May 3, 2011 - usually expressed in later development stages (Han, et al., 2006; Wang et al., 2011). ..... for a 3 min incubation at 56°C. The lysate was transferred into a ..... Pair 6F-6R did not result in good quality sequences in D. scabra.
CALIFORNIA STATE UNIVERSITY, NORTHRIDGE
EVOLUTION OF ANTHOCYANIDIN SYNTHASE IN HAWAIIAN SILVERSWORDS AND CALIFORNIA TARWEEDS
A thesis submitted in partial fulfillment of the requirements for the degree of Master of Science in Biology
by Ekaterina Kovacheva
May 2011
The thesis of Ekaterina Kovacheva is approved:
Michael Summers, Ph.D.
Date
Stan Metzenberg, Ph.D.
Date
Virginia berholzer Vandergon, Ph.D., Chau
Date
California State University, Northridge 11
TABLE OF CONTENTS
Signature Page
11
List ofFigures
IV
Abstract
v
Introduction
1
Materials and Methods
9
Results
15
Discussion
19
Conclusion
24
References
25
Appendix A: Figures
31
Appendix B: Coding Sequences and Multiple Sequence Alignments
49
111
LIST OF FIGURES Figure Al: A generalized scheme ofthe flavonoid biosynthetic pathway
31
Figure A2: Proposed mechanism of action of anthocyanidin synthase
32
Figure A3. Comparison of the action of iron and 2-0G dependent oxygenases ANS and FLS
33
Figure A4: Structure of ANS
34
Figure AS: Map of ANS and primers used
35
Figure A6: Primers that successfully produced results
36
Figure A7: Species used for pylogenetic analysis
37
Figure A8: PCR Protocol for all reactions
39
Figure A9: Cycling conditions for PCR
40
Figure AlO: Number of forward and reverse clones
41
Figure All: Intron sizes and sections of exons 1 and 2 that were sequenced from each copy of ANS
42
Figure Al2: Maximum likelihood tree generated with MEGA 5
43
Figure A13: Bayesian tree generated with MrBayes 3.1.2
45
Figure Al4: Ka/Ks values for selected Madiinae
47
Figure Al5: Ka/Ks ratios for various structural and regulatory genes found in Madiinae
48
Figure B 1: Coding sequences obtained in this study
49
Figure B2: Protein sequence aligmnent of all sequences used in this study
52
Figure B3: Protein sequence alignment of the sequences, excluding the short Holocarpha sp.
54
Figure B4: DNA alignment of all sequences used in this study
56
Figure B5: DNA aligmnent of the longest sequences used in this study
60
lV
ABSTRACT
EVOLUTION OF ANTHOCYANIDIN SYNTHASE IN HAWAIIAN SILVERSWORDS AND CALIFORNIA TARWEEDS
By
Ekaterina Kovacheva
Master of Science in Biology
One of the most spectacular examples of insular adaptive radiation can be found among the Hawaiian silverswords (Asteraceae-Madiinae). This monophyletic group was transported to the Hawaiian archipelago about five million years ago, and today comprises 32 species in three genera. Having to adapt to very varied and dynamic environments, the plants display stunning morphological and physiological diversity. However, their genotypic diversity is low, as evidenced by the ease with which species of different genera form hybrids in nature and in the lab. This discrepancy can be explained by differences in just a few key loci, which could modify entire pathways. The closest relatives of the silverswords are thought to be the tarweeds (Asteraceae-Madiinae) on the west coast ofNorth America. Two tarweed ancestors hybridized to form the allotetraploid ancestor of all known silverswords, which was then transported to the Hawaiian islands, most likely by a bird. The pathway I am concemed with is the anthocyanin-producing pathway, in particular anthocyanidin synthase (ANS), a late gene in the pathway. Anthocyanins are pigments responsible for blue, red and purple color in flowers. However, due to their many other functions, such as light attenuation in chloroplasts, I expect the genes of this pathway, including ANS, to be intact in most silverswords, even though only one species has red flowers. I also expect to find two copies in silverswords due to tetraploidy. Degenerate primers were designed in conserved regions of ANS and PCR was done to obtain the gene from two tarweeds (Madia elegans, Madia gracilis) and two silverswords (Dubautia linearis, Wilkesia gymnoxiphium). Gene sequences were spliced and edited with BioEdit, and were aligned with ClustalW 1.8. A maximum likelihood tree was made with MEGA 5, and a bayesian tree was made with MrBayes 3 .1.2. A Z-test of selection and a Ka/Ks test were done using MEGA 5. A RACE reaction wasperfonned on mRNA from the flowers of M elegans. The entire gene (except 20bp of primers in the flanking regions) of two exons and one intron was sequenced from all species studied. The RACE in M elegans produced the entire gene, and showed there was some expression in the floral tissues of this plant. As expected, two copies were found in the silverswords and only one in each of the tarweeds. No premature stop codons were found within coding sequences. Phylogenetic
v
analysis groups the Madiinae separately from other Asteraceae, and the tarweeds separately from the silverswords. Within the siverswords, copy 1 from both species is separate from copy 2 from both species. Interestingly, intron size seems to be conserved within the tarweeds and copy 2 (547bp), and also within copy 1 (485bp). Purifying selection was detected in the silversword copies, while no positive selection was found in any copy. The positions of all copies studied and their relative position to other Asteraceae support the allopolyploid origin of the silverswords. Each copy in the silverswords most likely came from each of the two mainland ancestors. Given the overall similarity of the sequences within all Madiinae, it is reasonable to conclude that the gene starts and ends in the same position in all Madiinae. The lack of premature stop codons indicates that the gene may be at the very least functional, although we have no knowledge of its level of expression within the floral or other tissues. The flower color of all plants in this study is yellow, indicating no accumulation of anthocyanins, although they may be found in other tissues. The lab is cunently investigating the sequences of other genes in the pathway, and future studies will focus on sequencing the promoter region and known regulators of ANS expression. This will give us a more complete picture of the evolution of the pathway, helping us understand the genetic mechanisms behind adaptive radiations.
Vl
INTRODUCTION
The anthocyanin biosynthetic pathway The anthocyanin biosynthetic pathway (ABP) is most well-known for its role in coloration and other important functions of plants, as well as for plants' health benefits to humans, and as such it has been very well-studied (Grotewold, 2006; Koes, Quattrocchio, & Mol, 1994; Winkel-Shirley, 2001). The earliest known research focused on anthocyanins (ANs) as pigments, and included studies ofthe effects of acids and bases on pigment colors (Winkel, 2006; Winkel-Shirley, 2001) and on the inheritance pattems of flower and seed color (MendelWeb, 1997). In the 1910s it was found that pigments in many plants are derived from three basic anthocyanidins: pelargonidin, cyanidin and delphinidin (Winkel, 2006). At that time it was also established that these compounds were chemically related to the flavonols quercetin, kaempferol and myricetin, and that they sometimes contained sugar or methoxy groups (Winkel, 2006; Winkel-Shirley, 2001 ). All of these compounds belong to the group of chemicals known as flavonoids; ABP is part of the larger flavonoid biosynthetic pathway. The 1950s and 1960s saw an increase in the understanding of flavonoid structures and in the genetic and enzymatic data of genes involved in their biosynthesis (Winkel, 2006). By the 1990s, the major enzymes in the pathway, including anthocyanidin synthase, the focus of this study, had been indentified and their structures solved (Saito, et al., 1999). An overview of the generalized flavonoid biosynthetic pathway is shown in Figure Al. Chalcone synthase (CHS) catalyzes the first committed step by condensing three molecules ofMalonyl-CoA with a molecule ofp-Coumaroyl-CoA to produce tetrahydroxychalcone (Grotewold, 2006; T. A. Holton & Comish, 1995). Chalcone isomerase (CHI) then isomerizes the chalcone by closing the C-ring, producing the flavanone naringenin. Flavanone 3-hydroxylase (F3H) converts naringenin to dihydrokaempferol (DHK), which is then converted to leucopelargonidin (a leucoanthocyanidin) by dihydroflavonol reductase (DFR). Leucoanthocyanidins are converted to colored anthocyanidins by anthocyanidin synthase (ANS, synonym: leucoanthocyanidin dioxygenase, LDOX). The resulting anthocyanidins are unstable; glycosylation at C-3 and/or C-5 converts them to anthocyanins and stabilizes them. Finally, glutathione S-transferase (GST) mediates the transport of ANs to the vacuole. As seen in Figure A1, there is multiple branching of the pathway. DHK can be hydroxylated by flavonoid 3 '-hydroxylase (F3 'H) to make dihydroquercetin (DHQ), and both DHK and DHQ can be converted to dihydromyricetin (DHM) by flavonoid 3 '5 'hydroxylase (F3 '5 'H). Subsequently, DHQ leads to cyanidin, and DHM to delphinidin. DFR is known to have some substrate specificity (Helariutta, et al., 1993; Katsumoto et al., 2007; Meyer, et al., 1987), but no such specificity has been observed in ANS. The resulting anthocyanidins differ by the number of hydroxyl groups on the B-ring and by their color; pelargonidins are typically brick red, cyanidins are purple, and delphinidins are blue. Some of the precursors of anthocyanidins lead to other important flavonoid compounds. All of the substrates ofDFR can be utilized by flavonol synthase (FLS) to produce flavonols, and flavone synthase (FNS) can produce flavones from naringenin. 1
