ANÁLISIS DE LA APLICACIÓN DEL LABORATORIO EN EL COLEGIO JEAN

the biochemi cal and molecular aspects of flower colour development in that species. Therefore, characterising the expression patterns of the fl avonoid biosynthetic genes is central to a molecular investigation of flower colour development.

The control of anthocyanin structural gene expression is usually reflected in their temporal expression pattern. The temporal expression patterns of flavonoid biosynthetic genes have been documented for several plant species, such as antirrhinum (Martin et aI., 1 99 1 ; lackson et aI . , 1 992), petunia (Quattrocchio et aI ., 1 993), maize (Dooner, 1 983), lisianthus

(Davies et ai., 1 993 ; Oren-Shamir et aI ., 1 999), arabidopsis (Pelletier and Shirley, 1 996), Vitis vinifera (grapes) (Boss et ai., 1 996) and peri lla (Gong et aI., 1 997).

For most dicots investigated, the cascade of genes involved in anthocyanin biosynthesis appear to be separated into two subsets that represent the division of the pathway into two independently regulated units. One group consists of the early biosynthetic genes (EBGs) and the other is comprised of the late biosynthetic genes (LBGs) (Beld et aI. , 1 989; lackson et ai., 1 992; Martin and Gerats, 1 993; Quattrocchio et aI., 1 993; Mol et aI ., 1 998). It is the

LBG expression, which peaks later than the EBGs, that is correlated with pigment production. In antirrhinum, CHS and CHI constitute the EBGs and F3H, DFR, ANS the LBGs (Jackson et aI., 1 992). In petunia, a similar pattern exists except that F3H i s coordinately regulated with CHS and CHI and the LBGs comprise the genes from DFR onward (van Tunen et aI., 1 988; Quattrocchio et aI., 1 993 ; Weiss et aI ., 1 993; Quattrocchio et ai., 1 998). S imilar patterns to these traditional dicot models are found in lisianthus and arab idopsis. In lisianthus, CHS and CHI form the EBGs, whi le F3H is coordinately regulated with the anthocyanin specific genes, and in arabidopsis, LBGs include DFR and

ANS, whi le CHS, CHI and F3H constitute the EBGs.

There are dicot species, such as grape and peri lla where this pattern does not hold true. I n acyanic grape berries, all genes have reduced expression levels, whereas, prior to pigment accumulation in red berries, transcripts for all genes, from CHS to ANS, are strongly expressed. Anthocyanin production is concomitant with UFGT expression (Boss et aI .,

1 996). While it is thought that the absence of UFGT expression is responsible for white grapes, it is not known if this is due to mutation(s) in the structural or regulatory gene(s) (Boss et aI., 1 996). In peril la, no distinct subdivision into LBGs and EBGs is observed as the transcript abundance of all structural genes examined was coordinately regulated (Gong et aI., 1 997).

The only monocot where simi lar research has been conducted is m aize. I n this case all the structural genes involved in anthocyanin biosynthesis were coordinately induced (Dooner,

structural genes involved in anthocyanin production in the maize kernel have the same temporal distribution pattern (reviewed in Mol et aI., 1 998).

Comparing maize to the traditional dicot models, where modular control of the pathway is observed, inferences can be made as to the evolutionary significance of discrete regulatory units ( Martin et aI., 1 99 1 ). I n maize, the pigmentation of the kernel may primarily be associated with attracting animals for seed dispersal . Therefore, there may be no selective advantage in modular control of anthocyanin biosynthetic genes in this tissue. However, in flowers, fl avones and flavonols absorb UV l ight and may also play a role as insect

attractants. Therefore, modular control of anthocyanin production allows for regulated control of the biosynthesis of these pigments separate from that of anthocyanin production. On this basis, the regulatory control in maize may be very different from that in the leaf or flower of another monocot species and may be of considerable relevance to pigment production in anthurium.

