Introduction
There are numerous examples showing that hopeful monsters might have contributed to evolution by mutations in key regulatory genes (Bharathan et al., 2002; Colosimo et al., 2005; Sucena et al., 2005). The model for such genes is the homeobox genes which play a key role in the specification of the animal body plan in both development and evolution. For example Pax-6 Hox gene containing a paired-box and homeobox, from mice (which triggers eye formation) can induce the formation of fly eyes all over the body, even on the wings in the fruitfly Drosophila (Quiring et al., 1994; Halder et al., 1995a; Walther and Gruss., 1991). Remarkably, Pax-6 helps to organize compound eyes in flies and camera eyes in both squid and vertebrates (Nancy et al., 1997). Mutations in Pax-6 gene, Small eye (Sey) result in a reduction of external eye size, a characteristic iris hypoplasia, and at later stages, corneal opacification and cataracts (Hogan et al. 1988). Aniridia in man has also been shown to be caused by heterozygous mutations of PAX6 and is characterized by a varying degree of iris hypoplasia, corneal opacification, cataracts, and glaucoma (Tom et al., 1992; Tim et al., 1992). Hox gene, tinman, induces heart formation in both insects and vertebrates (Rolf., 1993; Harvey., 1996; Komuro and Izumo, 1993; Lints et al.,1993; Evans et al., 1998). Distal-less controls the development of fly legs, fish fins and the tube feet of sea urchins (Stephen et al., 1989; Marie et al., 1994; Nicolas et al., 1996).
And floral symmetry is mainly controlled by 1 gene, namely Cycloidea (Da, Lau et al., 1995).
Interspecific transgenic experiments involve the moving of a single gene (whose function needs to be tested) from a donor species to replace the function of the endogenous gene in a recipient species. If there is divergence in gene regulation or protein function then this ortholog would yield phenotypes not normally seen in the recipient species. For example species-specific Yellow expression patterns were retained in D. melanogaster transformants carrying the D. subobscura and D. virilis yellow genes, indicating that sequence evolution within the yellow gene underlies the divergence of Yellow expression (Wittkopp et al. 2002). Drosophila males modulate the interpulse intervals produced during their courtship songs, which are altered by mutations in the clock gene period, that exhibit a species-specific variation to facilitate mating (Wheeler et al. 1991).
However when a protein from one species can complement a mutant or produce a similar phenotype in a second organism, even when the two species have been separated for long evolutionary periods, suggests the conservation in the function of proteins over long periods of evolutionary time. One of the most excellent examples is the ability of the mouse Small-eye (Pax-6) gene, which controls eye formation, to induce ectopic eye formation in Drosophila, indicating that the function of this protein has been conserved for the 500 million years since the divergence of arthropods and vertebrates (Halder et al., 1995). In another example, the chicken gHoxb-1 gene can substitute for a Drosophila Hox mutant (lab), which functions in head involution, when linked to the appropriate Drosophila regulatory sequences (Lutz et al., 1996). The Drosophila paired (prd) gene can rescue a gooseberry (gsb) mutant when the gsb control region is fused with prd coding regions, despite the fact that these two homeobox genes serve distinct roles in Drosophila development (Li and Noll, 1994). Similarly, the cdc2 homolog of maize complements the cdc28 mutant of Saccharomyces, despite a divergence time of at least 600 million years (Colasanti et al., 1991). Finally, the DEF protein of snapdragon fully complements an ap3-1 mutation and partially complements the ap3-3 mutation of Arabidopsis (Irish and Yamamoto, 1995). Evidence for the conservation of protein function over long evolutionary periods notwithstanding, proteins do evolve in function, and change in protein function is a crucial component of evolution. Indeed, there are a few cases in which the functional evolution of proteins involved in morphogenesis has been documented. For example, among members of the MADS box gene family of Arabidopsis, differences in three separate regions of the protein are involved in conferring the functional specificities that distinguish family members (Krizek and