Role of PHABULOSA in Arabidopsis root
vascular differentiation
Un-Sa Lee
Degree project in biology, Master of science (2 years), 2012
Examensarbete i biologi 45 hp till masterexamen, 2012
Biology Education Centre and Department of Organismal Biology, Uppsala University
Supervisor: Annelie Carlsbecker
External opponent: Ana Elisa Valdés
Abstract
The root of Arabidopsis has been widely used for studying tissue patterning in plants by virtue of its
relatively simple structure and compatibility in genetic analysis. This study particularly focuses on the
vascular developmental aspects in the Arabidopsis roots in connection to PHABULOSA (PHB), a
member of the class III transcription factors of the Homeodomain-Leucine Zipper family (HD-Zip III).
Utilizing a β-estradiol inducible XVE system, we show that formation of metaxylem is mediated by PHB
in a dosage-dependent manner in the vascular system, which suggests the direct involvement of PHB in
the regulatory mechanism of cell type patterning, and analyses of loss-of-function ARGONAUTE (AGO)
mutants reveal involvement of AGO1 and AGO10 in the same process, wherein AGO proteins are known
to play crucial roles in microRNA regulation. Performed global transcriptome analyses could not identify
potential downstream targets of HD-Zip III transcription factors due to inconsistency of data sets, and
comparison with a previously published microarray data proves treatment of β-estradiol is not the source
of observed discrepancy. In addition, we suggest that phenotype of phb-7d, a gain-of-function of PHB
mutant, could be mediated through increased level of cytokinin signaling, and it is possible that
ARABIDOPSIS HISTIDINE PHOSPHOTRANSFER PROTEIN 6 (AHP6)-independent pathways in
protoxylem formation exist.
Keywords: Arabidopsis thaliana, ARGONAUTE, Auxin, Class III HD-Zip, Cytokinin, Differentiation,
Metaxylem, Microarray, MicroRNA, Pattern formation, Protoxylem, Root, Vasculature, XVE system
*About cover image: (Top) Light microscope images of the root of 4-day-old pCRE1(WOL)::XVE>>phb-1d in wild type
background seedlings with and without 10 µM β-estradiol treatment. Without induction, protoxylem forms next to
metaxylem that is normal to wild type seedlings, whereas inducing phb-1d alters the pattern of xylem cell type acquisition
exhibiting differentiation of metaxylem in the place of protoxylem. (Bottom) Confocal microscope images of the root of 4day-old pCRE1(WOL)::XVE>>phb-1d in wol mutant background seedlings with and without 10 µM β-estradiol treatment. wol
mutant seedlings exhibit an all protoxylem phenotype due to compromised cytokinin signaling, but inducing phb-1d in this
background results in a metaxylem-only phenotype wherein metaxylem forms in the place of protoxylem. Filled arrowhead
indicates metaxylem, and unfilled indicates protoxylem.
2
Contents
I. Introduction ......................................................................................................................................... 5
I.1. Arabidopsis thaliana, the model organism ..................................................................................... 5
I.2. Root vascular development in Arabidopsis ..................................................................................... 6
I.3. Transcription factors of the Class III HD-Zip family........................................................................ 10
I.4. Role of HD-Zip III transcription factors in Arabidopsis ................................................................... 12
I.5. Regulation of HD-Zip III transcription factors by plant hormones.................................................. 15
I.6. Arabidopsis ARGONAUTE1 and ARGONAUTE10............................................................................ 16
I.7. Aim of this study .......................................................................................................................... 18
II. Materials and Methods...................................................................................................................... 19
II.1. Plant materials and growth condition ......................................................................................... 19
II.2. Transgenic lines and the β-estradiol inducible XVE system .......................................................... 19
II.3. β-estradiol induction and collection of seedlings ......................................................................... 20
II.4. Histological techniques and analysis of xylem phenotype............................................................ 21
II.5. Microarray data analyses ............................................................................................................ 21
II.6. other molecular techniques - GUS staining and GFP analysis ....................................................... 22
III. Results .............................................................................................................................................. 23
III.1. Verifying the role of PHB in root xylem cell type acquisition by utilizing the β-estradiol inducible
XVE system ........................................................................................................................................ 23
III.2. Global transcriptome analysis of the HD-Zip III transcription factor PHB with additional
comparison to other microarray data sets ......................................................................................... 36
III.3. Vascular patterning and cell division problem in phb-7d in relation to plant hormones auxin and
cytokinin............................................................................................................................................ 39
III.4. Loss-of-function mutant analyses of Arabidopsis AGO proteins with focus on root vascular cell
type patterning.................................................................................................................................. 44
IV. Discussion ........................................................................................................................................ 46
V. Conclusion......................................................................................................................................... 52
VI. Acknowledgements .......................................................................................................................... 53
VII. References ...................................................................................................................................... 54
3
Table of Figures
Figure 1 Photo images of Arabidopsis. ..................................................................................................... 5
Figure 2 Root vascular structure of Arabidopsis. ...................................................................................... 6
Figure 3 Schematic drawing of the bidirectional cell-to-cell communication for xylem cell patterning
(from Furuta et al., 2012, with permission from Elsevier)......................................................................... 8
Figure 4 Biochemical structure of homeodomain (HD) and leucine zipper (Zip) motifs with distinctive
domains exhibited by the HD-Zip subfamilies of Arabidopsis. ................................................................ 11
Figure 5 Phylogenetic classification of Arabidopsis AGO proteins in three clades (from Vaucheret, 2008,
with permission from Elsevier). ............................................................................................................. 17
Figure 6 A schematic diagram of the XVE vector (from Zuo et al., 2000, with permission from John Wiley
and Sons). ............................................................................................................................................. 20
Figure 7 Xylem phenotype analysis results of 4-day-old β-estradiol inducible transgenic line
pCRE1(WOL)::XVE>>phb-1d in wol mutant background upon 48-hour and constitutive induction. ......... 25
Figure 8 Longitudinal image of the entire root of a PHB-induced transgenic line in wol mutant
background. .......................................................................................................................................... 26
Figure 9 Xylem phenotype analysis results of the β-estradiol inducible transgenic lines
pCRE1(WOL)::XVE>>phb-1d and pCRE1(WOL)::XVE>>MIR165A in wild type background upon various
induction times. .................................................................................................................................... 29
Figure 10 Light and confocal microscope images of 4-day-old β-estradiol inducible transgenic lines
showing various xylem phenotypes upon inductions. ............................................................................ 30
Figure 11 Relative expression of phb-1d in the inducible transgenic line pCRE1(WOL)::XVE>>phb-1d in
wild type background upon induction with β-estradiol. ......................................................................... 32
Figure 12 Xylem phenotype analysis results of 4-day-old β-estradiol inducible transgenic lines
pCRE1(WOL)::XVE>>phb-1d and pCRE1(WOL)::XVE>>MIR165A in wild type background upon 48-hour
and constitutive induction. .................................................................................................................... 34
Figure 13 Analysis of root xylem phenotype in Arabidopsis wild-type Col-0 and phb-7d mutant seedlings
upon treatment of plant hormones or inhibitor of hormone. ................................................................. 40
Figure 14 Analysis results of transgenic lines containing CYCB1:GUS construct in wild type Col-0 and phb7d mutant backgrounds upon treatment of plant hormones or inhibitor of hormone. ........................... 41
Figure 15 Xylem phenotype analysis results of 4-day-old and 6-day-old loss-of-function ago mutants. .. 45
4
I. Introduction
I.1. Arabidopsis thaliana, the model organism
Over the past few decades, enormous achievement has been made in understanding the fundamental
molecular mechanism underlying plant development, especially thanks to Arabidopsis thaliana, the most
popular model organism in the research field of plant biology (Figure 1). Arabidopsis, a common thale
cress native to Europe, Asia, and northwestern Africa, has a rapid life cycle and one of the smallest
genomes among land plants making it useful for genomic studies. Arabidopsis can complete its entire life
cycle
within
six
weeks,
and
has
five
chromosomes with a genome size of 157 Mbps
(Bennett et al., 2003), whereas size of the
human haploid genome, for example, reaches
over three billion base pairs (International
Human
Genome
Sequencing
Consortium,
2001). It is strikingly interesting that the
number of genes in Arabidopsis is estimated to
be around 27,000 in comparison to the fact that
around 23,000 protein-coding genes are found
in the human genome (International Human
Genome Sequencing Consortium, 2001).
Since the whole sequencing was completed in
the year of 2000 by the Arabidopsis Genome
Initiative (The Arabidopsis Genome Initiative,
2000), honoring Arabidopsis to become the
first plant species to be ever sequenced, a new
era in plant biology has begun with a focus on
the
fundamental
genetic
and
Figure 1 Photo images of Arabidopsis.
Shown on the left are vegetative stages before flowering and
growth of the inflorescence, and at center is an adult plant at
full flowering and seed set. Flower, apical part of
inflorescence and seeds are shown on the right. White bars
represent 1 cm, except for flower and seeds: 1 mm.
Image courtesy of Institut Jean-Pierre Bourgin (http://wwwijpb.versailles.inra.fr/en/arabido/arabido.htm).
molecular
mechanisms of plant development, promising a bright future in connection to not only the evolutionary
aspects but also to the potential application in agricultural studies.
5
I.2. Root vascular development in Arabidopsis
This study particularly focuses on the vascular developmental aspects in Arabidopsis roots. The
Arabidopsis root has been widely used for studying tissue patterning in plants by virtue of its relatively
simple structure and compatibility in genetic analysis (Figure 2A), wherein pioneering studies in this field
date up to the 90s (Benfey et al., 1993; van den Berg et al., 1995). With the root stele in the center, this
stele is surrounded by single layers of different tissues that are endodermis, cortex, and epidermis (Dolan
et al., 1993). These tissue layers are positioned in a symmetrical manner along the radial axis of the root,
and the endodermis and cortex are together called as ground tissue (reviewed in Scarpella and Helariutta,
2010). The pericycle is positioned at the outermost layer of the root stele surrounding the inner vascular
bundle, and this vascular tissue consists of phloem and xylem tissues that are separated by the
meristematic cells, procambium (reviewed in Ye, 2002; reviewed in Carlsbecker and Helariutta, 2005;
reviewed in Scarpella and Helariutta, 2010) (Figure 2A).
Figure 2 Root vascular structure of Arabidopsis.
(A) Schematic representations of the root meristem and stele (from Carlsbecker et al., 2010, with permission from Nature
Publishing Group). Cells in the endodermis tissue layer adjacent to the stele are marked with asterisks. (B) Confocal laser
scanning micrograph of basic fuchsin-stained xylem of a 4-day-old wild type Col-0 seedling. Filled arrowhead indicates
metaxylem, and unfilled indicates protoxylem. Scale bar, 10 µm.
6
Xylem is composed of water-transporting tracheary elements and xylem fibers that provide necessary
support to the growing tissue (Nieminen et al., 2004). The tracheary elements typically undergo a
sequential differentiation program to coordinate different phases of xylem maturation, that involves cell
expansion, lignin deposition of secondary cell wall, cell death, and autolysis of cell contents, which are
mediated by recently identified NAC domain transcription factors (reviewed in Vera-Sirera et al., 2010;
reviewed in Bollhoner et al., 2012). During this process, the immature xylem cells acquire different
secondary cell wall morphologies and form two different xylem types that are metaxylem and protoxylem,
wherein metaxylem exhibits pitted secondary cell wall thickenings that is readily distinguishable from the
spiral lignin deposition pattern of protoxylem (Carlsbecker et al., 2010; reviewed in Vera-Sirera et al.,
2010) (Figure 2B; Figure 3A).
It has been known that this vascular differentiation process is closely related to the regulation of plant
hormones auxin and cytokinin which is not striking as auxin and cytokinin are involved in the majority of
crucial developmental steps in Arabidopsis. Studies have shown that auxin can induce differentiation of
the vasculature (reviewed in Scarpella and Helariutta, 2010), and the woodenleg (wol) mutant which is
compromised in cytokinin signaling exhibits an altered pattern of cell type differentiation in the root
vascular system showing an all protoxylem phenotype (Mahonen et al., 2000; Ueguchi et al., 2001;
Higuchi et al., 2004).
