TYPE II MADS-BOX GENES ASSOCIATED WITH POPLAR APICAL BUD DEVELOPMENT AND ...

188 Pages·2008·9.28 MB·English

ABSTRACT
Title of Dissertation: TYPE II MADS-BOX GENES ASSOCIATED WITH
POPLAR APICAL BUD DEVELOPMENT AND
DORMANCY
Kuang-Yu Chen, Doctor of Philosophy, 2008
Dissertation Directed By: Associate Professor, Gary D. Coleman, Department of
Plant Sciences and Landscape Architecture
MADS-box transcription factors regulate the development of vegetative and
reproductive organs in plants. Little is known about the role of MADS-box genes in
tree development. Using phylogenetic analysis, 57 putative type II MADS-box genes
representing 14 functional classes were identifi ed in the Populus trichocarpa genome.
cDNA sequencing of the poplar type II MADS-box genes indicates that 28.1% of the
transcripts differed in the intron-exon structures predicted in the genome database and
19.3% of the transcripts appear to be alternatively spliced. The majority of the poplar
type II MADS-box genes were expressed in a wide variety of tissues including shoot
apices, leaves, bark, xylem, root, and fl oral tissues and in shoot apices during bud
development and dormancy. These results indicate that poplar MADS-box genes have
diverse regulatory roles in a broad range of tissues and developmental processes.
Six poplar FLC-like genes, PtFLC1–PtFLC6, were identifi ed in the poplar
genome and expression of all six genes was detected in poplar shoot apices. The
expression of one gene, PtFLC2, declined in apical buds during SD photoperiod and
low temperature induced dormancy development suggesting a role in bud dormancy
and may represent an analogous regulatory mechanism to the down-regulation of
FLC during vernalization in Arabidopsis. In addition, several PtFLC2 splice isoforms
(PtFLC2as1–9) were identifi ed that were associated with the later stages of bud
dormancy. Overexpression of the PtFLC2as1 isoform delayed photoperiod induced
apical bud development and bud dormancy, growth cessation, and leaf senescence while
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overexpression of the PtFLC2as2 isoform appeared to accelerate bud development and
dormancy and reduce the amount of chilling required to overcome dormancy.
These fi ndings suggest that PtFLC2, unlike Arabidopsis FLC, could be an
integration point for both photoperiod and cold signals that regulate bud development
and dormancy. These results also suggest that in addition to transcriptional regulation,
that cold-mediated production of PtFLC2 splicing isoforms may have an important
regulatory role in bud dormancy. The regulated production of splicing isoforms could
regulate bud dormancy either by dominate negative interactions, by forming different
protein complexes or regulating different pathways that regulate growth, dormancy, and
dormancy release.
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TYPE II MADS-BOX GENES ASSOCIATED WITH POPLAR APICAL BUD
DEVELOPMENT AND DORMANCY
By
Kuang-Yu Chen
Dissertation submitted to the Faculty of the Graduate School of the
University of Maryland, College Park, in partial fulfi llment
of the requirements for the degree of
Doctor of Philosophy
2008
Advisory Committee:
Professor Gary D. Coleman, Chair
Professor Zhongchi Liu
Professor Stephen M. Mount
Professor Margaret Pooler
Professor Joseph H. Sullivan
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© Copyright by
Kuang-Yu Chen
2008
Dedication
This dissertation is dedicated to my husband Tsyr-Huei Chiou who regularly tells me that
nothing is beyond reach, my parents Chih-Hsuan Chen and Shu-Ching Chen who raised
me to who I am, and my country Taiwan which has enriched and empowered my life.
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Acknowledgements
Firstly, I would like to thank my advisor and mentor, Dr. Gary Coleman, who
inspired me to study on tree dormancy and gave 100% trust and support for me to
develop, design and carry out my project even in the diffi cult time of our lab.
I would like to acknowledge my committee for their continued support, advice,
help and encouragement of my project and study over the past fi ve years.
Many aspects of this project would not have been possible without the help and
input of other people. Dr. Charles Delwiche drew my focus on MADS-box genes. Dr.
