Ribosomal Competence and Spore Germination in Fusarium solani

Extracts prepared from macroconidia of Fusarium solani f. sp. phaseoli are capable, under defined conditions, of incorporating phenylalanine into polypeptide with exogenous polyuridylic acid as messenger. Extracts from ungerminated and germinated spores have approximately the same activity. With endogenous template, leucine incorporation occurs, but in this reaction extracts from germinated spores have about 10 times more activity than do those from ungerminated spores. It is suggested that the low rate in ungerminated spores is attributable to a relative deficiency in the number of ribosomes which are organized into polysomes.     

Effect of temperature on glycerol metabolism in membranes and on phospholipases C and D of germinating pea embryos

The effect of temperature of imbibition on the synthesis and turnover of membrane phosphatidyl choline was studied. Pea seeds (Pisum sativum cv. Alaska) were imbibed in [U-¹⁴C]glycerol and then germinated. Seeds were kept constantly either at 5° or 25°, or were imbibed at one temperature and then germinated at the other one. Glycerol incorporation into phosphatidyl choline in the ER and the plasma membrane, obtained from the embryonic axes after germination, and the glycerol pool were measured. Embryos from seeds kept constantly at 25° showed a rapid incorporation of glycerol into membranes followed by a loss of label; in embryos from seeds kept at 5° incorporation was much lower. Embryos from seeds transferred from 25° to 5° behaved as if continuously kept at 25°, while the behaviour of the embryos from seeds transferred from 5° to 25° resembled embryos from seeds maintained at 5°. The glycerol content of the axes rose during imbibition and fell thereafter. The activities of phospholipases C and D also responded to the initial temperature of imbibition, but the two activities changed differently. The results are discussed in relation to the effect of transient exposure to temperature changes in the seed membranes and the possible way in which such changes are sensed.     

Newly Synthesized mRNA Is Translated during the Initial Imbibition Phase of Germinating Maize Embryo

RNA synthesis is activated in the cells of the plant embryo very soon after the start of seed imbibition. We previously reported that mainly heterogeneous nuclear RNA is synthesized in the radicle of Zea mays embryo during the first hours of germination. The present study was undertaken in order to detect the time of appearance of the newly synthesized messenger RNA in the polysomes of germinating maize axes.

Free polysomes were prepared from embryonic axes rehydrated for 2 hours in the presence of radioactively labeled uridine. These polysomes were shown to be labeled and to contain labeled particles sedimenting, after dissociation with EDTA, in the 10S to 40S region of a sucrose gradient. The labeled polysomal RNA migrates heterogeneously in a gel with a mean size corresponding to about 16S, and 60% of these molecules are polyadenylated.

The data indicate that the newly synthesized RNA associated with the polysomes after 2 h of germination consists of messenger RNA molecules. Analysis of the polysomes prepared 0.5 and 1 h after the start of imbibition suggests that translation of the newly synthesized messenger RNA probably occurs within the 1st hour of imbibition of the isolated axis, thus well before the completion of the initial water uptake.

     

Possible involvement of the cytoskeleton in the regulation of barley caryopsis dormancy and germination

This study was conducted on barley cv. Ars. caryopses collected at full ripeness and divided into two batches. From one batch (dormant caryopses) polysomes were isolated from embryos immediately after harvesting and after two days of germination. From the other batch (non-dormant caryopses) the same was done after eight months storage in a dry state. A low ionic strength cytoskeleton-stabilizing buffer was used for the isolation of polysomes. Four different fractions of polysomes were examined: free polysomes (FP), membrane-bound polysomes (MBP), cytoskeleton-bound polysomes (CBP) and cytoskeleton-membrane-bound polysomes (CMBP). In germs grown from non-dormant caryopses, the first two fractions (FP + MBP) made up about 78 % of the total ribosomal material, whereas in embryos of dormant, imbibed caryopses, two last fractions (CBP + CMBP) made up about 71 %. The percentage of polysomes after 48 hours of imbibition of dormant caryopses in the FP, MBP and CBP was only about 13 % (i.e., 87 % monosomes), whereas a greater proportion (19.4 %) was found in the CMBP. The highest incorporation of ³H-uridine and ¹⁴C-amino acids (after 48 hours of germination and 0.5, 3 and 6 hrs incubation with precursors) took place in trhc CMBP both in dormant and non-dormant caryopses The major amount of the two polysome fractions associated with the cytoskeleton (CBP and CMBP) and the higher activity of CMBP in protein synthesis in embryos of dormant, imbibed triticale caryopses may indicate a significant role for polysomes associated with the cytoskeleton in the control of protein synthesis in dormant and germinating caryopses.     

