Functional characterization of the stunt lemma palea 1 mutant allele in rice

The rice stunted lemma/palea 1 (slp1) mutant displays a dwarf, shortened panicle length, degenerated lemma and palea, and sterility. A previous study suggested that a missense mutation at the sixth amino acid of the OsSPL16 protein was likely to be responsible for the slp1 mutant phenotype. The current study shows that the overexpression of the wild-type OsSPL16 allele in slp1/slp1 and Slp1/slp1 mutants was unable to convert the slp1 mutant phenotype to normal. However, the introduction of the mutant OsSPL16 allele into a normal rice cultivar led to the slp1 mutant phenotype in transgenic plants. These results indicated that the missense mutation in OsSPL16 creates a neomorphic allele, which affects plant height and the development of the inflorescence and spikelet.     

Differential gene expression during seed germination in barley (Hordeum vulgare L.)

A barley cDNA macroarray comprising 1,440 unique genes was used to analyze the spatial and temporal patterns of gene expression in embryo, scutellum and endosperm tissue during different stages of germination. Among the set of expressed genes, 69 displayed the highest mRNA level in endosperm tissue, 58 were up-regulated in both embryo and scutellum, 11 were specifically expressed in the embryo and 16 in scutellum tissue. Based on Blast X analyses, 70% of the differentially expressed genes could be assigned a putative function. One set of genes, expressed in both embryo and scutellum tissue, included functions in cell division, protein translation, nucleotide metabolism, carbohydrate metabolism and some transporters. The other set of genes expressed in endosperm encodes several metabolic pathways including carbohydrate and amino acid metabolism as well as protease inhibitors and storage proteins. As shown for a storage protein and a trypsin inhibitor, the endosperm of the germinating barley grain contains a considerable amount of residual mRNA which was produced during seed development and which is degraded during early stages of germination. Based on similar expression patterns in the endosperm tissue, we identified 29 genes which may undergo the same degradation process. Electronic Publication     

Utilization of F1 monosomics for genetic analyses involving awn expression,glume color,seed setting,and seed abortion in crosses of tetraploid and hexaploid wheats

Summary F₁ plants, monosomic for chromosomes 1A to 7B, from crosses of three lines of Triticum durum var. Khapli with the Chinese series were investigated together with their backcrosses to normal Chinese Spring. The three Khapli lines were designated K1-A, K1-B, and K1-D. Five parameters were analyzed: awn development, glume color, degree of selfing, crossing ability, and seed abortion.Morphological examination of F₁ monopentaploid plants revealed that, in the three lines, chromosomes 5A, 1B, 3B, 4B, 5B, and 6B had promotor genes and 2A, 6A, and 2B had inhibitor genes for awn development. Results on glume color suggested the presence of at least three inhibitor genes on 1B, 5B, and 7B, and three promotor genes on 3A, 6A, and 6B chromosomes of Chinese Spring.The first backcross of interspecific hybrids seemed to indicate that some chromosomes (1A, 1B, and 4B) originating from the Khapli lines possessed promotor genes, others (2B and 4A) carried inhibitor genes for seed setting. Similarily, the genetic factor (s) carried by chromosome 5A increased seed abortion whereas those on 2B and 6B reduced it.Self fertility in K1-D hybrids was probably the result of the inhibitor factor (s) on 7A and promotor genes on 3B, 4B, 5B, and 6B chromosomes coming from K1-D.     

The promoter of the asi gene directs expression in the maternal tissues of the seed in transgenic barley

The bifunctional alpha-amylase/subtilisin inhibitor (BASI) is an abundant protein in barley seeds, proposed to play multiple and apparently diverse roles in regulation of starch hydrolysis and in seed defence against pathogens. In the Triticeae, the protein has evolved the ability to specifically inhibit the main group of alpha-amylases expressed during germination of barley and encoded by the amyl gene family found only in the Triticeae. The expression of the asi gene that encodes BASI has been reported to be controlled by the hormones abscisic acid (ABA) and gibberellic acid (GA). Despite many studies at the gene and protein level, the function of this gene in the plant remains unclear. In this study, the 5'-flanking region (1033 bp, 1033-asi promoter) and the 3'-flanking region (655 bp) of the asi gene were isolated and characterised. The 1033-asi promoter sequence showed homology to a number of ciselements that play a role in ABA and GA regulated expression of other genes. With a green fluorescent protein gene (gfp) as reporter, the 1033-asi promoter was studied for spatial, temporal and hormonal control of gene expression. The 1033-asi promoter and its deletions direct transient gfp expression in the pericarp and at low levels in mature aleurone cells, and this expression is not regulated by ABA or GA. In transgenic barley plants, the 1033-asi promoter directed tissue-specific expression of the gfp gene in developing grain and germinating grain but not in roots or leaves. In developing grain, expression of gfp was observed specifically in the pericarp, the vascular tissue, the nucellar projection cells and the endosperm transfer cells and the hormones ABA or GA did not regulate this expression. In mature germinating grain gfp expression was observed in the embryo but not in aleurone or starchy endosperm. However, GA induced gfp expression in the aleurone of mature imbibed seeds from which the embryo had been removed. Expression in maternal rather than endosperm tissues of the grain suggests that earlier widespread assumptions that the protein is expressed largely in the endosperm may have been largely based on analysis of mixed grain tissues. This novel pattern of expression suggests that both activities of the protein may be primarily involved in seed defence in the peripheral tissues of the seed.     

