Genetic Engineering, Volume 25. Edited by J.K. Setlow
Kluwer Academic/Plenum Publishers, 2003 (pp. 113-141). FUNCTIONAL ANALYSIS OF PROMOTER ELEMENTS IN PLANTS Slavko Komarnytsky and Nikolai Borisjuk Biotech Center, Cook College Rutgers University 59 Dudley Rd., New Brunswick, NJ 08901-8520 INTRODUCTION Plant growth and development involve temporal and spatial expression oI the speciIic genes subsets in response to various physiological and environmental Iactors mediated by complex signal transduction pathways. A minimal gene regulatory network typically consists oI an input signal receptor and a transduction pathway linking the extracellular environment with intracellular targets; a core complex oI transacting regulatory proteins and relevant cis-acting DNA sequences; and a subsequent molecular output (RNA and protein) Irom the target genes. Generally, the result oI the activation oI such a regulatory pathway is stimulation or repression oI expression oI the genes coding Ior structural, metabolic, and behavioral capacities oI the plant cell. In addition, such networks oIten 2 include dynamic Ieedback loops that provide Ior Iurther regulation oI the network architecture and output. Central to speciIic activation are DNA recognition sequences which interact with basic transcription initiation complexes and numerous transcription Iactors. In plants, these are usually 5'-Ilanking modules that include a core promoter, proximal regulatory elements, and upstream enhancer sequences located close to the structural portion oI the gene (1). Regulatory elements can oIten be positioned quite Iar Irom the transcription initiation site (TIS) in mammalian genomes, whereas in yeast and plants they are located within a Iew thousands base pairs oI the TIS (2). The control regions oI plant genes may include multiple cis-acting elements that contribute to the complex expression proIile oI that particular gene. Moreover, the same transcription Iactors can act as activators or repressors depending on their concentration and the presence oI interacting partner proteins (3). Earlier in the Genetic Engineering series, GuilIoyle (4) published an excellent review Iocused on the basic structure oI plant promoters and the conservation oI speciIic cis-acting elements within promoters that respond similarly. While many oI these regulatory elements are well deIined today, there is little logic apparent in the organization oI multiple regulatory elements, and even less in the way that they interact to regulate gene expression. ThereIore, this chapter is aimed at illustrating the diversity and intricacies oI plant regulatory sequences, and highlighting how their interactions govern the structural and Iunctional interplay oI signal perception pathways. We also attempt to provide a deeper insight into the regulatory Iunction oI AT- rich sequences Iound in plant promoters. 3 TRANSCRIPTIONAL ACTIVITY IN PLANT NUCLEUS The eukaryotic nucleus contains three diIIerent classes oI RNA polymerases (pol). RNA pol I is responsible Ior the transcription oI ribosomal DNA genes, and is active only in the nucleolus region. RNA pol II transcribes all protein-encoding genes and several small nuclear RNAs; while RNA pol III transcribes 5S ribosomal RNA genes, transIer RNA genes, and some small nuclear RNAs. There is strong evidence that Iunctional separation between all three major classes oI polymerases is Iurther enhanced by spatial compartmentalization oI their activity in the diIIerent domains oI the eukaryotic nucleus (5). Plant genes transcribed by RNA pol II typically contain common core elements recognized by general transcription Iactors, and gene-speciIic DNA elements recognized by regulatory Iactors, which in turn modulate the Iunction oI the general initiation Iactors (6, 7). The availability oI whole genome data and sophisticated microarray technology has opened up new avenues Ior the analysis oI gene regulation and expression. Unraveling the regulatory network(s) that inIluence expression oI a given gene or gene Iamily is typically based on the a priori assumption that co-regulated genes usually have common regulatory elements. Eunctional dissection oI the promoter region usually requires identiIication and characterization oI a minimal promoter, location oI the putative binding sites oI known and unknown transcription Iactors, and sorting out the eIIects oI distant enhancer- and repressor-like modules. Local chromatin states, and the availability oI scaIIold attachment regions and DNA methylation sites, may Iurther contribute to the activity oI the 5`-Ilanking region. 4 The primary strategy Ior the Iunctional analysis oI any promoter is a computer- assisted scan over the entire sequence by a consensus approach, which relies on known or conserved DNA elements. This is generally achieved by querying the sequence oI interest against the reIerential databases using scanning soItware that does not require download and installation (Table 1). This inIormation allows preliminary assessment oI promoter strength, speciIicity, and regulation. Subsequently, a series oI wet -lab experiments must be perIormed to dissect the individual and combinatorial activities oI the putative motiIs both in vitro and in vivo (see below Ior recent reports and reIerences). D promoter Iusion to a reporter gene (8) D Iusion to a minimal promoter (9) D transient or stable expression assay (10) D promoter deletion and base substitution (11) D insertional mutagenesis (12) D linker-scanning mutagenesis (13) D high-throughput SELEX-SAGE (14) D one-hybrid yeast system (15) D DNA-protein crosslinking (16) D electrophoresis mobility shiIt assay (17) D primer extension (18) D DNase I Iootprinting (19) D RNase protection assay (20) D in vitro transcription systems (21) CORE PROMOTER Eukaryotic transcription is regulated by two main classes oI transcription Iactors: general transcription Iactors (GTEs) and sequence-speciIic transcription Iactors (reviewed in 22). GTEs bind to the core promoter close to the TIS and, together with RNA pol II 5 assemble in an ordered Iashion to Iorm the pre-initiation complex. It has been suggested that approximately 15 oI the genes oI Arabidopsis encode Ior transcription-related proteins comprising more than 1500 transcription Iactors, 45 oI which are speciIic to plants (23). About one-third oI all plant genes are expressed throughout the entire plant, albeit at variable levels. Another one-third may have their products present in a Iew organs, and the rest may belong to a unique organ as Iar as expression is concerned (24). Core promoter elements are deIined as minimal DNA elements that are necessary and suIIicient Ior accurate initiation oI transcription by RNA pol II in a reconstituted cell -Iree system (6). Several major elements, acting independently or collectively, mediate the direct binding oI the TEIID complex to a proper TIS (25). The Iirst element described as regulating this process was a classical TATA box, TATA(A/T)A, located 25 to 30 base positions upstream oI TIS (26). However, subsequent studies suggested that AT-rich sequences completely unrelated to the TATA-box stimulate transcription with equal or increased eIIiciency (27). Eurthermore, although the Iirst step oI transcripti on initiation is highly speciIic, TEIID also binds with high aIIinity to several TATA elements that do not match the consensus sequence and is active in promoting transcription in vitro Irom these elements (28). Another weakly conserved 'initiator element was described in the direct vicinity oI the TIS (29). This element anchors the TEIID complex in the absence oI the TATA-box, and may Iunction synergistically in TATA-mediated initiation (30). Mammalian initiator elements share a consensus PyPyAN(T/A)PyPy core (31). A similar pyrimidine rich initiator motiI has been described in the promoter oI the nuclear psaDb gene encoding the Ierredoxin-binding subunit oI photosystem I, although the overall 6 Irequency oI TATA-less promoters