Comprehensive Summaries of Uppsala Dissertationsfrom the Faculty of Science and Technology 580

_____________________________ _____________________________

Functional Characterization of the Pointed Cotyledon Subclass

of HDZip Genes in Arabidopsis thaliana

BY

JOHANNES HANSON

ACTA UNIVERSITATIS UPSALIENSISUPPSALA 2000

Dissertation for the Degree of Doctor of Philosophy in Physiological Botany presented at Uppsala University in 2000

ABSTRACT

Hanson, J. 2000. Functional Characterization of the Pointed Cotyledon Subclass of HDZip genes in Arabidopsis thaliana. Acta Universitatis Upsaliensis. Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology 580. 56 pp. Uppsala. ISBN 91-554-4846-1.

Genes encoding homeodomain leucine zipper, HDZip, transcription factors constitute a large gene family in Arabidopsis thaliana. In this thesis the isolation and characterization of four HDZip genes (ATHB3, -13, -20 and –23) is described. These genes are similar in sequence and form a distinct subclass within the HDZip gene family. Since the genes cause similar alterations in cotyledon shape when expressed constitutively, we refer to the members of this subclass as the pointed cotyledon HDZip genes.

To determine the biological functions of the genes, the phenotypes of plants constitutively expressing the genes have been analysed. Each of the genes specifically inhibits lateral cell expansion in cotyledons and leaves, and thereby causes them to be abnormally narrow. Detailed expression analysis shows that only ATHB23 is expressed in the entire leaf and cotyledon from early stages of development while ATHB20 is predominantly expressed in the root cortex. ATHB13 is expressed in basal parts of mature leaves and floral organs and ATHB3 in root and stem cortex. The ATHB13 protein acts within a signalling pathway that mediates a response to sucrose that specifically regulates the expression of specific sugar-regulated genes. ATHB3 specifically inhibits primary root development without affecting the development of secondary roots when constitutively expressed.

Reduced expression of ATHB3 by antisense suppression results in increased expression of ATHB13, indicating that ATHB3 acts as a repressor of ATHB13 expression in the wild type.

This thesis also reports the isolation of seven new genes of HDZip class I and reviews available functional information on the genes in this class. One conclusion is that HDZip I proteins that are closely related phylogenetically are also functionally related, in most cases. Seven different mutations in HDZip I genes were identified. The lack of phenotypic deviations from wild type of these mutants suggests that these HDZip proteins act in a redundant fashion in the plant.

Johannes Hanson, Department of Physiological Botany, Evolutionary Biology Centre, Villav. 6, SE-752 36, Uppsala, Sweden

© Johannes Hanson 2000

ISSN 1104-232XISBN 91-554-4846-1

Printed in Sweden by University Printers, Uppsala 2000

The homeobox is a 180 base pair sequence motif…

This thesis is based on the following manuscripts, which will be referred to in the text

by their respective Roman numerals:

I Hanson, J., Johannesson, H., and Engström, P. (2000). Sugar dependent alterations in cotyledon and leaf development in transgenic plants expressing the HDZip gene ATHB13. Plant Mol. Biol. In press

II Hanson, J., Regan, S., and Engström, P. (2000). ATHB13 is highly expressed in the vascular tissue at the base of petioles in both Arabidopsis and hybrid aspen. (manuscript)

III Hanson, J., and Engström, P. (2000). Constitutive expression of each of four closely related homeobox genes in transgenic Arabidopsis causes similar pointed cotyledon phenotypes. (manuscript)

IV Hanson, J., and Engström, P. (2000). ATHB3 represses ATHB13 expression, and, when constitutively expressed, specifically affects primary root development in Arabidopsis thaliana. (manuscript)

V Johannesson, H., Hanson, J., Söderman, E., Wang, Y., and Engström, P. (2000) HDZip proteins in Arabidopsis thaliana: a case of functional conservation and redundancy within a family of transcription factors. (manuscript)

Manuscript I has been reprinted with the kind permission of Kluwer Academic Publishers.

5

TABLE OF CONTENTS

ABBREVIATIONS................................................................................................... 6PREFACE ................................................................................................................. 7INTRODUCTION .................................................................................................... 8 Arabidopsis – a useful weed ................................................................................ 8 Root Development ............................................................................................... 9 Post-embryonic development of the root........................................................ 9 The pattern for post-embryonic development of the root is laid down during embryogenesis ........................................................ 10 Initiation and formation of secondary roots ................................................. 12 Cotyledon and leaf development........................................................................ 13 Life history of a leaf ..................................................................................... 13 The control of leaf expansion and leaf shape ............................................... 13 The cotyledon ............................................................................................... 14 Sugar sensing ..................................................................................................... 16 HDZip transcription factors ............................................................................... 19 The pre-history of the HDZip domain.......................................................... 19 HDZip transcription factors are encoded by a large gene family in Arabidopsis .................................................... 20 HDZip proteins act as dimeric transcription factors..................................... 21 HDZip proteins are involved in a wide range of processes in plants ........... 24RESULTS AND DISCUSSION Isolation and characterization of novel HDZip genes........................................ 26 Novel genes distantly related to previously known HDZip I genes ............. 26 Pointed cotyledon-HDZip genes .................................................................. 28 Functional characterization of the poc-HDZip genes ........................................ 30 Constitutive expression of poc-HDZip genes results in pointed cotyledons and serrated leaves .................................................. 30 Poc-HDZip genes are differentially expressed ............................................. 31 ATHB13 affects cotyledon and leaf development in a sucrose dependent manner............................................................... 33 ATHB3 specifically affects primary root development when constitutively expressed................................................................ 35 ATHB3 antisense gene expression increases ATHB13 expression to higher levels ....................................................................................... 36 Lack of phenotypic deviations caused by HDZip I gene mutations indicates functional redundancy within the gene family ....................................... 37 Concluding remarks ........................................................................................... 38SUMMARY IN SWEDISH – POPULÄRVETENSKAPLIG SAMMANFATTNING .................................. 40ACKNOWLEDGEMENTS .................................................................................... 42REFERENCES ....................................................................................................... 44

6

ABBREVIATIONS

2dGlc 2-deoxy glucose35S::ATHB3 line constitutively expressing the gene ATHB335S::POC line constitutively expressing one of the poc-HDZip genes3-O-mGlc 3-O-methyl glucose6dGlc 6-deoxy glucoseABA abscisic acidACC amino-cyclopropane-carboxylic acid (ethylene precursor)AGI Arabidopsis Genome InitiativeATHB Arabidopsis thaliana homeoboxATP adenosine triphosphateBAP 6-benzylaminopurine (cytokinin)bp base pairbZip basic leucine zippercDNA complementary DNADNA deoxyribonucleic acidHDZip homeodomain leucine zipperHXK hexokinaseHXT hexose transporterIAA 3-indoleacetic acid (auxin)mRNA messenger RNAPCR polymerase chain reactionpoc-HDZip pointed cotyledon HDZip RING really interesting new geneRNA ribonucleic acidSAM shoot apical meristemSUT sucrose transporterT-DNA transferred DNA

The following conventions have been followed in this thesis:

Names of genes are written in italicized upper-case letters, e.g. ATHB13

Names of proteins are written in non-italicized upper-case letters, e.g. ATHB13

Names of mutants and mutations are written in italicized lower-case letters,

e.g. athb13-1

7

PREFACE

In 1993, I joined the HDZip group of Peter Engström at the department of physiological

botany, Uppsala University. At that time, only four HDZip genes had been cloned in

the lab and I thought we were going to show that these homeobox genes regulated

similar processes to their animal counterparts. I now know that this was a naive belief

of a young developmental biologist who was unaware of how plants live and develop.

Over the years here in Uppsala I have gradually gained more and more understanding

of plant physiology and development. In the beginning I was very disappointed and

desperately tried to fit plants into my preconceptions of development, based on what

I had learned about the development of the fly Drosophila melanogaster and other

animal models. I have now realized that the cute little plant Arabidopsis thaliana

is not just a simplified fruit fly, but rather an elegant survivor that has evolved its

own fascinating systems to cope with an ever-changing environment. However, this

thesis does not reflect my journey in plant biology. It is just a snapshot of my present

location. I would like to dedicate this snapshot to the species Heliathus tuberosus.

Uppsala, 2000

Johannes Hanson

8

INTRODUCTION

Arabidopsis – a useful weedArabidopsis thaliana (L.) Heynh, commonly known as thale cress, mouse-ear cress

or wall cress, but usually referred to as Arabidopsis in the scientic literature, is

a plain plant that only attracts the eyes of researchers (Figure 1). It belongs to the

mustard family (Brassicaceae or Cruciferae) and is related to various economically

important crops such as rape and broccoli. It is widely distributed in the temperate

climate zone of the Northern Hemisphere and is usually found in poor and exposed

habitats such as roadsides (Price et al. 1994). In nature, it grows as a winter annual.

The seed germinates and grows during the autumn, the plant survives the winter as a

rosette, then owers and sets seeds in the spring (Rédei 1992).

Arabidopsis has become the favourite model organism for plant research. It was

rst recognized as an organism suited for genetic investigations in the middle of the

twentieth century (the early history of Arabidopsis as a scientic model is reviewed

by Rédei 1992). Geneticists found it convenient for many reasons: it has a short

generation time of about six weeks, it is small in size (its rosette diameter being

approximately 5 cm and its inorescence height about 30 cm) and can be grown in

large quantities, it produces large numbers of seeds (up to 5000 per plant), it is self

pollinating but can be cross-pollinated with ease and it is easy to mutagenize by either

chemicals or radiation (Koncz et al. 1992). When the breakthrough of molecular

genetics came in the 1980’s there was at rst no consensus on which organism

to work on as a model. Investigations were focused on many different organisms

including tomato, petunia, pea, rice, barley, maize, snapdragon and tobacco - all of

which have different advantages and disadvantages. Although an impressive amount

of information was collected, advances in many disciplines were limited because

information was scattered, comparison of results from different organisms was often

difcult and efforts were sometimes duplicated as the same information was collected

from many organisms (Meinke et al. 1998). A search for a new organism suited

to the new molecular and classical genetical methods was initiated and, through a

gradual process, Arabidopsis was chosen. The new generation of molecular geneticists

favoured Arabidopsis for the same reasons as the previous generation. The newcomers

also beneted from the work that had already been done on the organism, like the

genetic maps and hundreds of characterized mutants (Meinke et al. 1998; Somerville

and Meyerowitz 1994). Arabidopsis was also found to be easily transformed by means

of Agrobacterium tumefaciens (Bechtold et al. 1993). Furthermore, it has a very

small genome of only approximately 120 million base pairs (bp) and a low level of

repetitive DNA (Meyerowitz 1992). The Arabidopsis genome contains a gene every

5,000 bp on average and is estimated to contain approximately 20,000 genes (Bevan

et al. 1998). In 1996, the Arabidopsis Genome Initiative, AGI, was established to

9

INTRODUCTION

facilitate the sequencing of the whole Arabidopsis genome (Bevan et al. 1997), a

task that will be completed before the end of the year 2000. Thousands of mutants

have been generated by means of insertion mutagenesis. These mutants are easily

screened by means of PCR so, within a few years, knockout mutants for virtually

every Arabidopsis gene will be available to the scientic community (Krysan et al.

