[Advances in Cellular and Molecular Biology of Plants] Cellular and Molecular Biology of Plant Seed Development Volume 4 || Seed Maturation and Control of Germination

15. Seed Maturation and Control of Germination

JOHN J. HARADASection ofPlant Biology, Div ision ofBiological Sciences , University ofCalifornia, OneShields Avenu e, Davis, CA 956/6, USA

ABSTRACT. Seed maturation is a critical period of seed development during which many ofthe unique processes required for seed formation occur. The major events that characterizeseed maturation include the arrest of embryo morphogenesis, the synthesis and accumulationof storage reserves , the acquisition of desiccation tolerance, the desiccation of seed and theaccompanying induction of metabolic quiescence, and, in some species, the establishment ofembryo dormancy. Although this period of seed development has been studied extensively atseveral levels, a foundation for understanding the processes that control seed maturation is justbeginning to emerge . Information derived from studies of the regulation of seed protein genes,of the effects of the phytohormone abscisic acid on cultured embryos, of mutants defective inabscisic acid accumulation and perception, and of mutations that affect other aspects of seeddevelopment do not implicate a single master regulator of seed maturation but, rather, provideevidence that multiple regulatory pathways are each involved in controlling specific facets ofseed maturation. Factors that have been identified as regulators of seed maturation includeabscisic acid, the VIVIPAROUSI/ABA INSENSITIVE3 class of transcriptional regulatoryproteins , and the products of the Arabidopsis LEAFY COTYLEDON genes. Coordination ofthe activities of these and, perhaps, other regulators ultimately underlies the processes thatoccur during seed maturation .

Abbreviations: ABA, abscisic acid; aba ,ABA accumulation; abi , aha insensitive;emb, embryodefective;fus,fusca; lee , leafy cotyledon; vp, viviparous.

1. Introduction

Seed are complex structures that are bounded by a maternally-derived coatand that contain, generally, a mature and fully formed embryo and extra-embryonic tissues that are desiccated, arrested developmentally, and in aquiescent state . Simplistically, seed development can be divided into twoconceptually distinct phases. One is a period of morphogenesis during whichthe embryo's body plan is established and the embryonic organs and tissuesare formed (reviewed by Goldberg et aI., 1994;Juergens, 1994;Meinke, 1995;West and Harada, 1993). The other phase, which is the focus of this review, isconcerned with the maturation of seed. In this chapter, a broad interpretationof the seed maturation phase will be used that includes essentially all of theevents that occur after cell division has ceased within the embryo (Bewley

B.A. Larkins and IX. Vasil ieds .), Cellular and Molecular Biology of Plow Seed Development. 545-5 92.@ 1997 Kluwer Academic Publishers.

546 John 1. Harada

and Black, 1995; Galau et aI., 1991; Kermode, 1990; Koornneef and Karssen,1994; Walbot, 1978). By this definition, seed maturation temporally occupiesroughly one-half to two-thirds of seed development and includes the arrest oftissue and organ formation , the accumulation of nutrient reserves, the suppres-sion of precocious germination, the acquisition of desiccation tolerance, thedesiccation of seed , and, in some species, the induction of dormancy (Koorn-neef and Karssen, 1994). In mature seed without substantial endosperm tissue,seed maturation is also designated as late embryogenesis (Galau et aI., 1991).Germination and postgerminative growth represent the phase during whichmetabolic and morphogenetic reactivation of the quiescent seed occurs (Hara-da et aI., 1988). Although there are fundamental differences in the processesthat occur during seed maturation and during germination/postgerminativegrowth, there is an intimate relationship between the two phases. The mor-phological and physiological processes that occur during both of these phaseshave been studied and described extensively, however, a great deal remains tobe learned of the mechanisms controlling seed maturation and the transitionto germination and postgerminative growth.As a prelude to considering questions about the control of seed maturation,

it is instructive to consider the origin of this phase. The ability to make seedis not a universal characteristic of plants but , rather, it is limited to seed plantsof the divisions Cycadophyta, Coniferophyta, Ginkophyta, Gnetophyta , andMagnoliophyta (Gifford and Foster, 1989). Non-vascular plant s and seedle ssvascular plants do not make seed, and , thus, they do not undergo seed matu-ration (Steeves and Sussex, 1989). Thus, the ability to make seed, known asthe seed habit, is one of the defining characteristics of higher plants.Several aspects of this unique reproductive strategy have contributed to the

success of seed plants, particularly the angiosperms (Crouch, 1987; Steeves,1983). For example , the megagametophyte is enclosed within maternal tissueaffording some degree of protection to the female gametophytic generation.Fertil ization occurs within the megagametophyte and within maternal tissuesenabling reproduction to occur efficiently on land in the absence of an aqueousenvironment. Endosperm formation obviates the need for a large femalegametophyte to support the developing embryos nutritionally. Furthermore,embryo development occurs within the protective confines of the maternalplant, providing a consistent environment for embryogenesis and increasingthe chances for the embryos' survival.The seed represents an elegantly designed and an efficient storage and

dispersal unit for several reasons. First, the seed coat provides mechanicalprotection to the embryo and can serve as a permeability barrier for waterand gases. Second, macromolecular reserves stored within the seed such asstorage proteins , lipids, and carbohydrates, provide the seedling with a nutri-ent source that is used until it can grow autotrophically. Third, desiccation-induced metabolic quiescence enables the embryos to survive periods ofadverse environmental conditions and provides a mean s by which embryos

Seed Maturation and Control of Germination 547

can remain viable for extended periods with minimal aging damage. Fourth,many seed are dormant, and, therefore, they are constrained from germinatinguntil specific environmental conditions are present that are conducive for theresumption of plant growth and development.It has been argued that seed maturation leading to developmental arrest is

an imposed condition that is not required for the formation of a viable embryo(Walbot, 1978 ). A ration ale for this proposal is that most lower plants do notundergo developmental arrest and metabolic quiescence and, therefore, thereis no defined end to embryogenes is nor a distinct beginning to postembryonicdevelopment in these plants (Steeves and Sussex, 1989). The implication isthat seed plants have incorporated processes related to seed maturation , dor-mancy, and germination into the continuous mode of morphogenetic develop-ment characteri stic of many lower plants. In support of this viewpoint, thereare many naturally occurring and mutan t higher plant s that are defective inmany aspects of seed maturation yet they can give rise to apparently normalvegetative plants (see Section 4). A corollary of this hypothesis is that seedmaturation comprises a set of processes that evolved uniquely in the seedplants and, therefore, that the regulatory programs controlling this phase arelikely to be novel.Seed maturation and germination/postgermination have been studied at

the morphological, physiological, genetic, and molecular levels, and infor-mation of the regul atory mechanisms controlling these phases is beginningto emerge (reviewed by Bewley and Black, 1995; Crouch, 1987; Galau et aI.,1991 ; Goldberg et aI., 1989; Goldberg et aI., 1994; Harada et aI., 1988; Hil-horst, 1995 ; Karssen et aI., 1983; Kermode, 1995; Kermode, 1990; Koomneefand Karssen , 1994; McCarty, 1995; Quatrano , 1986; Thomas, 1993; Walbot ,1978). Thi s chapter focuses primarily on the regulation of seed maturationand, as a consequence, on the control of germin ation at a mechanistic level.It is not intended , however, to be an encyclopedic review of seed maturationand germination because it is impossible to provide an exhaustive discussionof all top ics related to these phases. Rather, the following questions serve asthe framework for this chapter.What initiates the pha se transition from a period of embryo morphogenesis

to seed maturation?How is the phase of seed maturation maintained?What mechanisms underlie the inhibition of germination during seed devel-

opment ?Thi s chapter's majo r message is that there does not appear to be a single

master regulator of either seed maturation and/or germination. Rather, a vari-ety of factors appears to control distinct aspects of these phases. Althoughthe growth regulator, abscisic ac id (ABA), is implicated to play significantregul atory roles, it is clearly not the only factor involved in regulating seedmaturation and germination.

548 John J . Harada

2. General Description of Seed Maturation

Before discussing studies of the control of seed maturation, I will first brieflyreviewgeneral information about seed structure and development. Other chap-ters in this volume are devoted to specific aspects of the structure, biochem-istry, and development of seed. Readers are advised to consult these chapters,as needed, to supplement the information presented in this review.Angiosperm seed are derived from ovules as a consequence of double

fertilization events although examples of non-sexual seed production exist(Koltunow, 1993). Typically, at least four structures are present in seed atsome point during their development (reviewed by Bewley and Black, 1995;Boesewinkel and Bouman, 1984). 1) The mature embryo, which arises fromthe fertilization of the egg cell by a sperm, consists of two embryonic organsystems: the axis, which will form the body of the mature plant, and thecotyledon(s) or the scutellum, which often functions as a storage organ formacromolecular reserves that are utilized by the growing seedling. Early inembryogenesis, the embryo proper is attached to the suspensor, an ephemeralstructure that is thought to act as a conduit for the transport of nutrients andgrowth regulators from maternal tissues (Meinke and Yeung, 1993).2) Theendosperm is formed from a distinct fertilization event in which the two polarnuclei of the embryo sac's central cell fuse with a sperm nucleus . This extra-embryonic tissue serves, primarily, a nutritional role for either developingembryos or postembryonic seedlings, and, therefore, this structure has eithera transient or persistent existence in the seed. Endospermic seed, such as thoseof cereals, the Solanaceae , and castor bean, contain considerable endospermtissue in the mature seed. Alternatively, plants with only a few layers ofendosperm tissue, such as oilseed rape, lettuce, and Arabidopsis, or withessentially no viable endosperm, such as soybean and peanuts, are designatedas non-endospermic (Bewley and Black, 1995). The cotyledons are usually theprimary storage organ in non-endospermic plants. 3) The perisperm derivesfrom the nucellar tissue of the ovule and, therefore, is maternal in genotype.The perisperm is usually a transient structure but it serves as a storage tissue ina few species. 4) The seed coat or testa is maternal in origin as it differentiatesfrom the integument(s) of the ovule. Virtually all viable seed contain a seedcoat although there are a few examples of coatless seed (Boesewinkel andBouman, 1984).As indicated in Figure 1, the period of seed maturation, as defined in

this chapter, begins following the termination of cell division in the embryoroughly one-third to one-half through seed development. Significant changesin embryo size and in fresh and dry weights are not observed until after theonset of seed maturation when the accumulation of storage reserves begins.The principal macromolecular storage reserves in a mature seed are pro-teins, lipids, and carbohydrates although other minor constituents, such asphytin, are often present (reviewed by Bewley, 1995). The most abundant

Seed Maturation and Control ofGermination 549

MORPHOGENESIS EMBRYO DESICCATIONMATURATION GERMINATION

,,//"II ,

/ '--- ',,III

60 0 1 2

DAYS POSTIMBIBITION

-.

50

,,,20 30 40

DAYS POSTANTHESIS

- - - - ,,/\,\ /"

\ #' \~r:/II

10o

1 6

~ Oio Eoil I- 4~J:

I ~~ ~ 2

~a::u,

Fig. 1. Physiological Parameters Divide Seed Development into Distinct Stages. Changes infresh and dry weights and in fraction water content of developing embryos and postgerminativeseedlings are shown. Bars indicate stages of seed development and germination. Data are foroilseed rape embryos and seedlings and are taken from Comai and Harada, 1990.

seed proteins, designated storage proteins, are generally packaged into pro-tein bodies which are modified vacuoles or extensions of the endoplasmicreticulum. Lipids, primarily in the form of triacylglycerols, are sequesteredinto oil bodies that contain an abundant oil body membrane protein, oleosin.A variety of storage carbohydrates, ranging from starch, which accumulatesin amyloplasts, to the hemicelluloses mannans and xyloglucans, which accu-mulate in cell walls, are also synthesized and stored in seed. These reservesare synthesized during the early to middle period of seed maturation, andthey accumulate primarily in either the endosperm or the embryonic cotyle-dons although some accumulation occurs in the embryonic axis (Lopes andLarkins, 1993; Mansfield and Briarty, 1992; Olsen et al., 1992; Tykarska,1982; Tykarska, 1987 a, b). Late in seed development, the water content of theseed drops dramatically as desiccation occurs. This decrease in water contentpresumably results from the severing of the vascular connection between theseed and the fruit and evaporative drying. Immediately preceding this peri-od of drying, a group of moderately abundant Late embryogenesis-abundant(Lea) proteins accumulate which have been hypothesized to playa role inconferring desiccation tolerance to the seed (Galau et al., 1986; Roberts etal., 1993). As shown in Figure 1, these physiological changes define threestages of seed development: the cotyledon stage, a particularly intense periodof cell division during which the cotyledons are fonned in dicot embryos, theembryo maturation stage during which embryo weight increases dramatically,and the desiccation stage to indicate the striking decrease in water content.As will be discussed, other parameters of seed development, such as geneexpression patterns, correlate with the existence of these stages.

