CELLULOSE, CELL WALLS, AND CELL WALL DEPOSITION AND THE COBRA FAMILY OF GLYCOSYLPHOSPHATIDYLINOSITOL-ANCHORED CELL WALL PROTEINS IN ARABIDOPSIS THALIANA
by Susie Hudson
A thesis submitted in partial fulfillment of the requirements for the degree of BACHELOR OF SCIENCE in FOREST SCIENCES
Faculty of Forestry The University of British Columbia (Vancouver)
FRST 498 APRIL 2014
FRST 498 Susan Hudson
Summary
An increasing global shift towards sustainable fuels has placed emphasis on the use of trees as potential bioenergy crops. For this to be economical, trees must be bred or designed to grow rapidly, efficiently, and to be easily processed into bioproducts. Cell walls are the components in trees that form the bulk of the raw materials that are converted to bioenergy products; therefore, understanding the genetic basis of the function, composition, and biosynthesis of cell walls is paramount for tree breeding for bioenergy and bioproduct production. An aspect of this strategy involves understanding the biosynthetic pathways that result in cell wall formation, including the involvement of several key proteins, such as those in the COBRA family.
This paper reviews the importance of the cell wall to plants, details their structure and synthesis, as well as the role of the COBRA family of proteins in this process. Previous studies have shown that in Arabidopsis thaliana COBRA is associated with primary cell wall synthesis. Mutants with loss-of-function alleles in COBRA (cob-1) exhibit conditional root expansion defects, while mutants with null alleles (cob-4) exhibit sterility and stunted growth, suggesting the COBRA gene is integral to normal plant development in Arabidopsis, and specifically in cell elongation. A homolog of COBRA in Arabidopsis, COBRA-LIKE 4, has also been identified as having function restricted to secondary cell wall formation.
To characterize the functional specialization and localization patterns of AtCOBL4, a promoter swapping experiment was proposed. This would involve the secondary wall-specific gene under regulation of the primary wall promoter, followed by an attempted recovery of the primary cell wall (COBRA) phenotypes in cob-1 and cob-4 mutants. Furthermore, a fluorescent protein fusion to AtCOBL4 was engineered to observe the protein localization under the regulation of the native promoter (PAtCOB) and visualization using laser microscopy. Localization of COBRA family orthologs such as PtCOBRA-3 (Populus trichocarpa) under the regulation of PAtCOB can also be observed using fluorescent protein fusion constructs. To perform these experiments, the following specific constructs were generated using a variety of molecular cloning techniques:
PAtCOB::GFP, PAtCOB::AtCOBL4, PAtCOB::AtCOBL4-YFP, and PAtCOB::PtCOB3-YFP. These constructs were introduced into Arabidopsis wild-type, cob-1 and cob-4 mutants, all in the Columbia-0 background, and putative transgenic seed was generated. Future work will include genotyping, phenotyping, and confocal laser microscopy of the transgenic plants generated.
Key Words: cellulose synthesis; cell wall deposition; COBRA; COBRA-LIKE 4, Arabidopsis thaliana; promoter swap
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Contents
Summary...... 1 Key Words ...... 1
Introduction ...... 3 The importance of cell walls ...... 3 The structure of cell walls ...... 3 Cellulose and the formation of cell walls ...... 4 The COBRA family in Arabidopsis thaliana ...... 6 Experimental objectives ...... 9
Methods ...... 9 Plant growth ...... 9 Molecular cloning of genetic constructs for use in promoter swapping ...... 11 Plant transformation: introduction of constructs to Arabidopsis ...... 17
Future work...... 18
References ...... 21
Appendix: Oligonucleotide primer sequences ...... 24
Index of Figures and Tables
Figure 1: Photos of AtCOBRA promoter amplification using polymerase chain reaction...... 11
Figure 2: Diagram showing constructs produced for AtCOB/AtCOBL4 promoter swap experiment. Restriction sites that were used in ligation cloning are displayed...... 14
Table 1: Outline of the Agrobacterium tumefaciens-mediated plant transformations performed, including the constructs introduced (all vectors contained a Hygromycin B resistance marker) and the genotype of the plants that were transformed...... 18
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Introduction
The importance of cell walls
Cell walls are among the most important structures found in plants, and the evolution of land plants was dependent on the emergence of these structures. Cell walls provide plants with rigidity, support, and defence; they prevent excessive water loss; and, they allow for cell-cell communication, allowing plants to function successfully as multicellular organisms (Cosgrove
2005; Popper et al. 2011). The presence of a cell wall allowed plants to develop at least 35 different cell types, which lead to differentiation of plant cells into organs, including leaves, roots, seeds, pollen, and vascular tissue; these organs facilitate the effective survival of plants
(Kenrick and Crane 1997; Cosgrove 2005). Ultimately, this resulted in significant diversification within the plant kingdom, allowing plants to occupy and dominate a wide variety of environmental niches (Popper et al. 2011). In addition, cell walls form the majority of the wood biomass that is ultimately converted to bioenergy, timber, and pulp products (Cosgrove 2005;
Andersson-Gunnerås et al. 2006). Therefore, our understanding of cell wall ultrastructure and chemistry, and its biosynthesis is crucial to develop more effective techniques for crop production.
