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First published online January 16, 2009; 10.1104/pp.108.133777 Plant Physiology 149:1261-1276 (2009) © 2009 American Society of Plant Biologists OPEN ACCESS ARTICLE
Chloroplast NADPH-Thioredoxin Reductase Interacts with Photoperiodic Development in Arabidopsis1,[W],[OA]Department of Biology, University of Turku, FI–20014 Turku, Finland (A.L., S.K., M. Keränen, E.R.); Agrifood Research Finland, FI–21500 Piikkiö, Finland (E.-M.L.); Faculty of Biosciences, Department of Biological and Environmental Sciences, Genetics, University of Helsinki, FI–00014 Helsinki, Finland (G.B.); and Faculty of Biosciences, University of Joensuu, FI–80101 Joensuu, Finland (N.S., M. Keinänen)
Chloroplast NADPH-thioredoxin reductase (NTRC) belongs to the thioredoxin systems that control crucial metabolic and regulatory pathways in plants. Here, by characterization of T-DNA insertion lines of NTRC gene, we uncover a novel connection between chloroplast thiol redox regulation and the control of photoperiodic growth in Arabidopsis (Arabidopsis thaliana). Transcript and metabolite profiling revealed severe developmental and metabolic defects in ntrc plants grown under a short 8-h light period. Besides reduced chlorophyll and anthocyanin contents, ntrc plants showed alterations in the levels of amino acids and auxin. Furthermore, a low carbon assimilation rate of ntrc leaves was associated with enhanced transpiration and photorespiration. All of these characteristics of ntrc were less severe when plants were grown under a long 16-h photoperiod. Transcript profiling revealed that the mutant phenotypes of ntrc were accompanied by differential expression of genes involved in stomatal development, chlorophyll biosynthesis, chloroplast biogenesis, and circadian clock-linked light perception systems in ntrc plants. We propose that NTRC regulates several key processes, including chlorophyll biosynthesis and the shikimate pathway, in chloroplasts. In the absence of NTRC, imbalanced metabolic activities presumably modulate the chloroplast retrograde signals, leading to altered expression of nuclear genes and, ultimately, to the formation of the pleiotrophic phenotypes in ntrc mutant plants.
Thiol redox regulation is a universal mechanism to control biochemical processes in living cells. Among thiol redox regulators, the thioredoxin superfamily consists of regulatory proteins that mediate dithiol-disulfide exchange of Cys residues, thereby modulating the activity of their target proteins. Members of the thioredoxin superfamily include thioredoxins, glutaredoxins, protein disulfide isomerases, as well as a group of thioredoxin-like proteins with mostly unknown function (Buchanan and Balmer, 2005  ; Gelhaye et al., 2005; Holmgren et al., 2005; Meyer et al., 2005, 2006). Members of the thioredoxin superfamily are characterized by the presence of a conserved redox-active site, where two Cys residues are separated from each other by two amino acids (Lemaire, 2004; Meyer et al., 2005). Thioredoxins with the redox-active site WC(G/P)PC constitute the best-characterized subclass of the thioredoxin superfamily and are found in all cellular compartments (Meyer et al., 2005). Numerous studies have revealed crucial functions for thioredoxins in the regulation of developmental and acclimation processes in plants (for review, see Buchanan and Balmer, 2005), and the vital roles for thioredoxin superfamily members in oxidative stress responses have also become evident (Hirt et al., 2002; Perez-Ruiz et al., 2006; Vieira Dos Santos and Rey, 2006).
