Präsident der Humboldt-Universität zu Berlin
Prof. Dr. Christoph Markschies

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. P-sensitive 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 Wurzelhaar-Position 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 per2-ähnlichen 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.

• 1 INTRODUCTION

  • 1.1  Iron homeostasis in plants

    • 1.1.1  Acquisition and uptake of iron from the soil

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

    • 1.1.3 Iron uptake by leaf cells, iron storage, and phloem-transport into sink organs

    • 1.1.4 Regulation of iron homeostasis

    • 1.1.5 Linking of iron homeostasis to the homeostasis of other nutrients

  • 1.2 Phosphate homeostasis in plants

    • 1.2.1  Acquisition of phosphate from the soil

    • 1.2.2 Phosphate transport

    • 1.2.3 Xylem loading of phosphate and distribution within the plant 

    • 1.2.4 Metabolic adaptations to P shortage

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

    • 1.2.6 Further regulators of Pstarvation responses

    • 1.2.7 Involvement of hormones in the regulation of phosphate homeostasis

  • 1.3 Root hair development

    • 1.3.1  Root hair specification

    • 1.3.2 Epidermal cell polarity and root hair initiation

    • 1.3.3 Root hair initiation and bulge formation

    • 1.3.4 Root hair tip growth 

    • 1.3.5 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

    • 3.2.1  Regulation of root hair development under Fe deficiency

    • 3.2.2 Regulation of root hair development under P deficiency

  • 3.3 The influence of Fe and P availability on the stages of root hair development

    • 3.3.1  Analysis of mutants with defects in root hair specification

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

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

    • 3.3.4 Differential cytoplasmic staining of rhizodermal cells in adaptation to Fe and P supply

    • 3.3.5 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 

    • 3.4.1  Mutant screening and genetic analysis

    • 3.4.2 Microscopic analysis of the per2 root hair phenotype

    • 3.4.3 The per2 mutation lead to a constitutively high lateral root number

    • 3.4.4 The per2 mutant showed and increased anthocyanin accumulation under P deficiency

    • 3.4.5 The per2 mutantion did not influence the phosphorus content or biomass production

    • 3.4.6 The impaired root hair elongation of per2 was rescued by the phosphate analogon phosphite

    • 3.4.7 The per2 mutation showed an additive genetic interaction with gl2 and erh3

    • 3.4.8 Map-based cloning of the per2 locus

• 4 DISCUSSION

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

  • 4.2 Split-root experiments: local or systemic control?

    • 4.2.1  A local or systemic Fe sufficiency signal is dominant in regulating root hair branching

    • 4.2.2 A local or systemic P deficiency signal is dominant in regulating the root hair number 

  • 4.3 Which stage of root hair development is influenced by Fe and P?

    • 4.3.1  Analysis of mutants with defects in root hair specification

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

    • 4.3.3 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

• Publikationen

• Erklärung

• Table 1 : Affinity of several components involved in iron homeostasis to divalent metals other than iron.

• Table 2 : Genotypes of the mutants used in the experiment.

• Table 3 : Primers for SNaPshot analysis

• Table 4 : Primers for sequencing

• Table 5: The effect of Fe and P deficiency on root hair formation in the primary roots of 3-week-old Arabidopsis wildtype and mutant plants. Values represent the number (mean ± S_E) of the indicated cell type per cell layer. Ten roots were scored for each genotype and treatment.

• Table 6 : The number of root hairs or root hair bulges per mm. The plants were cultivated under sufficient nutrient supply (control) or in media without Fe (Fe) or P (P). Root hairs were counted in the root hair zone of root tip within the 2^nd mm behind the apex. n = 20.

• Table 7 : The percentage of H and N cells showing GUS staining. Twelve-day-old GUS plants were cultivated in the presence and absence of either Fe or P. The spatial GUS expression was analyzed in cross-sections from the late meristematic region of the root. n = 6.

• Table 8 : The statistical data from the primary and secondary mutant screening.

• Table 9 : The SNPs between Cvi and Col-0 identified by sequence analysis.

• Table 10 : The candidate genes for the per2 mutation.

