The Relationship of Protein Fractions and Individual Proteins to Seedling Vigour in Wheat

Foliar applications of urea were applied to winter wheat (Triticumaestivum L. cv. Logan) to study the relationships between seedsize, total, fractional, and individual protein contents andseedling vigour. Seedling vigour was found to be closely relatedto the total protein content of the whole seed and endosperm.Seedling vigour was also related to the salt-insoluble and salt-solublefractions of the endosperm. The majority of individual proteinsas separated by SDS gel electrophoresis from both the salt-solubleand -insoluble fractions of the endosperm were positively correlatedwith seedling vigour. In growth analysis studies, while the high-protein seeds lostweight and protein more rapidly than low-protein seeds, no appreciabledifferences in net assimilation rate, relative growth rate orleaf area ratio were detected. The ratio of insoluble to soluble proteins did not change withseed size but was increased by urea applications. The nitrogencontent of the gliadin fraction was increased by nitrogen fertilizer,foliar applications of urea and sub-toxic levels of herbicideswhile concomitantly the nitrogen content of the glutenin fractiondecreased.     

The Role of Maturation Drying in the Transition from Seed Development to Germination: III. INSOLUBLE PROTEIN SYNTHETIC PATTERN CHANGES WITHIN THE ENDOSPERM OF Ricinus communis L. SEEDS

Kermode, A. R., Gifford, D. J. and Bewley, J. D. 1985. The roleof maturation drying in the transition from seed developmentto germination. III. Insoluble protein synthetic pattern changeswithin the endosperm of Ricinus communis L. seeds.—J.exp. Bot. 36: 1928–1936. Immature seeds of Ricinus communisL. cv. Hale (castor bean) removed from the capsule at 30 or40 days after pollination (DAP) can be induced to germinateby being subjected to drying. This desiccation–inducedswitch from development to germination is mirrored by a change,upon subsequent rehydration, in the pattern of insoluble proteinsynthesis within the endosperm storage tissue. During normaldevelopment from 25–40 DAP there is rapid synthesis ofthe insoluble (11S) crystalloid storage protein. At later stagesof development (45 and 50 DAP), crystalloid protein synthesisdeclines markedly and synthesis of new insoluble proteins commences.Following premature drying at 30 or 40 DAP, the pattern of insolubleprotein synthesis upon rehydration is virtually identical tothat following imbibition of the mature seed. Proteins synthesizedduring normal late development (at 45 and 50 DAP) are producedup to 48 h after imbibition; a subsequent change in the patternof insoluble protein synthesis occurs between 48 and 72 h. Thus,in contrast to the rapid switch in the pattern of soluble proteinsynthesis induced by drying, insoluble protein syntheses withinthe endosperm are redirected towards those uniquely associatedwith a germination/growth programme only after a considerabledelay following mature seed imbibition, or following rehydrationof the prematurely dried seed. Nevertheless, these results supportour contention that drying plays a role in the suppression ofthe developmental metabolic programme and in the permanent inductionof a germination/growth programme. Key words: Desiccation, crystalloid storage proteins, castor bean, seed development, seed germination     

The Structure of Mature Rye Endosperm

Endosperm tissue of mature kernels of rye (Secale cereale L.)cv. Dominant was examined by light and transmission electronmicroscopy. It was found that storage protein in sub-aleuronecells occupies up to 35 per cent of the cell volume and formsa continuous matrix in which starch grains and cytoplasmic remnantsare embedded. In the prismatic endosperm, the storage proteinis present as a fine network interspersed between the numeroustype A and B starch grains. Protein bodies are not found inthe prismatic endosperm; only a few, less than 1 µm indiameter, are observed in pockets of disorganized cytoplasmin the sub-aleurone tissue. Thick cell walls and intercellularmaterial may contribute to the high pentosan content of ryeendosperm. Secale cereale L., rye, endosperm, protein matrix, ultrastructure     

Accumulation of Metabolites during Seed Development in Trifolium repens L.