The promoter of the asi gene directs expression in the maternal tissues of the seed in transgenic barley

The bifunctional -amylase/subtilisin inhibitor (BASI) is an abundant protein in barley seeds, proposed to play multiple and apparently diverse roles in regulation of starch hydrolysis and in seed defence against pathogens. In the Triticeae, the protein has evolved the ability to specifically inhibit the main group of -amylases expressed during germination of barley and encoded by the amy1 gene family found only in the Triticeae. The expression of the asi gene that encodes BASI has been reported to be controlled by the hormones abscisic acid (ABA) and gibberellic acid (GA). Despite many studies at the gene and protein level, the function of this gene in the plant remains unclear. In this study, the 5-flanking region (1033 bp, 1033-asi promoter) and the 3-flanking region (655 bp) of the asi gene were isolated and characterised. The 1033-asi promoter sequence showed homology to a number of ciselements that play a role in ABA and GA regulated expression of other genes. With a green fluorescent protein gene (gfp) as reporter, the 1033-asi promoter was studied for spatial, temporal and hormonal control of gene expression. The 1033-asi promoter and its deletions direct transient gfp expression in the pericarp and at low levels in mature aleurone cells, and this expression is not regulated by ABA or GA. In transgenic barley plants, the 1033-asi promoter directed tissue-specific expression of the gfp gene in developing grain and germinating grain but not in roots or leaves. In developing grain, expression of gfp was observed specifically in the pericarp, the vascular tissue, the nucellar projection cells and the endosperm transfer cells and the hormones ABA or GA did not regulate this expression. In mature germinating grain gfp expression was observed in the embryo but not in aleurone or starchy endosperm. However, GA induced gfp expression in the aleurone of mature imbibed seeds from which the embryo had been removed. Expression in maternal rather than endosperm tissues of the grain suggests that earlier widespread assumptions that the protein is expressed largely in the endosperm may have been largely based on analysis of mixed grain tissues. This novel pattern of expression suggests that both activities of the protein may be primarily involved in seed defence in the peripheral tissues of the seed.     

Environmental and transgene expression effects on the barley seed proteome

The barley (Hordeum vulgare) cultivar Golden Promise is no longer widely used for malting, but is amenable to transformation and is therefore a valuable experimental cultivar. Its characteristics include high salt tolerance, however it is also susceptible to several fungal pathogens. Proteome analysis was used to describe the water-soluble protein fraction of Golden Promise seeds in comparison with the modern malting cultivar Barke. Using 2D-gel electrophoresis to visualise several hundred proteins in the pH ranges 4-7 and 6-11, 16 protein spots were found to differ between the two cultivars. Eleven of these were identified by mass spectrometric peptide mass mapping, including an abundant chitinase implicated in defence against fungal pathogens and a small heat-shock protein. To enable a comparison with transgenic seed protein patterns, differences in spot patterns between field and greenhouse-grown seeds were analysed. Four spots were observed to be increased in intensity in the proteome of greenhouse-grown seeds, three of which may be related to nitrogen availability during grain filling and total protein content of the seeds, since they also increased in field grown seeds supplied with extra nitrogen. Finally, the fate of transgene products in barley seeds was followed. Spots containing two green fluorescent protein constructs and the herbicide resistance marker phosphinothricin acetyltransferase were observed in 2D-gel patterns of transgenic seeds and identified by mass spectrometry. Phosphinothricin acetyltransferase was observed in three spots differing in pI suggesting that post-translational modification of the transgene product had occurred.     

Design, expression and characterisation of lysine-rich forms of the barley seed protein CI-2

Chymotrypsin inhibitor CI-2 is a small (84 residue) barley seed protein that has been used extensively to study protein folding. It also contains eight lysine residues, making it an attractive target for expression in transgenic plants to increase their lysine contents. We have designed three lysine-enriched forms of CI-2 and compared their structures and properties with that of the wild type protein. One mutant containing three additional lysine residues in the inhibitory loop shows high stability to denaturation and reduced inhibitory activity, indicating its suitability for use in genetic engineering.     

Early gene expression response of barley root tip to toxic concentrations of cadmium

Plant Molecular Biology - Already a short-term Cd treatment induces changes in gene expression in barley root tips via IAA and ROS signaling during mild and severe Cd stress, respectively. Even a...     

Overlap of gametophytic and sporophytic gene expression in barley

Summary The overlap of gametophytic and sporophytic gene expression in barley was studied by means of enzyme electrophoresis. Of the isozymes found, 60% were expressed in both gametophyte and sporophyte, 30% were sporophyte specific, and 10% were gametophyte specific. A considerable amount of the barley genome is thus potentially amenable to gametophytic selection. The estimated sizes of the common, sporophytic and gametophytic domains in barley gene expression correspond with the estimates obtained in other plant species.