Biomedical subjects

M J Giroux

Publications and source records attributed to M J Giroux.

12 recordsLinked to original sources

PubMed2006

Complementation of the pina (null) allele with the wild type Pina sequence restores a soft phenotype in transgenic wheat.

The tightly linked puroindoline genes, Pina and Pinb, control grain texture in wheat, with wild type forms of both giving soft, and a sequence alteration affecting protein expression or function in either giving rise to hard wheat. Previous experiments have shown that addition of wild type Pina in the presence of mutated Pinb gave intermediate grain texture but addition of wild type Pinb gave soft grain. This raises questions as to whether Pina may be less functional than Pinb. Our goal here was to develop and characterize wheat lines expressing the wild type Pina-D1a sequence in hard wheat with the null mutation (Pina-D1b) for Pina. Three transgenic lines plus Bobwhite were evaluated in two environments. Grain texture, grain protein, and kernel weight were determined for the transgenic lines and Bobwhite. The three transgenic lines had soft phenotype, and none of the transgenic lines differed from Bobwhite for grain protein or kernel weight. The soft phenotype was accompanied by increases in Pina transcript accumulation. Total Triton X-114 extractable PINA and PINB increased from 2.5 to 5.5 times those from a soft wheat reference sample, and friabilin, PINA and PINB bound to starch, increased from 3.8 to 7.8 times those of the soft wheat reference. Bobwhite showed no starch bound PINA, but transgenic lines had levels from 5.3 to 13.7 times those of the soft wheat reference sample. Starch bound PINB in transgenic lines also increased from 0.9 to 2.5 times that for the soft wheat reference sample. The transgenic expression of wild type Pina sequence in the Pina null genotype gave soft grain with the characteristics of soft wheat including increased starch bound friabilin. The results support the hypothesis that both wild type Pin genes need to be present for friabilin formation and soft grain.

Alleles↗

PubMed2006

Increased puroindoline levels slow ruminal digestion of wheat (Triticum aestivum L.) starch by cattle.

Starch is the primary nutrient in ruminant diets used to promote high levels of performance. The site of starch digestion alters the nature of digestive end products (VFA in the rumen vs. glucose in the small intestine) and the efficiency of use. Cereal grain endosperm texture plays a major role in the rate and extent of starch degradation in ruminants. Wheat grain texture is regulated by the starch surface protein complex friabilin that consists primarily of puroindoline (PIN) A and B. Soft kernel texture in wheat is a result of both PIN genes being in the wild type active form and bound to starch. The objective of this study was to investigate the effect of varying PIN content in wheat on the rate of starch digestion in the rumen of beef cattle. In Exp. 1, 6 transgenic soft pin a/b isolines created in a hard wheat background, and 2 hard wheat controls were milled to yield a wide range of mean particle sizes across all lines. Milled samples were incubated in situ for 3 h. Increased expression of both PINA and PINB decreased DM digestibility (DMD) by 29.2% (P < 0.05) and decreased starch digestibility by 30.8% (P < 0.05). Experiment 2 separated the effects of particle size and total PIN content on digestion by milling the hardest and softest lines such that the mean particle size was nearly identical. Increased PIN decreased DMD by 21.7% (P < 0.05) and starch digestibility by 19.9% (P < 0.05) across particle sizes smaller than whole kernel. Experiment 3 addressed the time course of PIN effects in the rumen by observing ground samples of the hardest and softest lines over a 12-h in situ period. Increased PIN decreased DMD by 10.4% (P < 0.05) and starch digestibility by 11.0% (P < 0.05) across all time points. Dry matter and starch digestibility results demonstrated that increased expression of PIN was associated with a decreased rate of ruminal digestion independent of particle size. Puroindolines seem to aid in the protection of starch molecules from microbial digestion in the rumen, potentially increasing the amount of starch entering the small intestine.

Animals↗

PubMed2003

Wheat puroindolines interact to form friabilin and control wheat grain hardness.

