Respiration and Utilization of Storage Materials in Wild and Cultivated Pea Seeds during Germination

Breakdown of storage materials, oxygen uptake, respiratory control and ADP/O ratios in the cotyledons of the garden pea P. sativum and in the wild pea P. elatius were compared. Starch and protein degradation was slower in P. elatius than in P. sativum. Embryo growth began later in the wild pea. However, in the garden pea the mitochondria were uncoupled after about 48 h of germination, while in P. elatius the ability to carry out oxidative phosphorylation was maintained for 4 days. The respiratory control ratio was higher in the wild pea at all stages of germination and a steady level of oxygen uptake was maintained in the cotyledons for at least 3 days. The findings are discussed in relation to the ecological requirements for germination in the two species.     

Galactosidases in cultivated and wild peas

The level of α- and β-galactosidase was followed in the cotyledons and embryos of germinating seeds of Pisum sativum and P. elatius. α-Galactosidase is preformed in the cotyledons but its activity increases during germination in the embryos. β-Galactosidase activity in embryos increases during germination but shows little change in cotyledons. The possible function of α- and β-galactosidase is discussed.     

Influence of Endogenous Cytokinins on Reverse Mobilization in Cotyledons of Cicer arietinum L: Reproduction of Endogenous Levels of Total Cytokinins, Zeatin, Zeatin Riboside, and Their Corresponding Glucosides

The embryonic axis plays an essential role in the mobilization of the main reserves of the cotyledons of seeds of Cicer arietinum L. cv Castellana. This control by the axis of the metabolism of the storage products of the cotyledons largely takes place through the cytokinins, which are transported from the embryonic axis to the cotyledons where the mobilization of reserves begins. The principal regulatory role of the endogenous cytokinins concerns the metabolism of carbohydrates and proteins; there is less influence on lipid metabolism. However, each cytokinin seems to have a different role in the mobilization processes. The glucosides, glucosyl zeatin riboside, and glucosyl zeatin act only as storage forms of the hormones. Zeatin riboside affects mainly the mobilization of carbohydrates and has less effect on protein mobilization. Zeatin regulates both the mobilization of carbohydrates and that of proteins and is more marked in the latter case.     

The Influence of Axis Removal on Protein Metabolism in Cotyledons of Pisum sativum L

The protein metabolism of cotyledons attached to the embryonic axis has been compared with that in cotyledons removed from the axis at the initiation of a 6-day imbibition. Total protein declined in the attached but not in the detached cotyledons. Concurrent with the decline in protein level in the intact cotyledons there was an increased capacity to incorporate exogenously supplied leucine into protein. In contrast, detached cotyledons showed a restricted capacity for protein synthesis. It was demonstrated that ribosomal preparations from cotyledons of intact seedlings contained an increasing proportion of polyribosomes as germination progressed and such ribosomes were active in in vitro amino acid incorporation. Ribosomal preparations from detached cotyledons contained few polyribosomes and had a restricted capacity to incorporate amino acids in vitro. The in vitro incorporation of phenylalanine was stimulated by polyuridylic acid with the stimulation being greatest in ribosomal preparations from detached cotyledons. The results suggest that an axis component may regulate the availability of messenger RNA in the cotyledons during germination.     

NAD pattern and NADH oxidase activity in pea (Pisum sativum L.) under cadmium toxicity

Seeds of pea (Pisum sativum L.) were germinated for 5 days by soaking in distilled water or 5 mM cadmium chloride. Compared to the control, cadmium (Cd) caused a reduction in percent germination and embryo growth. Pyridine nucleotide coenzyme concentrations were determined in cotyledons and embryonic axis. Nicotinamide adenine dinucleotide (NADH) oxidase activity was examined. Cd treatment caused a restriction in levels of reduced coenzyme form in the mitochondria and the post-mitochondrial fraction of cotyledons, and embryonic axis. The oxidized coenzyme form has been accumulated by Cd-treated mitochondria of both tissues. It was also found that NADH oxidase activity was stimulated. The relationship between coenzyme levels, seed germination, pea growth, and Cd stress has been reported.     

