Early Cotyledonary Stage Research Articles

Patterns of inheritance for cotyledon resistance against Sclerotinia sclerotiorum in Brassica napus

Sclerotinia rot, caused by the fungal pathogen Sclerotinia sclerotiorum, is a devastating disease on oilseed rape and mustard worldwide. While the focus to effectively control yield losses from Sclerotinia has been on locating stem resistance, resistance to leaf and to early (cotyledon) stage resistance to this pathogen are also important, both not only limiting additional plant damage, but also inoculum build up and spread onto stems. Three Brassica napus breeding populations developed in India, C2 (NC-8 × RQ-001-NCA-8 NC2-7), C5 (cv. Charlton × RQ-001-NCA-8 NC2-7) and C6 (cv. Charlton × NC4-5), were screened for cotyledon resistance (based on lesion diameter) under controlled environmental conditions to investigate the inheritance of disease resistance. Each population consisted of parents (P1 and P2), F1, F2, BC1P1 and BC2P2, except for population C5 which lacked BC1P1. Moderate broad sense heritability of 0.42, 0.31 and 0.49 for cotyledon resistance was found in populations C2, C5 and C6, respectively, and there was mostly non-additive genetic control of resistance. Analyses of generation means and variances indicated that the additive-dominance model was adequate to explain genetics of cotyledon resistance in population C2. There was heterosis for susceptibility (i.e., larger lesion diameter). The dominance × dominance digenic epistasis explained genetic control in population C6, with heterosis towards resistance (i.e., smaller lesion diameter). This information is critical for breeding for resistance to this important pathogen in Australia, India and elsewhere.

Plant regeneration through somatic embryogenesis and shoot organogenesis from immature zygotic embryos of Sapium sebiferum Roxb.

Sapium sebiferium Roxb. is a highly valued ornamental plant species in tropical and subtropical regions. Here we present an efficient protocol for plant regeneration from immature zygotic embryos (IZEs) of S. sebiferium through direct somatic embryogenesis and shoot organogenesis for the first time. The results showed that mechanical damage, plant growth regulators (PGRs) and light regimes had great influence on in vitro morphogenesis. High frequency of somatic embryogenesis was obtained when the mechanical damaged IZEs were cultured on the Murashige and Skoog (MS) medium containing 1.0mgl−1 indole-3-acetic acid (IAA) under continuous darkness for two weeks followed by a light/dark photoperiod cycle for four weeks. The induction frequency of somatic embryogenesis was up to 90.0%. Somatic embryos (SEs) at early cotyledonary stage showed highest proliferation frequency (81.7%) and 80.0% of secondary somatic embryos (SSEs) germinated on plant growth regulators (PGRs)-free MS medium. For direct shoot induction pathway, the best shoot regeneration rate (63.3%) was obtained on the medium supplemented with 1.0mgl−1 6-benzyladenine (6-BA). The plantlets derived from two ways were successfully acclimatized in the greenhouse with more than 90% survival rate. This study will be beneficial for the rescue of immature embryo, inheritance of heterosis as well as the genetic improvement of S. sebiferium.

Protocols for Obtaining Zygotic and Somatic Embryos for Studying the Regulation of Early Embryo Development in the Model Legume Medicago truncatula.

Early embryogenesis starting from a single cell zygote goes through rapid cell division and morphogenesis, and is morphologically characterized by pre-globular, globular, heart, torpedo and cotyledon stages. This progressive development is under the tight regulation of a complex molecular network. Harvesting sufficient early embryos at a similar stage of development is essential for investigating the cellular and molecular regulation of early embryogenesis. This is not straightforward since early embryogenesis undergoes rapid morphogenesis in a short while e.g. 8 days for Medicago truncatula to reach the early cotyledon stage. Here, we address the issue by two approaches. The first one establishes a linkage between embryo development and pod morphology in helping indicate the stage of the zygotic embryo. This is particularly based on the number of pod spirals and development of the spines. An alternative way to complement the in vivo studies is via culturing leaf explants to produce somatic embryos. The medium includes an unusual hormone combination - an auxin (1-naphthaleneacetic acid), a cytokinin (6-benzylaminopurine), abscisic acid and gibberellic acid. The different stages can be discerned growing out of the callus without dissection.

