《种子学》课程教学资源（文献资料）Germination—Still a mystery

Plant5 cience179(2010)574-581 Contents lists available at ScienceDirect Plant Science ELSEVIER journal homepage:www.elsevier.com/locate/plantsci Germination-Still a mystery Hiroyuki Nonogaki,George W.Basselb.J.Derek Bewley ARTICLE INFO ABSTRACT prepa nt s ng the ay2010 vity initially lve com ed ithn the mature drys gt en thisand th d within the embry to physicall oth.Whileth ed that of the are limited opp s at prese r improving germ what is.and occurs dur g.germination of the ination has aseed cor vith th course ofv ith th arance of the ase 1)un allof the matrices an d cell contents are ful seed e l1)which remains anedin seeds that do not com her than get minati dling growth is n ith Phase lll is initially and bried as t req and though this f th et of seeds under the conditions to which they While the use of these Phases s is convenient gronomical sense.because this isa pract the er seeds ding even follow ng emerge mtsmgoehi6fnfeemetaboacaentsocurmingwithinthe state.edu (H.Nonogakil

Plant Science 179 (2010) 574–581 Contents lists available at ScienceDirect Plant Science journal homepage: www.elsevier.com/locate/plantsci Germination—Still a mystery Hiroyuki Nonogaki a,∗, George W. Bassel b, J. Derek Bewleyc a Department of Horticulture, Oregon State University, Corvallis, OR 97331, USA b Division of Plant and Crop Sciences, University of Nottingham, Sutton Bonington Campus, Loughborough, Leics, LE12 5RD, UK c Department of Molecular and Cellular Biology, University of Guelph, Guelph, Ontario N1G 2W1, Canada article info Article history: Received 1 November 2009 Received in revised form 9 February 2010 Accepted 16 February 2010 Available online 23 February 2010 Keywords: Embryo Endosperm Germination Seed Transcription Translation abstract Germination is a complex process during which the seed must quickly recover physically from matura￾tion drying, resume a sustained intensity of metabolism, complete essential cellular events to allow for the embryo to emerge, and prepare for subsequent seedling growth. Early following the start of imbibi￾tion of the dry seed there is re-establishment of metabolism; restitution of the chemical and structural integrity of cells requires the co-participation of synthetic and protective events. Protein synthesis and respiratory activity initially involve components stored within the mature dry seed, although transcrip￾tion and translation commence early during imbibition, as shown by transciptome and metabolome analyses. Increases or modifications to hormones, especially GA, play an important role in achieving the completion of germination, at least in intact seeds. Removal or deactivation of ABA is also important; interactions between this and GA play a regulatory role. A restraint on the completion of germination in seeds of some species is imposed by the surrounding structures, e.g. the endosperm, and thus there is a requirement either for it to be enzymically weakened to allow the radicle to emerge, or for sufficient force to be generated within the embryo axis to physically break through, or both. While there is much information with respect to changes in gene expression during germination, no key event(s) has been identified that results in its completion. The downstream effects of the observed hormone changes are not known, and given the multipart nature of the seed, the requirements imposed upon it (repair, main￾tenance, preparation for seedling growth) in addition to completing germination (which involves only a limited number of cells), the challenge to identify ‘germination-completion’ genes is large. Hence there are limited opportunities at present for improving germination through genetic manipulation. © 2010 Elsevier Ireland Ltd. All rights reserved. 1. Overview: what is, and occurs during, germination? By definition, germination of a seed commences with the uptake of water and is completed with the appearance of the embryo, in most species radicle first, through the surrounding structure(s). Thereafter, germination is completed, and the seed is regarded as having germinated (sometimes termed ‘visible germination’), rather than germinating, and seedling growth is now underway. Failure to follow the definition has too frequently led to claims of cellular and molecular events occurring during germination, when in fact they are part of seedling growth; therefore, care must be taken to define clearly the time course of germination for a particular set of seeds under the conditions to which they are sub￾jected. Seedling emergence from the soil is sometimes referred to as ‘germination’ in an agronomical sense, because this is a practi￾cal indication of establishment; however, in this review the strict experimental definition of germination is used. ∗ Corresponding author. E-mail address: Hiro.nonogaki@oregonstate.edu (H. Nonogaki). A useful depiction of the progress of germination has evolved around the time course of water uptake by a germinating seed (Fig. 1). Initially there is rapid imbibition of water by the dry seed (Phase I) until all of the matrices and cell contents are fully hydrated. This is followed by a period of limited water uptake (Phase II), which remains unchanged in seeds that do not com￾plete germination, such as in dormant or dead seeds. The increase in water uptake associated with Phase III is initially, and briefly, related to completion of germination, although this is proportion￾ately a low amount. The slight increase in water content is followed by a much larger uptake as the cells of the growing radicle, and sub￾sequently the rest of the seedling, increase due to mitotic divisions and cell expansion. While the use of these Phases is convenient to illustrate the events taking place in germinating seeds, in some species they are not so well defined; for example in larger seeds water uptake into the reserve-laden cotyledons still may be pro￾ceeding even following emergence of the radicle. Also, the Phases do not temporally define the metabolic events occurring within the germinating seed, for these are confluent. The cellular contents of a dry seed are laid down during prior development, which allows for the resumption of metabolic events 0168-9452/$ – see front matter © 2010 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.plantsci.2010.02.010

H.Nonogaki etal/(1)574-58 575 Germination sensu stricto Post-germination Phase I Phase ll Phase IlⅢ ranscription and translation of new mRNAs M Cell divisio DNA synthesis piration,mitoch Time rapidly upon reintroduction to water.whether or not the seed is Foraconsiderable number of seeds,there isan additional barrie the r by synth eliberation as to whether the mbryo de ops suthc nt thrus Drying.and ted impenetrability.or both nd 2.Rehydration- ntial process seed onsible n at the duetemnoisturecoi peningin dry storage,resu 厂 and loss o tial for germination to commence events esser and the radicle. otterntcemswoudapeartonceasehechalenge sults in the hydration ould they be limited to such a relatively small Theincrease uptake poeLFig-1)i embryo xis pror to the completion of ge nation.includi of an inc g hy ored during i co seed

H. Nonogaki et al. / Plant Science 179 (2010) 574–581 575 Fig. 1. Time course of physical and metabolic events occurring during germination (Phases I and II) and early seedling growth (Phase III). The time taken for these events to occur varies between species and is influenced by germination conditions. The curve shows a stylized time course of water uptake. Modified from [74,91]. rapidly upon reintroduction to water, whether or not the seed is dormant. How long these components are utilized during germi￾nation is the subject of debate. But, it appears that, for example, all that is required for the resumption of protein synthesis is present in the dry seed, while there is a gradual replacement, augmented by synthesis of new RNAs, as germination proceeds. Drying, and rehydration of a seed from the dry state, impose considerable stresses upon the component cells; upon imbibition there is leakage of solutes indicative of temporary membrane dam￾age. Organelles such as mitochondria, vital for efficient respiration and energy metabolism, are damaged and reduced in number; their repair and replacement must occur during germination. Even DNA does not escape the severity of drying and rehydration without some damage, and repair is an early priority. Thus, it is anticipated that following imbibition the synthesis of enzymes and compounds to limit and repair cellular damage is prevalent. As germination proceeds, presumably metabolism of the seed becomes engaged in those processes vital for the emergence of the radicle. Signaling to and within those cells which are responsible for this terminating event must occur, including the synthesis of key proteins. But which cells are responsible for the initial emer￾gence of the embryo? In Arabidopsis, the only seed for which data are available, it is not the radicle itself that undergoes elongation in order to pass through the surrounding structures (endosperm and testa). Rather it is a region immediately behind this, the lower hypocotyl and transition zone between this and the radicle, in which a few cells (less than ten cells in length) elongate and push out the radicle. This would appear to increase the challenge in iden￾tifying the specific cellular events induced to ensure the completion of germination, should they be limited to such a relatively small number of cells. Although there is an increase in DNA content in the embryo axis prior to the completion of germination, including by endoreduplication, cell division is exclusively a post-germination event. For a considerable number of seeds, there is an additional barrier which must be overcome in order for germination to be completed – the restraint imposed on the embryo by its surrounding struc￾tures. Of these, the endosperm, or in rarer