Role of ABA and Components of the ABA Signalling Pathway

(1) Promotion of Developmental Processes, Prevention of Precocious Germination and Induction of Dormancy During Seed Development

Whether a seed is dormant or quiescent at maturity, its quality and vigour rely heavily on processes that occurred during seed development: reserve deposition (accumulation of storage proteins and storage lipids or starch), regulation of precocious germination, and development of stress tolerance. Control of seed maturation in turn is mediated by key interactions between different hormone signalling pathways and other regulatory cues provided by the seed environment.

Embryos excised from developing seeds at certain stages often germinate when placed on water or on a minimal culture medium. Thus, even though they have not completed their development, embryos germinate precociously, but often fail to establish viable seedlings. Therefore, an important role of the seed environment is to maintain embryos in a developmental mode until they are fully formed and have accumulated sufficient reserves to permit successful germination and subsequent seedling establishment. Studies to elucidate the mechanisms whereby the seed environment retains the embryo in a developmental mode and suppresses germination have implicated several regulatory factors of which two are ABA and restricted water uptake. One component of the chemical environment surrounding the developing embryo is the osmotic environment. This specialised environment maintains a highly negative osmotic potential during development, which in turn is thought to suppress precocious germination of embryos by restricting water uptake.

Earlier studies implicating ABA as a regulatory factor were largely based on the effects of exogenous ABA on immature embryos excised from the seed and on the patterns of accumulation of endogenous ABA during seed development. Isolated embryos treated with ABA are inhibited from germinating precociously and continue maturation processes, including the synthesis of storage proteins and storage lipids (reviewed in Kermode, 1990, 1995). Typically the level of ABA in seeds is low during early development (i.e. during the histodifferentiation and early pattern formation stage); it increases thereafter and usually peaks around mid-maturation. ABA levels decline precipitously during late development, particularly during the maturation drying phase (Bewley and Black, 1994; Meinke, 1995; Kermode, 1995; Bewley, 1997 and references therein). These developmental changes in endogenous ABA correlate well with the pattern of expression of the gene encoding the ABA biosynthesis enzyme, zeaxanthin epoxidase (reviewed in Seo and Koshiba, 2002; see references therein). However, several factors add to the complexity of the pattern of ABA increase and decline, including developmental changes in the sensitivities of embryo and seed tissues to ABA and the differential thresholds for the initiation and maintenance of developmental events (e.g. the expression of certain developmental genes and the duction and maintenance of dormancy) (Xu and Bewley, 1991; Jiang et al., 1996; Kermode, 1995). Moreover, the environment during seed development (e.g. light, temperature and water availability) can strongly influence the ABA content and sensitivity of the mature seed. In wheat, low temperatures can effect changes in ABA content or sensitivity which in turn influence the degree of dormancy during development and in the mature grain (Walker-Simmons, 1990; Garello and LePage-Degivry, 1999). Likewise, water stress imposed during the development of Sorghum bicolor seeds decreases both their ABA content and sensitivity, and the seeds have an increased capacity for germination during development (Benech-Arnold et al., 1991).

More definitive evidence towards elucidating the role of ABA in controlling seed and embryo maturation processes (including the inception of dormancy) has come from the generation and analysis of mutants (e.g. of tomato, maize and Arabidopsis) that are either deficient in ABA, or exhibit a relative insensitivity to ABA (the so-called response mutants). These latter mutants have defects in ABA signalling pathways and are characterised by reduced dormancy, which is generally accompanied by disruption of seed maturation and precocious expression of germinative/post-germinative genes. Screens to identify suppressors of ABA signalling mutants have helped to define some of the components of ABA signalling that interact with other signalling pathways and thus form the basis of hormonal networks that control the dormancy-to-germination transition.

ABA not only plays an important role in the deposition of storage reserves, but it is also important for the acquisition of desiccation tolerance, prevention of vivipary, and induction of primary dormancy. Vivipary is characterised by germination of the embryo within the fruit on the parent plant. There is an uninterrupted progression from embryogenesis to germination with little or no cessation of growth (quiescence) and in most cases, little or no dehydration. Vivipary is a normal occurrence in Rhizophora mangle (the mangrove), in which seeds and seedlings exhibit a relative insensitivity to ABA (Sussex, 1975). The ABA-deficient sitiens mutant of tomato has approximately 10% the ABA content of its wildtype counterpart. This mutant exhibits reduced dormancy and seeds germinate viviparously in overripe fruits (Groot, 1987; Groot et al., 1991). However, low endogenous ABA may not directly contribute to vivipary in this case, since the embryos also exhibit a reduced sensitivity to the highly negative osmotic potential of surrounding seed tissues. ABA-deficient mutants of maize, Arabidopsis, and tobacco (viviparous (vp)5 or vp14, aba1 and aba2, respectively), exhibit reduced dormancy (Arabidopsis and tobacco) or vivipary (maize) (Karssen et al., 1983; Tan et al., 1997; Frey et al., 1999; White et al., 2000). Severe mutants of Arabidopsis exhibiting relative ABA insensitivity (i.e. some of the abi3 mutants) produce seeds that are intolerant of desiccation (Ooms et al., 1993; Nambara et al., 1995). Seed viability is not altered in ABA-deficient (aba) and the less severe ABA-insensitive (abi3) mutants of Arabidopsis, yet seeds of double mutants exhibiting these two traits do not undergo desiccation on the parent plant, are intolerant of artificial desiccation, and fail to produce some of the late abundant proteins (LEAs) (Koornneef et al., 1989; Meurs et al., 1992). These double-mutant seeds accumulate only low amounts of the major storage proteins and are deficient in several low molecular weight polypeptides, both soluble and bound, some of which are heat soluble. During development (14 to 20 days after pollination) the low amounts of various maturation-specific proteins are degraded. Seeds of the abi3-7 mutant exhibit reduced dormancy and sensitivity to ABA and have undetectable Em6 mRNA and protein and reduced transcripts for two 2S albumin proteins (Bies-Etheve et al., 1999).