The molecular basis of cell type patterning in Arabidopsis root is well understood, wherein two plantspecific GRAS family transcription factors, SHORT-ROOT (SHR) and SCARECROW (SCR), together
play crucial role in generating positional information for vascular cells to be able to acquire different cell
identities (Sena et al., 2004; Cui et al., 2007; Miyashima et al., 2009; Carlsbecker et al., 2010; Miyashima
et al., 2011; reviewed in Furuta et al., 2012). It has been shown that SHR is transcribed in the stele and
moves to the endodermis, where it activates the transcription of SCR and forms a nuclear localized
protein complex with SCR (Nakajima et al., 2001; Gallagher et al., 2004; Cui et al., 2007) (Figure 3B).
The SHR-SCR protein complex then not only regulates the asymmetric division of the undifferentiated
cortex/endodermis initial (CEI) daughter cells but also the expression of a number of endodermis-specific
genes including SCR itself (Di Laurenzio et al., 1996; Helariutta et al., 2000; Wysocka-Diller et al., 2000;
Heidstra et al., 2004; Sena et al., 2004), thereby moderating the cell fate of CEI and specifying a single
layer of endodermis (Sena et al., 2004; Cui et al., 2007; Miyashima et al., 2009).
The mobile transcription factors SHR and SCR also activate expression of genes encoding microRNAs
(miRNAs), MIRNA165A (MIR165A), MIRNA166A (MIR166A) and MIRNA166B (MIR166B), that are
responsible for the post-transcriptional down-regulation of the class III transcription factors of the
7
Homeodomain-Leucine Zipper (HD-Zip) family (HD-Zip III) (Carlsbecker et al., 2010; Miyashima et al.,
2011; reviewed in Furuta et al., 2012), which involves PHABULOSA (PHB), PHAVOLUTA (PHV),
REVOLUTA (REV), CORONA/ATHB15/INCURVATA4 (CNA), and ATHB8 (Talbert et al., 1995; Baima et
al., 2001; McConnell et al., 2001; Ohashi-Ito et al., 2005; Prigge et al., 2005).
Figure 3 Schematic drawing of the bidirectional cell-to-cell communication for xylem cell patterning (from Furuta et
al., 2012, with permission from Elsevier).
(A) Transverse section of the root meristematic region. (B) A molecular model of SHR and miR165/166 movement.
SHR is transported to the pericycle and endodermis cells and activates miR165/166 when it forms a complex with SCR
in the endodermis. Endodermally expressed miR165/166 then moves to the pericycle and protoxylem cells where it
suppresses the level of PHB transcripts making a gradient of PHB activity. PD = plasmodesmata. (C) A model of
symplastic SHR and miR165/166 transport through the PD.
8
The two related miRNAs, miR165/166, with only a single nucleotide difference in their sequence, target
and down-regulate the mRNAs of HD-Zip III transcription factors thereby restricting their expression
domain in the shoot and root of Arabidopsis (McConnell and Barton, 1998; Emery et al., 2003; Tang et
al., 2003; Lee et al., 2006). Mutations in the miRNA target sites of PHB and PHV mRNAs result in a
gain-of-function mutation by restricting miRNAs ability to degrade the transcripts of PHB and PHV
(Mallory et al., 2004), and similar effect was observed when genes involved in the miRNA processing,
such as ARGONAUTE1 or SERRATE, were mutated that provides further support for the miRNAmediated down-regulation of HD-Zip III transcription factors (Kidner and Martienssen, 2004; Grigg et al.,
2005).
It has been shown that the endodermally produced miR165/166 acts non-cell-autonomously to degrade
its target mRNA that encodes the class III HD-Zip transcription factors (Carlsbecker et al., 2010;
Miyashima et al., 2011) (Figure 3B), which results in a differential distribution of the target mRNAs in
the xylem-forming procambial cells. This differential distribution of HD-Zip III transcription factors, in
turn, determines the either metaxylem or protoxylem cell fate of the xylem tissue in a dosage-dependent
manner (Carlsbecker et al., 2010; reviewed in Scarpella and Helariutta, 2010).
In other words, the patterning of xylem cell type is modulated by a bidirectional cell-to-cell
communication between the vascular cylinder and its surrounding ground tissue, that is mediated by the
mobile transcription factor SHR and miR165/166 (reviewed in Furuta et al., 2012), wherein the stele
produced SHR moves to the endodermis activating expression of miR165/166, and the endodermally
expressed miR165/166 move to procambial cells modulating level of HD-Zip III expression (Figure 3B).
Recent studies have suggested that both SHR and miR165/166 move through the plasmodesmata, i.e. via
the symplastic pathway (Vaten et al., 2011; reviewed in Furuta et al., 2012), in which plasmodesmata is a
small channel in the cell wall that connects the cytoplasm of neighboring plant cells (Figure 3C).
However, up to now the direct or indirect role of the class III HD-Zip transcription factors and miRNAs
on determining the identity of xylem cells in the root vasculature is not yet fully explored, and in this
regard, this study is dedicated to enhance our knowledge of how the development and patterning of the
vasculature is coordinated by the HD-Zip III transcription factors and miR165/166, and also to explore
the HD-Zip III transcription factors potential downstream targets in order to better understand their
elaborate working mechanism during the whole process.
9
I.3. Transcription factors of the Class III HD-Zip family
One important aspect that makes the genetic studies complicated is the fact that more than 65% of the
Arabidopsis genes are members of gene families (The Arabidopsis Genome Initiative, 2000), so that it is
essential to study and understand plant genome in relation to this context. Among the various gene
families that have been intensively studied, HD-Zip is one of the most well-known gene families
(reviewed in Elhiti and Stasolla, 2009) (Figure 4). The homeodomain (HD) is a DNA-binding protein
domain of conserved 60-amino acid motif encoded by homeobox (HB) genes (Figure 4A), in which HB is
named after the homeotic effect in a Drosophila mutant, and found in all eukaryotic organisms (reviewed
in Ariel et al., 2007).
Although the leucine zipper motif (Zip) is also present in other eukaryotic organisms mainly acting as a
dimerization motif (Lee and Chun, 1998) (Figure 4B), its association with the HD forming together a
single protein is specific to the plant kingdom (Schena and Davis, 1992), where Zip domain is found
immediately downstream of HD in case of members of the HD-Zip family. It has been shown that this Zip
domain is crucial for the DNA-binding ability of HD (Tron et al., 2004).
Among the four different subfamilies of HD-Zip proteins, which are grouped on the basis of different
gene structure and unique domains and functions (reviewed in Ariel et al., 2007), the class III HD-Zip
transcription factors have been shown to play crucial roles in different developmental stages during the
entire life cycle of Arabidopsis (see below), which are distinguished from other groups by their four
additional amino acids between HD and Zip in the binding domain (Schrick et al., 2004) (Figures 4C).
Other conserved domains are also found such as steroidogenic acute regulatory (StAR) protein-related
lipid transfer (START) domain and START-adjacent domain (SAD), followed by the MEKHLA domain
at the C-terminus (reviewed in Ponting and Aravind, 1999; reviewed in Stocco, 2001; Schrick et al., 2004;
Mukherjee and Burglin, 2006), although direct roles of these domains have not yet been well understood
in plants. StAR proteins are responsible of cholesterol transfer in animals playing a common role in lipid
transport and metabolism (reviewed in Ponting and Aravind, 1999; reviewed in Stocco, 2001), and in
plants, a recent study has shown that soluble receptors of abscisic acid (ABA), PYRABACTIN
RESISTANCE/PYRABACTIN-LIKE (PYR/PYL) or REGULATORY COMPONENTS OF ABA
RECEPTOR (RCAR), have a high homology to START proteins (Joshi-Saha et al., 2011), in which
binding of ABA to PYR/PYL/RCAR triggers structural changes in the receptors.
10
Figure 4 Biochemical structure of homeodomain (HD) and leucine zipper (Zip) motifs with distinctive domains
exhibited by the HD-Zip subfamilies of Arabidopsis.
(A) The Antennapedia HD protein from Drosophila melanogaster is bound to a fragment of DNA, wherein the
recognition helix and unstructured N-terminus are bound in the major and minor grooves of the DNA, respectively.
Image
courtesy
of
Opabinia
Regalis.
GNU
Free
Documentation
License
(http://www.wikipremed.com/image.php?img=040101_68zzzz247350_Homeodomain-dna-1ahd_68.jpg&image_id=247350). (B) The
Zip motif. Blue colored Zip is bound to DNA, and the leucine residues are colored red. Image courtesy of Thomas
Splettstoesser. GNU Free Documentation License (http://www.wikipremed.com/image.php?img=040401_68zzzz284200_514pxLeucine_zipper_68.jpg&image_id=284200). (C) Schematic representation of the distinctive domains exhibited by the HD-Zip
subfamilies (from Ariel et al., 2007, with permission from Elsevier). HD, homeodomain; LZ, leucine zipper; MEKHLA
domain, named after the goddess of lightning, water and rain; SAD, START adjacent domain; START, steroidogenic acute
regulatory (StAR) protein-related lipid transfer domain.
The MEKHLA domain is named after Mekhla (with various other spellings), the goddess of lightning,
water, and rain, as it shares significant similarity with the PAS domain that has been shown to be
involved in light, oxygen, and redox potential sensing (Schrick et al., 2004). PAS domains are found in all
kingdoms of life, and named after their original discovery in the Drosophila period (Per) protein, the
11
vertebrate Arnt proteins, and the Drosophila single-minded (Sim) protein (Hoffman et al., 1991; Nambu
et al., 1991; Mukherjee and Burglin, 2006). Since the PAS domains have been indicated to be able to
dimerize (Card et al., 2005), the MEKHLA domain was also speculated to be involved in dimerization as
three-fourth of the MEKHLA domain at the carboxy-terminal is deemed to have the same structure as the
PAS domain (Mukherjee and Burglin, 2006), and it has been shown that the AP2 domain of two
paralogous
proteins,
DORNRÖSCHEN
(DRN)
(also
known
as
ENHANCER
OF
SHOOT
REGENERATION1; ESR1) and DRN-LIKE (DRNL) (also known as ESR2), can interact with the
MEKHLA domain enabling both DRN and DRNL to heterodimerize with members of the class III HDZip family in Arabidopsis (Chandler et al., 2007).
I.4. Role of HD-Zip III transcription factors in Arabidopsis
The five members of the HD-Zip III family often exhibit overlapping and antagonistic functions in
various developmental aspects in Arabidopsis (Prigge et al., 2005), in which they have been shown to act
as key developmental regulators during embryogenesis and postembryonic apical meristem initiation as
well as in auxin transport (McConnell and Barton, 1998; McConnell et al., 2001; Otsuga et al., 2001;
Emery et al., 2003; Prigge and Clark, 2006; Izhaki and Bowman, 2007; Ochando et al., 2008; Carlsbecker
et al., 2010; Duclercq et al., 2011), and best described is their role in specifying the adaxial identity of
lateral organs (McConnell et al., 2001; Emery et al., 2003; Hawker and Bowman, 2004). A semidominant gain-of-function mutant of PHB, phb-1d, exhibits adaxialization of lateral organs and ectopic
meristem formation undersides of leaves (McConnell and Barton, 1998), and gain-of-function mutants of
PHV also exhibit leaf polarity defects (McConnell et al., 2001; Tang et al., 2003).
It has been shown that REV and PHB together regulate maintenance of shoot apical meristem and
initiation of lateral organs (Otsuga et al., 2001), and REV, PHB, and PHV together with KANADI genes
control the abaxial-adaxial patterning of lateral organs (Emery et al., 2003; reviewed in Bowman and
Floyd, 2008; reviewed in Elhiti and Stasolla, 2009), in which the loss-of-function phb-6 phv-5 rev-9 triple
mutant exhibits radialized abaxialized cotyledons and complete loss of functional shoot apical meristem
(Emery et al., 2003; Hawker and Bowman, 2004). Although KANADI genes are not thought to be required
for a proper meristem function, they act antagonistically to HD-Zip III transcription factors in patterning
of lateral organs (Hawker and Bowman, 2004), wherein uniform expression of KANADI genes in
12
developing lateral organs results in a complete abaxialization (Eshed et al., 2001; Kerstetter et al., 2001;
Emery et al., 2003). Expression patterns of the five HD-Zip III transcription factors are also consistent
with each of their hypothesized role in determining organ polarity (McConnell and Barton, 1998;
McConnell et al., 2001; Emery et al., 2003), and a recent study has suggested that the HD-Zip III and
KANADI transcription factors control the cambium activity as well (Ilegems et al., 2010), in which
KANADI transcription factors might act by inhibiting auxin transport and HD-Zip III transcription factors
by promoting xylem differentiation.
