Seed dormancy and germination Maarten Koornneef*,Leonie Bentsink and Henk Hilhorst Seed dormancy and germination are comnlex adantive traits of This review focuses on recent progress made in seed higher plants that are influenced by a large number of genes dormancy/germination research,especially through the use tudies of ge etics and physiology of molecula ant ho s abscisi is favore n the i excelle nt sul mination More recently the use of quantitative gen etics are similar to thes and mutant approaches has allowed the further genetic physiological seed research.However.some cereal species dissection of these traits and the identification of previously unknown comp and especially ded in a ngid en o for the analysis of seed dormancy and the progress made in underst germination.These tools preferentially use Arabidopsis thaliana ceae and ac mancy research Gen of wild p PSae,fuimeaedplantsTieswbstantialinfenceot environmental effects on the expression of germinatior ma mak typ Current Opinion in Plant Biology 2002,5:33-36 OTL analysis f he test sic acid rgenumberotgeeicilidecnclseese,Cecds,a ras snosteroe the same RIL)in different environmental conditions response1 has s that OTL identified for whe aeybutri Wild specief -1.3-glucanase show stronger dorm ncy than cultivated genotypes,making OTL lowed by Introduction Such studies have been initiated in barley [9.10]and he seec tsin is e plant cmb and whi ne t suc alo the by map-based cloning.However.the cloning of such the next generation.The dry dormant sced is well dormancy O'TL has not vet been reported. equipped to su ive exter periods infavor s te Mutants in dorm id (ABA) conditions [and is controlled by several environmental deficient,and signaling mutants tomato factors,such as light.temperature and the duration of Istorag ripening).Dorma erminat Wellthe requrement for GA for germination we the tis ans which surrounding it.Bewley [1]concluded in his recent review defective in a zeaxanthin epoxidase gene(encoding one of that little is known about the mechanism of dormancy and germination
33 Seed dormancy and germination are complex adaptive traits of higher plants that are influenced by a large number of genes and environmental factors. Studies of genetics and physiology have shown the important roles of the plant hormones abscisic acid and gibberellin in the regulation of dormancy and germination. More recently, the use of quantitative genetics and mutant approaches has allowed the further genetic dissection of these traits and the identification of previously unknown components. Molecular techniques, and especially expression studies and transcriptome and proteome analyses, are novel tools for the analysis of seed dormancy and germination. These tools preferentially use Arabidopsis thaliana because of the molecular genetic resources available for this species. However, Solanaceae and cereals also provide important models for dormancy research. Addresses Laboratory of Genetics (MK and LB) and Plant Physiology (HH), Department of Plant Sciences, Wageningen University, Arboretumlaan 4, 6703 BD Wageningen, The Netherlands *e-mail: maarten.koornneef@genetics.dpw.wau.nl Current Opinion in Plant Biology 2002, 5:33–36 1369-5266/02/$ — see front matter © 2002 Elsevier Science Ltd. All rights reserved. Abbreviations ABA abscisic acid abi ABA insensitive BR brassinosteroid ctr1 constitutive triple response1 cts comatose Dof DNA-binding with one finger ein2 ethylene insensitive2 fus3 fusca3 GA gibberellin βGlu I β–1,3-glucanase lec leafy cotyledon QTL quantitative trait loci sly1 sleepy1 Introduction The seed is the structure in which a usually fully developed plant embryo is dispersed, and which enables the embryo to survive the period between seed maturation and seedling establishment, thereby ensuring the initiation of the next generation. The dry dormant seed is well equipped to survive extended periods of unfavorable conditions. Seed dormancy is defined as the failure of an intact viable seed to complete germination under favorable conditions [1] and is controlled by several environmental factors, such as light, temperature and the duration of seed storage (after ripening). Dormancy and germination are determined by the co-action of the growth potential of the embryo and the restraints imposed by the tissues surrounding it. Bewley [1] concluded in his recent review that little is known about the mechanism of dormancy and germination. This review focuses on recent progress made in seed dormancy/germination research, especially through the use of molecular genetics. Arabidopsis is developing as a favored model in this field because of its excellent suitability for genetic and molecular studies, and because its germination responses are similar to those of many species used in physiological seed research. However, some cereal species and Solanaceae species such as tobacco and tomato, in which the embryo is embedded in a rigid endosperm [2], are also suitable models and have contributed significantly to the progress made in understanding germination biology. The genetic analysis of ‘natural’ differences in seed dormancy and germination characteristics Genetic variation for seed dormancy within species is present both among accessions of wild plants and among varieties of cultivated plants. The substantial influence of environmental effects on the expression of germination characteristics and the involvement of many genes make dormancy a typical quantitative trait. Such traits are becoming more amenable to genetic analysis because the position of individual quantitative trait loci (QTL) and the relative contribution