Download The role of the PXY-CLE signalling pathway in regulating cell

The role of the PXY-CLE signalling
pathway in regulating cell division
during wood formation in Poplar
A thesis submitted to the University of
Manchester for the degree of MPhil
Biotechnology in the
Faculty of Life Sciences
2016
Laxmi Mishra
Table of Contents
Abstract ....................................................................................................................... 5
Declaration .................................................................................................................. 6
Published work ........................................................................................................... 6
Note on Copyright ...................................................................................................... 6
Acknowledgements ..................................................................................................... 8
Abbreviations ............................................................................................................. 9
List of Figures ........................................................................................................... 11
List of Tables ............................................................................................................ 13
Chapter 1: Introduction............................................................................. 14
1.1 Why Woody biomass? ............................................................................................. 14
1.2 Models for the studying wood formation .............................................................. 14
1.2.1 Arabidopsis thaliana (annual weed) .............................................................. 14
1.2.2 Poplar .................................................................................................................. 15
1.3 Vascular tissue .......................................................................................................... 16
1.3.1 Procambium and Vascular Cambium ............................................................. 17
1.3.2 Xylem .................................................................................................................. 19
1.3.3 Phloem ................................................................................................................ 20
1.3.4 Interfascicular fibers.......................................................................................... 22
1.4 Primary growth and vascular pattern ..................................................................... 23
1.5 Secondary growth function of Vascular Cambium .............................................. 25
1.6 Regulatory mechanisms of Vascular cambium during secondary development27
1.7 Novel Peptide-receptor signalling pathways: radial vascular patterning .......... 30
1
Aims and objectives: ................................................................................................ 35
Chapter 2: Material and Methods ....................................................... 36
2.1 Materials .................................................................................................................... 36
2.1.1 Chemicals and reagents .................................................................................... 36
2.1.2 Plant Material ..................................................................................................... 36
2.1.3 Bacterial strains.................................................................................................. 36
2.1.4 DNA Vectors ...................................................................................................... 36
2.1.5 Oligonucleotide primers ................................................................................... 37
2.1.6 Software .............................................................................................................. 37
2.2 Plant Material and Growth Conditions .................................................................. 40
2.2.1 Arabidopsis thaliana plants.............................................................................. 40
2.2.2 In vitro propagation of Populus tremula x
Populus tremuloides T89 (hybrid aspen) ................................................................ 40
2.3 Generating and analysing DNA constructs ........................................................... 41
2.3.1 Plasmid isolation protocol ................................................................................ 41
2.3.2 Polymerase chain reaction (PCR).................................................................... 41
2.3.3 Restriction Digest .............................................................................................. 42
2.3.4 Agarose gel electrophoresis ............................................................................. 43
2.3.5 Gel extraction ..................................................................................................... 43
2.3.6 DNA Sequencing reaction ................................................................................ 43
2.3.7 Construction of Entry clone ............................................................................. 44
2.3.8 Construction of a Gateway® destination vector............................................ 44
2.3.9 Restriction Based cloning ................................................................................. 46
2.4 Transformation of Electro-competent Agrobacterium tumefaciens ................... 47
2.4.1 Preparation of Electro-competent Agrobacterium tumefaciens .................. 47
2.4.2 Transformation of Electro-competent Agrobacterium tumefaciens ........... 48
2
2.5 Transformation of Plants ......................................................................................... 48
2.5.1 Arabidopsis thaliana plants by floral dipping ................................................ 48
2.5.2 Screening Transgenic Arabidopsis thaliana plants ....................................... 49
2.5.3 Hybrid aspen T89 plants by co-cultivation .................................................... 49
2.5.4 Screening transgenic hybrid aspen T89 plants .............................................. 50
2.5.5 Transplanting into Greenhouse ........................................................................ 50
2.6 Histological analysis................................................................................................. 51
2.7 Growth characteristics analysis of transgenic plants ........................................... 52
2.7.1 Transgenic Arabidopsis thaliana plants ......................................................... 52
2.7.2 Transgenic hybrid aspen T89 plants ............................................................... 53
2.8 Β-glucuronidase (GUS) marker gene expression ................................................. 53
2.9 Gene Expression Analysis ....................................................................................... 54
2.9.1 RNA extraction .................................................................................................. 54
2.9.2 First strand synthesis ......................................................................................... 54
2.9.3 Semi-quantitative and Q-PCR to determine gene expression levels .......... 55
Chapter 3: Results ......................................................................................... 56
3.1 Generating PttPXY and PttCLE41 over-expression vectors ............................... 56
3.2 Effects of over-expression of PttPXY and PttCLE41 genes in Arabidopsis ..... 60
3.3 Cloning the binary vector rolD::PttPXY 35S::PttPXY ........................................ 63
3.4 Identification and cloning promoters for tissue–specific overexpression vectors
........................................................................................................................................... 65
3.5 Generating transgenic Poplars for function analysis of poplar PXY and CLE in
trees. .................................................................................................................................. 70
3.6 Generating transcriptional reporter clones and function analysis of promoter
PttANT and PtPP2........................................................................................................... 71
3.7 Analysis of ectopic expression of PttPXY and PttCLE41 in hybrid aspen
(Populus tremula x P. tremuloides) .............................................................................. 73
3
3.8 Tissue-specific over-expression of PXY and CLE41 genes in hybrid aspen
(Populus tremula x P. tremuloides) enhance wood formation and deposition. ...... 75
3.9 Growth characteristics of hybrid aspen overexpressing PttCLE41/PttPXY...... 76
3.10 PttCLE41 and PttPXY expression analysis in PtPP2::PttCLE41PttANT::PttPXY over-expressor lines .......................................................................... 85
3.11 Clonal propagation of PtPP2::PttCLE41-PttANT::PttPXY transgenic hybrid
aspen.................................................................................................................................. 88
Chapter 4: Discussion.................................................................................. 90
Strategies to Increase tree growth ....................................................... 95
References ........................................................................................................... 98
Word Count: 20 260
4
Abstract
Focus on biomass production has increased due to the depletion of the nonrenewable sources of energy. Trees are the most important source of biomass. Being
perennial by nature, they produce the majority of terrestrially available biomass.
Therefore, there is a vital need for a good understanding of tree development. Using
Arabidopsis, previous studies in our lab have identified the influence of the receptor
kinase PXY and ligand CLE signalling pathways in regulating vascular cell division.
It has been shown that manipulating PXY-CLE signalling pathway increases
vascular cell division in Arabidopsis. PXY-CLE functions towards increasing the
stem cell population in the procambium/cambium of the Arabidopsis. In the current
study, we have altered the expression of poplar homologues of PXY and CLE41 in
attempt to increase cambial cell division. The poplar homologues of PXY and CLE41
genes were cloned from hybrid aspen (Populus tremula x P. tremuloides), and will
be referred to as PttPXY and PttCLE41. Using tissue-specific over-expression of
these PttPXY and PttCLE41 genes we were able to significantly increase the rate of
tree growth and biomass above ground. Not only did the poplars exhibit a two-fold
increase in the rate of wood formation, but they were also taller and possessed larger
leaves compared to the untransformed controls. Together these results suggest that
engineering PXY-CLE41 signalling offers an opportunity to dramatically increase
commercial tree productivity.
5
Declaration
No portion of the work referred to in this thesis has been submitted in support of an
application for another degree or qualification of this or any other university or other
institute of learning.
Published work
The bulk of this research has been published as a manuscript entitled "Wood
formation in trees is increased by manipulating PXY-regulated cell division" in the
journal Current Biology on April 20, 2015. The experiment performed by the coauthors was generating the SUC2::AtCLE41 Arabidopsis thaliana lines (Peter
Etchells),
generating
the
promoter
constructs
for
PttANT::PttPXY
and
PtPP2::CLE41 and amino acid alignment of the Arabidopsis and Poplar PXY and
CLE41 genes (Manoj Kumar) and Cellprofiler analysis (Liam Campbell). The entry
clones of PXY and CLE41 were generated by a lab colleuge before I started to work
on the project. Those entry clones were later modified by me due to the absence of
the stop codon in the gene sequence. The work contributed in the paper by the coauthors and mentioned in this thesis have been represented and cited as (Etchells et
al. 2015).
Note on Copyright
The author of this thesis (including any appendices and/or schedules to this thesis)
owns certain copyright or related rights in it (the “Copyright”) and s/he has given
The University of Manchester certain rights to use such Copyright, including for
administrative purposes.
6
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Reproductions described in it may take place is available in the University IP Policy
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Library,
The
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Library’s
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(see
http://www.manchester.ac.uk/library/aboutus/regulations) and in The University’s
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7
Acknowledgements
I would like to show my gratitude to Prof. Simon Turner to give me the opportunity
to do an MPhil under his guidance. I really appreciate the help from all the members
in the Turner Lab. I would especially like to thank Liam Campbell, Manoj Kumar
and Sophie Mogg who helped me a lot during thesis writing. I would like to thank
Peter Etchells and Manoj Kumar who taught me working with Arabidopsis and
gateway cloning.
Above all, this thesis would have not happened without the support of my parents,
husband, and my son Abhay and the Almighty.
8
Abbreviations
% wt
Percentage Wild-type
pc
procambium
°C
Degree Centigrade
ph
Phloem
Pi
pith
PNH
PINHEAD
PP2
Phloem Protein 2
PRE
REV pop REVOLUTA
Pt
Populus Tricocarpa
Amphivasal vascular
ABV1
AHK2/3
ANT
ATHB-15
bundle 1
ARABIDOPSIS HIS
KINASES 2/3
AINTEGUMENTA
Arabidopsis homeoboxleucine zipper protein-8
AtIPT3, 5
Arabidopsis
and 7
isopentenyltransferases
Adenosine
ATP/ADP
triphosphate/ adenosine
IPTs
diphosphate
Ptt
Populus tremula ×
Populus tremuloides
isopentenyltransferases
bHLH
cDNA
CIM
PXC1,
basic helix-loop-helix
2, 3
PXY-correlated 1, 2, 3
PHLOEM
Complementary
PXY
deoxyribonucleic acid
INTERCALATED WITH
XYLEM
Callus induction media
RAM
Root apical meristems
RIM
Root induction media
CLAVATA3/EMBRYO
CLE41/44
SURROUNDING
REGION-RELATED
41/44
CLV1
CLAVATA 1
RNA
Ribonucleic acid
Col
Columbia
rpm
Rotation per minute
COV-1
continuous vascular
RT
Room temperature
cr
cortex
SAC51
cz
cambial zone
SACL
SAC51-LIKE
dH20
Distilled water
SAM
Shoot apical meristems
9
SUPPRESSOR OF
ACAULIS 51
EDTA
Ethylenediaminetetraacetic
acid
SE-CCC
Sieve element-companion
cell complex
Ep
epidermis
SHR
SHOOT-ROOT
EtOH
Ethanol
SIM
Shoot induction media
GFP
Green flourescent protein
STM
SHOOTMERISTEMLESS
GUS
β- glucuronidase
SUC2
Sucrose Proton Symporter
2
Tracheary element
hca
high cambial activity
TDIF
differentiation inhibitory
factor
HD-ZIP III
If
ifl1/rev
KNOX
class III homeodomainluecine zipper
interfascicular region
TDR
TMO5
interfascicular
fiberless1/revolute
class I knotted-like
homeobox gene
Vb
Vascular bundle
Weight/ volume
w/v
LHW
LONESOME HIGHWAY
WOX4
mRNA
MS
Receptor Like Kinase
Messenger ribonucleic
acid
Murashige and Skoog
10
MONOPTEROS5
volume/volume
Luria-Bertani medium
Leucine-Rich Repeat-
TARGET OF
v/v
LB
LRR-RLK
TIDF-Receptor
WUSCHEL-like
HOMEOBOX 4
WPM
Woody plant media
WT
Wild-type
x
Xylem
ZeHB-
Zinnia homeobox-leucine
13
zipper protein- 13
List of Figures
Chapter 1
Figure 1.1: Schematic representation of plant vascular tissue .................................. 16
Figure 1.2: Vascular Patterning in Arabidopsis thaliana stem stained with Toluidine
blue. ............................................................................................................................ 17
Figure 1.3: Schematic representation of vascular bundle patterning ........................ 23
Figure 1.4: Transverse Arabidopsis thaliana stems section showing the initiation of
secondary growth induced by CLE41 expression ...................................................... 26
Figure 1.5: Transverse section of hybrid aspen ........................................................ 27
Figure 1.6: Resin-embedded transverse section of the wild-type and pxy mutant
hypocotyls and stem (vascular bundle). ..................................................................... 30
Figure 1.7: Resin-embedded transverse section of Arabidopsis thaliana showing the
phenotype in plants where CLE41 and PXY are over-expressed. ............................. 31
Chapter 3
Figure 3.1: Sequence alignment of amino acid sequences of PXY and CLE41.. ..... 58
Figure 3.1.1: 35S poplar PttPXY and PttCLE41 over-expression vectors made by
the LR clonase reaction. ............................................................................................. 59
Figure 3.2: The Poplar orthologues of CLE41 and PXY genes are functional in
Arabidopsis. ............................................................................................................. 60
Figure 3.2.1: Growth characteristics of Arabidopsis plants overexpressing poplar
PXY and CLE genes .................................................................................................. 62
Figure 3.3: Summary of generating pDONR-PttCLE41 vector. ............................... 64
Figure 3.3.1: Summary of generating rolD::PttPXY 35S::PttPXY vector. ............... 64
11
Figure 3.4: Summary of cloning PttPXY and PttCLE41 entry clones in custommade Gateway destination vectors pVX31 and pVX33 ............................................ 67
Figure 3.4.1: Maps for transcriptional reporters…………………………………...67
Figure 3.4.2: Generating the tissue-specific over-expressor PtPP2::PttCLE41PttANT::PttPXY ........................................................................................................ 69
Figure 3.5: Summary of the steps involved in production of transgenic poplars ..... 70
Figure 3.6: GUS staining of reporter gene lines ....................................................... 72
Figure 3.7: Phenotype of Populus tremula x P. tremuloides constitutively overexpressing PXY and CLE41. ..................................................................................... 73
Figure 3.8: Phenotypes of hybrid aspen with tissue-specific overexpression of
PttCLE41 and PttPXY ............................................................................................... 75
Figure 3.9: Growth characteristics of hybrid aspen lines in tissue culture 3 weeks
post-rooting ................................................................................................................ 79
Figure 3.9.1: Growth characteristics of hybrid Aspen (Populus tremula x Populus
tremuloides) lines constitutively overexpressing PttCLE41/PttPXY ........................ 80
Figure 3.9.2: Growth characteristics of trees with tissue-specific PttCLE41/PttPXY
overexpression............................................................................................................ 81
Figure 3.9.3: Characteristics of the morphology of hybrid aspen engineered with
tissue-specific PXY and CLE41 expression .............. Error! Bookmark not defined.