Due to the overlapping functions of the five members, rev and cna loss-of-function mutants are the only
single mutants that exhibit a phenotype among the HD-Zip III transcription factors (Otsuga et al., 2001;
Green et al., 2005; Prigge and Clark, 2006; Ochando et al., 2008; Duclercq et al., 2011). Loss-of-function
rev mutant exhibits defects in the development of the shoot and leaves in addition to its abnormal vascular
development and problem with auxin transport (Talbert et al., 1995; Otsuga et al., 2001; Prigge et al.,
2005; Prigge and Clark, 2006), and gain-of-function rev-10d mutant has been shown to cause
radialization of vascular bundles in the stem and polarity defects in the leaves (Emery et al., 2003).
Expression of REV can be detected at the very earliest stages of lateral shoot meristem and floral
meristem formation suggesting its role in lateral meristem initiation (Otsuga et al., 2001), in which REV is
thought to be acting at lateral positions to activate the expression of other known meristem regulators.
On the other hand, CNA has been shown to be expressed in developing vascular tissue and loss-offunction cna mutant exhibits subtle defects in meristem development in the Arabidopsis ecotype
Landsberg erecta (Ler) background (Green et al., 2005), whereas the gain-of-function icu4-1 allele has
been shown to stimulate production of the vascular tissue in another Arabidopsis ecotype background,
Enkheim-2 (En-2) (Ochando et al., 2008). Recently, a previously described hoc mutant in the ecotype
C24 background (Catterou et al., 2002), that exhibits high organogenic capacity for shoot regeneration
and hence a bushy phenotype, has been also identified as another allele of CNA, wherein the observed
bushy phenotype is suspected to be a result of mutation on protein functions, but not due to the loss-offunction of CNA (Duclercq et al., 2011).
Although, single loss-of-function mutant of ATHB8, another member of the five HD-Zip III
transcription factors, does not display any phenotypic alteration compared to wild type, ectopic
expression of ATHB8 has been shown to result in xylem overproduction suggesting its possible role in
xylem differentiation (Baima et al., 1995; Baima et al., 2001), and it has been also shown that ATHB8
controls the early events of procambial development in the leaf veins of Arabidopsis (Kang and Dengler,
2002). While analyzing the phenotype of all possible combinations of double, triple, quadruple, and
13
quintuple mutants of the HD-Zip III transcription factors (Prigge et al., 2005), ATHB8, together with CNA,
have been shown to antagonize functions of REV in the lateral shoot meristem and floral meristem.
Interestingly, the rev phb phv cna athb8 quintuple mutant results in a lethal terminal phenotype,
exhibiting a radially symmetric apical structure which is due to embryonic shoot meristemlessness
(Prigge et al., 2005).
Together, observed defects in vascular development of these mutants could provide crucial clues of the
HD-Zip III transcription factors role on xylem differentiation (Otsuga et al., 2001), wherein
overexpression phenotype of ATHB8 and its expression patterns have further enhanced this speculation
(Baima et al., 1995; Baima et al., 2001). As previously described, Carlsbecker A and Lee JY have been
recently able to provide evidence that HD-Zip III transcription factors determine xylem cell types in a
dosage-dependent manner in the vascular system of Arabidopsis roots through a regulatory mechanism
conducted by microRNAs (miRNAs) (Carlsbecker et al., 2010), and it has been also shown that
differentiation of the pericycle and ground tissue patterning are also mediated by the miRNA dependent
suppression of PHB (Miyashima et al., 2011).
Although, leaf polarity defects are also often accompanied by vascular defects in the leaf and stem of
the HD-Zip III gain- or loss-of-function mutants, in a manner such as gain-of-function mutants exhibit
amphivasal (xylem surrounding the phloem) vascular bundles whereas multiple loss-of-function mutants
demonstrate amphicribal (phloem surrounding the xylem) vasculature (McConnell et al., 2001; Zhong and
Ye, 2004), in roots, changes in the collateral arrangement of xylem and phloem could not be detected
(Hawker and Bowman, 2004; Ilegems et al., 2010). However, corresponding alterations can be instead
observed in the central-peripheral dimension of the root vasculature, wherein the arrangement of
protoxylem and metaxylem is strongly affected by mutations of the HD-Zip III transcription factors
(Carlsbecker et al., 2010). While the gain-of-function mutant of PHB, phb-1d, displays ectopic
metaxylem formation in protoxylem position, no metaxylem is observed in quadruple mutants where
instead protoxylem forms in metaxylem position (Carlsbecker et al., 2010). Surprisingly, quintuple
mutant of the HD-Zip III transcription factors fail to form any xylem that further suggests their role in de
novo xylem formation as well (Carlsbecker et al., 2010).
14
I.5. Regulation of HD-Zip III transcription factors by plant hormones
Another important aspect concerning HD-Zip III transcription factors role on various developmental
stages in Arabidopsis is their close relation with the plant hormone auxin. Studies have shown that
treating with a polar auxin transport inhibitor can mimic phenotypes of rev mutants (Mattsson et al., 1999;
Zhong and Ye, 2001), and expression of ATHB8 is dependent upon the activity of auxin response factors
(ARFs), which together suggest direct or indirect regulation of the auxin signaling pathway on expression
of the HD-Zip III transcription factors (Baima et al., 1995; Baima et al., 2001; Kang and Dengler, 2002;
Sawa et al., 2002; Mattsson et al., 2003; Zhao et al., 2003). Further studies have identified that this ARFsdependent ATHB8 expression promotes vascular differentiation in Arabidopsis leaves by stabilizing
preprocambial cell specification during vein formation (Donner et al., 2009; reviewed in Donner et al.,
2010), and transcripts of PHV, CNA, and REV have also been shown to be induced by auxin treatment in
addition to ATHB8 (Zhou et al., 2007).
Crosstalk between auxin and cytokinin is highly crucial during various developmental stages in plants
that include, among others, the emergence of lateral roots and maintenance of root meristem activity in
Arabidopsis (reviewd in Moubayidin et al., 2009; reviewed in Bishopp et al., 2011a), although it has been
suggested that a feedback loop involving cytokinin-mediated auxin regulation may not occur during
embryonic root development (Muller and Sheen, 2008; reviewd in Bishopp et al., 2011a). While auxin is
required for the initiation and development of lateral root primodia (Laskowski et al., 1995; Himanen et
al., 2002; Benkova et al., 2003; reviewed in Casimiro et al., 2003), cytokinin negatively regulates
formation of the lateral roots (Li et al., 2006; Laplaze et al., 2007; Kuderova et al., 2008), and whereas
auxin maintains the root meristem by promoting cell division (Sabatini et al., 1999; Blilou et al., 2005;
Vanneste and Friml, 2009), cytokinin represses both the signaling and transport of auxin thereby
promoting cell differentiation in the meristem (Dello Ioio et al., 2007; Dello Ioio et al., 2008; reviewd in
Benkova and Hejatko, 2009).
Owing to this fact, efforts have been made to identify the role of cytokinin in vascular developmental
aspects as well (Mahonen et al., 2006; Bishopp et al., 2011b; Bishopp et al., 2011c), and studies have
shown that an ARABIDOPSIS HISTIDINE PHOSPHOTRANSFER PROTEIN 6 (AHP6)-mediated
mutual inhibitory interaction between auxin and cytokinin specifies the vascular patterns in the root
meristem (Mahonen et al., 2006; Bishopp et al., 2011b), in which AHP6 allows formation of protoxylem
by counteracting cytokinin signaling, and signaling of cytokinin negatively regulates the spatial
expression domain of AHP6 (Mahonen et al., 2006).
15
It has been also shown that cytokinin regulates the bisymmetric localization of the PIN-FORMED (PIN)
auxin efflux proteins in a relatively direct manner (Pernisova et al., 2009; Ruzicka et al., 2009; Bishopp et
al., 2011b), and high auxin conversely induces transcription of AHP6, the cytokinin signaling inhibitor,
together forming an interactive feedback loop during vascular patterning in the root meristem (Bishopp et
al., 2011b). However, although it has been suggested that the phloem-mediated long distance transport of
cytokinin might play a crucial role during this process (Bishopp et al., 2011c), up to now relatively less is
known about the molecular basis of cytokinin transport in comparison to the fairly well understood
transport mechanism of auxin (reviewed in Moubayidin et al., 2009), and most definitely even less in
understanding its direct or indirect relation with the HD-Zip III transcription factors.
I.6. Arabidopsis ARGONAUTE1 and ARGONAUTE10
While also found in bacterial and archaeal species (Kitamura et al., 2010; Wei et al., 2012), proteins of
the ARGONAUTE (AGO) family are highly conserved among eukaryotes and shares a conserved Cterminal region consisting of PAZ, MID, and PIWI domains acting as integral players in all known small
RNA-mediated regulatory pathways (Song et al., 2004; Rivas et al., 2005; Tolia and Joshua-Tor, 2007;
Hutvagner and Simard, 2008; Voinnet, 2009; Frank et al., 2010). The PAZ domain is responsible in
recognizing the 3’ end of small RNAs, whereas the MID domain binds to the 5’ phosphate of small RNAs
(Hutvagner and Simard, 2008; Frank et al., 2010), and the PIWI domain has been shown to carry out
endonuclease activity in the AGO-centered RNA-induced silencing complexes (RISCs) by forming an
RNaseH-like fold structure that cleaves single-stranded RNAs (ssRNAs) (Song et al., 2004; Rivas et al.,
2005; Voinnet, 2009).
The Arabidopsis genome contains ten AGO proteins belonging to three different phylogenetic clades
(reviewed in Vaucheret, 2008) (Figure 5), and among all the ten members, functions of AGO1 and
AGO10, that belong to the same clade, have been particularly well described. AGO1 preferentially
recruits small RNAs with a 5’-terminal uridine, and plays a major role in the miRNA pathway as most of
the miRNAs harbor a terminal 5’ uridine (Mi et al., 2008; Montgomery et al., 2008; Takeda et al., 2008;
Mallory and Vaucheret, 2009), which is consistent with the severe phenotype of loss-of-function ago1
mutants in comparison to the relatively limited or no obvious developmental defects of other single
mutants in the same family (Bohmert et al., 1998; Vaucheret et al., 2004; Yang et al., 2006).
16
It also needs to be emphasized that, owing to its
crucial role in the working mechanism of miRNAs,
defects of ago1 mutants strongly resemble that of the
gain-of-function mutants of PHB and PHV since
transcripts of PHB and PHV are both down-regulated
by miR165/166 as described before (Kidner and
Martienssen, 2004). Moreover, Miyashima et al. have
proposed that AGO1 mediates a post-transcriptional
regulatory pathway independent of SHR and SCR in
maintaining the root ground tissue pattern (Miyashima
et al., 2009), but their recent discovery of linkage
between SHR/SCR and miR165 highly suggests that
AGO1 might not act independently (Miyashima et al.,
2011).
Figure 5 Phylogenetic classification of Arabidopsis
AGO proteins in three clades (from Vaucheret,
2008, with permission from Elsevier).
Protein sequences were aligned using MultiAlin
(http://prodes.toulouse.inra.fr/multalin/multalin.h
tml), and PAM indicates the point accepted
mutation.
AGO10 was previously identified as PINHEAD
(PNH) and ZWILLE (ZLL), wherein both of the names
were originally coined from their loss-of-function
mutant phenotypes that exhibit abnormal shoot apical
meristem (SAM) development (Endrizzi et al., 1996;
Barton, 1998; Moussian et al., 1998; Lynn et al., 1999;
Newman, 2002; Moussian et al., 2003). PNH and ZLL
were later revealed to be allelic based on map-based cloning, and therefore PINHEAD/ZWILLE was
eventually renamed as AGO10 after the discovery of the AGO gene family in the Arabidopsis genome
(reviewed in Vaucheret, 2008), although the respective mutants pnh and zll are still referred to their
original names in order to prevent confusion with other mutants of the same gene. AGO10 has been
shown to maintain undifferentiated cells in SAM and establish polarity in leaves through specific
interaction with miR165/166 (Liu et al., 2009; Mallory et al., 2009), and a recent study has shown that
AGO10 and AGO1 might play counteracting roles in relation to the regulation of miR165/166 (Zhu et al.,
2011).
