A Model for Seed Transmission of a Plant Virus: Genetic and Structural Analyses of Pea Embryo Invasion by Pea Seed-Borne Mosaic Virus

Pea seed-borne mosaic virus (PSbMV), a seed-transmitted virus in pea and other legumes, invades pea embryos early in development. This process is controlled by maternal genes and, in a cultivar that shows no seed transmission, is prevented through the action of multiple host genes segregating as quantitative trait loci. These genes control the ability of PSbMV to spread into and/or multiply in the nonvascular testa tissues, thereby preventing the virus from crossing the boundary between the maternal and progeny tissues. Immunocytochemical and in situ hybridization studies suggested that the virus uses the embryonic suspensor as the route for the direct invasion of the embryo. The programmed degeneration of the suspensor during embryo development may provide a transient window for embryo invasion by the virus and could explain the inverse relationship between the age of the mother plant for virus infection and the extent of virus seed transmission.     

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.     

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     

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.     

Necrosis in field beans (Vicia faba) induced by interactions between bean leaf roll, pea early-browning and pea enation mosaic viruses

Mixed infections with two or three viruses - bean leaf roll (BLRV), pea early-browning (PEBV) and pea enation mosaic (PEMV) - were detected in plants showing leaf curling, stunting and necrosis in a crop of field beans grown for seed in 1980. In glasshouse tests, field bean plants infected with any one of these viruses showed no necrosis, and plants infected with PEBV and PEMV together showed symptoms of PEMV only. However, mixed infection with BLRV and PEMV almost invariably induced severe stunting and leaf necrosis, and infection with BLRV and PEBV often induced both leaf and stem necrosis and sometimes caused early death. Thus it seems that the necrotic symptoms seen in the field were induced by interactions between BLRV and the other viruses. No transmission of PEBV was detected through seed harvested from the crop, but up to 5% transmission was detected through seed from experimentally-infected plants. The infected seedlings were symptomless.     

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     

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.     

Protein patterns associated with <Emphasis Type="Italic">Pisum sativum</Emphasis> somatic embryogenesis

Total protein patterns were studied in the course of development of pea somatic embryos using simple protocol of direct regeneration from shoot apical meristems on auxin supplemented medium. Protein content and total protein spectra (SDS-PAGE) of somatic embryos in particular developmental stages were analysed in Pisum sativum, P. arvense, P. elatius and P. jomardi. Expression of seed storage proteins in somatic embryos was compared with their accumulation in zygotic embryos of selected developmental stages. Pea vegetative tissues, namely leaf and root, were used as a negative control not expressing typical seed storage proteins. The biosynthesis and accumulation of seed storage proteins was observed during somatic embryo development (since globular stage), despite of the fact that no special maturation treatment was applied. Major storage proteins typical for pea seed (globulins legumin, vicilin, convicilin and their subunits) were detected in somatic embryos. In general, the biosynthesis of storage proteins in somatic embryos was lower as compared to mature dry seed. However, in some cases the cotyledonary somatic embryos exhibited comparatively high expression of vicilin, convicilin and pea seed lectin, which was even higher than those in immature but morphologically fully developed zygotic embryos. Desiccation treatments did not affect the protein content of somatic embryos. The transfer of desiccated somatic embryos on hormone-free germination medium led to progressive storage protein degradation. The expression of true seed storage proteins may serve as an explicit marker of somatic embryogenesis pathway of regeneration as well as a measure of maturation degree of somatic embryos in pea.     