550 John 1. Harada

3. Seed Maturation Is Uncoupled from Morphogenesis and Partitionedinto Distinct Programs

The events that occur during seed development are exceedingly diverse, rang-ing from embryo morphogenesis to desiccation tolerance. A key to under-standing the processes involved in making a seed is to define the regulatoryprograms that constitute seed development and to understand how these dis-tinct programs are coordinated. One view of higher plant development is thatthe imposition of seed maturation into embryogenesis is a relatively recentevolutionary event. In this section, the question of how seed maturation hasbeen integrated into the higher plant life cycle is discussed.

3.1. Distinct programs regulate different aspects of seed maturation

The strategy of studying temporal and spatial patterns of gene expressionto monitor transitions in developmental programs has been used extensive-ly to study seed development, particularly in relationship to seed matura-tion (Crouch, 1987; Dure, 1985; Goldberg et aI., 1989; Morton et al., 1995;Thomas, 1993). A global picture of the mRNA populations present in seedhas been obtained with RNA/DNA solution hybridization experiments (Galauand Dure , 1981;Goldberg et al., 1981 ; Pernollet, 1984). These studies showedthat similar numbers of genes, ranging from 12,000 to 18,000, are expressed inembryonic cotyledons, in embryonic axes , and in endosperm. A comparablenumber of diverse mRNAs are also pre sent in cotyledon stage embryos and inmature embryos (Goldberg et aI., 1981) . Furthermore, studies measuring over-laps in mRNA populations showed that at least 85% of the mRNAs present inmaturation stage embryos are also present in cotyledon stage embryos and inmature embryos (Goldberg et aI., 1981). Together, these results indicate thatthe genetic complexity of seed at these different stages does not differ greatlyfrom one another, suggesting that relatively few genes underlie the distinctprocesses that occur during seed development.Analyses of the changes in gene expression patterns that occur during seed

development have contributed clues about the regulatory programs governingthis period. Two studies in particular have been informative in providing anoverview of these programs. In one study, both endogenous proteins andin vitro translation products of mRNA from developing cotton cotyledonswere analyzed at sequential stages of seed development and postgerminativegrowth. As summarized in Figure 2, seven distinct accumulation patternswere identified in this study. In addition to a set that was present at all stagesstudied , different classes of gene products accumulated at specific periodsof seed development and postgerminative growth (Chlan and Dure, 1983;Dure, 1985; Dure et aI., 1981). These results suggest the existence of discreteregulatory programs during seed development.

MORPHOGENESIS


EMBRYO DESICCATIONMATURAnON GERMINATION

CONSTITUTIVE

EMBRYO-SPECIFIC ,/

EARLYEMBRYOGENESIS

IGERMINATION-5PECIFIC

>-oc0 roo0<5 Iotns:

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0I

10I20

, /

\I '-II

/I

/30 40

\\

I

50 60 0

,

/

1 2

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~ ~z 6--l Z

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DAYS POSTANTH ESIS DAYS POSTIMBIBITION

Fig . 2. Gene Expression Patterns Divide Seed Development into Specific Stages . A conceptualrepresentation of the accumulation pattern of seven mRNA sets that are present during seeddevelopment and postgerm inative growth. Hatched bars indicate the period during which theindicated mRNA set is detected. For points of reference, mRNA accumulation patterns areoverlayed on Figure I. Adapted from Dure, 1985 and Goldberg et al., 1989.

The observation that distinct sets of genes are expressed at different stagesof seed development was confirmed and extended in a more detailed analysisof gene expression in developing cotton cotyledons that categorized the accu-mulation of forty-seven moderately abundant and abundant mRNAs (Hughesand Galau, 1989). As shown in Figure 3, these mRNAs could be categorizedinto five different groups based upon their accumulation patterns during seeddevelopment and postgerminative growth. Most mRNAs could be placed ina single group although some mRNAs displayed accumulation kinetics thatwere characteristic of two or more mRNA sets. The functions of the geneswithin each set, where known, corresponded well with the physiologicalprocesses that characterize a given period of seed maturation. For example,all storage protein mRNAs displayed similar patterns of accumulation duringthe middle period of seed development, Lea mRNAs accumulated late in seeddevelopment after the presumed abscission of the seed 's vascular connectionto the ovary wall , a subset of Lea mRNAs that responds to ABA treatmentduring embryogenesis defined a separate ABA program, and mRNAs encod-ing enzymes involved in storage reserve mobilization during postgerminativegrowth, including isocitrate lyase and malate synthase, accumulated primar-ily after germination (Galau et aI., 1986; Hughes and Galau, 1989, 1991;

552 John J.Harada

CotyledonPostabsclsslon

I Germination

Embryo Maturation Desiccation

PA

lGrm

I'.',,,,II,

I,_...J.- __,•II,

•I•I•I..----I

Mat ..----,/ I

• 1I I

· II

Cot

10I

20 30 40

DPA

Fig . 3. Modular Organization of Seed Development. Analyses of the levels of forty-sevenmRNAs during cotton seed development and postgerminative growth implicate the existenceof five regulatory programs : ABA, cotyledon (Cot), maturation (Mat) , postabscission (PA), andgermination (Grm).These temporal programs indicate the modular nature of seed development,and they define the developmental stages depicted by the bars . Adapted from Hughes and Galau,1989. DPA, days postanthesis; DPT, days postimbibition.

Jakobsen et aI., 1994). Based upon these results, five temporal stages of seeddevelopment have been designated: cotyledon, embryo maturation, I postab-scission, desiccation, and germination. These results indicate the temporallymodular organization of seed development.

I This component of seed development was designated as the maturation program. To avoidconfusion with seed maturation, the term embryo maturation will be used in this review.


3.2. Embryo morphogenesis and seed maturation are regulatedindependently

The early events of seed development are involved largely with establishingthe plant's body plan and with forming the embryo's tissue and organ systems(reviewed by Goldberg et al., 1994; Juergens, 1994; Meinke, 1995; West andHarada, 1993) . Histogenesis and organogenesis are repetitive processes thatare initiated during embryogenesis and that largely continue throughout thelife cycle. An exception in higher plants is that morphogenesis is arrested dur-ing seed maturation. Thus, there are fundamental differences in the events thatcharacterize the morphogenetic and seed maturation phases of development.Studies described in the previous section showed that distinct sets of genes

are expressed during the phases of morphogenesis and seed maturation, indi-cating that different regulatory programs operate during these periods (Chlanand Dure, 1983; Dure, 1985; Dure et al., 1981; Hughes and Galau , 1989).mRNAs that accumulated during the cotyledon stage also accumulated duringthe postgerminative stage, consistent with the expression pattern expected ofgenes involved in morphogenesis. Genes known to function in morphogenet-ic processes such as Knotted} and SHOOTMERISTEMLESS, which are bothinvolved in shoot meristem function, are expressed both early in embryogen-esis and postembryonically (Long et aI., 1996; Smith et aI., 1995). Similarly,a gene whose expression correlates with cortex differentiation in roots, isexpressed both early in seed development and during postembryonic devel-opment (Dietrich et al., 1989; Dietrich et al., 1992).Given differences in the processes that characterize the morphogenetic and

the seed maturation phases and given the assumption that seed maturation isa more recent adaptation of higher plants, it is not surprising that these phasesare regulated by distinct regulatory programs. An unexpected finding was thatthe initiation of seed maturation can occur independently of morphogenesis.That is, it is not necessary that the morphogenetic phase be completed beforeat least some aspects of seed maturation begin. This conclusion derives fromstudies of embryo defective (emb) mutants of Arabidopsis. emb mutationsidentify a large number of genes that are essential for embryonic development,and they result in diverse groups of mutant phenotypes (Meinke, 1986, 1995;Yadegari et al., 1994). A set of emb mutants has been characterized thatarrest in their development with the morphological phenotypes of early stageembryos such as those at the preglobular, globular, transition, and heart stagesof embryogenesis. The terminal phenotypes of these embmutants are similarto those of wild type embryos at similar morphological stages. However, themutants differ in that they often have enlarged suspensors atypical of wildtype, and they have convex protuberances on the embryo's surface. Detailedanalyses of some of these mutants revealed that although they arrest with theoverall morphology of an embryo at an early stage of seed development, theyexpress markers of seed maturation (Devic et al., 1996; Schwartz et aI., 1994;

554 John J. Harada

Yadegari et aI., 1994; T.A. Lotan, M.A.L. West, and J.J . Harada, unpublishedresults). For example, mutant embryos appear to possess each of the threeembryonic tissue systems, they express genes that are normally active duringseed maturation such as those encoding storage proteins, lipid body protein s,and Lea proteins, and the convex protuberances on their surfaces are similarto those seen on epidermal cell s of wild type embryos at late embryonicstages. These traits are characteristic of many but not all emb mutants. Timecourse experiments demonstrated that storage protein genes are activatedconcurrently in mutant and wild type embryos (T.A. Lotan, M.A.L. West,and J.J. Harada, unpubli shed results). Thu s, initiation of seed matura tionremain s correctly regulated in these mutants sugges ting that morphogenesisis not a prerequisite for the onset of seed maturation. The mechanisms thatmediate the temporal control of seed maturation in the absenc e of cell divisionand embryo morphogenesis are unknown. The uncoupling of these phases isconsistent with the view that seed maturation evolved independently of themorphogenetic processes of embryogenesis. A major que stion is, what arethe signals that control the timing of seed maturation?In summary, the discrete stages of seed development defined on the basis of

morphological and physiological criteria correlate well with stages designatedon the basis of gene expression patterns. Thi s correspondence suggests thatdifferent aspects of seed development are controlled by distinct regulatoryprograms. Although the completion of morphogenesis is not a requirementfor the initiation of seed maturation , it is clear that these program s must beintegrated to permit the production of a viable yet developmentally arre stedseed. A major unresolved problem is to define the mechanisms that permitthese diverse programs to be coordinated.

4. Regulation of Seed Maturation - Initiation and Maintenance

A longstanding prob lem that has been the focus of intense efforts is under-standing the processes that initiate and maintain seed maturation . The transi-tion from embryo morphogenesis to seed maturation repre sent s a major shiftin developmental phase during a plant 's life cycle . Little is known of thesignal s that initiate and maintain this shift to seed maturation . Neither a spec-ified number of cell divisions nor the completion of embryo morphogenesisappears to be the time-keeping mechanism that activates seed maturation . Inthis section, information about the initiation and maintenance of seed matu-ration is reviewed.