Meyerowitz, 1996). In this case, homologous proteins cannot substitute for other members of the same protein family. Because these different functions are required for normal flower development, one might infer that floral evolution has involved changes in protein function. When one observes full complementation of function by a protein from another species, the interpretation is clear. Function has been conserved. When partial or noncomplementation is observed, the interpretation is more complex. One possibility is that the protein has specifically evolved to interact with a different set of partners or bind to a different set of downstream genes in ways that alter the phenotype. A second possibility is that the protein has changed in "specificity" but not in "function as related to the phenotype." For example, a pair of interacting proteins in one species may have coevolved on a particular path, whereas in a second species the orthologous pair of proteins has evolved independently along a different path. These pairs may fulfill the same function (e.g., they may activate the same set of downstream genes) in both species, but the individual proteins of one species may not be able to complement the "function" of their orthologs in the other species because of changes that specify the interaction with the particular partner. For example LEAFY controls specific aspects of the life cycle in a basal plant, the moss Physcomitrella patens while LEAFY has more specialized functions in angiosperms, where it specifically induces floral fate during the reproductive phase. The strong sequence conservation of the DNA binding domain suggested that the molecular function of LFY is conserved as well. However when moss genes PpLFY1 and PpLFY2 cDNAs were linked to the Arabidopsis LFY promoter and introduced into a strong lfy mutant; in transgene they were inactive. This indicates that LFY function has diverged during evolution. However Angiosperm genes fully complement lfy mutant, whereas gymnosperm genes provide only partial rescue. Among homologs from the most basal group, the fern genes have some rescue ability, although less than the gymnosperm genes. This gradient of complementation reflects the phylogenetic distance from angiosperms and suggests that a continuum of discrete and nonneutral changes, rather than a sudden modification, is responsible for changes in function (Maizel et al. 2005).
Further the direct transformation of Arabidopsis thaliana with genes from other species has provided evidence to support a role for KNOX genes in the evolution of leaf morphology (Hay and Tsiantis, 2006) and of LFY in the evolution of plant architecture where introduction of L. crassa LFY gene into an A. thaliana lfy-6 background rescued the production of petal and stamen (Yoon and Baum, 2004).
Two class I KNOX homologues (CRKNOX1 and CRKNOX2) from Ceratopteris richardii were expressed in the SAM and in the incipient leaf primordia, as was observed for another fern, Anogramma charophylla (Bharathan et al,. 2002; Sano et al,. 2005). In order to reveal the function of CRKNOX1 and CRKNOX2, over expression experiments on these genes were conducted in Arabidopsis. The resulting phenotypes resembled the KNAT1 over expression phenotype, suggesting that these proteins can function similarly to their angiosperm counterparts in meristem development and leaf architecture (Sano et al. 2005).
Expression of C. hirsuta AS1 under the control of the broadly expressed CaMV 35S promoter complemented the A. thaliana as1 mutant phenotype and repressed expression of the KNOX gene in as1 leaves, indicating that the function of the two proteins is conserved. The function of either LjPHANa or LjPHANb (Lotus japonicus ARP genes ortholog) was the same as other ARP genes orthologs at protein level as the transgenic plants typically displayed elongated leaves with narrower, longer blades and longer petioles as compared with the leaves of wild type Landsberg erecta (Jiang et al., 2005). These transgenic phenotypes mimicked to the ones of a previous study, in which the overexpression of RS2 (maize ARP genes ortholog) and AS1 in Arabidopsis caused narrower leaves with longer petioles (Theodoris et al., 2003). SkARP1 (Selaginella kraussiana ARP genes ortholog) was also functionally equivalent to eudicot ARP genes as it complemented as1 leaf phenotypes and repressed the KNOX genes in Arabidopsis (Harrison et al, 2005).