In tobacco, over-expression of the gene encoding the ABA biosynthesis enzyme zeaxanthin epoxidase (i.e. ABA2) produces transgenic seeds with more pronounced dormancy than that of the wildtype; conversely, the down-regulated expression of this gene by an antisense approach results in reduced levels of ABA and in reduced dormancy (Frey et al., 1999). Sequestration of ABA within developing transgenic tobacco seeds (effected by expression of an anti-ABA single chain variable fragment antibody) leads to a marked disruption of storage reserve deposition. Reserve accumulation is reduced to the extent that the storage parenchyma cells of the seed more closely resemble plant vegetative cells (Phillips et al., 1997). The transgenic seeds exhibit other features of seedlings, including green-chloroplast-containing cotyledons, desiccation intolerance and premature activation of the shoot apical meristem.

It is important to note that ABA synthesis in the ABA-deficient mutants (and in some transgenic plants), while decreased, is generally not completely abolished. Mutants are often ‘leaky’, allowing some biosynthesis of ABA and redundant genes/pathways often exist to compensate for defective genes. ABA contents of the vp14 mutant of maize are about 30% of that found in wild-type seeds (Tan et al., 1997); hence, the ABA in these mutant seeds is sufficient for allowing normal development to proceed (with respect to reserve deposition), and only the later maturation stages are altered (e.g. vp14 seeds exhibit an enhanced capacity for precocious germination).

As intimated above, the phenotypes of the ABA-insensitive mutants are also variable, depending upon the severity of the defective allele. Only the null alleles of the ABI3 gene, for example, lead to a failure to complete seed maturation, producing mutant seeds that are green, reduced in their storage reserve content and desiccation-intolerant.

Characterisation of the ABA-insensitive mutants of maize and Arabidopsis led to the cloning of genes involved in ABA signalling, some of which are transcription factors. To date six classes of transcription factors have been identified that appear to be essential for ABA- or seed-specific gene expression: ABI3/VP1, ABI4, ABI5, LEC1, LEC2 and FUS3 (reviewed in Finkelstein et al., 2002). Lesions in the genes encoding these factors underlie some of the phenotypic characteristics of mutants that are disrupted in seed maturation. For example, the VP1 gene of cereals and the ABI3 gene of Arabidopsis encode novel transcription factors that regulate the expression of ABA-responsive genes during seed development (e.g. storage-protein and LEA genes) (McCarty et al., 1991; Giraudat et al., 1992). The encoded proteins are transcription factors of the B3 domain family. Since their isolation from these species, other ABI3/VP1 orthologues have been identified in many plant species, including rice, wheat, carrot, bean (Phaseolus vulgaris), the resurrection plant and poplar (Hattori et al., 1994; Bobb et al., 1995; Jones et al., 1997; Chandler and Bartels, 1997; Shiota et al., 1998; Rohde et al., 1998; Bailey et al., 1999). All contain four conserved domains: an acidic activation domain and three basic domains, B1, B2 and B3 (Figure 33.3). The abilities of ABI3/VP1 proteins to transactivate the expression of ABA-responsive developmental genes in seeds or embryos have been demonstrated in different systems by transient gene expression assays and by in vivo expression studies. Ectopically expressed ABI3 protein (effected by stable transformation of Arabidopsis with a chimeric 35S-ABI3 gene) leads to the re-activated expression of seed-specific genes in vegetative tissues and seedlings (Parcy and Giraudat, 1997).

VP1 and ABI3 likely interact with other transcription factors to effect gene activation/repression. The promoter elements of the rice Osem gene (an Em-type gene) required for regulation by VP1 have been identified (Hattori et al., 1995). These include TACGTGTC (an ABA-responsive element or ABRE), a small sequence located just downstream of the ABRE, and a quantitative element (the sph box/RY repeat), conserved in many seed-specific gene promoters.

VP1 and ABI3 can also repress gene expression, particularly that of post-germinative genes (McCarty, 1995; Nambara et al., 1995; Nambara et al., 2000; Paek et al., 1998; Hoecker et al., 1995, 1999). As noted earlier, in some instances in which ABI3/VP1 genes are defective, the mutant seeds show an altered or premature activation of post-germinative gene expression (e.g. genes encoding the chlorophyll a/b binding protein and isocitrate lyase in the Arabidopsis abi3 mutant and genes encoding malate synthase and isocitrate lyase in the maize vp1 mutant). VP1 also inhibits the induction of αα-amylase gene expression in aleurone layer cells of developing maize seeds (Hoecker et al., 1995); derepression of the expression of a chimeric αα-amylase (5′)-GUS gene depends on the presence of a viviparous embryo (Hoecker et al., 1999). The repressor function of VP1 is localised between the B1 and B2 domains. As noted above, VP1 and ABI3 likely interact with other transcription factors to effect gene activation/repression. Thus, the B1–B2 region of the protein may create the appropriate context for protein–protein interactions, rather than containing inherent repressor activity (Hoecker et al., 1995). 14-3-3 proteins are suggested to facilitate interactions between VP1/ABI3 and other proteins due to their chaperone/scaffolding functions; some of these functions in turn may rely on the phosphorylation status of the 14-3-3 protein (Schultz et al., 1998; Finnie et al., 1999; Fu et al., 2000).

Recently, an ABI3 gene orthologue (CnABI3) was cloned from the gymnosperm Chamaecyparis nootkatensis (yellow-cedar) (Lazarova et al., 2002). The yellow-cedar ABI3 gene encodes a protein of 794 amino acids. Comparison of the deduced amino acid sequence of CnABI3 to other ABI3/VP1 proteins by multiple alignment indicates that the yellow-cedar protein has all four regions that are typically conserved (Figure 33.4). The conifer ABI3/VP1 protein shares a similar role to its angiosperm counterparts in relation to the control of seed developmental gene expression (Zeng et al., 2003). For example, GUS chimeric genes driven by storage-protein gene promoters (those of the napin and vicilin genes) are activated in transgenic tobacco leaves by the ectopic expression of the CnABI3 gene and ABA is able to enhance this expression (Zeng et al., 2003).