Whereas AGO1 is a key player in the regulatory mechanism of miRNAs, it has been suggested that
AGO10 sequesters miR165/166 from their binding to AGO1 through acting as a decoy by taking
advantage of its stronger binding affinity to miR166, thereby up-regulating the transcript level of HD-Zip
III family genes (Zhu et al., 2011). This is consistent with the fact that mutations in AGO10 result in
17
reduction of PHB, PHV, and REV transcripts in stems and leaves (Liu et al., 2009), and it has been also
shown that expression levels of PHB, PHV, and CNA are down-regulated in the roots of zll-3 mutants
(Iyer-Pascuzzi et al., 2011). Interestingly, the pnh and zll mutants were both identified in Ler background,
but ago10 mutations in Col-0 background do not exhibit severe defects in SAM development (reviewed
in Vaucheret, 2008; Liu et al., 2009). It is possible that there may be a factor in Col-0 preventing the
incorporation of AGO1 into the RISC of miR165/166, as making miRNA166 more accessible to AGO1
than AGO10 leads to similar phenotypes in both of the ecotypes (Zhu et al., 2011).
I.7. Aim of this study
In this study, I have focused on PHB among all the five members of the class III HD-Zip family, and βestradiol inducible systems of PHB and miR165 were used in order to shed light on their specific role on
xylem differentiation, by analyzing changes in cell pattern of the root vasculature after inducing the
corresponding constructs (Experiment I). Vascular structure was also analyzed in the roots of different
ago mutants to identify AGOs possible influence on xylem cell type patterning (Experiment IV).
In addition, a global transcriptome analysis was previously performed utilizing the same inducible
system mentioned above to identify potential downstream targets of HD-Zip III transcription factors, and
I did comparisons with previously published microarray data to examine relevance of the employed
system (Experiment II).
Finally, changes in root xylem pattern and meristem cell number of phb-7d, a dominant overexpression
mutant of PHB, were analyzed upon treatment with the plant hormones auxin and cytokinin or inhibitor
of auxin transport, in an effort to better understand the direct or indirect relation of the class III HD-Zip
transcription factors with these hormones and to address cell division problems in the root meristem upon
the applied chemical treatments (Experiment III).
18
II. Materials and Methods
II.1. Plant materials and growth condition
All Arabidopsis seeds were surfaced sterilized for 25 minutes in a solution containing 25% of the
commercial bleach ‘klorENT’, and rinsed with 70% EtOH for 1 minute before washing 5 times with
sterile distilled water. Seeds were kept in darkness at 4ºC for 2 days for stratification, and then plated on a
solid 0.5x MS-medium (Murashige and Skoog, 1962) containing 1% (w/v) agar at pH 5.8, and 1% (w/v)
sucrose. All plates were positioned vertically in a light chamber under long-day condition with a light
regime of 16h light/8h darkness at 22ºC.
phb-7d harbors a point mutation in the miR165/166 target site thereby rendering the PHB transcript
resistant to miRNA-mediated degradation (Carlsbecker et al., 2010). ago1-3 and zll-3 are ethyl
methanesulfonate (EMS) induced mutants in the Arabidopsis ecotype Col-0 and Landsberg erecta (Ler)
backgrounds, respectively (Bohmert et al., 1998; Moussian et al., 1998; Moussian et al., 2003).
For treatment of hormones or inhibitor of hormone transport, stock solutions of 1-Naphthaleneacetic
acid (NAA) (DUCHEFA, N0904), 1-N-Naphthylphthalamic acid (NPA) (DUCHEFA, B0904), and 6Benzylaminopurine (BAP) (DUCHEFA, N0926) were prepared as recommended by the chemical
supplier and diluted in the MS-medium described above with a concentration of 100 nM, 5 µM, and 100
nM, respectively. Seedlings were germinated and grown on plates containing these chemical-added
growth media.
II.2. Transgenic lines and the β-estradiol inducible XVE system
pCRE1(WOL)::XVE>>phb-1d (Roberts C, Lehesranta S, Helariutta Y, Carlsbecker A, unpublished
results) is a two-step inducible construct, in which the stele-specific promoter of CYTOKININ
RESPONSE 1 (CRE1) (Inoue et al., 2001), the same gene as WOODENLEG (WOL) and ARABIDOPSIS
HISTIDINE KINASE 4 (AHK4) (Mahonen et al., 2000; Ueguchi et al., 2001; Higuchi et al., 2004), drives
the expression of a β-estradiol inducible XVE system (Zuo et al., 2000). The XVE system is a
combination of the DNA binding domain of LexA, the transcriptional activation domain of VP16 and the
regulatory region of the human estrogen receptor (Zuo et al., 2000) (Figure 6).
19
Figure 6 A schematic diagram of the XVE vector (from Zuo et al., 2000, with permission from John Wiley and Sons).
Only the region between the right and left borders is shown (not to scale). PG10−90, a syntheMc promoter controlling XVE;
XVE, DNA sequences encoding a chimeric transcription factor containing the DNA-binding domain of LexA (residues 1–87),
the transcription activation domain of VP16 (residues 403–479) and the regulatory region of the human estrogen
receptor (residues 282–595); TE9, rbcS E9 poly(A) addition sequence; Pnos, nopaline synthase promoter; HPT, hygromycin
phosphotransferase II coding sequence; Tnos, nopaline synthase poly(A) addition sequence; OLexA, eight copies of the
LexA operator sequence; −46, the −46 35S minimal promoter; MCS, mulMple cloning sites for target genes; T3A, rbcsS3A
poly(A) addition sequence. Arrows indicate the direction of transcription (Zuo et al., 2000).
Upon β-estradiol treatment, the receptor functions as a transcription factor and binds to its promoter
thereby activating transcription of phb-1d (McConnell and Barton, 1998; McConnell et al., 2001), which
is another miRNA-resistant allele of PHB that is stronger than the previously described phb-7d
(Carlsbecker et al., 2010). Construct had been transformed into wol mutant background, and the acquired
transgenic line, pCRE1(WOL)::XVE>>phb-1d in wol background, had been crossed to Col-0 as well in
order to get the transgene into wild type background.
Additionally, a β-estradiol inducible miRNA165a construct (pCRE1::XVE>>MIR165A) had also been
prepared in the same way as described above and transformed into wild type Col-0 background (Roberts
C and Carlsbecker A, unpublished results).
II.3. β-estradiol induction and collection of seedlings
20 mM stock solution of β-estradiol was prepared in 100% EtOH. β-estradiol containing MS-medium
was prepared by adding according amount of stock solution to the before described MS-medium as to
make the final concentration either at 5 or 10 µM. In addition, a thin sterilized-mesh was directly placed
on the top of the β-estradiol containing MS-medium and seeds were plated on top of it. Induction of βestradiol was performed by transferring the mesh with seedlings grown on it from normal MS-medium to
estradiol-containing MS-medium upon various time points. In order to collect all seedlings at the same
age, induction was performed in a way such as the 48-hour-induction was performed by transferring the
mesh 48 hours before collection.
20
II.4. Histological techniques and analysis of xylem phenotype
4-5 days old seedlings were stained with basic fuchsin immediately after collection (Mahonen et al.,
2000). After first incubating in an acidified methanol solution (20% methanol with 4% of concentrated,
37% HCl) for 15 min at 55°C, seedlings were transferred to a basic solution containing 7% NaOH in 60%
ethanol for clearing and kept there for 15 min at room temperature.
A re-hydration step followed by replacing the incubation solution first to 40% ethanol, and then to 20%
and 10%, incubating for 10 min in each solution. Seedlings were stained for 5 min in 0.01% basic fuchsin
solution (in H2O) before de-staining was performed for 5 min in 70% ethanol. Same re-hydration step as
above was repeated and equal volume of 50% glycerol was added after re-hydrating in 10% ethanol. After
incubating overnight for stabilization, seedlings were finally mounted on an objective glass slide in 50%
glycerol. If not otherwise mentioned, all steps were performed under room temperature.
Light and confocal microscopes were used to analyze and photograph images of root vasculature. Leica
DM-RX and Zeiss Axioplan were used for light microscopy, and images were taken using digital cameras
Leica DFC490 and Leica DFC295, respectively. The imaging software Leica Application Suite V3
(ver.3.5.0) was used for both of the microscopes. Confocal laser scanning microscopy (CLSM) images
were taken on Leica DMI4000, an inverted epifluorescence microscope, using differential interference
contrast (DIC) settings. Composite images of the vascular bundle were acquired by projecting optical
sections together and acquired images were further processed with Adobe® Photoshop® CS3 (ver.10.0).
II.5. Microarray data analyses
Two independent microarray experiments, with three biological replicates each, had been previously
performed using the pCRE1(WOL)::XVE>>phb-1d -inducible transgenic line in wol mutant background
(Roberts C, Carlsbecker A, unpublished results). Different β-estradiol induction schemes were applied for
the two experiments, which were 2 and 6 hours for one experiment and 1, 1.5, and 2 hours for the other.
RNA was collected from root tips of 5-day-old seedlings.
Produced raw data files (.CEL files) were analyzed using the Affymetrix® Expression ConsoleTM
(ver.1.1) software for normalizing and filtering of the data (Detection P-value < 0.05). Expression levels
of genes upon each time points were compared in log scale to 0h, and only genes with significantly
21
altered expression were chosen to make lists of either up- or down-regulated. Genes exhibiting more than
0.495 log fold change in their expression were regarded as up-regulated, and less than -0.495 as downregulated.
A previously published microarray data set from Liu et al., which is available as the accession number
GSE6954 (not GSM6954 as it is misspelled in the original reference) in the Gene Expression Omnibus
(http://www.ncbi.nlm.nih.gov/geo), was compared to our data (Liu et al., 2008). Liu et al. used the same
XVE system for induction of AGAMOUS-LIKE 24 (AGL24), a MADS-box transcription factor (Liu et al.,
2008). Lists of up- or down-regulated genes were made based on data set comparison of β-estradiol
treated wild type seedlings (GSM158703) with mock treated transgenic lines (GSM158701), in which
they have used dimethyl sulfoxide for the mock treatment. Data files were analyzed the same way as
described above in order to compare the two data sets each other. Venn diagrams were made using the
online tool VENNY (http://bioinfogp.cnb.csic.es/tools/venny/index.html) (Oliveros, 2007).
II.6. other molecular techniques - GUS staining and GFP analysis
β-glucuronidase (GUS) activity was analyzed by staining collected 5-day-old seedlings overnight at
37°C in a solution containing 0.5 mg/ml 5-bromo-4-chloro-3-indolyl ß-D-glucuronide (X-gluc) dissolved
in N,N-dimethylformamide, 0.1% Triton X-100, 0.5 mM potassium ferrocyanide (K4Fe(CN)6⋅3H2O), 0.5
mM potassium ferricyanide (K3Fe(CN)6), and 50 mM sodium phosphate buffer (NaPO4, pH 7.5) (Vitha et
al., 1995). After rinsing with distilled water, seedlings were washed with 70% ethanol before mounting on
objective glass slides in 50% glycerol, and number of cells expressing GUS activity was counted in the
root meristem. P-values were calculated by two-tailed Student’s t-test for determining the significance in
difference between compared samples.
Signal of green fluorescent protein (GFP) was assessed under a conventional fluorescence stereo
microscope, Leica MZ-FLIII, equipped with an Hg lamp.
22
III. Results
III.1. Verifying the role of PHB in root xylem cell type acquisition by utilizing
the β-estradiol inducible XVE system
Induction of PHB converts protoxylem to metaxylem in absence of cytokinin signaling
It has been previously shown that the expression level of HD-Zip III transcription factors determines
xylem cell types in a dosage-dependent manner in the vascular system of Arabidopsis roots through a
regulatory mechanism conducted by miRNAs, wherein miR165/166 negatively regulates the expression
level of HD-Zip III transcription factors (Carlsbecker et al., 2010). In their study, Carlsbecker et al. have
indicated that the semi-dominant gain-of-function mutant of PHB, phb-1d, in which the phb-1d transcript
is resistant to miRNA degradation thereby constitutively active, exhibits an altered pattern of cell type
acquisition that forms metaxylem ectopically in the place of protoxylem (Carlsbecker et al., 2010).
In order to create a tool by which xylem cell fate can be converted, a construct in which the miRNAresistant
phb-1d
protein
can
be
stele-specifically
induced
had
been
prepared,
i.e.
pCRE1(WOL)::XVE>>phb-1d (Roberts C, Beste L, Lehesranta S and Carlsbecker A, unpublished results).
As previously mentioned, the wol mutant is compromised in cytokinin signaling that results in an all
protoxylem phenotype in the root vasculature (Mahonen et al., 2000; Ueguchi et al., 2001; Higuchi et al.,
2004). The above described transgene had been hence transformed into the wol mutant background in
order to see whether induction of phb-1d can alter the pattern of cell type acquisition of root xylem cells
resulting in formation of metaxylem in this mutant background (Roberts C, Beste L, Lehesranta S and
Carlsbecker A, unpublished results).
