Henk W. M. Hilhorst

Structure and Composition

Seeds are very diverse in their shape and size. In the mature state they contain an embryo, the next generation of plant, surrounded by a protective structure (the seed and/or fruit coat) and, in species in which the nutritive reserves are not stored within the cotyledons, by an alternative storage tissue (endosperm, perisperm, or megagametophyte). Most seeds contain large and characteristic quantities of polymeric reserves. The major ones are carbohydrates, oils, and proteins, with minor amounts of phosphate-rich phytin. Starch, a polymer of glucose, is the most common form of stored carbohydrate, contained within cytoplasmic starch granules; less common are the hemicelluloses, stored in secondary cell walls, usually as mannan polymers. Oils are triacylglycerols, each composed of glycerol and three fatty acids that are specific to the oil; these are present within oil bodies. Storage proteins, of which there are three types, albumins, globulins, and prolamins, are sequestered in protein storage vacuoles. These reserves are vital components of human and animal diets, and their production in crops is a basis of agriculture.

A critical update on seed dormancy. I. Primary dormancy (review).

The emphasis of modern dormancy research is almost entirely on the form of dormancy that is acquired during seed development, primary dormancy. Abscisic acid (ABA) appears to be intimately involved in its regulation. The action of abscisic acid has also been implied in many other developmental processes. The coincidence of developmental events, such as dehydration and completion of maturation, with the acquisition of primary dormancy suggests that dormancy is influenced by these processes. Germinability, both during development and after maturation, is sometimes directly correlated with ABA content. The lack of such a correlation may be explained by assuming a decisive role for the responsiveness to ABA or other overriding factors. ABA has been detected in all seed components. The different seed tissues may all contribute, to various extents, to the degree of whole seed dormancy. It is concluded that ABA action in dormancy regulation is not restricted to the embryo but is also located in endospermic tissue. In addition, a role of ABA in the morphological development of germination modifying seed tissues is proposed. The mechanism for ABA action appears to be associated with cell wall properties.

Identification of Reference Genes for RT–qPCR Expression Analysis in Arabidopsis and Tomato Seeds

Quantifying gene expression levels is an important research tool to understand biological systems. Reverse transcription-quantitative real-time PCR (RT-qPCR) is the preferred method for targeted gene expression measurements because of its sensitivity and reproducibility. However, normalization, necessary to correct for sample input and reverse transcriptase efficiency, is a crucial step to obtain reliable RT-qPCR results. Stably expressed genes (i.e. genes whose expression is not affected by the treatment or developmental stage under study) are indispensable for accurate normalization of RT-qPCR experiments. Lack of accurate normalization could affect the results and may lead to false conclusions. Since transcriptomes of seeds are different from other plant tissues, we aimed to identify reference genes specifically for RT-qPCR analyses in seeds of two important seed model species, i.e. Arabidopsis and tomato. We mined Arabidopsis seed microarray data to identify stably expressed genes and analyzed these together with putative reference genes from other sources. In total, the expression stability of 24 putative reference genes was validated by RT-qPCR in Arabidopsis seed samples. For tomato, we lacked transcriptome data sets of seeds and therefore we tested the tomato homologs of the reference genes found for Arabidopsis seeds. In conclusion, we identified 14 Arabidopsis and nine tomato reference genes. This provides a valuable resource for accurate normalization of gene expression experiments in seed research for two important seed model species.

Abscisic acid controls embryo growth potential and endosperm cap weakening during coffee (Coffea arabica cv. Rubi) seed germination.

The mechanism and regulation of coffee seed germination were studied in Coffea arabica L. cv. Rubi. The coffee embryo grew inside the endosperm prior to radicle protrusion and abscisic acid (ABA) inhibited the increase in its pressure potential. There were two steps of endosperm cap weakening. An increase in cellulase activity coincided with the first step and an increase in endo-β-mannanase (EBM) activity with the second step. ABA inhibited the second step of endosperm cap weakening, presumably by inhibiting the activities of at least two EBM isoforms and/or, indirectly, by inhibiting the pressure force of the radicle. The increase in the activities of EBM and cellulase coincided with the decrease in the force required to puncture the endosperm and with the appearance of porosity in the cell walls as observed by low-temperature scanning electronic microscopy. Tissue printing showed that EBM activity was spatially regulated in the endosperm. Activity was initiated in the endosperm cap whereas later during germination it could also be detected in the remainder of the endosperm. Tissue printing revealed that ABA inhibited most of the EBM activity in the endosperm cap, but not in the remainder of the endosperm. ABA did not inhibit cellulase activity. There was a transient rise in ABA content in the embryo during imbibition, which was likely to be responsible for slow germination, suggesting that endogenous ABA also may control embryo growth potential and the second step of endosperm cap weakening during coffee seed germination.

The regulation of secondary dormancy: the membrane hypothesis revisited.