in non-photosynthetic plant genes is less than 10 (32). Two more elements that contribute to the basic transcription initiation were described recently. The 'downstream promoter element, (A/G)G(A/T)CGTG, located at position 30 bp downstream oI the TATA-less TIS, was able to direct transcription initiation in Drosophila (33) and inIluence the activity oI the tungro bacilliIorm virus promoter in transient expression assays in rice protoplasts (34). Einally, a Iorth class oI potentially active core promoter elements with little consensus homology other than GC-rich composition, has been reported in the mammalian system (35). Earlier, a homologous conserved GC-rich element was described immediately upstream oI the TATA-box in the barley pr1 promoter (36). These Iindings strongly suggest that even though most oI the knowledge oI the core promoter elements was initially derived Irom yeast and mammalian models, the major principles oI the regulation oI gene expression are conserved in plants as well. A possible explanation Ior the observed di versity in the core promoter elements could be their contribution to the combinatorial gene regulation, including the restriction oI enhancer stimulatory capacity to a speciIic set oI promoters (37). Two more proximal regulatory elements, not universally required Ior the activity oI the core eukaryotic promoters, when present, signiIicantly aIIect their initiation Irequency. These sequences are located close to TIS (around 100 bp) and appear to assist in the recruitment oI basic transcription Iactors to the TIS sites oI housekeeping and TATA-less genes. The GC-box, GGGCGG, plays an important regulatory role in the core promoter regions oI mammalian genes (38). Co-transIection oI expression cassettes 7 together with the GC-box binding protein usually results in a Iour-, to ten-Iold increase in GC-box-regulated promoter activity (39). GC-boxes have also been localized in the upstream promoter regions oI many plant genes (40- 42). Another well conserved element, the CCAAT-box, is oIten present at 80 to 150 bp upstream oI TIS, and may operate cooperatively with other putative conserved motiIs (43). However, no uniIying expression pattern Ior plant genes containing putative CCAAT elements has been observed (44, 45). Moreover, multiple copies oI the genes coding Ior the subunits oI the CCAAT-binding protein exist in Arabidopsis, suggesting the potential Ior multiple alternative Iorms oI these complexes in plants (46). UPSTREAM REGULATORY ELEMENTS In plant promoters, all the necessary inIormation to direct proper gene expression is generally located in a very compact region, less than 1000 bp upstream oI the TIS. Only Iew plant promoters are known to be constitutively expressed in most plant cells. They include promoters Irom housekeeping genes like ubiquitin (47) or actin (48). Interaction oI the upstream regulatory elements with sequence-speciIic transcription Iactors (SSTEs) determines the time, place and level oI activity oI all genes within a set oI highly coordinated expression networks. LIGHT-RESPONSIVE ELEMENTS (LREs) 8 Higher plants have developed a complex biochemical system to perceive and respond to light, which is based on three classes oI photoreceptors: phytochromes, blue-light receptors, and receptors Ior UV-light. Light signals absorbed by these photoreceptors regulate transcription oI a large number oI genes that encode proteins involved in photosynthesis and various developmental processes through a complex signal transduction cascade (49). It is generally believed that diIIerent light-activated pathways target diIIerent LREs and transcription Iactors within the promoter region, but they may also target common LREs (50). Extensive mutagenesis and deletion analysis oI the promoter regions oI light-induced genes resulted in the identiIication oI a number oI cis- acting elements involved in the control oI light-regulated transcription. Several oI these motiIs, such as the GT1-box (GGTTAA), I-box (GATAAAGR), G-box (CACGTG) and H-box, ACCTA(A/C)C(A/C), have been experimentally shown to be important components in the light response. Although similar LREs are present in many diIIerent promoters and are assembled in a variety oI combinations, no universal element has been Iound in all photoregulated promoters. Some oI the LREs are present in promoters that are not light-regulated (51). Eurthermore, the same LRE is Irequently Iound in promoters with opposite light responsiveness, as was shown Ior the GT1-box (52). None oI the LREs identiIied so Iar has been shown to conIer light responsiveness by itselI. Eor example, while a tetramer oI the GT1-binding element was able to conIer light regulation on a minimal (90 bp) 35S promoter containing the as-1 element, it Iailed to act in the same way when combined with a shorter version oI the 35S promoter (46 bp) lacking as-1 (53). It has also been shown that only artiIicial sequences composed oI paired 9 combinations oI tetrameric repeats oI G- and GATA-boxes, or GT1- and GATA-boxes, but not multimers oI a single motiI, can Iunction as LREs (54, 55). These and many other experimental data resulted in a general hypothesis that plant LREs are actually composite elements, i.e. combinations oI cis-regulatory sequences in which their overall activity is the result oI synergetic interactions between cognate transcription Iactors (56, 51). The modular component oI LREs is composed oI two general elements, the light-speciIic element, and a coupling element. Light-speciIic elements are targeted by transcription Iactors that are regulated by light. Coupling elements bind protein Iactors that direct the light stimulus to transcription in a spatial or temporal manner, or determine the relative strength oI the light-induced gene expression. Numerous protein Iactors that bind to cis-elements containing GATA- and GT1-core sequences have been identiIied and extensively characterized (51, 57). Although in many studies clear diIIerences in speciIicities and suggestive changes in activity in response to light were described, the conclusive Iunctions oI these DNA-binding proteins in light- regulated transcription has yet to be determined (58). The only two transcription Iactors, Ior which there is strong evidence suggesting their importance in light -mediated regulation, are Arabidopsis Iactors HY5 (59) and PIE3 (60), which interact with the G- box. The G-box-bound PIE3 was shown to speciIically bind phytochrome B Iollowing light-stimulated conversion oI the photoreceptor to its biologically active Iorm. HY5 was shown to be required Ior light induction oI the minimal light-responsive module CMA5, a native 52-bp Iragment oI tobacco rbcS 8B promoter, which contains I- and G-box motiIs. As HY5 itselI does not have an activation domain, it is assumed that it could aIIect transcriptional activity through interaction with other Iactors (60). 