AS1 and AS2 mutations on formation of Arabidopsis thaliana leaves

By: Elijah Meador

Abstract
The AS1 and AS2 mutants of Arabidopsis display different phenotypes with multiple
genes. Expression of a number of genes such as KNOX genes are enhanced with mutations. We
attempted to identify the phenotypic structures of AS1 and AS2 mutants that were within the
Arabidopsis thaliana plant family. When the KNOX genes are expressed the size of the leaves,
sepals, and petals were reduced. When the AS1 and AS2 mutants are present, the KNOX genes
are causing the mutations in the size and structure of the leaves. There are some features though
that are not caused by the KNOX genes, such as asymmetric formation which may be caused by
other genes enhanced by the AS1 and AS2 mutants.

Introduction
An important model system for identifying different genes and their functions within
plants is the Arabidopsis thaliana (Ikezaki et al. 2009). These plants were the first plants to be
have the entire genome sequenced and any mutations in the plants genome are easy to be
observed (The Arabidopsis Information Resource 2014). Arabidopsis leaves are very good for
the study of leaf morphology because they have a simple, stable form and their genome has
already been sequenced.
When studying plants, leaves are usually the top feature that the plant is identified by.
While observing leaves, one characteristic that stands out is the symmetry of the leaves. From
the petiole there is a near mirror symmetry of the leaf going left to right (Semiarti et al. 2001).

During leave development, leaves grow from a shoot apical meristem causing the left to right
symmetry upon the adaxial-abaxial axes (Semiarti et al. 2001). There are different mutants that
can cause leaves to not grow symmetrical; these mutations can alter the leaf morphology causing
change in shape and size of the plants (Semiarti et al. 2001).
AS1 encodes a domain protein that regulates knotted-class homeobox genes (Chua et al.
2000). These genes are very important in leaf development, ensuring the continuous production
of mature leaves (Chua et al. 2000). AS1 genes are also what regulate the adaxial-abaxial
polarity in leaves (Chua et al. 2000). AS2 genes of the Arabidopsis are what establish the leaf
venation system which includes: the midvein and the development of a symmetric lamina
(Iwakawa et al. 2002). The AS2 genes limit the appearance of class 1 knox homeobox genes in
leaves.

Materials and Methods

DNA was isolated from Arabidopsis plants. The isolation of the DNA will amplify the
as1 and as2 genes. To perform the DNA isolation a small amount of plant tissue was placed in a
1.5 mL tube. 400 uL of extraction buffer (200 mM Tris-Cl, pH 7.5; 250 mM NaCl; 25 mM
EDTA; 0.5% SDS) was then added and using a blue micropestle, the plant was mashed up into
the buffer. The tube was then centrifuged for 10 minutes at maximum speed to get a supernatant
material that was then was placed into 300 uL of isopropanol. After centrifuged again for 10
minutes, the pellet was added to 200 uL of 70% ethanol. The ethanol was removed after
centrifuged again for 5 minutes and the dna was allowed to air dry. 100 uL of TE (10 mM TrisCl, pH 8.0; 1 mM EDTA) was added to the pellet and centrifuged for one minute to give us the
product needed for the PCR amplication.