• Figure 1 : A model for iron transport through dicotyl plants. The apoplast is marked in grey, the symplast in white. Iron uptake starts in the rhizodermis (Rh). The Fe³⁺ ions are mobilized by the secretion of phenolics (P), organic acids (OA), and protons (H⁺) into the rhizosphere. Free or chelated iron diffuses or precipitates within the apoplastic space and is detained by the casparian band. Uptake into the rhizodermal cells takes place after reduction of Fe³⁺ chelates by FRO2 through IRT1. Inside the root cells, iron is chelated by NA and reaches the cortical (C), endodermal (En), pericycle (Per) and xylem parenchyma cells of the root (Xpr) via the symplastic way through plasmodesmata. NA accumulates in the vacuoles of the endodermis. VIT1 mediates iron sequestration into vacuoles. Xylem loading is mainly accomplished by the xylem parenchyma. The main iron transport species within the xylem is Fe³⁺ citrate. A prerequisite for xylem transport of iron is the excretion of citrate (Cit) into the apoplast catalyzed by FRD3. For the reoxidation of iron during the transfer from Fe²⁺-NA towards Fe³⁺ citrate, no molecular component has been identified so far. It is not clear, whether the iron is oxidized before or after release into the apoplast. FRO3 and YSL2 are induced in the endodermis, pericycle, and the xylem parenchyma cells of root (Xpr) and shoot (Xps), but their function is unknown. From their localization, a role in xylem loading or exchange processes along the pathway to the shoot could be assumed. The vacuolar metal transporter NRAMP also occurs in the shoot xylem parenchyma and may be involved in the remobilization of iron from vacuolar iron pools. After release of Fe³⁺ citrate into the leaf apoplast, iron uptake by the leaf cells again depends on FRO action. An acidification of the apoplast is also important. The symplastic transport within the leaf cells again occurs as Fe²⁺-NA. Transport into plastids is mediated by PIC1. The main iron storage component in plastdis is ferritin that has ferroxidase activity. Phloem transport towards sink organs mainly occurs in the Fe³⁺ state, probably bound to ITP. Phloem parenchyma is not shown; it expresses FRD3 and FRO3. The loading of iron into sink organs is not well understood. A function of Fe²⁺-NA as the transport form within the cells would comprise a further reduction step catalyzed by FRO gene family members. IRT1, YSL1, and YSL2 are found in sink organs. An important iron storage component within seeds beneath NA is ferritin.

• Figure 2 : Root tips (a-c) and hand-cut sections (d-f) of 18-day-old Col-0 plants grown under sufficient nutrient supply (a, d), Fe deficiency (b, e), or P deficiency (c, f). Bar = 250 µm (a-c) and 20 µm (d-e). The arrow head indicates a branched root hair out of focus.

• Figure 3 : Root hair patterns of the primary roots of 18-day-old Arabidopsis plants grown under sufficient nutrient supply (control), without Fe (-Fe) or P (-P) for seven days, or without both nutrients for four days (-Fe & -P). (a) % of root hairs in the H position. 100% correspond to the total number of epidermal cells that had contact to two underlying cortical cells. (b) % of root hairs in the N position. 100% correspond to all epidermal cells with contact to one underlying cortical cell. (c) % of branched root hairs. 100% corresponds to the sum of root hairs in the H and N position. n = 20 plants.

• Figure 4 : Cryo-scanning electron microscope images of Fe-sufficient (a) and Fe-deficient (b, c) Arabidopsis roots. Bar = 20 µm.

• Figure 5 : Ferric-chelate reductase activity in the roots of plants that were split two days on +/+Fe, -/-Fe, or +/-Fe agar media. The reductase was measured as an end-point determination. Results are an average of three independent experiments, each with 10 plants. Treatment for three days gave the same results.

• Figure 6 : Split-root experiments related to Fe nutrition. Roots of 25-day-old Arabidopsis were divided into two approximately equal parts and grown on +/+Fe, /Fe, or +/Fe agar medium as indicated in the x-axis of each bar graph. The mean percentage ± standard error of root hairs in the H position and of branched root hairs was determined after two (a, b) and seven days (c, d). The split-root experiment was also conducted with the frd3-1 mutant that has an Fedeficient shoot (e, f). The occurrence of root hairs in the N position was below 1% in all treatments. Numbers above the bars in the charts indicate the P value that results from the respective ttest comparing either +/+Fe with /Fe or the split+ with the split- halves of the root. n = 10.

• Figure 7 : Split-root experiments related to P nutrition. Plants were grown on +/+P, /P, or +/P agar media. +/+P is the same as +/+Fe in Fig. 2, but was determined separately. The mean percentage ± standard error of root hairs in the H position root hairs in the N position, and branched root hairs was determined after two (a, b, c) and seven days (d, e, f). (g, h, i) Split-root experiments with the pho1 mutant that had a P-deficient shoot and (j, k, l) with wildtype plants and (m, n, o) pho1 whose shoot was removed. Numbers above the bars in the graph indicate the P values that results from the respective t-test comparing either +/+P with -/-P or the split+ with the split- halves of the root.

• Figure   8 : Root tips of the cpc and ttg mutants grown under control conditions or in the absence of Fe or P. (a) cpc control, (b) cpc –Fe, (c) cpc –P, (d) ttg control, (e) ttg –Fe, (f) ttg –P. Bar = 250 µm.

