Changes in protein profiles associated with somatic embryogenesis in peanut

The somatic embryogenesis potential of zygotic embryo axes of peanut (Arachis hypogaea L. cv. DRG-12) at different stages of development was evaluated by culturing on MS medium with 18.1 μM 2,4-dichlorophenoxyacetic acid (2,4-D). A 100 % frequency with 18.3 somatic embryos per explant was observed from 4 mm long immature zygotic embryo axes collected 31 – 40 d after pollination. Medium supplemented with 16.6 μM picloram resulted in slow development of somatic embryos whereas in the presence of 21.5 μM α-naphthaleneacetic acid (NAA), the explants underwent maturation with induction of roots after 30 d. The changes in protein profiles in zygotic embryo axes at different stages of development correlated with their potential to form somatic embryos. Immature zygotic embryo axes exhibited high frequency somatic embryogenesis in the stage preceding abundant accumulation of 22 and 65 kDa proteins. The content of 22 and 65 kDa proteins decreased immediately after culture on medium fortified with 18.1 μM 2,4-D and increased again after 12 d of culture coinciding with the development of somatic embryos on the explants. The content of 22 and 65 kDa proteins was low at 15 d of culture on medium supplemented with 16.6 μM picloram possibly due to slow development of the somatic embryos on the explant. On maturation medium containing 21.5 μM NAA, a marked increase in the content of 22 and 65 kDa proteins in 15 d-old cultures was observed.     

Occurrence of phosphorylated proteins and kinase activity in coconut tissues cultured in vitro in a mediumthat induces somatic embryogenesis

The presence of tyrosine kinase and tyrosine-phosphorylated proteins was investigated in coconut tissues cultured in vitro. In order to study this phenomenon, plumular explants were taken from mature zygotic embryos and cultured in a medium that induces somatic embryogenesis. Immunoblot analyses of soluble proteins of coconut cultured tissues with a recombinant monoclonal antibody against phosphotyrosine detected protein bands with molecular masses ranging from 170 to 27 kDa. The highest response was exhibited by plumule-forming callus, which decreased both in number and intensity of bands with a longer time of in vitro culture. The specific immunodetection was corroborated by incubating the membranes with anti-phosphotyrosine antibody in the presence of 1 mM phosphotyrosine. Tyrosine phosphorylated proteins was also suggested by the presence of phosphoproteins resistant to alkaline treatment. In plumule, plumular callus and callus with globular embryos and shoots, a 41-kDa protein remained phosphorylated after alkaline treatment. In plumule, most [³²P]-proteins remained phosphorylated after alkaline treatment. Phosphoaminoacid analysis in protein hydrolysates from [³²P]-labelled 41-kDa protein showed the presence of [³²P]-tyrosine and [³²P]-threonine. Evaluation of tyrosine kinase activity in these tissues by the use of RR-SRC, a synthetic peptide substrate (derived from the amino acid sequence surrounding the phosphorylation site), showed that the activity was highest in plumule forming callus and initial explant, whereas in other tissues, tyrosine kinase activity decreased to values close to zero. Genistein, a specific tyrosine kinase inhibitor, diminished the ability of soluble extracts from coconut tissues cultured in vitro to incorporate ³²P into RR-SRC. These results suggest the presence of tyrosine phosphorylated proteins and tyrosine kinase activity in coconut tissues that have been cultured in vitro.     

Evaluation of somatic embryos of interior spruce. Characterization and developmental regulation of storage proteins

Storage proteins of interior spruce ( Picea glauca engelmanii complex) somatic embryos were compared to those of zygotic embryos by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting. Somatic embryos contain the same storage proteins as zygotic embryos based on similarities of molecular weight, isoelectric variants, solubility characteristics and disulfide linkages. Storage protein levels varied among different somatic embryo genotypes; however, all genotypes tested accumulated significant amounts of storage proteins. Zygotic and somatic embryos display a similar developmental accumulation of storage proteins. The 22, 24, 33 and 35 kDa proteins appear in early stage embryos, while the 41 kDa protein begins to accumulate during mid cotyledon development. The 22, 24 and 41 kDa proteins accumulate continuously during cotyledon development in somatic embryos cultured on abscisic acid. In contrast, zygotic embryos display a more rapid and transient accumulation of these proteins.     

