Efforts to elucidate the genetic basis of biosynthetic pathways tend to result in the identification of structural genes, i.e. genes encoding enzymes that catalyze the conversion of intermediates in a specific pathway. This is in part due to the relatively high expression of the structural genes, which results in abundant representation in expressed sequence tag (EST) libraries, or high probability of identification when cDNA subtraction methods or other methods that rely on differential expression are used.
In order to fully understand the biochemical pathway, it is also important to identify the regulatory mechanisms that control both the timing (developmental stage, environmental cues) and the location (cells, tissues or organs) of the biosynthesis of a particular compound or class of compounds. The regulation of biosynthetic pathways is mediated by regulatory genes that encode transcription factors and repressors that can enhance and inhibit the expression of the structural genes, and that are under control of internal (e.g. developmental) and/or external (e.g. environmental) cues. The identification of regulatory genes is generally much more difficult, in part because the expression of regulatory genes tends to be much lower than the expression of the structural genes.
Figure 3-8. Biosynthesis of anthocyanins and condensed tannins. The enzymes involved in this pathway are: (a) anthocyanidin synthase (E.C. 126.96.36.199), (b) anthocyanin 3-glycosyl transferase, and (c) BANYULS.
Furthermore, there is considerable sequence similarity between transcription factors, even though they may operate in different biosynthetic pathways. Hence, based on sequence similarity it is often difficult to identify the target genes of a given transcription factor.
Interestingly, regulatory genes involved in the biosynthesis of anthocyanins were identified relatively early on. This was possible because of a number of mutants that accumulated high levels of anthocyanins, or that accumulated anthocyanins in tissues where they were normally not found, in combination with the easily scorable phenotype of these mutants.
The Red color (R) and Booster (B) genes are regulatory genes that control the tissue-specific deposition of anthocyanins in maize. These two genes were shown to independently activate the same target gene Bz1 (Dooner and Nelson, 1977). In most maize lines the R gene is actually represented by a small family of homologous genes that map closely together and that are thought to have arisen through gene duplication and divergence. A functional R gene is required for the pigmentation of all plant tissues. More than 50 naturally occurring R alleles have been identified, which differ from each other in the spatial and temporal patterns of anthocyanin accumulation. The particular pattern of pigmentation displayed by a given plant is the result of the combined expression of all R family members that it contains. The standard R locus is responsible for pigmentation of the aleurone, anthers, and coleoptile. This phenotype is due to the expression of two tightly linked members of the R gene family, S and P. The S gene controls pigmentation of the aleurone of the kernel, whereas the P gene controls pigmentation of the anthers and coleoptile of the plant. Another member of the R gene family is the Lc gene, which conditions the pigmentation of the leaf midrib, the ligule and auricle (both of these tissue are at the boundary of the leaf blade and leaf sheath), several tissues in the male inflorescence (glume, lemma, palea), and the seed pericarp. The R-nj gene was cloned by transposon tagging (Dellaporta et al., 1988) and used to isolate genomic and cDNA clones of the Lc member of the R gene family. The Lc gene was shown to encode a transcriptional activator of the myc class. This transcription factor is required for the accumulation of transcripts of the chalcone synthase (C2) and dihydroflavonol4-reductase (A1) genes in the anthocyanin biosynthetic pathway (Ludwig et al., 1989). The R-r gene complex was characterized in detail at the molecular level by Robbins et al. (1991). It was shown to contain three complete R transcription units P, S1, S2, and one incomplete unit, Q. The P gene controls plant pigmentation, whereas the S genes control the pigmentation of the aleurone layer in the seed.