cases the perisperm, is the major limiting tissue, rather than the testa. There is some deliberation as to whether the embryo develops sufficient thrust to break through the barrier, and emerge, i.e. if there is an increase in growth potential, or if there is hormone-mediated induction of wall-weakening enzymes in the limiting tissues to reduce their impenetrability, or both. In the following sections, this series of events occurring during germination is elaborated upon. 2. Rehydration—an essential process Seeds are metabolically inactive (quiescent) in the mature dry state although they undergo some physiological and chemical changes even at their low water contents (∼7–14% moisture con￾tent). The consequences of these changes due to, for example after-ripening in dry storage, result in changes in germination capacity due to dormancy release [1,2], or they can cause dete￾rioration and loss of viability [3]. However, rehydration through imbibition is essential to set in motion the metabolic events essen￾tial for germination to commence. Dry seeds absorb water rapidly and the kinetics of uptake are influenced by the structure of a seed, in that it may not enter all regions equally [4]. Initially, imbibition results in the hydration of matrices, such as the cell walls and reserve polymers within the cells. The increase in water uptake with time (Phase I, Fig. 1) is due to an increasing number of cells within the seed becoming hydrated, rather than as a result of an increase in the hydration of all cells uniformly. The use of proton nuclear magnetic resonance (NMR) micro-imaging, a non-destructive method, allows for the pattern of hydration to be monitored during imbibition: in tobacco seeds

576 H.Nonogaki et aL Plant Science 179 (2010)574-581 water first reaches the micropylar endosperm and the tip of radi- PIMT1.one of the genesencoding thisenzyme,reduces the accumu- uptake through the both seed ne in harvested dry yledons at the of the seed are the first to hydrate as well as the 3.Resumption of energy metabolism and cellular repair While lular lo uptake by a dis rapid.re v and o duri Phase Imbibed seeds generate reactiv species(ROS)during in n 20-31 o for sever radicals b e.dehydr rbate reduct g the completion of imbi bition,during erat dtuningimbibito y may b art o an antioxidativ t patterns of mitoch ent.In starc ds s 4.Reawakening of protein synthesis upon imbibition-old of n w mitoc oil-storing seeds such as peanut and new messages nd n 5-13 also Whether or not dry seeds are capable of transcribing DNA and tempo ayanaerobicconditionsduringorfolon ominates w The glycoly than 12.00 mRNA speci structresaval ted pe RNAs 35 mRN are en mit the dry ingagreater gaaaembe nesis RNAs yme of gluc ponsive(ABREs)with the core motif ACGT The rte protein synt elow the PH2112 olandirectwincrel dry seeds (whea pathways.such asth ribo somes be RNAs during germ tio id (ABA 23](se meta olism al inhibi Amanitin an inhibitor or RNA rotein nts radicle em ne structures that are mainta ned in a ge these d to the d 24.5om e memb for germinat gh stored mRNAS of solutes from the ce)Damage to proteins any. for the completion of germi taking place ytransfera ant role in at six HA carlier arried out artylresidestoyftayzingtheconvc urs in hoth no nd do mant(Cape Verde Islands:Cvi)lines and ceases around three HAl

576 H. Nonogaki et al. / Plant Science 179 (2010) 574–581 water first reaches the micropylar endosperm and the tip of radi￾cle [5], presumably leading to a rapid resumption of metabolism in these regions. A similar pattern of water uptake through the micropyle occurs in many other seeds, including those of wheat [6], but there are exceptions, e.g. western white pine, in which the cotyledons at the chalazal end of the seed are the first to hydrate [7]. 3. Resumption of energy metabolism and cellular repair While water uptake by a seed is rapid, resumption of steady state metabolism is more gradual. An early event during Phase I imbibition is the resumption of energy metabolism. Mature dry seeds contain mitochondria with poorly differentiated inner mem￾branes. Functional enzymes necessary for respiration such as those of the Krebs cycle, and terminal oxidases, are present which likely provide sufficient ATP from oxidative phosphorylation for several hours after the start of imbibition (HAI) [8]. These enzymes may be protected in the dry seed by mitochondrion-specific late embryo￾genesis abundant (LEA) proteins [9,10]. Mitochondrial oxidation reactions increase following the completion of imbibition, during Phase II, as the mitochondria undergo repair and replication. There are two distinct patterns of mitochondrial development. In starch￾storing seeds such as pea, mung bean and cowpea, there is repair and reactivation of pre-existingmitochondria; however, biogenesis of new mitochondria is typical of oil-storing seeds such as peanut, castor bean and pumpkin [11–13]. Two other respiratory pathways, glycolysis and the pentose phosphate pathway (PPP), are also active in imbibed seeds. Many seeds experience temporary anaerobic conditions during or follow￾ing imbibition leading to ethanol production [14]. The glycolytic pathway predominates when mitochondrial ATP production is restricted by low oxygen availability, often due to limited per￾meability of the structures surrounding the embryo. In contrast, when mitochondria become active, the PPP predominates [15,16]. Nitrogen-containing compounds such as nitrate and nitrite release seeds from dormancy [17–19], hypothetically by acting as electron acceptors, affecting the rate of electron transport, or by causing the oxidation of NADPH, thus allowing a greater carbon flow through the PPP [16,20]. The oxidized pyrimidine nucleotide NADP is a coen￾zyme of glucose-6-phosphate dehydrogenase, which is a key link between the glycolysis pathway and the PPP. The effectiveness of nitrate in promoting germination is exerted through its reduction to nitrite, hydroxylamine or nitric oxide (NO) [17]. NO is recog￾nized as an important signal transduction component, and might accelerate the metabolic flux through PPP by indirectly increasing the oxidation of NADPH [21], although it could stimulate germina￾tion through other pathways, such as those involved in hormone metabolism, e.g. NO promotes gibberellin (GA) biosynthesis in Arabidopsis [22]. Nitrate (hence NO) could act by deactivating abscisic acid (ABA [23] (see later for further discussion of hormone metabolism). Although proteins and membranes are protected in the mature dry seed, damage still occurs during maturation drying and imbi￾bition. Cellular membrane structures that are maintained in a gel phase during maturation drying undergo a transition to a liq￾uid crystalline phase when rehydrated [24]. Some membranes are damaged during this phase transition, which results in the leakage of solutes from the cells [25]. Damage to proteins could occur due to the formation of abnormal amino acids during desiccation and/or aging during seed storage. These could result in misfolding of pro￾teins, causing a reduction in, or loss of protein function. The protein l-isoaspartyl methyltransferase (PIMT) plays an important role in the repair of damaged proteins in seeds by catalyzing the conver￾sion of abnormal l-isoaspartyl residues to their normal l-aspartyl form [26,27]. Overexpression in mutagenized Arabidopsis seeds of PIMT1, one of the genes encoding this enzyme, reduces the accumu￾lation of abnormal residues in proteins which results in increases in both seed vigor and longevity. In contrast, reduced expression of this gene increases the accumulation of l-isoaspartyl residues in proteins within freshly harvested dry mature seeds, leading to reduced seed vigor under stressed conditions and hypersensitiv￾ity to aging treatments [27]. PIMT2 is targeted to endomembrane systems, such as in mitochondria and chloroplasts, as well as the cytoplasm [26], suggesting this enzyme is involved in protein repair in several subcellular locations. Seed vigor and longevity have not been intensively targeted in breeding programs, but with the iden￾tification of relevant genes there are prospects for improving these traits through molecular breeding. Imbibed seeds generate reactive oxygen species (ROS) during water uptake [28]. A positive role of ROS in releasing seeds from dormancy has been proposed [29–31]. However, in non-dormant seeds, free radicals could be harmful to proteins and other cell components. Dry pea seeds contain superoxide dismutase, catalase, ascorbate peroxidase, dehydroascorbate reductase and glutathione reductase which are involved in the scavenging of free radicals gen￾erated during imbibition [28]. They may be part of an antioxidative complex in seeds and contribute to the repair and/or protection of proteins and other polymeric compounds during imbibition. 4. Reawakening of protein synthesis upon imbibition—old and new messages Whether or not dry seeds are capable of transcribing DNA and synthesizing proteins in the dry state, e.g. during prolonged after￾ripening, remains to be demonstrated unequivocally. Transient, low-level transcription and translation may occur in air-dry, low￾hydrated seeds [32]. More than 12,000 mRNA species are present in dry seeds of Arabidopsis [33] and barley [34]; dry rice seeds contain over 17,000 mRNAs [35]. These mRNAs are called stored or residual messages [36], and are carried over from seed development, sur￾viving cell desiccation. The location of these messages in the dry seed is unknown, but they could be sequestered in mRNPs (mes￾senger ribonucleoprotein complexes) within the cytoplasm, such as occurs during alfalfa embryogenesis [37]. Many of the genes encoding residual mRNAs contain abscisic acid-responsive elements (ABREs) with the core motif ACGT in their promoter regions, and are typically activated during seed maturation [33]. It is possible that stored mRNAs support de novo protein synthesis before transcriptional activity is resumed and new messages become available during early stages of imbibition (see below). In dry seeds (e.g. wheat embryos), all of the compo￾nents needed for the resumption of protein synthesis are present [3,38]. Within minutes of rehydration, ribosomes become recruited into polysomal protein-synthesizing complexes, utilizing extant mRNAs [3]. Involvement of stored mRNAs during germination is supported by experiments on Arabidopsis using transcriptional and translational inhibitors. -Amanitin, an inhibitor of RNA poly￾merase II does not inhibit germination, while cycloheximide, a protein synthesis inhibitor clearly prevents radicle emergence; these observations have led to the controversial contention that de novo protein synthesis utilizing stored mRNAs is sufficient to allow for the completion of germination [39]. Although stored mRNAs may be utilized during early imbibition, it is unknown which, if any, are essential for the completion of germination. A number of studies show there is considerable transcription taking place, especially during late Phase I, and Phase II, and it is possible this is required for germination to be completed. Tran￾script profiling at six HAI and earlier has been carried out using Arabidopsis seeds. Phase I is characterized by seed swelling, which occurs in both non-dormant (Columbia:Col-0 ecotype) and dor￾mant (Cape Verde Islands: Cvi) lines and ceases around three HAI

