2024 年 12 巻 p. 79-92
The non-coding regions of genes contain DNA sequences which are binding sites for factors that regulate the genes. Transcription of some genes involves simultaneous binding of regulatory proteins at different regions which interact based on prevailing signals. Identifying regions bound by various factors and testing their function has been a longstanding area of research. In most cases, signal multiplicity in the cell cannot be fully mediated through a single element; rather, shared responses by composite elements direct transcription. This review summarises experimentally proven cis-regulatory element combinations and how they regulate light, cold, hormones, pathogens, and wounding signals. We highlight the untapped potential of motif combinations in driving stimulus-specific trait enhancement in plants. Arranging regulatory elements adjacent to each other unveils their complex interplay, providing avenues for enhancing the transcriptional regulation of genes implicated in developmental processes and responses to environmental cues.
In living organisms, regulation of mRNA is imperative for nearly all biological processes. Sequence elements in noncoding regions often control gene expression and knowing a gene’s place in the larger regulatory network is essential to understanding its function [1]. The transcriptional regulation of genes is mainly controlled via the interactions between gene promoters and transcription factors (TFs), and between TFs and chromatin-modifying machinery [2]. These interactions are complex and the binding of TFs for transcriptional regulation of genes is dependent on intrinsic event and regulatory motifs specificity. Traditionally, deconstructive approaches to promoter structure/function analysis, such as 5’ or 3’ promoter deletion, linker or deletion scanning, and mutagenesis of putative cis-acting elements, have been employed to analyse promoter structure [3]. In recent decades, advanced bioinformatic tools and databases have made prediction of putative cis-regulatory elements more flexible, as only core motifs are predicted and tested rather than promoter fragments.
Promoter complexity is simplified with synthetic promoter, since only chimeric construct containing specific cis-regulatory element(s) (CRE) is designed and introduced into plants. Fusing non-native minimal promoter (MP) to combination of CREs in a synthetic promoter and ectopically expressing in a cell helps reduce the complexity of expression pattern of native promoter, thus delineating the role of composite elements. This has been used to determine the activity of cis-acting elements [4, 5, 6] and enhancers [7]. Discussion about the usefulness of synthetic promoters have been reviewed in Ali and Kim [8]. Synthetic promoter effectiveness to induce specific gene expression patterns in response to stimulus in non-native promoter contexts validates the functionality of composite elements within such promoters.
The transcriptional initiation by RNA polymerase II in eukaryotic cells is accompanied by convergence of multiple cis- and trans- acting regulatory mechanisms [9], such that, regulation of gene is hinged on the specificity of perceived stimuli and recruitment of suitable factors to the promoter. Eukaryotes are known to employ combinatorial strategies to generate a variety of expression patterns from a relatively small set of regulatory motifs [10] and exploit motif geometry as another dimension of combinatorial power for regulating transcription [11]. The CREs orientation, specificity, spacing, location, copy number and composite elements of the synthetic promoter can alter its function in the cell, see review by Venter [12]. Promoter with multiple CREs is transactivated when stimuli that can simultaneously activate the elements are induced. However, stimuli that efficiently activate only a subset of elements may not induce promoter response.
CREs identification and confirmation through DNA footprinting, gel mobility shift assay (EMSA), chromatin immunoprecipitation (ChIP), methylation interference and other techniques are active areas of research, but outside the scope of this review. It is commonly accepted that promoter activity in vivo represents a combinatorial interaction of the functional DNA elements contained within the promoter [3]. CREs integrate signals from multiple TFs resulting in combinatorial control, and function in crosstalk between different signals. Delineating elements required for gene regulation has been part of the frontline initiative in elucidating on transcription and expression of genes. In this review, we discuss experimentally proven combinatory effects of cis-acting elements as pairwise and/or multiple combinations of different elements in non-native promoter contexts. Signal transduction and responsiveness of combined cis-acting elements are summarised.