PCR products were sequenced to show if the mutant leaf phenotypes were due to
mutations in the as1 and/or as2 genes. Two PCR products were used: AS2F and AS2R primers.
These primers were given as a 5uM stock, but the reaction contained 0.2 uM. The final volume
of the whole PCR was 25 uL. The PCR contained 2X stock concentration of the PCR mix (Taq
polymerase, Mg++, buffer and dNTPs) that needed to be 1X concentration, primer 1, primer 2,
genomic template DNA, and water. For the total volume to be 25 uL, there was 12.5 uL of the
2X PCR mix, 1 uL of the 5 uM primer 1, 1 uL of the 5 uM primer 2, 1 uL of the genomic
template DNA, and 9.5 uL of water. The reaction was set up in order from water, to PCR mix, to
primer 1, to primer 2, to DNA. It was centrifuged then placed on ice.
Gel electrophoresis is a method used to separate the DNA according to molecular size.
The molecules are pushed by an electrical field through a gel that contains small pores. The
molecules travel through the pores in the gel and the small DNA molecules will travel a greater
distance through the gel than will the larger ones. The PCR product was run on the gel with a 6X
loading dye. The amount of loading dye used was 5 uL.
To set up the restriction enzyme digest 2.1 buffer was used at a temperature of 37 degrees
Celsius. The buffers are supplied at a 10X concentration, so the amount the buffer was diluted
was 1/10. In the restriction digest the total volume was 20 uL the components were 2 uL of
plasmid DNA, 0.5 uL of the restriction enzyme, 2 uL of the restriction enzyme buffer, and 15.5
uL of water.
ICM Pro was used to create the molecular models for the as1 and as2 genes. The structure
of the proteins were displayed and mutations were then identified. When the mutants were
superimposed, it was easily seen in what areas the proteins lined up and where they did not.

Results
AS1

Wild Type

The

AS2

Figure 1: Phenotypic representations of the AS1 mutation, AS2 mutation and the
normal wild type

The main purpose of this study was is to determine investigate phenotypic effects caused
by mutation in the AS1 and AS2 genes. Phenotypes of the AS1 and AS2 mutations are seen
above in figure 1 where we can see that the leaves are significantly different as compared to the
normal wild type. The average lengths of leaves for the class population of each plant are shown
below in Figure 2. Petiole lengths were measured in millimeters. As the data below shows, each
mutant is significantly different from the other.

Leaf Length in mutants

AS2 Leaf
AS1 Leaf
WT Leaf
0

10

Petiole Length (mm)

Figure 2: Average petiole lengths of A thaliana. wild type, AS1 mutant,

and AS2 mutant (mm)

12

AS1 Wild Type Sequence Alignment

Figure 3 below shows DNA base sequence alignments of AS1 wild type, AS1 and AS2
mutations. Figure 4 below shows DNA base sequence alignments of AS2 wild type, AS1 and
AS2 Mutants. Mutations are indicated by dashes in place of base letter and are highlighted.

AAAGCGGTTAGGGAAGTGGTGGGAAGTGTTTAAGGAGAAGCAACAGAGAGAAGAGAAAGAAS1WT
AAAGCGGTTAGGGAAGTGGTGGGAAGTGTTTAAGGAGAAGCAACAGAGAGAAGAGAAAGAAS2
AAAGCGGTTAGGGAAGTGGTGGGAAGTGTTTAAGGAGAAGCAACAGAGAGAAGAGAAAGAAS1

************************************************************
GAGTAACAAGAGAGTTGAGCCTATTGACGAGAGTAAGTACGATCGGATTCTCGAGAGTTT
GAGTAACAAGAGAGTTGAGCCTATTGACGAGAGTAAGTACGATCGGATTCTCGAGAGTTT
GAGTAACAAGAGAGTTGAGCCTATTGACGAGAGTAAGTACGATCGGATTCTCGAGAGTTT

************************************************************
CGCTGAGAAGCTTGTCAAAGAGCGGTCTAACGTTGTCCCTGCTGCTGCCGCTGCTGCAAC
CGCTGAGAAGCTTGTCAAAGAGCGGTCTAACGTTGTCCCTGCTGCTGCCGCTGCTGCAAC
CGCTGAGAAGCTTGTCAAAGAGCGGTCTAACGTTGTCCCTGCTGCTGCCGCTGCTGCAAC

************************************************************
GGTTGTGATGGCTAATTCGAATGGAGGGTTTTTACATTCTGAACAACAAGTTCAGCCTCC
GGTTGTGATGGCTAATTCGAATGGAGGGTTTTTACATTCTGAACAACAAGTTCAGCCTCC
GGTTGTGATGGCTAATTCGAATGGAGGGTTTTTACATTCTGAACAACAAGTTCAGCCTCC