Development of Mitochondrial Activity in Pea Cotyledons Following Imbibition; Influence of the Embryonic Axis

The development of mitochondria in cotyledons during the initialstages following imbibition and the subsequent degenerationwere faster when the embryonic axis was attached. This was moreclearly demonstrated when mitochondrial activity was determinedusing malate or -oxoglutarate as substrate rather than NADHor succinate. Cycloheximide did not inhibit the initial developmentof mitochondria in attached cotyledons, although it inhibitedthe incorporation of ¹⁴C-amino acid into protein. Abscisic acidinhibited almost completely the initial increase in mitochondrialactivities of attached cotyledons. The activities of severalenzymes in mitochondrial fractions were almost the same in attachedand detached cotyledons during 4 d after, imbibition. The degenerationof mitochondria was not accompanied by the loss of enzymes.It was inferred that the initial development and the subsequentdegeneration of mitochondria in cotyledons during 4 d afterimbibition was brought about by the structural improvement anddisorganization of mitochondria, respectively. The initial differencesin the development of mitochondrial activities between attachedand detached cotyledons were suggested to be attributable todifferences in the development of the activities of electrontransfer pathway from endogenous NADH to ubiquinone.     

Diamine Oxidase in Cotyledons of Pisum sativum Develops as a Result of the Supply of Oxygen through the Embryonic Axis during Germination

The activity of diamine oxidase (EC 1.4.3.6.) in pea, Pisum sativum cv Alaska, cotyledons was studied. The rapid hydration caused by soaking seeds in water, the excision of the embryonic axis, and the suppression of the elongation of the embryonic axis by indoleacetic acid generate anaerobic conditions in these cotyledons that suppress diamine oxidase activity. These results show that oxygen is essential for the induction of diamine oxidase activity in pea cotyledons. During germination cotyledonary diamine oxidase develops as a result of the supply of oxygen through the embryonic axis of the intact pea seedling.     

The influence of the embryonic axis and cytokinins on reserve mobilization in germinating lupin seeds

The influence of the embryonic axis and cytokinins (CKs) onreserve mobilization has been examined in yellow lupin (Lupinusluteus L. cv. JSG 6167) seed during germination and during earlygrowth for up to 9 d in the dark. The study included determinationof starch, soluble sugars, proteins, and amino acid content.Amylolytic and proteolytic enzyme activity was also measuredin untreated cotyledons with intact embryo (attached) or detachedcotyledons (embryo removed), and in detached cotyledons followingtreatment with CKs namely, dihydrozeatin, (diH)Z, and 6-benzylaminopurine,BAP. Generally, the detached cotyledons showed reduced mobilizationand decreased enzymatic activity in comparison to attached cotyledons,indicating the importance of the embryonic axis in this process.However, a rise in protease activity and free amino acid contentwas detected in 9-d-old detached cotyledons suggesting thatthe end products do not necessarily inhibit enzyme activity.While (diH)Z was partially effective in inducing reserve mobilizationand enzymatic activity in detached cotyledons, the effect ofBAP was more pronounced and appeared to replace the embryonicaxis. The embryonic axis of this species has recently been shownto synthesize CKs which are transported to the cotyledons, arehighly stabe and induce cotyledon expansion and chlorophyllsynthesis. The results of the present investigation and previousstudies from this laboratory collectively indicate that theregulation of reserve mobilization in yellow lupin seeds appearsto be mediated, at least in part, by a stimulus, probably aCK, emanating from the embryonic axis. Key words: Lupinus luteus, cytokinins, benzylaminopurine, dihydrozeatin, embryonic axis, lupin seeds, reserve mobilization     

Influence of the axis on the development of mitochondrial activity in imbibed black gram cotyledons

The respiration rate of mitochondria from detached Vigna mungo L. cotyledons (without the embryonic axis) doubled during the first day after imbibition, whereas that of mitochondria from attached ones increased by only 50%. Contrary to the respiration rate, the respiratory control ratio was higher in attached cotyledons. The activities of enzymes in the mitochondrial fraction from detached cotyledons increased by about 30% during the first day, while those in mitochondria from attached ones changed little. Cycloheximide did not inhibit the development of mitochondrial respiration in either attached or detached cotyledons, although it almost completely inhibited the incorporation of [¹⁴C]-leucine into mitochondrial proteins. Cycloheximide did not retard the increase in the activities of mitochondrial enzymes in detached cotyledons. It is inferred that the development of mitochondria in black gram cotyledons is brought about by repair or activation of mitochondria present in dry seeds and that the axis affects this repair process.     