Oxidized thioredoxins become rereduced via the action of thioredoxin reductases that, together with the target thioredoxins, constitute the cellular thioredoxin systems. Plants differ from other organisms in having a wide spectrum of possible thiol redox mediators. Recent analyses of Arabidopsis (Arabidopsis thaliana) gene sequence databases revealed more than 100 genes encoding members of the thioredoxin superfamily (Buchanan and Balmer, 2005
The light-activated ferredoxin/thioredoxin system has been linked to the regulation of primary photosynthetic processes during diurnal dark/light transitions of plants (Buchanan, 1991
In chloroplasts, NADPH can be produced via photosynthetic electron transfer reactions in light and via NADP dehydrogenase activity in darkness. These different pathways provide alternative interactions for NADPH-dependent thioredoxin systems and metabolic pathways in chloroplasts. Thus, it is intriguing that Arabidopsis ntrc knockout mutants showed distinctly reduced growth, and a shift of plants to continuous darkness further pronounced the mutant phenotype (Perez-Ruiz et al., 2006 In this paper, we report that NTRC has a critical role in the regulation of photoperiod-dependent metabolic and developmental processes in Arabidopsis. Transcript and metabolite profiling of plants grown under short-day (8 h of light/16 h of dark) and long-day (16 h of light/8 h of dark) photoperiods revealed that ntrc plants display severe photoperiod- and age-dependent developmental disorders. The absence of NTRC induced alterations in chloroplast biogenesis, which in turn interfered with photoperiodic development in Arabidopsis. Thus, apart from being a source of energy, functional chloroplasts also contribute to the regulation of plant development in response to changing environmental cues.
Identification of the ntrc Knockout Mutants and Accumulation of NTRC in Different Plant Organs Homozygous ntrc plants were identified from the SALK institute's T-DNA insertion mutant collection (SALK_096776 and SALK_114293). To confirm the absence of the NTRC enzyme in ntrc plants, a polyclonal antibody was raised against amino acids 475 to 488 of Arabidopsis NTRC. In total leaf extracts isolated from wild-type plants, the antibody specifically recognized NTRC, which was detected as a polypeptide with an apparent molecular mass of 60 kD on SDS-PAGE (Fig. 1B ). NTRC was also present in the cotyledons and in the stem of the inflorescence but not in the roots of wild-type plants (Fig. 1B). Clearly, ntrc knockout plants showed no accumulation of NTRC (Fig. 1B).
The ntrc Mutants Show Photoperiod-Dependent Phenotypes Growth of plants under various light rhythms with daily 8-h, 16-h, or 24-h illumination periods revealed distinct photoperiod-dependent alterations in the growth rate, pigmentation, and flowering time of ntrc plants as compared with wild-type plants (Fig. 2 ; Table I ; Supplemental Fig. S1; Supplemental Table S1). The most profound mutant phenotype was observed when ntrc plants were grown under a short 8-h photoperiod. Cotyledons of ntrc plants, however, were visually indistinguishable from those of the wild-type plants, and the clear mutant phenotype became evident upon the emergence of the first true leaves. During the first month of growth, ntrc plants formed small rosettes with pale green leaves (Fig. 2). In 4-week-old ntrc rosette leaves, the total concentration of chlorophyll per leaf area was 50% lower than in the wild-type plants (Table I). Upon aging, however, the pale green ntrc leaves started to green (Fig. 2; Table I) and the ntrc rosette gained the size typically observed for mature wild-type plants (Fig. 2). Intriguingly, the greening of ntrc leaves occurred gradually via a transient phase of the reticulate phenotype, with dark green cells surrounding the vascular tissue (Fig. 2). Furthermore, the life cycle of ntrc plants was significantly extended with delayed flowering time (Table II ).
Under a long 16-h photoperiod or under continuous light, the ntrc mutant phenotype was less distinct and the biomass production by the ntrc line was partially restored (Fig. 2; Table I). Both the ntrc and wild-type plants responded to long photoperiods by increasing the total chlorophyll concentration per leaf area and the chlorophyll a/b ratio (Table I). Moreover, the transition to flowering took place approximately at the same time in ntrc and wild-type plants (Table II). Imaging of chlorophyll autofluorescence by confocal microscopy revealed that the retarded growth and diminished chlorophyll content of ntrc plants were accompanied by structural changes in the mesophyll tissue. In plants grown under the short photoperiod, the mesophyll cells of the diminutive ntrc leaves were significantly smaller and contained fewer chloroplasts than wild-type leaves (Fig. 2). Notably, the greening of ntrc leaves during aging was accompanied by the accumulation of chloroplasts in the mesophyll cells, while the small cell size was still retained despite the expansion of the leaves (Fig. 2). In plants grown under the long photoperiod or under continuous light, both the mesophyll cell size and the number of chloroplasts per cell were partially restored (Fig. 2). The accumulation of anthocyanins was dramatically reduced in ntrc leaves under both short-day and long-day conditions (Table I; Supplemental Table S1). When the plants were grown in the short photoperiod under low temperature of 10°C, the difference in pigmentation was even visually observable (Fig. 2). Wild-type plants responded to low temperature by enhancing the accumulation of anthocyanins, whereas in ntrc leaves, only modest anthocyanin accumulation became evident (Fig. 2). Morphologically, the cold-acclimated 3-month-old ntrc plants were quite similar to wild-type plants (Fig. 2).