These flavonoids have a myriad of other functions in plants, but more importantly they can contribute to color stability by serving as co-pigments for anthocyanins (Grotewold, 2006). Evolution ofFlavonoids Flavonoids most likely appeared in stages as plants migrated to land (Koes, et al., 1994; Stafford, 1991). Chalcones, flavanones and flavonols first appeared in the ancestors of modem Bryophytes, about 500 million years ago (mya), although not all modem Bryophytes contain flavonoids. These compounds are today found across the entire plant kingdom (Stafford, 1991). Proanthocyanidins appeared in ferns 370mya (Koes, et al., 1994; Stafford, 1991). Anthocyanins did not appear until120mya, and are today found in some gymnosperms and in all angiosperms (Stafford, 1991), except the Caryophyllales (Stafford, 1994). Although UV protection is a widespread function of flavonoids that would have been particularly helpful in land transition, Stafford (1991) argues that early flavonoids may have functioned as regulatory and signaling molecules instead. When flavonoid pathway enzymes first appeared, they were likely very inefficient and the flavonoid levels produced were not sufficient to provide the UV protection shield that plants needed (Stafford, 1991). Regulation or signaling, on the other hand, need very low levels of the necessary molecules in order to work. There is evidence that the enzymes producing these secondary compounds have evolved from enzymes of the primary metabolism. Large parts of the sequences of plant DFR genes have been found to have high homology with mammalian 3b-hydroxysteroid dehydrogenases, bacterial cholesterol dehydrogenases and some viral ORFs (Baker & Blasco, 1992). A phylogenetic analysis confinns a common origin of all these enzymes (Baker & Blasco, 1992). Anthocyanidin Synthase ANS has been shown tln·ough sequence similarity (Martens et al., 2003) and biochemical reactions (Saito, et al., 1999; Turnbull et al., 2004) to be a member of the 2oxogluratate (2-0G) and iron-dependent family of oxygenases, which include other enzymes in the flavonoid pathway, such as FNS I (Britsch, 1990a), F3H (Britsch, 1990b), and FLS (Holton, Brugliera, & Tanaka, 1993). Other 2-0G dioxygenases are involved in many other biosynthetic pathways, such as those producing collagen, modified amino acids and peptides, and ~-lactam antibiotics (Schofield & Zhang, 1999). In ABP, ANS has been suggested to catalyze the last step of the pathway, converting colorless leucoanthocyanidins to colored anthocyanidins (Menssen et al., 1990). The reaction is thought to be: leucoanthocyanidin + 2-0G + 0 2 anthocyanidin (flavylium ion)+ succinate+ C02 (Saito, et al., 1999). The exact mechanism of the reaction has not been elucidated, and its investigation is complicated by the instability of the resulting anthocyanidin. The anthocyanidin can exist as a colored flavylium ion, but that structure is highly prone to hydration at the C-2 position, turning it to a colorless pseudobase (Figure A2). The pH level can affect the likelihood of this reaction, but the levels typically found in plants (3-6) tend to favor the hydration of the umnodified 2
anthocyanidins (Francisco, 1995). Glycosylation of the anthocyanidin at C-3 and/or C5 by glycosyl transferases (GT) contributes the most to the stabilization of the molecules, and allows for further chemical modifications by the addition of acyl, methyl, and hydroxyl groups, some of which have further effect on stability (Francisco, 1995; Grotewold, 2006). The glycosylated anthocyanidin derivatives are called anthocyanins and are the relatively stable compounds commonly found in flower petals, seeds, or other structures of anthocyanin-producing plants. Stability of anthocyanins is achieved in several ways, but the overall mechanism is that of shielding the flavylium ion from attacks by water (Francisco, 1995). Anthocyanins can stack on top of each other and thus prevent attacks, and stacking is easier when 3,5-glycosylated ANs are involved (Hoshino, et al., 1982). Anthocyanin clusters (anthocyanic vacuolar inclusions, AVIs) have been observed in vivo in plant vacuoles, in structures that are thought to contribute to color modification and stability as well (Markham et al., 2000; Pecket & Small, 1980). Interestingly, colorless flavones can act as co-pigments by performing the same function- flavones and anthocyanins can stack on top of one another in a similar fashion, with glucose moieties greatly improving the stability of the structure (Harborne, 1988). Acyl groups attached through glucose to the main anthocyanin also have a role in stability by positioning themselves on both sides ofthe flavylium ion (Harborne, 1988). Although ANS has been established as the link from colorless leucoanthocyanidins to the colored anthocyanidins, this enzyme appears to have a role in other reactions and processes as well. As a member of the 2-0G oxygenase family, it has been shown to exhibit FLS activity in several plant species (Stracke et al., 2009; Turnbull et al., 2000; Xu et al., 2008), and in some cases anthocyanidins are a minor product (Turnbull et al., 2000). Some enzymes in the ABP - ANS, FLS, F3H and FNS I- all act on the C-2 and/or C-3 portion of their substrates, possibly allowing for some overlap in functions (Figure A3; Saito, 1999). Phylogenetic analyses of2-0G dependent oxygenases in A. thaliana and P. crispum (parsley) show ANS to be closer to FLS than to any other enzymes in the family, providing some explanation for the FLS activity of ANS (Martens, 2003; Owens, 2008). Anthocyanidin synthase seems to have yet another role in the flavonoid producing pathway. In A. thaliana, ANS is necessary for proanthocyanidin production and normal vacuole development (Abrahams, 2003).
3
From Pathway to Flower Color Factors that can affect flower color are even more complex than it seems at first glance, and are not limited to the composition of the pathway itself. The eventual flower color can be affected by substrate specificity, enzyme expression and function, spatial and temporal localization ofboth enzymes and pigments, co-pigmentation, and other factors. In order to produce the flower pigments, enzymes need to be expressed in the right place at the right time. Early enzymes, such as CHS, or F3 '5 'H can be turned off in the developing and mature flowers without compromising the production of pigments and the resulting flower color (Han et al., 2006). In contrast, late genes, such as DFR are usually expressed in later development stages (Han, et al., 2006; Wang et al., 2011). In some cases, pigments can be produced in tissues other than the flower, with no effect on floral color. Streisfeld and Rausher (2009) showed various amounts and types of anthocyanins in the stems of two Ipomoea species, even though the flowers sometimes accumulated pigments that differed from the pigments found in the stems of the same plant. Anthocyanins have been known for providing some protection from excessive light to the photosynthetic machinery (Neill & Gould, 2003), which may be why they are found in high quantities in leaves as well. Therefore, the genomic presence of an enzyme does not always mean expression or activity in the expected tissue. Due to the branching ofthe pathway at the dihydroflavonollevel, after enzyme F3H, substrate specificity can determine what pigment is made. The enzyme DFR acts on any of the 3 dihydroflavonols- DHK, DHQ and DHM, which differ by the presence or absence ofOH groups at 3' and 5' positions ofthe B-ring, and the type(s) ofDFRpresent in any given species can act more efficiently on one substrate than on others. This phenomenon has been seen in many plant species (Johnson et al., 1999; Katsumoto, et al., 2007), demonstrating that DFR specificity has a significant effect on the biosynthesis of a particular anthocyanin. Once anthocyanidins of any of the basic types are produced, biochemical modifications can further change their color. Anthocyanidins are almost never found in their unmodified form due to their instability. Glycosylation of the 3 and/or 5 position stabilizes the molecule, and makes it avaliable for futiher chemical changes; the resulting compound is known as anthocyanin and is the commonly ocurring pigment found in most higher plants. Aromatic or aliphatic acylation of the glycosyl groups is common, and over 65% ofknown anthocyanins are acylated (Andersen & Jordheim, 2006). In total, hydroxylation, methylation, glycosylation and acylation of anthocyanidins produces over 400 known anthocyanins, with many more discovered every year (Kong, et al., 2003). These modifications not only lead to variety, but also affect the color of the pigment. Pelargonidin derivatives fi·om strawberries, radishes and other plants show bathochromic or hypsochromic shifts when acylated or glycosylated, respectively (Giusti, RodriguezSaona, & Wrolstad, 1999). In addition, glycosylation and acylation may contribute to stabilization of the color, and acylation has been linked to vacuolar transportation (Conn, Zhang, & Franco, 2003). It should be noted that the entire range of anthocyanins is rarely found within the same species or family of plants, and it is possible that some classes of the pigments are mutually exclusive or redundant. Of the 3 main anthocyanins, many 4
plant taxa lack one or two because of substrate specialization ofDFR or lack ofF3'5'H activity (Rausher, 2006). Nevertheless, there are usually many possible outcomes for an anthocyanidin in any given plant once it is synthesized by ANS. The various anthocyanins' ability to produce color is further enhanced by copigmentation with flavonols, flavones, carotenoids and metal ions (Forkman, 1991; Harbome, 1988). Efficiency of anthocyanin transport to the vacuole, and pH inside it are other factors that can affect the shade and stability of the resulting color. Evidently, the genomic presence of a functional anthocyanin pathway does not warrant presence of color in any plant structure. Anthocyanins have a variety of essential functions in plants and it is important to note their complex role when further investigating the evolution of these compounds and the enzymes that produce them.