Storage protein accumulation during zygotic and somatic embryo development in Picea abies (Norway spruce)

Total protein was extracted from zygotic embryos and from somatic embryos of Picea abies (L.) Karst. (Norway spruce) cultured in vitro at different times during their development. An analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 2-dimensional gel electrophoresis of the protein extracts showed that protein composition and the temporal changes in protein abundance were very similar in the two embryo types. Both zygotic and somatic embryos accumulated storage proteins in abundance during their maturation phase of growth; the somatic embryos when cultured on medium containing 90 m M sucrose and 7.6 μ M ABA. The major storage proteins are composed of polypeptides with molecular masses of about 22, 28, 33 and 42 kDa and they are identical in both embryo types according to their molecular mass and average isoelectric points. These proteins are also the most abundant proteins in the female gametophytic tissue of the mature seed.     

Patterns of storage protein and triacylglycerol accumulation during loblolly pine somatic embryo maturation

Conifer somatic embryo germination and early seedling growth are fundamentally different than in their zygotic counterparts in that the living maternal megagametophyte tissue surrounding the embryo is absent. The megagametophyte contains the majority of the seed storage reserves in loblolly pine and the lack of the megagametophyte tissue poses a significant challenge to somatic embryo germination and growth. We investigated the differences in seed storage reserves between loblolly pine mature zygotic embryos and somatic embryos that were capable of germination and early seedling growth. Somatic embryos utilized in this study contained significantly lower levels of triacylglycerol and higher levels of storage proteins relative to zygotic embryos. A shift in the ratio of soluble to insoluble protein present was also observed. Mature zygotic embryos had roughly a 3:2 ratio of soluble to insoluble protein whereas the somatic embryos contained over 5-fold more soluble protein compared to insoluble protein. This indicates that the somatic embryos are not only producing more protein overall, but that this protein is biased more heavily towards soluble protein, indicating possible differences in metabolic activity at the time of desiccation.     

Protein phosphorylation in Streptomyces albus

The phosphorylated proteins of Streptomyces albus, radioactively labeled with [32P]orthophosphate have been analyzed by gel electrophoresis and autoradiography. More than 10 protein species were found to be phosphorylated. With [32P]ATP as substrate cell free extracts phosphorylated endogenous proteins in vitro which were predominantly phosphorylated in vivo. From cell extract which exhibited active phosphorylated in vitro, a protein kinase has been partially purified. The kinase activity was identified in fractions corresponding to a 90 kDa protein.     

The conserved mitotic kinase polo is regulated by phosphorylation and has preferred microtubule-associated substrates in Drosophila embryo extracts.

The Drosophila gene polo encodes a protein kinase required for progression through mitosis. Wild-type polo protein migrates as a tight doublet of 67 kDa which is converted to a single band by phosphatase treatment, which also inactivates the kinase. We have determined putative polo substrates in a cell-free system derived from mutant embryos. Exogenous polo protein kinase phosphorylates proteins of sizes 220 kDa, 85 kDa and 54 kDa, to a greater extent when added to extracts of polo(1)-derived embryos compared with extracts of wild-type embryos, which in both cases have been subject to mild heat treatment to inactivate endogenous kinases. Proteins of the same size are predominantly phosphorylated by the endogenous kinases present in wild-type extracts, and are either not phosphorylated or are poorly phosphorylated in extracts of polo(1)-derived embryos. We show that a specific monoclonal antibody to beta-tubulin precipitates the phosphorylated 54 kDa protein together with an associated 85 kDa protein also phosphorylated by polo protein kinase. Moreover polo binds to an 85 kDa protein which is enriched in microtubule preparations. We discuss the extent to which these in vitro phosphorylation results reflect the effects of mutations in polo on microtubule behaviour during the mitotic cycle.     