H.Nonogaki et al.Plant Science 179(2010)574-58 577 e HAI are also high tion:many have he t of th yme GA 3-oxidase lyzes th ncluding th e for A】 of e do hle-k first to b water uptake.These ger nthesis during ge mination.invo with the A n of ge r with activation er hich co active for fGA to inactiv to CA teins are n the see eds 40 thus on germination ormati ana ysis of g Ara ge which catalyze the cl e of 9'- taining Up-1 ACCCTA ed by lation of the c 42 ctor that ABA applied ABA the ment and replenishment ke of an ABA g vent during minatio BA-GE ABA affe 151 for rice with ination Thus naly that 76 d be and thre 54 very r to wl rt to he ted with ing the ucc ema ng pr a clear program switch bet 10- HAl and n of ke of im hibi ng ARA and are de ads 14? tive removal of some transcripts epo id d s)in is th This ge eds 5.Hormone metabolism eed es in their lett pa nodulate the m sm of these sigr ing molecule ortant featur fGA and ABA me 5m5 inte in the the vents.An in tion [571.thu s modul 581g I59.n oke[ ally influence the appropriate pathways is still meta any of the transcripts encoding enzymes involved in hormone Dolsm are d by de novo transcription during germ

H. Nonogaki et al. / Plant Science 179 (2010) 574–581 577 [40]. Transcripts of genes up-regulated by three HAI are also high at six HAI but decline by 12 HAI [33,40]; therefore, they may have specific roles during early imbibition. Most of the genes up￾regulated around three HAI are those associated with primary metabolism, including those for the PPP [40], providing supporting evidence that the resumption of respiratory activity is one of the first to be initiated upon water uptake. These genes are expressed in both Col and Cvi seeds, and thus are not specifically associ￾ated with the completion of germination per se, but rather with the essential re-establishment of basal metabolism common to dormant and non-dormant seeds. Genes encoding ribosomal pro￾teins are up-regulated exclusively in the non-dormant seeds [40], suggesting that ribosome biogenesis is a necessary step in the com￾pletion of germination. The potential importance of this during early imbibition was suggested from previous studies [41,42]. Using bioinformatic analysis of genes up-regulated during Arabidopsis germination, cis regulatory elements enriched in the 5 upstream sequences containing Up-1 (GGCCCAWW) and Up-2 (AAACCCTA) motifs were identified, which are integral to genes encoding ribo￾somal proteins [42]. AtTCP14 (named from TEOSINTE BRANCHED1, CYCLOIDEA and PCF), a bHLH transcription factor that targets a cis element and is almost identical to the Up-1 motif is also expressed at three HAI [40]. Seeds carrying a mutation in this gene show a delay in the completion of germination and hypersensitivity to applied ABA. Thus, the establishment and replenishment of key components of the protein synthesis machinery appears to be a key event during germination. Transcriptome data (for one and three HAI) are also available for rice seeds during early imbibition, and are integrated with a metabolome analysis [35]. Gene expression analysis using the Rice GeneChip indicates that 1469 and 1276 transcripts are up￾and down-regulated between one and three HAI, while only 59 and four transcripts are up- and down-regulated during the first hour of imbibition [35]. This is very similar to what was observed in Arabidopsis. In contrast to the limited transcriptional activity occurring during the first hour of imbibition, there are changes to 25 out of 126 metabolites over the same time period, and these are predominantly associated with carbohydrate metabolism. Fru- 6-P, Glc-6-P, and glycerate-3-phosphate increase during the first hour, and thereafter, as do tricarboxylic acid (TCA) cycle intermedi￾ates 2-oxoglutarate, aconitate, fumarate, malate and succinate. This suggests there is an immediate increase in the activity of glycolysis and the TCA cycle to facilitate early, energy-demanding processes [35]. The combined metabolome and transcriptome analyses also identified a clear program switch between 0–1 HAI and 1–3 HAI, indicative of a change in activity during imbibition. Down-regulation of key transcripts may be also of importance during germination. For example, several genes encoding proteins that inhibit germination, including ABA-response genes ABI4, ABI5, ABI8, and that of the GA receptor GID1A, are down-regulated in germinating Arabidopsis seeds [43]. Therefore, regulation of germi￾nation may require both the selective removal of some transcripts and the de novo synthesis of others. 5. Hormone metabolism Hormones have a profound influence over the regulation of seed germination and dormancy. In turn, the genes encoding proteins that modulate the metabolism of these signaling molecules (espe￾cially those involved in the biosynthesis and deactivation of GA and ABA) are major genetic regulators of these events. An informative review article on the interactions of the hormone metabolic path￾ways and environmental signals during germination is available [44]. Many of the transcripts encoding enzymes involved in hormone metabolism are produced by de novo transcription during germi￾nation; many have been identified in Arabidopsis. GA biosynthesis is required for Arabidopsis seed germination [45]. The GA biosyn￾thesis enzyme GA 3-oxidase catalyzes the conversion of inactive forms of the hormone to active forms (GA9 to GA4, or GA20 to GA1). Seeds of the double-knockout mutants ga3ox1 ga3ox2 do not germinate without the application of GA [46], demonstrating the requirement for GA biosynthesis during germination, involv￾ing expression of these genes. GA2ox encodes GA 2-oxidase, a GA deactivation enzyme which converts active forms of GA to inactive forms (GA4 to GA34, or GA1 to GA8) [44,47]. The GAMT gene encodes a GA methyltransferase, which converts GA4 to GA4-methyl ester, thus inactivating it. These enzymes probably play a role in pre￾venting germination during seed development by removing active forms of GA [48]. NCED6 and NCED9 encode seed-specific 9-cis epoxycarotenoid dioxygenases which catalyze the cleavage of 9 -cis-neoxanthin to xanthoxin, a rate-limiting process in ABA biosynthesis [44]. ABA is deactivated by hydroxylation of the C-8 position by ABA 8 - hydroxylase encoded by the gene CYP707A; 8 -hydroxy ABA is sub￾sequently converted to phaseic acid and dihydrophaseic acid [49]. CYP707A2 expression controls the rapid decrease in ABA during Arabidopsis seed imbibition. cyp707a2 mutants exhibit hyperdor￾mancy [50]. Thus there is a correlation between a decline in ABA and the ability of the seed to germinate. ABA can be deactivated also through the formation of an ABA glucose ester (ABA-GE), which results in germination being promoted. In contrast, hydrolysis of ABA-GE can reactivate ABA and negatively affect germination [51]. Hormone metabolism has a considerable impact on the regula￾tion of Arabidopsis seed germination. Thus genes involved in GA and ABA biosynthesis and deactivation, for example, can be viewed as potential targets for improvement of germination in other species. Orthologues of such genes have been characterized in rice [52], sorghum [53] and barley [54], and thus results from research on seeds of the model species Arabidopsis might usefully be applied to those of commercial interest. Discovery of these genes has allowed for a start to be made in answering questions important to these and other crop species. An example relates to failure of seeds to ger￾minate at super-optimal temperatures. When seeds are imbibed at higher than optimal temperatures, they go though the metabolic events occurring during Phase I and probably much of II, but do not enter Phase III, i.e. radicle protrusion does not occur (ther￾moinhibition). Analysis of the expression of hormone metabolism genes in thermoinhibited Arabidopsis seeds reveals that high tem￾peratures stimulate ABA biosynthesis and repress GA biosynthesis [55]. Lettuce (Lactuca sativa ‘Salinas’) seeds also fail to germi￾nate when imbibed above 25 ◦C. However, seeds of Lactuca serriola (UC96US23) do not exhibit thermoinhibition up to 37 ◦C. Compar￾ative genetics, physiology and gene expression studies reveal that temperature sensitivity of the expression of the LsNCED4 gene, a lettuce NCED, may determine the upper temperature limit for germination [56], as do the equivalent genes (AtNCED, encoding 9- cis-epoxycarotenoid dioxygenases) in Arabidopsis [55]. This gene is up-regulated at the higher temperature only in the thermoinhib￾ited seeds of ‘Salinas’ lettuce, which also contain increased amounts of ABA compared to L. serriola. This genetic marker thus has the potential to be used by seed companies in their lettuce-breeding programs, for it may be applicable to other temperature-sensitive species. An important feature of GA and ABA metabolism is their inter￾action. For example, ABA down-regulates GA biosynthesis and up-regulates GA deactivation [57], thus modulating seed ABA con￾tent [44]. Responses of seeds to environmental factors such as cold [58], light [59], nutrition [23] and smoke [60] are also well explained by changes in hormone metabolism [44,47], although how they specifically influence the appropriate pathways is still being elucidated

