Biomedical subjects

Brian A Larkins

Publications and source records attributed to Brian A Larkins.

17 recordsLinked to original sources

PubMed2006

The maize Mucronate mutation is a deletion in the 16-kDa gamma-zein gene that induces the unfolded protein response.

Mucronate (Mc) was identified as a dominant maize (Zea mays L.) opaque kernel mutation that alters zein storage protein synthesis. Zein protein bodies in Mc endosperm are misshapen and are associated with increased levels of ER Lumenal Binding Protein (BiP). Using GeneCalling to profile endosperm RNA transcripts, we identified an aberrant RNA in Mc that encodes the 16-kDa gamma-zein protein. The transcript contains a 38-bp deletion (nucleotides 406-444 after the initiation codon) that creates a frame-shift mutation and an abnormal sequence for the last 63 amino acids. Genetic mapping revealed the Mc mutation is linked with the locus encoding the 16-kDa gamma-zein, and two-dimensional gel electrophoresis confirmed the 16-kDa gamma-zein protein is altered in Mc. The mutant protein exhibited changes in solubility properties and co-immunoprecipitated with the molecular chaperone, BiP. Transgenic maize plants expressing the Mc 16-kDa gamma-zein manifested an opaque kernel phenotype with enhanced levels of BiP in the endosperm, similar to the Mc mutant. Unlike the wild-type protein, the Mc 16-kDa gamma-zein interacted only weakly with the 22-kDa alpha-zein when expressed in the yeast two-hybrid system. These results indicate that the Mc phenotype results from a frame-shift mutation in the gene encoding the 16-kDa gamma-zein protein, leading to the unfolded protein response in developing endosperm.

Alleles↗

PubMed2006

Grasses like mammals? Redundancy and compensatory regulation within the retinoblastoma protein family.

The retinoblastoma (RB) protein family plays a conserved and inhibitory role in cell cycle progression in higher eukaryotes. In mammals, this family includes, in addition to RB, the related (RBR) proteins p107 and p130, which appear to have both specific and redundant functions compared to those of the prototypical RB protein. Whereas most plant species seem to possess only one RBR gene, a recent study has shown that in maize there are two types of distinctly regulated RBR proteins, RBR1 and RBR3. Expression of RBR3 RNA is controlled by the RBR1-E2F pathway, and it is upregulated upon inhibition of RBR1 activity by the wheat dwarf virus RepA protein in tissue culture, indicating the presence of a specific compensatory mechanism sustaining high pocket protein activity. Database mining and phylogenetic analyses suggest the presence of two distinct RBR genes to be a unique feature of grasses among plants, which might help to explain their recalcitrance to genetic transformation.

Animals↗

PubMed2005

RBR3, a member of the retinoblastoma-related family from maize, is regulated by the RBR1/E2F pathway.

Retinoblastoma-related (RBR) proteins regulate cell division in higher eukaryotes by controlling the adenovirus E2 promoter binding factor (E2F)/dimerization partner (DP) family of transcription factors that regulate expression of many genes involved in cell-cycle progression. We identified a previously undescribed member of the maize RBR family, RBR3, which has the characteristic structure and binding activities of pocket proteins, where interaction depends on a LxCxE motif in the partner proteins and a critical cysteine within the B pocket domain. Like other RBR proteins, RBR3 appears to be regulated by phosphorylation mediated by cyclin-dependent kinases. During endosperm development, RBR3 expression is restricted to the mitotic stage preceding the onset of endoreduplication. This finding suggests a role distinct from RBR1, which is constitutively expressed. Two sites in the RBR3 promoter bind to complexes containing maize E2F1 and DP proteins. Expression of wheat dwarf virus RepA protein, which blocks RBR1 activity and stimulates cell proliferation, dramatically up-regulates RBR3, but not RBR1, RNA in embryogenic maize calli. The results indicate that RBR3 expression is controlled by RBR1 through the activity of E2F/DP and that RBR3 is the maize equivalent of mammalian p107. Furthermore, maize and related grasses might have evolved a compensatory mechanism among distinct types of RBR proteins to ensure robust control of pocket protein activity.

Amino Acid Motifs↗

PubMed2005

Cyclin-dependent kinase inhibitors in maize endosperm and their potential role in endoreduplication.