Metabolite deposition during seed development was examined histochemicallyin Trifolium repens by light- and fluorescence microscopy. Allendosperm haustorium at the chalazal pole of the embryo sacand wall protrusions in cell walls of the suspensor and theembryo sac suggest that transfer of metabolites from maternalto offspring tissue takes place primarily at these sites. Thisis further supported by prominent cutinization of the interpolarregion of the embryo sac wall, accumulation of starch in integumentaltissue at the embryo sac poles, and breakdown of interpolarendothelial cells. Decomposition of osteosclereid starch isfollowed by accumulation in the cellular endosperm and subsequentlyin the embryo parallel to endosperm degradation. The starchaccumulates gradually inward from the subepidermal cells ofthe embryo to the stele. Protein bodies are formed in the vacuolesalong the tonoplast, later to be cut off in vesicles releasedinto the cytoplasm. At maturity the embryo is packed with proteinand starch, but without lipid reserves. Phytin is observed inthe protein bodies. The mature embryo is surrounded by a proteinand starch containing aleurone layer which originates from theendosperm.Copyright 1994, 1999 Academic Press White clover, protein, starch, cuticle, embryo sac wall     

Role of Chitinase and Chitin Oligosaccharides in Lignification Response of Cultured Carrot Cells Treated with Mycelial Walls

Chitinase activity was induced in cultured carrot cells by incubationwith mycelial walls of a fungus, Chaetomium globosum. Both intra-and extracellular chitinases were resolved into four componentsby gel filtration chromatography. The extracellular enzymesliberated soluble oligosaccharides of different sizes from insolublechitin, suggesting that these carrot chitinases are endo-hydrolases.The solubilized chitinase digests obtained from insoluble mycelialwalls of C. globosum and chitin were fractionated by gel filtrationchromatography, and the elicitor activity of each fraction forthe accumulation of phenolic acids in cultured carrot cellswas determined. In both solubilized fragments of fungal wallsand of chitin, elicitor-active oligosaccharides were distributedin many fractions, however, potent activity for inducing phenolicacid synthesis was observed in the high molecular weight fractions. (Received October 5, 1987; Accepted February 12, 1988)     

CHANGES IN THE CARBOHYDRATES, PROTEINS AND NUCLEIC ACIDS IN THE OVULES OF ZEPHYRANTHES LANCASTERI AT DIFFERENT STAGES OF MATURATION

The major changes in the levels of soluble and insoluble carbohydrates,nitrogenous compounds and nucleic acids were investigated atdifferent stages of seed development in Zephyranthes lancasteri.The activities of amylases, glutamic-alanine transaminase, ribonucleaseand deoxyribonuclease were also studied. The alcohol-solublenitrogen and carbohydrates attain their maximal levels priorto the elongation ofthe cotyledon. Both of these decrease markedlyduring further maturation of the seed. The accumulation of totalnitrogen in the ovule follows a sigmoid pattern. The glutamic-alaninetransaminase activity appears to be exclusively localized inthe endosperm and is absent from the embryo. The embryo seemsto derive its organic nitrogen fromthe endosperm. The peak inthe level of DNA per ovule is attainedprior to the elongationof the cotyledon while that of RNA is foundsoon after. ¹Present address: AEC Plant Research Laboratory, Michigan StateUniversity, East Lansing, Mich., U.S.A.     