Figure 3.9.4: Biomass of hybrid Aspen tissue-specific overexpression of PXY and
CLE41 ........................................................................................................................ 83
Figure 3.10: PttCLE41 and PttPXY expression analysis in PtPP2::PttCLE41PttANT::PttPXY lines ................................................................................................. 86
Figure 3.10.1 Gene expression analysis by Q-PCR .................................................. 87
Figure 3.11. Analysis of clonal PtPP2::PttCLE41- PttANT::PttPXY lines ............... 88
12
List of Tables
Chapter 2
Table. 2.1. DNA vectors used in this study ............................................................... 37
Table 2.2. PCR primers used in the study ................................................................. 38
Chapter 3
Table 3.1. Analysis of wood morphology from hybrid aspen with tissue-specific
expression of PXY and CLE41 .................................................................................. 84
13
Chapter 1: Introduction
1.1 Why woody biomass?
Trees provide wood for numerous applications such as building houses, ships,
furniture, pulp for paper and also used as a source of fuel. However, the utilisation of
woody biomass can be considered controversial. While it is a renewable natural
resource, it is also exhaustible. Despite the fact that no energy resource or raw
material is ideal, wood from trees may be a practical option. Trees are perennial and
produce the majority of terrestrial biomass (Bhalerao et al. 2003), which makes
wood a candidate for a renewable source of energy. The demand for wood as
biomass has increased, leading to further climate change and other environmental
problems (Popp et al. 2014). The solution has been to try to grow wood in a similar
way to how we grow agricultural crops like wheat, barley and maize. Demand for
forests and forest products in the future will largely depend on the rotation of fast
growing tree species. This can be achieved by generating genetically improved trees
by various biotechnological methodologies, including gene transfer, in vitro
propagation and molecular breeding (Merkle and Dean 2000).
1.2 Models for studying wood formation
1.2.1 Arabidopsis thaliana (annual weed)
For over two decades Arabidopsis thaliana have been the organism of choice for
plant biology research (Koornneef and Meinke 2010). Important features like a short
generation time, small size and abundant seed formation by self-pollination makes it
easy to use for a range of experimental studies. Arabidopsis thaliana is used for
14
molecular genetics and studying vascular differentiation and patterning. The
identification, characterisation and availability of Arabidopsis thaliana mutants have
created opportunities to understand the molecular fundamentals of vascular tissue
development, (reviewed in (Ye 2002)).
1.2.2 Poplar
Populus is an appropriate model organism to study tree and woody perennial plant
biology. Populus trichocarpa (black cottonwood) genome sequencing (Tuskan et al.
2006) has opened new avenues for research in tree genetics and developmental
biology. The availability of the genome sequence has revealed details about genome
duplications and the organisations of gene families. Approximately 8000 pairs of
duplicated genes and 45,000 potential protein coding genes have been identified
(Tuskan et al. 2006). The ease of transforming poplar (hybrid aspen) has also
provided an opportunity to study tree genetics and biochemistry. Populus is, like
Arabidopsis, is found in the angiosperm Eurosids I clade. As a close relative, it is
likely to have conserved many gene functions (Jansson and Douglas 2007).
Populus has provided an opportunity to explore essential plant processes which are
not fully understood or cannot be studied in Arabidopsis, such as wood formation,
autumn senescence, flowering, determination of sex, and interactions with other
organisms (Jansson and Douglas 2007).
15
Figure
1
Figure 1.1: Schematic representation of plant vascular tissue. A schematic
representation of two embryo stages (mid-globular and early-heart) showing two and
four initial cells of the procambium (A), vascular tissue organization of the stem,
showing the xylem (blue), cambium (red) and phloem (orange) (B) and tissue
organization of primary stem showing xylem, cambium and phloem tissue from the
vascular bundle, showing transport of nutrients indicated by arrows (C). (Modified
from Boundless Biology 2016; Jouannet et al. (2015) )
1.3 Vascular tissue
Vascular tissues are essential for plant growth and survival. Vascular
tissue comprises of a complex conducting tissue made up of more than one cell type.
These networks of cells are capable of connecting vascular tissue to the leaves, the
shoots and the roots facilitating the transport of nutrients through the plant organs.
Xylem and phloem are the two fundamental units of the plant vascular system as
seen in (Figure 1.1C). The xylem transports and stores water, nutrients, plant
hormones and provides mechanical support to the plant, whereas the phloem
distributes photosynthetic molecules and is a key player in translocating sugars and
16
nutrients as well as proteins and transcripts that are essential for plant growth and
development (reviewed in (Ye 2002)).
Figure 2
Figure 1.2: Vascular patterning in an Arabidopsis thaliana stem stained with
Toluidine blue. Transverse section of an Arabidopsis wild-type stem showing the
collateral arrangement of the vascular bundles (A). Close-up of a single vascular
bundle (B). Section were stained with toluidine blue. The (x) xylem, (ph) phloem,
(if) interfascicular region, (pc) procambium, (cr) cortex, (ep) epidermis, (Pi) pith and
(Vb) Vascular bundle are indicated. Scale bars represent 50 µm (A) and 200 µm (B).
1.3.1 Procambium and Vascular Cambium
Root and shoot apical meristems namely RAM and SAM respectively are created
during the process of plant embryo development. The four initial cells of the
17
procambium are formed as early as the globular embryo stage (Figure 1.1A)
(reviewed in (Jouannet et al. 2015)). However, the vascular cambium, also known as
lateral meristem, appears at later stages of plant development due to the influence of
the hormone-triggered cellular differentiation mechanisms (Figure 1.1B) (reviewed
in (Schuetz et al. 2013)). During the early growth of stems and roots, procambial
initials originating from the apical meristem produce primary xylem and phloem
(Chaffey 2003). However, subgroups of cells present in the procambium are left in
an undifferentiated form, sandwiched between the differentiating xylem and phloem
tissues (Chaffey 2003). The undifferentiated cells function as vascular stem cells and
facilitate the continued development of vascular cambium that can further give rise
to the secondary growth in plants (reviewed in (Schuetz et al. 2013)). The vascular
cambium zone consists of a few layers of narrow, elongated and thin walled juvenile
cells (initials), which further divide into xylem and phloem mother cells. The
increased and continuous division of the xylem mother cells give rise to more xylem
cells than phloem cells which is the reason for the significant disproportion present
between the xylem and phloem tissues (Plomion et al. 2001) (Figure 1.2).
The newly formed shoot and root tissues are derived by these two meristems (RAM
and SAM) but the formation of the new organs is tightly co-ordinated with the
procambium formation and the organisation of the vascular tissues (Bayer et al.
2009). Later in development, especially in the woody plants (trees), the vascular
cambium initials originate from the procambium and parenchyma cells during plants
secondary growth (Savidge 2001). These give rise to secondary xylem, also called
wood and to secondary phloem. In woody plants the vascular cambium is
characteristically comprised of two types of fusiform initials that generate tracheary
18
elements and xylary fibres along the longitudinal axis of wood, and ray initials that
make ray parenchyma cells along the transverse axis of wood (reviewed in (Ye
2002)). The cambium is fundamental for the radial expansion in the angiosperms and
gymnosperms, especially the wood produced in trees. The cambium is a key player
in maintaining the perennial life cycle of trees by regularly adding xylem and
phloem (Plomion et al. 2001).
1.3.2 Xylem
Xylem cells are found in all the vascular plants, including the angiosperms and the
gymnosperms.
Mature xylem tissue consists of conducting and non-conducting
elements. In gymnosperms, water conducting cells are known as tracheids whereas
in angiosperm (hardwood) trees the water conducting cells are known as vessels. The
water conducting cells (vessels and tracheids) are collectively referred as tracheary
elements (T.E’s) (Zhao et al. 2008). The vessels in angiosperms are interspersed with
non-conducting cells composed of either xylary fibres of xylem parenchyma
(Nieminen et al. 2015). In angiosperms, the xylem vessels are connected to one
another by perforation plates forming a continuous hollow column (reviewed in
(Schuetz et al. 2013)). The key processes in xylem development are: the formation
of procambium and vascular cambium, initiation of xylem differentiation, cell
elongation, secondary wall thickening, and cell death (reviewed in (Schuetz et al.
2013)). Programmed cell death is crucial for the differentiation of xylem vessels.
Although there is huge pressure from surrounding cells in conjunction with the
negative pressure produced by the flow of water through the xylem, T.E’s are
capable of preserving their shape due to secondary cell wall thickenings (Pockman et
al. 1995). During early plant development two different types of TE’s are formed
19
called the protoxylem and metaxylem. The protoxylem characteristically appears to
be contained as helical secondary cell wall thickenings, while the meta-xylems are
characterised by their complex reticulate thickenings (Larson 1976). In contrast, the
short, flat ended xylem parenchyma cells do not undergo cell death. The conversion
from primary growth to secondary growth follows an acropetal pattern of
development (Dengler 2001).
1.3.3 Phloem
The procambial or vascular cambial stem cells are considered to be the source of
phloem predecessor cells that differentiate into particular phloem cell types. A fully
developed phloem tissue comprises of conducting sieve tube elements, nonconducting cells called as parenchyma cells, the phloem companion cells, and, in
many cases, the phloem fibres (reviewed in (Ye 2002; Schuetz et al. 2013)). The
function of the sieve tube elements is to carry metabolites from the source tissues,
like leaves, to metabolism sites, namely the meristems (roots and shoots). While
differentiating sieve tube elements undergo extensive morphological and intracellular alterations, during development they lose their organelles such as the
nucleus, yet interestingly they are still physiologically alive. The sieve tube elements
are attached to the phloem companion cells via a number of plasmodesmata that
form cytoplasmic bridges allowing the companion cells capable of maintaining the
metabolic activities of the sieve tubes (Kempers and van Bel 1997). Together these
cells form the sieve element-companion cell complex (SE-CCC) (reviewed in
(Turner and Sieburth 2003)). The companion cells when in contact with the phloem
parenchyma cells they form cell wall in growths. This organisation allows the cargo
to be loaded in the companion cells and delivered via the long-distance transport in
20
the sieve elements (reviewed in (Turner and Sieburth 2003)). Compared to the
current advancements in understanding xylem development, not much is known
about the precise governing factors that are involved in the developmental processes
of phloem and phloem cell fates, such as the formation of sieve tube or companion
cells (reviewed in (Schuetz et al. 2013)). The first genes identified that plays an
important role in phloem development was ALTERED PHLOEM DEVELOPMENT
(APL) (Bonke et al. 2003). APL is an MYB coiled-coil transcription factor that is
expressed during the early stages of phloem development in which it is crucial for
the differentiation of sieve elements and the companion cells (Bonke et al. 2003).
Mutation in apl demonstrated aberrations in phloem differentiation. The apl mutants
roots possessed cells similar to xylem cells, that were found at the position of the
phloem cells. The authors also demonstrated that ectopic expression of APL can
suppress xylem differentiation in the vascular bundle (Bonke et al. 2003). Further
studies on apl mutant during early phloem development by Truernit et al. (2008)
revealed the absence of abnormalities in cell elongation and cell wall thickening.
Work published by Mouchel et al. (2006), Scacchi et al. (2009) and Scacchi et al.
(2010) have showed that BREVIS RADIX (BRX) expressed in the protophloem was a
direct target of MONOPTEROS (MP). The authors also demonstrated that BRX is
important for protophloem differentiation. Further studies were carried out on the
short-root phenotype of brx by Depuydt et al. (2013). The authors showed that
mutating the second site in BARELY ANY MERISTEM3 (BAM3) can suppress the
short-root phenotype of brx. BAM3 is expressed in the roots in which the expression
is restricted in the differentiating phloem region that also, overlaps with the BRX
expression domain (Depuydt et al. 2013). They also demonstrated that on
exogenously applying CLAVATA3/ENDOSPERM SURROUNDING REGION 45
21
(CLE45) leads to arrest in root growth and aberrations in the
protophloem
differentiation (Depuydt et al. 2013).
1.3.4 Interfascicular fibers
The interfascicular fibres are assembled to form lignified arcs throughout the stem,
separating the vascular bundles (Figure 1.2). Although, the interfascicular fibres are
not considered to be vascular tissue their differentiation is absolutely fundamental to
vascular arrangement and patterning (Figure 1.2) (Altamura et al. 2001). By the
identification of the Arabidopsis mutant interfascicular fiberless1/revolute (ifl1/rev)
in it is clear that interfascicular fibres play an important role in vascular patterning
(Zhong et al. 1997; Zhong and Ye 1999; Zhong and Ye 2004). The ifl1 mutants
exhibits an absence of the interfascicular fibres between the vascular bundles and
reduced secondary xylem. It has been shown that the ifl1/rev gene encodes a class III
homeodomain-luecine zipper (HD-ZIP III) protein, which is involved in regulating
cell differentiation in Arabidopsis inflorescences stems (Zhong and Ye 1999). The
author also showed that IFL1 gene is expressed in the interfascicular region where
the fibers differentiate and the vascular regions. This finding is consistent with the
role of IFL1 gene of controlling interfascicular fiber differentiation (Zhong and Ye
1999). Further studies have demonstrated that ifl1/rev can influence the auxin polar
flow that is essential for fibre and vascular differentiation (Zhong and Ye 2001).
22
1.4 Primary growth and vascular pattern
Figure 3
Figure 1.3: Schematic representation of vascular bundle patterning. The phloem
surrounds the xylem is amphicribral (A), the xylem surrounds the phloem is
amphivasal (B) and with phloem on the outside and xylem in the centre is collateral
(C).
The common vascular patterns are amphicribral as seen in Figure 1.3A (phloem
surrounds the xylem), amphivasal as seen in Figure 1.3B (in which xylem
surrounding the phloem), and as collateral bundles as seen in Figure 1.3C. The latter
is the most common, in which the xylem and the phloem surrounding the pith in the
centre and phloem on the margin of the stem as in Arabidopsis Figure 1.2A. The
procambial cells give rise to highly organised vascular cells presenting the phloem at
the outer ring and xylem towards the centre of the stem. Figure 1.2B shows a section
of a mature five week old plant in which the vascular bundles comprise mainly of
files of xylem and phloem cells and comparatively fewer procambial cells (Turner
23
and Somerville 1997) Each vascular bundles are separated from one another by the
interfascicular region.
Several mutants have been identified that affect vascular cell organisation. A semidominant mutation in the gene AVB1 in Arabidopsis leads to a replacement of the
collateral phloem and xylem in a vascular bundle with amphivasal organisation. The
avb1 mutant shows a phenotype of disrupting the ring-like organization in the stem
and also exhibits a prototype indicative of monocot stems that includes several
vascular bundles that penetrate into the pith (Zhong et al. 1999). Further studies by
Parker et al. (2003) have reported that the COV-1 (Continuous Vascular-1) gene
mutation in Arabidopsis promotes vascular tissue development in the interfascicular
region that normally separates the vascular bundles. In cov1 mutant, differentiated
vascular tissue forms a continuous ring-like pattern of the phloem and xylem, with
almost negligible interfascicular tissue and therefore no vascular bundle pattern.
COV1 is likely to be a membrane protein or a signalling molecule that negatively
regulates the differentiation and development of vascular tissue in the Arabidopsis
stem (Parker et al. 2003). Another study has identified high cambial activity (hca) as
a mutant that has increased xylem in the stems. The mutant exhibits increased
secondary growth resulting into a continuous ring of vascular tissues. hca mutants
have reduced sensitivity to auxin and cytokinin compared to the wild-type (Pineau et
al. 2005).
In addition to the mutants above mentioned, many other gene candidates have been
identified, which influence vascular cell proliferation and differentiation. The HDZip III genes ATHB-8 and ZeHB-13 (Zinnia)/ATHB-15 are considered as key
regulators of xylem and phloem proliferation (Baima et al. 2001; Ohashi-Ito and
24
Fukuda 2003). ATHB-8 and ATHB-15 in Arabidopsis are expressed in the
procambium region (Baima et al. 2001; Ohashi-Ito and Fukuda 2003). Further
studies revealed that the expression of ATHB-8 regulates early vascular development
(Baima et al. 2001). Although the formation of the vascular tissue was not affected in
the athb-8 mutant, overexpression of ATHB-8 in the Arabidopsis plants increased the
xylem proliferation and differentiation leading the authors to conclude that
expression of ATHB-8 regulates early vascular development (Baima et al. 2001).