Secondary dormancy is predominantly associated with seed behaviour in soil seed banks. Periodic changes in secondary dormancy may explain seasonal emergence of weedy species. Temperature and possibly soil water potential appear to be the predominant factors that determine the annual cycling of dormancy. Dormancy cycling is paralleled by modulations of germination responsiveness to germination stimulants, such as light and nitrate and to the width of the germination temperature window. Membranes have been proposed to be the primary target for the perception of temperature. It is hypothesized that alterations in properties of cellular membranes are involved in the regulation of dormancy. Possible mechanisms are discussed.

Endo-β-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.

Dose-Response Analysis of Factors Involved in Germination and Secondary Dormancy of Seeds of Sisymbrium officinale II. Nitrate

The role of nitrate as a promoter of germination of Sisymbrium officinale seeds was examined in optimal light conditions. It was shown that the requirement for nitrate was absolute. This was true for all seed lots used. The probit of germination in water was log-linearly related to the level of endogenous nitrate. Preincubation at 15 degrees C resulted in an immediate decrease in germination, whereas in 25 millimolar KNO(3) the decrease was delayed. The decline of germination in water was strongly correlated with the rate at which nitrate leached from the seeds. The germination response to a range of KNO(3) concentrations was followed during preincubation at 24-hour intervals. During the entire 264-hour preincubation period increasingly higher nitrate concentrations were required to maintain a response. This resulted in a right-hand shift of the dose-response curve parallel to the x axis. After 120 hours the high maximum germination level started to decline. The dose-response curves could be simulated by an equation from the receptor-occupancy theory. It is proposed that induction of secondary dormancy is a result of a decrease of the number of nitrate receptors. After 24 and 48 hours of preincubation, the nitrate-response curves were biphasic. The biphasic character could be related to the level of endogenous nitrate and to a differential requirement for nitrate of two fractions of the seed population. Similarities with the behavior of fluence-response curves after prolonged dark incubation led to the hypothesis that phytochrome and nitrate share the same site of action.

Interaction between parental environment and genotype affects plant and seed performance in Arabidopsis

Highlight text The genotype-by-environment interactions of five parental environments with seed and plant performance are mediated by distinct genetic and molecular pathways, and the selective pressures that have shaped their natural variation.

The Re-Establishment of Desiccation Tolerance in Germinated Arabidopsis thaliana Seeds and Its Associated Transcriptome

The combination of robust physiological models with “omics” studies holds promise for the discovery of genes and pathways linked to how organisms deal with drying. Here we used a transcriptomics approach in combination with an in vivo physiological model of re-establishment of desiccation tolerance (DT) in Arabidopsis thaliana seeds. We show that the incubation of desiccation sensitive (DS) germinated Arabidopsis seeds in a polyethylene glycol (PEG) solution re-induces the mechanisms necessary for expression of DT. Based on a SNP-tile array gene expression profile, our data indicates that the re-establishment of DT, in this system, is related to a programmed reversion from a metabolic active to a quiescent state similar to prior to germination. Our findings show that transcripts of germinated seeds after the PEG-treatment are dominated by those encoding LEA, seed storage and dormancy related proteins. On the other hand, a massive repression of genes belonging to many other classes such as photosynthesis, cell wall modification and energy metabolism occurs in parallel. Furthermore, comparison with a similar system for Medicago truncatula reveals a significant overlap between the two transcriptomes. Such overlap may highlight core mechanisms and key regulators of the trait DT. Taking into account the availability of the many genetic and molecular resources for Arabidopsis, the described system may prove useful for unraveling DT in higher plants.

ABA Inhibits Embryo Cell Expansion and Early Cell Division Events During Coffee (Coffea arabica ‘Rubi’) Seed Germination

BACKGROUND AND AIMS Coffee seed germination represents an interplay between the embryo and the surrounding endosperm. A sequence of events in both parts of the seed determines whether germination will be successful or not. Following previous studies, the aim here was to further characterize the morphology of endosperm degradation and embryo growth with respect to morphology and cell cycle, and the influence of abscisic acid on these processes. METHODS Growth of cells in a fixed region of the axis was quantified from light micrographs. Cell cycle events were measured by flow cytometry and by immunocytochemistry, using antibodies against beta-tubulin. Aspects of the endosperm were visualized by light and scanning electron microscopy. KEY RESULTS The embryonic axis cells grew initially by isodiametric expansion. This event coincided with reorientation and increase in abundance of microtubules and with accumulation of beta-tubulin. Radicle protrusion was characterized by a shift from isodiametric expansion to elongation of radicle cells and further accumulation of beta-tubulin. Early cell division events started prior to radicle protrusion. Abscisic acid decreased the abundance of microtubules and inhibited the growth of the embryo cells, the reorganization of the microtubules, DNA replication in the embryonic axis, the formation of a protuberance and the completion of germination. The endosperm cap cells had smaller and thinner cell walls than the rest of the endosperm. Cells in the endosperm cap displayed compression followed by loss of cell integrity and the appearance of a protuberance prior to radicle protrusion. CONCLUSIONS Coffee seed germination is the result of isodiametric growth of the embryo followed by elongation, at the expense of integrity of endosperm cap cells. The cell cycle, including cell division, is initiated prior to radicle protrusion. ABA inhibits expansion of the embryo, and hence subsequent events, including germination.