Structure and In Vitro Growth of Zygotic Embryos of Taro (Colocasia esculenta var. antiquorum)

Zygotic embryos of taro, Colocasia esculenta var. antiquorumwere examined using both light and scanning electron microscopyand cultured on Linsmaier-Skoog (LS) medium without the additionof growth regulators. Embryos present within mature seed consistof a hypocotyl-root axis and an undeveloped cotyledon and aresurrounded by two major types of endosperm cells, aleurone andstarchy endosperm. Embryos cultured on LS medium developed intomature plants only in the presence of endosperm tissue. Excisedembryos turned green after 2–4 d in culture and reacheda rapid growth period between days 4 and 6. Culture of taroembryos leading to viable plantlet development depends upon(1) removal of the outer and inner integument, and (2) the presenceof endosperm tissue (including an intact aleurone layer). Colocasia esculenta var. antiquorum, Araceae, taro, embryo culture, integument, endosperm     

Effects of Auxin and Phenobarbital on Morphogenesis and Production of Digitoxin in Digitalis Callus

Pieces of callus obtained from seedlings of Digitalis purpureawere grown on solid Murashige-Skoog's medium supplemented with1 mg liter^–1 BA and 0.1 mg liter^–1 IAA or NAA, withor without phenobarbital (40 mg liter^–1). The replacementof the natural auxin IAA by the synthetic auxin NAA increasedcallus growth and inhibited organogenesis, whereas the additionof phenobarbital had the opposite effect. Morphometric measurementsrevealed a high ratio of vacuole to cytoplasm (v/v) in calluscells. This ratio was affected by the different treatments inthe same way as the fresh weight. The activity of mitochondrialcytochrome P450scc (the enzyme that provides the precursor,pregnenolone, for the biosynthesis of cardenolide in foxgloveplants) was detected in the relevant fraction of callus grownunder all experimental conditions, and its activity was increasedby the addition of phenobarbital. The different treatments testedincreased the cardenolide content and quantifiable amounts ofdigitoxin were detected in all callus tissues. It is of specialinterest that phenobarbital added to the culture medium increasedthe accumulation of digitoxin. The mechanism affecting the developmentand production of cardenolide in callus tissues of D. purpureaby phenobarbital and the replacement of IAA by NAA is discussed. (Received July 18, 1994; Accepted December 14, 1994)     

Germination Preventing Mechanisms in Iris Seeds

The different germination behaviour of the seeds of two irises,Iris lorteti and I. atropurpurea was found to be due to thedifferent mechanical resistance of the integument, at the micropylarend, to radicle protrusion. A pressure of 135 atm was necessaryin l. lorteti seeds for radicle protrusion, while in I. atropurpurea77 atm was sufficient. In contrast Pancratium maritimum requireda pressure of only 10 atm. The outer integument of seeds ofI. lorteti was found to contain a compound which was toxic tothe germinated embryo but did not act as a germination inhibitor.Extracts of the endosperm also had a slight germination inhibitingeffect. An interaction between this weak inhibitor and the effectof the testa could not be ruled out completely. A test assayfor germination using excised embryos was developed. A methodfor germination of Iris seeds, by cutting off the outer integumentat the micropylar end, was developed and is being exploitedcommercially. Iris lorteti, Iris atropurpurea, germination, germination inhibition, embryo culture, seed coat mechanical resistance     

Predator-prey interactions between Isochrysis galbana and Oxyrrhis marina. II. Release of non-protein amines and faeces during predation of Isochrysis

During the growth of Isochrysis galbana, several non-proteinamines may be detected in the growth medium. Of these, one (termed‘TTl’) accumulates in proportion to the numbersof cells present. The concentrations of ‘TTl’, andof another (termed ‘TA’), are 3–5 times higherin cultures in which Isochrysis is predated by Oxyrrhis marina.The lowest estimates of the concentration of extracellular ‘TTl’are an order of magnitude higher than those of any protein aminoacid. Of the protein amino acids, some like glycine are utilizedduring predation while others, like histidine, accumulate inthe medium Because of the unknown N-content and reactivity ofthe non-protein amines during HPLC, it is not possible to sayif these compounds (together with other components of dissolvedorganic N) form a significant proportion of the unaccountedfor N in the system after predatory activity. During predationin the absence of detectable free ammonium (when Isochrysismay be expected to be N-deprived), particles accumulate in themedium. Most of these are <2.5 µ.m in diameter andare suggested to be remains of digested prey. There is evidenceof a reassimilation of these particles by prey-deplete Oxyrrhis.     