Analysis of global gene expression profiles to identify differentially expressed genes critical for embryo development in Brassica rapa.

Embryo development represents a crucial developmental period in the life cycle of flowering plants. To gain insights into the genetic programs that control embryo development in Brassica rapa L., RNA sequencing technology was used to perform transcriptome profiling analysis of B. rapa developing embryos. The results generated 42,906,229 sequence reads aligned with 32,941 genes. In total, 27,760, 28,871, 28,384, and 25,653 genes were identified from embryos at globular, heart, early cotyledon, and mature developmental stages, respectively, and analysis between stages revealed a subset of stage-specific genes. We next investigated 9,884 differentially expressed genes with more than fivefold changes in expression and false discovery rate ≤ 0.001 from three adjacent-stage comparisons; 1,514, 3,831, and 6,633 genes were detected between globular and heart stage embryo libraries, heart stage and early cotyledon stage, and early cotyledon and mature stage, respectively. Large numbers of genes related to cellular process, metabolism process, response to stimulus, and biological process were expressed during the early and middle stages of embryo development. Fatty acid biosynthesis, biosynthesis of secondary metabolites, and photosynthesis-related genes were expressed predominantly in embryos at the middle stage. Genes for lipid metabolism and storage proteins were highly expressed in the middle and late stages of embryo development. We also identified 911 transcription factor genes that show differential expression across embryo developmental stages. These results increase our understanding of the complex molecular and cellular events during embryo development in B. rapa and provide a foundation for future studies on other oilseed crops.

Plant regeneration through direct somatic embryogenesis from immature zygotic embryos of the medicinal plant, Paris polyphylla Sm.

A protocol for induction of direct somatic embryogenesis and subsequent plant regeneration for the medicinally important and endangered plant Paris polyphylla Sm. has been developed for the first time. Immature zygotic embryos (IZEs) were cultured on different media namely Gamborg (B5), ½ B5, Murashige and Skoog (MS), ½ MS, Chu et al. (N6), ½ N6, Schenk and Hildebrandt (SH) and ½ SH. Highest frequency of somatic embryogenesis (32.6 %) and mean number of somatic embryos (SEs) per explant (28.7 ± 1.7) were obtained on ½ MS medium directly without an intermediate callus phase. The frequency of SE induction was significantly increased to 40.7 % when ½ MS medium was solidified with gelrite compared to agar (32.6 %). Secondary somatic embryos (SSEs) appeared on the primary SEs in a repetitive way on plant growth regulator-free ½ MS medium but with a gradual decrease in embryogenic potential during subsequent subcultures. Plasmolyzing pre-treatment of SSEs with 1.0 M mannitol for 12 h effectively maintains its embryogenic capacity. Primary embryos at the elongated dimpled and early cotyledonary stage displayed the highest embryo forming capacity of 26.94 and 27.87, respectively. High frequency of SE germination (94.0 %) occurred on ½ MS medium with 0.5 mg/l gibberellic acid. Highest percentage of seedling to plantlet conversion was observed in the medium supplemented with 0.05 mg/l 6-benzylaminopurine and 0.1 mg/l α-naphthalene acetic acid. Regenerated plants displayed morphological characteristics similar to that of the wild plants. Flow cytometry analysis showed ploidy stability of the regenerated plants.

Initiation and development of microspore embryogenesis and plant regeneration of Brassica nigra

Brassica nigra is generally regarded as a recalcitrant species for microspore culture among Brassica crops. Conditions for reliable induction of microspore embryogenesis of B. nigra were studied in this context. Flower bud length and microspore developmental stage were correlated with further embryogenesis. The optimal bud size range was 2.0–2.5 mm for the highest proportion of totipotent, late uninucleate microspore and the highest frequency of microspore embryogenesis. Treatment of a short heat shock by incubating the microspore culture at 32°C for 24 h was suitable for the microspore survival, sustained cell divisions, and further induced embryogenesis. Subsequently, the use of NLN medium with the addition of 13% sucrose and 0.1% activated charcoal (AC) provided the optimal conditions for the development of microspore-derived embryos (MDEs). The early cotyledonary (EC) stage embryos cultured on MS medium fortified with 4.6 μM zeatin (ZT) and 0.12 μM indole-3-acetic acid (IAA) resulted in the most efficient rates of plantlet regeneration. The ploidy levels of regenerated plants of B. nigra were determined by flow cytometry, revealing that 50.6% were diploid. The results enable the advancement of breeding programs and genetic studies in B. nigra.

