CAPITULO IV DESARROLLO DE LA INVESTIGACIÓN
4.1.2. Contextualización del objeto de investigación
5.2.1 Northern analysis
The procedure for northern blotting is described in Chapter 2 (Section 2. 14. 1 and 2. 14.2). RNA from the three genetically defined lines was isolated in Professor Kuehnle's
laboratory during a two-week visit. Because of the limited supply of tissue, samples could not be obtained for all stages in each cultivar. Northern analysis was performed using the full-length anthurium cDNA clones for CHS, F3H, DFR and ANS. Blots were compared with separate blots of RNA from Altar spathe tissue.
5.2.2 F3'H assay procedure
Due to difficulties encountered in isolating a F3'H cDNA clone, biochemical assays were conducted to determine ifF3'H protein activity is present in anthurium spathe. The assay is based on the procedure for the rapid isolation of microsomal fraction using Mg2+
precipitation (Diesperger et al., 1974).
This assay was performed with 1 g of freshly picked tissue. For anthurium (Altar), this comprised a mix of spathe tissue from Stages 2 to 4. For petunia, the positive control, flower limb tissue at Stages 2 and 3 was used. The petunia flower stages were as described in Froemel et al. ( 1 985). The petunia cultivar used was BR1 40 described in Chapter 3, as being recessive for F3'H but dominant for F3'j'H. Petunia F3'5'H was shown to work with naringenin as substrate in a similar assay (Menting et al., 1 994) and so was considered a valid positive control, as evidenced by the results.
Each extraction was performed with 10
mL
of extraction buffer comprised of the following ingredients:Extraction Buffer 0. 1 M Tris-HCI pH 7.S 0.02 M 2� ME
5 Ilg mL-1 pepstain A 1 mM PMSF 0.25 M sucrose 0.25 M mannitol 0.001 M EDTA. 0.5% (w/v) BSA
A bulk stock of 200 mL of extraction buffer (without the 2� ME, PM8F and pepstain A) was made up and stored at room temperature. The remaining ingredients were added at the time when the extraction was being performed. Once the buffer was made, 0. 1 g of polyclar AT and 0.5 g of acid-washed sand were added to a pre-cooled (4 °C) mortar. The tissue was added (1 g), along with 5 mL of extraction buffer, and crushed into a
homogenous suspension. The homogenate was then filtered through one layer ofMira cloth into a sterile 15
mL
Corex tube. This was followed by a 5 min spin at 5,000 rpm in a 80rval 88-34 rotor. The resulting supematant was transferred to a fresh Corex tube and centrifuged again at 10,000 rpm for 20 min. Once the spin was completed, the supematant wastransferred to a Corex tube and IM MgCh was added to a fmal concentration of 30
mM
to precipitate the microsomal fraction. Tubes were left on ice for 1 0 min, and then centrifuged at 12,000 rpm for 20 min. The microsomal pellet was then resuspended in 200 JlL of assay buffer.buffer: 0. 1 M Tris-HCI 0.000 1 M PMSF 0.001 M 2� ME
Four .replicate assay reactions were set up for each species, using the fresh extract, and having the necessary negative and positive controls. The assay was set up as follows:
1 00 ilL of resuspended microsomal pellet 5 ilL naringenin (1 mg mL-1)
5 ilL 50 mM NADPH (resuspended in assay buffer) 90 ilL assay buffer
The reaction was allowed to
run
for 2 h and in some instances, overnight, at room temperature. To terminate the reaction two sequential ethyl acetate (500 J.lL) extractions were performed. The extracts were then dried under 02-free N2 and the pellet resuspended in 1 0 J.lL of 80% (v/v) methanol. The entire sample was then loaded onto TLC cellulose plates, 2 cm from the base and 3 cm apart from the next sample. Once loaded, samples were dried and ascending chromatography against naringenin and eriodictyol standards was performed with chloroform/acetic acid/water (CA W) 10:9 : 1 (v/v/v).5.3 RESULTS
5.3.1 Northern analysis of genotypically defined white lines
Although transcripts for all four genes were detected in the white lines, for two lines they were at reduced levels compared to the cyanic cultivar Altar (Figure 5 . 1 ). This pattern was also observed for Acropolis (Figure 4.7). Cultivar 1250 deviates from this pattern with transcript levels that are similar to those of Altar for CHS, F3H and DFR. ANS transcript levels were lower than Altar but higher than in 1244 or 1 349.
