An Analysis of Seed Development in Pisum sativum: IV. COTYLEDON CELL POPULATIONS IN VIVO AND IN VITRO

A method for quantifying changes in the cell population of Pisumsativum cotyledons during development is described. The methodis based on determining the frequency distribution for cellarea following the random sampling of a single-cell suspensionof cotyledon cells. The population profile of these cells changedprogressively and systematically from a single population, similarin size to meristematic cells, found in embryos less than 3.0mg in fresh weight, to a bimodal population in embryos greaterthan 100 mg fresh weight. This method was used to compare embryosof similar size from two genotypes near-isogenic except forgenes at the r locus. No significant differences were foundbetween the cell population profiles of embryos up to 30 mgfresh weight. However, a significant difference was found betweenembryos with fresh weights of 100 mg, the wrinkled (rr) linehaving a higher mean and maximum cell area (2 951 µm²and 9 240 µm² respectively) than the round (RR) line (2591µm²and 6470 µm²respectively). Comparisons were alsomade between cotyledon cell populations from round (RR) embryosgrown in vivo and in vitro. The most obvious differences werethe higher mean and maximum cell size of the large cell populationof in vitro grown embryos which were twice those found in vivo.Embryos grown to either 30 mg or 100 mg fresh weight in vitrohad a much greater proportion of large cells in the populationwith a corresponding reduction in total cotyledon cell number,compared with similar sized embryos grown in vivo. These data suggest that comparisons between different genotypes,or, between cultured and in vivoembryos, based on morphologicalsimilarities between embryos, may be invalid and subject tomisinterpretation. Key words: Pea, seed development, cell population     

Mutant alleles at the rugosus loci in pea affect seed moisture sorption isotherms and the relations between seed longevity and moisture content

Pea (Pisum sativum L.) mutant near-isogenic lines (RRrbrb, rrRbRb, rrrbrb) with lower starch but higher lipid contents, brought about by lesions in the starch biosynthetic pathway, had seed moisture sorption isotherms displaced below that of the wild type (RRRbRb). The negative logarithmic relationship between seed longevity and seed storage moisture content (%, f.wt basis), determined in hermetic storage at 65 degrees C, also differed: longevity in the mutant near-isogenic lines was poorer and less sensitive to moisture content than in the wild type (i.e. C(W) was lower). The low-moisture-content limit (m(c)) to this relation also differed, being lower in the mutant near-isogenic lines (5.4-5.9%) than in the wild type (6.1%). In contrast, all four near-isogenic lines showed no difference (P >0.25) in the negative semi-logarithmic relationship between equilibrium relative humidity (ERH) and seed longevity. It is concluded that the effect of these alleles at the r and rb loci on seed longevity was largely indirect; a consequence of their effect on seed composition and hence on moisture sorption isotherms. However, this explanation could not be invoked at moisture contents below m(c) where differences in longevity remained substantial (RRRbRb double that of rrrbrb). Hence, these mutant alleles affected seed longevity directly at very low moisture contents.     

Comparative proteomic analysis of apomictic monosomic addition line of <Emphasis Type="Italic">Beta corolliflora</Emphasis> and <Emphasis Type="Italic">Beta vulgaris</Emphasis> L. in sugar beet

Apomixis refers to a process in which plants produce seed without fertilization through female syngamy that produces embryos genetically identical to the maternal parent. In sugar beet, interspecific hybrids between diploid Beta vulgaris and tetraploid Beta corolliflora were established and monosomic addition line M14 was selected because of the apomictic phenotype. By using two-dimensional electrophoresis gels we identified the proteins which were differently expressed between the M14 and B. vulgaris. A total of 27 protein spots which varied expressed between lines were isolated and successfully identified with MALDI-TOF MS. Among them five protein spots were found to be only presented in M14 and two protein spots only expressed in Beta. According to their functional annotations described in Swissprot database, these proteins were, respectively, involved in important biological pathways, such as cell division, functionally classified using the KEGG functional classification system. The result may be useful for us to better understand the genetic mechanism of apomixes.     