5
The Hawaiian Silversword Alliance (HSA) One of the most spectacular examples of adaptive radiation is the Hawaiian Silversword Alliance (HSA: Asteraceae-Madiinae) (Carlquist, 1980).This plant group consists of 32 perennial species in three genera- Argyroxyphium, Dubautia and Wilkesia (Can, 1985; Robichaux, et al., 1990). The group is monophyletic, migrated to Hawaii only 5.2 +!- 0.8 million years ago (Baldwin 1998), and has utilized a wide range ofliving conditions. This process has resulted in a stunning diversity in morphological and physiological features among the HSA (Baldwin, 1997; Baldwin and Robichaux, 1995; Robichaux, et al., 1990). Within the six islands that the HSA make their home one can find mat plants, shrubs, shrubs with sessile or elevated rosettes, trees and a Iiana, with leaf sizes ranging fi·om 5mm to 500mm (Can, 1985; Robichaux et al. 1990). Flower size and arr-angements vary across species, and flower color can be red, yellow, tan or white (Can, 1985); Can et al., 1989; (Robichaux, et al., 1990). The plants occupy six of the eight high islands of the Hawaiian archipelago, and only five species can be found on more than one island (Can, 1985; Robichaux, et al., 1990). Almost all ofthe diversity in habitat of the islands is utilized by the silverswords; members of the alliance can be found in bogs, wet forests, dty scrub and lava (Can, 1985; Robichaux, et al., 1990). Although the origin of the HSA has been contested, an extensive survey of morphology suggested that the closest relatives were the North American tarweeds, found in the Califomia Floristic Province (Carlquist, 1959). Work with hybrids and chloroplast DNA (Baldwin, et al., 1991), ITS data (Baldwin, 1992) and allozyme research (Witter & Can, 1988) confirm Carlquist's hypothesis. These results indicate that the HSA migrated over a distance of3900km in order to reach the Hawaiian islands. Fmther support for this idea is provided by the presence of fme bristles in the seeds of Dubautia scabra, which allow them to attach to bird feathers and thus be transpmted over long distances (Carlquist, 1980). Continental Madiinae are a group of 89 species in 21 genera found exclusively in the Califomia Floristic Province (Carlquist, Baldwin, & Can, 2003). The group consists of mostly annual or ephemeral herbs that are well adapted to prolonged periods of drought (Carlquist, et al., 2003). While some species escape drought by producing seeds in early spring, others accumulate pectin-like polysaccharides in their leaves as a means of water conservation, a feature found in dry-living Hawaiian Madiinae as well (Carlquist, 1965; Carlquist, et al., 2003). The name tarweed is derived from the glandular exudate, or tar, that the continental plants produce to avoid herbivory, and is found to a much lesser extent in Hawaiian relatives due to the lower incidence of herbivores in Hawaii (Carlquist, 1965; Carlquist, et al., 2003). The migration ofthe HSA ancestor coincides with the age ofKaua'I (5.1 million years, my), the oldest island they inhabit (Carson & Clague, 1995). The radiation proceeded as new islands appeared to the south-east: O'ahu (3.7my), Moloka'i, Maui and Lana'i (1.9my), and Hawaii (0.4my) (Carson & Clague, 1995). Although establishment was as difficult and perilous as the joumey itself (Carlquist, 1980), the few species that made it had a better chance of taking hold in an area relatively free of competitors (Carlquist, 1965). This may have given early visitors the opportunity to occupy more niches, leading to more diversity. One notable difference between the continental and Hawaiian species is the presence of woodiness in some of the silverswords, whereas none of the tarweeds (except 6
some Hemizoans) are woody. Arborescence seems to be common in island radiations, and Hawaii is not an exception. One reason could be the increased opportunities for colonists mentioned earlier, with niches for shrubs and trees still largely unoccupied at anival (Carlquist, 1980). Since herbaceous seeds are usually better suited for long-range dispersal, it is likely that most plants anived on Hawaii as herbs, so arborescence necessarily occuned in order to become shrubs and trees (Carlquist, 1980). Only perennials and a few biennials are found among Hawaiian flora, which means there is a general tendency towards a longer life cycle, driven by conditions on the archipelago. Arborescence is one way to achieve this (Carlquist, 1980). Lack of seasonal extremes has also been linked to a shift to shmbs or trees in herbaceous plants (Carlquist, 1980). Diversity of habitats can be a main factor in the diversity of adapting organisms. The Hawaiian silverswords are found in elevations ranging from 75m to 3750m, no two species have the same range, and many species are found within a portion of that range (Can·, 1985; Robichaux, et al., 1990). The plants are also exposed to varied rainfall ranging from the low 400mm to 12,300mm, which is among the highest in the world (Can, 1985; Robichaux, et al., 1990). Some of the most notable differences among silverswords are those between wet-dwelling and dry-dwelling species. Among Dubautia species, leaf turgor maintenance capacity is much higher in dry scrub habitat species than in wet habitat ones (Robichaux & Canfield, 1985), thus allowing them to maintain high turgor pressure when water availability is low. Dry living Dubautias also tend to have smaller leaves to reduce water evaporation (Robichaux, et al., 1990). In the Argyroxiphium genus, one of the tme silverswords resides on Mt. Haleakala on Hawaii, at the 10,000ft elevation, where it experiences drought and sunlight without the thick cover of clouds (Carlquist, 1965). Argyroxiphium sandwicense has leaves covered with tiny silvery hairs that reflect the sunlight and protect it fi·om overheating and water loss (Carlquist, 1965). Water loss in this species is also avoided by the presence of the same extracellular mucilage found in tarweeds (Carlquist, 1965). Chromosomal reanangements seem to have aided this rapid diversification of the Hawaiian Madiinae. Most members of the HSA have 14 pairs of chromosomes, but some Dubautia species have 13 (Can, Baldwin, & Kyhos, 1996). Island distribution, higher diversity and hybrid studies suggest that the 14-paired cytotype is older, and some species subsequently lost one pair (Can, Baldwin, & Kyhos, 1996). Cytogenetic studies reveal seven different chromosomal anangements within the 14-paired group, where hybridization between species with different genomes is more limited than hybridization within the same genome (Can, Witter, & Kyhos, 1989). The limitation across groups and the readiness with which species with the same genome hybridize provided further variety in possible gene combinations and has likely contributed to the diversity of the Hawaiian silverswords. Aneuploid reduction in the 13-paired species has contributed to morphological and physiological evolution by providing them with additional adaptations. Higher turgor capacity and smaller leaves are characteristic of the 13-paired Dubautias, which make them better adapted to the d1y scmb areas they tend to inhabit (Carr, et al., 1989; Robichaux & Canfield, 1985; Robichaux, et al., 1990). Another factor in the silverswords' genetic ability to diversify could be their allopolyploid origin. Phylogenetic analysis of the floral homeotic genes ASAP1 and ASAP3/TM6 shows that the closest relatives are the mainland tarweeds Raillardiopsis muirii ( Carlquista muirii) and Raillardiopsis scabrida (Anisocarpus scabridus), and 7
points to an ancestor that was a hybrid between members of these two lineages (BaiTier et al. 1999). The hybridization resulted in a tetraploid condition in the putative progenitor, as has been observed in all oftoday's Hawaiian silversword species (CalT, Baldwin, & Kyhos 1996; CalT and Kyhos, 1986; Kyhos, CalT, & Baldwin 1990; Witter & CalT, 1988), while most tarweeds are diploids (Baldwin 1996). Polyploidy effectively duplicates all genes in the genome and allows for each copy to follow a different evolutionary path. Many phenotypic differences between polyploid and diploid taxa have been documented (reviewed in Lawton-Rauh, Robichaux, & Purugganan, 2003), and some changes have been observed just a few generations after polyploidization (LawtonRauh, Robichaux, & Purugganan, 2003; Rong et. al., 2010). Within the HSA, different patterns of nucleotide variation have been observed between homeologous copies of the ASAP1 and ASAP3/TM6 genes in A. sandwicense and D. ciliolata (Lawton-Rauh, Robichaux, & Purugganan, 2003). Limited genotypic variation (CalT, Baldwin, & Kyhos, 1996; Witter & CalT, 1988) means that very few loci are responsible for the observed phenotypic diversity. This makes sense in the context of pathways, such as the anthocyanin producing pathway, where a single mutation in one gene can result in a change or complete loss of color in the plant. Given the variety of vital functions of anthocyanins, however, silenced expression in floral tissues would be favored over loss of gene function. Furthermore, evolution of structural genes alone still does not account for the discrepancy between molecular and morphological evolutionary rates. It has been suggested that evolution in adaptive radiations is driven mostly by changes in regulatory genes, and to a lesser extent in structural genes. The regulatory floral homeotic genes mentioned above were found to have higher incidence in nonsynonymous/synonymous substitutions compared to the structural ASCAB9 gene in Hawaiian species from the three genera (BalTier, Robichaux, & Purugganan, 2001). In this study we have sequenced a putative anthocyanidin synthase gene from three silverswords and three tarweeds. We look at phylogenetic placements ofthe sequences and the evolution rates among them to try to elucidate the role of ANS in this adaptive radiation.