The Role of Maturation Drying in the Transition from Seed Development to Germination: II. POST-GERMINATIVE ENZYME PRODUCTION AND SOLUBLE PROTEIN SYNTHETIC PATTERN CHANGES WITHIN THE ENDOSPERM OF Ricinus communis L. SEEDS

Kermode, A. R. and Bewley, J. D. 1985. The role of maturationdrying in the transition from seed development to germination.II. Post–germinative enzyme production and soluble proteinsynthetic pattern changes within the endosperm of Ricinus communisL. seeds.—J. exp. Bot. 36: 1916–1927. Immature seedsof Ricinus communis L. cv. Hale (castor bean) removed from thecapsule at 30 or 40 d after pollination (DAP) do not germinateunless first subjected to a desiccation treatment. This changefrom development to germination elicited by premature desiccationis also mirrored by a change, upon subsequent rehydration, inthe pattern of soluble protein synthesis within the endospermstorage tissue. Following rehydration of prematurely dried 30or 40 DAP seeds, soluble proteins characteristic of developmentcease to be synthesized after 5 h of imbibition, and those uniquelyassociated with germination and growth are then produced. Apattern of soluble storage protein breakdown comparable to thatfound in endosperms from mature seeds following imbibition isalso observed. In contrast, hydration of 40 DAP seeds immediatelyfollowing detachment from the mother plant results in a continuationof the developmental pattern of protein synthesis. Prematuredesiccation at 40 DAP elicits the production within the endospermof enzymes involved in protein reserve breakdown (leucyl ß–naphthylamidase;LeuNAase) and lipid utilization (isocitrate lyase; ICL) to levelscomparable to those observed in mature–hydrated endosperms.It is proposed that drying plays a role in redirecting metabolismfrom a developmental to a germinative mode; it also appearsto be a prerequisite for the induction of hydrolytic enzymesessential to the post–germinative (growth) phase of seedlingdevelopment. Key words: Desiccation-tolerance, germinability, seed development, castor bean     

Cytochemical Localization of Reserves during Seed Development in Arabidopsis thaliana under Spaceflight Conditions

Successful development of seeds under spaceflight conditionshas been an elusive goal of numerous long-duration experimentswith plants on orbital spacecraft. Because carbohydrate metabolismundergoes changes when plants are grown in microgravity, developingseed storage reserves might be detrimentally affected duringspaceflight. Seed development in Arabidopsis thaliana plantsthat flowered during 11 d in space on shuttle mission STS-68has been investigated in this study. Plants were grown to therosette stage (13 d) on a nutrient agar medium on the groundand loaded into the Plant Growth Unit flight hardware 18 h priorto lift-off. Plants were retrieved 3 h after landing and siliqueswere immediately removed from plants. Young seeds were fixedand processed for microscopic observation. Seeds in both theground control and flight plants are similar in their morphologyand size. The oldest seeds from these plants contain completelydeveloped embryos and seed coats. These embryos developed radicle,hypocotyl, meristematic apical tissue, and differentiated cotyledons.Protoderm, procambium, and primary ground tissue had differentiated.Reserves such as starch and protein were deposited in the embryosduring tissue differentiation. The aleurone layer contains alarge quantity of storage protein and starch grains. A seedcoat developed from integuments of the ovule with gradual changein cell composition and cell material deposition. Carbohydrateswere deposited in outer integument cells especially in the outsidecell walls. Starch grains decreased in number per cell in theintegument during seed coat development. All these characteristicsduring seed development represent normal features in the groundcontrol plants and show that the spaceflight environment doesnot prevent normal development of seeds in Arabidopsis. Arabidopsis ; spaceflight; embryo; endosperm; seed coat; storage reserves     