10 METABOLIC REGULATION Hormones play a key role in regulating plant growth and development. Recent evidence has suggested that sugars control gene expression and developmental processes in a manner similar to classical plant hormones (61). The question is: how do relatively simple molecules regulate a variety oI responses within an assortment oI cells, tissues, and organs oI plants? The answer to this question requires an understanding oI hormone and sugar perception and signal transduction as a part oI the metabolic regulation oI gene expression in plants. Auxin-Responsive Elements (AuxREs) Auxin plays an important role in root Iormation, apical dominance, tropism, and senescence at the organism level, while acting as a signal Ior division, extension, and diIIerentiation inside the plant cell. Auxin-mediated responses at the gene level can be detected as early as two to Iive minutes aIter auxin application (62, 63). The most extensively studied auxin-responsive plant gene promoters are those Irom the pea PS- IAA4/5 gene (64), the soybean GH3 gene (65, 66), and the soybean Small Auxin-Up RNA, SAUR15A gene (67). These studies led to the identiIication oI the cis-acting motiIs (G/T)GTCCCAT, within an AuxRE oI the pea PS-IAA4/5 promoter (64), and TGTCTC, within the three small AuxREs oI the soybean GH3 promoter (65, 66). An AuxRE oI the SAUR15A promoter contained both types oI these motiIs (67). A Iunctional study oI AuxREs in the soybean GH3 promoter showed that the TGTCTC element requires a 11 closely associated constitutive or coupling DNA element to Iunction as an AuxRE (66). Similarly, TGTCTC and TGTCCCAT elements in the soybean SAUR15A promoter and the pea PS-IAA4/5 promoter may also Iunction as composite AuxREs that contain diIIerent coupling elements than those Iound in composite AuxREs oI the soybean GH3 promoter (63). Within the composite AuxREs, auxin-responsive elements repress the transcription-stimulating activity oI the adjacent or overlapping constitutive element when auxin level is low. When auxin level is high, the repression is released and the composite element is activated (63). Depending on the nature oI the constitutive or coupling elements oI diIIerent AuxREs, they could potentially conIer a variety oI tissue- speciIic and developmentally regulated expression patterns (68). Both TGTCTC and TGTCCCAT elements can Iunction as AuxREs in the absence oI any coupling element when multimerized with appropriate spacing and orientation (64, 69, 70). Properly spaced and oriented, TGTCTC AuxREs are several Iold more active than natural AuxREs (69, 70). A highly active palindromic repeat oI the TGTCTC element was used as bait in a yeast one-hybrid system to identiIy the Iirst member oI the auxin responsive Iactors, ARE1, Irom Arabidopsis (69). Both the ARE Iamily and the Aux/IAA proteins have been identiIied as key regulators oI auxin-modulated gene expression (71), however, the DNA-binding activity oI the Aux/IAA proteins has not been conclusively demonstrated (72). Aux/IAA proteins are believed to regulate transcription by modulating the activity oI AREs (63). Another AuxRE that has received considerable attention is the ocs/as-1 element (73). Originally Iound in the CauliIlower Mosaic Virus (CaMV) 35S promoter, activation sequence-1 mediates both salicylic acid- and auxin-inducible transcription activation, and 12 consists oI two imperIect TGACGTCA palindromes (74). Whereas the sequence can deviate quite substantially Irom its consensus, the12 bp space between the two palindromic centers is conserved in all osc/as-1 elements that respond to auxin (75). This element was also detected in several plant promoters, including the auxin-inducible tobacco Par genes (73, 76). A nuclear protein complex, ASE-1, that binds to the TGACGTCA palindrome was identiIied by using electrophoretic mobility shiIt assay (74). A number oI cDNAs encoding as-1-binding TGA proteins have been described in tobacco (77, 75). Gibberellin-Responsive Elements (GAREs) The mechanism by which the gibberellic acid (GA) signal is perceived and transduced in plants has been studied extensively using aleurone-speciIic expression as a model (78). Analysis oI GA regulation oI ,-amylase promoters uncovered cis-acting elements that are suIIicient Ior gibberellin responsiveness (79). These included the GARE motiI (TAACA(A/G)A), TATCCAC-box, and pyrimidine box (C/T)CTTTT(C/T). Eurther Iunctional analysis oI the barley ,-amylase gene promoter showed that TAACA(A/G)A is very similar to the c-Myb consensus binding site. Subsequently, a novel MYB-related clone (GAmyb) was isolated Irom a barley aleurone cDNA library. When the ,-amylase promoter was Iused to the uidA reporter gene (GUS), GAMyb was the sole gibberellin-regulated transcription Iactor required Ior transcriptional activation oI the expression cassette in the absence oI GAs (80). Similarly, synthesis oI EPB-1, a cysteine proteinase responsible Ior the degradation oI seed endosperm storage proteins in barley, is induced by GAs and repressed by ABA. 13 Eunctional analysis oI the EPB-1 promoter revealed that the GARE, a pyrimidine box, and an upstream element are all necessary Ior GA induction. Constitutive expression oI the GAMyb transcriptional Iactor, in the absence oI GA, led to the transactivation oI EPB-1 expression in a dosage dependent manner with the highest level comparable to that in Iully GA-induced tissue (81). Eunctional analysis oI another GA-regulated wheat ,-amylase promoter, ,-Amv2/54, has also indicated that three elements are essential Ior GA-induced expression. They include the proposed GARE module and a new putative element GATTGACTTGACC (82). It is interesting to note that the GA-responsive ,- amylase gene was reported to be sugar-repressed in rice embryos, and that both GARE and the pyrimidine box have been partially involved in sugar-induced gene repression (83). Abscisic Acid-Responsive Elements (ABREs) Multiple ABREs are located upstream oI the ABA-induced genes. The G-box with the sequence (C/G/T)ACGTGGCG, is probably the most studied element subject to ABA regulation (84). Multiple tandem copies oI this module conIer ABA responsiveness when Iused upstream oI the minimal 35S promoter (79). While the isolated G-box Iailed to conIer ABA responsiveness to the minimal promoter, a complex module comprising the G-box and a coupling element Iunctioned as an ABRE in vivo (85). Multiple ABREs were identiIied in the 5` Ilanking region oI the wheat Em gene (86), and shown to be activated in the presence oI VP1 transcriptional activator (87). Typically, the presence oI VP1 enhances ABA induction oI late embryogenesis genes, but also suppresses germination speciIic genes (88). As with auxins, organ- and species-speciIic activation oI 14 ABA-responsive genes is achieved only by the cooperative action oI several cis-acting elements (89). Ethylene-Responsive Elements (EREs) The best-known eIIect oI ethylene is the promotion oI Iruit ripening. Other notable processes regulated by ethylene include seed germination, senescence, and responses to stress Iactors such as Ilooding, wounding or pathogen attack (90). In climacteric Iruits such as tomatoes, apples, bananas and avocados, the initiation oI ripening is associated with a burst in ethylene biosynthesis, and signiIicant alteration in the expression proIile oI many genes (91). A Iunctional ERE was identiIied in the tomato Iruit ripening genes E4 and E8 (92, 93). Transcription oI E4 is rapidly activated by ethylene in both leaves and Iruit, but E8 is activated by ethylene only in Iruit, and is additionally regulated by separate Iruit ripening developmental signals. Sequences required Ior both ethylene responsiveness and Iruit ripening regulation were identiIied within the 161 bp upstream oI the E4 transcription start, and the sequence Irom 85 to 140 was shown to be essential Ior ethylene-responsive gene transcription, but not suIIicient to conIer ethylene- responsiveness to the minimal 35S promoter (94). It has been concluded that at least two cooperative cis-acting sequences, an upstream (150 to 121) and a downstream (40 to 65) regulatory elements, are required Ior ethylene-responsive regulation oI E4 (94). A nuclear protein that binds to the 5`-Ilanking regions oI both E4 and E8 genes, E4/E8BP, was identiIied by gel shiIt assay and DNase I Iootprinting, and subsequently cloned (95). A truncated version oI E4/E8BP-1 was shown to transactivate the E4 promoter in a 15 transient assay, demonstrating that this DNA-binding protein can activate the E4 promoter to speciIically enhance gene transcription in vivo. The senescence oI carnation Ilower petals involves an increased biosynthesis oI ethylene. A 126 bp sequence within the promoter region oI the carnation glutathione-S- transIerase gene was shown to be necessary and suIIicient Ior ethylene regulation during petal senescence (96). A protein that interacts with that sequence was identiIied and shown to have speciIic DNA-binding activity in pre-senescent petals, senescent petals and petals treated with ethylene. DNase I Iootprinting deIined the DNA sequence between 510 and 488 within the region speciIically protected by bound proteins. This protein-protected region shares an 8-bp sequence A(T/A)TTCAAA with the protein binding sequence oI the E4 promoter, raising the possibility that Ilower petal senescence and Iruit ripening may have some common mechanisms Ior ethyl ene gene regulation (93). Sugar-Responsive Elements (SUREs) Biochemical, molecular, and genetic experiments all support a central role Ior sugars in the control oI plant metabolism, growth, and development, and have revealed interactions that integrate light, stress, hormone signaling, and coordinate carbon and nitrogen metabolism (61, 97). It has been proposed that sugar transporter-like proteins and some extracellular sugar-binding proteins can serve as sugar sensors to perceive and transmit sugar-mediated signals which alternatively may be triggered by hormones, light, and other environmental stimuli that cross-talk with sugar signaling pathways (61, 98). 