Degradation of the endosperm cell walls of Lactuca sativa L., cv. Grand Rapids

The timing of mobilisation of lipid, sucrose, raffinose and phytate in lettuce seeds (achenes) (cv. Grand Rapids) has been examined. These reserves (33%, 1.5%, 0.7%, 1.4% of achene dry weight, respectively) are stored mostly in the cotyledons. Except for a slight degradation of raffinose and increase in sucrose, there is no detectable reserve mobilisation during germination. The endosperm (8% of seed dry weight), which has thick, mannan-containing cell walls (carbohydrate, 3,4% of seed dry weight), is completely degraded within about 15h following germination. Mannanase activity increases about 100-fold during the same period and arises in all regions of the endosperm. Also during this period sucrose and raffinose are degraded and fructose and glucose accumulate in the embryo. The endosperm hydrolysis products are taken up by the embryo, and are probably used as an additional reserve to support early seedling growth. However, endosperm cell-wall carbohydrates, such as mannose, are not found as free sugars. Lipid and phytate are degraded in a later, second phase of mobilisation. Low levels of sucrose are present in the embryo, mostly in the cotyledons, and large amounts of fractose and glucose (14% of seedling dry weight at 3 days after sowing) accumulate in the hypocotyl and radicle. It is suggested that sucrose, produced in the cotyledons by gluco-neogenesis, is translocated to the axis and converted there to fructose and glucose.     

Degradation of the endosperm cell walls of Lactuca sativa L., cv. grand rapids in relation to the mobilisation of proteins and the production of hydrolytic enzymes in the axis,cotyledons and endosperm

The timing of changes in total nitrogen and soluble amino nitrogen content, and in the activities of proteinase (pH 7.0), isocitrate lyase, catalase, phytase, phosphatase (pH 5.0), -galactosidase and -mannosidase were studied in extracts from the cotyledons, axis and endosperms of germinating and germinated light-promoted lettuce seeds. The largest amount of total nitrogen (2.7% seed dry weight) occurs within the cotyledons, as storage protein. As this decreases the total nitrogen content of the axis increases and the soluble amino nitrogen in the cotyledons and axis increases. Proteinase activity in the cotyledons increases coincidentally with the depletion of total nitrogen therein. Enzymes for phytate mobilisation and for gluconeogenesis of hydrolysed lipids increase in activity in the cotyledons as the appropriate stored reserves decline. Beta-mannosidase, an enzyme involved in the hydrolysis of oligo-mannans released by the action of endo--mannase on mannan reserves in the endosperm, arises within the cotyledons. This indicates that complete hydrolysis of mannans to the monomer does not occur within the endosperm. Mobilisation of all cotyledon reserves occurs after the endosperm has been degraded, providing further evidence that the endosperm is an early source of food reserves for the growing embryo.Abbreviations HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulphonic acid - EDTA ethylenediamine tetraacetic acid disodium salt - TCA trichloroacetic acid Part 2 of a series, of which the first was published in Planta 139, 1–8 (1978)     

Action spectra for light-induced germination in dormant lettuce seeds

Fluence response curves for red light-induced germination of thermodormant (TD) seeds of Lactuca sativa L. show two regions that differ in their light sensitivity. In the region of high sensitivity, the germination responses differ between seed batches and can be altered by dark storage or far red irradiation. Induction of germination in far red dormant (FRD) seeds requires far higher fluences. Action spectra for induction to 60% germination were determined for these various response types. Spectra for the regions of low sensitivity response are similar for TD and FRD seeds. In comparison, the action spectrum for the highly sensitive response in TD seeds is significantly shifted to longer wavelengths. Analogous differences exist in the action spectra for far red reversal of the red induced germination responses. Germination induction in the low sensitivity region shows repeated red-far red reversibility. Far red reversal of red induction in the high sensitivity region does not saturate even at the highest far red fluences available and requires increased red fluences for subsequent reinduction. A model quantitatively accounting for these observations is presented. It is pointed out that action spectra of processes involving photoreversible pigments with partly overlapping absorption spectra in general are not identical with the absorption spectra of the partners. They should depend upon the degree of phototransformation required to elicit a given physiological response. In the case of induction of lettuce seed germination the observed action spectra can be interpreted as reflecting different requirements for P _fr of the various response types. Our results do not necessitate the assumption of spectroscopically different forms of phytochrome in these seeds.Abbreviations TD thermodormant - FRD far red dormant - P phytochrome - P _r red absorbing form of P - P _fr far red absorbing form of P     

Interaction of light and temperature on seed germination of Rumex obtusifolius L.

