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«Dissertation zur Erlangung des akademischen Grades doctor rerum naturalium (Dr. rer. nat.) im Fach Biologie eingereicht an der ...»

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Arabidopsis root hair development in adaptation to

iron and phosphate supply

Dissertation

zur Erlangung des akademischen Grades

doctor rerum naturalium

(Dr. rer. nat.)

im Fach Biologie

eingereicht an der

Mathematisch-Naturwissenschaftlichen Fakultät I

der Humboldt-Universität zu Berlin

von

Diplom-Biologin Margarete Müller

geboren am 13.7.1973 in Leer

Präsident der Humboldt-Universität zu Berlin

Prof. Dr. Christoph Markschies

Dekan der Mathematisch-Naturwissenschaftlichen Fakultät I Prof. Dr. Christian Limberg Gutachter: Prof. Dr. Thomas J. Buckhout PD Dr. Wolfgang Schmidt Tag der mündlichen Prüfung: 27. 02. 2007

ABSTRACT

Limitation of immobile nutrients, such as iron (Fe) and phosphate (P), induces the development of additional root hairs that lead to an increase of the absorptive surface of the root. The increased root hair frequency of Fe- and P-deficient Arabidopsis was realized by different strategies. Phosphate-deficient plants increased the number of root hairs while in Festarved plants root hairs were branched. The Fe and P starvation responses in plants are thought to be regulated by a systemic signaling mechanism that communicates the nutrient status of the shoot to the root and by a local signaling mechanism that perceives the Fe or P availability in the soil. The influence of local and systemic signals on the respective root hair phenotype was investigated in split-root experiments. This treatment was combined with either a nutrient-sufficient or -deficient shoot. The root hair branching typical of Fe-deficient plants only occured in the presence of both a local and a systemic Fe-deficiency signal. As a consequence, an Fe sufficiency signal acted dominantly to any deficiency signal, independent of its origin. The increased number of root hairs in P-deficient plants, conversely, was activated through either a local or a systemic P deficiency signal. Thus, the P deficiency signal acted dominantly to any sufficiency signal. To determine, which stage of root hair development was influenced by iron and phosphate, mutants with defects in different stages of root hair development were investigated for their root hair phenotype. Mutants affected in the early stages of root hair development, such as specification, displayed marked changes in the number and localization of root hairs. However, the nutritional signal was perceived and translated in this group of mutants. This indicates that the specification genes are involved in the nutrient-sensitive root hair formation, but may not be the direct targets. Early cell characteristics of root hairs in the late meristematic region of the root, like the expression of marker genes, were unaltered in plants adapted to Fe or P deficiency. This suggested the nutritional signal modulates root hair development after these characteristics have been established. Mutants with defects in the later stages of root hair development, such as root hair elongation, showed short or deformed root hairs in the proper position and frequency and were, thus, impaired independent of the Fe or P supply. Thus, the nutritional signal may enter the root hair developmental pathway around the stage of root hair initiation and bulge formation. Finally, six mutants were screened that did not form root hairs under P deficiency but developed normal, when the plants were transferred to P-sufficient medium. One of these mutants, per2 (phosphate deficiency root hair defective2), was characterized phenotypically and genetically. In addition to the impaired root hair growth, the per2 mutant displayed a constitutively high lateral root number and accumulated an increased amount of anthocyanins under P starvation. Epistatic analysis revealed that per2 action is independent of early cell specification genes. The per2 mutation was mapped to a 87.6 kbp region on the upper arm of chromosome 3 containing 19 genes. The per2 phenotype has not been described before. Thus, PER2 is a potential new gene involved in root hair development under phosphate deficiency.

ZUSAMMENFASSUNG

Pflanzenwurzeln reagieren auf Phosphat- oder Eisenmangel mit einer vermehrten Wurzelhaarbildung, was eine Vergrößerung der absorptiven Oberfläche bewirkt. Die erhöhte Anzahl an Wurzelhaaren wird dabei auf verschiedene Weise gebildet. Phosphat-defiziente Arabidopsis-Pflanzen erhöhen die Anzahl an Wurzelhaarzellen, während sich unter Eisenmangel verzweigte Wurzelhaare entwickeln. Die Fe- und P-Homöostase wird durch systemische und lokale Signalwege reguliert. Der Einfluss dieser Signale auf die Fe- bzw. Psensitive Wurzelhaarentwicklung wurde mithilfe von split-root-Experimenten untersucht, die mit einem systemischen Mangel- oder Suffizienzsignal kombiniert wurden. Die Verzweigung der Wurzelhaare Fe-defizienter Pflanzen wurde durch ein dominantes Suffizienzsignal reprimiert, unabhängig von seiner lokalen oder systemischen Herkunft. Die Erhöhung der Wurzelhaarzahl bei P-Mangelpflanzen wurde durch ein dominantes Defizienzsignal induziert.