Plants require light as a source of energy, but the spectrum of light, duration, and light intensity control developmental processes. Light signals are mediated through gene regulation. The photoreceptors - phytochromes, cryptochromes and UV light receptors have evolved to mediate light-dependent gene regulation in plants. Light induces the expression of many nuclear-encoded photosynthetic genes, such as small subunit of ribulose-1,5-bisphosphate carboxylase (RBCS) and light-harvesting chlorophyll a/b binding proteins (LHCB) which belong to photosynthesis-associated nuclear genes (PhANGs) [13, 14]. Extensive studies of promoter fragments of light-responsive genes have identified and functionally characterised a plethora of CREs and their trans-acting factors (see reviews by [15, 16, 17]). For instance, light responsive elements - GT1 sites, I-box (GATA motif), G-box, ASF1 motif, Z-motif and several others have been identified in PhANGs, reviewed in Terzaghi and Cashmore [16]. Quite a few reports have functionally characterised these cis-acting elements in heterologous minimal promoter using sequence-specific motifs rather than promoter fragments obtained from numerous distal and/or proximal deletions. The regulatory motifs are relatively shorter sequence and can be bound by specific TF, however, promoter fragments are large enough to interact with multiple different TFs. Light regulatory elements (LREs) for many genes are still emerging and it is hypothesised that plants light regulatory units (LRUs) are made up of composite elements, i.e. aggregate of cognate sequences that are target sites for different TFs, which interact to regulate gene expression [16, 18]. This hypothesis is supported by promoter fragments composed of GCGC-box and SORLIP1 (RBCSZm1) [19], G-box and I-box (RBCS-1A) [20], (CMA5) [21].
In transgenic Arabidopsis, pairwise combinations of either GT1 or Z-motif or G-box with GATA motif constitute a LRU, and high-irradiance can directly modulate the activity of the promoters containing the LRE pairs but not those with individual LRE [4]. The LRE pairs (G-box – GATA, GT1 – GATA, Z – GATA) are responsive to a broad spectrum of light - white light, red and far-red light, and blue light [4, 22, 23]. GT1 motif or Z-motif fused alone to non-light responsive minimal promoter is dark activated and repressed by high light, but coupling of GATA motif to either of these motifs altered promoter activity. Thus, switching promoter function into dark repressed and light inducible [4]. Transitioning from dark activators to light inducible promoters when paired is an indication that GATA acts to reverse dark phase derepression of single element promoters. Agreeably, it could be said that light inducibility of unpaired Z and GT1 motif is dependent on GATA for high light signalling. One could have anticipated that signals mediated through Z – GATA or GT1 – GATA combination to be silenced - since unpaired GATA motif has contrasting function both in dark and light condition, but this was not the case. Probably, GATA binding TF exerts dominant action in the protein-protein interaction through TF silencing thereby suppressing transactivation of the partner motif in the dark. Combinatorial interactions between transcription factors bound at a promoter may also qualitatively change transcription factor behaviour, in extreme cases affecting whether a signal-regulated transcription factor activates or represses transcription [24]. Curiously, synergistic or antagonistic interaction of pairwise combination of GT1 and Z motif on modulating a non-light responsive minimal promoter remains to be experimentally validated.
Promoter study of the ELIP2 gene of Arabidopsis has characterised the functional unit that mediates multiple stress signals (high light, UV-B and cold) in heterologous minimal promoter. Singly, cis-acting elements A, B and C identified in ELIP2 promoter are non-responsive to high light. But pairing element A and B constitute minimal LRU (B + A) required for response to high irradiance white light in transgenic plants [6]. In vitro and mutant analyses revealed that ELONGATED HYPOCOTYL 5 (HY5) protein recognizes element B [6]. Chattopadhyay et al. [25] also showed that the high light response of the G-box – GATA motif LRE pair requires HY5 for its full induction. HY5 involvement in light regulation of element B and G-box promoter seems somewhat exclusive, because absence of G-box motif in GT1 – GATA promoter had no effect on light-activated expression of transgene [25]. Distal element C of ELIP2 promoter acts as a repressor of high light response when coupled with element A, B or in tripartite combination with A and B [6]. Perhaps, trans-factor bound to C in synthetic promoters act as a repressor of transcription, thus suppressing transactivation of LREs.