Further studies on a British form of pea early-browning virus

An isolate of pea early-browning virus from Britain (PEBV (B)) has tubular particles most of which are either about 103 or 212 mμ long with sedimentation coefficients of 210 and 286 S respectively. Both types show cross-banding at intervals of 2.5 mμ. Virus preparations containing only the shorter particles were not infective. PEBV (B) was transmitted to pea seedlings by both adult and juvenile Trichodorus primitivus (de Man) (Nematoda) and persisted for 32 days in T. primitivus kept without plants. In two experiments T. primitivus failed to transmit a Dutch isolate (PEBV (D)), which is distantly related serologically to PEBV (B). PEBV (B) was transmitted by nematodes to cucumber roots more readily in soil at 20d? than at 24d? C., and more readily at 24d? than at 29d? C. When transmitted by inoculation of sap, PEBV (B) and PEBV (D) caused similar symptoms in some pea varieties but differed in virulence towards others. Thirty-one varieties resistant to natural infection with PEBV in The Netherlands were susceptible to PEBV (B) when manually inoculated with sap or when grown in naturally infested soil from one site; twenty-six of these varieties did not become infected in soil from a second site, in which several other varieties that are susceptible in The Netherlands were infected. Varieties should therefore be tested for resistance by growing them on many infested fields. All but one of the pea varieties resistant to PEBV in The Netherlands became infected with the English form of tomato black ring virus when grown in soil containing infective Longidorus attenuatus Hooper.     

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.     

An Analysis of Seed Development in Pisum sativum: VI. ABSCISIC ACID ACCUMULATION

The abscisic acid (ABA) content of wrinkled (rr) pea seed tissueshas been quantified during development using multiple-ion-monitoringcombined gas chromatography-mass spectrometry and a deuteratedinternal standard. The level of ABA in the embryo generallyincreased with increasing cotyledon fresh weight while thatin the testa showed a distinct maximum at the time of maximumendosperm volume and the slowing in the growth of the testa.Pods contained relatively little ABA on a fresh weight basis.The total seed ABA content showed a biphasic distribution, thefirst maximum following the maximum growth rate of the testaand the second that of the embryo. The biphasic distributionof ABA in the pea seed was confirmed using a second pea genotype,near-isogenic to the first except for the r locus, and by theanalysis of individual seeds using a radioimmunoassay for ABA.The first maximum was composed mainly of a testa component andthe second mainly of an embryo component. When plants were grownin different environments, wrinkled seeds were found to containslightly more ABA than round (RR) but this was only significantlate in development. Immature seeds were capable of metabolizing17'-deoxy ABA to ABA, as determined by incorporation of either³H or ²H, and the metabolite was present mainly in the testa.The production of ABA in pea seeds is discussed in relationto the development of the different seed tissues. Key words: Abscisic acid, peas, seed development     

Effects of storage temperature on viability, germination and antioxidant metabolism in Ginkgo biloba L. seeds.

The behaviour of the Ginkgo biloba L. seeds was studied during storage at 4 and 25 degrees C. When stored at 25 degrees C, all the seeds died in 6 months. Cold temperatures preserved seed tissue viability for 1 year but did not preserve their capability to germinate, since such capability decreased after 6 months. A significant increase in lipid peroxidation occurred in the seed both in the embryo and in the endosperm. During storage a progressive deterioration of the endosperm tissues was evident. The two major water soluble antioxidants, ascorbate (ASC) and glutathione (GSH), showed different behaviour in the two conditions of storage and in the two main structures of the seed, the embryo and the endosperm. The ASC content of embryos and endosperms remained quite unchanged in the first 9 months at 4 degrees C, then increased. At 25 degrees C a significant decrease in the ASC content in the embryos was evident, whereas it remained more stable in the endosperm. The GSH pool decreased at both storage temperatures in the embryos. As far as the ASC-GSH redox enzymes are concerned, their activities decreased with storage, but changes appeared to be time-dependent more than temperature-dependent, with the exception of the endosperm ascorbate free radical (AFR) reductase (EC 1.6.5.4), the activity of which rapidly decreased at 25 degrees C. Therefore overall the antioxidant enzymes were scarcely regulated and unable to counteract oxidative stress occurring during the long-term storage.     

