Characterization and Complexity of Wheat Developing Endosperm mRNAs

Free and membrane-bound (MB) polysomes and the corresponding polyadenylated RNAs (polyA⁺ RNAs) have been isolated from developing wheat endosperm (Triticum aestivum L.) Free and MB poly(A)⁺ RNAs, analyzed on isokinetic sucrose gradient with [³H]polyuridylic acid [poly(U)] hybridization detection, appear to be 11S to 12S in size with a 7% poly(A) tail for MB RNAs. cDNAs synthesized using both of these mRNA populations in presence of a potent RNase inhibitor (RNasin), have been used for hybridization kinetics experiments. The mean square fitting analysis of the hybridization kinetics between MB cDNA and its template reveals the presence of two abundance classes representing roughly ⅔ and ⅓ of the MB poly(A)⁺ RNAs and containing the information for approximately 75 superabundant species (21,000 copies per cell) and 750 intermediate species (530 copies per cell), respectively. The mRNA population extracted from free polysomes is divided into three abundance classes. The first one is composed of superabundant sequences which would correspond to the MB superabundant mRNAs. The free mRNAs consist of about 11,000 diverse sequences, most of them being rare sequences. Heterologous hybridizations of MB cDNAs to free mRNAs have shown that some mRNAs are common to both populations. This could be explained either by a partial contamination or by free polysomes en route to their membrane destination. Contrary to the low number of diverse mRNAs corresponding to the legume seed storage proteins, the wheat endosperm superabundant mRNAs consist of about 75 different sequences which would encode most of the seed storage proteins, especially gliadins.     

Proteome analysis of embryo and endosperm from germinating tomato seeds

Proteome analysis of embryo and endosperm tissues from germinating tomato seed was conducted using 1-DE, 2-DE, and MS. Mobilization of the most abundant proteins, which showed similar profiles in the two tissues, occurred first in the endosperm. CBB R-250 staining of 2-DE gels revealed 352 and 369 major protein spots in the embryo and endosperm, respectively, at 0 h. Of these, 75 major spots were selected, excised, in-gel digested with trypsin, and analyzed by MALDI-TOF-MS and/or LC-ESI-Q/TOF-MS/MS. Peptide MS and MS/MS data were searched against publicly available protein and EST databases, and 47 proteins identified. Embryo-specific proteins included a BAC19.13 homologue, whereas four proteins specific to the endosperm were tomato mosaic virus coat proteins related to defense mechanisms. The most abundant proteins both in the embryo and endosperm were seed storage proteins, i.e., legumins (11 spots), vicilins (11 spots), albumin (2 spots). Housekeeping enzymes, actin-binding profilin, defense-related protein kinases, nonspecific lipid transfer protein, and proteins involved in general metabolism were also identified. The roles of some of the proteins identified in the embryo and endosperm are discussed in relation to seed germination in tomato.     

Characterization and cloning of rat dorsal prostate mRNAs. Androgen regulation of two closely related abundant mRNAs

Electrophoresis of rat dorsal prostate mRNAs on agarose gels containing methyl mercury hydroxide indicates the presence of several highly abundant mRNAs. In vitro translation of the total mRNAs in a cell-free system, followed by polyacrylamide gel electrophoresis, yields protein products including two intense bands corresponding to 23,000 and 21,000 Da. Following castration of rats, these in vitro translation products of dorsal prostate mRNAs are absent. However, the dorsal prostate levels of these two proteins are returned to normal in castrated rats which have received testosterone. In order to investigate these abundant mRNAs of the dorsal prostate, we have constructed double-stranded cDNA clones using poly(A+) RNA extracted from that rat tissue. Clones containing sequences complementary to abundant mRNAs were selected kinetically by colony hybridization with 32P-labeled dorsal prostate cDNA. Further characterization of individual clones was accomplished by restriction mapping and Northern blot analysis. One clone, pM-40, was found to be near full length and was used for further studies. Interestingly, in hybrid-arrested cell-free translation, clone pM-40 completely arrests the translation of both the 23,000- and 21,000-Da protein products indicating close sequence homology between these two proteins. Furthermore, dot hybridization experiments demonstrate that, in the dorsal prostate, the pM-40-specific mRNAs decrease following castration and are restored by testosterone administration. However, the low levels of the same mRNAs in the ventral prostate are not altered by androgen manipulation. Thus, two closely related, androgen-dependent tissues maintain differential regulation of the pM-40 gene(s). This system provides an opportunity to study in two tissues the differential regulation of a gene that may be duplicated or that may code for two separate proteins.     