Ribonucleic acid synthesis and loss of viability in pea seed

Summary RNA synthesis and protein synthesis in embryonic axis tissue of viable pea (Pisum arvense L. var. N.Z. maple) seed commences during the first hour of germination. Protein synthesis in axis tissue of non-viable pea seed is barely detectable during the first 24 h after the start of imbibition. Nonviable axis tissue incorporates significant levels of [³H]uridine into RNA during this period but the level of incorporation does not increase significantly over the first 24 h of imbibition. In axis tissue of non-viable seed during the first hour of imbibition most of the [³H]uridine was incorporated into low molecular weight material migrating in advance of the 4S and 5S RNA species in polyacrylamide gels but some radioactivity was incorporated into a discrete species of RNA having a molecular weight of 2.7×10⁶. After 24 h, non-viable axis tissue incorporates [³H]uridine into ribosomal RNA, the low molecular weight material migrating in advance of the 4S and 5S RNA peak in polyacrylamide gels and a heterogeneous RNA species of molecular weight ranging from 2.2×10⁶ to 2.7×10⁶. No 4S or 5S RNA synthesis is detectable after 24 h of imbibition in non-viable axis tissue. Axis tissue of viable pea seed synthesises rRNA, 4S and 5S RNA, the low molecular weight material migrating in advance of the 4S and 5S RNA peak in polyacrylamide gels and the rRNA precursor species at both periods of germination studied. Loss of viability in pea seed appears to be accompanied by the appearance of lesions in the processing of rRNA precursor species and a significant loss of RNA synthesising activity.Abbreviations rRNA ribosomal RNA - TCA trichloroacetic acid - SLS sodium lauryl sulphate - PPO 2,5 Diphenyloxazole - POPOP 1,4-Bis-2-(4-methyl-5-penyloxazolyl)-benzene     

Phospholipid metabolism of wheat grains: Enzymes of the CDP-diglyceride phospholipid biosynthetic pathway

Summary Enzymes of the CDP-diglyceride pathway of phospholipid synthesis, CDP-diacylglycerol synthetase, CDP-diacylglycerol: glycerol 3-phosphate phosphatidyl-transferase and enzymes of phosphatidylserine formation were initially of relatively high specific activities in aleurone cells of wheat and declined upon imbibition. Enzyme activity of phosphatidylinositol synthesis was not detected in dry grains but was present upon imbibition. CDP-diacylglycerol: glycerol 3-phosphate phosphatidyltransferase shifted during imbibition from 85% of the activity in the supernatant of aleurone layers from dry seeds to 98% associated with large particle fractions after 36 hours of imbibition. Phosphatidylserine formation shifted from a dominant location in the 1,500 x g fraction in the dry seed to a predominantly mitochondrial location after 36 hours of imbibition. The subcellular distribution of CDP-diacylglycerol synthetase did not change appreciably upon imbibition from that of the dry seed, 75 to 80% of the activity was found in the supernatant. Only CDP-diacylglycerol: glycerol 3-phosphate phosphatidyltransferase showed increased specific activity late in the imbibition period. GA₃ accelerated the decrease of already declining activities of the CDP-diglyceride enzymes and the changes in their patterns of distribution, augmented the activities of the phosphatidylinositol synthesizing enzyme, and both accelerated and augmented the increase in the activity of the enzyme of phosphatidylglycerol synthesis which occurred late in imbibition.Committee on Institutional Cooperation Travelling Scholar from the University of Chicago.     