578 H.Nonogaki et aL Plant Science 179 (2010)574-581 &Cmnnnthembnolkadigtothecompkeioaofcd In growing Arabidopsis roots the primary osmotically-active solute germi de bed but king fortheirino ement in the ofgermin tion a Pha to eith ting toma Phase lll is regulated is not und ngation zone ed on the obs ation that seeds unable to produc nes have been ized that are ble o n sor of GA The vith ho and lig use the that a re RGL ion in 61 otein is no of ce xpansion.suchas the water-channel pr ins and cell may be use the e are DELLA-inde proteir are coded by large m i-gene fam and functiona eed germinatio uld be out Therefore in the a ence of a it is difficul tiva require GA for th .Systems level anal ol ge n the do not need the data ndana pron ng approa to elucida g stru ghts int n on v whereas the embryo in the intact seed mus udy investigating gene expression in the ti sues 7 an intact seed ph enon. nduce mecha wall n cal/b d by the s mination.andh are fore n as radicle emergence cell walls ncellular tur h uptake th mmencing Phase )The nd of phase ll the em are unknown sins,xyloglucan endotra plex com sition the this D odifying enzym ns are In th n of elonga the radicle per se but in region in the adjacent tra nsiti enc Iwalls,th cells adja reas r DNA within the e mbry cells of cel that are ready toeo and canr nd g0 osis 65 th potential m chanical resi way ation that radicle em ent the ans by increa gence from the ng.ho eve acilitated b Il are not well understo hra Protein tance of the sur unding tissues(e.g.the micn There are many studie esta gh and radicle em 7778 9.80 oh6A ater teins and is there antiale potential is required to stimulate water uptake into a growing cell gest that endosperm weakening is effected by cell wall hydrolases

578 H. Nonogaki et al. / Plant Science 179 (2010) 574–581 6. Changes in the embryo leading to the completion of seed germination Following the initial uptake of water into the seed (imbibition; Phase I; Fig. 1) there is little increase during Phase II, but this is when environmental cues are integrated with the physiological state of the seed to determine whether or not it will enter into Phase III, a final phase of increased water uptake driving cell expansion lead￾ing to the completion of germination. The majority of characterized genetic regulators of germination act during Phase II to either pro￾mote or inhibit the entry of seeds into the final Phase. How the transition from Phase II to Phase III is regulated is not understood, although it is clear that GA plays a key role, at least in the intact seed. This is based on the observation that seeds unable to produce GA do not complete germination. The DELLA protein RGL2 is a key repressor of GA responses in Arabidopsis seeds because those that are GA-deficient com￾plete germination in the absence of a functional RGL2 gene [61]. A decrease in transcripts for, and removal of, the RGL2 protein is not required, however, for the completion of Arabidopsis seed germi￾nation [62,63]. This may be because there are DELLA-independent GA signaling pathways regulating the GA-mediated completion of seed germination, or there could be proteolysis-independent deac￾tivation, rather than degradation, of this DELLA protein [64]. It is noteworthy that embryos which require GA for the comple￾tion of germination in the intact seed do not need the presence of this hormone to achieve this when they are separated from their surrounding structures. Isolated embryos of GA-deficient (gib-1) mutants of Arabidopsis and tomato, for example, will complete ger￾mination on water, whereas the embryo in the intact seed must be supplied with the hormone. Hence the requirement for GA is an intact-seed phenomenon, perhaps to induce mechanisms to overcome mechanical restraints or other inhibitions (e.g. chemi￾cal/hormonal) imposed by the surrounding structures. Embryo (radicle) cell expansion to complete germination is facilitated through a combination of decreases in the restraining forces of the enclosing cell walls and a rise in cellular tur￾gor pressure driving increases in cell volume through increased water uptake (thus commencing Phase III). The specific modifi- cations facilitating cell wall loosening to promote embryo growth are unknown, although expansins, xyloglucan endotransglycosy￾lase/hydrolase (XTH) and cellulase may be involved. Given the complex composition of the cell wall, it is likely that the concerted action of several modifying enzymes and proteins contribute its loosening. As noted earlier, the initial region of elongation of Arabidopsis embryos to effect the completion of germination is not located in the radicle per se, but in a small region in the adjacent transition zone and lower hypocotyl [65]. Elongation is due exclusively to cell expansion, following an increase in cellular DNA within the embry￾onic axis (including by endoreduplication, which is characteristic of cells that are ready to elongate and cannot undergo mitosis [65]). That the completion of germination is the result of cell elongation and not division is reinforced by the observation that radicle emer￾gence from Medicago trunculata seeds can occur in the presence of oryzalin, a compound that disrupts the cytoskeleton and inhibits mitosis [66]. The mechanisms of water uptake by the embryo during Phase III are not well understood. Plasma membrane Intrinsic Proteins (PIPs) and Tonoplast Intrinsic Proteins (TIPs) function as channels that regulate the passage of water across membranes. Treatment of Arabidopsis seeds with mercury (HgCl2), a water-channel inhibitor, results in a delay of testa rupture by 8–9 h, and radicle emergence by 25–30 h [67] which is suggestive that the induction of Phase III involves these water channel proteins. A decrease in osmotic potential is required to stimulate water uptake into a growing cell. In growing Arabidopsis roots the primary osmotically-active solute is potassium [68], but whether this ion is involved in water uptake by germinating embryos is unknown. Fluctuations in a range of low-molecular-weight metabolites during germination have been described, but evidence is lacking for their involvement in the req￾uisite changes in osmotic potential, which will be important in, if not confined to, cells in the discrete region of cell elongation. Using enhancer traps, distinct gene expression patterns have been identified within the elongation zone of the embryo at the time of completion of germination [65,69]. In germinating tomato seeds, the expansin gene LeEXP8 is expressed in a region similar to the Arabidopsis elongation zone, suggestive of its involvement in localized cell wall modification to facilitate cell expansion [70]. Numerous genes have been characterized that are capable of regulating the transition from Phase II to Phase III in Arabidopsis seeds [2]. These tend to be associated with hormonal and light responses, and include ABI3, ABI5, RGL2, EIN3 and PIL5. But how these regulators influence gene expression of downstream targets of cell expansion, such as the water-channel proteins and cell wall￾modifying enzymes, is unknown. A confounding issue is that these proteins are encoded by large multi-gene families, and functional redundancy likely masks mutant phenotypes of single-gene knock￾out plants. Therefore in the absence of a phenotype, it is difficult to designate a given gene as being a downstream target that is involved in the completion of germination. Systems-level analyses of germination-related data are a promising approach to elucidat￾ing these complex and redundant regulatory networks. Insights into the biological processes occurring specifically in the embryo and endosperm have emerged from a transcriptomic study investigating gene expression in these tissues [71]. Genes encoding components of protein synthesis, ribosomes, photo￾synthetic genes, DNA synthesis, cell cycle, cell wall remodeling enzymes, and major water channel proteins are all enriched in the embryo during germination, and therefore play some role in this event, leading to its completion as radicle emergence. 7. Relationships between the embryo and its surrounding structures in regulating the completion of germination At the end of Phase II the embryo, usually the radicle, emerges from the seed, marking the completion of germination. To achieve this, it has to break through surrounding tissues such as the endosperm (or perisperm) and the testa (seed coat). In some species these structures are barriers to emergence, imposing physical dor￾mancy. In others, such as hard-seeded legumes (carob, Chinese senna, fenugreek), asparagus, coffee and date, while the endosperm is impenetrable in the region surrounding the cotyledons because of the presence of thick mannan-rich cell walls, those cells adja￾cent to the radicle have thin walls (or in the case of date, a ring of thin-walled cells) allowing it to penetrate to complete germination [72,73]. In all seeds that must penetrate the surrounding structures, embryo growth potential must exceed the mechanical resistance of these tissues [74]. One way of changing the balance between these opposing forces is to augment the former, perhaps by increasing the concentration of small metabolites that lead to a decrease the osmotic potential of the embryo [75]; conclusive evidence for this is lacking, however. Radicle emergence could be facilitated by reducing the resis￾tance of the surrounding tissues (e.g. the micropylar endosperm) through enzymatic or physical weakening of their cell walls. There are many studies on endosperm weakening by hydrolases such as xyloglucan endotransglycosylase/hydrolase (XTH) [76], - glucanases [77,78] and endo--mannanase [79,80], or by other cell wall proteins such as expansins (EXP)[70]. However, only in tomato and Datura seeds is there other than circumstantial evidence to sug￾gest that endosperm weakening is effected by cell wall hydrolases;