Mutations in the ABI4 and ABI5 gene loci have similar effects on seed development and ABA sensitivity as those associated with the abi3 mutant; however, null mutations in the ABI3 gene locus result in more severe phenotypes than those in the ABI4 or ABI5 gene loci (Parcy et al., 1994; Finkelstein et al., 1998; Finkelstein and Lynch, 2000a, 2000b). In comparison to the ABI5 gene, whose expression occurs late in the seed maturation programme (i.e. 17–20 days post-anthesis, DPA), ABI4 gene expression is detected from 8 DPA onwards. It is the highest in seeds where it is confined to the embryo, but there are some ABI4 gene transcripts present at low levels in vegetative tissues (Söderman et al., 2000). Abi4 mutant seeds exhibit significantly reduced expression of LEA genes (e.g. AtEm6 and PAP140). Likewise, abi5 mutant seeds exhibit reduced expression of certain LEA genes (AtEm1, AtEm6 and LeaD34), while the expression of some developmental genes is not altered (e.g. the storage-protein gene vicilin and the gene encoding the integral oil body protein, oleosin). ABI5 gene transcripts are down-regulated in the abi1, abi2, abi3, abi4, and aba1 mutant backgrounds of Arabidopsis, suggesting that the ABI5 gene is regulated by ABA and these abi loci (Finkelstein and Lynch, 2000a).

The encoded proteins (ABI4 and ABI5) contain putative DNA binding and protein interaction domains (reviewed in Finkelstein et al., 2002). ABI4 is closely related to the APETALA2 domain family of transcription factors (within the AP2 domain); however, the cis-elements of genes necessary for ABI4-regulated expression have not yet been identified (reviewed in Finkelstein et al., 2002). The ABI5 family of proteins all share three conserved charged domains (present in the N-terminal half of the protein and containing potential phosphorylation sites) and a bZIP domain at the C-terminus. Proteins showing the greatest similarity to ABI5 of Arabidopsis are DPBF-1, a Dc3-promoter binding factor family protein of sunflower, HvABI5 of barley, wheat TaABF and TRAB1, a rice protein that interacts with VP1 (Kim et al., 1997; Hobo et al., 1999; Johnson et al., 2002; Casaretto and Ho, 2003). Like VP1/ABI3, ABI5 can either activate or repress gene expression and ABI3 and ABI5 may have antagonistic or synergistic effects on gene expression, depending on the gene (Finkelstein and Lynch, 2000a; Delseny et al., 2001).

There appears to be extensive cross-regulation of expression among ABI3, ABI4 and ABI5 genes (Söderman et al., 2000). Ectopic expression of any of these transcription factors results in ABA hypersensitivity of vegetative tissues which is at least partly dependent on increased ABI5 expression (Parcy et al., 1994; Söderman et al., 2000; Lopez-Molina et al., 2001). Thus, the three transcription factors likely participate in combinatorial control of gene expression, possibly by forming a regulatory complex mediating seed-specific and/or ABA-inducible expression (Finkelstein et al., 2002). ABI3 and ABI5 interact directly via the B1 domain of ABI3 and two of the conserved charged domains in ABI5 that contain putative phosphorylation residues (Nakamura et al., 2001). ABI5 binding to ABRE elements may tether ABI3 to target promoters and facilitate the interaction of ABI3 with RY elements and transcription complexes (Finkelstein et al., 2002). In contrast, ABI4 does not appear to interact physically with either ABI3 or ABI5 (Nakamura et al., 2001).

(2) Multiple Factors Function in Concert to Control Seed Development

Along with ABI3, ABI4 and ABI5, other proteins act in a concerted fashion to promote events that are critical to the later stages of seed development, including the acquisition of desiccation tolerance and induction of dormancy. At the same time, they appear to repress germinative and post-germinative functions (Holdsworth et al., 1999).

Arabidopsis LEAFY COTYLEDON (LEC) genes are defined by mutations at three loci, LEC1, LEC2 and FUSCA3 (FUS3). Recessive mutations at these gene loci lead to various abnomalities during early-, mid- and late-embryogenesis (reviewed in Harada, 2001). During the morphogenesis phase, the LEC genes are required to maintain suspensor cell identity; in the lec mutants, the normally single-celled layer comprising the suspensor, becomes multilayered as a result of abnormal subdivisions (Lotanet al., 1998). Specification of cotyledon identity is another early function of the LEC genes and in lec mutants, the absence of LEC gene activity leads to partial reversion of the cotyledons to more leaf-like organs (Meinke et al., 1994). The later functions of the LEC genes are manifested in various defects including a loss of dormancy and failure to acquire desiccation tolerance (e.g. all known alleles of the lec1 mutation and strong fus3 mutant alleles). Synthesis and accumulation of storage proteins and lipids (and other gene products associated with reserve deposition) are highly reduced in lec1 and fus3 embryos. Interestingly, the mutant embryos appear to compensate by accumulating starch grains, unlike wild-type embryos (Keith et al., 1994; Meinke et al., 1994: Bäumlein et al., 1994; Kirik et al., 1996; Parcy et al., 1997). In lec mutants, the rate of development is altered and some characteristics of lec mutant embryos are seedling-like, indicating that post-germinative development is elicited precociously. For example, in Arabidopsis, the embryonic shoot apical meristems become activated during embryogenesis and more closely resemble post-germinative meristems. Thus, there is a defect in maturation processes that normally inhibit precocious germination and the expression of germinative- and post-germinative genes (Harada, 1997, 2001). Lec1 and fus3 mutant embryos occasionally exhibit vivipary (Meinke, 1992; West et al., 1994; Keith et al., 1994; Nambara et al., 2000).

Some of the effects of LEC gene defects on ABA biosynthesis and response have been examined. Lec1 mutant seeds are not sensitive to ABA (Parcy et al., 1997; but see also Meinke et al., 1994). Immature fus3 mutant siliques accumulate about one-third of the ABA level characteristic of wild-type seeds; however, by maturity, fus3 siliques have an equivalent amount of ABA to that of the wildtype (Nambara et al., 2000).

Both FUS3 and LEC1 encode putative transcription factors. Thus, they are thought to exert their effects by protein-DNA and/or protein-protein interactions, although the precise interactions have yet to be identified (see subsequent discussion). The LEC1 gene encodes a protein with sequence similarity to the HAP3 subunit of CCAAT binding factors (Lotan et al., 1998). In mammalian cells, these binding factors, which are heterotrimeric, enhance the transcription of a large number of genes (reviewed in Harada, 2001; see references therein). The pattern of expression of the Lec1 gene is not confined to the embryo, suggesting that its role is a global one—establishing an environment that promotes embryogenesis; this cellular environment likely coordinates the morphogenesis and maturation phases of development (Harada, 2001).