of these loci can now be determined. QTL analysis for seed dormancy requires permanent or immortal mapping populations, such as recombinant inbred lines (RILs), because these allow the testing of a large number of genetically identical seeds (i.e. seeds from the same RIL) in different environmental conditions. QTL analysis of seed dormancy has been reported for Arabidopsis thaliana [3], barley [4], rice [5] and wheat [6]. It appears that QTL identified for wheat co-locate with barley QTL but not with rice QTL [6]. Wild species often show stronger dormancy than cultivated genotypes, making crosses between wild and cultivated genotypes useful for QTL analysis [7,8]. QTL analysis can be followed by the study of individual genes (or chromosome regions) containing specific dormancy QTL and by fine mapping. Such studies have been initiated in barley [9,10] and Arabidopsis (L Bentsink, unpublished data). It is expected that the study of such QTL will allow the molecular identification of genes that affect dormancy in these species by map-based cloning. However, the cloning of such dormancy QTL has not yet been reported. Mutants in dormancy and germination research Studies of gibberellin (GA)-deficient, abscisic acid (ABA)- deficient, and signaling mutants in Arabidopsis and tomato have identified the crucial role of ABA in seed dormancy, as well as the requirement for GA for germination [11,12]. The isolation of a Tos17-transposon-induced viviparous (non-dormant) mutant in rice, which was shown to be defective in a zeaxanthin epoxidase gene (encoding one of the enzymes of the ABA-synthetic pathway) [13], showed that ABA is also important in dormancy control in cereals. Seed dormancy and germination Maarten Koornneef*, Leónie Bentsink and Henk Hilhorst
34 Growth and developmen Manipulation of seed ABA content by genetic modification suppressors of the ABA-insensitive/mutant.This of tobacco has shown that overexpression of z xanthin mutant strongly resembles the GA auxotrophs.Howeve dor wh kn h cannot be by GA vields phenorypes that are less dormant 4.The observation GA rec ion I231 Another mutant with a marked reductio that inhibitors of ABA biosynthesis,such as norfluorazon in germination potential is comatose(cs).Although the the mainten at mature seeds deABA synthesis.as has also been found for that ct&promotes increased germination potential role of during sced-sm In addition to these mutants affecting the embryo prope In addition to the whic)and mutants have been selected that control dormancy throug he oat or o or se T251 mutants of edcaeeaomae2saehelponhc 0 idopsis are also hype ABA [16",17].This cture as a constraint to radicle ent w 13 apparen sed by y the of the (mutant.which is characterized by a both GA-deficient mutants 12]and accessions that have among mutants s ancers th n-dorman mutant of the Dof AFFECTING GERMINATIONI (DAGD phenotype of the double that gene, the of cr s-tall to those of other reduced dor 127.28 between sugar signaling and ethylene signaling is s this phenotypic effect is determined by the matera by the sugar-insensitive phenotype c cr/[18]and genotype.This maternal in neritance is consistent with the that e npatcncecio which is genctically regulation of germination and carly seedling growth.This derived from the mother plant 126"1. interaction is further supported by the obse that Ge s with ern correlated cy and siatRoat a subclass of the ABA-insensitive mutants 20. In addition toth n and subsequent cloning o es through the ng s Detailed analysis of the de and has shown that they basis of their e This unbiased search of genes with and oci prob b regulat on ge nes with assumed function owth in immature Examples of genes identified by the latter me sm o prevent germin 10 ase gene controllin nthes by LECI and FUS3 1211. ling a dormancy-specific NADP phatase which has a higher activity in dormant seeds than in non-dormant Mutants have alse BR mutants de-etiolated2 (det2)and inseusitivel During seed maturation the exp sion of many genes is and specific classes of mRNAs such as those of o GAs s are not he ESIS-ABUNDANT A)ge function in seed dormaney.Although it appears that seed Recently,sleepyl (),an Arabidopsis mut ant that has severe germination defect,was selected in a screen for highly
Manipulation of seed ABA content by genetic modification of tobacco has shown that overexpression of zeaxanthin epoxidase results in increased dormancy, whereas ‘knocking out’ the gene encoding this enzyme by antisense techniques yields phenotypes that are less dormant [14]. The observation that inhibitors of ABA biosynthesis, such as norfluorazon, promote germination [12] indicates that the maintenance of dormancy in imbibed seeds is an active process involving de novo ABA synthesis, as has also been found for Nicotiana plumbaginifolia [15•]. These findings complement those of earlier studies that emphasized the role of ABA during seed development. In addition to the well-known ABA insensitive (abi) and enhanced response to ABA (era) mutants, which all have a seed germination phenotype, it was recently found that the ethylene insensitive2 (ein2) and ethylene response (etr) mutants of Arabidopsis are also hypersensitive to ABA [16•,17]. This finding is consistent with the fact that ein2 mutants were isolated as suppressors of the abi1 mutant. The constitutive triple response1 (ctr1) mutant, which is characterized by a constitutive ethylene response, was among mutants selected as enhancers of the ABA-insensitive mutant abi1-1. The ctr1 monogenic mutants are also slightly ABA resistant [16•]. These findings, in combination with the non-dormant