2.2 Phytochrome cis-regulatory responsePhytochrome (PHY) photoreceptors mediate red light and far-red light signalling in plants. In Arabidopsis, five members (namely PHY-A, -B, -C, -D, -E) constitute the PHY gene family. PHYB is necessary for continuous red light (R) perception, others (A, C, D and E) are neither necessary nor sufficient for R. However, PHYA is required for continuous far-red light (FR) perception, reviewed in Quail et al. [26]. Response to red light signals has been shown to require a combination of cis-elements for signal transduction. LRE pairs (G-box – GATA, Z-box – GATA and GT1 – GATA) modulate phytochrome regulation in plants (pulse red light [4], high irradiance R and FR [22, 23]). Uncoupled G-box, GATA, Z and GT1 have been reported to derepress promoter activity causing protein accumulation in etiolated plants. But paired LREs reportedly mimicked CAB1 promoter which repressed transcription in the dark [4], thus conferring skotomorphogenesis responsiveness to the LRUs. PhANGs require light for their activation, transcript induction of unpaired LRE in dark indicates the lack of partner element necessary for phytochrome regulation and dark repression. Similar observation was reported by Degenhardt and Tobin [27] where they demonstrated with a promoter fragment of L. gibba LHCB2-1 gene that mutation of either CCAAT or GATA element increased promoter activity in dark-treated plants and suppressed phytochrome regulation. The TFs bound to LRE pairs in etiolated seedlings act somewhat concertedly to repress promoter activity, but the dynamic changes when light is introduced. The reversibility of inactive Pr to active Pfr in LRE pairs reaffirms their involvement in phytochrome signalling in plants. However, mutation of hy5 in transgenic plant harbouring G-box – GATA pair eliminated both induction and reversibility of Pr to Pfr [25].
Mutating PHYA (phyA) or PHYB (phyB) locus caused repression of G-box – GATA, Z-box – GATA and GT1 – GATA LRE pairs, thus entrenching the validity of far-red and red-light photoreceptors in light signalling of each regulatory unit. The sensitivity of these LRUs to R and FR light in mutants were abolished under white light conditions [22, 23]. The redundancy of phytochrome mutation in white light suggests that LRE pairs can maintain signal specific transactivation. phyB mutation compromised Pfr and Pr induction of Z-box – GATA pair [23]. Beside their redundant and antagonistic function, phytochromes A and B have overlapping functions in FR and R light signalling pathway [26]. Pfr induction of the G-box – GATA pair in myc2 mutant was suppressed in constant far-red light, although transcript activation in dark and other light spectrums were derepressed [28]. MYC2 is a negative regulator that interacts with the Z-box of CAB1 and G-box of RBCS-1A promoters [29]. Perhaps, TF bound to the GATA-motif in the LRE pair might have a silencing effect, as evidenced by the compensation of phytochrome signalling in the uncoupled G-box by the myc2 mutation.
2.3 Cryptochrome cis-regulatory responsePlants are sensitive to blue light which is mediated by blue light (BL) receptors. In Arabidopsis, cryptochromes – CRY1, CRY2 [30] and phototropins – PHOT1, PHOT2 [31] are blue light specific photoreceptors. Synthetic promoters containing LRE pairs (G-box – GATA motif, GT1 – GATA motif, Z-box – GATA motif) confer responsiveness to high irradiance of blue light in stable Arabidopsis transformants. Mutation of blue light photoreceptor - cry1 in plants transformed with these LRE pairs was shown to attenuate reporter gene responsiveness to blue light induction [22, 23]. G-box – GATA combination in cry1 mutant plants also attenuated red light response [22], thus suggesting that both cryptochromes and phytochromes have overlapping functions [26]. Gangappa et al. [28] demonstrated that the G-box – GATA pair is further induced by blue light when G-box repressor MYC2 is mutated. Aside from CRY1 involvement in BL induction, it is also required for optimal RL induction in addition to the PHYB photoreceptor [22] and this observation is reportedly exclusive to G-box containing LRE pair.
2.4 UV-B cis-regulatory responseUV-B light induces photodamage in plants and is recognized by UVR8, a UV-B photoreceptor. This spectrum of light induces DNA damage and repair [32] and alters the transcript levels of several genes [33]. Attempts at delineating the cis-regulatory elements involved in UV-B response in plants have identified ACE and MRE (in Arabidopsis CHS, CFI, F3H, FLS promoter) [34, 35], G-box and MRE (in parsley CHS promoter) [18], MRE and UVBox (in Arabidopsis ANAC13 promoter) [36]. These reports and several others identified UV regulatory units required for UV response within native promoter context but could not query their combinatorial function in heterologous minimal promoter.