The structure of cell walls
There are two types of cell walls in plants: primary cell walls and secondary cell walls. Primary cell walls are deposited as cells expand; secondary cell walls are deposited between the primary cell wall and the plasma membrane once cell expansion has completed (Cosgrove 2005; Taiz and
Zeiger 2010). Compared to primary cell walls, secondary cell walls are much more rigid and strong; as a result, secondary cell walls are generally formed only in cells that require additional
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support and defense, such as tracheids and fibers, while primary cell walls are found in all plant cells (Taiz and Zeiger 2010).
All cell walls are composed of a variety of biomolecules, including, hemicellulose, pectins, and cellulose, though the proportions of each of these molecules varies between primary and secondary walls, and also varies depending on the tissue in which the cell is found. In addition, the secondary wall contains the hydrophobic polyphenol composed of phenylpropanoid subunits called lignin (Taiz and Zeiger 2010). The dry-weight biomass of both primary and secondary cell walls consists of 25-50% cellulose, making it a major component of cell walls (Andersson-
Gunnerås et al. 2006; Taiz and Zeiger 2010). Thus, in order to understand the properties of cell walls, we must understand cellulose synthesis and the processes that lead to the incorporation of this polymer into the cell wall matrix.
Cellulose and the formation of cell walls
Cellulose is a biopolymer that consists entirely of D-glucose. Two molecules of glucose are joined in a β-1,4 linkage to form a subunit called cellobiose, which when repeated and elongated forms the cellulose elementary chains (Taiz and Zeiger 2010). The angle of the cellobiose linkages is such that multiple aligned glucan chains can form hydrogen bonds with each other, and when six glucan chains become hydrogen-bound in one bundle, this forms one fibril
(Somerville 2006; Endler and Persson 2011). Six fibril strands then hydrogen-bond together, forming a microfibril; thus, up to thirty-six glucan chains are required to form a single microfibril (Somerville 2006).
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Cellulose synthesis is carried out by a group of glycosyltransferase enzymes known as the cellulose synthases (CesAs) (Richmond and Somerville 2000; Taiz and Zeiger 2010). CesAs are believed to be highly important to plant development, evidenced by the severity of loss-of- function of various CesA mutants (Turner and Somerville 1997; Yin et al. 2009). Individual
CesA proteins are synthesized in the cell’s endoplasmic reticulum, and multiple CesA proteins aggregate into a structure known as a cellulose synthase complex (CSC); this CSC is then delivered to and embedded in the plasma membrane by the Golgi apparatus (Somerville 2006;
Joshi and Mansfield 2007; Mizrachi et al. 2012). It is believed that approximately 36 CesA subunits combine to form the rosette structure that is the CSC (Somerville 2006; Joshi and
Mansfield 2007). Thus, when a CSC is active, the ~36 glucan chains being formed by each CesA subunit of the CSC collectively form one cellulose microfibril. As these microfibrils form, they are incorporated into the cell wall matrix in an organized fashion, and additional matrix polysaccharides are added to further solidify the growing cell wall (Endler and Persson 2011).
This organized deposition of cellulose is due, in part, to the tracking of CSCs along cortical microtubules found in the cytoskeleton, and leads to anisotropic cell expansion (Wasteneys
2004; Wasteneys and Fujita 2006; Taiz and Zeiger 2010).
