Protein Synthesis During Rehydration, Germination and Seedling Growth of Naturally and Precociously Matured Soybean Seeds (Glycine max)

Soybean seeds [Glycine max (L.) Merr.] synthesize de novo andaccumulate several non-storage, soluble polypeptides duringnatural and precocious seed maturation. These polypeptides havepreviously been coined ‘maturation polypeptides’.The objective of this study was to determine the fate of maturationpolypeptides in naturally and precociously matured soybean seedsduring rehydration, germination, and seedling growth. Developingsoybean seeds harvested 35 d after flowering (mid-development)were precociously matured through controlled dehydration, whereasnaturally matured soybean seeds were harvested directly fromthe plant. Seeds were rehydrated with water for various timesbetween 5 and 120 h. Total soluble proteins and proteins radio-labelledin vivo were extracted from the cotyledons and embryonic axesof precociously and naturally matured and rehydrated seed tissuesand analyzed by one-dimensional PAGE and fluorography. The resultsindicated that three of the maturation polypeptides (21, 31and 128 kDa) that had accumulated in the maturing seeds (maturationpolypeptides) continued to be synthesized during early stagesof seed rehydration and germination (5–30 h after imbibition).However, the progression from seed germination into seedlinggrowth (between 30 and 72 h after imbibition) was marked bythe cessation of synthesis of the maturation polypeptides followedby the hydrolysis of storage polypeptides that had been synthesizedand accumulated during seed development. This implied a drasticredirection in seed metabolism for the precociously maturedseeds as these seeds, if not matured early, would have continuedto synthesize storage protein reserves. Glycine max (L.) Merr, soybean, cotyledons, maturation, germination/seedling growth     

Changes in Seed Constituents During Germination and Seedling Growth of Precociously Matured Soybean Seeds(Glycine max)

During 7 d of precocious maturation of soybean seed (Glycinemax), the starch content declined and soluble sugar levels increasedin patterns similar to natural seed dehydration and maturation.Total seed protein content and total seed dry weight increasedwhereas oil content remained relatively unchanged. Overall,the proportions of the constituents in precociously maturedseeds were comparable to naturally mature seeds. Precociouslymatured soybean seeds showed much the same germination and seedlinggrowth frequency patterns as naturally matured seeds. Duringgermination and seedling growth of precociously matured seeds,starch, soluble sugar, protein and oil levels followed patternssimilar to naturally mature, germinating seeds and seedlings.Therefore, precocious maturation may be used as a model systemto investigate the control of the physiological and biochemicalevents occurring during seed maturation which lead to germinationand subsequently, seedling growth. Glycine max (L.) Merr., soybean, cotyledons, maturation, germination/seedling growth     

Changes in RNA and Protein Metabolism Associated with Alterations in the Germination Efficiency of Sunflower Seeds

Precise knowledge of seed quality after harvest and during storageis of particular importance for seed producers. We analyseddifferent sunflower seed lots (Helianthus annuusL.) characterizedby extremes of germination ability. We used RNA analysis tostudy possible changes in gene expression in seeds unable togerminate. Total RNA content was very small in dry seeds showinga low germination ability. Capacity for total RNA synthesisat the onset of imbibition was also reduced in these seeds.In addition, correlations were found between these parametersand germination ability at 19 °C. We demonstrated a highcorrelation between the amount of total RNA in the dry seed,the capacity of RNA synthesis at the onset of imbibition andthe seed moisture content at the time of the harvest. The abilityof dry seed mRNAs to be translatedin vitrowas also reduced andseven polypeptides, from stored mRNAs, were characteristic ofthe cotyledons from high germinability seeds. Germination canthus be affected at several levels including membrane, enzymaticand nucleic acid deteriorations. Gene expression; germination ability; Helianthus annuusL.; marker; protein; RNA; seed; sunflower     

Metabolic Changes in Axes of Germinating Vigna unguiculata Seeds as Related to Effects of Removal of Cotyledons