Interspecies transformation studies have both strengths and weaknesses when used to study the genetic basis of species differences. On the positive side, studying genes from different species can reveal fundamental mechanisms that are obscured in traditional mutant or reverse-genetic studies (Harrison et al, 2005; Jiang et al., 2005; Yoon, 2003; Hay and Tsiantis, 2006; Theodoris et al., 2003; Yoon and Baum, 2004). On the negative side, transgenic experiments that do not yield perfect conversion of the recipient species into the phenotype of the donor species may provide neither rigorous rejection nor support of a prior hypothesis (Sliwinski et al., 2007).
Gene duplication has long been recognized as a major source of new genes and functions. Until recently, it was generally assumed that duplicate genes were free to evolve new functions ('neofunctionalization') because the original function was maintained by the other copy (Taylor and Raes, 2004). However, several recent case studies and comparisons of genome content have suggested that most new genes do not have novel functions (Prince and Pickett, 2002). Instead, paralogous gene pairs are often 'subfunctionalized' with two or more functions being partially or completely subdivided between the two genes after gene duplication.
Work in the previous chapter described the isolation of two ARP like genes from Begonia luxurians. To check whether expression of these two Begonia ARP genes (BARP1 and BARP2) could complement the as1 phenotype and to see the effects of BARP1 and BARP2 overexpression on leaves, I generated Arabidopsis plants expressing AS1, modified AS1 (BARP2 like AS1) and BARP1 from 35S promoter of cauliflower mosaic virus. The rationale for theses experiments was that if BARP1 and BARP2 are functionally equivalent to other ARP genes then these should rescue Arabidopsis as1 mutant phenotypes and over expressed lines should generate plants with narrower leaves and longer petioles in wild type Arabidopsis plants (Theodoris et al., 2003). However if duplicated BARP genes has undergone subfunctionalization, neither of the BARP genes copy should complement as1 mutants alone. In case of neofunctionalisation one of the copies should rescue mutant phenotype and other copy which could have acquired new function should not complement.
Experimental Design
Cloning of AS1 and BARP1 genes
ASYMMETRIC LEAVES1 (AS1) cloned in pBluescript vector (named pPOD#12) was kindly provided by Professor Andrew Hudson. I confirmed the presence of insert with double digestion (AflII and HindIII) as shown in figure 5.1. AS1 gene was recloned into pGEM T easy vector system (named pSU68) as pBluescript vector provides fewer restriction enzyme choices for later manipulation of the vector. The insert was confirmed with colony PCR followed by sequencing.
Complete AS1 and BARP1 CDS were amplified with primers having adapters for directional pENTRE dtopo cloning as described in 2.4 from Arabidopsis and B. luxurian respectively. Fresh PCR products of AS1 and BARP1 were column purified and cloned into pENTRE D-TOPO vector (figure 5.2 and figure 5.3) and plasmids were named pSU64 and pSU65 respectively (figure 5.4 A & B). Restriction analysis was done to select the desired clones (figure 5.4 C & D) for sequencing with M13 forward and reverse primers. DNA sequences were aligned with already sequenced AS1 and BARP1 and 100% homology was found.
A
C
LR recombination reactions were carried out with Gateway® LR Clonase â„¢ II Enzyme mix using 150ng of destination vector (pB7WG2.0) and 150ng of entry clone (pSU65.16/pSU64.3) as described in 2.4.5. The resultant vectors were named pSU69 and pSU70 carrying AS1 and BARP1 genes respectively. The inserts were confirmed by sequencing using gene specific primers. The flow chart of the LR reaction is shown in figure 5.5.
Figure 5. LR reaction facilitates recombination of an attL substrate (entry clone) with an attR substrate (destination vector) to create an attB-containing expression clone (see diagram below). This reaction is catalyzed by LR Clonaseâ„¢ II enzyme mix
Construction of modified AS1
As described in 4.4 the difference between BARP1 and BARP2 is an 18 bp deletion (six amino acid) in the myb domain2 of BARP2. In order to characterize the function of BARP2 from Begonia I modified AS1 from Arabidopsis by deleting the same 18 bp from the myb domain2 of AS1. 250bp fragment of AS1 gene with 18 bp deletion was synthesized by gene Synthesizer Company DNA 2.0. The plasmid was rescued from the filter as described in 2.4.3. The yield of plasmid was very low and plasmid was retransformed into TOP 10 cells and named pSU66. Presence of the insert was confirmed through restriction analysis followed by sequencing. The physical map of plasmid is shown in figure 5.6 A.