1999).

The post-genome era of Arabidopsis research will surely be as successful as

the rst fty years and certainly more and more of the information collected from

Arabidopsis will nd uses in new improvements of more economically important

species.

Root DevelopmentPost-embryonic development of the rootArabidopsis has been shown to be an excellent system for studying post-embryonic

development of the root, as it has a fairly uncomplicated cellular organization (Dolan

et al. 1993) in which the developmental history of every cell has been determined

(Scheres et al. 1994), and its roots are nearly transparent. The Arabidopsis root

consists of a few different cell types organized in a radially symmetrical pattern

along the length axis. The cell types of each layer are continuously produced in les

from progenitor cells, the initials, similar to a conveyor belt, as the root elongates

(Figure 2). Only four cells of the Arabidopsis root meristem, the quiescent centre,

are fully undifferentiated and these cells rarely divide under normal conditions. The

cells around the quiescent centre are the initials for the different cell-types of the

Figure 1

Arabidopsis thaliana var. Ler .

Drawing by Dr. Eva Sundberg.

10

INTRODUCTION

root (the epidermis, cortex, endodermis, etc., see Figure 2). The initials divide to

form one cell that takes on the fate the mother cell and a second cell that is destined

to differentiate into a certain type (epidermis, cortex, endodermis, etc.). Mutations

affecting these processes in different cell types have been identied, for example

the shortroot mutation that blocks the formation of endodermis cells (Scheres et al.

1995). The fate of each initial cell is determined by its position in the root: positional

signals pass from differentiated cells to undifferentiated cells rather than from the

initials (van den Berg et al. 1995). Thus, cells differentiate like their more basal

daughters. The molecular nature of these positional cues is not known but mapping

the plasmodesmata in the root has shown that connections are preferentially made to

cells of the same type in the root (van den Berg et al. 1998 and references therein).

The contacts between the initials and the quiescent centre cells keep the initials in an

undifferentiated state since ablation of the cells in the quiescent centre causes initials

to stop dividing and to differentiate according to cues from the daughter cells in the

same le (van den Berg et al. 1997).

The pattern for post-embryonic development of the root is

laid down during embryogenesisThe cellular organization of the root is laid down during embryogenesis, and the

ontogeny of each cell has been determined through the use of genetically marked

embryonic cells, Figure 2 (Scheres et al. 1994). The rst division of the zygote

generates an apical and a basal cell and the root is formed from derivatives of both

of these cells. The basal cell generates the quiescent centre and the columella root

cap initials. The rest of the root cells, including the initials, are derivatives of the

apical cell (reviewed in Scheres and Heidstra 1999). The apical-basal pattern is laid

down early in embryogenesis as dened by the rst visible developmental deviations

detected in the monopteros, mp, mutant (Berleth and Jurgens 1993). The capacity

of mp mutant plants to make adventitious roots indicates that mp plants can make

largely normal apical structures. All organs, however, display defective vascular

strands and impaired auxin transport (Przemeck et al. 1996). This indicates that MP

promotes axialization and cell le formation, processes that are important for both

embryonic axis formation and vascular system development, rather than specifying

the apical-basal pattern. The determination of cell fates along the apical-basal axis

also involves the HOBBIT, HBT gene, as the hbt mutant is unable to form a root

meristem (Willemsen et al. 1998). From very early development the cells that will

give rise to the quiescent centre and the columella initials (Figure 2) divide aberrantly

in the hbt mutant, consistent with the hypothesis that the gene has a specic function

11

INTRODUCTION

-

AB

C

D

Estele

endod.cortex

epid.

l.r.c.

col.

cot.cot.

Figure 2

Embryonic and post-embryonic root development of Arabidopsis

Simplified schematic drawings of cellular arrangements (transverse sections) during root

development. Cortex and endodermis cells are depicted in dark grey and the stele (internal), columella

and lateral root cap (peripheral) is shown in light grey. The quiescent centre and epidermis of the root

are in white, as are all other cells of the non-root lineage. The figures are adopted from Scheres et al.

1994 and van den Berg et al. 1998, with kind permission from the authors.

A, Early heart stage embryo. B, Late heart stage embryo. C, Seedling. cot., cotyledon. D, Root apex

of seedling. E, Close up of central region in root meristem shown in D. Initial cells for all the different

cell types surrounding the quiescent centre. endod., endodermis; epid., epidermis; l.r.c., lateral root

cap; col., columella. Arrows indicate the direction of daughter cell displacement.

12

INTRODUCTION

in these cells (Willemsen et al. 1998). The radial axis of the root is laid down after

the apical-basal axis, since the rst deviations are detectable in mutants specically

affected in stages of radial pattern formation that appear later in embryogenesis

than those altered in hbt and mp (Scheres et al. 1995). It is reasonable to believe

that embryonic root formation shares extensive similarity to post-embryonic root

development as hbt and mutations affecting the radial pattern of the root also cause

the same deviations in secondary roots (Malamy and Benfey 1997a).

Initiation and formation of secondary rootsThe primary root of the adult Arabidopsis plant is only a minor part of its root system,

which is largely composed of secondary roots formed from the primary root or other

tissue, and further secondary roots formed from them, and so on. The secondary roots

perform and develop like primary roots, but are not initiated during embryogenesis.

Secondary roots are initiated from differentiated tissue and the frequency of initiation

is highly inuenced by environmental conditions (Charlton 1996). This plasticity in

the formation of the root system is one way in which plants overcome their inability

to move away from poor soils and towards nutrients. The lateral roots of Arabidopsis

are initiated from cells in the pericycle (an internal layer adjacent to the stele, Figure

2) of the root. A small number of pericycle cells start dividing and eventually form

the lateral root primordium. The lateral root primordium forms a structure identical

to the root meristem through a series of dened stages of development, and emerges

through the outer cell layers of the root (Laskowski et al. 1995; Malamy and Benfey

1997b). Most mutations identied in Arabidopsis that affect the development of the

primary root also affect the lateral roots. This indicates that the formation of the

lateral root meristem shares many regulatory mechanisms with those of primary root

initiation during embryogenesis (reviewed by Malamy and Benfey 1997a; Scheres

and Heidstra 1999).

The induction of lateral roots is dependent on the hormone auxin, which is

transported from aerial parts of the plant. This has been demonstrated by the

application of both auxin (Torrey 1950) and inhibitors of auxin transport (Reed et

al. 1998). Mutations with reduced sensitivity to auxin exhibit a reduction or loss of

lateral roots (Celenza et al. 1995; Estelle and Sommerville 1987) and the extensive

production of lateral roots in the mutant alf1 (Celenza et al. 1995), which is allelic

to superroot and rooty, has been linked to elevated auxin levels (Boerjan et al.

1995; King et al. 1995). In screens for mutants with altered frequencies of lateral

root initiation in Arabidopsis, only one mutation, alf4-1, has been shown to inhibit

this induction without affecting auxin perception or synthesis (Celenza et al. 1995),

demonstrating the importance of the hormone in the process. However, the initiation

13

INTRODUCTION

of secondary roots in other species, originating from other tissues, may be regulated

by other hormones, as illustrated by the ethylene dependence of adventitious root

formation (i.e. secondary root production from non-root tissue) from stem cuttings of

tomato and petunia (Clark et al. 1999).

Cotyledon and leaf developmentLife history of a leafAll the leaves of a plant originate from a small group of cells in the apical parts of

the shoot, the shoot apical meristem (SAM). This group of cells forms a dome that

undergoes extensive cell division in a highly regular manner (Lyndon 1970). As new

cells are formed the older ones are displaced to the periphery of the dome. These

cells will later form the primordia of leaves (Lyndon and Cunninghame 1986). The

shoot apical meristem continuously produces leaf primordia at its anks in a spiral

pattern. The earliest histological evidence of primordium initiation is a change in the

orientation of cell divisions in the region where the primordia will form (Lyndon

1970). At the molecular level this is correlated with a repression of knox genes in

maize and Arabidopsis. These genes are expressed in the central parts of the SAM and

have been shown to be important for maintaining the undifferentiated state of these

cells (Reiser et al. 2000).

During its initiation or early development the leaf primordium is divided into

discrete domains along the basic axes of the future leaf, in other words, a polarity

is established. The cells in the different positions along these axes/polarities then

develop according to different fates. The phantastica mutation in Antirrhinum majus

abolishes the formation of both the lateral and the dorsoventral axis as the leaves

of the mutant plants have complete radial symmetry (Waites and Hudson 1995).

Regulation of pattern along the proximodistal axis has been extensively studied in

maize where the distal and proximal parts of the leaf differ in many aspects of their

cellular differentiation (Sylvester et al. 1990). At least 15 different gene products

have been shown to specically affect structures along this axis in maize (reviewed

in Sylvester et al. 1996).

The control of leaf expansion and leaf shapeMuch of the diversity of leaf shapes seen in nature is caused by variation in the

amount of expansion within the leaf (Dale 1988). Variations in leaf expansion causing

different shapes of leaves may occur early, as shown in snapdragon, Antirrhinum

majus, where variation in leaf shape along the length of the shoot is due to variation

in the growth of the lamina early in development (Harte and Meinhard 1979a, b). In

14

INTRODUCTION

Arabidopsis, which has leaves with a shape similar to those of snapdragon, the genes

ANGUSTIFOLIA and ROTUNDIFOLIA act after leaf blade formation to control the

length and width of the leaf (Tsuge et al. 1996). The leaves and leaf cells of the

angustifolia (an) mutant are signicantly narrower than wt leaves (Rédei 1962) but

the leaf length is not affected by the mutation (Tsuge et al. 1996). Cell shapes in

plants are determined by the direction-specic inhibition of expansion exerted by

cellulose microbrils, while the driving force is the positive turgor pressure of the

cell (Taiz and Zeiger 1991). This means that inhibition of expansion in one direction

by cell wall microbrils leads to expansion in the other directions. AN affects cell

width, and secondarily leaf width, by this mechanism as an leaves and cells are

signicantly thicker than wt cells (Tsuge et al. 1996). AN also affects the orientation of

microtubules and, thus, most likely the orientation of cellulose microbrils (Tsukaya

et al. 1999). This mechanism also seems to occur in species other than Arabidopsis

since the fat mutant from tobacco, Nicotiana sylvestris, also has thick, narrow leaves

(McHale 1993).