[¹⁴C]-Labeled amino acids and sucrose were fed to Vigna unguiculataseeds through cut-ends of cotyledons, and incorporations ofradioactivity into trichloroacetic acid- and 80% ethanol-insolublefractions of axes, respectively, were followed during 48 h ofthe post-imbibition development. The results of these studies,together with determinations of changes in dry weight and proteincontents after the onset of imbibition, indicated that the reservematerials stored in cotyledons were available for active growthof axes only after 12 h of post-imbibition. However, pulse-labelingexperiments, where [³H]-labeled leucine and uridine were feddirectly to axes attached to or detached from cotyledons, indicatedthat synthesis of protein and RNA in both axes was very pronouncedeven at earlier stages (2–8 h) of post-imbibition. Albuminand globulin proteins of axes disappeared most rapidly duringthe 6–12 h period of post-imbibition. Cycloheximide, -amanitinand cordycepin added to imbibing axes inhibited the degradationof major globulin proteins, whereas the inhibitors had littleeffect on the degradation of major albumin proteins. Both proteolyticand amylolytic activities were found to occur in embryonic axesof ‘dry’ seeds, and increased to higher levels asthe germination proceeded. Axes at early stages of germinationmay degrade the self-sustained reserve proteins and utilizethem for the synthesis of new proteins. (Received June 11, 1983; Accepted August 16, 1983)     

Synthesis of 125I-ubiquitin conjugates in extracts of Lemna minor

Low levels of ubiquitin conjugating activity are typically detectedin the green tissues of plants, an observation that may at leastpartially explain why no method to purify multi-ubiquitinatedproteins from photosynthetic cells has been reported in theliterature. The present paper provides a contribution to improvethe available methodology for the isolation of an efficientubiquitin conjugating system from photosynthetic cells. We haveselected Lemna minor L. as a plant system and have developeda simple and rapid methodology to synthesize and purify highmolecular mass ubiquitin-protein conjugates, formed with endogenoussubstrates and exogenous ¹²⁵I-ubiquitin, using small amounts(<2g) of green tissue. It is demonstrated that L. minor possessesan ATP-dependent activity capable of forming ubiquitin conjugateswith endogen ous proteins in vitro. Anion exchange chromatographyon diethylaminoethyl-cellulose provides a simple and rapid techniqueto remove endogenous ubiquitin and to concentrate and partiallypurify the enzyme system responsible for ubiquitin conjugatingactivity. This enriched fraction has therefore been utilizedto syn thesize high molecular mass ¹²⁵I-ubiquitin conjugatesformed with L. minor proteins. These conjugates were subsequentlypurified by directly loading the reaction mixture on a SephacrylS-300 gel filtration column, with no requirement for additionalconcentration or purification steps. This methodology is highlyreproducible. Key words: Ubiquitin, ubiquitin-protein conjugates, synthesis, purification, Lemna minor     

Transport and Metabolism of Dihydrozeatin Riboside in Germinating Lupin Seeds

[³H]-dihydrozeatin riboside was applied selectively to the embryonicaxes or to the cotyledons of germinating lupin (Lupinus luteusL. cv. Weiko III) seeds 6 h following the start of imbibition.There was little transport of dihydrozeatin riboside from embryoto cotyledons up to 6 h after the application, but a substantialamount of radioactivity had moved into the cotyledons at theend of the 10 h incubation period. However, there was no detectablemovement of [³H]-dihydrozeatin riboside from the cotyledonsto the embryonic axis. This indicated a highly polarized movementof cytokinins during the early stages of seed germination. Exogenouslyapplied [³H]-dihydrozeatin riboside was found to be very stable,both when applied to the embryonic axes and cotyledons of intactseed, or following excision, and there was little metabolismwith only small amounts of radioactivity found associated withdegradative metabolites. The embryonic axis of this specieshas recently been found to synthesize cytokinins within 12 hfrom the start of imbibition, and the results of this studyindicate that the embryo-derived cytokinin is probably transportedto the cotyledons where it accumulates and subsequently participatesin the control of cotyledon function. Key words: Lupinus luteus, cytokinin transport and metabolism, dihydrozeatin riboside, seed germination     