I tried to get modified AS1 gene fragment from pSU66 through double digestion (AflII and HindIII) but yield was very low. So I carried out the infusion PCR reaction. The modified AS1 fragment was amplified from pSU66 with Infusion F and R primers (figure 5.8). pSU68 (pGEM T easy vector with AS1) was digested with AflII and Xho1 enzymes (figure 5.7). Infusion reaction was carried out as described in chapter 2 and resulting plasmid was named pSU80. Colony PCR followed by sequencing was done to select positive clone (figure 5.9).
Infusion PCR was carried out to transfer modified AS1 from pGEM Teasy vector to pENTRE D-TOPO vector (figure 5.10). The Infusion804F and Infusion 804R primers were used to amplify modified AS1 from pGEM Teasy vector (figure 5.12 B), and pSU64.3 was digested with Xho1 and Not1 enzymes (figure 5.12 A) to perform infusion reaction. Infusion reaction was carried out as described in chapter 2 and positive clone was selected with colony PCR (figure 5.13) for sequencing with M13F and R primers.
LR recombination reactions were carried out with Gateway® LR Clonase â„¢ II Enzyme mix using 150ng of destination vector (pB7WG2.0) and 150ng of entry clone (pSU82.1) as described in 2.4.5. The resultant vector was named pSU95 carrying modified AS1 gene. The insert was confirmed through sequencing using gene specific primers.
pSU69, pSU70 and pSU95 were transformed into Agrobacterium strain GV3101 as described in 2.4.6 and resultant vectors were named as pSU96, pSU97 and pSU98 respectively. The transformation of Arabidopsis was done according to the floral dipping method with pSU96, pSU97 and pSU98 as described in 2.4.7. Arabidopsis transformants were selected on ½ MS plates with 50ug/ml kanamycin as described in 2.4.8. Kanamycin resistant plants with true leaves and extended root system were then transferred into 9 cm pots and phenotypes of plants were evaluated.
Results
AS1, BARP1 and BARP2 like AS1 rescued as1 mutant plants
The development of leaves is disrupted in asymmetric leaves1 (as1) mutants (figure 5.14). The mutant leaves have increased width to length ratio (shorter petiole and wider leaves). The lamina has prominent lobes. Later leaves have more lobes as compared to the early leaves (Byrne et al., 2000). Expression of AS1, BARP1, and modified AS1 (BARP2 like AS1) in an as1 mutant were able to complement the as1 leaf development phenotype in (compare figure 5.14 with 5.15. 5.16 & 5.17) suggesting that BARP1, modified AS1 and AS1 are functionally interchangeable and that this pathway is functionally conserved. Compared with wild-type and as1 controls, the phenotype of transformed plants varied from as1- like to wild type like plants. The ratio for rescued to non rescued plants were 2:1, 1:1 and 2:1 for AS1, BARP1, and modified AS1 transgenics respectively as described in table 5.1.
Over expression of AS1, BARP1 and BARP2 like AS1 in Arabidopsis
Over expression of the genes should increase gene expression in leaves and should produce dominant leaf phenotypes. Whereas loss of AS1 resulted in plants with shorter petioles and wider leaves (increased width to length ratio), 35S expression of either AS1 or BARP1 in wild-type plants resulted in the opposite effect while producing elongated leaves with narrower blades and longer petioles (decreased width to length ratio) (compare figure 5.18 with 5.19 & 5.20). However when the modified AS1 was over expressed in wild type plants it produced as1 like plants (compare figure 5.18 with 5.21). This suggests that modified AS1 is a negative dominate regulator.