Cis-regulatory elements A, B and C identified in ELIP2 promoter analysis have been characterised with respect to their UV-B responsiveness in transgenic Arabidopsis. Trimer of each element fused individually to MP is non-responsive to UV-B signals [6]. Coupling element B with A (i.e. B + A) synergistically activated UV-B responsiveness of the synthetic promoter. This combination retained in planta functionality when element position was interchanged or with sequence reorientation [6]. Reordering the sequence of B + A units could not cause steric hindrance of TFs even when the geometry of protein binding to DNA sequence was reoriented. HY5 protein recognizes element B and is involved in UV-B signalling through UVR8 [6]. Element C acts as a repressor either pairwise with a single element or in combination with A and B. More so, in its distal position in C + B + A tripartite combination, element C suppressed the transactivation of downstream partner elements in transgenic plants [6].
2.5 Cell and tissue specificity of light regulatory combinationsCoupling cis-regulatory elements can confer cell or tissue specificity in plants and numerous studies on motif combination have been shown through GUS-staining or bioluminescence assay. The expression pattern achieved by a single element in a promoter can be reversed or complemented when paired with another element. Puente et al. [4] reported that paired-LREs (G-box – GATA, Z-motif – GATA, GT1 – GATA) confer tissue and cell specificity, also responsiveness to chloroplast developmental state in cotyledons of light-grown seedlings. The light-independent expression for paired-LRE was mediated in a way different from the single element. The specificity conferred by LRE pairs are novel as supposed to simple additive effects of individual elements [4]. Conversely, LRE pairs (GT1 – GATA, G-box – GATA) transformed into cop1 and det1 mutant lines reversed light-dependent expression, rather the expression in cotyledon was induced in dark-grown seedlings [22]. Mutation of cop1 and det1 genes of transgenic LRE pairs exhibit seedling development in complete darkness that mimics that normally induced by light. The LRE pairs sufficiently responded to light signals, developmental control, and light-independent regulation in cop1 and det1 mutants in a manner similar to several native light-inducible promoters [22]. COP1 gene also acts to suppress the induction of Z – GATA LRE pairs in the cotyledon of dark grown plants [23]. The weak expression of LRE pairs in cotyledons and leaves of mutated hy5 transgenes in light grown plants demonstrates that HY5 protein is required for optimum activation of promoters [23, 25]. The LRE pair (B + A) characterised in ELIP2 promoter conferred green tissue specific expression upon illumination of seedling. Hyper accumulation of protein in cotyledons of transgenes harbouring the paired-LRE upon exposure to light stresses mimics the response observed in native promoter. Addition of Element C (C + B + A) suppressed the total stress responses by differential change of the stress response among tissue [6]. The summary of signal-dependent light regulation in plants is presented in Table 1.
Dehydration-responsive element (DRE) or C-repeat (CRT) with G/ACCGAC core motif is present in promoters of cold induced genes and genes responsive to other stresses [see 37, 38, 39 40]. In addition to the DRE/CRT, cold regulated genes have ABA-responsive element (ABRE), G-box, low temperature responsive element (LTRE), induction of CBF expression region 1 and 2 (ICEr1, ICEr2), evening element (EE), and other elements enriched in their promoters [41, 42, 43]. Elucidating the functionality of these CREs individually or in combinations when fused to heterologous minimal promoter is underreported.
Mikkelsen and Thomashow [44], presented evidence that a pairwise combination of EE motif (AAAATATCT) and ABRE-like (ACGTG) constitute a cold responsive unit (CRU) of BBX29 and COR27 genes in transgenic plants. Loss of function analysis carried out in their assay confirmed that mutating either of the composite elements abolished cold responsiveness. Thus, suggesting that both elements act cooperatively to mediate cold induction of genes. Cold responsive elements in Arabidopsis ELIP2 promoter have been identified and tested in transgenic plants. The same cis-regulatory unit (B + A) identified in high light and UV-B response is also involved in cold signalling. Individually, the cis-acting elements are non-functional, rather pairing A with B sufficiently mediates cold activation [6]. Combining element C to other elements (i.e. C + B + A) represses promoter response to cold [6], suggesting that this element is bound by a repressor TF.
