Glabrousness In Gl 1 Locus Of Arabidopsis Lyrata Biology Essay

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Trichomes are adaptive traits of plants which help them in variety of ways. They are basically hairy epidermal outgrowths of plants which act as defensive structures and protect plants from various abiotic stresses like drought etc. The transcription factor GLABRA 1 (GL1) is responsible for regulating the genes necessary for trichome production. Mutations in those genes which are responsible for expression of this transcription factor make plants without trichomes and are called as glabrous. In this study, molecular analysis of trichome formation was analyzed in Arabidopsis lyrata to find out which polymorphism was responsible for glabrous phenotype. This was done by transforming mutant lines of gl-1 gene with pCAMBIA constructs carrying polymoprhisms in different combinations. It was concluded that it is the SNP is the causative but not Ins or Del in GL-1 gene that is responsible for glabrous phenotype of Arabidopsis lyrata.

Arabidopsis thaliana is a small weed belonging to the mustard family often considered as laboratory mice because of the ease with which it can be handled. It requires very small space to grow, has tiny genome and completes its life cycle in few weeks (Campbell & Reece, 2008). This self-pollinating angiosperm (Meinke et al., 1998) has 125 mega base of sequence which encodes approximately 26,700 genes (Roberts, 2007). In recent years large information was accumulated on this plant because of growing importance of this organism as model plant to study not only plants but commonly many eukaryotes. Increased international cooperation among research communities, well established stock centers in U.S.A and U.K which provide ready accessibility of many different ecotypes and mutant lines of Arabidopsis is one more reason to say why this organism is favorite among plant biologists (Campbell & Reece, 2008). A. lyrata is a close relative of A. thaliana. It is an outcrossing, perennial herb (JGI, 2010). As it is out crossing, over a period time there may be many natural polymorphisms accumulated in its genome which makes Arabidopsis lyrata to use in this study about molecular analysis of Trichome formation.


Trichomes are aerial projections of epidermis. Generally trichome distribution varies between species of plants. Young Arabidopsis thaliana possesses trichomes on true leaves. The first true leaves possess them only on adaxial side of leaves which are generally oval shaped. Then, the subsequent leaves possess them on both sides. Other than leaves stem, sepals also possess trichomes in plants (David, 1997). Trichomes are involved in variety of functions of plants. Some control leaf surface temperature by reflecting light and some act as defensive structures and protect plants from various abiotic stresses like drought etc. Some may even help plants from herbivores by secreting a variety of substances. They also help in controlling transpiration rate. They also come in variety of structure in which some are branched and some are not branched. (Roberts, 2007)

At molecular level many genes regulate the development of trichome formation. Three genes which are essential for trichome formation include GL-1 (GLABROUS1), TTG (TRANSPARENT TESTA GLABRA) and TRY (TRIPTYCHON). Other genes include CAPRICE (CPC), COTYLEDON TRICHOME 1 (COT1) (Roberts, 2007). GL-1 (GLABROUS1), and TTG1 (TRANSPARENT TESTA GLABRA1) are essential for trichome formation as null mutants are completely glabrous (Hauser et al, 2001). The gl1 mutant lacks only trichomes but the ttg1 mutant lacks not only trichomes but also anthocyanin pigments which results in yellow seed which is transparent rather than reddish brown wild type seeds and it also lacks polysaccharides which are normally found in outer layer of seed testa (David,1997). This is a perfect example of plieotrophy. GL-1 gene encodes myb related transcription factor which are normally involved in growth related process of plants. TTG-1 encodes a protein with Wd40 repeats which are involved in G protein signal transduction pathway (Roberts. 2007). Both gl-1 and ttg-1 possess trichomes on margin of cauline and rosette leaves which in turn clearly shows that there are many genes involved in trichome formation other than GL-1and TTG (David, 1997).