To gain insights into possible photoperiod-dependent adjustments in photosynthetic light reactions, thylakoid membrane protein complexes of ntrc and wild-type plants were studied by blue-native gel electrophoresis (Fig. 3 ). When equal amounts of chlorophyll were loaded in the wells, the pattern of thylakoid protein complexes in ntrc was comparable to that of the wild-type plants under both short-day and long-day conditions (Fig. 3A). In fact, the amount of trimeric PSII light-harvesting antenna (LHCII) complexes was even slightly higher in ntrc than in wild-type plants (Fig. 3A). Consistently, the ntrc plants showed a slightly increased level of the LHCII protein Lhcb2 (Fig. 3B) and a lower chlorophyll a/b ratio than the wild-type plants (Table I). No significant adjustments were observed in the levels of PsaD and D1 proteins, representatives of PSI and PSII core complexes, respectively.
In contrast to the basic structures of the light reactions that were not affected, ntrc plants showed a decrease in the rate of net CO2 assimilation under low and ambient CO2 concentrations. CO2 assimilation varied in individual ntrc rosettes (Supplemental Fig. S2). However, the CO2 compensation point was distinctly higher in all analyzed short-day ntrc plants than in the wild-type plants. The ntrc plants also suffered from enhanced photoinhibition of PSII, measured as a decrease in Fv/Fm (see "Materials and Methods"), when grown under the short photoperiod (Table III ). Under the long photoperiod, the differences in the net CO2 assimilation, in the CO2 compensation point, and in PSII photoinhibition between ntrc and wild-type plants were less distinct (Table III; Supplemental Fig. S2B). Modeling of the response of net CO2 assimilation to increasing atmospheric CO2 concentrations revealed only slight differences in photosynthetic parameters between ntrc and wild-type plants, regardless of the length of the photoperiod (Table III). Indeed, the calculations indicated that maximal electron transport rate (Jmax) and maximal carboxylation rate of Rubisco (Vcmax) were not significantly impaired in ntrc leaves.
Next, we studied whether the absence of NTRC had influenced the activities of two thioredoxin-regulated enzymes of chloroplasts, the chloroplast fructose-1,6-bisphosphatase (FBPase) and the malate dehydrogenase (MDH; Buchanan and Balmer, 2005 ). Chloroplast FBPase is a Calvin cycle enzyme, while MDH controls the export of reducing equivalents through the chloroplast envelope via the malate shuttle. The ntrc and wild-type plants showed no differences in the estimated activation state of FBPase (data not shown), and also the activation state of MDH was only slightly lower in ntrc as compared with wild-type plants (Table III). Thus, both the unchanged FBPase activity and the unaltered values of Jmax and Vcmax (Table III) indicated that the low net CO2 assimilation rate of ntrc leaves under the short photoperiod was not due to an imbalance in Calvin cycle reactions. Instead, the low net assimilation of CO2 in short-day ntrc could be explained by the low number of chloroplasts in leaves (Fig. 2) and, importantly, by enhanced respiration (Rd), which was distinctly higher in ntrc than in wild-type plants (Table III). Finally, we explored whether the retarded growth of ntrc plants under short-day conditions was associated with imbalances in diurnal starch metabolism. The accumulation or degradation of starch in ntrc leaves did not differ to a large extent from the cycling of starch in wild-type leaves (Supplemental Fig. S3). Indeed, iodine staining of intact leaves indicated only slightly lower accumulation of starch in ntrc plants grown under the short photoperiod, probably due to the lower number of chloroplasts in mesophyll cells (Fig. 2; Supplemental Fig. S3). Moreover, both the ntrc and wild-type plants were capable of degrading starch during the following dark period. Gas exchange and transpiration through stomata are also crucial determinants of photosynthetic performance. Notably, wild-type plants showed distinct photoperiod-dependent adjustments in stomatal density, with a clear increase in stomatal index upon growth under the long photoperiod (Table II). Moreover, the stomatal density was higher in ntrc than in wild-type plants, especially in short-day conditions (Table II). Accordingly, ntrc plants lost significantly more water from excised rosettes than the wild-type plants during the light period (Fig. 4 ). At the end of the diurnal dark period, before the onset of illumination, water loss from excised rosettes of both ntrc and wild-type plants was significantly reduced, indicating that both wild-type and ntrc plants were capable of closing the stomata during the dark period (data not shown).