8
MATERIALS AND METHODS
Genomic DNA Genomic DNA was extracted from leaf tissues from Madia elegans and Madia gracilis, using a modified Doyle and Doyle protocol (1987). DNA was previously extracted from Holocarpha sp., Dubautia linearis, Dubautia scabra and Wilkesia gymnoxiphium using the same method. Approximately 1g of leaf tissue was ground into a fine powder in liquid nitrogen with a mmiar and pestle. The ground tissue was placed in a 50 ml polypropylene tube, cooled to -20°C. Seven milliliters (7 ml) of room temperature extraction buffer #1 [2.0% w/v CTAB (cetyltrimethylammonium bromide), 0.1 M TrisHCl, pH8, 0.02 M EDTA, 1.4 M NaCl, 0.08 )lg/ml RNase, 0.2% 2-mercaptoethanol] was added, the tube was vortexed vigorously and then kept in a 65°C water bath for one hour. The tube was vortexed every 15 minutes during that hour. Five milliliters (5 ml) of chlorofonn:isoamyl alcohol (24:1) was added, the solution was mixed well, and the cap was loosened to release the accumulated gas. The tube was centrifuged for 10 minutes at 8000 x g and 4°C. The supernatant was transferred to a clean 50 ml polypropylene tube, and 10% v/v CTAB/NaCl solution, equal to 1/9th ofthe transferred volume, was added. Five milliliters (5 ml) of chlorofonn:isoamyl alcohol (24: 1) was added and the tube was again centrifuged for 10 minutes at 8000 x g and 4°C. Supernatant was transferred to a clean tube. Five milliliters (5 ml) of ice cold isopropyl alcohol was added and the tube was kept at -20°C for one hour. The sample could also be kept overnight at the same temperature. The tube was centrifuged at 8000 x g and 4°C for 30 minutes to pellet the DNA. The supernatant was carefully poured off, pulse-spun with the pellet facing up/outwards, and the rest ofthe liquid was pipetted out. The pellet was air-dried at 37°C for 10-30 minutes, until liquid had evaporated. One and a half milliliters (1.5 ml) ofnpH20 (nanopure water) was added and the tube was kept in a 37°C water bath for 10-30 minutes. The tube was flicked every few minutes to help the pellet come off and resuspend. Seven milliliters (7 ml) of extraction buffer 2 [2.0% w/v CTAB, 0.1 M TrisHCl, pH8, 0.02 M EDTA, 1.4 M NaCl, 10 mg/ml caylase powder] was added to the tube and kept in a 65°C water bath for 30 minutes. Five milliliters (5 ml) of chlorofonn:isoamyl alcohol (24:1) was added, the tube was vortexed and then centrifuged for 10 minutes at 8000 x g and 4 °C. The procedure was repeated and the supernatant was again transferred to a new tube. Five milliliters (5 ml) of ice cold isopropyl alcohol was added, the tube was rocked gently to precipitate DNA and then was kept at -20°C for one hour. The tube was centrifuged at 8000 x g and 4°C for 30 minutes to pellet the DNA. The supernatant was poured off and 5 ml of solution (7 6% ethanol, 10 mM ammonium acetate) was added, the tube was swirled and allowed to sit for 10 minutes. The supernatant was poured off, the rest of the liquid was pipetted out and the pellet was airdried at 37°C. The pellet was resuspended in 200 )11 TE, warmed up to 37°C. Suspended DNA was transferred to 1.5 ml tubes and stored at -20°C for up to 5 years. Floral RNA extraction
9
RNA extraction was carried out using the Plant RNeasy Kit (Qiagen). Less than 1OOmg of pre- and post-anthesis flowers from M elegans were frozen in liquid nitrogen, ground in a mortar and pestle and decanted into a microtube with 450uL ofRLT buffer for a 3 min incubation at 56°C. The lysate was transferred into a provided QIAshredder spin column and centrifuged for 2 minutes at full speed. The flow through was collected and 0.5 J..LL 100% ethanol was added. The sample was transferred into a RNeasy spin column and centrifuged at 8000 x g for 15 sand the flow through discarded. Then 700 J..LL ofRW1 buffer was added to the spin column and centrifuged at 8000 x g for 15 sand the flow through discarded. Then 500 J..LL RPE buffer was added to the spin column and centrifuged for 8000 x g for 15 sand the flow through discarded. The RPE buffer membrane wash was repeated once more. The column was placed into a new microtube and the RNA eluted with 30 J..LL nuclease-fi:ee water by centrifuging at 8000 x g for 1 min. The eDNA synthesis reaction was carried out using a high capacity RNA-to-eDNA kit (Applied Biosystems). The eDNA synthesis reaction contained 10 J..LL 2X RT Buffer, 1 J..LL 20X Enzyme Mix, and 9 J..LL RNA sample. A no enzyme negative control was used. Tubes were pulse centrifuged then incubated at 37°C for 60 min. The reaction was tenninated by heating up to 95° C for 5 min and holding indefinitely at 4°C. Preparation ofRACE-ready eDNA RACE preparation protocol was done using a GeneRacer ™ kit with a SuperScript™ III RT module (Invitrogen, L1502-01) as described below. Total RNA from Madia elegans was used. All other solutions, except ethanol, were provided in the kit. RNA dephosphorylation A reaction was set up on ice, in a 1.5 ml tube with the following: 1 J..Ll10X CIP buffer, 1 )..Ll RN aseOut TM, 1 J..Ll CIP (calf intestinal phosphatase), 7 J..Ll total RNA (345ng/J..Ll). It is recommended that 1-5 )..Lg of total RNA are used; 2.41 )..Lg were used here. The tube was mixed gently and incubated for 1 hour in a water bath set at 50°C. The reaction was centrifuged briefly to collect the fluid and placed on ice after incubation. RNA precipitation Ninety microliters ofDEPC water and 100 J..Ll ofphenol:chloroform:isoamyl alcohol (25 :24:1) solution were added to the dephosphmylated RNA and the tube was vortexed vigorously for 30 seconds. The tube was centrifuged at 14,000rpm for 5 minutes at room temperature, then the top layer c~ 100 J..Ll) was transferred to a new 1.5 ml tube. 2 J..Ll10 mg/ml mussel glycogen and 10 J..Ll3M sodium acetate, pH 5.2 were added and the reaction was mixed well. 220 J..Ll95% ethanol was added and the tube was vortexed briefly. The tube was fi·ozen on dry ice for 10 minutes and centrifuged at 14,000rpm for 20 minutes, at room temperature (centrifuging at 4°C is recommended). The supernatant was removed by pipetting, without disturbing the pellet. 500 J..Ll 70% ethanol was added, the reaction was mixed by inverting 3-4 times and by vortexing, and centrifuged at 14,000rpm for 2 minutes at room temperature. The supernatant was discarded, the tube 10
was centrifuged and the remaining ethanol was removed with a pipet. The pellet was airdried for 2 minutes at room temperature and suspended in 7 ).ll DEPC water. mRNA cap removal The precipitated RNA was immediately used in the decapping reaction by adding 1 ).ll TAP buffer, 1 ).ll RNaseOut and 1 ).ll TAP (tobacco acid pyrophosphatase) to the tube. The reaction was mixed gently and placed at 37°C for 1 hour. After incubation, the tube was .centrifuged and placed on ice. Precipitation was done as described above, with the RNA suspended in 7 ).ll DEPC water. Ligation of RNA oligo to decapped mRNA The decapped mRNA (7 ).ll) was transfeiTed to a tube with the lyophilized GeneRacer ™ RNA Oligo (0.25 ).lg). The solution was mixed by pipeting to help resuspend the RNA oligo. The tube was incubated at 65°C for 5 minutes and placed on ice for 2 minutes. 1 ).ll10mM ATP, 1 ).ll RNaseOut ™, 1 ).ll10X ligase buffer and 1 ).ll T4 RNA ligase were added to the tube. The reaction was placed in 37°C for 1 hour, RNA was precipitated as previously described but this time it was suspended in 10 ).ll DEPC water. Reverse transcription of mRNA The provided GeneRacer ™ Oligo dT Primer (Figure A6) was used to reverse transcribe the RNA. One microliter of the primer, 1 ).ll dNTP mix and 1 ).ll dH20 were added to the 10 ).ll of ligated RNA and incubated at 65°C for 5 minutes, then chilled on ice for 1 minute. The following was added to the mixture: 4 ).ll 5X First strand buffer, . 1 ).ll 0.1 M DTT, 1 ).ll RNaseOut TM, 1 ).ll SuperScript ™ III Reverse Transcriptase. The reaction was transfeiTed to a 0.2 ml microcentrifuge tube and placed in the BioRad thermal cycler with the following conditions: 50°C for 45 minutes, 70°C for 15 minutes. At completion, the reaction was placed on ice for 2 minutes, 1 ).ll RNase H was added to it and it was kept at 37°C for 20 minutes. The RACE-ready eDNA was stored at -20°C and used for amplification within a month.