The basic aim of this practical is to do molecular analysis of trichome formation in Arabidopsis lyrata a close relative of A.thaliana and to draw a causative relationship between genotype and phenotype. Arabidopsis lyrata naturally possesses three genetic polymorphisms in GL-1gene. They are Single nucleotide polymorphism (SNP), insertion and deletion. The hypothesis is that there should be a correlation between genotype and phenotype in trichome development, to find out whether single mutation or many mutations affect trichome development. To test this hypothesis, Genes containing these mutations in different combinations are cloned in to pCAMBIA vector and transformed in to Arabidopsis lyrata gl-1 plants. If these Genes are able to rescue the gl-1 mutant by helping them to form the trichomes, then it is considered as not harmful. But, if a particular mutation results in loss of trichome formation then it can be concluded as a candidate gene for trichome formation whose mutation resulted in loss of trichomes.

All the mutated plants are glabrous clearly concluding that GL-1 gene is essential for trichome development. In addition to that ttg1-1 and ttg2-1 have yellow seed coat which was a result of pleiotropic effect which was observed initially when the seeds were sterilized and stratified. Thus, the mutants in ttg1-1 and ttg2-1 are not only glabrous but also are victims for other phenotypic characters like yellow seed coat.

In the C-DNA samples, weak band or no band was found in Lanes 6, 11, 16, 17, 18, 19, 20, 22 {fig 2(B,C), fig 3(B,C)} suggesting the absence of C-DNA of GL-1 or due to contamination which was same even for CHS gene which was used as positive control. The negative controls of both C-DNA and Chalcone synthase (CHS) amplification respectively gave unexpected results with weak smear bands which can be attributed to contamination during PCR setup.


The primary objective of this study is to find out the candidate polymorphism responsible for glabrous nature in A.lyrata. To do this, pCAMBIA constructs carrying polymorphisms in all possible combinations were transformed in to gl-1plants of A. lyrata. The phenotypical analysis done by scoring trichomes under light microscope concludes that all the mutant lines carrying gl-1 gene lack trichomes suggesting the role of GL-1 in formation of trichomes. Some samples harboring constructs of both Ins and Del showed reduced number of trichomes. All the lines harboring constructs with an SNP mutation in conjunction with Ins or Del or both are completely glabrous on their oval rosette leaves. But constructs carrying only Ins or Del or both without an SNP have trichomes with three or four branches on their leaves. This shows that constructs carrying Ins or Del or both without an SNP rescued the gl-1 line of A.lyrata and can be concluded as not harmful (Hauser et al, 2001). But as SNP in any combination or independently caused glabrous nature, which can in turn be concluded as candidate gene responsible for glabrous phenotype in A. lyrata (Hanna, 2006). Thus, there exists a causative relationship between genotype and phenotype in trichome formation and the proposed hypothesis in turn can be accepted for now. It can also be further concluded that this SNP is reason behind glabrous phenotype of all the mutants which carried band during RNA extraction except gl-1 which completely lacked any band. This is the same case even in ecotypes as only Li-2 carried band and trichomes.

In the gene expression analysis, after the RNA extraction bands were absent in wild type Ler which is inconclusive. The phenotypic analysis of Ler revealed presence of trichomes in very low number. This can be concluded by saying that there is a very weak expression of GL-1 gene in it or due to contamination during the experiment. The gl-1 mutant stands as perfect example of correlation between genotype and phenotype with no band during C-DNA amplification but a small one during RNA extraction and Chalcone Positive control analysis.

Interestingly, pGL1 Ins Del genotype showed a weak band in both C-DNA analysis as well as in Chalcone synthase gene amplification and that may be due to weak or low level of RNA expression due to which some samples carrying those constructs produce relatively low trichomes. But, still further comparative analysis should be carried in different types of Arabidopsis population and different ecotypes to deduce a final conclusion on varying nature of trichome density in some samples as well as on glabrous nature of leaves.

Though the present study throws some light on genetics of trichome formation, further analysis with different population combined with genomic approaches will show us further insights about development of these structures in plants.