Development of the quiescent center in maturing embryonic radicles of pea (Pisum sativum L. cv. Alaska)

Maturing embryos of pea (Pisum sativum L. cv. Alaska) were treated with an aqueous solution of tritiated thymidine for 1 h, sectioned, and processed for autoradiography. An analysis of the distribution of labelled nuclei and mitotic figures demonstrated the presence of a quiescent center (QC) in the radicles of developing embryos. The QC developed in the radicle during the growth of the embryo. Immature radicles that did not contain a well-formed zone of root-cap initials did not show a QC. In the latter stages of seed ripening, the pattern of arrest of DNA synthesis and mitosis was tissue-specific. Cells within the QC remained inactive. The region lacking labelled nuclei and mitotic figures progressively expanded to include the root cap initials and then the provascular cylinder. Mitosis was arrested before DNA synthesis in the embryonic cortex. Cells within the QC synthesized DNA during the first stages of seed germination.Abbreviations [³H]TdR tritiated thymidine - QC quiescent center     

Developing embryos of Sesbania sesban have unique potential to photosynthesize under high osmotic environment

This study has been carried out to investigate the photosynthetic activities in developing embryos of Sesbania sesban under a highly osmotic environment. In S. sesban, the embryo turns green/chlorophyllous at the early heart shape stage. Interestingly, despite being deeply embedded within the supporting tissues (several layers of pod wall, seed coat and endosperm) and developing in a highly osmotic environment, the growing embryo of the developing seed showed the presence of various components of photosynthetic machinery besides being chlorophyllous. The shade-adaptive nature of the photosynthetic machinery of the embryo is evident from (a) low chlorophyll a/b ratio, (b) photosystem (PS) II attaining maximal activity at low photon flux density and (c) lesser plastoquinone pool. The photosynthetic potential of the growing embryo seems to contribute towards seed filling as it has the potential not only to harvest light energy but also to fix CO2 as efficiently as other photosynthetic parts of S. sesban. In fact, ribulose-1,5 bisphosphate carboxylase purified from embryos manifested subunit composition similar to that of leaves. The PS II activity in leaves, cotyledonary leaves and pod wall declined sharply with increase in the level of NaCl and sucrose above 150 and 300 mmol, respectively. Amazingly, PS II activity in developing embryos was maximal in the presence of 250 mmol NaCl or 500 mmol sucrose and remained high even when NaCl and sucrose levels were increased to 500 and 1000 mmol, respectively. We hypothesize that the developing embryos have some factor(s) which protect(s) the photosynthetic machinery in an environment of high osmotic strength.     

Somatic embryogenesis in pea (Pisum sativum L.): effect of explant, genotype and culture conditions

In this study 16 cultivars of pea (Pisum sativum L.) were screened in vitro for the formation of somatic embryos which were dependent on the genotype, culture conditions and explant source used. The cultivars Stehgolt, Maro and Progreta showed the highest tendency to form somatic embryos (c. 25%) while Alaska, Rondo and Ascona did not show any embryo production. Using the cultivar Belman, the highest embryo production was achieved by using nodal explants of shoots (10.6%) and a cotyledon-free embryo as explant source (14.1%) in the light (15.8%) compared to using apices as explants (1.8%) and a seedling as the explant source (9.4%) in the dark (3.3%). Media containing picloram (0.75 mg/litre) followed by BA (1 mg/litre) or kinetin (1 mg/litre), each for four weeks gave the highest somatic embryo production. The development of embryos to whole plants was unreliable and some 90% of the embryos induced did not develop any further, died, recallused or formed secondary embryos. The size of the embryo at separation and the timing of the separation from the original callus were important factors determining success for complete development to whole plant. Regeneration of 184 plants was achieved with ensuing flowering, pod formation and viable seed production from the techniques described.     

A pea seed mutant affected in the differentiation of the embryonic epidermis is impaired in embryo growth and seed maturation

During legume seed development the epidermis of the embryos differentiates into a transfer cell layer which mediates nutrient uptake during the storage phase. This specific function of the epidermal cells is acquired at the onset of embryo maturation. We investigated this process in the pea seed mutant E2748. The epidermal cells of the mutant embryo, instead of turning into transfer cells, enlarge considerably and become vacuolated and tightly associated with adjacent seed tissues. Expression of a sucrose transporter gene that is upregulated in wild-type transfer cells decreases in the mutant and changes its spatial pattern. This indicates that the outermost cell layer of mutant cotyledons cannot acquire transfer cell morphology but loses epidermal cell identity and does not function as a sucrose uptake system. Seed coat growth as well as composition, concentration and dynamics of sugars within the endospermal vacuole are unchanged. The loss of epidermal identity has severe consequences for further embryo development and is followed by disruption of the symplast within the parenchyma, the breach of the developmental gradient, lower sucrose and starch levels and initiation of callus-like growth. It is concluded that the E2748 gene controls differentiation of the cotyledonary epidermis into transfer cells and thus is required for the regional specialisation with a function in embryo nutrition.     