HISTOLOGICAL ANALYSIS OF SOMATIC EMBRYOGENESIS INDUCED IN TROPICAL LADY'S SLIPPER ORCHID (PAPHIOPEDILUM NIVEUM)

Indirect somatic embryogenesis, an asexual process of plant propagation, is an acceptable tool for cell differentiation and developmental studies. This study was conducted to elucidate the developmental pathway of a somatic embryo in the Paphiopedilum niveum orchid. The callus-derived somatic embryo was obtained by using modified Vacin and Went (VW) medium supplemented with 0.1 mg L-1 naphthalene acetic acid (NAA) and 0.5 mg L-1 thidiazuron (TDZ) as well as 10 g L-1 sucrose. Developmental phases were characterized by histological analysis. The callus-derived somatic embryos developed progressively through the early globular, globular and cotyledonary stages. After a month of culture, a proembryonic mass (or early globular-shape stage) originated on the surface of the callus and differentiated rapidly to a globular somatic embryo with a well-demarcated protoderm. These somatic embryos developed protrusions that indicated the cotyledonary stages after two months of culture. The organized structures of plantlets were eventually obtained when protocorm-like bodies (PLBs) involving somatic embryogenesis were transferred to modified Murashige and Skoog (MS) medium supplemented with 50 g L-1 homogenated banana. Histological analysis also revealed that the pattern of somatic embryo formation of P. niveum showed the same process as the developmental pathway of monocot species.

Manifestation of embryogenic potential in culture of zygotic embryos of Quercus robur L.

For the initiation of somatic embryogenesis early cotyledonary stage of zygotic embryo explants (from 15th July until late August) was suitable. The highest frequency of differentiation of somatic embryos was obtained on cotyledons of zygotic embryos cultured on basal modified medium MS (with 1/2 concentration macronutrients) or WPM medium containing 500 mg•l<sup>-1</sup> glutamine, proline and casein hydrolysate and supplemented with 2,4-D (1,0-2,0 mg•l<sup>-1</sup>) and BAP (0,5-1,0 mg•l<sup>-1</sup>). The development of somatic embryos was direct and indirect and the process was continuous over a long period. Primary somatic embryos were able to produce secondary embryos. Repetitive somatic embryogenesis led to the proliferation of a large number of new somatic embryos on their cotyledons, hypocotyl or radicula. The process of embryo differentation is asynchronous - various stages of somatic embryos could be observed in embryogenic culture. A somatic embryo conversion was rare on tested media. Embryo germination occured on medium containing BAP (0,1 mg•l<sup>-1</sup>) or on medium with ABA and GA<sub>3</sub> (each 0,2 mg•l<sup>-1</sup>) after a previous culture on WPM medium without plant growth regulators supplemented with sorbitol (6%). The embryo germination occurred also on WPM medium with 0.2 mg•l<sup>-1</sup> BAP when cultures were mantained at 2<sup>o</sup>C for 4 weeks. Only 8 somatic embryos developed into plantlets. Their transplantation to <em>in vivo</em> conditions was unsuccessful.