5.3.2 F3'H assay
Whereas F3'H activity was consistently observed in the petunia positive control, with the conversion of naringenin to eriodictyol, no such activity was observed for the anthurium (Altar) microsomal fraction, even though the procedure was repeated several times with different stages of spathe tissue (Figure 5.2). In addition, no naringenin was detected in the anthurium assays and neither was any seen in the control assay with denatured petunia protein. Additional control experiments were performed without substrate and without protein, both of which had negative results. Finally, an assay was set up with a mixture of protein extracts from petunia and antirrhinum and this efficiently converted naringenin to eriodictyol (data not shown).
Figure 5.1. Northern analysis for testing genetic model. The blots show a comparison of
transcript levels in genotypically defined white anthurium cultivars (1 349, 1 244 and 1 250 each with a reported genotype oom-) with that Altar. Membranes were blotted separately but probed simultaneously in the same probing solution. Radiolabeled full-length cDNA clones were used as probes. 20 J,1g total RNA was loaded for each lane and a 25/268 ribosomal RNA probe (PTIP6) from asparagus (King and Davies, 1 992) was used as the loading control.
Altar (wildtype red)
- - - - -
Stages I 2 3 4 5 6
Genotypically defined white anthurium cultivars
2 3 4 6 4 5 6 4 1 349 1 244 CHS F3H DFR ANS pTIP6
Figure 5.2. Chromatogram for F3'H assay. AI-A4 are the anthurium replicates (using Altar
spathe tissue), while P I and P2 are the petunia positive controls. The positions of the two flavonoid standards S I (eriodictyol) and S2 (naringenin) are indicated by green arrows and red" arrows point to the eriodictyol formed in the petunia assay.
naringenin
5.4 DISCUSSION
5.4.1 Investigating the nature of the 0 locus in anthurium
There was a marked decline in transcripts for all four genes (CHS, F3H, DFR and ANS) over the various stages of spathe development in Acropolis (Figure 4.7) and cultivars 1 349 and 1244 (Figure 5.1) when compared with Altar, although significant levels of CHS
transcripts are still present. Therefore, the white phenotypes observed in Acropolis, 1 349 and 1 244 are most likely due to a mutation in a regulatory gene for anthocyanin
biosynthesis. Given that 1349 and 1244 are recessive for the 0 locus, the data suggest that this locus could correspond to the affected regulatory gene. Although 1 349 and 1244 may be homozygous recessive at the M locus, mm does not eliminate pigment formation, as cultivars that are O-mm have orange/coral spathe (Kamemoto et al., 1 988). Therefore, the results suggests that the 00 genotype in white lines results in reduced expression levels of several flavonoid genes in the spathe, and is possibly the main reason for the absence of pigment in these lines. However, this is contradicted by the 1250 data, if the genotyping is correct.
If 0 is a regulatory gene controlling the expression of all the flavonoid biosynthetic genes in the anthurium spathe, then its mutation should result in a reduction in flavone levels in white anthurium. Interestingly, the absence of anthocyanins does not result in either a decrease or a greater production of flavones in white lines, because in related work, similar quantities of flavones were detected in anthurium whites as in coloured lines (David Lewis, personal communication). Although the genetic make up of the lines tested was not known, one interpretation of the data is that for anthurium the regulation of flavone biosynthesis is separate from that of anthocyanins. Therefore, the existence of more than one regulatory gene controlling flavonoid accumulation in anthurium is likely. In fact, in other species the regulators of anthocyanin biosynthesis do not regulate other branches of the pathway. As a result the regulation of flavonoid biosynthesis in anthurium may have similarities to species such as maize (Grotewold et al., 1994) and antirrhinum (Moyano et al., 1 996) with
independent regulators for the different branches of the flavonoid pathway.