Soya Bean Seed Growth and Maturation by In Vitro Pod Culture

Immature Glycine max (L.) Merr. seeds initially at 50–70mg fresh weight were successfully grown and matured in vitroin detached pods. Surface sterilized pods were floated in aliquid medium containing 5 per cent sucrose, minerals, and glutaminein 125 ml Erlenmeyer flasks and incubated at 25 °C under350–400 µE m^–1 s^–1 white light. Seedswhich were matured in vitro increased tenfold in dry weight,were visually similar to commercial seeds of the same size,were tolerant to desiccation and germinated with normal seedlinggrowth. Excised pods transported dye from the pedicel to thegrowing seed within 120 min. Soya bean pod culture is a usefultechnique to study the influence of single or combinations ofchemical or environmental parameters on regulation of seed growth,seed maturation, and subsequent germination events without theconfounding interactions with the mother plant. Glycine max (L.) Merr., soya bean, pod culture, seed culture, seed growth, seed maturation, germination     

Some Effects of Potassium Deficiency on Seedling Development

Seedlings with leaf-like photosynthetic cotyledons such as Cucumissativus, Sinapis alba and Trifolium pratense have higher growthrates and a greater requirement for an external supply of potassiumthan hypogeal species (Pisum sativum, Vicia faba) or epigealspecies with very fleshy cotyledons (Phaseolus vulgaris, Lupinusalbus, Lupinus angustifolius). The initial stages of seedlingdevelopment in the latter two groups involve mainly transferof reserves from cotyledons to axis and a relatively small increasein total seedling dry weight takes place during the first 2to 3 weeks. Potassium may be drawn either from the supply already presentin the cotyledons or from an external source. Species with expandingphotosynthetic cotyledons utilize the reserve potassium duringcotyledon development and it is not transported to the epicotyl.Thus, in these species an external supply of potassium is essentialfor full development of the photosynthetic system and the roots.In species whose cotyledons are not major photosynthetic organsthe reserve potassium is transported from the cotyledons tothe developing axis. In these species culture in a nutrientsolution deficient in potassium or in distilled water increasesroot dry weight but reduces plumule weight (although to a lesserextent than in species with leaf-like cotyledons). In general,the response of seedlings to culture in a nutrient solutiondeficient in potassium was very similar to culture in distilledwater only. Thus, although all the species studied had similar levels ofpotassium in the seed (µg potassium/mg dry weight) itssite of utilization differs. It appears that seedlings withleaf-like cotyledons are more susceptible during early seedlinggrowth to deficiencies in external factors, such as potassium,than are species with fleshy cotyledons.     

Effects of Nutrient Supply on Pre-emergence Growth and Nutrient Absorption in Wheat (Triticum aestivumL.) and Sugarbeet (Beta vulgarisL.)

Nutrient absorption in wheat and sugarbeet was studied duringpre-emergence growth by adding 0, 7, 10.5 or 14 mol m^-3nitrogen(N) to the growth medium. Seedling growth and carbon, N and¹⁵Ncontents of the seedling parts were measured. Differences betweenthe natural abundance of¹⁵N in seeds and in nutrient solutionwere used to determine the proportion of N in the organs originatingfrom seed reserves and from absorption. Absorption began laterfor wheat than for sugarbeet and had less effect on seedlinggrowth. The absorbed N was found mainly in roots. Compared towheat, sugarbeet seedling N content was greatly altered andthe hypocotyl showed increased elongation when nutrients wereadded. Most of the absorbed N was found in the radicle and hypocotylwith less in the cotyledons. Sugarbeet seedling emergence andearly growth could be decreased by adverse conditions occurringafter sowing by affecting mineral availability in the soil orthrough altered root absorption.Copyright 1998 Annals of BotanyCompany Triticum aestivumL., wheat,Beta vulgarisL., emergence, natural isotopic composition, seedling, seed reserves     

