Contrasting patterns in the spread of two seed-borne viruses in pea embryos

The temporal and spatial patterns of pea seed-borne mosaic potyvirus (PSbMV) and pea early browning tobra-virus (PEBV) accumulation in pea embryos were analyzed using in-situ hybridization and immunohistochemistry. For PSbMV, which infects embryos after fertilization, the distribution changed as the embryo developed and some tissues remained free of virus infection. In contrast, embryos were uniformly infected with PEBV from the earliest stages of embryo development, and PEBV was detected in the egg cell and pollen grains, indicative of gametic transmission into the embryo. These observations suggest that gametically transmitted viruses may be appropriate as potential vectors for the ectopic and uniform expression of novel genes in embryonic tissues. Functional complementarity in the two processes of embryo invasion was tested following co-inoculation with PSbMV and PEBV. Instead of complementation, interference in PSbMV seed transmission by PEBV was observed; PEBV seed transmission remained unaffected by PSbMV.     

Pea seed-borne mosaic virus seed transmission exploits novel symplastic pathways to infect the pea embryo and is, in part, dependent upon chance

Summary. Seed transmission of pea seed-borne mosaic virus (PSbMV) depends upon symplastic transport of the virus from infected maternal cells to the embryo. Such transport pathways have not been identified in higher plants. To identify these pathways, we have studied the ultrastructure of the tissues and cells around the micropyle of young developing seeds and compared transmitted and nontransmitted virus isolates. A characteristic of PSbMV infection was the presence of cylindrical inclusions positioned over plasmodesmal openings. The presence of cylindrical inclusions on the testa–endosperm boundary wall, together with immunogold labelling for virus-specific products on the wall and in the endosperm, indicated that symplastic connections existed at this interface. Close examination of the endosperm–suspensor boundary at the base of the suspensor revealed discontinuities in the suspensor sheath wall as porelike structures, which the virus might pass through en route to the embryo. A nontransmitted PSbMV isolate was able to invade the maternal tissues of the developing seed but was excluded from the embryo, although it was detected at a low level in the endosperm. Since the endosperm did not support virus replication, it appeared that passive accumulation determined the amount, timing, and location of the virus relative to the base of the suspensor. Rarely, therefore, could the nontransmitted virus isolate reach the correct location in the endosperm at the correct time for embryo infection via the suspensor to occur.Present address: Institute of Genetics, Chinese Academy of Sciences, Beijing, People's Republic of China.Correspondence and reprints: Department of Disease and Stress Biology, John Innes Centre, Norwich Research Park, Colney, Norwich NR4 7UH, United Kingdom.Received January 7, 2003; accepted May 19, 2003; published online September 23, 2003     

Desiccation Alters the Stability and Distribution of Pea Seed-borne Mosaic Virus in Pea (Pisum sativum) Cotyledon Cells

As a seed transmitted pathogen, pea seed-borne mosaic vires (PSbMV) not only replicates in embryonic cells but can also withstand seed desiccation. To understand the mechanism of PSbMV tolerance to seed desiccation, the authors compared the stability of viral coat protein (CP) and the distribution of viral particles in the cotyledon cells of pea ( Pisum sativum L. ) embryos collected before and after the dehydration process. Before dehydration, when the embryo was fresh and immature, degradation of CP was observed and a predominantly perinuclear distribution of viral particles in the cotyledon cells was evident. After dehydration, when the embryo was dry and mature, degradation of CP did not occur and the perinuclear viral distribution disappeared. Instead, aggregates containing PSbMV CP were found in the cytoplasm. Electron microscopy showed that these aggregates were composed of PSbMV particles. The formation of PSbMV particle aggregates is apparently triggered by seed dehydration and may be favorable to the virus survival in the desiccated embryonic cells.     