1.5 Secondary growth function of Vascular Cambium
Previous studies in Arabidopsis have shown that secondary growth in the hypocotyl
and root can occur just only after a week of germination (Busse and Evert 1999). The
appearance of the anticlinal and preclinal divisions in the procambium and pericycle
cells are the first indication of secondary growth (Busse and Evert 1999). These
divisions are responsible for the formation of the cambium that increases the
diameter of the vascular tissue. After the continuous ring of cambium has been
formed, secondary xylem differentiates towards the inside of the hypocotyl and the
cells that are present outside give rise to secondary phloem (Chaffey et al. 2002).
Due to existence of several cell types, the molecular mechanisms controlling
vascular cell development and organisation is complex. Studies involved in
understanding vascular organisation are often focussed on xylem and phloem
differentiation. As the focus here is to understand the development of trees to
increase their yield for biomass production, understanding the formation of the
vascular cambium is essential. The cambium is the progenitor of all cells in the
25
vascular tissue, and it is central to wood growth during secondary growth. Therefore,
manipulation of radial growth is reliant upon a good understanding of the factors
regulating cambial activity. The work by Etchells and Turner (2010) showed that by
tissue-specific expression of CLE41 by the SUC2 promoter leads to an increase in
the radius of the shoot by the production of secondary xylem and phloem (Figure
1.4). However understanding the nature of the molecular signals that mediate the
initiation of procambium cells and promote their division is essential towards
understanding radial growth.
Figure 4
Figure 1.4: Transverse Arabidopsis thaliana stems section showing the initiation of
secondary growth induced by CLE41 expression. Wild-type Arabidopsis (A), Plant
over-expressing CLE41 driven by promoter SUC2 (B). Box indicates the
interfascicular region. The (x) xylem and (ph) phloem are indicated. Arrow in (B)
showing the increased cell divisions that are absent in (A). Scale bars represent 200
µm (A) and 100 µm (B).
26
1.6 Regulatory mechanisms of Vascular cambium during secondary
development
Figure 5
Figure 1.5: Transverse section of hybrid aspen (Populus tremula × Populus
tremuloides) stem, showing the organization and regulatory components in cambium
and secondary xylem and phloem. Modified from (Ursache et al. 2013)
To increase our understanding about the underlying mechanisms for secondary
growth, Arabidopsis has proven to be very helpful. Global expression analysis
suggests that several transcriptional regulators are involved during secondary growth
are also in association with the shoot apical meristem (Schrader et al. 2004).
Amongst the genes expressed in both the SAM and the cambial region are orthologs
of the Arabidopsis thaliana class I knotted-like homeobox (KNOX) gene
SHOOTMERISTEMLESS (STM); class III homeodomain- leucine zipper (HD ZIP)
27
genes PHAVOLUTA⁄ PHABULOSA and ATHB-15; KANADI1; SHOOT-ROOT
(SHR); and potential orthologs of AINTEGUMENTA (ANT) and PINHEAD (PNH)
(Spicer and Groover 2010) (Figure 1.5) (Ursache et al. 2013). These genes are key
players in the SAM in which ANT controls cell proliferation, PNH regulates
determinate versus indeterminate shoot growth, class III HD ZIPs and KANADI1
that influence vascular development and SHR specifying tissue identity (Spicer and
Groover 2010). HD-ZIP III are also fundamental regulators of stem cell production
and xylem formation. Previous studies have shown that a Populus orthologue of
REV, popREVOLUTA (PRE) is expressed in woody stems of Poplar and is
expressed during secondary growth and probably plays a vital role in the initiation of
cambium and in founding the secondary vascular tissues (Robischon et al. 2011).
Studies have revealed that the vascular cambium is the stem cell niche and the
procambium is the stem cell initials (Lau et al. 2010). The regulation of stem cells is
very well studied in Arabidopsis embryogenesis (Lau et al. 2010) and at the
cotyledons (Mähönen et al. 2000) stage. Several genes have been identified that
regulate stem cell maintenance, proliferation and signalling (Reviewed by
(Miyashima et al. 2013)). Key players in maintaining the stem cell population in the
vascular cambium are the homeobox transcription factor WOX4 (WUSCHEL
HOMEOBOX RELATED 4), Leucine-Rich Repeat- (LRR) -receptor kinase,
PHLOEM INTERCALATED WITH XYLEM (PXY)/TDIF RECEPTOR (TDR) and
its peptide ligand CLAVATA3/ESR-related 41/44 (CLE41/44) (Fisher and Turner
2007; Ito et al. 2006; Hirakawa et al. 2008; Whitford et al. 2008; Etchells and Turner
2010; Hirakawa et al. 2010). Further studies have highlighted the significance of
gene WOX4 (WUSCHEL HOMEOBOX RELATED 4 gene) in maintaining the cell
28
division activity of vascular stem cells during secondary growth in the Arabidopsis
hypocotyls (Hirakawa et al. 2010; Suer et al. 2011).
In recent decades, several studies have indicated that plant hormones are also among
the key players in developing and maintaining the cambium (reviewed in (Wang and
Irving 2011)). The plant hormones, namely the auxin, cytokinin, gibberellin,
ethylene, jasmonate and brassinosteroids are involved in the initiation and
preservation of cambium and/or the development of secondary vascular tissues
(reviewed in (Wang and Irving 2011)). Secondary growth of the vascular cambium
is also controlled by the hormones auxins and cytokinins (Dubrovsky and Rost
2012).
Auxin is one of the important plant hormones that influences cambium development
and secondary tissue formation. Auxin also performs a significant role in secondary
xylem differentiation from the vascular cambium (Nieminen et al. 2015). Previous
work by Tuominen et al. (1997) has confirmed a strong correlation in developing
woody stems of poplar between the localized auxin concentration and the initiation
of secondary vascular differentiation from the vascular cambium (Tuominen et al.
1997). Down-regulating the expression of auxin efflux carrier genes PIN3 and PIN4,
resulted in lowered auxin polar flow and further inhibiting vascular cambium activity
at the basal parts of the Arabidopsis inflorescence stems (Zhong and Ye 2001).
Further evidence suggests that auxin transporter genes, AUX1-like of influx and
PIN1-like efflux carriers, are expressed at the Populus cambium, perhaps regulating
the distribution of this hormone in the cambium (Schrader et al. 2004). Furthermore,
auxins have shown a positive effect on homeobox transcription factor WOX4
expression in the cambium (Suer et al. 2011). Works by Suer et al. (2011) have
29
revealed that auxin dependent vascular cambium stimulation requires WOX4. They
also demonstrated that PXY is essential for stable auxin dependent increase in WOX4
mRNA abundance and stimulation of vascular cambium activity. These findings
suggest that PXY and WOX4 are the key players controlling the vascular cambium
activities (Suer et al. 2011). Studies have also illustrated the role of cytokinins in
regulating the cell propagation activities in the vascular cambium during secondary
growth (Matsumoto-Kitano et al. 2008). The genes responsible for the biosynthesis
of the biologically important cytokinins are ATP/ADP isopentenyl transferases
(ATP/ADP IPTs) of which AtIPT3, 5 and 7 are the most highly expressed (Miyawaki
et al. 2006). To evaluate the effect of the cytokinins the quadruple ipt mutant (ipt1 3
5 7) was generated and lower levels of some types of cytokinins were observed, and
showing defects in procambium proliferation and absence of any secondary growth
(Miyawaki et al. 2006; Hejátko et al. 2009). Loss of function of two cytokinin
receptors, AHK2 and AHK3 also led to a significant decrease in secondary vascular
growth in the Arabidopsis stem (Hejátko et al. 2009).
1.7 Novel
patterning
Peptide-receptor
signalling
pathways:
radial
vascular
Figure 6
Figure 1.6: Resin-embedded transverse section of the wild-type and pxy mutant
hypocotyls and stem (vascular bundle). Wild-type (A and B) and pxy (C and D),
30
hypocotyl (A and C) and stem (B and D) are shown. The arrows in (C), indicate the
xylem interspersed with the phloem indicative of the phenotype of the pxy mutant. In
stem the pxy vascular bundles appeared more flattened around the stem, the sieve
plates of the phloem appeared to be adjacent to, or interspersed with, the xylem as
indicated by arrows in the pxy mutant (D). The (x) xylem and (ph) phloem are
indicated. Scale bars represent 200 µm.
Figure 7
Figure 1.7: Resin-embedded transverse section of Arabidopsis thaliana showing the
phenotype in plants where CLE41 and PXY are over-expressed. Transverse sections
of a hypocotyl (left hand panel) and stem (right hand panel) of a wild-type (A),
35S::CLE41 (B), SUC2::CLE41 (C), SUC2::CLE41- 35S:: PXY (D) plants. Note
that Arabidopsis transformed with 35S::CLE41 have disorganised hypocotyl, but this
is not in the case for plant expressing SUC2::CLE41 (C) or SUC2 :: CLE41 - 35S::
PXY (D). The (x) xylem and (ph) phloem are indicated. Scale bars represent 100 µm.
The balance of cell propagation and differentiation plays a vital role in maintaining
stem cell populations. Signalling via interaction between secreted mobile peptides
31
and receptor like kinases is responsible for the maintenance of stem cell niche
(Sablowski 2011). In the SAM, the LRR receptor CLV1 interacts with the CLV3
peptide
ligand
to
determine
stem
cell
fate
(Brand
et
al.
2000).
CLAVATA3/EMBRYO SURROUNDING REGION-RELATED (CLE) genes
encode small peptides with conserved carboxyl termini that acts as a signalling
molecule between cells in the plants vascular tissue (Hirakawa et al. 2008). Ito et al.
(2006) showed that CLE peptides inhibit tracheary element differentiation in the
xylogenic cell culture (Zinnia elegans L) model. The extracellular factor that inhibits
tracheary element differentiation was isolated and named tracheary element
differentiation inhibitory factor (TDIF). TDIF was characterized as a dodecapeptide
with two hydroxyproline residues that regulates vascular and meristem formation
(Sawa et al. 2006). Fisher and Turner (2007) identified the PHLOEM
INTERCALATED WITH XYLEM (PXY) mutant where the orientation of vascular
cell division was lost (Figure 1.6C and D) and the xylem and phloem are partially
interspersed. The PXY gene encodes for the (RLK) receptor-like kinase that is
responsible for the functionality of the RLKs in the meristem to maintain the cell
polarity essential for the orientation of cell division during plant vascular
development (Fisher and Turner 2007).
Hirakawa et al. (2008) showed that non-cell autonomous signals control the fate of
vascular stem cells. They identified that the receptor for TIDF, that they called TDIF
receptor (TDR), was the same previously identified receptor PXY. CLE41 functions
synergistically with CLE6 to control the rate of cell division in the vascular meristem
(Whitford et al. 2008). PXY is confirmed as the receptor for the CLE peptide (TDIF)
where the effects of CLE41 overexpression are dependent upon PXY (Figure 1.6 and
32
1.7) (Etchells and Turner 2010). The question was further addressed by Etchells and
Turner (2010) who investigated how the signalling between CLE41 and PXY genes
controls the organization plane of cell division in the vascular meristem. PXY is
expressed in the dividing cells of the procambium while CLE41 is secreted from
adjacent phloem cells. Increasing the CLE41 expression under the 35S (cauliflower
mosaic virus) promoter (35S::CLE41) resulted in loss of cell division orientation,
leading to a loss of ordered vascular tissue development (Figure 1.7B). In contrast,
increasing expression of CLE41 specifically in the phloem via the SUC2 (Sucrose
Proton Symporter 2) promoter (SUC2::CLE41) resulted in increasing the number of
cell divisions in the procambium region while maintaining the orientation of cell
(Figure 1.7C). Over expression of the receptor PXY together with its ligand results in
striking phenotypes depending on where CLE41 is produced. Most significantly for
increased biomass applications, by over expressing CLE41 and PXY (SUC2::CLE41
35S::PXY) an increased cell number in both the vascular bundle and in the
interfascicular region was observed, leading to considerably enhanced secondary
growth (Figure 1.7D). A reduction in xylem differentiation along with the presence
of undifferentiated cells is also observed. However, despite the increase in the
number of cell divisions, their correct orientation is maintained. This study provides
strong genetic evidence that the interaction between PXY and CLE41 is sufficient to
regulate the orientation of cell division and proliferation in the vascular tissue by
increasing the cell population in the procambium (Etchells and Turner 2010) (Figure
1.7 C and D).
WOX4 (WUSCHEL HOMEOBOX RELATED 4 gene) is a mediator of cell division
in the vascular cambium downstream of the CLE41–PXY signalling pathway
33
(Hirakawa et al. 2010). WOX4 is expressed in the procambium and cambium under
the influence of CLE41. In a wox4pxy double mutant the number of cell division in
the cambium is reduced significantly in conjunction with PXY (Hirakawa et al.
2010). Hirakawa et al. (2010) also demonstrated that growing the pxy mutant and
wox4pxy double mutant on TDIF supplemented media resulted in the xylem cells
forming adjacent to the phloem tissues. These finding were consistent with the
observation by Hirakawa et al. (2008) who showed that deficiency in CLE peptide or
TDIF perception promotes xylem cell differentiation of procambial cell during the
postembryonic development. The results strongly imply that mutation in PXY leads
to the suppression of cambial cell proliferation and increases xylem differentiation
but mutation in WOX4 can only cause suppression of cambial cell proliferation
(Hirakawa et al. 2010). This supports the idea that the CLE–PXY–WOX4 signalling
pathway is a general determinant of secondary growth of the vascular meristem
(Hirakawa et al. 2010). On prediction that manipulating the expression of the poplar
homologs of CLE, PXY and WOX4 in the cambium region will lead to the similar
pattern to that in Arabidopsis (Figure 1.6 and 1.7). Furthermore, Wang et al. (2013)
identified three Arabidopsis LRR-RLK genes (PXY-correlated; PXC1, 2, 3). The insilico and functional clustering analysis identified that PXY-correlated genes have a
strong co-relation with the other key regulators of vascular development like PXY,
CLE41, REV and HB-8. The promoter-reporter analysis of all the PXC1, 2, 3
demonstrated their expression in the vascular tissues of the shoot apex, the
cotyledons, leaves, hypocotyls and the roots. It was also observed that PXC1
(At2g36570) expression overlapped the PXY expression in the vascular bundle
confirming a strong correlation between them. Further to investigate the
characteristics of PXC1, the pxc1 mutants were grown in long day conditions and
34
then transferred to short day growth condition. The loss of function pxc1 mutants
exhibited a severe reduction in the secondary wall formation in xylem fibers. The
qPCR analysis of the pxy, pxc1, cle41, cle44 and wox4 mutants indicated a
correlation between the pathways mediated by PXC1 and CLE–PXY–WOX4
(Figure 1.6) (Wang et al. 2013)
Aims and objectives:
The current study focuses on two aims firstly to investigate whether the PXY-CLE41
pathways is conserved in trees. Secondly, whether we can exploit PXY-CLE41
pathway in trees for production of more cells in the vascular meristem and increase
the biomass. The objectives are first to establish whether the poplar orthologues of
PXY and CLE41 genes are functional in Arabidopsis, secondly if the genes are
functional then to generate transgenic hybrid aspen (Populus tremula x Populus
tremuloides) (T89 clone). The transgenic hybrid aspen will be overexpressing poplar
PXY and CLE genes. These transgenic hybrid aspen will be further investigated to
prove, if the PXY-CLE41 pathways are conserved in trees. Once confirmed then the
transgenic hybrid aspen will be used to perform biomass analysis to conclude that
manipulating PXY-CLE41 pathway can increase the biomass of the trees for
biotechnological applications.