Zhongchi Liu encouraged my interest on plant development and genetics and guided
me in the study of Arabidopsis. Dr. Steve Mount brought to my attention on the study
of alternatively spliced variants. Dr. Cecile Parmentier-Line guided me through the
experimental world of molecular and genetic biology. Mr. Shaun Faulkner ensured that
all the growth chambers and microcosms were functioning normally with plenty supplies
for me to carry out my experiments. Dr. Kuang-Chi Chen offered her expertise and
assisted on statistical analysis. Dr. Tsyr-Huei Chiou took almost all the photos, worked
together with me in histology, and discussed with me daily whenever I wanted.
Finally I would like to thank my family, friends, and members in Dr. Coleman’s
lab who have given me endless support, love, and encouragement.
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Table of Contents
Dedication ...........................................................................................................................ii
Acknowledgements ............................................................................................................iii
Table of Contents ...............................................................................................................iv
List of Tables .....................................................................................................................vii
List of Figures ..................................................................................................................viii
List of Abbreviations ............................................................................................................x
Chapter 1: Literature review ................................................................................................1
Dormancy .........................................................................................................................1
Tree growth cycle and Populus ....................................................................................1
Apical bud formation, development, and dormancy ....................................................3
Photoperiod and vegetative bud development and dormancy .....................................4
Chilling requirement and vegetative bud development and dormancy .......................8
MADS-box genes ...........................................................................................................11
Transcription factors ..................................................................................................11
Type I and type II MADS-box genes .........................................................................12
Functions of type II MADS-box genes ......................................................................13
Development of fl ower and apical bud ..........................................................................19
ABCE fl oral quartet model ........................................................................................19
Flowering time control and FLC class genes .............................................................21
Vernalization and epigenetics ....................................................................................23
Alternative splicing in plants .........................................................................................24
Alternative splicing (AS) ...........................................................................................24
Biological roles ..........................................................................................................28
AS and fl owering time control: FCA and FLC ..........................................................30
Questions........................................................................................................................33
Chapter 2: Molecular and Phylogenetic Analyses of the Type II MADS-box Gene
Family in Poplar .........................................................................................................34
Abstract ..........................................................................................................................34
Introduction ....................................................................................................................34
Results ............................................................................................................................38
Gene identifi cation and nomenclature .......................................................................38
Gene structure and splicing variants of poplar type II MADS-box genes .................41
Phylogenetic analysis of the Populus trichocarpa MADS-box gene family .............45
Expression of type II MADS-box gene in various tissues and dormant buds of
poplars ........................................................................................................................48
Dormancy cycle and chilling requirement .................................................................54
Discussion ......................................................................................................................55
Annotation of the poplar MADS-box gene family ....................................................55
Phylogenetic and functional classifi cation of poplar MADS-box genes ...................58
Alternative splicing in poplar type II MADS-box genes ...........................................59
Expression of poplar type II MADS-box genes .........................................................60
Materials and Methods ...................................................................................................62
Sequence collection from databases and phylogenetic analysis ................................62
Plant materials ............................................................................................................67
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RNA extraction and reverse transcription ..................................................................68
Gene-specifi c PCR primer design, cloning and sequence analysis ............................69
PCR analysis ..............................................................................................................70
Chilling requirement and dormancy measurement ....................................................70
Chapter 3: Characterization of a poplar FLC-like 2 gene (PtFLC2) regulating bud
development, dormancy release, and leaf senescence in Populus alba x tremula .....71
Abstract ..........................................................................................................................71
Introduction ....................................................................................................................72
Results ............................................................................................................................77
Identifi cation and structure of PtFLC genes in the poplar genome ...........................77
PtFLC2 is down-regulated and PtFLC4 is up-regulated during vegetative bud
dormancy ....................................................................................................................81
Characterization of transgenic poplars transformed with PtFLC2 overexpression
and RNAi chimeric genes ..........................................................................................84