An eclipse of pea seed-borne mosaic virus in vegetative tissue of pea following repeated transmission through the seed

Four isolates of pea seed-borne mosaic virus (PSbMV) representing pathotypes P1 (isolates US and Q) and P4 (isolates S4 and S6), and groups III (US and Q) and V (S4 and S6) have been used in a study of the survival and partitioning of PSbMV under conditions of continuous seed transmission in the commercial pea cultivar Dundale. Assays suitable for detecting virus in small tissue samples were developed, and included dot-immunobinding assay with antisera to both PSbMV and cytoplasmic inclusion body (CIB) protein, and dot hybridisation assay (DHA) with cDNA transcribed from virus RNA. Under the conditions of our experiments, seed transmission occurred at rates exceeding 90% for all virus isolates. Virus was detectable by serology and symptoms in inoculated plants, and in all vegetative tissue of second generation plants raised from seed of the inoculated plants. However, in the third, fourth and fifth sequential generations raised from seed, all plants were symptomless. Neither virus nor CIB were detectable in leaf, stem or roots by serology, but both were readily detectable in some floral parts, and in immature and mature seed. Mature seed contained virus and CIB antigen in the testa, cotyledon and embryo. Inoculum prepared from whole seeds was infectious. The testa was shown not to be involved in transmission between generations, thus implicating the embryo alone in vertical transmission. Virus antigen could not be detected in the emerging cotyledons of germinating seed and all true leaves by serology, but the leaves contained PSbMV RNA detectable by DHA. These results show that PSbMV infection can be transferred through the vegetative phase at a subliminal level, and reaches relatively high concentrations in floral parts and seeds. Thus PSbMV may be maintained at a high level of infection in seed in the absence of any apparent symptoms in the plant, and without a requirement for horizontal transmission between plants by vectors. Such a mechanism may explain the high levels of infection commonly reported in pea breeding lines.     

种子脱水改变豌豆种传花叶病毒在子叶细胞中的稳定性与分布方式

作为种传病原物,豌豆种传花叶病毒（ＰＳｂＭＶ） 必须能承受种胚的干燥脱水过程方能在胚细胞中存活并种传。为了研究ＰＳｂＭＶ承受种胚干燥脱水的机制,比较了该病毒在豌豆（ Ｐｉｓｕｍ ｓａｔｉｖｕｍ Ｌ．） 新鲜胚与干燥胚子叶细胞中的稳定性与分布方式。在新鲜的、未成熟胚的子叶细胞中,ＰＳｂＭＶ的外壳蛋白（ＣＰ） 受到部分降解,该病毒粒体及其ＣＰ在细胞质内呈环核分布。在干燥、成熟的胚的子叶细胞中,ＰＳｂＭＶ的外壳蛋白未受到任何降解,其粒体和ＣＰ不再呈环核分布,而是存在于位于细胞质边缘的多聚体中。免疫金标记电镜检查证明这类多聚体中含有ＰＳｂＭＶ的粒体。很明显,种胚的干燥脱水过程可改变ＰＳｂＭＶ在子叶细胞中的稳定性与分布方式,粒体多聚体的形成可能有助于ＰＳｂＭＶ在干燥脱水的胚细胞中的稳定与存活     

Narrow Bottlenecks Affect Pea Seedborne Mosaic Virus Populations during Vertical Seed Transmission but not during Leaf Colonization

The effective size of populations (Ne) determines whether selection or genetic drift is the predominant force shaping their genetic structure and evolution. Populations having high Ne adapt faster, as selection acts more intensely, than populations having low Ne, where random effects of genetic drift dominate. Estimating Ne for various steps of plant virus life cycle has been the focus of several studies in the last decade, but no estimates are available for the vertical transmission of plant viruses, although virus seed transmission is economically significant in at least 18% of plant viruses in at least one plant species. Here we study the co-dynamics of two variants of Pea seedborne mosaic virus (PSbMV) colonizing leaves of pea plants (Pisum sativum L.) during the whole flowering period, and their subsequent transmission to plant progeny through seeds. Whereas classical estimators of Ne could be used for leaf infection at the systemic level, as virus variants were equally competitive, dedicated stochastic models were needed to estimate Ne during vertical transmission. Very little genetic drift was observed during the infection of apical leaves, with Ne values ranging from 59 to 216. In contrast, a very drastic genetic drift was observed during vertical transmission, with an average number of infectious virus particles contributing to the infection of a seedling from an infected mother plant close to one. A simple model of vertical transmission, assuming a cumulative action of virus infectious particles and a virus density threshold required for vertical transmission to occur fitted the experimental data very satisfactorily. This study reveals that vertically-transmitted viruses endure bottlenecks as narrow as those imposed by horizontal transmission. These bottlenecks are likely to slow down virus adaptation and could decrease virus fitness and virulence.     