Primer design Over 30 known ANS/LDOX genomic or mRNA sequences downloaded from GenBank (NCBI) were aligned with ClustalW 1.8 available on Biology Workbench (SDSC) using the default settings. Degenerate primers were designed in conserved regions (see Figures A5 and A6 for primer positions and sequences). Once the initial6F6R primer pair resulted in a sequence from all species of interest, species-specific primers were designed for RACE in M elegans and were used with the GeneRacer™ kit's 5' and 3' primers. Primer pair 5' and ME-R3 were used, followed by a nested PCR with 5'Nested and ME-R4 to produce the sequence of the first exon. Similarly, ME-F3 and 3 ', and ME-F4 and 3'Nested were used for the second exon. After the entire coding 11
sequence was obtained from M elegans, degenerate primers ANS-F1, ANS-F3s, ANSR1C/G and ANS-R2 were designed based on an alignment of M elegans and other Asteraceae species (C. chinensis, Gerbera hybrid, Saussurea medusa, Chrysanthemum; see Figure A7 for accession numbers). Primers ANS-F1 and ME-R2 were used to amplify the first exon in all other species. Primer ME-R2 is species-specific forM elegans, and was initially designed for inverse PCR, but was found to work in other species as well. Primers ANS-F3s and ANS-R1C/G or ANS-R2 were used to obtain the second exon. Finally, ANS-4F2 and ANS-6R were used to get enough overlapping sequence within exon 1.
PCR and subcloning PCR was performed with degenerate primers based on sequences specified in Figure A7, as well as with species-specific primers. Protocols for reaction mixes and thermocycler conditions for each primer pair are described in Figures A8 and A9. Eppendorfthermal cyclers, either a Mastercycler®, or an Mastercycler® gradient were used in the early stages of the experiment, and a BioRad thermal cycler was used later. PCR products were stored at 4°C for up to 2 days before screening and cloning. Screening was done on 0.8% TAE (Tris base, glacial acetic acid, EDTA) agarose gel, with a 'A Hindiii marker (Promega, G 1711) running for comparison. Six to eight microliters ofPCR product were loaded into a well, and l).ll of loading dye (Promega, G190A; Fisher, 57-50-1) unless the loading dye was already present in the PCR buffer. Gels were stained in a 0.5% ethidium bromide solution for 5-10 minutes and screened on a Fisher FBTIV-816 UV transilluminator. If the marker bands were too pale, or the whole gel seemed orange under UV light, it was further stained in ethidium bromide, or destained in H 2 0, respectively. The ethidium bromide solution tends to weaken after several uses or over time, so staining time had to be adjusted constantly. When multiple bands resulted fi·om PCR and optimizing the protocol did not improve this, PCR products were run on 0.8% low-melting agarose (Sigma, A-4018), bands of interest were cut out with a razor blade, and DNA was extracted using a gel extraction kit (Qiagen, 28704). In early stages of the experiment when gene size was not known or gel extraction was inefficient, PCR products with two or three bands were cloned and used for minipreps or lysis, then clones with inserts of desired size were sequenced. PCR products or gel-extracted bands were cloned into a TOPO-TA vector (Invitrogen, 45-0641) as follows: 0.5).ll TOPO™ vector, 0.5J.ll salt solution, 1-2).ll PCR product, 0-1 J..Ll water, for a total of 3 J..Ll. Reactions were incubated at room temperature for 10-20 minutes and either used immediately in transformation or kept at -20°C overnight (15-20 hours). Two or three microliters of the cloning reaction were transferred to a vial of OneShot TM TOP 10 competent cells (Invitrogen, ), previously thawed on ice for 10-15 minutes. Vials were incubated on ice for 10-15 minutes, heat-shocked by placing in a 42°C water bath for 30 seconds, and placed ilmnediately on ice. Each vial had 250ml of the kit's SOC medium added to it, and was placed in a shaking incubator at 37°C, 200rpm. Forty microliters ofX-gal [40mg of 5-bromo-4-chloro-3-indolyl-beta-Dgalactopyranoside (C 1Jl1sBrClN0 6) per 1ml dimethylforamide (DMF: (CH3) 2NC(O)H] 12
and 50-lOOml of cells were antiseptically added to LB agar plates with 50mg/ml kanamycin; usually two plates with different volumes of cells were done to ensure proper spacing between colonies. Plates were kept at 37°C for 14-18 hours, or longer if colonies were too small. Between 10 and 20 white colonies were picked fi·om each original PCR product, and each colony was grown in 4ml ofLB broth with 50mg/ml kanamycin at 37°C, 200rpm, for 16-20 hours. qRT-PCR Primer pairs for the subsequent PCR are shown in Figure A6. RT-PCR reactions were done by Brett Smith on an Applied Biosystems 7300 Real-Time PCR System. The reaction is as follows: 12.5 IlL of Maxima® SYBR Green/ROX qPCR Master Mix, 0.75!-LL of 10 !lM primer stock, 1 IlL of template, 10!-LL ultra pure nuclease free water. Tubes were mn in duplicate. Standards included 10\ 102, 104, 10 5, and 107 gene copies. A second unknown sample diluted 1OX was also mn in tandem. Control reactions contained no enzyme. PCR cycles were, 1 cycle 95° C for 10 minutes, 40 cycles of 95° C for 15 s then 55° C for 1 min, 1 cycle 95° C for 15 s then 60° C for 30 s then 95° C for 15 s. Cycle threshold numbers were analyzed automatically by the Applied Biosystems Sequence Detection System software. Plasmid Isolation Two methods were employed to isolate the plasmids from the LB-kanamycin cultures. One and a half milliliters (1.5ml) ofLB-kanamycin culture was transfened to a clean 1.5ml Eppendorfmicrotube and spun at lO,OOOg for 10 minutes. The supernatant was decanted and the pellet resuspended in 200!-Ll GTE solution (50 mM glucose, 25 mM Tris, and 10 mM EDTA). Four hundred microliters (400!-Ll) fresh lysis solution (0.2 M NaOH, 1% sodium dodecyl sulfate) was added to the tube, mixed gently and the tube was incubated on ice for 5 minutes. Three hundred microliters (300)..1.1) of ice-cold 5M potassium acetate was added and the tube was again mixed and incubated on ice for 5 minutes. The mictrotube was centrifuged at maximum speed for 5 minutes and the supernatant was transfened to a clean tube. A volume of isopropanol equal to 0.6 times the volume of the transfened supernatant (~540)..1.1) was added to the tube, mixed, incubated at room temperature and centrifuged at maximum speed for 2 minutes. The supernatant was discarded and the pellet was washed with 70% ethanol, which was then decanted. The pellet was allowed to air dry for up to 10 minutes and was finally resuspended in 50).!1 TE buffer, pH 7.5 or in 50).!1 distilled water. The other method for plasmid isolation was to use the Promega SV Miniprep Wizard kit. A restriction enzyme digest was used to test for the presence ofPCR insert in the plasmids. Five microliters of reaction was mixed with 1Oml of digestion master mix (1.5!-LL lmg/ml bovine semm albumine, 1.5!-LL lOX Buffer H [500mM Tris-HCl, lOOmM MgC}z, lOmM DTT, 1M NaCl], 0.5!-LL of 5U/).!L EcoRI and 6.5!-LL nanopure water). The digestion was allowed to proceed for 2-3 hours at 37°C waterbath or in a thermocycler. The products were mn on 0.8% agarose gels as described above. Plasmids that were positive for PCR inserts were tested for concentration with a NanoDrop spectrophotometer and sent to the CSUN Sequencing Facility for sequencing. 13
Sequencing and editing Sequencing was perfonned at the CSUN DNA Sequencing Facility using an ABI Prism 377 DNA sequencer. Primers used were forward and reverse Ml3 primers, which were suitable for the plasmids employed by our laboratmy. The plasmid sequences were omitted from each clone sequence before performing a search through BLAST. Sequences that matched known ANS genes were saved and then aligned with ClustalW on either Biology Workbench or BioEdit. Ifnecessmy, sequences were inve1ted using Reverse Complement (2010). A consensus sequence was generated for each fragment and each species (see Figure A10). Evolutionary analysis Known ANS sequences from over 50 gymnospenns, dicots and monocots were obtained from GenBank . A multiple sequence alignment of these was generated with ClustalW (see Figure A7 for a list of sequences and accession numbers). The alignment was done with gap open/extension penalties of 50/5.0 for both the pairwise and multiple aligmnents; this produced gaps of multiples of3 (3, 6, 9, etc), which reflect inse1ted or deleted residues in the proteins. All Madiinae sequences were assumed to be the same length, so gaps that did not occur between a Madiinae and any other sequence were replaced with a question mark (?), which most software programs recognize as missing data rather than a gap. A model test was run on Jmodeltest (Posada, 2008), which estimated the GTR+G+I (general time-reversible, garmna distributed, with invariant sites) method to be the best model for a maximum likelihood (ML) analysis. A ML tree was constructed with MEGA 5 (Tamura et al., 2007) with the suggested GTR+G+I model of substitution, and 1000 bootstraps to test the phylogeny. MEGA 5 estimated the gamma parameter to be 0.9484 and invariable sites were 15.7% of all sites. A Bayesian tree was generated with MrBayes 3.1.2 (Huelsenbeck & Ronquist, 2001) with GTR+G+I for 3,000,000 generations and sampling every 100 generations. The first 25% of the samples were discarded and not included in the automatic analysis. Propmtions ofnonsynonymous (Ka) and synonymous (Ks) substitutions among eight of the ten Madiinae (Holoca1pha sp. sequences were too short to give accurate estimates) were determined with MEGA 5. The Ka/Ks ratios were calculated using Excel. Tests of selection (neutral, positive or purifying) were performed with the Z-test and Fisher's exact test, both available on MEGA 5. Protein alignments were performed with ClustalW 1.8, available on Biology Workbench (2010; Subraminiam, 1998) with the default settings (input order; Gonnet series; accurate alignment method; pairwise alignment: gap open/extension: 10/0.1, Ktuple size= 1; multiple alignment: gap open/extension: 10/0.2; delay divergent sequences: 30; residue-specific and hydrophilic gap penalties: on).