H.Nonogaki et al.Plant Science 179(2010)574-58 579 as occurs in germin suf ent number s for use as experimental d o eorhiza egio as t at seeds,in which the larg gest volume o ttgtoaeaationor nativ he s t but this is very smal much of the 861.and based cloning hasr netabolism ation,pr withi ntile ap ncy t eds tha ot rer mant sion of germination tha that of the co eoptile <I orth CYP7 ional cha at occ urs du whic throug stive of germination con this tiss the ant rmination to be effe on ther nic crop SITIVES (ABIS) ant c nt of path 8 te be trolled in the end ditions (e. s).wheble tcom for tho nducible.but ar re not suppressed by ABA.Indeed.initial micropyla limatic and soil condition particular treat nts employe eed viahility and vigo nects of seed qualit 8.Germination-pe and im rspectives and applications nditions to ture seeds a ould be of valu the qualit ng.Se ral vi germination. use germir ion nd ent eve ding of the r and moled changes c during seed ge ologies to improve cellular nation to be com seeds.the cruci ownstream targets There arei as to why it has been so difficult ifywhat are the key hin ents required to th omplet t is a multi-cellular which the major cell mas References the axis can be removedand usedas xperiment nd Bu ear to could be asked by the

H. Nonogaki et al. / Plant Science 179 (2010) 574–581 579 in these species, endo--mannanase [81]. It is also possible that cell walls are chemically modified by hydroxyl radicals (•OH)− in vivo, as occurs in germinating cress (Lepidium sativum) seeds [82]. Some embryonic tissues in cereals, such as the epiblast, and the coleorhiza that covers the radicle, may play an important role in germination control [83,84]; the coleorhiza is a potential barrier to radicle emergence that must be modified to allow for completion of germination. Changes include an increase in poros￾ity of the cell walls and more intercellular spaces, which signify cell separation. Mobilization of proteins from storage vesicles in the epiblast/coleorhiza regions of rice seeds during germination results in vacuole formation, and a decline in lipid bodies [83]; similar changes occur in the micropylar endosperm of the seeds of the dicot species such as tomato prior to penetration by the radicle [85]. Quantitative trait loci (QTL) analysis of rice seed germination under cold conditions has identified a relevant chromosome region, qLTG3-1 (quantitative trait locus for low-temperature germinability) [86], and map-based cloning has revealed a gene encoding a plant￾specific protein of unknown function. The promoter of qLTG3-1 is activated in imbibed rice seeds in the epiblast, coleoptile and the coleorhiza [83]. Thus the surrounding structures may influence the completion of germination of rice seeds, at least under cold conditions. The ABA content of the coleorhiza is better correlated with sup￾pression of germination than that of the coleoptile, scutellum, or other embryonic tissues [84]. Expression of HvABA8 OH (Hordeum vulgare ABA 8 -hydroxylase), an orthologue of CYP707A2 occurs exclusively in the coleorhiza of after-ripened, 6-h-imbibed barley seeds [54], suggestive of germination control by this tissue. For ABA to be effective in controlling germination there must be an effective signal transduction pathway. In Arabidopsis ABA INSEN￾SITIVE5 (ABI5) is an important component of this pathway, and studies using a ABI5:GUS reporter gene indicate that it is activated in the endosperm region surrounding the radicle [71,87]. In tomato seeds, endosperm-cap specific, cell-wall associated genes are GA￾inducible, but are not suppressed by ABA. Indeed, initial micropylar endosperm weakening can occur in the presence of low concentra￾tions of ABA [67]. Modification of the tissues adjacent to the radicle appears to be an important part of the termination of germination and could be the final determinant as to whether or not it occurs. 8. Germination—perspectives and applications Germination is a complex process, frequently involving recovery from maturation drying, the re-establishment of basal metabolism, in some species the overcoming of dormancy, and preparation for the completion of germination. Because germination and post￾germination are confluent events distinguished as before and after embryo (radicle) emergence, there are presumably metabolic changes occurring during the former in preparation for subsequent seedling establishment. The difficulty is in distinguishing between what is key to germination and what is not. Despite much research it is still not possible to answer definitively the question....what cellular event (or events) is essential for germination to be com￾pleted? Although there is strong evidence for the involvement of hormones, at least in intact seeds, the crucial downstream targets influenced by them are unknown. There are likely several reasons as to why it has been so difficult to identify what are the key events required to the completion of germination. Some reasons relate to the nature of the seed itself. It is a multi-cellular organism in which the major cell mass is storage tissue. From larger seeds, e.g. some legumes and cereals, the axis can be removed and used as experimental material. But even these contain many cells, of different types, and since only a relatively few cells appear to be involved in elongation to effect radicle emergence, changes within these could be masked by the larger mass of unchanging cells. This issue is amplified in smaller seeds such as of the dicot Arabidopsis, from which removal of a sufficient number of axes for use as experimental material is very challenging, and obtaining an appropriate quantity of cells in a limited region such as that undergoing elongation is almost impossible. Thus at least some of the changes recorded during germination of Arabidopsis seeds, in which the largest volume of tissue is not the axis, but the storage cotyledons, may be integral to the latter, which could be in preparation for post-germinative events, rather than related to radicle emergence per se. There is limited mobilization of reserves during germination, perhaps to provide a source of sugars and amino acids, but this is very small compared to reserve utilization following germination. Related to this is the question of how much of the metabolism occurring during germination is actually necessary for this phe￾nomenon to be completed. Lettuce seeds subjected to long-term dormancy in the hydrated state (skotodormancy) reduce their metabolism (respiration, protein synthesis) substantially within weeks, presumably to conserve their limited nutrient resources [88,89]. When eventually released from dormancy they complete germination, but the amount of metabolism they undertake, e.g. respiration, is less than half of that in seeds that were not rendered dormant before germination. At the present time we do not have a complete picture of what occurs during seed germination. Nonetheless, recent progress has provided much information for the transcriptional and transla￾tional changes that occurs during germination which, through genetic manipulation, may help in its improvement. There are challenges to enhance the speed and percentage of germination in horticultural and agronomic crops, and in those of forest species, and they have been met for some species using priming and pellet￾ing/coating technologies. Success has been achieved particularly on a small scale, for seeds planted in controlled environmental conditions (e.g. glasshouses), whereas the outcomes for those planted in the field can be variable, depending on prevailing climatic and soil conditions. The particular treatments employed for improving germination are the results of empirical trials, rather than through identifying and manipulating specific metabolic processes, for example. Seed viability and vigor are important aspects of seed quality, and important in determining the success of a planted crop. Many factors influence these attributes, not least of which are the storage conditions to whichmature seeds are subjected. It would be of value to producers and distributors, as well as purchasers to know what is the quality of a seed batch prior to planting. Several vigor tests are in use [90], although more information is needed to identify particular biochemical or molecular markers of deterioration that can predict poor germinability. Thus, while our understanding of the cellular and molecular events occurring during seed germination is broadening, which events are critical for its completion remain to be elucidated, as a prelude to the application of molecular technologies to improve and control this vital step in the plant life cycle. Acknowledgements This work was supported by NSF grant IBN-0237562 (to H.N.) and NSERC Discovery grant 044191 (to J.D.B.). G.W.B was the recip￾ient of an NSERC Postdoctoral Fellowship. References [1] L. Russell, V. Larner, S. Kurup, S. Bougourd, M. Holdsworth, The Arabidopsis COMATOSE locus regulates germination potential, Development 127 (2000) 3759–3767. [2] M.J. Holdsworth, L. Bentsink, W.J.J. Soppe, Molecular networks regulating Ara￾bidopsis seed maturation, after-ripening, dormancy and germination, New Phytol. 179 (2008) 33–54.