Somatic embryogenesis in Alnus glutinosa (L.) Gaertn

Induction of somatic embryos and plant regeneration was demonstrated for the first time in Alnus glutinosa. Somatic embryos were initiated from zygotic embryos collected 1–3 weeks post-anthesis (WPA), i.e., when they were at globular or early cotyledonary stage and were 0.5–1 mm in length. Induction frequency (16.6 %) and the mean number of somatic embryos (4.5 embryos/explant) were highest after culture of zygotic embryos, collected at 3 WPA, on Murashige and Skoog medium (MS) supplemented with 0.9-μM 2,4-dichlorophenoxyacetic acid and 2.22-μM benzyladenine (BA). No embryogenic induction was observed on medium with BA alone. Initial somatic embryos differentiated indirectly from callus tissue formed at the surface of the zygotic embryos. Embryogenic competence was maintained by secondary embryogenesis, which was affected by explant type, plant growth regulators and genotype. Secondary embryogenesis was induced by culture of small groups of whole somatic embryos or isolated cotyledon explants on medium consisting of MS medium (half-strength macronutrients) supplemented with 0.44-μM BA. Histological study of isolated cotyledon explants revealed that secondary embryos developed directly from differentiated embryogenic tissue on the surface of cotyledons. Somatic embryos at successive stages of development, including cotyledonary-stage embryos with shoot and root meristems, were evident. For plantlet conversion, somatic embryos were transferred to maturation medium supplemented with 3 % maltose, followed by 6 weeks of culture in Woody Plant Medium supplemented with 0.44-μM BA and 0.46-μM Zeatin (Z). This novel protocol appears promising for mass propagation, conservation and genetic transformation of black alder.

In vitro plant regeneration from immature zygotic embryos and repetitive somatic embryogenesis in kohlrabi (Brassica oleracea var. gongylodes)

A simple and efficient protocol for direct somatic embryogenesis and plant regeneration of kohlrabi (Brassica oleracea var. gongylodes) was developed. Somatic embryos were induced from immature zygotic embryos at different developmental stages cultured on Murashige and Skoog medium supplemented with 0, 0.5, 1.0, or 1.5 mg/l 2,4-dichlorophenoxyacetic acid. Zygotic embryos at the early cotyledonary stage, which were cultured for 4 wk on plant growth regulator-free (PGR-free) medium, displayed the highest percentage of somatic embryogenesis (80.7%). Embryogenic tissue could be subcultured on the same medium for over 1 yr. Embryogenic lines derived from early cotyledonary stage zygotic embryos displayed the highest intensity of secondary embryogenesis (highest mean number of new somatic embryos per responsive somatic embryo explant). Histological analyses confirmed the direct origin of the secondary somatic embryos. Prolonged culturing of embryogenic tissue on PGR-free medium led to somatic embryo development into plantlets that were successfully acclimated in the greenhouse with a survival rate of 72.5%. Flow cytometry analysis showed no ploidy variation in 96.7% of the acclimated plants.

Characterization and systematic implications of the diversity in timing of programmed cell death of the suspensors in Leguminosae

In angiosperm seeds, the developing embryo acquires nutrients via a suspensor that typically undergoes programmed cell death (PCD) at the early cotyledon stage. However, in Leguminosae (the third largest angiosperm family), the suspensors can disappear at the heart-shaped stage (i.e., prior to the cotyledon stage) or still persist at the cotyledon stage. Here, in a comprehensive survey of legume suspensors and embryos, the variation and the evolutionary direction of timing of suspensor PCD in Leguminosae were characterized, and systematic implications were evaluated. Suspensor development and morphology for 66 leguminous species from 49 genera, 21 tribes, and 3 subfamilies were comparatively studied using standard paraffin sectioning and light microscopy. Three patterns of suspensor PCD were observed at the early cotyledon stage. (A) The suspensor persisted. (B) The suspensor separated from the wall of the embryo sac and persisted as a vestige at the radicle apex. (C) The suspensor disappeared completely, and the absorption of nutrients by embryo was carried out via a "contact zone" between the embryo and the endosperm. Pattern C of early suspensor PCD was found only in the tribe Fabeae. An ancestral character reconstruction revealed that the long-lived suspensors of pattern A represented a plesiomorphic condition in Leguminosae and that the suspensors of pattern C evolved only once in the common ancestor of Fabeae. In Leguminosae, short-lived suspensors have thus evolved multiple times from long-lived suspensors. It remains largely unknown, however, how the embryo acquires nutrients after the early suspensor PCD.