Um herauszufinden, welches Entwicklungsstadium von dem jeweiligen Nährstoff beeinflusst wird, wurden Mutanten mit Defekten in frühen und späten Wurzelhaarentwicklungsstadien untersucht. Mutanten mit beeiträchtigter Wurzelhaar-Spezifikation wichen in ihrer Wurzelhaarzahl und –lokalisation vom Wildtyp ab, zeigten aber eine Fe- oder P-sensitive Veränderung. Die Gene aus frühen Entwicklungsstadien sind demnach essentiell für die Reaktion, sind aber nicht das direkte Ziel der Mangelsignale. Frühe Zelleigenschaften in der meristematischen Region waren durch die Eisen- oder Phosphatverfügbarkeit nicht verändert, was darauf hindeutet, dass die Wurzelhaarbildung erst in einem späteren Entwicklungsstadium durch die Nährstoffe beeinflusst wird. Mutanten mit Defekten in späteren Entwicklungsstadien zeigten kurze oder verformte Wurzelhaare unabhängig von der Nährstoffversorgung. Das Fe- oder P-Signal mündet also vor der Wirkung dieser Komponenten in die Wurzelhaarbildung ein. Das heisst, nachdem die korrekte WurzelhaarPosition und -Anzahl in Anpassung an das Fe- oder P-Angebot festgelegt wurde, werden die Wurzelhaare unter allen Wachstumsbedingungen von einer gemeinsamen Maschinerie elongiert. Zur Identifikation potentiell neuer Gene, die die Wurzelhaarbildung in Anpassung an P-Mangel regulieren, wurden sechs Mutanten isoliert, die keine Wurzelhaare bei P-Mangel bilden, aber nach dem Transfer auf P-suffizientes Medium nicht beeinträchtigt waren. Eine dieser Mutanten, per2, wurde phänotypisch und genetisch charakterisiert. Neben der veränderten Wurzelhaarbildung zeigte per2 auch eine konstitutiv erhöhte Lateralwurzelbildung und eine erhöhte Anthozyan-Akkumulation bei P-Mangel. Laut epistatischen Analysen gehört die per2 Mutante zu einem Signalweg, der unabhängig von frühen Zellspezifikationsgenen wirkt. Der per2-Locus wurde innerhalb eines 87,5 kpb großen Abschnittes auf dem oberen Arm von Chromosom 3 kartiert. Mutanten die einen per2ähnlichen Phänotyp zeigen, wurden bisher nicht beschrieben. Daher handelt es sich bei PER2 möglicherweise um ein neues Gen, das die Wurzelhaarbildung bei Phosphatmangel reguliert und weitere P-Mangelreaktionen beeinflusst.





TABLE OF CONTENT

Abstract

ZUSAMMENFASSUNG

1 INTRODUCTION

1.1 Iron homeostasis in plants

Acquisition and uptake of iron from the soil

Iron release into the xylem and long-distance transport through the xylem

Iron uptake by leaf cells, iron storage, and phloem-transport into sink organs............... 10 Regulation of iron homeostasis

Linking of iron homeostasis to the homeostasis of other nutrients

1.2 Phosphate homeostasis in plants

Acquisition of phosphate from the soil

Phosphate transport

Xylem loading of phosphate and distribution within the plant

Metabolic adaptations to P shortage

Integration of local and systemic signals by the PHR1/PHO2/At4 pathway

Further regulators of Pstarvation responses

Involvement of hormones in the regulation of phosphate homeostasis

1.3 Root hair development

Root hair specification

Epidermal cell polarity and root hair initiation

Root hair initiation and bulge formation

Root hair tip growth

Cell wall components involved in root hair tip growth

1.4 Aim of the work

2 MATERIALS AND METHODS

2.1 Plant material

2.2 Growth conditions

2.3 Hand-cut sections, counting of root hairs, and photography

2.4 GUS assay, histology, and differential cytoplasmic staining

2.5 Cryo-SEM

2.6 Ferric-chelate reductase activity

2.7 Mutant screening

2.8 Crossing and analysis of double mutants

2.9 Map-based cloning

2.10 DNA-isolation

2.11 PCR, SNaPshot analysis, and sequencing

2.12 Anthocyanin measurement

2.13 Inductively coupled plasma emission spectroscopy (ICP)

3 RESULTS

3.1 Root hair patterns of the Arabidopsis wildtype adapted to the Fe and P availability

3.2 Split-root experiments

Regulation of root hair development under Fe deficiency

Regulation of root hair development under P deficiency

3.3 The influence of Fe and P availability on the stages of root hair development....... 45 Analysis of mutants with defects in root hair specification

