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There is a great debate about either "Evolution of form is very much a matter of teaching very old genes new tricks" (Carroll, 2005) as Carroll believes or the evolution of form is very much a matter of teaching old genes to make new genes (Coyne, 2005).
According to Carroll, the main source of evolutionary changes is in the switches that control proteins instead of change in protein coding sequence. These switches are the promoters and enhancers in DNA that regulate transcription. They promote evolution by causing existing genes to be expressed at new times and places. Carroll also claims that proteins are resistant to evolutionary change because they are often involved in many pathways, and therefore a change in protein sequence, while enhancing one aspect of the protein's many functions, could damage several others. In contrast, changing an enhancer or promoter can affect the expression of a single protein without altering its structure, so such changes are more likely to be adaptive. He denies the idea of new genes causing diversity of most animal groups and deduces that changes of the expression pattern in same set of genes between different animal species enable them to be made using essentially the same tool kit.
Carroll explains that dissimilar species can in fact be genetically similar. For example mice and humans are identical at about 25,000 genes sets and chimps and humans are almost 99 percent identical at the DNA level. Since the sets of genes are so widely shared, the differences arise through the evolution of non coding regulatory elements (Carroll, 2005). So whether you are a man or a mouse apparently depends solely on your promoters and enhancers (Carroll, 2005). The studies of natural species also reveal the correlations between the spatial patterns of gene expression and phenotype. For example, expression of Distal-less domains is linked with the formation of eye spots on butterfly wings (Brakefield et al., 1996), so a change in the spatial pattern of Distal-less expression could confer a change in the size and distribution of eye spots. Comparison of Hox gene expression in primitive wingless and advanced winged insects suggests that genes involved in wing formation acquired cis-regulatory elements that brought them under the negative control of Hox genes in some body segments (Carroll et al., 1995). Changes in Hox gene expression patterns are also associated with the transformation of thoracic limbs into feeding appendages in crustaceans (Averof and Patel, 1997).
On the other hand Coyne describes that changes in proteins are the cause of diversity of form on earth (Coyne, 2005). He argues that humans have about 32,000 protein-coding genes while fruitflies only 13,000 and between 40% and 50% of humans protein-coding genes have no known homologues in flies. Clearly, the difference between these species involves the origin of new proteins. Further humans and chimps have different amino-acid sequences in at least 55% of their proteins, a figure that rises to 95% for humans and mice. Thus we can't exclude protein-sequence evolution as an important reason of evolution of form. He rejects the idea of Carroll that "change in protein-coding sequence can destroy its one of several functions" by describing the processes of protein evolution which does not have any injurious side effects. These include gene duplication and whole genome duplication events. Extra copies of a gene can arise by unequal crossing over or by reverse transcription, allowing one copy to retain its function while the other assumes a new function. This process has been a major force in evolution (Ohno, 1970; Zhenglong et al., 2001). A large fraction of genes are members of families derived from repeated duplications and diversification of ancestral genes, a process that has yielded many evolutionary novelties. These families include the globins; immunoglobulins; opsins (which led to colour vision in Old World primates); and olfactory receptors (almost certainly involved in the evolution of a keen sense of smell in land animals). Lactalbumin, which helps to produce milk in mammals, resulted from a duplication of lysozyme, and the crystallins of our eye lenses are ultimately derived from heat-shock genes.
It is possible that gene function evolved, giving rise to distinct morphological traits in different species, either by changes to upstream elements or by changes to the properties of the gene product. Also, recruitment of new target genes could change the output of the original gene and, as a result give rise to new phenotypes.
Changes in the expression pattern of key regulators are important in plants as well. An excellent example is genetic regulation of flower development which is quite well understood in Arabidopsis and snapdragon. The ABC model posited that floral organ identity is controlled by three gene functions, A, B and C that act in combination; A-function alone specifies sepal identity, A- and B-functions together control petal identity; B- and C-functions together control stamen identity; C-function alone specifies carpel identity (Coen and Meyerowitz, 1991; Bowman et al., 1991). Few mutations in one of the ABC genes can cause homeotic conversions of floral organs. Homeotic conversions of stamens into petals or vice versa are quite common in some lineages (Ronse et al., 2003). Two classes of transcription factors are regulating the anthocyanin production in various species. One class, the R family, includes maize genes R and B, snapdragon Delila, and petunia An4 genes. The second class C, includes maize C1 and P1 genes, snapdragon Myb305, and petunia An2 genes (Peter et al., 1998).
