genetic analysis of two tomato mutants affected in the regulation of iron metabolism

Mol Gen Genet (1996) 252:87-92 © Springer-Verlag 1996

Hong-Qing Ling • Axel Pich Giinter Scholz • Martin W. Ganal

Genetic analysis of two tomato mutants affected in the regulation of iron metabolism

Received: 15 January 1996/Accepted: 9 May 1996

Abstract Iron is one of the most important micronutrients for plants. Like other organisms, plants have developed active mechanisms for the acquisition of sufficient iron from the soil. Nevertheless, very little is known about the genetic mechanisms that control the active uptake. In tomato, two spontaneously derived mutants are available, which are defective in key steps that control this process. The recessive mutation chtoronerva (chtn) affects a gene which controls the synthesis of the non-protein amino acid nicotianamine (NA), a key component in the iron physiology of plants. The root system of the recessive mutant fer is unable to induce any of the characteristic responses to iron deficiency and iron uptake is thus completely blocked. We present a characterization of the double mutant, showing that the fer gene is epistatic over the chln gene and thus very likely to be one of the major genetic elements controlling iron physiology in tomato. In order to gain access to these two genes at the molecular level, both mutants were precisely mapped onto the high density RFLP map of tomato. The chln gene is located on chromosome 1 and the fer gene is on chromosome 6 of tomato. Using this high-resolution map, a chromosome walk has been started to isolate the fer gene by map-based cloning. The isolation of thefer gene will provide new insights into the molecular mechanisms of iron uptake control in plants.

Key words Lycopersicon esculentum • Genetic mapping • RFLP • RAPD • Plant nutrition

Introduction

The uptake of iron as an essential micronutrient is very difficult for plants. In the soil, iron is present almost

Communicated by R. Hagemann

H.-Q. Ling • A. Pich • G. Scholz • M.W. Ganal ( I~) Institut ffir Pflanzengenetik und Kulturpflanzenforschung, Corrensstr. 3, D-06466 Gatersleben, Germany

exclusively in its oxidized form as Fe 3+, which has a very low solubility. Ferrous iron (Fe 2÷) is basically not available in the soil because it is immediately oxidized to ferric iron.

Like other organisms, plants use sophisticated mechanisms for iron uptake from the soil. These mechanisms are induced by iron deficiency and are switched off when sufficient iron is available in the cell. All plants, except Gramineae use an uptake mechanism known as strategy I (Marschner et al. 1986), which is functionally similar to the iron uptake mechanism in yeast. Under iron stress, strategy I plants show an increased level of proton extrusion from the root into the rhizosphere in order to mobilize ferric iron by lowering the external pH. Mobilized Fe 3 ÷ is then reduced to Fe 2+ by the rhizodermal cells using an Fe3÷-chelate reductase ('Turbo' reductase) that is located in the plasmalemma (Bienfait 1985). Furthermore, in many plants these processes are accompanied by the morphological development of specialized rhizodermal transfer cells. The reduced iron is transferred into the cytoplasma via a presumed channel or active transporter and is then available for physiological purposes.

The graminaceous plants (including barley, wheat, rye, maize, and rice) have adopted a different strategy for iron acquisition, which is called strategy II (Mar- schner et al. 1986) and is similar to bacterial systems for iron acquisition. These plants synthesize and secrete phytosiderophores, powerful Fe 3 +-chelating agents, into the rhizosphere. There, these chelators, which be- long to the mugeneic acid class, complex with Fe 3 ÷ and mobilize iron in this way. These Fe3÷-chelates are subsequently taken up by the root and reduced in the cell.

Whereas much is known about the physiology and biochemistry of iron uptake in plants, very little is known about the molecular biology and genes involved in this process. Furthermore, only a limited number of mutants for this process are available and characterized. Two of the most informative mutants have been

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described in tomato (Lycopersicon esculentum) and have helped to elucidate some of the key components of strategy I in plants.

