Bryan C. Gibbon

Altered starch structure is associated with endosperm modification in Quality Protein Maize

The biochemical basis of modified kernel texture in Quality Protein Maize (QPM) is poorly understood. Proteomic analysis of several QPM lines indicated increased levels of granule-bound starch synthase I in the soluble nonzein protein fraction of these genotypes. Increased extraction of this enzyme reflected a change in starch structure, which was manifested as shorter amylopectin branches and increased starch-granule swelling. In mature kernels, these alterations in starch structure were associated with interconnections between starch granules that resulted in a vitreous kernel phenotype. Understanding the molecular basis for this previously uncharacterized starch structure will accelerate the development of QPM.

Genetic analysis of opaque2 modifier loci in quality protein maize

Quality protein maize (QPM) was created by selecting genetic modifiers that convert the starchy endosperm of an opaque2 (o2) mutant to a hard, vitreous phenotype. Genetic analysis has shown that there are multiple, unlinked o2 modifiers (Opm), but their identity and mode of action are unknown. Using two independently developed QPM lines, we mapped several major Opm QTLs to chromosomes 1, 7 and 9. A microarray hybridization performed with RNA obtained from true breeding o2 progeny with vitreous and opaque kernel phenotypes identified a small group of differentially expressed genes, some of which map at or near the Opm QTLs. Several of the genes are associated with ethylene and ABA signaling and suggest a potential linkage of o2 endosperm modification with programmed cell death.

Identification and Characterization of Endoplasmic Reticulum-Associated Degradation Proteins Differentially Affected by Endoplasmic Reticulum Stress

The disposal of misfolded proteins from the lumen of the endoplasmic reticulum (ER) is one of the quality control mechanisms present in the protein secretory pathway. Through ER-associated degradation, misfolded substrates are targeted to the cytosol where they are degraded by the proteasome. We have identified four maize (Zea mays) Der1-like genes (Zm Derlins) that encode homologs of Der1p, a yeast (Saccharomyces cerevisiae) protein implicated in ER-associated degradation. Zm Derlins are capable of functionally complementing a yeast Der1 deletion mutant. Such complementation indicates that the Der1p function is conserved among species. Zm Derlin genes are expressed at low levels throughout the plant, but appear prevalent in tissues with high activity of secretory protein accumulation, including developing endosperm cells. Expression of three of the four Zm Derlin genes increases during ER stress, with Zm Derlin1-1 showing the strongest induction. Subcellular fractionation experiments localized Zm Derlin proteins to the membrane fraction of microsomes. In maize endosperm, Zm Derlin proteins were found primarily associated with ER-derived protein bodies regardless of the presence of an ER stress response.

Characterization of opaque2 modifier QTLs and candidate genes in recombinant inbred lines derived from the K0326Y quality protein maize inbred

Quality protein maize (QPM) is a high lysine-containing corn that is based on genetic modification of the opaque2 (o2) mutant. In QPM, modifier genes convert the starchy endosperm of o2 to the vitreous phenotype of wild type maize. There are multiple, unlinked o2 modifier loci (Opm) in QPM and their nature and mode of action are unknown. We previously identified seven Opm QTLs and characterized 16 genes that are differentially up-regulated at a significant level in K0326Y QPM, compared to the starchy endosperm mutant W64Ao2. In order to further characterize these Opm QTLs and the genes up-regulated in K0326Y QPM, we created a population of 314 recombinant inbred lines (RILs) from a cross between K0326Y QPM and W64Ao2. The RILs were characterized for three traits associated with endosperm texture: vitreousness, density and hardness. Genetic linkage analysis of the RIL population confirmed three of the previously identified QTLs associated with o2 endosperm modification in K0326Y QPM. Many of the genes up-regulated in K0326Y QPM showed substantially higher levels of expression in vitreous compared with opaque RILs. These included genes associated with the upstream regulation of the ethylene response pathway, and a gene encoding a regulatory subunit of pyrophosphate-dependent fructose-6-phosphate 1-phosphotransferase, an adaptive enzyme of the glycolytic pathway.

