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     

Temporal and nutritional factors modulate responses to abscisic acid and osmoticum in their regulation of storage protein synthesis in developing seeds of alfalfa (Medicago sativa L.)

Storage protein synthesis during alfalfa (Medicago sativa L.)seed development displays a temporal pattern and is regulatedboth by ABA and low osmotic potentials. Deposition of the 2S,7S and 11S storage proteins occurs maximally at the mid–to late stages of development. Osmoticum, but not ABA alonecould effectively induce storage protein synthesis at earlystages of development in isolated alfalfa embryos placed onwater for several days. Neither ABA nor osmoticum alone couldmaintain storage protein synthesis in isolated late-stage embryosplaced on water. On the other hand, isolated developing embryoscultured on a nutrient (Murashige and Skoog) medium could maintainstorage protein synthesis in the presence of ABA and osmoticum,alone or in combination, for up to 14 d after being dissectedfrom the pod. Of the components of this medium, the inorganicsalts appeared to be the most important. The response to ABAand osmoticum varied with the time of isolation during developmentwith the greatest enhancement of storage protein synthesis inisolated embryos coinciding with the time of maximum synthesiswithin the seed when in planta. Addition of ABA and osmoticumafter the time of maximum storage protein synthesis did notelevate the amount of synthesis, but rather prolonged the timeover which it could take place. Thus, while the amplitude ofstorage protein synthesis could be modified by ABA or osmoticum,the inherent temporal pattern, with maximum synthesis occurringonly at mid– to late stages of embryo development, couldnot. Key words: Embryogenesis, Medicago sativa L., alfalfa, abscisic acid, osmotic potential, storage protein synthesis, nutrient supply     

Identification and Characterization of the Seed Storage Proteins from Alfalfa (Medicago sativa)

The major seed storage proteins in alfalfa are medicagin (alegumin-like globulin), alfin (a vicilin-like globulin) anda family of Lower Molecular Weight albumins (LMW₁₃). These comprise30%, 10% and 20%, respectively, of the total extractable proteinfrom cotyledons of mature seeds. Alfin is a heterogeneous oligomericprotein (Mr 150 kD) composed of polypeptides ranging in sizefrom Mr 50 to 14 kD (₁,-₆; 50, 38, 32, 20, 16 and 14 kD, respectively).Medicagin is also a high molecular weight oligomeric protein,but requires high concentrations of salt for solubilization.It is comprised of a family of individually distinct subunits,each composed of an acidic polypeptide (A₁–A₉; Mr 49 to39 kD) linked via disulphide bond(s) to a basic polypeptide(B₁, B₂, B₃; Mr 24, 23 and 20 kD, respectively). This pairingis highly specific and two families are recognizable on thebasis of the B polypeptide (B₃ or B₁/B₂). Subunits (M_r 50–65kD) are assembled as trimers (8S) or larger oligomers (12S–15S)in mature seeds. The lower molecular weight albumins (LMW₁–₃)are acidic (pl<6), and consist of sets of disulphide-bondedpolypeptides (Mr 15 and 11 kD). Key words: Medicago sativa, seed storage proteins, alfin, medicagin     

Cold Acclimation, Freezing Resistance and Protein Synthesis in Alfalfa (Medicago sativaL. cv. Saranac)