4.1. Analyses of seed protein gene regulation suggest that seve ral pathwaysare involved in controlling seed maturation

Seed proteins which include storage proteins, Lea proteins , and other proteinswith potential roles in plant defense such as protease inhibitors and lectins,


accumulate to high levels in developing seeds (Chrispeels and Raikhel, 1991;Galau et aI., 1986; Shotwell and Larkins, 1989). Collectively, these proteinshave long been the subject of intense research in plant biology because oftheir agricultural importance and because the abundance of these proteins andtheir mRNAs facilitated early studies of plant gene structure, organization,and expression. Although seed protein gene expression is often influencedat the posttranscriptional, translational, and/or posttranslational level, tran-scriptional control processes generally play a major role in regulating theexpression of these genes. Thus, a frequently used strategy to understand thecontrol mechanisms that operate during seed maturation has been to definethe cis-acting DNA regulatory sequences that are involved in controlling seedprotein gene activity and to identify the DNA binding proteins that interactwith these sequences to activate the genes. The rationale of this approachis that the DNA binding proteins may represent transcription factors thatcontrol the expression of batteries of genes. Understanding the regulation oftranscription factor activity provides insight into the pathways that underliespecific aspects of seed maturation. In this section, work will be discussedshowing that seed protein genes are regulated combinatorially, i.e., a numberof different DNA elements and their associated factors act in combination tocontrol the transcriptional activity of a gene, and that different seed proteingenes appear to be regulated by distinct sets of factors. Overall , the resultsindicate that multiple factors are involved in controlling seed protein geneexpression.Specific DNA sequences in the 5' flanking regions of a number of seed

protein genes have been identified that appear to be involved in activatingtranscription specifically in seed (reviewed byGoldberg et al., 1989;Morton etaI., 1995; Thomas, 1993; Wobus et aI., 1995). In many cases, DNA sequencessufficient for seed-specific expression are located in the region immediatelyupstream of the transcription initiation site, designated the proximal promoterregion . This observation has led to the proposal that promoters of seed proteingenes have a bipartite arrangement of cis-acting regulatory sequences inwhichthe proximal promoter region determines the seed and/or tissue specificity oftranscription, and other sequences located farther upstream in the 5' flankingregions contain elements that modulate seed-specific transcription (Thomas,1993; Thomas et aI., 1991). It has not been established whether this bipartiteorganization reflects a fundamental characteristic of promoter organization, acommon origin of these seed protein promoters, or coincidence.Table 1 lists seed protein genes with proximal promoter regions that are

sufficient to activate transcription in developing seed (Bustos et aI., 1991;Chamberland et aI., 1992; Croissant-Sych and Okita, 1996; de Pater et aI.,1993; Goldberg et al., 1989; Nunberg et aI., 1994, 1995; Voelker et aI.,1987). These regions often contain sequence motifs that have been shown orimplicated to play roles in transcriptional activation. In most cases, however,it is not known whether these motifs correspond to the regulatory elements

556 John J. Harada

TABLE I

Proximal Promoter Regions of Seed Protein Genesa

Seed Protein Gene

Carrot Lea gene DcG3

French bean lectin gened/ec2

French bean phaseolin gene

Rice glutelin gene Gt3Soybean lectin gene LeISoybean glycinin gene GyI

Soybean ,B-conglycinina t-subunit gene CgyI

Sunflower helianthin in genesHaG 3-A & HaG 3-D

Proximal PromoterRegion '

-117 to +26

- 125 to +1

- 295 to +20

- 181 to +7-77 to -I-66 to -I- 140 to +13

- 116 to +24

Reference

Thomas, 1993Voelker et al., 1987

Bustos et al., 1991Croissanl-Sych and Okita, 1996

Goldberg et al., 1989Goldberg et aI., 1989Lessard et al., 1993

Nunberg et al., 1994

I Distance relative to the transcription initiation site.

that are responsible for seed-specific expression (Thomas, 1993; Thomas etal., 1991). Although all of these proximal promoter regions specify seed-specific expression, each of these region s appears to be distinct. For example,the proximal promoter region of the gene encoding the a' subunit of thej3-conglycinin storage protein of soybean contains the following potentialregulatory elements: a CCAAAT box, a RY or Sph motif, and, pos sibly,a vicilin box (Chamberland et aI., 1992; Chen et aI., 1986, 1988; Lessardet aI., 1993). The RY[Sph motif is the core of the legumin box sequencethat is found upstream of 12S legume seed globulins, and the vicilin box ispresent in the upstream regions of many 7S vicilin genes (Baumlein et aI.,1986; Gatehouse et aI., 1986). By contrast, DNA sequences responsible forseed-specific expression of the Gyl gene encoding another soybean storageprotein, glycinin, does not contain any of these potenti al elements nor anyothermotifs implicated to playa role in seed protein gene regulation (Goldberget aI., 1989). Similarly, a WS motif which is similar to the con sensus bindingsite for steroid hormone receptors and a CCAAAT box are present in theproximal promoter region of two sunflower helianthinin gene s whereas acarrot Lea gene, Dc3, does not possess these elements but , rather, contains Ebox motifs which bind basic helix-loop-helix transcription factors in animal s(Nunberg et al., 1994, 1995). Although the precise elements responsiblefor seed-specific expression remain to be identified, these result s suggestthat many distinct DNA sequence elements are involved in activating genetranscription specificall y in seeds.


In addition to DNA elements located in proximal promoter regions, otherDNA sequences located further upstream of seed protein genes act to mod-ulate transcriptional activity spatially, temporally, and quantitatively and inresponse to ABA. There are several examples of DNA sequences that modifythe spatial expression of seed protein genes. For example, regions of the 5'flanking regions of both high molecular weight and low molecular weightwheat glutenin storage protein genes and of a rice glutelin storage proteingene have been identified that are sufficient for endosperm-specific expres-sion in transgenic tobacco (Colot et aI., 1987; Croissant-Sych and Okita,1996; Halford et aI., 1989).A number of studies have elegantly demonstrated that distinct and specific

DNA sequences in the 5' flanking regions of seed protein genes activatetheir transcriptional activity in different parts of a developing embryo (Bustoset aI., 1991; da Silva Conceicao and Krebbers, 1994; Goldberg et aI., 1994;Nunberg et aI., 1995; Perez-Grau and Goldberg, 1989). For example , separateDNA sequences have been shown to direct gene activity in cotyledons, inhypocotyls, and in radic1es of transgenic tobacco and Arabidopsis althoughthe specific elements responsible for activating transcription have not beenidentified. These results suggest that the apparent constitutive expression ofa seed protein gene throughout the embryo results from discrete regulatoryevents that activate the gene in at least three different embryonic domains.As discussed by Goldberg et aI. (1994), these three transcriptional regionscorrespond roughly with three embryonic domains, apical, central, and basal,that are established early in embryogenesis and that have been implicated tofunction in the regional specification of embryos (Mayer et aI., 1991).DNA sequences that quantitatively modulate the transcriptional activity of

a number of seed protein genes have been identified (Baumlein et aI., 1992;Bustos et aI., 1989, 1991; da Silva Conceicao and Krebbers, 1994; Goldberget aI., 1989; Jordano et aI., 1989; Marcotte et aI., 1989; Nunberg et aI., 1994;Voelker et aI., 1987). In some cases, Arr rich DNA sequences have beenshown to be involved in modulating transcription quantitatively (Baumleinet aI., 1992; Bustos et aI., 1989; Goldberg et aI., 1989; Jordano et aI., 1989;Marcotte et aI., 1989). Transcriptional repression also appears to contributeto the seed-specific expression of some of these genes. Deletion of specificDNA sequences from the 5' flanking regions of a number of seed proteingenes results in their inappropriate activation in mature plant organs (Burowet aI., 1992; Bustos et aI., 1991; Croissant-Sych and Okita, 1996; Lessardet aI., 1993). A novel repressor from French bean that affects seed proteingene activity named regulator ofMAT2 (ROM2) has recently been described(Chern et aI., 1996). This repressor binds to the upstream region of phy-tohemagglutinin genes and ,B-phaseolin genes and represses transcriptionalactivation of the former promoter late in seed development. ROM2 has beenproposed to participate in the temporal regulation of phytohemagglutinin and,perhaps, other seed protein genes late in embryogenesis .

558 John J. Harada

As will be discussed, ABA is a key factor involved in controlling seeddevelopment, and, therefore, the role of ABA in regulating gene activity hasreceived considerable attention (Bray and Beachy, 1985; Finkelstein et aI.,1985; Giraudat et aI., 1994; Hetherington and Quatrano, 1991; Quatrano etaI., 1993; Rivin and Grudt, 1991; Skriver and Mundy, 1990; Thomas, 1993;Thomas et aI., 1991). Initial identification of a DNA element mediating ABAresponsiveness of seed proteins came from studies of the wheat Lea gene,Em. The Em gene is expressed in wheat embryos during the late stagesof seed development, in seedlings that are water stressed or treated withABA, and in cultured cells treated with ABA (Marcotte et aI., 1989; Morriset aI., 1990). Dissection of the Em promoter identified the DNA sequence,GGACACGTGGC, as being both necessary and sufficient to mediate ABA-induced transcriptional activation (Guiltinan et aI., 1990; Marcotte et aI.,1989). The core ACGT motif is found upstream of many other seed proteingenes in regions that have been shown or postulated to be involved in ABAregulation although a mutated promoter with a single base change in this coremotif has also been shown to remain responsive to ABA (Goupil et aI., 1992;Lam and Chua, 1991; Mundy et aI., 1990; PIa et aI., 1993; Shen and Ho,1995; Thomas, 1993). Other ABA responsive elements that do not consistsolely of the ACGT core motif have been identified (Hattori et aI., 1992).In one case, two distinct DNA elements including one containing this coremotif is required to confer ABA responsiveness (Shen and Ho, 1995). It isinteresting to note that this ACGT motif is also present in the upstream regionof a number of other genes including those encoding zein storage proteins,other seed proteins, light regulated proteins, alcohol dehydrogenase, andhistones (DeLisle and Fed, 1990; Giuliano et aI., 1988; Schulze-Lefert et aI.,1989;Tabata et aI., 1991). It is possible that a common DNA binding proteinmay bind with this sequence element and that specificity is determined bya transcription factor that interacts with this binding protein. Others haveshown that sequences flanking this motif determine the specificity of theelement (lzawa et aI., 1993).A rationale for defining cis-acting elements controlling seed protein genes

is that this information is needed to identify trans-acting factors that regu-late the genes' transcriptional activities. A number of studies have identifiednuclear proteins that bind with the 5' flanking regions of seed protein genes(reviewed by Goldberg et aI., 1989; Morton et aI., 1995; Thomas, 1993;Wobus et aI., 1995). In some cases, the nuclear proteins were shown tobind with DNA sequences that are either demonstrated or implicated to playroles in transcriptional regulation. For example, proteins that bind specificallywith functionally defined ABA responsive elements have been identified andisolated and shown to possess basic leucine zipper motifs that are presentin transcription factors from organisms ranging from yeast to humans andplants (Guiltinan et aI., 1990; Oeda et aI., 1991). Similarly, several highmobility group-like nuclear proteins have been identified that bind with Arr


rich regions implicated to control transcription quantitatively (Bustos et al.,1989; Jofuku et aI., 1987; Jordano et aI., 1989; Vellanowethand Okita, 1993).The precise role, if any, of these proteins in regulating seed protein genetranscription has not yet been determined.One of the best characterized transcription factors controlling seed protein

gene expression is defined by the opaque-2 mutation of maize. Major conse-quences of the opaque-2 mutation are the partial or nearly total eliminationfrom maize endosperm of the 22 kDa class of zein storage proteins and of theribosome inactivating protein b-32 and the reduction in levels of other seedproteins (reviewed by Mueller et aI., 1995; Schmidt, 1993). The effects ofthe opaque-2 mutation are mediated through transcriptional inactivation of itstarget genes (Kodrzycki et aI., 1989). Isolation and analysis of the wild typeOpaque-2 gene revealed that the predicted protein contains a basic leucinezipper motif that is characteristic of transcription factors, including those thatbind with ABA responsive elements (Hartings et aI., 1989;Motto et aI., 1988;Schmidt et aI., 1987, 1990). The finding that Opaque-2 binds with the DNAsequence TCCACCTAGA in the 5' flanking region of a 22 kDa zein geneand with the upstream region of the b-32 gene suggests that the protein actsas a transcription factor (Lohmer et aI., 1991; Schmidt et aI., 1992). Thesefindings provide several clues about the control of seed maturation . First,the opaque-2 mutation does not affect the expression of all zein genes inmaize sugge sting that Opaque-2 regulates only a subset of these genes. Sec-ond, the Opaque-2 gene is expressed specifically in endosperm at the timeof zein synthesis (Schmidt et aI., 1987). This result suggests that Opaque-2gene expression is itself modulated and that an upstream regulatory processultimately controls the timing and location of zein protein synthesis. Third,Opaque-2 can bind with its target sequence either as a homodimer or as aheterodimer with another basic leucine zipper protein, OHP1, which appearsto be present in virtually all plant organs (Pysh et aI., 1993). Thus, Opaque-2activity may be controlled by its interactions with other proteins.