The leaves and cells of the rotundifolia3 (rot3) mutant are shorter than those of the

wt, but the width and thickness of the cells are not altered (Tsuge et al. 1996). This

proves that plants can inhibit leaf cell expansion in one direction without affecting

expansion in the other directions. ROT3 encodes a cytochrome P450 with similarities

to animal steroid hydroxylases (Kim et al. 1998). How it affects the length of the

leaf is not currently known, but the mechanism is distinct from that of AN as the

arrangement of microtubili is not altered in the rot3 mutant (Tsukaya et al. 1999).

Over-expression of ROT3 in transgenic plants causes the development of longer

leaves compared to wt, indicating that the gene can both positively and negatively

regulate leaf length (Kim et al. 1999). Over-expression of the rot3-2 gene (with

encodes a ROT3 protein with an amino acid substitution) causes the rot3 phenotype

(short leaves) to appear in wt plants, indicating that the ROT3-2 protein dominantly

inhibits the wt protein (Kim et al. 1999).

The cotyledon Unlike the leaf, the cotyledon does not originate from a meristem. The cotyledons

are initiated during embryogenesis (Figure 2). The Arabidopsis cotyledons contain

stored reserves (lipids, proteins and starch) that are broken down and mobilized

during germination and early phases of seedling establishment (Manseld and Briarty

1996). Approximately 60 hours post-imbibition the Arabidopsis seedling switches to

auxotrophic growth and starts to photosynthesise. After this transition the cotyledons

15

INTRODUCTION

physiologically act as leaves (Manseld and Briarty 1996). When the rosette leaves

are fully developed, the small cotyledons are effectively shaded from the light and

senesce.

The Arabidopsis mutants extra cotyledon1 and -2 (xtc1 and –2) and altered

meristem programming 1, amp1, frequently have more than two cotyledons (Conway

and Poethig 1997). The position and timing of emergence of these extra cotyledons

indicate that the rst leaves of these mutants develop according to cotyledon fate.

However, these cotyledon-like leaves are initiated prematurely during embryogenesis

by aberrant timing of SAM initiation, and develop like wt leaves if the timing of

their emergence is restored to wt (Conway and Poethig 1997). These observations

indicate that during embryogenesis a factor that suppresses vegetative development

and/or promotes embryo specic development is expressed in a manner allowing it

to affect the prematurely developed leaf primordia. The gene LEAFY COTYLEDON1

(LEC1) is possibly one such factor, as it is expressed in the whole embryo during

early embryogenesis. When ectopically expressed during vegetative development

LEC1 causes the plant to develop according to embryo specic programs, including

the expression of storage proteins and the development of ectopic embryos on

vegetative tissue (Lotan et al. 1998). Mutant lec1 cotyledons develop into leaf-like

organs, which lack storage proteins and oil bodies present in wt cotyledons, while

developing trichomes and vascular characteristics of wt leaves. Mutant lec1 seeds

are intolerant to desiccation and, at a low frequency, germinate viviparously (Meinke

1992). However, the embryonic development of lec1 mutants is not fully transformed

into the vegetative program, possibly indicating that LEC1 acts partly redundantly

with other factors (Lotan et al. 1998).

The development of the cotyledon after the mobilization of storage reserves is

similar to that of leaves, although cells of Arabidopsis cotyledons do not divide after

the seed is mature (Tsukaya et al. 1994). The mechanisms that regulate the shape of

the cotyledon are only partly shared with those of leaves, as an cotyledons develop

an abnormally narrow shape (Tsukaya et al. 1994) while the shape of rot3 cotyledons

is indistinguishable from that of wt cotyledons (Tsuge et al. 1996).

16

INTRODUCTION

Sugar sensingSugars are important molecules in all organisms, both as carriers of stored chemical

energy and as raw materials for the synthesis of other molecules. Sugars are therefore

of great importance during all stages of the plant life cycle and it is not surprising that

changed sugar concentrations affect the expression of a large number of genes (Koch

1996). Continuous sensing of sugar levels occurs at the level of the individual cells

and three major sensing mechanisms/receptors have been suggested to be present

in plants, hexokinase (HXK) sensing, hexose transport (HXT) sensing and sucrose

(SUT) sensing (Smeekens and Rook 1997).

Hexokinase sensing has been shown to control diverse processes and metabolic

pathways in plants such as the sugar induced feedback regulation of photosynthesis

and the mobilization of storage reserves from seeds (Smeekens and Rook 1997). The

sensor is hexokinase (HXK), the rst enzyme of glycolysis, and phosphorylation of

hexoses by HXK induces the enzyme to initiate a signalling cascade (Jang et al.

1997). Two HXK genes have been cloned from Arabidopsis and transgenic plants

with enhanced or reduced expression of these genes have conrmed the importance

of the proteins in sugar signalling (Jang et al. 1997). HXK can phosphorylate

the non-metabolizable sugar analogues 2-deoxy glucose (2dGlc) and mannose, and

consequently start signalling in response to the presence of these molecules. HXK

cannot phosphorylate two other analogues, 3-O-Methyl Glucose (3-O-mGlc) and

6-deoxy glucose (6dGlc), and these sugars are therefore unable to trigger HXK

signalling (Smeekens and Rook 1997). By the use of specic HXK inhibitors Pego

et al. (1999) have shown that the signalling properties of HXK, and not the depletion

of ATP or phosphates, is responsible for the inhibition of germination in response to

these analogues in Arabidopsis.

Sugar analogues that are taken up by the cells but are not phosphorylated by HXK

such as 3-O-mGlc and 6dGlc can affect gene expression, but clearly by a second

mechanism. Genes encoding extracellular invertase and sucrose synthase are induced

by sucrose and glucose in suspension cultures of Chenopodium rubrum cells and this

induction can be mimicked by 6dGlc (Roitsch et al. 1995). The sugar and amino

acid-induced promoter PAT(33B) can also be induced by 6dGluc and 3-O-mGlc in

transgenic Arabidopsis (Martin et al. 1997). The effect of these analogues has been

attributed to Hexose transporter (HXT) signalling in plants (Figure 3), in analogy

with the function of HXT in yeast (Özcan et al. 1996). HXT proteins in yeast, like

HXK in yeast and plants, have dual functions, both transporting hexoses over the

membrane and signalling. However, none of the cloned members of the HXT gene

family from Arabidopsis has yet been proven to be involved in signalling (reviewed

in Buttner and Sauer 2000).

17

INTRODUCTION

Sucrose

HXT

SUT

HXK

Sucrose

Hexose

Hexose

cell membrane

GlycolysisVacuole

Hexose

Nucleus

Figure 3

Sugar signalling receptors in plants

Schematic drawing of simplified plant cell showing the three suggested sugar receptors

(grey) as well as their suggested positions in the cell. The interconversion of sucrose to

hexose is indicated.

Sucrose is an important sugar transport and storage molecule in plants and most

likely also has a signalling function. Several observations indicate that sucrose can

be sensed in plants. The patatin and rolC genes have been shown to be specically

induced by sucrose (Jefferson et al. 1990; Wenzler et al. 1989; Yokoyama et al.

1994), and the expression of a bZip transcription factor gene, ATB2, is specically

repressed by sucrose (Rook et al. 1998). Since sucrose is readily hydrolysed to

glucose and fructose, both inside the cell membrane and in the apoplast, it is difcult

to determine if such effects are directly mediated by the sucrose molecule. However,

in the above cases, combinations of glucose and fructose are less efcient than sucrose

in mediating the response, indicating that sucrose is the specic mediator. A sucrose

transporter protein, SUT (Figure 3) is suggested to be involved in signalling but as

yet there is no experimental support for this hypothesis. A recently cloned gene from

Arabidopsis encodes a protein with sequence similarity to both sucrose transporters

and signal transducing cytoplasmic domains of yeast sugar sensors (Barker et al.

2000). This protein might act as a sucrose sensor in plants as it lacks transport activity,

like yeast sugar sensors (Barker et al. 2000).

18

INTRODUCTION

How the signal is transduced from the receptors to the responses in plants is

presently unknown. However, in yeast many genes and enzymes are repressed by

glucose. This repression has been shown to be controlled by a complex signalling

cascade, in which the protein kinase SNF1 plays a central role (Ronne 1995). SNF1-

like protein kinases have been isolated from many plant species including rye, barley,

tobacco, soybean, potato, Arabidopsis and others (reviewed in Halford and Hardie

1998). Expression of the tobacco SNF1 homologue in yeast causes constitutive

expression of a glucose repressible gene (Muranaka et al. 1994) indicating that there

is functional homology between the yeast and plant proteins and, possibly, in their

signalling cascades. An independent indication of the involvement of SNF1-like

proteins in sugar signal transduction is provided by the PRL1 protein. PRL1, when

mutated, confers sugar hyper-sensitivity to the plant (Nemeth et al. 1998) possibly

by interacting with SNF1-like kinases, as recently shown in vitro (Bhalerao et al.

1999).

Sugar signals are also integrated with signals from other signal transduction

pathways in the plants. Mutations identied on the basis of altered sugar responses

have also been shown to affect hormone-mediated responses. This is a strong

indication of a close connection between the signalling pathways. Crosstalk between

sugar and ethylene (Zhou et al. 1998), abscisic acid (Arenas-Huertero et al. 2000;

Huijser et al. 2000; Laby et al. 2000), and cytokinin, ethylene, abscisic acid and auxin

(Nemeth et al. 1998) signalling pathways has been demonstrated by this method. It

has also been shown that a gene involved in brassinolide biosynthesis is repressed

by sugars (Szekeres et al. 1996). Sugar signals form one among many types of

input monitored by the highly integrated regulatory network controlling the onset of

owering in Arabidopsis. Sucrose availability to the aerial parts of the plants promotes

owering in the dark (Roldan et al. 1999). In the light, high concentrations of sugars

inhibit owering, possibly through repression of the regulatory gene, LEAFY (Otho

et al. 1998). Many more examples of crosstalk between sucrose signalling and other

signalling pathways are likely to be revealed in the future.