Mohapatra, S. S., Poole, R. J. and Dhindsa, R. S. 1987. Coldacclimation, freezing resistance and protein synthesis in alfalfa(Medicago sativa L. cv. Saranac).—J. exp. Bot. 38: 1697–1703. Changes in freezing resistance (percent survival at —10°C), pattern of protein synthesis and translatable mRNApopulation during cold acclimation of alfalfa (Medicago sativaL. cv. Saranac) have been examined. Two days of cold acclimationat 4 °C increased freezing resistance from about 6% to 40%,protein content by 200% and total RNA content by 100%. Acclimationfor longer periods did not cause further increases in freezingresistance, protein content or RNA content. Examination of proteinchanges by sodium dodecyl sulphate-polyacrylamide gel electrophoresis(SDS-PAGE) coupled with protein staining, and by fluorographyof in vivo labelled proteins separated by SDS-PAGE, showed thatseveral proteins are increasingly or newly synthesized duringcold acclimation. Analysis of in vitro translation productsby SDS-PAGE and fluorography shows changes in the populationof translatable mRNAs. It is concluded that in this varietyof alfalfa cold acclimation for only 2 d is sufficient to confermaximum freezing resistance, and that changes in proteins duringcold acclimation are regulated most probably at the transcnptionallevel. Key words: Freezing resistance, protein synthesis, cold acclimation, SDS-PAGE, Medicago sativa L.     

Purification and Characterization of Proteins from the 2S Fraction from Seeds of the Brassicaceae Family

The 2S protein fractions from Brassica rapa, Brassica oleracea,and Brassica napus seeds have been obtained and their componentspurified and characterized. These albumins represent about 13%of the total seed protein extracted with saline buffer. Thecalculated molecular weights of the eleven purified proteinsare very similar, about 14 500 Daltons, although two componentsisolated from B. napus exhibit a lower molecular weight. Theamino acid compositions of the isolated proteins present a seriesof common features: a high content of Cys and basic residuesas well as a very high amount of Pro (15%) and Glx (30%). Theeleven purified proteins cross-react with antibodies againstSin a I, the 2S protein from yellow mustard seeds. The obtainedresults suggest the existence of homology between the 2S albuminsof Brassicaceae seeds. Key words: Brassicaceae, seeds, storage protein     

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.)     

{beta}-Amylase Activity as an Index for Germination Potential in Rice

Seeds of different vigour also differ in their germination ability.In rice (Oryza sativa), this difference was correlated withthe level of incorporation of ³⁵S-methionine into 25-60% ammoniumsulphate precipitable material that was rich in amylase proteins.This protein fraction, from dry seeds, contained no -amylaseactivity. In contrast, ß-amylase activity was presentin all seed stocks capable of 99% germination, although thelevel was lower in seeds that grew slowly when germinated. Inlow viability low vigour stock (i.e. extensively deterioratedseeds) ß-amylase activity was absent. Alpha-amylaseactivity in all stocks was detected only after 24 h from thestart of imbibition. These results indicate that ß-amylaseactivity is reliable indicator of the germination ability ofrice seed stocks and of their vigour during germination.Copyright1995, 1999 Academic Press Rice (Oryza sativa L.,), germination, ß-amylase, -amylase, seed vigour     

The role of abscisic acid in germination, storage protein synthesis and desiccation tolerance in alfalfa (Medicago sativa L.) seeds, as shown by inhibition of its synthesis by fluridone during development

Abscisic acid and osmoticum maintain maturation and proteinsynthesis of developing alfalfa embryos, individually and incombination. The in situ environment of developing alfalfa zygoticembryos is rich in ABA and low in osmotic potential. When ABAsynthesis was inhibited by treating the pods with fluridoneat an early stage of development, the seeds which subsequentlydeveloped contained low amounts of ABA, but had a similar osmoticpotential as untreated control seeds. The reduced ABA in seedsfrom fluridone-treated pods did not change the morphology exceptthe colour of seeds, nor did it induce viviparous germinationor affect storage protein synthesis. However, two nonstorageproteins which were synthesized in control seeds during earlyto mid-development were absent from fluridone-treated seeds.Control seeds containing these two proteins were desiccation-tolerant,whereas the fluridone-treated seeds which lacked them were desiccation-intolerant,at least until the deposition of storage proteins was nearlycomplete. Culture of isolated embryos on nutrient medium inducedgermination and curtailed storage protein synthesis in the embryos.Addition of either ABA or osmoticum to the nutrient medium preventedgermination and maintained storage protein synthesis. When fluridonewas added along with osmoticum, germination occurred, but storageprotein synthesis was maintained. Key words: Embryogenesis, Medicago sativa L., alfalfa, ABA, osmotic potential, fluridone, desiccation, storage protein synthesis     

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.     