Changes in mRNA of Vigna mungo Cotyledons during Seed Germination

Quantitative and qualitative changes of mRNA in Vigna mungocotyledons during seed germination have been investigated. TotalRNA is higher in dry cotyledons and declines during germination.Poly(A)⁺ RNA also is present at a relatively high level in drycotyledons, increases slightly during the first day of germination,and then decreases. Polysomal RNA is very low in dry cotyledonsbut increases rapidly during the first day of germination, andthen declines. The translational activity of the mRNA in a wheatgerm cell-free system is low on day 0 but increases rapidlyon day 1 of germination. Two-dimensional gel electrophoresisof in vitro translation products reveals that many new peptidesare synthesized on day 1 of germination. Synthesis of most ofthese polypeptides continue throughout 5 days of germination. Change in the mRNA population during germination has been investigatedusing cDNA against poly(A)⁺ RNA from 3-day-old cotyledons. Withtotal RNA of day 3 and 5, the cDNA strongly hybridized withRNA similar in size to 25 S ribosomal RNA, but no specific bandsare detected with samples of day 0 or 1. With poly(A)⁺ RNA ofday 5 or 1, the cDNA tends to hybridize with RNAs of relativelysmall molecular size. Cordycepin and -amanitin prevent the increasein poly (A)⁺ RNA content and the appearance of new mRNAs duringthe first day of germination. ¹Present address: Division of Regulation of Macromolecular Function,Institute for Protein Research, Suita City, Osaka 565, Japan. (Received January 13, 1986; Accepted June 10, 1986)     

Degradation of Protein Bodies in Germinating Seeds of Brassica campestris L. var. sarson Prain

Histochemical investigations were carried out on dry and germinatingseeds of Brassica campestris var. sarson, to study the degradationof protein bodies with globoidal inclusions. During germination,a wave of protein body degradation sets in from the radicularend of the embryo, passing through the hypocotyl and shoot apex,and ending in the cotyledons. The digestion of protein bodiesis of the internal type. The various isolated parts of the embryoshowed a similar pattern of protein body digestion to that ofthe whole embryo, except that in some cells the globoids persistedeven after complete digestion of protein bodies; the rate wasfaster in the comparatively more expanded part of the isolatedorgans. No specific factor controlling the initiation of thewave of protein body digestion could be ascertained. Brassica campestris var. sarson, yellow sarson, seed, germination, protein bodies, degradation     

Response Breadths on Environmental Gradients of Germination and Seedling Growth in Two Dominant Forest Tree Species of Central Himalaya

The responses of Pimis roxburghii (chir pine), an early successionalspecies, andQuercus leucotrichophora(banj oak), a late successionalspecies were compared in terms of seed germination and seedlinggrowth. Seed germination was observed on single-factor gradientsof temperature, water stress, seed desiccation and light, andseedling growth on gradients of shade and soil moisture. Thesuppression of germination caused by continuous dark and far-redwas greater in Pinus than in Quercus. Pinus seed germinationshowed wider response breadth on gradients of water stress andseed desiccation, and narrower response breadth on the gradientof light quality, compared withQuercus. In terms of seedlinggrowth, the response breadth of Pinus was wider on the gradientof soil moisture and narrower on the gradient of shade comparedwith Quercus. Under higher soil moisture stress, the seedlingsof both species attained similar heights, butQuercus seedlingsachieved higher dry weights, a higher root: shoot ratio andlower leaf weight ratio than Pinus.     

Proteolytic Processes Underlying Molt-Induced Claw Muscle Atrophy in Decapod Crustaceans