14
RESULTS DNA Extraction and PCR Extractions of genomic DNA from leaf tissues from M gracilis and M elegans yielded products with concentrations between 250 ng/1-Ll and 500 ng/1-Ll. Genomic DNA was previously extracted in the lab from Dubautia linear is, Dubautia scabra, Wilkesia gymnoxiphium andHolocarpha macradenia between the years of2004 and 2008. Total RNA extracted from developing and mature floral tissue fi·om M elegans was between 250 ng/1-Ll and 400 ng/1-Ll. Gene Sequences Degenerate primers designed in conserved regions of anthocyanidin synthase were used in PCR to amplify gene fragments. A total of four overlapping fi·agments were sequenced from two tarweeds and two silverswords, and a few additional fragments were sequenced from the silverswords to help resolve the multiple copies in each species (Figure A1 0). Only one fragment was isolated from Holocarpha macradenia and Dubautia scabra. In total, 10 putative ANS sequences were isolated from all species investigated in this study. The single intron was sequenced from all species and was used to help distinguish the genomic copies in the tetraploid species. Primers ANS-6F and ANS-6R (pair 6F-6R) were designed based on an alignment of ANS sequences fi·om species marked in Figure A7. This segment includes 63bp of exon 1, the intron, and 153bp of exon 2 (Figure A5). The very first putative ANS sequence obtained in this lab was 6F-6R from Madia elegans, and eventually these primers worked on DNA from all three tarweeds and three silverswords included in this study. These sequences allowed the design of more specific primers to use in other PCR protocols to get the flanking fragments. One gene copy was found in M elegans and M gracilis, and two copies in H macradenia, W gymnoxiphium and D. linearis. Pair 6F-6R did not result in good quality sequences in D. scabra. Instead, primer ANS-F1 was used with ANS-6R to obtain 478bp of the first exon, the variable intron and 153bp of the second exon. This intron was 484bp in Dscabra1 and 539bp in Dscabra2. Fragments as long as this (over 1OOObp) get lower quality sequencing towards the end of the procedure, so the ends of the forward and reverse sequences were not considered reliable. A RACE protocol was performed with M elegans, which yielded the flanking regions of the coding sequence, as well as parts of the 5' and 3' untranslated regions of the mRNA. The RACE kit's primer 5' and species-specific primer ME-R4 resulted in a segment of 450bp from the start codon to close to the end of exon 1. The position of an ATG site was confinned with over 20 forward and reverse sequences (Figure A10). The 3' RACE similarly showed an in-frame stop codon (TGA), confirmed by 9 forward and reverse sequences. After splicing all overlapping pieces for M elegans and cutting out the intron, exon 1 of the putative ANS gene came out to 503bp, and exon 2 was 565bp, including the stop codon.
15
Primer ANS-Fl was designed at the start codon based on the sequences of M elegans and three of the closest Asteraceae (Callistephus chinensis, Dahlia variabilis and Gerbera hybrida). This primer, paired with ME-R2 (pair Fl-R2b), specific forM elegans, produced a fragment of 502 bases, or almost the entire first exon from M gracilis, D. linearis and W gymnoxiphium. Because primer ANS-Fl was designed at the very beginning of the gene, and primer sequences were not included in any analysis, none of these sequences contain the ATG codon. Since the overlap of segments Fl-R2 and 6F-6R was only 41 bases, primer ANS4F2 was designed in the middle of exon 1 and paired with 6R (pair 4F2-6R). The resulting fragment contains 274bp of exon 1, the intron and 17lbp of exon 2. This fragment also was too long to provide enough overlap of forward and reverse sequences in the intronic region. As a result these sequences were less useful in confinning the introns; however, the forward clones provided the needed overlap between Fl-R2 and 6F-6R and allowed for the copies within the same species to be resolved. Exon 2 was initially tried with primers ANS-F3s and ANS-RlC or ANS-RlG, which extend to the stop codon, but no sequences were obtained in this manner. Primer ANS-R2 was designed 61 bases from the expected end of the gene, and combined with ANS-F3s produced 485bp of exon 2 from M gracilis and W gymnoxiphium. The five clones obtained for W gymnoxiphium were too similar to each other to be considered to belong to different genomic copies, and at this point it is not known which copy this sequence is from. Therefore, only the first exon and the 171 bp of the second exon previously obtained were considered for phylogenetic and evolutionary analysis. For D. linearis, primers ANS-F4 and ANS-R2 were used instead. Primers ANS-Fl and ANS-RlG produced the entire gene from W gymnoxiphium, and the sequence was used to get some more support for the exons. This pair was tried as part of a technique to be used in the future (see discussion). Intron Sequences A review of over 50 known ANS sequences from dicots and monocots (Figure A 7) revealed a coding sequence of 1000-1100 nucleotides and the presence of a single intron nested in the codon immediately following a highly conserved tyrosine. In the Madiinae, this tyrosine was found at position 167, based on the putative length of the gene as isolated from M elegans, which was 355 amino acids. All introns have the characteristic GT and AG flanking nucleotides. Intron sizes seemed to be mostly conserved across species; much larger differences were found between the two copies within a species. Introns in copy 1 from all species except D. scabra were 485bp, introns from the Madias, copy 2 fi·om W gymnoxiphium and D. linearis were 547bp, and copy 2 from H macradenia was 53 7bp (Figure All). The introns in D. scabra were 484bp in copy 1 and 539bp in copy 2. The introns were the only regions where size differences occurred; all other portions of the gene were the same size in all Madiinae. Therefore, introns were the primary clue in distinguishing between the two copies in each species. Gene Identity and Characteristics
16
A total of eight putative ANS copies from three tarweeds (Madia elegans, Madia gracilis, Holocarpha macradenia) and two silverswords (Dubautia linearis and Wilkesia gymnoxiphium) were isolated and sequenced (See Figure Bl). All sequences were evaluated with BLAST to confinn their identity. Coding regions of the eight Madiinae sequences showed 86-88% similarity to coding regions of ANS from Dahlia variabilis and 82-87% to Callistephus chinensis ANS (both Asteraceae). A domain conserved in all iron-dependent 2-oxoglutarate dioxygenases was identified at residue positions 100-300 (based on residue position in M elegans) with protein BLAST. Residues His-234, Asp236 and His-290, which are putative iron coordination sites, and Arg-300, required for 20G binding, were found in M elegans, M gracilis and both copies of D. linearis (Figure B2, B3). These residues were also found in the downstream portions of D. scabra and W gymnoxiphium (data not shown, see discussion). No premature stop codons, deletions or insertions were detected within the coding sequence of any copy. Evolutionary Analysis A total of 60 ANS complete coding sequences from dicots, monocots and gymnospenns from GenBank (Figure A 7), plus the 10 obtained Madiinae sequences were used to construct a maximum likelihood (ML) tree with MEGA 5 (Tamura et al., 2007) and a Bayesian tree with MrBayes 3.1.2 (Huelsenbeck & Ronquist, 2001). The trees are shown in Figures Al2 and A13, respectively. Both trees group all the Madiinae together, with good support for most branches in that clade, except for the branches leading to Holocarpha. Among the silverswords (Wilkesia and Dubautia), copies of similar intron lengths are closer to each other than to other copies in the same species, as expected from allotetraploidy. The two Madia species are separated from both Hawaiian copies. Holocarphal and Holocarpha2 seem to be closer to copy 1 of the silverswords than to the other tarweeds, however, the support for those branches in the ML tree is