Characterization of Seed Protein Fractions from Castanea spp

Seed proteins of Castanea sativa and C. crenata were extractedby sequential procedures. Albumins accounted for over 20% ofthe total protein. Globulins were the main storage proteinsand represented close to half of the total protein. Additionof 2-mercaptoethanol was essential for maximum extraction ofthe globulins, a fraction with an amino acid composition similarto that of the 1 IS storage protein from legumes. Negligibleamounts of prolamins (<3%) were present in the seeds. Glutelinsrepresented just under 30% of the total protein. The proteinfractions were characterized by electrophoresis in polyacrylamidegels. The glutelin fraction contained unextracted globulinseven when 2-mercaptoethanol was used. Key words: Castanea spp, seeds, storage protein     

Auxin and heat shock activation of a novel member of the calmodulin like domain protein kinase gene family in cultured alfalfa cells

A calmodulin like domain protein kinase (CPK) homologue wasidentified in alfalfa and termed MsCPK3. The full-length sequenceof cDNA encoded a 535 amino acid polypeptide with a molecularweight of 60.2 kDa. The deduced amino acid sequence showed allthe conserved motifs that define other members of this kinasefamily, such as serine-threonine kinase domain, a junction regionand four potential Ca²⁺-binding EF sites. The recombinant MsCPK3protein purified from E. coli was activated by Ca²⁺and inhibitedby calmodulin antagonist (W-7) in in vitro phosphorylation assays.The expression of MsCPK3 gene increased in the early phase ofthe 2,4-D induced alfalfa somatic embryogenesis. Heat shockalso activated this gene while kinetin, ABA and NaCl treatmentdid not result in MsCPK3 mRNA accumulation. The data presentedsuggest that the new alfalfa CPK differs in stress responsesfrom the previously described homologues and in its potentialinvolvement in hormone and stress-activated reprogramming ofdevelopmental pathways during somatic embryogenesis. Key words: Medicago sativa, CPK, stress, 2,4-D, phosphorylation, somatic embryogenesis.     

Control of Pratylenchus penetrans and Meloidogyne hapla and Yield Response of Alfalfa Due to Oxamyl Seed Treatments

Alfalfa (Medicago sativa L. cv. Saranac) seed were soaked for 20 minutes in water, acetone, or methanol containing 10 or 50 mg/ml of oxamyl (Vydate L) or coated with a 2% aqueous cellulose solution containing the same amounts of oxamyl. Seed were analyzed for oxamyl by HPLC immediately after treatment and after 9 and 26 months of storage. Oxamyl content of alfalfa seed did not decline after 26 months of storage. The effects of seed treatment on growth of alfalfa and nematode control were examined using soils infested with Pratylenchus penetrans and Meloidogyne hapla. Germination was not affected by any of the seed treatments. Twenty-one days after sowing, the total growth of alfalfa seedlings grown from seed treated with 50 mg/ml of oxamyl in P. penetrans-infested soils had increased by 62% over controls. Nodulation per pot increased by as much as 267%, and the densities of P. penetrans per gram of root were reduced by as much as 73% compared to control plants. In M. hapla-infested soils, increases in plant growth (32%) and nodulation (71%) also occurred with oxamyl-treated seeds. Root gall reduction (86%) was also substantial due to oxamyl seed treatment.     