19

INTRODUCTION

HDZip transcription factorsThe pre-history of the HDZip domainHomeosis is dened as an anomaly where one part of the body develops into the

likeness of another part (Bateson 1894). Mutations causing this kind of anomaly have

been observed in many organisms including plants and animals and are referred to

as homeotic mutations. When the rst genes corresponding to this type of mutants

in Drosophila were cloned they were found to share a 180 bp sequence motif that

was called the homeobox and the 60 amino acid domain it encoded was named

the homeodomain (McGinnis et al. 1984; Scott and Weiner 1984). Homeodomain

proteins were rst suggested to act as transcription factors, as the amino acid sequence

of the homeodomain has slight similarity to bacterial, viral and yeast transcriptional

regulators of the helix-turn-helix type (Laughon and Scott 1984; Shepherd et al.

1984). This was later proven to be correct experimentally, by demonstrations that the

factors bind DNA, and affect the transcription levels of nearby genes upon binding,

both in vitro (Johnson and Krasnow 1990) and in vivo (Driever and Nüsslein-Volhard

1989). The homeodomain was also found to have a three-dimensional structure similar

to that of helix-turn-helix proteins, when the structure of the ANTENNAPEDIA

homeodomain was resolved (Qian et al. 1989).

Homeobox genes were also found in other segmented animals and were shown to

control processes such as determination of segment identity during embryogenesis.

It was therefore surprising when homeobox genes were found in the non-segmented

animal Ceanorhabditis elegans by screening genomic libraries with a degenerate

nucleotide pool designed to match all possible codons of the most highly conserved

part of previously known homeobox genes (Bürglin et al. 1989). Inspired by Bürglin

et al. three different groups independently took the same approach to search for

homeobox genes in Arabidopsis (Mattsson et al. 1992; Ruberti et al. 1991; Schena

and Davis 1992). All of the groups found homeobox genes, but of a different

type to those present in animals. They found genes encoding proteins having two

separate domains (the homeodomain and the leucine-zipper domain), known from

different types of transcription factors, fused in single proteins. This arrangement

was novel and since then has only been found in plant genes, although the complete

genomes of other species, including Saccharomyces cerevisiae (Goffeau et al. 1996),

Drosophila melanogaster (Adams et al. 2000), Ceanorhabditis elegans (The C.

elegans sequencing consortium 1998), and Homo sapiens have been sequenced

(Clinton 2000). Thus, this arrangement, the homeodomain leucine zipper, HDZip,

domain is most likely plant specic.

20

INTRODUCTION

HDZip transcription factors are encoded by a large gene family

in ArabidopsisAfter isolation of the rst genes encoding Homeodomain leucine zipper (HDZip)

proteins many more were isolated from Arabidopsis on the basis of sequence similarity

(I; III; V; Carabelli et al. 1993; Lee and Chun 1998; Schena and Davis 1994; Söderman

et al. 1994). The HDZip genes of Arabidopsis are grouped into four families HDZip

I – IV (Sessa et al. 1994) based on sequence similarity as well as other sequence

criteria (summarized in Figure 4). The genes of HDZip I and II appear to share a

common origin while members of HDZip III and IV are more distantly related (Chan

et al. 1998) and differ in their arrangement in the homeodomain compared to HDZip

I and II and to the ANTENNAPEDIA class of animal homeodomains (Figure 4).

The HDZip I and II genes are not clustered on the chromosomes like some

animal homeobox genes (Graham et al. 1989). Instead, they are dispersed among

all Arabidopsis chromosomes (Figure 5). There is no clear correlation between

sequence similarity and chromosomal location, which indicates that most of the gene

duplications from which these genes presumably originate are ancient. This is also

HELIX1 HELIX2 HELIX3 LEUCINE ZIPPER

a b c d e f

Class Length of Introns in Extra amino acids mRNA homeodomain* in HDZip domain

HDZip I approx. 1500 bases a or none None

HDZip II approx. 1500 bases c and e None

HDZip III approx. 3300 bases d Four (between helix 2 and 3) and four (between helix 3 and leucine zipper)HDZip IV approx. 3000 bases b and f Seven (between helix 3 and leucine zipper)

* According to the schematic drawing in A

A

B

Figure 4

Summary of differences between the four different classes of HDZip genes in Arabidopsis

A, Schematic drawing of the primary sequence of the HDZip domain. The putative helical

structures are indicated by boxes. Triangles indicate intron positions.

B, Distinguishing characters of the four different classes of HDZip genes in Arabidopsis.

Adopted from Sessa et al. (1994).

21

INTRODUCTION

HAT3ATHB12

ATHB20

mi357

mi456

ATHB1

150

125

100

75

50

25

0cM

ATHB13

ATHB23

mi106

mi443

ATHB54

I III

HAT1ATHB2

ATHB16

ATHB20

mi87

mi123

HAT22

IV

ATHB53ATHB5

ATHB52

HAT2

ATHB33

HAT14

ATHB51mi121

mi184

V

ATHB21ATHB6HAT9

ATHB22

ATHB4ATHB7

mi421

mi79a

IIATHB17

Figure 5

Chromosomal locations of HDZip I and II genes

Chromosomal locations of 26 HDZip I and II genes (V; Sessa et al. 1994). The relative

positions of ten RFLP markers (mi79a to mi443) are indicated (Liu et al. 1996).

true for HDZip IV genes (Tavares et al. 2000). An ancient origin of the gene family

is also supported by the fact that genes encoding HDZip proteins are found in a wide

range of land plant species including: tomato (Meissner and Theres 1995), sunower

(Chan and Gonzalez 1994), soybean (Moon et al. 1996a), resurrection plant (Frank et

al. 1998), poplar (Sterky et al. 1998), carrot (Kawahara et al. 1995; Mattsson 1995),

Phalaenopsis (Nadeau et al. 1996), Pimpinella brachycarpa (Moon et al. 1996b), rice

(Meijer et al. 1997), maize (Ingram et al. 1999) and ferns (Aso et al. 1999).

HDZip proteins act as dimeric transcription factorsMuch of our knowledge of HDZip proteins is deduced from the vast mass of

information concerning the biochemical properties of animal homeodomains and

leucine-zipper proteins. Homeodomains form a globular structure consisting of three

alpha helices. When the factors bind to DNA the third helix is positioned in the

major groove. Basic leucine-zipper, bZip, proteins also bind DNA by an alpha-helical

structure positioned in the major groove, which is enriched with basic residues. This

basic region of bZip proteins extends into a long alpha helix in which every seventh

22

INTRODUCTION

amino acid is a leucine residue. These residues form a hydrophobic face of the helix

that mediates interaction with similar proteins. bZip proteins bind DNA as dimers.

The leucine zipper parts of the protein orientate the basic regions in the major groove

of the DNA. HDZip proteins bind DNA as homeodomain proteins and dimerise as

bZip proteins in accordance with the fact that they consist of a fusion of these two

domains. A simplied model of how HDZip proteins may be oriented when bound to

DNA is shown in Figure 6.

Dimers of two identical HDZip proteins have been shown to bind to a pseudo-

palindromic DNA sequence, CAATNATTG, consisting of two overlapping identical

half-sites (Johannesson et al. 2000; Meijer et al. 2000; Sessa et al. 1993). The homeo-

domains of HDZip proteins bind to DNA in a similar fashion to their monomeric

counterparts from animals as indicated by mutational analysis of ATHB2. Exchanging

residues important for DNA binding of homeodomain proteins in ATHB2 abolishes

its DNA binding capacity (Sessa et al. 1997). The bases in the central position

of the optimal DNA binding site differ between different HDZip proteins, ATHB2

favours a G/C pair while ATHB1 prefers an A/T pair (Sessa et al. 1993; Sessa et

al. 1997). This specicity has been attributed to the amino acids Arg55, Glu46 and

Thr56, and unspecied residues outside helix 3 (Sessa et al. 1997). In contrast to

ATHB1 and ATHB2, the HDZip I proteins ATHB5, ATHB6 and ATHB16 do not show

middle position specicity, and can interact with both sites (Johannesson et al. 2000).

ATHB12 has been demonstrated to interact with a different site (TCAATTAATTGA)

composed of the same two half sites but with a different spacing (Chun and Lee

1999). As ATHB5, -6, -12 and –16 are identical to ATHB1 in the positions in helix 3

that are postulated to be important for specicity (positions 46, 55 and 56) (Sessa et al.

1997), the alternative DNA binding preferences of these proteins must be determined

by residues somewhere else in the proteins. This hypothesis is also supported by the

nding that some HDZip I and II proteins from rice can interact with both types of

site in yeast (Meijer et al. 2000).

The dimerisation specicity of bZip proteins has been extensively studied (Vinson

et al. 1993) and the three-dimensional structures of bZip homo- and hetero- dimers

bound to DNA (Ellenberger et al. 1992; Glover and Harrison 1995) have been

determined. The formation of salt bridges between basic and acidic residues in

different proteins is apparently important (Vinson et al. 1993; Glover and Harrison

1995). Assuming that HDZip proteins form dimers like the bZip proteins, HDZip I

proteins are very similar in the amino acids facing the putative dimerisation surface,

but distinct from HDZip II proteins. In agreement with this notion, all proteins so

far tested can form homodimers in solution (Johannesson et al. 2000; Meijer et al.

23

INTRODUCTION

Figure 6

HDZip domain binding to DNA

Theoretical model of HDZip domain binding to DNA based on the three-dimensional structures

of the Drosophila ENGRAILED homeodomain (Kissinger et al. 1990) and the yeast GCN4

leucine zipper motif (Ellenberger et al. 1992). DNA is shown in white and the HDZip domain

(secondary structure) in grey. (Johansson, K., Johannesson, H. and Söderman, E. unpublished).

2000; Sessa et al. 1993). Some HDZip proteins from rice and resurrection plants are

able to form heterodimers with members of the same class (Frank et al. 1998; Meijer

et al. 2000). For example, ATHB5 forms heterodimers with ATHB6 and ATHB16

but not with ATHB1 in vitro (Johannesson et al. 2000). This indicates that there are

limitations to the promiscuity of dimerisation among the proteins of the two classes.