3.2 Abscisic acid regulationThe phytohormone abscisic acid (ABA) is involved in several physiological, cellular and molecular processes as well as response to several environmental stresses. More than twenty different functional cis-elements that are involved in ABA-mediated regulation have been identified in ABA-responsive promoters, see review by [45]. Several reports illustrate that a single copy of ABRE is not sufficient for ABA induction but only multimerized synthetic ABRE can drive ABA response [5, 46, 47, 48, 49, 50]. ABA-inducible promoters are expected to consist of several elements since a single element is not believed to provide sufficient specificity for regulation of transcription in eukaryotes [45, 51]. In some promoters, ABRE acts together with a partner element to mediate transcriptional response to ABA treatment. For instance, barley HVA22 promoter analysis showed that ABRE3 (GCCACGTACA) and coupling element1 (CE1, TGCCACCGG) constitute ABA response complex 1 (ABRC1), and inducibility depends on HVA22 intron present downstream of heterologous minimal promoter. The ABRC is orientation independent, yet sensitive to the 20-bp nucleotide spacer separating ABRE3 and CE1 [48]. However, the enhanced induction of ABA by the intron situated downstream of minimal AMY64 promoter suggests the presence of another functional element. Because transient assay by Schmidt et al. [52] affirmed that coupling ABRE3 with CE1 did not confer ABA induction in minimal 35S promoter fusion. Introns can alter tissue-specific expression patterns when fused to heterologous promoter and validated in plants [53, 54].
Another ABRC (named ABRC3: ABRE2 - CCTACGTGGC and CE3 – ACGCGTGTCCTC) was reported in barley HVA1 promoter, in which ABRE2 is located immediately downstream of cis-acting element CE3. The ABRE element present in both ABRC1 and ABRC3 can confer aleurone and vegetative tissue responsiveness provided they are coupled with a distal or proximal coupling element, namely CE1 and CE3. Synergistic interaction between CE1 and CE3 conferred additive ABA induction when combined with ABRE2 in barley aleurone [5]. Hobo et al. [55] also demonstrated that ABRC3 (ABRE: TACGTGTC and CE3: GACGCGTGTC) identified in OsEM promoter conferred ABA responsiveness in the transient expression system. Pairwise combination of ABRE and CE3 can mediate responsiveness to VIVIPAROUS-1 (VP1). The ABRC as a unit cooperatively modulates VP1-dependent ABA signal transduction pathway. The two ABRCs (ABRC1: ABRE – CE1 and ABRC3: ABRE – CE3) are qualitatively different in terms of VP1 responsiveness [5, 55]. The VP1 transactivation of ABRC3 tends to rely solely on the specificity of the coupling element in the ABA response complex. Although both elements are functionally equivalent, interchangeable and capable of in vitro binding to TRAB1 factor [55]. The combinatorial configuration of elements necessary for VP1 transactivation is dependent on sequence specificity of ABRE and interaction with coupling element3. Rice OsEM ABRC has a contrasting arrangement of composite elements, and a spacer sequence which is lacking in barley HVA1 ABRC. However, the signal transduction of both ABRCs is unaffected by elements position and spacer.
3.3 Auxin responsePlant developmental processes such as cell division, cell elongation, cell differentiation, root formation, apical dominance and tropism are modulated through auxin regulation of their constitutive genes. Ulmasov et al. [56] characterised two composite cis-elements which constitute auxin response element (AuxRE) in D1 and D4 domains of auxin inducible promoter fragment of soybean GH3 gene. Both domains consist of a constitutive element and a TGTCTC element which are separated from each other in D4, but overlap in D1 domain. The constitutive elements in D1 and D4 do not share sequence similarity, suggesting that TGTCTC might be able to confer auxin inducibility on heterologous constitutive elements. Multimer of D1 11-bp auxin response element (AuxRE: CCTCGTGTCTC) fused to heterologous minimal promoter confers tissue and organ specific auxin responsiveness in transgenic tobacco seedlings (Table 1). Although, CCTCGTG element alone can induce high basal expression with or without auxin, coupling of TGTCTC represses basal expression and activates auxin inducibility when paired [56]. Perhaps, the interaction between the AuxRE pair might result from competition of TFs for specific DNA binding sites or from steric hindrance between TFs and the basal transcriptional machinery [58, 59]. This assumption is based on the fact that TGTCTC repressed activity of the constitutive elements and reactivates auxin response upon exogenous application.