The role of the persistent fruit wall in seed water regulation in Raphanus raphanistrum (Brassicaceae)

Background and Aims

Dry fruits remain around the seeds at dispersal in a number of species, especially the Brassicaceae. Explanations for this vary, but usually involve mechanisms of innate dormancy. We speculate that, instead, a persistent fruit may give additional protection through control of dehydration, to species growing in arid or Mediterranean environments where water is sporadic.

Methods

X-rays and weight measurements were used to determine the extent to which Raphanus raphanistrum seeds within mature fruits imbibe water, and germination tests determined the roles of the fruit and seed coat in seed dormancy. Rates of water uptake and desiccation, and seedling emergence were compared with and without the fruit. Finally, germinability of seeds extracted from fruits was determined after various periods of moist conditions followed by a range of dry conditions.

Key Results

Most seeds rapidly take up water within the fruit, but they do not fully imbibe when compared with naked seeds. The seed coat is more important than the dry fruit wall in maintaining seed dormancy. The presence of a dry fruit slows emergence from the soil by up to 6–8 weeks. The fruit slows the rate of desiccation of the seed to a limited extent. The presence of the fruit for a few days during imbibition somehow primes more seeds to germinate than if the fruit is absent; longer moist periods within the pod appear to induce dormancy.

Conclusions

The fruit certainly modifies the seed environment as external conditions change between wet and dry, but not to a great extent. The major role seems to be: (a) the physical restriction of imbibition and germination; and (b) the release and then re-imposition of dormancy within the seed. The ecological significance of the results requires more research under field conditions.     

Mobilisation of the major stored reserves in the embryo of fenugreek (Trigonella foenum-graecum L., Leguminosae), and correlated enzyme activities

Changes in total nitrogen, soluble amino nitrogen, lipid and phytate contents, and in the activities of proteinase (pH 7.0), isocitrate lyase and phytase were followed in the endosperm, cotyledons, and axis during germination of fenugreek seeds and subsequent growth of the seedlings. The endosperm is comprised largely of cell-wall galactomannans: the majority of the seed total nitrogen, lipid and phytate (5%, 8%, 0.44% of seed dry weight respectively) is localised within the cotyledons as stored reserves. Germination is completed after 10–14 h from the start of imbibition, but the major reserves are not mobilised during the first 24 h. Then the total nitrogen content of the cotyledons starts to decrease and that of the axis increases; there is a concomitant accumulation of soluble amino nitrogen in both cotyledons and axis. An increase in proteinase activity in the cotyledons correlates well with the depletion of total nitrogen therein. Depletion of lipid and phytate reserves in the different seed tissues constitutes a late event, occurring after 50 h from the start of imbibition, and is coincident with the final disintegration of the endosperm tissue. The depletion of phytate and stored lipids is accompanied by an increase in phytase and isocitrate lyase activity. It appears that the products of lipid hydrolysis are converted by gluconeogenesis to serve as the major source of sugars for the growing axis after the endosperm galactomannan has been completely mobilised.     

Light-induced changes of enzyme activities in parsley cell suspension cultures: Increased rate of synthesis of phenylalanine ammonia-lyase

When dark-grown cell suspension cultures of parsley (Petroselinum hortense) were illuminated for increasing periods of time, increasing amounts of phenylalanine ammonialyase activity were obtained 5 hr after the onset of light.Pulses of [³⁵S]methionine of varying duration from 1 to 150 min were given to cell cultures in the dark period subsequent to a light period of 2.5 hr. The cells were harvested 5 hr after the onset of light. Analysis of the soluble proteins by polyacrylamide gel electrophoresis revealed a distinct peak of radioactivity coinciding with the activity of phenylalanine ammonia-lyase. The results of experiments in which radioactive methionine was administered for 10 min to dark-grown or light-induced cells at different times after the light period were compared. An efficient incorporation of radioactivity into the fractions possessing the enzyme activity was observed 5 hr after induction, while no significant labeling was detected either after 1.5 or 25 hr, or in extracts from nonilluminated cells. The radioactive fractions containing the enzyme activity were further analyzed by sodium dodecyl sulfate-disc gel electrophoresis. Significant amounts of radioactivity at the molecular weight of the subunits of phenylalanine ammonia-lyase (84,000) were found only in the extracts from cells which had been labeled 5 hr after induction. These results suggest that the light-induced increase in phenylalanine ammonia-lyase activity is due to de novo synthesis, but not to an activation of preformed, inactive enzyme.     