Growth of wildtype and PtFLC2 transgenic poplars .................................................85
PtFLC2as1 overexpression impinges on bud formation, leaf senescence, leaf
abscission, and chilling requirements ........................................................................90
Expression of type II MADS-box genes, PtFT1, PtFRI, and PtVIN3 during
dormancy ....................................................................................................................97
PtFLC2 induces early fl owering in Arabidopsis ......................................................101
Discussion ....................................................................................................................104
Overexpression PtFLC2as1 alters photoperiod response and delays growth cessation
and bud development ...............................................................................................104
Overexpression of PtFLC2as1 alters bud dormancy ...............................................105
PtFLC2 may not regulate the expression of poplar homologs of SOC1, AP1,
and FT genes ............................................................................................................107
PtFLC2 induces early fl owering in late fl owering Arabidopsis (FRI-Sf2) ..............108
PtFLC2 is an essential component of bud dormancy ..............................................110
Materials and Methods .................................................................................................111
Plant materials and growth conditions .....................................................................111
Isolation of PtFLC2 and RT-PCR analysis ..............................................................112
PtFLC2 overexpression and RNAi constructs .........................................................114
Production of transgenic poplars .............................................................................116
Production of transgenic Arabidopsis ......................................................................117
Measurement of growth cessation and leaf senescence ...........................................118
Chilling requirement and dormancy measurement ..................................................119
Histology and light microscopy ...............................................................................119
Statistical analysis ....................................................................................................120
Chapter 4: Analysis of the alternatively spliced transcripts of the poplar FLC-like 2
gene (PtFLC2) .........................................................................................................121
Abstract ........................................................................................................................121
Introduction ..................................................................................................................122
Results ..........................................................................................................................126
The PtFLC2 transcript is alternatively spliced ........................................................126
Expression of PtFLC2 alternatively spliced variants during bud dormancy ...........131
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PtFLC2as1 and PtFLC2as2 induce early fl owering in Arabidopsis FRI-Sf2 .........131
Overexpression of PtFLC2 splicing variants did not affect RNA levels of genes
downstream of FLC in Arabidopsis FRI-Sf2 ...........................................................137
Growth of wildtype and PtFLC2 splicing variants transgenic poplars ....................137
PtFLC2as1 and PtFLC2as2 overexpression affects bud formation and chilling
requirement in poplar ...............................................................................................139
Discussion ....................................................................................................................145
Identifi cation of PtFLC2 alternatively spliced transcripts .......................................145
Effect of PtFLC2as on Arabidopsis .........................................................................146
Effect of PtFLC2as2 on poplar ................................................................................148
Materials and Methods .................................................................................................152
Plant materials and growth conditions .....................................................................152
Isolation of PtFLC2 alternative splicing variants and RT-PCR analysis .................152
Overexpression PtFLC2as constructs and Agrobacterium transformation .............154
Segregation and RNA analysis of transformed Arabidopsis ....................................154
Measurement of growth cessation, chilling requirement and dormancy status .......154
Chapter 5: Summary .......................................................................................................155
Appendices .......................................................................................................................162
Bibliography ....................................................................................................................165
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List of Tables
Table 1-1. Summary of Arabidopsis and poplar MADS-box genes .................................14
Table 1-2. Alternative splicing of MADS-box genes of Arabidopsis ...............................32
Table 2-1. Locations and alignment differences in intron-exon structure between
predicted JGI genes and cloned cDNA sequences .....................................................42
Table 2-2. Summary of alternative splicing (AS) events in poplar type II MADS-box
genes ..........................................................................................................................43
Table 2-3. Dormancy development of Populus trichocarpa Nisqually-1, P. alba x tremula
717-1B4, and P. deltoides x trichocarpa 545-4183 ...................................................49
Table 2-4. Summary of expression of poplar type II MADS-box genes ..........................53
Table 2-5. Dormancy quantifi cation in Populus trichocarpa clone Nisqually-1, Populus
alba x tremula clone 717-1B4, and Populus deltoides x trichocarpa clone
545-4183 ....................................................................................................................56
Table 4-1. Cluster analysis and one-way ANOVA multiple mean comparison
of fl owering time in Arabidopsis (FRI-Sf2) transformed with PtFLC2 alternative
splice variants ..........................................................................................................136
Table 4-2. Ectopic expression of PtFLC2 alternatively spliced transcripts induced early
fl owering under long days in transgenic Arabidopsis (FRI-Sf2) .............................138
Table A-1. PCR conditions used for specifi c primer combinations of poplar type II
MADS-box genes ....................................................................................................162
Table A-2. ANOVA tables of growth cessation, leaf senescence, and dormancy
measurement ............................................................................................................163
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