Lec2 is able to establish a cellular environment sufficient to initiate embryo development; its ectopic expression during the post-embryonic phase induces the formation of somatic embryos and other organ-like structures and confers embryonic characteristics to seedlings (Stone et al., 2001).

Fus3 mRNA accumulates primarily in developing siliques and is at maximal levels during mid-maturation (Leurssen et al., 1998). FUS3 and LEC1 modulate the abundance of ABI3 protein in seeds and synergistic interactions between the three proteins (ABI3, FUS3 and LEC1) are thought to control various key events, including accumulation of chlorophyll and anthocyanins, sensitivity to ABA and expression of individual members of the 12S storage protein gene family (Parcy et al., 1997). Interestingly, part of FUS3 (a continuous stretch of 100 amino acids) shows significant similarity to the B3 domain of ABI3 and VP1 proteins (Luerssen et al., 1998), a domain which interacts with the RY cis promoter motif of several seed proteins (Figure 33.3). In fact, in transient transcription assays, FUS3 activates the transcription of maturation-specific genes containing the RY motif (Reidt et al., 2000). Thus, both FUS3 and ABI3 may be essential components of a regulatory network acting in concert through the RY-promoter element to control gene expression during late embryogenesis and seed development.

None of the ABI transcription factors (ABI3, ABI4 or ABI5) appear to interact directly with either FUS3 or LEC1 in a yeast two-hybrid assay (Brocard-Gifford et al., 2003). Analyses of abi fus3 and abi lec1 double mutants indicate that despite the lack of physical interaction and disparities in peak expression of the corresponding genes, ABI4 and ABI5 interact genetically with both LEC1 and FUS3 in controlling the expression of genes associated with mid- and late-embryogenesis (Brocard-Gifford et al., 2003). Processes central to those developmental stages that are especially affected in the double mutants include pigment accumulation and suppression of vivipary. However, also uncovered by the analyses of the double mutants were cryptic effects of individual loci that were not detectable by analyses of single mutants and it is not clear to what extent the double mutant analyses reflect in vivo interactions between these factors.

Figure 33.5 illustrates a model showing some of the factors that interact with ABI3 to control maturation events and prevent a premature switching to a germination and growth programme (Bonetta and McCourt, 1998). The actions of FUS3 and LEC1 are shown upstream of ABI3; this is based on the findings that FUS3 and LEC1 can regulate the amount of ABI3 protein and generally, defects within these genes cause more widespread phenotypic consequences on developmental processes than those caused by ABI3 gene mutations.

(3) Maintenance of Dormancy at Seed Maturity

The putative role of the VP1/ABI3 gene product as a regulator of dormancy in mature seed has been examined (Jones et al., 1997, Fukuhara et al., 1999; Nakamura and Toyama, 2001; Carrari et al., 2001; Zeng et al., 2003). In Avena fatua (wild oat), expression of the VP1 gene is correlated with the degree of embryo dormancy and may be important for maintaining ABA-controlled metabolism in the imbibed seed (Jones et al., 1997). For example, in embryos of A. fatua seeds transcripts are higher in seeds that are stored at 4°C and are still dormant, but they decline in seeds that are fully after-ripened at 24°C (Jones et al., 2000, 1997). A VP1 gene orthologue cloned from sorghum (Sorghum bicolor) has been examined in relation to its potential involvement in pre-harvest sprouting resistance (Carrari et al., 2001). In pre-harvest sprouting, there is germination of physiologically mature grain while still on the parent plant; this phenomenon is particularly prevalent when maturation of the grain takes place under low temperature and high-humidity conditions (Bewley and Black, 1994). In wheat, barley and sorghum cultivars that are susceptible to pre-harvest sprouting, there is a relative insensitivity of the embryo to ABA as compared to resistant cultivars, generally in the absence of any substantial differences in ABA content, or a higher ABA content in the susceptible cultivars (Walker-Simmons, 1987; Steinbach et al., 1995; Benech-Arnold et al., 1999). Interestingly, the expression pattern of the sorghum VP1 gene is different in two cultivars/genotypes exhibiting differential resistance to pre-harvest sprouting at physiological maturity. More specifically, in the embryos of the susceptible cultivar, transcripts encoding VP1 peak at a relatively early stage of grain development (20 days after pollination), while the peak in the resistant cultivar occurs at much later developmental stages when seed maturation is almost complete (Carrari et al., 2001).

The role of ABI3/VP1 proteins in the maintenance of dormancy of imbibed mature seeds needs to be more thoroughly addressed in a wider range of species and studies are clearly needed that examine expression at both the mRNA and protein levels. Maintenance of dormancy in the conifer species yellow-cedar, whose seeds are deeply dormant at maturity, appears to involve ABI3 (Zeng et al., 2003). The protein (CnABI3) is present in the megagametophyte and embryo of dormant mature and warm stratified yellow-cedar seeds, but declines during subsequent moist chilling, a treatment effective in breaking dormancy. In contrast, the protein is preserved (albeit in lower amounts) in seeds subjected to a control treatment (12 weeks in warm moist conditions) that is ineffective in breaking dormancy. A decline in CnABI3 gene transcripts is also positively correlated with dormancy breakage, but does not occur during moist chilling itself, but rather during subsequent germination. Regulation of the CnABI3 gene in relation to dormancy control is likely not restricted to the transcriptional level and may additionally occur at the level of stability of the CnABI3 protein or involve some other post-translational change (Zeng et al., 2003).