Water Stress and Foliar Boron Application Altered Cell Wall Boron and Seed Nutrition in Near-Isogenic Cotton Lines Expressing Fuzzy and Fuzzless Seed Phenotypes

Our previous research, conducted under well-watered conditions without fertilizer application, showed that fuzziness cottonseed trait resulted in cottonseed nutrition differences between fuzzy (F) and fuzzless (N) cottonseed. Under water stress conditions, B mobility is further limited, inhibiting B movement within the plant, affecting seed nutrition (quality). Therefore, we hypothesized that both foliar B and water stress can affect B mobility, altering cottonseed protein, oil, and mineral nutrition. The objective of the current research was to evaluate the effects of the fuzziness seed trait on boron (B) and seed nutrition under water stress and foliar B application using near-isogenic cotton lines (NILs) grown in a repeated greenhouse experiment. Plants were grown under-well watered conditions (The soil water potential was kept between -15 to -20 kPa, considered field capacity) and water stress conditions (soil water potential between -100 and -150 kPa, stressed conditions). Foliar B was applied at a rate of 1.8 kg B ha^-1 as H₃BO₃. Under well-watered conditions without B the concentrations of seed oil in N lines were higher than in F lines, and seed K and N levels were lower in N lines than in F lines. Concentrations of K, N, and B in leaves were higher in N lines than in F lines, opposing the trend in seeds. Water-stress resulted in higher seed protein concentrations, and the contribution of cell wall (structural) B to the total B exceeded 90%, supporting the structural role of B in plants. Foliar B application under well-watered conditions resulted in higher seed protein, oil, C, N, and B in only some lines. This research showed that cottonseed nutrition differences can occur due to seed fuzziness trait, and water stress and foliar B application can alter cottonseed nutrition.     

The reaction of near-isogenic lines of flax to the rust fungus Melampsora lini. II. Biochemical studies

The nature of resistance to rust fungus Melampsora lini (Pers.) Lev. was studied in two near-isogenic lines of flax, the susceptible Redwing (rr) and the immune S-66-53 (RR). Proteins of theses lines were immunologically and electrophoretically similar. Hydrolytic enzymes which release phenolic moities, i.e., -glucosidase and esterase(s), and phenol-oxidizing enzymes, i.e., peroxidase and polyphenol oxidase(s), did not appear to be involved in resistance or susceptibility as controlled by the R locus. Catalase activity was found to be higher in younger tissues of Redwing than in S-66-53 or their immune F₁ hybrid (Rr). Older tissues in both lines, whether inoculated or not, did not show any detectable catalase activity. A hypothesis to explain the genetic control of resistance and susceptibility in these lines before and after inoculation is suggested.This work has been supported by the National Research Council of Canada.     

Isoelectrofocusing Electrophoretic Analysis on Soluble Protein of Germinated Embryos and Young Shoots from Different Cytoplasmic Male Sterile Lines and Their Maintainers in Wheat

The soluble proteins of germinated embryos and young shoots in Wheat were studied by means of isoelectrofocusing (IEF) electrophoresis. The materials used were three species of cytoplasmic male-sterile (CMS) lines (Type E, T and A) and their maintainers. The protein quantity of pI 4. 9 in male-fertile materials is higher than the corresponding male-sterile ones; the protein of pI 6.85 is probably the result of gene expression in Timopheevi cytoplasm; the protein of pI 7.6 is a typical band of Jinfeng male-sterile line. Evident differences on soluble protein chromatograms of IEF electrophoresis were observed both in germinated embryos and young shoots which were from different kinds of CMS line, these differences could be used as indexes identifying various CMS lines.     