There are also several cases in the evolution of physiological traits in which specific adaptations are correlated with changes in the pattern of gene expression. Flowers of Clarkia breweri which emit a strong sweet scent has evolved from an extant nonscented species, C. concinna. The scent is controlled by the production of S-linalool, an acyclic monoterpene. Lis, the gene encoding S-linalool synthase, is highly expressed in C. breweri, as compare to in the non scented C. concinna. The differential expression of Liz regulates the scent emission in these species (Dudareva et al., 1996). Expression patterns of a suite of enzymes, normally used for housekeeping functions, are altered in C4 species which is accompanied by alterations in internal histology and chloroplast structure of the leaf. Expression patterns common to all C4 lineages are central to the evolution and development of the pathway and patterns that vary are lineage specific (Sinha and Kellogg, 1996).
Changes in the expression pattern of ARP and KNOX genes are reported to be associated with leaf form in a range of species-examples followed. Mutations at the phantastica (phan) locus of Antirrhinum majus has described that subtle changes in the level or pattern of phan activity can give rise to a variety of organ morphologies including needle like leaves, cup shaped leaved and peltate leaves (Waites and Hudson, 1995). Expression domain of ARP in a range of species with compound leaves correlates with the type of compound leaf i-e pinnate, palmate or peltate palmate (Kim et al., 2003a; Kim et al., 2003b). This suggests that the convergent evolution of ARP expression may be responsible for leaf shape variation in species.
In tomato, expression of a homeobox-containing gene in the leaf primordia is associated with the formation of compound rather than simple leaves (Hareven et al., 1996). An up regulation of homeobox-containing gene LeT6 resulted in the conversion of unipinnately compound leaves into three- or four fold pinnately compound leaves. (Chen et al., 1997). Species-level differences in leaf form in the native tomatoes of Galapagos Islands are also due to changes in the KNOX (PETROSELINUM (PTS)) genes expression where the expression of the KNOX genes is up regulated in the leaves of highly dissected Solanum galapagense in comparison to its expression levels in the less dissected sister species Solanum cheesmaniae (Kimura et al., 2008). The compound leaf character of Elaeis guineensis (palms) is found to be dependant on the expression of KNOX1 genes (Stefan et al., 2007) and reactivation of KNOX genes expression after leaf formation in the basal meristem of Welwitschia mirabilis is responsible for the generation of its leaves over 400 to 1500 years (Pham and Sinha, 2003).
Differential expression of KNOX genes between pinnately compound leaved Cardamine hirsute and simple leaved Arabidopsis thaliana correspond to the natural variation in the leaf shape of these two closely related species. The difference in the expression pattern of KNOX genes between these two species is driven by the variation in the promoter region of KNOX genes between two species (Hay & Tsiantis, 2006).
A model is purposed to show the correlation of expression pattern of ARP and KNOX genes with leaf form (figure 6.1 and 6.2), where expression of ARP genes along adaxial domain of developing leaves mostly generates simple leaves, ARP expression confined to distal regions of developing leaves results in the formation of peltate leaves and ARP expression along the boundary of adaxial domain depict the leaflet placement in compound leaf formation. And lack of KNOX genes expression in developing leaves mostly generate simple leaves and KNOX expression reactivation in developing leaves results in the formation of compound leaves (Champagne et al. 2004; Kim et al., 2003).
I have tested above hypothesis in Begonia section Gireoudia species through in situ hybridization in a range of species. This section has a number of simple, peltate and compound leaved sister species making it an excellent model to test the inter specific variation of leaf form.
Fixation of vegetative buds
Vegetative buds of different species were fixed and sectioned as described in chapter 2. I tried to fix the vegetative buds of the same age from all the species in order to compare the expression pattern of genes at the same stage of development. In order to make the judgement about the age and orientation of vegetative buds to be used for in situ hybridization, I initially did the disection of vegetative buds of different species extensively under disection microscope. Close observation of different species gave a general idea about the time differences in the initiation of two successive primordia which range between 13 to 22 days for different species. The smallest time difference in the initiation of two successive primordia was for B. mazae and longest was for B. thiemei. This could have dependant on number of leaves each species generate in a specific time period or could be due to the size of leaves. B. mazae clearly generates more number of leaves during a given time period as compare to B. thiemei and produces smaller leaves then B. thiemei as well.
For hybridization I harvested the buds of same sizes from different species which were approximately of the same age. I did quick disection of vegetative buds to remove larger and unwanted parts while they were still attached to the plant and directly fixed them into fixative after harvesting. As older stipules wrapped up the developing leaves very tightly and could hinder the fixation of enclosing material, so I dissected the material after 2 hours of fixation with fine needles and forceps to reveal the youngest stipules enclosing youngest primordia and shoot apical meristem under disection microscope. Begonia species have large amount of trichomes which have created problems during sectioning. I have tried to remove them after buds had undergone ethanol and histoclear treatments but doing this was leaving material unintegrated. So only way to find good fixed material was disection of lots of buds at the same time.