Germination of Rumex obtusifolius L. seeds (nutlets) is low in darkness at 25° C. Germination is stimulated by exposure to 10 min red light (R) and also by a 10-min elevation of temperature to 35° C. A 10-min exposure to far-red light (FR) can reverse the effect of both R (indicating phytochrome control) and 35° C treatment. Fluence-response curves for this reversal of the effect of R and 35° C treatments are quantitatively identical. Treatment for 10 min with light of wavelenght 680, 700, 710 and 730 nm, after R and 35° C treatment, demonstrates that germination induced by 35° C treatment results from increased sensitivity to a pre-existing, active, far-red-absorbing form of phytochrome (Pfr) in the seeds.Abbreviations FR far-red light - P phytochrome - Pr red-absorbing form of P - Pfr far-red-absorbing form of P - R red light     

Interaction of light and temperature on the germination of Rumex obtusifolius L.

Seeds (nutlets) of Rumex obtusifolius L. fail to germinate in darkness at 25° C, but are stimulated by short exposure to red light (R) the effectiveness of which can be negated by a subsequent short exposure to far red light (F) indicating phytochrome control. Short periods of elevated temperature treatment (e.g. 5 min at 35° C) can induce complete germination in darkness. Although short F cannot revert the effect of 35° C treatment, cycling the phytochrome pool by exposure to short R before short F results in reversion of at least 50% of the population. Prolonged or intermittent F can also revert the germination induced by 35° C treatment. The effect of elevated temperature treatment is interpreted on the basis of two possible models; (i) that it increases the sensitivity of the seeds to a low level of pre-existing active form of phytochrome (Pfr) (ii) that it induces the appearance of Pfr in the dark. In both cases it is envisaged that elevated temperature treatment and Pfr control germination at a common point in the series of reactions that lead to germination.Abbreviations D Dark - F far red light - P phytochrome - Pr red absorbing form of P - Pfr far red absorbing form of P - R red light     

Growth physics and water relations of red-light-induced germination in lettuce seeds

Red light (R) and gibberellins (GA) each induce a water potential decrease in the axes of lettuce (Lactuca sativa L.) embryos resulting in germination of intact seeds (achenes) or an increase in growth of the axes of isolated embryos. The fruit coat and endosperm are a substantial barrier to the penetration of exogeneous GA. Isolated embryos take up 35 times as much [³H]GA₁ as the embryos of intact seeds and respond to less than 1·10^-10 M GA₃ or GA₄₊₇. We calculated that only 1·10^-8 M of either GA₃ or GA₄₊₇ would result in 50% germination if the GA were able freely to penetrate the fruit coat. Exogenous GA₃ or GA₄₊₇, at concentrations insufficient to cause germination, result in an apparent synergistic promotion of germination when suboptimal R is applied. Yet suboptimal concentrations of exogenous GA₃ or GA₄₊₇ and suboptimal R result in only additive increases in the growth response in axes of isolated embryos. Dose-response curves demonstrate quantitative increases in the growth response of the isolated axes after R or GA treatments insufficient to induce germination in intact seeds, indicating that a threshold potential must be achieved by the embryonic axes before germination can occur.Abbreviations FR far=red light - GA gibberellin - PEG poly-ethylene glycol 4000 - P_fr far-red-absorbing phytochrome - R red light III.=Carpita et al. 1979b; IV.=Carpita et al. 1979c     