************************************************************
TAACCCAGTGATCCCGCCTTGGTTAGCTACTTCTAACAATGGGAACAATGTTGTTGCAAG
TAACCCAGTGATCCCGCCTTGGTTAGCTACTTCTAACAATGGGAACAATGTTGTTGCAAG
TAACCCAGTGATCCCGCCTTGGTTAGCTACTTCTAACAATGGGAACAATGTTGTTGCAAG

************************************************************

GCCTCCCTCGGTAACTTTGACATTATCGCCTTCCACAGTGGCTGCAGCTGCGCCTCAACC
GCCTCCCTCGGTAACTTTGACATTATCGCCTTCCACAGTGGCTGCAGCTGCGCCTCAACC
GCCTCCCTCGGTAACTTTGACATTATCGCCTTCCACAGTGGCTGCAGCTGCGCCTCAACC

************************************************************
GCCAATCCCGTGGCTGCAGCAGCAACAGCCTGAGAGAGCAGAGAACGGTCCAGGGGGACT
GCCAATCCCGTGGCTGCAGCAGCAACAGCCTGAGAGAGCAGAGAACGGTCCAGGGGGACT
GCCAATCCCGTGGCTGCAGCAGCAACAGCCTGAGAGAGCAGAGAACGGTCCAGGGGACT

***********************************************************

Single base pair

deletion of
guanine is
located at the
highlighted area

TGTGTTAGGGAGTATGATGCCGTCTTGTAGTGGGAGTAGCGAGAGTGTGTTCTTGTCAGA
TGTGTTAGGGAGTATGATGCCGTCTTGTAGTGGGAGTAGCGAGAGTGTGTTCTTGTCAGA
TGTGTTAGGGAGTATGATGCCGTCTTGTAGTGGGAGTAGCGAGAGTGTGTTCTTGTCAGA

************************************************************
GCTTGTGGAGTGTTGTAGAGAGTTGGAGGAAGGGCACCGAGCTTGGGCAGACCATAAGAA
GCTTGTGGAGTGTTGTAGAGAGTTGGAGGAAGGGCACCGAGCTTGGGCAGACCATAAGAA
GCTTGTGGAGTGTTGTAGAGAGTTGGAGGAAGGGCACCGAGCTTGGGCAGACCATAAGAA

************************************************************
AGAGGCTGCATGGAGGCTAAGAAGGCTGGAGCTGCAGCTAGAGTCAGAGAAGACGTGTAG
AGAGGCTGCATGGAGGCTAAGAAGGCTGGAGCTGCAGCTAGAGTCAGAGAAGACGTGTAG
AGAGGCTGCATGGAGGCTAAGAAGGCTGGAGCTGCAGCTAGAGTCAGAGAAGACGTGTAG

************************************************************
ACAAAGGGAGAAGATGGAGGAGATTGAGGCAAAGATGAAAGCTCTTAGGGAAGAGCAGAA
ACAAAGGGAGAAGATGGAGGAGATTGAGGCAAAGATGAAAGCTCTTAGGGAAGAGCAGAA
ACAAAGGGAGAAGATGGAGGAGATTGAGGCAAAGATGAAAGCTCTTAGGGAAGAGCAGAA

************************************************************
GAACGCAATGGAGAAGATCGAAGGAGAGTACAGAGAACAGCTCGTTGGTTTGAGGCGAGA
GAACGCAATGGAGAAGATCGAAGGAGAGTACAGAGAACAGCTCGTTGGTTTGAGGCGAGA
GAACGCAATGGAGAAGATCGAAGGAGAGTACAGAGAACAGCTCGTTGGTTTGAGGCGAGA