6.2.2 Transcription of probes
C- terminal domain (721 bp) of BARP1 genes, which is down stream of Myb domains, is used to make probes for detection of RNA by DIG in insitu hybridization. The reason for using C-terminal domain only is that Myb domain of ARP genes show significant similarity with most plant Myb proteins and the use of whole BARP1 genes as a probe would have mislead results by binding with other myb proteins. For example Myb domains of ARP genes in snapdragon had only one amino acid upstream of the first repeat, and the first repeat was 2 or 3 amino acids longer than that of other MYB proteins (Waites and Hudson, 1998).
PCR based sense and antisense probes were made using BARP1-C-TerT3, BARP1-C-TerT7 and BARP1-C-TerSP6 primers to amplify c-terminal domain of BARP1 genes with High Fidelity Taq polymerase as described in chapter2. DNA of different species were used as templates and amplified fragments were sequenced for confirmation of the region.
For KNB1 and KNB2 genes specific primers were used for making probes as described in chapter 2. KNB1 and KNB2 have high homology so a region in the homeodomain which was less conserved between KNB1 and KNB2 was selected. Species specific probes were used for hybridization.
Expression of BARP1 in Begonia
In B. mazae BARP1 is expressed in the shoot apical meristem, leaf primordia, vascular bundles, stipule primordia and at the distal tips of developing lamina and developing stipules. BARP1 is also expressed in adaxial region of petiole in B. mazae. There is strong expression of BARP1 in the dormant axillary meristem and there was no BARP1 expression in the active meristem in B. mazae (figure 6.1 A). Similarly in B. kellemanii and B. heracleifolia BARP1 is expressed in the shoot apical meristem, leaf primordia, stipule primordia and vascular bundles. BARP1 is expressed all over in the younger leaf primordia and expression gets confined to the tips of developing leaves in B. kellemanii and B. heracleifolia (figure 6.1 B & C). BARP1 is also expressed on the adaxial side of petiole in B. heracleifolia (figure 6.1 B). In B. carolineifolia BARP1 is expressed at the tips of leaflets, vascular bundles and at the tips of developing stipules (figure 6.1 D).
Expression of KNB1 in Begonia
KNB1 expression is present in shoot apical meristem, tips of developing leaves and in the developing stipules of B. mazae (figure 6.2 A). KNB1 is expressed in SAM, developing leaves and everywhere in the developing stipules of B. kellemanii (figure 6.2 B). KNB1 expression is detected in SAM, leaf primordia, at the tips of developing stipules, on the adaxial side of developing stipules and at dorsal tips of developing leaves of B. heracleifolia (figure 6.2 C). In B. carolineifolia KNB1 expression is very low or absent in leaflet primordia and very strong expression is present at the tips of developing leaflets (figure 6.2 D).
Expression of KNB2 in Begonia
KNB2 is expressed everywhere in B. mazae and B. heracleifolia (figure 6.3 A & C). KNB1 expression is stronger then KNB2 in B. mazae (figure 6.3 A). KNB2 is expressed in SAM, leaf primordia, vascular bundles, at the tips of developing leaves and in stipules of B. kellemanii (figure 6.3 B). KNB2 is expressed everywhere in B. carolineifolia as well (figure 6.3 D). In B. thiemei KNB2 is expressed at the tips of leaflet primordia (figure 6.3 E).
Unlike in maize, Anthirrinum and Arabidopsis, tomato ARP (LePHAN) and KNOX transcripts are co-localized within the shoot apex (Koltai and Bird, 2000). In the compound leaved plants Senna actinophylla, Acacia hindisii, Vitex cannabifolia, Dizygotheca elegantissima, Oxalis regnellii, Koelreuteria paniculata, Aquilegia formosa and Pachira aquatica ARP genes are found in the shoot apical meristem, stem and leaf vascular traces (Kim et al., 2003). In barley KNOX genes are expressed in SAM and young leaves (Muller et al., 2001). Similarly BARP1 and KNOX genes are co expressed in shoot apical meristem, stem and vascular bundles in Begonia section Gireoudia species.
ARP genes are required for the establishment of dorsal identity in Antirrhinum majus as it is responsible for regulating all aspects of dorsoventrality in leaves, bracts and petals: from specifying the position of laminal initiation early in organ development, to determination of dorsal cell types at a later stage in this plant (Waites and Hudson, 1995). Presence of BARP1 expression on the adaxial side of petiole of B. mazae and B. heracleifolia indicates that BARP1 is specifying the position of laminal initiation and indicates the presence of ab adaxiality in Begonia petioles earlier in the development which later on become completely abaxialized. The presence of a notch at P1 of peltate species of Begonia also indicates that establishment of peltateness is a late event in Begonia leaf development. The presence of BARP1 expression on adaxial side of petioles may indicate that BARP1 is promoting the dosoventrarlity earlier in development and its confinement to the distal tips of laminae later may be the cause of establishing peltateness as restriction of the adaxial domain to the distal end of the leaf primordium in antiLePHAN tomato plants has resulted in the production of peltately palmate leaves. Further ARP expression was confined to the distal region of the leaf primordium in a range of peltate compound-leafed species (Kim et al., 2003). Likely BARP1 is expressed at the distal tips of laminae in Begonia species and all Begonia section Gireoudia species are peltate to some degree (abaxialized petioles and laminar outgrowth at the lamina- petiole attachment point).
