Download S3. Computational Molecular Modeling- AS1 AS2

S3-Background information
AS1 AND AS2 REPRESS KNOX GENE EXPRESSION
In plants, the shoot apical meristem (SAM) is 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
differentiated into leaf cells. In Arabidopsis thaliana, the presence of proteins encoded by a family of
genes known as the class I KN1-like Homeobox (KNOX) genes maintains an undifferentiated cell fate (1).
Cells are recruited from the SAM and differentiate into leaf tissue when the KNOX genes expression is
suppressed (1). While the mechanism that initially silences KNOX genes and initiates differentiation
remains unknown, the ASYMMETRIC LEAVES1 (AS1) and ASYMMETRIC LEAVES2 (AS2) proteins
are required to maintain the KNOX gene silencing in the developing leaves (2, 3). The AS1 and AS2
proteins are known to physically interact (4) and this physical interaction is required for the AS1/2
complex to bind KNOX gene promoters (5).
In wild type plants, AS1 is expressed in the cotyledons of the embryos, but not in the SAM (Byrne et al.
2000). This expression pattern is consistent with the role of AS1 maintaining repression of the KNOX
genes in the developing leaves. Phenotypically, mutations in the Arabidopsis AS1 gene cause leaves to be
wrinkled, curled under and have shortened petioles (figure 1C). This mutant phenotype is observed
because when the AS1 gene is mutated, KNOX gene expression is reactivated in the developing leaf tissue
(3). The AS1 gene encodes an R2-R3 myb domain transcription factor (3). In Arabidopsis, the R2-R3
Myb domain proteins are members of a large gene family. The R2-R3 Myb proteins are proteins that
typically bind DNA and function in a wide variety of cellular processes (6). Several experiments
demonstrate that complexes containing AS1 bind DNA (5, 7); however, experiments with ROUGH
SHEATH2 (RS2), the maize homologue of AS1, failed to demonstrate that RS2 directly binds DNA (8).
Therefore, it is possible that in AS1 containing complexes shown to bind DNA, AS1 is not the component
that contacts DNA.
In wild type plants, AS2 is expressed on the adaxial side of the cotyledons in the embryos but not in the
SAM (9). Mutations in the Arabidopsis AS2 gene cause plants to have wrinkled leaves with leaflet
structures that form on some leaves (figure 1b; (2)). As with as1 mutants, the as2 mutant phenotype is
due to the reactivation of KNOX gene expression in the developing leaf tissue (10). The AS2 gene is a
member of a large gene family and encodes a protein that containing both a leucine-zipper like and zinc
finger motif (11). Several members of this gene family, including AS2, have been shown to bind DNA
(11).
A)
B)
C)
F IGURE 1 P HENOTYPES OF WILD TYPE (A), AS2 MUTANT (B) AND AS 1 MUTANT (C) PLANTS.
P HOTOS WERE TAKEN BY UNDERGRADUATE STUDENTS ENROLLED IN THE B IOLOGY 231
(GENETICS) COURSE AT R ADFORD UNIVERSITY.
While it is clear from previous experiments that AS1 binds to AS2 and that the AS1-AS2 complex binds
DNA and maintains KNOX gene silencing during leaf development in Arabidopsis much still remains to
be discovered about AS1 and AS2. For example, none of the previous experiments reveal the 3-D
structure of AS1 or AS2 and therefore, the exact regions of the AS1 and AS2 that bind together and how
this complex binds DNA remains to be determined. In addition, other proteins that bind AS1 and/or AS2
have already been identified but it is likely that more have yet to be discovered. An understanding of the
3-D structure of all of these proteins and how these proteins physically interact with each other and with
DNA will help understand the exact mechanism of how this complex maintains repression of KNOX gene
expression during leaf development.