ve1y low. One way to detennine what kind of selection is acting on a gene is to look at the ratio ofnonsynonymous (Ka) and synonymous (Ks) substitutions among coding sequences of different species. An equal ratio suggests there is no selection because nonsynonymous changes are not selected for or against. Lower and higher ratios would suggest presence of purifying and positive selection, respectively. A Z-test of selection on the Madiinae and two Asteraceae as an outgroup using MEGA 5 showed there was purifying selection in most copies (Figure Al4). Fisher's exact test, more accurate for sequences similar to each other, also confnn1s the presence of purifYing selection (data not shown). No positive selection was detected anywhere within these Asteraceae, however, purifYing selection was detected by a Z-test for most copies (Figure Al4). Nonsynonymous to synonymous substitution ratios (Ka/Ks) were calculated for all sequences except the ones from Holocarpha, which were too short (Figure Al4). The mean averages for copy 1 (Wilkesial, Dlinearisl, Dscabral), copy 2 (Wilkesia2, Dlinearis2 and Dscabra2), the tarweeds (Melegans, Mgracilis) as well as other known structural and regulatory silversword genes are summarized in Figure Al5. A higher Ka/Ks value for copy 2 of ANS (0.405) shows a little less constraint than in copy 1 (0.296), and both are less contrained than in tmweeds (0.114). Structural genes chalcone synthase (CHS) and chlorophyll AlB binding protein 9, as well as the regulatory GAl homologue have very similar ratios; the only known exceptions are the floral relulatory 17
genes ASAPl and ASAP3/TM6, where silversword ratios are much higher. All genes, structural and regulatory, exhibited higher ratios in the silverswords than in the tarweeds. Gene Expression Quantitative RT -PCR was performed on eDNA from developing and mature floral tissue from M elegans. Results showed very low levels of expression of ANS, with slightly more ANS in the buds than in the mature flowers (data not shown). Presence of ANS mRNA was also confirmed by the sequences obtained through RACE :fi·om mature M elegans flowers.
18
DISCUSSION Gene Sequences Up to four different fragments per species were spliced to produce the final sequences (Figure A15). When editing each fragment, caution had to be taken to distinguish between sequencing e1rors and polymorphisms. With some silversword fragments, all clones seemed too similar even when two copies were already found in that species. In D. scabra and W gymnoxiphium, segment F3s-R2e had several clones, but but it was hard to say which copy that segment belonged to, which is why these parts were not included in any sequence analysis. To avoid some of the difficulties with editing, future studies will be employing an additional technique. As described in results, primers ANS-F1 and ANS-R1G were used to amplify the entire gene, exons and intron, from W gymnoxiphium. This pair can be used to amplify and clone any sequence as described in the methods, and then do PCR targeting shmier segments on plasmids extracted from individual clones. This will ensure that the clones are coming from the same initial sequence, so it can be assumed that any differences between clone sequences are due to sequencing errors and are not allelic or gene/genomic copy differences. This should reduce the overall differences between clones as well. Although this tactic has not yet been tried in our laboratory, the successful insertion in a plasmid and subsequent sequencing of the fragment seems promising. Anthocyanidin Synthase The eight sequences isolated from the three tarweeds and three silverswords in this study are putative ANS sequences. The sequences obtained from Madia elegans through regular PCR and RACE contain start and stop codons, and regular PCR techniques revealed an intron at positions consistent with those in known ANS sequences. The putative coding sequences in all Madiinae show over 80% identity with ANS genes from Asteraceae species, and over 70% identity with over 100 ANS sequences found through BLAST. Furthennore, the only sequences that produce significant alignments with BLAST are confirmed ANS genes. The protein stmcture of ANS has been solved for Arabidopsis thaliana (Figures A4-B, A4-C) (Wilmouth et al., 2002). The domain common to all genes in the irondependent 2-oxoglutarate oxygenase family was identified through PFAM, PROFILE (data not shown) and BLAST (Figure A4-A) in all sequences where the second exon was almost entirely sequenced (M elegans, M gracilis, both copies of D. linearis). Although copies could not be distinguished for most of the second exon in W gymnoxiphium and D. scabra, a protein sequence formed from merging either copy with segment F3s-R2e resulted in detection of the same domains. Residues His-234, Asp-236 and His-290, which are putative iron coordination sites, and Arg-300, required for 2-0G binding (Saito, et al., 1999; Wilmouth, et al., 2002), were similarly found in all sequences. Other enzymes of the flavonoid producing pathway belong in this oxygenase family as well, and flavonol synthase (FLS) is the closest relative of ANS (Martens, et al., 2003). Both ANS and FLS act on C2 and C3 of the C-ring of their substrates (Figure A3) and ANS is known to have FLS activity in some plants (Stracke et al., 2009; 19
Turnbull et al., 2000; Xu et al., 2008) while no ANS activity has been reported for FLS. These studies suggest that the ANS function appeared later, and originally ANS was performing only the activity ofFLS. Phylogeny The phylogenetic placement of the isolated ANS copies is consistent with the known history of the Madiinae. Anthocyanidin synthase is not known to be a gene family and as expected only one copy was found in the Madia tarweeds. The presence of two copies in each silversword provides further evidence of the tetraploid condition of the Hawaiian species (Barrier, et al., 1999; Kyhos, Carr, & Baldwin, 1990). The copies segregate by copy type, not by species, and both copies are grouped separately from the Madia lineage. This topology confirms the copies appeared after the divergence of the silverswords from these tarweeds, but before the Hawaiian speciation occurred. The Hawaiian progenitor was a tetraploid hybrid of two tarweed ancestors (Barrier, et al., 1999), so the two copies we see in each silversword are most likely due to tetraploidy and not gene duplications. It has been demonstrated that the Madia lineage is more closely related to one of the tarweed ancestors (Carlquista muirii) (BatTier, et al., 1999), and as a result its genes cluster closer to one of the two copies in the silverswords. In previous publications concerning other genes in Madiinae, copy B is the one closest to Madia, and copy A is more closely related to the other ancestor, Anisocarpus scabridus (Barrier, et al., 1999); in this study the copies are designated copy 2 and copy 1, respectively. It is surprising that the Holocarpha copies are so close to copy 1 of the silverswords, as this tm-weed was originally included as an outgroup. The closest silversword relatives are found within the Madia!Raillardiopsis lineage, which does not include Holocarpha (Barrier, et al., 1999). It is possible that the much shorter sequence (252bp) used for Holocarpha created some bias in the aligmnent, and a tree based on the whole gene may produce a very different topology. Because of this, and the somewhat low branch support in that clade, it is not possible to say when the duplication in Holocarpha occurred. It is even more surprising to find two copies in Holocmpha in the first place, as this was one of the species with the most clones obtained, confirming the sequence several times, and errors in the sequences were much less likely. This species is thought to be diploid, with some individuals having fewer than the original base chromosome number of6 due to dysploidy (Carlquist, et al., 2003). We have no infonnation about the exact chromosome number of the individual plants that provided the DNA for this experiment. A gene duplication within Holocarpha seems most probable given these data. The Madiinae clade is closest to sequences from the Asteraceae family with somewhat low support on most branches. The Madiinae subtribe is part of the Heliantheae alliance, Asteraceae family. The only other species in the same alliance is Dahlia variabilis, which places closest to Madiinae in all trees (Figures A12, A13). Evolution Rates
20