The CesA family has been studied extensively in bacteria, algae, and land plants, and 10 members of the gene family have been identified in Arabidopsis thaliana (Pear et al. 1996;
Fagard et al. 2000; Richmond and Somerville 2000; Taylor et al. 2003; Zhong et al. 2003;
Persson et al. 2005; Desprez et al. 2007; Yin et al. 2009; McDonnell 2010; Wightman and
Turner 2010; Morgan et al. 2013). The importance of CesA genes to normal plant growth and development has been demonstrated through study of Arabidopsis plants with mutations in various CesA genes. For example, Taylor et al. (1999) found that irregular xylem 3 mutants
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exhibited a collapsed xylem phenotype due to a severe reduction in cellulose, resulting in significantly reduced growth. Zhong et al. (2003) found that fragile fiber 5 mutants had reduced fiber cell wall thickness and decreased mechanical strength in the stem due to a decrease in cellulose. While there appears to be some level of redundancy between members of this gene family, it has been found that multiple AtCesA genes are required to produce a single CSC, and thus there is a biological requirement for the existence of more than one AtCesA gene (Cosgrove
2005; Somerville 2006). In addition, AtCesA genes appear to have relatively different levels of importance to cellulose production and normal plant growth; for example, in primary cell walls,
AtCesA1 and 3 are imperative, whereas others (such as AtCesA2, 6 and 9) appear to have more redundant function (Somerville 2006).
While CesAs are responsible for the formation of cellulose, the main component of cell walls, there are a number of additional proteins that interact in a coordinated fashion with CSCs that assist in the translocation and positioning of cellulose in the cell wall matrix (Somerville 2006;
Joshi and Mansfield 2007; Sampathkumar et al. 2013). Some examples in Arabidopsis include
KORRIGAN, MICROTUBULE ORGANIZATION 1, COBRA, and COBRA-LIKE 4 (Brown et al. 2005; Roudier et al. 2005; Maloney and Mansfield 2010; Fujita et al. 2011). These proteins have been identified as imperative to normal plant development, and when absent or non- functional, the cell wall properties were significantly altered and plant development stunted. This demonstrates that several other proteins are highly involved in cellulose synthesis and cell wall formation.
The COBRA family in Arabidopsis thaliana
The COBRA gene family has been shown to play a critical role in cellulose deposition in plant cell walls and in anisotropic cell expansion in Arabidopsis thaliana. COBRA (COB) was
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identified in Arabidopsis by examining mutant cobra (cob) plants that exhibited conditional abnormal root expansion; it was determined that when COBRA function was inhibited or absent, the direction of cell expansion in the root was mis-regulated and occurred radially rather than longitudinally (Benfey et al. 1993; Hauser et al. 1995; Schindelman et al. 2001). Based on the analysis of cDNA and amino acid sequences, it was determined that proteins in the COBRA family contain glycosylphosphatidylinositol (GPI) anchors (Schindelman et al. 2001). These proteins undergo a post-translational modification that results in the addition of the GPI-anchor at the C-terminus, putatively allowing for positioning of the protein at the cell membrane
(Schindelman et al. 2001; Orlean and Menon 2007).
Subsequent studies have isolated additional COBRA mutant alleles, such as cob-5 and cob-6, loss-of-function alleles that resulted in abnormal growth and increased stress response, and cob-
4, a null allele that resulted in stunted growth and sterility (Roudier et al. 2005; Ko et al. 2006).
Analysis of the cob-4 null allele suggested that COBRA appears to possess a significant role in the orientation of cellulose microfibrils and anisotropic expansion, specifically in root cells
(Roudier et al. 2005). Furthermore, COBRA was found to be highly co-expressed with primary wall genes AtCesA1, 3, and 6, suggesting a high level of involvement in the formation of cellulose in primary cell walls (Persson et al. 2005).
In Arabidopsis, there are 12 members in the COBRA family split into two subgroups, based on alignment of DNA and amino acid sequences. The first subgroup contains the original AtCOBRA
(COB), as well as AtCOBRA-LIKE (COBL) 1-6; the second subgroup contains AtCOBL7-11.
With the exception of AtCOBL6 and AtCOBL9, most family members have overlapping expression patterns, as well as similar intron/exon and proposed protein structure, suggesting some level of functional redundancy (Roudier et al. 2002). While this functional redundancy is
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believed to be responsible for the conditional occurrence of the root expansion phenotypes observed in COBRA mutants (Hauser et al. 1995; Schindelman et al. 2001; Roudier et al. 2002), the redundancy is poorly understood, and requires further focussed examination of both localization and specialization of the individual COBL genes in Arabidopsis.
