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First published online January 28, 2009; 10.1104/pp.109.135277

Plant Physiology 150:157-166 (2009)
© 2009 American Society of Plant Biologists

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DEVELOPMENT AND HORMONE ACTION

The Barley Magnesium Chelatase 150-kD Subunit Is Not an Abscisic Acid Receptor1,[OA]

André H. Müller and Mats Hansson*

Carlsberg Laboratory, DK–2500 Valby, Copenhagen, Denmark


   ABSTRACT
TOP
 ABSTRACT
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 LITERATURE CITED
 
Magnesium chelatase is the first unique enzyme of the chlorophyll biosynthetic pathway. It is composed of three gene products of which the largest is 150 kD. This protein was recently identified as an abscisic acid receptor in Arabidopsis (Arabidopsis thaliana). We have evaluated whether the barley (Hordeum vulgare) magnesium chelatase large subunit, XanF, could be a receptor for the phytohormone. The study involved analysis of recombinant magnesium chelatase protein as well as several induced chlorophyll-deficient magnesium chelatase mutants with defects identified at the gene and protein levels. Abscisic acid had no effect on magnesium chelatase activity and binding to the barley 150-kD protein could not be shown. Magnesium chelatase mutants showed a wild-type response in respect to postgermination growth and stomatal aperture. Our results question the function of the large magnesium chelatase subunit as an abscisic acid receptor.

Chloroplast development and the development of the entire plant are entirely dependent on chlorophyll biosynthesis. Magnesium chelatase, the first committed enzyme in the chlorophyll pathway, can therefore be expected to regulate not only chlorophyll biosynthesis but also the development of the plant in a larger context. Magnesium chelatase is a complex enzyme consisting of six 40-kD subunits, six 70-kD subunits (Elmlund et al., 2008Go), and an unknown number of 150-kD subunits. The enzyme exists in homologous forms in anoxygenic (gene names bchI, bchD, and bchH) and oxygenic (chlI, chlD, and chlH) phototrophs (Willows and Hansson, 2003Go). In barley (Hordeum vulgare) the 40-, 70-, and 150-kD subunits are called XanH, XanG, and XanF, respectively, as they are encoded by the genes Xantha-h, -g, and -f (von Wettstein et al., 1971Go). The two smaller subunits form a double hexameric ring complex typical of an AAA protein (ATPase associated with various cellular activities; Fodje et al., 2001Go; Reid et al., 2003Go; Willows et al., 2004Go; Axelsson et al., 2006Go; Elmlund et al., 2008Go). The diverse AAA proteins are a superfamily involved in processes such as folding, assembly, and disassembly of protein complexes (Neuwald et al., 1999Go; Iyer et al., 2004Go). Usually they undergo large conformational changes during their functional cycle (Vale, 2000Go; Hanson and Whiteheart, 2005Go). The 150-kD subunit has been suggested to be the catalytic subunit as it binds the protoporphyrin IX substrate and most likely also the Mg2+ substrate (Willows et al., 1996Go; Jensen et al., 1998Go; Willows and Beale, 1998Go; Karger et al., 2001Go). A structural model of Rhodobacter capsulatus BchH at 25 Å has recently been established based on electron microscopy and single-particle reconstruction (Sirijovski et al., 2008Go). The polypeptide folds into three lobe-shaped domains connected at a central point. Upon protoporphyrin IX binding two of the lobes fuse at their termini. It is probable that the 150-kD subunit can be regarded as a substrate of the double hexameric AAA complex.