Upon induction, it had been observed that metaxylem formed in the place of protoxylem, and this line
had therefore been used in a global transcriptome analysis to assess changes in gene expressions upon
xylem cell type alteration (Roberts C, Beste L, Lehesranta S and Carlsbecker A, unpublished results). In
this study, this transgenic line, i.e. pCRE1(WOL)::XVE>>phb-1d in wol mutant background, was
analyzed in depth so that to confirm inducing expression of PHB in the stele can alter the pattern of cell
type acquisition causing the stele cells to differentiate as metaxylem instead of protoxylem.
23
Using the transgenic line pCRE1(WOL)::XVE>>phb-1d in wol mutant background, induction with βestradiol was performed upon various time points, and detailed phenotypic analyses of the vascular
structure under light and confocal microscopes were followed, focusing on the phenotypic changes in root
xylem cell type patterning in comparison to wild type (Figure 7). Results suggest that constitutive
expression of phb-1d could induce metaxylem differentiation in the vascular system of roots in wol
mutant background that confirms what was observed before (Figure 7A, 7B, 7C, 7D and 7E).
However, interestingly, seedlings treated with β-estradiol for 48 hours have not exhibited a clear
alteration in cell type patterning (Figure 7F), rather showing an intermediate type of xylem structure
which was hard to distinguish between proto- and metaxylem. This weak level of phenotypic change upon
48-hour-induction is surprising as seedlings were only 4-day-old upon collection, which means 48 hours
were basically half of the seedlings life time. Similar phenotype could be also observed at the upper part
of roots when seedlings were germinated on β-estradiol containing growth media (Figure 8B).
In addition, I observed that the vasculature starts to form more shootwards from the root tip in
correlation to the duration or level of induction, which was therefore the most significant when seedlings
were germinated on β-estradiol containing growth media (Figure 7C; Figure 8A). However, this observed
trend has not yet been fully measured and quantified, and hence further analysis is required for
confirmation. Interestingly, although constitutive induction of phb-1d is supposed to inhibit formation of
protoxylem, in regions higher up along the roots, protoxylem formation could still be identified (Figure
8C). In other words, despite of the constitutive phb-1d induction, the root vascular phenotype of these
seedlings was similar to the non-induced seedlings in the region near hypocotyl, wherein wol seedlings
exhibit the all protoxylem phenotype (Figure 8C).
Together, despite of the fact that the level of change in cell type patterning was weak upon lower
induction times, it is still possible to conclude that induction of phb-1d can convert protoxylem to
metaxylem in the wol mutant background as constitutive expression of phb-1d could clearly induce
formation of metaxylem. Since the wol mutant is compromised in cytokinin signaling, this result suggests
that phb-1d can direct differentiation of metaxylem in the absence of cytokinin signaling.
24
Figure 7 Xylem phenotype analysis results of 4-day-old β-estradiol inducible transgenic line pCRE1(WOL)::XVE>>phb-1d in
wol mutant background upon 48-hour and constitutive induction.
Phenotypic alteration resulted from10 µM β-estradiol treatments of pCRE1(WOL)::XVE>>phb-1d in wol mutant background
(A) with light and confocal microscope images of observed phenotypes shown below (B-F). Constitutive induction of phb-1d
in wol mutant background results in formation of metaxylem in the place of protoxylem (B, C, D, and E). No xylem could be
observed near root tips upon constitutive induction of phb-1d (C) and intermediate xylem phenotypes could be observed
upon 48-hour-induction (F). mx, metaxylem; px, protoxylem. Horizontal axis on (A) represents hours of β-estradiol induction
with ‘n’ indicating number of seedlings analyzed. Filled arrowhead indicates metaxylem, and unfilled indicates protoxylem;
*, intermediate xylem phenotype. Scale bar, 10 µm.
25
Figure 8 Longitudinal image of the entire root of a PHB-induced transgenic line in wol mutant background.
Shown is a 4-day-old seedling of pCRE1(WOL)::XVE>>phb-1d in wol background germinated on a 10 µM β-estradiol
containing growth medium that results in constitutive expression of PHB. 37 light microscope images were taken in row
and presented are artificially combined images in order to show the entire root of the seedling; 14, 14, and 9 images
from left to right. Total root length was 14.8 mm. (A) 3.6 mm from root tip where xylem starts to form (metaxylem
forming in the place of protoxylem). (B) 7.2 mm from root tip where an intermediate type of xylem structure could be
observed which is hard to distinguish between proto- and metaxylem. (C) 10 mm from root tip exhibiting all protoxylem
phenotype that is normal in the root of wol mutant seedlings. Filled arrowhead indicates metaxylem, and unfilled
indicates protoxylem; *, intermediate xylem phenotype. Scale bar, 10 µm. c.f. Longitudinal image of entire root was
only made from this line in wol mutant background since roots of other transgenic lines in wild type background are
much longer than this.
26
Introducing phb-1d inducible transgene into wild type Col-0 background by crossing
For a better understanding of how xylem specification is directed by PHB in wild type, the transgenic
line, pCRE1(WOL)::XVE>>phb-1d in wol background, had been crossed to Col-0 in order to get the
transgene into wild type background. All offspring of an F3 plant, line 1:20, showed resistance to BASTA,
i.e. selection marker of the transgene. As the line was segregating with wol phenotypes, which is readily
distinguishable as wol seedlings exhibit short root and tiny shoots, F4 plants showing wild type phenotype
were collected individually to determine segregation in the F5 generation (Table 1). Four lines were
determined as non-segregating with wol phenotypes (Table 1).
Table 1 Segregation ratio of line 1:20.
pCRE1(WOL)::XVE>>phb-1d in wol background was backcrossed to wild type Col-0, and all offspring of an F3 plant,
line 1:20, showed resistance to the selection marker BASTA. As the line was segregating with wol phenotypes, 20 of
the F4 plants showing wild type phenotype were collected individually to determine segregation on F5. Phenotypes of
30 to 40 seedlings were observed upon each F5 lines when seedlings were 17-day-old, and four lines, i.e. 11, 15, 17
and 19, were determined as non-segregating with wol phenotypes.
Line 1:20
wol/total
Line 1:20
wol/total
1
2
3
4
5
6
7
8
9
0.26
0.36
0.36
0.16
0.14
0.26
0.18
0.34
0.16
11
12
13
14
15
16
17
18
19
0
0.26
0.24
0.33
0
0.15
0
0.25
0
10
contamination
20
0.27
27
Comparison between inductions of phb-1d and miR165 in wild type background suggests
low efficiency of phb-1d induction in xylem cell type conversion
Two transgenic lines in which either phb-1d or miR165 can be induced in wild type Col-0 background
were analyzed, in order to compare the consequences of inductions in root xylem cell type patterning. We
expect to see an all metaxylem phenotype by inducing expression of phb-1d in wild type background, and
the opposite effect of it with miR165 induction as miR165 negatively regulates expression of the HD-Zip
III transcription factors that includes PHB. In other words, inducing miR165 in this transgenic line by
treating with β-estradiol is assumed to result in a phenotype where all stele cells differentiate as
protoxylem, exhibiting a similar phenotype as the wol mutant in the root vasculature.
Upon induction of miR165, it had been observed that protoxylem formed in the place of metaxylem, and
this line had therefore been also used in a global transcriptome analysis to assess changes in gene
expressions upon xylem cell type alteration (Roberts C and Carlsbecker A, unpublished results). In this
study, this transgenic line, i.e. pCRE1(WOL)::XVE>>MIR165A in wild type background, was analyzed in
depth for comparison with the phb-1d inducible transgenic line in wild type background. After treating
the transgenic lines with β-estradiol upon various time points, detailed phenotypic analyses of the
vascular structure under light and confocal microscopes were followed, focusing on the phenotypic
changes in root xylem cell type patterning in comparison to wild type (Figure 9; Figure 10).
28
Figure 9 Xylem phenotype analysis results of the β-estradiol inducible transgenic lines pCRE1(WOL)::XVE>>phb-1d
and pCRE1(WOL)::XVE>>MIR165A in wild type background upon various induction times.
(A) pCRE1(WOL)::XVE>>phb-1d in wild type Col-0 background exhibits double protoxylem phenotype in a dosagedependent manner upon β-estradiol inductions. (B) pCRE1(WOL)::XVE>>MIR165A in wild type Col-0 background
exhibits protoxylem-only phenotype in a dosage-dependent manner upon β-estradiol inductions. In general,
phenotypic alteration caused by inducing miR165 is much stronger when compared to induction of phb-1d. px,
protoxylem; mx, metaxylem. Horizontal axis represents hours of β-estradiol induction, and (>50%) or (<50%)
represents whether more or less than half of the corresponding phenotype was observed along each of the xylem
axis among the seedlings analyzed with ‘n’ indicating the number of seedlings. c.f. 48*, seedlings were treated with
5 µM of β-estradiol which was otherwise 10 µM for all the other experiments.
29
Figure 10 Light and confocal microscope images of 4-day-old β-estradiol inducible transgenic lines showing various xylem
phenotypes upon inductions.
Light and confocal microscope images of transgenic lines pCRE1(WOL)::XVE>>phb-1d and pCRE1(WOL)::XVE>>MIR165A in
wild type background upon induction with 10 µM β-estradiol. (A, C, and G) Without treatment, normal formation of
protoxylem can be observed as it is in wild type. Constitutive induction of phb-1d results in formation of metaxylem in the
place of protoxylem (B and H), whereas constitutive induction of miR165 results in protoxylem-only phenotype (F and I). (D)
No xylem could be observed near root tip when phb-1d was constitutively induced, and only metaxylem starts to form
further up along the root axis. (E) Double protoxylem phenotype that resulted from 6-hour-induction of phb-1d. Filled
arrowhead indicates metaxylem, and unfilled indicates protoxylem; *, no xylem formation. Scale bar, 10 µm.
30
Induction of phb-1d by 10 µM β-estradiol exhibited frequent double protoxylem formation in the root
vasculature of 4-day-old seedlings in wild type background, wherein an additional protoxylem strand
forms next to the normal protoxylem (Figure 9A; Figure 10A and 10E). On the other hand, when treated
in the same way, induction of miR165 resulted in a protoxylem-only phenotype (Figure 9B; Figure 10C,
10F and 10I), in which protoxylem forms in the place of metaxylem as it has been shown before (Roberts
C and Carlsbecker A, unpublished results), in addition to formation of double protoxylem (Figure 9B). In
both of the cases, alteration in xylem cell type differentiation has demonstrated a dosage-dependent
manner upon different levels of inductions, i.e. when different durations of β-estradiol treatment were
applied (Figure 9A and 9B), and the varying levels of phb-1d induction were confirmed by real-time
qPCR (Figure 11A and 11B) (Roberts C and Carlsbecker A, unpublished results).
Figure 11 (Continued)
31
Figure 11 Relative expression of phb-1d in the inducible transgenic line pCRE1(WOL)::XVE>>phb-1d in wild type
background upon induction with β-estradiol.
(A) RNA was collected from the root tips (2 mm) of 4-day-old seedlings treated with 10 µM of β-estradiol for 24 and 48
hours in order to see the relative change in expression of phb-1d by real-time qPCR analysis (Roberts C and Carlsbecker
A, unpublished results). Results indicate induction of phb-1d upon treatment with β-estradiol. **, p-value < 0.01. Error
bars indicate standard error. (B) Xylem phenotype analysis result of seedlings that were used for the real-time qPCR
experiment. Almost no phenotypic change in xylem cell type patterning could be identified which suggests weaker level
of induction in comparison to other performed experiments. px, protoxylem; mx, metaxylem. Horizontal axis represents
hours of β-estradiol induction with ‘n’ indicating number of seedlings analyzed.
Although the same XVE system was applied for all the constructs prepared, induction of miR165
resulted in a more significant phenotypic alteration compared to induction of phb-1d in wild type
background (Figure 9A and 9B). For example, when treated with β-estradiol for 24 hours, although
similar degree of phenotypic alteration was expected, changes caused by inducing phb-1d were around
1.8 times less than that of miR165; approximately 72% and 41% of the seedlings showed any phenotypic
variation form wild type in the inducible miR165 and phb-1d transgenic lines in wild type background,
respectively. Furthermore, in wild type background, metaxylem forms in the place of protoxylem only
when phb-1d is induced for more than 48 hours and not at 24 hours (Figure 9A; Figure 12A), whereas
induction of miR165 results in a protoxylem-only phenotype already upon 16 hours of induction, with
more than 30% of seedlings exhibiting the phenotype after 24-hour-induction (Figure 9B).
