Kyle Wingfield

Bio 231

Regulation of Boundary-Specifying Genes to Promote Sepal and Petal

Development by Arabidopsis Wildtype and Mutant Genes AS1 and AS2
By Kyle Wingfield

Abstract
In this experiment, it was shown that the Arabidopsis thaliana asymmetric leaves 1 and 2 (as1 and
as2) genes function in the sepal and petal structure in order to repress boundary-specifying genes for
normal development of the organs. The methods used to obtain this data were DNA extraction that
provided DNA with purity suitable for a PCR based detection method. The abnormal sepals and petals
exhibited abnormal overexpression of the boundary-specifying genes Petal Loss (PTL) and cup shaped
cotyledons 1 and 2 (CUC1 and CUC2). Loss of PTL or CUC1 and CUC2 functions in the as1 jag back
round could partially rescue the tiny sepal and petal phenotypes, supporting the theory that the tiny
sepal/petal phenotypes are caused in part by abnormal expression of boundary-specifying genes. Together,
this data revealed a previously unrecognized regulation by which AS1, AS1 and JAG act to define sepal
and petal from their boundaries. In plants, the shoot apical meristem (SAM) is self a self-renewing group
of cells that differentiates into the above ground plant tissues. Several genes encode proteins required to
regulate the balance between maintaining the undifferentiated meristem cell population and recruiting
cells from the SAM that will differentiate into leaf tissue. In wildtype plants, AS1 is expressed in
cotyledons of the embryos and AS2 is expressed on the adaxial side of the cotyledons in the embryo but
not in the SAM. AS1 is a repressor that encodes an R2-R3 MYB domain transcription factor and AS2 is a
repressor that encodes a protein containing both leucine-zipper like and zinc finger motif. AS1 binds to
AS2 and AS2 binds to DNA and they repress the function of the KNOX gene. When AS1 and AS2 are
mutated they do not bind to the operator and turn it off therefore transcription occurs and the wrinkled
leaves phenotype is shown.

Introduction
This experiment describes the validation of a small-scale DNA extraction protocol to extract
genomic DNA from plant leaves for qualitative PCR based detection methods. The purpose of the DNA
extraction was to provide DNA with purity suitable for PCR based detection methods. Arabidopsis plants
are easily manipulated, genetically tractable and a lot is already known about them which makes them a
good model genetic system. Arabidopsis plants were used in this experiment to gain comprehensive
knowledge of plant life and because they offered the ability to test our hypothesis quickly and more
efficiently and there is also much knowledge that can be gained from using the model plant Arabidopsis.
Each protein is folded, causing the linear chain of amino acids forming the protein to attain a defined
three-dimensional structure. These folded protein structures can help to explain the function of the
corresponding gene because the structures are highly specific and related to each protein's functions.
As1 and As2 mutants have overall similar phenotypes with both abnormal leaves and floral
organs. AS1 encodes an R2-R3 MYB-domain protein whereas AS2 encodes a plant-specific lateral organ
boundary domain containing a transcription factor that associates with AS1 (1). In wildtype leaves, AS1
and AS2 are known to be required for repression of meristematic genes and for leaf adaxial-adaxial
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polarity establishment, whereas their roles in flower development are unknown. (Knox Gene) (2) JAG
encodes a C2H2 transcription factor and promotes morphogenesis in several types of lateral organs. It
appears that AS1/AS2 and JAG act in parallel to their boundaries. Developmental regulation of cellular
growth emerges as a crucial component in regulation of leaf form with TCP and CUC2 transcription
factors playing a key role in this process (3).
The CLAVATA2 (CLV2) gene regulates both meristem and organ development in Arabidopsis.
The CLV2 gene is isolated and found that it encodes a receptor-like protein (RLP), with a presumed
extracellular domain composed of leucine-rich repeats similar to those found in plant and animal
receptors. RLPs lacking cytoplasmic signaling domains have not been shown to regulate development in
plants. However, prior work has demonstrated that the CLV1 receptor-like kinase (RLK) is present as a
disulfide linked multimer. CLV1 is required for the normal accumulation of CLV1 protein and its
assembly into protein complexes, indicating that CVL2 may transduce extracellular tissues (4). Also, the
chromosomal region in which CLV2 is located contains an extremely high rate of polymorphism.
Arabidopsis was originally adopted as a model organism because of its usefulness for genetic
experiments. Import features including a short generation time, small size that limits the requirement for
growth facilities, and prolific seed production through self-pollination contribute to why they are a model
organism (5).
In Arabidopsis plants, class I KNOTTED1-like homeobox (KNOX) gene suppression and leaf
polarity establishment are two processes crucial for leaf morphogenesis.
The Arabidopsis genes, ASYMMETRIC LEAVES1 and 2(AS1 and AS2), are required for repressing the
class I KNOX genes and promoting leaf adaxial cell fates. In addition, the RNA-DEPENDENT RNA
POLYMERASE6 (RDR6) gene acts simultaneously with AS1 and AS2 to specify the adaxial polarity and
repress the KNOX genes in leaves. It is known that RDR6 is one of the key components in plant posttranscriptional gene silencing (PTGS), and is likely to function with other silencing components in a
genetic pathway in regulating leaf patterning. Here we report phenotypic analyses of double mutants
combining as1 or as2 with other mutations relating to different RNA silencing pathways.
The expression and functions of photoreceptors and key signaling molecules are highly
coordinated and regulated at multiple leves of the central dogma in molecular biology (6). Light activates
gene expression through actions of positive transcriptional regulators and relaxation of chromatin by
histone acetylation. Small rRNAs help reduce the expression of light-responsive genes. Alternative
splicing, protein phosphorylation/dephosphorization, the formation of diverse transcriptional complexes,
and selective protein degradation all contribute to proteasome diversity and can therefore change the
functions of individual proteins.