S ince flavonoid biosynthetic genes are controlled at the level of transcription, then the temporal distribution of the genes serves as a window to the pattern of regulation of anthocyanin biosynthesis in that species. Regulatory proteins involved in anthocyanin biosynthesis have been defined in only a few plant model systems and are discussed in the fol lowing sections.

1. 10 GENE TRANSCRIPTION

Regulating the expression of genes is fundamental to an organism's ability to control its biological processes. M ost regulation of expression occurs at the level of transcription and is mediated by transcription factors that either activate or repress transcription of the gene. M uch of our knowledge in this area has come from studies of eukaryotic transcription activators and these studies have revealed a highly intricate system that is tightly regulated at m any levels. Regulation of transcription is effected by sequence specific bin ding of the transcription factor to eis-acting elements in the promoter or enhancer regions of the target gene. This binding facilitates the assembly of the transcription factor complex, which includes several basic transcription factors and RNA polymerases, to initiate mRNA

synthesis. Consistent with their function, transcription factors have a DNA binding domain and a regulatory activation domain (or a repressor domain for factors that repress

transcription). These domains are functionally separable and can be repositioned within the protein or exchanged between proteins without loss of function (Ptashne, 1 988). Further to this, it is the activation domain that is thought to interact with the basic transcription machinery (Ptashne, 1 988). Several fami lies of transcription factors have been identified both in animals and plants, based on characteristic structural DNA-binding motifs (e.g. Myb, bZI P, bHLH, MADS-box) (Meshi and Iwabuchi, 1 995). While the DNA binding domain tends to be specific and highly conserved for a certain class of transcription factor, the activating regions are less precisely defined, and exhibit extensive variation within a given class.

There is a growing body of knowledge on the precise steps involved in the transcription of a gene. The ability to perform in vitro transcription reactions (Buratowski et aI., 1 989) has shed light on the general order of assembly for the transcription factor complex. While several enzymes and a growing number of cofactors (Inostroza et aI., 1 992; Nakaj ima et aI.,

1 997) are involved at various levels of gene transcription, for protein-encoding genes, the key enzyme involved in eukaryotic systems is RNA polymerase II (RNA Pol II). Though structural ly complex (it is made up 1 2-sub units), it cannot recognise its target promoters directly. Added to which, it is required to modulate production of the RNA transcripts of individual genes in response to developmental and environmental signals (Nikolov and B urley, 1 997). To accomplish these two functions it requires interaction with variety of proteins that comprise the basal transcription machinery which includes TAT A-binding protein (TBP), general transcription factors (TFI IA, TFI IB , TFI ID, TFIIE, TFIIF, TFIIH), TBP-associate factors (TAFs), and Switch/Snif (SWN/SNF) proteins which are involved in chromatin remodeling events. The process of gene transcription can be summarised into three phases. The first is initiation, which involves binding to the TA TA-box in the target

DNA by TFIlD and the assembly of the initiation complex. This is followed by the

unwinding of the DNA through the helicase activity of TFIIH and subsequent synthesis of an RNA transcript by RNA Pol n, in the presence of nucleoside triphosphates.

last exon. This signal is used to add a series of adenylate residues during RNA

processing. Transcription often terminates at 0.5-2 kb downstream of the poly (At signal.

Two models have been advanced to explain the many protein-protein interactions associated with the transcription of a gene. The first is a step-wise assembly developed from in vitro transcription factor studies (Buratowski et al., 1 989). This model assumes an ordered sequential assembly of the transcription pre-initiation complex. However, such a process appears to be inefficient given the extraordinarily large size of the complex. In addition, it is hard to envisage the assembly of such a complex within the time frame necessary at each promoter (Lemon and Tj ian, 2000). The competing model is at the other end of the spectrum and is described as the pre-assembly model. Founded on the discovery that RNA Pol I I existed as a holoenzyme, complexed with several members of the basal transcription machinery, such as SWIISNF, the model assumes the recruitment of a completely assembled RNA Pol II pre-initiation complex (Koleske and Young, 1 994; Parvin and Young, 1 998). However, the validity of this model is also being challenged as new information becomes available (Zawel et al., 1 995; Kimura et al., 1 999). In addition to these two models, there is the possibility that genes may undergo an early chromatin arrangement involving interactions with transcription factors and chromatin-associated remodeling factors that render the gene competent for activation prior to binding of the initiation complex (Cosma et al ., 1 999; Krebs et al. , 1 999).