Brown et al. (2005) examined the function of a member of the COBRA family in Arabidopsis,
AtCOBL4. Despite being evolutionarily close to and, at the protein level, structurally similar to the primary cell wall COB, COBL4 function was found to be restricted to secondary cell wall biosynthesis. BRITTLE CULM 1 (BC1), a homolog in rice (Oryza sativa) with >72% identity with AtCOBL4, was found to be more highly expressed in vascular tissue with thick secondary cell walls, and plants deficient in BC1 protein had a reduction in cellulose and mechanical strength (Qian et al. 2001; Li et al. 2003). Similarly, Arabidopsis mutants lacking COBL4 called irregular xylem 6 showed no visible difference in growth habit, but had severely weakened stems and a reduction in cellulose content (Brown et al. 2005). AtCOBL4 was also found to be highly co-regulated with the cellulose synthase genes most involved in secondary cell wall formation in
Arabidopsis, CesA4, 7, and 8 (Persson et al. 2005). This co-expression combined with the irregular xylem 6 phenotype observed in the absence of AtCOBL4 is suggestive of the functional specialization of AtCOBL4 to the deposition of secondary cell walls, and by extension, the structural properties of plants. In poplar (Populus spp.), PtCOBRA3 was identified as a possible ortholog of AtCOBL4, as it showed much higher expression in stem tissues than leaves, bark, roots, or shoot tips (Ye et al. 2009). The purpose of COBRA family proteins specialized for secondary cell wall biosynthesis is unclear, and further investigation of the exact function and regulation of these proteins is of interest.
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Experimental objectives
For my undergraduate thesis project, I intended to investigate the spatial and temporal regulation of the AtCOBRA-LIKE 4 (COBL4) protein to broaden our understanding of its functional specialization in Arabidopsis thaliana. To accomplish this, a promoter-swap experiment involving the AtCOB promoter and AtCOBL4 gene-coding sequence was designed. With this experiment, I planned to attempt recovery of cob-1 and cob-4 mutant phenotypes using the secondary cell wall-specific COBL4 under regulation of the primary cell wall-specific promoter, using the construct PAtCOB::AtCOBL4. In addition, fluorescent protein fusions (PAtCOB::AtCOBL4-
YFP and PAtCOB::PtCOB3-YFP) were designed to be used for observation of the spatial and temporal localization of secondary wall proteins under regulation of the primary wall promoter using confocal laser microscopy.
Methods
Plant growth
Direct-to-soil method, for Columbia-0 Wild Type (WT)
Arabidopsis thaliana seeds were vernalized by suspending them in a 0.01% agar solution and placed in the dark at 4oC for 48 hours. This suspension was spread directly on top of potting soil in 10×10×10cm pots and grown in a growth chamber at 21oC, exposed to 16h-light days, and bottom-watered approximately once every 3 days.
Plate selection method, for cob-1 and cob-4 mutants and T1 seed
Confirmed cob-1 and cob-4 mutant A. thaliana seeds were kindly provided by Dr. Miki Fujita.
These seeds required selective propagation. This was done under aseptic conditions, using half- strength Murashige and Skoog (MS) agar media and the necessary selective agent. The cob-1
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mutants were expected to express a conditional phenotype under high-sucrose (4.5% w/v) concentrations, and cob-4 mutants were expected to be glufosinate-ammonium (BASTA) resistant (Schindelman et al. 2001; Roudier et al. 2005). As cob-4 is a null allele, the homozygous mutants were sterile, so were propagated in heterozygous lines for transformation using the Agrobacterium-mediated floral dipping method.
First, seeds were sterilized:
1) Desired seeds were aliquoted into a 1.5mL microcentrifuge tube. 2) 1 mL of 70% ethanol was added to the tube, allowed to soak for 3-5 minutes to wash, and drained. 3) 1 mL of 10% chlorine bleach solution was added to the tube, allowed to soak for 10-15 minutes to sterilize, and drained. 4) 1 mL of sterile distilled water was added to rinse seeds, and drained. This was repeated 4 more times. 5) Seeds were suspended in approximately 1mL sterile 0.01% agar solution to allow seeds to be easily spread onto agar media.
These suspended sterilized seeds were then spread evenly onto half-strength MS media plates containing 0.1% (w/v) Phytagel, 0.6% (w/v) agar, and the desired selection agent, either 4.5% w/v sucrose, 25mg/L BASTA, or 25mg/L Hygromycin B (all reagents from Sigma-Aldrich). The plates were left open to allow the agar suspension solution to evaporate, adhering the seeds to the media. The plates were then sealed using plastic film and placed in the dark at 4oC for 2 days to vernalize, then transferred to a culture room with 16h-light days. After 14 days, positive seedlings were identified visually, transferred to potting soil, and grown in the growth chamber.
