Bruce R. Thomas

Three cis-elements required for rice α-amylase Amy3D expression during sugar starvation

Expression of α-amylase genes during seedling development plays a key role in production of sugar from the starch stored in the cereal seed. Rice α-amylase Amy3D promoter/GUS constructs in transgenic rice cell lines were studied to identify cis elements in the promoter of this metabolite-regulated gene. Three sequences having the greatest effects on Amy3D gene expression included the amylase element (TATCCAT), the CGACG element, and a G box-related element (CTACGTGGCCA). These promoter cis elements are needed for high-level expression of Amy3D under conditions of sugar starvation. The involvement of G box cis-elements in environmental stress responses suggest a link between the nutrient stress and the environmental stress responses of the plant.

Metabolite Signals Regulate Gene Expression and Source/Sink Relations in Cereal Seedlings

Our knowledge of the genetic structures and regulatory mechanisms active during cereal seedling development has increased dramatically in recent years. For this reason, a more integrated view of the molecular aspects of seedling development and physiology is needed. This review presents the Carbon Metabolite Signal Hypothesis, which attempts to explain how regulatory interactions between metabolite signals and hormonal signals participate in controlling gene expression, growth, and metabolism of the cereal seedling.

Differential expression of rice α-amylase genes during seedling development under anoxia

The unique capability of rice (Oryza sativa L.) seedlings to grow under anoxic conditions may result in part from their ability to express α-amylase and maintain the supply of sugar needed for energy metabolism. Previous studies have demonstrated that under aerobic conditions the Amy1 and Amy2 subfamily genes are regulated primarily by phytohormones while the Amy3 subfamily genes are induced during sugar starvation. The expression patterns for these α-amylase genes were considerably different in anoxic vs. aerobic rice seedlings. The level of total α-amylase mRNA under anoxic conditions was decreased in aleurone layers while it increased in the embryo. Anoxic conditions greatly diminished the expression of the Amy1A gene in aleurone. Conversely, expression of many Amy3 subfamily genes was up-regulated and prolonged in embryo tissues under anoxic conditions.

Metabolic regulation of α-amylase gene expression in transgenic cell cultures of rice (Oryza sativa L.)

Expression of two genes in the α-amylase gene family is controlled by metabolic regulation in rice cultured cells. The levels of RAmy3D and RAmy3E mRNAs in rice cultured cells are inversely related to the concentration of sugar in the culture medium. Other genes in the rice α-amylase gene family have little or no expression in cultured cells; these expression levels are not controlled by metabolic regulation. A RAmy3D promoter/GUS gene fusion was metabolically regulated in the transgenic rice cell line 3DG, just as the endogenous RAmy3D gene is regulated. An assay of GUS enzyme activity in 3DG cells demonstrated that RAmy3D/GUS expression is repressed when sugar is present in the culture medium and induced when sugar is removed from the medium. The 942 bp fragment of the RAmy3D promoter that was linked to the coding region of the GUS reporter gene thus contains all of the regulatory sequences necessary for metabolic regulation of the gene.

Characterization of rice endo-β-glucanase genes (Gns2-Gns14) defines a new subgroup within the gene family

Thirteen new beta-glucanase-encoding genes have been identified in the rice genome. These genes, together with other monocot beta-glucanases, have now been classified into four subfamilies based on the structure and function of the genes. Two tandem gene clusters, Gns2-Gns3-Gns4 and Gns5-Gns6, were classified in the defense-related Subfamily A. Growth-related 1,3;1,4-beta-glucanase Gns1 was classified in Subfamily B. Gns7 and Gns8, together with the barley genes GVI and Hv34, represent Subfamily C. Gns9 and a beta-glucanase gene from wheat were grouped in Subfamily D. Genes in Subfamilies C and D have structures that are distinct from those of the other subfamilies, but there are very little data available on the biochemical or physiological roles of these genes. Gene expression in growing tissues and lack of gene induction in response to disease-related treatments suggest that Subfamilies C and D may function in control of plant growth.