16 Sucrose-responsive elements were Iound in the promoters oI genes coding Ior cereal ,-amylases (99, 100), potato tuber storage proteins (101), sweet potato vegetative storage proteins (10, 102, 103), potato sucrose synthase (104), bean photosynthesis protein RBCS2 (105), and the cucumber malate synthase (106). No conserved cis-acting element (common to all sugar-regulated promoters) has been reported, indicating perhaps the complex nature oI sugar signaling pathways. A sugar response sequence (SRS) Iound in the promoter oI a sugar starvation-inducible rice amylase gene, Amv3, was shown to conIer sugar responsiveness to a minimal promoter. The SRS contains three essential motiIs: a GC-box, a G-box, and a TATCCA element, identiIied at 90 to 150 bp in all promoters oI ,-amylase genes isolated Irom cereals (107). The TATCCA element bound three structurally related rice OsMYBS proteins in yeast one-hybrid assays, and two oI them, OsMYBS1 and OsMYBS2, were able to transactivate a TATCCA-containing promoter in vivo (100). Analysis oI the --amylase promoter oI sweet potato, which is induced by metabolizable sugars such as sucrose, glucose and Iructose, detected a TGGACGC element that is important Ior sugar-inducible expression oI both sporamin and --amylase genes (10). Sucrose-responsive elements SURE1 (AATAGAAAA) and SURE2 (AATACTAAT) were Iound in the promoter region oI patatin (103). A similar SP8 motiI (TACTATT) was present in the promoters oI the sweet potato sporamin and --amylase genes (102, 103). The SP8 element speciIically binds protein Iactor SPE1, which is a negative regulator that is transcriptionally repressed by sucrose (102). A putative homolog oI SPE1 that encodes a WRKY domain transcription Iactor, has been also Iound in cucumber and Arabidopsis (108). Both the W-box (wound-responsive) and G-box (ABA- 17 responsive) elements have been reported in sugar-responsive amylase promoters (109, 110). Exogenous application oI ABA was shown to activate both the --amylase (111) and --phaseolin promoters, the latter being modulated by externally supplied sucrose at the same time (112). Together, these data provide strong evidence that sugar, hormone, and deIense signaling may converge in the transcriptional control oI diverse promoters through activation oI the W- and G-box elements. ABIOTIC STRESS Heat-Stress Responsive Elements (HSEs) The heat-shock response is widely conserved in all living cells (reviewed in 113), and heat stress transcription Iactors are the central proteins in this process. Despite their general variability in sequence and size, their structure and promoter recognition sequences are remarkably conserved among the eukaryotes. All oI them comprise an N- terminal DNA-binding domain, the hydrophobic core oI which ensures the precise location oI the central helix-turn-helix motiI at the HSE. This element contains a repetitive pattern oI palindromic binding motiIs, nGAAnnTTCnnGAA (114), and plays a major role in the heat-shock response in tomato (115), soybean (116), and sunIlower (117). Eor example, expression oI the ascorbate peroxidase gene is oIten induced by both oxidative stress and a subsequent heat-shock response, the latter resulting Irom the accumulation oI hydroxyl and superoxide radicals, and hydrogen peroxide (118). Sequence comparison oI the promoter regions upstream oI the ascorbate peroxidase gene Irom pea (119) and Arabidopsis (120) revealed the presence oI only one region oI high 18 homology that is located around the TATA box, and contains several sequence motiIs characteristic oI the HSE identiIied in promoters oI all heat-shock-inducible genes. This putative HSE was recognized by the tomato heat-shock Iactor in vitro, and contributes partially to the induction oI the ascorbate peroxidase gene by oxidative stress (121). Some plant genes use more than a HSE to regulate their thermo-induced expression. Eor example, the soybean heat shock gene Gmhsp17.3-B is regulated via the classical HSE, but Iull promoter activity requires additional sequences located upstream oI HSE. Structural Ieatures within this putative enhancer region include a run oI simple sequences which are also present upstream oI the HSE-like sequences oI other soybean heat shock genes, and three perIect CCAAT boxes located immediately upstream Irom the most distal HSE oI the promoter (122). While the AT-rich domain oI the 5' Ilanking region was unable to direct transcription Irom the TATA box oI a truncated 35S promoter, heat - inducible transgene activity was detectable when additional sequences Irom the native promoter, containing three CCAAT boxes and a single HSE, were present in the constructs. Oxidative-Stress Responsive Elements Increasing evidence indicates that H 2 O 2 Iunctions as a stress signal in plants, mediating adaptive responses to various stresses by modulating expression oI many genes, including those coding Ior antioxidant enzymes and modulators oI H 2 O 2 production (123, 124). A global microarray analysis oI gene expression in response to H 2 O 2 showed that approximately 1 oI the Arabidopsis transcriptome is regulated by H 2 O 2 . OI these genes, some were also regulated by various stimuli such as UV-light, 19 elicitor treatment, and drought stress (125). Despite the Iact that H 2 O 2 -responsive promoters were identiIied in that study, neither signaling pathway(s) nor transcription Iactors or H 2 O 2 -regulatory DNA sequences have yet been isolated and characterized. However, several promoter elements such as W-, G-, and H-boxes, and the ethylene- responsive GCC element, which are present in the oxidative stress-responsive part oI plant promoters, have been put Iorward as candidates Ior H 2 O 2 -responsible motiIs (126). There is strong evidence that ethylene, ABA, SA, and calcium contribute to the combinatorial regulation oI the response against to oxidative damage in Arabidopsis. Both inhibitor and genetic data suggested that ethylene, SA, and ABA are actually used by Arabidopsis in vivo to protect plant tissues against heat-induced oxidative stress (127). The Iuture challenge will be to determine what signaling pathways these Iour components are involved in, and to identiIy the other signaling components in these pathways. Cold-, Drought-, and Osmotic Stress- Responsive Elements When exposed to low temperatures, plant cells encounter three major problems: changes in the spatial organization oI biological membranes, retardation oI biochemical and chemical reactions, and alterations in the availability and status oI water. The latter physiological state is oIten induced by the direct eIIect oI drought and osmotic stress as well. Consequently, tolerance oI Ireezing and drought may have certain prospective mechanisms in common, because the majority oI Ireezing injuries usually result Irom Ireeze-induced dehydration oI plants. Several cold-responsive promoters were shown to contain the dehydration response element (DRE) with a CCGAC conserved motiI (C- repeat) repeated several times (128). The 5' region oI the cor15a gene oI Arabidopsis 20 harboring the C-repeat element between 305 and 78 base position is inactive, or very weakly active, in most oI the tissues and organs oI plants grown at normal temperature but becomes activated throughout most oI the plant in response to low temperature. Gene Iusion experiments indicated that this region, in addition to imparting cold-regulated gene expression, can impart ABA- and drought-regulated gene expression (129). More recent eIIorts have deIined an Arabidopsis DRE transcription activator, CBE1 (130). Mutation oI the core pentamer, CCGAC, oI two putative low temperature responsive elements in the 5'-proximal region oI the winter Brassica napus cold-induced gene BN115 resulted in complete loss oI low-temperature regulation by the promoter. This indicates that the CCGAC sequence is critical to the low-temperature response in the BN115 gene. In contrast, mutation oI two G-boxes, CACGTG, staggered between these elements in the same region oI the promoter did not alter cold-inducible gene expression (131). Similarly, a G-box in combination with an anaerobic response element is required Ior cold stress induction oI the Arabidopsis aldehyde dehydrogenase promoter, but ABA appears to play no role in cold-responsive expression oI this gene (132). Thus many oI the changes in the gene expression that occur in response to low temperature and drought appear to require ABA signaling via a second, independent signal transduction pathway. Drought and cold stress inducible genes that are activated in this pathway usually contain a potential ABRE, (T/C)ACGTGGG, in their promoter regions. A similar correlation can be observed in the closely related Arabidopsis rd29A and rd29B genes. Even though these genes are closely linked in the Arabidopsis genome, they are diIIerentially induced under the conditions oI dehydration, low temperature, high salinity, or treatment with exogenous ABA. It appears that rd29A has at least two cis-acting elements, one involved 21 in the ABA-associated response to dehydration, and the other induced by changes in osmotic potential, and that rd29B contains at least one cis-acting ABRE that is involved in ABA-responsive, slow induction (128). The maize rab28 gene has also been identiIied as ABA-inducible in embryos and vegetative tissues, and it is activated by exposure to water stress in young leaves. The proximal promoter region oI this gene contains a well - conserved ABRE module. Transient expression assays in rice protoplasts indicated that a 134 bp Iragment (194 to 60) Iused to a truncated 35S promoter was suIIicient to conIer ABA-responsiveness upon the gus reporter gene (133). These data suggests that complex molecular responses to various dehydration- and cold-related stresses may be mediated by both regulatory systems, where the ABA-signalling pathway does not induce an immediate response, but plays an important role in prolonged, long-term adaptive response to cold stress induced by dehydration oI plant tissues (134). Anaerobic-Responsive Elements (AREs) The low-oxygen response oI higher plants is complex and involves induction oI speciIic gene sets. The primary target oI this response is a pathway leading to increased expression oI the alcohol dehydrogenase gene (Adh) and ethanolic Iermentation (135). The Adh gene Irom Arabidopsis can be induced by dehydration and cold, as well as by hypoxia stress. Deletion mapping oI the 5' end and site-speciIic mutagenesis identiIied Iour regions oI the promoter essential Ior expression under all three stress conditions (136). The most critical region essential Ior expression oI the Adh promoter under anaerobic conditions contained sequences homologous to the GT motiI, (T/C)GGTTT, and the GC motiI, GCC(G/C)C, reported in maize (137). On the other hand, both G-box 22 regions close to the ARE did not aIIect expression under hypoxic conditions, but signiIicantly reduced induction by cold stress and, to a lesser extent, by dehydration stress (136). The Iunctional properties oI the ARE positioned in the maize Adh1 gene have been analyzed using a transient expression assay in electroporated maize protoplasts. ARE Iunctioned in both orientations, and the promoter activity under anaerobic conditions was proportional to the number oI complete ARE sequences nested in the Adh1 promoter (137). The MYB-related transcription Iactor, AtMYB2, was shown to bind to two separate motiIs in the promoter oI the Arabidopsis Adh1 gene (138). When driven by a constitutive promoter, AtMYB2 was able to transactivate Adh1 expression in transient assays in both Arabidopsis and tobacco protoplasts, while mutation oI the GT- motiI abolished binding oI AtMYB2 and caused a loss oI activity oI the Adh1 promoter. BIOTIC STRESS AND WOUNDING Wounding oI plants triggers a number oI diIIerent deIense reactions in order to develop resistance throughout the plant. Like many other biological processes, pathogen and wound-induced signal transduction pathways in plants oIten converge in the cell nucleus (139). There is a large number oI known pathogen-inducible genes, and their promoters are among the best studied in plants. Upstream regulatory sequences nested in these promoters Iorm a complex combinatorial regulation network and respond to a variety oI agents, including ethylene, salicylic acid (SA), jasmonic acid (JA), methyl jasmonate (MeJA), ABA, and various bacterial and Iungal elicitors. 23 JA and MeJA are Iatty acid derivatives which are considered to be global signals oI deIense gene expression since many deIense-related genes have been shown to respond to jasmonates (140). Either pathogen elicitors or exogenous application oI JA activates de novo synthesis oI phytoalexins (141). At the same time, JA and ethylene act synergistically in inducing members oI the PR1 and PR5 gene Iamilies oI pathogenesis- related proteins (142), and Arabidopsis plants impaired in JA perception or biosynthesis are unable to mount appropriate deIense responses (143). A MeJA-responsive region (JARE) has been identiIied in the promoter oI the vspB gene which is stimulated by MeJA and sugars in soybean (144). A DNA domain that mediates the MeJA response was localized between 535 and 585 bp, and contained a G-box and a C-rich element. Similar regions containing the bZIP protein-binding TGACG motiIs or G-boxes, are present in the promoters oI barley lipoxygenase 1 (145), potato Pin2 proteinase inhibitor II (146), and nested in the as-1-like elements (147). More recently, Menke and co-workers (148) described the elicitor-induced strictosidine synthase (str) gene in Catharanthus that required JA as a second messenger. A 42 bp region in the str promoter contained a GCC-box-like element, and was both necessary and suIIicient Ior JA- and elicitor-responsive expression. Typically, the GCC box commonly Iound in the 5' Ilanking regions oI ethylene-inducible deIense genes is believed to be the core sequence Ior ethylene responsive transcription (149). The ERE (ethylene-responsive element binding Iactor) proteins were Iirst isolated as GCC box binding proteins Irom tobacco (150). ERE2 and ERE4 enhanced the GCC box-mediated transcription oI a reporter gene in tobacco protoplasts, suggesting that they act as transcriptional activators, while ERE3 Iunctions as a repressor (151). Over-expression oI 24 Pti4, a tomato transcription Iactor that belongs to the ERE Iamily, in transgenic Arabidopsis plants induced a more than 2.5-Iold higher expression oI twenty eight PR genes containing a GCC box (152). Ethylene is also required in the transduction pathway leading Irom injury, and ethylene and JA seem to act together to regulate proteinase inhibitor gene expression during the wound response (153). On the other hand, GCC box- mediated transcription oI deIense genes does not always requires ethylene (154), thereIore, it appears that minor variations in the core sequences oI the GCC-element impart