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580 H. Nonogaki et al. / Plant Science 179 (2010) 574–581 [3] J.D. Bewley, M. Black (Eds.), Seeds: Physiology of Development and Germina￾tion, 2nd edition, Plenum Press, New York, 1994, p. 445. [4] H. Nonogaki, Seed germination and reserve mobilization, in: Encyclopedia of Life Sciences, John Wiley & Sons, Ltd., Chichester, 2008, www.els.net, doi:10.1002/9780470015902.a0002047.pub2. [5] B. Manz, K. Muller, B. Kucera, F. Volke, G. Leubner-Metzger, Water uptake and distribution in germinating tobacco seeds investigated in vivo by nuclear mag￾netic resonance imaging, Plant Physiol. 138 (2005) 1538–1551. [6] J.R. Rathjen, E.V. Strounina, D.J. Mares,Water movement into dormant and non￾dormant wheat (Triticum aestivum L.) grains, J. Exp. Bot. 60 (2009) 1619–1631. [7] V.V. Terskikh, J.A. Feurtado, C. Ren, S.A. Abrams, A.R. Kermode, Water uptake and oil distribution during imbibition of seeds of western white pine (Pinus monticola Dougl. ex D. Don) monitored in vivo using magnetic resonance imag￾ing, Planta 221 (2005) 17–27. [8] A. Hourmant, A. Pradet, Oxidative phosphorylation in germinating lettuce seeds (Lactuca sativa) during the first hours of imbibition, Plant Physiol. 68 (1981) 631–635. [9] J. Grelet, A. Benamar, E. Teyssier, M.-H. Avelange-Macherel, D. Grunwald, D. Macherel, Identification in pea seed mitochondria of a late-embryogenesis abundant protein able to protect enzymes from drying, Plant Physiol. 137 (2005) 157–167. [10] D. Tolleter, M. Jaquinod, C. Mangavel, C. Passirani, P. Saulnier, S. Manon, E. Teyssier, N. Payet, M.-H. Avelange-Macherel, D. Macherel, Structure and func￾tion of a mitochondrial late embryogenesis abundant protein are revealed by desiccation, Plant Cell 19 (2007) 1580–1589. [11] Y. Morohashi, J.D. Bewley, Development of mitochondrial activities in pea cotyledons: influence of desiccation during and following germination of the axis, Plant Physiol. 66 (1980) 637–640. [12] Y. Morohashi, J.D. Bewley, E.C. Yeung, Biogenesis of mitochondria in imbibed peanut cotyledons. II. Development of light and heavy mitochondria, Plant Physiol. 68 (1981) 318–323. [13] Y. Morohashi, Patterns of mitochondrial development in reserve tissues of ger￾minated seeds: a survey, Physiol. Plant. 66 (1986) 653–658. [14] R.A. Kennedy, M.E. Rumpho, T.C. Fox, Anaerobic metabolism in plants, Plant Physiol. 100 (1992) 1–6. [15] E.H. Roberts, A survey of the effects of chemical treatments on dormancy in rice seed, Physiol. Plant. 17 (1964) 30–43. [16] E.H. Roberts, The distribution of oxidation–reduction enzymes and the effects of respiratory inhibitors and oxidising agents on dormancy in rice seed, Physiol. Plant. 17 (1964) 14–29. [17] S.B. Hendricks, R.B. Taylorson, Promotion of seed germination by nitrate, nitrite, hydroxylamine, and ammonium salts, Plant Physiol. 54 (1974) 304–309. [18] M.A. Cohn, D.L. Butera, J.A. Hughes, Seed dormancy in red rice. III. Response to nitrite, nitrate, and ammonium ions, Plant Physiol. 73 (1983) 381–384. [19] H.W.M. Hilhorst, C.M. Karssen, Nitrate reductase independent stimulation of seed germination in Sisymbrium officinale L. (hedge mustard) by light and nitrate, Ann. Bot. 63 (1989) 131–137. [20] L.M. Roberts, J.M. Lord, Developmental changes in the activity of messenger RNA isolated from germinating castor bean endosperm, Plant Physiol. 64 (1979) 630–634. [21] P.C. Bethke, I.G.L. Libourel, R.L. Jones, Nitric oxide in seed dormancy and ger￾mination, in: K.J. Bradford, H. Nonogaki (Eds.), Seed Development, Dormancy and Germination, Blackwell Publishing, Oxford, 2007, pp. 153–175. [22] P.C. Bethke, I.G.L. Libourel, N. Aoyama, Y.-Y. Chung, D.W. Still, R.L. Jones, The Arabidopsis aleurone layer responds to nitric oxide, gibberellin, and abscisic acid and is sufficient and necessary for seed dormancy, Plant Physiol. 143 (2007) 1173–1188. [23] T. Matakiadis, A. Alboresi, Y. Jikumaru, K. Tatematsu, O. Pichon, J.-P. Renou, Y. Kamiya, E. Nambara, H.-N. Truong, The Arabidopsis abscisic acid catabolic gene CYP707A2 plays a key role in nitrate control of seed dormancy, Plant Physiol. 149 (2009) 949–960. [24] J.H. Crowe, B.D. McKersie, L.M. Crowe, Effects of free fatty acids and transition temperature on the stability of dry liposomes, Biochim. Biophys. Acta (BBA) – Biomembranes 979 (1989) 7–10. [25] L.O. Copeland, M.B. McDonald (Eds.), Principles of Seed Sceince and Technology, 2nd edition, Burgess Publishing Company, Minneapolis, 1985, p. 321. [26] R.D. Dinkins, S.M. Majee, N.R. Nayak, D. Martin, Q. Xu, M.P. Belcastro, R.L. Houtz, C.M. Beach, A.B. Downie, Changing transcriptional initiation sites and alter￾native 5 - and 3 -splice site selection of the first intron deploys Arabidopsis PROTEIN ISOASPARTYL METHYLTRANSFERASE2 variants to different subcellu￾lar compartments, Plant J. 55 (2008) 1–13. [27] L. Oge, G. Bourdais, J. Bove, B. Collet, B. Godin, F. Granier, J.-P. Boutin, D. Job, M. Jullien, P. Grappin, Protein repair l-isoaspartyl methyltransferase1 is involved in both seed longevity and germination vigor in Arabidopsis. Plant Cell 20 (2008) 3022–3037. [28] L. Wojtyla, M. Garnczarska, T. Zalewski, W. Bednarski, L. Ratajczak, S. Jurga, A comparative study of water distribution, free radical production and activa￾tion of antioxidative metabolism in germinating pea seeds, J. Plant Physiol. 163 (2006) 1207–1220. [29] K. Oracz, H. El-Maarouf-Bouteau, J.M. Farrant, K. Cooper, M. Belghazi, C. Job, D. Job, F. Corbineau, C. Bailly, ROS production and protein oxidation as a novel mechanism for seed dormancy alleviation, Plant J. 50 (2007) 452–465. [30] K. Oracz, H. El-Maarouf-Bouteau, I. Kranner, R. Bogatek, F. Corbineau, C. Bailly, The mechanisms involved in seed dormancy alleviation by hydrogen cyanide unravel the role of reactive oxygen species as key factors of cellular signaling during germination, Plant Physiol. 