Stipules are attached to the main stem in pea and are flattened laminae that are conventionally described as lateral organs of the pea compound leaf (Sachs, 1972) and the CRI (ARP orthologue in pea) regulates stipule initiation in pea (Tattersall et al., 2005). Presence of BARP1 expression in stipule primordia and developing stipules may indicate a role of BARP1 in stipule initiation and stipule development in Begonia.
KNOX genes are linked with indeterminacy (Long et al 1996; Volbrechet et al, 2000). Their expression is deactivated in simple leaved species but reactivated in compound leaved species during leaf development for leaflet formation (Shani et al, 2009; Hay & Tsiantis, 2006; Harevan et al, 1996). KNOX expression patterns corresponded to the developmental stage of the leaf primordia and not necessarily with the final leaf morphology (Bharathan et al., 2000). KNOX expression is correlated with complex leaf primordia such as in Lepidium oleraceum KNOX proteins are expressed in the complex leaf primordium which undergoes secondary morphogenesis to form simple leaves (Bharathan et al., 2000). KNOX genes are expressed in the leaf primordia and developing leaves of Begonia section Gireoudia species. Their expression in leaves may be required for the formation of laminae outgrowth later in leaf development which may have required indeterminate environment as peltateness is established later in leaf development in Begonia.
KNOX genes are present as a multigene family. KNOX genes have evolved by sub functionalisation and through the acquisition of new roles in plant morphogenesis. KNB1 and KNB2 have different expression pattern in Begonia section Gireoudia. KNB1 is present only in SAM, developing leaves, developing stipules and vascular bundles while KNB2 expression is detected everywhere. This may indicate a case of neofunctionalisation of KNOX genes in Begonia which is not known yet.
KNOX genes are not controlling compound leaf formation in Begonia. But KNOX independent mechanism of compound leaf formation has been reported for pea where UNIFOLIATA (ortholog of Arabidopsis LEAFY) regulates compound leaf formation (Gourlay et al., 2000). And NO APICAL MERISTEM/ CUP-SHAPED COTYLEDONS3 (NAM/CUC3) family are required for proper expression of KNOX/UFO like genes during compound leaf formation in Solanam lycopersicon, Cardamine hirsuta and Pisum sativum (Blein et al. 2008). These genes may be the key regulators for controlling compoundness in Begonia.
I have optimized the protocol of in situ hybridization for Begonia section Gireoudia species as described in chapter 2. I have used PCR based probes for hybridization. Firstly PCR optimization for primers with T3, T7 and SP6 adapters was time consuming. I did different PCR based techniques and in my hands touch own PCR worked best for longer primers. Secondly correct amount of RNA probes for hybridization was different for different genes and also vary for different species so I tested several concentrations of every RNA probe (200, 300, 400, 600, 800 and 1000 ng/slide) as described in chapter2. I did get signals in sense probes at some times and sometimes these signals were at the same places as were antisense and sometimes at random places. It may be because of using higher amount of RNA probes for Begonia as compare to other species. Every time fresh PCR products were used to transcribe probes and 4 PCR reactions (each in 50ul total volume) were pooled to get 800 ng of RNA probes for hybridization. I did not sequence all the PCR reactions each time and there is possibility that sometimes non specific products can have generated and used for making probes which gave signals in sense probes.
Finding the correct orientation of Begonia vegetative buds of fixed material was difficult task. For me transverse sections have worked better. In longitudinal sections material was coming in unintegrated parts through microtome which may be due to poor fixation. Describing different regions of Begonia vegetative buds sections was challenging as known literature is available for Begonia histology.
BARP1 and KNB1 expressions are co localized in SAM, leaf primordia, developing stipules, developing leaves and vascular bundles while KNB2 is expressed everywhere in simple, peltate and compound leaves of Begonia section Gireoudia. BARP1 may be the key regulator for controlling peltate leaf form in this section because it is expressed in the distal tips of developing leaves which has been reported for most of peltate leaved species. This is supported by the association mapping studies where BARP1 is a major locus for controlling peltateness. KNOX genes are not linked with compound leaf form in Begonia. LEAFY and CUC genes are good candidates to look at for understanding the regulation of compoundness in this section.