Spatio-temporal accumulation and activity of calcium-dependent protein kinases during embryogenesis, seed development, and germination in sandalwood

Western-blot analysis and protein kinase assays identified two Ca(2+)-dependent protein kinases (CDPKs) of 55 to 60 kD in soluble protein extracts of embryogenic cultures of sandalwood (Santalum album L.). However, these sandalwood CDPKs (swCDPKs) were absent in plantlets regenerated from somatic embryos. swCDPKs exhibited differential expression (monitored at the level of the protein) and activity in different developmental stages. Zygotic embryos, seedlings, and endosperm showed high accumulation of swCDPK, but the enzyme was not detected in the soluble proteins of shoots and flowers. swCDPK exhibited a temporal pattern of expression in endosperm, showing high accumulation and activity in mature fruit and germinating stages; the enzyme was localized strongly in the storage bodies of the endosperm cells. The study also reports for the first time to our knowledge a post-translational inhibition/inactivation of swCDPK in zygotic embryos during seed dormancy and early stages of germination. The temporal expression of swCDPK during somatic/zygotic embryogenesis, seed maturation, and germination suggests involvement of the enzyme in these developmental processes.     

Effects of cAMP- and cGMP-dependent protein kinases, and calmodulin on Ca2+ uptake by highly purified sarcolemmal vesicles of vascular smooth muscle

Sarcolemmal fractions of vascular smooth muscles were prepared from porcine thoracic aortae by differential and sucrose density gradient centrifugation. In these fractions, there was a high activity of 5'-nucleotidase, a putative marker enzyme of plasma membrane, and a low activity of rotenone insensitive NADH-cytochrome c reductase a marker of sarcoplasmic reticulum. In these fractions, the Ca2+ uptake was ATP-dependent. A low concentration of saponin which inhibited Ca2+ uptake by the plasma membrane but not by the sarcoplasmic reticulum, inhibited 65% of the Ca2+ uptake of this fraction. The Ca2+ uptake of this fraction was enhanced by cAMP- and cGMP-dependent protein kinases, and by calmodulin. The cAMP-dependent protein kinase enhanced the phosphorylation of 28 and 22 kDa proteins, while the cGMP-dependent protein kinase phosphorylated the 35 kDa protein. The phosphorylation of 100, 75, 65, 41 and 22 kDa proteins was enhanced by Ca2+ and calmodulin. These results indicate that cAMP- and cGMP-dependent protein kinases as well as calmodulin play important roles in Ca2+ transport in the sarcolemma, and that the phosphorylated proteins may be associated with an enhancement of Ca2+ transport in the sarcolemma.     

Possible endogenous substrate proteins for cytosolic protein-tyrosine kinase from porcine spleen.

1. Endogenous phosphate acceptor proteins by cytosolic protein-tyrosine kinase from porcine spleen (CPTK-40) were studied using subcellular fractions of porcine spleen and supernatant fraction of rat various tissues. 2. At least 13 phosphate acceptor proteins ranging from 94 to 26 kDa were observed in all but mitochondrial subcellular fractions. 3. Among the supernatant fraction of rat tissues, brain, testis and spleen contained many phosphate acceptor proteins. 4. The most heavily phosphorylated band of around 55 kDa which was commonly recognized among various tissues was confirmed as tubulin by the immunoreactivity with anti-tubulin antibody. 5. The results obtained in this paper indicate that CPTK-40 has the potential to catalyze the phosphorylation of numerous endogenous proteins including tubulin.     