• Figure   9 : Root tips of the trh1, tip1, and rhd3 mutants grown under control conditions or in the absence of Fe or P. (a) trh1 control, (b) trh1 Fe, (c) trh1 –P, (d) tip1 control, (e) tip1 –Fe, (f) tip1 –P, (g) rhd3 control, (h) rhd3 Fe, (i) rhd3 –P. Bar = 250 µm.

• Figure 10 : Roots of the rhd1 mutant grown in P deficient medium. (a) The primary root did not form root hairs. (b) Epidermal cells of lateral roots exhibited large bulges and normally shaped root hairs. Bar = 100 µm.

• Figure 11 : (a-c) Schematic representation of one transverse cell layer from the Arabidopsis root hair zone with only cortical and epidermal cells. Root hairs in the H position are marked in blue. (a) In five-day-old seedlings nearly all eight epidermal cells in the H position form a root hair. (b) In roots older than five days that are cultivated under sufficient nutrient supply or Fe deficiency, only three of eight H positions develop a root hair in a randomly distributed manner. (c) Pdeficient roots older than five days have on average five root hairs in the H position per cell layer. (d-f) Differential cytoplasmic staining with toluidine blue is shown in cross-sections from the late meristematic region of three-week-old Arabidopsis. (d) control, (e) Fedeficient, and (f) Pdeficient roots. The differential cytoplasmic staining is visible in the epidermis, which is surrounded by cells of the lateral root cap. The lower intensity of staining in (e) is due to a lower thickness of the cross-section (2µm). (g-l) Shown are cross-sections from the late meristematic region of three-week-old GL2-GUS and CPC-GUS plant roots adapted to Fe and P nutrition. (g) control GL2-GUS, (h) Fe-deficient GL2-GUS, (i) P-deficient GL2-GUS, (j) control CPC-GUS, (k) Fe-deficient CPC-GUS, and (l) P-deficient CPC-GUS plants. Bar = 20 µm.

• Figure   12 : The root hair phenotype of the per2 mutant. (a, b) Stereo microscope images (a) per2, -P, (b) per2 +P. (c, d) Micrographs of hand-cut sections from the root hair zone. (c) per2, P, (d) Col-0, P. (e-m) Cryo-SEM images. (e) Col0, control, (f) Col0, Fe, (g) Col0, P, (h) per2, control, (i) per2, Fe, (j) per2, P, (k) per2, control, (l, m) per2, P. Bars = 250 µm (a, b), 100 µm (e, g), 50 µm (c, d, h, i, j, l, m), and 30 µm (f, k).

• Figure 13 : The percentage of wildtype root hairs and per2 bulges in the H position. 14-day-old plants were germinated in the presence of P (grey bars) or without P (black bars).

• Figure 14 : The root system architecture of the per2 mutant. The primary root length (a) and the number of lateral roots (b) of per2 and the Col-0 wildtype grown in vertical culture for 15 days in the presence (grey bars) or absence (black bars) of P. Any lateral root outgrowth that was visible without magnification was counted as a lateral. Also included in the experiment were F3 plants that were the progeny of F2 lines from a backcross with Co0 that had been selected for the per2 root hair phenotype. (c) The increased lateral root development of the per2 mutant on +P medium is shown in an image of 16dayold plants. Bar = 1 cm. Two repetitions of the experiment gave the same results.

• Figure 15 : The anthocyanin content of the per2 mutant grown vertical culture for 20 days in the presence (grey bars) or absence (black bars) of P. Also included in the experiment were F3 plants that were the progeny of F2 lines from a backcross with Co0 that had been selected for the per2 root hair phenotype. Repetition of the experiment gave the same result.

• Figure 16 : (a, b) The phosphorus content of per2 and Col-0 that had been cultivated on Petri dishes (a) or in liquid culture medium (b). (a) Two independent experiments were conducted. In the first experiment, per2 and Col-0 were treated for 20 days. The second experiment includes also the backcross (bc). During this repetition, the climate conditions had been changed by others to a continuous light period and 23°C. Because direct competion of the replicas was not impossible, the culture period was then extended to 27 days to increase potential effects of P deficiency on the P content of the mutant. (b) The plants were also grown in liquid culture. Also a replenishment study was included. One part of the plants were harvested after 7 days of treatment with P deficiency together with the sufficient controls. The remaining plants were replenished with medium containing sufficient amounts of P and were harvested after one day. Two experimental replicas were conducted. During the second experiment, plant material was limiting and was only sufficient to conduct the replenishment experiment.

• Figure 17 : The effect of the phosphate analogon phosphite on the root hair elongation of per2. The plants were germinated on medium containing phosphite instead of phosphate and were analyzed after 14 days. Bar = 250 µm.