Functions of the Cell Wall in the Interactions of Plant Cells: Analysis Using Carrot Cultured Cells

Interactions between cells and between tissues are importantin the development and morphogenesis of higher plants. Attemptsto characterize the role of the cell wall in such interactionshave benefited from the use of carrot (Daucus carota L.) culturedcells in vitro as a model system. The development of carrotcells in culture can be divided into three processes: the acquisitionof embryogenic competence; the development of the embryo; andthe maturation and dormancy of the embryo. Induction of non-embryogeniccallus is accompanied by weakened intercellular attachment,decreased levels of endogenous ABA and a decrease in responsivenessto exogenous ABA. Cell wall polysaccharides are known to beinvolved in various developmental and morphogenetic events.In carrot cultured cells, possible roles in intercellular attachmenthave been proposed for arabinan and xylose in the neutral sugarregions of pectins, and various extracellular proteins havebeen shown to be involved in somatic embryogenesis in vitro.Some of these proteins are also present around and/or in zygoticembryos, possibly being involved in the formation and functionsof zygotic embryos and seeds. A 57-kDa extracellular solubleglycoprotein that binds to insulin-like peptides and an 18-kDaextracellular insoluble cystatin that inhibits the proteinasesof germinating seeds of carrot might be involved in cellularsignal transduction and inter-tissue interaction, respectively,in carrot seeds. ¹ Recipient of the JSPP Young Investigator Award, 1997     

Formation of Protein Bodies in the Starchy Endosperm of Rice (Oryza sativa L.): A Re-investigation

The mechanism of protein body formation in the starchy endospermis described for two rice varieties, one of normal protein contentand the other high in protein. Three types of protein bodieswere found in both rices. While each protein body type was depositeddifferently, the mechanism of formation for the same type inthe two varieties was analogous. First secreted was the largespherical protein body. It was deposited within rough endoplasmicreticulum, had a dense centre and a concentric ring appearance,and was bounded by a single membrane. The second protein bodyto form was crystalline, was found in vacuoles, and was secretedvia the Golgi apparatus. The third type, the small sphericalprotein body, was secreted late in development, was depositedin vesicular rough endoplasmic reticulum, and lacked dense centresand concentric rings. The high protein rice had a thicker sub-aleuronelayer than the normal protein rice. Oryza sativa L., rice, protein bodies, starchy endosperm, ultrastructure     

Early Stages in Wheat Endosperm Formation and Protein Body Initiation

The early stages of endosperm formation and protein body initiationare described for hard red winter wheat using light and transmissionelectron microscopy. Two days after flowering (DAF) the endospermwas a thin layer of coenocytic cytoplasm lining the embryo sac.By 4 DAF the endosperm had cellularized and completely filledthe embryo sac. Enough differentiation had occurred by 6 DAFto distinguish cells destined to become the aleurone layer,sub-aleurone region and central endosperm. Protein bodies wereinitiated at about 6–7 DAF and were first found near theGolgi apparatus. Wheat was ready for combine harvest at 34 DAF.Enlargement of the small protein bodies near the Golgi apparatusoccurred by several mechanisms: (1) fusion with one or moreof the dense Golgi vesicles or fusion with other protein bodies,(2) fusion with small electron-lucent Golgi-derived vesicles,(3) pinocytosis of a portion of the adjacent cytoplasm intothe developing protein body and (4) fusion of large proteinbodies with one another at later stages of grain development.Of the four mechanisms described, the pinocytotic vesicles andfusion of protein bodies were the most frequent and consistentprocesses observed. Direct connections between rough endoplasmicreticulum (RER) and protein bodies were not observed. The resultssuggest a rôle for the Golgi apparatus in the initiationof protein bodies. Also, the lack of RER derived vesicles suggestsa soluble mode of secretion of storage proteins involved inthe enlargement of protein bodies. Triticum aestivum, wheat endosperm, protein bodies Golgi apparatus     

Composition, Degradation and Utilization of Endosperm During Germination in the Oil Palm (Elaeis guineensis Jacq.)