Class I beta-1,3-glucanase and chitinase are expressed in the micropylar endosperm of tomato seeds prior to radicle emergence.

beta-1,3-Glucanase (EC 3.2.1.39) and chitinase (EC 3.2.1.14) mRNAs, proteins, and enzyme activities were expressed specifically in the micropylar tissues of imbibed tomato (Lycopersicon esculentum Mill.) seeds prior to radicle emergence. RNA hybridization and immunoblotting demonstrated that both enzymes were class I basic isoforms. beta-1,3-Glucanase was expressed exclusively in the endosperm cap tissue, whereas chitinase localized to both endosperm cap and radicle tip tissues. beta-1,3-Glucanase and chitinase appeared in the micropylar tissues of gibberellin-deficient gib-1 tomato seeds only when supplied with gibberellin. Accumulation of beta-1,3-glucanase mRNA, protein and enzyme activity was reduced by 100 microM abscisic acid, which delayed or prevented radicle emergence but not endosperm cap weakening. In contrast, expression of chitinase mRNA, protein, and enzyme activity was not affected by abscisic acid. Neither of these enzymes significantly hydrolyzed isolated tomato endosperm cap cell walls. Although both beta-1,3-glucanase and chitinase were expressed in tomato endosperm cap tissue prior to radicle emergence, we found no evidence that they were directly involved in cell wall modification or tissue weakening. Possible functions of these hydrolases during tomato seed germination are discussed.     

A Comparative Analysis of Embryo and Endosperm Proteome from Seeds of Jatropha curcas

Jatropha curcas is an important economic plant for biodiesel, which is extracted mainly from the endosperm of its mature seeds. Despite the morphological and functional differences between the embryo and endosperm, proteomic characteristics of the two tissues are not yet known. Similar proteomic profiles were observed in the two-dimensional gel electrophoresis maps from the two tissues. There were 380 and 533 major protein spots in the embryo and endosperm, respectively. Fourteen identical spots, showing a notable change, were selected and identified by tandem mass spectrometry. Among these proteins, dihydrolipoamide acetyltransferase (spot 27) participates in tricarboxylic acid cycle, which is an amphibolic pathway. The two parts both included proteins related to stress (spots 8, 115, 118, 125, 130) and signal transduction (spots 7, 100, 108). According to the volume percentage of proteins in embryo and endosperm, the proteins in endosperm (spots 54, 61, 73) were catabolism-related enzymes and reserves to provide the nutrition for seed germination; the proteins in embryo (spots 27, 62, 122) were inclined to anabolism and utilized the nutrition from the endosperm to generate a new life.     

小麦种子基因的表达序列标签分析

以授粉后12 d的小麦(Triticum aestivum L.)种子为材料,构建起cDNA文库.从中随机挑选10 000个克隆,利用Biomek 2000核酸工作站制成高密度cDNA阵列.然后分别以未受精子房、胚和胚乳中提取的RNA为模板,反转录合成探针与膜杂交,进行差示筛选.根据筛选结果,选取800个在胚、胚乳或胚和胚乳中表达的克隆进行表达序列标签(EST)分析,鉴定出216个不同的基因序列.其中24个ESTs属于已知的小麦基因;122个ESTs为推测的小麦新基因,它们编码的产物与种子贮藏蛋白或与生化代谢、发育等其他的生物学过程有关;70个ESTs的序列特征尚未确定.本研究为研究种子发育和小麦品质改良等提供了基础资料.     

Heterosis in early seed development: a comparative study of F1 embryo and endosperm tissues 6 days after fertilization

Heterosis specifies the superior performance of heterozygous individuals and although used in plant breeding the underlying molecular mechanisms still remain largely elusive. In this study, we demonstrate the manifestation of heterosis in hybrid maize embryo and endosperm tissue 6 days after fertilization in crosses of several inbred lines. We provide a comparative analysis of heterosis-associated gene expression in these tissues by a combined approach of suppression subtractive hybridization and microarray hybridizations. Non-additive expression pattern indicated a trans-regulatory mechanism to act early after fertilization in hybrid embryo and endosperm although the majority of genes showed mid-parental expression levels in embryo and dosage dependent expression levels in endosperm. The consistent expression pattern within both tissues and both inbred line genotype combinations of genes coding for chromatin related proteins pointed to heterosis-related epigenetic processes. These and genes involved in other biological processes, identified in this study, might provide entry points for the investigation of regulatory networks associated with the specification of heterosis.     