A two-hybrid screen in yeast has identified three proteins that interact specifically with VP1 of A. fatua (Jones et al., 2000); these proteins (referred to as AfVIP1, AfVIP2 and AfVIP3) may play specific roles in AfVP1-mediated regulation of the dormancy to germination transition. AfVIP1 and AfVIP2 (encoding a ring-type zinc finger domain protein) are more highly expressed in dormant embryos, while expression of AfVIP3 (encoding a paralogue of the human C1 transcription factor involved in cell cycle control) is greater in germinated seeds (Jones et al., 2000). Yeast two-hybrid screens using B2 and B3 domains of the Arabidopsis ABI3 protein as bait identified four putative interacting proteins: a CONSTANS-related factor, a protein related to the Drosophila transcription factor GOLIATH (involved in mesoderm formation) and the RPB5 subunit of RNA polymerase II (Kurup et al., 2000). Yeast two-hybrid screens using the intact CnABI3 protein of yellow-cedar as bait identified three putative interacting proteins, a zinc-finger transcription factor, a putative transcription factor, and a heat shock protein binding factor (Zeng and Kermode, unpublished). Further characterisation of the various VP1/ABI3-interacting proteins uncovered by yeast two-hybrid screens, including the implementation of reverse genetics approaches will aid in elucidating their specific roles. The functions of the CnABI3 protein of yellow-cedar appear to be similar but not identical to that of ABI3-like proteins of angiosperms (Zeng and Kermode, unpublished). The characterisation and functional attributes of proteins that interact with CnABI3 will contribute to an understanding of the functions of ABI3-like proteins in seeds of gymnosperms, particularly in relation to the maintenance of deep dormancy.

(4) Other Components of ABA Signal Transduction

As with any classical signal transduction pathway, ABA signalling is presumed to start with the reception of the hormone signal by a specific receptor and there is evidence that ABA perception occurs both intracellularly as well as at the extracellular face of the plasma membrane (reviewed in Lovegrove and Hooley, 2000; see references therein). Purification of ABA-binding proteins (e.g. from broad bean leaf epidermal cells; Zhang et al., 2002) is a major step toward isolating ABA receptors. Interaction between the hormone and its receptor(s) then triggers a signal transduction cascade involving an array of intermediary proteins and secondary signals. The end-point of the process is the up-/down-regulation of expression of certain target genes (e.g. those involved in the maintenance or termination of dormancy). However, the concept of a simple (linear) signal transduction chain has been replaced by the more realistic view that signaling networks involving interacting components likely control complex processes including the dormancy-to-germination transition and the germination-to-growth transition. Thus, regulation of seed developmental processes involves considerable cross-talk between different hormone signalling pathways and gene expression may be controlled by parallel pathways involving both ABA-dependent and ABA-independent steps.

As noted above, characterisation of mutants that have altered sensitivity to ABA and the cloning of the wild-type genes involved in the underlying processes that are disrupted in the mutants (e.g. resulting in enhanced or reduced dormancy) has led to the identification of certain components of the ABA signalling pathway. Many are transcription factors and hence are likely part of the later stages of signal transduction. Other proteins that have been identified—either as positive or negative modulators of ABA signalling—are kinases, phosphatases and farnesyl transferases—enzymes that are capable of effecting post-translational modifications of proteins. The targets of these modifying enzymes likely include proteins that act as intermediaries in signal transduction cascades. Changes in the phosphorylation status of a protein can in turn modulate its function and stability or half-life; farnesylation of proteins is also a key modulator of protein function by allowing proteins to insert into membranes. Still other proteins that play some part in the ABA signal transduction pathway are RNA-binding/processing proteins, GTP-binding proteins, enzymes of phospholipid or phosphoinositide metabolism, and proteins regulating vesicle trafficking or subcellular localisation of proteins. Only selected examples of these will be discussed.

Evidence that reversible protein phosphorylation plays a role in ABA signalling has come in part from the analyses of the abi1 and abi2 mutants of Arabidopsis, which lead to relative ABA insensitivity of seeds as well as of vegetative tissues (Koornneef et al., 1984; Finkelstein and Somerville, 1990). The ABI1 gene of Arabidopsis, encodes a member of the 2C class of serine/threonine protein phosphatases (PP2Cs), and its activity is regulated by proton and Mg2+ ions, but not by Ca2+ ions (Leube et al., 1998). The ABI2 gene of Arabidopsis also encodes a 2C class protein phosphatase and it is likely that a family of these phosphatases act as redundant negative regulators of ABA signalling (Gosti et al., 1999), a contention supported by analysis of the Arabidopsis genome. There are about 70 proteins with homology to members of the family in Arabidopsis, several of which are related to ABI1 and ABI2 proteins (The Arabidopsis Genome Initiative, 2000). In each of the respective mutants (i.e. abi1 and abi2), the gene encoding the PP2C has a point mutation that results in an amino acid substitution in the catalytic domain, thus diminishing the activity of the enzyme (Leung et al., 1994, 1997; Meyer et al., 1994; Rodriguez et al., 1998); the mutant alleles of these genes are dominant. The mutant seeds exhibit reduced dormancy and leaves of the mutant plants are prone to wilting.

Even though the abi1 mutant is characterised by reduced sensitivity to ABA, ABI1 is indeed a negative regulator of ABA signalling (Gosti et al., 1999). Protein phosphatase 2C activity is absent in intragenic revertants of abi1 and abi2 mutants and the revertants are characterised by ABA hypersensitivity (Gosti et al., 1999; Merlot et al., 2001). Over-expression of ABI1 in maize protoplasts decreases the ABA-mediated activation of a chimeric gene driven by an ABA-inducible promoter and this, along with other evidence (Tahtiharju and Palva, 2001) is consistent with the role of ABI1 as a negative regulator of ABA signalling (Sheen, 1998).

Expression of the ATHB6 gene is up-regulated by ABA and drought stress (Söderman et al., 1999). This gene encodes a homeodomain transcription factor of the HD-Zip class; the protein interacts with ABI1 and is likely a key regulator of ABA signalling (Himmelbach et al., 2002). The ATHB6 protein contains a phosphatase domain and a DNA-binding site at its N-terminus; both are required for its interaction with ABI1. The transcription factor acts as a negative regulator in ABA signalling and acts downstream of ABI1; its constitutive expression in transgenic Arabidopsis plants leads to ABA insensitivity in a subset of ABI1-dependent responses, one of which is manifested by a three- to five-fold reduction in ABA responsiveness as far as germination is concerned (Himmelbach et al., 2002).

A gene encoding a PP2C has been isolated from beechnut (Fagus sylvatica) (designated FsPP2C1). Expression of the gene is up-regulated by ABA. Further, FsPP2C1 gene transcripts increase during a two-week imbibition of dormant seeds and they decline throughout a dormancy-breaking moist chilling treatment, especially after 6 weeks of moist chilling, a treatment that elicits ~50% germination (Lorenzo et al., 2001).