************************************************************

CGCAGAGGCCAAAGACCAGAAACTGGCTGATCAATGGACCTCTAGGCATATCAGACTCAC
CGCAGAGGCCAAAGACCAGAAACTGGCTGATCAATGGACCTCTAGGCATATCAGACTCAC
CGCAGAGGCCAAAGACCAGAAACTGGCTGATCAATGGACCTCTAGGCATATCAGACTCAC

************************************************************
CAAGTTTCTTGAACAACAAATGGGTTGCAGATTAGACCGCCCCTGAACCGTCTCCTAAAC

CAAGTTTCTTGAACAACAAATGGGTTGCAGATTAGACCGCCCCTGAACCGTCTCCTAAAC
CAAGTTTCTTGAACAACAAATGGGTTGCAGATTAGACCGCCCCTGAACCGTCTCCTAAAC

************************************************************
T
T
T
*
Figure 3: AS1 wild type and AS1, AS2 PCR product DNA sequence
alignment. Mutation is indicated by highlighted area.

AS2 Wild Type Sequence Alignment

GCATCTTCTTCAACAAACTCACCATGCGCCGCTTGCAAATTCCTCCGGCGAAAATGTCAAAS2WT
GCATCTTCTTCAACAAACTCACCATGCGCCGCTTGCAAATTCCTCCGGCGAAAATGTCAAAS1
GCATCTTCTTCAACAAACTCACCATGCGCCGCTTGCAAATTCCTCCGGCGAAAATGTCAAAS2

************************************************************
CCGGAATGTGTATTCGCGCCCTATTTCCCACCGGACCAGCCACAAAAATTCGCAAACGTT
CCGGAATGTGTATTCGCGCCCTATTTCCCACCGGACCAGCCACAAAAATTCGCAAACGTT
CCGGAATGTGTATTCGCGCCCTATTTCCCACCGGACCAGCCACAAAAATTCGCAAACGTT

************************************************************
CACAAAGTGTTTGGAGCAAGTAACGTGACAAAGCTCCTCAACGAGCTTCACCCTTCACAA
CACAAAGTGTTTGGAGCAAGTAACGTGACAAAGCTCCTCAACGAGCTTCACCCTTCACAA
CACAAAGTGTTTGGAGCAAGCTCCTCAACGAGCTTCACCCTTCACAA
***********************************************
CGTGAAGACGCAGTGAACTCTTTGGCCTATGAAGCCGACATGCGCCTCCGTGACCCTGTC
CGTGAAGACGCAGTGAACTCTTTGGCCTATGAAGCCGACATGCGCCTCCGTGACCCTGTC
CGTGAAGACGCAGTGAACTCTTTGGCCTATGAAGCCGACATGCGCCTCCGTGACCCTGTC

************************************************************
TACGGCTGCGTCGGCGTCATCTCTCTCCTCCAACATCAGCTTCGTCAGCTTCAGATAGAT
TACGGCTGCGTCGGCGTCATCTCTCTCCTCCAACATCAGCTTCGTCAGCTTCAGATAGAT
TACGGCTGCGTCGGCGTCATCTCTCTCCTCCAACATCAGCTTCGTCAGCTTCAGATAGAT

************************************************************
CTCAGCTGTGCTAAATCTGAGCTCTCTAAGTACCAAAGCCTCGGTATCCTCGCCGCCACT
CTCAGCTGTGCTAAATCTGAGCTCTCTAAGTACCAAAGCCTCGGTATCCTCGCCGCCACT
CTCAGCTGTGCTAAATCTGAGCTCTCTAAGTACCAAAGCCTCGGTATCCTCGCCGCCACT

Here we see a 13
base pair deletion
of both pyrimidines
and purines

************************************************************
CATCAGAGTCTTGGCATCAACTTACTCGCCGGAGCAGCAGATGGAACAGCCACCGCCGTG
CATCAGAGTCTTGGCATCAACTTACTCGCCGGAGCAGCAGATGGAACAGCCACCGCCGTG
CATCAGAGTCTTGGCATCAACTTACTCGCCGGAGCAGCAGATGGAACAGCCACCGCCGTG