| Promoter | Gene | Context | Function | Assay | Localization | Plant | Ref |
|---|---|---|---|---|---|---|---|
| Light regulation | |||||||
| G-box + GATA | LHCB, RBCS | NOS101 | High light induction of gene, phytochrome signalling expression | 〇 | Light: cotyledons (mesophyll, guard cell); Dark: root, hypocotyl; Mature plant: sepals | A. thaliana | [4] |
| Z + GATA | LHCB, RBCS | NOS101 | High light induction of gene, phytochrome signalling expression | 〇 | Light: cotyledons (mesophyll, guard cell); Dark: root, hypocotyl; Mature plant: sepals | A. thaliana | [4] |
| GT1 + GATA | LHCB, RBCS | NOS101 | High light induction of gene, phytochrome signalling expression | 〇 | Light: cotyledons (mesophyll, guard cell); Dark: cotyledons (weak); Mature plant: sepals | A. thaliana | [4] |
| B + A | ELIP2 | 35S CaMV | High light and UV-B light responsive unit | 〇 | Cotyledons | A. thaliana | [6] |
| C + B + A | ELIP2 | 35S CaMV | Element C repression of high light and UV-B responsive B+A unit | 〇 | Apical meristem | A. thaliana | [6] |
| G-box + GATA | LHCB | NOS101 | Dark, phytochrome and cryptochrome regulation in myc2 mutant transgene | 〇 | Cotyledons Mature plant: Leaf, stem |
A. thaliana | [28] |
| G-box + GATA | LHCB | NOS101 | Mutation of hy5 suppresses the transactivation of G-box containing LRE pair. | 〇 | Cotyledons (weak) Mature plant: leaves (weak) |
A. thaliana | [25] |
| Z + GATA | LHCB | NOS101 | Mutation of hy5 suppresses the transactivation of Z-box containing LRE pair. | 〇 | Cotyledons (weak) Mature plant: leaves (weak) |
A. thaliana | [23] |
| GT1 + GATA, G-box + GATA | LHCB, RBCS | NOS101 | Phytochrome and cryptochrome signalling, cop1 and det1 mutational transactivation of LRE pair. | 〇 | Dark: cotyledons Light: cotyledons (weak) |
A. thaliana | [22] |
| Cold Response | |||||||
| EE + ABRE-Like | BBX29, COR27 | 35S CaMV | Cold regulatory unit | 〇 | n.a. | A. thaliana | [44] |
| B + A | ELIP2 | 35S CaMV | Minimal cold responsive unit | 〇 | Cotyledons | A. thaliana | [6] |
| C + B + A | ELIP2 | 35S CaMV | Element C repression of cold responsive B+A unit | 〇 | Apical meristem | A. thaliana | [6] |
| ABA response | |||||||
| ABRE2 + CE3 | HVA1 | AMY64(i) | Exogenous ABA induction and VP1 transactivation of gene | × | Aleurone and vegetative tissue | H. vulgare | [5] |
| ABRE + CE3 | OsEM | 35S CaMV(i) | Exogenous ABA induction, VP1 transactivation and in vitro binding of TRAB1. | × | O. sativa | [55] | |
| CE3 + ABRE2 | HVA1 | 35S CaMV | Exogenous ABA induced transactivation of ABRC. | 〇 | Leaf blades | B. vulgaris | [52] |
| Auxin Response | |||||||
| AuxRE (D1, D4) | GH3 | 35S CaMV | Tissue and organ specific auxin induction | 〇 | Hypocotyl, roots | N. tabacum | [56] |
| AuxRE (DR5) | 35S CaMV | Synthetic auxin responsive unit | 〇 | Cotyledons, hypocotyl, root | A. thaliana | [57] | |
| Circadian Response | |||||||
| B + A | ELIP2 | 35S CaMV | Circadian rhythm responsive unit | 〇 | n.a | A. thaliana | [6] |
n.a. – not applicable. (i) – Intron included downstream of minimal promoter. o – in planta assay, × – transient expression.
Pathogen-inducible cis-regulatory elements are conserved among plant species [60], and classified according to their response to defence signalling molecules like salicylic acid, methyl jasmonate and ethylene or based on their core motifs such as W-boxes, GCC-box and several others which have been reviewed [61, 62]. Response to wounding stress also involves several hormones, with the most ubiquitous signal being jasmonic acid [63]. Most pathogen-inducible cis-acting elements are also induced by wounding and mechanical damages [60, 64]. Numerous reports have identified cis-acting elements that confer responsiveness to elicitors, wounding and pathogens [62].