The 1-MD magnesium chelatase complex is 20 times larger than the monomeric or dimeric ferrochelatase, which catalyzes a very similar kind of reaction—the insertion of Fe2+ into protoporphyrin IX generating heme (Dailey and Dailey, 2003Go). It is therefore likely that the magnesium chelatase could harbor several domains involved in other activities such as regulation but not affecting the chelatase reaction per se. Magnesium chelatase has been implicated in chloroplast-to-nucleus signaling (Mochizuki et al., 2001Go; Strand et al., 2003Go; Ankele et al., 2007Go) but whether the product Mg-protoporphyrin IX is the signal molecule is under debate (Mochizuki et al., 2008Go; Moulin et al., 2008Go). Recently magnesium chelatase has also been reported as an abscisic acid receptor (Shen et al., 2006Go). Abscisic acid is a phytohormone regulating various processes in plants, among them abscission of leaves, stomatal aperture, seed development, germination, and postgermination development. It is involved in adaptation to abiotic stresses such as salt, cold, and drought stress through regulation of gene expression (Christmann et al., 2006Go). Several proteins of abscisic acid signaling in plants have been identified recently. The barley aleurone protein ABAP1 was found to be an abscisic acid-binding protein (Razem et al., 2004Go). The homologous Arabidopsis (Arabidopsis thaliana) RNA-binding protein Flowering Time Control Protein A (FCA) was identified as an abscisic acid receptor controlling flowering time (Razem et al., 2006Go). A G-protein-coupled receptor (G-protein-coupled receptor 2 [GCR2]) was found to mediate all known abscisic acid responses in Arabidopsis (Liu et al., 2007Go). Also the 150-kD magnesium chelatase subunit H (CHLH) was specifically indicated to be affected by abscisic acid (Shen et al., 2006Go). Initial studies were done on broad bean (Vicia faba) ABAR (Zhang et al., 2002Go), but more extensive analyses were performed with Arabidopsis CHLH (Shen et al., 2006Go). Magnesium chelatase activity of Arabidopsis was reported to be enhanced by abscisic acid. Arabidopsis mutants underexpressing magnesium chelatase showed phenotypes insensitive to exogenous abscisic acid in germination and postgermination growth, whereas overexpression mutants showed hypersensitive phenotypes.

In this work, we analyzed the effects of abscisic acid on magnesium chelatase-deficient barley. We produced recombinant XanF and scrutinized the effects of abscisic acid on chelatase activity and whether abscisic acid was bound by the subunit.


   RESULTS
TOP
 ABSTRACT
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 LITERATURE CITED
 

Postgermination Development

Retardation of growth postgermination is one of the fundamental effects of abscisic acid (Himmelbach et al., 1998Go). To find an abscisic acid concentration effectively inhibiting root and shoot growth, wild-type barley seeds were imbibed in abscisic acid solutions of different concentrations. The seeds were then germinated on paper soaked with the same solution. The assays were kept in the dark due to the light sensitivity of abscisic acid. However, they were daily exposed to ambient light when the lengths of roots and shoots were measured. The experiment showed that barley root and shoot development is effectively arrested by abscisic acid in a concentration-dependent manner (Fig. 1 ). Barley treated with 10 µM (±)-abscisic acid had a root length after 3 d that was less than 25% of the water-treated control. Shoot length after 3 d was only 15% of the control.


Figure 1
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  		Figure 1. Abscisic acid inhibits root (A) and shoot (B) growth in a concentration-dependent manner in wild-type barley. Root and shoot lengths were measured of seedlings germinated in water (black) and in 1 µM (dark gray), 2 µM (gray), 6 µM (lighter gray), 10 µM (light gray), and 20 µM (white) (±)-abscisic acid. Time in days (d).

 
The experiment was repeated with 10 µM (±)-abscisic acid on seeds of the barley mutants xantha-f.10, -f.58, -g.44, -h.57, and -l.81. All the mutants were inhibited in their development by abscisic acid to comparable extents (Fig. 2 ). The behavior of the Xantha-f mutants deviated neither from the wild type nor the other mutants.


Figure 2
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  		Figure 2. The postgermination development of different Xantha mutants is effectively inhibited by abscisic acid. The root lengths of seedlings germinated in 10 µM (±)-abscisic acid (A) and water (B) and their shoot lengths in 10 µM (±)-abscisic acid (C) and water (D) were measured over 4 d (day 1: black bars; day 2: dark-gray bars; day 3: light-gray bars; day 4: white bars). The mutants used were xantha-f.10, -f.58, -g.44, -h.57, and -l.35. Segregating seedlings with wild-type phenotype are marked wt, and homozygous mutant seedlings are marked mut. The number of seedlings (n) with the respective phenotype measured is given; bars represent SD.

 

Abscisic Acid-Induced Stomatal Closure

Mutant and wild-type leaves of xantha-f.10 barley were floated on abscisic acid solutions in ambient light. After 2 h the opening status of 119 to 259 stomata on the lower side of the leaf was determined. The stomata were noted as either closed or open. The results showed a clear abscisic acid-induced stomatal closure. The percentage of closed stomata increased from approximately 25% in the control to approximately 85% in 10 µM (±)-abscisic acid (Fig. 3A ). This was true for the wild-type and the mutant leaves. Thus, both wild-type and mutant stomata closed under the influence of the phytohormone. The mutant stomata were generally closed to a slightly higher extent, probably due to the higher stress in chlorophyll-deficient leaves.