1 . 1 1 Myb TRANSCRIPTION FACTORS

Myb proteins play a key role in the regulation of structural gene expression for anthocyanin biosynthesis. They are a unique family of DNA binding proteins that bind DNA as

monomers, unlike other sequence-specific DNA binding proteins (Howe et al., 1 990), and induce a bend in the target sequence (Saikumar et al., 1 994; S olano et al., 1 995a,b). The cellular proto-oncogene c-Myb and its truncated version v-Myb, isolated from the avian myeloblastosis virus, were the first Myb proteins discovered and characterised as DNA binding proteins acting as transcription factors (Klempnauer et al., 1 982; B iedenkapp et al.,

1 988).

1. 11. 1 Myb distribution and diversity of fun ction

Myb proteins have been found in a wide range of eukaryotes such as humans (Nomura et ai., 1 988), chicken, mouse (Howe et aI., 1 990; Lipsick, 1 996), Xenopus (Amaravadi and King, 1 994), Drosophila (Peters et aI ., 1 987), fungi (Tice-Baldwin et ai., 1 989; Ohi et ai.,

1 994; Wieser and Adams, 1 995), slime mould (Stober-Grasser et aI., 1 992; Guo et aI. , 1 999), a wide selection of angiosperms including both dicots and monocots (Jackson e t ai., 1 99 1 ; Avila et aI ., 1 993 ; Baranowskij et aI., 1 994; Lin et aI., 1 996; Romero et aI., 1 998), gymnosperms such as black spruce (Charest et aI., 1 994) and mosses, for example

Physcomitrella patens (Leech et aI ., 1 993).

The first plant Myb gene to be identified was the maize anthocyanin regulator C l (Paz-Ares et aI., 1 987). S ince that time, the number of Myb proteins in plants has expanded

enonnously with arabidopsis alone assumed to have in excess of 1 00 Myb genes (Romero et aI., 1 998). A similarly l arge family is suggested for the maize genome (Rabinowicz et aI.,

1 999), petunia (Avila et aI., 1 993) and tomatoes (Lin et aI ., 1 996). In fact, for plants, Myb proteins constitute a large gene family of transcription factors that play a significant role in the transcription control of a diverse range of p lant processes (Table 1 . 1 ).

Thi s pattern seen in plants, is completely unlike that in vertebrates, where the Myb gene fami ly is small and their function seems l imited to controll ing cellular proliferation and differentiation (Weston, 1 998). The large size of the Myb family in plants, can in part, be explained by genetic redundancy and overlapping functions, such that structurally similar Mybs have similar functions, recognising the same target genes, and are involved in similar processes but are expressed in different tissues. AN2 is an R2R3 Myb related protein that acts as the main determinant of colour differences in the petunia corolla (Quattrocchio,

1 994). AN2 regulates anthocyanin production primaril y in the l imb, although limited expression can be detected in the tube and pistil (de Vlaming et aI . , 1 984, Quattrocchio et aI., 1 993). The paralogous AN4 appears to control anthocyanin production in a separate tissue, anthers, and it is the likely candidate to substitute for AN2 activity in tissues where

Table 1 . 1 The range of plant processes controlled by Myb proteins.

Plant process

Phenylpropanoid metabol ism

Plant defense

Cell ular differentiation Cell shape

Circadian clock

Stress response

Seed development and germination Leaf polarity

Trichome development Lateral meristem initiation

Reference

Romero et ai. , 1 998; Jin and M artin, 1 999 and several references throughout the thesis Yang and Klessig, 1 996

Oppenheimer et ai., 1 99 1 ; Wada et ai., 1 997 Noda et aI., 1 994; Mur, 1 995.