Transcript Profiling of ntrc Plants under Different Physiological and Developmental Stages To identify the metabolic processes that contribute to the age- and photoperiod-dependent ntrc phenotype, we carried out comparative transcript profiling of ntrc and wild-type plants. Ten-day-old seedlings and expanded rosettes of plants grown under short-day or long-day conditions were used for microarray analysis. A total of 621 genes with higher (>1.8-fold and P < 0.05) or lower (<0.55-fold and P < 0.05) transcript levels in ntrc mutants relative to wild-type plants were chosen for further analysis. This revealed ntrc-specific, age-specific, and photoperiod-dependent alterations in gene expression of ntrc plants (Fig. 5 ; Table IV ; Supplemental Fig. S4; Supplemental Table S2). Under long-day conditions, the transcriptomic adjustments of ntrc plants were much more pronounced than under short-day conditions. However, in short-day ntrc rosette leaves, 68% of the differentially expressed genes were up-regulated, whereas in long-day ntrc rosette leaves, the proportion of up-regulated genes was only 4%. Furthermore, the transcript profile of short-day ntrc rosette leaves comprised a unique combination of induced genes, which differed from the transcript profiles of both the young short-day seedlings and all of the long-day-grown ntrc plants (Fig. 5; Supplemental Fig. S4).
Gene Expression Changes Associated with the Morphogenic Phenotypes of ntrc Plants
Two genes belonging to families that regulate photoperiodic growth in plants, CRYPTOCHROME2 (CRY2) and the FAR-RED-IMPAIRED RESPONSE REGULATOR (FRS3), were down-regulated in ntrc plants (Table IV). CRY2 is a blue-light receptor that mediates the inhibition of hypocotyl growth and the timing of flowering in Arabidopsis (for review, see Li and Yang, 2007
The ntrc plants also showed lowered accumulation of transcripts for genes assigned to the development of structural components in leaves (Table IV). Expression of DYNAMIN-RELATED MEMBRANE REMODELING-LIKE (FZL), STOMATAL DENSITY AND DISTRIBUTION1-1 (SDD1-1), and EPIDERMAL PATTERING FACTOR1 (EPF1) was repressed in ntrc plants under all conditions tested. Of these genes, FZL encodes a protein that controls the organization of grana and stroma thylakoids in chloroplasts (Gao et al., 2006 ). SDD1-1 and EPF1 encode putative plasma membrane proteins that control the early signaling steps in stomatal development (Casson and Gray, 2008). Accordingly, sdd1-1 mutants showed increased stomatal density, which was also the case in ntrc plants (Table II; Von Groll et al., 2002).