Auxins induce alpha-amylase activity in pea cotyledons

The report documents how the development of α-amylase activity in detached cotyledons of Pisum sativum cv Alaska is accelerated 2- to 12-fold during incubation with 1 micromolar to 10 micromolar 2,4-dichlorophenoxyacetic acid, 2,4,5-trichlorophenoxyacetic acid, or with 4-chloroindoleacetic acid, an endogenous auxin from Pisum sativum. It seems probable that auxin from the embryonic axis induces α-amylase in the attached cotyledons during germination.     

Increased Na+ and Cl− accumulation induced by NaCl salinity inhibits cotyledonary reserve mobilization and alters the source-sink relationship in establishing dwarf cashew seedlings

We studied the NaCl-induced changes in cotyledons and the embryonic axis of establishing dwarf cashew (Anacardium occidentale) seedlings. The salt stress reduced the growth of dwarf cashew seedlings, and this response was related to the inhibition of cotyledonary reserve depletion. Lipid mobilization was inhibited by NaCl due to reduced lipase activity in the emerging and establishing seedlings. Additionally, there was reduced transient starch accumulation in the cotyledons of the salt-stressed seedlings that was associated with lower starch synthase activity at the early developmental stages and inhibited amylolytic and starch phosphorylase activities at the established seedling stage. The NaCl-induced changes in lipid and starch metabolism influenced the soluble sugar content in the cotyledons. Protein mobilization was inhibited by NaCl, and we observed the accumulation of amino acids and the inhibition of proteolytic activity in the cotyledons of the salt-stressed established seedlings. Salinity significantly reduced the free amino acid and reducing sugar contents in the embryonic axes of both emerged and established seedlings, whereas the non-reducing sugar content was affected by this stress only in the established seedlings. The Na⁺ and Cl^? contents progressively increased in the cotyledons and embryonic axis of the seedlings as the salinity increased. We conclude that salt stress inhibits dwarf cashew seedling establishment by inhibiting the mobilization of reserves, an inhibition that was related to increased Na⁺ and Cl^? accumulation in the cotyledons. Additionally, these toxic ions reduced the sink strength of the embryonic axis with regard to the products of cotyledonary reserve mobilization.     

Imunohistochemical Localization of alpha-Amylase in Cotyledons of Vigna mungo Seedlings

We studied the localization of α-amylase with indirect fluorescence microscopy in transversely sectioned cotyledons of Vigna mungo seedlings. Tissue sections were fixed in periodate-lysine-paraformaldehyde and treated with anti-α-amylase immunoglobulin G followed by fluorescein isothiocyanate labeled goat anti-rabbit immunoglobulin G. α-Amylase appeared in the cells farthest from vascular bundles on the second day of growth and appeared gradually closer to the vascular bundles as growth progressed. The pattern of α-amylase appearance was similar in detached cotyledons, indicating that attachment of the embryonic axis has no effect on this pattern. However, in attached cotyledons, α-amylase disappeared from the regions where starch grains had been digested, but in detached cotyledons there was no disappearance of α-amylase, and digestion was slower than in intact cotyledons.     

No influence of the embryonic axis on the development of diamine oxidase in pea cotyledons

A reexamination has been made for the supposed regulation of pea (Pisum sativum cv Alaska) cotyledonary diamine oxidase (EC 1.4.3.6) activity by the embryonic axis. When dry cotyledons from which the embryo and testa have been removed surgically are imbibed by soaking in water, there is little increase of the enzyme activity during subsequent incubation on filter paper. However, if the dry cotyledons are imbibed and maintained on filter paper from the first, the increase of the enzyme activity is similar to that in the intact seedling. Thus, rapid imbibition of the isolated dry cotyledons is responsible for repression of enzyme development, and a role for the axis need not be invoked.     

