The maize zmsmu2 gene encodes a putative RNA-splicing factor that affects protein synthesis and RNA processing during endosperm development

We characterized two maize (Zea mays) mutants, zmsmu2-1 and zmsmu2-3, that result from insertion of a Mutator (Mu) transposable element in the first exon of a gene homologous to the nematode gene, smu-2, which is involved in RNA splicing. In addition to having a starchy endosperm with reduced levels of zein storage proteins, homozygous zmsmu2-1 mutants manifest a number of phenotypes, including defective meristem development. The zmsmu2 mutants have poor seedling viability and surviving plants are sterile. The gene encoding ZmSMU2 is expressed in the endosperm, embryo, and shoot apex, which explains the pleiotropic nature of the mutation. We found that proper expression of Zmsmu2 is required for efficient ribosomal RNA processing, ribosome biogenesis, and protein synthesis in developing endosperm. Based on the pleiotropic nature of the mutations and the known function of animal Zmsmu2 homologs, we propose a possible role for ZmSMU2 in the development of maize endosperm, as well as a mechanism by which misregulation of zmsmu2 causes the mutant phenotypes.     

Genetic regulation of storage protein content in maize endosperm

Sodium dodecylsulfate-polyacrylamide gel electrophoresis reveals that zein prepared from normal maize inbred (Zea mays L.) contains six separable components. Z1 and Z2 are the predominant species, with molecular weights of 21,800 and 19,000 daltons. Amino acid analysis of these two components shows that both are rich in glutamic acid, leucine, and proline, but low in lysine. Of the four minor bands, Z3, Z4, Z5, and Z6, the latter two exist only in trace amounts. A mutation at the opaque-2 locus severely suppresses the synthesis of Z1. The nonallelic mutant, opaque-7, strongly suppresses the synthesis of Z3 and Z4, while slightly reducing Z2. On the other hand, the floury-2 mutant appears to reduce the synthesis of these six proteins in the same relative proportion. In the double mutant combinations, opaque-2 apparently is epistatic to opaque-7 and floury-2 in the synthesis of zein components. The glutelin fraction shows a more complex banding pattern; however, qualitative differences are not apparent among the mutant lines examined.This research was supported in part by a grant from the Lilly Endowment.Journal Paper No. 6100 of the Purdue University Agricultural Experimental Station.     

OsBC1L4 encodes a COBRA-like protein that affects cellulose synthesis in rice

Plant morphogenesis is highly dependent on the regulation of cell division and expansion. The organization of the cellulose microfibrils in the cell wall is a key determinant of cell expansion. Previously, a dwarf mutant with fewer tillers, Osbc1l4 (Oryza sativa brittle culm 1 like 4), was identified by screening a rice T-DNA insertion mutant library. It is reported here that OsBC1L4 encodes a COBRA-like protein that exhibits typical structural features of a glycosylphosphatidylinositol-anchor protein. The T-DNA insertion in OsBC1L4 results in abnormal cell expansion. A decrease in cellulose content but the increase in pectin and starch contents was identified in Osbc1l4 mutants by measuring the content of wall components. OsBC1L4 was expressed in all tissues/organs examined, with a low level in leaves. OsBC1L4 protein is mainly located in the cell wall and plasma membrane. Correlation analysis indicated that the expression of OsBC1L4 was highly correlated to that of several primary wall-forming cellulose synthase genes (CESAs). Moreover, the expression level of several cellulose-related genes is increased in Osbc1l4 mutants, which suggests that a feedback mechanism may exist during cellulose synthesis.     

OsPKpa_1 encodes a plastidic pyruvate kinase that affects starch biosynthesis in the rice endosperm

Pyruvate kinase(PK) is a key enzyme in glycolysis and carbon metabolism. Here, we isolated a rice(Oryza sativa) mutant, w59, with a white-core floury endosperm. Map-based cloning of w59 identified a mutation in OsPKpa_1, which encodes a plastidic isoform of PK(PKp). OsPKpa_1 localizes to the amyloplast stroma in the developing endosperm, and the mutation of OsPKpa_1 in w59 decreases the plastidic PK activity, resulting in dramatic changes to the lipid biosynthesis in seeds. The w59 grains were also characterized by a marked decrease in starch content. Consistent with a decrease in number and size of the w59 amyloplasts, large empty spaces were observed in the central region of the w59 endosperm, atthe early grain-filling stage. Moreover, a phylogenetic analysis revealed four potential rice isoforms of OsPKp. We validated the in vitro PK activity of these OsPKps through reconstituting active PKp complexes derived from inactive individual OsPKps, revealing the heteromeric structure of rice PKps, which was further confirmed using a protein–protein interaction analysis. These findings suggest a functional connection between lipid and starch synthesis in rice endosperm amyloplasts.     

