Seed dormancy and germination Koornneef,Bentsink and Hihorst 35 sed in seeds The analysi later stages of seed development (reviewed in 3D). ng n in among the ph. mutants:and To study genes that are activated during the radicle protrusion Many of thes being involved mm: the ldr on (e.g.in the m d with hes s that were embryo development n my of n tabol enzyme Conclusions and perspectives traits tha in rowh.the activation of roection mechanisms (such as those moound mebolm depen The latte Germinatio in toma and tob cco ontrolled may inc compounds at are imported rom the mothe ng of the the has identified the crucial role of ABA in seed dor well as the T34 ed in the A targets of ABA and GA.or whether they affect sees upt ponse to plan on in an promote radicle ed tha ortant.as will the identification of more arge of this enzyme in germination was supported by the genes.Using whole ranscriptome and proteome approache with a se will be the most efficient way to identify target genes. sed BGlu I act ents increased endosperm rupture [36].In tomato.BGlu expressed specifica in the endosperm cap ne and endosperm we n as the References and recommended reading 37].In addition to th 0 dosnnSieresy 1. egermination and dormancy.Pant Cl9 Gene or prom ne such a B-glucuronic ion. 39 olated gene traps they identified an in tion close to PR/.This gene in the 5. :2600 ed in developi 1. These microarrays revealed many genes of unknown ndddomanYTe e0.10expressed after germination are also expressed during the later stages of seed development (reviewed in [31]), suggesting that some aspects of post-germination growth are initiated during maturation. The onset of early germi￾nation is also obvious in some of the Arabidopsis maturation mutants: lec, fus3 and abi3. To study genes that are activated during late embryo development and germination, mRNAs from immature siliques of the abi3 fus3 double mutant were compared with those from wildtype siliques using a differential display [32]. The genes that were identified as being active during late embryo development and germination encode a variety of metabolic enzymes, regulatory proteins and a number of ribosomal proteins. Cellular processes involved in growth, the activation of protection mechanisms (such as those involved in protection against oxidative stress), and storage-compound metabolism are expected to be related to germination. Germination in tomato and tobacco is controlled by interactions between the embryonic radicle tip and the enclosing endosperm cap. Weakening of the endosperm cap, by enzymatic hydrolysis, is required to allow radicle protrusion. Enzymes involved in this process are expansin [33] and endo-β-mannanase [34], which are specifically expressed in the endosperm cap of tomato. A close correla￾tion between class I β–1,3-glucanase (βGlu I) induction and endosperm rupture in response to plant hormones and environmental factors in tobacco suggested that βGlu I may also promote radicle protrusion [35]. The involvement of this enzyme in germination was supported by the observation that transgenic plants with a sense construct of the gene encoding βGlu I under control of an ABA inducible promoter had both increased βGlu I activity and increased endosperm rupture [36•]. In tomato, βGlu I was also expressed specifically in the endosperm cap [37]. However, a correlation between the expression of this gene and endosperm weakening could not be shown as the activity of βGlu I was inhibited by applied ABA, which did not inhibit endosperm weakening [37]. In addition to this, Toorop et al. [38] demonstrated that endosperm cap weak￾ening in tomato is a biphasic process and that inhibition of germination by ABA occurs exclusively at the second step in this process. Gene or promoter trapping with a reporter gene, such as β-glucuronidase (GUS), may identify genes with a specific expression. Dubreucq et al. [39•] isolated gene traps that are expressed during seed germination, among which they identified an insertion close to AtEPR1. This gene encodes an extensin-like protein, is specifically expressed in the endosperm during seed germination and is under control of GAs [39•]. The use of genomics and proteomics in seed research Microarrays containing 2600 genes expressed in developing Arabidopsis seeds were described by Girke et al. [40•]. These microarrays revealed many genes of unknown function that are highly expressed in seeds. The analysis of protein patterns by 2D gel electrophoresis and the subsequent identification of a number of those proteins, showed that among the 1300 seed proteins detected, 74 changed in abundance during the imbibition phase or during the radicle protrusion of Arabidopsis. Many of these proteins had previously been described as being involved in germination (e.g. in the mobilization of food reserves). In addition, proteins not previously associated with these processes were identified [41•]. Conclusions and perspectives Dormancy and germination are complex traits that are controlled by a large number of genes, which are affected by both developmental and environmental factors. Seed dormancy and germination depend on seed structures, especially those surrounding the embryo, and on factors affecting the growth potential of the embryo. The latter may include compounds that are imported from the mother plant and also factors that are produced by the embryo itself, including several plant hormones. Genetic analysis has identified the crucial role of ABA in seed dormancy, as well as the requirement for GAs for germination. QTL and mutant analyses are identifying additional genes. Whether these genes with unknown functions are downstream targets of ABA and GA, or whether they affect seed dormancy/germination in an independent way is currently not known. The molecular identification of all these genes will be important, as will the identification of more target genes. Using whole transcriptome and proteome approaches will be the most efficient way to identify target genes. Acknowledgements LB was supported by the Earth and Life Sciences Foundation, which is subsidized by The Netherlands Organization for Scientific Research. References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as: • of special interest ••of outstanding interest 1. Bewley JD: Seed germination and dormancy. Plant Cell 1997, 9:1055-1066. 2. Hilhorst HWM, Groot SPC, Bino RJ: The tomato seed as a model system to study seed development and germination. Acta Bot Neerl 1998, 47:169-183. 3. Van der Schaar W, Alonso-Blanco C, Léon-Kloosterziel KM, Jansen RC, Van Ooijen JW, Koornneef M: QTL analysis of seed dormancy in Arabidopsis using recombinant inbred lines and MQM mapping. Heredity 1997, 79:190-200. 4. Han F, Ullrich SE, Clancy JA, Jitkov V, Kilian A, Romagosa I: Verification of barley seed dormancy loci via linked molecular markers. Theor Appl Genet 1996, 92:87-91. 5. Lin SY, Sasaki T, Yano M: Mapping quantitative traits loci controlling seed dormancy and heading date in rice, Oryza sativa L., using backcross inbred lines. Theor Appl Genet 1998, 96:997-1003. 6. Kato K, Nakamura W, Tabiki T, Miura H, Sawada S: Detection of loci controlling seed dormancy on group 4 chromosomes of wheat and comparative mapping with rice and barley genomes. Theor Appl Genet 2001, 102:980-985. 7. Cai HW, Morishima H: Genomic regions affecting seed shattering and seed dormancy in rice. Theor Appl Genet 2000, 100:840-846. Seed dormancy and germination Koornneef, Bentsink and Hilhorst 35