Members of the homeodomain and bZip transcription factor families have been

shown to either activate or repress (or both) transcription upon DNA binding

(Latchman 1998), and this also applies to different HDZip proteins. ATHB1 activates

transcription from a promoter with HDZip binding sites, CAAT(A/T)ATTG, (Aoyama

et al. 1995) while experiments with ATHB2 indicate that ATHB2 can act as a negative

regulator of transcription (Steindler et al. 1999). A fusion construct between the strong

24

INTRODUCTION

transcriptional activation domain of VP16 from the herpes simplex virus (Dalrymple

et al. 1985) and ATHB2 affects transgenic plants in the same way as a reduction in

the activity of the wt protein (Steindler et al. 1999). This is a strong indication that

ATHB2 acts a transcriptional repressor in wt Arabidopsis. In rice suspension cells

the HDZip proteins Oshox1 and –3 (HDZip II) repress transcription, while Oshox4

and –5 (HDZip I) activate it (Meijer et al. 2000). Possibly, HDZip I proteins may

generally act as activators and HDZip II proteins as repressors. However, the level of

sequence similarity between the different HDZip proteins outside the HDZip domain

is low, indicating that different HDZip I and II proteins use different mechanisms to

affect transcription.

HDZip proteins are involved in a wide range of processes in

plantsThe functions of only three HDZip genes; GL2, IFL1 and ANL2, have been identi-

ed by analysis of mutant phenotypes. The glabra2, gl2, mutation causes disturbed

trichome and root hair development and the mutant also lacks seed coat mucilage

(Di-Cristina et al. 1996; Rerie et al. 1994). The gene INTERFASCICULAR FIBER-

LESS1, IFL1, regulates interfascicular ber differentiation (Zhong and Ye 1999) and

the gene ANTHOCYANINLESS2, ANL2, affects anthocyanin and root development

in Arabidopsis (Kubo et al. 1999). ATHB8 is expressed in procambial cells of the

embryo and adult plants and is suggested to be a regulator of vascular development in

Arabidopsis (Baima et al. 1995). These four genes belong to HDZip classes III (IFL1

and ATHB8) and IV (GL2 and ANL2).

Altered expression levels of the HDZip II gene ATHB2 (also named HAT4) in

Arabidopsis result in phenotypes that suggest it has a role in light signalling (Schena

et al. 1993; Steindler et al. 1997). Elevated levels of ATHB2, by means of transgene

expression, affect cell expansion in cotyledons and hypocotyls, inhibit secondary

growth of the vascular system in roots and inhibit lateral root formation (Steindler et

al. 1999). Some of the phenotypic effects can be reversed by application of the plant

hormone auxin. Reduced levels of ATHB2 expression by antisense suppression cause

reciprocal effects, indicating that the phenotypic effects caused by elevated expression

reect the wt function of the gene (Steindler et al. 1999). ATHB2 expression is

regulated by far-red-rich light (Carabelli et al. 1993) and the effects caused by altered

expression levels of the gene indicate that ATHB2 has a role in shade avoidance

responses (Steindler et al. 1999). ATHB4 expression is regulated in the same manner

as ATHB2, by far-red-rich light (Carabelli et al. 1993), indicating that ATHB4 has a

role similar to that of ATHB2. Two HDZip II genes from resurrection plant, CPHB1

25

INTRODUCTION

and –2, are regulated by drought and may be involved in gene regulation in response

to drought (Frank et al. 1998). Putative homologues of CPHB1 and –2 are present in

the Arabidopsis genome and may be involved in similar responses.

Data on the function of HDZip I genes are sparse. Indirect evidence from

expression studies indicates that the genes ATHB6, ATHB7 and ATHB12 regulate

gene expression in response to both the plant hormone abscisic acid, ABA, and to

environmental conditions known to increase endogenous levels of the hormone, such

as water deciency (Lee and Chun 1998; Söderman et al. 1999; Söderman et al.

1996). The expression of ATHB7 and ATHB12 is specically and strongly induced

by applications of ABA and by treatments known to cause increased endogenous

levels of the hormone. The induced expression of ATHB7 is impaired in the ABA-

insensitive mutant abi1, suggesting that ATHB7 regulates responses in the ABA

signal transduction pathway downstream of the ABI1 gene (Söderman et al. 1996).

ATHB6 is expressed in developing organs and up-regulated by ABA treatments, and is

suggested to have a function related to cell division and/or differentiation (Söderman

et al. 1999). The expression of the tomato HDZip I gene H52, is up-regulated during

pathogen infection. Suppressed expression in transgenic plants results in a miss-

regulation of cell death control and regulation of defence genes, which indicates

that H52 is involved in limiting the spread of programmed cell death after infection

(Mayda et al. 1999). Another tomato HDZip I gene, VaHox1, is expressed in the

phloem during secondary growth and may participate in the regulation of processes

specic to secondary phases of phloem development (Tornero et al. 1996). The

functions of four HDZip I genes, ATHB3, -13, -20 and –23, are discussed in this

thesis.

26

RESULTS AND DISCUSSION

Isolation and characterization of novel HDZip genesNovel genes distantly related to previously known HDZip I

genesIn extensive searches for Arabidopsis HDZip I and II genes in available databases we

found, in total, 26 genes. Seven of these were novel and named ATHB21, -22, -40,

-51, -52, -53 and 54 (V). We think these 26 genes represent all the HDZip I and II

genes in the Arabidopsis genome, as more than 98 % of the estimated Arabidopsis

genome has now been sequenced (September 2000). All novel genes encoded a

HDZip domain similar to those previously isolated. The deduced sequence of all

HDZip domains could be aligned without creating gaps in the alignment, with the

exception of ATHB22 (which aligned after creating a gap in the alignment between

helixes 1 and 2 in the other sequences, see Figure 7). These amino acids may

form a loop in the turn between the two helices, as has been shown for the yeast

homeodomain MATα2, which has an insertion of three residues at a similar position

(Hall and Johnson 1987). The three-dimensional structure of MATα2 (Wolberger et

al. 1991) is very similar to the homeodomains of Drosophila proteins ENGRAILED

and ANTENNAPEDIA, indicating that the extra residues in ATHB22 do not affect

the structure or DNA-binding properties of the domain.

A homeodomain consensus sequence has been dened on the basis of a compilation

of 346 homeodomain sequences from a range of different eukaryotes (Bürglin 1994).

The homeodomains of all HDZip proteins, including the newly identied forms,

are highly similar to this homeodomain consensus sequence (Figure 7). The 26

HDZip domains are also very similar at positions 7, 8, 54, and 61. In addition to the

conserved homeodomain, all HDZip proteins, including the newly identied HDZip

proteins, contain leucine zipper motifs in identical positions, towards the C-terminal

from the homeodomain (Figure 7). The sequence similarity between the proteins was

signicantly lower in the leucine zipper region compared to the homeodomain.

To investigate if the classication suggested by Sessa et al. (1994) was also

applicable to the novel genes, the amino acid sequences of the entire HDZip domain

were phylogenetically analysed. The analysis resulted in four equally parsimonious

trees with similar branching patterns, one of which is depicted in Figure 8. The four

trees all resolve HDZip I and II, indicating that the classication suggested by Sessa

et al. (1994) reects the evolutionary history of the gene family. Related genes in

the tree have the same or similar intron patterns, further supporting the evolutionary

signicance of the depicted tree.

27

RESULTS AND DISCUSSION

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28

RESULTS AND DISCUSSION

Some groups of functionally related genes form well-separated clades in the tree

(V) e.g. ATHB3, -13, -20 and –23, which cause similar phenotypes when constitutively

expressed (III), and ATHB7/12, both of which are induced by ABA (Lee and Chun

1998; Söderman et al. 1996). The seven novel genes, however, are not closely

assosiated with any previously characterized gene. Hence their function cannot be

predicted on the basis of their phylogenetic relationship with other HDZip proteins.

Pointed cotyledon-HDZip genesThree genes ATHB13, ATHB20 and ATHB23, were isolated on the basis of their

similarity in sequence to the ATHB3 gene (Mattsson et al. 1992). ATHB13 and

ATHB20 were isolated by use of the ATHB3 cDNA as a probe in low stringency

screens of cDNA and genomic libraries (I, III). ATHB23 was rst identied as

an Expressed Sequence Tag or EST (Newman et al. 1994), that showed extensive

sequence similarity to ATHB13 (III). All four genes have now been sequenced by

the Arabidopsis Genome Initiative, AGI, (acc. nr. AL353993, AC013289, AC008261,

AC005508) and the sequences used in our studies are identical to those presented by

the AGI.

A comparison of the proteins encoded by the four genes, ATHB3, ATHB13,

ATHB20 and ATHB23, shows that these genes encode very similar proteins (III),

with a HDZip domain (Figure 7). The four genes belong to HDZip I class of HDZip

proteins (Sessa et al. 1994) as all of them have HDZip I specic amino residues at the

positions that discriminate between HDZip I and II. These four proteins are identical

to each other, but different from all other HDZip I and II proteins in the HDZip

domain at positions 2, 23 and 37, and thus form a subclass of HDZip I proteins. This

is in agreement with phylogenetic analyses of the HDZip domain sequences in which

this subclass forms a separate clade within the HDZip I, Figure 8 (V).

Motifs that are found in each of the four genes but not in other HDZip proteins

are also present outside of the HDZip domain. Two such motifs are located in the

N-terminal part of the proteins: Motif 1, M A F X X X N/G F M X Q X X H E D,

which begins approximately 20 residues from the N terminus and Motif 2, E E X X

S D D G, which is situated directly N-terminal of the homeodomain. The carboxy-

terminal ends of the proteins are also very similar. This motif is 34 to 44 amino acids

long, with stretches of identical amino acids occurring along the whole motif (III).

The two motifs are present in putative homologues of these genes in sunower and

carrot (Chan and Gonzalez 1994; Kawahara et al. 1995). Shared sequence elements

outside the HDZip domain are also found among other HDZip I proteins, but only

in proteins that have very similar HDZip domains e.g. ATHB7/12 and ATHB6/16.