150 (2009) 494–505. [31] P. Schopfer, Hydroxyl radical-induced cell-wall loosening in vitro and in vivo: implications for the control of elongation growth, Plant J. 28 (2001) 679–688. [32] G. Leubner-Metzger, -1,3-Glucanase gene expression in low-hydrated seeds as a mechanism for dormancy release during tobacco after-ripening, Plant J. 41 (2005) 133–145. [33] K. Nakabayashi, M. Okamoto, T. Koshiba, Y. Kamiya, E. Nambara, Genome-wide profiling of stored mRNA in Arabidopsis thaliana seed germination: epigenetic and genetic regulation of transcription in seed, Plant J. 41 (2005) 697–709. [34] N. Sreenivasulu, B. Usadel, A. Winter, V. Radchuk, U. Scholz, N. Stein, W. Weschke, M. Strickert, T.J. Close, M. Stitt, A. Graner, U. Wobus, Barley grain maturation and germination: metabolic pathway and regulatory network com￾monalities and differences highlighted by new MapMan/PageMan profiling tools, Plant Physiol. 146 (2008) 1738–1758. [35] K.A. Howell, R. Narsai, A. Carroll, A. Ivanova, M. Lohse, B. Usadel, A.H. Millar, J. Whelan, Mapping metabolic and transcript temporal switches during ger￾mination in rice highlights specific transcription factors and the role of RNA instability in the germination process, Plant Physiol. 149 (2009) 961–980. [36] N. Ishibashi, T. Minamikawa, Molecular cloning and characterization of stored mRNA in cotyledons of Vigna unguiculata, Plant Cell Physiol. 31 (1990) 39–44. [37] S.K. Pramanik, J.D. Bewley, Post-transcriptional regulation of protein synthesis during alfalfa embryogenesis: proteins associated with the cytoplasmic polyso￾mal and non-polysomalmRNAs (messenger ribonucleoprotein complex), J. Exp. Bot. 47 (1996) 1871–1879. [38] A. Marcus, J. Feeley, T. Volcani, Protein synthesis in imbibed seeds. III. Kinetics of amino acid incorporation ribosome activation, and polysome formation, Plant Physiol. 41 (1966) 1167–1172. [39] L. Rajjou, K. Gallardo, I. Debeaujon, J. Vandekerckhove, C. Job, D. Job, The effect of -amanitin on the Arabidopsis seed proteome highlights the distinct roles of stored and neosynthesized mRNAs during germination, Plant Physiol. 134 (2004) 1598–1613. [40] J. Preston, K. Tatematsu, Y. Kanno, T. Hobo, M. Kimura, Y. Jikumaru, R. Yano, Y. Kamiya, E. Nambara, Temporal expression patterns of hormone metabolism genes during imbibition of Arabidopsis thaliana seeds: a comparative study on dormant and non-dormant accessions, Plant Cell Physiol. 50 (2009) 1786–1800. [41] P.E. Toorop, R.M. Barroco, G. Engler, S.P.C. Groot, H.W.M. Hilhorst, Differen￾tially expressed genes associated with dormancy or germination of Arabidopsis thaliana seeds, Planta 221 (2005) 637–647. [42] K. Tatematsu, K. Nakabayashi, Y. Kamiya, E. Nambara, Transcription factor AtTCP14 regulates embryonic growth potential during seed germination in Arabidopsis thaliana, Plant J. 53 (2008) 42–52. [43] G.W. Bassel, P. Fung, T.-F.F. Chow, J.A. Foong, N.J. Provart, S.R. Cutler, Elucidating the germination transcriptional program using small molecules, Plant Physiol. 147 (2008) 143–155. [44] M. Seo, E. Nambara, G. Choi, S. Yamaguchi, Interaction of light and hormone signals in germinating seeds, Plant Mol. Biol. 69 (2009) 463–472. [45] M. Koornneef, J.H. van der Veen, Induction and analysis of gibberellin sensi￾tive mutants in Arabidopsis thaliana (L.) Heynh, Theor. Appl. Genet. 58 (1980) 257–263. [46] M.G. Mitchum, S. Yamaguchi, A. Hanada, A. Kuwahara, Y. Yoshioka, T. Kato, S. Tabata, Y. Kamiya, T.-P. Sun, Distinct and overlapping roles of two gibberellin 3-oxidases in Arabidopsis development, Plant J. 45 (2006) 804–818. [47] S. Yamaguchi, Y. Kamiya, E. Nambara, Regulation of ABA and GA levels dur￾ing seed development and germination in Arabidopsis, in: K.J. Bradford, H. Nonogaki (Eds.), Seed Development, Dormancy and Germination, Blackwell Publishing, Oxford, 2007, pp. 224–247. [48] M. Varbanova, S. Yamaguchi, Y. Yang, K. McKelvey, A. Hanada, R. Borochov, F. Yu, Y. Jikumaru, J. Ross, D. Cortes, C.J. Ma, J.P. Noel, L. Mander, V. Shulaev, Y. Kamiya, S. Rodermel, D. Weiss, E. Pichersky, Methylation of gibberellins by Arabidopsis GAMT1 and GAMT2, Plant Cell 19 (2007) 32–45. [49] E. Nambara, A. Marion-Poll, Abscisic acid biosynthesis and catabolism, Annu. Rev. Plant Biol. 56 (2005) 165–185. [50] T. Kushiro, M. Okamoto, K. Nakabayashi, K. Yamagishi, S. Kitamura, T. Asami, N. Hirai, T. Koshiba, Y. Kamiya, E. Nambara, The Arabidopsis cytochrome P450 CYP707A encodes ABA 8 -hydroxylases: key enzymes in ABA catabolism, EMBO J. 23 (2004) 1647–1656. [51] S.D. Chiwocha, A.J. Cutler, S.R. Abrams, S.J. Ambrose, J. Yang, A.R. Ross, A.R. Kermode, The etr1-2 mutation in Arabidopsis thaliana affects the abscisic acid, auxin, cytokinin and gibberellin metabolic pathways during mainte￾nance of seed dormancy, moist-chilling and germination, Plant J. 42 (2005) 35–48. [52] G. Zhu, N. Ye, J. Zhang, Glucose-induced delay of seed germination in rice is mediated by the suppression of ABA catabolism rather than an enhancement of ABA biosynthesis, Plant Cell Physiol. 50 (2009) 644–651. [53] L. Perez-Flores, F. Carrari, R. Osuna-Fernandez, M.V. Rodriguez, S. Enciso, R. Stanelloni, R.A. Sanchez, R. Bottini, N.D. Iusem, R. Benech-Arnold, Expression analysis of a GA 20-oxidase in embryos from two sorghum lines with contrast￾ing dormancy: possible participation of this gene in the hormonal control of germination, J. Exp. Bot. 54 (2003) 2071–2079. [54] A.A. Millar, J.V. Jacobsen, J.J. Ross, C.A. Helliwell, A.T. Poole, G. Scofield, J.B. Reid, F. Gubler, Seed dormancy and ABA metabolism in Arabidopsis and barley: the role of ABA 8 -hydroxylase, Plant J. 45 (2006) 942–954. [55] S. Toh, A. Imamura, A. Watanabe, K. Nakabayashi, M. Okamoto, Y. Jikumaru, A. Hanada, Y. Aso, K. Ishiyama, N. Tamura, S. Iuchi, M. Kobayashi, S. Yamaguchi, Y. Kamiya, E. Nambara, N. Kawakami, High temperature-induced abscisic acid biosynthesis and its role in the inhibition of gibberellin action in Arabidopsis seeds, Plant Physiol. 146 (2008) 1368–1385.