Deterioration of western redcedar (Thuja plicata Donn ex D. Don) seeds: protein oxidation and in vivo NMR monitoring of storage oils

Deterioration of conifer seeds during prolonged storage hasa negative impact on reforestation and gene conservation efforts.Western redcedar (Thuja plicata Donn ex D. Don) is a speciesof tremendous value to the forest industry. The seeds of thisspecies are particularly prone to viability losses during long-termstorage. Reliable tools to assess losses in seed viability duringstorage and their underlying causes, as well as the developmentof methods to prevent storage-related deterioration of seedsare needed by the forest industry. In this work, various imagingmethods and biochemical analyses were applied to study deteriorationof western redcedar seeds. Seedlots that exhibited poor germinationperformance, i.e. those that had experienced the greatest lossesof viability during prolonged storage, exhibited greater abundanceof oxidized proteins, detected by protein oxidation assays,and more pronounced changes in their in vivo ¹³C NMR spectra,most likely due to storage oil oxidation. The proportion ofoxidized proteins also increased when seeds were subjected toaccelerated ageing treatments. Detection of oxidized oils andproteins may constitute a reliable and useful tool for the forestindustry. Key words: Conifer seeds, in vivo NMR spectroscopy, MRI, oil peroxidation, protein carbonylation, seed deterioration, seed storage, storage lipids, western redcedar Received 23 May 2007; Revised 28 November 2007 Accepted 17 December 2007     

Turnover of Storage Protein in Seeds of Soya Bean and Pea

We were interested in determining whether the low protein contentof pea seeds (Pisum sativum L.) as compared to soya bean seeds(Glycine max L. Merrill) might be due to faster degradationof the pea storage proteins during development of the seed.Pea and soya bean cotyledons were subjected to a ‘pulse-chase’experiment using [³H]glycine in in-vitro cultures. In peas,legumin had a half-life of 146 days, while vicilin had a half-lifeof 39 days. There was no measureable degradation of soya beanstorage proteins. Even with the pea storage proteins, the half-liveswere so much longer than the maturation time of seeds that degradationof storage proteins could not account for the lower proteincontent of peas as compared to soya beans. The validity of theseresults was indicated by the finding that non-storage proteinshad much shorter half-lives and that omission of a carbon ora nitrogen source greatly accelerated degradation. Labelledglycine was found to be a good probe for protein turnover studiesbecause it was very rapidly metabolized. Glycine max L. Merrill, soya bean, Pisum sativum, L. pea, protein turnover, storage proteins, legumin, vicilin     

Seed Longevity of Rice Cultivars and Strategies for their Conservation in Genebanks

Changes in seed quality during ripening were studied in sixteencultivars of rice, representing the three ecogeographic racesofOryza sativa, and one cultivar ofO. glaberrima, grown duringone dry season (Nov. –May) 1992 –1993 at Los Baños, Philippines. Mass maturity (defined as the end ofseed filling period) among the cultivars was attained between18.5 and 21.6d after anthesis (DAA). The seed moisture contentat mass maturity varied between 24 and 40%. Germination abilityof seeds in the early stages of development varied significantly,but as mass maturity approached, germination increased to themaximum and no significant differences were found among cultivars.The seeds were stored hermetically at 35 °C with 15±0.2%moisture content and the resultant seed survival data were analysedby probit analysis. Potential longevity (quantified by the valueof seed lot constantK_iof the seed viability equation) was greatestbetween 33 and 37 DAA, i.e. about 2 weeks after mass maturity.The stage during development at which seeds achieve maximumpotential longevity is described by the term storage maturity.Lowlandjaponicacultivars, large seeded accessions (seed mass40mg) andO. glaberrimahad shorter storage longevity ( , standarddeviation of the frequency of seed deaths in time=1.47 weeks)while cultivars with purple pericarp survived longer than othercultivars ( =2.33 weeks). The initial germination of thejaponicacultivarsat storage maturity was high (99 –100%) and the estimatesof maximum potential longevity (K_i) which ranged between 3.3(Shuang cheng nuo) and 4.4 (Minehikare) were close to thoseof theindicacultivars. This research suggests that seed production environment betweenNov. and May at Los Ba ños is benign for the temperatejaponicacultivars.The implications of these results on management of rice geneticresources are discussed. Oryza sativaL.; rice; germplasm conservation; seed production environment; seed development; seed longevity     

In vivo Studies on Protein Synthesis in Developing Seeds of Canavalia gladiata D.C.