Light effects upon dry lettuce seeds

Germination of certain dry seeds (achenes) of Lactuca sativa L. cv. Grand Rapids was increased to ca. 75% after irradiation with 665 nm red light (R; 1x10³ J m^-2); this response was eliminated by far-red light (FR) following the R. The response of dry seeds required an order of magnitude more light than that of wetted seeds, and was not maximal until 48 h after irradiation. Other seeds, which could not be stimulated by R in dry state, showed a partial response after 10 min hydration. Irradiation of dry seeds (or seeds wetted 1 h) with FR (1x10³ J m^-2) reduced dark germination from 26% to 2%. Seeds dehydrated in an oven (60°C, 90 min) showed a decrease in germination if irradiated with R (1x10⁵ J m^-2) before wetting. The results show that phytochrome is present in dry lettuce seeds (and functional in some seed lots) prior to wetting; and that in other seed lots the molecule becomes functional within minutes after wetting the seeds. Transformation of the FR absorbing from of phytochrome (P_FR) to the inactive from (P_R) occurs at lower seed moisture content than the reverse reaction. It appears that dormancy in seeds ripened in sunlight might be assured during seed drying and maturation by the more effective transformation of P_FR to P_R than vice versa as phytochrome is dehydrated.Abbreviations FR far-red - R red - CAL seeds from California - NC seeds from North Carolina (see text)     

An unusual effect of the far-red absorbing form of phytochrome: Photoinhibition of seed germination inBromus sterilis L.

Seeds ofBromus sterilis L. germinated between 80–100% in darkness at 15° C but were inhibited by exposure to white or red light for 8 h per day. Exposure to far-red light resulted in germination similar to, or less than, that of seeds maintained in darkness. Germination is not permanently inhibited by light as seeds attain maximal germination when transferred back to darkness. Germination can be markedly delayed by exposure to a single pulse of red light following 4 h inhibition in darkness. The effect of the red light can be reversed by a single pulse of far-red light indicating that the photoreversible pigment phytochrome is involved in the response. The response ofB. sterilis seeds to light appears to be unique; the far-red-absorbing form of phytochrome (Pfr) actually inhibiting germination.Abbreviations Pr red absorbing form of phytochrome - Pfr far-red absorbing form of phytochrome     

Interaction of [3H]gibberellin A1 with a sub-cellular fraction from lettuce (Lactuca sativa L.) hypocotyls

The relationship between elongation growth and the incorporation of [³H]gibberellin A₁ ([³H]GA₁) into a 2,000g pelletable (2KP) fraction from lettuce (Lactuca sativa L., cv. Arctic) hypocotyl sections has been examined. Sections were loaded with incremental amounts of GA₁ under conditions where growth was arrested (5° C) or permitted (30° C) and, after 16 h, all were transferred to a GA-free medium at 30° C. Growth and 2KP radioactivity were measured at this point and after a further 24 h in the chase medium. Uptake was reduced by 80% at 5° C, as compared to 30° C, but 2KP labelling and protein synthesis were only reduced by half. The growth rate of the 5° C pretreated sections during the chase period was comparable to that observed during the pulse in the 30° C material but the dose/response relationship was flatter. Low temperature sections incorporated a much higher percentage of GA₁ uptake into the 2KP fraction (27% at maximum) but the absolute levels of labelling at this temperature were lower than those measured at 30° C. The data are interpreted as showing that 2KP labelling is not a consequence of growth. It must either precede response or be an unconnected concurrent process.     

A structural study of germination in celery (Apium graveolens L.) seed with emphasis on endosperm breakdown

Germination of celery seed occurred after 6 d of imbibition in light. During this time the embryo enlarged at the expense of the adjacent endosperm cells and at the time of germination was 2–3 times as long as in the dry seed. Breakdown of the endosperm cells near the root cap preceeded radicle emergence. None of these changes occurred in darkness.Endosperm digestion began adjacent to the embryo and spread radially. In degrading cells, the aleurone grains often became larger and fewer in number. The cell walls were modified and appeared to undergo partial degradation. Ultimately the cells seemed to lose their contents. In cells adjacent to the root cap, similar changes occurred except there was a transient appearance of starch grains. Radial progression of endosperm breakdown also occurred in isolated endosperm treated with gibberellin A₄₊₇.The results indicate that (1) the stimulus for breakdown of celery endosperm emanates from the embryo in response to light; (2) the stimulus may be a gibberellin because changes in endosperm cells and the sequence of endosperm digestion during germination resemble the responses of isolated endosperm to gibberellin; and (3) the radial progression of endosperm breakdown during germination may be the result of a sequential response of cells to a uniformly applied stimulus rather than the result of gradual embryo expansion.     