Analysis of proteome and frost tolerance in chromosome 5A and 5B reciprocal substitution lines between two winter wheats during long-term cold acclimation

Dynamics of cold tolerance and crown proteome composition has been analysed in a set of two winter wheat cultivars Mironovskaya 808 and Bezostaya 1 and four reciprocal substitution lines with interchanged chromosomes 5A and 5B during a long-term cold-acclimation (CA) treatment. Proteome analysis has revealed 298 differently abundant spots during experiment. Most of them (260) were changed due to CA process and only 52 spots displayed differences between genotypes. Two hundred and seven protein spots were successfully identified by tandem mass spectrometry. Comparison of samples before and after vernalization fulfillment by a combination of ANOVA and Student' T-test displayed ten differentially abundant protein spots (e.g. chopper chaperones). However, differences in the accumulation of these spots did not reflect differences in vernalization requirement of genotypes. Therefore, our results indicate that vernalization process has not influenced total proteome of CA wheat crowns. A few protein spots (14 spots; e.g. malate dehydrogenase) revealed differential accumulation levels between the individual genotypes or their groups possessing chromosome 5A or 5B from Mironovskaya 808 versus Bezostaya 1. The study has shown the effect of chromosome 5A on physiological traits and also proteome in winter wheat. Putative candidate protein markers for cold tolerance in wheat are discussed.     

Comparative proteomics analysis reveals the mechanism of fertility alternation of thermosensitive genic male sterile rice lines under low temperature inducement

Thermosensitive genic male sterile (TGMS) rice line has made great economical contributions in rice production. However, the fertility of TGMS rice line during hybrid seed production is frequently influenced by low temperature, thus leading to its fertility/sterility alteration and hybrid seed production failure. To understand the mechanism of fertility alternation under low temperature inducement, the extracted proteins from young panicles of two TGMS rice lines at the fertility alternation sensitivity stage were analyzed by 2DE. Eighty‐three protein spots were found to be significantly changed in abundance, and identified by MALDI‐TOF‐TOF MS. The identified proteins were involved in 16 metabolic pathways and cellular processes. The young panicles of TGMS rice line Zhu 1S possessed the lower ROS‐scavenging, indole‐3‐acetic acid level, soluble protein, and sugar contents as well as the faster anther wall disintegration than those of TGMS rice line Zhun S. All these major differences might result in that the former is more stable in fertility than the latter. Based on the majority of the 83 identified proteins, together with microstructural, physiological, and biochemical results, a possible fertile alteration mechanism in the young panicles of TGMS rice line under low temperature inducement was proposed. Such a result will help us in breeding TGMS rice lines and production of hybrid seed.     

Major differences in isoforms of starch-branching enzyme between developing embryos of round- and wrinkled-seeded peas (Pisum sativum L.)

In order to determine whether round-and wrinkled-seeded peas (Pisum sativum L.) differ in the activity and properties of starch-branching enzyme (1,4--D-glucan, 1,4--D-glucan-6-glycosyl transferase; EC 2.4.1.18) in their developing embryos, essentially isogenic lines of peas, differing only at the r (rugosus) locus that confers the round (RR, Rr) or wrinkled (rr) phenotype, were studied. Activity of the enzyme rises rapidly from an early stage of development in embryos of round peas, but only at later stages in embryos of wrinkled peas. The purified enzyme from mature embryos of round peas can be resolved into two isoforms that differ in molecular weight and in their ability to branch amylose. The purified enzyme from mature embryos of wrinkled peas is a single protein with the same molecular weight and branching properties as one of the isoforms from embryos of round peas. The difference in activity of starch-branching enzyme between embryos of round and wrinkled peas is likely to be due to the absence from embryos of wrinkled peas of one of the isoforms occurring in embryos of round peas.Abbreviations DEAE diethylaminoethyl - FW fresh weight - kDa kilodalton - SDS sodium dodecyl sulfate     

小麦不同细胞质雄性不育系及其保持系萌动胚及幼芽可溶性蛋白等电聚焦电泳分析

用等电聚焦电泳分析的方法,测定了小麦（Ｔｒｉｔｉｃｕｍ ａｅｓｔｉｖｕｍ ）３ 种细胞质雄性不育类型（Ａ 型、Ｅ型、Ｔ型）及其相应同核保持系萌动胚及幼芽可溶性蛋白． 发现雄性可育系等电点（ｐＩ）为４．９０ 的蛋白质合成数量高于相应的不育系;ｐＩ为６．８５ 的蛋白质可能是Ｔ 型细胞质基因表达的结果;ｐＩ为７．６ 的蛋白质可能为津丰Ａ 不育系特有的区带．表明细胞质来源不同的不育类型,其萌动胚及幼芽可溶性蛋白等电聚焦电泳图谱差异明显,有可能作为鉴别它们的依据     