Most pathogen-inducible cis-acting elements found in promoters of responsive genes are reported to function alone and do not require a partner element for transactivation, [see 60, 62, 65, 66, 67]. The core motifs are usually tested with flanking short DNA sequences. However, few of these identified motifs have been empirically proven in planta, to determine their responsiveness in heterologous minimal promoter. Rushton et al. [60] revealed that box-S, box-D and box-W2, when fused individually to heterologous 35S minimal promoter conferred responsiveness to peptide elicitor (pep25) in a transient expression system. In planta study by the same group demonstrated that combining these cis-acting elements (W – S – D and W – S) is inducible by a range of pathogens tested, with low basal expression (summarised in Table 2). Combining two or more pathogen-inducible cis-acting elements can expand the scope of responsiveness achieved with a single element, such that combinatorial action of elements can mediate optimum expression to host and non-host pathogens. Pairwise combination of two pathogen-inducible elements (E17 – F) conferred additive response to Rhizoctonia solani and showed higher pathogen biotrophic sensitivity in canola leaves. Both elements are pathogen-inducible in their native promoters, fast responding, low background expression and are non-responsive to wounding and mechanical damages [67]. The additive response suggests the complementary action of TFs bound to each element to mediate fungal response in canola. Abscisic acid response complex3 (ABRC3: CE3 – ABRE2) identified in barley HVA1 promoter [5] is responsive to Cercospora beticola in sugar beet. The ABRC3 is activated locally and not systematically during the plant–pathogen interaction at the developing necrotic spots [52], thus conferring site specific induction. Elicitor – benzo-(1,2,3)-thiadiazole-7-carbothioic acid S-methyl ester (BTH), which induced systemic acquired resistance in plants, has been reported to induce ABA signalling of ABRC3 [52]. The activation of the ABA regulatory pair confirms its involvement in pathogenic response signalling.
| Promoter | Gene | Context | Pathogen | Function | Assay | Localization | Plant | Ref |
|---|---|---|---|---|---|---|---|---|
| Pathogenic response | ||||||||
| Box W2 + Box S | PR1 (W2), ELI7 (S) | 35S CaMV | P. parasitica, P. syringae, E. cichoracearum, B. graminis |
Responsiveness to host and non-host pathogenic attacks | 〇 | Leaves | A. thaliana | [60] |
| Box W2 + Box S + Box D | PR1 (W2), ELI7 (S), PR2 (D) | 35S CaMV | P. parasitica, P. syringae, E. cichoracearum, B. graminis |
Responsiveness to host and non-host pathogenic attacks | 〇 | Leaves | A. thaliana | [60] |
| E17 + F | ELI17 (E17), AtCMPG1 (F) | 35S CaMV(i) | Rhizoctonia solani | Additive biotrophic responsiveness | 〇 | Leaves | B. napus | [67] |
| CE3 + ABRE2 | HVA1 | 35S CaMV | Cercospora beticola | Pathogenic induced response of ABRC | 〇 | Leaves (necrotic region) | B. vulgaris | [52] |
| Wounding response | ||||||||
| Box W2 + Box S | PR1 (W2), ELI7 (S) | 35S CaMV | n.a. | Responsiveness to wounding stress | 〇 | Leaves | A. thaliana | [60] |
| Box W2 + Box S + Box D | PR1 (W2), ELI7 (S), PR2 (D) | 35S CaMV | n.a. | Responsiveness to wounding stress | 〇 | Leaves | A. thaliana | [60] |
| Inhibitor/elicitor response | ||||||||
| Z + GATA, G-box + GATA, GT1 + GATA | LHCB, RBCS | NOS101 | n.a. | Norflurazon-induced promoter repression and chloroplast photooxidation | 〇 | A. thaliana | [4] | |
| CE3 + ABRE2 | HVA1 | 35S CaMV | n.a. | Benzo-(1,2,3)-thiadiazole-7-carbothioic acid S-methyl ester (BTH) induction of systemic acquired resistance | 〇 | B. vulgaris | [52] | |
| GST1 + W-box + HSRE | GST1, AtNPR1, HSR203 | 35S CaMV | n.a. | Additive response to probenazole-induced systemic acquired resistance | 〇 | A. thaliana | [77] | |
n.a. – not applicable. (i) – Intron included downstream of minimal promoter. o – in planta assay.