A tonoplast intrinsic protein (TIP) is present in seeds, roots and somatic embryos of Norway spruce (Picea abies)

Many plant species contain a seed-specific tonoplast intrinsic protein (TIP) in their protein storage vacuoles (PSVs). Although the function of the protein is not known, its structure implies it to act as a transporter protein, possibly during storage nutrient accumulation/breakdown or during desiccation/imbibition of seeds. As mature somatic embryos of Picea abies (L.) Karst. (Norway spruce) contain PSVs, we examined the presence of TIP in them. Both the megagametophyte and seed embryo accumulate storage nutrients, but at different times and we therefore studied the temporal accumulation of TIP during seed development. Antiserum against the seed-specific a-TIP of Phaseolus vulgaris recognized an abundant 27 kDa tonoplast protein in mature seeds of P. abies. By immunogold labeling of sectioned mature megagametophytes we localized the protein to the PSV membrane. We also isolated the membranes of the PSVs from mature seeds and purified an integral membrane protein that reacted heavily with the antiserum. A sequence of 11 amino acid residues [AEEATHPDSIR], that was obtained from a polypeptide after in-gel trypsin digestion of the purified membrane protein, showed high local identity to a-TIP of Arabidopsis thaliana and to a-TIP of P. vulgaris. The greatest accumulation of TIP in the megagametophytes occurred at the time of storage protein accumulation. A lower molecular mass band also stained from about the time of fertilization until early embryo development. The staining of this band disappeared as the higher molecular mass (27 kDa) band accumulated in the megagametophyte during seed development. Total protein was also extracted from developing zygotic embryos and from somatic embryos. In zygotic embryos low-levels of TIP were seen at all stages investigated, but stained most at the time of storage protein accumulation. The protein was also present in mature somatic embryos but not in proliferating embryogenic tissues in culture. In addition to the seed tissue material, the antiserum also reacted with proteins present in extracts from roots and hypocotyls but not cotyledons from 13-day-old seedlings.     

Development of white spruce somatic embryos: I. Storage product deposition

Summary The growth and development of white spruce somatic embryos was followed from the filamentous immature to the mature cotyledonary embryo stage. Histochemical examination of the various stages of embryo development showed that lipids, proteins, and polysaccharides were produced to varying degrees during the process. During early stages (1 to 2 wk on ABA), mostly polysaccharide was produced, whereas during later stages, polysaccharides, lipids, and protein accumulated. Electron microscopy indicated that lipid deposition in somatic embryos started during the first week after transfer to ABA-containing medium. Deposition of the storage products began at the basal end of the embryonal mass and within the proximal zone of the suspensors. Accumulation continued to the peripheral regions and then inward toward the cortex of the developing embryo. In all cases, polysaccharide accumulated first, followed by lipid and lastly, protein. Quantitatively, cotyledonary stage somatic embryos had less lipid and protein and more starch when compared to zygotic embryos at the same developmental stage. Total protein profiles elucidated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that the majority of proteins were similar in zygotic and somatic embryos. Prominent protein bands were found at 30, 20, 19.5, 15, 14.4, 12, and 10 Kd. However, protein bands at 40, 15, and 12 Kd in total protein from somatic embryos were either absent or highly underexpressed.     

Regeneration of Acacia mangium through somatic embryogenesis

 Somatic embryogenesis and whole plant regeneration were achieved in callus cultures derived from immature zygotic embryos of Acacia mangium. Embryogenic callus was induced on MS medium containing combinations of TDZ (1–2 mg/l), IAA (0.25–2 mg/l) and a mixture of amino acids. Globular embryos developed on embryogenic callus cultured on the induction medium. Nearly 42% of embryogenic cultures with globular embryos produced torpedo- and cotyledonary-stage embryos by a two-step maturation phase. The first stage occurred on 1/2-strength MS basal medium containing 30 g/l sucrose and 5 mg/l GA₃ followed by the second stage on 1/2-strength MS basal medium containing 50 g/l sucrose. Of the cotyledonary-stage somatic embryos, 11% germinated into seedlings that could be successfully transferred to pots. Light- and scanning electron microscopy showed that the somatic embryos originated from single cells of the embryogenic callus. Further, a single cell layer could be detected beneath the developing somatic embryos that appeared to be a demarcation layer isolating the somatic proembryonic structure from the rest of the maternal callus. A suspensor-like structure connected the globular embryos to the demarcation layer. This is the first successful report of plant regeneration through somatic embryogenesis for this economically important tropical forest species. Received: 20 January 2000 / Revision received: 28 September 2000 / Accepted: 29 September     