From embryo sac to oil and protein bodies: embryo development in the model legume Medicago truncatula

• The cell and developmental biology of zygotic embryogenesis in the model legume Medicago truncatula has received little attention. We studied M. truncatula embryogenesis from embryo sac until cotyledon maturation, including oil and protein body biogenesis. • We characterized embryo development using light and electron microscopy, measurement of protein and lipid fatty acid accumulation and by profiling the expression of key seed storage genes. • Embryo sac development in M. truncatula is of the Polygonum type. A distinctive multicellular hypophysis and suspensor develops before the globular stage and by the early cotyledon stage, the procambium connects the developing apical meristems. In the storage parenchyma of cotyledons, ovoid oil bodies surround protein bodies and the plasma membrane. Four major lipid fatty acids accumulate as cotyledons develop, paralleling the expression of OLEOSIN and the storage protein genes, VICILIN and LEGUMIN. • Zygotic embryogenesis in M. truncatula features the development of a distinctive multicellular hypophysis and an endopolyploid suspensor with basal transfer cell. A clear procambial connection between the apical meristems is evident and there is a characteristic arrangement of oil bodies in the cotyledons and radicle. Our data help link embryogenesis to the genetic regulation of oil and protein body biogenesis in legume seed.

Proteomic analysis of seed development in Chinese fir (Cunninghamia lanceolata)

Seed development is a complex process governed by highly coordinated changes in the expression of a large protein set. DIGE (Difference Gel Electrophoresis)-based proteomics was used to study developing Chinese fir seeds. 153 spots were obtained by using the analysis of DeCyder software (v. 6.5). Cluster analysis showed that they could be joined into three main groups. Eleven spots, more actively expressed at early cotyledonary stage of developing seeds, were identified by LC/MS/MS (tandem MS). Ten spots were identified by searching NCBInr or EST databases. They included two legumin-like storage proteins, LEA protein, small heat-shock protein, PR10-1.13, a protein similar to eukaryotic translation initiation factor, a protein similar to maternal effect embryo arrest 51, ORF115, a protein similar to monodehydroascorbate reductase, and unknown proteins. The potential function of these proteins during the precotyledonary stage of seed development was discussed.

Somatic embryogenesis and plant regeneration in Pterocarpus marsupium Roxb.

Somatic embryogenesis (SE) has been achieved from hypocotyl-derived callus culture in Pterocarpus marsupium. Ninety percent of hypocotyl explants (excised from 12-day-old in vitro germinated axenic seedlings) produced callus on Murashige and Skoog medium supplemented with 5 μM 2,4-dichlorophenoxyacetic acid and 1 μM a 6-benzyladenine (BA). Induction of SE occurred after transfer of callus clumps (200 ± 20 mg fresh mass) to MS medium supplemented with BA at 2.0 μM, where a maximum of 23.0 ± 0.88 globular stage embryos per callus clump were observed after 4 weeks of culture. Subculturing of these embryos on MS medium supplemented with 0.5 μM BA, 0.1 μM α-naphthalene acetic acid and 10 μM abscisic acid significantly enhanced the maturation of somatic embryos to early cotyledonary stage, where 21.4 ± 0.32 embryos per callus clump were recorded after 4 weeks of culture. Of 30-well developed somatic embryos, 16.6 ± 0.33 germinated and subsequently converted into plantlets on half-strength MS medium supplemented with 1.0 μM BA. The morphologically normal plantlets with well-developed roots were first transferred to 1/4-liquid MS medium for 48 h and then to pots containing autoclaved soilrite and acclimatized in a culture room. Thereafter, they were transferred to a greenhouse, where 60% of them survived.

Polyamine profiles and biosynthesis in somatic embryo development and comparison of germinating somatic and zygotic embryos of Norway spruce