Two maize (Zea mays) cyclin-dependent kinase (CDK) inhibitors, Zeama;KRP;1 and Zeama;KRP;2, were characterized and shown to be expressed in developing endosperm. Similar to the CDK inhibitors in Arabidopsis (Arabidopsis thaliana) and tobacco (Nicotiana tabacum), the maize proteins contain a carboxy-terminal region related to the inhibitory domain of the mammalian Cip/Kip inhibitors. Zeama;KRP;1 is present in the endosperm between 7 and 21 d after pollination, a period that encompasses the onset of endoreduplication, while the Zeama;KRP;2 protein declines during this time. Nevertheless, Zeama;KRP;1 accounts for only part of the CDK inhibitory activity that peaks coincident with the endoreduplication phase of endosperm development. In vitro assays showed that Zeama;KRP;1 and Zeama;KRP;2 are able to inhibit endosperm Cdc2-related CKD activity that associates with p13(Suc1). They were also shown to specifically inhibit cyclin A1;3- and cyclin D5;1-associated CDK activities, but not cyclin B1;3/CDK. Overexpression of Zeama;KRP;1 in maize embryonic calli that ectopically expressed the wheat dwarf virus RepA protein, which counteracts retinoblastoma-related protein function, led to an additional round of DNA replication without nuclear division.

Amino Acid Sequence↗

PubMed2005

Molecular genetic approaches to developing quality protein maize.

Since its development more than two decades ago, Quality Protein Maize (QPM) has been adopted for cultivation in many regions of the developing world. Given the potential benefits of widespread use of QPM, research to better understand the genetic and biochemical mechanisms responsible for its altered kernel texture and protein quality is important. Recent investigations into the improved protein quality of the opaque2 mutant and the genetic mechanisms that can suppress its starchy kernel phenotype provide new insights to support the continued improvement of QPM. Chief among these developments are the use of transgenic approaches to improve nutritional quality and the discovery that an important component of modified endosperm texture in QPM is related to altered starch granule structure.

Chromosomes, Plant↗

PubMed2004

A model for estimating joint maternal-offspring effects on seed development in autogamous plants.

We present a statistical model for testing and estimating the effects of maternal-offspring genome interaction on the embryo and endosperm traits during seed development in autogamous plants. Our model is constructed within the context of maximum likelihood implemented with the EM algorithm. Extensive simulations were performed to investigate the statistical properties of our approach. We have successfully identified a quantitative trait locus that exerts a significant maternal-offspring interaction effect on amino acid contents of the endosperm in maize, demonstrating the power of our approach. This approach will be broadly useful in mapping endosperm traits for many agriculturally important crop plants and also make it possible to study the genetic significance of double fertilization in the evolution of higher plants.

Algorithms↗

PubMed2004

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 alpha-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.

Cytoskeletal Proteins↗

PubMed2004

A dominant negative mutant of cyclin-dependent kinase A reduces endoreduplication but not cell size or gene expression in maize endosperm.

Cells in maize (Zea mays) endosperm undergo multiple cycles of endoreduplication, with some attaining DNA contents as high as 96C and 192C. Genome amplification begins around 10 d after pollination, coincident with cell enlargement and the onset of starch and storage protein accumulation. Although the role of endoreduplication is unclear, it is thought to provide a mechanism that increases cell size and enhances gene expression. To investigate this process, we reduced endoreduplication in transgenic maize endosperm by ectopically expressing a gene encoding a dominant negative mutant form of cyclin-dependent kinase A. This gene was regulated by the 27-kD gamma-zein promoter, which restricted synthesis of the defective enzyme to the endoreduplication rather than the mitotic phase of endosperm development. Overexpression of a wild-type cyclin-dependent kinase A increased enzyme activity but had no effect on endoreduplication. By contrast, ectopic expression of the defective enzyme lowered kinase activity and reduced by half the mean C-value and total DNA content of endosperm nuclei. The lower level of endoreduplication did not affect cell size and only slightly reduced starch and storage protein accumulation. There was little difference in the level of endosperm gene expression with high and low levels of endoreduplication, suggesting that this process may not enhance transcription of genes associated with starch and storage protein synthesis.

Cell Cycle↗

PubMed2004

Characterization of the maize endosperm transcriptome and its comparison to the rice genome.

The cereal endosperm is a major organ of the seed and an important component of the world's food supply. To understand the development and physiology of the endosperm of cereal seeds, we focused on the identification of genes expressed at various times during maize endosperm development. We constructed several cDNA libraries to identify full-length clones and subjected them to a twofold enrichment. A total of 23,348 high-quality sequence-reads from 5'- and 3'-ends of cDNAs were generated and assembled into a unigene set representing 5326 genes with paired sequence-reads. Additional sequencing yielded a total of 3160 (59%) completely sequenced, full-length cDNAs. From 5326 unigenes, 4139 (78%) can be aligned with 5367 predicted rice genes and by taking only the "best hit" be mapped to 3108 positions on the rice genome. The 22% unigenes not present in rice indicate a rapid change of gene content between rice and maize in only 50 million years. Differences in rice and maize gene numbers also suggest that maize has lost a large number of duplicated genes following tetraploidization. The larger number of gene copies in rice suggests that as many as 30% of its genes arose from gene amplification, which would extrapolate to a significant proportion of the estimated 44,027 candidate genes of its entire genome. Functional classification of the maize endosperm unigene set indicated that more than a fourth of the novel functionally assignable genes found in this study are involved in carbohydrate metabolism, consistent with its role as a storage organ.

