S.P.C. Groot

Proteomics of Arabidopsis Seed Germination. A Comparative Study of Wild-Type and Gibberellin-Deficient Seeds

We examined the role of gibberellins (GAs) in germination of Arabidopsis seeds by a proteomic approach. For that purpose, we used two systems. The first system consisted of seeds of the GA-deficient ga1 mutant, and the second corresponded to wild-type seeds incubated in paclobutrazol, a specific GA biosynthesis inhibitor. With both systems, radicle protrusion was strictly dependent on exogenous GAs. The proteomic analysis indicated that GAs do not participate in many processes involved in germination sensu stricto (prior to radicle protrusion), as, for example, the initial mobilization of seed protein and lipid reserves. Out of 46 protein changes detected during germination sensu stricto (1 d of incubation on water), only one, corresponding to the cytoskeleton component α-2,4 tubulin, appeared to depend on the action of GAs. An increase in this protein spot was noted for the wild-type seeds but not for thega1 seeds incubated for 1 d on water. In contrast, GAs appeared to be involved, directly or indirectly, in controlling the abundance of several proteins associated with radicle protrusion. This is the case for two isoforms of S-adenosyl-methionine (Ado-Met) synthetase, which catalyzes the formation of Ado-Met from Met and ATP. Owing to the housekeeping functions of Ado-Met, this event is presumably required for germination and seedling establishment, and might represent a major metabolic control of seedling establishment. GAs can also play a role in controlling the abundance of a β-glucosidase, which might be involved in the embryo cell wall loosening needed for cell elongation and radicle extension.

Proteome-Wide Characterization of Seed Aging in Arabidopsis : A Comparison between Artificial and Natural Aging Protocols

A variety of mechanisms have been proposed to account for the extension of life span in seeds (seed longevity). In this work, we used Arabidopsis (Arabidopsis thaliana) seeds as a model and carried out differential proteomics to investigate this trait, which is of both ecological and agricultural importance. In our system based on a controlled deterioration treatment (CDT), we compared seed samples treated for different periods of time up to 7 d. Germination tests showed a progressive decrease of germination vigor depending on the duration of CDT. Proteomic analyses revealed that this loss in seed vigor can be accounted for by protein changes in the dry seeds and by an inability of the low-vigor seeds to display a normal proteome during germination. Furthermore, CDT strongly increased the extent of protein oxidation (carbonylation), which might induce a loss of functional properties of seed proteins and enzymes and/or enhance their susceptibility toward proteolysis. These results revealed essential mechanisms for seed vigor, such as translational capacity, mobilization of seed storage reserves, and detoxification efficiency. Finally, this work shows that similar molecular events accompany artificial and natural seed aging.

Analysis of Natural Allelic Variation of Arabidopsis Seed Germination and Seed Longevity Traits between the Accessions Landsberg erecta and Shakdara, Using a New Recombinant Inbred Line Population

Quantitative trait loci (QTL) mapping was used to identify loci controlling various aspects of seed longevity during storage and germination. Similar locations for QTLs controlling different traits might be an indication for a common genetic control of such traits. For this analysis we used a new recombinant inbred line population derived from a cross between the accessions Landsberg erecta (Ler) and Shakdara (Sha). A set of 114 F9 recombinant inbred lines was genotyped with 65 polymerase chain reaction-based markers and the phenotypic marker erecta. The traits analyzed were dormancy, speed of germination, seed sugar content, seed germination after a controlled deterioration test, hydrogen peroxide (H2O2) treatment, and on abscisic acid. Furthermore, the effects of heat stress, salt (NaCl) stress, osmotic (mannitol) stress, and natural aging were analyzed. For all traits one or more QTLs were identified, with some QTLs for different traits colocating. The relevance of colocation for mechanisms underlying the various traits is discussed.

Gibberellin induced hydrolysis of endosperm cell walls in gibberellin deficient tomato seeds prior to radicle protrusion.

The weakening of the mechanical restraint of the endosperm layer in tomato (Lycopersicon esculentum Mill.) seeds, a prerequisite for germination, has been studied with the use of seeds of the gibberellin (GA)-deficientgib-1 mutant. Incubation ofgib-1 endosperms, including part of the testa, in 10 μM GA4+7, resulted within 12 h in the release of fructose, glucose, galactose and mannose into the incubation medium. Only small amounts of sugars diffused out of thegib-1 endosperms during incubation in water. Chemical hydrolysis of endosperm cell walls ofgib-1 seeds showed that they are mainly composed of mannose, and smaller quantities of glucose and galactose. Treatment with GA4+7 induced in the endosperms the production of endo-β-mannanase activity that was not detectable during incubation in water, and also increased the activities of mannohydrolase and α-galactosidase as compared with the water controls. No cellulase activity was found. It is concluded that in tomato seeds the weakening of endosperms prior to radicle protrusion is mediated by a GA-induced enzymatic degradation of the mannan-rich cell walls.