Based on genetic and additional biochemical data, one of the key substances in iron metabolism is the non-protein amino acid nicotianamine (NA). This substance is synthesized from S-adenosyl methionine and is a central component of both strategy I and strategy II (Scholz et al. 1992). In strategy II plants, NA is the precursor of the phytosiderophores (Kawai et al. 1988; Shojima et al. 1989). In strategy I plants, the role of NA is very different and is illustrated by the phenotype of the spontaneous tomato mutant chIoronerva (chtn), which lacks the ability to synthesize this substance (Rudolph and Scholz 1972), probably due to a non- functional NA synthase. The chln mutant is unable to switch off all strategy I iron-deficiency responses. As a consequence, chln accumulates large amounts of iron and other trace elements, but, in spite of this, exhibits morphological and physiological symptoms of iron deficiency. All these characters can be normalized by exogenous supply of NA (Scholz et al. 1988; Stephan and Griin 1989) or by grafting on wild-type rootstock (Bbhme and Scholz 1960). It has been hypothesized that in strategy I, NA is directly or indirectly involved in iron-dependent metabolic processes, as well as in the regulation of iron-deficiency response mechanisms at the genetic level, through its ability to selectively form complexes with Fe 2÷ (Pich et al. 1991).

A second tomato mutant, T3238fer (fer), is deficient in another key step in the iron-deficiency response. This mutant is completely unable to switch on strategy I iron-deficiency responses, including the development of transfer cells, enhanced extrusion of protons and in- crease in Fe 3 +-reductase. Thus, this mutant is unable to survive on Fe 3 ÷ in soil. However, if supplied with the iron complex FeHEDTA at high concentrations, or grafted onto wild-type rootstock, the plant develops normally (Brown and Ambler 1974; Brown et al. 1971; Landsberg 1982, 1984).

For an understanding of the molecular mechanisms and the genetic control of strategy I responses in plants, the molecular isolation of genes involved in, or controlling, this response is an absolute necessity. The two key mutants make tomato an ideal system for the isolation of such genes by a map-based cloning approach (Tanksley et al. 1995). In this paper, we report a genetic and physiological characterization of the chin andfer mutants and the corresponding double mutant and the fine-structure mapping of these genes onto the genetic map of tomato.

and of the wild-type T3238FER and its mutant fer were obtained from the seed collection at Gatersleben. L. escutentum cv. Moneymaker and L. penneIlii LA 716 were originally provided by the Tomato Genetics Stock Center, Davis, Calif., USA.

Plant culture

All wild-type plants were grown under standard conditions in soil or a hydroponic system (Pich et al. 1991). For crosses, the mutants were germinated and young seedlings with emerged cotyledons were grafted onto wild-type tomato root stocks (cv. Bonner Beste or Moneymaker) according to B6hme and Scholz (1960). Successful grafting reverts the mutants completely to wild-type phenotypes so that they flower and set fruits normally. These grafted plants were used as female parents for the interspecific crosses to L. penneltii or reciprocally for the generation of the double mutants. For some physiological analyses, mutant plants were rescued in the following way: chloronerva plants were supplied with 50 or 500 gM nicotianamine in aqueous solution (pH 6.0) by brushing the leaves five times a day, starting on day 12 of plant growth. Over a period of 14 days, each mutant plant received an amount equivalent to approximately 0.44.0 gM nicotianamine.fer plants were transferred 12 days after seed imbition into 2.5-1 vessels containing aerated nutrient solution with 10 or 100 gM Fe-N-hydroxyethylethylene-diaminetetraacetic acid (FeHEDTA) or iron was directly added to the soil as a 100 gM FeHEDTA solution.

Visualization of proton extrusion and reductase activity

For visualization of Fe 3 +-chelate reduction and proton extrusion under iron-deficiency conditions, the agar method described by Marschner et al. (1982) was used as detailed below.