spn-F encodes a novel protein that affects oocyte patterning and bristle morphology in Drosophila

The anteroposterior and dorsoventral axes of the Drosophila embryo are established during oogenesis through the activities of Gurken (Grk), a Tgfalpha-like protein, and the Epidermal growth factor receptor (Egfr). spn-F mutant females produce ventralized eggs similar to the phenotype produced by mutations in the grk-Egfr pathway. We found that the ventralization of the eggshell in spn-F mutants is due to defects in the localization and translation of grk mRNA during mid-oogenesis. Analysis of the microtubule network revealed defects in the organization of the microtubules around the oocyte nucleus. In addition, spn-F mutants have defective bristles. We cloned spn-F and found that it encodes a novel coiled-coil protein that localizes to the minus end of microtubules in the oocyte, and this localization requires the microtubule network and a Dynein heavy chain gene. We also show that Spn-F interacts directly with the Dynein light chain Ddlc-1. Our results show that we have identified a novel protein that affects oocyte axis determination and the organization of microtubules during Drosophila oogenesis.     

Identification of quantitative trait loci that affect endoreduplication in maize endosperm

Endoreduplication in maize endosperm precedes the onset of starch and storage protein synthesis, and it is generally thought to influence grain filling. We created four backcross populations by reciprocally crossing the F₁ progeny of a cross between Sg18 and Mo17 to the parental inbreds, which differ in endoreduplication by two parameters—mean ploidy and percentage of endoreduplicated nuclei. This four-backcross design allowed us to estimate and test the additive and dominant genetic effects of quantitative trait loci (QTLs) affecting endoreduplication. An analysis of endosperm from the four backcross populations at 16 days after pollination using a modified triploid mapping approach identified three endosperm QTLs influencing mean ploidy and two endosperm QTLs affecting the percentage of endoreduplicated nuclei. Some of these QTLs may manifest their effects on endoreduplication via expression in the embryo. The QTLs detected display strong dominance or over-dominance and interacted epistatically with an embryo-expressed QTL. This helps to explain the genetic basis for transgressive segregation in the backcross progeny. Although the favorable alleles that increase mean ploidy and percentage of endoreduplicated nuclei can be contributed by both parents, the Mo17-derived alleles for endoreduplication were often dominant or over-dominant to the Sg18-derived allele. One QTL on chromosome 7 that may be expressed in both the embryo and endosperm exerted a pleiotropic effect on two different parameters of endoreduplication. The results from this study shed light on the regulation of endoreduplication in maize endosperm and provide a marker-assisted selection strategy for potentially improving grain yield. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. C. M. Coelho and S. Wu contributed equally to this work and should be considered as first authors.     

Regulation of maize lysine metabolism and endosperm protein synthesis by opaque and floury mutations.

The capacity of two maize opaque endosperm mutants (o1 and o2) and two floury (fl1 and fl2) to accumulate lysine in the seed in relation to their wild type counterparts Oh43+ was examined. The highest total lysine content was 3.78% in the o2 mutant and the lowest 1.87% in fl1, as compared with the wild type (1.49%). For soluble lysine, o2 exhibited over a 700% increase, whilst for fl3 a 28% decrease was encountered, as compared with the wild type. In order to understand the mechanisms causing these large variations in both total and soluble lysine content, a quantitative and qualitative study of the N constituents of the endosperm has been carried out and data obtained for the total protein, nonprotein N, soluble amino acids, albumins/globulins, zeins and glutelins present in the seed of the mutants. Following two-dimensional PAGE separation, a total of 35 different forms of zein polypeptides were detected and considerable differences were noted between the five different lines. In addition, two enzymes of the aspartate biosynthetic pathway, aspartate kinase and homoserine dehydrogenase were analyzed with respect to feedback inhibition by lysine and threonine. The activities of the enzymes lysine 2-oxoglutate reductase and saccharopine dehydrogenase, both involved in lysine degradation in the maize endosperm were also determined and shown to be reduced several fold with the introduction of the o2, fl1 and fl2 mutations in the Oh43+ inbred line, whereas wild-type activity levels were verified in the Oh43o1 mutant.     