Notably, despite the stunted phenotype of ntrc plants, the mutant phenotype was not accompanied by any significant induction of marker genes related to hydrogen peroxide-dependent (Vanderauwera et al., 2005
Short-day ntrc rosette leaves with the most severe mutant phenotype also showed a distinct transcript profile, reflecting the unique metabolic state of the ntrc leaves under these conditions (Fig. 2; Supplemental Fig. S4). Related to the pale green leaves and the low net CO2 assimilation of short-day-grown ntrc leaves, two distinct metabolic processes came up from the transcript profiling: chlorophyll biosynthesis and photorespiration (Table IV). Two genes encoding enzymes of the chlorophyll biosynthesis pathway, glutamyl-tRNA reductase (HEMA1) and the H subunit of the Mg-chelatase (GUN5), were among the most up-regulated genes in short-day-grown ntrc leaves (Table IV). Slightly increased accumulation of the transcript for GUN5 was also observed in long-day-grown ntrc rosette leaves. The increased accumulation of GUN5 transcripts in ntrc leaves was verified by northern blotting (Supplemental Fig. S5). Furthermore, transcripts for CHLOROPLAST BIOGENESIS6 (CLB6) accumulated in short-day-grown ntrc rosette leaves. CLB6 is an enzyme of plastid isoprene biosynthesis, which provides phytol chains for chlorophyll molecules.
Consistent with the high CO2 compensation point of ntrc leaves (Supplemental Fig. S2), transcript levels of six photorespiratory genes were induced in short-day-grown ntrc rosette leaves: peroxisomal CATALASE2, ALANINE-2-OXOGLUTARATE AMINOTRANSFERASE, and NAD+-HYDROXYPYRUVATE REDUCTASE, mitochondrial GLYCINE DECARBOXYLASE P SUBUNIT and SERINE HYDROXYMETHYL TRANSFERASE, and plastidial FERREDOXIN-DEPENDENT GLUTAMATE SYNTHASE (Table IV). Thus, enhanced photorespiration may account for draining of excess light energy in the slowly growing ntrc leaves, thereby hindering more severe damage to photosynthetic structures (Kozaki and Takeba, 1996
It is worth noting that CATALASE2 and GLUTATHIONE PEROXIDASE7 were the only hydrogen peroxide metabolism-linked genes that were differentially expressed in short-day-grown ntrc plants (Table IV). However, eight genes related to various stress responses were markedly induced in short-day ntrc leaves. Four of them belong to heat shock proteins (HSP70, HSP81-1, HSP17.6-CII, and HSP17.6A) that were also induced by high-light treatment of catalase-deficient plants (Vanderauwera et al., 2005
Chloroplast-derived metabolites serve as precursors for biosynthesis of the growth hormone auxin (indole-3-acetic acid [IAA]) and the growth inhibitor abscisic acid (ABA). The auxin content is highest in the first true leaves of Arabidopsis seedlings after 8 to 10 d of germination (Ljung et al., 2001
Photoperiod-Dependent Accumulation of Amino Acids in Young ntrc and Wild-Type Plants
The clear deficiency in the accumulation of auxin and anthocyanins in short-day ntrc plants (Table I; Fig. 7) prompted us to address possible photoperiod-dependent alterations in the accumulation of amino acids in the wild-type and ntrc plants. Aromatic amino acids, which serve as precursors for the biosynthesis of auxin and flavonoids, are synthesized in chloroplasts via the shikimic acid pathway (Herrmann, 1995
Seedling Development on a Medium Supplemented with External Auxin and Amino Acids As demonstrated above, the absence of NTRC resulted in photoperiod-dependent imbalances in both amino acid and auxin metabolism in the developing ntrc seedlings. Thus, we tested whether the small size and the pale green phenotype of ntrc observed under short-day conditions could be rescued by the addition of external auxin or amino acids on the growth medium. When grown on Murashige and Skoog agar plates, the ntrc phenotype was somewhat less distinct than in ntrc grown on soil (Supplemental Fig. S6). Addition of external auxin did not significantly enhance the growth of ntrc seedlings (Supplemental Fig. S6A). An enlargement of mesophyll cell size and an increase in the number of chloroplasts per cell, however, were observed when the growth medium was supplemented with aromatic amino acids (Supplemental Fig. S6B). The most distinct recovery of cell size and a particularly enhanced accumulation of chloroplasts were observed when ntrc seedlings were supplemented with Ile (Supplemental Fig. S6B). Notably, the level of this chloroplast-derived amino acid was reduced in ntrc seedlings (Fig. 8). A similar but slightly less pronounced effect was observed upon the addition of Phe (Supplemental Fig. S6B). The enlargement of ntrc cell size was also obtained by the addition of Trp in the growth medium (Supplemental Fig. S6B), but its impact on the recovery of cell size was not systematic on every plate. Moreover, it is worth emphasizing that none of the externally added amino acids fully restored the greening process of ntrc leaves. The addition of Ala had no effect on the growth and development of ntrc plants (Supplemental Fig. S6B). The external supply of auxin or amino acids did not significantly modulate the growth and development of wild-type seedlings.