It is clear that a number of distinct cis-acting DNA sequences participate inthe control of seed protein gene expression. Each seed protein gene appears tobe regulated by several cis-acting regulatory elements and their correspondingtrans-acting factors . These factors act in combination to confer the propertemporal and spatial expression pattern of each gene. This combinatorialmode of regulation emphasizes the complexity of control proces ses that occurduring seed maturation. One interpretation of the multiplicity of elementsand factors involved in controlling these genes is that numerous regulatoryprograms operate in parallel to control seed maturation. This theme willemerge repeatedly in this chapter.

560 John 1. Harada

CotyledonPostabscission

I Gennlnatlon

Embryo Maturation Desiccation

PA

!GnnMat ..----,I I

"j \ I',, I

,j 2 I ABA Level \

, =tl, (1)

j iii", I , eo<

g I , (1)

I 30 I =tlE.s , z»Qj I r> I

(1)Q) <...

1 !P.-clD-c

403020

01.-_--L-_---l__....L-_--l-_.L-....L.---oI

10I

DPA

Fig. 4. ABA Accumulati on Pattern during Seed Development. Changes in ABA levelsduring seed development in cotton are indicated by the thick line. For point s of reference ,the accumulation patterns of embryo maturat ion (Mat), postabsci ssion (PA), and germination(Grm) mRNAs are shown. Adapted from Galau et aI., 1987. DPA, days postanthesis; DPI, dayspostimbibition.

4.2. Does ABA playa role in the initiation and/or maintenance of seedmaturation?

ABA has long been proposed to play key roles in several aspects of seeddevelopment, including the control of seed maturation and the suppressionof germination (reviewed by Black, 1991; Crouch, 1987; Galau et al., 1991;Giraudat et al., 1994; Quatrano, 1986) . The former topic, which is controver-sial, is discussed in detail in this section while the latter will be discussed insection 5.2.


A number of observations have been cited as evidence that ABA regulatesdevelopmental programs during seed maturation. In some species, ABA levelsduring seed development are high at the time of maximal reserve accumula-tion (Ackerson, 1984; Hsu, 1979; King, 1976; Quebedeaux et al., 1976). Asdiagrammed in Figure 4, ABA levels are generally low early in seed devel-opment, become maximal during the early period of seed maturation , anddecline to a low value in mature seeds (Black, 1991). The total ABA contentof the seed represents contributions from both maternal and zygotic sources;the hormone is synthesized in fruits, seed coats, embryos, and endosperm .However, precise comparisons of ABA accumulation kinetics and of seedprotein gene expression do not support the hypothesi s that ABA level aloneregulates seed protein accumulation. For example, as shown in Figure 4, thedecrease in ABA levels during seed maturation does not result in a concomi-tant drop in the levels of storage protein mRNAs , and Lea mRNA levels areinduced at a time when ABA levels have declined from their maximum duringseed development (Finkelstein et aI., 1985; Galau et aI., 1987; Harada et al.,1989). ABA sensitivity does not appear to increase late in seed maturationto compensate for the decline in hormone level. Studies of ABA's role insuppressing precocious germination indicate that many embryos become lesssensitive to ABA during the late stages of seed maturation (Eisenberg andMascarenhas, 1985; Finkelstein and Crouch, 1986). Thus, ABA cannot be thesole factor responsible for storage protein and Lea protein accumulation.Another observation cited in support of a role for ABA in the control of

seed maturation is the hormone's promotion of seed protein gene expressionin cultured embryos. As will be discu ssed, developing embryos removed fromseed and cultured on basal medium without ABA generally germinate pre-cociously (reviewed by Crouch, 1987; Galau et al., 1991; Quatrano, 1986;see section 5.2). Upon culture, embryos from some species switch immedi-ately to the germination program, others appear to complete seed maturationbefore germinating, and some germinate and express genes characteristic ofboth seed maturation and germination (Dure and Galau, 1981; Eisenberg andMascarenhas, 1985; Finkelstein and Crouch, 1984; Finkelstein et aI., 1985;Hughes and Galau, 1991; Jakobsen et aI., 1994; Kermode and Bewley, 1988;Long et aI., 1981; Quatrano, 1986; Stinissen et al., 1984). This variation mayreflect inherent species-specific differences in response to culturing or simplydifferences in the stages at which embryos were excised and/or in the condi-tions in which they were cultured. Embryos cultured on ABA generally donot germinate prematurely but they still undergo a varied set of responses.Immature wheat embryos develop with a normal morphology and accumulateseed proteins including wheat germ agglutinin and the Lea protein, Em, whencultured on ABA medium (Quatrano, 1986). Oilseed rape (Brassica napusL.) embryos cultured with ABA continue to accumulate cruciferin and napinstorage proteins at rates similar to those of embryos grown in planta (Crouchand Sussex, 1981; Finkelstein et al., 1985). Furthermore, the relative tran-

562 John J. Harada

scriptional activities of these storage protein genes and the levels of storageprotein mRNAs are higher in embryos treated with ABA than those culturedon basal media, although ABA treatment does not restore these values to inplanta levels (DeLisle and Crouch , 1989; Finkelstein et aI., 1985). Immaturesoybean embryos cultured on ABA continued to grow, and they appear tomaintain many aspects of their seed maturation program as indicated by thecontinued accumulation of mRNAs for both ,B-conglycinin and glycinin stor-age proteins (Eisenberg and Mascarenhas, 1985). It was also observed thatABA caused an increase in mRNA encoding the ,B subunit of ,B-conglycininbut not those for the ex' or ex subunits (Bray and Beachy, 1985). Thus, ABAcan affect storage protein accumulation differentially.These effects of ABA on seed protein gene expression in cultured embryo s

have also been interpreted to indicate that ABA does not playa major rolein initiating or maintaining seed protein gene expression primarily for tworeasons (reviewed by Galau et aI., 1991; Quatrano, 1986). First, seed proteinsaccumulate at a low level in cultured embryos in the presence or absenceof ABA. Although exogenous ABA enhances the expression of several seedprotein genes, many of these gene products are detected at low levels inembryos cultured without ABA, including the storage proteins and the Leaproteins of wheat, cotton, tobacco , oilseed rape, and soybean (Eisenberg andMascarenhas, 1985; Finkelstein and Crouch, 1984; Finkelstein et aI., 1985;Hughes and Galau, 1991; Jakobsen et aI., 1994; Quatrano, 1986). This basallevel of seed protein synthesis does not appear to result from an increasein endogenous ABA levels in cultured embryos (Ackerson, 1984; Bray andBeachy, 1985; Finkelstein et aI., 1985). Expression of these seed proteingenes in the absence of ABA suggests that the hormone cannot be solelyresponsible for their activation. Second, Galau and his colleagues arguedthat cultured embryos are actually seedlings physiologically and, thus, arenot appropriate models to study the regulation of seed protein genes duringembryogenesis (Galau et aI., 1991; Hughes and Galau, 1991; Jakobsen et aI.,1994). As will be discussed, their analyses of seed protein gene expressionin cultured cotton embryos suggest that excision and culture of the embryoinduces the postabscission and germination programs simultaneously (seesection 5.3). Because young seedlings exposed to water stress are generallytolerant of desiccation and because Lea proteins are postulated to serve asdesiccation protectants , they suggest that the induction of Lea gene expressionin cultured embryos represents a seedling response to an environmental stress.This interpretation is reasonable based upon the data obtained with cotton butit does not explain the induction of seed proteins by ABA in other plantspecies.Studies of mutants defective in ABA synthesis have also provided infor-

mation relevant to this question . Accumulation mutants with reduced lev-els of seed ABA have been identified in several species including maize(viviparous2 (vp2), vp5, vp7, vp8, vp9), Arabidopsis (aba), tomato tfiacca .


notabilitis, sitiens), pea (wilty), and barley (nar2i) (Koomneef et a!., 1982;Neill et a!., 1986; Robertson, 1955; Tal and Nevo, 1973; Walker-Simmons eta!., 1989; Wang et a!., 1984; reviewed by Giraudat et a!., 1994; Zeevaart andCreelman, 1988). Analyses of these mutants suggest that ABA is requiredfor some but not all aspects of seed maturation. For example, most aspectsof seed maturation including the accumulation of the 2S and 12S storageproteins do not appear to be compromised in two ABA-deficientArabidopsismutants that each possess less than 4% of the ABA content of wild type seed(Karssen et a!., 1983; Koornneef et a!., 1989). As will be discussed, however,seed dormancy was affected by the mutation indicating that there are differ-ent thresholds for seed ABA responses (see section 5.5). Maize viviparousmutants which are deficient in ABA display more striking defects in seedmaturation. Mutant embryos accumulate some but not all seed proteins, theygerminate precociously on the plant, and they are intolerant of desiccation(Kriz et a!., 1990; Neill et aI., 1986; PIa et a!., 1991). Furthermore, many of theviviparous mutants fail to accumulate carotenoid pigments although it is notclear whether this phenotype results from the lesion in the ABA biosyntheticpathway (Zeevaart and Creelman, 1988). Thus, either specific facets of seedmaturation have different threshold requirements for ABA or the hormone isrequired for only a subset of these processes. By contrast, mutation s affect-ing ABA perception have profound effects on seed development as will bediscussed in section 4.3 .Evidence that maternal ABA can play important roles in embryo matura-

tion has come from analysis of Arabidopsis ABA synthesis and perceptionmutants.ABA iNSENSiTIVE3 (ABl3) is a gene required for ABA perception inArabidopsis seed (Koornneef et aI., 1984). Embryos homozygous for a weakmutant allele, abi3-1, and for the ABA accumulation mutation, aba-l , willsurvive desiccation if they are produced on maternal plants that are homozy-gous for abi3 -i but heterozygous for aba. However, digenic mutant seed aredesiccation intolerant if they are produced on maternal plants homozygousfor both defective loci (Koomneef et a!., 1989). Because the aba mutation isrecessive, this result implies that maternal ABA is sufficient to induce des-iccation tolerance in double mutant embryos. The role of maternal ABA inconferring desiccation tolerance may differ among species because hormoneprovided by the parental plant does not prevent vivipary of maize viviparousmutants defective in ABA accumulation (Neill et al., 1987).An obvious conclusion of this discus sion is that ABA cannot be the only

factor involved in initiating and/or maintaining seed maturation. Others havehypothesized that ABA does not playa role in controlling seed maturationbut, rather, that it acts solely to inhibit germination during seed developmentin dicots (Galau et a!., 1991; Hughes and Galau, 1991; Jakobsen et a!., 1994).However, studies of ABA's effects on seed maturation can also be interpretedto indicate that ABA is necessary to initiate and/or maintain some aspectsof seed maturation but that other facets can occur either in its absence or at

564 John J. Harada

exceedingly low hormone levels. For example, ABA treatment of culturedembryos induces the accumulation of some but not all seed protein s. Thisresult does not appear to be consistent with the interpretation that the ABAinduction of seed proteins in cultured embryos represents a seedling responsebecause storage protein s are normally synthesized only in embryos. The lackof accumulation of some seed proteins in mutants deficient in ABA alsosuggests that the hormone may mediate some aspects of seed maturation.The manner in which ABA fulfills this postulated role in seed maturation isnot clear. Based on the observation that low osmotic potential can sometimessubstitute for ABA in maintaining seed protein synthesis, the suggestion hasbeen made that ABA's role in inhibiting cell elongation typ ical of germinatingseedlings is the physiological process that maintains seed maturation (Black,1991; Finkelstein et aI., 1985; Hilhorst, 1995). Thi s hypothesis will requirefurther evaluation because the maize viviparous mutants and , as discus sedbelow, the Arabidopsis leafy cotyledon mutants express seed protein genes,and they germinate precociously.