Interestingly, when the result of 24-hour-induction by 10 µM β-estradiol is compared to that of the 48hour-induction by 5 µM β-estradiol (Figure 9A), it is obvious that the change in xylem cell type
patterning is more affected by the absolute concentration of β-estradiol rather than the duration of
treatment.
In order to assess the effect of phb-1d induction over a longer time period, seedlings were also
germinated on β-estradiol containing growth media for constitutive expression of the constructs (Figure
10; Figure 12). Constitutive induction of phb-1d in wild type background resulted in a metaxylem-only
phenotype wherein metaxylem forms in the place of protoxylem (Figure 10A, 10B, 10G and 10H; Figure
12A), and on the contrary, constitutive induction of miR165 exhibited a protoxylem-only phenotype
wherein protoxylem forms in the place of metaxylem, as it was expected (Figure 10C, 10F and 10I;
Figure 12B).
32
Figure 12 (Continued)
33
Figure 12 Xylem phenotype analysis results of 4-day-old β-estradiol inducible transgenic lines pCRE1(WOL)::XVE>>phb-1d
and pCRE1(WOL)::XVE>>MIR165A in wild type background upon 48-hour and constitutive induction.
Phenotypic alteration resulted from 10 µM β-estradiol treatments of inducible transgenic lines pCRE1(WOL)::XVE>>phb-1d
and pCRE1(WOL)::XVE>>MIR165A in wild type background (A and B) with light and confocal images shown below (C and D).
Constitutive induction of phb-1d in wild type backgrounds results in ectopic metaxylem formation and no xylem could be
observed near root tips in severe cases, whereas constitutive induction of miR165 results in a protoxylem-only phenotype.
In general, phenotypic alteration caused by inducing miR165 is much stronger when compared to induction of phb-1d.
Despite of the constitutive induction of phb-1d in wild type background, formation of protoxylem could be observed at
higher regions of the root (C and D). mx, metaxylem; px, protoxylem. Horizontal axis of (A) and (B) represents hours of βestradiol induction, and (>50%) or (<50%) represents whether more or less than half of the corresponding phenotype was
observed along each of the xylem axis among the seedlings analyzed with ‘n’ indicating the number of seedlings. Filled
arrowhead indicates metaxylem, and unfilled indicates protoxylem. Scale bar, 10 µm.
As it was seen upon phb-1d induction in wol mutant background, the vasculature starts to form more
shootwards from the root tip in correlation to the duration or level of induction upon phb-1d induction in
wild type background as well, and hence no xylem formation could be observed near the root tip in severe
cases (Figure 10D; Figure 12A). Likewise, as it was in the wol mutant background, non-altered xylem
phenotypes could be observed at regions near hypocotyl in wild type background as well when phb-1d
was constitutively active from the time of germination, wherein the wild type seedlings exhibit normal
metaxylem formation next to protoxylem (Figure 12C and 12D). This was not observed upon induction of
miR165, in which the changed vascular phenotype is consistent throughout the root axis when miR165
was constitutively induced (Figure 10F and 10I).
In summary, results suggest that inducing expression of phb-1d in wild type Col-0 background causes
ectopic metaxylem formation in a dosage-dependent manner in the root vascular system of Arabidopsis,
but the degree of phenotypic change was weaker than what was expected indicating the low efficiency of
phb-1d induction in xylem cell type conversion. This observation is consistent to what Carlsbecker et al.
have shown before (Carlsbecker et al., 2010), and result of this study further supports the direct
involvement of PHB in the regulatory mechanism of cell type patterning, which means formation of
metaxylem is directly mediated by PHB in the vascular system of Arabidopsis roots.
On the other hand, induction of miR165 exhibits a protoxylem-only phenotype as it has been observed
before (Roberts C and Carlsbecker A, unpublished results), which is similar to the all protoxylem
phenotype of the wol mutant seedlings. However, in wol mutant seedlings, all the stele cells differentiate
as protoxylem and number of stele cells that exhibit the all protoxylem phenotype is also fewer in
comparison to wild type as it has been shown by Mähönen et al. (Mahonen et al., 2000), whereas
induction of miR165 in wild type background causes protoxylem differentiation in the place of
metaxylem, but not in the place of procambium or phloem cells, while exhibiting not only more stele cells
34
but also more cells per xylem axis compared to the non-induced seedlings (Roberts C and Carlsbecker A,
unpublished results).
35
III.2. Global transcriptome analysis of the HD-Zip III transcription factor PHB
with additional comparison to other microarray data sets
Major discrepancy found between data sets
In an effort to analyze the global transcriptome changes in the root meristem of the wol mutant upon
phb-1d induction and to further identify the potential downstream targets of HD-ZIP III transcription
factors as well, two independent microarray analyses with biological triplicates had been performed
(Roberts C, Beste L, Lehesranta S and Carlsbecker A, unpublished results). The previously described
transgenic line in which the miRNA resistant allele of PHB, phb-1d, could be induced in the stele of the
wol mutant, i.e. pCRE1(WOL)::XVE>>phb-1d in wol mutant background, that causes the stele cells to
differentiate as metaxylem instead of protoxylem upon induction, was used for the experiments with
different induction schemes. One microarray experiment included induction times of 0, 2 and 6 hours, and
the other included 0, 1, 1.5 and 2 hours of inductions.
However, it has been found that there is a large discrepancy of the two microarray experiments as the
lists of significantly up- or down-regulated genes were inconsistent between the two analyses upon the
same 2 hours induction, when a cutoff value of log fold change at 0.495 was applied to define upregulated genes and -0.495 for down-regulated genes (p-value < 0.05) (Table 2). Although we expected to
see a high similarity between the two gene lists as same growth conditions and induction time were used,
only 9 genes were common out of the 181 and 188 up-regulated genes of the two microarray data sets,
and only 7 out of the 112 and 171 down-regulated genes (Table 2).
Comparison with other microarray data reveals β-estradiol treatment has no major
influence on discrepancy
In order to check the potential influence of the applied chemical β-estradiol itself on the observed
discrepancy, I compared our data with a previously published microarray data from Liu et al., in which a
similar β-estradiol induction method was used (Liu et al., 2008). Liu et al. used the same XVE system for
induction of AGAMOUS-LIKE 24 (AGL24), a MADS-box transcription factor (Liu et al., 2008). In their
experiment, seedlings were induced for 12 hours with 10 µM of β-estradiol and RNA was collected from
9-day-old seedlings (Liu et al., 2008).
36
Lists of up- or down-regulated genes upon treatment with β-estradiol in wild type were made based on
comparison of two data sets; data of β-estradiol treated wild type seedlings and data of mock treated
transgenic lines wherein they have used dimethyl sulfoxide for the mock treatment. Same cutoff value as
above was applied on all data, and results of the comparison indicate that only a minor fraction of up- or
down-regulated genes are overlapping with our data (Table 2).
Table 2-1 Comparisons of the lists of up- or down-regulated genes of three independent microarray data sets indicating
number of overlapping genes with results also shown on Venn diagrams.
A, inducible phb-1d in wol mutant background with 0, 2, and 6 hour inductions; B, inducible phb-1d in wol mutant background
with 0, 1, 1.5, and 2 hour inductions; C, previously published data of β-estradiol treated wild type seedlings with induction
time of 12 hours (Liu et al., 2008). Comparison of data sets A and B does not exhibit significant overlap of the 2-hour
inductions from each experiment indicating inconsistency of data, and both the data sets A and B have only a minor fraction
of genes overlapping with data set C suggesting that the influence of applied β-estradiol itself is not significant. Cutoff value
of log fold change at 0.495 was applied to define up-regulated genes and -0.495 for down-regulated genes in all data sets (pvalue < 0.05).
Altered
expression
upon 2hourinduction
Altered
expression
upon all
inductions
(combined)
Data sets
Number of up-regulated genes
(with proportions in %)
Number of down-regulated
genes (with proportions in %)
A
181
112
B
188
171
A∩B
9
7
A
242
158
B
241
245
C
385
394
A∩C
7
1
B∩C
27
16
(A∩C)/A
2.9
0.6
(B∩C)/B
11.2
6.5
37
Table 2-2 Comparisons of the lists of up- or down-regulated genes of three independent microarray data sets indicating
number of overlapping genes with results also shown on Venn diagrams.
Up-regulated
Down-regulated
Upon
2-hourinduction
Upon all
inductions
(combined)
In case of the microarray data with 0, 2 and 6 hours induction scheme, although all the either up- or
down-regulated genes of both the 2 and 6 hours inductions were combined, only 2.9% of the genes
overlap among the up-regulated and 0.6% among the down-regulated genes (Table 2). In case of the other
microarray data with the 0, 1.5, and 2 hours induction scheme, 11.2% of the genes overlap among the upregulated and 6.5% among the down-regulated genes (Table 2). Although ratios of overlapping genes are
higher in the latter, no genes are overlapping when the three arrays are compared altogether suggesting
that the higher ratios are not due to β-estradiol treatment (Table 2). Together, results suggest that βestradiol treatment did not contribute to the changes of gene expression to a major extent that enables us
to safely exclude the possibility of the potential influence of chemical treatment.
38
III.3. Vascular patterning and cell division problem in phb-7d in relation to
plant hormones auxin and cytokinin
In order to investigate the poorly understood direct or indirect relation of the HD-Zip III transcription
factors with plant hormones auxin and cytokinin on root cell type patterning, I did various experiments
using the phb7-d mutant (Carlsbecker et al., 2010), which harbors a point mutation in the microRNA
target site that results in overexpression of PHB by inhibiting its degradation (Figure 13; Figure 14; Table
3). As described before, for exogenous application of hormones or inhibitor of hormone transport,
seedlings were germinated and grown on plates containing NAA, NPA, or BAP-added growth media.
Inhibition of auxin transport recovers protoxylem formation in phb-7d and causes loss of
protoxylem phenotype in wild type
The phb-7d mutant harbors a point mutation in the microRNA target site resulting in overexpression of
PHB by inhibiting its degradation. Without treatment, metaxylem forms in the place of protoxylem due to
this reason, and exogenous application of NAA, a synthetic auxin, could slightly recover formation of
protoxylem (Data not shown), whereas no phenotypic change in xylem structure was observed upon the
same treatment on wild type seedlings (Figure 13). The pattern of protoxylem recovery upon NAA
treatment on phb-7d was, however, difficult to quantify with light microscopy and hence further
characterization is required. It needs to be emphasized that, interestingly, treatment of NPA, an inhibitor
of auxin transport, could also slightly recover formation of protoxylem in phb-7d mutant seedlings
(Figure 13).
On the other hand, in wild type seedlings, treating with NPA resulted in a similar phenotype as
exogenous application of BAP, a synthetic cytokinin, which is consistent to the recent study of Bishopp et
al. (Bishopp et al., 2011b) (Figure 13). Bishopp et al. have shown that NPA treatment causes a dosagedependent loss of protoxylem phenotype in wild type seedlings (Bishopp et al., 2011b).
Xylem pattern of phb-7d is unresponsive to exogenous application of cytokinin
In phb-7d seedlings, protoxylem strands could not be observed in most parts of the root which is
consistent to the previous study of Carlsbecker et al. (Carlsbecker et al., 2010) (Figure 13), wherein they
39
show that phb-7d leads to ectopic metaxylem formation due to overexpression of PHB that causes altered
gradient of HD-Zip III transcripts level in the root vasculature (Carlsbecker et al., 2010). Loss of
protoxylem phenotype, which is highly similar to the phenotype of phb-7d mutants, could be frequently
observed upon exogenous application of BAP on wild type Col-0 seedlings, whereas phb-7d seedlings did
not exhibit any changes in xylem phenotype upon the same treatment (Figure 13). However, by only
analyzing the longitudinal axis, it is difficult to confirm whether this pattern in wild type is due to
additional formation of metaxylem or just simply due to loss of protoxylem, and Mähönen et al. had
previously shown that treatment of BAP on wild type seedlings rather leads to inhibition in protoxylem
formation that is different to ectopic metaxylem formation (Mahonen et al., 2006).
Figure 13 Analysis of root xylem phenotype in Arabidopsis wild-type Col-0 and phb-7d mutant seedlings upon
treatment of plant hormones or inhibitor of hormone.