Methods and Materials

The principles of the DNA extraction consists of first releasing the DNA present in the tissue in
aqueous solution and then purifying the DNA from PCA inhibitors. The method started with a lysine step
followed by two precipitations. Afterwards, two other precipitations were performed. RNA was then
removed by digestion with RNase followed by precipitation again .The presence of inhibitors in genomic
DNA extracted from leaves was evaluated by PCR using as a template a dilution series of Arabidopsis
genomic DNA. A fragment of the Arabidopsis gene was chosen as an amplification target for the
Arabidopsis genome. Suitable primers (Table 1) were used for the PCR analysis. The Arabidopsis
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genomic DNA sample was reduced to a concentration of 30 ug/ul with TE buffer. The measurements
shown for the PCR reaction mixture totaled a volume of 25 ul.
To isolate the DNA from the wild type, AS1, and AS2 Arabidopsis plants, a tissue sample was
collected from each set of plants and placed in separate 1.5 mL micro centrifuge tubes. 400 uL of
extraction buffer was added to the tubes and the tissues were mashed using blue Pellet Pestles until they
had been completely broken down. These samples were then placed in a balanced centrifuge and allowed
to run for 10 minutes. Once this was complete the supernatant was pipetted off into a clean micro
centrifuge tube and mixed with 300 uL of isopropanol by inverting, this mixture was then placed in a
balanced centrifuge for 10 minutes. Once this was completed, the supernatant was discarded carefully in
order to not pour out the DNA pellet. 200 uL of 70% ethanol was then added to the micro centrifuge tube
containing the pellet and placed in the centrifuge for 5 minutes. The remaining ethanol was poured out
and the DNA pellet was allowed to air dry for 10 minutes. Once it had dried the cells were suspended
using 100 uL of water and a pipette, then spun for one minute in a balanced centrifuge. The micro
centrifuge tube was placed on ice. In another tube ingredients (in this order) 9.5 uL water, 12.5 uL 2X
PCR mix, 1 uL 5uM primer R, 1 uL 5uM primer L, and 1 uL of our DNA were added together, spun in the
centrifuge, labelled and placed on ice.
Table 1: Sequence of Primers for amplifying the Arabidopsis mutant leaves
Name
AS1 Forward Primer
AS1 Reverse Primer
AS2 Forward Primer
AS2 Reverse Primer