From this point forward, they were grown and transformed in the same method as described for starting plants from seed above.
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Molecular cloning of genetic constructs for use in promoter swapping
AtCOB reporter constructs
To test the functionality of the AtCOB promoter, two reporter constructs were generated for two promoter lengths. Two lengths were cloned to see if there was a difference in promoter function between the two sizes. For the ‘short’ promoter (PSAtCOB), a -1623 +86 fragment upstream of the
AtCOB start codon was amplified from genomic DNA (extracted from Columbia-0 Wild Type) using iProof High-Fidelity DNA Polymerase (BioRad). For the ‘long’ promoter (PLAtCOB), a -
2350 to +86 fragment upstream of the AtCOB start codon was amplified using the same method as for the short promoter. These fragments were amplified using the protocol provided by
BioRad for 50µL reactions, using primer pairs 5-CCTAGGGGTGCTTTGCTTGAAGGAGG-
3/5-CACCCTGCAGGAAGCTACCGAGGGCAATGA-3 and 5-
CCTAGGGGTGCTTTGCTTGAAGGAGG-3/5-
CACCCTGCAGGTGTGGTCATGAGGCGTTCA-3 respectively.
The PCR products were
run on a 0.8% agarose
gel (Figure 1), excised,
and purified, and then
subcloned into the
pENTR vector
(Invitrogen) using
Figure 1: Photos of AtCOBRA promoter amplification using polymerase directional TOPO chain reaction for A) PSAtCOB, the shorter promoter length with an expected size of 1709bp; B) PLAtCOB, the longer promoter length with an expected size of 2436bp. For both gels, the loading order was 1) amplified PCR product; 2) cloning with a PstI site H2O PCR control; 3) 1Kb DNA ladder (Invitrogen). PCR products were run on 0.8% agarose gel at 130V for 40 minutes, and imaged using ethidium bromide and ultraviolet light.
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at the 5-terminus and either a KpnI or AvrII site at the 3-terminus (Figure 2). Correct amplification was confirmed by aligning sequencing data against the expected sequence obtained from Phytozome (locus At5g60920) using APE (A Plasmid Editor) software.
Gateway cloning
To generate the entry vector, the promoter fragment was amplified off of the confirmed pENTR plasmid containing the promoter sequence using iProof High-Fidelity DNA Polymerase
(BioRad). The primers used were specially designed to amplify the sequence flanked with attB1/attB2 cloning sites using primer pairs
5-GGGGACAAGTTTGTACAAAAAAGCAGGCTGAAGCTACCGAGGGCAATGA-3/
5-GGGGACCACTTTGTACAAGAAAGCTGGGTGGTGCTTTGCTTGAAGGAGG-3
(PSAtCOB) and
5-GGGGACAAGTTTGTACAAAAAAGCAGGCTGTGTGGACATGAGGCGTTCA-3/
5-GGGGACCACTTTGTACAAGAAAGCTGGGTGGTGCTTTGCTTGAAGGAGG-3
(PLAtCOB). Once amplified, these PCR products were run on a 0.8% agarose gel to confirm the length, then excised and purified and subcloned into pDONR221-Zeo using BP Gateway
Clonase II (Invitrogen). Using an LR Gateway Clonase II kit (invitrogen), PAtCOB in pDONR221-
Zeo as an entry vector, and pMDC107 (provided by Grant McNair) as a destination vector, the
PSAtCOB::GFP and PLAtCOB::GFP constructs were generated (Figure 2). Correct amplification and orientation of the constructs was sequence-confirmed.