29

RESULTS AND DISCUSSION

ATHB51

ATHB22ATHB40

ATHB53

ATHB21

ATHB12

ATHB7

ATHB1ATHB16

ATHB6

ATHB5

ATHB23 ATHB13ATHB20 ATHB3

ATHB52ATHB54

HAT22HAT9

ATHB2 HAT1

HAT2HAT3

ATHB4HAT14

ATHB17

5 changesFigure 8

Phylogeny of the HDZip I and II domains

Unrooted phylogenetic tree of 26 Arabidopsis HDZip I and II sequences. The analysis was

performed using PAUP (Swofford 2000) based on an amino acid alignment of HDZip I and II

sequences. The presented tree is one of four equally parsimonious trees, which only differ in

the branch-points separating HAT1/HAT2, HAT9/HAT22, HAT3/ATHB4, HAT14 and ATHB2,

respectively.

This indicates that no extensive exon shufing has recently occurred between HDZip

genes and that the phylogenetic analysis based on the sequence of the HDZip domain

reects the evolutionary history of the gene family (V).

Based on the extensive sequence similarity among these four genes as well as the

results from the phylogenetic analysis we conclude that they form a distinct subclass

within HDZip I that we have named pointed cotyledon, or poc-HDZip genes, due to

the phenotypic deviations caused by the constitutive expression of any one of these

genes in transgenic Arabidopsis (III).

30

RESULTS AND DISCUSSION

Functional characterization of the poc-HDZip genesConstitutive expression of poc- HDZip genes results in pointed

cotyledons and serrated leavesTo approach the function of ATHB3, ATHB13, ATHB20 and ATHB23, we expressed

each of the genes in transgenic plants under the control of the strong constitutive 35S

promoter from Cauliower Mosaic Virus (Odell et al. 1985). The transgenic plants

(denoted 35S::ATHB3, 35S::ATHB13, 35S::ATHB20 and 35S::ATHB23, respectively)

showed very similar developmental deviations from wt plants. All genes specically

affected the cotyledon and leaf shape when constitutively expressed (I, III). The

abnormal cotyledons were signicantly narrower than wt cotyledons (Figure 9) due

to the inhibition of lateral cell expansion, as shown by the shape of the epidermal cells

(III). We therefore named the genes belonging to this subclass pointed cotyledon, poc-

HDZip genes. The leaves of the adult 35S::POC plants were also different from wt

control plants in that the leaf margins were markedly serrated (III). The plants most

affected by transgene expression (which also had the highest transgene expression

levels) were severely dwarfed and their reproductive development was delayed (III).

The cotyledons of 35S::POC plants resemble the cotyledons of the angustifolia, an,

mutant (Tsukaya et al. 1994). In contrast to poc-HDZip genes, AN affects leaf and

cotyledon thickness, the number of cell-layers in the cotyledons, the development

of trichomes and the siliques, causing the latter to twist as they develop. The leaf

margins of an plants are not serrated, in contrast to the leaves of 35S::POC plants,

which are similar to the leaves of the serrate, se, mutant (Rédei and Hirono 1964).

Furthermore, se cotyledons are narrow like cotyledons constitutively expressing poc-

HDZip genes. However, in contrast to 35S::POC plants, se plants develop aerial

rosettes on the inorescence, have aberrant phyllotaxy and deviant timing of phase

changes compared with wt (Clarke et al. 1999). As these mutants share similarities

with 35S::poc plants it is possible that the phenotypic deviations in these mutants are

partly mediated by enhanced expression of poc-HDZip genes. This hypothesis has

been partly falsied, since an mutants have been subjected to micro array analysis

and the expression of ATHB3 was not affected by the mutation (Tsukaya et al. 2000).

However, the other poc-HDZip genes were not included in the array.

Leaf length can be regulated independently of the width and thickness dimensions

in Arabidopsis, as illustrated by the short leafed mutant rot3, which has leaves of wt

width and thickness (Tsuge et al. 1996). Poc-HDZip genes, in contrast, affect leaf

width without affecting length or thickness (I, III), unlike the narrow leafed mutant

an, which has leaves that are both abnormally narrow and thick (Tsuge et al. 1996;

31

RESULTS AND DISCUSSION

Figure 9

Pointed cotyledon phenotype

Ten day old seedling constitutively expressing ATHB3, 35S::ATHB3 (right) compared with

wild type, wt (left).

Tsukaya et al. 1994). The phenotypes of constitutive expressers of poc-HDZip genes

thus both support and extend the hypothesis that the dimensions of leaf expansion in

Arabidopsis are independently controlled, as suggested by Tsuge et al. (1996).

Poc-HDZip genes are differentially expressedConstitutively expression of genes in transgenic plants can result in two different

types of effects depending on where the gene is expressed in the wild type, wt. Either

the effect of the transgene expression is present in cells where the endogenous gene

is expressed, denoted effects of over-expression, or in cells where it is not expressed,

denoted effects of ectopic expression or ectopic effects. This distinction is crucial

for interpretation of the phenotypic deviations caused by the transgene, especially

when expressing members of a gene family. If the constitutive expression of a gene

results in ectopic effects, the phenotype caused by the transgene might reect the

activity of another family member. Flowers of plants constitutively expressing the

MADS box genes SPH1 and SPH2 show a phenotype similar to owers from plants

constitutively expressing the very similar MADS box gene AG, namely carpelloid

sepals and staminoid petals (Liljegren et al. 2000). The expression pattern of SPH1

together with the phenotype of double null mutants of the genes clearly shows

that their function is to control fruit dehiscence and not to regulate organ identity

(Liljegren et al. 2000). AG regulates organ identity within the ower, and most likely

SPH1 and –2 take on the functions of AG when ectopically expressed in the ower.

The differentially expressed Arabidopsis RING nger genes SHI, SRS1 and SRS2

also cause the same phenotypic effects when constitutively expressed, indicating that

these genes are also able to functionally substitute for each other when constitutively

expressed (Fridborg 2000).

32

RESULTS AND DISCUSSION

In order to determine which of the above-mentioned situations apply to constitutive

expression of poc-HDZip genes we analysed the expression patterns of the genes using

transgenic Arabidopsis transformed with reporter gene constructs. These constructs

were designed so that the promoters of the poc-HDZip genes directed the expression

of the GUS gene. The location of the promoter-driven expression of the GUS gene

can easily be assayed by histochemical staining of GUS protein activity (Jefferson

et al. 1987). The staining patterns obtained suggest that the poc-HDZip genes

are differentially expressed in Arabidopsis (II, III). Only ATHB23 is expressed in

developing cotyledons and leaves, organs that are affected by the constitutive

expression of the genes. Thus, it is possible that the phenotypic deviations caused

by ATHB23 in leaves and cotyledons might reect the function of the gene in

the wt. The genes ATHB3, -13 and –20 cause ectopic effects in cotyledons and

leaves that constitutively express the transgene. This indicates that these genes

functionally substitute for ATHB23 when expressed in cotyledons and leaves, and

that the pointed cotyledon phenotype is not a reection of their function in the wt. If

so, the experimental demonstration that expression of truncated versions of ATHB13

can cause the same developmental deviations as the full-length protein may indicate

that ATHB23 acts as a transcriptional repressor when inhibiting lateral cell expansion.

The truncated proteins and full-length proteins other than ATHB23 might then exert

their activity in leaves and cotyledons by binding to the ATHB23 binding sites and

impairing the wt activation of the gene or genes.

The expression patterns are informative about the function of the individual genes.

The root and cortex specic expression, respectively, of ATHB20 (III) and ATHB3

(Söderman et al. 1994) strongly suggest that the genes have a specic function

in these cells. ATHB13 is expressed in the vasculature in the basal parts of all

leaf-like organs, cotyledons, rosette and cauline leaves and the ower organs (II).

The expression pattern of ATHB13 is strikingly similar to that of the gene VSP2,

which encodes vegetative storage protein (Utsugi et al. 1998) and ATHB13 may

regulate VSP2 expression in response to sucrose (see below). Other genes that have

some similarities in expression pattern to ATHB13 include the GA1 gene, which is

expressed like ATHB13 in the ower (Silverstone et al. 1997) and the ACS2 gene,

which is expressed like ATHB13 in the silique (Ecker and Theologis 1994). These

genes encode biosynthetic enzymes for the plant hormones, gibberelic acid and

ethylene, respectively. The overlaps in expression patterns, however, are unlikely to

be of biological signicance, as no other data suggest that ATHB13 plays any part in

the regulation of hormone biosynthesis (II).

33

RESULTS AND DISCUSSION

For ATHB13, we also studied its promoter-directed expression in a heterologous

species, hybrid aspen. The staining pattern in hybrid aspen was strikingly similar to

that observed in Arabidopsis (II), indicating that the mechanisms regulating ATHB13

expression are conserved between the two species. The use of aspen also revealed

that it was strongly expressed in the leaf gap area, a feature that was later conrmed

in Arabidopsis upon closer inspection of the staining pattern (II). The functional

signicance of the expression in the leaf gap is difcult to deduce as the leaf gap

area consists of parenchymous tissue dened only by its non-vascular character (Fahn

1990). To our knowledge ATHB13 is the rst gene shown to be specically expressed

in this tissue. The presence of leaf gap-specic gene expression implies a specic

physiological function for the leaf gap tissue, differing from that of the surrounding

cortex.

ATHB13 affects cotyledon and leaf development in a sucrose

dependent mannerConstitutive expression of ATHB13, ATHB3 and ATHB20 results in alterations in

cotyledon and leaf development (I, III). These effects are most likely ectopic as

none of the genes are expressed in these organs (III, Söderman et al. 1994). In

the case of ATHB13, this developmental effect is dependent on the presence of

metabolizable sugars in the medium (I), while ATHB3 and ATHB20 affect cotyledon

development independently of sugar (III). We have determined that the effect of

sugars on 35S::ATHB13 seedlings is independent of the osmotic activity of the

sugars, and is due to their signalling or metabolic properties. The effect on cotyledon

development seen in the 35S::ATHB13 plants is mediated through sucrose specic

signalling, as sucrose causes stronger effects on the cotyledons of 35S::ATHB13

seedlings than glucose, fructose or a combination of these two sugars (I). Further,

results from experiments using sugar analogues capable of activating other sugar

signalling receptors indicate that signals from these receptors are not responsible

for the sugar dependent effects seen in 35S::ATHB13 plants. Enhanced or reduced

hexokinase (HXK) activity, caused by transgene expression, did not affect the

magnitude of the effects on development caused by expression of ATHB13 in

experiments by Jang et al. (1997), further supporting our conclusion that HXK

signalling does not mediate the effect of ATHB13 on development in 35S::ATHB13

plants (I). We also showed that two sugar regulated genes, ATß-Amy and VSP,

encoding ß-amylase and vegetative storage protein, respectively, are signicantly

hyper-regulated in response to sucrose in 35S::ATHB13 seedlings, while general

sucrose sensitivity is unaltered compared to wt seedlings (I). It is possible that

34

RESULTS AND DISCUSSION

ATHB13 directly regulates the expression of these genes, as sites that ATHB13

specically binds in vitro (Johannesson et al. 2000), are present in the promoters of

VSP and ATß-Amy. The expression patterns of the genes also have similarities to that

of ATHB13 (II; Utsugi et al. 1998; Wang et al. 1995), further indicating that ATHB13

might regulate the expression of these genes in vivo. The functions of vegetative

storage proteins and ß-amylase in Arabidopsis have not been resolved, but ATß-Amy

may possibly be expressed in the phloem, and could prevent the accumulation of

highly polymerized polysaccharides at high sucrose concentrations (Wang et al. 1995).