Gene Expression Programs during Brassica oleracea Seed Maturation, Osmopriming, and Germination Are Indicators of Progression of the Germination Process and the Stress Tolerance Level

During seed maturation and germination, major changes in physiological status, gene expression, and metabolic events take place. Using chlorophyll sorting, osmopriming, and different drying regimes, Brassica oleracea seed lots of different maturity, stress tolerance, and germination behavior were created. Through careful physiological analysis of these seed lots combined with gene expression analysis using a dedicated cDNA microarray, gene expression could be correlated to physiological processes that occurred within the seeds. In addition, gene expression was studied during early stages of seed germination, prior to radicle emergence, since very little detailed information of gene expression during this process is available. During seed maturation expression of many known seed maturation genes, such as late-embryogenesis abundant or storage-compound genes, was high. Notably, a small but distinct subgroup of the maturation genes was found to correlate to seed stress tolerance in osmoprimed and dried seeds. Expression of these genes rapidly declined during priming and/or germination in water. The majority of the genes on the microarray were up-regulated during osmopriming and during germination on water, confirming the hypothesis that during osmopriming, germination-related processes are initiated. Finally, a large group of genes was up-regulated during germination on water, but not during osmopriming. These represent genes that are specific to germination in water. Germination-related gene expression was found to be partially reversible by physiological treatments such as slow drying of osmoprimed seeds. This correlated to the ability of seeds to withstand stress.

The role of the cell cycle machinery in resumption of postembryonic development.

Cell cycle activity is required for plant growth and development, but its involvement in the early events that initiate seedling development remains to be clarified. We performed experiments aimed at understanding when cell cycle progression is activated during seed germination, and what its contribution is for proper seedling establishment. To this end, the spatial and temporal expression profiles of a large set of cell cycle control genes in germinating seeds of Arabidopsis (Arabidopsis thaliana) and white cabbage (Brassica oleracea) were analyzed. The in vivo behavior of the microtubular cytoskeleton was monitored during Arabidopsis seed germination. Flow cytometry of Arabidopsis germinating seeds indicated that DNA replication was mainly initiated at the onset of root protrusion, when germination reached its end. Expression analysis of cell cycle genes with mRNA in situ localization, β-glucuronidase assays, and semiquantitative reverse transcription-polymerase chain reaction showed that transcription of most cell cycle genes was detected only after completion of germination. In vivo green fluorescent protein analysis of the microtubule cytoskeleton demonstrated that mitosis-specific microtubule arrays occurred only when the radicle had started to protrude, although the assembly of the microtubular cytoskeleton was promptly activated once germination was initiated. Thus, seed germination involves the synthesis and/or activation of a reduced number of core cell cycle proteins, which only trigger DNA replication, but is not sufficient to drive cells into mitosis. Mitotic divisions are observed only after the radicle has protruded and presumably rely on the de novo production of other cell cycle regulators.

Differentially expressed genes associated with dormancy or germination of Arabidopsis thaliana seeds

Differential display analysis using dormant and non-dormant Arabidopsis thaliana (L.) Heynh seeds resulted in a set of genes that were associated with either dormancy or germination. Expression of the germination-associated genes AtRPL36B and AtRPL27B, encoding two ribosomal proteins, was undetectable in the dry seed, low in dormant seed, and high under conditions that allowed completion of germination. Expression of these genes was also found to be light-regulated and to correlate with germination speed. Expression of the dormancy-associated genes ATS2 and ATS4, encoding a caleosin-like protein and a protein similar to a low-temperature-induced protein respectively, was high in the dry seed and decreased during germination. Expression of ATS2 and ATS4 was high in primary and secondary dormant seed but low in after-ripened or chilled seed. The expression of both genes was also light-regulated, but no relationship with temperature-dependent germination speed was found.