Wheat grain is sold based upon several physiochemical characteristics, one of the most important being grain texture. Grain texture in wheat directly affects many end use qualities such as milling yield, break flour yield, and starch damage. The hardness (Ha) locus located on the short arm of chromosome 5D is known to control grain hardness in wheat. This locus contains the puroindoline A ( pina) and puroindoline B ( pinb) genes. All wheats to date that have mutations in pina or pinb are hard textured, while wheats possessing both the 'soft type' pina-D1a and pinb-D1a sequences are soft. Furthermore, it has been shown that complementation of the pinb-D1b mutation in hard spring wheat can restore a soft phenotype. Here, our objective was to identify and characterize the effect the puroindoline genes have on grain texture independently and together. To accomplish this we transformed a hard red spring wheat possessing a pinb-D1b mutation with 'soft type' pina and pinb, creating transgenic isolines that have added pina, pinb, or pina and pinb. Northern blot analysis of developing control and transgenic lines indicated that grain hardness differences were correlated with the timing of the expression of the native and transgenically added puroindoline genes. The addition of PINA decreased grain hardness less than the reduction seen with added PINB. Seeds from lines having more 'soft type' PINB than PINA were the softest. Friabilin abundance was correlated with the presence of both 'soft type' PINA and PINB and did not correlate well with total puroindoline abundance. The data indicates that PINA and PINB interact to form friabilin and together affect wheat grain texture.

Blotting, Northern↗

PubMed2003

Puroindoline A-gene expression is involved in association of puroindolines to starch.

Puroindolines largely influence cereal grain hardness. In order to understand how they exert this influence, we carried out a molecular analysis of the pina and pinb genes of many Italian wheat cultivars. On the basis of their pin genotypes they could be divided into three groups: Pina-D1a/Pinb-D1a; Pina-D1a/Pinb-D1b; and Pina-D1b/Pinb-D1a. Five cultivars from each group were chosen to be studied to examine the quantity of puroindolines associated with starch (friabilin) and the amount not associated with starch. In addition, the level of pina expression was measured using RT-PCR. Soft cultivars ( Pina-D1a/Pinb-D1a) exhibited the highest level of expression of pina; among the hard cultivars, those with the Pina-D1a/Pinb-D1b genotype showed a lower level of expression, while those with the Pina-D1b/Pinb-D1a genotype did not express pina. Total puroindoline and friabilin content was then measured by flow cytometry. Soft Pina-D1a/Pinb-D1a cultivars displayed high puroindoline content that was primarily starch associated. Hard Pina-D1b/Pinb-D1a cultivars had very low puroindoline content with no puroindoline bound to starch. Hard Pina-D1a/Pinb-D1b cultivars were highly heterogeneous with respect to both the content of puroindolines and the level of association with starch. The accurate quantification of puroindolines in starch-bound and not starch-bound forms in association with molecular analysis, indicates that pina expression and presence controls the abundance of total puroindoline and its association with starch.

Base Sequence↗

PubMed2002

Hordoindolines are associated with a major endosperm-texture QTL in barley (Hordeum vulgare).

Endosperm texture has a tremendous impact on the end-use quality of wheat (Triticum aestivum L.). Cultivars of barley (Hordeum vulgare L.), a close relative of wheat, also vary measurably in grain hardness. However, in contrast to wheat, little is known about the genetic control of barley grain hardness. Puroindolines are endosperm-specific proteins found in wheat and its relatives. In wheat, puroindoline sequence variation controls the majority of wheat grain texture variation. Hordoindolines, the puroindoline homologs of barley, have been identified and mapped. Recently, substantial allelic variation was found for hordoindolines among commercial barley cultivars. Our objective was to determine the influence of hordoindoline allelic variation upon grain hardness and dry matter digestibility in the 'Steptoe' x 'Morex' mapping population. This population is segregating for hordoindoline allele type, which was measured by a HinA/HinB/Gsp composite marker. One-hundred and fifty lines of the 'Steptoe' x 'Morex' population were grown in a replicated field trial. Grain hardness was estimated by near-infrared reflectance (NIR) and measured using the single kernel characterization system (SKCS). Variation attributable to the HinA/HinB/Gsp locus averaged 5.7 SKCS hardness units (SKCS U). QTL analysis revealed the presence of several areas of the genome associated with grain hardness. The largest QTL mapped to the HinA/HinB/Gsp region on the short arm of chomosome 7 (5H). This QTL explains 22% of the SKCS hardness difference observed in this study. The results indicate that the Hardness locus is present in barley and implicates the hordoindolines in endosperm texture control.

Hordeum↗

PubMed2001

Expression of wheat puroindoline genes in transgenic rice enhances grain softness.