Improvements in the detection of pea seed-borne mosaic virus by ELISA

The detection by ELISA of pea seed-borne mosaic virus (PSbMV) in pea leaves and seeds was improved by the addition of cellulase or Triton X-100 to the extraction fluid, probably because the additives aided the release of virus particles from host materials. With leaf extracts the additives were most effective at 0.1%. In initial tests cellulase was used with macerozyme, but the latter enzyme was then shown to decrease the effectiveness of cellulase. Triton X-100 was as effective as cellulase and the absorbance values obtained in ELISA of infected leaf extracts, diluted to 1/10 in extraction fluid containing the additive, were about six times greater than those of infected extracts diluted in normal extraction fluid. Five named isolates of PSbMV, in addition to the homologous isolate, were readily detected in infected leaves extracted in fluid containing Triton X-100. In tests on seeds and seedlings of seven infected seed lots of pea cv. Waverex, using Triton X-100 in the extraction fluid, PSbMV was detected in five times as many seeds as seedlings, probably mainly because in many infected seeds the virus was in the testa and not in the embryo. About 9% of infected seedlings were without recognisable symptoms 4 wk after emergence.     

Morphology and Cytology of Pea Embryos During Histogenesis

Cytological examination showed that day 3 pea embryo cells wereundifferentiated in terms of morphological or gross cytologicalappearance. Histogenesis had commenced by day 4 and was accompaniedby cytological differentiation with a gradient in vacuolationapparent along the root/shoot axis. Day 3 embryonic cells werecytologically different from meristematic (day 4 and 5) cellsof the shoot apex. Cells of the embryo base appeared to havean intimate association with the middle suspensor cells. Pisum sativum L. cv. ‘Alaska’, pea, morphology, cytology, histogenesis, development     

Studies on resistance of pea to pea seed borne mosaic virus and new pathotypes

Studies were carried out to search for virulent pathotypes of pea seed borne mosaic virus (PSbMV) on pea and to explore new sources of resistance in French and Indian pea collections. A virulent pathotype, PSbMV-Pi, capable of partially overcoming the recessively resistant gene sbm 1, was identified for the first time in an Indian pea line. PSbMV-Pi did not produce visible symptoms on sbm 1 lines, on which it had a reduced multiplication and could no longer be detected by ELISA seven wk after inoculation. However, it multiplied normally on the susceptible cultivars and could be differentiated from other strains on a set of strain differentials. Also, another strain, PSbMV-Pv, of the virulent pathotype, that appeared to multiply slightly better than PSbMV-Pi on sbm 1 lines, was recovered from the local strain of PSbMV.
Five new sources of resistance to PSbMV were screened from the Indian pea collection by a rapid screening based on the assumption that, in germplasm collections, the virus is generalised to all susceptible lines by aphid vectors. The initial step of testing a few testas of each germplasm line in ELISA and the rejection of positive lines eliminated 85% of the germplasm before further testing in the field. The inheritance studies on four of the five resistant lines lead to reidentification of the sbm 1 gene. The sbm 1 lines behaved fairly well under heavy inoculum pressure in the pea fields in 1989.     

In silico identification and characterization of putative differentially expressed genes involved in common bean (<Emphasis Type="Italic">Phaseolus vulgaris</Emphasis> L.) seed development