Figure 3
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  		Figure 3. Abscisic acid determines the opening of the stomata. A, The percentage of closed stomata depending on abscisic acid concentration; lines represent stomata from wild-type leaves, dashed lines those of mutant leaves. The graphs marked by squares and those marked by triangles represent two measurements using different batches of xantha-f.10 plants. B, An infrared photograph shows the similar rise in leaf temperature in xantha-f.10 wild-type (first five seedlings to the left) and mutant leaves (five seedlings to the right; see insert) placed in water and 1, 2, 5, and 10 µM (±)-abscisic acid.

 
A second approach was used to analyze the abscisic acid-induced stomatal closure. Infrared photographs were taken of wild-type and mutant leaves that had been placed in different abscisic acid solutions in ambient light. The leaf temperature is determined by the amount of transpiration, which is in turn a function of the degree of opening of the stomata. The photograph (Fig. 3B) illustrates and proves the dependence of leaf temperature and thus stomatal closure on the concentration of exogenous abscisic acid.


Obtaining a Recombinant XanF

The barley Xantha-f gene was cloned into the Escherichia coli expression vector pET15b from which a protein with an N-terminal His6 tag can be obtained. As the Xantha-f gene is large (6,252 bp encoding 1,381 amino acid residues) we used a strategy where Xantha-f cDNA was amplified by reverse transcription-PCR in three pieces and then assembled into a complete gene downstream of the inducible T7 promoter of pET15b. Primers were designed according to the Xantha-f DNA sequence of Olsson et al. (2004)Go. The initial 43 amino acid residues were not included in the construct as they were estimated to constitute the chloroplast transit peptide according to ChloroP 1.1 (Emanuelsson et al., 1999Go). The final plasmid construct was named pET15bXanF. Expression of Xantha-f from pET15bXanF was tested in E. coli BL21 (DE3), E. coli BL21 (DE3) pLysS, and E. coli Rosetta (DE3) pLysS (Novagen). Two parameters were varied to optimize the yield of soluble XanF: (1) growth temperature (37°C, 30°C, 22°C, 16°C) and (2) culture density at the addition of isopropyl-β-D-thiogalactopyranoside (OD600 = 0.2, 0.4, and 0.7). Most soluble XanF was obtained if E. coli Rosetta (DE3) pLysS/pET15bXanF was grown at 30°C to OD600 = 0.7 before isopropyl-β-D-thiogalactopyranoside was added to a final concentration of 1 mM. The culture was moved to 16°C and subsequently incubated for 40 h before harvest. Cells were pelleted by centrifugation, washed and lysed with a French press (SLM Aminco). After one freeze-thaw cycle the His6-tagged XanF protein was purified from soluble fractions by Ni2+-affinity chromatography (HisTrap HP, Amersham Biosciences). XanF typically eluted at an imidazole concentration of 450 to 500 mM. As magnesium chelatase activity was sensitive to imidazole in the elution solution of the recombinant XanF protein, a better storage solution for XanF was sought. Initially, we tried the solution (50 mM Tricine-NaOH, pH 8.0, 15 mM MgCl2, 4 mM dithiothreitol [DTT], 6% glycerol) described for the homologous enzyme BchH from R. capsulatus (Sirijovski et al., 2006Go) but the recombinant protein precipitated during desalting (NAP-5 column; GE Healthcare). Next, the amounts of NaCl and imidazole, which were present at 500 mM in the elution solution, were varied individually and together (Fig. 4 ). A NaCl concentration of 250 mM was necessary to maintain XanF solubility while the effect of the imidazole concentration on the solubility was comparatively negligible. The final XanF storage solution was composed of 50 mM Tricine-NaOH, pH 8.0, 250 mM NaCl, and 50 mM MgCl2. In this solution XanF was stable on ice for 4 weeks. The protein was not stable in freeze-thaw cycles or at room temperature.


Figure 4
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  		Figure 4. XanF solubility is dependent on the NaCl concentration. Lanes 1, 4, and 7 show polyacrylamide gel bands of XanF in elution solution (500 mM NaCl, 500 mM imidazole) after purification. In lanes 2 and 3 the NaCl concentration was decreased to 250 mM and 100 mM, respectively; imidazole was present at 500 mM. In lanes 5 and 6 the imidazole concentration was decreased to 250 mM and 100 mM, respectively; NaCl was present at 500 mM. In lanes 8 and 9 both NaCl and imidazole were decreased to 250 mM and 100 mM, respectively. Lane 10, XanF in elution solution; lane 11, XanF desalted into the final storage solution (see text).