For Fe 3+-chelate reduction, roots were excised from plants on day 21 after germination, rinsed with distilled water and submerged in an agar solution containing bathophenanthrolinedisulfonic acid, BPDS, kept at 35 ° C. Upon further cooling, the agar solidified around the roots. After an incubation time of 15-60 rain in the dark, the staining pattern typical of formation of the Fe 2 +-BPDS complex developed along those root segments where active Fe 3 +-reduction occurred.

For analysis of proton efflux, roots of intact plants were embed- ded in agar containing bromocresol purple, as above, and incubated under continuous illumination. After 10-t2 h, typical color changes indicative of alterations in pH occur along the respective regions of the roots. The actual pH value was estimated by comparison with calibrated standards of bromocresol purple.

DNA extraction

DNA was extracted from the plants and analyzed using standard techniques (Bernatzky and Tanksley 1986). In most cases, a modified micropreparation protocol was used as described in Messegner et al. (1991). This protocol allowed the isolation of sufficient DNA even from the cotyledons offer plants, which died soon after the development of the first true leaves if not supplied with FeHEDTA.

Material and methods

Plant material

Seeds from the wild-type tomato Lycopersicon escuIentum cv. Bon- her Beste and its spontaneously derived mutant chtoronerva (chin)

RAPD analysis

The initial mapping of the chloronerva andfer genes was performed by bulking (Giovannoni et al. 1991; Michelmore et aL 1991) the different phenotypes (wild type/mutant). Some 5-10 phenotypicaUy scored plants were extracted together and the bulk DNA was used for standard RAPD (random amplified polymorphic DNA)

reactions (Martin et al. 1991) using primers from Operon Techno- logies (Alameda, Calif., USA). PCR products were separated on agarose gels and differences between the two pools were confirmed on another set of pools. F inn confirmation that the fragments were indeed linked to the genes of interest was obtained by the analysis of 50 phenotypically scored plants. The respective fragments were then excised and purified from gels (GeneClean, Biol01) and used as hybridization probes on DNA derived from the standard mapping population of tomato (Tanksley et al. 1992).

Genetic mapping

Restriction digests, gel separation, blotting and hybridization were performed according to standard procedures (Bernatzky and Tan- ksley 1986; Tanksley et al. 1992). Genetic maps were established using the MAPMAKER program (Lander et al. 1987). CentiMorgan distances were calculated using the Kosambi function of the program. For the mapping of the RAPD fragments linked to the two genes, 43 plants from the mapping population described by Tan- ksley et al. (1992) were used.

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and Fe 3 ÷-reduction, double mutants were constructed. For this, the mutants were grafted onto wild-type rootstock in order to normalize them phenotypically. Then, they were crossed reciprocally to each other to exclude maternal effects. The F1 hybrids of both crosses grew normally to maturity and displayed the normal strategy I iron responses. This indicates that chln andfer are recessive mutations in two different genes. In the F2 generation, three phenotypes were observed, based on plant morphology, iron reduction by complex formation with BPDS and proton extrusion: wild-type plants, chln and fer. The segregation ratios in both crosses are displayed in Table 2 and Chi-square tests indicate a segregation ratio of wild type :fer:chtn of 9:4:3 (P <0.05). These data demonstrate that fer is epistatic over chin and controls the expression of proton extrusion and Fe a +-chelate reduction in tomato roots.

Isolation of yeast artificial chromosome (YAC) clones and inverse PCR

YACs were isolated from the tomato YAC library described by Martin et al. (1992) using a PCR screening method (Ganal et al. 1995). The primers used for the YAC screening with TG 590 were 5 ' -GTGAACTGGTTCAAACCAAACTTC-3' and 5 ' -GGCGTGC- TGCTGTTTGATTCTCCT-3 ' . For the YAC library screening with TG 118, the primers 5 ' -GGAAGTAGATGTGTCAACCTTAAG-3' and 5 ' -CTGGGTGAACCCAAACGTTGTGCT-3 ' were used. In- verse PCR (IPCR) was performed according to procedures described by Ochman et al. (1988).