Promoter swap constructs: T4 ligation cloning
All of the constructs were generated using the same Restriction Enzyme (RE) digest and T4
Ligation cloning method. The destination vector was a modified version of pCAMBIA 1390,
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called pSM3, and was kindly provided by Grant McNair. This vector contained multiple restriction sites for use in cloning, as well as a 35S cauliflower mosaic virus promoter driving a
Hygromycin B resistance marker to aid in selection of transformed plants. One version of the vector contained the AtCOBL4 (locus At5g15630) gene sequence, and another contained the
PtCOB3 gene sequence. The remainder of the protocol was as follows:
1) RE digest of both the destination vector and the vector containing the desired insert fragments was completed using the following reagent mixture: 1 mg purified plasmid, 1 uL of each required FastDigest enzyme (Thermo Scientific), 5 µL FastDigest buffer
(Thermo Scientific), nanopure H2O to fill the reaction volume to 50 µL. This mixture was incubated at 37oC for 10 minutes shows the REs used for each construct. In addition,
when digesting PLAtCOB from pENTR, ApaL1 was used in addition to the bordering enzymes to further digest the vector backbone, otherwise both the insert and backbone fragments would have been the same length and been difficult to excise from a gel. 2) Isolation of fragments: The reaction mixtures were run on a 0.8% w/v agarose gels in 1M TAE buffer (40mM TRIS-HCl, 1mM EDTA, 20mM acetate) using an electrophoresis. The desired bands were visualized using ethidium bromide and ultraviolet light, and excised and collected in a 1.5mL microcentrifuge tube. 3) Gel purification of fragments: The excised gel bands were weighed in milligrams. The equivalent number in µL was considered one volume of gel. Three volumes of Buffer QG (Qiagen) was added to the tube, which was incubated at 55oC using a heat block to solubilize the gel fragments. One volume of 100% isopropanol was added to the mixture, which was inverted several times. This mixture was left to incubate at room temperature for 5-8 minutes. The mixture was then spun at 13,000 RPM for one minute through a binding column (Qiagen) in 700µL increments. 500µL of Buffer PE (Qiagen) was also spun through the binding column as a wash buffer and spun at 13,000 RPM for 30
seconds. This was repeated two more times. 25µL of nanopure H2O was added to the binding membrane to resuspend the product, and allowed to incubate at room temperature for 5 minutes. The binding column was then placed into a clean recovery tube and spun at 8,000 RPM for 2 minutes to collect the purified fragments. The final product was
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measured for concentration of DNA using a NanoDrop Lite Spectrophotometer (Thermo Scientific). 4) T4 ligation reaction: using the established (by spectrophotometer) concentrations of vector and insert fragments, a 5:1 molar ratio of insert:vector was calculated. These amounts were added to the following reaction mixture: 2 µL of T4 Ligase buffer (Thermo Scientific), x µL of gel purified product (insert - as calculated), x µL of gel purified
product (vector - as calculated), 1 µL of T4 Ligase (Thermo Scientific), Nanopure H2O to fill the total reaction volume to 20 µL. The reaction mixture was allowed to incubate at room temperature for 16-24 hours. 5) Transformation: 5µL of the reaction mixture was transformed into E. coli, screened, and sequence confirmed as described in the next section.
Figure 2: Diagram showing constructs produced for AtCOB/AtCOBL4 promoter swap experiment. Restriction sites that were used in ligation cloning are displayed.
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Transformation of cloned plasmids into E. coli competent cells
This protocol was the same for all types of cloning. Specifically, competent E. coli cells
(subcloning-efficiency strain DH5α; Invitrogen) were used, and the transformation protocol recommended by the manufacturer. The transformation reaction was plated onto Lysogeny Broth
(LB) media containing 1.5% w/v agar and 50 mg/L kanamycin or 25 mg/L Zeocin as a selective agent for the target plasmid. After 16-24h of incubation at 37oC, single colonies were cultured in isolation in 5 mL of liquid LB containing 50 mg/L kanamycin or 25 mg/L Zeocin to select positive resistant plasmids; the target plasmid was then isolated using the mini-prep plasmid purification protocol, as follows:
1) Culture was spun at 5,000 RPM to pellet the bacterial cells. 2) Supernatant was discarded, and the pellet was resuspended in 250 µL of resuspension solution (50mM Tris-HCl, 10mM EDTA, 0.1 mg/mL RNase A) by vortexing. This resuspension was transferred to a 1.5 mL microcentrifuge tube. 3) 250 µL of lysis buffer (200mM NaOH, 1% SDS) was added to the resuspension and mixed well, and left to incubate at room temperature for 5-8 minutes. 4) 350 µL of neutralization buffer (4.2M guanidine hydrochloride, 0.9M potassium acetate, pH to 4.8 with glacial acetic acid) was added to the mixture, the tube was inverted several times. The tube was spun at 13,000 RPM for 10 minutes. 5) 750 µL of supernatant was transferred to a binding column (Qiagen) and allowed to incubate at room temperature for 2 minutes. 6) The spin column was spun at 13,000 RPM for 1 minute. The flow-through was discarded. 7) 500 µL of wash buffer (60mM potassium acetate, 0.83% Tris-HCl, 40mM EDTA, 60% ethanol) was added to the column. The column was spun at 13,000 RPM for 1 minute, and the flow through was discarded. This wash step was repeated, and the column was spun at 13,000 RPM for 2 minutes to dry the column’s filter. The collection tube was discarded. 8) 50 µL of nanopure water was added carefully to the spin column’s surface. The column was left to incubate at room temperature for 2-5 minutes. 9) The column was spun at 8,000 RPM into a clean 1.5mL microcentrifuge recovery tube for 2 minutes. 10) The final product was measured using a NanoDrop Lite Spectrophotometer (Thermo Scientific) to determine the purity and concentration of the purified plasmid.