Storage protein dynamics in zygotic and somatic embryos of white fir

Composition and accumulation patterns of storage proteins in female gametophyte and embryos of the white fir (Abies concolor) were investigated during embryogenesis and germination of mature seeds using SDS-PAGE and immunological approach. Altogether 9 major and minor protein components with molecular masses of 14, 16, 22, 24, 27, 30, 35, 38, and 43 kDa were detected in female gametophytes and 9 protein bands in the embryos with the molecular sizes of 14, 16, 22, 24, 25, 27, 34, 38, and 43 kDa. The species seems to deviate in this respect from other representatives of Pinaceae. A conspicuous increase of storage protein synthesis was observed at the stage of fully cellularized female gametophytes and at the cotyledonary stage of embryo development. There exists a high degree of similarity between storage protein profiles of white fir zygotic and somatic embryos. Successive stages of somatic embryogenesis exhibited a high degree of similarity of storage proteins except for cotyledonary stage when a noticeable increase in storage protein synthesis was registered. Conversely, during germination of somatic embryos, an overwhelming majority of storage proteins was depleted.     

Cloning and characterization of the pknA gene from Streptomyces coelicolor A3(2), coding for the Mn2+ dependent protein Ser/Thr kinase

A gene pknA, coding for an eukaryotic-type protein Ser/Thr kinase, was cloned from the Streptomyces coelicolor A3(2) chromosome. The PknA protein kinase, containing the C-terminal eukaryotic-type kinase domain with an N-terminal extension, was expressed in Escherichia coli and Streptomyces lividans. The affinity purified MBP-PknA fusion protein was assayed for kinase activity that showed its ability to autophosphorylate in vitro in the presence of [gamma-32P]ATP. The activity was Mn2+ dependent. The preautophosphorylated kinase phosphorylated at least two proteins (sizes 30 and 32 kDa) in the S. coelicolor J1501 cell-free extracts of all developmental stages. The larger of them was also phosphorylated in vitro by an endogenous protein kinase in late stages extracts, but not earlier. Although Mn2+ dependent protein phosphorylation has previously been described in Streptomyces, this is the first report of a gene encoding such an enzyme in this genus.     

Distribution of Cytosolic mRNAs Between Polysomal and Ribonucleoprotein Complex Fractions in Alfalfa Embryos : Stage-Specific Translational Repression of Storage Protein Synthesis during Early Somatic Embryo Development

Cell-free translational and northern blot analyses were used to examine the distribution of storage protein messages in the cytoplasmic polysomal and mRNA-protein complex (mRNP) fractions during development of somatic and zygotic embryos of alfalfa (Medicago sativa cv Rangelander RL-34). No special array of messages was identified in the mRNP fraction; however, some messages were selectively enriched in either the polysome or mRNP fractions, and their distribution pattern varied quantitatively during development of the embryos. During the earliest stages of somatic embryo development, storage protein messages already were present, but there was no detectable accumulation of the proteins. Selective enrichment of messages for the 11S, 7S, and 2S storage proteins occurred in the mRNP fraction during the globular, heart, and torpedo stages of somatic embryogenesis, but the distribution pattern was shifted toward the polysomal fraction at the beginning of cotyledon development. Thus, there was translational repression of storage protein synthesis at the early stage of somatic embryo development that was relieved later. During the cotyledonary development stages in the somatic and zygotic embryos, storage protein synthesis and distribution of the messages were similar in that these specific messages were predominantly in the polysomal fraction.     