4.3. Genes involved in ABA perception are major regulators of seedmaturation

A hormone's effect on physiological processes can be mediated throughchanges in its concentration or through changes in the plant 's sensitivity tothe hormone. The role of ABA in seed maturation has also been addressedby studying plants with an altered perception of ABA. These studies have notonly confirmed a role for ABA in seed maturation but they have also revealedthat protein s involved in ABA perception are themselve s major regulators ofthis phase.Many plants have been identified that have reduced sensitivity to ABA. For

example, the mangrove,Rhizophora mangle , is normally viviparous. Growthof the prematurely germinated seedlings could not be inhibited by physiolog-ical concentrations of ABA, suggesting that the plant is relati vely insensitiveto the hormone (Sussex, 1975). Of greater significance for experimental pur-poses, mutations resulting in reduced ABA sensitivity have been identifiedin several model plant species. The maize gene involved in ABA perception,Yiviparousl (Vpl), was identified as the only viviparous mutant that is notABA deficient (Neill et aI., 1986; Robertson , 1952). The mutant 's reducedsensitivity to ABA was indicated by embryo growth experiments in cultureand by the fact that the mutation does not affect ABA synthes is, tran sport ,or metabolism (Robichaud and Sussex, 1986, 1987). Similar to viviparousmutants deficient in ABA, kernel s containing severe vp l mutant alleles donot become desiccati on tolerant but they germinate precociously. A uniquecharacteristic of most vpl mutant alleles is that they also cause defects inanthocyanin biosynthesis and aleurone development resulting in colorlessseed (Dooner, 1985; Robertson , 1955). Five Arabidopsis genes controlling


ABA sensitivity have been identified genetically as mutations that permitseeds to germinate in the presence of ABA: ABIl, AB!2 , AB/3, AB/4, andAB!5 (Finkelstein, 1994; Koomneef et al., 1984). Mutant abi embryos havenormal or enhanced ABA levels in seed (Koomneef et al., 1984). Of thesegenes, AB!3 ,4 and 5 appear to function primarily during seed developmentalthough all of the abi mutations affect seed germination. AB!l and AB!2have only a minor effect on seed development; they primarily affect vegeta-tive processes suggesting that they function in parallel processes with AB!3 ,4and 5 (Finkelstein and Somerville, 1990; Koomneef et al., 1984). Addition-ally, a pea mutant whose stomata do not respond to treatment with exogenousABA appears to have a defect in hormone perception (Raskin and Ladyman ,1988).Extensive characterizations ofmaize vpl and the Arabidopsis abi3 mutants

have revealed that the two genes have similar though not identical roles duringseed maturation (reviewed by Giraudat et al., 1994; McCarty, 1995). Pheno-typic and molecular analyses indicate that Vpl and AB!3 function primarily, ifnot exclusively, during seed development (McCarty et al., 1989; Parcy et al.,1994). Severe alleles of both mutations result in embryos that do not becometolerant of desiccation, an indication that there are defects in seed maturation.Both mutations affect the expression of many but not all seed protein genes.For example, the vpl mutation causes defects in the expression of at least twoembryo storage protein genes, Glbl and Glb2, and a Lea gene , Em, but it hasno effect on a different Lea gene, MLG3 (Kriz et al., 1990; Paiva and Kriz,1994; Rivin and Grudt, 1991; Thomann et al., 1992). Similarly, the expres-sion of many storage protein genes, including those encoding cruciferin A,cruciferin C, 2S napin-like protein, and several Lea proteins, are affected bysevere mutant alleles ofAB/3 (Finkelstein, 1993; Nambara et al., 1995; Parcyet al., 1994; Vilardell et al., 1994). However, the expression of other seedprotein genes, such as the cruciferin B gene , is unaffected. Thus, vp! andabi3 mutations affect many but not all aspects of seed maturation, suggestingthat the corresponding genes cannot be the sole regulators of this phase.

vpl and abi3 mutations have heterochronic effects on seed maturation ,i.e., they cause changes in the rate of development. The ability of mutantvpl embryos to germinate in planta indicate s that the wild type gene isrequired to suppress germination. Although abi3 mutants are not viviparous,three phenotypic characteristics indicate that postgerminative developmenthas been initiated prematurely during seed development (Nambara et al.,1995). First, strong abi3 mutations cause premature activation of the shootapex. The shoot apices ofwild type Arabidopsis embryos are normally flat andlack detectable leaf primordia but they are activated in seedlings in that theshoot apical meristems are domed and flanked by leaf primordia (Barton andPoethig, 1993; Medford, 1992). The shoot apices of abi3 mutant embryos aremore similar to those of wild type seedlings than embryos in that the apiciesare domed and possess leaf primordia. Second, vascular differentiation typical

566 John J. Harada

691

VP1

ABI3720

Fig. 5 . Viviparous I and ABA INSENSITIVE3 are Homologous Proteins. Diagramatic rep-resentation of the two proteins. Filled bars indicate regions of amino acid sequence identity,and hatched bars represent acidic regions. Numbers indicate amino acid residues in eachpolypeptide.

of postgerminative development occurs prematurely in abi3 mutant embryos.Third, the promoter of the chlorophyll alb binding protein gene is more activein abi3 mutant embryos than in wild type. Normally, this promoter doesnot become active until after seeds have germinated. Together, these resultsindicate that mutations of the Vp l and AB13 genes cause at least some aspectsof postgerminative growth to occur prematurely.Both the Vpl gene and the ABl3 gene have been isolated (Giraudat et

aI., 1992; McCart y et aI., 1989). Analyses of these genes have indicated thebasis for similarities in the mutant phenotypes and have provided insightinto the mechanisms by which these genes part icipate in regulating seedmaturation (discussed in Giraudat et aI., 1994; McCarty, 1995). Comparisonsof gene structures and of deduced amino acid sequences indicate that Vpland ABl3 are homologous genes that are likely to be orthologous, i.e., theyfulfill the same roles in their respective organisms. Both genes contain sixexons, and introns interrupt each gene at similar locations (Giraudat et aI.,1992; McCarty et aI., 1991). Moreover, these genes share four region s withsignificant sequence identity as shown in Figure 5 (Bobb et aI., 1995; Giraudatet aI., 1992). One is a region relatively rich in serine residu es, two are basicregions, and the other is an extensive region of sequence identity comprising11 8 carboxyl-terminal amino acids .The finding that these regions of sequenceidentity are also present in the predicted polypeptides of the rice and theFrench bean homologs, Osvpl and PvAlf, respectively, suggests that theseconserved regions are important for Vpl /AB13 function (Bobb et aI., 1995;Hattori et aI., 1994).Several lines of evidence indicate that the VPI/ABI3 class of proteins

function as transcriptional regul ators (Giraudat et aI., 1992; McCarty et aI.,1991). First, both proteins possess features characteristic of transcriptionfactors. They contain acidic region s at their amino termini although theseregions do not share significant sequence ident ity. The carboxyl-terminalregions of extensive sequence identity are similar to the DNA binding anddimerization region s in the human transcription factor, CTF/NFI (Mermod et


aI., 1989). However, no evidence has been reported indicating that VPI andABI3 bind DNA directly, leading to the proposal that the proteins serve as co-activators that interact with a DNA binding protein (McCarty, 1995). In thisregard, VPI has been shown to enhance the binding of another transcriptionfactor, EmBP-I, with a VPI target gene (Hill et aI., 1996). Second, VPI/ABI3proteins have been shown experimentally to act as transcriptional activators(Bobb et aI., 1995; Hattori et aI., 1992; McCarty et aI., 1991 ; Parcy et aI.,1994). Vpl expression in maize protoplasts activated the promoter of the Leagene, Em, greater than IOO-fold and resulted in a significant increase in theABA responsiveness of this promoter (McCarty et aI., 1991). Similar resultshave been obtained with the Osvpl gene (Hattori et aI., 1995). PvAlf wasshown to activate the promoters of the storage protein phaseolin gene and thelectin PHA-L gene in particle bombardment experiments with both embryosand leaves (Bobb et aI., 1995). The amino-terminal acidic regions of Vp1and PvAlf have been shown to function as a transcriptional activation domainwhen fused with a DNA binding protein . Third, ABI3 's role in transcriptionalactivation has been demonstrated in transgenic plants. ABA treatment oftransgenic leaves ectopically expressing the gene caused the accumulation ofseed protein mRNAs encoding the storage proteins At2S3 and cruciferin-C,and the Lea AtEm I (Parcy et aI., 1994). Consistent with this result, expressionof these seed protein genes is disrupted in abi3 mutant seed. Together, theseresults sugge st that the VP 1/ABI3 class of proteins serve to control seedmaturation through their roles as transcriptional regulators.Although Vpl and AB13 share many functions, they also exhibit specif-

ic differences. The abi3 mutation compromises dormancy in Arabidopsis(Koornneef et aI., 1984; see section 5.5). Plants with weak abi3 mutant alle-les can produce viable dry seed that, unlike wild type, do not require eithercold or light treatments for seed germination . Because maize kernels aremetabolically quiescent but non-dormant, VPI cannot serve a similar role.Another difference is that VPI has been shown to repress the promoter activityof a-amylase genes in maize aleurone cells (Hoecker et aI., 1995). a-Amylaseis a starch hydrolytic enzyme whose activity is normally induced transcrip-tionally by gibberellic acid during and following germination of cereal seeds.This result suggests that VP 1 acts to repress at least some facets of the ger-mination program during seed maturation. No similar function for ABI3 hasbeen reported. The VP I region that mediates repression, which is distinctfrom the transcriptional activation region, is not conserved with ABI3. vpland abi3 mutations also differ in their effects on anthocyanin biosynthesis .Anthocyanin normally accumulates in the aleurone of the endosperm and inthe scutellum of the embryo during maize seed maturation. At least eightgenes , including three regulatory genes, Cl, Rl, and Vpl, are required foranthocyanin accumulation in seed (reviewed by Dooner et aI., 1992). Vpl hasbeen shown to activate Cl expression, and Cl is thought to interact with Rlto activate genes encoding enzymes in the anthocyanin biosynthetic pathway

568 John 1. Harada

(Hattori et al., 1992). By contrast, the abi3 mutation does not appear to abol-ish anthocyanin synthesis in Arabidopsis but, rather, it appears to cause theinappropriate coloring of embryonic cotyledons (Nambara et al., 1995).While it is clear that Vpl and AB!3 are required for seed ABA responses,

there is also evidence to indicate that the genes are involved in several dis-tinct ABA signaling pathways. The Cl promoter is regulated by ABA, and,therefore, Vpl may regulate Cl through its role in mediating ABA respons-es (Hattori et al., 1992). However, Cl activation by VPl appears to requirelower levels of ABA than those necessary to induce desiccation toleranceand seed protein gene activation. This observation suggests that ABA respon-sive processes have different ABA threshold levels (McCarty et al. , 1989).Evidence for parallel ABA regulatory pathways has also been obtained fromgenetic analyses of Arabidopsis abi mutations (Finkelstein and Somerville,1990).Several features of the Vp 1 and Abi3 mutant phenotypes indicate that

both genes also control ABA independent events. First, defects in seed pro-tein gene expression are more severe in vpl and abi3 mutants than in theABA accumulation mutants (Finkelstein, 1993; Paiva and Kriz , 1994; PIaet al., 1991). Second , strong Arabidopsis aba mutants are desiccation tol-erant while strong abi3 mutant s are not (Koomneef et al., 1982; Nambaraet al. , 1992). Third , molecular analyses demonstrated that VPl acts throughboth ABA respon sive and non-ABA responsive cis-acting DNA regulatorysequences to regulate the Em and Cl gene s (Hattori et al. , 1992; McCartyet al., 1991 ). Furthermore, VP I overexpression in protoplasts in the absenceof ABA strongly activates the Em and C1 promoters. Thu s, VP I and ABl3appear to function in regulatory processes that do not involve ABA.The phenotypes of the vpl and abi3 mutations indicate that these genes

play key roles in controlling seed maturation but that they are not the onlyregulators of this phase of development. Other genes that have been implicatedas regulators of seed maturation have been identified, and they are discussedin the next section.