After confirming promoter constructs using sequencing, the purified plasmids were transformed into Agrobacterium tumefaciens for plant transformation.
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Transformation of cloned plasmids into Agrobacterium tumefaciens competent cells
Competent cells of Agrobacterium tumefaciens strain GV3101 (made in-lab) were thawed for 30 minutes on ice. 500ng of purified plasmid was added to the cells and mixed gently. The mixture was incubated on ice for 30-60 minutes, then heat shocked at 37oC in a water bath for 5 minutes.
950 µL of liquid Yeast Extract Peptone (YEP) broth was added and the mixture was incubated at
28oC for approximately 4 hours while shaking at 200 RPM. 100-200 µL of the reaction was plated on solid YEP media plates containing 1.5% w/v agar, 25mg/L rifampicin (to prevent growth of E. coli), and 50mg/L kanamycin (as a selective agent for the target plasmid). After 40-
72h of incubation at 28oC, single colonies were cultured in isolation in 5mL of liquid YEP containing 25mg/L rifampicin and 50mg/L kanamycin to select positive resistant plasmids. This culture was used to perform a Polymerase Chain Reaction (PCR) to confirm successful transformation of the desired construct plasmid, as plasmid isolation and sequencing of
Agrobacterium cultures is not possible. For this PCR, the following reagent mixture was used: 5
µL water-resuspended Agrobacterium culture, 2 µL 10× buffer, 0.5µL dNTP solution mixture,
25µM F primer, 25µM R primer, 0.2 µL Taq polymerase (primers from Integrated DNA technologies, sequences listed in Appendix; all other reagents from New England Biolabs). The following program was used in a PCR thermocycler:
1) 95oC for 2 minutes (initial denature) 2) 95oC for 45 seconds (denature) o 3) ‘Ta’ C for 30 seconds (anneal primers) 4) 72oC for ‘Y’ seconds (DNA extension) 5) Repeat steps 2) through 5) 30-35 times 6) 72oC for 3 minutes (final extension) 7) Hold at 4oC forever (chill products until needed)
‘Ta’ is the primer annealing temperature, determined from the melting temperature provided for each primer by the manufacturer.
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‘te’ is the extension time, which was approximated as 30 seconds per 500 base pairs (bp) expected in the amplified product. The PCR products were run on a 1% agarose gel and imaged using ethidium bromide and ultraviolet light to confirm the presence of each construct.
Glycerol stocks
After successful sequence confirmation (E. coli) or PCR confirmation (Agro) of constructs, a glycerol stock of the live culture was made for long-term storage. The positive colony used in cloning was streaked onto a fresh agar media plate, and a single colony was grown in a 5 mL liquid culture containing selective antibiotics. 1 mL of this liquid culture (LB for E. coli, YEP broth for Agro) was diluted with 250 µL sterile LB, and glycerol was added to obtain a final concentration of ~17% glycerol. This mixture was promptly flash frozen using liquid nitrogen and stored at -80oC.
Plant transformation: introduction of constructs to Arabidopsis
The Agrobacterium tumefaciens-mediated transformation of Arabidopsis plants was done using the floral dip method detailed by Zhang et al. (2006). The following modifications were made: the soil was not covered (step 2), the plants were grown only in long-day conditions (step 3),
YEP broth was used instead of LB to grow Agrobacterium cultures (steps 6-7), SYLGARD 309
(Dow Corning) was used in place of Silwet L-77 (step 9), and plant aerial parts were immersed horizontally in a petri dish rather than inverting into a beaker (step 10). Table 1 outlines which constructs were introduced into which plant genotypes. All constructs contained the Hygromycin
B resistance gene as a selective marker, so all transformants were screened on plate media using
Hygromycin B at a concentration of 25mg/L as a selective antibiotic using the same method as described earlier.
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Table 1 Outline of the Agrobacterium tumefaciens-mediated plant transformations performed, including the constructs introduced (all vectors contained a Hygromycin B resistance marker) and the genotype of the plants that were transformed. All plants used in this experiment are in the Columbia-0 background.