In addition to PP2Cs that act as negative regulators of ABA signalling, there also appear to be phosphatases (PP2As) that function early in ABA signal transduction and act as positive regulators (Kwak et al., 2002). One such protein is RCN1 and rcn1 mutant seeds are characterised by reduced ABA inhibition of germination and a reduction in the ABA-induced transcripts encoding RD29A.

Characterisation of the protein kinase, PKABA1 provides further support for a role of reversible protein phosphorylation in ABA signalling (Anderberg and Walker-Simmons, 1992). PKABA1 is an ABA-responsive protein kinase whose synthesis is up-regulated in the dormant mature-imbibed cereal grain. Transient expression of PKABA1 in cereal aleurone layer cells is able to suppress the GA-induced expression of post-germinative genes (those encoding αα-amylases and a cysteine protease) (Gomez-Cadenas et al., 1999). However, the antagonistic effect of ABA on GA-mediated induction of αα-amylase is not altered by inhibiting PKABA1 gene expression using double-stranded RNA interference technology (i.e. transient expression of a PKABA1 RNAi). Thus, two independent ABA signalling pathways may lead to the suppression of αα-amylase in barley aleurone layer cells, one dependent and the other independent of PKABA1 (Zentella et al., 2002).

What are some of the proteins subject to phosphorylation by the wheat kinase? A wheat protein (TaABF, a homologue of ABI5) binds to PKABA1 in yeast two-hybrid assays. The nature of this interaction is specific, i.e. TaABF is a phosphorylation substrate of the kinase and interactions do not occur with a mutant form of PKABA1 that lacks the nucleotide-binding domain. Further, PKABA1 synthesised in transgenic cell lines is able to phosphorylate synthetic peptides representing three specific regions of TaABF (Johnson et al., 2002). PKABA1 gene expression is not exclusive to the seed, but is also responsive to exogenous ABA and to stress within vegetative tissues. In contrast, TaABF gene expression is seed specific. It is suggested that TaABF serves as a physiological substrate for PKABA1 in the ABA signal transduction pathway during grain maturation, dormancy maintenance and ABAsuppressed gene expression (Johnson et al., 2002).

Disruption of ABA signal transduction can lead to enhanced seed dormancy. Examples of this are the era (enhanced responsiveness to ABA) mutants which exhibit both increased sensitivity to ABA and enhanced primary dormancy (Cutler et al., 1996). The ERA1 locus encodes a ββ-subunit of a farnesyl transferase (Cutler et al., 1996). Farnesyl transferases are key enzymes in signal transduction as they effect a post-translational modification (attachment of a 15-C farnesyl pyrophosphate to proteins containing a CaaX motif). Farnesylation and other lipid modifications (e.g. geranylgeranylation, myristoylation, and palmitoylation) mediate the localisation of proteins to membranes (Yang, 2002). Membrane localisation in turn is a prerequisite for the correct functioning of several signal transduction proteins, including those belonging to the small GTPase superfamily. It is quite possible that negative protein regulators of ABA signalling require farnesylation in order to function (e.g. to attenuate ABA receptor activation).

Some evidence for this has come from an examination of ROP10, a member of the Arabidopsis Rop subfamily of RHO GTPases implicated in ABA signalling (Zheng et al., 2002). Despite normal ABA levels, and normal responses to other hormones, the null rop10 mutant is hypersensitive to ABA as far as germination is concerned. Freshly harvested rop10-1 seeds germinate at significantly lower rates than wild-type seeds, but after a4-day moist chilling period, the germination rates of mutant and wild-type seeds are similar. The ABA hypersensitivity of rop10 mutants also extends to root elongation and stomatal closure and ABA is able to specifically down-regulate the synthesis ROP10-encoding transcripts in roots. Constitutive expression of the normal ROP 10 gene in transgenic plants reduces ABA inhibition of seed germination. The negative regulation of ABA signalling by ROP 10 depends on the protein being correctly localised to the plasma membrane and the protein contains a putative farnesylation site to effect its targeting to this subcellular locale (Zheng et al., 2002).

In addition to protein modification, RNA processing has come to be viewed as a modulator of ABA signalling. A reporter system (the luciferase gene coding region linked to an ABA-inducible promoter) was exploited to identify mutations resulting in increased ABA sensitivity by identifying plants that showed abnormal bioluminescence in response to exogenous ABA (Xiong et al., 2001a). A further characterisation of one of the mutants uncovered (sad1) indicated that it was indeed supersensitive to ABA (although it also displayed reduced ABA biosynthesis), and one manifestation of this was an increased ability of ABA to inhibit seed germination and root growth and enhance expression of some ABA-responsive genes (particularly ABA-dependent phosphatase type 2C genes, previously implicated as negative regulators of ABA signalling (see above, Sheen, 1998). Interestingly, the SAD1 gene encodes a protein with similarity to the Sm-like proteins structurally related to Sm proteins contained in snRNPs that function in RNA splicing (reviewed in McCourt, 2001). Sm proteins (e.g. of animals and yeast) are multifunctional; most of their functions are related to the control of RNA processing (splicing, nuclear export and degradation). Another essential modulator of ABA signalling is ABH1, an Arabidopsis homologue of a nuclear mRNA cap-binding protein (Hugouvieux et al., 2001). Analyses of era1-2 and abh1 double mutants (both characterised by ABA hypersensitivity), suggest that ERA1 and ABH1 do not modulate the same negative regulator in ABA signalling (Hugouvieux et al., 2002). These and other studies intimate that RNA metabolism (e.g. mRNA turnover) is essential for the modulation of ABA signalling and may in fact directly modulate ABA receptor function (McCourt, 2001).