Identification and characterization of lysine-rich proteins and starch biosynthesis genes in the opaque2 mutant by transcriptional and proteomic analysis

BackgroundThe opaque2 mutant is valuable for producing maize varieties with enhanced nutritional value. However, the exact mechanisms by which it improves protein quality and creates a soft endosperm texture are unclear. Given the importance of improving nutritional quality in grain crops, a better understanding of the physiological basis for these traits is necessary.ResultsIn this study, we combined transcript profiling and proteomic analysis to better understand which genes and proteins are altered by opaque2 in the W64A inbred line. These analyses showed that the accumulation of some lysine-rich proteins, such as sorbitol dehydrogenase and glyceraldehyde3-phosphate dehydrogenase, was increased in mature kernels and may contribute substantially to the lysine content of opaque2 endosperm. Some defense proteins such as beta-glucosidase aggregating factor were strongly down regulated and may be regulated directly by opaque2. The mutant also had altered expression of a number of starch biosynthesis genes and this was associated with a more highly crystalline starch.ConclusionsThe results of these studies revealed specific target genes that can be investigated to further improve nutritional quality and agronomic performance of high lysine maize lines, particularly those based on the presence of the opaque2 mutation. Alteration of amylopectin branching patterns in opaque2 starch could contribute to generation of the soft, starchy endosperm.

Cytoskeletal Proteins Are Coordinately Increased in Maize Genotypes with High Levels of eEF1A

The opaque2 (o2) mutation increases the Lys content of maize (Zea mays) endosperm by reducing the synthesis of zein storage proteins and increasing the accumulation of other types of cellular proteins. Elongation factor 1A (eEF1A) is one of these proteins, and its concentration is highly correlated with the amount of other Lys-containing proteins in the endosperm. We investigated the basis for this relationship by comparing patterns of protein accumulation and gene expression between a high (Oh51Ao2) and a low (Oh545o2) eEF1A inbred, as well as between high and low eEF1A recombinant inbred lines obtained from their cross. The content of α-zein and several cytoskeletal proteins was measured in high and low eEF1A inbred lines, and the levels of these proteins were found to correlate with that of eEF1A. To extend this analysis, we used an endosperm expressed sequence tag microarray to examine steady-state levels of RNA transcripts in developing endosperm of these genotypes. We identified about 120 genes coordinately regulated in association with eEF1A content. These genes encode proteins involved in several biological structures and processes, including the actin cytoskeleton, the endoplasmic reticulum, and the protein synthesis apparatus. Thus, higher levels of eEF1A in o2 mutants may be related to a more extensive cytoskeletal network surrounding the rough endoplasmic reticulum and increased synthesis of cytoskeleton-associated proteins, all of which contribute significantly to the Lys content of the endosperm.

eEF1A Isoforms Change in Abundance and Actin-Binding Activity during Maize Endosperm Development

Eukaryotic elongation factor 1A (eEF1A) appears to be a multifunctional protein because several biochemical activities have been described for this protein, in addition to its role in protein synthesis. In maize (Zea mays) endosperm, the synthesis of eEF1A is increased in o2 (opaque2) mutants, and its concentration is highly correlated with the protein-bound lysine content. To understand the basis of this relationship, we purified eEF1A isoforms from developing endosperm and investigated their accumulation and their functional and structural properties. Formation of three isoforms appears to be developmentally regulated and independent of the o2 mutation, although one isoform predominated in one high lysine o2 inbred. The purified proteins differ in their ability to bind F-actin in vitro, suggesting that they are functionally distinct. However, they share similar aminoacyl-tRNA-binding activities. Tandem mass spectrometry revealed that each isoform is composed of the four same gene products, which are modified posttranslationally by methylation and phosphorylation. The chemical differences that account for their different actin-binding activities could not be determined.

Actin monomer-binding proteins and the regulation of actin dynamics in plants

Plants are uniquely adapted to respond to environmental and developmental signals for survival. Many signals result in dramatic changes of cell shape or cytoplasmic organization that are dependent on the actin cytoskeleton. The dynamic nature of the actin cytoskeleton is conferred by a wide variety of actin-binding proteins. One class of these proteins is capable of binding to free actin monomers and thereby regulates the polymerization of actin filaments. Two such proteins have been identified in plants: profilin and actin-depolymerizing factor (ADF). These proteins comprise multigene families in plants and the isoforms of each protein have unique developmentally and spatially regulated expression patterns. Biochemical analysis of the plant monomer-binding proteins indicates that they are able to both stimulate and inhibit actin polymerization in vitro. Furthermore, microinjection of these proteins into cells reveals that simple models for the interaction of monomer-binding proteins with actin are inadequate. The complex effects on actin in vitro and in vivo are due to the ability of profilin and ADF to interact with a number of other ligands, such as regulatory proteins and polyphosphoinositide lipids. The monomer-binding proteins also respond to changes in cytosolic Ca 2+ and pH. Regulation of these proteins by phosphorylation adds an additional level of complexity for the study of their role in coordinating actin reorganization in plant cells. A model of actin filament assembly in tip-growing cells that incorporates the activities of profilin and ADF is presented.