35
Chapter 2: Material and Methods
2.1 Materials
2.1.1 Chemicals and reagents
All chemicals and reagents were procured from Sigma-Aldrich Company Ltd.,
Melford Laboratories Ltd. (Ipswich, UK), VWR International Ltd., or Starlab (UK)
Ltd., unless otherwise mentioned.
2.1.2 Plant Material
The Arabidopsis thaliana ecotype Columbia-0 (Col-0) was used for all experiments.
For all the poplar experiments the hybrid aspen Populus terumla x Populus
tremuloides clone T-89 (a gift from Bjorn Sundberg) was used.
2.1.3 Bacterial strains
Competent Escherichia coli strain (genotype: F- mcrA Δ( mrr-hsdRMS-mcrBC)
Φ80lacZΔM15
Δ lacX74 recA1 araD139
Δ( araleu)7697 galU galK rpsL
(StrR) endA1 nupG) One Shot® TOP10 cells Invitrogen were used for E.
coli
transformations. The Agrobacterium tumefaciens GV3101 (Koncz and Schell 1986)
was used for plant transformations.
2.1.4 DNA Vectors
The vector backbone (Invitrogen) and DNA constructs were obtained from suppliers
or constructed by gateway ® cloning in the lab. For details see Table (2.1)
36
2.1.5 Oligonucleotide primers
Unmodified DNA oligonucleotides were obtained from Eurofins MWG, Ebersberg,
Germany. The sequence of all primers used in the study are shown in Table (2.2)
2.1.6 Software
The Arabidopsis sequences were compared against TAIR 10 and Phytozome v9.1.
Bioinformatics analyses were performed on GeneDoc, Vector NTI® software Life
Technologies, Snapgene® and Serial Cloner 2.6. Statistical analyses were performed
in Microsoft® Excel 2013 and SPSS 20 for Windows. Xylem cell counting and
section analysis was performed on CellProfiler 2.1.0 (revision 0c7fb94) software. All
images were processed in COREL Paint Shop Pro Photo X2 and Gimp2.8 software.
Table 1
Construct
Entry vector
Entry
Cloning
Destination
Destinatio
vector
Method
vector
n vector
supplier
35S::PttCLE41
pENTR-D-
Invitrogen
topo
35S::PttPXY
pENTR-D-
rolD::PttPXY
PtPP2::PttCLE41
PttCLE41 to
Invitrogen
Invitrogen
Invitrogen
topo
PttANT::PttPXY
pENTR-D-
PttANT::PttPXY
PttANT::GUS-GFP
pENTR-D-
Gateway
VIB-PSB
Ghent
pK2GW7
VIB-PSB
Ghent
Gateway
pK7M34GW2
VIB-PSB
cloning
-8M21GW3
Ghent
Gateway
pVX33
Cambia
pVX31
Cambia
Restriction
pVX33/pVX3
Cambia
based cloning
1
pVX33
cloning
Invitrogen
topo
PtPP2::PttCLE41
pK2GW7
cloning
pENTR P3P4
pENTR-D-
Gateway
cloning
topo
35S::PttCLE41
supplier
Gateway
cloning
Invitrogen
topo
pK2GWFS7,
VIB-PSB
Gateway
0
Ghent
cloning
37
Cambia
PtPP2::GUS-GFP
pK2GWFS7,
VIB-PSB
Gateway
0
Ghent
cloning
pVX31
Cambia
Table. 2.1 DNA vectors used in this study
Table 2
Primer Name
Primer Sequence
PttCLE41
PttCLE41_F
5'-CACCTAGCTAGCCTTGGTGCTGGT-3'
gene
PttCLE41_R
5'-CAACCCTCTCACAGACGACA-3'
PttPXY
PttPXY_F
5'-CACCATGAAACTCCCTTTTCTTTT-3'
gene
PttPXY_R
5'-ACATTCGACTGCAGGCTTTT-3'
PttPXY
Ptt_PXY_D
5'-CACCCCATAACAACCATGAA-3'
TOPO
TOPO_F
Cloning
PttPXY STOP_R
5'-cgatcGTCATTATCAACATTCGACTG-3'
Ptt_PXY_TOPO_F-
5'-CACCCCCATAACAACCATGAA-3'
1
PttPXY STOP_R1
5'-cgatcGTTATCAACATTCGACTGCA-3'
Promoter
pPttANT_F
5’-atcgggcccCCGAAGTTGCTCACTTC-3'
Primers
pPttANT_R
5’-atcactagtGACAAGCTGAGAGACTG-3'
pPtPP2_F
5’-atccctaggcctgcaggTAAGCTATGTACGTTTTGG-3'
pPtPP2_R
5’-atcactagTCTTGAGAGCAAATAAATTCTAGTGTTA-3'
Rold prom _F
5’-CCTCCAAGCAGCCCATATAA-3'
35S prom_ F
5'-CGCACAATCCCACTATCCTT-3'
PtPXY
PtPXY_26_F
5'-TGAAACTCCCTTTTCTTTTCTTTC-3'
internal
PtPXY_252_F
5'-GCCACTGCTCAAATCACATC-3'
sequencing
PtPXY_723_F
5'-TCCACTCCTATCAGGCAATGT-3'
primers
PtPXY_1260_F
5'-AGCAAACTGCACCTCTTTGTC-3'
PtPXY_1731_F
5'-TTACTCACCGGTTCCATTCC-3'
PtPXY_2058_F
5'-GATGAACGAGAGATCGGACC-3'
PtPXY_2249_F
5'-ACTGTGGGGTAAGCACAAGG-3'
Pt PXY_2565_F
5'-ATGGAGGCTAGAGTGGCAGA-3'
PtPXY_2731_F
5'-ATGGGGTGGTGTTAATGGAG-3'
PtPXY_2835_F
5'-TGCAGTGAAGAACTGGATCG-3'
PtPXY_3375_F
5'-CATACTTGATTTTCCAAATGCA-3'
PtPXY_3870_F
5'-GGACGCTCTCCTTATTATT-3'
PtPXY_150_R
5'-GGAAGGTCTTAATGGAGAGAAGA-3'
PtPXY_581_R
5'-GTAGCTCCCACCAAGGTTGA-3'
PtPXY_1097_R
5'-TCCGGTTAGGTTGTTGTTCC-3'
PtPXY_1608_R
5'-ATGGAATGCTGCCATTGAAC-3'
PtPXY_2098_R
5'-GGTCCGATCTCTCGTTCATC-3'
38
PtPXY_2603_R
5'-TGCCACTCTAGCCTCCATCT-3'
PtPXY_3076_R
5'-GCAATAGCCCCATCAACAGT-3'
Pt PXY_3495_R
5'-CCAAGTGCATCGAGAAGTCA-3'
PtPXY_3494_R
5'-CGATCCAGTTCTTCACTGCA-3'
PttCLE41
PttCLE41_F
5'-CTCTTGGGGGTGGTTTCTTT-3'
internal
PttCLE41_R
5'-GCTCTCAAGGGCAGTACGAG-3'
attB site
attB3RP
5'-ATAGAAAAGTTGCTCTTGGGGGTGG-3'
flanked
attB4FP
5'-GTATAATAAAGTTGGCTCTCAAGGG-3'
Primers
attB3RS
5'-GGGGCAACTTTGTATAGAAAAGTTG-3'
attB4FS
5'-GGGGCAACTTTGTATAGAAAAGTTG-3'
attB-PttPXY_F
5'-
sequencing
primers
ggggacaagtttgtacaaaaaagcaggctggATGAAACTCCCTTTTCTT3'
attB-PttPXY_R
5'-ggggaccactttgtacaagaaagctgggtaTTATCAACATTCGAC-3'
attB-PttCLE41_F
5'ggggacaagtttgtacaaaaaagcaggctggATGTTAAGTTGGTGCTTA3'
attB-PttCLE41_R
5'-ggggaccactttgtacaagaaagctgggtaTTACCTGTTTGAAATAG3'
RT-PCR
PttCLE41_F
5'-ATGGCAACACCAAAAACACAGT-3'
Primers
PttCLE41_R
5'-TTACCTGTTTGAAATAGGGTTTG-3'
PttCLE41_geno_F
5'-CACCCTTGTGGGCTCTTG-3'
Ptt CLE41_geno_R
5'-AGAAGTAAGGCTGAGGATGATG-3'
Alpha-Tubulin_F
5'-ATGAGGGAGATAATAAGCATACA-3'
Alpha-Tubulin_R
5'-TGAGGAGAAGGATAGATGGTGA-3'
PttPXY_F
5'-TAAGCCTAGTAATATATTATTGGAC-3'
PttPXY_R
5'-CTATTATCAACATTCGACTGCA-3'
Table 2.2. PCR primers used in the study. (The uppercase in the primer sequence
indicates the gene specific sequences whereas the lowercase indicates the sequence
of the vector backbone or added restriction site for cloning.)
39
2.2 Plant Material and Growth Conditions
2.2.1 Arabidopsis thaliana plants
Wild-type and transgenic Arabidopsis thaliana seeds were surfaced sterilised by
shaking for 10-15 minutes in sterilizing solution [30% sodium hypochlorite v/v), 1%
Triton-X100 (v/v)] followed by 5 washes in sterile dH20 for five minutes each. Post
sterilisation, the seeds were suspended in 0.1% sterile agar solution and plated by
pipetting on ½ MS (Murashige and Skoog media added with B5 vitamins and 1.0%
(w/v) agar) plates (Murashige and Skoog 1962). The plates were sealed with 3M
Millipore® micropore tape and then stratified by incubation in the dark at 4°C for 48
to 72 hrs. Post stratification the plates were maintained (vertically) in the growth
cabinet (Sanyo MLR) at 22°C with a light intensity of 120-140 µmol m-2s-1 for one
week. Following this the seedlings were transplanted on a medium consisting of
Levington-F2 (Everris) compost: vermiculite: perlite (3:1:1) in a square 10 cm
plastic pots. The soil mixture was initially watered with 0.2 g.1-1 Intercept 70WG.
The plants were maintained under continuous light (120-150 µmol m-2s-1) in the
growth cabinet (Percival, Ohio, USA) at 22°C.
2.2.2 In vitro propagation of Populus tremula x Populus tremuloides T89 (hybrid
aspen)
In vitro propagation of T89 (hybrid aspen) was performed according to Meilan and
Ma (2007) with some modifications as described below: 4 to 6 weeks-old in-vitro
grown plants shoot tips were excised and propagated on propagation media (Woody
plant media - WPM pH 5.6 (Lloyd and McCown 1980), phytagel, 250 mg/L
cefotaxmine). The culture was maintained in polypropylene Microbox© Combiness
40
in the growth cabinet (Sanyo MLR) at 22°C ± 2°C, 16 h photoperiod with a light
intensity of 120-140 µmol m-2s-1 for three weeks.
2.3 Generating and analysing DNA constructs
2.3.1 Plasmid isolation protocol
A single E.coli colony was inoculated in a 5ml LB media containing appropriate
antibiotics (50 µg/ml kanamycin and/or 25 µg/ml spectinomycin) and incubated in a
shaker (200rpm) at 37oC overnight. The recombinant plasmids were purified using
the Qiagen® QIAprep Spin Mini-prep Kit (Qiagen, Crawley, UK), as per the
manufactures protocol. Briefly, the culture is centrifuged at 8000 rpm and the pellet
lysed by resuspending in alkaline lysis buffer. The plasmid DNA is bound to the
silica membrane in high salt binding buffer. The RNA, proteins and low molecular
weight contaminating molecules and salt were removed by the washing buffer. The
plasmid DNA is subsequently eluted in a low salt buffer.
2.3.2 Polymerase chain reaction (PCR)
For routine screening, My Taq Red-Mix (Bioline ™) was used. A 20 µl reaction
consist of 10 µl of 10x My Taq Red-Mix, 50 pmol of forward and reverse primers, 1
µl of template and dH2O to make up the total volume. Unless specified otherwise,
the PCR programme on Bio-Rad® Thermal cycler for screening is as follows:
94°C, 2 min
94°C, 30 sec
55°C, 30 sec
34 cycles
41
72°C, 1 min
final extension at 72°C for 7 min
hold at 8°C
The PCR reactions were stored at 4oC before being visualised in 1% agarose gels by
electrophoresis.
To generate high-fidelity PCR products that were used in further cloning, Phusion®
High-Fidelity DNA Polymerase (Thermo scientific) was used. A 20 µl reaction
consist of 10 µl of 5X Phusion® HF Buffer 50 pmol of forward and reverse primers,
dNTP mix (to a final concentration of 200 µm), 1 µl of template and dH2O to make
up the total volume.
94°C, 2 min
94°C, 30 sec
55-58°C*, 30 sec
39 cycles
72°C, 1 min
final extension at 72°C for 7 min
hold at 8°C
The PCR reactions were stored at 4oC before being analysed in 1% agarose gels by
electrophoresis. The annealing temperature was dependent on the primer sequences
used in the High-Fidelity PCR.
2.3.3 Restriction Digest
42
Restriction digests were set up with up to 100ng plasmid DNA; appropriate
restriction buffer New England Biolabs ® Inc (NEB), up to 5 U of restriction
enzyme and dH2O to make up to the total volume of 20 µl. For double digest the
suitability of buffer was determined on the NEB website double digest finder. The
restriction digest reaction was incubated at 37oC for 1 to 2 hrs and further analysed
on 1% agarose gels by electrophoresis.
2.3.4 Agarose gel electrophoresis
As a routine practice 1% (w/v) agarose gels were used to analyse the DNA
fragments. A 1L 50X TAE buffer stock solution was prepared (242 g Tris-Base, 57.1
ml glacial acetic acid and 100 ml 50 mM EDTA) that was diluted to a 1X
concentration with dH2O. For UV visualization, SafeView® (NBS biological,
Huntingdon, UK) was added to the molten agarose gels to 0.01% (v/v) final
concentration. The DNA fragments to be analysed were mixed with 5 µl of Bioline
TM
5x DNA Loading Buffer Blue before loading. Standard molecular marker (5 µl
Bioline Hyperladder I) was loaded in the first well. The agarose gel was run in 1X
TAE buffer at 120V for approximately 45 mins. The gels were visualized using
RedTM UV trans-illuminator (Alpha Innotech).
2.3.5 Gel extraction
Bands were excised from the agarose gel under UV-trans-illumination and the DNA
extracted using a QIAquick Gel Extraction Kit as per the manufacturer’s protocol.
2.3.6 DNA Sequencing reaction
43
The DNA sequencing reaction mixture was prepared as per the BigDye® Terminator
v3.1 Cycle Sequencing Kit (Applied Biosystems®) protocol provided by the Faculty
of Life Sciences DNA sequencing facility, University of Manchester. The mixture
consists of plasmid DNA up to 100 ng, 4µmol sequencing primers and dH2O to
make up to a final volume of 10 µl.
2.3.7 Construction of Entry clone
The PttPXY entry clone were generated by inserting the PttPXY fragment into a
gateway cassette, p-ENTR dTOPO® (Life Technologies). Prior to setting up TOPO
reaction, genomic DNA was extracted according to (Csaikl et al. 1998) from Populus
tremula x tremuloides clone T89 (hybrid aspen). Following PCR amplification of the
genomic DNA and gel extraction the TOPO reaction was set up using 2 µl PCR
product, 1 µl of salt solution, 1 µl TOPO recombinase enzyme and TOPO® vector
with sterile water adding up to a final volume of 6 µl. This reaction was incubated at
room temperature for 30 mins and transformed into competent E. coli One Shot®
TOP10 cells as per manufactures protocol, using kanamycin to select positive
colonies. Restriction digest with MluI (NEB) of the entry clone was performed to
check for the presence of insert. Clones were also sequenced with sequencing
primers (Table 2.2) to determine the presence of the stop codon and confirm the
absence of any point mutation or frame shifts. The PttCLE41 entry clone was
amplified with primers (Table 2.2) flanked with attB sites by High-Fidelity PCR.