Endo-1,3;1,4-β-glucanase from coleoptiles of rice and maize: role in the regulation of plant growth

The Matrix Polymer Hydrolysis Model for regulation of growth in plants is based on the simultaneous hydrolysis and incorporation of new glucans into the cell wall observed in growing plant tissues. The inhibition of growth in rice coleoptile tissues treated with glucanase antibodies confirms similar results observed previously in maize coleoptiles and provides direct evidence for a role of glucanase in control of plant growth. Analysis of two-maize coleoptile endo-glucanase ESTs shows that these sequences are not related to any other previously known family of glycosyl hydrolase. Thus, the coleoptile endo-glucanase enzyme should be classified as a new enzyme group (E.C. 3.2.1.xx). These discoveries enable new initiatives for further investigation of the glucanase role in control of plant growth.

Molecular analysis of the plant gene encoding cytosolic phosphoglucose isomerase

The gene encoding a cytosolic isozyme of phosphoglucose isomerase (PGI, EC 5.3.1.9) was isolated from Clarkia lewisii, a wild flower native to California, and the structure and sequence of the entire coding region determined. PGI catalyzes an essential step in glycolysis and carbohydrate biosynthesis in plants. Spanning about 6 kb, the gene has 23 exons and 22 introns, the highest number yet reported in plants. The exons range in size from 43 to 156 nt and encode a protein of 569 amino acids. The protein is about 44–46% identical to the inferred protein sequences of pig, Escherichia coli and Saccharomyces cerevisiae. All of the introns are bordered with the consensus 5′-GT...AG-3′ dinucleotides. The longest intron includes a large stem-loop structure bounded by a perfect 9 nt direct repeat. We cloned the PGI gene from a genomic library prepared from a single plant of known PGI genotype. The locus and allele of the clone were identified by matching restriction fragments to fragments from genetically defined genomic DNAs by Southern hybridization.

New perspectives on the endo-beta-glucanases of glycosyl hydrolase Family 17

Isozymes of glycosyl hydrolase Family 17 hydrolyze 1,3-beta-glucan polysaccharides found in the cell wall matrix of plants and fungi, enabling these plant enzymes to serve diverse roles in plant defense and plant development. Fourteen genes from Family 17 have been characterized in the genome of rice. A sequence dendrogram analysis divided these genes into four subfamilies. The recombinant GNS1 enzyme from subfamily B had 1,3;1,4-beta-glucanase activity, suggesting a role for this isozyme in plant development.

Characterization of rice α-amylase isozymes expressed by Saccharomyces cerevisiae

Two rice α-amylase isozymes, AmylA and Amy3D, were produced by secretion from genetically engineered strains of Saccharomyces cerevisiae. They have distinct differences in enzymatic characteristics that can be related to the physiology of the germinating rice seed. The rice isozymes were purified with immunoaffinity chromatography. The pH optima for amy3D (pH optimum 5.5) and Amy1A (pH optimum 4.2) correlate with the pH of the endosperm tissue at the times in rice seedling development when these isozymes are produced. Amy3D showed 10–14 times higher reactivity to oligosaccharides than Amy1A. Amy1A, on the other hand, showed higher reactivity to soluble starch and starch granules than Amy3D. These results suggest that the isozyme Amy3D, which is expressed at an early stage of germination, produces sugars from soluble starch during the early stage of seed germination and that the isozyme Amy1A works to initiate hydrolysis of the starch granules.

Kinetic parameters of two rice α-amylase isozymes for oligosaccharide degradation

Abstract The rice α-amylase isozymes Amy1A and Amy3D have distinct kinetic parameters and product profiles. For Amy3D, Michaelis constants ( K m ) decrease, and molecular activities ( k cat ) increase with the chain lenght of the substrates from maltose to maltoheptaose. Although Amy1A has a lower K m than Amy3D, the k cat of Amy1A was much lower than that of Amy3D. Amy1A produces only negligible amounts of glucose, while Amy3D produces glucose as a major product. Glucose regulates the expression of a number of genes in developing seedlings, so Amy3D may have a unique regulatory role in control of seedling development.