Should a separate regulator exist in anthurium spathe for flavones, it may target CHS and
CHI directly. The fact that the CHS transcript levels are reduced in the spathe along with
DFR and ANS suggests the existence of common regulatory elements. However, significant levels of CHS transcripts are still produced, which may indicate the activity of other
regulatory proteins. The availability of an anthurium CHI cDNA clone would be useful in exploring this possibility. From this preliminary effort, it appears that the actual genetics of flower colour inheritance in anthurium tissue appears more complex than the dihybrid inheritance model suggested by Kamemoto et a1. (1 988).
Higher transcript levels are detected in line 1250 for CHS, F3H and DFR than in lines 1 349 and 1 244 (Figure 5. 1 ), and only ANS transcript levels appear to be markedly reduced
compared to Altar. It would be useful to monitor transcript levels in more than one stage for 1 250. However, the high transcript levels of CHS, F3H and DFR seems to suggest that this cultivar may be a structural mutant in one of the genes acting downstream from DFR such as ANS, UFGT or GST.
It may also be possible that the genotype for 1250 is incorrect. However, if 1 250 is in fact 00 homozygous recessive, then it seems unlikely that 0 can encode a regulatory factor necessary for CHS, F3H or DFR expression. Thus, the regulatory mutation in some white cultivars may be in a locus distinct from O. Alternatively, they may carry distinct 0 alleles, which have markedly differing effects in their various mutant forms. In this regard the 0 locus for anthurium may be a complex loci as seen with the antirrhinum anthocyanin regulator ROSEA (Schwinn, 1999). Again, because white lines accumulate flavones, CHI
activity is assumed. Although the presence of the transcript does not always correlate to a functional protein, post-transcriptional modification does not appear to be a significant mechanism for regulating the expression of genes involved in phenylpropanoid
biosynthesis (Martin et al., 1 991). Therefore, it is likely that the transcripts being detected for CHS, F3H, DFR and ANS for 1250 do encode functional proteins. Of course a point mutation affecting protein function cannot be ruled out.
Further work is needed to make conclusive statements on the identity of the gene product coded for by the 0 locus. The cloning of the anthurium anthocyanin regulatory genes would greatly assist this objective. The primary limitation to this experimental effort was the location of stock plants in Hawaii. This meant limited RNA samples prevented experiments from being repeated or developed further.
5.4.2 Investigating the nature of the M locus in anthurium
To test the proposal that M encodes the F3 'H, an anthurium F3 'H clone was needed, as well as lines genotypically defined for the M locus. The first phase of the strategy was to isolate an anthurium F3'H cDNA clone and perform a comparative northern or PCR analysis with orange-coloured anthurium lines having an 0_ mm genotype and red-coloured lines of the 0_ MM or 0_ Mm genotype. If the model held true then, when probed with anthurium F3 'H,
there should be no (or at least very little) transcript in orange lines with genotype 0_ mm
compared to levels in the red (0 _M_ ) coloured spathe. Of course this approach to test the model assumes the predicted mutation in F3 'H is a null mutation and therefore does not allow a full-length transcript to be produced.
As discussed in Chapter 3, no cDNA clone encoding the anthurium F3'H protein was isolated from the cDNA library or by PCR approaches. Difficulty in securing F3'H clones has been encountered for other species (Kevin Davies, personal communication).
As an alternative, experiments were performed to determine if F3'H enzyme activity existed in anthurium using tissue from Altar spathe. When compared to the petunia positive
control, no definitive F3'H protein activity was detected for anthurium spathe tissue (Figure 5.2). It would, however, be useful to compare the amount of the protein in the extracts of both anthurium and petunia to determine that the extraction procedure was effective for anthurium tissue. Naringenin substrate was not detected for the anthurium assays, and was only found in the boiled controls, suggesting that the absence of naringenin is related to enzyme activity. Therefore, one possibility explaining the absence of naringenin in anthurium assay reactions is conversion to flavones by FNS or F2H as both are