Construct Vector Genotype of plants transformed
PAtCOB::PtCOB3-YFP pSM3 Wild Type
PAtCOB::GFP pMDC107 Wild Type
PAtCOB::AtCOBL4 pSM3 cob-1; cob-4
PAtCOB::AtCOBL4-YFP pSM3 cob-1; cob-4
Future work
Due to technical difficulties during the molecular cloning stage, I was unable to perform phenotyping on my plants. I was ultimately successful in performing all of the molecular cloning and plant transformations as detailed in the methods, and I am currently waiting to collect T1 seeds to move forward with selective propagation, genotyping, confocal imaging, and phenotyping of successful transformants. For one transformation, PAtCOB:: PtCOB3-YFP, I was able to obtain T2 seed. I will be completing these final phenotyping observations as a student research associate in the Mansfield lab this summer (May – August, 2014).
Genotyping of transformed plants using Polymerase Chain Reaction
After selective propagation and seedling transfer to soil, PCR screening can be done on T1 plants to confirm successful insertion of the generated constructs. This can be done using the same
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reagent mixture as detailed previously for colony PCR. The primers to be used for this genotyping work are listed in the Appendix.
Seven putative positive T1 lines were identified using Hygromycin B selection and propagated in soil. T2 seeds were collected from these lines; these seeds can be genotyped using PCR and germinated for visualization using laser microscopy.
Confocal laser visualization
PAtCOB::GFP was introduced into Columbia-0 wild type as a reporter construct. T1 seeds were generated and harvested, and screened using the protocol described previously. Putative positive transformant seedlings can be selected based on presence of true leaves and lateral roots, and transferred to soil and grown under growth chamber conditions. These putative positive lines require genotyping by PCR (see Appendix), and once confirmed, T2 seeds can be collected from these plants and germinated for use in confocal laser microscopy experiments.
PAtCOB::PtCOB3-YFP was also introduced into Columbia-0 wild-type plants as a reporter construct. This was generated as an alternative to the GFP construct in the event that either plant transformation or visualization of that construct was unsuccessful.
PAtCOB::AtCOBL4-YFP was also transformed into cob-1 and cob-4 mutants to observe localization of AtCOBL4.
Phenotyping of transformed cob mutants to observe putative complementation
PAtCOB::AtCOBL4 was transformed into cob-1 and cob-4 lines. T1 seeds were generated, and can be screened for positive transformants and propagated. Once positive T1 lines are confirmed using selective propagation and PCR genotyping, T2 seeds can be generated. These T2 seeds for
19 FRST 498 Susan Hudson
PAtCOB::AtCOBL4 can be plated on MS media, and observing recovery of the condition root expansion (for cob-1) or sterility (for cob-4) phenotypes. Thus far, cob mutant phenotypes have not been recovered successfully by COBRA orthologs or homologs, so the results of this study could provide insights into the importance and role of COBRA family members in plant development.
20 FRST 498 Susan Hudson
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23 Appendix: Oligonucleotide primer sequences
The following table details the primer pairs that are to be used for genotyping of Agrobacterium transformed Arabidopsis plants using Polymerase Chain Reaction. The same primer pairs were also used for colony PCR to confirm the sequence from Agrobacterium liquid cultures, as described in the methods section. The expected amplified product size, annealing temperature (Ta), and extension time (te) used for amplification are also shown for each primer pair. Note that the primer pairs do not differ between the short and long promoter size as the regions amplified by these primer pairs will be found in both the short and long promoter fragments.
Product size, T , Construct Forward primer Reverse primer a and te
AtCOB_3F GFP 5R 302bp P ::GFP AtCOB 5- TCAAATCATCGTTCCCGGCT-3 5-ATCAGGGTAACGGGAGAAGC-3 52oC, 30s PtCOB3YFP rt-F PtCOB3YFP rt-R 99bp P ::PtCOB3-YFP AtCOB 5- CTGTGTCAAGAGTAACTCGAAGGAA-3 5-GGGCACATATGATGTGTGCA-3 54oC, 10s AtCOB_3F AtCOBL4 rt-R 992 bp P ::AtCOBL4 AtCOB 5- TCAAATCATCGTTCCCGGCT-3 5- GTGGACTCTAACAGGGCACATG-3 52oC, 60s AtCOBL4 rt-F YFP 5R 610 bp P ::AtCOBL4-YFP AtCOB 5- GAGCTGCGTCAAGGCTGAT-3 5’- CAGCTTGCCGTAGGTGGCAT-3 53oC, 45s