Nonsynonymous to synonymous substitution ratios (Ka/Ks) can reveal a lot about the evolution of a gene. After speciation or a gene duplication, the two sequences go through changes that reflect the level of selection, or constraint, on each sequence. Within a coding sequence, nonsynonymous substitutions are usually either deleterious or advantageous to the gene's function(s), while synonymous substitutions tend to have no effect. In an adaptive radiation, where species are adapting to diverse habitats, we expect to see low Ka/Ks ratios, indicating purifying selection. At the same time, slightly higher ratios in the Hawaiian clade (silverswords) would indicate faster evolution as they establish their niches, compared to the North American clade (tarweeds). With ANS, Ka/Ks was much lower in tarweeds compared to silverswords, indicating constraint is more relaxed in the Hawaiian Madiinae (Figure A15). This trend is obvious in all genes currently sequenced from a variety ofMadiinae, listed in Figure A15. Purifying selection in most copies of ANS was detected by the Z-test in MEGA 5 (Figure A14). In some cases selection was more relaxed in one genomic copy than in the other. Anthocyanidin synthase and the GAl homologue both have two copies in the silverswords, and these copies have been established as due to allotetraploidy (Remington & Purugganan, 2002). Copies 1 and 2 of ANS correspond to copies A and B in the other studies listed in Figure A15, based on their phylogenetic placement in relation to Madia species. It seems that ANS-1 has higher constraint than ANS-2, and GAI-B has higher constraint than GAI-A. Ratios for genomic copies of ASAPl, ASAP3/TM6 and ASCAB9 did not differ significantly (Barrier, et al., 1999). No genomic copies have been distinguished for CHS, and the two copies presented here are the result of an ancient duplication that happened long before the diversification of the Madiinae; therefore differences between genomic copies could not be established (Rodriguez, personal cmmnunication). The different ratios between genomic copies of ANS and the GAl homologue suggest the copies are under different selective pressures. Polyploidization has been especially important in the evolution of flowering plants. It is estimated that at least half of all flowering plants have experienced at least one polyploidization event in their evolutionary history (Leitch & Bennett, 1997). On a genomic level, genes duplicated by polyploidization may take different evolutionary paths. One copy could duplicate again, be deleted, evolve faster or become a pseudogene (Wendel, 2000). In Hawaiian silverswords, ASAP3/TM6 genomic copies were found to have different levels and pattems of nucleotide polymorphisms in Dubautia cilia lata (Lawton-Rauh, Robichaux, & Purugganan, 2003). In addition, there is evidence suggesting that one copy of the gene has been deleted in a portion ofthe D. ciliolata individuals examined (Lawton-Rauh, Robichaux, & Purugganan, 2003). So far, it seems that ANS, ASAP3/TM6 and the GAl homologue are undergoing differential evolution of their genomic copies, suggesting that polyploidy is playing a role in this adaptive radiation. The studies mentioned in Figure A15 sought to investigate the possibility of regulatory genes evolving faster than structural genes, especially in adaptive radiations. So far there is not much support for this hypothesis among the silverswords, as all structural genes and one regulatory gene (GAl) have similar evolution rates (Figure A15). The only exception are the floral regulators, ASAP 1 and ASAP3/TM6, which have much higher rates (0.980 and 0.790, respectively). All regulatory genes regulate pathways related to growth (GAl homologue) or floral morphology (ASAPl, ASAP3/TM6), both highly variable traits among the silverswords. We would therefore expect to see higher 21
rates in genes from these pathways, even though rates in individual genes within the same pathway may vary. It is worth noting that Remington et al. (2002) found variable evolution rates in GAI promoter regions, as opposed to the consistently low rates in coding regions, suggesting the gene may be evolving through changes in regulatory regions instead. The structural gene ASCAB9 is responsible for chlorophyll binding and is thus relatively highly conserved across taxa, explaining the higher constraint in this gene. Chalcone synthase and anthocyanidin synthase are both involved in producing anthocyanins, and CHS is ve1y early in the pathway (Figure Al). Variety of flower color among all known silverswords is limited to white, tan, yellow and red flowers, and the species covered here are either white (D. scabra) or yellow (all others). Based on the limited visible diversity of pigments, these species are not expected to have very divergent CHS and ANS sequences, which is supported by the data. Although data seem to support the hypothesis of faster evolution in regulatory genes, part of the pattern can be explained by the expected variability of each gene. At this point the bigger picture of the fates of regulatory and structural genes in adaptive radiations is incomplete. A comparison of rates within the same pathway or simply a larger sample of genes could shed more light on this phenomenon. The importance of CHS early in a pathway that produces not only anthocyanins but also other essential compounds (lignins, proanthocyanins, flavones, flavonols) may explain the higher constraint in one of its copies compared to ANS. Gene Expression and Function The RACE reaction on eDNA from Madia elegans indicates at least some expression of ANS in the mature flowers of this tarweed. The qRT-PCR results could not establish the exact amount of mRNA in M elegans, however, there seems to be a ve1y small amount ofmRNA in this species (data not shown). The flowers in both have a yellow color, M elegans also having red in parts of the anthers, and the base of the petals. Among flavonoids, chalcones and aurones have yellow color, and carotenoids also could be responsible for the yellow flower color in the species covered in this study. No studies have been done on anthocyanin content in any Madiinae to this date, and it is unknown what kinds of pigments contribute to the flower colors in any of them. Although information about pigments is not available, it is likely that the red colors in M elegans are due to anthocyanins. There are three known types of plant pigments anthocyanins, betalains and carotenoids. Betalains are found exclusively in the Cmyophyllales order, and carotenoids could produce light yellow to orange colors, but not the red found in M elegans. Given the integrity and expression of ANS in this species, it can be assumed that it produces a functional ANS enzyme, which in tum synthesizes anthocyanins. Although anthocyanins have been extensively studied as pigments and antioxidants, they seem to have other functions in plants as well. Some types of anthocyanins are found only in the stems or leaves of a given plant, while other types are found in floral tissues of the same plant (Streisfeld & Rausher, 2009). Other studies suggest a function of anthocyanins as light attenuators, protecting the photosynthetic machinery of the cell fi·mn excessive solar radiation (Neill & Gould, 2003). Together these findings suggest that anthocyanins could be synthesized even in plants that do not 22
exhibit them as floral pigments. Since no premature stop codos were found in any of the ANS sequences obtained in this study, it is likely that the machinery to produce anthocyanins is intact in the Madiinae, despite the lack of orange, red or blue flower color in most of them. Regardless of the actual mechanism providing the red color in M elegans, the same process likely is responsible for the red color in the endangered Argyroxiphium sandwiscense, the only Hawaiian silversword with red flowers.
23
CONCLUSION A putative anthocyanidin synthase (ANS) genomic sequence was isolated from three tarweeds (Madia elegans, Madia gracilis, Holocmpha sp.) and three silverswords (Wilkesia gymnoxiphium, Dubautia linearis, Dubautia scabra). There was some mRNA expression in M elegans, and no premature stop codons were found in any of the other species. These data suggest that the ANS gene is potentially functional in all species. The presence of two copies in the silverswords and their phylogenetic position on maximum likelihood and Bayesian trees support the allotetraploid origin of the silverswords, and the tarweeds in the North American west coast as their closest relatives. Evolution was much more relaxed in the Hawaiian silverswords, consistent with expectations and previous findings about this rapidly diversifying lineage. Differential evolution rates in the genomic copies of silverswords suggest that polyploidy may have played a role in speciation and diversification. The fmdings are consistent with a hypothesis of faster evolution in regulatory genes, as evolution rates in ANS were low compared to those of some regulatory genes in this clade. Studies on ANS promoter regions and regulatory genes are currently under way to try and elucidate the mechanisms through which pathways evolve and their role in adaptive radiations.
24
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30
R
R
(x
OH
2 ~; R~
'HO
ss
#
,(
HO
H
4
R 2-0xoglutarate, 02
HO
\,
--~-'\--...~...ANS l
3 OH
OH
Succinate,
co2,H2o
Leucoanthocyanidin (Ravan-3,4-cis-dlol) Colorless
(2-Fiaven-3,4-diol)
R
R
OH
OH
HO
R
Acidic
HO
R
Basic H
Anthocyanidin {Fiavylium ion)
Pseudobase (3-Fiaven-2,3-diol) Colorless
RED
Figure A2. Proposed mechanism of action of anthocyanidin synthase (Saito et al., 1999).
32
R
HO
, ,•
R
OH
CX
HO
R
R
Anthocyanidin synthase (ANS)
OH
OH
Leucoanthocyanidin f