We have interpreted the developmental deviations caused by constitutive expression

of ATHB13 as being due to ectopic effects, possibly it functionally substituting

for ATHB23 (III), and suggest that ATHB13 is a component of a sucrose-specic

signalling pathway, active at points close to the targets of the signal transduction (I).

When constitutive expression of similar transcription factors causes the same

deviation from wt development, the simplest and most likely explanation is that the

factors affect the same set of genes when constitutively expressed. We, therefore,

conclude that ATHB13 activity most likely requires the interaction with a sucrose

dependent factor or a direct modication of ATHB13 by such a factor to be able to

functionally substitute for ATHB23 in cotyledons and leaves. We previously suggested

that a gene downstream of ATHB13 is affected by a sucrose dependent factor (I).

This is now thought unlikely as the sucrose dependent phenotypic deviations caused

by constitutive expression of ATHB13 are identical to those caused by other poc-

HDZip genes (III). The C- and N- terminal domains of ATHB13 are dispensable for

ATHB13 with respect to its effects on the development of cotyledons and leaves since

constitutive expression of genes encoding truncated versions of the ATHB13 protein

cause the same phenotypic deviations from wt development as the full-length protein

(III). That only the HDZip domain is necessary for ATHB13 to affect development

indicates that the putative sucrose dependent factor interacts with this domain. The

HDZip domain of ATHB13 is very similar, but not identical, to that of other poc-

HDZip proteins and to other HDZip I proteins demonstrated to cause other phenotypic

deviations from wt when constitutively expressed, Figure 7 (III). Only two putative

residues, Ala77 and Leu81, are specic to the HDZip domain of ATHB13 and they

may be important in interactions with a sucrose dependent factor. These two residues

are located close to each other in the leucine zipper and oriented away from the

dimerising surface, possibly towards the solution, a location well suited for interaction

with another factor.

SHP1 and –2 functionally substitute for a closely related gene when ectopically

expressed in the ower (Liljegren et al. 2000), in a manner similar to our interpretation

of the way ATHB13 causes abnormal cotyledon development. However, plants

35

RESULTS AND DISCUSSION

constitutively expressing SHP1 and –2 also show phenotypic deviations in the

dehiscence zone, which appear to be indicative of their functions in the plant (Liljegren

et al. 2000). Thus, the specic phenotypes caused by constitutive expression is the

most relevant indicator of the function of a gene, as shown by the effects of SHP1, –2

and ATHB13.

ATHB3 specically affects primary root development when

constitutively expressedBesides affecting cotyledons and leaves, like all other poc-HDZip genes, ATHB3

inhibits primary root development when constitutively expressed (IV). Adventitious

roots are initiated prematurely in 35S::ATHB3 seedlings. Lateral roots in Arabidopsis

are induced by auxin transported from the aerial parts of the plants (Reed et al. 1998).

The process of adventitious root formation in Arabidopsis is similar to that of lateral

roots induced by auxin (King and Stimart 1998). Thus, the premature formation of

adventitious roots in 35S::ATHB3 seedlings might depend on a putative accumulation

of auxin, transported from the aerial parts of the plants, in the basal end of the hypocotyl

when the auxin sink is removed. Surgical removal of the primary root of wt plants

causes premature induction of adventitious roots, as occurs in 35S::ATHB3 seedlings

(IV; Torrey 1950). The growth and development of the premature adventitious roots

of the 35S::ATHB3 seedlings are comparable to primary and adventitious roots of wt

plants (IV). This indicates that initiation of the primary root is the only developmental

anomaly in 35S::ATHB3 seedlings. Hormones like auxins, cytokinins and ethylene

are known to affect the growth of both primary and secondary roots (Meyerowitz

and Sommerville 1994). Adventitious roots and aerial parts of 35S::ATHB3 plants

respond to the hormones IAA, BAP and the ethylene precursor ACC in a wt like

manner (IV), indicating that ATHB3 does not affect the hormone sensitivity of the

plant.

Radicle elongation during germination is a passive process and cell division starts

in the wt root after radicle emergence (Bewley 1997). Thus, two possible ways

in which ATHB3 may affect primary roots can be envisaged. ATHB3 may either

act before germination, and affect the formation of the structures needed for post-

germinative growth of the root, or after germination, specically interfering with the

onset of activity in the meristem of the primary root. Analysis of mutants impaired

specically in root development has shown that most of the processes of embryonic

root development are shared with the development of lateral and adventitious roots

(Malamy and Benfey 1997a; Scheres and Heidstra 1999). Adventitious and lateral

root development is unaffected by constitutive expression of ATHB3. The mutations

36

RESULTS AND DISCUSSION

monopteros, mp, and bodenlos, bdl, specically affect the development of primary

roots (Hamann et al. 1999; Przemeck et al. 1996). Altered root development is

detectable at early stages during embryo development in these two mutants (Berleth

and Jürgens 1993; Hamann et al. 1999). Mutants affecting both primary roots

and lateral roots affect embryo development at later stages (Scheres et al. 1995;

Scheres and Heidstra 1999). It is thus possible that a process during early stages

of embryogenesis is specic for primary roots, and ATHB3 might specically affect

primary root development by interfering with this putative process.

The second possibility is that ATHB3 specically interferes with the onset of

activity in the meristem of the primary root when constitutively expressed. The root

meristem less mutations specically affect cell proliferation in the root meristem

(Cheng et al. 1995). These mutations affect both primary and lateral roots, indicating

that the onset of activity in the meristem of both types of roots is governed by

the same regulatory mechanisms (Cheng et al. 1995). However, the deviations of

35S::ATHB3 plants have not yet been characterized at the cellular level, so we cannot

presently discriminate between the two possibilities.

ATHB3 antisense gene expression induces ATHB13 expres-

sion to higher levelsTrangenic plants designed to have reduced expression of ATHB3 by means of antisense

suppression (35S::antiATHB3 plants) express ATHB13 at higher levels than wt plants

(IV). This effect on ATHB13 expression suggests that ATHB3 acts as a repressor

of ATHB13 expression in the wt. 35S::antiATHB3 plants are indistinguishable from

plants constitutively expressing ATHB13 in that the cotyledons of the seedlings are

signicantly narrower than wt cotyledons, when metabolizable sugars are present in

the media (IV). The phenotypic deviations of 35S::antiATHB3 plants are caused by

increased expression of ATHB13, as the athb13-1 null mutation fully suppresses the

deviations caused in the antisense ATHB3 transgene (IV).

However, expression of the ATHB20 gene is also affected by the antisense

expression, albeit in a different fashion (IV). The antisense ATHB3 mRNA corresponds

to the whole coding sequence, including introns and untranslated parts of the ATHB3

gene. The introns and untranslated regions are not similar to ATHB20, but some parts

of the coding regions are extremely similar. Possibly, the level of sequence similarity

between the genes is sufciently high at some parts of the genes for the antisense

transcript to suppress the expression of ATHB20 (IV). However, we consider this

less likely than the second possibility: that ATHB3 affects the transcription of both

genes in the wt in opposite directions. The effects on transcription of c-MYB and

37

RESULTS AND DISCUSSION

v-MYB proteins binding to DNA depend on both the DNA sequence and the presence

of different co-factors (Ganter et al. 1999). It is conceivable that ATHB3 may have

a similarly variable action in Arabidopsis. The N-terminus of the Oshox1 proteins is

also shown to both repress and activate transcription depending on promoter context

(Meijer et al. 2000). The induction of ATHB13 expression may also be mediated by

the combined effect of reduced activity of both the ATHB3 and ATHB20 proteins.

To resolve this question, null mutants in ATHB3 are needed. We have screened for

such mutants by PCR on DNA from collections of plants harbouring T-DNA and

transposon insertions, but so far we have not been successful (V). Reducing the

activity of ATHB20 by means of mutations or antisense suppression does not affect

the expression of ATHB13, since such plants lack phenotypic deviations from wt

when grown on media with or without sucrose (IV).

Lack of phenotypic deviations caused by HDZip I gene muta-

tions indicates functional redundancy in the gene familyStudying plants with reduced activity of a gene product is the best method for gaining

understanding of the function of a gene. Antisense RNA expression approaches,

although proven to be efcient in some cases, have limitations when suppression

of individual members of a family of closely related genes is wanted. Knock-out

mutations are generally recessive, allowing studies of genes that cause lethal

effects when mutated, which is not possible with constitutively expressed antisense

transcripts. We therefore screened available collections of insertion mutants by means

of PCR, using gene-specic primers directed towards each of the genes in combination

with a primer corresponding to the inserted element (Krysan et al. 1996). In total,

ve insertion mutations were found in HDZip I genes; athb5-1, athb6-1, athb7-1,

athb40-1 and athb51-1, in a collection of T-DNA mutants (V). We also screened a

collection of mutations caused by En-1 transposon insertions, described by Baumann

et al. (1998) and found two mutations, athb13-1 and athb20-1 (I, II).

None of these lines exhibited any visible phenotypic alterations compared to

the wild type, either during seedling growth or as adult plants grown in soil (V).

We also assayed the athb13-1 and athb20-1 mutants for phenotypic deviations on

media containing sucrose, and for abnormal root development on vertical agar plates

supplemented with different hormones (I, III and IV), without detecting any deviations

from wt development. One explanation for the lack of distinguishable phenotypic

effects associated with the mutations in these lines is that HDZip genes might only

be functional under specic physiological conditions. If so, no phenotypic deviations

from wt would be visible unless the mutant plant was subjected to conditions in which

38

RESULTS AND DISCUSSION

the gene is essential. A second explanation could be gene redundancy, as shown in

another family of transcription factors in Arabidopsis (Ferrandiz et al. 2000; Pelaz et

al. 2000).