Once the entry clones were ready the Gateway® cloning was applied to generate the
expression vector.
2.3.8 Construction of a Gateway® destination vector
44
The 35S poplar overexpression vectors were constructed by inserting the entry
clones in the Gateway® destination vector pK2GW7, an 11135bp plasmid with
single cloning site for site-specific recombination, 35S promoter and antibiotic
resistance for spectinomycin in bacteria and Kanamycin in plants (Fig 1A). The LR
clonase recombination reaction mixture consisted of 2 µl of 5X LR ClonaseTM II
enzyme mix and 1 µl destination vector (pK2GW7), 1 µl of the entry clone and 3 µl
of TE buffer, pH 8.0. The reaction was incubated at RT overnight and transformed
into competent E. coli One Shot® TOP10 cells using spectinomycin to select the
transformed colonies. To construct a double overexpression binary vector, the
pDONR- PttCLE41 clone was also made by Gateway® recombination. The
Gateway® recombination reaction was set up with the 1 µl attB site flanked PCR
product, 1 µl of vector (pDONR), 1 µl of 5X BP Clonase and 2 µl of TE buffer, pH
8.0 by incubating at RT for overnight. The purified pDONR- PttCLE41 clone was
checked by sequencing with the PttCLE41 forward and reverse primers. To combine
the PttCLE41 and PttPXY sequences in a single vector for co-expression, a
recombination reaction with pDONR- PttCLE41, PttPXY entry clone and the
pK7m34GW2-8m21GW3 destination vector was set up. In order to carry out tissuespecific overexpression PttPXY and PttCLE41 was cloned in custom-made
Gateway® destination vectors pVX31 and pVX33 by LR clonase recombination.
The promoter sequences were identified from the publically available poplar
expression data. The PttANT (Potri.002G114800) promoter sequences were used for
cambium specific expression and for PtPP2 (Potri.015G120200) promoter sequences
were used for phloem specific expression (Etchells et al. 2015). For amplifying
PtPP2 promoter sequence from P. trichocarpa primers PtPP2-F and PtPP2-R see
Table 2.2 were used to generate a fragment of 1999 base pairs upstream of the start
45
codon. For amplifying PttANT promoter sequence from P. tremula x P. tremuloides
primers PttANT-F and PttANT-R see Table 2.2 were used to generate a fragment of
1156 base pairs upstream of start codon to 904 base pairs downstream of the start
codon. Although the promoter sequence did include the gene sequence, it was able to
drive the expression in the vascular tissue. Further, the tissue-specific double over
expressor was constructed by excising SbfI-pPtPP2-PttCLE41- t35S-SbfI cassette
and cloning into SbfI site of ApaI-pPttANT1-PttPXY-t35S-SbfI (Etchells et al.
2015). The transcriptional reporter clones for PttANT and PtPP2 were generated by
inserting eGFP-GUS fusion from pKGWFS7 into pVX31 and pVX33 by LR clonase
recombination reaction resulting PttANT:: eGFP-GUS and PtPP2::eGFP-GUS
constructs.
2.3.9 Restriction Based cloning
The tissue-specific double overexpression vector was generated by restriction based
cloning. The PttANT::PttPXY and PtPP2::PttCLE41 was treated with restriction
enzyme SbfI-HF® (NEB).
The enzyme linearizes PttANT::PttPXY and from
PtPP2::PttCLE41 a 3000 bp fragment containing of PtPP2/PttCLE41/T35S of
approximately was separated (see Figure 3.4). The linearized PttANT::PttPXY was
treated by alkaline phosphatase (FastAP Thermosensitive Alkaline Phosphatase) in
order to remove the 5’ phosphate and prevent self-ligation of the construct. The SAP
reaction mixture consisted of 50 µl of linearized fragment, 6 µl of each FastAP
enzyme and buffer. The reaction mixture was incubated in the thermo cycler at 37oC
for 30 mins and the enzyme was deactivated by heating the reaction mixture for 5
mins at 75oC. The fragment from PtPP2::PttCLE41 was ligated into the FastAP
treated
PttANT::PttPXY
linearized
fragment
46
to
generate
PttANT::PttPXY
PtPP2::PttCLE41. The ligation was carried out in a 1.5 ml Eppendorf tube by
combining the 5 µl insert (digested fragment from PtPP2::PttCLE41), 3 µl vector
(FastAP treated PttANT::PttPXY linearized fragment), 1 µl T4 DNA ligase (Roche)
and 1 µl DNA ligase buffer. The mixture was transformed into competent E. coli
cells One Shot® TOP10 cells using kanamycin to select positive colonies. The
transformed clones were identified by restriction digest with Fermentas® restriction
enzyme EcoRI and BamHI and with internal sequencing primers (Table 2.2).
2.4 Transformation of Electro-competent Agrobacterium tumefaciens
2.4.1 Preparation of Electro-competent Agrobacterium tumefaciens
Stocks of electro-competent Agrobacterium tumefaciens cells were produced with a
few modifications as described by (den Dulk-Ras and Hooykaas 1995).
Agrobacterium strain GV3101 was streaked onto gentamycin selective LB agar
plates and incubated overnight at 30oC. A single colony was picked by the pipette tip
to inoculate a starter culture (5ml of LB liquid medium, 50 µg/ ml gentamycin) and
incubated overnight at 30oC with shaking at 250 rpm. This starter culture was then
used to inoculate 250 ml of medium that was incubated with shaking at 250 rpm at
30oC for approximately 4-5 hrs until the OD600 of cells was between 0.5-1.0 (1010
cells/ml). Cells were aseptically transferred to ice cold 50 ml propylene tube. Cells
were pelleted by centrifuging at 5000 rpm for 15 minutes in a bench top refrigerated
centrifuge machine at 4oC. After decanting the supernatant, pellet was re-suspend in
50 ml of sterile cold ddH2O. Using the same conditions mentioned above the cells
were centrifuged twice and re-suspended in 25 ml then in 10 ml sterile cold ddH2O
containing sterilized cold 10% glycerol. This suspension was again pelleted and
47
finally re-suspend in 1-1.5 ml filter sterilized cold 10% glycerol, divided 50 μl
aliquots that were flash frozen in liquid nitrogen and stored at -80oC until further
use.
2.4.2 Transformation of Electro-competent Agrobacterium tumefaciens
Transformation of electro-competent GV3101 Agrobacterium was carried out
according to a modified version of a Weigel and Glazebrook (2005) protocol. A 50
μl aliquot of GV3101 cells was thawed on ice, 1 µl of destination vector plasmid was
added and the contents carefully mixed. This mixture was transferred to a pre-chilled
Gene Pulser® 0.2cm electrode cuvette (Bio-Rad) and tapped on the bench to make
sure that all the contents were at the bottom. The cuvette was incubated on ice for
approximately 20 minutes and then placed into a Gene Pulser (Bio-Rad®) set to 25
µF; 2.5 kV; 600Ω and a pulse length of 13 milliseconds. After the pulse, 1ml icecold LB was added to the transformed cells with gentle mixing and the suspension
incubated at 30°C for 4 hrs. After incubation the cells were centrifuged at 8000 rpm
for 2 mins, the supernatant was removed and the pellet re-suspended in 100 µl of LB.
The cells were spread on antibiotic selective LB agar plates and incubated at 30°C
for 48 to 72 hrs.
2.5 Transformation of Plants
2.5.1 Arabidopsis thaliana plants by floral dipping
Transgenic Arabidopsis plants were generated by a floral dipping method modified
from Clough and Bent (1998). A single Agrobacterium colony containing the
48
destination vector plasmid was chosen and inoculated in 5 ml antibiotic selective LB
medium. The culture was incubated at 30°C, shaking at 250 rpm overnight. From
this starter culture 1ml was used to inoculate 500 ml of antibiotic selective LB
medium. This was incubated at 30°C and shaken at 250 rpm for approximately 16-18
hrs until the OD600 of cells was between 0.5-0.8. The cells were then centrifuged at
5000 rpm for 20 mins at 4°C, the supernatant was decanted and the pelleted cells
were re-suspended in infiltration medium (500ml of full strength MS solution
containing 5% sucrose (w/v) and 0.05% silwet L-77 (v/v). For transformation, pots
containing 9 healthy, 4-5 weeks old Arabidopsis plants were used. The pot was
carefully held upside down in the Agrobacterium solution so that it fully covers all
immature flower buds. Plants were held in this position for 1 min under vacuum and
then carefully transferred to a large polythene bag to maintain humidity. The plants
were transferred in these bags to a growth chamber, and after 24 hrs removed from
the bags and grown as normal until maturity.
2.5.2 Screening Transgenic Arabidopsis thaliana plants
The T1 seeds were harvested from the mature Agrobacterium transformed
Arabidopsis plants. The seeds were sterilized as described above, plated on antibiotic
selective ½ MS medium and incubated until green leaves and long roots were visible
on the transformants, which were then transferred to soil.
2.5.3 Hybrid aspen T89 plants by co-cultivation
Agrobacterium and T89 (hybrid aspen) co-cultivation for poplar transformation was
performed according to the Meilan and Ma (2007) with some modifications as
described. A single Agrobacterium colony containing the destination vector plasmid
49
was picked and inoculated in 30 ml antibiotic selective LB medium. The culture was
incubated at 30°C, shaking at 250 rpm overnight. The cells were then centrifuged at
5000 rpm for 20 mins at 4°C, the supernatant discarded and the pelleted cells resuspended with sterile ½ MS liquid medium containing 2% sucrose (w/v) and 25μM
acetosyringone to an OD600nm = 0.4 to 0.6. Sterile Populus explants (30-40 leaf
discs and wounded 3-5 mm stems) were added to a 50 ml Falcon tube containing 3040 ml diluted Agrobacterium (Figure 3.5B). This culture was incubated at RT,
shaking at 150-200 rpm for approximately 2 hours. Excess Agrobacterium
suspension was removed from the explants by blotting on sterile paper, and the
explants cultivated on fresh CIM callus induction media (Figure 3.5C) (full strength
MS Medium 4.4g/l, 0.85% Agar, Sucrose 2%, BAP 1mg/l, IBA 0.1mg/l and TDZ
1mg/l) without antibiotic selection in the dark for 2 days.
2.5.4 Screening transgenic hybrid aspen T89 plants
After cultivation on CIM without antibiotic selection for 2 days, the explants were
moved onto CIM with kanamycin 100mg/l and incubated for 2-3 weeks in darkness.
The calli were sub-cultured on fresh CIM every 2 weeks. After 4-6 weeks callus
were transferred onto SIM shoot induction media with antibiotic selection ( see
Figure 3.5D) (WPM, Sucrose 2%, BAP 1mg/l, IBA 0.1mg/l, and Phytagel). Callus
was subculture on fresh SIM every 2-3 weeks until shoots were formed. The
elongated shoots were then transferred to RIM root induction media ( see Figure
3.5E) (WPM, 2%, Sucrose. 0.28% Phytagel w/v) maintaining the antibiotic selection
pressure causing only the true transformants to root.
2.5.5 Transplanting into Greenhouse
50
Approximately 4-6 weeks old in-vitro propagated plants of about 5-6 cm in height
were removed from polypropylene Microbox© Combiness and excess agar washed
off gently with clean tap water. The plants were potted in small pots with potting soil
(Perlite: Peat moss 2:1). The pots were transferred into re-sealable polythene bag
5x7.5" with 100 ml water for about 3 weeks in a growth room at 25 ± 1˚C under 16 h
photoperiod. The bags were gradually opened every day for increasing time intervals
to allow for acclimatization. In due course the bags were removed and the plants
maintained in a Greenhouse.
2.6 Histological analysis
Stem and hypocotyls of 5-6 weeks old Arabidopsis plants, stems of four weeks tissue
culture grown poplars and 32 weeks Firs Botanical ground grown poplars were
embedded in plastic resin for histological analysis. The plant material was collected
in freshly prepared FAA (50% absolute ethanol, 10% Formaldehyde solution (36.538% in H2O) 5% Acetic acid, all v/v). The tissue was fixed in FAA at 4°C overnight.
Post fixing, the tissue was dehydrated at RT in series of alcohol dilution (50%, 70%,
90% and 100%) for 1-2 hrs at each dilution. Finally the tissue was dehydrated in
100% alcohol twice. Post dehydration the tissue samples were embedded using a JB4 Embedding Kit (Polysciences, Inc) as per a few modifications to the manufactures
protocol. Explants were initially treated with infiltration solution (a mixture of JB-4
Solution A (Monomer) 100.00ml and Benzoyl Peroxide, Plasticized (Catalyst)
1.25gm). The samples were infiltrated in increasing concentration of infiltration
solution in ethanol. Finally the tissue was treated in 100% infiltration solution for a
minimum of 3 changes prior to embedding. For poplar tissue fixing, alcohol
dehydration and infiltration steps were performed under vacuum. The embedding
51
solution consisted of freshly prepared 25 ml infiltration solution and 1ml of JB-4
Solution B (Accelerator), vortexed to mix well. The tissue sample was carefully
dabbed on tissue roll to get rid of the excess infiltration solution and placed in the
embedding mould. 1.5 ml of fresh embedding solution was added to the mould and
covered with an air tight film (Parafilm® M) to maintain an anaerobic exothermic
reaction. The blocks were allowed to polymerize overnight. Post polymerization the
blocks were removed from the mould and allowed to harden in the presence of silica
gel (Type II, 3.5 mm bead size Sigma-Aldrich) for 5 to 6 days. The hardened blocks
were sectioned (3-5 µm sections) using glass knifes generated in-house. The sections
were stained with 0.05% aqueous toluidine blue for 30 seconds, mounted in cytoseal and were observed under bright field illumination on a Leica DM5500
microscope and photographed with a SPOT Xplorer 4MP camera (Diagnostic
Instruments).
2.7 Growth characteristics analysis of transgenic plants
2.7.1 Transgenic Arabidopsis thaliana plants
The T2 lines seeds of confirmed transformants were sterilized and plated on ½ MS
plates until ready to be transplanted on soil.
50-60 seedlings (wild-type and
transformants) were transplanted on soil on the same day. When the plants were 5-6
weeks old, 40 whole plants were selected randomly and measured for height and wet
weight (biomass analysis). The plants were bagged in glassine bags and allowed to
dry at 50oC for approximately 2 to 3 weeks, following which dry weight was
recorded. Stems and hypocotyls were collected from the remaining 10 to 15 plants
52
and embedded for sectioning for microscopic analysis (morphology/phenotype and
cell count of the vascular bundle).
2.7.2 Transgenic hybrid aspen T89 plants
Confirmed transformants and controls were synchronously re-rooted by excising the
shoot tips of healthy tissue-culture grown poplar plants and transferring them onto
propagation medium. Once top shoot tips were excised the middle internodes were
collected
and
embedded
for
sectioning
for
microscopic
analysis
(morphology/phenotype and xylem cell count). 3 weeks post-rooting, whole plants
were measured for dry weight (biomass analysis) as described above. Additionally,
for plants that were grown in Firs Botanical grounds, stem diameter at base (2cm
above soil) and whole plant height were measured over a period of three, five and
six months after transfer to soil. At 8 months after transfer, 8 plants from 15 were
harvested at the 50th internode for leaf phenotype/area measurement, microscopic
and gene expression analysis. At twelve months, the wet and dry weight of 10 cm of
material taken from the base (2cm above soil), 50th internode and top of the stem was
also determined.