DNA, Complementary↗

PubMed2003

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.

Amylopectin↗

PubMed2003

A defective signal peptide in a 19-kD alpha-zein protein causes the unfolded protein response and an opaque endosperm phenotype in the maize De*-B30 mutant.

Defective endosperm* (De*)-B30 is a dominant maize (Zea mays) mutation that depresses zein synthesis in the developing endosperm. The mutant kernels have an opaque, starchy phenotype, malformed zein protein bodies, and highly increased levels of binding protein and other chaperone proteins in the endosperm. Immunoblotting revealed a novel alpha-zein protein in De*-B30 that migrates between the 22- and 19-kD alpha-zein bands. Because the De*-B30 mutation maps in a cluster of 19-kD alpha-zein genes, we characterized cDNA clones encoding these proteins from a developing endosperm library. This led to the identification of a 19-kD alpha-zein cDNA in which proline replaces serine at the 15th position of the signal peptide. Although the corresponding gene does not appear to be highly expressed in De*-B30, it was found to be tightly linked with the mutant phenotype in a segregating F2 population. Furthermore, when the protein was synthesized in yeast cells, the signal peptide appeared to be less efficiently processed than when serine replaced proline. To test whether this gene is responsible for the De*-B30 mutation, transgenic maize plants expressing this sequence were created. T1 seeds originating from the transformants manifested an opaque kernel phenotype with enhanced levels of binding protein in the endosperm, similar to De*-B30. These results are consistent with the hypothesis that the De*-B30 mutation causes a defective signal peptide in a 19-kD alpha-zein protein.

Amino Acid Sequence↗

PubMed2003

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.

Actins↗

PubMed2002

Stimulation of the cell cycle and maize transformation by disruption of the plant retinoblastoma pathway.

The genome of the Mastreviruses encodes a replication-associated protein (RepA) that interacts with members of the plant retinoblastoma-related protein family, which are putative cell cycle regulators. Expression of ZmRb1, a maize retinoblastoma-related gene, and RepA inhibited and stimulated, respectively, cell division in tobacco cell cultures. The effect of RepA was mitigated by over-expression of ZmRb1. RepA increased transformation frequency and callus growth rate of high type II maize germplasm. RepA-containing transgenic maize calli remained embryogenic, were readily regenerable, and produced fertile plants that transmitted transgene expression in a Mendelian fashion. In high type II, transformation frequency increased with the strength of the promoter driving RepA expression. When a construct in which RepA was expressed behind its native LIR promoter was used, primary transformation frequencies did not improve for two elite Pioneer maize inbreds. However, when LIR:RepA-containing transgenic embryos were used in subsequent rounds of transformation, frequencies were higher in the RepA+ embryos. These data demonstrate that RepA can stimulate cell division and callus growth in culture, and improve maize transformation.

Cell Cycle↗

PubMed2002

An improved genetic model generates high-resolution mapping of QTL for protein quality in maize endosperm.

The genetic mapping of polymorphic markers in a cross between two inbred plant lines has proven to be a powerful method for detecting quantitative trait loci (QTL) underlying complex traits. However, existing methods of QTL mapping were developed for disomic inheritance of both marker and QTL loci in a diploid population. To map QTL influencing traits expressed in the endosperm, a triploid embryo-nourishing tissue resulting from double fertilization, existing QTL mapping models require modification to consider the trisomic inheritance of the endosperm and the generation difference between the mapping population and the endosperm. Such a model requires simultaneous use of two successive generations, which theoretically can lead to an increase in resolution for QTL mapping compared with the use of a single backcross or F(2) generation. Using a newly developed model based on these considerations, we demonstrate the improved resolution of QTL, influencing protein quality traits in maize endosperm. The increased resolution made possible with this approach makes identified QTL accessible to positional cloning.

Chromosome Mapping↗

PubMed2002

Genetic analyses of endoreduplication in Zea mays endosperm: evidence of sporophytic and zygotic maternal control.