The B gene was cloned by Chandler et al. (1989) based on postulated sequence homology to the R1 gene. A probe derived from the cloned R1 gene was used to screen Southern blots obtained from a population of recombinant inbred lines in which two B alleles (b and B) were segregating. In addition to a strong hybridization signal corresponding to the R1 gene itself, a weak hybridization signal was detected, the size of which varied depending on whether the b or the B allele was present. The B-Peru allele, conferring a blotched pigmentation pattern to the leaf sheaths, was subsequently cloned from a sub-genomic library. The coding sequence and the direction of transcription were determined. Chandler et al. (1989) also showed a direct correlation between the expression level of the B gene and the expression of the target genes A1 and Bz1. This was based on expression analyses of maize plants homozygous for the b null allele, the weakly expressed B-Peru allele, and the highly expressed B-I allele. The B-Peru gene and the corresponding cDNA were subsequently sequenced (Radicella et al., 1991) and shown to encode a protein with similarity to the myc transcription factors. Comparison of the B-Peru allele with the sequence of the B-I allele indicated that the variation in expression pattern was the result of sequence variation in the promoter and the 5' part of the gene (Radicella et al., 1992).
The maize C1 (Colorless1) gene regulates the expression of the structural genes C2, A1, Bz1, Bz2, and A2 in the seed. Presence of a dominant C1 allele, in combination with functional copies of the abovementioned target genes results in seeds with colored (purple) aleurone. The C1 gene was cloned independently by Cone et al. (1986) and Paz-Arez et al. (1986) using a transposon-tagging strategy, and shown to encode a transcription factor of the myb class (Paz-Ares et al., 1987). Aside from a number of mutant c1 alleles that reduce or abolish the expression of the gene, a dominant mutant allele C1-I was identified. The protein encoded by the C1-I allele lacks 21 amino acid residues at the carboxy terminus of the protein and contains a mutation that results in an amino acid substitution (Paz-Ares et al., 1990). As a result of these two changes, the mutant C1-I protein can bind to the promoter regions of its target genes, but not activate transcription, and hence acts as a dominant inhibitor of the functional C1 protein. The similarity between the R and B proteins, both at the protein sequence level, and in molecular complementation studies, prompted Cone et al. (1993a) to investigate whether a similar relationship existed between the C1 and Purple plant (Pl) genes. The C1 gene determines the color of the aleurone, whereas the Pl gene determines the color of the vegetative and reproductive parts of the plant. Evidence in support of this hypothesis came from the existence of alleles for both genes that were only expressed after exposure to light. Cone et al. (1993a)
were thus able to clone the maize Pl gene with a DNA fragment from the C1 locus as a probe to screen genomic and cDNA libraries. Based on sequence comparison of the Pl and C1 cDNA's, more than 90% of the amino acids at the amino and carboxyl terminal domains that are important for the regulatory function of the C1 protein were shown to be identical. The difference between the light-dependent expression of the pl allele and the essentially constitutive expression of the Pl allele was subsequently shown to reside in the promoter (Cone et al., 1993b). The pl allele was expressed at very low levels, but was not a null allele. Rather, Cone et al. (1993b) proposed a threshold model, whereby the amount or concentration of the Pl protein had to be above a minimum in order for the structural anthocyanin biosynthetic genes to be activated.
In summary, anthocyanin biosynthesis in maize requires a combination of R1 and C1 or B and Pl, in addition to functional structural genes. The R1 and C1 combination is necessary for anthocyanin biosynthesis in the seeds, whereas the B and Pl combination stipulates anthocyanin synthesis in the vegetative parts of the plant. A range of modifications to this general model, such as tissue-specific deposition or light-dependent deposition, exists as a consequence of the availability of numerous mutant alleles.
In Arabidopsis the TT2, TT8, TTG1 and TTG2 genes have been shown to be regulatory genes controlling the biosynthesis of flavonoids. Walker et al.
(1999) cloned the TTG1 gene using map-based cloning and showed that this gene encodes a protein with four 'WD40' repeats. WD40 proteins have diverse roles in intracellular signaling, including control of the cell cycle and vesicular trafficking. The TTG1 protein is thought to act as a signaling molecule that activates transcription factors of the myc class. Nesi et al.