The Impact of Chlorophyll-Retention Mutations,d1d2 and cyt-G1, during Embryogeny in Soybean

The ultrastructural, physiological, and molecular changes in developing and mature seeds were monitored in a control line (Glycine max [L.] Merr., cv Clark) that exhibited seed degreening and two mutant lines (d1d2 and cyt-G1) that retained chlorophyll upon seed maturation. Ultrastructural studies showed that the control line had no internal membranes, whereas stacked thylakoid membranes were detected in the green seed from the mutant lines. Pigment analyses indicated that total chlorophyll was lowest in the mature seeds of the control line. Mature d1d2 and cyt-G1 seed had elevated Chl a and Chl b levels, respectively. In both control and mutant lines, Lhcb1, Lhcb2, and RbcS mRNAs were abundant in embryos prior to cotyledon filling, declined after the onset of storage protein accumulation, and were barely detectable or undetectable in all later stages of seed development. Therefore, the chlorophyll-retention phenotype must be a result of the alteration of a process that occurs after translation of photosynthesis-related mRNAs to stabilize apoprotein and pigment levels. Furthermore, different elements controlling either the synthesis or turnover of Chl a and Chl b must be impaired in the d1d2 and cyt-G1 lines. No reproducible differences in total leaf, embryonic, and chloroplast protein profiles and plastid DNAs could be correlated with the mutations that induced chlorophyll retention.     

Genetics of hybrid incompatibility between Lycopersicon esculentum and L. hirsutum

We examined the genetics of hybrid incompatibility between two closely related diploid hermaphroditic plant species. Using a set of near-isogenic lines (NILs) representing 85% of the genome of the wild species Lycopersicon hirsutum (Solanum habrochaites) in the genetic background of the cultivated tomato L. esculentum (S. lycopersicum), we found that hybrid pollen and seed infertility are each based on 5-11 QTL that individually reduce hybrid fitness by 36-90%. Seed infertility QTL act additively or recessively, consistent with findings in other systems where incompatibility loci have largely been recessive. Genetic lengths of introgressed chromosomal segments explain little of the variation for hybrid incompatibility among NILs, arguing against an infinitesimal model of hybrid incompatibility and reinforcing our inference of a limited number of discrete incompatibility factors between these species. In addition, male (pollen) and other (seed) incompatibility factors are roughly comparable in number. The latter two findings contrast strongly with data from Drosophila where hybrid incompatibility can be highly polygenic and complex, and male sterility evolves substantially faster than female sterility or hybrid inviability. The observed differences between Lycopersicon and Drosophila might be due to differences in sex determination system, reproductive and mating biology, and/or the prevalence of sexual interactions such as sexual selection.     

Proteomic analysis of seed viability in maize

To identify specific proteins related to maize seed viability, seeds of Zhengdan 958 (one of the high-yield maize hybrids in China) were sorted based on viability evaluation with triphenyltetrazolium chloride (TTC) assay and used for comparative proteomic analysis. After TTC staining, embryos of high-viability seeds were deep red (R type), while embryos of dead seeds were white (W type). Proteomic analysis revealed that 28 protein spots identified were differently expressed significantly between R and W embryos, of which 20 were up-regulated and 8 down-regulated in R embryos. Among them were proteins involved in stress response, protein folding, and stabilization, as wells as proteins related to nutrient reservoir and metabolism. Prominently, small heat shock proteins, late embryogenesis abundant (LEA) proteins, and antioxidant enzymes were highly up-regulated, while two proteases were highly down-regulated in R embryos compared to W embryos. One of LEA proteins was EMB564, which declined in abundance during artificial aging of seeds. Our results suggested an association of EMB564 with maize seed viability. It would be of interest to use these small proteins to develop quick tests for seed quality.     