Two genotypes of common bean (Phaseolus vulgaris L.) were studied to determine the structural cause of seed abortion in this species. In the non-abortive control (wild-type, cultivar BAT93), the histological analysis revealed a classical pattern of seed development and showed coordinated differentiation of the embryo proper, suspensor, endosperm tissue and seed coat. In contrast, the ethyl methanesulfonate (EMS) mutant (cultivar BAT93) showed disruption in the normal seed development leading to embryo abortion. Aborted embryos from these degenerate seeds showed abnormalities in suspensor and cotyledons at the globular, heart, torpedo and cotyledon stages. Exploring the feasibility of incorporating the available online bioinformatics databases, we identified 22 genes revealing high homology with genes involved in Arabidopsis thaliana embryo development and expressed in common bean immature seeds. The expression patterns of these genes were confirmed by RT–PCR. All genes were highly expressed in seed tissues. To study the expression profiles of isolated genes during Phaseolus embryogenesis, six selected genes were examined by quantitative RT–PCR analysis on the developing embryos of wild-type and EMS mutant plants. All selected genes were expressed differentially at different stages of embryo development. These results could help to improve understanding of the mechanism of common bean embryogenesis.     

Maternal,Single-Gene Regulation of Assimilate Partitioning in Pea

Assimilate partitioning has been identified as a key process in the control of yield. Although the role of reproductive structures in this process has received intensive study, our understanding of the role of the maternal plant is limited. We suggest that the Sn gene of pea (Pisum sativum L.) is a potentially valuable genetic tool for studying maternal regulation of partitioning. In this study, nearly isogenic lines differing at the Sn locus were compared with respect to seed-filling characteristics and carbon assimilation. Lines with the Sn gene had a slower rate and shorter duration of seed growth than the line recessive for this gene, and these traits could not be ascribed to reduced carbon assimilation. Flowers of the two nearly isogenic lines were manually pollinated to control the genotype of the developing embryo independently of the maternal genotype. The final dry weight of the seed was determined by the genotype of the maternal plant and not by the genotype of the embryo, supporting the hypothesis that the Sn gene acts in the vegetative plant to regulate the partitioning of assimilates between vegetative and reproductive growth. Although the Sn gene has been noted for delaying apical senescence, it also delayed leaf senescence in this study; leaves of the Sn line continued to photosynthesize long past the time that leaves of the recessive line had senesced and after the seeds and pods were dry.     

EMBRYO GROWTH AND SEED SIZE IN RAPHANUS SATIVUS: MATERNAL AND PATERNAL EFFECTS IN VIVO AND IN VITRO

Theories on the evolution of the angiosperm seed disagree as to the effects of different plant tissues on embryo growth. To examine the relative contributions of maternal and paternal genes on embryo growth, we conducted controlled crosses in the greenhouse with wild radish plants (Raphanus sativus), looked for maternal, paternal, and interaction effects on embryo development, and compared the performance of embryos within fruits and in embryo culture. Maternal plant identity affected fruit set, seeds per fruit, embryo developmental stage, and mean seed weight. In embryo culture, maternal effects were found for cotyledon size and embryo weight. Paternal effects were fewer or smaller in magnitude than maternal effects. The identity of the pollen donor affected embryo developmental stage and mean seed weight. In culture, paternal effects were detected for cotyledon size and embryo weight. Our results demonstrate that both maternal and paternal elements affect embryo growth. The fact that maternal effects are greater than paternal effects on embryo development in culture may result from cytoplasmic elements or maternal nuclear genes. Embryo performance in vivo compared to that in vitro varied among maternal plants. The interaction between an embryo and its endosperm and maternal tissues may be either positive or negative, depending upon the maternal plant and the embryo's developmental stage.     

Embryo development in the lady's slipper orchid, Paphiopedilum delenatii, with emphasis on the ultrastructure of the suspensor

BACKGROUND AND AIMS: Owing to large-scale collecting, the lady's slipper orchid, Paphiopedilum delenatii, is under threat of extinction. Asymbiotic germination provides a useful way to re-establish plants in the wild and for commercial propagation. A detailed study of embryo development would provide information on subsequent germination events and aid in the propagation of the species. METHODS: Developing capsules were collected for histochemical and ultrastructural studies by using both light and transmission electron microscopy. KEY RESULTS: The suspensor of this species consists of three vacuolated cells. During the early globular stage of embryo development, structural differentiation occurs, revealing an abundance of smooth endoplasmic reticulum cisternae and wall ingrowths within the suspensor cells. These features are not present in cells of the embryo proper. Furthermore, the results of Nile red staining demonstrate that a cuticular layer is present only in the embryo proper, but absent from the suspensor. Cuticular material is also present in the inner walls of the seed coat, and persists through seed maturation. CONCLUSIONS: The morphological features of the transfer cell and the absence of cuticular material in the suspensor cell wall corroborate the hypothesis that the suspensor is the major nutrient uptake site for the developing embryo in the lady's slipper orchid. The absence of an endosperm and presence of cuticular material in the inner walls of the seed coat enclosing the embryo proper further support the notion that nutrient uptake by the embryo is confined to the micropylar end of the seed through the suspensor.     