Heterotrimeric G proteins and G-protein-coupled receptors are early components of signal transduction pathways that control growth and differentiation in eukaryotes. Activation of G-protein-coupled receptors as a result of the binding of an extracellular ligand, triggers a transduction cascade that is mediated by a G protein; GTP binds to the αα-subunit of the G-protein, causing a conformational change that allows the αα-subunit to dissociate from the ββγγ subunit. Activation of the G protein in this manner then activates downstream components (ion channels and adenylate cyclase) that lead to increases in intracellular mediators (Ca2+ and cAMP, repectively). Ultimately, changes in cellular functions including the activation/repression of genes are effected. This particular pathway has not diversified over the course of plant evolution nearly to the extent of that in animals (Colucci et al., 2002 and references therein). Recently a gene of Arabidopsis encoding a putative G-protein coupled receptor (i.e. CGR1) was isolated. Overexpression of this gene in Arabidopsis creates transgenic plants that have an early flowering phenotype and produce seeds that lack dormancy. The transformants behave as if they have altered sensitivity to endogenous ABA and/or GA and seeds are less sensitive to exogenous ABA than are wildtype seeds as far as their germination is concerned. Overexpression of the CGR1 gene also induces the expression of the MYB65 gene, an Arabidopsis homologue of the GAMYB gene of barley, whose encoded product regulates GA-mediated αα-amylase induction in aleurone layer cells (Colucci et al., 2002).

Ca2+ is a major component of many signal transduction pathways in animals and plants and is involved as a downstream component of G-protein-linked signal transduction pathways. In animal cells there are two major mechanisms for the release of Ca2+ from intracellular stores. The first is the inositol 1,4,5-triphosphate (IP3)-dependent mobilisation of Ca2+ by activation of the inositol 1,4,5-triphosphate receptor. The second is the cyclic ADP ribose (cADPR)-mediated release of stored Ca2+ by the ryanodine receptor (Sanchez and Chua, 2001 and references therein). ABA treatment of plant cells causes a transient increase in the levels of cADPR and inositol 1,4,5-triphosphate; the precise role of the increased amounts of these molecules is unknown, but it is presumed that both may control occillations in Ca2+ that in turn are important for ABA signal transduction. Control of ABA responsiveness and attenuation of ABA signalling is suggested to involve turnover of phosphoinositols; one enzyme mediating this turnover is an inositol polyphosphate 1-phosphatase, a negative regulator of ABA signalling in seed and seedling tissues (Xiong et al., 2001b). The phenotype of the fiery1 mutant which contains a defective inositol polyphosphate 1-phosphatase is characterised by enhanced ABA sensitivity as far as seed germination is concerned; seedling growth is also more affected by ABA and by stresses such as drought, freezing and salt stress. Upon ABA treatment, there is a greater accumulation of inositol 1,4,5-triphosphate (IP3) in mutant plants than in wild-type plants (Xiong et al., 2001b). Inducible suppression and over-expression of genes encoding enzymes involved in phosphoinositol production and metabolism (phospholipase C1 and inositol 1,4,5-triphosphate 5-phosphatase) was effected by use of a glucocorticoid-inducible system in transgenic Arabidopsis (Sanchez and Chua, 2001). Enhanced phospholipase C1 activity and increased inositol 1,4,5-triphosphate (IP3) levels are necessary for maximal gene induction by ABA; however, overexpression of the phospholipase C1 gene itself is not sufficient to trigger the expression of ABA-responsive genes in vegetative tissues (including RD29a, KIN2 and RD22 genes). Phospholipase C1 and inositol 1,4,5-triphosphate may be involved as secondary signals that potentiate the primary ABA response, while cADPR may mediate the primary ABA response (Sanchez and Chua, 2001).

(5) Dormancy Termination Is Accompanied by Changes in ABA Biosynthesis, Turnover and Sensitivity

In general, whether a seed is dormant or quiescent at maturity, both the amount of ABA and the sensitivity of the embryo to ABA decline during seed development, especially during late maturation and desiccation (Kermode et al., 1989; Xu and Bewley, 1991; Kermode, 1990, 1995; Jiang et al., 1996; Bewley, 1997; Schmitz et al., 2000, 2002a, 2001). Genetic studies have clearly demonstrated a function for ABA in dormancy imposition during seed development (see discussion above) (Karssen et al., 1983; Kermode 1995; Bewley, 1997; Phillips et al., 1997; Foley, 2001). Moreover, de novo synthesis of ABA appears to be necessary for dormancy maintenance following seed imbibition (e.g. in seeds of sunflower, barley, beechnut, lettuce, and Douglas-fir) (Le Page-Degivry and Garello, 1992; Wang et al., 1995; Bianco et al., 1997; Le Page-Degivry et al., 1997; Yoshioka et al., 1998). For instance, when tobacco seeds are imbibed, there is an accumulation of ABA in dormant seeds but not in seeds that have been allowed to afterripen. The carotenoid- and ABA-biosynthesis inhibitor fluridone, when used in conjunction with gibberellic acid (GA3), is effective in breaking dormancy; exogenous application of both chemicals to seeds inhibits accumulation of ABA during imbibition (Grappin et al., 2000). Similar to tobacco, GA3 and fluridone are effective in breaking dormancy of yellow-cedar seeds in the absence of any additional treatment (Schmitz et al., 2001). These seeds normally require a three-month dormancy-breaking treatment consisting of one month of warm, moist conditions followed by two months of moist chilling (Ren and Kermode, 1999). However, fluridone alone (i.e. with no GA3) is much less effective in eliciting germination, indicating that a decline in ABA amount alone is not sufficient to break dormancy and other changes (e.g. synthesis of gibberellins) may also be necessary.

In Pinus sylvestris (Scots pine) seed, dormancy-breaking treatments that include either white or red light decrease ABA levels prior to radicle protrusion; seeds subjected to a far-red light pulse after red light do not exhibit as great a decline in ABA, nor is dormancy relieved (Tillberg, 1992). During dormancy breakage of yellow-cedar seeds, there is about a 2-fold reduction of ABA in the embryo; in the megagametophyte, ABA does not change; however, the embryos exhibit a change in both ABA turnover and in their sensitivity to ABA (see subsequent discussion) (Schmitz et al., 2000, 2002a). ABA decreases in both the megagametophyte and embryo during moist chilling of Douglas fir seeds; in the former, ABA declines 4-fold during 7 weeks of moist chilling (Corbineau et al., 2002). The longer the duration of moist chilling, the faster the rate of ABA decline during subsequent germination (Corbineau et al., 2002).

