Earl Taliercio

Expression of genes associated with carbohydrate metabolism in cotton stems and roots

BackgroundCotton (Gossypium hirsutum L) is an important crop worldwide that provides fiber for the textile industry. Cotton is a perennial plant that stores starch in stems and roots to provide carbohydrates for growth in subsequent seasons. Domesticated cotton makes these reserves available to developing seeds which impacts seed yield. The goals of these analyses were to identify genes and physiological pathways that establish cotton stems and roots as physiological sinks and investigate the role these pathways play in cotton development during seed set.ResultsAnalysis of field-grown cotton plants indicated that starch levels peaked about the time of first anthesis and then declined similar to reports in greenhouse-grown cotton plants. Starch accumulated along the length of the stem and the shape and size of the starch grains from stems were easily distinguished from transient starch. Microarray analyses compared gene expression in tissues containing low levels of starch with tissues rapidly accumulating starch. Statistical analysis of differentially expressed genes indicated increased expression among genes associated with starch synthesis, starch degradation, hexose metabolism, raffinose synthesis and trehalose synthesis. The anticipated changes in these sugars were largely confirmed by measuring soluble sugars in selected tissues.ConclusionIn domesticated cotton starch stored prior to flowering was available to support seed production. Starch accumulation observed in young field-grown plants was not observed in greenhouse grown plants. A suite of genes associated with starch biosynthesis was identified. The pathway for starch utilization after flowering was associated with an increase in expression of a glucan water dikinase gene as has been implicated in utilization of transient starch. Changes in raffinose levels and levels of expression of genes controlling trehalose and raffinose biosynthesis were also observed in vegetative cotton tissues as plants age.

Leaf expansion of soybean subjected to high and low atmospheric vapour pressure deficits

Summary Genotypic differences were found in decrease of leaf expansion with exposure to high vapour pressure deficit. Changes in leaf expansion were associated with down-regulation of expansin and extensin genes.

Cotton benzoquinone reductase: Up-regulation during early fiber development and heterologous expression and characterization in Pichia pastoris

Benzoquinone reductase (BR; EC 1.6.5.7) is an enzyme which catalyzes the bivalent redox reactions of quinones without the production of free radical intermediates. Using 2D-PAGE comparisons, two proteins were found to be up-regulated in wild-type cotton ovules during the fiber initiation stage but not in the fiberless line SL 1-7-1. These proteins were excised from the gel, partially sequenced and identified to be BR isoforms. PCR was used to amplify both full length coding regions of 609bp and once cloned, the restriction enzyme HindIII was used to distinguish the clones encoding the BR1 (one site) and BR2 (two sites) isoforms. Both deduced protein sequences had 203 residues which differed at 14 residues. The molecular mass and pIs were similar between the measured protein (2D-PAGE) and the theoretical protein (deduced). Heterologous proteins BR1 and BR2 were produced for further study by ligating the BR1 and BR2 clones in frame into the alpha-factor secretion sequence in pPICZalphaA vector and expressed with Pichia pastoris. Both BR1 and BR2 were approximately 26.5kDa and did enzymatically reduce 2,6-dimethoxybenzoquinone similar to the fungal BR.

Identification of transcripts translated on free or membrane-bound polyribosomes by differential display

Differential display has been widely and successfully used to identify differentially expressed genes based on physiological treatments or genetic variation. We used differential display to identify genes based on their site of translation. Identification was made based on the fractionation of RNA from soybean leaves into total RNA, free polyribosomal RNA, and membrane-bound (MB) polyribosomal RNA. Sequences were identified representing RNAs uniquely translated on free or MB polyribosomes. The compartmentalization was confirmed on RNA blots. Differential display of free and MB polyribosomal RNA from genetic mutants or physiological studies has 2 potential advantages. First, the sensitivity of the method is increased. Second, localization of mRNAs to the free or MB compartments may identify genes that are controlled at the level of translation or that switch compartments in response to a treatment.

Characterization of an ADP-glucose pyrophosphorylase small subunit gene expressed in developing cotton ( Gossypium hirsutum ) fibers

ADP-glucose pyrophosphorylase (ADPGp, EC 2.7.7.27) is a tetrameric protein composed of two small and two large subunits that catalyzes the biosynthesis of ADP-glucose from glucose-phosphate which is used to provide the glucose subunits for starch biosynthesis. A second cotton gene encoding an ADPGp small subunit has been cloned and characterized. The gene contains eight introns similar to previously reported potato and cotton ADPGp small subunit genes. The deduced translation of the gene contained a poorly conserved transit peptide and well conserved catalytic and regulatory elements typical of other plant ADPGps. The 5′ end of the mRNA was cloned and sequenced to identify the transcriptional start site (TSS). The promoter region upstream of the TSS did not contain the core promoter sequence in the typical positions indicating this gene may not use a standard core promoter. Other sequence motifs associated with tissue specific expression and phytohormone response were present. Reverse transcription (RT)-PCR with gene specific primers identified the sites of expression of this gene. Expression was most abundant in the meristem region, and immature stem and relatively lower in starch accumulating roots demonstrating that this gene has a different pattern of expression than the previously reported cotton ADPGp small subunit gene. Additionally this gene was differentially expressed in cotton fibers. The presence of starch was confirmed in developing cotton fibers suggesting that starch metabolism plays a role in cotton fiber development.