2.8 β-glucuronidase (GUS) marker gene expression
The plant tissues were incubated in 90% acetone (v/v) for 1 hr on ice under vacuum.
Following this, the acetone was decanted and the tissue samples were washed with
GUS staining buffer (100mM sodium phosphate (pH 7.2), 10 mM EDTA, 0.5 mM
ferricyanide, 0.5 mM ferrocyanide) under vacuum. Post washing, the tissue samples
are incubated in GUS staining buffer plus 1mM X-GlcA under vacuum until the
tissue samples sank and then incubated at 37 oC for 16 to 18 hrs. Samples are then
53
cleared in by incubating in increasing concentration of EtOH (20 % to 100%) for 1-2
hrs at each concentration (Rodrigues-Pousada et al. 1993). The stem tissue were
further sectioned and photographed.
2.9 Gene Expression Analysis
2.9.1 RNA extraction
For RNA extraction, samples of T89 hybrid aspen grown in the Fir’s Botanical
ground were stored at -80°C. Total RNA was extracted from stem material using the
CTAB- LiCl purification method (Xu et al. 2009). The purified total RNA was
subjected to clean-up by using the Qiagen RNeasy® Plant Mini Kit and stored at
-80°C prior to first strand synthesis. RNA samples were quantified using a Nanodrop
spectrophotometer (ND-1000, Labtech).
2.9.2 First strand synthesis
First strand synthesis was performed as per the manufacturer’s protocol and
Untergasser (2008). A volume equivalent to 1 µg of RNA was added to 1 µl of RQ1
DNase I enzyme and buffer (Promega) and nucleases free sterile H2O to a final
volume of 10 µl. The sample was then incubated at 37°C for 30 minutes. Afterwards,
1 µl of DNase stop solution (Promega) was added and the mixture incubated at 65°C
for 10 min. The DNase treated RNA was prepared for annealing by adding 3 µl 50
µm Random Hexamers Ambion® and nucleases free sterile H2O to a final volume of
20 µl, followed by incubation at 70°C for 10 mins and then 25°C for 10 mins . To
this preparation was added the cDNA master mix consisting 8 µl of 5x First Strand
Buffer, DDT (100 mM) 4 µl, dNTP (10 mM each) 2 µl, SUPERase inhibitor
Ambion® 20 U, SuperScript II® Invitrogen 200 U and nucleases free sterile H2O 4
54
µl. 20 µl of this mix was add to each reaction and incubated in the thermocycler
C1000 (Bio-Rad) at 25 °C for 10 min, 37 °C for 45 min, 42 °C for 45 min, 70 °C for
15 min. The cDNA was stored at -20°C until needed.
2.9.3 Semi-quantitative and Q-PCR to determine gene expression levels
Semi-quantitative RT-PCR was performed using Thermocycler C1000 (Bio-Rad).
PCR programme used 24 cycles for housekeeping gene α-tubulin and 34 cycles for
gene of interest. PCR reactions were performed in a volume of 20 µl containing 2 µl
cDNA, 10 µl 2X My Taq Redmix™ (Bioline Reagents Ltd, UK), 50 pmol oligonucleotide primers (Table 2.2) (Eurofins MWG, Ebersber, Germany). The levels of
cDNA were normalized to the expression of housekeeping gene. The RT-PCR
product was electrophoresed on 1.5% agarose gel and the relative band intensity was
measured using Image Lab 5.1 software (Bio-rad).
55
Chapter 3: Results
3.1 Generating PttPXY and PttCLE41 over-expression vectors
The poplar orthologs of PXY and CLE41 were identified and sequenced from hybrid
aspen, here after referred as PttPXY and PttCLE41. The primers used were designed
against the Populus trichorpa PXY paralogue (Potri.003G107600) and CLE41
paralogue (Potri. 012G019400). Arabidopsis and P. trichocarpa sequence data were
obtained from Phytozome v9.1. (http://www.phytozome.net/). Figure 3.1A shows an
alignment of amino acid PXY sequences from Arabidopsis (AtPXY, AT5G61480),
P. trichocarpa (PtPXY, Potri.003G107600) and hybrid aspen PttPXY. Figure 3.1B
shows a similar alignment of amino acid CLE41 sequences from Arabidopsis
(AtCLE41, AT3G24770), P. trichocarpa (PtCLE41, Potri.012G019400) and hybrid
aspen PttCLE41 (Etchells et al. 2015).
Initially the entry clones carrying the PttPXY (pENTR-D-TOPO-PttPXY) and
PttCLE41 (pENTR-D-TOPO-PttCLE41) genes were a gift from a lab colleague. To
generate the expression vectors the entry clones were sequenced to check for error.
It was found that the stop codon was missing in the pENTR-D-TOPO-PttPXY.
Therefore to generate pENTR-D-TOPO-PttPXY, primers were designed against the
Populus trichorpa PXY paralogue (Potri.003G107600). In this case the reverse
primer (Table 2.2), that contained the stop codon, was tagged with a MluI restriction
site 5’—ACGCGT—3’ to generate an easy means of checking for the presence of
the stop codon. The pENTR-D-TOPO-PttCLE41 was amplified with primers (Table
2.2) flanked with attB sites by High-Fidelity PCR. Once the entry clones had been
constructed and verified by sequencing they were used in a recombination reaction to
56
generate the PXY and CLE expression vectors (Figure 3.1.1) to be further
transformed in Arabidopsis to check the PXY-CLE functionality. These 35S driven
expression vectors (35S::PttCLE41 and 35S::PttPXY) were generated by cloning the
pENTR-D-TOPO-PttPXY and pENTR-D-TOPO-PttCLE41 in binary expression
vector pK2GW7 behind the constitutive 35S promoter (Figure 3.1.1). The
35S::PttCLE41 and 35S::PttPXY constructs were checked by restriction digestion
using the restriction enzyme MluI and subsequently were transformed in
Agrobacterium for Arabidopsis transformation. These expression vectors were
generated to investigate the role of PXY and CLE41 in poplar.
57
Figure 8
Figure 3.1: Sequence alignment of amino acid sequences of PXY and CLE41. PXY
(A) and CLE41 (B) GenBank accession Numbers for PXY is KP682331 and for
CLE41 is KP682332 (Etchells et al. 2015).
58
Figure 9
Figure 3.1.1: 35S poplar PttPXY and PttCLE41 over-expression vectors made by the
LR clonase reaction. Recombination of the attL1/attR1 and attL2/attR2 take place
during the reaction and replaces the ccdb cassette with the PXY sequence to generate
35S::PttPXY or CLE41 sequence to generate 35S::PttCLE41.
59
3.2 Effects of over-expression of PttPXY and PttCLE41 genes in
Arabidopsis
Figure 10
Figure 3.2: The Poplar orthologues of CLE41 and PXY genes are functional in
Arabidopsis. Transverse section of a stem vascular bundle (upper) and a hypocotyl
(lower) are shown for each genotype taken from five to six week old plants. Upper
panel (A-C) are sections from inflorescence stem vascular bundles and lower panel
(A-C) are the sections from Arabidopsis hypocotyls (5-6 weeks old plants).
Representative images are shown for wild-type (A), 35S::PttCLE41 (B), pxy mutant
(C), a pxy mutant complemented with 35S::PttPXY (D), SUC2::AtCLE41 in wt (E)
60
and 35S::PttPXY
in SUC2::AtCLE41 (F). The (x) xylem and (ph) phloem are
indicated. Scales bars represent 50 µM (A-F).
The PXY-CLE41 receptor and ligand pair can control the rate and orientation of the
vascular cell division in Arabidopsis (Etchells and Turner 2010). To test the
functional capacity of the PXY and CLE41 orthologous they were transformed into
Arabidopsis. 35S::PttCLE41 Arabidopsis transformants demonstrated a disrupted
vasculature phenotype that was observed in 8 of 10 representative lines (Figure
3.2B). It also showed increased cell numbers per vascular bundle (Figure 3.2.1A)
and decreased plant height (Figure 3.2.1C) similar to previous observations using
AtCLE41 by Etchells and Turner 2010. Six of ten 35S::PttPXY lines transformed into
a pxy mutant background showed a restoration of wild-type vascular patterning
(Figure 3.2D) and plant height was increased and restored to wild-type levels (Figure
3.2.1C). 35S::PttPXY SUC2::AtCLE41 over-expressors produced large numbers of
vascular cells (Figure 3.2.1A) and strong secondary growth in the interfascicular
region compared with the wild-type and SUC2::AtCLE41 while maintaining a wellorganised vasculature and defined xylem (Figure 3.2E and F). These observations
are consistent with the findings of Etchells and Turner (2010) who used the
Arabidopsis genes. The over-expression of the poplar PXY and CLE gene in
Arabidopsis also increased plant biomass (Figure 3.2.1B). We therefore concluded
that the 35S::PttCLE41 and 35S::PttPXY constructs are functional in Arabidopsis
and are functional orthologues of the Arabidopsis genes.
61
Figure 11
Figure 3.2.1: Growth characteristics of Arabidopsis plants overexpressing poplar
PXY and CLE genes. Cell count per vascular bundle (A), dry weight (biomass
analysis) (B) and plant height (C) of wild-type or mutant Arabidopsis lines
62
expressing the constructs indicated. The p values were determined using ANOVA
and a post-hoc test (LSD), N =10 (A) and N=40 (B and C). Error bars are standard
error (SE).
3.3 Cloning the binary vector rolD::PttPXY 35S::PttPXY
A previous study by Etchells and Turner (2010) reported reduction in xylem
differentiation in Arabidopsis 35S::PXY 35S::CLE41 lines. Therefore to evaluate the
function of expressing the PXY and CLE genes together in poplar, a constitutive
double overexpression binary vector rolD::PttPXY 35S::PttPXY was generated. We
aimed to examine whether expressing the PXY and CLE genes together increases the
number of cell divisions in Populus. If rolD::PttPXY 35S::PttPXY transgenic poplar
lines produce similar phenotypes to 35S::AtPXY 35S::AtCLE41 lines, it would
indicate presence of similar pathways in trees. This would have potential industrial
applications, such as increasing tree biomass. For rolD::PttPXY 35S::PttPXY, the
Agrobacterium rhizogenes rolD promoter was identified as being able to produce
strong ectopic expression of variety of genes (Leach and Aoyagi 1991). It was
appropriate to use the rolD promoter instead of using two 35S promoters in order to
prevent gene silencing. Therefore for generating rolD::PttPXY 35S::CLE41
construct, PttCLE41 was sub-cloned into pDONRP4-P3 (Figure 3.3) which was
further combined with pENTR-D-TOPO-PttPXY and pK7m34GW2-8m21GW3
(Figure 3.3.1) using an LR clonase recombination reaction.
63
Figure 12
Figure 3.3: Summary of generating pDONR-PttCLE41 vector. Recombination of the
attP1/attP3 and attP2/attP4 during the BP clonase recombination reaction replaces
the ccdb cassette with the CLE41 sequence and generates the pDONR-PttCLE41
vector.
Figure 13
64
Figure 3.3.1: Summary of generating rolD::PttPXY 35S::PttPXY vector.
Recombination of attL1/attL2 of pENTR-D-TOPO-PttPXY, attb1/ attb2 of pDONRPttCLE41 with attR1/attR2 and attR3/attR4 of pK7M34GW2-8M21GW3 was crucial
for generating the double overexpression binary vector rolD::PttPXY 35S::PttPXY.
The ccdb cassette is replaced with the PXY and CLE41 sequences at the respective
site during the LR clonase recombination reaction.
3.4
Identification
and
cloning
promoters
for
tissue–specific
overexpression vectors
Etchells and Turner (2010) have already shown by in situ hybridisation that CLE41
is expressed in phloem cells adjacent to dividing cells in the cambium region
where PXY is expressed. To study the function of phloem expressed CLE41 in
Populus, the PP2 (Phloem Protein 2) was identified referring to the poplar
expression database. The PP2 promoter was identified and was used to check
whether it confers phloem specific expression making it suitable for driving the
expression of CLE41. The PtPP2 promoter was cloned from Populus trichocarpa
and used to generate the construct (PtPP2::PttCLE41). In order to study the effects
of tissue-specific PXY expression in Populus, publicly available expression data was
screened and the AINTEGUMENTA (ANT)
promoter was identified.
Transcriptomic data revealed that the ANT promoter is highly expressed, localised
exclusively in the young actively dividing cells of the cambium (Schrader et al.
2004), making it suitable for driving the expression of PXY. The PttANT promoter
ANT was cloned from hybrid aspen Populus tremula x tremuloides and used to drive
expression of PttPXY (PttANT::PttPXY). Both were generated in a pCambia2300
65
backbone and the primers used are listed in Table 2.2. Prior to the LR clonase
recombination reaction, the PttPXY and PttCLE41 entry clones were digested with
restriction enzyme EcoNI to deactivate the kanamycin resistance in the vector
backbone, as the destination vector carried kanamycin gene as well for bacterial
antibiotic selection. After the LR clonase recombination reaction PttANT::PttPXY
and PtPP2::PttCLE41 were generated (Figure 3.4). The constructs were checked by
digest with EcoRI and BamHI and by sequencing with the M13 forward and reverse
sequencing primers. On checking the latest annotation of the Populus tremula
cambium cDNA library EST name: UB11CPH11, GenBank: BU820600.1, it was
observed that the PttANT promoter fragment consisted of the both upstream and
downstream sequences of the potential transcriptional start site. Therefore it was
absolutely crucial to generate the transcriptional reporter clones to determine the
function of the promoters used in the study. The transcriptional reporters
pPttANT::eGFP-GUS and pPtPP2::eGFP-GUS, (Figure 3.4.1 A and B) were
generated by cloning a fragment encoding eGFP-GUS into pVX31 and pVX33,
carrying the promoters pPttANT and pPtPP2 respectively.
A second double over-expresser PtPP2:: PttCLE41- PttANT::PttPXY was generated
by digesting PtPP2::PttCLE41 with SBf1 to yield a fragment consisting of the PtPP2
promoter, CLE41 gene sequence and the T35S terminator and cloning this into SBf1
linearized PttANT::PttPXY
(Figure 3.4.2). The tissue-specific overexpression
vectors were transformed in Agrobacterium. These Agrobacterium carrying the
expression vector were used to transform poplars to study the phenotype and growth
characteristics in the over-expressing poplar lines.
66
Figure 14
Figure 3.4: Summary of cloning PttPXY and PttCLE41 entry clones in custom-made
Gateway destination vectors pVX31 and pVX33 respectively. Recombination of the
attL1/attR1 and attL2/attR2 during the LR clonase recombination reaction replaces
the ccdb cassette with the CLE41 sequence and generates PtPP2::PttCLE41 whereas
ccdb cassette is replaced with PXY sequence generating PttANT::PttPXY.
67
Figure 3.4.1: Maps for transcriptional reporters. pPttANT::eGFP-GUS (A) and
pPtPP2::eGFP-GUS (B), showing the promoters pPttANT and pPtPP2, eGFP-GUS
and the T35S terminator.
68
Figure 15
Figure 3.4.2: Generating the tissue-specific over-expressor PtPP2::PttCLE41PttANT::PttPXY. Summary of cloning PtPP2::PttCLE41 into PttANT::PttPXY to
generate PtPP2::PttCLE41- PttANT::PttPXY using the restriction enzyme SBf1.