responsiveness to diIIerent environmental stimuli. There is increasing evidence that W-boxes are a major class oI cis-acting elements responsible Ior the pathogen inducibility oI many plant genes. The importance oI W- boxes was illustrated recently by studies oI the Arabidopsis transcriptome during systemic acquired resistance, SAR (155). The signiIicant over-representation oI W-box motiIs, and their clustering, on PR1 subset gene promoters suggests that W-box binding proteins, WRKY Iactors, are crucial in co-regulation oI these genes (156). Similarly, the cognate W-box sequence was present in upstream regions oI all the SA-responsive receptor-like protein kinase genes examined (157). Indeed, both GCC-like elements and WRKY transcription Iactors have been implicated in gene expression in response to wounding as well (158), suggesting that wound- and pathogen-induced signaling consists oI networks with some shared components (159). ORGAN-SPECIFIC EXPRESSION Seeds 25 A number oI promoter elements including the RY-repeat motiI (CATGCATG), ACGT motiI, E-box (CACCCTG), AACA motiI (AACAAACTCAATC), the GCN4 motiI (TGAGTCA), and the Prolamin-box (TGTAAAG) have been shown to be involved in the seed-speciIic expression oI many seed storage protein (SSP) genes (160, 161). The RY-repeat motiI is widely distributed in dicot and monocot SSP genes and comprises a portion oI the 28-bp legumin box (162, 163). In a number oI studies it has been shown that the sequence is important Ior expression oI SSP genes in coordination with other cis- acting elements (164, 165). Deletion oI the RY repeat element not only dramatically reduced expression directed by legumin or glycilin promoters in seeds but, in the case oI the legumin LeB4 promoter induced expression in leaves (162). In a number oI plant promoters the CATGCATG motiI interacts with the conservative B3-domain oI the transcriptional activators JP1 oI maize (166), and fus3 and abi3 proteins oI Arabidopsis (167, 168). Both the nucleotide sequence and the alteration oI purine and pyrimidine nucleotides were shown to be essential Ior the activity oI the CATGCATG motiI (167). The sequence TGTAAAG, commonly named the prolamin box (P-box), was initially identiIied on the basis oI both its highly conserved nucleotide sequence and location ( 300 bp) relative to TIS oI prolamin genes. It was recognized as a strong candidate Ior coordinating the expression oI many SSPs, because it is present within the promoters oI all zein genes in maize (169), as well as many SSP genes Irom related cereals. In many prolamin genes, P-box and GCN4 motiIs are coupled with each other with only a Iew nucleotides separating them. This tandem module is designated as the biIactorial endosperm box (170). A third motiI, AACA, conserved in rice glutelin genes, is also involved in controlling endosperm-speciIic expression (171, 172). These three promoter 26 motiIs are recognized by speciIic DNA binding proteins. The AACA motiI is recognized by MYB proteins (173); the P-box is recognized by member oI the DOE class oI zinc Iinger proteins, called PBE (174), and GCN4 is recognized by the bZIP protein Opaque- 2 and its homologues (175, 176). Recently, Wu et al., (177) perIormed an extensive Iunctional analysis oI the rice storage protein gene promoter GluB-1, which contains AACA, P-box, GCN4 and ACGT motiIs in its 197 bp promoter region. To gain insight into the combinatorial interplay among these motiIs, a series oI constructs containing various combinations and modiIications oI these motiIs was examined in transgenic rice. Multiple copies oI GCN4 conIerred an endosperm expression pattern when Iused to the minimal 35S-promoter Iollowed by the GUS-reporter gene, while tandem repeated copies oI any oI the other three motiIs were unable to direct expression in transgenic rice plants. The data indicate that the GCN4 motiI is essential Ior determining endosperm-speciIic expression, whereas the AACA, ACGT and P-box contribute to the quantitative regulation oI the GluB-1 gene. Fruit A number oI Iruit-speciIic genes that are activated during ripening have been isolated Irom plant species with either climacteric or non-climacteric Iruits (178). Although Iruit- speciIic promoters have been isolated and analyzed Ior a number oI species (179- 181), tomato has long served as the primary model Ior the investigation oI Iruit - and ripening- speciIic promoters (182-184). It has also served as a heterologous system to test the Iunction oI putative promoter sequences isolated Irom other Iruit species, such as apple (179) and pepper (180). 27 Eruit-speciIic promoters, such as tomato polygalacturonase (182) and E8 (183) promoters, have attracted much interest because oI their practical use in the manipulating Iruit metabolism and the production oI valuable pharmaceutical proteins such as antibodies, and edible vaccines in genetically engineered Iruits (185, 186). However, the detailed mechanisms oI Iruit-speciIic transcription are poorly understood, and many oI the essential cis-elements have not been identiIied. Recently, a novel cis-acting element that determines Iruit-speciIic, high-level expression oI cucumisin was identiIied and Iunctionally characterized in melon (181). Gain-oI-Iunction experiments revealed that the 20-bp sequence, GACACGTGTCACAACCTAAT (which contains the perIect palindromic G-box in its Iirst halI) includes a Iruit-speciIic enhancer element (TGTCACA). Eour tandem repeats oI the Iull module in either orientation were suIIicient to direct Iruit-speciIic expression when Iused to a minimal promoter. Stressing the importance oI the TGTCACA motiI, gel mobility shiIt assays showed that the enhancer element itselI, but not the G-box, was an essential target Ior a Iruit-speciIic protein binding to that region oI the cucumisin promoter. No ethylene-responsive elements, such as the GCC-box that is conserved in the promoters oI many ripening-induced genes (150), have been Iound in the cucumisin promoter, indicating that its expression is probably regulated in a developmental and organ-speciIic manner. This Iinding demonstrates that cis-elements responsible Ior Iruit speciIicity could be successIully separated Irom those that mediate ripening-associated developmental and ethylene- mediated regulation. Pollen 28 A number oI genes speciIically expressed during various stages oI pollen development and germination have been isolated and characterized both Ior dicot and monocot plants. The pollen-related genes are divided into early and late groups, according to their maximal expression beIore or aIter pollen mitosis I, when there is a transition Irom microspore development to pollen maturation (187). Promoter analysis oI genes that are coordinately expressed during pollen development in tomato, tobacco and maize revealed enhancer sequences and shared regulatory elements required Ior pollen- speciIic transcription (8, 188, 189). Promoter deletion analysis in transgenic plants demonstrated that relatively short proximal regions are required Ior developmentally regulated expression in pollen, and in speciIic cell types oI the sporophyte. Cis-acting regulatory elements oI three tomato late gene promoters LAT52, LAT59 (188, 190) and LAT56 (191), tobacco genes g10 (8) and NTP303 (192) and the monocot pollen-speciIic promoter ZM13 (189) Irom maize were characterized in detail using transient and stable expression analysis. There is a striking architectural similarity between the promoters Irom maize ZM13, tomato LAT52, LAT59 and the tobacco NTP303 gene. All oI them contain a 30-32 bp module located 30-50 bp upstream Irom the TATA box responsible Ior pollen-speciIic expression. In addition, short elements required Ior the enhancement oI pollen-speciIic expression: the Q-element, AGGTCA, and an AAATGA motiI, have been identiIied in the maize ZM13 and tobacco NTP303 promoters, respectively. The maize Q-element was reported to positively modulate the expression oI the proximal promoter region but showed no ability to cause expression in pol len on its own. A similar enhancer-like, non-speciIic activity oI the short cis-elements has been reported Ior the pollen-speciIic expression in tomato (190), Arabidopsis (193) and Brassica (194). 