Combined metabolomic and genetic approaches reveal a link between the polyamine pathway and albumin 2 in developing pea seeds

Several legume seed proteins that are potentially allergenic, poorly digested by farm animals, and/or have undesirable functional properties, have been described. One of these is the albumin protein in pea (Pisum sativum) called PA2. A naturally occurring mutant line that lacks PA2 has been exploited in studies to determine the biological function of this nonstorage protein in seed development. The mutant, which has a small seed, a tall plant phenotype, and lacks most of the PA2-encoding genes, has been crossed with a standard cultivar, 'Birte,' which contains PA2 to give rise to a recombinant inbred (RI) population. An F(3) line carrying the mutation and having a short plant phenotype has been used to generate backcross (BC) lines with 'Birte.' Despite having a lower albumin content, seeds from the mutant parent and RI lines lacking PA2 have an equivalent or higher seed nitrogen content. Metabolite profiling of seeds revealed major differences in amino acid composition and polyamine content in the two parent lines. This was investigated further in BC lines, where the effects of differences in seed size and plant height between the two parents were eliminated. Here, differences in polyamine synthesis were maintained as was a difference in total seed protein between the BC line lacking PA2 and 'Birte.' Analysis of enzyme activities in the pathways of polyamine synthesis revealed that the differences in spermidine content were attributable to changes in the overall activities of spermidine synthase and arginine decarboxylase. Although the genes encoding spermidine synthase and PA2 both localized to the pea linkage group I, the two loci were shown not to be closely linked and to have recombined in the BC lines. A distinct locus on linkage group III contains a gene that is related to PA2 but expressed predominantly in flowers. The results provide evidence for a role of PA2 in regulating polyamine metabolism, which has important functions in development, metabolism, and stress responses in plants.     

An Analysis of Seed Development in Pisum sativum II. The Effect of the r-Locus on the Growth and Development of the Seed

The r-locus is one of the few genetic loci known to affect thestorage product composition and the morphology of pea (Pisumsativum) seeds. Lines which are near-isogenic except for ther-locus have been developed to study the effects of this locuson seed development. The plant phenotypes of these lines werevery similar except for those characteristics previously attributedto the r-locus. The seed development of the two lines followedsimilar patterns until the endosperm was absorbed by the embryo.The fresh weight of the rr line then increased more rapidlydue to the overall effect of a higher rate of water uptake anda lower rate of dry weight increase of the rr embryos comparedwith embryos homozygous for the R-allele. The effect of the r-locus on the relationship between embryofresh weight and dry weight suggests that the alleles may beaffecting the osmotic regulation of the developing embryo. Pisum sativum, pea, seed development, r-locus, genetic variation, growth analysis     

AAP1 regulates import of amino acids into developing Arabidopsis embryos

The embryo of Arabidopsis seeds is symplasmically isolated from the surrounding seed coat and endosperm, and uptake of nutrients from the seed apoplast is required for embryo growth and storage reserve accumulation. With the aim of understanding the importance of nitrogen (N) uptake into developing embryos, we analysed two mutants of AAP1 (At1g58360), an amino acid transporter that was localized to Arabidopsis embryos. In mature and desiccated aap1 seeds the total N and carbon content was reduced while the total free amino acid levels were strongly increased. Separately analysed embryos and seed coats/endosperm of mature seeds showed that the elevated amounts in amino acids were caused by an accumulation in the seed coat/endosperm, demonstrating that a decrease in uptake of amino acids by the aap1 embryo affects the N pool in the seed coat/endosperm. Also, the number of protein bodies was increased in the aap1 endosperm, suggesting that the accumulation of free amino acids triggered protein synthesis. Analysis of seed storage compounds revealed that the total fatty acid content was unchanged in aap1 seeds, but storage protein levels were decreased. Expression analysis of genes of seed N transport, metabolism and storage was in agreement with the biochemical data. In addition, seed weight, as well as total silique and seed number, was reduced in the mutants. Together, these results demonstrate that seed protein synthesis and seed weight is dependent on N availability and that AAP1-mediated uptake of amino acids by the embryo is important for storage protein synthesis and seed yield.