Digesting PtPP2::PttCLE41 with SBf1 yields a fragment consisting the PtPP2
promoter, CLE41 gene sequence and the T35S terminator whereas SBf1 linearizes
PttANT::PttPXY. The digested fragment was ligated into the linearized
PttANT::PttPXY
to
generate
PtPP2::PttCLE41-
PttANT::PttPXY.
Map
of
PtPP2::PttCLE41- PttANT::PttPXY, the tissue-specific double overexpression
vector. The map shows the promoter PttANT sequence followed by the PttPXY gene
sequence and the T35S terminator followed by the promoter PtPP2 and then
PttCLE41 gene sequence and the T35S terminator.
69
3.5 Generating transgenic Poplars for function analysis of poplar PXY
and CLE in trees.
Figure 16
Figure 3.5: Summary of the steps involved in production of transgenic poplars.
Typical wild-type aspen growing in tissue culture (A). Co-cultivation of
agrobacterium with internodes explants (B) Incubation of co-cultured internode
explants on callus induction media (CIM) (C). Calli on shoot induction medium
(SIM) (D). Shoots with forming roots on root induction media (E).
Poplar transformation was performed using a modified version of the protocol of
Meilan and Ma (2007) and is outlined in Figure 3.5. In vitro grown, four to five
weeks old hybrid aspen were used as the starting material for transformation (Figure
3.5A). Explants, in this case the stem internodes, were co-cultured in the dark to
induce calli formation. The calli were visible at three to four weeks (Figure 3.5D).
70
The calli then were moved to shoot induction media where the shoots eventually
regenerated at approximately seventeen weeks. The nascent shoots were allowed to
elongate by moving them on to fresh shoot induction media before the shoots were
excised and then moved on to the root induction medium. The shoot elongation took
approximately four weeks. The shoots that rooted in the presence of the antibiotic
selection were considered as transformants (Figure 3.5). The process of rooting took
approximately four to five weeks. During the process chimeras also formed as well
as false positives. In order to check the transgene insertion the plants were
propagated in the presence of the antibiotic selection for 10 to 12 weeks. The
transformants were transferred to soil and gradually allowed to acclimatize and
harden before being used for evaluating growth characteristics and gene expression
studies. The process of hardening takes two to three weeks with approximately 95%
of plants surviving. The hardened transgenic poplars are grown in the greenhouse to
evaluate growth characteristics for up to 1 year.
3.6 Generating transcriptional reporter clones and function analysis of
promoter PttANT and PtPP2
The reporter clones were transformed in the poplars and assayed for β-glucuronidase
activity of the marker gene. GUS analysis of PtPP2::GUS revealed that the promoter
gave excellent phloem-specific expression in stems and also the expression was
confined to the vascular region in the leaf (Figure 3.6 B and E) in comparison to the
wild-type (Figure 3.6 A and D) . GUS analysis of PttANT::GUS confirmed the GUS
expression in the dividing cambium region of the stem and also the expression was
only confined to the vascular region in the leaf (Figure 3.6 C and F) suggesting that
although the PttANT fragment included sequences both upstream and downstream of
71
the supposed transcriptional start site the promoter still functioned to give tissuespecific expression.
Figure 17
Figure 3.6: GUS staining of reporter gene lines. Wild-type control (A and D),
PtPP2::GUS (B and E), and PttANT::GUS (C and F) plant material strained for
GUS activity and cleared prior to being photographed. The upper panels shows
transverse stem sections, while the lower panels are leaves. The (x) xylem, (c)
cambium and (ph) phloem are indicated. Scale bars represent 100 µm (upper panels)
and 200 µm (lower panels).
72
3.7 Analysis of ectopic expression of PttPXY and PttCLE41 in hybrid
aspen (Populus tremula x P. tremuloides)
Figure 18
Figure 3.7: Phenotype of Populus tremula x P. tremuloides constitutively overexpressing PXY and CLE41. Sections from tissue culture-grown plantlets 3 weeks
post-rooting (A) and representative images of greenhouse-grown plants 13 weeks
after transfer to soil (B). Where two sections are shown, they were selected to show
the range of phenotypes observed. Red asterisks demonstrate examples of ordered
files of cells. The phloem (ph) and xylem (x) are indicated. Scale bars indicate 200
µM (upper panels) and 50 µM (lower panels).
73
After confirming that the poplar orthologues of PXY and CLE41 are functional in
Arabidopsis, we examined the outcome of constitutive over-expression of PXY and
CLE41 genes in hybrid aspen (Populus tremula x P. tremuloides). 35S::PttPXY,
35S::PttCLE41 constructs individually or both genes together in a single binary
plasmid that contained 35S::PttCLE41 and rolD::PttPXY cassettes were transformed
in hybrid aspen. 35S::PttCLE41 poplar plants displayed a varying degree of
disrupted vasculature phenotype in all 15 lines similar to the vascular patterning of
Arabidopsis 35S::PttCLE41 (Figure 3.7A, 3.2B ). Decreased plant height was
observed by 35S::PttCLE41 poplar plants in comparison to the untransformed
controls (Figure 3.7B). All 10 lines of 35S::PttPXY poplar plants also showed some
loss of organization in the xylem, but to a much lesser extent in comparison to the
35S::PttCLE41 poplar plants (Figure 3.7A). Furthermore the rolD::PttPXY
35S::PttCLE41 hybrid aspen also exhibited loss of organization. 7 out of 15
rolD::PttPXY 35S::PttCLE41 lines exhibited a wild-type poplar phenotype whereas
the remaining 8 displayed varying degrees of tissue disorganization (Figure 3.7A).
35S::PttCLE41, 35S::PttPXY and rolD::PttPXY 35S::PttCLE41 produced transgenic
trees that demonstrated no increases in tree growth. 35S::PttCLE41 poplar lines were
considerably shorter than wild-type controls. These 35S::PttCLE41 poplar lines also
demonstrated several growth aberrations like stunned growth and smaller leaves
compared to the untransformed controls. (Figure 3.7B). The results confirms that
PXY-CLE41 influences vascular organisation and development in perennial trees.
74
3.8 Tissue-specific over-expression of PXY and CLE41 genes in hybrid
aspen (Populus tremula x P. tremuloides) enhance wood formation and
deposition.
Figure 19
Figure 3.8: Phenotypes of hybrid aspen with tissue-specific over-expression of
PttCLE41 and PttPXY. Sections from tissue culture grown plantlets 3 weeks postrooting (A). Greenhouse grown plants 13 weeks after transfer to soil (B). The xylem
(x) and phloem (ph) are indicated. Arrows indicate the disrupted xylem. Scale bars
represent 200 μm.
75
On observing the consequence of constitutively over-expressing PXY and CLE gene
in poplar, we hypothesised that the tissue-specific expression of both the genes might
be vital for increasing vascular cell division while retaining vascular tissue
organization. Consequently, we transformed the hybrid aspen with constructs with
tissue-specific over-expression constructs containing PXY and CLE genes. In
contrast to 35S::PttCLE41 fourteen independent PtPP2::PttCLE41 lines showed
highly organized tissue vasculature (Figure 3.8A). 7 out of 15 PttANT::PttPXY lines
did exhibit minor xylem tissue disruptions (Figure 3.8A; arrow) analogous to those
detected in 35S::PttPXY poplar lines (Figure 3.7A). All lines over-expressing
PtPP2::PttCLE41 and PttANT::PttPXY together demonstrated highly organized
tissue vasculature analogous to that of wild-type controls (n=12) (Figure 3.8A). After
3 weeks post-rooting in vitro the plants with tissue-specific over-expression all
exhibited increases in the number of vascular cells (Figure 3.9A) and increased dry
weight (Figure 3.9B).
3.9
Growth
characteristics
of
hybrid
aspen
overexpressing
PttCLE41/PttPXY
To further analyse the growth characteristics of hybrid aspen overexpressing
PttCLE41/PttPXY, we transferred the plants to soil and maintained them in the
greenhouse. The transgenic hybrid aspen were measured to determine the plant
height and stem diameter at three intervals; 15 weeks, 26 weeks and 33 weeks after
transfer to soil/following acclimation in soil. The hybrid aspen over-expressing
35S::PttCLE41, 35S::PttPXY and rolD::PttPXY 35S::PttCLE41 did not demonstrate
increase in plant height or stem diameter throughout the period of 33 weeks in
comparison to the wild-type plants (Figure 3.9.1A and B). Similar to the Arabidopsis
76
35S::PttCLE41, poplar 35S::PttCLE41 lines were shorter (Figure 3.7B). In contrast
PtPP2::PttCLE41, PttANT::PttPXY and PtPP2::PttCLE41 PttANT::PttPXY plants
all appeared to grow normally (Figure 3.8B) and were consistently larger than the
wild-type control plants with both greater plant height and stem diameter (Figure
3.9.2A and B). The tissue-specific over-expressor when measured throughout the
three time point’s demonstrated increase in plant height and diameter in comparison
to the constitutive over-expressor and untransformed controls (Figure 3.9.2A and B).
The most consistent performer was the PtPP2::PttCLE41- PttANT::PttPXY double
over expressor. At all three time points this line displayed greater plant height and
stem diameter in comparison to the wild-type controls and the PtPP2::PttCLE41 and
PttANT::PttPXY lines (Figure 3.9.2A and B). The next best performing lines were
the PtPP2::PttCLE41 plants, displaying significant increase in stem diameter and
PttANT::PttPXY lines, displaying significant increases in plant height in comparison
to the wild-type hybrid aspen (n=15) (Figure 3.9.2A and B).
It was evident that PtPP2:: PttCLE41- PttANT::PttPXY transgenic hybrid aspen
were taller than the other tissue-specific over-expressor due to the faster growth rate.
In order to establish the basis of the increased growth rate we measured both the
number
of
internode
and
average
internode
length.
PtPP2::PttCLE41-
PttANT::PttPXY plants had significantly larger numbers of internodes (90)
as
compared to the wild-type controls trees (n=15) (Figure 3.9.3A). The double over
expressor also displayed a significant increase in internode length compared to the
wild-type control (n=15) (Figure 3.9.3B). Although the transgenic hybrid aspen
displayed normal morphology (Figure 3.8A and B), the double over expressor
PtPP2::PttCLE41- PttANT::PttPXY lines also demonstrated 2 fold increases in leaf
77
area (Figure 3.9.3D). To gain a better insight about the cause of increases in the
stem diameter in PtPP2::PttCLE41- PttANT::PttPXY lines, 8 independent
PtPP2::PttCLE41- PttANT::PttPXY lines were harvested to perform biomass
analysis. The stem material was harvested from the 50th internode in order to monitor
for changes in growth rate. The wet and dry weight of the double over-expressor
lines was almost twice that of the wild-type controls (Figure 3.9.4A and B). The
measurements obtained at the base (2cm above soil level), 50th internode (middle)
and at the top of the stem confirmed that PtPP2::PttCLE41- PttANT::PttPXY lines
showed significant increase in the dry weight (Figure 3.9.4B) compared to the other
over-expressor used in the study. At the base and the middle of the tree, the dry
weight of PtPP2::PttCLE41- PttANT::PttPXY stem pieces were more than twice the
dry weight of the wild-type control plants. Further, to investigate that is it possible to
enhance wood formation without modifying xylem cell morphology, the software
Cell profiler was used to determine various morphological features of the xylem cells
(Etchells et al. 2015). The analysis by the cell profiler software demonstrated that
there were no significant alterations to average cell size and lumen size, area of the
cell wall and density of vessels in the double over expressor in comparison to the
wild-type control lines (Table 3.1) (Etchells et al. 2015). Therefore we can conclude
that the enhanced wood production is not at the expense of modified wood
morphology.
78
Figure 20
Figure 3.9: Growth characteristics of hybrid aspen lines in tissue culture 3 weeks
post-rooting. Xylem cell number (A) Dry weight (B) of the PttPXY/PttCLE41 over
expressing hybrid aspen lines in tissue culture 3 weeks post-rooting. The p values
were determined using ANOVA and a post-hoc test (LSD), N=7, ** indicates p<0.05
and * indicates p=0.011. The error bars are standard error (SE).
79
Figure 21
Figure 3.9.1: Growth characteristics of hybrid aspen (Populus tremula x P.
tremuloides) lines constitutively overexpressing PttCLE41/PttPXY. Mean tree height
(A) and tree diameter (B) measurements of the transgenic poplar lines (N=15) grown
in soil. Trees were transplanted in soil in April and were measured at three intervals,
15 weeks (July), 26 weeks (August) and 33 weeks (October). The p values were
determined using ANOVA and a post-hoc test (LSD), N = 15. Error bars are
standard error.
80
Figure
Figure 22
Figure 3.9.2: Growth characteristics of trees with tissue-specific PttCLE41/PttPXY
overexpression. Mean tree height stem diameter (A) and stem diameter (B)
measurements from hybrid aspen overexpressing PttCLE41/PttPXY grown in soil are
shown. Trees were transplanted in soil in April and were measured at three intervals,
15 weeks (July), 26 weeks (August) and 33 weeks (October). The p values were
determined using ANOVA and a post-hoc test (LSD), N = 15. Error bars are
standard error.
81
Figure 3.9.3: Characteristics of the morphology of hybrid aspen engineered with
tissue-specific PXY and CLE41 expression. 26 weeks old PtPP2::PttCLE41,
PttANT::PttPXY and PtPP2::PttCLE41- PttANT::PttPXY hybrid aspen were
analysed for number of internodes (A), mean length of 50th internode (B), xylem
cell number in a sector of a stem transverse section taken from the 50th internode
(C) and leaf area calculated from measurements of five leaves from around the 50th
internode (D). Error bars are standard error.
82
Figure 23
Figure 3.9.4: Biomass of hybrid aspen tissue-specific overexpression of PXY and
CLE41. Dry weight (A) Wet weight (B). Tissue samples were taken from the base
(2cm above soil), middle (50th internode) and top, except for 35S::PttCLE41 lines
which had less than 50 internodes in which case sections were taken halfway
between the top and bottom for analysis. The p values were determined using
ANOVA and a post-hoc test (LSD), N=7 * means p < 0.05; ** means p < 0.001. The
error bars are standard error.
83
Control
PtPP2::CLE41- PttANT::PXY
Average cell size
4.46 ±0.095
4.24 ±0.13
Average lumen size
2.29 ±0.095
2.54 ±0.066
Average cell wall 1.94 ±0.17
area
Vessels
cells
per
1.91 ±0.11
1000 0.36 ±0.014
0.36 ±0.044
Table 3
Table 3.1. Analysis of wood morphology from hybrid aspen with tissue-specific
expression of PXY and CLE41. The analysis was carried out on transverse stem
sections from the 50th internode. Mean values are derived from 5 independent
PtPP2::PttCLE41- PttANT::PttPXY lines and are shown with the standard error. The
area measurements are presented in µm. The statistical analysis performed by using a
t-test that revealed no significance differences (Etchells et al. 2015).
84
3.10 PttCLE41 and PttPXY expression analysis in PtPP2::PttCLE41PttANT::PttPXY over-expressor lines
The tissue were harvested around the 50th internode for PXY/CLE gene expression
analysis (Figure 3.10A and B). The results demonstrate PttCLE41 gene expression is
closely correlated with the xylem cell count whereas PttPXY was much less
correlated with the xylem cell count (Figure 3.10C and D). For consistency, qPCR
analysis was performed on the same samples used for semi-quantitative method
(Figure 3.10B and 3.10.1A). As seen in Figure 3.10.1A, qPCR data confirms
PttCLE41
and
PttANT::PttPXY
PttPXY
lines.
expression
The
in
expression
8
of
independent
PttCLE41
PtPP2::PttCLE41
and
PttPXY
in
PtPP2::PttCLE41- PttANT::PttPXY over-expressor lines was calculated as (% wt).
