dollar crop of the U.S. is fed to livestock, but about one- third is manufactured into malt.     

Assessment of Triazole Growth-Retarding Activity in an α-Amylase Bioassay Using Spring Barley Endosperm

The possibility to assess the growth-retarding activity of triazole compounds, such as paclobutrazole, uniconazole, and azovite, by an express bioassay based on the evaluation of the -amylase activity, which hydrolyzes starch into soluble sugars in the aleurone layer of barley (Hordeum vulgare L.), was investigated. The retarding effects of triazoles, when evaluated by the inhibition of amylase activity, decreased in the following order: uniconazole > azovite > paclobutrazole chlorocholine chloride. Among barley cultivars, cv. No-sovskii 9 was the most sensitive to the tested triazole compounds. At the concentrations of 10^–7 to 10^–3 M, these chemicals manifested notable growth-retarding activity in the bioassays with the seedlings of long stemmed pea cv. Torsdag and spring barley, cvs. Nosovskii 9, Moskovskii 2, and Zazerskii 85. All these express bioassays were validated for assessing the triazole growth-retarding activity.     

γ-Irradiation of Barley Seeds and Its Effect on the Phytohormonal Status of Seedlings

We investigated the effect of γ-irradiation (4–50 Gy) of barley seeds (Hordeum vulgare L., cv. Nur) on the content of endogenous phytohormones–stimulators of plant growth and development: indol-3-acetic acid (IAA), indolyl-3-butyric acid (IBA), zeatin and abscisic acid (ABA). The ratio (IAA + IBA + zeatin)/ABA from the third to the seventh day of germination has been measured. It was shown that the changes in the content of phytohormones as a function of the radiation dose were nonlinear. In the dose range of 4–20 Gy, phytohormones balance was changed due to increased content of growth stimulators and decreased ABA content. Using a dose of 50 Gy led primarily to a decrease in the content of growth stimulators and an increase in ABA content, and the ratio (IAA + IBA + zeatin)/ABA shifted toward ABA content.     

The Effect of Ultracentrifugation on Fine Structure and α-Amylase Production in Barley Aleurone Cells

Ultracentrifugation of barley aleurone cells results in the stratification of organelles thus allowing for a quantitation of those organelles. Gibberellic acid (GA₃)-stimulated α-amylase production in stratified cells is reduced by centrifugation at gravitational forces greater than 40,000g. Forces below 30,000g do not affect GA₃-stimulated α-amylase production although stratification of organelles occurs at these forces. The ability of centrifuged cells to respond maximally to GA₃ by producing α-amylase is related to the degree of redistribution of organelles within these cells. Thus, recovery of cells from centrifugation at forces below 30,000g is rapid, while recovery from forces above 40,000g is slow.     

Cell-wall Polysaccharides of Immature Barley Plants. I. Isolation and Characterization of a β-d-Glucan

A β-d-glucan was isolated on fractionation of a 4% potassium hydroxide extract (hemi-celluloses) of immature barley plants (Hordeum distichum L.). Most of the glucose residues in the extract were found to be derived from the glucan. Methylation analysis and enzyme degradation studies showed that the glucan had (l-→3)-and (1-→4)-linked d-glucopyranosyl residues in an approximate molar ratio of 1.0:2.3. The molecular weight of the glucan was estimated to be 1.8 x 10⁵ by gel filtration on Sepharose CL-6B.     

Is The Barley Endosperm a Water Reservoir for the Embryo When Germinating Seeds Are Dried?

The water content of germinating seeds fluctuates in response to water potential changes in the surrounding environment. We tested the hypothesis that the endosperm functions as a water reservoir when imbibed seeds experience drying, and we characterized water uptake and movement within barley (Hordeum vulgare cv. Triumph) caryopses (hereafter referred to as seeds). Water movement into and through germinating barley seeds during imbibition and drying was determined gravimetrically and with the fluorescent dye trisodium 8-hydroxy-1,3,6-pyrenetrisulfonate (PTS). During imbibition, embryo tissues hydrated more rapidly and reached a higher water content (g H20/g dry weight) than did the endosperm, although the endosperm eventually contained nine times as much total water. When barley seeds that had imbibed for 12 h were exposed to moderate (-4 MPa) drying, PTS solution moved from the endosperm into the shoot meristem, radicle, and scutellum, but not vice versa. Radicle emergence and elongation proceeded for up to 8 h. With harsh (-150 MPa) drying, PTS concentrated almost exclusively in the radicle. These data illustrate that the endosperm is at least a temporary water storage compartment external to the embryo itself. We speculate that water supplied by the endosperm may be important in reducing the harmful effects of drying during the critical transition period when a germinating seed changes from a desiccation-tolerant to a desiccation-intolerant organism.