phenotype of the ein2 abi3-4 double mutant, indicate that ethylene may suppress seed dormancy by inhibiting ABA action [16•]. In addition, the presence of cross-talk between sugar signaling and ethylene signaling is suggested by the sugar-insensitive phenotype of ctr1 [18] and the sugar-hypersensitive phenotype of etr [19]. Apparently, ABA, ethylene and sugar signaling strongly interact during the regulation of germination and early seedling growth. This interaction is further supported by the observation that many sugar-signaling mutants turn out to be ABA-biosynthesis mutants or alleles of abi4 and abi5, which represent a subclass of the ABA-insensitive mutants [20•]. Detailed analysis of the seed-maturation mutants leafy cotyledon (lec), fusca3 (fus3) and abi3 has shown that they differ in the time at which they can undergo premature germination. The LEC1 and FUS3 loci probably regulate developmental arrest, as mutations in these genes cause a continuation of growth in immature embryos. Control of dormancy by ABA (via ABA and ABI) might represent a different mechanism to prevent germination, which occurs later and is additive to the developmental arrest controlled by LEC1 and FUS3 [21]. Mutants have also been useful in establishing the role of brassinosteroids (BRs) in seed germination. The Arabidopsis BR mutants de-etiolated2 (det2) and brassinosteroid insensitive1 (bri1) show reduced germination but eventually germinate without BR, indicating that, in contrast to GAs, BRs are not absolutely required for germination [22]. Recently, sleepy1 (sly1), an Arabidopsis mutant that has a severe germination defect, was selected in a screen for suppressors of the ABA-insensitive abi1-1 mutant. This mutant strongly resembles the GA auxotrophs. However, the lack of germination of sly1 cannot be rescued by GA, therefore, SLY1 has been postulated to be a key factor in GA reception [23]. Another mutant with a marked reduction in germination potential is comatose (cts). Although the morphology of cts plants is not altered, mature cts seeds do not respond to gibberellin. It has therefore been suggested that CTS promotes increased germination potential, represses embryo dormancy and might be involved in seed-specific GA signaling [24]. In addition to these mutants affecting the embryo proper, mutants have been selected that control dormancy through the seed coat or other maternal factors. A number of seedcoat or testa mutants [25•] have a maternally inherited reduced seed dormancy. This indicates the importance of the testa structure as a constraint to radicle emergence. In Arabidopsis, dormancy is apparently imposed by the seed coat because removal of the testa allows the germination of both GA-deficient mutants [12] and accessions that have a very strong dormancy (L Bentsink, unpublished data). Evidently, lack of germination may also be due to a reduced growth potential of the embryo. A knockout mutant of the Dof AFFECTING GERMINATION1 (DAG1) gene, which encodes a Dof (DNA-binding with one finger) transcription factor, caused reduced dormancy [26•]. In contrast to those of other reduced dormancy mutants [27,28], this phenotypic effect is determined by the maternal genotype. This maternal inheritance is consistent with the expression pattern of the DAG1 gene in the vascular tissue that enters the developing seeds, which is genetically derived from the mother plant [26•]. Genes with an expression pattern correlated to dormancy and germination In addition to the identification and subsequent cloning of genes through the use of mutants, genes controlling seed dormancy and germination can also be identified on the basis of their expression pattern. This may involve an unbiased search of genes with germination-specific expression or may focus on genes with assumed functions that are related to seed germination. Examples of genes identified by the latter strategy include a 3 β hydroxylase gene controlling gibberellin biosynthesis in a light-induced and seed-specific way [29], and the gene encoding a dormancy-specific NADP+ phosphatase, which has a higher activity in dormant seeds than in non-dormant seeds of Avena sativa [30]. During seed maturation the expression of many genes is altered and specific classes of mRNAs such as those of the LATE-EMBRYOGENESIS-ABUNDANT (LEA) genes appear. However, none of these genes has a proven specific function in seed dormancy. Although it appears that seed maturation and post-germination growth have a distinct gene-expression profile, some genes that are highly 34 Growth and development
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 e0.10
expressed 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 germination 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 correlation 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 weakening 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. 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Plant J 2000, 23:643-652. The authors demonstrate the use of gene trapping for the identification of seed-germination-specific genes. 40. Girke T, Todd J, Ruuska S, White J, Benning C, Olrogge J: Microarray • analysis of developing Arabidopsis seeds. Plant Physiol 2000, 124:1570-1581. The use of microarrays is an emerging technique in seed research. This paper illustrates the use of a seed-development-specific microarray. 41. Gallardo K, Job C, Groot SPC, Puype M, Demol H, Vandekerckhove J, • Job D: Proteomic analysis of Arabidopsis seed germination and priming. Plant Physiol 2001, 126:835-848. A demonstration of the use of proteomic techniques in seed research. 36 Growth and development