Changes in ABA concentration within embryos and surrounding seed tissues can contribute to dormancy inception, maintenance, and termination (Cutler and Krochko, 1999; Schmitz et al., 2000, 2002a). However, it is abundantly clear that levels of ABA and ABA catabolites in plant cells and tissues are under constant flux as a result of the opposing forces of biosynthesis versus degradation (reviewed in Cutler and Krochko, 1999; Seo and Koshiba, 2002). Thus, ABA amount is not likely to be immediately indicative of changes in dormancy status; rather, the capacity for biosynthesis versus catabolism (metabolic flux) is a superior indicator of whether a seed will or will not terminate dormancy (i.e. germinate). Increased ABA catabolism is associated with dormancy termination of seeds of yellow-cedar, beechnut, Douglas fir and barley (Le Page-Degivry et al., 1997; Schmitz et al., 2000, 2002a; Corbineau et al., 2002; Jacobsen et al., 2002).

The degradation or inactivation of ABA occurs via oxidation and conjugation. The major pathway by which ABA is catabolised is through hydroxylation at the 8′ position to form 8′hydroxy-ABA, which reversibly cyclises to phaseic acid (PA) (Figure 33.6). Further reduction of PA can take place at the 4′ position to form dihydrophaseic acid (DPA). ABA and ABA metabolites (PA and DPA) can also become conjugated with glucose forming an ester or glucoside. Other minor pathways include formation of 7′hydroxy-ABA (7′OH-ABA) and ABA 1′,4′diols (Cutler and Krochko, 1999; Zeevaart, 1999)

in yellow-cedar seeds, dormancy-termination is accompanied by a change in the ability of the embryo to metabolise ABA, in which 8′-hydroxylation becomes rate-limiting (Schmitz et al., 2002a). Changes in ABA metabolism as a result of dormancy-breakage, were determined by examining the effects of a metabolism-resistant ABA analogue S-(+)-d6-ABA (S-[8′, 8′, 8′, 9′, 9′, 9′]-hexadeuteroabscisic acid) on the germination of isolated yellow-cedar embryos as compared to natural S-(+)-ABA. The analogue contains deuterium atoms in place of hydrogens in six positions of the natural ABA molecule, generating a hexadeuteroabscisic acid with deuterium-labelled ring methyl groups that render it more resistant to oxidation by 8′-hydroxylase and thus more persistent and possessing greater activity. During an effective dormancy-breaking treatment imposed upon intact yellow-cedar seeds, the excised embryos become increasingly less sensitive to the metabolism-resistant ABA analogue as far as their germination is concerned. These embryos also become less sensitive to natural S-(+)-ABA, and yet, there is an isotope effect, such that the analogue consistently slows the germination rate to a greater extent. Thus, the conversion of the C–H bond to the C–OH bond becomes rate limiting as a result of yellow-cedar dormancy-breaking treatments indicating that a change in ABA metabolism is associated with dormancy termination. These changes in metabolism, together with reduced embryo sensitivity to ABA are important for eliciting high germinability of whole seeds (Schmitz et al., 2002a). In cress seeds, a deuterated analogue slows germination to a greater extent than S-(+)-ABA when applied in equivalent concentrations (Lamb et al., 1996), clearly indicating the importance of ABA metabolism and ABA 8′-hydroxylase in the modulation of germination rates in vivo. For yellow-cedar, effective dormancy termination (caused by subjecting seeds to moist chilling in conjunction with other treatments) may have a direct effect on the 8′-hydroxylase enzyme, as well as cause changes that affect ABA reception or possibly downstream signal transduction events (Schmitz et al., 2002a).

Effective dormancy breakage likely stimulates ABA metabolism, but at the same time may reduce ABA biosynthesis. Moist chilling of western white pine seeds is accompanied by a significant decrease in ABA in both the embryo and megagametophyte (Figure 33.7 and Feurtado et al., unpublished). More importantly, the decline of ABA after different durations of moist chilling correlates well with an increased capacity of seeds to germinate following their transfer to germination conditions. The decline of ABA (due to enhanced breakdown and/or reduced synthesis) continues during germination (Figure 33.7). Thus, moist chilling not only effects a decline in ABA as chilling proceeds by stimulating ABA metabolism but, perhaps equally important, it seems to increase the capacity for ABA catabolism, particularly when the seeds are subsequently placed in germination conditions. Over time in conditions that are not effective for breaking dormancy, ABA increases in western white pine seeds but there is a decrease of PA, 7′OH ABA, and DPA (or maintenance at steady-state amounts) suggesting a slower rate of ABA breakdown.

Studies to monitor changes in the amount of ABA in seed tissues should be extended to analyses of the nature of the ABA metabolites. For example, the importance of ABA catabolism via 8′-hydroxylation versus other pathways of catabolism (e.g. those involving 7′-hydroxylation of ABA) varies between species and even between different tissues and different developmental stages of the same species (Cutler and Krochko, 1999; Zeevaart, 1999). This is the case in western white pine seeds. In the megagametophyte, ABA is metabolised through 8′-hydroxylation, generating PA and DPA; both metabolites increase during a 98 day moist chilling period. In the embryo, ABA is metabolised through both the 8′- and 7′-hydroxylation pathways as well as by conjugation to glucose (Feurtado et al., unpublished).

Despite these more detailed studies to examine the role of ABA metabolism in dormancy termination and germination, several questions remain to be addressed. One pertains to the distribution of ABA metabolites in different parts of the seed and whether this changes during germination. Does ABA-GE represent an irreversibly inactivated form of ABA, or can it later generate free ABA? It is generally assumed that ABA-GE is not a storage form of ABA and that the conjugation is irreversible, with the conjugate being sequestered in the vacuole (Kleczkowski and Schell, 1995; Zeevaart 1999). While it is clear that ABA is metabolised through various pathways, it is not known what other pathways exist or what the end-points of catabolism are. Further, it is not known what the biological roles of the initial catabolites of ABA are (i.e. those of 7′OH ABA and 8′OH ABA). It is generally is assumed that ABA glucose ester, PA and DPA are inactive, while the hydroxy ABA catabolites (7′- and 8′-hydroxy-ABAs) can act as signalling molecules (Zuo et al., 1995; Hill et al., 1995).

Mechanisms that underlie the fine-tuned balance between ABA biosynthesis and its catabolism and potential transport within and between seed tissues need to be elucidated. Changes in ABA sensitivity (and sensitivity to certain metabolites) undoubtedly also play a role in the transition of seeds from a dormant to non-dormant state.

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