Seedlings were germinated and grown on plates containing NAA, NPA, or BAP-added growth media. In wild type,
whereas NAA treatment does not induce any change on patterning of xylem cells in the root vasculature, exogenous
application of BAP results in a loss of protoxylem phenotype. A similar pattern can be observed upon treatment of NPA
as well although phenotypic change is much weaker. In phb7-d mutant seedlings, metaxylem forms in the place of
protoxylem without treatment. Exogenous application of NPA can slightly recover formation of protoxylem, and BAP
treatment does not exhibit any phenotypic changes. mx, metaxylem; px, protoxylem. (>50%) or (<50%) represents
whether more or less than half of the corresponding phenotype was observed along each of the xylem axis among the
20-25 seedlings analyzed. c.f. Root xylem phenotype of NAA treated phb-7d mutant seedlings were hard to distinguish
whether formation of metaxylem in the place of protoxylem occurs more or less than 50% along the xylem axis, but in
general, a slight recovery of protoxylem formation could be observed upon all the seedlings analyzed.
40
Cell division problem in phb-7d mutant seedlings
phb-7d mutant seedlings have very small root meristems. In order to assess if this is because of a
reduction in cell division, the cell cycle marker CYCB1:GUS (Colon-Carmona et al., 1999), which is
expressed when individual cells are mitotically active in division, was previously crossed into both wild
type Col-0 and phb-7d mutant backgrounds (Roberts C and Carlsbecker A, unpublished results). Number
of cells expressing GUS activity was counted in the root meristem of 5-day-old seedlings, and results
indicate significantly reduced number of actively dividing cells in phb-7d compared to wild type
seedlings (p-value = 4.49011E-06) (Figure 14). This result suggests that PHB inhibits cell division.
Figure 14 Analysis results of transgenic lines containing CYCB1:GUS construct in wild type Col-0 and phb-7d
mutant backgrounds upon treatment of plant hormones or inhibitor of hormone.
Number of cells expressing GUS activity was counted in the root meristem of 5-day-old seedlings of CYCB1:GUScontaining transgenic lines. phb-7d mutant seedlings exhibit significantly reduced number of actively dividing
cells in comparison to wild type seedlings. Exogenous application of BAP results in a reduced number of dividing
cells in wild type seedlings, and both NAA and NPA slightly recovers the cell division problem of phb-7d. *, pvalue < 0.05; **, p-value < 0.01. Error bars indicate standard deviation.
41
Both exogenous application of auxin and inhibition of auxin transport can recover cell
division problem in phb-7d
Similar to the above results of xylem phenotype analysis (Figures 13), exogenous application of BAP
resulted in a reduced number of dividing cells in wild type seedlings as it is in phb-7d (p-value =
1.48567E-09) (Figure 14), and both NAA and NPA could recover this cell division problem in phb-7d
mutant seedlings (p-value = 0.0480 for NAA; p-value = 0.0027 for NPA) (Figure 14). It is possible that
problem in auxin transport, likely an increased transport of auxin out of the root meristem, is more crucial
in comparison to the auxin synthesis problem in phb-7d, as treatment with NPA exhibits stronger effect
than NAA (Figure 14). However, this is difficult to prove with our current knowledge since the
biologically relevant concentration of auxin that reaches into the root meristem is yet unknown.
Non-detectable AHP6 expression in phb-7d upon treatment with plant hormones or inhibitor
of hormone
AHP6 allows formation of protoxylem by counteracting cytokinin signaling, and cytokinin signaling
negatively regulates the spatial domain of AHP6 expression (Mahonen et al., 2006). It has been also
reported that AHP6 is directly downstream of auxin signaling (Bishopp et al., 2011b). In order to identify
whether the recovery of protoxylem in phb-7d mutant is mediated through AHP6, previously prepared
transgenic lines containing AHP6:GFP construct in wild type and phb-7d mutant backgrounds were
analyzed (Table 3). It has been shown that expression of AHP6:GFP is reduced or undetectable in phb-7d
mutants (Carlsbecker et al., 2010), and although AHP6 was normally expressed in wild type background
upon treatments with NAA or NPA, GFP signal was not observed in phb-7d mutants when NAA or NPA
were exogenously applied (Table 3). Since cytokinin inhibits the expression of AHP6, signal could be
detected in neither Col-0 nor phb-7d mutant seedlings upon BAP treatment (Table 3).
42
Table 3 GFP analysis results of transgenic lines containing AHP6:GFP in wild type Col-0 and phb-7d mutant
backgrounds upon treatment with plant hormones or inhibitor of hormone.
Shown is whether GFP signal could be detected or not in the roots of 5-day-old seedlings containing AHP6:GFP
in wild type Col-0 and phb-7d mutant backgrounds. AHP6 is normally expressed in wild type background upon
treatments other than BAP, but no GFP signal could be detected in phb-7d mutants. +, expression of AHP6:GFP;
-, no expression of AHP6:GFP.
Treatment
Col-0
phb-7d
GM
+
-
+ 100nM NAA
+
-
+5µM NPA
+
-
+ 100nM BAP
-
-
43
III.4. Loss-of-function mutant analyses of Arabidopsis AGO proteins with focus
on root vascular cell type patterning
AGOs potential influence on root vascular cell differentiation
The Arabidopsis HYPONASTlC LEAVES 1 (HYL1) protein is involved in the processing of primary
miRNA precursors (Han et al., 2004), and hyl1-1 mutant has been reported to display metaxylem
formation in the place of protoxylem due to increased level of HD-Zip III expression in this microRNA
deficient line (Carlsbecker et al., 2010). On the other hand, it has been also shown that zll-3 mutant
occasionally exhibits two protoxylem strands per pole that is similar to the phenotype observed when
expression of HD-Zip III is suppressed (Carlsbecker et al., 2010).
Interestingly, compared to wild type, we find a small increase in AGO10 expression in our two
additional microarray data of which one is from induction of miR165 and the other from analyses of the
quadruple loss-of-function mutant cna phb phv rev (Roberts C, Beste L, Carlsbecker A, unpublished
results). Therefore, it is possible that the HD-Zip III transcription factors might repress expression of
AGO10 forming a possible feedback loop between each other which further motivates our intention of
analyzing ago mutants.
It has been known that mutations in either AGO1 or AGO10 lead to changes in the expression level of
HD-Zip III transcription factors that is mediated by regulation of miRNAs (Kidner and Martienssen, 2004;
Liu et al., 2009), and hence it is possible that this might induce altered cell identities in the vascular
system. Since AGO1 is a crucial component of the RISC complex of miRNAs, mutation in the gene could
hinder the degradation of HD-Zip III transcripts in the vasculature resulting in a phenotype similar to that
of the gain-of-function mutant of PHB showing ectopic metaxylem formation, and vice versa, mutation in
the counteracting partner AGO10 leading to a phenotype similar to that of the loss-of-function mutants of
HD-Zip III transcription factors causing altered arrangement of protoxylem and metaxylem.
To test the hypothesis that the AGO proteins might have potential influence on root vascular cell
differentiation, xylem phenotypes were analyzed in two different mutant lines at different ages; ago1-3
and zll-3 (Bohmert et al., 1998; Moussian et al., 1998; Moussian et al., 2003), in which zll-3 is a loss-offunction allele of AGO10. Comparisons were made with each of the mutants corresponding Arabidopsis
ecotypes, and two independent analyses were preformed when seedlings were either 4-day-old or 6-dayold (Figures 15).
44
Figure 15 Xylem phenotype analysis results of 4-day-old and 6-day-old loss-of-function ago mutants.
Mutants are shown with their corresponding Arabidopsis wild types; ago1-3 is in Col-0 background, and zll-3 is in Ler
background. Results indicate that protoxylem forms in the place of metaxylem more often in the roots of mutant seedlings.
px, protoxylem; mx, metaxylem. (>50%) or (<50%) represents whether more or less than half of the corresponding
phenotype was observed along each of the xylem axis among the seedlings analyzed with ‘n’ indicating the number of
seedlings.
Among different Arabidopsis wild types, a pattern of forming protoxylem next to protoxylem in the
place of metaxylem could be identified more often in the Ler ecotype in comparison to Col-0 (Figure 15).
However, even in this regard, zll-3, which is in Ler background, still exhibited a significant difference
where majority of the seedlings have shown ectopic protoxylem formation in the place of metaxylem, and
a more severe pattern could be observed in ago1-3 seedlings (Figure 15).
Together, although mutation in AGO1 does not exhibit ectopic formation of metaxylem as assumed, zll3 indicates altered cell type patterning in the root vasculature which is consistent with the above
suggested hypothesis. However, as both ago1-3 and zll-3 exhibit a similar xylem phenotype of forming
two protoxylem strands per xylem pole, it is hard to conclude if the observed phenotype of zll-3 is due to
repressed expression level of HD-Zip III transcription factors. This finding of two protoxylem strands per
pole in zll-3 may indicate either repressed level of HD-Zip III transcription factors or elevated level of
PHB as it was previously observed upon induction of phb-1d, and further study needs to be done in order
to define the interaction between HD-Zip III and AGO10.
45
IV. Discussion
Direct involvement of PHB in the regulatory mechanism of cell type patterning and
comparison of efficiency between phb-1d and miR165 inducible lines
This study has corroborated our previous knowledge that formation of metaxylem is mediated by PHB
in the vascular system of Arabidopsis roots by showing the direct involvement of PHB in the regulatory
mechanism of cell type patterning. However, compared to induction of miR165, the degree of phenotypic
change after inducing phb-1d was milder than what was expected. It is possible that other members of the
HD-Zip III transcription factors regulate the process together with PHB, thereby inducing solely phb-1d
might not have been enough to fully convert the pattern of cell type acquisition unless phb-1d was
constitutively expressed from the time of germination. Correspondingly, the strong phenotypic alteration
caused by inducing miR165 can be explained in the same context as miR165/166 down-regulates not only
PHB but also other members of the class III HD-Zip family.
Another hypothesis is that this could be also due to the dimerization pattern of HD-Zip III members. It
has been known that plant HD-Zip proteins efficiently bind DNA by forming homo- or heterodimers
between members in the same family or with other proteins (Johannesson et al., 2001; Chandler et al.,
2007). Therefore, solely inducing only one of the components could be not sufficient to result in
significant alterations if the HD-Zip III members work as heterodimers.
However, it is also possible that the phb-1d inducible transgenic lines in both wild type and wol mutant
backgrounds are relatively weaker than the miR165 inducible line. In case of the miR165 inducible
transgenic line, three independent lines were previously analyzed and the strongest line was chosen for
following experiments, whereas only one single line was assessable for the phb-1d inducible transgenic
lines in both wild type and wol mutant backgrounds. In this reason, the pCRE1(WOL)::XVE>>phb-1d
construct has been re-transformed into both Col-0 and wol mutants in order to acquire more lines for
comparison, and analyzing these new lines would provide further information of which of the above
suggested hypothesis is more likely.
After all, verifying PHBs relation with other members of the HD-Zip III transcription factors in the
regulatory mechanism is a remaining question that still needs to be addressed (Prigge et al., 2005). To
explore to what extend their roles are overlapping, and to identify each of their specific roles during the
process of vascular development might be another major challenge that has not yet been approached.
Furthermore, as this study have provided information about the performance of the newly prepared
46
transgenic line wherein expression of PHB is inducible in wild type background, promising further
studies can be followed by utilizing this line that can shed lights on the specific role of PHB in other
developmental aspects as well.
Trend of forming two protoxylem strands per pole and observation of normal growth
pattern near hypocotyl despite of the constitutive induction of phb-1d
Although it has been shown that constitutive induction of phb-1d and miR165 results in a metaxylem
and protoxylem-only phenotype, respectively, there is a tendency to form an additional protoxylem strand
next to the normal protoxylem, if the induction is not strong enough to fully convert the pattern of cell
type differentiation. It is possible to interpret this additional protoxylem strand formation as an
intermediate type of phenotypic alteration that could be induced when abnormality in the regulatory
pathway had been detected by the xylem-forming procambial cells during differentiation. In other words,
induction of phb-1d could affect the patterning of xylem cell type differentiation, but not strong enough to
lead the cells to differentiate as metaxylem, eventually resulting in a milder alteration that causes the cells
to exhibit abnormal protoxylem formation. Interestingly, it has been previously shown that similar pattern
of forming additional protoxylem strands can be also seen in double and triple mutants of the HD-Zip III
members (Carlsbecker et al., 2010).