The growth physics and water relations of red-light-induced germination in lettuce seeds

Using lettuce (Lactuca sativa L., cv. Grand Rapids) embryos in osmotica, we have demonstrated that when the growth rates of the embryonic axes of seeds treated with red (R) or far-red (FR) light are equalized, the axes of R-treated seeds develop a 3.4-bar decrease in water potential (paper No. III).As axial growth begins, reserve protein and phytin decrease rapidly, concomitant with increases in reducing sugars, -amino nitrogen, and inorganic and esterified soluble phosphates. However, no differences between the axes of R-and FR-treated seeds are found with respect to the changes in these compounds, indicating that these changes arise as a result of growth and are not under immediate phytochrome control. Little change in the total lipid content is found in either treatment. The axes of FR-treated seeds hydrolyze endogenous sucrose at a greater rate thant those of R-treated seeds. Axes of R-treated seeds accumulate K⁺ and Na⁺ to a greater extent than those of FR-treated seeds. When potassium salts are added to the incubation medium, R induces increased K⁺ uptake by the axis and greater medium acidification by the axis. Malate and other organic acids and acidic amino acids increase at equal rates in both treatments, indicating that inorganic anions may also be taken up to balance the ionic charges. The results are compatible with the assumption that changes in the osmotic and pressure potentials of the embryonic axes of R-treated seeds are the result of a phytochrome-stimulated proton pump which, in whole dormant seeds, would initiate water-potential changes allowing the embryos to overcome the mechanical restraint of the surrounding seed layers, resulting in germination.Abbreviations FR far-red light - PEG polyethylene glyeol 4000 - Pfr far-red-absorbing form of phytochrome - R red light III=Carpita et al. 1979     

Disappearance of stored polyadenylic acid and mRNA during early germination of radish (Raphanus sativus L.) embryo axes

Polyadenylic acid [poly (A)] is detected, characterized and quantitated in dry radish embryo axis RNA using a ³H poly (U) probe. The amount of poly (A) gradually decreases after the onset of soaking, and, after a few hours, recovers to the initial level. This variation is shown to result from the addition of two opposed phenomena: the decay of stored poly (A) and the accumulation of newly synthesized poly (A). Stored poly (A), as well as the in vivo protein synthesis coded for by preformed mRNA, decreases during early germination with a half-life of two hours. As a whole, these results demonstrate that at least a fraction of the stored mRNA is translated as soon as the seed is soaked and that its role is rapidly taken over by newly-made mRNA.Abbreviations Poly (A) (+) RNA polyadenylated RNA - Poly (A) polyadenylic acid - Poly (U) polyuridylic acid     

Interaction of [3H]gibberellin A1 with a sub-cellular fraction from lettuce (Lactuca sativa L.) hypocotyls

The relationship between protein synthesis and the incorporation of [³H]gibberellin A₁ ([³H]GA₁) into a 2,000xg pelletable (2KP) fraction from lettuce (Lactuca sativa L.) hypocotyl sections has been investigated. Concentrations of D-2-(4-methyl-2,6-dinitroanilino)-N-methylpropionamide (MDMP) between 10^-7 M and 10^-4 M caused increasing inhibition of growth, 2KP labelling and incorporation of [¹⁴C]leucine into soluble protein. Growth and 2KP radioactivity were highly correlated (r=0.996). Transfer to MDMP early or late in the course of GA response caused reductions in both growth and incorporation into the 2KP fraction. Exposure to the inhibitor had more effect at 4 h than at 20 h. The proportions of alkali-soluble and insoluble radioactivity in the 2KP fraction were also altered by this treatment. The implications of these findings are discussed.Abbreviations GA₁ gibberellin A₁ - MDMP D-2-(4-methyl-2,6-dinitroanilino)-N-methylpropionamide - 2KP a2,000xg pelletable fraction     

Effects of high temperature on the germination of maize (Zea mays L.)