5- AAAGCGGTTAGGGAAGTGGT-3
5-AGTTTAGGAGACGGTTCAGG-3
5-GCATCTTCTTCAACAAACTCACC-3
5CACCAAAACCCTAAAATCTCA-3

Plant material and growth conditions of the Mutant alleles of as1 and as2
were obtained. DNA extraction and manipulation were carried out using standard
protocols. To sequence EMS-induced mutations, DNA from mutant plants was
amplified with primer pairs containing the exon region KNAT1. PCR products were
sequenced with internal primers, using dye terminator cycle sequencing. Following
treatment with DNAse, complementary DNA was synthesized using the forward and
reverse primers given (Table 1 above). RT-PCR reactions were performed with these
gene-specific primers. KNAT2 primers span the exon3/exon 4 junction.

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Results

Figure 1: Shows the Wild type plant phenotype. The white scale bar is set to one
centimeter. This phenotype is characterized by long petioles, which are the stalks
leading from the leaf to the base of the plant. The plants appear very large and
spread out compared to the mutated plants.

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Figure 2: Shows the AS1 mutant plant. The white scale bar is set to one centimeter. This
phenotype is characterized by very short petioles, which give the plant its very small
compacted look. These plants are the smallest of those being tested.

Figure 3: Shows the AS2 mutant plant phenotype. The white scale bar is set to one centimeter. This
phenotype has long petioles similar to the Wild phenotype. The leaves are significantly smaller than
the Wild phenotype though, which cause it to have a smaller less spread out appearance.

Average length n mm of Arabidopsis leaves

wt leaf 1 (mm)

as1 Leaf1

as2 leaf 1

10
0

Length in mm

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Figure 4: Bar chart containing class averages (in mm) of petiole lengths of the
leaves of
as1, as2 and the wild type of Arabidopsis plants.
Sequence Alignment of AS1 PCR product amplified DNA results isolated from wild type, as1 and
as2 plants. The mutation is the highlighted G at 512, with a one base pair deletion.
AS1_AS1_plant
AS1_AS2_plant
AS1_wild

AAAGCGGTTAGGGAAGTGGTGGGAAGTGTTTAAGGAGAAGCAACAGAGAGAAGAGAAAGA
AAAGCGGTTAGGGAAGTGGTGGGAAGTGTTTAAGGAGAAGCAACAGAGAGAAGAGAAAGA
------------------------------------------------------------

AS1_AS1_plant
AS1_AS2_plant
AS1_wild

GAGTAACAAGAGAGTTGAGCCTATTGACGAGAGTAAGTACGATCGGATTCTCGAGAGTTT
GAGTAACAAGAGAGTTGAGCCTATTGACGAGAGTAAGTACGATCGGATTCTCGAGAGTTT
------------------------------------------------------------

AS1_AS1_plant
AS1_AS2_plant
AS1_wild

CGCTGAGAAGCTTGTCAAAGAGCGGTCTAACGTTGTCCCTGCTGCTGCCGCTGCTGCAAC
CGCTGAGAAGCTTGTCAAAGAGCGGTCTAACGTTGTCCCTGCTGCTGCCGCTGCTGCAAC
------------------------------------------------------------

AS1_AS1_plant
AS1_AS2_plant
AS1_wild

GGTTGTGATGGCTAATTCGAATGGAGGGTTTTTACATTCTGAACAACAAGTTCAGCCTCC
GGTTGTGATGGCTAATTCGAATGGAGGGTTTTTACATTCTGAACAACAAGTTCAGCCTCC
------------------------------------------------------------

AS1_AS1_plant
AS1_AS2_plant
AS1_wild

TAACCCAGTGATCCCGCCTTGGTTAGCTACTTCTAACAATGGGAACAATGTTGTTGCAAG
TAACCCAGTGATCCCGCCTTGGTTAGCTACTTCTAACAATGGGAACAATGTTGTTGCAAG
------------------------------------------------------------