Epitopes from two soybean glycinin subunits are antigenic in pigs

BACKGROUND Glycinin is a seed storage protein in soybean (Glycine max) that is allergenic in pigs. Glycinin is a hexamer composed of subunits consisting of basic and acidic portions joined by disulfide bridges. There are five glycinin subunit isoforms designated Gy1-Gy5. The purpose of this study is to identify epitopes from selected glycinin subunits that are antigenic in pigs. RESULTS Twenty-seven out of 30 pigs had antibodies against glycinin in their sera. Ten of these sera had immunoglobulin G (IgG) against the Gy4 (A5A4B3) or Gy1 (A1aBx) subunit. Three sera recognised overlapping regions between the two subunits tested, though no serum stained both A5A4B3 and A1aBx. Two sera stained a highly conserved region between A5A4B3 and A1aBx, though again neither serum stained both peptides. The basic part of the A1aBx subunit was not recognised by any of the sera tested even though immunoblot data indicated that the basic and acidic subunits of glycinin are nearly equally antigenic. CONCLUSION Two antigenic regions of A5A4B3 and A1aBx were identified that bound antibodies in half of the sera that reacted with these two proteins. Half of the sera reacted with unique regions of A5A4B3 and A1aBx. The failure of the basic portion of A1aBx to bind pig antibodies may indicate that it is less antigenic than the basic portion of A5A4B3 and other glycinin subunits.

Comparisons of the Effects of Elevated Vapor Pressure Deficit on Gene Expression in Leaves among Two Fast-Wilting and a Slow-Wilting Soybean

Limiting the transpiration rate (TR) of a plant under high vapor pressure deficit (VPD) has the potential to improve crop yield under drought conditions. The effects of elevated VPD on the expression of genes in the leaves of three soybean accessions, Plant Introduction (PI) 416937, PI 471938 and Hutcheson (PI 518664) were investigated because these accessions have contrasting responses to VPD changes. Hutcheson, a fast-wilting soybean, and PI 471938, a slow-wilting soybean, respond to increased VPD with a linear increase in TR. TR of the slow-wilting PI 416937 is limited when VPD increases to greater than about 2 kPa. The objective of this study was to identify the response of the transcriptome of these accessions to elevated VPD under well-watered conditions and identify responses that are unique to the slow-wilting accessions. Gene expression analysis in leaves of genotypes PI 471938 and Hutcheson showed that 22 and 1 genes, respectively, were differentially expressed under high VPD. In contrast, there were 944 genes differentially expressed in PI 416937 with the same increase in VPD. The increased alteration of the transcriptome of PI 416937 in response to elevated VPD clearly distinguished it from the other slow-wilting PI 471938 and the fast-wilting Hutcheson. The inventory and analysis of differentially expressed genes in PI 416937 in response to VPD is a foundation for further investigation to extend the current understanding of plant hydraulic conductivity in drought environments.

Characterization of two cotton (Gossypium hirsutum L) invertase genes

Two cotton vacuolar-invertase genes were identified and sequenced. Both genes had seven exons, including an unusually small second exon typical of acid invertases. These genes encode peptides with many features shared by acid invertases from other species including, leader sequences that probably target the peptide to the vacuole, active site motifs and substrate binding motifs. Expression analyses indicated that one of the genes was expressed in roots during the starch filling stage of development. However, expression of the same gene fluctuated during the starch utilization stage of development. Therefore this gene was unlikely to play a role in determining sink strength of this tissue. Both genes were expressed in elongating fibers where they were likely to play a role in cell expansion. The invertase gene uniquely expressed in fiber had a simple sequence repeat (SSR) in the third intron that was polymorphic among various cotton species. An EST was identified with an expansion of the SSR that included the third intron indicating this SSR is associated with a splice variant. The polymorphic SSR may be useful in investigating the function of this gene in fiber development.

The DNA sequence of a gypsy element from Gossypium hirsutum L. and characterization of gypsy elements in three Gossypium species.

A strategy was developed to isolate a complete gypsy-element from Gossypium hirsutum L. based on the sequence of a RAPD that was polymorphic in near isoganic lines of cotton that varied in leaf shape. A 5998 nt clone was isolated and its gene order and sequence confirmed it was a gypsy-type retroelement. Sequences homologous to the gag portion of this clone were also found in Gossypium herbaceum L. and Gossypium raimondii L. A portion of the open reading frame of the integrase gene was amplified from three Gossypium species under investigation. PCR products of the expected size were amplified from all three Gossypium species. G. raimondii sequences were statistically more degenerate than sequences from either G. hirsutum or G. herbaceum. Finally, analysis of the open reading frames of selected integrase clones from the three Gossypium species revealed a second gypsy element based on similarity of the deduced amino acid sequence.

Identification of a second major antigenic epitope in the α-subunit of soy β-conglycinin

Soybean meal in the post-weaning diet of pigs results in a growth lag correlated with Immunoglobulin G (IgG) production against soybean proteins, including the seed storage protein β-conglycinin. We screened sera from 30 pigs for IgG directed against β-conglycinin. Analysis of the same sera on protein blots identified sera that had IgG against the α-subunit of β-conglycinin. A tiled peptide array (16 amino acids with an 11 amino acid overlap) of the α-subunit of β-conglycinin was screened to identify the epitopes of the α-subunit that bind IgG. One major IgG binding epitope previously identified was confirmed. A second major IgG binding epitope was identified that bound IgG from half of the animals tested. Minor epitopes that bound IgG from one or two other sera were also identified. Binding of IgG to β-conglycinin in an enzyme-linked immunosorbent assay (ELISA) was inhibited by a synthetic peptide representing one of the minor epitopes.