The claw muscles of large-clawed decapod crustaceans undergoa programmed atrophy in preparation for molting, or ecdysis.This is mediated by five cytosolic proteinases organized intotwo proteolytic pathways: calcium-dependent and ubiquitin/proteasome-dependent.The calcium-dependent system consists of four calcium-dependentcysteine proteinases (CDPs I, IIa, IIb, and III; native masses310, 125, 195, and 59 kDa, respectively) that completely degrademyofibrillar proteins and are activated in atrophic muscles.Immunological analysis shows that the active-site sequence inCDP IIa (60-kDa subunit mass) is similar to that in mammalianCDPs (calpains), and that CDP IIb is homologous to a calpain-likegene isolated from Drosophila cDNA libraries. Increased intracellullarCa²⁺ stimulates proteolysis in situ, indicating CDPs play animportant role in muscle protein catabolism. The ubiquitin/proteasome-dependentsystem involves the ATP-dependent conjugation of multi-ubiquitinchains to protein by ubiquitin-conjugating enzymes. This actsas a signal for substrate degradation by the 26S proteasome,a multi-subunit complex consisting of a 20S proteasome catalytic"core" and two PA700 (19S) regulatory complexes. PolyubiquitinmRNA, ubiquitin-protein conjugates, and 20S proteasome are elevatedabout 5-, 8-, and 2-fold, respectively, during atrophy. A heat-inducedform of the 20S proteasome hydrolyzes myosin, troponin, andtropomyosin to large fragments in vitro. Biochemical studiesidentified the branched-chain amino acid-preferring (BrAAP)activity, one of six distinct catalytic components in the complex,as the activity that carries out these initial cleavages. Theseresults indicate that the ubiquitin/proteasome pathway is involved,but its precise role remains to be resolved.     

Cytoplasmic HSP70 homologues of pea: differential expression in vegetative and embryonic organs

Eukaryotes express several cytoplasmic HSP70 genes, and their encoded proteins participate in diverse cellular processes. Three cDNAs encoding highly expressed cytoplasmic HSP70 homologues from Pisum sativum were cloned and characterized. They were designated PsHSP71.2, PsHSC71.0, and PsHSP70b. These HSP70 genes have different expression profiles in leaves: PsHSP71.2 is observed only in response to heat stress, PsHSC71.0 is present constitutively, and PsHSP70b is weakly constitutively expressed, but induced strongly in response to heat stress. In addition to being heat induced, the PsHSP71.2 mRNA is also expressed in zygotic, but not maternal organs of developing pea seeds, while PsHSC71.0 and PsHSP70b mRNAs are present in maternal and zygotic organs throughout seed development. Immunoblot analysis of parallel protein samples detects a 70 kDa polypeptide in all samples, and a 72 kDa polypeptide that corresponds to the PsHSP71.2 gene product is observed in cotyledons beginning at mid-maturation and in axes beginning between late maturation and desiccation. This polypeptide is not detected in the seed coat. The 72 kDa polypeptide remains abundant in both cotyledons and axes through germination, but declines substantially between 48 and 72 h after the onset of imbibition. Differential control of HSP70 expression during heat stress, seed maturation, and germination is consistent with the hypothesis that there are functional distinctions between cytoplasmic HSP70s.     

Cytokinins in Germinating Seeds of Phaseolus vulgaris L. II. Transport and Metabolism of 8[14C]t-Zeatin Applied to the Radicle

The application of 8[¹⁴C]t-zeatin to the radicle tips of germinatingPhaseolus vulgaris seeds revealed that cytokinins are transportedrapidly from the embryonic axis to the cotyledons, and are utilizedand metabolized extensively in these organs. The informationobtained on the transportation between the different parts ofthe embryo is consistent with the view that the mobilizationof food reserves from the cotyledons is controlled by cytokininswhich originate in the embryonic axis. Tentative identificationof the radioactive metabolites formed indicate that the appliedzeatin was altered by side-chain cleavage and by substitutionto the adenine ring. Phaseolus vulgaris, bean, germination, cytokinins, transport, radicle     

Seed Water Relations and the Regulation of the Duration of Seed Growth in Soybean