4.4. LEAFY COTYLEDON genes define a novel class ofseed maturationregulators

The preceding discussion indicated that ABA and gene s involved in ABAperception play significant roles in controlling seed maturation although itis clear that they are not the only regulators. Another class of Arabidopsisgenes has been identified that is essential for seed maturation. Aside fromtheir neces sity for the completion of seed maturation, these LEAFY COTYLE-DON gene s have been shown to function in many diverse processes duringembryogenesis. The LEAFY COTYLEDON genes may playa major role incoordinating many facets of seed development.


TABLE 2

Embryonic Phenotypes of Arabidopsis leafy cotyledon and abi3 Mutants '

wild type lecl lee2 Jus3 abii

Desiccation Tolerance/ Tolerant Intolerant Partially Intolerant Intolerant

IntolerantExpression of Seed

Protein Genes" Normal Abnormal Abnormal Abnormal AbnormalShoot Apex" Not Activated Activated Activated Activated ActivatedActivation of

Postgerminative Normal Premature ND6 ND Premature

Gene Expression"

Trichomes on

Cotyledons7 Absent Present Present Present AbsentABA Sensitivity" Sensitive Sensitive Sensitive Sensitive Insensitive

I Summarized from : Baumlein et aI., 1994; Keithet aI., 1994; K.L. Matsudaira Yee, J. Danao,and J.J. Harada, unpublished results; Meinke, 1992; Meinke et aI., 1994; West et aI., 1994.2 Tolerance indicates the abilit y of seed to withstand desiccation . Only parts of lee2 embryosare desiccation intolerant.3 Abnormal indicates that some seed protein genes that are expressed in wild type embryosare not active in mutant embryos.4 Shoot apices of wild type Arabidops is embryos are not activated in that they are relativelyflat and do not contain leaf primord ia. Activated apicies, such as those found in postembryonicplants, are domed and possess leaf primordia.Genes characteristic of postgerminative growth include those encoding isocitrate lyase,

lipid transfer protein , and light harvesting chlorophyll alb binding protein .6 Not determined.7 Presence of trichomes on the adaxial surface of cotyledons.8 Sensitive seed do not germinate in the presence of ABA.

Three LEAFY COTYLEDON genes have been described: LEAFY COTYLE-DONi (LECi ), LEC2 , and FUSCA3 (FUS3) (Baumlein et aI., 1994; Keithet aI., 1994; Meinke , 1992; Meinke et aI., 1994; West et aI., 1994) . The fus3mutant was originally grouped in the fusca clas s of mutants because it, likeall of the other leafy cotyledonmutants, accumulates anthocyanin during seedmaturation (Ba umlein et aI., 1994; Keith et aI., 1994) . However, the majorityof FUSCA genes are involved primarily in mediating plant responses to envi-ronmental signals, particularly to light (Castle and Meinke , 1994; Misera etaI., 1994). The Fus3 mutant phenotype, as described below, indicates th.1t thegene is of the LEAFY COTYLEDON rather than the FUSCA class. Prelimi-nary reports suggest that another potential leafy cotyledon mutant,fuscaiO,has been identified (Misera et aI., 1994). As summarized in Table 2, threeout standing characteristics of these mutants indicate their roles in diverseembryonic processes.

570 JohnJ. Harada

First, LEAFY COTYLEDON genes are clearl y requ ired for the completionof seed maturation . Severe mutant alleles ofLEC1 and FUS3 cause the embryoto become desiccation intolerant resultin g in embryo lethality (Baumlein etaI., 1994; Keith et aI., 1994; Meinke, 1992; Meinke et aI., 1994; West et aI.,1994). Although lec2 mutant embryos are capabl e of germinating followingseed desiccation , regions of the cotyledons are destroyed following severedrying suggesting that desiccation intolerance may be confined to specificregions of the embryo (Meinke et aI., 1994; J.A. Danao and J.J. Harada,unpubli shed results). Only one lec2 mutation has been described , and it isnot clear if the strength of the mutant allele accounts for the embryo's partialintolerance to desiccation. Another indication that LEAFY COTYLEDONgenes play critical roles in seed maturation is the finding that the mutantsare defective in the expression of specific sets of seed protein genes. Genesencoding the storage protein cruciferin A, the lipid body protein oleosin, andthe Lea protein DC-8 are expressed in leel mutant embryos but the promoterof the gene encoding the 0;' subunit of the 7S storage protein ,B-conglycinin isnot active (West et aI., 1994). Similarly, a functional FUS3 gene is requ ired toactivate the legumin B4 storage protein and the 2S albumin promoters but it isnot requi red for the activities of the seed protein promoters from the USP andthe Lea DC8 genes (Baumlein et aI., 1994). These defects in storage proteingene expression correlated with decreased levels of protein bodies in leel andJus3 mutant embryos (Keith et aI., 1994 ; Meinke, 1992). Simil ar to Vpl andAB13, the LEAFY COTYLEDON genes appear to play esse ntial but not globalroles in controlling seed maturation.Second, phenotypes of the leafy cotyledon mutants also indicate that the

genes are necessary to inhibit germination (Baumlein et aI., 1994; Keith etaI., 1994; Meinke , 1992; Meinke et aI., 1994 ; West et aI., 1994). Embryosfrom each of the mutants display some characteristics of postgerminativeseedlings. Unlike wild type embryos but similar to abis mutant embryos, theshoot apices of leel , lec2, andJus3 mutant emb ryos are activated in that theyall possess leaf primordi a much like that of wild type seedlings (Keith et aI.,1994; Meinke, 1992; Meinke et aI., 1994; West et aI., 1994; M.A.L. West,K.L. Matsudaira Yee, and J.J. Harada, unpublished result s). Consistent withthese morphological indicators, genes normally expressed primarily duringpostgerminative growth are activated prematurely in mutant leel embryos(West et aI., 1994). Thus, leafy cotyledon mutations cause at least someaspec ts of postgerminative development to occur prematurely during seedmaturation and, therefore, they are heterochronic mutation s (Keith et aI.,1994; West et aI., 1994).The third and, perhaps, the most striking example of the pleiotropic nature

of these mutations is that leafy cotyledon mutants possess trichomes on theupper surfaces of their cotyledons (Keith et al., 1994; Meinke, 1992; Meinkeet aI., 1994; West et aI., 1994). Trichome formation is not normally observedon wild type Arabidopsis cotyledons. These epidermal hairs have only been


detected on cotyledons in transgenic plants overexpressing the GLABROUSlgene, in cyp90mutants with defects in brassinosteriod synthesis, and in a new-ly identified mutant with characteristics of leafy cotyledon andfusca mutants(Larkins, 1994; Szekeres, 1996; K. Yamagishi and J.J. Harada, unpublishedresults) .Based on the mutant phenotypes, two alternative interpretations of the

roles of these genes have been proposed. One explanation is that the LEAFYCOTYLEDON genes function primarily during seed development in the spec-ification of cotyledon identity. Because trichomes are a leaf characteristic andbecause these mutations are recessive, this model suggests that the LEAFYCOTYLEDON genes are required to specify cotyledon identity (Keith et al.,1994; Meinke, 1992; Meinke et al., 1994; West et al., 1994). In the absenceof a functional gene , cotyledons attain more leaf-like characteristics. In thisview, a mutation in any of the LEAFY COTYLEDON genes must result inonly a partial loss of cotyledon identity because some seed protein genescontinue to be expressed in these mutants . Seed protein genes are not nor-mally expressed in leaves. In this regard, it has been shown that the anatomyof lecl mutant cotyledons is intermediate between that of a cotyledon anda leaf (West et al., 1994). An alternative to this explanation is that trichomeformation on cotyledons results from the premature induction of postger-minative development in these mutants (Baumlein et al., 1994; Keith et al.,1994; Meinke, 1992; Meinke et al., 1994; West et al., 1994). Studies to bedescribed in detail in section 5.1 demonstrated that the organ identity of leafprimordia is not determined at the time of their inception. Depending uponthe physiological state of the oilseed rape embryo, leaf primordia flankingthe embryonic shoot apical meristem can develop into leaves or they can beconverted into cotyledon-like organs that lack trichomes and that synthesizestorage proteins (Finkelstein and Crouch, 1984). Although this same phenom-enon has not been reported in Arabidopsis, two Arabidopsis mutations , extracotyledonsl and 2, have been described that cause a similar conversion of thefirst leaf pair to cotyledon-like organs (Conway and Poethig, 1993). The fateof cotyledon primordia may be similarly influenced. The premature initiationof postgerminative development may induce cotyledons to be converted intoleaf-like organs. Thus, heterochronic effects of the leafy cotyledon mutationsmay influence the specification of cotyledon identity (West et al., 1994).Unlike Vpl andAB13, the isolation of a LEAFY COTYLEDON gene has not

yet been reported. Thus, the mechanistic role of these genes in controllingseed maturation is unclear. Nevertheless, identification and analysis of thesegenes have provided insight into the complexity of the regulatory circuitriesthat control seed maturation.

572 John J. Harada

4 .5. Mul tiple programs control seed maturation

Genetic analyses implicate three classes of genes as prob able regulatorsof seed maturation: ABA accumulation genes, ABA perception genes, andLEAFY COTYLEDON genes. Analyses of digenic mutant s sugges t that eachof these gene classes operate in parallel pathways (Finkelstein, 1993; Finkel-stein and Somerville, 1990; Keith et aI., 1994; Koomneefet aI., 1989; Meinkeet aI., 1994; West et aI., 1994). For example, Arabidopsis aha ahi3 doublemutants display additive phenotypes as do leeI ah i3 double mutants. A some-what surprising result is that two LEAFY COTYLEDON genes, LEC1 andFUS3 , do not display epistasis. lecl Jus3 digenic mutants exhibit an additivephenotype, suggesting that the genes do not function in series in the samegenetic pathways (West et al., 1994). These results emphasize that multipleand, apparently, independent pathways are involved in controlling seed matu-ration . Consistent with this result is the finding that the Arahidops is lee1,Jus3,aha , and ahi3 mutations and the maize vp mutations each affect the expre s-sion of different seed protein genes . The observation that seed protein genesare regulated by different cis-acting regulatory DNA sequences is consistentwith this model.

5. Control of the Transition to Germination and PostgerminativeGrowth

Developing embryos do not genera lly germinate unt il after seed have des-iccated and entered a period of quiescence and , in some cases, dormancy.There is evidence that germination is actively suppressed during seed matu-ration. As discussed above, isolated immature embryos of many plants will"germinate" precociously when they are removed from the seed and cultured(reviewed by Crouch, 1987; Kermode, 1990, 1995; Quatrano , 1986). Becausethese embryos are not enclosed by a seed, germination here is defined by rootand hypocotyl extension , by cotyledon expansion, and, in some cases, bythe suppression of maturati on-specific genes and by the activation of genesexpressedprimarily during germination (Crouch, 1987). Freshly isolated seedcontaining immature embryos that are cultured usually do not germinate sug-gesting that the seed environment promotes maturation and inhibits germina-tion (Kermode, 1995). This intimate relationship between the suppression ofgermination and the maintenance of seed maturation will be explored below.