The polyamine (PA) contents and activities of PA biosynthetic enzymes in Norway spruce somatic embryos [Picea abies L. (Karst.), genotype AFO 541] were studied in relation to anatomical changes during their development, from proliferation to germination, and changes in these variables associated with the germination of mature somatic and zygotic embryos were compared. Activities of PA biosynthetic enzymes steadily increased during the development of somatic embryos, from embryogenic suspensor mass until early cotyledonary stages. In these stages, the spermidine (Spd) level was significantly higher than the putrescine (Put) level, and the increases coincided with the sharp increases in S-adenosylmethionine decarboxylase activity in the embryos. The biosynthetic enzyme activity subsequently declined in mature cotyledonary embryos, accompanied by sharp reductions in PA contents, especially in cellular Put contents in embryos from 6 weeks old through the desiccation phase (although the spermine level significantly increased during the desiccation phase), resulting in a shift in the Spd/Put ratio from ca. 2 in early cotyledonary embryos to around 10 after 3 weeks of desiccation. In mature zygotic embryos, Spd contents were twofold lower, but Put levels were higher, than in mature somatic embryos, hence their Spd/Put ratio was substantially lower (ca. 2, in both embryos and megagametophytes). In addition, the PA synthesis activity profiles in the embryos differed (ornithine decarboxylase and arginine decarboxylase activities predominating in mature somatic and zygotic embryos, respectively). The start of germination was associated with a rise in PA biosynthetic activity in the embryos of both origins, which was accompanied by a marked increase in Put contents in somatic embryos, resulting in the decline of Spd/Put ratio to about 2, similar to the ratio in mature and germinating zygotic embryos. The accumulation of high levels of PAs in somatic embryos may be causally linked to their lower germinability than in zygotic embryos.

Plant regeneration in Stone pine (Pinus pinea L.) by somatic embryogenesis

Regeneration of plants by somatic embryogenesis (SE) was achieved in Stone pine (Pinus pinea), one of the most characteristic tree species of the Mediterranean ecosystem. The initial explants were megagametophytes containing zygotic embryos from five selected half-sib families collected at different dates over 2 consecutive years. Rates of extrusion and initiation of SE differed in both years. However, qualitative patterns were very similar: for most families, the responsive developmental window was from late cleavage polyembryony to early cotyledonary stage. The highest overall mean frequencies of extrusion and SE initiation (7 and 0.9%, respectively, for the five families and the eight 2006 collections) were obtained on a modified Litvay’s medium with 9 μM 2,4-D and 4.5 μM BAP, supplemented with L-glutamine and casein hydrolysate. Families showed large differences in frequencies of SE initiation from year to year. Only seven embryogenic lines were induced in 2005, representing three of the five families tested, whereas 34 lines from all the families were obtained in 2006. Proliferation of embryonal masses (EM) was significantly improved when they were subcultured after dispersing in liquid medium and collected on filter paper disks, instead of being subcultured as small clumps. This effect showed a significant interaction with genotype. Several preconditioning treatments and culture media combinations were tested for embryo development and maturation. The high proliferation rate of EM hampered somatic embryo development. However, up to 42 mature embryos from different lines of three of the five families were obtained, 23 of them germinated and seven converted into somatic seedlings.

Identification and characterization of a matrix metalloproteinase (Pta1-MMP) expressed during Loblolly pine (Pinus taeda) seed development, germination completion, and early seedling establishment

Extracellular matrix (ECM) modifications occur during plant growth, development, and in response to environmental stimuli. Key modulators of ECM modification in vertebrates, the extracellular matrix metalloproteinases (MMPs), have also been described in a few plants. Here, we report the identification of Loblolly pine (Pinus taeda) Pta1-MMP and its characterization during seed development and germination. Pta1-MMP protein has the structural characteristics of other plant MMPs, the recombinant protein exhibits Zn(2+)-dependent protease activity, and is inhibited by EDTA and the active site-binding hydroxamate inhibitor GM6001. The Pta1-MMP gene is expressed in both embryo and megagametophyte, with transcript levels increasing in both during the period from proembryo to early cotyledonary stage, then declining during late embryogenesis and maturation drying. Protein extracts exhibited similar developmental-stage MMP-like activity. Seed germination was stimulated by GA(3) and inhibited by ABA, and the timing of germination completion was mirrored by the presence of MMP-like protease activity in both water- and GA(3)-imbibed embryos. Pta1-MMP gene transcript levels increased in association with radicle protrusion for both GA(3)- and water-treated embryos, in agreement with MMP-like activity. In contrast, by 11 days after imbibition, Pta1-MMP gene transcripts in ABA-treated embryos were at levels similar to the other treatments, although MMP-like activity was not observed. The application of GM6001 during Loblolly pine seed germination inhibited radicle protrusion. Our results suggest that MMP activity may be involved in ECM modification, facilitating the cell division and expansion required during seed development, germination completion, and subsequent seedling establishment.