Wang et aI ., 1 997; Wang and Tobin, 1 998; M izoguchi et aI., 2002 ; Carre and Kim, 2002 Urao et ai., 1 996; Itturriaga et aI., 1 996; Lu et ai., 2002

G ubler et aI ., 1 99 5 ; S uzuki et aI ., 1 997

Waites et ai., 1 998; G alego and Almeida, 2002 Oppenheimer et aI., 1 99 1 ; Sawa, 2002

Schmitz et ai . , 2 002

AN2 is not found (Quattrocchio et aI., 1 993 ; H uits et ai., 1 994; Quattrocchio et aI., 1 998). S imilarly in maize, two Myb proteins identified as C l (Paz-Ares et ai., 1 986, 1 987) and purple leaf (PL) (Cone et ai., 1 993) have been definitively characterised as transcriptional activators of the structural genes for anthocyanin biosynthesis (Sainz et ai., 1 997a). The Cl and PI gene products sharing 80% identity are functionally equivalent regulating the same genes but in different tissues (Cone et ai., 1 993). C l is expressed in the aleurone of the kernel and the embryo and controls pigment production in these tissues (Chen and Coe,

1 977; Coe, 1 985) while PL is required for pigmentation of m ost of the plant body (Cone et ai., 1 993). Further evidence for their functional simi larity was provided by the PL-Bh allele, that can substitute for C I activity in the kernel (Cocciolone and Cone 1 993).

However, it is possible for plant Mybs with very similar DNA binding domains to be involved in very different physiological processes. For example, GLABRA 1 (GL 1 ) and ROSEA l are classed in the same Myb subgroup based on the similarity of their M yb domain (Jin and M artin, 1 999) but GL 1 i s involved i n trichome specification and probabl y

mediates i ts activity by modifying cell size, while ROSEA 1 regulates anthocyanin biosynthesis (Oppenheimer et aI., 1 99 1 ; Schwinn, 1 999). Therefore, while some

redundancy may exist, diversity and flexibility are accomplished in having a large gene family. The overlapping function and apparent redundancy of M yb proteins in plants may represent a unique feature by plants to selectively use M yb transcription factors to regulate their specific physiological processes (Martin and Paz-Ares, 1 997).

1.11.2 Defining features of Myb proteins: The Myb domain

Myb proteins have a modular structure consisting of an N -tenninal DNA binding domain , and a C-terminal transactivation domain (only for transcriptional activators). Animal Mybs may also have a repressor domain in the C-terminus. The distinguishing feature of all M yb proteins i s the DNA binding domain that specifically binds to sequences of DNA in the promoter of target genes (Frampton et aI . , 1 989; Sakura et aI ., 1 989; H owe et aI ., 1 990). The transcriptional activation domain, in the C-tenninal end, is believed to interact with the target DNA in such a way as to promote the initiation of transcription (Ptashne, 1 988).

1.11. 2.1 Three repeat Mybs

The DNA binding domain of the prototypic vertebrate cel lular proto-oncogene c-Myb comprises three imperfect repeats (R 1 , R2 and R3 ) and regularly spaced tryptophan

residues, which provide a hydrophobic core (Anton and Frampton, 1 988; Ogata et aI . , 1 992, 1 994). E ach repeat adopts a helix-helix-turn-hel ix configuration for interaction with the maj or groove of DNA (Ogata et aI., 1 994). The first repeat i s not essential for DNA binding (B iedenkapp et aI., 1 988) and cooperative action between R2 and R3 is the suggested mechanism for binding, as neither of the two repeats can bind DNA independently

(Tanikawa et aI., 1 993). There is evidence for direct interaction at specific contact points to fonn the cooperative unit that recognises the target sequence in the maj or grooves of the DNA (Ogata et aI., 1 994). It is the R2 and R3 domains that are required for sequence specific binding, with the third helix in each repeat serving as the recognition helix (Ogata et aI ., 1 994). Plant proteins with three M yb motifs have recently been discovered i n arabidopsis, where they control the cel l cycle (Braun and G rotewold, 1 999; Kranz e t a I . ,

2000; Ito et aI ., 200 1 ; Lu et aI ., 2002). I n fact, they have been observed in all maj or evolutionary lineages in plants from bryophytes to mono cots ( Kranz et aI., 2000).