The insoluble carbohydrate and lipid fractions, and -D-galactosidase,ß-D-mannosidase and isocitrate lyase activities werestudied in the various tissues of oil palm (Elaeis guineensisJacq.) kernels prior to and during germination. In ungerminatedkernels insoluble carbohydrate and lipid constituted 36 and47% of endosperm dry weight respectively. During germinationthe thick endosperm cell walls became markedly thinner, concurrentwith a significant decrease in the percentage of insoluble carbohydrateand an increase in -galactosidase and ß-mannosidaseactivity in both degraded and residual endosperm. The proportionof lipid in degraded endosperm also increased significantly.The insoluble carbohydrate appears to be a galactomannan locatedin the secondary walls of the endosperm. No galactomannan wasdetected in oil palm embryos or haustoria. Isocitrate lyasewas present in, and confined to, tissues of the haustorium ofgerminating kernels. The enzyme was not active in endospermat any stage of germination, nor was it active in embryos beforeor at the end of imbibition. The results suggest that galactomannan is the second largestcomponent of oil palm endosperm and that it is utilized morerapidly than lipid during the early stages of germination. Thefact that isocitrate lyase activity is confined to the haustoriumsuggests that in Elaeis gluconeogenesis, the conversion of triglycerideto carbohydrate, takes place entirely within the cotyledon ofthe seed. Elaeis guineensis, galactomannan, galactosidase, germination, isocitrate lyase, mannosidase, oil palm     

Studies on the Proteinaceous Subcellular Particles in Rice Endosperm: Electron-Microscopy and Isolation

Electron-microscopic examination of rice endosperm revealed the existence of protein-aceous subcellular particles, 1 to 4 µ in diameter and spherical or oval in shape. Isolation of the particles was effected by differential centrifugation in density gradient medium after mechanical or enzymic disintegration of endosperm cells. The isolated particles were predominantly composed of protein, and residual constituents were mainly lipid and carbohydrate. Their shape and behaviors were similar to those found in the endosperm. These facts show that the subcellular particles concerned are “protein bodies” There seemed to be several kinds of protein bodies different with respect to their protein and lipid contents.     

Dormancy in Dioscorea: Rapid Germination of Detached Embryos from Dormant Seeds of D. tokoro

The intact dormant seeds of Dioscorea tokoro germinate slowlyif at all between 11-23°C; for full and rapid germinationthey require prior chilling treatment [Okagami and Kawai (1982)Bot. Mag. Tokyo 95: 155]. The germination abilities of zygoticembryos detached from dormant seeds of this species were studiedunder various nutritional and temperature regimes. For germinationof embryos, the minimum nutritional components in Murashigeand Skoog's (1962) medium that were required were sucrose andNO^–₃ or SO^2–₄. As the source of carbohydrate forgermination of detached embryos, sucrose, mannose and maltosewere effective; glucose and fructose were less effective; andrhamnose was entirely unable to support germination. Embryos detached from dormant seeds, incubated with the sucroseplus KNO₃, germinated more rapidly with increasing temperatureup to 35°C. However, application of sucrose and KNO₃ didnot induce germination of intact seeds above 26°C. Therefore,it is very possible that the endosperm exerts an inhibitoryfunction on germination at such high temperatures. When seeds were incubated after a cut was made over a smallpart of the edge of the endosperm in which the radicle of theembryo is encased, germination occurred rapidly but the increasein germination percentage was slight. This result suggests thatthe endosperm suppots part of the germination inhibition bymeans of a mechanical barrier or its impermeability to wateror gases. Physiological features of the endosperm alone or interactionsbetween the embryo and endosperm may contribute significantlyto the characteristics of dormancy of intact seeds of this species. (Received May 30, 1988; Accepted January 11, 1989)