The puroindoline genes (pinA and pinB) are believed to play critical roles in wheat (Triticum aestivum L.) grain texture. Mutations in either gene are associated with hard wheat. No direct evidence exists for the ability of puroindolines to modify cereal grain texture. Interestingly, puroindolines appear to be absent in cereal species outside of the tribe Triticeae, in which the dominant form of grain texture is hard. To assess the ability of the puroindolines to modify cereal grain texture, the puroindolines were introduced into rice (Oryzae sativa L.) under the control of the maize ubiquitin promoter. Textural analysis of transgenic rice seeds indicated that expression of PINA and/or PINB reduced rice grain hardness. After milling, flour prepared from these softer seeds had reduced starch damage and an increased percentage of fine flour particles. Our data support the hypothesis that puroindolines play important roles in controlling wheat grain texture and may be useful in modifying grain texture of other cereals.

Amino Acid Sequence↗

PubMed2001

Wheat puroindolines enhance fungal disease resistance in transgenic rice.

Antimicrobial peptides play a role in the immune systems of animals and plants by limiting pathogen infection and growth. The puroindolines, endosperm-specific proteins involved in wheat seed hardness, are small proteins reported to have in vitro antimicrobial properties. Rice, the most widely used cereal crop worldwide, normally does not contain puroindolines. Transgenic rice plants that constitutively express the puroindoline genes pinA and/or pinB throughout the plants were produced. PIN extracts of leaves from the transgenic plants reduced in vitro growth of Magnaporthe grisea and Rhizoctonia solani, two major fungal pathogens of rice, by 35 to 50%. Transgenic rice expressing pinA and/or pinB showed significantly increased tolerance to M. grisea (rice blast), with a 29 to 54% reduction in symptoms, and R. solani (sheath blight), with an 11 to 22% reduction in symptoms. Puroindolines are effective in vivo in antifungal proteins and could be valuable new tools in the control of a wide range of fungal pathogens of crop plants.

Gene Expression Regulation, Plant↗

PubMed2001

Maize genes encoding the small subunit of ADP-glucose pyrophosphorylase.

Plant ADP-glucose pyrophosphorylase (AGP) is a heterotetrameric enzyme composed of two large and two small subunits. Here, we report the structures of the maize (Zea mays) genes encoding AGP small subunits of leaf and endosperm. Excluding exon 1, protein-encoding sequences of the two genes are nearly identical. Exon 1 coding sequences, however, possess no similarity. Introns are placed in identical positions and exhibit obvious sequence similarity. Size differences are primarily due to insertions and duplications, hallmarks of transposable element visitation. Comparison of the maize genes with other plant AGP small subunit genes leads to a number of noteworthy inferences concerning the evolution of these genes. The small subunit gene can be divided into two modules. One module, encompassing all coding information except that derived from exon 1, displays striking similarity among all genes. It is surprising that members from eudicots form one group, whereas those from cereals form a second group. This implies that the duplications giving rise to family members occurred at least twice and after the separation of eudicots and monocot cereals. One intron within this module may have had a transposon origin. A different evolutionary history is suggested for exon 1. These sequences define three distinct groups, two of which come from cereal seeds. This distinction likely has functional significance because cereal endosperm AGPs are cytosolic, whereas all other forms appear to be plastid localized. Finally, whereas barley (Hordeum vulgare) reportedly employs only one gene to encode the small subunit of the seed and leaf, maize utilizes the two genes described here.

Base Sequence↗

PubMed1998

Wheat grain hardness results from highly conserved mutations in the friabilin components puroindoline a and b.

"Soft" and "hard" are the two main market classes of wheat (Triticum aestivum L.) and are distinguished by expression of the Hardness gene. Friabilin, a marker protein for grain softness (Ha), consists of two proteins, puroindoline a and b (pinA and pinB, respectively). We previously demonstrated that a glycine to serine mutation in pinB is linked inseparably to grain hardness. Here, we report that the pinB serine mutation is present in 9 of 13 additional randomly selected hard wheats and in none of 10 soft wheats. The four exceptional hard wheats not containing the serine mutation in pinB express no pinA, the remaining component of the marker protein friabilin. The absence of pinA protein was linked inseparably to grain hardness among 44 near-isogenic lines created between the soft variety Heron and the hard variety Falcon. Both pinA and pinB apparently are required for the expression of grain softness. The absence of pinA protein and transcript and a glycine-to-serine mutation in pinB are two highly conserved mutations associated with grain hardness, and these friabilin genes are the suggested tightly linked components of the Hardness gene. A previously described grain hardness related gene termed "GSP-1" (grain softness protein) is not controlled by chromosome 5D and is apparently not involved in grain hardness. The association of grain hardness with mutations in both pinA or pinB indicates that these two proteins alone may function together to effect grain softness. Elucidation of the molecular basis for grain hardness opens the way to understanding and eventually manipulating this wheat endosperm property.