 

Effect of Abscisic Acid on XanF Activity

The magnesium chelatase reaction requires the activity of the three proteins XanF, XanH, and XanG. All three subunits are present in a preparation of lysed greening barley etioplasts. Addition of up to 10 µM (±)-abscisic acid to lysed plastids did not influence the magnesium chelatase activity significantly (Fig. 5A ). With the availability of recombinant XanF we could also test the specific effect of abscisic acid on this subunit. XanF is the first barley magnesium chelatase subunit to be purified. To analyze if recombinant XanF contributed to magnesium chelatase activity we utilized lysed plastids of barley mutants xantha-f.10 and -f.26, which contain wild-type amounts of XanH and XanG (Olsson et al., 2004Go). However, the amount of XanF is much reduced in xantha-f.10. Mutant xantha-f.26 has wild-type level of XanF protein. Lysed plastids of xantha-f.10 show no magnesium chelatase activity, whereas those of xantha-f.26 have 9% of the wild-type activity (Olsson et al., 2004Go). It has previously been shown that magnesium chelatase activity can be reconstituted in vitro if lysed plastids of various barley magnesium chelatase mutants are combined (Hansson et al., 1999Go). Therefore, we expected that a mixture of recombinant XanF and lysed barley mutant plastids deficient in this subunit would show magnesium chelatase activity. This was also found. Recombinant XanF (5 and 10 µg) was added to lysed plastids of xantha-f.26. Magnesium chelatase activity increased with an increasing amount of added XanF protein (Fig. 5B). To test the effect of abscisic acid on the XanF subunit, 4.8 µg of recombinant XanF were preincubated with a 5.3-fold molar excess of (±)-abscisic acid on ice or at room temperature for 5 min. The mixtures were then added to the rest of the assay components. The magnesium chelatase activity was not influenced significantly by abscisic acid treatment (Fig. 5C).


Figure 5
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  		Figure 5. Magnesium chelatase activity measurements. A, Abscisic acid does not effect wild-type magnesium chelatase significantly. Activity of wild-type lysed plastids (line) and with 1 µM (dashed line), 5 µM (dotted line), or 10 µM (±)-abscisic acid (dash dotted line) added. B, Activity of recombinant XanF. Five micrograms XanF (dashed line) or 10 µg XanF (dotted line) were added to mutant xantha-f.26 lysed plastids (line). C, Abscisic acid does not influence the activity of recombinant XanF significantly. Activity of mutant xantha-f.10 lysed plastids alone (line), with 4.8 µg XanF added (dashed line), and with 4.8 µg XanF preincubated with a 5.3-fold molar excess of (±)-abscisic acid for 5 min on ice (dotted line) or at room temperature (dash dotted line). Values are averages of two or three (A) or two (C) individual assays for each condition (only one assay for xantha-f.10 lysed plastids alone in C). The bars represent SDs of the emissions at 577 nm. a.u., Arbitrary units.

 

Abscisic Acid Binding to XanF

The activity of barley magnesium chelatase was not influenced by abscisic acid. In addition, mutants of Xantha-f, as well as other tested chlorophyll biosynthetic mutants, showed a wild-type response to the plant hormone with respect to postgermination development and stomatal closure. Those results, however, did not necessarily exclude an interaction between the XanF protein and the phytohormone.

Therefore, the binding of radioactively labeled abscisic acid to the putative receptor protein XanF was studied. Tritiated (+)-abscisic acid was incubated with XanF. After equilibration of the mixture unbound abscisic acid was removed by charcoal and the radioactivity remaining in the supernatant was determined. Three different methods were used that were modifications of the protocols of Razem et al. (2004)Go and Shen et al. (2006)Go. The three methods differed in the composition of the assay solution, the ratio of hormone to protein, and the incubation conditions.

The activity determined in the samples containing XanF was similar to the activity determined in the samples containing the denatured polypeptide or no protein, indifferent of the method used (Table I ). Assuming one binding site per polypeptide as found by Shen et al. (2006)Go for Arabidopsis CHLH, the amount of activity to be retained in the supernatant in case of binding was calculated to be 515 min–1 following methods A or C and 465 min–1 following method B. These values correspond to 67% and 100% of the activity of the added tritiated (+)-abscisic acid, respectively. In the assay following methods A or C, the phytohormone was present in excess compared to the polypeptide. The polypeptide was present in excess following method B.


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  		Table I. Results of the abscisic acid-binding assays

The activities determined in the supernatant after sedimentation of the charcoal (±SD) are shown. Samples contained XanF, denatured XanF, or no protein. Blank is scintillation solution only.