************************************************************
AGAGACCACTATCACCACCACCAGTTTTTTCCTAGAGAACAAATGTTTGGTGGCTTGGAT
AGAGACCACTATCACCACCACCAGTTTTTTCCTAGAGAACAAATGTTTGGTGGCTTGGAT
AGAGACCACTATCACCACCACCAGTTTTTTCCTAGAGAACAAATGTTTGGTGGCTTGGAT

************************************************************
GTTCCGGCCGGTAACAACTACGACGGTGGGATTCTTGCCATTGGACAGATCACTCAGTTT
GTTCCGGCCGGTAACAACTACGACGGTGGGATTCTTGCCATTGGACAGATCACTCAGTTT
GTTCCGGCCGGTAACAACTACGACGGTGGGATTCTTGCCATTGGACAGATCACTCAGTTT

************************************************************
CAGCAGCCGAGAGCCGCCGCTGGAGATGATGGTCGCCGTACTGTTGATCCGTCTTGAGAT
CAGCAGCCGAGAGCCGCCGCTGGAGATGATGGTCGCCGTACTGTTGATCCGTCTTGAGAT
CAGCAGCCGAGAGCCGCCGCTGGAGATGATGGTCGCCGTACTGTTGATCCGTCTTGAGAT

************************************************************
TTTAGGGTTTTGGTGG
TTTAGGGTTTTGGTGG
TTTAGGGTTTTGGTGG
****************
Figure 4: AS2 wild type and AS1,AS2 PCR product DNA sequence
alignment. Mutation is indicated by highlighted area

Protien Alignment Sequence AS1

Figure 5 below shows amino acid sequence alignments between the AS1 wild type
protein and mutant protein. Figure 6 below shows amino acid sequence alignment of AS2 wild
type and mutant proteins. . Mutations are indicated by dashes in place of base letter and are
highlighted.
MKERQRWSGEEDALLRAYVRQFGPREWHLVSERMNKPLNRDAKSCLERWKNYLKPGIKKGAS1MUT

MKERQRWSGEEDALLRAYVRQFGPREWHLVSERMNKPLNRDAKSCLERWKNYLKPGIKKGAS1WT

************************************************************
SLTEEEQRLVIRLQEKHGNKWKKIAAEVPGRTAKRLGKWWEVFKEKQQREEKESNKRVEP
SLTEEEQRLVIRLQEKHGNKWKKIAAEVPGRTAKRLGKWWEVFKEKQQREEKESNKRVEP

************************************************************
IDESKYDRILESFAEKLVKERSNVVPAAAAAATVVMANSNGGFLHSEQQVQPPNPVIPPW
IDESKYDRILESFAEKLVKERSNVVPAAAAAATVVMANSNGGFLHSEQQVQPPNPVIPPW

************************************************************
LATSNNGNNVVARPPSVTLTLSPSTVAAAAPQPPIPWLQQQQPERAENGPGDLC
LATSNNGNNVVARPPSVTLTLSPSTVAAAAPQPPIPWLQQQQPERAENGPGGLVLGSMMP

***************************************************.*

Here we see
AS1 mutant is
very different
from the wild
type due to a
mutation in the
DNA alignment

SCSGSSESVFLSELVECCRELEEGHRAWADHKKEAAWRLRRLELQLESEKTCRQREKMEE

IEAKMKALREEQKNAMEKIEGEYREQLVGLRRDAEAKDQKLADQWTSRHIRLTKFLEQQM

GCRLDRP
Figure 5: AS1 wild type and mutant amino acid sequence alignment.
Mutation is indicated by highlighted area