Poor emergence of maize seedlings, due to high soil temperatures, is a major limitation of crop potential in the lowland tropics. Ability to germinate at high temperature (>c. 37° C) is related to the temperature sensitivity of the embryo, and there is considerable genotypic variation for this character.Respiration and mitochondrial phosphorylation proceed normally in seeds imbibing at 41° C, and ATP levels are adequate for germination. However, the specific activities of several important enzymes are lower, and the rate of protein synthesis is severely reduced compared with seeds imbibing at 28° C. The depression of the rate of protein synthesis in the embryos of several tropical hybrids imbibing at high temperature correlated with their known temperature sensitivity. It is concluded that protein synthesis is an especially temperature sensitive process in germinating maize embryos, and that this is the principal reason for the sensitivity of germinating maize seeds to high temperature.Abbreviations ADP adenosine-5-diphosphate - ATP adenosine-5-triphosphate - BSA bovine serum albumin - EDTA ethylenediaminetetra-acetic acid - HEPES N-2-hydroxyethylpiperazinc-N-2-ethanesulphonic acid - NADH nicotinamide-adenine dinucleotide, reduced form - PPO 2, 5-diphenyloxazole - PVP polyvinylpyrrolidone - SEM standard error of the mean - tris tris (hydroxymethyl)-methylamine     

Mannanase production by the lettuce endosperm

Endo--mannanase (EC 3.2.1.78) is produced and secreted by the cells of the endosperm of lettuce (lactuca sativa L.) seeds (achenes). In imbibed intact seeds, production is prevented by inhibitors. If the endosperm is incubated alone, these inhibitors can be removed by leaching, allowing mannanase production. Abscisic acid, a component of lettuce seeds, inhibits the production of mannanase in the isolated endosperm, and may be involved in regulation of mannanase production in intact seeds. During germination the inhibition is removed, beginning 4–8 h after red-light irradiation, which was given 4 h from sowing. The cotyledons participate in this process, and are controlled by events occuring in the axis within 4 h from red-light irradiation. This control by the axis apparently depends on the exchange of diffusible substances. Both benzyladenine and gibberellic acid can replace the influence of the axis if the latter is removed, and may therefore be involved in the control by the axis of the rest of the seed.Abbreviations ABA abscisic acid - BA 6-benzyladenine - GA₃ gibberellic acid - IAA indol-3-yl acetic acid - MES 2-(N-morpholino)ethane sulfonic acid - R red light Part of this work was carried out by P. Halmer at the Department of Biology, Washington University, St. Louis, MO 63130, USA (his present address)     

Expression of glutamine synthetase during the anaerobic germination of Oryza sativa L.

Glutamine synthetase (GS; EC.6.3.1.2.) occurs as cytosolic (GS₁) and plastidic (GS₂) polypeptides. This paper describes the expression of GS isoenzymes in coleoptile during the anaerobic germination of rice (Oryza sativa L.) and the influence of exogenous nitrate on this. By immunoprecipitation with anti-GS serum, two polypeptides of 41- and 44-kDa were detected of which the former was predominant. After fractionation by ion-exchange chromatography, the 41 and 44 kDa bands were identified as GS₁ and GS₂, respectively. Northern blot analysis with specific probes showed the presence of mRNA for cytosolic GS but not for the plastidic form. The presence of exogenous nitrate did not alter the activity and expression of GS in the coleoptile. The role of GS during the anaerobic germination of rice seems to induce the re-assimilation of ammonia rather than the assimilation of nitrate.Abbreviations GS glutamine synthetase - GS₁ cytosolic glutamine synthetase - GS₂ platidic glutamine synthetase We are grateful to Dr. Julie V. Cullimore for providing GS anti-serum and clones. The research was supported by the National Research Council of Italy, special project RAISA, sub-project N. 2 paper N. 1586.     

Plastid development in seedlings of Echinochloa crus-galli var. oryzicola under anoxic germination conditions

Plastid development in the primary leaf of Echinochloa crus-galli (L.) Beauv. var. oryzicola (Vasing.) Ohwi was followed during 5 d of anoxic germination and growth. Plastids develop slowly from simple spheroidal proplastids into larger pleomorphic plastids with several stromal membranes and many peripheral membrane vesicles. A small prolamellar body is present at 96 h with perforated (pro)thylakoids extending into the stroma. Changes in starch grains and plastoglobuli are evidence of carbohydrate and lipid metabolism. Plastid division is indicated by dumbbell plastid profiles after 4 d of anoxia. These results demonstrate that plastids not only maintain their integrity during anaerobic germination but also show developmental changes involving an increase in internal membrane complexity, although to a lesser extent than in etiolated shoots.Abbreviation PLB prolamellar body Scientific paper No. 6167. College of Agriculture, Washington State University, Pullman     