Interaction between maternal effect and zygotic effect mutations during maize seed development

Double fertilization of the embryo sac by the two sperm cells of a pollen grain initiates seed development. Proper development of the seed depends not only on the action of genes from the resulting embryo and endosperm, but also on maternal genes acting at two stages. Mutations with both sporophytic maternal effects and gametophytic maternal effects have been identified. A new maternal effect mutation in maize, maternal effect lethal1 (mel1), causes the production of defective seed from mutant female gametophytes. It shows reduced pollen transmission, suggesting a requirement in the male gametophyte, but has no paternal effect on seed development. Interestingly, the defective kernel phenotype of mel1 is conditioned only in seeds that inherit mel1 maternally and are homozygous for the recessive allele (endogenous to the W22 inbred line) of either of two genes, sporophyte enhancer of mel1 (snm1) or snm2, suggesting redundancy between maternally and zygotically required genes. Both mel1 and snm1 map to the short arm of chromosome 2, and snm2 maps to the long arm of chromosome 10. The mode of action of mel1 and the relationship between mel1 and snm1 and snm2 are discussed.     

D-type cyclins control cell division and developmental rate during Arabidopsis seed development

Seed development in Arabidopsis is characterized by stereotypical division patterns, suggesting that coordinated control of cell cycle may be required for correct patterning and growth of the embryo and endosperm. D-type cyclins (CYCD) are key cell cycle regulators with roles in developmental processes, but knowledge regarding their involvement in seed development remains limited. Here, a family-wide gene expression, and loss- and gain-of-function approach was adopted to reveal additional functions for CYCDs in the development of seed tissues. CYCD genes have both discrete and overlapping tissue-specific expression patterns in the seed as revealed by GUS reporter gene expression. Analysis of different mutant combinations revealed that correct CYCD levels are required in seed development. The CYCD3 subgroup is specifically required as its loss caused delayed development, whereas overexpression in the embryo and endosperm of CYCD3;1 or a previously uncharacterized gene, CYCD7;1, variously leads to induced proliferation, abnormal phenotypes, and elevated seed abortion. CYCD3;1 overexpression provoked a delay in embryonic developmental progression and abnormalities including additional divisions of the hypophysis and suspensor, regions where CYCD3 genes are normally expressed, but did not affect endosperm development. Overexpression of CYCD7;1, not normally expressed in seed development, promoted overgrowth of both embryo and endosperm through increased division and cell enlargement. In contrast to post-germination growth, where pattern and organ size is not generally related to division, results suggest that a close control of cell division through regulation of CYCD activity is important during seed development in conferring both developmental rate and correct patterning.     

Cytokinin Content of Pea Seeds during their Growth and Development

Seed development in Pisum arvense L. cv. New Zealand Maple has been studied in relation to changes in level of total endogenous cytokinins. Growth of both the whole seed and the embryo is diauxic, having two phases of active growth separated by a lag period. The two maxima in the growth rates of the whole seed and the embryo occur about the 23rd and 31st days after anthesis. The lag period occurs between days 26 and 28. Cell division is thought to have ceased prior to day 19 and the changes in total cytokinin content in pea seeds after this time are believed to be largely independent of cell division. The three maxima in the cylokinin content per gm. fresh weight of seed and per seed were found to be coincident with the maximum volume of endosperm per seed and the two maxima in the growth rates of the whole seed and the embryo.     