 
The binding of abscisic acid to XanF could thus not be shown in these experiments.


   DISCUSSION
TOP
 ABSTRACT
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 LITERATURE CITED
 
Identification of plant hormone receptors has been a key issue to dissect the signaling pathways of plant hormones. A genetic approach was often successfully used to identify receptors of classical plant hormones such as ethylene, cytokinins, brassinosteroids, gibberellins, and auxin (McCourt and Creelman, 2008Go). In those studies mutants of Arabidopsis that did not respond to the hormone of interest were isolated and the affected gene was identified by map-based positional cloning. Concerning abscisic acid, three receptors have recently been identified by a more biochemical approach (Razem et al., 2006Go; Shen et al., 2006Go; Liu et al., 2007Go). The three proteins FCA, CHLH, and GCR2 all showed high affinity to abscisic acid, which was explored during the identification of these receptors. Our previous experience from biochemical and structural studies of magnesium chelatase encouraged us to initiate a study of this enzyme and its relation to abscisic acid. In addition, our collection of 20 barley magnesium chelatase mutants, of which eight are in the Xantha-f gene encoding the CHLH, could complement the in vitro analysis.

However, our conclusion is that the barley CHLH does not qualify as an abscisic acid receptor in contrast to the Arabidopsis protein. Transgenic Arabidopsis RNAi lines underexpressing CHLH, clearly showed an abscisic acid-insensitive phenotype in terms of seed germination and postgermination growth. Furthermore, Arabidopsis lines overexpressing CHLH displayed an abscisic acid-hypersensitive response (Shen et al., 2006Go). In our barley mutant collection we have access to missense mutants of the Xantha-f locus deficient in active magnesium chelatase. The defects are due to changes of amino acid residues in subunit H although there are wild-type levels of the protein. In addition, there are other mutants with no CHLH present (Olsson et al., 2004Go). In this study we included both kinds of barley Xantha-f mutants. Barley wild type, mutants with deficient magnesium chelatase subunits D and I, and barley mutants with a deficient cyclase were used as controls. No differences in postgermination development could be seen in any of the different barley mutant strains. Shoot and root growth rates were recorded over a 4-d period in water as well as in 10 µM (±)-abscisic acid. No Xantha-f mutant showed any behavior typical of an abscisic acid-insensitive phenotype. If barley subunit H was a receptor of the phytohormone, at least the plants completely lacking the protein should develop independently of exogenously supplied abscisic acid. Neither were the other magnesium chelatase subunits nor the Xantha-l-encoded cyclase component implicated as abscisic acid receptors. Abscisic acid inhibition of postgermination development was clearly apparent in all the mutants.

It was further reported that the Arabidopsis line underexpressing CHLH was less drought resistant, whereas the overexpressing line was more resistant to dehydration (Shen et al., 2006Go). The difference in drought tolerance was correlated to stomatal aperture, which is regulated by an abscisic acid response. In our wild-type barley material we saw clear correlation between stomatal aperture and added amount of abscisic acid. In contrast to the results for Arabidopsis the barley magnesium chelatase mutant Xantha-f plants showed a wild-type abscisic acid response as well.

We also performed analyses on protein level. The barley magnesium chelatase gene Xantha-f was cloned and expressed in E. coli. The recombinant H subunit was purified as a His-tagged protein and shown to be active in magnesium chelatase assays. The other subunits of magnesium chelatase were provided by lysed plastids of barley Xantha-f mutant seedlings (containing only the other two chelatase subunits), which were added to the assay mixtures. The activity of magnesium chelatase was neither influenced by abscisic acid added to an assay of the wild-type enzyme nor by preincubating recombinant H subunit with abscisic acid prior to mixing with the other assay components. Thus, we could not detect an increased barley magnesium chelatase activity upon addition of abscisic acid as reported for the Arabidopsis system (Shen et al., 2006Go).

Finally, we also performed binding studies of abscisic acid to barley CHLH. The protein, active in magnesium chelatase assays, could not be shown to bind abscisic acid. This is in strong contrast to the Arabidopsis subunit H, which showed saturation kinetics with a likely binding ratio of one abscisic acid molecule per H subunit and an equilibrium dissociation constant of 32 nM.