The maize gene maternal derepression of r1 encodes a DNA glycosylase that demethylates DNA and reduces siRNA expression in the endosperm

Demethylation of transposons can activate the expression of nearby genes and cause imprinted gene expression in the endosperm; this demethylation is hypothesized to lead to expression of transposon small interfering RNAs (siRNAs) that reinforce silencing in the next generation through transfer either into egg or embryo. Here we describe maize (Zea mays) maternal derepression of r1 (mdr1), which encodes a DNA glycosylase with homology to Arabidopsis thaliana DEMETER and which is partially responsible for demethylation of thousands of regions in endosperm. Instead of promoting siRNA expression in endosperm, MDR1 activity inhibits it. Methylation of most repetitive DNA elements in endosperm is not significantly affected by MDR1, with an exception of Helitrons. While maternally-expressed imprinted genes preferentially overlap with MDR1 demethylated regions, the majority of genes that overlap demethylated regions are not imprinted. Double mutant megagametophytes lacking both MDR1 and its close homolog DNG102 result in early seed failure, and double mutant microgametophytes fail pre-fertilization. These data establish DNA demethylation by glycosylases as essential in maize endosperm and pollen and suggest that neither transposon repression nor genomic imprinting is its main function in endosperm.

Demethylation by DNA glycosylases is important for endosperm development, but only a subset of the affected loci are imprinted, suggesting demethylation may have additional functions.

IN A NUTSHELL Background: In 1970, Jerry Kermicle reported that maize kernels could have dramatically different pigmentation depending on which parent the r1 gene is inherited from. This was the first discovery of many genomically imprinted genes that are selectively expressed from the maternal genome in endosperm. Later, Kermicle also discovered a mutant with poor maternal r1 expression. He hypothesized that the normal function of the mutated gene would be to derepress maternal r1; hence the name maternal depression of r1 (mdr1). The identify of mdr1 has remained unknown since then, but studies using Arabidopsis thaliana have revealed that DNA demethylation by enzymes called DNA glycosylases is important for expression of some maternally inherited genes in endosperm. Question: We wanted to identify the mdr1 gene. We hypothesized that mdr1 would reveal insights into molecular mechanisms of genomic imprinting in maize. Findings: We discovered that mdr1 encodes one of two DNA glycosylases with high expression in endosperm. We found that at least one of the two must be functional for endosperm to develop normally, but the one encoded by mdr1 is expressed higher. Surprisingly, most of the genes the mdr1 DNA glycosylase demethylates do not appear to be genomically imprinted, and about half the DNA it demethylates is not even near genes. These findings suggest that DNA glycosylases also have an undiscovered function unrelated to genomic imprinting in endosperm. Next steps: We want to know how specific regions in the genome are targeted for demethylation. What distinguishes these regions from other regions in endosperm? And what keeps them from being demethylated in other tissues? On the flip side, little is known about the effect of demethylation in endosperm, other than genomic imprinting. We want to know what effect DNA demethylation by DNA glycosylases has on chromatin structure and why it is important.     

The maize (Zea mays L.) roothairless 3 gene encodes a putative GPI-anchored, monocot-specific, COBRA-like protein that significantly affects grain yield

The rth3 ( roothairless 3 ) mutant is specifically affected in root hair elongation. We report here the cloning of the rth3 gene via a PCR-based strategy (amplification of insertion mutagenized sites) and demonstrate that it encodes a COBRA-like protein that displays all the structural features of a glycosylphosphatidylinositol anchor. Genes of the COBRA family are involved in various types of cell expansion and cell wall biosynthesis. The rth3 gene belongs to a monocot-specific clade of the COBRA gene family comprising two maize and two rice genes. While the rice ( Oryza sativa ) gene OsBC1L1 appears to be orthologous to rth3 based on sequence similarity (86% identity at the protein level) and maize/rice synteny, the maize ( Zea mays L.) rth3-like gene does not appear to be a functional homolog of rth3 based on their distinct expression profiles. Massively parallel signature sequencing analysis detected rth3 expression in all analyzed tissues, but at relatively low levels, with the most abundant expression in primary roots where the root hair phenotype is manifested. In situ hybridization experiments confine rth3 expression to root hair-forming epidermal cells and lateral root primordia. Remarkably, in replicated field trials involving near-isogenic lines, the rth3 mutant conferred significant losses in grain yield.