29 While many pollen-speciIic promoters are interchangeable among a wide variety oI plant species, the 30-32 bp cis-elements essential Ior the expression in pollen show no or little sequence similarities to each other (190, 193). Eor example, the tomato LAT52 promoter is active in several other dicot plants studied (191, 195, 196). Eurthermore, the maize ZM13 promoter Iunctions well when stably transIormed into tobacco (197) and Arabidopsis (189). This data suggest that there should be some conserved Ieatures in the various pollen promoters that is not yet apparent, at least in terms oI consensus sequences. Extensive analysis oI the 100 to 72 bp region in the tomato pollen-speciIic LAT52 promoter revealed a core PBI motiI, TGTGGTT, a putative binding target Ior the GT-1 related transacting Iactors. Mutation oI the central GG residues in PBI reduced pollen- speciIic expression approximately ten-Iold (188). The PBI motiI, together with two other regulatory elements, GAAA and TCCACCATA, builds a strong pollen-speciIic transcriptional activation unit. Eurther mutagenesis and Iunctional combinatorial analysis demonstrated that PBIGAAA and GAAATCCACCATA Iunctional pairs could act in a pollen-speciIic manner, while the PBITCCACCATA unit was not active. This data together with the Iunctional analysis oI other pollen-speciIic promoters suggests the interplay oI several transcription Iactors, one or more oI which is responsible in establishing pollen-speciIic expression. Although several cDNAs encoding putative transcription Iactors speciIically expressed in anthers and/or pollen have been isolated (198-202), their Iunctional activity and target promoter cis-elements remain to be characterized. 30 QUANTITATIVE TRANSCRIPTION ENHANCERS An enhancer is deIined as a cis-acting module capable oI stimulating gene expression when placed, in either orientation, upstream or downstream oI the gene. Eirst described in animal viruses Ior their remarkable ability to dramatically increase gene expression upon SV40 sequences acting in cis to the beta-globin gene (203), enhancers were shown to act over considerable distances in the genome (204). While many enhancer-like sequences oIten direct tissue-speciIic or regulated expression (see above), numerous plant genes have been reported to include non-tissue-speciIic upstream regulatory elements with quantitative enhancer-like qualities. Dean et al. (205) have reported that sequences downstream oI the coding region contribute to quantitative diIIerences in expression oI two petunia rbcS genes. Similar A/T-rich sequences have long been observed to direct quantitative expression enhancement due to the potential combinatorial eIIect oI multiple cis elements. Bustos et al. (206) have studied a 0.8 kb Iragment Irom the 5'-Ilanking region oI a Erench bean beta-phaseolin gene. Gel retardation and Iootprinting assays using nuclear extracts Irom immature bean cotyledons revealed strong binding oI the nuclear proteins to an upstream region (628 to 682) that contained two inverted A/T- rich motiIs. Eusion oI a 103-base pair Iragment, or a 55-base pair synthetic oligonucleotide containing these motiIs, to a minimal 35S promoter/GUS cassette yielded high gus expression in several tissues. In another report, a 33 bp double-stranded oligonucleotide homologous to two AT-rich sequences located upstream (907 to 889, and 843 to 826) to the TIS oI the soybean heat shock gene Gmhsp17.5E stimulated transcription when placed 5' to a truncated (140) maize Adh1 promoter (207). Structural 31 Ieatures within this putative enhancer region included a run oI AT-rich sequences, and three perIect CCAAT boxes located immediately upstream Irom the most distal HSE oI the promoter (44). The A/T-rich positive regulatory region (444 to 177) oI the pea plastocyanin gene promoter conIers enhanced gus expression in leaves oI transgenic tobacco plants when Iused in either orientation to a minimal pea promoter (208). In many cases, the enhancer-like activity oI the AT-rich sequences in the upstream promoter module oI the gene can be related to the presence oI the matrix attachment region (MAR) at this location. MARs appear to organize the eukaryotic genome into large loops Iormed by the binding oI dispersed AT-rich sequences to non-histone proteins oI the nuclear scaIIold (reviewed in 209). Individual DNA loops are likely to deIine Iunctional units as well as topological domains, contributing to the regulation oI gene expression and DNA replication in general. Comparison oI the large number oI the MAR sequences showed no clear relationships among them and no strict consensus sequences (210). However, they share the property oI being asymmetrically AT-rich and contain dA/dT stretches which are responsible Ior a distinctively narrow minor groove to the double helix (211). There is also a clear size requirement Ior interaction oI MAR sequences with the nuclear scaIIold (Ior example, tight binding requires nearly 300 bp oI AT-rich DNA in Drosophila (212). In addition to the DNA-benting properties, the AT- rich regions readily become base-unpaired and are oI a great biological signiIicance to the MAR Iunction (213). A set oI other cis-acting elements characteristic oI MARs provide the binding sites Ior anchoring the core promoter and origin oI replication complexes to the nuclear matrix (214). These include A- and T-boxes (210), bent DNA motiI (215), topoisomerase II box (216), unwinding tract motiI (217) and autonomously 32 replicating sequences (218). In the intact plant nucleus, the MARs deIine individual loops and can be isolated using low concentrations oI chaotropic agents to achieve selective extraction under conditions in which precipitation artiIacts are minimized (219). The distribution oI the diIIerent classes oI DNA within this continuum, with respect to the predicted structural loops, reveals an interesting correlation: the long stretches oI mixed classes oI highly repetitive DNAs are oIten segregated into topologically sequestered units, whereas low-copy-number DNAs are positioned in separate loops (220). MARs increase reporter gene expression both in stably transIormed plant cells (221) and transgenic plants (222), and are widely used to minimize transgene silencing (reviewed in 223). The non-random distribution oI MARs according to localization oI transcription units, and their co-mapping with DNA sequences supporting the origin oI replication in yeast (212) and pea (224), strengthens the idea that MARs play a role in replication mechanisms as well. The potential relationship between anchorage oI Drosophila ribosomal DNA to a nuclear substructure and replication mechanisms in the enhancer - like upstream region oI the ribosomal DNA promoter have been suggested (225). Indeed, the 520 to 80 AT-rich region immediately upstream oI the tobacco ribosomal DNA promoter comprises a variety oI cis-acting elements with a typical quantitative enhancer- like module. This ampliIication-promoting sequence (aps) signiIicantly increased the copy number and expression activity oI the adjacent reporter genes when cloned in both upstream and downstream position in regards to the expression cassette (226). Similarl y, an 'upstream Sal repeats (USR) sequence located upstream Irom the ribosomal DNA promoter oI Arabidopsis thaliana was tested Ior its inIluence on the in vivo activity oI 33 nearby protein coding genes. On average, the presence oI the USR element led to a Iour- Iold increase in the expression oI a reporter gene (227). CONCLUSIONS AND PERSPECTIVES Over the last Iew years, a considerable body oI evidence has accumulated on the structural organization and regulation oI plant promoters, many aspects oI which have been previously reviewed (4, 24, 228). In this