Results

Physiological characterization of chtoronerva andfer and construction of double mutants

The ability of plant roots to reduce Fe 3 ÷ to Fe 2 + can be assessed by measuring complex formation with BPDS (Marschner et al. 1982). In roots of wild-type plants under iron-deficiency conditions, a red colored zone develops due to reduction of iron specifically in the 0-2 cm zone of the root. Under iron-sufficient conditions, the reduction activ~ity is switched off in wild- type plants. The mutantfer is unable to reduce Fe 3 ÷ in the medium under iron-deficiency conditions. In contrast, the mutant chln is able to reduce iron not only under iron-deficiency conditions but also, in contrast to wild type, under iron-sufficient conditions. The applica- tion of exogenous NA to the chln plants through the leaves leads to a partial restoration (phenotypic nor- malization) of this response, such that the reduction activity is drastically reduced under iron-sufficient conditions. A similar response is found for the acidification of the rhizosphere by proton extrusion (Table 1).

Since, physiologically, chln and fer display opposite phenotypes with respect to the extrusion of protons

Isolation of RAPD markers linked to chin and fer

Genetic mapping of the two mutants was performed in a two-step process. In the first step, plants from the

Table 1 Effects of iron nutrition and deficiency on wild-type and mutant plants. A summary of the effects of iron nutrition and nicotianamine (NA) supply on iron-deficiency response reactions of tomato wild-type plants T3238FER and Bonner Beste (BB) and their mutants T3238fer and chloronerva (chln) is presented. Standard growth medium (iron nutrition conditions) included 10gM FeEDTA (BB, chln) or 10 gM FeHEDTA (FER, fer) in the nutrient solution. Iron deficiency conditions included no iron in the nutrient solution. In the reversion experiment, chin plants were supplied with 500 gM NA to the leaves, n.d. not determined

Plant Iron nutrition Iron deficiency

Fe 3 +- Proton Fe 3 +- Proton reduction extrusion reduction extrusion

T3238FER - - + + T3238fer . . . . BB - - + + chln + + + + chln +NA - - n.d. n.d. chln/fer . . . .

Table 2 Segregation ratios observed in F2 progeny of crosses between chln and f er

Cross Plants Wild f er chln )~2 type

chln x f e r 146 73 48 25 4.79 a f er x chin 200 107 60 33 2.81 a

a The Chi-square tests indicate that the observed ratios do not deviate significantly from a 9 (wild-type,): 4 (fer): 3 (chln) ratio (P < 0.05). Plants were scored with respect to their morphology, iron reduction by complex fotTnation with BPDS, and proton extrusion measured with the pH indicator bromocresol purple

90

segregating F2 populations chlnxL, pennellii and fer x L. pennellii were generated. In both cases, the phenotype of the homozygous mutant plants was phenotypically identifiable in an unambiguous manner and the ratio of segregation was specific for a single recessive gene (3: 1, data not shown). Pooled samples of 5-10 wild-type and mutant plants from each population were prepared and used for RAPD analysis.

For the chtn gene, 200 random decamer primers were tested and 11 primers amplified fragments that were polymorphic between the wild-type and mutant pools. One of the 10 polymorphic primers (OPB 12) was very tightly linked to the chtn gene and revealed only one recombination in 50 analyzed gametes. After elution from the gel, the polymorphic fragment was used as a hybridization probe for mapping onto the standard tomato mapping population (L. esculentum × L. pennel- Iii). The amplified fragment was single copy and mapped to the long arm of chromosome 1 in the vicinity of RFLP marker TG 245. This indicated that the chtn gene is located in this region of chromosome 1 of tomato.