Novel Phenotypes
As described in table 1, some novel phenotypes were observed among over expressed transgenic plants with 35S AS1, BARP1 and modify AS1 in wild type background. Most of these plants have an upward bending of leaves, resulting from growth of the lower side. This is usually due to the change in stability of dorsoventral polarity (Waites and Hudson, 1995). More growth from lower side means ectopic abaxial cells. Some of the transgenic lines have filamentous leaves. Some of these plants showed lack of apical dominance (figure 5.22).
Col-0
as1 Col-0
as1
wild
o/e
Novel phenotypes
as1
wild
Novel phenotypes
pSU96
(35S AS1)
0
15
46
2!
15
26
1!
pSU97
(35S BARP1)
0
14
25
4*
54
55
4!
pSU98
(35S BARP2)
58
2
0
0
15
32
0
Table 5. Scoring of transgenic plants for leaf form. as1 means plants looking like as1 mutants, o/e is abbreviation for over expressed lines, * indicates plants showing phenotypes like over expressed REVOLUTA genes and ! represents plants lacking apical dominance.
Discussion
Previous work has shown that ARP genes function is conserved between lycophytes (Selaginella kraussiana), monocots (Zea mays) and eudicot plants (Arabidopsis thaliana, Cardamine hirsuta, Lotus japonicus) (Harrison et al, 2005; Theodoris et al., 2003; Hay and Tsiantis, 2006; Jiang et al., 2005). Likewise both BARP1 and modified AS1 rescued as1 mutant phenotypes indicates that BARP1 and modified AS1 are functionally equivalent to other ARP genes. These phenotypes of transformed plants varied from as1- like to wild type like plants. In Selaginella kraussiana the degree of phenotypic rescue was directly proportional to SkARP1 transcript levels (Harrison et al, 2005). The difference in BARP1 and modified AS1 genes transcript level may be the cause of variation in rescued phenotypes. This can be done by performing Quantitative PCR or northern blotting which were not done due to time constraints.
ARP genes are member of a small unique MYB-related gene family that are required for repressing expression of certain KNOX (KNOTTED1-like homeobox) genes in leaves and consists of two protein domains. Myb domain presumed to be involved in nucleic acid binding is at N-terminus. The Myb domain is highly diverged compared with that in other Myb proteins and the DNA recognition helix in Myb repeat R3 is completely unique. ARP genes binds with DNA and cofactors are required for nucleic acid interaction. The C- terminal domain involved in homodimerization and is highly conserved among ARP genes family and does not show homology to any other known protein sequence.
Transformation of wild type plants with modified AS1 yielded as1 like mutant plants which indicate that BARP2 is a negative dominate loci. AS1 and modified AS1 may have affinity to bind the similar nucleic acids and binding of modified AS1 with those nucleic acids may have distorted the function of ARP genes but this is unlikely as modified AS1 has complemented as1 mutants. Other possibility is that homodimerization of AS1 and modified AS1 may have yielded a protein structure which was unable to perform normal ARP genes function and yielded mutant like phenotypes as RS2 proteins do make dimmers with AS1 proteins. Further AS1 and modified AS1 may have various levels of interactions as modified AS1 over expressed lines have yielded a range of phenotypes ranging from as1 like plants to wild type like plants.
Class III HDZip genes have a fundamental role in the shoot in establishing a functional apical meristem and polarity in lateral organs (Bowman et al., 2004). The squit phenotypes observed among transgenic plants resembles those of HDZip III mutants. ARP genes work upstream of HDZip III genes. The fact that leaf form has altered in novel ways lends support to more complex models in which the ARP protein interacts with and possibly titrates factors that normally interact with HDZip III genes to modulate leaf morphology.
Conclusion
Both BARP1 and BARP2 like AS1 (modified AS1) genes are functionally equivalent to ARP genes for regulation of leaf morphology as both complements as1 mutant plants. Modified AS1 genes over expression in Arabidopsis have suggested that the modified AS1 is a negative dominant locus.