Protein Alignment Sequence AS2

MASSSTNSPCAACKFLRRKCQPECVFAPYFPPDQPQKFANVHKVFGASSSTSFTLHNVKTAS2MUT
MASSSTNSPCAACKFLRRKCQPECVFAPYFPPDQPQKFANVHKVFGASNVTKLLNELHPSAS2WT

************************************************.*.:.:
Q
Here we see a
QREDAVNSLAYEADMRLRDPVYGCVGVISLLQHQLRQLQIDLSCAKSELSKYQSLGILAA
drastic
*
difference
between AS2
mutant and AS2
wild type
because of
mutations in the


THQSLGINLLAGAADGTATAVRDHYHHHQFFPREQMFGGLDVPAGNNYDGGILAIGQITQ

FQQPRAAAGDDGRRTVDPS
Figure 6: AS2 wild type and mutant amino acid sequence alignment.
Mutation is indicated by highlighted area

AS1 3D Protein Structures

AS1 Superimposed

AS1 Mutant

AS1 Wild Type

Figure 8: AS1 3D protein structures are shown above to represent the

structure difference the mutation causes

AS2 3D Protein Structure

Figure 8: AS2 3D protein structures are shown above to represent the structure
AS2 Superimposed
AS2 Wild Type
difference
the mutation causes AS2 Mutant

Discussion
AS1 and AS2 cause phenotypic deformations in leaves which are due to inexpression of
genes. As seen in Figure 1, the leaves are visually significantly different from one another which
the data in Figure 2 supports. When looking at the protein structure of the AS1 and AS2 mutants
it is easy to see the mutations, and while one single base pair deletion doesnt seem significant in
the grand scheme of things when looking at alignment sequences The 3D structure is drastically
changed, causing the phenotypic differences that are plainly seen.
Using Arabidopsis thaliana as a genetic model is important on several different levels. It
is a great teaching and learning tool because of its well mapped genome, ease of mutating, high
production, and cost efficiency make them a great tool for genetic analysis. The information this
research provides about AS1 and AS2 is important. It indicates how seemingly minor changes
made at the molecular level such as a single base pair difference can result in a totally different
phenotype. More studies such as this can help us not only understand the genome of certain
organisms, but also alter them to further a species.

Works Cited

Chua, Y., Zgurski, J., Garcia, D., Hay, A., & Xu, L. (2002). AS1 The Transcrption Factor AS1
Arabidopsis Thaliana. Retrieved from Wikigenes:
https://www.wikigenes.org/e/gene/e/818340.html
Ikezaki, M., Kojima, M., Sakaibara, H., Kojima, S., Ueno, Y., Machida, C., & Machida, Y.
(2010). Genetic Networks regulated by AS1 and AS2 in leaf development in Arabidopsis
Thaliana. Plant J.
Iwakawa, H., Ueno, Y., Semiarti, E., Onouchi, H., Kojima, S., Tsukaya, H., . . . Ikezaki, M.
(2002). The Asymmetric Leaves 2 Gene of Arabidopsis Thaliana, Required for formation
of a symmetric flat leaf lamina. Public Med, 467-478.
Semiarti, E., Ueno, Y., Tsukaya, H., Iwakawa, H., Machida, C., & Machida, Y. (2001). The
Asymmetric Leaves 2 gene of Arabidopsis thaliana regulates formation of a symmetric
lamina, establishment of ventation and repression of meristem-related homeobox genes in
leaves. Development, 1771-1783.
The Arabidopsis Information Resource. (2014, July 14). Retrieved from Tair:
http://www.arabidopsis.org/
Tsukaya, H. (2013). Leaf Development. The Arabidopsis Book.
Xu, B., Li, Z., Wang, H., Ma, H., Dong, A., & Huang, H. (2008, February). Arabidopsis Genes
AS1, AS2, and JAG Negatively Regulate Boundary-Specifying Genes to Promote Sepal
and Petal Development. Retrieved from Plant Physiology:
http://www.plantphysiology.org/content/146/2/566.full