Seed storage proteins in developing somatic embryos of alfalfa: defects in accumulation compared to zygotic embryos

Somatic embryos of alfalfa (Medicago sativa L.) synthesizedall of the major storage proteins of zygotic embryos; an 11Sglobulin (medicagin), a 7S globulin (alfin), and a 2S albumin(LMW). In zygotic embryos (cotyledons and/or axis) these storageproteins accounted for 30%, 10%, and 20%, respectively, of thetotal extractable protein. In somatic embryos the 7S proteinwas predominant while the 11S (particularly subfamily I) and2S proteins were present in lower amounts. Analysis of cultivarsand selfed seed of the embryogenic clone (RL34) demonstratedthat these differences were predominantly physiologically, ratherthan genetically, based. The accumulated 7S and 11S storageproteins of somatic embryos were processed normally, aggregatedas oligomers, and were deposited in protein bodies. This wasnot the case for the 2S storage protein. In somatic embryosthat protein was localized in the cytoplasm rather than in proteinbodies, the site of deposition in zygotic embryos. Key words: Medicago (alfalfa), zygotic/somatic embryos (seeds), storage proteins, immunolocalization     

Post-transcriptional regulation of protein synthesis during alfalfa embryogenesis: proteins associated with the cytoplasmic polysomal and non-polysomal mRNAs (messenger ribonucleoprotein complex)

Cytoplasmic polysomal and non-polysomal mRNA-associated proteincomplexes were isolated from, and characterized in, developingsomatic and zygotic embryos of alfalfa (Medicago sativa L.).³⁵S-methionine-labelled intact embryos were irradiated withultraviolet light (UV) in situ to cross-link mRNA and proteinsoccurring within one bond length, and the polysomal and non-polysomalfractions were extracted. Then the mRNA-protein complexes wereisolated from the fractions and separated using two cycles ofaffinity chromatography on an oligo(dT)-cellulose column. Followingdigestion with RNase A and T1 and micrococcal nuclease, mRNA-associatedproteins were separated by SDS-PAGE. Several polypeptides of 15–150 kDa were associated withthe polysomal and non-polysomal (ribonucleoprotein, mRNP) fractionsof alfalfa embryos after UV-cross-linking. Many of the polypeptidesassociated with the polysomal and non-polysomal mRNAs were qualitativelysimilar, although their concentration in the two fractions wasdifferent. However, some developmentally stage-specific polypeptideswere found to be associated with the non-polysomal mRNA fractionduring the early stages of embryogenesis (precotyledonary) ofsomatic embryos. Thus the presence of mRNPs during embryogenesishas been demonstrated, and proteins intimately associated withthe mRNAs identified. Key words: Embryogenesis, translational control, protein synthesis, messenger ribonucleoproteins, alfalfa (Medicago sativa L.)     

Immunodetection and immunolocalization of globulin storage proteins during zygotic and somatic embryo development in Zea mays

Globulins (GLB) are storage proteins that accumulate to high levels during zygotic embryo development of Zea mays L. We visualized the distribution of GLB during zygotic embryo development by immunolabelling of polyethylene glycol sections with a GLB-specific antiserum and a fluorescent secondary antibody. In sections of embryos at 10 days after pollimation (DAP), GLB were detected in the scutellar node only. Sections of embryos of 17 DAP showed, besides the presence of GLB in the scutellar node, the presence of a low amount of GLB in the coleoptile and the leaf primordia. In 30-DAP embryos GLB were localized in the root, the coleorhiza, the leaf primordia, the coleoptile and in all cells of the scutellum with the exception of the epidermis and the pro-vascular tissues. The subcellular location of GLB was visualized by immunolabelling of ultrathin sections with anti-GLB and a gold-conjugated secondary antibody. Scutellum cells and root cortex cells of 30-DAP embryos were packed with protein storage vacuoles (PSV), which differed in electron density. GLB were either evenly distributed throughout the PSV or were localized in electron-dense inclusions within the PSV. SDS-PAGE and immunoblot analysis of total protein extracts indicated the presence of a low amount of the GLB1 processing intermediate proGLB1' in globular as well as mature somatic embryos. After maturation on an ABA-containing medium, somatic embryos showed the additional presence of the next GLB1 processing intermediate GLB1'. By immuno-electron microscopy it was possible to localize GLB in globular deposits in PSV in scutellum cells of these somatic embryos.