5.1. A switch between seed matu ration and germina tio n/postgerminativegrowth?

The events that characterize postgerminative growth differ substantially fromthose that occur during seed development , particularl y in relationship to


the metabolism of storage reserves and the water relations of embryos andseedlings (Bewley and Black, 1995; Harada et aI., 1988). Not surprisingly,differences in gene expression programs reflect changes in these physiolog-ical and morphological processes. In a previous section, evidence indicatingthat different batteries of genes are expressed specifically during seed mat-uration and during postgerminative growth was discussed (see section 3.1).Genes encoding enzymes involved in reserve mobilization such as a -amylase,isocitrate lyase , and malate synthase are expre ssed predominantly or specifi-cally after germination (Comai et aI., 1989; Harada et aI., 1988; Jacobsen andChandler, 1987).The differences in the processes that characterize seed maturation and

postgerminative growth and the finding that each phase is characterized byspecific gene expre ssion programs have led to speculation that the transitionbetween these two phases is controlled by a simple "switch" that terminatesseed maturation and induces germination. By this interpretation, one phasewould occur at the complete exclusion of the other. Several studie s haveprovided evidence con sistent with this idea of a simple switch mediating theshift from seed maturation to postgerminative growth . In one example, thetranscriptional activities of genes expre ssed specifically during seed matura-tion such as those encoding storage proteins and Lea proteins were comparedwith the activities of genes expressed primaril y during and after germin a-tion (Comai and Harada, 1990). Analyses of the transcriptional activitie s ofthese genes in isolated nuclei indic ated that seed maturation-specific geneswere active in nuclei from developing embryos, were present in a transcrip-tionally competent state in dry seed s, and were not transcribed in seedlingsimbibed for 15 hours. By contrast, genes expre ssed primarily in seedlings didnot become transcriptionally active until after imbibition. These results wereinterpreted to indicate that the seed maturation and germination phases donot overlap during normal development and that the switch occurs after seedhave imbibed.The conc ept of this simple switch controlling the transition between seed

maturation and germination is controversial. In particular, a number of obser-vation s have shown that varying degrees of overlap exist in the two programs.For example, several genes expressed at high levels during postgermina-tive growth are initially induced during the late stages of seed development,including isocitrate lyase and malate synthase which both function in themobilization of lipid s during postgerminative growth (Comai et aI., 1989;Harada et aI., 1988). One interpretation of this result is that at least someaspects of postgerminative growth are initiated during seedmaturation. How-ever, recent studies have shown that an isocitrate lyase gene is controlled bydifferent DNA regulatory sequences during late embryogenes is and duringpostgerminative growth, and similar results were obtained for the malate syn-thase gene (Zhang et aI., 1996; D.L. Laudencia-Chingcuanco and J.J. Harada,unpublished results). The finding that these genes are regulated independently

574 John J. Harada

during embryogenesis and during postgerminative growth is consistent withthe viewpoint that the two phases do not overlap .Another finding that suggested apparent overlaps between the two phases

is that precociously germinated immature oilseed rape embryos possess char-acteristics of both embryos and seedlings (Finkelstein and Crouch, 1984).Oilseed rape embryos have leaf primordia at their shoot apices (Fernandezet al., 1991). Mature embryos cultured on basal media germinate and giverise to morphologically and physiologically normal seedlings with leavesdeveloping from these primordia. When immature embryos are cultured, theyexhibit some signs of precocious germination in that radicle extension andcotyledon expansion occur. However, the primordia flanking the shoot apicalmeristem of these cultured embryos give rise to secondary cotyledons thatlack trichomes and synthesize storage proteins and that have the shape ofexpanded cotyledons . The seedlings' ability to undergo root extension andcotyledon expansion but also to generate an organ with characteristics of anembryonic cotyledon has often been interpreted to indicate that seed matu-ration and germination can occur simultaneously and, therefore, that thereis no simple switch between these two phases. More recently, however, ithas been shown that this apparent overlap in programs actually reflects thefact that processes characteristic of seed maturation and of postgerminativegrowth occur in different parts of these seedlings (Bisgrove et al., 1995).For example, maturation-specific genes are active in cotyledons at the sametime that marker genes for postgerminative growth are being expressed in thehypocotyls of the precociously germinated seedlings. These results imply thatthe seed maturation and germination programs do not overlap at the organand cellular level in these experimentally manipulated embryos.Genetic studies provide the most convincing evidence that overlaps

between the seed maturation and germination phases can occur. As discussedpreviously, the vp mutations of maize and the aba, abi3, and leafy cotyledonmutations of Arabidopsis cause the concurrent expression of facets of theseed maturation and germination phases (see section 4). In at least two cases,it has been shown that this apparent overlap results from the expression of thetwo phases in the same cells unlike the situation with prematurely germinatedoilseed rape embryos. Sectoring analyses suggested that the cell autonomousvpl mutation causes both defects in aleurone development and the prematureinduction of germination-specific hydro lases in the same cells (Hoecker etal., 1995). Additionally, analysis of leel mutants suggests that seed proteinmRNAs and mRNAs that serve as markers of the germination program accu-mulate in the same cells (West et al., 1994). Two conclusions emerge fromthese genetic analyses. First, although seed maturation and postgerminativedevelopment are normally separated in time and in space, mutations in specificgenes allow characteristics of both phases to occur simultaneously. Thus , theswitch(es) can be broken suggesting that these genes playa role in mediatingthis transition between seed maturation and postgerminative growth. Second,


the finding that mutations in several genes allow the simultaneous expres-sion of both programs suggests that there is not a simple switch but, rather,there are several switches controlling this phase transition. The relationshipbetween seed maturation and postgerminative growth will be more closelyanalyzed below.

5.2 . Suppression ofgermination and maintenance ofseed maturation

Although genetic analyses showed that aspects of seed maturation and post-germinative growth can occur simultaneously, the lack of overlap in thesephases during normal development implies that the developmental programsare tightly coordinated during this transition. Evidence that factors involvedin maintaining seed maturation also suppress germination/postgerminativegrowth provide support for this viewpoint. ABA has been implicated to playa role in both of these processes for several reasons. First, ABA levels aregenerally high during the middle stages of seed development when germina-tion is generally suppressed. Similarly, endogenous ABA levels at the time ofembryo excision often correlate with the time required for an isolated embryoto germinate in culture (Ackerson, 1984; Hsu, 1979; King, 1976; Prevostand Le Page-Degivry, 1985; Quebedeaux et al., 1976). Second, treatmentof cultured immature embryos with ABA is generally sufficient to inhibitprecocious germination (reviewed by Crouch, 1987; Kermode, 1990, 1995;Quatrano, 1986). In many but not all cases, embryos cultured on ABA willcontinue to synthesize at least some storage proteins (see section 4.2). Third,mutants deficient in ABA often germinate precociously, and they generallydisplay some defects in seed maturation (see section 4.2). Similar effects areobserved when seed ABA levels are depressed through application of theABA biosynthetic inhibitor fturidone (Fong et al., 1983; Oishi and Bewley,1992). Fourth, mutations that affect ABA perception, such as viviparous]and abi3, cause vivipary or they induce characteristics of postgerminativedevelopment in developing seeds although it is clear that these genes alsooperate through ABA-independent pathways (see section 4.3). As discussedpreviously, these mutations also have profound effects on seed maturation.Other factors, in addition to ABA, appear to play roles both in suppressing

germination and in maintaining seed maturation. Restricted water uptakewhich is usually achieved by culturing isolated embryos or seeds on highconcentrations of osmoticum such as sucrose, mannitol, or sorbitol has beenimplicated as such a factor (reviewed by Crouch, 1987;Kermode, 1990, 1995).For example, immature oilseed rape embryos cultured on medium of highosmolarity do not germinate prematurely, and they continue to accumulateat least some seed protein mRNAs (Finkelstein and Crouch, 1986). Theseosmotic effects do not appear to be mediated by ABA; it has been shownthat ABA levels do not increase in oilseed rape embryos cultured on highosmoticum (Finkelstein and Crouch, 1986). Furthermore, ABA treatment

576 John J .Harada

and restriction of water uptake can have distinct consequences on culturedembryos . Both ABA and high osmoticum prevented precocious germinationof vp5 mutant maize embryos but only ABA restored accumulation of Em seedprotein mRNA to wild type level (Butler and Cuming, 1993). Both osmoticumand ABA inhibit premature germination of cultured alfalfa embryos but seedprotein synthesis is differentially sensitive to ABA, osmoticum, and mediumcomposition (Xu and Bewley, 1995a, b; Xu et aI., 1990). ABA and highosmolarity also induce storage protein mRNA accumulation with differentkinetics in isolated oilseed rape embryos (Finkelstein and Crouch, 1986).These results have been interpreted to indicate that the role of ABA in seedmaturation is not merely to inhibit water uptake.Genet ic studies also implicate the existence of still other factors that sup-

press germination and maintain seed maturation. It is unlikely that VP1, AB/3,and the LEAFY COTYLEDON genes, which are involved in controlling seedmaturation and in suppressing germination during seed development, mediatetheir effects solely through ABA (see sections 4.3 and 4.4). The germinationof leafy cotyledonmutants remains sensitive to ABA, suggesting that the ABApathway remains intact in these seed even though maintenance of maturationand suppression of germination have been compromised. Furthermore, whileVPl and ABI3 are required for ABA perception, these proteins also havebeen shown to function in ABA-independent processes (see section 4.3).It is not known whether VP1, AB/3, and the LEAFY COTYLEDON genesare involved in controlling seed maturation and postgerminative growth byrestricting water uptake.Given the relationship between the maintenance of seed maturation and

the inhibition of germination/postgerminative growth, a critical step in under-standing the transition between the two phases is to define the factors thatterminate the seed maturation program. Two potential regulators of this eventare discussed.

5.3 . Are maternal factors involved in suppressing germination?

As discussed previously, detailed examination of mRNA accumulation pat-terns during cotton embryogenesis and postgerminative growth suggested theexistence of at least five regulatory programs, the cotyledon, the embryo mat-uration, the ABA, the postabscis sion, and the germination programs (Hughesand Galau, 1989; see section 3.1). Accumulation of these mRNAs was exam-ined in cultured embryos from cotton , oilseed rape, soybean, and tobacco toanalyze the factors that regulate these programs (Hughes and Galau, 1991;Jakobsen et al., 1994). Of particular interest were mRNAs that accumulat-ed during the embryo maturation and the postabscission stages, many ofwhich encode storage proteins and Lea proteins, respectively (see Figure 3).Embryos removed from the maternal plant and cultured on media displayeda characteristic decrease in the levels of embryo maturation mRNAs and a


simultaneous increase in the levels of postabscissionmRNAs and germinationmRNAs . These results were interpreted to indicate that a maternal factor(s)must be involved in maintaining the maturation program and that loss of thisfactor terminates this program. It has been proposed that termination of thematuration program and activation of the postabscission program is inducedboth by the presumed detachment of the seed's vascular connection throughabscission of the funiculus and by the excision of embryos from the mater-nal plant (Dure et al., 1981; Galau et al., 1987; Hughes and Galau, 1989).Although germination mRNAs and postabsci ssion mRNAs are induced simul-taneously in cultured embryos, they do not do so in embryos on the plant. Asdiscussed above, it is likely that restricted water uptake inhibits the germina-tion program in seed on the plant but not in embryos cultured on media. Thisresult suggests that water is the only factor required to induce the germinationprogram in non-dormant embryos (Comai and Harada, 1990; Galau et al.,1991; Hughes and Galau , 1991).The involvement of a maternal factor inmaintaining and, perhaps, initiating

the maturation program explains some but not all observations about seedmaturation. For example, maternal control of maturation could account forthe developmentally regulated activation of seed protein genes in Arabidopsisemb mutants in which seed maturation is uncoupled from morphogenesis (seesection 3.2). However, it is unclear how the maturation program is controlledduring somatic embryogenesis. Somatic embryos are generally derived fromcultured cells , and, therefore, they arise in the absence of maternal tissue. Yet,in many cases, somatic embryos synthe size and accumulate storage proteinsand Lea proteins in a pattern similar to zygotic embryos (Perez-Grau andGoldberg, 1989; Zimmerman, 1993).Although ABA enhances the accumulation of many postabscission

mRNAs, it is unlikely that the hormone is the positive inducer of this pro-gram. Studies have shown that endogenous ABA levels decline during embryoculture when postabscission genes are activated (Hughes and Galau, 1991).Furthermore, induction of the postabscission program is insensitive to theABA synthesis inhibitor, fturidone (Hughes and Galau, 1991). However, it isclear, as discussed previously, that ABA plays a significant role in the estab-lishment of desiccation tolerance (see section 4.2). Thus, it is possible thatABA is a maternal factor that maintains at least some aspects of the embryomaturation phase.