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H. Nonogaki et al. / Plant Science 179 (2010) 574–581 581 [56] J. Argyris, P. Dahal, E. Hayashi, D.W. Still, K.J. Bradford, Genetic variation for lettuce seed thermoinhibition is associated with temperature-sensitive expres￾sion of abscisic acid, gibberellin, and ethylene biosynthesis, metabolism, and response genes, Plant Physiol. 148 (2008) 926–947. [57] M. Seo, A. Hanada, A. Kuwahara, A. Endo, M. Okamoto, Y. Yamauchi, H. North, A. Marion-Poll, T.-P. Sun, T. Koshiba, Y. Kamiya, S. Yamaguchi, E. Nambara, Regulation of hormone metabolism in Arabidopsis seeds: phytochrome reg￾ulation of abscisic acid metabolism and abscisic acid regulation of gibberellin metabolism, Plant J. 48 (2006) 354–366. [58] Y. Yamauchi, M. Ogawa, A. Kuwahara, A. Hanada, Y. Kamiya, S. Yamaguchi, Activation of gibberellin biosynthesis and response pathways by low tempera￾ture during imbibition of Arabidopsis thaliana seeds, Plant Cell 16 (2004) 367– 378. [59] E. Oh, S. Yamaguchi, Y. Kamiya, G. Bae, W.-II, Chung, Won-Il, G. Choi, Light activates the degradation of PIL5 protein to promote seed germination through gibberellin in Arabidopsis, Plant J. 47 (2006) 124–139. [60] D.C. Nelson, J.-A. Riseborough, G.R. Flematti, J. Stevens, E.L. Ghisalberti, K.W. Dixon, S.M. Smith, Karrikins discovered in smoke trigger Arabidopsis seed ger￾mination by a mechanism requiring gibberellic acid synthesis and light, Plant Physiol. 149 (2009) 863–873. [61] S. Lee, H. Cheng, K.E. King, W. Wang, Y. He, A. Hussain, J. Lo, N.P. Harberd, J. Peng, Gibberellin regulates Arabidopsis seed germination via RGL2, a GAI/RGA-like gene whose expression is up-regulated following imbibition, Genes Dev. 16 (2002) 646–658. [62] G.W. Bassel, E. Zielinska, R.T. Mullen, J.D. Bewley, Down-regulation of DELLA genes is not essential for germination of tomato, soybean, and Arabidopsis seeds, Plant Physiol. 136 (2004) 2782–2789. [63] T. Ariizumi, C.M. Steber, Seed germination of GA-insensitive sleepy1 mutants does not require RGL2 protein disappearance in Arabidopsis, Plant Cell 19 (2007) 791–804. [64] T. Ariizumi, K. Murase, T.-P. Sun, C.M. Steber, Proteolysis-independent down￾regulation of DELLA repression in Arabidopsis by the gibberellin receptor GIBBERELLIN INSENSITIVE DWARF1, Plant Cell 20 (2008) 2447–2459. [65] E. Sliwinska, G.W. Bassel, J.D. Bewley, Germination of Arabidopsis thaliana seeds is not completed as a result of elongation of the radicle but of the adjacent transition zone and lower hypocotyl, J. Exp. Bot. 60 (2009) 3587– 3594. [66] C. Gimeno-Gilles, E. Lelievre, L. Viau, M. Malik-Ghulam, C. Ricoult, A. Niebel, N. Leduc, A.M. Limami, ABA-mediated inhibition of germination is related to the inhibition of genes encoding cell-wall biosynthetic and architecture: Modifying enzymes and structural proteins in Medicago truncatula embryo axis, Mol. Plant 2 (2009) 108–119. [67] C. Vander Willigen, O. Postaire, C. Tournaire-Roux, Y. Boursiac, C. Maurel, Expression and inhibition of aquaporins in germinating Arabidopsis seeds, Plant Cell Physiol. 47 (2006) 1241–1250. [68] L. Dolan, J. Davies, Cell expansion in roots, Curr. Opin. Plant Biol. 7 (2004) 33–39. [69] P.-P. Liu, N. Koizuka, T.M. Homrichhausen, J.R. Hewitt, R.C. Martin, H. Nonogaki, Large-scale screening of Arabidopsis enhancer-trap lines for seed germination￾associated genes, Plant J. 41 (2005) 936–944. [70] C.F. Chen, P. Dahal, K.J. Bradford, Two tomato expansin genes show divergent expression and localization in embryos during seed development and germi￾nation, Plant Physiol. 127 (2001) 928–936. [71] S. Penfield, Y. Li, A.D. Gilday, S. Graham, I.A. Graham, Arabidopsis ABA INSEN￾SITIVE4 regulates lipid mobilization in the embryo and reveals repression of seed germination by the endosperm, Plant Cell 18 (2006) 1887–1899. [72] X. Gong, G.W. Bassel, A. Wang, J.S. Greenwood, J.D. Bewley, The emergence of embryos from hard seeds is related to the structure of the cell walls of the micropylar endosperm, and not to endo--mannanase activity, Ann. Bot. 96 (2005) 1165–1173. [73] H.A. Williams, J.D. Bewley, J.S. Greenwood, R. Bourgault, B. Mo, The storage cell walls in the endosperm of Asparagus officinalis L. seeds during development and following germination, Seed Sci. Res. 11 (2001) 305–315. [74] H. Nonogaki, F. Chen, K.J. Bradford, Mechanisms and genes involved in germination sensu stricto, in: K.J. Bradford, H. Nonogaki (Eds.), Seed Devel￾opment, Dormancy and Germination, Blackwell Publishing, Oxford, 2007, pp. 264–304. [75] M.W. Nabors, P. Kugrens, C. Ross, Photodormant lettuce seeds: phytochrome￾induced protein and lipid degradation, Planta 117 (1974) 361–365. [76] F. Chen, H. Nonogaki, K.J. Bradford, A gibberellin-regulated xyloglucan endo￾transglycosylase gene is expressed in the endosperm cap during tomato seed germination, J. Exp. Bot. 53 (2002) 215–223. [77] G. Leubner-Metzger, C. Frundt, R. Vogeli-Lange, F. Meins Jr., I. Class, -1,3- glucanases in the endosperm of tobacco during germination, Plant Physiol. 109 (1995) 751–759. [78] G. Leubner-Metzger, C. Frundt, F. Meins Jr., Effects of gibberellins, darkness and osmotica on endosperm rupture and class I -1,3-glucanase induction in tobacco seed germination, Planta 199 (1996) 282–288. [79] S.P.C. Groot, C.M. Karssen, Gibberellins regulate seed germination in tomato by endosperm weakening: a study with gibberellin-deficient mutants, Planta 171 (1987) 525–531. [80] S.P.C. Groot, B. Kieliszewska-Rokicka, E. Vermeer, C.M. Karssen, Gibberellin￾induced hydrolysis of endosperm cell walls in gibberellin-deficient tomato seeds prior to radicle protrusion, Planta 174 (1988) 500–504. [81] J.D. Bewley, Breaking down the walls – a role for endo--mannanase in release from seed dormancy? Trends in Plant Sci. 2 (1997) 464–469. [82] K. Muller, A. Linkies, R.A.M. Vreeburg, S.C. Fry, A. Krieger-Liszkay, G. Leubner-Metzger, In vivo cell wall loosening by hydroxyl radicals during cress seed germination and elongation growth, Plant Physiol. 150 (2009) 1855–1865. [83] K. Fujino, H. Sekiguchi, Y. Matsuda, K. Sugimoto, K. Ono, M. Yano, Molec￾ular identification of a major quantitative trait locus, qLTG3-1, controlling low-temperature germinability in rice, Proc. Natl. Acad. Sci. U.S.A. 105 (2008) 12623–12628. [84] J.M. Barrero, M.J. Talbot, R.G. White, J.V. Jacobsen, F. Gubler, Anatomical and transcriptomic studies of the coleorhiza reveal the importance of this tissue in regulating dormancy in barley, Plant Physiol. 150 (2009) 1006–1021. [85] H. Nonogaki, M. Nomaguchi, N. Okumoto, Y. Kaneko, H. Matsushima, Y. Moro￾hashi, Temporal and spatial pattern of the biochemical activation of the endosperm during and following imbibition of tomato seeds, Physiol. Plant. 102 (1998) 236–242. [86] K. Fujino, H. Sekiguchi, T. Sato, H. Kiuchi, Y. Nonoue, Y. Takeuchi, T. Ando, S.Y. Lin, M. Yano, Mapping of quantitative trait loci controlling low-temperature germinability in rice (Oryza sativa L.), Theor. Appl. Genet. 108 (2004) 794–799. [87] U. Piskurewicz, Y. Jikumaru, N. Kinoshita, E. Nambara, Y. Kamiya, L. Lopez￾Molina, The gibberellic acid signaling repressor RGL2 inhibits Arabidopsis seed germination by stimulating abscisic acid synthesis and ABI5 activity, Plant Cell 20 (2008) 2729–2745. [88] A.D. Powell, D.W.M. Leung, J.D. Bewley, Long-term storage of dormant Grand Rapids lettuce seeds in the imbibed state: physiological and metabolic changes, Planta 159 (1983) 182–188. [89] A.D. Powell, J. Dulson, J.D. Bewley, Changes in germination and respiratory potential of embryos of dormant Grand Rapids lettuce seeds during long-term imbibed storage, and related changes in the endosperm, Planta 162 (1984) 40–45. [90] M. Black, J.D. Bewley, P. Halmer (Eds.), The Encyclopedia of Seeds. Science, Technology and Uses, CAB International, Wallingford, 2006, pp. 741–746. [91] J.D. Bewley, Seed germination and dormancy, Plant Cell 9 (1997) 1055–1066.