AS1_AS1_plant
AS1_AS2_plant
AS1_wild

GCCTCCCTCGGTAACTTTGACATTATCGCCTTCCACAGTGGCTGCAGCTGCGCCTCAACC
GCCTCCCTCGGTAACTTTGACATTATCGCCTTCCACAGTGGCTGCAGCTGCGCCTCAACC
------------------------------------------------------------

AS1_AS1_plant
AS1_AS2_plant
AS1_wild

GCCAATCCCGTGGCTGCAGCAGCAACAGCCTGAGAGAGCAGAGAACGGTCCAGGGG-ACT
GCCAATCCCGTGGCTGCAGCAGCAACAGCCTGAGAGAGCAGAGAACGGTCCAGGGGGACT
------------------------------------------------------------

AS1_AS1_plant
AS1_AS2_plant
AS1_wild

TGTGTTAGGGAGTATGATGCCGTCTTGTAGTGGGAGTAGCGAGAGTGTGTTCTTGTCAGA
TGTGTTAGGGAGTATGATGCCGTCTTGTAGTGGGAGTAGCGAGAGTGTGTTCTTGTCAGA
------------------------------------------------------------

AS1_AS1_plant
AS1_AS2_plant
AS1_wild

GCTTGTGGAGTGTTGTAGAGAGTTGGAGGAAGGGCACCGAGCTTGGGCAGACCATAAGAA
GCTTGTGGAGTGTTGTAGAGAGTTGGAGGAAGGGCACCGAGCTTGGGCAGACCATAAGAA
----------TGTTGTAGAGAGTTGGAGGAAGGGCACCGAGCTTGGGCAGACCATAAGAA
**************************************************

AS1_AS1_plant
AS1_AS2_plant
AS1_wild

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

AS1_AS1_plant
AS1_AS2_plant
AS1_wild

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

AS1_AS1_plant

GAACGCAATGGAGAAGATCGAAGGAGAGTACAGAGAACAGCTCGTTGGTTTGAGGCGAGA

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AS1_AS2_plant
AS1_wild

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

AS1_AS1_plant
AS1_AS2_plant
AS1_wild

CGCAGAGGCCAAAGACCAGAAACTGGCTGATCAATGGACCTCTAGGCATATCAGACTCAC
CGCAGAGGCCAAAGACCAGAAACTGGCTGATCAATGGACCTCTAGGCATATCAGACTCAC
CGCAGAGGCCAAAGACCAGAAACTGGCTGATCAATGGACCTCTAGGCATATCAGACTCAC
************************************************************

AS1_AS1_plant
AS1_AS2_plant
AS1_wild

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

AS1_AS1_plant
AS1_AS2_plant
AS1_wild

T
T
T
*

Sequence Alignment of AS2 PCR product amplified DNA results isolated from wild type, as1 and
as2 plants. The mutation highlighted is a thirteen base pair mutation.
AS2_PCR_as1
AS2_PCR_AS2
AS2_PCR_WT

GCATCTTCTTCAACAAACTCACCATGCGCCGCTTGCAAATTCCTCCGGCGAAAATGTCAA
GCATCTTCTTCAACAAACTCACCATGCGCCGCTTGCAAATTCCTCCGGCGAAAATGTCAA
GCATCTTCTTCAACAAACTCACCATGCGCCGCTTGCAAATTCCTCCGGCGAAAATGTCAA
***********************************************************

AS2_PCR_as1
AS2_PCR_AS2
AS2_PCR_WT

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

AS2_PCR_as1
AS2_PCR_AS2
AS2_PCR_WT

CACAAAGTGTTTGGAGCAAG-------------CTCCTCAACGAGCTTCACCCTTCACAA
CACAAAGTGTTTGGAGCAAG-------------CTCCTCAACGAGCTTCACCCTTCACAA
CACAAAGTGTTTGGAGCAAGTAACGTGACAAAGCTCCTCAACGAGCTTCACCCTTCACAA
********************
***************************