Although constitutive induction of phb-1d is supposed to inhibit formation of protoxylem, in regions
higher up along the roots, protoxylem formation could still be identified in both wild type and wol mutant
backgrounds. This possibly reflects a normal growth pattern of seedlings during early developmental
stages despite of the constitutive induction of phb-1d. Pre-determined cell identity during embryogenesis,
wherein the fate of xylem cells become already fixed before the induction of phb-1d begins in the
transgenic lines, could be a possible hypothesis in order to explain this observation. However, constitutive
induction of miR165 does not exhibit the same pattern, and hence it is possible that the identity of the
peripheral cells are established earlier than central cells, thereby making the central cells more easily
influenced when exposed to either phb-1d or miR165. This hypothesis can be further supported by the
fact that metaxylem normally differentiates later than protoxylem. Another possible explanation for this
observed trend would be the stronger effect of miR165 induction in comparison to phb-1d induction as it
was suggested before at the beginning of this discussion.
47
Boundary of proto- and metaxylem cell type acquisition
So far, it is believed that the xylem-forming procambial cells differentiate to either proto- or metaxylem
as they have readily distinguishable structures, wherein metaxylem exhibits pitted secondary cell wall
thickenings whereas protoxylem shows a spiral lignin deposition pattern (Carlsbecker et al., 2010;
reviewed in Vera-Sirera et al., 2010). Scarpella E and Helariutta Y describe this such as protoxylem and
metaxylem differentiates in a mutually exclusive manner (reviewed in Scarpella and Helariutta, 2010).
However, observations that might seek against this general belief has been found in this study, as xylem
structures exhibiting intermediate type of protoxylem and metaxylem could be seen upon analyses.
Although these xylem strands exhibit a spiral lignin deposition pattern that of the protoxylem, noncomplete pitted secondary cell wall structures are also detectable making these xylem strands difficult to
define as proto- or metaxylem. Nonetheless, merely with this observation it is not enough to contradict the
general belief of the mutually exclusive differentiation pattern of proto- and metaxylem. Further
investigation is required to verify if this observation is valid, and analyzing these xylem cells molecularly
as a mean of determining their identity would provide fruitful clues.
Global transcriptome analysis of the HD-Zip III transcription factor PHB
As potential downstream targets of HD-Zip III transcription factors were not identifiable due to the
inconsistency of our microarray data, additional comparison was made with a previously published
microarray data from Liu et al. in order to check whether β-estradiol treatment might have been the
source of the observed discrepancy (Liu et al., 2008). In our experiment, only the root tips of seedlings
were collected at the age of 4 days after germination (DAG), whereas Liu et al. had used the entire
seedlings for their experiment at the age of 9 DAG, and hence it is not surprising that not many genes are
overlapping between the compared gene lists (Liu et al., 2008). Additionally, induction time of Liu et al.
was 12 hours, which is longer than our experiments, and the way of treatment was also slightly different
as they had applied 10 µM of β-estradiol directly to the growth medium for induction (Liu et al., 2008),
whereas seedlings were transferred to β-estradiol containing growth media using a thin mesh in our
experiment.
48
However, these differences have relatively minor influence on our conclusion because the purpose of
comparison was to verify potential effects of the treatment itself regardless of the duration of treatment or
utilized methods. Therefore, although the comparison was not ideal, we can safely conclude that βestradiol application is not the major cause of gene expression changes observed in our experiments. As
our initial aim of identifying potential down-stream targets of the HD-Zip III transcription factors could
not be reached with this study, either repetition or alternative experiments would be required in order to
further achieve this goal. Utilizing the before described pCRE1(WOL)::XVE>>phb-1d in wild type
background for these experiments might be a good idea, and using Chromatin Immunoprecipitation (ChIP)
followed by ChIP-PCR assays with primers against candidates that are selected by analyzing the
combined array results would be another good approach in order to fulfill the purpose. ChIP-on-chip
(ChIP-chip) and ChIP-sequencing (ChIP-seq) are other alternative independent methods to identify direct
downstream targets of HD-Zip III transcription factors (Aparicio et al., 2004; Johnson et al., 2007; Jothi et
al., 2008).
Linkage of auxin transport problem in phb-7d mutant with cytokinin signaling and possible
AHP6-independent pathways of protoxylem formation
Auxin and cytokinin form complex feedback loops in regulating various developmental aspects of
Arabidopsis (reviewd in Moubayidin et al., 2009; reviewed in Bishopp et al., 2011a). Bishopp et al. have
recently shown that NPA treatment causes dose-dependent loss of protoxylem in wild type seedlings that
is similar to phenotypes observed upon treating wild type seedlings with BAP (Bishopp et al., 2011b), and
this is consistent with what was seen in this study. It has been suggested that auxin and cytokinin form a
feedback loop that is capable of specifying vascular patterning (Bishopp et al., 2011b), and phloemtransported cytokinin regulates polar auxin transport in order to maintain the vascular pattern in root
meristem (Bishopp et al., 2011c).
Upon exogenous application of BAP, wild type seedlings exhibit loss of protoxylem phenotype in the
vasculature and reduced number of actively dividing cells in the root meristem (Mahonen et al., 2006),
which is similar to the phenotype of phb-7d mutant seedlings. Therefore, it is possible that phenotypes of
phb-7d could be partially due to an increased level of cytokinin biosynthesis or signaling. However,
evidences for this hypothesis are not yet enough and further studies would be required for clarification.
For instance, crossing phb-7d to mutants with compromised cytokinin signaling, such as wol, would be
able to provide more clues in connection to this hypothesis.
49
Surprisingly, treatment with both NAA and NPA could recover not only the formation of protoxylem
but also the number of actively dividing cells in the roots of phb-7d mutant seedlings, and this suggests
that phb-7d might have problems in both auxin synthesis and transport. Regarding the degree of recovery
in cell division, treatment with NPA exhibits stronger effect than NAA. Hence, it is possible that problem
in auxin transport, likely an increased transport of auxin out of the root meristem, is more crucial than the
auxin synthesis problem in the phb-7d mutant. Again, as cytokinin plays a crucial role in auxin transport
by regulating bisymmetric localization of the PIN-FORMED (PIN) auxin efflux proteins in the root
vascular system (Pernisova et al., 2009; Ruzicka et al., 2009; Bishopp et al., 2011b), it is possible that this
problem in auxin transport could also occur due to altered level of cytokinin biosynthesis or signaling as
suggested above.
Although it has been shown that AHP6 allows formation of protoxylem by counteracting cytokinin
signaling (Mahonen et al., 2006), in this study, no signal of AHP6:GFP could be identified upon
treatment of NAA and NPA on phb-7d mutant seedlings despite of the observed recovery in protoxylem
formation. This might suggest the potential existence of other separate pathways in formation of
protoxylem that is not mediated by AHP6, which is further supported by the fact that protoxylem
formation can be observed in ahp6 mutants as well (Mahonen et al., 2006). It also needs to be pointed out
that, although it has been reported that AHP6 is directly downstream of auxin signaling (Bishopp et al.,
2011b), in this study, AHP6 was neither responding when NAA was exogenously applied on phb-7d
mutant seedlings.
Influence of AGO1 and AGO10 on root vascular differentiation and the potential redundancy
between AGO1 and AGO10
Xylem phenotype analysis of ago mutants revealed that mutation in AGO1 does not exhibit ectopic
formation of metaxylem, but rather a milder phenotypic alteration of forming two protoxylem strands per
xylem pole. The reason that loss-of-function AGO1 mutant does not exhibit more severe phenotypes
could be due to AGO1s potential redundancy with AGO10 in the RISC formation. Regarding the crucial
role of the miRNA-related regulatory mechanisms during the entire life cycle of Arabidopsis, it is not
difficult to assume that redundancy between members consisting RISC complex might exist so as to
enhance rate of survival in case problem occurs with one of the members in nature. Therefore, assessing
root xylem phenotype of ago1 ago10 double mutant might be a good approach in order to clarify this
hypothesis, wherein the disorganized shoot system of ago1 pnh double mutant was previously analyzed
that exhibits slender and filamentous organs in an unpredictable arrangement (Lynn et al., 1999).
50
As described above, the inducible phb-1d transgenic line exhibits formation of additional protoxylem
strands, in which protoxylem forms next to protoxylem in the place of metaxylem, when the induction is
not strong enough. Since a similar pattern could be observed in ago1-3 mutant seedlings, it is also
possible to interpret this result in the same context of what has been mentioned before regarding the phb1d induction results. It might be possible that mutation in AGO1 affects the patterning of xylem
differentiation, but could only induce a milder alteration that causes the xylem-forming procambial cells
to differentiate as abnormal protoxylem strands, not being able to cause stronger changes that can lead to
ectopic metaxylem formation. However, it needs to be emphasized that this can be also due to the
potential redundancy of AGO1 with AGO10.
Whereas AGO1 is a key player in the regulatory mechanism of miRNAs, AGO10 sequesters
miR165/166 from their binding to AGO1, thereby up-regulating the transcript level of HD-Zip III family
genes (Zhu et al., 2011). Therefore, mutation in AGO10 was assumed to lead to a phenotype similar to
that of the loss-of-function mutants of HD-Zip III transcription factors causing altered arrangement of
protoxylem and metaxylem, and zll-3 which is a loss-of-function allele of AGO10 exhibited altered
arrangement of protoxylem and metaxylem as expected. This indicates the possibility that AGO10 could
not successfully perform its role of sequestering miR165/166, thereby altering the gradient of HD-Zip III
transcript level that resulted more cells to differentiate as protoxylem. However, it is interesting that the
phenotype is more severe in 4-day-old seedlings than 6-day-old seedlings, which suggests a recovery
mechanism could be triggered as the seedlings get older. This might be possible by employing alternative
pathways of regulating miRNA activity after abnormality in cell type patterning has once occurred.
Together, results suggest certain influences of both AGO1 and AGO10 on vascular differentiation, and
further studies would be necessary for a more detailed verification of their direct or indirect regulatory
mechanism on vascular cell type patterning in relation to HD-Zip III transcription factors. Interestingly,
observations of xylem phenotype in different Arabidopsis ecotypes indicated that Ler exhibits altered cell
type differentiation more often than Col-0. It is possible that there might be a factor in Col-0 which could
render Col-0 more stable against inappropriate identity acquisition of xylem cells.
51
V. Conclusion
After all, using an inducible system, this study has shown that formation of metaxylem is mediated by
PHB in the vascular system of Arabidopsis roots which suggests the direct involvement of PHB in the
regulatory mechanism of cell type patterning. Inducing expression of PHB in both wild type and wol
mutant backgrounds causes ectopic metaxylem formation in a dosage-dependent manner in the root
vascular system, and induction of miR165 results in an opposite phenotype exhibiting a protoxylem-only
phenotype (Experiment I). Potential downstream targets of HD-Zip III transcription factors were not
identifiable through the performed global transcriptome analysis due to inconsistency of data sets, and
comparison with a previously published microarray data could prove that treatment of β-estradiol is not
the source of observed discrepancy (Experiment II).
Furthermore, by treating the gain-of-function of PHB mutant, phb-7d, with plant hormones auxin and
cytokinin or inhibitor of auxin transport, clues were suggested that observed phenotypes of phb-7d could
be mediated through increased level of cytokinin biosynthesis or signaling, and this study also suggests
the possible existence of AHP6-independent pathways in protoxylem formation (Experiment III). Last but
not least, analysis of loss-of-function AGO mutants indicated that both AGO1 and AGO10 are directly
involved in root vascular development as mutations in these genes result in altered xylem cell type
patterning, although mutation in AGO1 does no exhibit a severe phenotype that can be due to AGO1s
potential redundancy with AGO10 in the RISC formation (Experiment IV).
52
VI. Acknowledgements
First of all, I sincerely appreciate my supervisor Annelie Carlsbecker for her kind and generous, critical
guidance during the whole project. I thank Christina Roberts for her practical help and advices with
experiments, and also other members at the ‘Physiological Botany’ lab, Mattias Myrenås and Gun-Britt
Berglund, especially Ana-Elisa Valdés for being my opponent on my thesis defense. Great thanks to Peter
Engström for helping me with receiving the stipend ‘Törnlund, for the Faculty of Science and Technology
(1892)’ from Uppsala University, and also would like to say thanks to our group members at the Swedish
University of Agricultural Sciences, Eva Sundberg, Mattias Thelander, Jens Sundström, Katarina
Landberg, Emma Larsson, Tom Martin, Eric Pederson, Izabela Cierlik, and Andrea Claes for their helpful
comments upon group meetings.
I appreciate Maggie Cheung for her support during time of difficulties, and last but not least, I warmly
thank my parents for their trust and support during my study in Uppsala, Sweden.
53
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