This means the range of expression of the PXY/CLE gene per the range of gene
expression in the wild-type. Except for the lines 2 and 5 the expression pattern
determined by the Q-PCR and semi quantitative method remains the same i.e.
expression of CLE41 is higher whereas the expression of PXY is lowered. This data
shows a correlation to some extent between the Q-PCR and semi quantitative
method. Further a strong correlation between PttCLE41 expression (% wt) and
PttPXY expression (% wt) was observed (Figure 3.10.1B). This data suggest that
increase in CLE expression lowers the PXY gene expression in the
PtPP2::PttCLE41- PttANT::PttPXY over-expressor lines. These finding are
consistent with previous data shown by Etchells and Turner (2010) where high levels
of AtCLE41 expression results in suppression of PXY expression.
85
Figure 24
Figure 3.10: PttCLE41 and PttPXY expression analysis in PtPP2::PttCLE41PttANT::PttPXY lines.
RT-PCR showing expression levels in 8 independent
transgenic lines. RNA was extracted from stem material adjacent to the 50th
internode. α-tubulin was used as a control housekeeping gene (A). Relative intensity
of ng PCR product in (B). Intensity was determined using Image Lab 5.1 software
(Bio-Rad). Correlation between xylem cell number and PttPXY expression (C).
Correlation between xylem cell number and PttCLE41 expression (D).
86
Figure 25
Figure 3.10.1 Gene expression analysis by Q-PCR. Graph showing Q-PCR data
confirming PttCLE41 expression (% wt) and PttPXY expression (% wt) in 8
independent PtPP2::PttCLE41- PttANT::PttPXY lines (A). Correlation between
PttCLE41 expression and PttPXY expression (B).
87
3.11 Clonal propagation of PtPP2::PttCLE41- PttANT::PttPXY transgenic
hybrid aspen
Figure 26
Figure 3.11. Analysis of clonal PtPP2::PttCLE41- PttANT::PttPXY lines. Tree
diameter (A) and tree height (B) was measured every week starting at 4 weeks post
88
transfer to soil. Asterisks indicate p < 0.05 compared to the wild-type controls. The p
values were calculated by using ANOVA and a post hoc test (LSD); (n = 6) control;
(n = 5) 1, 3, and 9 PtPP2::PttCLE41- PttANT::PttPXY lines; (n = 4) for lines 2 and
4. Error bars are standard error.
In order to confirm that the differences that were observed between the double overexpressor and wild-type controls were reproducible we set-up a clonal propagation
experiment with 6 independent PtPP2::PttCLE41- PttANT::PttPXY lines. Plant
height and stem diameter measurements were recorded on weekly basis starting from
4 weeks after transfer to soil. As seen in Figure 3.11A the diameter of various clones
was significantly larger than the wild-type controls at all weeks monitored. There
was a degree of variation observed amongst the clones. For example,
PtPP2::PttCLE41- PttANT::PttPXY line 2 was both significantly taller and
demonstrated
larger
stem
diameter
as
compared
to
PtPP2::PttCLE41-
PttANT::PttPXY line 3 that was shorter and thinner at all-time points of
measurements (Figure 3.11A and B).
89
Chapter 4: Discussion
The study by Etchells and Turner (2010) demonstrated that in Arabidopsis, PXYCLE41 plays a crucial role in controlling both the rate of cell division and their
orientation in procambial stem. This system also inhibits xylem development
(Hirakawa et al. 2008; Hirakawa et al. 2010). Ectopic overexpression of CLE41 in
Arabidopsis can increase the number of cells in the vascular bundles. In turn, this
increase causes repression of xylem differentiation and a loss of vascular
organization. Moreover, as a consequence, a negative feedback loop is observed in
which high CLE41 expression leads to down regulation of PXY expression (Etchells
and Turner 2010).
In order to examine the function of PXY and CLE41 in trees the putative orthologs
of PXY and CLE41 genes were cloned from the hybrid aspen (Populus tremula x P.
tremuloides), referred to hereafter as PttPXY and PttCLE41. The 35S::PttCLE41
construct when transformed in Arabidopsis demonstrated loss of organisation similar
to the findings by Etchells and Turner (2010). The 35S::PttPXY construct restored
the wild-type phenotype by complementing the Arabidopsis pxy mutant (Figures
3.2D). Expressing 35S::PttPXY in SUC2::AtCLE41 Arabidopsis resulted in
increased plant biomass (Figure 3.2E) and suggested that PttPXY was able to
function with AtCLE41. Similar to their behaviour in Arabidopsis, constitutive overexpression of PttCLE41 and PttPXY genes in hybrid aspen, either individually or
together, frequently resulted in aberrant vascular organisation and plant growth and
did not significantly enhance wood formation. To overcome these problems
experiments were designed to restrict the up-regulation of PttCLE41 expression to
90
the phloem and PttPXY to the cambium zone. Using publicly available expression
data we selected the promoter of PtPP2 and PttANT to give phloem and cambialspecific expression respectively. The specificity of these promoters was confirmed
using GUS reporter genes (Figure 3.6). Tissue-specific over-expression of PttCLE41
and PttPXY individually and together did indeed lead to ordered vascular
organization and increase in plant biomass (Figures 3.8, 3.9.2 and 3.9.3). The
PtPP2::PttCLE41, PttANT::PttPXY and PtPP2::PttCLE41- PttANT::PttPXY lines
demonstrated a clear increase in the number of vascular cells three weeks post
rooting in tissue culture (Figure 3.8A). There were also some pleiotropic effects.
Over-expressing PttCLE41 and PttPXY together gave rise to much taller trees with
larger leaves and more biomass in comparison to the untransformed control plants
(Figures 3.9.2 and 3.9.3). When the transgenic hybrid aspen were moved to the
greenhouse and monitored for growth the PtPP2::PttCLE41, PttANT::PttPXY and
PtPP2::PttCLE41- PttANT::PttPXY lines showed both consistent increases in height
and diameter at all of the time points measured (Figure 3.9.2). Amongst the three
tissue-specific over-expressor lines, the double overexpressor PtPP2:: PttCLE41PttANT::PttPXY were both the tallest and showed the largest radial diameter (Figure
3.9.2). The increased height of PtPP2::PttCLE41- PttANT::PttPXY lines resulted
from both an increased number of nodes and increased internode length (Figure
3.9.3A and B). At 33 weeks, when the stem material from PtPP2::PttCLE41PttANT::PttPXY lines were harvested at the 50th internode, cell counts revealed a
strong correlation between the number of vascular cells, stem diameter and dry
weight (Figure 3.9.3C, 3.9.2B and 3.9.4). Analysis of xylem morphology using
CellProfiler
software
demonstrated
that
the
wood
that
is
formed
in
PtPP2::PttCLE41- PttANT::PttPXY lines is morphologically identical to wild-type
91
(Table 3.1) (Etchells et al. 2015). All this data indicates that from the first stage of
analysis, when the plants were first rooted, to the last stage nearly 1 year later, the
PtPP2::PttCLE41- PttANT::PttPXY plant consistently exhibited a 2 fold increase in
xylem formation (Figures 3.9.4 and 3.11). It seems likely that this pattern of growth
will continue during the lifetime of the tree although further studies are required to
confirm this. In particular, it will be important to look at the growth of these trees in
the field.
How PXY-CLE signalling acts to increase plant height and leaf size is not clear.
PXY and CLE could be acting directly on the apical meristem or the increased
vascular tissue could somehow feedback to the apical meristem. The increase in leaf
size seen in these plants may be due to the increased sink strength from the xylem or
direct signalling of PXY-CLE from within the leaf veins. Testing these hypotheses
will require further work (Etchells et al. 2015).
In recent times, our climate has undergone rapid changes and the effects of this can
be felt by rising temperatures and a decline of vegetation (Sykes 2009). What kind of
effect this will have on tree growth is not clear. By studying plants such as the PXYCLE41 engineered plants, we might be able to override the normal environmental
signals and also allow us to produce trees that are capable of maintaining high yield
even when exposed to adverse environmental conditions (Etchells et al. 2015). These
hypotheses need to be tested by exposing PXY-CLE41 engineered plants to
changing temperatures, drought conditions and ultimately to extensive field trials.
The results obtained in this study clearly establish that manipulating PXY and CLE41
expression is sufficient to enhance wood production under the conditions used here.
Even if a small fraction of the extent of enhanced wood production demonstrated in
92
this study is reproducible in a large-scale commercial forestry, the outcome will offer
future prospects of drastically increasing tree productivity and will ultimately
contribute towards meeting the growing demand for renewable resources (Etchells et
al. 2015).
The recent study by Wang et al. (2015) showed that Populus trichocarpa (Pt)
leucine-rich repeat receptor-like kinases (LRR-RLKs) are differentially expressed
across the vascular cambial zone in poplar. These LRR-RLKs, the largest sub-family
of receptor-like kinases (RLKs) in terrestrial plants, are key regulators of cell
signalling. Wang et al. (2015) selected seven poplar LRR-RLK genes and
determined cell specific expression by GUS (β-glucuronidase) reporter gene assay.
The hybrid aspen transformed with all the selected seven PtLRR-RLKpro:GUS
fusions individually showed high level of expression in the developing primary
xylem within the cambium zone. Ectopic over-expression of the selected seven Pt
LRR-RLK genes did not demonstrate any significant vascular developmental
changes. Although the lines of PtLRR-RLK-1, 2, 3 and 7 did demonstrate reduction
in height and an increased number of internodes for only the lines over-expressing
PtLRR-RLK-1, a homologue of PXY/TDR in Arabidopsis. On overexpressing
PtLRR-RLK-1, the transgenic plants also demonstrated reduced internode elongation
which is similar to our finding. When PttPXY was driven by the PttANT promoter,
the mean internode length was not significantly increased in comparison to the
untransformed controls. When we over-expressed PttPXY by CaMV 35S promoter,
some of the transgenic lines demonstrated phloem intercalated with xylem phenotype
in contrast to the study by Wang et al. (2015) where the 35S-PtLRR-RLK1
transgenic plants did not show any abnormal morphology. Nonetheless, in both the
93
works there was no significant increase in height. In our work we demonstrated that
simultaneous over expression of PttPXY and PttCLE41 by tissue-specific promoters
PtPP2 and PttANT respectively leads to trees that can grow two times faster and
have normal xylem morphology (Etchells et al. 2015) with some extent of similarity
to the work by Wang et al. The authors also observed ectopic lignin deposition in the
pith, enlarged secondary xylem development and an increased lignin content without
any noticeable effect on secondary cell wall thickness. These outcomes suggested
that PtLRR-RLK1 is a potential candidate for transducing signals during the process
of plants secondary growth and wood formation.
Other previous studies have demonstrated the role of WOX4 (WUSCHEL
HOMEOBOX RELATED 4 gene) as a mediator of cell division in the vascular
cambium, downstream of the CLE41–PXY signalling pathway (Hirakawa et al.
2010). WOX4 is expressed in the procambium and cambium under the influence of
CLE41. In a wox4pxy double mutant the number of cell division in the cambium is
reduced significantly (Hirakawa et al. 2010). In poplar, homologs of CLE, PXY and
WOX4 are expressed in the cambium region in a pattern similar to that in
Arabidopsis (Schrader et al. 2004). This supports the idea that the CLE–PXY–
WOX4 signalling pathway is a general determinant of secondary growth of the
vascular meristem in a variety of species.
Furthermore, a study by Vera-Sirera et al. (2015) have demonstrated that control of
vascular cell divisions and orientation in the root, hypocotyl, and leaf that are the
result of primary vascular tissue development. This study demonstrated that bHLH
transcription factor a heterodimer of TARGET OF MONOPTEROS5 (TMO5) and
LONESOME HIGHWAY (LHW) are key components of cell divisions that increase
94
the width of vascular tissue. Furthermore, they were able to identify ACAULIS5 an
antagonist to TMO5-LHW activity. ACAULIS5 encodes thermospermine synthase,
which promotes the accumulation of SAC51-LIKE (SACL) bHLH transcription
factors. These SACL proteins can hetero-dimerise with LHW in competition to
TMO5-LHW interactions thereby preventing the activation of the TMO5-LHW
target genes, and suppressing the excessive proliferation as a result of increased
TMO5/LHW activity. The regulatory components mentioned in this study control
the primary vascular tissues development in the root, hypocotyl, and leaf (VeraSirera et al. 2015). Although pervious works by Ito et al. (2006), Fisher and Turner
(2007), Hirakawa et al. (2008), Hirakawa et al. (2010) have identified various
genetic regulators that effect secondary growth, currently there is an absence of
strong evidence that suggests factors controlling secondary growth in Arabidopsis
are likely to act in primary vascular tissue development. However the possibility of
reactivation of the regulatory components of primary vascular tissue development
during secondary vascular tissue development cannot be ignored. Therefore, the
question still remains whether the factors controlling secondary growth in
Arabidopsis and Populus (CLE–PXY–WOX4) might be also involved in primary
vascular tissue development. It will be very interesting to determine which part of
TMO5-LHW ↔ SACL-LHM activity is employed to quantitatively control vascular
tissue growth during secondary growth.
Strategies to Increase tree growth
Previous work published in the past decade have focussed on alternative strategies to
increase tree growth. This could be by creating transgenic citrus trees that can
accelerate flowering time and maturation to increase produce (Peña et al. 2001) or by
95
producing semi-dwarf trees for applications in arboriculture, horticulture, and
forestry by targeting the GA 2-oxidase genes (Busov et al. 2003). Other reported
strategies suggest altering pathways of lignin deposition and wood formation (Hu et
al. 1999; Pilate et al. 2002). Work published by Eriksson et al. (2000) focused on
increasing tree growth and biomass by overexpressing the most important regulatory
gene in the biosynthesis of the plant hormone gibberellin (GA) in hybrid aspen
(Populus tremula x P. tremuloides). They reported these transgenic trees grew
faster, taller and wider with larger leaves, longer xylem fibers and increased biomass
compared to the untransformed control plants. The study also showed that GA
possesses an antagonistic effect on root initiation, which caused a major problem for
survival of the transgenic GA 20-oxidase plants when planting in soil (Eriksson et al.
2000). The authors also showed that increased levels of GA did not have any
negative effects on root growth at the later stages of tree growth. Although Eriksson
et al. (2000) claim that the production of longer fibers is beneficial for the production
of strong paper, this hypothesis has not been tested further. No published reports
were found confirming this hypothesis.
We have reported that PtPP2::PttCLE41- PttANT::PttPXY transgenic trees grew
faster, taller and wider with larger leaves and longer, more numerous internodes.
Further studies could determine the effect of manipulating the PXY-CLE on lignin
synthesis and cellulose content and formation. As our study confirmed that the PXYCLE signalling is conserved in the dicots (Arabidopsis and Poplar) it should be
further investigated if the pathway is conserved in the monocots. If the pathway is
conserved in the monocots, such as Oryza sativa and wheat, and eudicots, such as
rapeseed, then this finding maybe of wider agricultural and economic importance. To
96
further investigate the PXY-CLE signalling pathway, the tissue-specific overexpressor constructs could be expressed in Arabidopsis that is more amenable to
expression analysis using RNA sequencing, or other forms of expression analysis.
These experiments may help to identify some downstream targets of the PXY and
CLE genes. These targets can be further investigated to determine their role in wood
formation. It is clear that the outcome of our study can be the base for further
investigations which may produce results of biotechnological and economical
significance.
97
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