For thefer gene, 300 random decamer primers were analyzed with phenotypically specific pools. 10 revealed polymorphic fragments between the pools. One primer, OPP 06, revealed two polymorphisms, one of which was tightly linked to the fer gene and one that showed weak linkage in 50 analyzed gametes. Both fragments were eluted from gels and used as hybridization probe for mapping onto the standard tomato mapping population. Both of the fragments were of low copy number on Southern hybridizations and mapped to chromosome 6 of tomato. The fragment which showed tight linkage was located near the centromere of chromosome 6, between RFLP :markers CT 119 and TG 178, while the other with weak linkage was mapped to the vicinity of the RFLP marker TG 162 indicat- ing that thefer gene is located in the center of chromosome 6.

High-resolution mapping of the chln and fer genes in large populations

After the assignment of the chln and fer genes to their respective chromosomes, detailed fine-scale mapping was performed with all markers on the high density RFLP map (Tanksley et al. 1992) of the respective regions. Fine-scale mapping of the fer gene was performed in 1244 plants derived from a segregating population offer x L. pennellii plants. Since the RAPD analysis gives only an approximate position for the gene ( _+ 10 centiMorgan) on the genetic map (Michel- more et al. 1991), all markers from this region of chromosome 6 were used to determine the precise mapping position. Figure 1 shows the genetic map derived from this population. The RFLP markers TG 590 and TG 118 flank the gene on either side at a distance of 0.3 cM

Fig. 1 High-resolution mapping of the fer gene on chromosome 6 of tomato. In all 1244 phenotypically scored plants were used for high-resolution mapping of thefer gene on the long arm of chromosome 6. P6B indicates the mapping position of the RAPD fragment that was initially found to be linked to the fer gene. The two mapped YAC ends are indicated as 149AR and 356AL

Dist Marke[ dVl Name

0% o 7 e ,,o.o TG178

1 . 4 - -

TG232

2 . 6 ~

0.1 ~ TG590 ~ 149AR 0.2 \ for

1 . 6 - -

,, 356AL

0.4 - - TGl18

"rGI~ U

for TG 590 and 2.0 cM for TG 118. In the :map pub- lished by Tanksley et al. (1992), the genetic distance between TG 590 and TG 352 is 6.0 centiMorgan, while the distance in these experiments is 2.3 cM. This differ- ence is most likely to be due to the fact that in the mapping by Tanksley et al. (1992) only 67 F2 indi- viduals were used, while the data presented in this report are based on a much larger population.

Fine-scale mapping of the chtn gene was performed in an F2 population of 547 plants with all markers localized in the respective region of chromosome 1 of tomato. The results of this mapping experiment are shown in Fig. 2. The chln gene is located 1.0 cM from the RFLP marker CT 224 and 1.5 cM from CT 67, which flank this gene on both sides. All other markers located in this region (Tanksley et al. 1992) appear to be more distant from chtn, based on further segregation analysis.

Isolation of YACs for the markers flanking the fer gene

As a first step towards the isolation of thefer gene, we screened a tomato YAC library (Martin et al. 1992) with primers derived from the sequence of the two closest flanking markers. For TG 590, seven YACs could be isolated. For TG 118, six YACs were found in this library. Inverse PCR was performed on the purified YACs for each marker in order to isolate the terminal

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Fig. 2 High-resolution mapping of the chin gene on chromosome 1 of tomato. For clarity, only the relevant markers tightly linked to the gene on the long arm of chromosome 1 are shown. B12 is the RAPD marker that was initially found to be linked to the gene in bulked segregant analysis. The population of comprised 547 phenotypically scored plants. Other markers from the high density map of Tanksley et al. (1992) that are located in this region are further from chln than either CT224 or CT67

Dist Marl~r cM Name

TG83

6 . 6 ~

0.4 Z11_ ~ ras59 1.0 CT267 CT224

1 . 0 - chin

1 . 5 - - CT67

4.8

5.0 - - i TG16~B~2

sequences of the tomato YACs. While most of the end sequences that could be isolated in this way were re- petitive and could not be mapped, the largest yeast artificial chromosome (YAC 356) for the marker TG 118, with an insert size of 420 kb, extended with its left end over four recombination sites towards thefer gene. On the other side a YAC of 330 kb was investigated for TG 590 (YAC 149). The right end of this YAC extended past one recombinant breakpoint, or 0.1 cM, towards thefer gene, leaving two recombinant breakpoints that have to be crossed to reach the fer gene (see Fig. 1).