Most likely poc-HDZip proteins can activate the same set of genes when

constitutively expressed in Arabidopsis (III). ATHB3 and ATHB20 are also expressed

in the same cells in the root (III, Söderman et al. 1994). These data suggest that

ATHB3 may substitute for the missing ATHB20 in the root cortex in athb20-1 plants

and consequently that ATHB20 might be functionally redundant in the root cortex.

Other poc-HDZip genes may possibly functionally substitute for ATHB13 in other

parts of the plant such as the petioles of cotyledons where both ATHB13 and ATHB23

are expressed (III).

Future investigations on HDZip I genes would benet from the availability of

insertion-mutants in all the genes. Double mutants would probably resolve the

problems caused by genetic redundancy within the gene family. The collections of

T-DNA- and transposon-tagged mutations are steadily growing and within the near

future, these tools will be available for the coming generation of HDZip researchers.

Concluding remarksIn this thesis, I have described the characterization of four similar transcription

factor genes and our efforts to reveal their biological functions in the plant. Some

general conclusions can be drawn from the data presented. I want to emphasise the

following.

- Determination of gene function based on the phenotype of plants constitutively

expressing the gene is difficult. However, combined with information on the expression

pattern of the gene, more solid conclusions can be drawn. Constitutively expressed

members of a gene family may cause the same phenotypic effects. This is especially

evident for transcription factors that have their substrate in every cell. However, the

differences (possibly minor) in phenotypic effects caused by the different genes might

give important clues concerning the function of the individual proteins.

- Characterization of the phenotype of null mutants is the safest and most

straightforward method to identify its function. However, mutants may not show

an easily identifiable phenotype – possibly due to gene redundancy, even in the

Arabidopsis genome - one of the smallest genomes among angiosperms.

39

RESULTS AND DISCUSSION

Of the specific results gained from my studies on the genes ATHB3, ATHB13, ATHB20

and ATHB23, I think the following are most relevant to the field of Arabidopsis and

plant research.

- Sucrose signalling is just beginning to be deciphered in plants, and ATHB13 is

one of the first transcription factors shown to be specifically involved in sucrose sig-

nalling.

- The finding that ATHB3 acts as a repressor of ATHB13 expression is a first step

towards identifying the regulatory relations between the different HDZip proteins in

Arabidopsis.

- Cotyledon and leaf width can be regulated independently of thickness in

Arabidopsis, as evident from the phenotypic deviations caused by constitutive

expression of poc-HDZip genes.

These observations, and all the other information gained during my efforts to

understand the function of the four genes studied, have also raised many questions

that I or someone else may try to answer in the future – have fun.

40

SUMMARY IN SWEDISH

Alla levande organismer innehåller gener. Organismens genetiska uppsättning

bestämmer hur den kommer att utvecklas. Detta har varit känt sedan femtiotalet men

riktigt hur det går till är fortfarande inte helt klarlagt. Generna har två funktioner:

dels kan de kopieras till identiska kopior, vilket behövs för att kunna överföra dem

till nästa generation, dels fungerar generna som mallar för bildandet av proteiner.

Proteinerna utför sedan de olika funktioner som organismen behöver, exempelvis

fotosyntes eller sockernedbrytning. Man kan jämföra gener med ett kakrecept som

både skrivs av (kopieras till nästa generation) och bakas efter (gör proteiner).

Det långsiktiga målet med växtfysiologi – nämligen att förstå hur växter fungerar

- har underlättats betydligt sedan man upptäckte metoder att studera gener och deras

funktion. Vi forskare kan nu försöka svara på frågor angående enskilda gener, inte bara

studera hur organismen beter sig som helhet. Vissa funktioner behövs inte överallt i

växten. Exempelvis behövs inte fotosyntes i roten eller på natten och generna för de

proteiner som svarar for detta avläses inte heller i roten eller under natten. Genernas

avläsning regleras av andra proteiner vars enda funktion är att reglera avläsningen

– vi kallar dessa proteiner för transkriptionsfaktorer. Växten backtrav, Arabidopsis

thaliana, har ungefär 20 000 olika gener. Cirka fem procent av dessa gener ger

upphov till transkriptionsfaktorer .

Vi har försökt gå en genväg i vår forskning. Genom att studera

transkriptionsfaktorerna i sig hoppas vi förstå hur de reglerar andra gener och därmed

förstå hur växten fungerar. Jag har valt att studera fyra gener som avläses till fyra

olika transkriptions faktorer (ATHB3, ATHB13, ATHB20 och ATHB23) med hjälp av

två metoder. Först gör jag en s.k. uttrycksanalys d.v.s. jag tar reda på var i växten och

när dessa gener är aktiva och avläses till proteiner. En transkriptionsfaktor som nns i

bladen men ej i rötterna reglerar antagligen bladspecika processer (t. ex. fotosyntes).

Genen ATHB23 är exempelvis aktiv i blad. Detta kallas utrycksanalys. Dessutom kan

jag ta bort eller lägga till gener i växter och får därmed mer eller mindre av proteinerna

i fråga i växten. Jag kan därefter studera de förändrade växternas egenskaper och

på så sätt sluta mig till genernas funktion. Jag har exempelvis ökat aktiviteten av

genen ATHB3 i vissa växter (och därmed fått mer av transkriptions faktorn ATHB3

i växten). I gur 9 syns tydligt att plantan med för mycket ATHB3 (35S::ATHB3)

har smalare blad än den oförändrade vildtypen (wt). Jag kan därför sluta mig till

att proteinet ATHB3 kan påverka växters bladbredd. Genen ATHB23 ger i princip

samma effekt (vilket jag visar i manuskript III). I fallet ATHB23 stämmer resultaten

från båda metoderna mycket bra överens medan resultaten för ATHB3 är lite mer

svårtolkade. En regulator vars funktion är att reglera bladbredd borde vara aktiv i

bladen för att kunna utföra sin funktion.

41

POPULÄRVETENSKAPLIG SAMMANFATTNING

På detta sätt försöker jag förstå de enskilda genernas funktion. Nästa steg är

bland annat att ta reda på vilka gener ATHB23 reglerar - kanske är det en annan

transkriptionsfaktorsgen. Ofta styrs nämligen utvecklingen av former av kedjor av

regulatorer, som bonden på hunden på katten på råttan. Genom att börja med att

studera en av länkarna i kedjan kan vi kanske förstå vilka de andra länkarna är och på

så sätt förstå mer om hur t. ex. bladbredd bestäms i växter.

42

ACKNOWLEDGEMENTS

The creation of this thesis has been a collaborative effort. I would like to thank:

Peter Engström my co-author and supervisor. Without you, I would not know the importance of the clear logical line followed in every good scientific publication. You have also taught me how to dissect natural phenomena into discrete scientifically testable hypothesizes. I would also like to thank you for taking me on as a student and trusting me over the years.

Henrik, besides being the best of friends, you were the only one that kept listening during my worst sugar periods. When I got nice, interesting results, you were the one I showed them to first. From you I have learned the important word “mos” and that there is always some one more “mosig” than myself – an encouraging fact in dark times. I also spent many hours with you, discussing various important scientific matters – never has science been so much fun - schtizz.

Eva Söderman, as well as improving my manuscripts, your advice in the first years improved my laboratory skills and scientific thinking. I will always remember our trips to conferences together.

Jim, my previous room mate. You taught me a lot in the lab in the first years and even more over pizzas late at night. We were always arguing about which of the questions we raised was most important to answer – I do not fully know yet but what I know I learned from you.

Eva Sundberg for giving support during the tough end of my race and for all the effort you spent critically commenting on my manuscripts. Thank you for the drawing (Figure 1). Now, I can admit to you that I do think Ler is nicer than Ws-0.

The HDZip group, Mattias Hjellströn, Yan Wang and Anna Olsson, apart from sharing your results you have contributed valuable suggestions and comments on my work over the years. I wish you all luck with the, sometimes troublesome, HDZips in the future.

Many have contributed to this thesis with their professional skills during my doctoral years: Marie with the staff of Eva B., Afsaneh and Caisa made my life full of N-less sequences. Agneta made endless numbers of transgenic plants. Gunn-Britt made the competent cells used. Caisa supplied me with tons of media, and all the ethanol I needed. Marie Englund (former known as Svensson) taught me how to sequence in the hard days when deciphering 100 nice bp was a success. Charlotta Thornberg helped with RNA preparations and solutions. Anita and Birgitta paid my bills and arranged all the paperwork. Without Stefan and Gary the illustrations presented in this thesis would only exist digitally and not in prints. John for your corrections of this manuscript.

43

ACKNOWLEDGEMENTS

Jens, my fellow computer technician. Together, we have installed Microsoft Office at least 100 times and our discussions during the waiting times have made my work as “avsvarig” most enjoyable. May errors of type 2 never happen again.

I also want to thank Henrik, Mats and Ingela for valuable advice on technical aspects of thesis production.

I would also like to thank Birgitta for your strong and faithful support.

Life as a PhD student has been interesting in many ways. The doktorand gang of Ph.D. students has made lunches, fikas, parties, pub-evenings, dish-washing mornings, melons, clean-up parties, Tupperware™ nights, PCPs, late dinners in the lab, running, aerobic and journal club meetings events that I will remember and miss. Thank you Mats, Henrik, Jens, Katarina, Ingela, Annelie, Sandra, Eva, Yan, Mattias, Anna, Joel and Eva B.

I also like to thank all past and present members of the algal, fungal, and cellscape tribes for making my stay at the department enjoyable.

Tomas, Magnus, Mats and Malin my scientific friends from other fields, you have been listening to my wild plans without laughing (at least, if you did, I didn’t see it) and showed me that science is more than the department of psychological botany.

I also want to thank the grown-up Ph.D. students who now have left the department for teaching me how to behave properly in a laboratory. Thank you Lina, Marie and Anders.

My family, Staffan, Libban, Olof, Maria and Maria for listening and emotional support.

Simon, for forcing me to organize my work.

Jeanette, your support has been invaluable, especially during the writing period. You also convinced me to do more statistical analysis of my data (in agreement with some reviewers) than I would otherwise have done. However, most importantly, you constitutively make my life worth living.

44

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