Endosperm Cap Research Articles

Structure-activity Studies with ABA Analogs on Germination and Endo-β-Mannanase Activity in Tomato and Lettuce Seeds

Summary The effects of several analogs of ABA were tested on tomato ( Lycopersicon esculentum Mill.) and lettuce ( Lactuca sativa L.) seed germination and on the ability of these seeds to produce endo-β-mannanase (EC 3.2.1.78) during and following germination. The (+)-enantiomers of ABA and its analogs were more effective than the (−)-enantiomers in inhibiting the germination of seeds of both species. Endo-β-mannanase activity in the tomato endosperm cap during germination was not influenced by ABA or its analogs, regardless of whether the seeds completed germination or not. Thus, while there was a structure-activity relationship of the ABA analogs with respect to tomato seed germination, there was no such relationship with respect to endo-β-mannanase activity in the endosperm cap during this event. Post-germination enzyme activity in the tomato lateral endosperm region, on the other hand, was inhibited by the (+)-enantiomers of ABA and its analogs. (−)-Dihydroacetylenic ABA alcohol, a purported competitive inhibitor of ABA, did not reverse the inhibitory effects of (+)- or (−)-ABA on tomato seed germination; instead, it enhanced the inhibition. Lettuce seed endo-β-mannanase activity was suppressed by most analogs of ABA, and in the presence of (−)-dihydroABA and (+)-dihydroABA very low amounts of enzyme were produced in the endosperms of seeds that were able to complete germination. This observation supports the suggestion that there is no cause-and-effect relationship between lettuce seed germination and endo-β-mannanase production.

Internal anatomy of individual tomato seeds: relationship to abscisic acid and germination physiology

AbstractTomato seeds that have been dried, imbibed and redried (primed) develop internal free space between the embryo and endosperm. Seeds of the ABA-deficient sitiens(sitw) tomato mutant can exhibit internal free space at the completion of seed development even without priming. Both primed and sitwseeds germinate more rapidly than untreated wild-type seeds. To determine whether internal anatomy predicts germination physiology, individual sitwand primed wild-type seeds were sorted into three categories based upon the extent of internal free space observed non-destructively using X-radiography. Category 3 (C3, extensive free space present) sitw seeds completed germination more rapidly than all other seed categories and genotypes in water, in abscisic acid (ABA) or under far-red illumination. The force necessary to puncture the endosperm caps (and testa) of C3 sitw seeds was less, and the percentage of nuclei in C3 sitw radicle tips in the G2 stage of the cell cycle was greater than for all other seed categories. Wild-type seeds exhibited free space following long-term priming, but germination was still prevented by far-red light and ABA, and endosperm cap strength and nuclear DNA contents were not altered. Endo-β-mannanase activity of individual endosperm caps was not consistently related to their resistance to puncture. While internal free space is diagnostic for primed tomato seeds and occurs in a fraction of sitw seeds, it is not predictive of many aspects of germination physiology.

Endosperm cap weakening and endo-β-mannanase activity during priming of tomato (Lycopersicon esculentumcv. Moneymaker) seeds are initiated upon crossing a threshold water potential

AbstractThe relationship between endosperm cap weakening and endo-β-mannanase activity during priming and the time to germination after priming was studied in tomato (Lycopersicon esculentum) seeds. During priming of seeds in −0.4 MPa PEG, the mechanical restraint of the endosperm cap decreased while the endo-β-mannanase activity in the endosperm cap increased. There was no decrease in required puncture force and no increase in endo-β-mannanase activity in seeds during priming in −1.0 MPa PEG. Two classes of seeds could be distinguished during priming in −0.7 MPa PEG: one with decreased required puncture force and one without. A strong correlation was found between the lowering of the mechanical restraint and endo-β-mannanase activity. It was concluded that individual seeds have to cross a threshold water potential in order to develop enzyme activity and lower their mechanical restraint. A decrease in required puncture force and increase in endo-β-mannanase activity correlated with ice crystal-induced porosity in the endosperm cap cell walls in scanning micrographs. It was presumed that ice crystal-induced porosity reflects cell-wall hydrolysis. Germination time after priming correlated positively with required puncture force during priming, depending on the osmotic potential. Seeds in −1.0 MPa PEG improved their time to germination, without a decrease in the required puncture force. Therefore it was concluded that lowering of the endosperm restraint during priming positively affects the time to germination of primed seeds but is not a prerequisite for rapid germination.