AS2_PCR_as1
AS2_PCR_AS2
AS2_PCR_WT

CGTGAAGACGCAGTGAACTCTTTGGCCTATGAAGCCGACATGCGCCTCCGTGACCCTGTC
CGTGAAGACGCAGTGAACTCTTTGGCCTATGAAGCCGACATGCGCCTCCGTGACCCTGTC
CGTGAAGACGCAGTGAACTCTTTGGCCTATGAAGCCGACATGCGCCTCCGTGACCCTGTC
************************************************************

AS2_PCR_as1
AS2_PCR_AS2
AS2_PCR_WT

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

AS2_PCR_as1
AS2_PCR_AS2
AS2_PCR_WT

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

AS2_PCR_as1
AS2_PCR_AS2
AS2_PCR_WT

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

AS2_PCR_as1
AS2_PCR_AS2
AS2_PCR_WT

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

AS2_PCR_as1
AS2_PCR_AS2
AS2_PCR_WT

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

AS2_PCR_as1
AS2_PCR_AS2

CAGCAGCCGAGAGCCGCCGCTGGAGATGATGGTCGCCGTACTGTTGATCCGTCTTGAGAT
CAGCAGCCGAGAGCCGCCGCTGGAGATGATGGTCGCCGTACTGTTGATCCGTCTTGAGAT

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AS2_PCR_WT

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

AS2_PCR_as1
AS2_PCR_AS2
AS2_PCR_WT

TTTAGGGTTTTGGTGG
TTTAGGGTTTTGGTGG
TTTAGGGTTTTGGTGG
****************

Protein sequence alignment comparing wild type and Mutant AS1

as1
as1mutant

MKERQRWSGEEDALLRAYVRQFGPREWHLVSERMNKPLNRDAKSCLERWKNYLKPGIKKG
MKERQRWSGEEDALLRAYVRQFGPREWHLVSERMNKPLNRDAKSCLERWKNYLKPGIKKG
************************************************************

as1
as1mutant

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

as1
as1mutant

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

as1
as1mutant

LATSNNGNNVVARPPSVTLTLSPSTVAAAAPQPPIPWLQQQQPERAENGPGGLVLGSMMP
LATSNNGNNVVARPPSVTLTLSPSTVAAAAPQPPIPWLQQQQPERAENGPGDLC-----***************************************************.*

as1
as1mutant

SCSGSSESVFLSELVECCRELEEGHRAWADHKKEAAWRLRRLELQLESEKTCRQREKMEE
------------------------------------------------------------

as1
as1mutant

IEAKMKALREEQKNAMEKIEGEYREQLVGLRRDAEAKDQKLADQWTSRHIRLTKFLEQQM
------------------------------------------------------------

as1
as1mutant

GCRLDRP
-------

Protein sequence alignment comparing Wildtype and AS2

as2mutant
as2wild

MASSSTNSPCAACKFLRRKCQPECVFAPYFPPDQPQKFANVHKVFGASSSTSFTLHNVKT
MASSSTNSPCAACKFLRRKCQPECVFAPYFPPDQPQKFANVHKVFGASNVTKLLNELHPS
************************************************. *.: .
:

as2mutant
as2wild

Q----------------------------------------------------------QREDAVNSLAYEADMRLRDPVYGCVGVISLLQHQLRQLQIDLSCAKSELSKYQSLGILAA
*

as2mutant
as2wild

-----------------------------------------------------------THQSLGINLLAGAADGTATAVRDHYHHHQFFPREQMFGGLDVPAGNNYDGGILAIGQITQ

as2mutant
as2wild

------------------FQQPRAAAGDDGRRTVDPS

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Figure 5: The protein structure of the normal AS1 wildtype protein showing the amino acids K112, P223
and M1.

Figure 6: The protein structure of the mutant as1 protein

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Figure 7: 3D superimposed image of the protein structure of normal (wildtype) AS1 and the mutant AS1
Proteins.