Analysis of mutants with defects in root hair initiation and tip growth

Analysis of root hair mutants with defects linked to the cell wall

Differential cytoplasmic staining of rhizodermal cells in adaptation to Fe and P supply 52 Adaptation of GL2-GUS and CPC-GUS activities to Fe and P supply

3.4 Screening of the per2 mutant and phenotypical and genetical characterization...... 54 Mutant screening and genetic analysis

Microscopic analysis of the per2 root hair phenotype

The per2 mutation lead to a constitutively high lateral root number

The per2 mutant showed and increased anthocyanin accumulation under P deficiency. 57 The per2 mutantion did not influence the phosphorus content or biomass production... 57 The impaired root hair elongation of per2 was rescued by the phosphate analogon phosphite

The per2 mutation showed an additive genetic interaction with gl2 and erh3................ 59 Map-based cloning of the per2 locus

4 DISCUSSION

4.1 Root hair patterns of the Arabidopsis wildtype adapted to Fe and P availability.... 63 4.2 Split-root experiments: local or systemic control?

A local or systemic Fe sufficiency signal is dominant in regulating root hair branching 64 A local or systemic P deficiency signal is dominant in regulating the root hair number. 66 4.3 Which stage of root hair development is influenced by Fe and P?

Analysis of mutants with defects in root hair specification

Analysis of mutants with defects in root hair initiation and tip growth

Fe or P deficiency did not affect the pattern of early cell characteristics in the late meristematic region

4.4 The per2 mutant

LITERATURE

ACKNOWLEDGMENTS

Lebenslauf

Erklärung

INTRODUCTION

A single rye plant possesses a root surface of about 640 square metres (Dittmer 1937). During limitation of immobile nutrients, such as iron (Fe) and phosphate (P), plants increase their root surface by the development of extra root hairs to achieve an improved mobilization and uptake of the respective nutrient. In this work, root hair development adapted to phosphate and iron deficiency has been investigated. In this process, root hair formation is linked to iron and phosphate homeostasis.

1.1 Iron homeostasis in plants

As a transition metal, iron is able to change its oxidation state, making it an important component of biological redox systems such as photosynthesis or the respiratory chain.

Although iron is the fourth most abundant element in the lithosphere (http://www.matpack.de/Info/Nuclear/Elements/lithosphere.html), under aerobic conditions it is poorly available for plants, because Fe3+ is highly insoluble due to precipitation of iron hydroxide or iron phosphate. In anaerobic environments (e.g. in waterlogged soils) the concentration of the soluble Fe2+ increases. When Fe2+ is taken up in excess, it is toxic, because it reduces oxygen into superoxide radicals that generate hydrogen peroxide. In the Fenton reaction, hydroxyl radicals are produced. The hydroxyl radicals cause lipid peroxidation and other, non-selective, oxidative damage (Marschner 1995). Thus, in living organisms iron is chelated and its homeostasis underlies a strict regulation (Schmidt 1999, 2003, Hell & Stephan 2003).

Acquisition and uptake of iron from the soil

A visible iron deficiency symptom is an interveinal chlorosis in young leaves, as several steps of chlorophyll synthesis depend on iron (Marschner 1995). For improved iron mobilization and uptake, a variety of physiological and morphological adaptation reactions have evolved.

Morphological changes of the root system inducible by a low Fe availability are an enhanced lateral root development and an increased number of root hairs (Moog et al. 1995, Pinton et al. 1998, Landsberg 1996, Schmidt 1999). Also root tip swellings were observed (Landsberg 1996). In legume or Proteaceen species, proteoid roots, which are dense clusters of short laterals with a high root hair density, develop in response to a low Fe supply (White & Robson 1989, Arahou & Diem 1997). According to López-Bucio et al. (2003), the main processes affecting the overall root system architecture are the primary root meristem activity leading to changes in the primary root length, lateral root formation, which increases the exploratory capacity of the root system, and the development of additional root hairs, which increases the surface area of the root. The Fe-deficiency-responsive formation of rhizodermal transfer cells, which have labyrinth-like cell wall inversions, in a variety of species increases the apoplast-symplastic surface area if the root (Kramer et al. 1980, Schmidt 1999).



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