Presently, we cannot explain the differences in abscisic acid response between the barley and Arabidopsis magnesium chelatase H subunits. A close look at the primary sequences reveals that they are very similar with 82% identical residues. Both H subunits consist of 1,381 amino acid residues including an N-terminal chloroplast transit peptide. An alignment (Larkin et al., 2007Go) does not introduce any gaps longer than five residues (Fig. 6 ). Thus, it is hard to believe that the Arabidopsis subunit H could function as an abscisic acid receptor while the barley subunit H does not, even though Arabidopsis is a dicotyledonous plant while barley is monocotyledonous.


Figure 6
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  		Figure 6. Polypeptide sequence alignment of Arabidopsis CHLH (accession NM 121366) and barley XanF (accession AY039003) by the ClustalW algorithm 2.0.10 (Larkin et al., 2007Go). Asterisks denote identical residues, colons conserved substitutions, and periods semiconserved substitutions. The plastid transit peptide of XanF (Emanuelsson et al., 1999Go) is highlighted in black.

 
The Arabidopsis genome contains one CHLH gene encoding the CHLH. Careful analysis of the rice (Oryza sativa) genome reveals one CHLH gene on chromosome 3 (LOC_Os03g20700, http.//rice.plantbiology.msu.edu) and one truncated version of CHLH on chromosome 7 (LOC_Os07g46310). The polypeptide of the short gene is 761 amino acid residues and 94% identical to the C-terminal half of the full-length version (1,388 amino acid residues). The genome sequence of barley is not available, but Southern-blot analysis demonstrated the presence of a single subunit H encoding gene, Xantha-f, which is located on the short arm of chromosome 2H (Hansson et al., 1998Go). It is very likely that a truncated barley Xantha-f gene, similar to the gene in rice, would have been recognized in the study. Thus, there is no evidence of a second Xantha-f-like gene whose product could play the role of an abscisic acid receptor in barley.

Although chlorophyll biosynthesis is tightly linked to chloroplast development and thereby to the development of the plant in general, chlorophyll biosynthesis and chloroplast development have not been traditionally connected to abscisic acid responses. Therefore, the function of CHLH as an abscisic acid receptor could not have been foreseen from earlier physiological studies involving abscisic acid (McCourt and Creelman, 2008Go). It should be noted that there are also conflicting results concerning the suggested abscisic acid receptor GCR2 (Gao et al., 2007Go; Johnston et al., 2007Go). Very recently the publication suggesting FCA as an abscisic acid receptor was retracted (Razem et al., 2008Go; Risk et al., 2008Go). Our results question the idea that CHLH is an abscisic acid receptor.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
LITERATURE CITED
 

Plant Material

Barley (Hordeum vulgare ‘Svalöfs Bonus’) was used as wild-type barley. Magnesium chelatase mutants xantha-f.10, -f.26, -f.58, -g.44, and -h.57 and aerobic cyclase mutant xantha-l.81 were employed for various tests. All mutations are lethal. The mutation xantha-f.10 is a 3-bp deletion, which removes the conserved amino acid residue E424 of the XanF subunit. Lysed plastids of xantha-f.10 show no magnesium chelatase activity and the amount of XanF protein is very much reduced (Olsson et al., 2004Go). In contrast, xantha-f.26 is a leaky mutant that contains 8% to 10% of the chlorophyll accumulated in wild-type leaves (Henningsen et al., 1993Go). The missense mutation results in a M632R exchange, which does not seem to affect the stability of the protein as XanF is found in wild-type amount in a xantha-f.26 mutant leaf. In vitro, the magnesium chelatase activity is 9% of that of wild type (Olsson et al., 2004Go). In mutant xantha-f.58 the entire Xantha-f gene is deleted (Olsson et al., 2004Go). In xantha-g.44, a GTT codon has been changed to CTT (V390 to L; Axelsson et al., 2006Go). Only a trace amount of XanG is found in xantha-g.44, which is a slightly leaky mutant with 7% of the chlorophyll found in the wild type (Henningsen et al., 1993Go). The mutation in xantha-h.57 results in no XanH protein (Lake et al., 2004Go). Interestingly, the XanG protein is absent as well in this mutant. The aerobic cyclase catalyzes the formation of protochlorophyllide from Mg-protoporphyrin IX monomethyl ester. Xantha-l encodes one of the subunits of this enzyme complex. In the nonleaky mutant xantha-l.81, a GGG codon has been changed to GCG (G155 to E; Rzeznicka et al., 2005Go). The resulting XanL protein is inactive, but stably maintained in the plastid.


Postgermination Development

To find the abscisic acid concentration effectively inhibiting barley postgermination development, 20 wild-type seeds each were imbibed in (±)-abscisic acid (Sigma) solutions for 24 h. The seeds were transferred to paper soaked with the respective solution in plastic petri dishes and placed in the dark. The root and shoot lengths were recorded over a period of 3 d.