Protein synthesis in oocytes of Xenopus laevis is not regulated by the supply of messenger RNA

The control of protein synthesis in oocytes of Xenopus laevis has been investigated by injecting oocytes with mRNA and polysomes followed by labeling with ¹⁴C-amino acid mixtures. Contrary to previous reports in which injected oocytes were labeled with ³H-histidine, injected globin mRNA is found to decrease amino acid incorporation into endogenous proteins competitively at all concentrations tested. No increase in overall amino acid incorporation is detected when more mRNA is supplied. Similar results are obtained after labeling injected oocytes with leucine, methionine, proline or valine individually. An explanation is presented for the conflicting results obtained when histidine is used as a label.When reticulocyte polysomes are injected, rather than purified globin mRNA, incorporation of amino acids into endogenous proteins remains roughly constant and overall incorporation increases. Similarly, when encephalomyocarditis viral RNA is injected together with either globin mRNA or reticulocyte polysomes, the globin mRNA causes decreased amino acid incorporation into encephalomyocarditis proteins, but the polysomes do not do so. The results demonstrate that different types of mRNA compete for a strictly limited translational capacity which is saturated in the normal oocyte. The limiting component is present in polysomes and is not message-specific. The constraint on protein synthesis in the amphibian oocyte cannot be fully explained by masked mRNA.     

Measurement of protein synthesis in rat lungs perfused in situ

Compartmentalization of amino acid was investigated to define conditions required for accurate measurements of rates of protein synthesis in rat lungs perfused in situ. Lungs were perfused with Krebs–Henseleit bicarbonate buffer containing 4.5% (w/v) bovine serum albumin, 5.6mm-glucose, normal plasma concentrations of 19 amino acids, and 8.6–690μm-[U-¹⁴C]phenylalanine. The perfusate was equilibrated with the same humidified gas mixture used to ventilate the lungs [O₂/CO₂ (19:1) or O₂/N₂/CO₂ (4:15:1)]. [U-¹⁴C]Phenylalanine was shown to be a suitable precursor for studies of protein synthesis in perfused lungs: it entered the tissue rapidly (t_½, 81s) and was not converted to other compounds. As perfusate phenylalanine was decreased below 5 times the normal plasma concentration, the specific radioactivity of the pool of phenylalanine serving as precursor for protein synthesis, and thus [¹⁴C]phenylalanine incorporation into protein, declined. In contrast, incorporation of [¹⁴C]histidine into lung protein was unaffected. At low perfusate phenylalanine concentrations, rates of protein synthesis that were based on the specific radioactivity of phenylalanyl-tRNA were between rates calculated from the specific radioactivity of phenylalanine in the extracellular or intracellular pools. Rates based on the specific radioactivities of these three pools of phenylalanine were the same when extracellular phenylalanine was increased. These observations suggested that: (1) phenylalanine was compartmentalized in lung tissue; (2) neither the extracellular nor the total intracellular pool of phenylalanine served as the sole source of precursor for protein; (3) at low extracellular phenylalanine concentrations, rates of protein synthesis were in error if calculated from the specific radioactivity of the free amino acid; (4) at high extracellular phenylalanine concentrations, the effects of compartmentalization were negligible and protein synthesis could be calculated accurately from the specific radioactivity of the free or tRNA-bound phenylalanine pool.     

CHANGES IN POLYSOMES OF THE DEVELOPING RAT BRAIN

Abstract— Rat brain polysomes were prepared from a deoxycholate-treated postmito-chondrial supernatant in the presence of 2% bentonite and 1 mg/ml of yeast RNA to prevent partial degradation during preparation.