(2000) cloned the TT8 gene using T-DNA tagging. Sequence analysis revealed that this gene encodes a protein containing a basic helix-loop-helix at its C-terminus, with similarity to the maize R protein and other transcription factors of the myc class. Based on expression data obtained with quantitative PCR, Nesi et al. (2000) concluded that the TT8 protein is important for the expression of the structural genes DFR and BAN. They also provided data that TTG1 and TT2 are important regulators of these two genes.
The cloning of the TT2 gene by T-DNA tagging was reported by Nesi et al. (2001). This gene encodes an R2R3 myb protein that shows similarity to the maize C1 protein. In order to test whether the TT2 protein functions as a transcriptional activator, Nesi et al. (2001) were able to show that this protein is localized in the nucleus, that the spatio-temporal expression pattern is consistent with the production of condensed tannins in the seed coat, and that over-expression of TT2 in the presence of a functional TT8 protein results in ectopic expression (i.e. throughout the plant) of the BAN gene. Hence, TT2 and TT8 appear to work together to regulate the expression of BAN.
The TTG2 gene encodes a WRKY transcription factor (Johnson et al., 2002). This class of transcription factors is characterized by the presence of a WRKYGQK amino acid sequence (one-letter amino acid code) near the N-terminal region and a conserved C-X4.5-C-X22-23-H-X1-H sequence that resembles zinc finger motifs. Together, these motifs are referred to as the WRKY domain. WRKY transcription factors were initially implicated in the response to wounding or pathogen attack, but based on the phenotype of the ttg2 mutant, which includes developmental defects to the trichomes, it is clear that they can also function in plant development. Based on the lack of condensed tannins in the endothelial cells, the TTG2 gene is thought to play a role in the regulation of structural genes, possibly BAN, involved in the synthesis of condensed tannins.
The TT1 gene was cloned from a transparent testa mutant that was allelic to the original tt1 mutant. The new mutant originated from the insertion of the maize En transposon (see Section 3.2) that had been introduced in Arabidopsis via transformation. The insertion of the En element enabled the cloning of the TT1 gene (Sagasser et al., 2002). The deduced amino acid sequence of the TT1 protein revealed the presence of two zinc fingers, one near the N-terminus and one near the C-terminus of the protein, and an additional two zinc fingers in the C-terminal part of the protein. The presence of zinc fingers is indicative of a role as transcription factor. The TT1 sequence, however, revealed very limited homology with known proteins. TT1 and a small number of other plant proteins were classified as a novel group of transcriptional activators, the WIP subfamily of zinc finger proteins, where WIP refers to the first three conserved amino acids. The TT1 gene was shown to be expressed in the endothelial cells of the seed coat using a reporter gene construct with the GUS gene (see Chapter 1, Section 3.5). The expression of BAN was reduced, but not completely eliminated in the tt1 mutant, suggesting that TT1 is not as specific as TT8 in its regulation of BAN expression. TT1 may instead play a more general role in the differentiation of the endothelial tissue.
The maize P gene has been implicated in the regulation of phlobaphene synthesis. Phlobaphenes are red pigments that accumulate in the pericarp of the maize kernel, as well as various other parts of the plant, including the cob, husks, and tassel glumes. Phlobaphenes arise from the polymerization of flavan-4-ols, although the exact structure is not known (see Chapter 1, Section 3.14). Alleles of the P gene are typically referred to by a two-letter suffix that reflects their expression in the pericarp ad the cob. For example, the P-rr allele results in red pericarp and red cobs, whereas the P-rw allele results in red pericarp and white cobs. The P gene was isolated using a P-vv allele that resulted in variegated patterns of phlobaphene deposition in both pericarp and cob as a result of an Ac insertion in a P-rr allele (Lechelt et al., 1989). The P gene encodes two different transcripts that have the 5' exon and first intron in common, but that differ from each other at the 3' end (Grotewold et al., 1991). The proteins encoded by the P gene contain a DNA binding domain resembling that of the myb-class of transcriptional activators, and this domain of the P protein is similar to that of the R and Pl proteins that regulate anthocyanin biosynthesis. The functional P protein regulates the expression of the A1 (DFR) and the C2 (CHS) genes (Grotewold et al., 1991; 1994).
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