To investigate the effect of abscisic acid on different Xantha mutants, seeds of xantha-f.10, -f.58, -g.44, -h.57, and -l.81 were imbibed in water or 10 µM (±)-abscisic acid overnight and placed on paper soaked with the respective solution in petri dishes. The assays were kept in the dark. The individual shoot and root lengths were measured daily starting on the first day after sowing (day 1). The papers were wetted with the respective solution on days 1 and 4. After 4 d, the seedlings were transferred to ambient light to allow for phenotypic differentiation between wild-type and mutant seedlings.


Stomatal Closure

The influence of exogenous abscisic acid on the opening status of the stomata of xantha-f.10 plants (grown in vermiculite in ambient light for 8 d) was determined by two methods.

Primary leaves were floated lower-face down on 1 mM MES-NaOH (pH 6.0) containing 20 mM KCl in ambient light. To test the effect of abscisic acid on the stomatal closure, 1 µM, 5 µM, and 10 µM (±)-abscisic acid (Sigma) were added to the solution. After 2 h the opening status of more than 110 stomata on the lower face of the leaves was determined using a light microscope (400x magnification).

Primary leaves were placed in water and 1 µM, 2 µM, 5 µM, and 10 µM (±)-abscisic acid in ambient light in a circling air environment of 21.5°C. After 2 h, pictures of the leaves were taken with an infrared camera ThermaCAM T360 (FLIR).


Construction of a Xantha-f Expression Plasmid

PCR primers were designed according to the barley Xantha-f sequence deposited in the National Center for Biotechnology Information database under accession number AY039003 (Olsson et al., 2004Go). The primers were designed to omit the chloroplast transit peptide, which was determined to be 43 amino acid residues according to ChloroP 1.1 (Emanuelsson et al., 1999Go). A cleavage site for NdeI was inserted before the first codon following the transit peptide, and one for BamHI after the codon of the last amino acid. Total RNA treated with DNase was used as template. SuperscriptIII/Platinum Taq reverse transcriptase (Invitrogen) was used according to the manufacturer's instructions. Three primer pairs were employed to produce three overlapping fragments of Xantha-f: primer 5'-TAC TAC ATA TGT GCG CCG TGG CCG GGA ACG GGC-3' (bases 1,012–1,033; numbering according to the full-length Xantha-f sequence; NdeI site and upstream bases added shown in bold) and primer 5'-CTG GAA GAC CCT CGA C-3' (bases 3,299–3,314) generated the first fragment comprising bases 130 to 1,636 of the intron-free sequence. The primers 5'-AAC CTC CAG AAC TTC CTC AA-3' (bases 1,624–1,643) and 5'-CAG ACC ACA TGG CAG TAG-3' (bases 4,097–4,114) were used to produce the second fragment (bases 742–2,436 of the intron-free sequence). The third fragment was amplified using the primers 5'-AGC TCC CAG CCA ATG AGC-3' (bases 4,026–4,053) and 5'-TAC GGA TCC TCA CCG GTC AAT TCC TTC GAT CTT GTC-3' (bases 5,902–5,928; BamHI site and downstream bases added shown in bold type) and consisted of bases 2,348 to 4,146 of the intron-free sequence. The three fragments were initially ligated into the pGEM-T easy vector (Promega). They were then joined into the pET15b vector (Novagen) using intrinsic restriction sites of PsyI (base 4,049 of the full-length sequence, base 2,371 of the intron-free sequence) and SalI (base 1,987 of the full-length sequence, base 1,105 of the intron-free sequence) and the added restriction sites for NdeI and BamHI. The pET15bXanF construct was transformed into the bacterial expression strain Escherichia coli Rosetta (DE3) pLysS (Novagen).