Relationship of Endo-[beta]-D-Mannanase Activity and Cell Wall Hydrolysis in Tomato Endosperm to Germination Rates.

The endosperm tissue enclosing the radicle tip (endosperm cap) governs radicle emergence in tomato (Lycopersicon esculentum Mill.) seeds. Weakening of the endosperm cap has been attributed to hydrolysis of its mannan-rich cell walls by endo-[beta]-D-mannanase. To test this hypothesis, we measured mannanase activity in tomato endosperm caps from seeds allowed to imbibe under conditions of varying germination rates. Over a range of suboptimal temperatures, mannanase activity prior to radicle emergence increased in accordance with accumulated thermal time. Reduced water potential delayed or prevented radicle emergence but enhanced mannanase activity in the endosperm caps. Abscisic acid did not prevent the initial increase in mannanase activity, although radicle emergence was markedly delayed. Sugar composition and percent mannose (Man) content of endosperm cap cell walls did not change prior to radicle emergence under any condition. Man, glucose, and other sugars were released into the incubation solution by endosperm caps isolated from intact seeds during imbibition. Pregerminative release of Man was suppressed and the release of glucose was enhanced when seeds were incubated in osmoticum or abscisic acid; the opposite occurred in the presence of gibberellin. Thus, whereas sugar release patterns were sensitive to environmental and hormonal factors affecting germination, neither assayable endo-[beta]-D-mannanase activity nor changes in cell wall sugar composition of endosperm caps correlated well with tomato seed germination rates under all conditions.

A Single-Seed Assay for Endo-β-Mannanase Activity from Tomato Endosperm and Radicle Tissues

Completion of germination (radicle emergence) is an all-or-none developmental event for an individual seed. Variation in germination timing among seeds in a population therefore reflects variation among seeds in the rates or extents of physiological or biochemical processes prior to radicle emergence. For tomato (Lycopersicon esculentum Mill.) seeds, correlative evidence suggests that endo-[beta]-mannanase activity weakens the endosperm cap tissue opposite the radicle tip to permit radicle emergence. To test whether endo-[beta]-mannanase activity is causally related to germination rates, we have developed a sensitive assay suitable for use with individual radicle tips or endosperm caps. We show that endo-[beta]-mannanase activity varies at least 100-fold and often more than 1000-fold among individual inbred tomato seeds prior to radicle emergence. Other sources of variation (tissue size and experimental error) were evaluated and cannot account for this range of activity. Endo-[beta]-mannanase activity was generally 10-fold greater in leachates from endosperm caps than from radicle tips. Release of reducing sugars from individual endosperm caps also varied over a considerable (9-fold) range. These extreme biochemical differences among individual tomato seeds prior to radicle emergence indicate that results obtained from bulk samples could be misleading if it is assumed that all seeds exhibit the "average" behavior.

Endo-[beta]-Mannanase Activity from Individual Tomato Endosperm Caps and Radicle Tips in Relation to Germination Rates.

Endo-[beta]-mannanase is hypothesized to be a rate-limiting enzyme in endosperm weakening, which is a prerequisite for radicle emergence from tomato (Lycopersicon esculentum Mill.) seeds. Using a sensitive, single-seed assay, we have measured mannanase activity diffusing from excised tomato endosperm caps following treatments that alter the rate or percentage of radicle emergence. Most striking was the 100- to more than 10,000-fold range of mannanase activity detected among individual seeds of highly inbred tomato lines, which would not be detected in pooled samples. In some cases a threshold-type relationship between mannanase activity and radicle emergence was observed. However, when radicle emergence was delayed or prevented by osmoticum or abscisic acid, the initial increase in mannanase activity was unaffected or even enhanced. Partially dormant seed lots displayed a bimodal distribution of activity, with low activity apparently associated with dormant seeds in the population. Gibberellin- and abscisic acid-deficient mutant seeds exhibited a wide range of mannanase activity, consistent with their variation in hormonal sensitivity. Although the presence of mannanase activity in the endosperm cap is consistently associated with radicle emergence, it is not the sole or limiting factor under all conditions.