The three-repeat plant proteins were shown to be more similar to vertebrate c-Myb than to other plant Mybs (Braun and Grotewold, 1 999; Kranz et aI. , 2000). I n addition, the putative amino acid motif in the R2 repeat that determines recognition specificity and the target genes of three repeat Mybs is also conserved in the plant counterparts (Kranz et aI., 2000). This suggests R I R2R3 Mybs may have a conserved function in eukaryotes, that is cell cycle control and differentiation ( Kranz et aI., 2000), whereas R2R3 Mybs regulate plant specific processes that evolved during p lant speciation ( Kranz et aI., 2000).

1.11. 2. 2 Two-repeat Mybs

Unlike the three-repeat configuration in animal Mybs, most plant Mybs have two repeats (R2 and R3) which are most similar to the second and third repeats of the prototypic c-Myb (Will iams and Grotewold, 1 997). In fact, to date, plants appear to be the only source of R2R3 M yb genes (Riechmann et aI . , 2000). The R2R3 Myb group is the largest Myb group in plants and given the extensive range of processes that they regulate, it had been

suggested, that plant R2R3 M ybs were the equivalent of the R I R2R3 M ybs and that the three M yb-repeat DNA binding domain did not exist before the divergence of animal and plant l ineages (Rosinski and Atchley, 1 998). H owever, the discovery of the three-repeat Mybs throughout the plant kingdom, coupled with their strikingly similar gene intronlexon structure to those of animal three-repeat Mybs, suggest that the three-repeat Myb domain formed prior to the divergence of plant and animals.

The discovery of three-repeat Myb proteins across all plant l ineages does support the

hypothesis that the two-repeat Mybs may have evolved from loss of the first repeat (R 1 )

from an ancient R I R2R3 ancestor (Lipsick, 1 996). R I has been shown not to be essential

for M yb protein function (Biedenkapp et aI., 1 988). Therefore, the vast number of R2R3

Mybs may have resulted from the duplication of entire genes within plant systems (Lipsick, 1 996).

1 . 1 1 . 2. 3 Single repeat Mybs

A growing number of single-repeat Mybs (R I or R2) have been i dentified in plants ( Baranowskij et aI., 1 994; Kirik and Baumlein, 1 996; Feldbrugge et aI., 1 997 ; Wada et aI.,

1 997; Wang et aI., 1 997; Schaffer et aI., 1 998; Lu et aI., 2002; Sawa 2002). It has been suggested that one-repeat Mybs may have a different mechanism for DNA binding compared to that of two or three-repeat Mybs (l in and Martin, 1 999), but thi s is not supported by current research. NMR analysis of the DNA binding domain of the s i ngle­ repeat Mybs, TFR l and TFR2, bound to telomeric DNA has revealed three helices with architecture similar to the three repeats of c-Myb (Nishikawa et aI., 200 1 ). S equence specific binding is possible with only the single repeat and again, the third helix o f TFR l is the recognition helix sitting in the maj or grove of the target DNA, a configuration that paral lels that seen in three and two-repeat Mybs (B ianchi et aI ., 1 997; Nishikawa et aI., 200 1 ).

1. 1 1 . 2. 4 Myb domain features for plant and animal Mybs

Certain key differences within the DNA binding domain of plant R2R3 Myb distinguish them from their animal counterparts. One such difference is that i n most plant M ybs, there is a conservative substitution of the tryptophan residue in the third repeat (Martin and Paz­ Ares, 1 997). Additionally, there is an extra leucine residue between the second and third helices of the R2 repeat in plants, coupled with a glutamine to serine substitution in the R2 DNA recognition helix (Williams and Grotewold, 1 997). Such changes may alter the recognition specificities of the recognition helix as well as the co-operative activity