Figure 8: Superimposed image of the normal (wildtype) AS2 and the superimposed wild type and Mutant
AS2

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Figure 9: 3D Image of the Normal (wildtype) version of the AS2 protein with amino acids M1, E72, R96
and Q149 highlighted.

Figure 10: 3D Image of the Mutant version of the AS2 protein with amino acids M1, E72, R96 and Q149
Highlighted.

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`

Image 1: Fragmentation of the DNA aragose gel electrophorese of genomic DNA

samples. Lane 1: control. Lanes 2-3 showed results for the PCR around 800 Base
pairs while lane 4 did not yield any results.

Figures (5-10) represent the process of the analysis of the 3d protein structures of the Wildtype,
AS1 and AS2 proteins. Certain amino acids were highlighted in each one (figures 6 and 9). The
amino acids were analyzed to view the salt bridges from one amino acid to another in the
mutated sequences. It was found that there was capability of a salt bridge to form in the AS2
protein, the bonds were too far apart in the AS1 protein to have a bond to bond interaction and
the most dissimilarity was found in the c-terminal. By changing the amino acid sequence on the
3D images, the locations of the proteins in each sequence in the protein structure could be
changed in the way the bonds interact, changing the length of the structure and the appearance. It
could also change the folding of the proteins, the interaction of the bonds, the side chain
structures and the overall stability of the structure.
As1, As2 and wildtype Arabidopsis plants are displayed (Figures 1-3) to
show the difference in leaf phenotypes that is caused by the mutations in the
genes. Figure 1 represents the wildtype plant that shows the longer and
thinner leaves. The as1 mutant (figure 2) shows shorter leaves and smaller
plants while the as2 mutant (figure 3) shows longer leaves than the as1
mutant but shorter than the wildtype and smaller plants than the wildtype
plants.
The petiole lengths were measured by separate groups in the class,
and the data was collected as a whole with the average taken (Figure 4) to
represent the different petiole lengths of as1, as2 and the wild type. From the
data, it is easy to tell that the wildtype plant had the longest leaves with the
as1 having the shortest ones and as2 being somewhere in between.
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(Figures 1-3) The sequence alignments of the PCR product amplified DNA
results isolated from the wildtype, as1 and as2plants and display where the
mutations occurred in each sequence.
The protein sequence alignments display where the wildtype genes and the
mutant genes of AS1 and AS2 differ in the protein sequences.

The Data reported confirmed that the extraction method, applied to samples
of leaves, produces DNA that can be suitable for the quantity and quality for
subsequent PCR-methods. The size of the extracted DNA was evaluated by agarose
gel electrophorese (image 1); 10 ul of each solution was analyzed on an agarose
gel. Lane four did not yield any results and showed no bands. Causes for this can be
anywhere from forgetting an ingredient in the reaction mix to absence of the target
sequence in the template DNA.

Discussion
The goals of this lab were to explore the AS1 and the AS2 genes of Arabidopsis. The
overall goal of this experiment was to understand how changes in the DNA level can
result in the mutant phenotype in the leaves of the plants. The analysis of the
phenotype of as1 and as2 Arabidopsis mutants was observed by isolating DNA from
wildtype, as2 and/or as1 mutants, and PCR amplify the AS1 and AS2 genes. Table 1
shows the primers and the DNA fragments were estimated for the sizes of the PCR
products. The actual products (shown on image 1) were homozygous and around
800 Base pairs which was within close proximity of the estimates. The AS1 and AS2
products were sequenced and analyzed to use in determining how the DNA
sequence of the AS1 and AS2 gene varies in wildtype, as1 and as2 plants.

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Works Cited
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Tom Beeckman, Pascal Genschick, Martin Kuiper, Dirk Inze and Lieven De Veylder. 2005. The
Cyclin-Dependent Kinase Inhibitor KRP2 Controls the Onset of the Endoreduplication Cycle
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Weig, and Per Kjellbom. 2001. The Complete Set of Genes Encoding Major Intrinsic Proteins in
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5. Maarten Koorneef and David Meinke. 2009. The development of Arabidopsis
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