Soybean [Glycine max (L.) Merrill] seeds and cotyledons weregrown in an in vitro culture system to investigate the relationshipsbetween cell expansion (net water uptake by the seed) and drymatter accumulation. Seeds or cotyledons grown in a completenutrient medium containing 200 mol m^–3 sucrose continueddry matter accumulation for up to 16 d after in planta seedsreached physiological maturity (maximum seed dry weight). Seedor cotyledon water content increased throughout the cultureperiod and the water concentration remained above 600 g kg^–1fresh weight. These data indicate that the cessation of seeddry matter accumulation is controlled by the physiological environmentof the seed and is not a pre-determined seed characteristic.Adding 600 mol m^–3 mannitol to the medium caused a decreasein seed water content and concentration. Seeds in this mediumstopped accumulating dry matter at a water concentration ofapproximately 550 g kg^–1. The data suggest that dry matteraccumulation by soybean seeds can continue only as long as thereis a net uptake of water to drive cell expansion. In the absenceof a net water uptake, continued dry matter accumulation causesdesiccation which triggers maturation. Key words: Glycine max (L.) Merrill, solution culture, duration of seed growth, water content, dry matter accumulation     

Citrate metabolism in. soybean cotyledons

Citrate metabolism and the citrate cleavage enzyme were investigatedin soybean (Glycine max (L.), Merr.) cotyledons throughout developmentand during the first 5 days of germination. It was noted thatboth the lipid synthesizing and the acetyl CoA generating systemsare present when the soybean seed matures, and that the activityof these systems declines throughout development as citrateincreases. Citrate represents 1% of the cotyledon dry weightat seed maturity. During the first 24 hr of germination, therewas an activation of the citrate cleavage enzyme and a concomitantdrop of some 60% in citrate content. Accompanying the drop incitrate content is the de novo synthesis of fatty acids foruse in the production of phospholipids. All of the data areconsistent with the hypothesis that acetyl CoA for lipid synthesisis supplied by the citrate molecule via the citrate cleavageenzyme and that activity of this system is necessary both duringseed development and during germination. ¹ Research was supported by cooperative investigations of theAgricultural Research Service, United States Department of Agriculture,and Illinois Agricultural Experiment Station. ² This research represents partial fulfillment of Ph.D. degree. (Received September 20, 1976; )     

Growth Analysis of Soybean Seedlings During the Lifespan of the Cotyledons

Patterns of seedling growth of Glycine max in light and darknesswere compared during the period from germination to cotyledonabscission. Fitted growth curves and the derived functions,relative growth rate, unit leaf rate, leaf area ratio and specificleaf area, were used to assess the relative importance in seedlinggrowth of cotyledon storage reserves, cotyledon photosynthesisand leaf photosynthesis. The cotyledons are of an intermediatetype with a predominant storage and a minimal photosyntheticfunction. Cotyledon reserves support seedling growth until theprimary leaves expand, after which growth depends on leaf photosynthesis. Glycine max (L.) Merr., soybean, cotyledons, growth analysis, seedling development     

The r{beta}-Conglycinins of Soybean Remain Assembled in a 7S Conformation While Undergoing Proteolytic Cleavage During Germination and Early Seedling Growth

The degradation of the rß-conglycinins, the secondmost abundant seed storage protein complex of Glycine max, thatoccurs as a result of proteolysis during seed germination andearly seedling growth, was investigated. The rß-conglycininsof soybean are composed of a semi-random association of threedifferent subunits, a', a, and rß, in a trimeric complexwith a sedimentation coefficient of 7S. Western immunoblot analysisof the degradation products showed that proteolytic cleavageyields specific fragments as has been shown in other legumes.The proteolytic fragments produced in G. max, cv. Provar aredesignated here as FPI (62 kDa), FPII (57 kDa), FPIII (52 kDa),FPIV (31 kDa), and FPV (27 kDa). Comparison of the fragmentsfrom G. max cv. Keburi, a variant lacking the a' subunit, withthose from G. max cv. Provar showed that the FPIV fragment isderived from the a' subunit. All fragments stained with periodicacid-Schiff's reagent, indicating that exhaustive deglycosylationof these subunits is not a prerequisite for cleavage. All ofthe fragments detected in these experiments sediment in linearsucrose density gradients with sedimentation coefficients ofabout 7S, identical to that of intact rß-conglycinin.These results suggest that as proteolysis of the rß-conglycininsoccur during germination and early growth, the cleavage productsare retained within the holoprotein structure. Further proteolysiscleaves the polypeptides into smaller fragments leading to thedisintegration of the 7S storage protein structure.