chapter, we have tried to summarize both the well-known and newly described cis-active elements nested in the plant promoters, deliberately emphasizing the most recent studies that illuminate their Iunctional activity in the multi-level regulation oI plant gene expression. The organization oI plant promoters Iollows the general structure common Ior other eukaryotes: a 40 to 50 bp core promoter adjacent to the TIS, Iollowed by an upstream region oI about 1 kb, containing the proximal and upstream cis-acting elements, and the outside enhancer-like sequences (Eig. 1). While many plant core promoters share combinations oI well-deIined elements such as TATA-box, initiator, DPE, and CCAAT- box, their consensus sequences and spacing may vary signiIicantly, so that no two core promoters are identical. ThereIore, the composition oI the transcription-initiation complex that binds to the core promoter is variable, and this oIIers additional opportunities Ior the regulation oI basic transcription (229-231). Nevertheless, the major level oI transcriptional control is mediated by trans-acting Iactors binding to the upstream regulatory elements. In Arabidopsis, the regulation oI genome expression requires the products oI about 3,000 oI the predicted 25,498 genes, reIlecting the complexity oI this 34 transcription-regulation network (232). At any given time, a distinct set oI transcription Iactors is available to Iorm the higher-order nucleoprotein complexes at the active promoter (233). The composition oI individual trans-acting Iactors within these complexes may oIten change when a speciIic signal is perceived, so that a given transcription Iactor can play multiple roles, and aIIect multiple gene sets, depending on its local concentration and availability. It should be mentioned that one oI the most successIul strategies Ior Iunctional characterization oI many cis-acting elements is the gain-oI-Iunction Iusion oI deIined individual elements to a minimal plant promoter, thereby reducing the complexity oI the expression proIile (9). Recent advances in the development oI stable in vitro transcription systems based on rice whole-cell and tobacco nuclear extracts (234, 235), in combination with a range oI available plant natural and synthetic promoters, provides Iurther opportunities Ior the determination oI the molecular mechanisms underlying selective gene expression in response to various signals that could be modeled in vitro. Eunctional studies oI plant promoters have also given rise to a general concept that upstream regulatory elements are composite and not individual, where each cis-acting element contributes to the overall activity oI the module through synergistic interactions between cognate transcription Iactors (52, 66, 82, 100, 136). Stimuli-speciIic elements within a module are targeted by transcription Iactors that are regulated by signal - perception pathways, while coupling elements bind protein Iactors that determine spatial, temporal, or quantitative expression. Eor example, G-boxes serve as the coupling elements in many modular regulatory units that respond to a variety oI chemical and environmental signals. Such Ilexibility is achieved both by the diIIerent G-box Ilanking 35 sequences (236), and close interaction with immediate cis-and trans-acting Iactors. To understand the interplay oI transcriptional networks in plants, we ultimately need to know the expression patterns oI all trans-acting Iactors as well. IdentiIication and Iunctional characterization oI the cis-acting element is no longer enough only thorough and creative studies that can sort out the cross-talk oI pleiotropic or signal-speciIic eIIects in mutants will deIine the physiological roles oI diIIerent signals and establish connections between diIIerent pathways in the regulation oI plant gene transcription. The practical importance oI better understanding the regulation oI plant promoters is the potential to inIluence gene expression to manipulate plant metabolism (185), and achieve compartmentalized production oI valuable pharmaceutical proteins Irom seeds (237), leaves (238), roots (239), or Iruits (186). AKNOWLEDGEMENTS We wish to thank Peter Day, Michael Lawton, and Nir Yakoby Ior helpIul discussions and critical reading, and Alison Garvey Ior her assistance in preparation oI the manuscript. 36 Table 1. Online tools Ior structural and Iunctional analysis oI plant promoters. Online Resource Description PlantCARE http://oberon.rug.ac.be:8080/PlantCARE/ ReIerential database with 435 plant cis-acting elements describing 159 plant promoters. PLACE http://www.dna.aIIrc.go.jp/htdocs/PLACE/ Database oI cis-acting regulatory DNA elements reported in vascular plants. TRANSFAC http://transIac.gbI.de/TRANSEAC/ Database on eukaryotic cis-acting regulatory DNA elements and trans-acting Iactors. Eukaryotic Promoter Database, EPD http://www.epd.isb-sib.ch/ EPD is a specialized annotation database oI eukaryotic promoters Irom EMBL. Neural Network Promoter Prediction http://www.IruitIly.org/seqtools/promoter.html Prediction oI the putative promoters in prokaryotic and eukaryotic DNA sequences. Signal Scan http://bimas.dcrt.nih.gov/molbio/signal/ Homology search Ior published cis-elements based on TRANSEAC signal database. MAR-Wiz http://www.IuturesoIt.org/MAR-Wiz/ Detection oI putative matrix attachment regions in eukaryotic DNA sequences. 37 Table 2. Common cis-acting elements involved in temporal and/or spatial regulation oI gene expression in plants. Only transcription Iactors, Ior which there is strong evidence suggesting their importance in gene regulation are included in this table. Responses &LV-elements/Consensus 7UDQV-acting Factors L i g h t Light GT-1 box, GGTTAA I-box, GATAAAGR G-box, CACGTG H-box, ACCTA(A/C)C(A/C) HY5 (59), PIE3 (60) Auxin TGTCTC motiI TGTCCCAT box osc/as-1 element ARE1 (69) ASE1 (74) Gibberellin TAACA(A/G)A element TATCCAC element (C/T)CTTTT(C/T) element GAmyb (80) Abscisic Acid G-box, CCACGTGG VIP1 (87) Ethylene A(T/A)TTCAAA element E4/E8BP (95) M e t a b o l i c
R e g u l a t i o n Sugars TATCCA element GC-box, GCC(G/C)C G-box, CACGTG SURE, (AA)TACTA(A/T)T W-box, (T)TGAC(C/T) OsMYBS (107) SPE1 (102) Heat GAATTC element HSE (121) Oxidation G-box, CACGTG H-box, ACCTA(A/C)C(A/C) W-box, (T)TGAC(C/T) GCC element Cold, Drought C-repeat, CCGAC G-box, CACGTG CBE1 (130) Hypoxia GT motiI, (T/C)GGTTT GC motiI, GCC(G/C)C AtMYB2 (138) E n v i r o n m e n t a l
S t r e s s Pathogen, Wounding, Ethylene, 1A, SA G-box, CACGTG C-repeat, CCGAC GCC element, AGCCGCC W-box, (T)TGAC(C/T) ORCA2 (148), ERE1 (150) WRKY (155) Seed- RY motiI, CATGCATG G-box, CACGTG E-box, CACCCTG AACA motiI GNC4 motiI, TGAGTCA P-box, TGTAAAG EUS3 (167), ABI3 (168) OsMYB5 (173) Opaque2 (175) PBE (174) Fruit- TGTCACA motiI D e v e l o p m e n t Pollen- 32 bp motiI 38 REFERENCES 1. Katagiri, E. and Chua, N.-H. (1992) Trends Genet. 8, 22-27. 2. Chandler, V.L. and Vaucheret, H. (2001) Plant Physiol. 125, 145-148. 3. Roberts, S.G. and Green, M.R. (1994) Nature 371, 717-720. 4. GuilIoyle, T. (1997) Genetic Engineering: Principles and Methods 19, 15-47. 5. Spector, DL. (1993) Annu. Rev. Cell Biol. 9, 265-315. 6. Roeder, R.G. (1996) Trends Biochem. Sci. 21, 327-335. 7. Dvir, A., Conaway, J.W. and Conaway R.C. (2001) Curr. Opin. Genet. Dev. 11, 209-214. 8. Rogers, H.J., Bate, N., Combe, J., Sullivan, J., Sweetman, J., Swan, C., Lonsdale, D.M. and Twell, D. (2001) Plant Mol. 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These elements are oIten modular and require copuling oI a constitutive cis-element (e.g., G-box) and a signal-speciIic cis-element Ior a Iull-strength response. Bending and other modiIications to the local DNA structure Iacilitate the cross-talk interaction between the diIIerent modules and core promoter region, adding to the complex combinatorial regulation oI plant promoters. DPE INI TATA CAA GCC RNA Pol TFIID BTFs BTFs TBP SSTF SSTF DN Proximal Elements FLV1 Upstream Elements G-box G-box FLV FLV3 FLV2 Core Promoter