Intracellular pH of Cotton Embryos and Seed Coats during Fruit Development Determined by P Nuclear Magnetic Resonance Spectroscopy

The pH of the phosphate-containing compartments of developing cotton seed coat and embryo tissues was determined by means of ³¹P nuclear magnetic resonance spectroscopy. The pH values of these tissues varied as a function of developmental age. From 27 to approximately 38 days postanthesis, a strong pH differential existed between the two tissues; the seed coat was up to 1.4 pH units more acid than developing cotton embryos. The pattern of pH values found with this technique agrees with pH values of tissue homogenates in distilled water. The results confirm an earlier suggestion that seed coat cells are more acidic than embryo cells during key developmental stages of the seed. The pH differential between these two tissues causes abscisic acid to diffuse from seed coats to embryos against its apparent concentration gradient to prevent viviparous germination, despite a higher abscisic acid concentration in the embryo.     

Improvement of pea biomass and seed productivity by simultaneous increase of phloem and embryo loading with amino acids

The development of sink organs such as fruits and seeds strongly depends on the amount of nitrogen that is moved within the phloem from photosynthetic‐active source leaves to the reproductive sinks. In many plant species nitrogen is transported as amino acids. In pea (Pisum sativum L.), source to sink partitioning of amino acids requires at least two active transport events mediated by plasma membrane‐localized proteins, and these are: (i) amino acid phloem loading; and (ii) import of amino acids into the seed cotyledons via epidermal transfer cells. As each of these transport steps might potentially be limiting to efficient nitrogen delivery to the pea embryo, we manipulated both simultaneously. Additional copies of the pea amino acid permease PsAAP1 were introduced into the pea genome and expression of the transporter was targeted to the sieve element‐companion cell complexes of the leaf phloem and to the epidermis of the seed cotyledons. The transgenic pea plants showed increased phloem loading and embryo loading of amino acids resulting in improved long distance transport of nitrogen, sink development and seed protein accumulation. Analyses of root and leaf tissues further revealed that genetic manipulation positively affected root nitrogen uptake, as well as primary source and sink metabolism. Overall, the results suggest that amino acid phloem loading exerts regulatory control over pea biomass production and seed yield, and that import of amino acids into the cotyledons limits seed protein levels.     

Marker assisted pea breeding: <Emphasis Type="Italic">eIF4E</Emphasis> allele specific markers to <Emphasis Type="Italic">pea seed-borne mosaic virus</Emphasis> (PSbMV) resistance

Traditional plant breeding relies upon crosses and subsequent selection of genotypes to meet desirable traits. The incorporation of marker-assisted selection into breeding strategies would result in a reduction in the number of offspring to be propagated, selected and tested. In the case of pea (Pisum sativum L.), the testing of resistance to viral pathogens such as pea seed-borne mosaic virus (PSbMV) is included in the breeding process. Resistance to the common strains of PSbMV is conferred by a single recessive gene (eIF4E), localized on LG VI (sbm-1 locus). We have analyzed for variation in the eIF4E genomic sequences from 43 pea varieties and breeding lines, reported as donors of resistance. This enabled a comprehensive investigation of the eIF4E gene structure and mutations responsible for PSbMV resistance were identified. Subsequently, PCR-based and gene-specific single nucleotide polymorphism and co-dominant amplicon length polymorphism markers were developed. All together 60 accessions were analyzed using sequence data and/or allele specific DNA markers. Developed allele specific markers were reproducibly amplified across a broad spectra of pea varieties and breeding lines. These were found to be 100% accurate in detecting the presence of the respective alleles when compared to symptomology and ELISA, testing (74% reliable). Hence, these molecular markers will substantially speed-up PSbMV diagnosis and resistance breeding processes in pea.