Cytokinins in Germinating Seeds of Phaseolus vulgaris L. I. Changes in Endogenous Levels Within the Cotyledons

Ethanolic extracts from the cotyledons of mature dry Phaseolusvulgaris L. seed yielded cytokinin-like activity which co-chromatographedwith zeatin and ribosylzeatin. Under conditions which stimulatedgermination and cotyledon expansion, the level of these cytokininsdecreased rapidly in both intact embryos and excised cotyledons.In the excised cotyledons the decrease was continuous, resultingin very low levels of cytokinin being detected after 4 daysof incubation. With the embryonic axis present, however, theinitial decrease was arrested and reversed after 3 days. Thissuggests that the cotyledons do not synthesize cytokinins butthat these hormones are imported from the embryonic axis, particularlyonce radicle growth is well under way. Phaseolus vulgaris, bean, cotyledons, cytokinins, germination     

Permeability of seed coats to water as related to drying conditions and metabolism of phenolics

The seed coat of Pisum elatius is normally impermeable to water. When seeds are dried in the absence of oxygen their coats are totally permeable to water. Structural differences are observed between permeable and impermeable seed coats. In the genus Pisum, species with normally impermeable seed coats have a high content of phenolics and of catechol oxidase, while seed coats of P. sativum contain very little catechol oxidase and have a very low content of phenolics. Such differences are not noted in the cotyledons. We hypothesized that during dehydration of seeds, oxidation of phenolic compounds in seed coats through catalysis of catechol oxidase in presence of O₂ might render the seed coats impermeable to water.     

A soil-borne generalist pathogen regulates complex plant interactions

Background and aims

Seeds are inhabited by diverse bacterial and fungal taxa whose colonization patterns are little understood. We hypothesized, however, that specific niches within seeds host microbes.

Methods

In this study, the putative presence of bacteria, inhabiting the seed endosphere of an angiosperm, the melon Cucumis melo reticulatus group cv. ‘Dulce’, was examined by scanning electron microscopy (SEM) and confocal laser-scanning microscopy coupled with double labeling of oligonucleotide probes for fluorescence in situ hybridization (DOPE-FISH).

Results

SEM images showed microbial-like structures in different tissues and FISH revealed endophytic bacteria colonizing the outer and inner seed parts, on perisperm/endosperm envelope, inside the cotyledons as parts of the embryo, and, to a lesser extent, inside embryonic hypocotyl-root axis tissues. Alphaproteobacteria were shown to inhabit the seed coat and the envelope surrounding the embryonic hypocotyl-root tissues, but could not be seen in the cotyledons, whereas Betaproteobacteria were only detected in the outer seed coat. Some Gammaproteobacteria were also seen in the outer seed coat, but were mainly visualized in the cotyledons with a few inside the seed’s embryonic hypocotyl-root tissues, among other bacteria. Firmicutes were visualized inside the seed coat, but mostly inside the cotyledon tissues, on the perisperm/endosperm envelope and inside the embryonic hypocotyl-root axis tissues. Microscopy revealed Actinobacteria inside the inner and outer seed coat and inside the embryonic parts such as cotyledons, with a few inside the hypocotyl-root axis.

Conclusions

This is the first demonstration of niches for the most active groups of bacteria inhabiting different seed tissues of an angiosperm.

     

Reactive oxygen species release, vitamin E, fatty acid and phytosterol contents of artificially aged radish (Raphanus sativus L.) seeds during germination

Seeds of Raphanus sativus L. subjected to accelerated ageing were investigated for reactive oxygen species (ROS) release and for content of vitamin E (tocopherol, TOC, and tocotrienol, TOC-3), fatty acids and phytosterols in seed coats, cotyledons and embryonic axes during germination. In unaged seeds, ROS release occurred mainly in seed coats of non-imbibed seeds and in seedlings (48?h of imbibition). TOC and TOC-3 were mainly represented by the ??-isoform, abundant in embryonic axes. Fatty acids were mainly found in cotyledons. In seed coat and embryonic axis, phytosterols consisted mainly of sitosterols. The effects of ageing were mainly visible in embryonic axes at 48?h of imbibition. Deterioration was associated with a decrease in fresh weight increase percentage, germination percentage, ??-TOC and total fatty acid content. An increase in ROS release from seed coats and in ??-TOC, ??-TOC, ??-TOC-3 content in embryonic axis was also observed. The use of ??-TOC and total fatty acids in embryonic axis as parameters of seed quality evaluation during storage was suggested.     

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     

Isocitrate lyase in germinating seeds of Prunus dulcis

Isocitrate lyase activity increased in cotyledons of germinating seeds of Prunus dulcis (almond) which had been induced to germinate by either stratification or treatment with gibberellic acid (GA). Germination of embryos, growth of the embryonic axis and activity of isocitrate lyase increased with increasing concentrations of GA from 10^?7 to 10^?3 M.