Magnesium Chelatase Activity Assays

Barley was sown in vermiculite and grown in darkness for 8 d at 20°C. The plants were illuminated for 7 h, harvested, and ground in grinding solution (0.4 M D-mannitol, 20 mM Tricine-NaOH, pH 9.0, 1 mM DTT) using a modified kitchen blender (Kannangara et al., 1977Go). Grinding and all further procedures were carried out at 4°C. The suspension was filtered through two layers of nylon tissue (31 µm mesh) and centrifuged (5,000g, 5 min, 4°C) to separate the chloroplasts and cell debris from the soluble cytosolic components. The resulting pellet was resuspended in 7 mL grinding solution and overlaid on 5 mL Percoll solution (40% Percoll, 0.4 M D-mannitol, 20 mM Tricine-NaOH, pH 9.0, 1 mM DTT). Centrifugation (5,525g, 22 min, 4°C) pelleted the chloroplasts. These were washed in 1 mL grinding solution, pelleted again (13,000g, 3 min, 4°C), and resuspended in lysis solution (20 mM Tricine-NaOH, pH 9.0, 1 mM DTT, 1 mM phenylmethanesulfonyl fluoride). After 10 min of lysis on ice, the mixture was centrifuged (3,000g, 3 min, 4°C) to pellet the starch and the lysed plastids were aliquoted after addition of glycerol to a final concentration of 11%. The lysed plastids were stored at –80°C until use. A typical protein concentration of the lysed plastids was 16 µg/µL.

The activity of XanF was assayed as the insertion of Mg2+ into deuteroporphyrin IX. Typically 37 µL lysed plastids were used in a reaction mixture of in total 45 µL. A smaller volume of wild-type lysed plastids was sufficient and made up to volume with lysis solution. The assay also contained 16.8 mM MgCl2, 4.2 µM deuteroporphyrin IX, 3.4 mM adenosine triphosphate, 16.8 mM phosphocreatine, and 2.5 units phosphocreatine kinase. Recombinant XanF and/or abscisic acid were added in 3 µL storage solution (50 mM Tricine-NaOH, pH 8.0, 250 mM NaCl, 50 mM MgCl2).

The assay was mixed on ice and incubated in darkness at 37°C for 120 min. To stop the reaction and to precipitate proteins, 1 mL alkali acetone solution (acetone:water:25% ammonia, 80:20:1, v/v/v) was added. After centrifugation (13,000g, 2 min, 20°C), the supernatant was analyzed in a Fluoro-Max-2 fluorometer (Jobin-Yvon SPEX).

Mg-deuteroporphyrin IX emits light at 577 nm upon excitation at 408 nm. The magnesium chelatase activity was quantified by the fluorescence of the formed product.


Binding Studies

The binding studies employing 3H-labeled (+)-abscisic acid followed modified published protocols.

Method A: As described by Razem et al. (2004)Go, binding of abscisic acid was determined by adding 1 µg of affinity-purified XanF to 10 pmol (+)-[3H]abscisic acid in a total volume of 100 µL adjusted by a solution composed of 250 mM Suc, 5 mM MgCl2, 1 mM CaCl2, and 25 mM Tris-HCl (pH 7.3). The mixture was gently shaken at 4°C for 1 h. After that, 100 µL of a 1.2% charcoal (Dextran T70-Coated Charcoal; Sigma) solution were added and the charcoal was removed by centrifugation. The radioactivity in 100 µL supernatant was determined.

Method B: As described by Shen et al. (2006)Go, binding was determined by adding 2 µg of affinity-purified XanF to 6 pmol (+)-[3H]abscisic acid in a total volume of 200 µL adjusted using a solution composed of 250 mM mannitol, 2 mM MgCl2, 1 mM CaCl2, and 50 mM Tris-HCl (pH 7.0). The mixture was incubated at 25°C for 30 min before addition of 100 µL 0.5% charcoal solution. After removing the charcoal by centrifugation the radioactivity in 150 µL supernatant was determined.

Method C: Due to the high salt requirements in the storage solution for XanF, a solution containing 250 mM NaCl, 5 mM MgCl2, 1 mM CaCl2, and 25 mM Tris-HCl (pH 7.3) was used in a third set of binding assays. Other than that, the procedure of method A was followed.

An assay with heat-denatured XanF and a protein-free assay were performed as controls. The blank sample to measure background radiation was scintillation solution. The specific activity of (+)-[3H]abscisic acid was determined as 155 min–1 pmol–1 (SD 25 min–1 pmol–1) in a charcoal-free sample.


   ACKNOWLEDGMENTS
 
We are grateful to Dr. Robert Hill and his group for providing us with tritiated abscisic acid and technical help in the abscisic acid binding assays. We also thank Dr. Simon Gough for critically reading the manuscript.

Received January 7, 2009; accepted January 22, 2009; published January 28, 2009.


   FOOTNOTES
 
1 This work was supported by the Danish Natural Science Research Council. Back

The author responsible for the distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Mats Hansson (mats{at}crc.dk).

[OA] Open access articles can be viewed online without a subscription. Back

www.plantphysiol.org/cgi/doi/10.1104/pp.109.135277

* Corresponding author; e-mail mats{at}crc.dk.


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