Biosynthesis of nonspecific lipid transfer proteins in germinating castor bean seeds

The biosynthesis of nonspecific lipid transfer proteins (ns-LTPs) in germinating castor bean (Ricinus communis L.) seeds were investigated. Lipid transfer activities of ns-LTPs in the cotyledons, axis, and endosperm increased with growth after germination. The activity increases were accompanied by increased amounts of ns-LTPs in each tissue, as measured by immunoblot using anti-ns-LTP serum. These results suggest that the ns-LTPs are synthesized de novo in each tissue after germination and not activated from inactive proteins synthesized before germination. Comparison of the immunoblot products in each tissue from 4-day-old seedlings indicate the occurrence of tissue-specific isoforms of ns-LTPs; 9 kilodaltons (major) and 7 kilodaltons (minor) in the cotyledons, and 7 kilodaltons (major) and 9 kilodaltons (minor) in the axis, whereas only the 8-kilodalton ns-LTP is present in the endosperm. In vitro translation from poly(A)⁺ RNAs from three tissues of castor bean seedlings and the detection of immunoprecipitated products indicate that translatable mRNAs for ns-LTPs exist in the three tissues a day before the synthesis of ns-LTPs; the translation products, which are 3.5 to 4.0 kilodaltons larger than ns-LTPs, were processed to the mature ns-LTPs. The production of mature ns-LTPs from translatable mRNAs without any delay suggests that gene expression of ns-LTPs in castor bean seedlings is controlled at a step before the formation of translatable mRNAs.     

A genome-wide survey of imprinted genes in rice seeds reveals imprinting primarily occurs in the endosperm

Genomic imprinting causes the expression of an allele depending on its parental origin. In plants, most imprinted genes have been identified in Arabidopsis endosperm, a transient structure consumed by the embryo during seed formation. We identified imprinted genes in rice seed where both the endosperm and embryo are present at seed maturity. RNA was extracted from embryos and endosperm of seeds obtained from reciprocal crosses between two subspecies Nipponbare (Japonica rice) and 93-11 (Indica rice). Sequenced reads from cDNA libraries were aligned to their respective parental genomes using single-nucleotide polymorphisms (SNPs). Reads across SNPs enabled derivation of parental expression bias ratios. A continuum of parental expression bias states was observed. Statistical analyses indicated 262 candidate imprinted loci in the endosperm and three in the embryo (168 genic and 97 non-genic). Fifty-six of the 67 loci investigated were confirmed to be imprinted in the seed. Imprinted loci are not clustered in the rice genome as found in mammals. All of these imprinted loci were expressed in the endosperm, and one of these was also imprinted in the embryo, confirming that in both rice and Arabidopsis imprinted expression is primarily confined to the endosperm. Some rice imprinted genes were also expressed in vegetative tissues, indicating that they have additional roles in plant growth. Comparison of candidate imprinted genes found in rice with imprinted candidate loci obtained from genome-wide surveys of imprinted genes in Arabidopsis to date shows a low degree of conservation, suggesting that imprinting has evolved independently in eudicots and monocots.     

Endosperm-specific demethylation and activation of specific alleles of α-tubulin genes of Zea mays L.

We have investigated the methylation status of the α-tubulin genes, and the degree of accumulation of their mRNAs in endosperm, embryo and seedling tissues of Zea mays L. We have found that many of the α-tubulin genes are differentially demethylated in the endosperm relative to the embryo and seedling. However, only for tubα2 and tubα4 could a correlation between DNA demethylation and increased RNA accumulation be detected. By analyzing the inbred lines W64A and A69Y and their reciprocal crosses, we have also identified in the endosperm two α-tubulin genes, tubα3 and tubα4, that are differentially demethylated if transmitted by the maternal germline, but that remain hypermethylated when transmitted by the paternal germline.     

Polyadenylation state of abundant mRNAs during Drosophila development

We have used a two-dimensional gel analysis of cell-free translation products to determine whether individual mRNAs present in Drosophila melanogaster embryos, larvae, pupae, and adults are predominantly polyadenylated or nonadenylated. While the majority of the embryonic mRNAs we detected exist mainly in the polyadenylated form, these mRNAs become more evenly distributed between the poly(A)+ and poly(A)- RNA fractions during postembryonic development. Although DNA:RNA hybridization experiments have indicated that Drosophila RNA populations contain a large group of rare class mRNAs restricted to the poly(A)- RNA compartment, this is not true for the 150 more abundant mRNA species analyzed by our methods. The histone mRNAs are the only abundant mRNA species which appear to be exclusively in the poly(A)- RNA class.