• 1 The polysomal preparations had an absorption maximum at 260 mμ and an absorption minimum at 235 mμ. The ratio of absorption maximum to minimum and the RNA to protein ratio were 1·58 and 1·06 respectively in 6-day-old rat brain polysomes. The sedimentation patterns showed six distinct peaks with sedimentation coefficients of 235S, 185S, 173S, 135S, 100S and 80S, indicating that these preparations have the characteristics of pure heavy polysomes.

• 2 The rate of [¹⁴C]phenylalanine incorporation into brain polysomal protein was maximal at approximately 10 days of age and decreased thereafter. A similar progressive reduction with increasing age was found in the stimulation of phenylalanine incorporation by the addition of 60 μg/tube of polyuridylic acid. However, the incorporation of phenylalanine into young rat brain polysomes was usually greater even with the addition of polyuridylic acid than in the older animals.

• 3 The comparative studies on sucrose density gradient centrifugation of polysomes between young and adult rat brains showed a considerable decrease of heavy polysomes in the older animals.

• 4 The effect of various factors on the stability of brain polysomes from both ages has been studied. The rates of RNA, protein and acid-soluble phosphorus release from polysomes of the adult rat brains were usually greater in the presence of high salt concentration, ethylenediaminetetra-acetic acid and urea than those from the corresponding preparations of younger animals. On the basis of evidence obtained from the above results it suggested that the adult brain polysomes were more unstable than those of younger animals.

• 5 The amount of polysomal RNA linearly increased up to the first 20 days after birth and then levelled off. The ratio of G + C/A + U of polysomal RNA was less in the young rat brains, falling to 1·30 as compared to 1·50 in older animals. The differences were statistically significant at less than a 1% level of confidence.

• 6 Polysomal preparations also contained RNase, phosphomonoesterase, phospho-diesterase and 5′-nucleotidase activities which cannot be washed off. The specific activities of these enzymes were generally higher in young rat brains than those in the adult.

     

Biosynthesis of Starch in Proplastids of Germinating Ricinus communis Endosperm Tissue

Electron photomicrographs of endosperm tissue from germinating seed of Ricinus communis L. cv. Hale show proplastids which contain prominent starch grains. The content of starch in endosperm tissue increased from 500 micrograms per seed, in imbibed seed, to 1,100 micrograms per seed in 5-day-old seedlings. The maximum net rate of starch deposition was 1.1 nanomoles glucose incorporated per minute per seed. About 200 micrograms of starch remained in the endosperm 9 days after imbibition. Starch content followed the same developmental pattern as the content of sucrose, free reducing sugars, and other metabolic processes found in this tissue. Two key enzymes of starch synthesis, adenosine diphosphoglucose (ADPG) pyrophosphorylase and ADPG-starch glucosyl transferase (starch synthetase) exhibited maximum activities at 4 and 5 days after germination, respectively. The maximum activity of ADPG pyrophosphorylase was 8.17 nanomoles ADPG formed per minute per seed, whereas starch synthetase exhibited an activity of 125 nanomoles glucose incorporated per minute per seed. These levels of enzyme activity are sufficient to account for the starch synthesis observed. Other enzymes which may be involved in starch synthesis include 3-phosphoglycerate kinase which showed an activity of 8.76 units per seed, triose-P isomerase (2.56 units per seed), fructose-1,6-bisphosphate aldolase (0.99 units per seed), fructose-1,6-bisphosphatase (0.23 units per seed), phosphoglucose isomerase (12.6 units per seed), and phosphoglucomutase (9.72 units per seed). The activities of these enzymes were similar to previously reported values.

Starch synthetase was found in association with the fraction containing proplastids isolated from endosperm tissue. Of the total starch synthetase activity in the endosperm, 38% was particulate. Forty-four% of the total particulate activity of starch synthetase placed on sucrose gradients was associated with the band containing proplastids. The proplastids contained 98% of the ribulose 1,5-bisphosphate carboxylase carboxylase activity placed on the gradient.