• Figure 18 : The characterization of a potential genetic interaction of the per2 mutation with mutations in genes regulating epidermal cell specification. The per2 mutant was crossed with gl2 (a) or erh3 (b). The F2 progeny was germinated on –P medium and the phenotype was analyzed after 14 days. The percentage of plants exhibiting the indicated phenotype is given. (a) The F2 offspring of a cross between per2 and gl2 segregated into 4 different phenotypes. Plants that showed many and elongated root hairs represented the homozygous wildtype and the heterozygous mutant genotypes. The recessive gl2 mutation is phenotypically linked with the lack of trichomes (Koornneef et al. 1982). Thus, plants that showed many root hairs but no trichomes represented the homozygous gl2 mutants. One-quarter of the plants showing the per2 root hair phenotype also had no trichomes indicating that those mutants carrying a homozygous mutation in both loci have an additive phenotype. n = 910 plants. (b) The root system of the recessive erh3 mutant is stunted and the roots are radially expanded (Schneider et al. 1997, Burk et al. 2001). The F2 progeny of a cross between per2 and erh3 also segregated in four phenotypical groups. Those plants that exhibited the per2 phenotype segregated into plants with normal root growth and plants, which showed the erh3 root morphology. Thus, the homozygous per2erh3 genotypes showed both the per2 and the erh3 phenotypes suggesting an additive interaction. n = 684 plants.

• Figure 19 : The low resolution mapping of the per2 mutation with the SNP marker set described by Törjek et al. (2003). The SNPs were detected with the SNaPshot method. (a) Analysis of 213 F2 plants from a mapping population derived from a cross between per2 (Col-0 background) and C24 that showed the per2 root hair phenotype revealed a co-segregation with markers on the upper arm of chromosome 3. (b) Genetic map distances in cM between per2 and markers from the upper arm of chromosome 3 calculated with the Kosambi function.

• Figure 20 : The fine mapping of per2. The upper arm of chromosome 3 is shown ranging from nucleotide no. 6,500,000 to 8,000,000. The F2 plants from the low resolution mapping and 987 further plants exhibiting the per2 phenotype from a mapping population with Cvi were analyzed for recombinations between the markers MASC04279 and MASC02841. The location of these recombinations was then analyzed with a higher resolution of SNP markers that were either obtained from the NASC SNP database (http://www2.mpiz-koeln.mpg.de/masc/search_masc_snps.php) or identified by sequence analysis (^a). The name of the markers was chosen according to the BAC clone they were located on. The numbers below the SNP marker names indicate the chromosomal location of the polymorphic nucleotide. Three lines were heterozygous at the marker location F3H11-7.22 and homozygous at F3H111r, two lines were heterozygous at MQC12 and homozygous at MQC121, and one line was heterozygous at MQC121 and homozygous at K10D20-7.16. The frame indicates markers where no recombination was found.

• Figure 21 : Influence of local and systemic signals related to iron (a, b) and phosphate homeostasis (c, d) on root hair development and deficiency markers. (a) If the shoot is Fe sufficient, a local Fe limitation can not activate root hair branching (RHB). Also the Fe-deficiency marker ferric-chelate reductase (FCR) is inactive. (b) Only if a systemic Fe deficiency signal is combined with a local deficiency signal, RHB is induced. FCR is active also in the presence of local Fe. (c) Also in the presence of a sufficient shoot local P depletion leads to an increase in the root hair number (RHN). The P starvation markers Mt4/At4 are not induced under these conditions. (d) If the shoot is P deficient, RHN is increased and Mt4/At4 induced.

• Figure 22 : The involvement of genes from the root hair developmental pathway in root hair formation in Arabidopsis adapted to P- or Fe-deficient conditions (-P or Fe). In the group of genes from the root hair specification, those are mentioned, whose dysfunction caused a different root hair frequency in the H or N position or in the degree of branching (B) compared to the wildtype (P ≤ 0.01). The ERH3 gene product is required in all cell types independent of the Fe or P supply. The need for ERH1, WER, CPC, TTG, and GL2 gene function is different in the particular cell types of P- or Fe-deficient plants. The RHL genes and RHD6 are required for H cell specification and root hair initiation, irrespective of the Fe or P supply. TRH1 is only essentiell for root hair initiation and tip growth of Fe-deficient plants but is not required under P starvation. From the gene products required for root hair initiation, bulge formation, and tip growth, those are mentioned that differed in root hair length and shape. RHD1, TIP1, RHD2, RHD3, RHD4, KJK/CSLD, and LRX are required for root hair development independent of the nutrient supply. A different frequency of root hair branching during Fe deficiency is caused by mutation of ERH3, ERH1, CPC, TTG, GL2, TIP1, KJK, and LRX. The arrowheads mark possible entries of the Fe- or P-nutritional signal into the root hair developmental pathway.