Flow cytometry was used to assess the variability of endoreduplication in endosperms of maize inbred lines. Little variation was found between midwestern dent types, and high levels of endoreduplication were observed in popcorns. Endoreduplication is different between inbred lines by 13-18 days after pollination, and flow cytometric analysis of ploidy level was feasible until 20 DAP. To study the genetic regulation of endoreduplication, four inbreds were crossed to B73 and developing endosperms from both parental, reciprocal F(1), and backcross generations were subjected to flow cytometric analysis. Three measurements of endoreduplication were calculated from these data and analyzed as quantitative genetic traits. Multiple models of trait inheritance were considered including triploid, diploid, sporophytic maternal, and maternal and paternal zygotic nuclear inheritance. Maternal zygotic effects, often considered a form of parental imprinting, and maternal sporophytic effects were detected. To test the feasibility of introgressing a high endoreduplication phenotype into a midwestern dent inbred line, a backcross population was generated from B73 x Sg18. Parental and progeny endoreduplication levels were compared and heritabilities assessed. The heritabilities calculated from these data generally agree with the values calculated in the larger crossing experiments.

Analysis of Variance↗

PubMed2002

Maize opaque endosperm mutations create extensive changes in patterns of gene expression.

Maize starchy endosperm mutants have kernel phenotypes that include a brittle texture, susceptibility to insect pests, and inferior functional characteristics of products made from their flour. At least 18 such mutants have been identified, but only in the cases of opaque2 (o2) and floury2 (fl2), which affect different aspects of storage protein synthesis, is the molecular basis of the mutation known. To better understand the relationship between the phenotypes of these mutants and their biochemical bases, we characterized the protein and amino acid composition, as well as the mRNA transcript profiles, of nearly isogenic inbred lines of W64A o1, o2, o5, o9, o11, Mucuronate (Mc), Defective endosperm B30 (DeB30), and fl2. The largest reductions in zein protein synthesis occur in the W64A o2, DeB30, and fl2 mutants, which have approximately 35 to 55% of the wild-type level of storage proteins. Zeins in W64A o5, o9, o11, and Mc are within 80 to 90% of the amount found in the wild type. Only in the cases of o5 and Mc were significant qualitative changes in zein synthesis observed. The pattern of gene expression in normal and mutant genotypes was assayed by profiling endosperm mRNA transcripts at 18 days after pollination with an Affymetrix GeneChip containing >1400 selected maize gene sequences. Compared with W64A sugary1, a mutant defective in starch synthesis, alterations in the gene expression patterns of the opaque mutants are very pleiotropic. Increased expression of genes associated with physiological stress, and the unfolded protein response, are common features of the opaque mutants. Based on global patterns of gene expression, these mutants were categorized in four phenotypic groups as follows: W64A+ and o1; o2; o5/o9/o11; and Mc and fl2.

DNA-Binding Proteins↗

PubMed2002

Zein protein interactions, rather than the asymmetric distribution of zein mRNAs on endoplasmic reticulum membranes, influence protein body formation in maize endosperm.

Prolamin-containing protein bodies in maize endosperm are composed of four different polypeptides, the alpha-, beta-, gamma-, and delta-zeins. The spatial organization of zeins within the protein body, as well as interactions between them, suggests that the localized synthesis of gamma-zeins could initiate and target protein body formation at specific regions of the rough endoplasmic reticulum. To investigate this possibility, we analyzed the distribution of mRNAs encoding the 22-kD alpha-zein and the 27-kD gamma-zein proteins on cisternal and protein body rough endoplasmic reticulum membranes. In situ hybridization revealed similar frequencies of the mRNAs in both regions of the endoplasmic reticulum, indicating that the transcripts are distributed more or less randomly. This finding implies that zein protein interactions determine protein body assembly. To address this question, we expressed cDNAs encoding alpha-, beta-, gamma-, and delta-zeins in the yeast two-hybrid system. We found strong interactions among the 50-, 27-, and 16-kD gamma-zeins and the 15-kD beta-zein, consistent with their colocalization in developing protein bodies. Interactions between the 19- and 22-kD alpha-zeins were relatively weak, although each of them interacted strongly with the 10-kD delta-zein. Strong interactions were detected between the alpha- and delta-zeins and the 16-kD gamma-zein and the 15-kD beta-zein; however, the 50- and 27-kD gamma-zeins did not interact with the alpha- and delta-zein proteins. We identified domains within the 22-kD alpha-zein that bound preferentially the alpha- and delta-zeins and the beta- and gamma-zeins. Affinities between zeins generally were consistent with results from immunolocalization experiments, suggesting an important role for the 16-kD gamma-zein and the 15-kD beta-zein in the binding and assembly of alpha-zeins within the protein body.

Binding Sites↗