Characterization of green seed, an Enhancer of abi3-1 in Arabidopsis That Affects Seed Longevity

Seeds are usually stored in physiological conditions in which they gradually lose their viability and vigor depending on storage conditions, storage time, and genotype. Very little is known about the underlying genetics of seed storability and seed deterioration. We analyzed a mutant in Arabidopsis disturbed in seed storability. This mutant was isolated as a grs (green-seeded) mutant in an abi3-1 (abscisic acid 3) mutant background. Genetic and physiological characterization showed that the monogenic grs mutant was not visibly green seeded and mapped on chromosome 4. This enhancer mutation did not affect the ABA sensitivity of seed germination or seed dormancy but was found to affect seed storability and seedling vigor. Seed storability was assessed in a controlled deterioration test, in which the germination capacity of the mutant decreased with the duration of the treatment. The decrease in viability and vigor was confirmed by storing the seeds in two relative humidities (RHs) for a prolonged period. At 60% RH, the mutant lost germinability, but storage at 32% RH showed no decrease of germination although seed vigor decreased. The decrease in viability and vigor could be related to an increase in conductivity, suggesting membrane deterioration. This was not affected by light conditions during imbibition, expected to influence the generation of active oxygen species. During seed maturation, ABI3 regulates several processes: acquiring dormancy and long-term storability and loss of chlorophyll. Our results indicate that GRS is a common regulator in the latter two but not of dormancy/germination.

Seed storage at elevated partial pressure of oxygen, a fast method for analysing seed ageing under dry conditions

Background and Aims Despite differences in physiology between dry and relative moist seeds, seed ageing tests most often use a temperature and seed moisture level that are higher than during dry storage used in commercial practice and gene banks. This study aimed to test whether seed ageing under dry conditions can be accelerated by storing under high-pressure oxygen. Methods Dry barley (Hordeum vulgare), cabbage (Brassica oleracea), lettuce (Lactuca sativa) and soybean (Glycine max) seeds were stored between 2 and 7 weeks in steel tanks under 18 MPa partial pressure of oxygen. Storage under high-pressure nitrogen gas or under ambient air pressure served as controls. The method was compared with storage at 45 °C after equilibration at 85 % relative humidity and long-term storage at the laboratory bench. Germination behaviour, seedling morphology and tocopherol levels were assessed. Key Results The ageing of the dry seeds was indeed accelerated by storing under high-pressure oxygen. The morphological ageing symptoms of the stored seeds resembled those observed after ageing under long-term dry storage conditions. Barley appeared more tolerant of this storage treatment compared with lettuce and soybean. Less-mature harvested cabbage seeds were more sensitive, as was the case for primed compared with non-primed lettuce seeds. Under high-pressure oxygen storage the tocopherol levels of dry seeds decreased, in a linear way with the decline in seed germination, but remained unchanged in seeds deteriorated during storage at 45 °C after equilibration at 85 % RH. Conclusions Seed storage under high-pressure oxygen offers a novel and relatively fast method to study the physiology and biochemistry of seed ageing at different seed moisture levels and temperatures, including those that are representative of the dry storage conditions as used in gene banks and commercial practice.

Cell cycle events in developing neem (Azadirachta indica) seeds: are they related to intermediate storage behaviour?

Seeds of neem ( Azadirachta indica A. Juss.), a multipurpose tropical tree, have limited desiccation tolerance. Whether their intermediate storage behaviour might be caused by an elevated percentage of 4C nuclei in the embryo at maturity was investigated. Morphological development of neem seeds was monitored on selected trees in Ouagadougou, Burkina Faso. Developing seeds were tested for germinability, and cell cycle events were determined by using flow cytometry and analysing the level of β-tubulin. Germination could occur after 8 weeks of seed development, but normal seedlings resulted only after 10 weeks or more. The change in fruit colour from green to yellow began after approximately 12 weeks of seed development. Immature, 4-week-old embryos about 2 mm in length had 15% of cells in the G 2 phase and 60% in the G 1 phase of the cell cycle, as indicated by 4C and 2C nuclear DNA levels, respectively. During maturation, the proportion of G 2 cells declined to 3% at full maturity and slightly further after drying, and the proportion of G 1 cells increased to approximately 90%. A strong β-tubulin signal was observed in tips of young embryonic radicles and cotyledons, but a weak or non-detectable signal was found in 9-week-old ones and in those from green-mature and yellow fruits. Because DNA replication and β-tubulin level were almost negligible at seed maturity, as in orthodox tomato seeds, it is suggested that these factors are not involved in the intermediate storage behaviour of neem seeds.