Genetic analysis as a tool to investigate the molecular mechanisms underlying seed development in maize

BACKGROUND: In angiosperms the seed is the outcome of double fertilization, a process leading to the formation of the embryo and the endosperm. The development of the two seed compartments goes through three main phases: polarization, differentiation of the main tissues and organs and maturation. SCOPE: This review focuses on the maize kernel as a model system for developmental and genetic studies of seed development in angiosperms. An overview of what is known about the genetic and molecular aspects underlying embryo and endosperm formation and maturation is presented. The role played by embryonic meristems in laying down the plant architecture is discussed. The acquisition of the different endosperm domains are presented together with the use of molecular markers available for the detection of these domains. Finally the role of programmed cell death in embryo and endosperm development is considered. CONCLUSIONS: The sequence of events occurring in the developing maize seed appears to be strictly regulated. Proper seed development requires the co-ordinated expression of embryo and endosperm genes and relies on the interaction between the two seed components and between the seed and the maternal tissues. Mutant analysis is instrumental in unravelling the genetic control underlying the formation of each compartment as well as the molecular signals interplaying between the two compartments.     

Alisma embryogenesis: The development and ultrastructure of the suspensor

Summary The development of the suspensor (consisting of a basal cell and a few chalazal cells) inAlisma plantagoaquatica andA. lanceolatum was investigated using cytochemical methods, light and electron microscopy. The basal cell becomes differentiated during the first three days of embryo development. As a result of endopolyploidization the volume of the nucleus rapidly increases, as does the quantity of chromatin it contains and the size of the nucleolus. As basal cell grows, its cytoplasm increases in volume and the number of organelles increase, and wall ingrowths begin to form on the walls at the micropylar pole of the cell. The full development and functioning of the suspensor occurs during the next three days. The enormous basal cell then attains its maximum degree of differentiation: its nucleus reaches a ploidy of 256n or 512n, the micropylar transfer wall is fully developed, as is the cytoplasm, rich in proteins, ribonucleic acids (RNA) and organelles, particularly dictyosomes and long cisternae of the rough endoplasmic reticulum. The chalazal suspensor cells joining the embryo proper to the basal cell also become differentiated. In the seven-day embryo the suspensor begins to degenerate which coincides with the cellularization of the endosperm at the micropylar pole of the embryo sac. The senescence of the suspensor involves the degradation of the nucleus, increasing cytoplasmic vacuolization, and a distinct decrease in protein and RNA content, first in the basal cell, then in the chalazal suspensor cells. Analysis of the development and ultrastructure of the basal suspensor cell suggests that it plays the role of an active metabolic transfer cell, translocating nutrients from the maternal tissues via the chalazal suspensor cells to the growing embryo proper.     

Regeneration of zygotic-like microspore-derived embryos suggests an important role for the suspensor in early embryo patterning

The inaccessibility of the zygote and proembryos of angiospermswithin the surrounding maternal and filial tissues has hamperedstudies on early plant embryogenesis. Somatic and gametophyticembryo cultures are often used as alternative systems for molecularand biochemical studies on early embryogenesis, but are notwidely used in developmental studies due to differences in theearly cell division patterns with seed embryos. A new Brassicanapus microspore embryo culture system, wherein embryogenesishighly mimics zygotic embryo development, is reported here.In this new system, the donor microspore first divides transverselyto form a filamentous structure, from which the distal cellforms the embryo proper, while the lower part resembles thesuspensor. In conventional microspore embryogenesis, the microsporedivides randomly to form an embryonic mass that after a whileestablishes a protoderm and subsequently shows delayed histodifferentiation.In contrast, the embryo proper of filament-bearing microspore-derivedembryos undergoes the same ordered pattern of cell divisionand early histodifferentiation as in the zygotic embryo. Thisobservation suggests an important role for the suspensor inearly zygotic embryo patterning and histodifferentiation. Thisis the first in vitro system wherein single differentiated cellsin culture can efficiently regenerate embryos that are morphologicallycomparable to zygotic embryos. The system provides a powerfulin vitro tool for studying the diverse developmental processesthat take place during the early stages of plant embryogenesis. Key words: Brassica napus, microspore embryogenesis, pattern formation, polarity, suspensor, zygotic embryogenesis