Discussion

The uptake of iron as an important essential micronutrient is precisely regulated in plants. Yet very little is known about the molecular mechanisms and genes that control this process. The genes, chln andfer in tomato control key elements in the process of acquisition of iron from the soil. However, chin andfer mutants have different phenotypes. The mutant chln, which is most likely to be defective in the synthesis of the amino acid nicotianamine, simultaneously displays constitutive expression Of iron-deficiency responses in the form of enhanced proton extrusion and reductase activity. The mutant fer is unable to induce either deficiency response under iron stress.

Through the construction of the double mutant between fer and chln, we have shown that the Jet gene is epistatic over chln. Thus, the chtn mutant is unable to show its constitutive response under iron-sufficient conditions in the absence of a wild-typefer gene. As for fer alone, under iron-deficiency conditions, the double mutant is unable to induce the typical responses. These results are in agreement with the hypothesis that nicotianamine, which can form stable complexes only with Fe 2+ and not Fe 3+, could act as a kind of sensor (Stephan and Scholz 1993) for the iron status in a plant. FeZ+-nicotianamine complexes could, directly or via a signal transduction cascade, control the expression of the iron-deficiency responses. If the Fe 2 +-nicotianamine-receptor complex is not present, either because of a lack of nicotianamine synthesis (as in the mutant chln) or Fe2+-deficiency, signals are induced that lead to the expression of the typical deficiency responses in roots. The expression of this response is constitutive in chln because nicotianamine is perma- nently absent.

In fer plants, the expression of the iron deficiency responses, i.e. the differentiation of transfer cells, enhanced proton extrusion and reductase activity, is per- manently blocked. This block most probably, occurs at some point in the signal transduction or transcriptional regulation pathway that leads to the induction of the response genes. Thus, the double mutants show thefer phenotype irrespective of whether a wild-type or mutant chln allele is present.

In order to isolate these genes by a map-based cloning approach (Tanksley et al. 1995), we have initiated a chromosome walk to thefer gene using tightly linked markers from a high-density genetic map for tomato (Tanksley et al. 1992) and a high-resolution genetic map for this gene. The characterization of the first YACs containing flanking markers indicates that isolation of the fer gene is indeed feasible by a map-based cloning approach (Martin et al. 1993).

A molecular analysis of these two tomato mutants would open the door to understanding the molecular mechanisms controlling iron uptake in strategy I plants, since comparable mutations have not been described yet in the plant model system Arabidopsis (Yi et al. 1994). The isolation of the fer gene, in particular which is most likely to be involved in the signal transduction that leads to the induction of the typical iron deficiency responses, could answer the question whether the molecular mechanisms that control these processes in plants are the same as those of the well- characterized yeast Saccharomyces cerevisiae, which shows comparable iron deficiency responses (Dancis et al. 1990; Eide et al. 1992; Dix et al. 1994; Yuan et al. 1995). In the long term, this might also lead to a better understanding of general uptake mechanisms for micronutrients and some other heavy metals, such as copper, in plants (Pich et al. 1994).

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Acknowledgements We thank H. Haugk, D. Kriseleit and D. B6h- mert for technical assistance. Part of this work is supported by a grant from the DFG to G.S., as well as funds from the IPK and DFG to H.-Q.L. and M.G., which are gratefully acknowledged.

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genetic analysis of two tomato mutants affected in the regulation of iron metabolism

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