Metabolism of nicotinamide, adenine and inosine in developing microspore-derived canola (Brassica napus) embryos

The metabolic fate of [carbonyl-(14)C]nicotinamide, [8-(14)C]adenine and [8-(14)C]inosine was examined in microspore-derived canola (Brassica napus) embryos at different developmental stages: globular stage (day 10, stage 1), early cotyledonary stage (day 20, stage 2), late cotyledonary stage (day 25, stage 3), and fully developed stage (day 35, stage 4). Uptake of [8-(14)C]nicotinamide by the embryos was always rapid. A lower uptake rate was found for [8-(14)C]adenine and [8-(14)C]inosine, especially at stages 1 and 2. [Carbonyl-(14)C]nicotinamide was converted to nicotinic acid and further metabolized to pyridine nucleotides (NAD/NADP). Some radioactivity was also associated to nicotinic acid glucoside. [8-(14)C]adenine was efficiently utilized for the synthesis of adenine nucleotides and RNA. A small fraction of adenine was degraded to CO(2) via ureides. Up to 40% of [8-(14)C]inosine was salvaged to nucleotides and RNA, although degradation of [8-(14)C]inosine to CO(2) was pronounced. At stage 1, highest salvage activities of nicotinamide, adenine and inosine were observed. In contrast, the lowest purine salvage and highest purine catabolism were found in stage 3 embryos. These results suggest that both nicotinamide and purine salvage for NAD/NADP and purine nucleotides synthesis are extremely high in the globular stage (stage 1). These activities decrease gradually until the late cotyledonary stage (stage 3), before increasing again in the fully developed embryos (stage 4). Overall it appears that nicotinamide and purine salvage are required in support of active growth during the initial phases of embryogenesis and at the end of the maturation period, in preparation for post-embryonic growth.

Caspase-like proteases involvement in programmed cell death of Phaseolus coccineus suspensor

The suspensor of Phaseolus coccineus L. is a short-living tissue, which begins to degenerate at the early cotyledon stage of embryogenesis when it has become no more necessary for further embryo development. The degeneration of terminally differentiated suspensor is considered one of the most typical examples of developmental programmed cell death in plants. We report the occurrence of some hallmarks of animal apoptosis such a leakage of cytochrome C from the mitochondria to the cytosol and an induction of caspase-like proteases, which are sensitive to pH and to specific inhibitors but not affected by generic protease inhibitors.

Delayed embryo development in the ARABIDOPSIS TREHALOSE‐6‐PHOSPHATE SYNTHASE 1 mutant is associated with altered cell wall structure, decreased cell division and starch accumulation

The tps1 mutant, which is disrupted in the TREHALOSE-6-PHOSPHATE SYNTHASE 1 gene, has been previously characterized as a recessive embryo lethal. tps1 embryos do not develop past late torpedo or early cotyledon stage. We report here that at the ultrastructural, biochemical, and transcriptional levels tps1 exhibits many features typically associated with the maturation phase. The appearance of storage reserve transcripts and organelles follows the same temporal pattern in tps1 and wild-type (WT) embryos in the same silique as does accumulation of storage lipid and protein. The mutant plastids accumulate large starch granules that persist until the end of seed development, in contrast with WT plastids where starch accumulation is transient. The transcriptome of tps1 embryos shows a coordinate downregulation of genes involved in starch and sucrose degradation. Interestingly, genes involved in lipid mobilization and gluconeogenesis are induced in tps1 embryos. The cell walls of tps1 embryos show a remarkable degree of thickening at the ultrastructural level and immunodetection of cell wall components shows that altered deposition of pectins accounts for this altered morphology. Consistent with this at the transcriptome level, genes involved in sugar nucleotide and pectin metabolism are altered in the mutant. The frequency of cell division in tps1 embryos is half that of the wild type at the heart and torpedo stages. These results suggest that TPS1 may play a major role in coordinating cell wall biosynthesis and cell division with cellular metabolism during embryo development.