In successive stages of seed development of Canavalia gladiata,fruits were harvested and time-course changes of seed proteinaccumulation and SDS-gel electrophoretic patterns of the polypeptideswere examined. Albumin content increased gradually after thestage of 1.8 g fresh weight per seed, reached a peak, then decreasedat later stages. SDS-gel electrophoresis showed a clear changein albumin composition between the stages of 2.3 g and 3.6 gfresh weight per seed. Seed globulins were synthesized activelyafter the stage of 1.8 g fresh weight per seed, and the majorpolypeptides of seed globulins, including the main polypeptidesof canavalin, were first detected at this stage by gel electrophoresis.In contrast to the albumins, these polypeptides of globulinscontinued to accumulate until the maturation was completed.In vivo protein synthesis was also analyzed by SDS-gel electrophoresisand fluorography, after [³⁵S]methionine had been fed to cotyledonsections of maturing seeds at the stages of 3.4 g and 5.0 gfresh weight per seed. The results indicated that the synthesisof canavalin starts earlier than that of concanavalin A andalso ends earlier during seed development. (Received December 10, 1985; Accepted June 2, 1986)     

Energy-Level Model for Isothermal Seed Germination

We have been successful in building an energy-level model thatdescribes seed germination. We used the autocatalytic reactionrate equation to fit the germination rate for seed germination.The two parameters [A]₀ and [F]₀ were found by fitting the integratedgermination rate equation to the data. The values of [A]₀ and[F]₀ obey the Arrhenius equation and give activation energiesfor the second and third stage of the four-compartmental modelof seed germination. The thermodynamics of isothermal seed germinationis proposed and the enthalpy, entropy, and free energy are calculatedfor the transition from state A to state F. The time delay isa function of temperature and it leads to a rate constant thatcan be used to get the activation energy for the total germinationprocess. We believe the model is universal. It fits alfalfa(Medicago sativa), turnip (Brassica rapa), and lettuce (Lactucasativa) seeds. Key words: Seed germination, thermodynamics, kinetics, Arrhenius, energy-level model     

Quantal Response of Fruit and Seed Germination Rate in Quercus robur L. and Castanea sativa Mill, to Constant Temperatures and Photon Dose

A linear relationship between constant temperatures in the sub-optimaltemperature range and germination rate is shown in both Quercusrobur L and Castanea sativa Mill germinated under nominal darkconditions. The mean base temperature was interpolated for Qrobur as 0 8 ? or 2-4 ?, depending on seed lot provenance, andfor C. sativa as 1 -4? The optimum temperature for germinationin Q. robur was about 20? compared with around 28 ? in C. sativaOver the sub-optimal temperature range the distribution of thermaltimes was log-normal for each population studied their spreadvarying both between Q robur seed lots and between species However,in C. sativa germinated close to the mean base temperature,the distribution in thermal times was reduced Thermal timesto germination were decreased in Q. robur and C sativa by approximately0 3 and 0-5 log-units, respectively, when the pericarp was removed,i.e in the seeds, but the sensitivity of the response remainedrelatively unaltered In both species the germination rate was the same when nominaldark or safe green light conditions were employed during thegermination test. However, at 21 ? Q robur exhibited the highirradiance reaction (HIR) at photon doses above 30mmol m^–2d^–1. HIR first affected the germination rate by an inhibitionof radicle extension The sensitivity of the response to thermaltime was reduced as photon dose increased. This photo-inhibitionwas exacerbated at supra-optimal temperatures. In contrast,C. sativa germination rate at 26 ? was little influenced bylight at a photon dose of 752 mmol m^–2 d^–1 Key words: Seed germination rate, temperature, thermal time, light, photon dose