5.4 . A role for maturation drying in the switch from seed maturation togermination?

Desiccation is generally the terminal event in seed maturation that imme-diately precedes the embryo's entry into a state of metabolic quiescence .Seed generally are not able to survive desiccation early in development dur-ing the morphogenetic phase but, rather, tolerance is acquired during seed

578 John J . Harada

maturation (reviewed by Bewley and Black, 1995; Kermode, 1990; Vertucciand Farrant, 1995). Specific metabolic changes correlate with the onset ofdesiccation tolerance including changes in the levels of mono- and oligo-saccharides and of Lea protein, and some have speculated that these changesplaya mechanistic role in conferring tolerance (Bewley and Black, 1995;Kermode, 1990; Roberts et aI., 1993; Vertucci and Farrant, 1995). More rele-vant for this discussion is the finding that specific and reproducible changes ingene expression occur in response to seed drying. As discussed below, thesecorrelative change s are the basi s for the hypothesis that desiccation servesas a switch between seed maturation and germination (reviewed by Bewley,1995; Kermode, 1990,1995; Kermode et aI., 1986).The acquisition of desiccation tolerance generally occurs before the onset

of maturation drying (Dasgupta and Bewley, 1982; Kermode and Bewley,1985; Long et aI., 1981; Rosenberg and Rinne, 1986). Immature seed are ableto survive drying long before the precipitous decline in seed water contentis observed. Mature seed are able to survive rapid drying regimes, such asthat which occurs on the plant, but younger seed generally must be desiccatedslowly (Adams et aI., 1983;Kermode and Bewley, 1985). This observation hasbeen interpreted to indicate that the degree of desiccation tolerance increasesprogressively during seed maturation.Experimental manipulations that defined the acquisition of desiccation

tolerance also demonstrated that drying can promote seed germination. Forexample, freshly harvested castor bean seed are not normally able to ger-minate until approximately 50 days postanthesis. However, germination willoccur as early as 25 days postanthesis if developing seed are dried premature-ly (Kermode and Bewley, 1985). This relationship between premature dryingand seed germinability has been extended to show that , following prematuredesiccation and rehydration, proteins and mRNAs characteristic of seed mat-uration cease to accumulate and that they are replaced by those representativeof postgerminative growth (Dasgupta and Bewley, 1982; Kermode and Bew-ley, 1985, 1986;Misra and Bewley, 1985; Oishi and Bewley, 1992; Rosenbergand Rinne, 1988). The termination of seed maturation gene expression andthe induction of postgerminative gene s following premature desiccation hasbeen shown to occur in a variety of species. It has also been shown that pre-mature drying terminates the expression of some seed protein genes throughtranscriptional repression (Jiang et al. , 1995; Oliver et aI., 1993).Extensive drying of seed does not appear to be a requirement for germina-

tion. Only partial drying is sufficient to effect the switch from seed maturationto germination in seed that are normally substantially desiccated at maturit y.For example, conditions that caused only minimal water loss of immature cas-tor bean and French bean seed was sufficient to induce germination (Bewleyet aI., 1989). In these instances, at least partial drying appeared to be requiredbecause seed detached from plants and maintained in a fully hydrated statedid not germinate. However, the requirement for desiccation does not appear


to be universal. Seed from plants with fleshy fruits remain hydrated duringtheir development, at least as compared to other species that shed their maturedry seed. Even without partial drying treatments, these seed will germinateupon removal from the fruit (Berry and Bewley, 1991, 1992; Welbaum andBradford, 1989).Thes e results have been interpreted to indicate that seed desiccation , either

partial or complete, may be involved in terminating seed maturation and pro-moting postgerminative development in many but not all species. How doesdrying induce this switch in developmental programs? It has been suggestedthat the effects of drying may be mediated through changes in ABA responsesin two ways (Galau et a\. , 1991; Kermode , 1995). First, it has been observedin several species that seed drying results in a decline in ABA levels (Acker-son, 1984; King, 1976). A decrease in the level of this hormone may permitseed to germinate prematurely. Second, drying also has been shown to causea decrease in the plant's sensitivity to the hormone. Seed late in matura-tion are less sensitive to the hormone than are younger seed, and prematuredrying has been shown to cause a decrease in ABA perception by the seed(Eisenberg and Mascarenhas, 1985; Finkelstein et a\., 1985; Kermode et a\.,1989; Williamson et a\., 1985; Xu and Bewley, 1991). Thus, desiccation mayserve to reduce the influence of ABA on the seed. However, it is unclear howmaturation drying affects the other "switches" controlling the transition fromseed maturation to postgerminative growth.

5.5. Control of dormancy

Seed dormancy is defined as " the temporary failure of a viable seed to ger-minate after a specified length of time in a particular set of environment alconditions that later evoke germination when the restrictive state has beenterminated by either natural or artificial means" (Simpson, 1990 as cited byKoornneef and Karssen, 1994). As indicated by this definition, seed dormancyinvolves a complex interplay of responses to environmental and endogenouscues. Thus , in addition to the elaborate programs that are in place to suppressgermination during maturation, seed germination in many but not all speciesis also controlled after seed maturation.There are several forms of dormancy, two of the major categories being

primary and secondary dormancy (reviewed by Bewley and Black, 1995). Pri-mary dormancy forms during seed development, and, thus, seed are dormantas they emerge from the plant. Secondary dormancy is induced in mature seedthat are initially non-dormant. Dormancy may be coat-imposed, indicatingthat the isolated embryo can germinate but that other components of the seed,such as the seed coat or endo sperm, cause dormancy. Alternatively, embryo-imposed dormancy, as the name indicates, occurs when the embryo itselfis dormant. Moreover, there are a variety of causes of dormancy includinginterference of water uptake or gas exchange by impermeable seed coat and

580 John J. Harada

mechanical constraints caused by seed tissue that restrains the germinatingseedling from emerging from the seed (reviewed by Bewley and Black, 1995).In keeping with a theme of this chapter, I will focus on ABA's role in causingprimary dormancy.Because ABA is an inhibitor of germination during seed development,

substantial attention has been focused on the hormone as a mediator of dor-mancy. There does not appear to be a convincing correlation between ABAlevel in maturing or dry seed and the extent of dormancy (Bewley and Black,1995; Hilhorst, 1995). However, other lines of evidence provide substantialevidence for a role for ABA in inducing primary dormancy (reviewed byBewley and Black, 1995; Black, 1991; Hilhorst, 1995; Hilhorst and Karssen,1992; Karssen, 1995; Koornneef and Karssen, 1994) First, the aba mutant ofArabidopsis and the jiacca and sitiens mutants of tomato are deficient in ABAand are non-dormant (Groot and Karssen, 1992; Koornneef et aI., 1982). Itwas shown that maternal ABA produced by fruit tissue does not influencedormancy in Arabidopsis. Plants heterozygous for the recessive ABA accu-mulation mutation, aba, produce segregating aba : /aba: , ABA+/abar , andABA+/ABA+ embryos in maternal seed coat and fruit tissue that are het-erozygous for the mutation. Although ABA is provided to the embryo bythese maternal tissues, aba: [aba: seed do not become dormant suggestingthe necessity of embryonic ABA in the induction of dormancy. Additionally,exogenous ABA does not induce dormancy. Second, defects in ABA percep-tion also cause defects in dormancy. abil and abi3 mutants of Arabidopsishave reduced dormancy (Koomneef et aI., 1984). Furthermore, embryo ger-mination of a wheat cultivar that is resistant to premature sprouting is moresusceptible to ABA than is the germination of sprouting-suceptible cultivars,suggesting a correlation between ABA perception and the degree of dormancy(Walker-Simmons, 1987).It is clear that other factors besides ABA are involved in mediating dor-

mancy. The seed's osmotic environment and its osmotic sensitivity have beenimplicated in primary dormancy (Groot and Karssen, 1992). Arabidopsismutants with reduced dormancy have been identified that appear to havenormal levels of ABA and display normal sensitivity to the hormone (Leon-Kloosterziel et aI., 1996). This suggests that genes that are not involved inABA accumulation or perception playa role in dormancy.A variety of treatments, many of which involve environmental parame-

ters, will induce emergence from primary dormancy. Treatments that relievedormancy include the slow drying of dormant seed, known as after ripening,the chilling of imbibed seed, and the exposure of seed to specific light con-ditions (reviewed by Bewley and Black, 1995; Hilhorst and Karssen, 1992).Although the mechanisms that underlie the termination of primary dormancyare not well understood, some evidence suggests that these treatments resultin a decrease in ABA levels. Thus, after ripening may lead to degradation ofABA, prechilling may induce a leakage of ABA, and light may induce the


degradation or conjugation of ABA (Dulson et al., 1988; Groot and Karssen,1992; Toyomasu et al., 1994).

6. Summary

A long and extensive search has not uncovered a single "master regulator"of seed maturation. Although several candidates have been identified thatappear to be involved in initiating, maintaining, and/or terminating differentaspects of seed maturation, none of these have been shown to be the principalregulator. Thus, either there is no "master regulator" of this phase or the con-siderable studies of seed maturation have not yet revealed its existence . Theavailable evidence points to a multicomponent regulatory scheme in whichdifferent regulators control both common and distinct sets of seed matura-tion responses. For example, the acquisition of desiccation tolerance can besuppressed if the activities or accumulation of several different regulatorsare inhibited. However, each of these regulators appears to be involved incontrolling different aspects of seed maturation, suggesting that they operatein distinct regulatory programs. Moreover, detailed phenotypic analyses andtests of genetic interactions suggest that these regulators act in parallel path-ways to control seed maturation. Additional support for this interpretation isthe finding that several distinct pathways appear to be involved in regulatingthe expression of seed protein genes .What are the regulators of seed maturation? ABA has been implicated to

be one of the key regulators. Analy ses of ABA accumulation mutants, ofABA perception mutants, and of the effects of ABA on cultured embryosstrongly suggest a role for the hormone in suppressing germination and ininitiating or maintaining many facets of seed maturation. In some species,ABA also is involved in conferring dormancy. Although ABA clearly hasan important function in these processes, other pathways are involved inregulating this phase. The Vpl/AB!3 class of genes, which are required forthe acqui sition of desiccation tolerance, for the expression of many seedprotein genes, and for the suppression of germination, functions in bothABA-dependent and ABA-independent pathways. InArabidopsis, theLEAFYCOTYLEDON genes have been shown to be required for the completionof seed maturation, yet they do not appear to operate in ABA pathways.Evidence also suggests that a maternal factor(s), a postabscission factor, andthe restriction of water uptake by the embryo may also play roles in regulatingdistinct aspects of seed maturation. It is conceivable that other factors involvedin controlling seed maturation remain to be discovered. Major objectives areto identify these other regulators, to determine the mechanisms by which theseregulators operate, and to understand how these distinct regulatory pathwaysare coordinated to control seed maturation.

582 John J. Harada

Acknowledgements

I thank Bob Fischer, Tami Lotan, and Kazutoshi Yamagishi for their criticalreading of the manuscript. Work from my lab that is cited in this review wassupported by grants from the DOE and the NSF.

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[Advances in Cellular and Molecular Biology of Plants] Cellular and Molecular Biology of Plant Seed Development Volume 4 || Seed Maturation and Control of Germination