Over the decades, testing combinations of CREs in planta has progressed slowly. Cis-motifs combination holds multiple potential benefits for crop improvements through precision breeding and genome engineering. Stimulus specific activation of CREs combination can be harnessed to drive spatiotemporal expression of stress and developmental genes involved in morphological traits enhancement in plants. There are several examples of novel gene expression patterns that have translated to acquisition of improved traits vis-à-vis CREs modification and have become bases for breeding selection and domestication of crops. To our knowledge, most of these CREs-mediated crop improvements were achieved with either mutating cis-element site within native promoter context [extensively reviewed in 68, 69, 70] or coupling promoter fragments in a synthetic promoter [7, 17, 19, 20, 71, 72, 73, 74]. Misra and Ganesan [75] have summarised the utilisation of fragments from constitutive, inducible, and tissue-specific promoters to enhance genetic and phenotypic traits in plants.
The use of cis-elements combination has not been fully explored despite having greater precision in terms of organ and stimulus-specific inducibility over other conventional approaches. For instance, concatemers promoters (SynP15, SynP16 and SynP18) have been reported to direct root-specific drought-inducible expression in both soybean and Arabidopsis plants [76]. The synthesis of tissue-specific promoters proved the precision and reliability of the method and can be exploited to fine-tune plant development and responsiveness to environmental cues. The foundational research on the development of stress-inducible promoters [5, 6, 44, 48, 60, 77] offer the opportunity to engineer traits that provide phenotypic tolerance to stresses in plants. Overexpression of Arabidopsis CBF1 driven by ABRC1 of barley HVA22 gene lead to the development of transgenic tomato plants with enhanced tolerance to chilling, drought and salt stress and minimal effects on phenotype and yield [78]. Hsieh et al. [79, 80] reported that 35S CaMV promoter-driven overexpression of CBF1 in tomato also improved tolerance to the same stresses, but resulted in a dwarf phenotype, reduction in fruit set and seed number per fruit. The ABRC1 promoter, known for its inducibility under stress conditions and reversibility in the absence of stress, is particularly well-suited for driving genes related to abiotic stress in plants [78]. The suitability arises from its reported transcriptional and translational stability within the plant's genetic machinery. With CREs combination, gene regulation is tightly controlled by cis-acting regulatory motifs interactions and their corresponding TFs. Eliminating transcriptional noises or interferences common in fragment-driven promoters minimise the risk of unexpected promoter function in the cell.
The promoter structure of eukaryotes is complex and bound by TFs which act together with the pre-initiation complex to regulate transcription of genes. The combinatorial nature of many of the DNA binding interactions is mainly responsible for the diversity and complexity observed in eukaryotes [81]. Several efforts aimed at deciphering the complexity have in some ways revealed the regulatory network of genes and matrices of events with answers to what, when, how, where and which signals are transduced in the cell. Promoter strength and inducibility in response to stimulus are dependent on composite elements. Identifying CREs in promoters and testing their functions help to delineate DNA-protein and protein-protein interaction.
Cis-motifs are discrete, and each has the potential to function effectively when placed in the correct position and combination. The only method to validate an effective combination of cis-elements is by shuffling elements and testing their performance through a trial-and-error approach [76]. This process is laborious, time-consuming, and quite unpredictable. However, assessing elements’ function in promoter constructs is highly qualitative. CREs when combined may act synergistically, cooperatively and in some cases antagonistically. Promoter regions contain multiple binding sites for TFs, and it is believed that TFs act together to mediate signal-specific expression of genes. Placing CREs side by side in a heterologous minimal promoter reveals the intrinsic interaction of proteins bound to these elements. Several putative elements have been identified in promoters of plants, but relatively few research have delved into testing regulatory elements rather than DNA fragments. However, we must advance our knowledge about promoter structure of genes and how each element contributes to overall expression. The crosstalk of signals transmitted via multiple elements situated closely together within a synthetic promoter is more robust compared to what can be achieved with a single-element promoter.
Preliminary testing of elements in their combinatorial form can be done in a transient expression system. Agro-infiltration of leaves with agrobacterium cultures can be used for rapid evaluation of transient gene expression, but this system may not be useful for spatiotemporal promoters driving expression in other organs [82]. Interpreting the results of promoter responsiveness in transient expression systems should be done cautiously. For instance, combinatorial action of pathogen-inducible elements in transformed Arabidopsis seedlings gave contrasting responses when same constructs were transiently expressed in parsley protoplast [60]. It is imperative to validate results obtained from transient expression systems with a follow-up experiment using in planta assays to ensure the reproducibility of promoter activity in stable transformants.
I would like to thank Natsuki Hayami for a good discussion.