Endo-?-mannanase isoforms are present in the endosperm and embryo of tomato seeds, but are not essentially linked to the completion of germination

A current hypothesis is that endo-β-mannanase activity in the endosperm cap of tomato (Lycopersicon esculentum Mill. cv. Moneymaker) seeds is induced by gibberellin (GA) and weakens the endosperm cap thus permitting radicle protrusion. We have tested this hypothesis. In isolated parts, the expression of endo-β-mannanase in the endosperm after germination is induced by GAs, but the expression of endo-β-mannanase in the endosperm cap prior to radicle protrusion is not induced by GAs. Also, abscisic acid (ABA) is incapable of inhibiting endo-β-mannanase activity in the endosperm cap, even though it strongly inhibits germination. However, ABA does inhibit enzyme activity in the endosperm and embryo after germination. There are several isoforms in the endosperm cap and embryo prior to radicle protrusion that are tissue-specific. Tissue prints showed that enzyme activity in the embryo spreads from the radicle tip to the cotyledons with time after the start of imbibition. The isoform and developmental patterns of enzyme activity on tissueprints are unaffected when seeds are incubated in ABA, even though germination is inhibited. We conclude that the presence of endo-β-mannanase activity in the endosperm cap is not in itself sufficient to permit tomato seeds to complete germination.

Involvement of Endomannanase in the Control of Tomato Seed Germination under Low Temperature Conditions

The cause of differences in germination rates in a cold-tolerant tomato line (PI341988), a control line (UC82B), and six progeny lines stemming from crosses and backcrosses between the two parent lines was investigated. Pursuant to earlier work showing that differences in germination ability at 12°C are due to the barrier imposed by the endosperm layer, we analysed the activity of cell-wall-hydrolysing enzymes extracted from these lines. A significant increase in endomannanase activity was found in plant line PI341988 prior to germination at 12°C. Extracts of PI341988 seeds that had imbibed at either 12 or 25°C exhibited higher endomannanase activity than their counterparts from plant line UC82B. Moreover, a positive relationship was found between germination ability at low temperature and endomannanase activity in the six progeny lines. Analysis of endomannanase activity in sub-regions of the seed indicated that the increase in activity prior to germination was higher in the micropylar endosperm cap than in the rest of the seed. Exogenous application of mannanase originating from soil-borne bacteria increased germination rates under both moderate and low temperature conditions. Cellulase (endo-1,4-β-glucanase) activity was also found to be higher in plant line PI341988. However, the activity of this enzyme probably increases after germination and it is therefore not considered as a key enzyme controlling germination at low temperatures.

Morphological Studies on Amaranthus cruentus

ABSTRACTThe morphology of Amaranthus cruentus was studied using scanning election and light microscopy. The A. cruentus seed consists of a single layer of cells which is intimately associated with the perispeim. In the region of the embryo, the seed coat is attached to the endosperm remaining in the mature seed. The major portion of endosperm caps the root. Large, thick‐walled endosperm cells contain spherical bodies embedded in a spongy matrix. The cells of the campylotropous embryo contain material which has properties typical of lipid and lipid complexes as demonstrated by hexane extraction and by staining with osmium tetroxide. Spherical cell bodies, embedded in the lipid‐containing matrix are partially digested by trypsin, thus suggesting they are proteinaceous in nature. Perisperm in the center of the seed contains starch granules in the form of amylopectin, demonstrated by iodine‐potassium iodide stain and α‐amylase digestion.