PROJECT OVERVIEW
The overall goal of this project is to understand how changes in DNA sequence change the 3-D shape of
protein which in turn, changes the protein’s function resulting in a mutant phenotype. In week one, as1
and as2 mutant plants are compared to wild type plants. The goal of the week 1 activity is to document
and understand the phenotypic differences between the mutant and wild type Arabidopsis plants. In week
two, DNA is isolated from wild type and mutant plants and the AS1 and AS2 genes are PCR amplified. In
weeks three and four, the DNA sequence of the wild type and mutant AS1 and AS2 genes are compared in
order to determine what DNA mutation lead to the as1 and as2 mutants. In addition, the wild type AS1
and AS2 protein sequences are retrieved from a database. Once the DNA sequence mutation has been
determined, the information is used to modify the wild type protein sequence into the mutant protein
sequence. Both the wild type and mutant protein sequences are then submitted to a publically available
service that will make a 3-D model protein model based on the amino acid sequence. The goal of week’s
2-4 activities is to understand how mutations in the DNA change the amino acid sequence of the encoded
protein. In week five, molecular modeling software is used to compare the structure of the wild type and
mutant AS1 and AS2 proteins. The goal of the week five activity is to understand how changes in amino
acid sequence cause changes in the 3-D protein structure. At the end of the five weeks a laboratory report
will be written. In the report, the data collected throughout the five week series of activities will be
presented and explained. The report should clearly articulate how DNA sequence influences protein
structure/function and influences phenotype.
References
1.
Hake S, Smith HMS, Holtan H, Magnani E, Mele G, Ramirez J. 2004. The role of knox genes
in plant development. Annu Rev Cell Dev Biol 20:125–151.
2.
Semiarti E, Ueno Y, Tsukaya H, Iwakawa H, Machida C, Machida Y. 2001. The
ASYMMETRIC LEAVES2 gene of Arabidopsis thaliana regulates formation of a symmetric
lamina, establishment of venation and repression of meristem-related homeobox genes in leaves.
Development 128:1771–1783.
3.
Byrne ME, Barley R, Curtis M, Arroyo JM, Dunham M, Hudson a, Martienssen R a. 2000.
Asymmetric leaves1 mediates leaf patterning and stem cell function in Arabidopsis. Nature
408:967–971.
4.
Xu L, Xu Y, Dong A, Sun Y, Pi L, Xu Y, Huang H. 2003. Novel as1 and as2 defects in leaf
adaxial-abaxial polarity reveal the requirement for ASYMMETRIC LEAVES1 and 2 and
ERECTA functions in specifying leaf adaxial identity. Development 130:4097–4107.
5.
Guo M, Thomas J, Collins G, Timmermans MCP. 2008. Direct repression of KNOX loci by the
ASYMMETRIC LEAVES1 complex of Arabidopsis. Plant Cell 20:48–58.
6.
Dubos C, Stracke R, Grotewold E, Weisshaar B, Martin C, Lepiniec L. 2010. MYB
transcription factors in Arabidopsis. Trends Plant Sci 15:573–581.
7.
Iwasaki M, Takahashi H, Iwakawa H, Nakagawa A, Ishikawa T, Tanaka H, Matsumura Y,
Pekker I, Eshed Y, Vial-Pradel S, Ito T, Watanabe Y, Ueno Y, Fukazawa H, Kojima S,
Machida Y, Machida C. 2013. Dual regulation of ETTIN (ARF3) gene expression by AS1-AS2,
which maintains the DNA methylation level, is involved in stabilization of leaf adaxial-abaxial
partitioning in Arabidopsis. Development 140:1958–69.
8.
Theodoris G, Inada N, Freeling M. 2003. Conservation and molecular dissection of ROUGH
SHEATH2 and ASYMMETRIC LEAVES1 function in leaf development. Proc Natl Acad Sci U S
A 100:6837–6842.
9.
Iwakawa H, Ueno Y, Semiarti E, Onouchi H, Kojima S, Tsukaya H, Hasebe M, Soma T,
Ikezaki M, Machida C, Machida Y. 2002. The ASYMMETRIC LEAVES2 gene of Arabidopsis
thaliana, required for formation of a symmetric flat leaf lamina, encodes a member of a novel
family of proteins characterized by cysteine repeats and a leucine zipper. Plant Cell Physiol
43:467–478.
10.
Ori N, Eshed Y, Chuck G, Bowman JL, Hake S. 2000. Mechanisms that control knox gene
expression in the Arabidopsis shoot. Development 127:5523–5532.
11.
Husbands A, Bell EM, Shuai B, Smith HMS, Springer PS. 2007. Lateral organ boundaries
defines a new family of DNA-binding transcription factors and can interact with specific bHLH
proteins. Nucleic Acids Res 35:6663–6671.