Journal Article↗

PubMed1996

A single mutation that increases maize seed weight.

The maize endosperm-specific gene shrunken2 (Sh2) encodes the large subunit of the heterotetrameric starch synthetic enzyme adenosine diphosphoglucose pyrophosphorylase (AGP; EC 2.7.7.27). Here we exploit an in vivo, site-specific mutagenesis system to create short insertion mutations in a region of the gene known to be involved in the allosteric regulation of AGP. The site-specific mutagen is the transposable element dissociation (Ds). Approximately one-third (8 of 23) of the germinal revertants sequenced restored the wild-type sequence, whereas the remaining revertants contained insertions of 3 or 6 bp. All revertants retained the original reading frame 3' to the insertion site and involved the addition of tyrosine and/or serine. Each insertion revertant reduced total AGP activity and the amount of the SH2 protein. The revertant containing additional tyrosine and serine residues increased seed weight 11-18% without increasing or decreasing the percentage of starch. Other insertion revertants lacking an additional serine reduced seed weight. Reduced sensitivity to phosphate, a long-known inhibitor of AGP, was found in the high seed-weight revertant. This alteration is likely universally important since insertion of tyrosine and serine in the potato large subunit of AGP at the comparable position and expression in Escherichia coli also led to a phosphate-insensitive enzyme. These results show that single gene mutations giving rise to increased seed weight, and therefore perhaps yield, are clearly possible in a plant with a long history of intensive and successful breeding efforts.

Base Sequence↗

PubMed1994

De novo synthesis of an intron by the maize transposable element Dissociation.

The mechanisms by which introns are gained or lost in the evolution of eukaryotic genes remain poorly understood. The discovery that transposable elements sometimes alter RNA splicing to allow partial or imperfect removal of the element from the primary transcripts suggests that transposons are a potential and continuing source of new introns. To date, splicing events that precisely restore the wild-type RNA sequence at the site of insertion have not been detected. Here we describe alternative RNA splicing patterns that result in precise removal of a Dissociation (Ds) insertion and one copy of its eight-nucleotide host site duplication from an exon sequence of the maize shrunken2-mutabe1 (sh2-m1) mutant. In one case, perfect splicing of Ds was associated with aberrant splicing of an intron located 32 bp upstream of the insertion site. The second transcript type was indistinguishable from wild-type mRNA, indicating that Ds was spliced like a normal intron in about 2% of the sh2-m1 transcripts. Our results suggest that the transposition of Ds into sh2 in 1968, in effect, marked the creation of a new intron in a modern eukaryotic gene. The possibility of precise intron formation by a transposable element demonstrated here may be a general phenomenon of intron formation, since consensus intron splice sites can be explained by insertions that duplicate host sequences upon integration. A model is presented.

Alleles↗

PubMed1994

ADP-glucose pyrophosphorylase in shrunken-2 and brittle-2 mutants of maize.

The Shrunken-2 (Sh2) and Brittle-2 (Bt2) genes of maize encode subunits of the tetrameric maize endosperm ADPglucose pyrophosphorylase. However, in all sh2 and bt2 mutants so far examined, measurable ADPglucose pyrophosphorylase activity remains. We have investigated the origin of the residual activity found in various sh2 and bt2 mutants as well as tissue specific expression and post-translational modification of the Sh2 and Bt2 proteins. Sh2 and Bt2 cDNAs were expressed in Escherichia coli and antibodies were prepared against the resulting proteins SH2 and BT2 specific antibodies were used to demonstrate that SH2 and BT2 are endosperm specific, are altered or missing in various sh2 or bt2 mutants, and have a mol. wt. of 54 and 51 kDa respectively in the wild type. The Sh2 and Bt2 transcripts are also endosperm specific. Ten sh2 and eight bt2 mutants show varying severity of phenotypes expressed at transcript, protein subunit and kernel level. Synthesis of multiple transcripts and proteins commonly occurs as a result of sh2 or bt2 mutation. While all mutants produce detectable enzymic activity, not all produce detectable transcripts and proteins. To examine the origin of the apparent non-SH2/BT2 endosperm enzymic activity, homologs of Sh2 and Bt2, designated Agp1 and Agp2 respectively, were isolated from an embryo cDNA library and found to hybridize to endosperm transcripts distinct from those of Sh2 and Bt2. Thus Agp1 and Agp2 or closely related genes may be responsible for the residual activity in some sh2 and bt2 mutants. Surprisingly, no evidence of post-translational modification of the SH2 and BT2 protein subunits was detected.

Animals↗