     

Studies on the protein-synthesizing activity of the ribosomes of rat liver. The activity of free polysomes

1. The activities of microsome fractions from the liver of adult and 5-day-old rats for the incorporation of [(14)C]phenylalanine into protein were similar in the presence and absence of polyuridylic acid. 2. The activity of a light-microsome fraction from adult liver was greater than that of a heavy-microsome fraction, and the light-microsome fraction was also more markedly stimulated by the presence of polyuridylic acid. 3. The light-microsome fraction, when analysed by density-gradient centrifugation, contained a higher ratio of free ribosomes to bound ribosomes, whereas the reverse was true for the heavy-microsome fraction. Similar results were obtained for liver from adult and 5-day-old rats. 4. When the light-microsome fraction was incubated under conditions in which amino acid was incorporated into protein there was only a small increase in the ratio of free to bound ribosomes. When such a fraction was incubated with [(14)C]leucine and was then subjected to density-gradient centrifugation the fraction with the highest specific activity based on RNA had a density between that of the bound and free ribosomes. Treatment of the incubated fraction with ribonuclease shifted the radioactivity towards the free ribosome peak. These properties are consistent with the presence of active free polysomes. Such a component appeared also to be present when the heavy-microsome fraction was incubated under similar conditions. 5. The effect of the presence of polyuridylic acid on the incorporation of [(14)C]phenylalanine by the light-microsome fractions from liver of adult and 5-day-old rats was greatest in the region of the free ribosomes, but it is probable that some small polysomes containing polyuridylic acid are formed. 6. Polyuridylic acid also stimulated the bound ribosomes to a small extent when the heavy-microsome fraction from the liver of young rats was incubated with [(14)C]phenylalanine. 7. The results are discussed in terms of the various morphological constituents in liver now known to play a role in the synthesis of protein for export and for the internal activity of the cell.     

Role of seed coat in imbibing soybean seeds observed by micro-magnetic resonance imaging

Background and Aims

Imbibition of Japanese soybean (Glycine max) cultivars was studied using micro-magnetic resonance imaging (MRI) in order to elucidate the mechanism of soaking injury and the protective role of the seed coat.

Methods

Time-lapse images during water uptake were acquired by the single-point imaging (SPI) method at 15-min intervals, for 20 h in the dry seed with seed coat, and for 2 h in seeds with the seed coat removed. The technique visualized water migration within the testa and demonstrated the distortion associated with cotyledon swelling during the very early stages of water uptake.

Key Results

Water soon appeared in the testa and went around the dorsal surface of the seed from near the raphe, then migrated to the hilum region. An obvious protrusion was noted when water reached the hypocotyl and the radicle, followed by swelling of the cotyledons. A convex area was observed around the raphe with the enlargement of the seed. Water was always incorporated into the cotyledons from the abaxial surfaces, leading to swelling and generating a large air space between the adaxial surfaces. Water uptake greatly slowed, and the internal structures, veins and oil-accumulating tissues in the cotyledons developed after the seed stopped expanding. When the testa was removed from the dry seeds before imbibition, the cotyledons were severely damaged within 1·5 h of water uptake.

Conclusions

The activation of the water channel seemed unnecessary for water entry into soybean seeds, and the testa rapidly swelled with steeping in water. However, the testa did not regulate the water incorporation in itself, but rather the rate at which water encountered the hypocotyl, the radicle, and the cotyledons through the inner layer of the seed coat, and thus prevented the destruction of the seed tissues at the beginning of imbibition.Key words: Dry seeds, Glycine max, MRI, seed coat, soaking injury, soybean, testa, role of inner layer of seed coat, water uptake     

Protein synthesis in the embryo ofPinus thunbergii seed III.