Lipases in the storage tissues of peanut and other oil seeds during germination

The castor-bean endosperm-the best-studied material of reserve lipid hydrolysis in seed germination-was previously shown to have an acid lipase and an alkaline lipase having reciprocal patterns of development during germination. We studied oil seeds from 7 species, namely castor bean (Ricinus communis L.), peanut (Arachis hypogaea L.), sunflower (Helianthus annus L.), cucumber (Cucumis sativus L.), cotton (Gossypisum hirsutum L.), corn (Zea mays. L.) and tomato (Lycopersicon esculentum Mill.). The storage tissues of all these oil seeds except castor bean contained only alkaline lipase activity which increased drastically during germination. The pattern of acid and alkaline lipases in castor bean does not seem to be common in other oil seeds. The alkaline lipase of peanut cotyledons was chosen for further study. On sucrose gradient centrifugation of cotyledon homogenate from 3-d-old seedlings, about 60% of the activity of the enzyme was found to be associated with the glyoxysomes, 15% with the mitochondria, and 25% with a membrane fraction at a density of 1.12 g cm^-3. The glyoxysomal lipase was associated with the organelle membrane, and hydrolyzed only monoglyceride whereas the mitochondrial and membrane-fraction enzymes degraded mono-, di- and triglycerides equally well. Thus, although the lipase in the glyoxysomes had the highest activity, it had to cooperate with lipases in other cellular compartments for the complete hydrolysis of reserve triglycerides.     

Phytochrome-mediated germination control of maize caryopses

Maize caryopses sown in water germinate equally well either in darkness or under any light regime. However, when they are imbibed in mannitol solutions, continuous far-red light proves to be strongly inhibitory on the final germination as compared to darkness. Similar but less pronounced inhibition is also exhibited by continuous red or blue light. Intermittent far-red light can partially substitute for continuous far-red light in inhibiting maize caryopsis germination, and its effect is reversed to the intermittent red light level when red light is given immediately after each far-red illumination. These results are interpreted as a proof of existence and involvement of phytochrome in the germination control of maize caryopses, though its manifestation is realized only under osmotic stress.Abbreviations D darkness - FR far-red - R red - B blue - c-FR, c-R, c-B continuous FR, R, B, resp. - i-FR, i-R intermittent FR, R, resp.     

The nutritional status of the apical meristem of Lactuca sativa as affected by NaCl salinization: An electron-probe microanalytic study

A volume of tissue of lettuce (Lactuca sativa L.) plants extending 2 mm basipetally from the apical meristem and including leaf primordia and young expanding leaves was surveyed using electron-probe microanalysis (EPMA) on both frozen-hydrated and freeze-dried samples. This analysis was carried out either 2 or 5 d following NaCl salinization of the medium from the 10 mol · m^–-3 control level up to 80 mol · m^–-3. The objective was the investigation of possible changes in the nutritional status of the apical meristem that might account for some aspects of salt-induced growth inhibition. Sodium and chloride increased significantly in tissues basal to the apical meristem, while both phosphorus and potassium decreased in the same region. These changes were evident in specimens collected just 2 d after the commencement of salinization (20 h after completion of the salinization) and were not exacerbated by an additional 3 d of treatment; they were present in tissue as close as 100 m to the meristem and extending down to 500 m. The apical 10–50 m were relatively protected from both the increase in sodium and chloride and the decrease in phosphorus and potassium that occurred in more basal regions. Young leaves (up to 1.5 mm in length) appear to control their own mineral nutrient levels when challenged by salinization of the medium, presumably because of altered growth. A decrease in the concentration of total Ca as a result of salinization was significant in cells 500 m basal to the meristem, but was evident as a tendency in the data even within the first 50 m. Using an improved automatic method for the analysis of calcium by EPMA, it was found that total Ca was reduced by salinization, especially in basal regions (500 m below the apex) and also in young leaves (1–1.5 mm in length). We suggest that the nutrition of the shoot apical meristem may be disturbed soon after salinization and that the shoot meristem might be the source of a signal to expanding leaves, as well as exerting its own direct influence over leaf emergence.Abbreviation EPMA electron-probe microanalysis This work was supported by U.S. Department of Agriculture grant 87-CRCR-1-2462.