Dissociability of the monomer ribosomes prepared from dry and imbibed pine (Pinus thunbergii) seed embryos was analyzed in sucrose density gradient containing a high salt buffer. Abnormal dissociation into the subunits was observed with the ribosome preparation from dry seed embryos when compared with that from imbibed seed embryos, i.e. each subunit peak was broader and localized at a lower site in sucrose density gradient. This indicates some change(s) in ribosomes during imbibition of seeds. These ribosomal changes also progressedin vitro. That is, after incubation of ribosome preparation from dry seed embryos in a high salt buffer for 5 min at 30 C or in a low salt buffer for 15 hr at 0 C, complete dissociation into the normal subunits was observed. No difference was found between polyacrylamide gel electrophoresis patterns of ribosomal RNA from dry and imbibed seed embryos. These results suggest some alteration in the protein components of ribosome during imbibition of pine seeds. This paper is dedicated to Prof. Shyogo Sawamura, Utsunomiya University on his retirement in March, 1979.     

Localization of major endopeptidase activities in maize endosperms

Using a tissue print method, major endopeptidase activitieswere observed in the aleurone layer and along parts of scutellumsurface 1 d after imbibition. By day 2 the zone of activityhad spread into the subaleurone and starchy parenchyma cellsof the endosperm. Three days later, activity was detected throughoutthe endosperm tissue, but not in the embryo. Endosperm tissues,aleurone layers and scu-tella were dissected from the seedlingsat different stages after imbibition and endopeptidase activitywas analysed by an activity stain after native PAGE. At leastten different endopeptidase activities were detected in theendosperm tissues during the initial 5 d. Activities similarto these ten enzymes were also detected in aleurone layers.These results suggest that the main source of these endopeptidasesin the endosperm is the aleurone layer. The scutellum had adifferent spectrum of endopeptidases. One of these alternativeendopeptidases, which was detected on the first day after theaddition of water, was a metallo-enzyme with electrophoreticproperties similar to an activity found in endosperm tissueshortly after imbibition. Key words: Zea mays, endopeptidase localization, seed germination     

Weakening of muskmelon perisperm envelope tissue 4

Muskmelon (Cucumis melo L.) embryos are enclosed in an envelopeof tissue consisting of a layer of endosperm and a multi-cell-layeredperisperm that the radicle must penetrate for germination tooccur. The force and energy required to penetrate the perispermenvelope tissue were measured using an Instron universal testingmachine at a crosshead speed of 5 mm min^–1 after 0, 10,15, 22, 23, and 25 h of imbibition at 25C. The cellular structureof perisperm envelope tissue surrounding the radicle was observedafter 10, 15, 20, 25, and 48 h of imbibition using scanningelectron microscopy. The force required to puncture 5-mm-long,micropylar seed pieces declined steadily from 1.65 N in driedseeds to 0.65 N after 21 h of imbibition. The penetration energydeclined from 3.0 N mm in dry seeds to 1.1 N mm at 21 h afterthe start of imbibition when the first seeds germinated. Theforce and energy required to penetrate germinated seed pieceswere 0.55 N and 0.9 N mm, respectively, so the net punctureforce and energy needed to rupture the micropylar region ofthe perisperm envelope was roughly 0.10 N and 0.2 N mm at radicleemergence, respectively. Instron measurements of penetrationforce and energy decreased dramatically at crosshead speedsless than the 5 mm min^–1. Crosshead speeds greater than5 mm min^–1 may overestimate the pressure needed to ruptureperisperm and endosperm tissues. Intracellular cracks were firstobserved in SEM images 15 h after the start of imbibition, andafter 20 h cracking was apparent throughout the micropylar regionof the perisperm envelope. The perisperm envelope ruptured inone of two ways, coincident with radicle emergence. In approximately85% of muskmelon seeds, a large crack formed in the perispermenvelope adjacent to the radicle, while in roughly 15 % a circulararea of the perisperm envelope detached during radicle emergence.In dead seeds, the penetration force remained constant from10–24 h after the start of imbibition, and there wereno visible signs of tissue degradation. Cellular degradationand weakening of the perisperm envelope tissue precedes radicleemergence in muskmelon seeds. Key words: Seed, Instron, turgor, cell wall, electron microscopy, Cucumis melo