Plants possess two main types of thioredoxin reductases, the universal NTRs and the plastidial FTR, which is unique for photosynthetic organisms (Hirt et al., 2002 ; Dai et al., 2004). The family of NTRs includes NTRA and NTRB, dually localized in cytosol and mitochondria, and chloroplastic NTRC (Hirt et al., 2002). Functional analysis of the Arabidopsis single mutants ntra and ntrb revealed no phenotypic deficiencies under standard growth conditions (Reichheld et al., 2007). Surprisingly, the ntra ntrb double knockout mutant also was viable, even though the absence of the two important regulatory enzymes, NTRA and NTRB, led to a notable reduction in growth rate (Reichheld et al., 2007). In this paper, we demonstrate that single mutants deficient in NTRC display severe photoperiod- and age-dependent developmental disorders, indicating that the two types of plastidial thioredoxin reductases, FTR and NTRC, are functionally nonredundant. Moreover, the pleiotropic phenotype of ntrc plants suggests that NTRC contributes to multiple important metabolic processes in chloroplasts, such as the biosynthesis of chlorophyll and amino acids. NTRC is present solely in green tissues (Fig. 1; Perez-Ruiz et al., 2006), emphasizing its impact on light-dependent functions of plastids. Loss of NTRC, however, does not interfere with the primary photosynthetic reactions (Fig. 3; Table III), implying that the FTR-dependent ferredoxin/thioredoxin system acts as the primary regulator of photosynthetic electron transfer reactions and the Calvin cycle in chloroplasts (Buchanan and Balmer, 2005).
The ntrc lines showed substantial reduction of chlorophyll content and of the number of chloroplasts per cell, particularly under short-day conditions. In line with the pale green phenotype, microarray analysis revealed a specific up-regulation of genes related to chlorophyll biosynthesis in ntrc leaves: the genes encoding glutamyl-tRNA reductase (HEMA1), the H subunit of Mg-chelatase (GUN5), an enzyme of plastid isoprene biosynthesis (CBG6), and chloroplast clip protease (CLPC1; Table IV). Glutamyl-tRNA reductase and Mg-chelatase catalyze the important regulatory steps of tetrapyrrole biosynthesis (Tanaka and Tanaka, 2007
The activity of Mg-chelatase is regulated via thioredoxin-mediated disulfide/dithiol exchange in the CHL-I subunit (Ikegami et al., 2007
Reduced activity of chlorophyll biosynthesis enzymes may promote signals that contribute to the attenuation of chloroplast biogenesis in ntrc leaves (Nott et al., 2006
Interestingly, a null mutant for chloroplast ATP/ADP transporters (NTTs) also resembles ntrc with respect to chlorophyll biosynthesis and photoperiod-dependent reduction in growth (Reinhold et al., 2007
Regulation of developmental processes by light intensity is well documented in plants. We found that short-day-grown wild-type Arabidopsis plants possess thin leaves, low chlorophyll a/b ratio, and low stomatal density typical of shade-grown plants, whereas the opposite was observed for long-day-grown plants with adaptations typical of high-light-acclimated leaves (Tables I and II). Thus, the daily light period and the light intensity seem to be comparable in terms of developmental regulation.
Both the photoperiodic and photomorphogenic development are regulated by phytochromes and cryptochromes in Arabidopsis (Li and Yang, 2007
Together, the defects in light receptor-mediated signaling, altered expression of chlorophyll biosynthesis genes, and lower number of chloroplasts in short-day ntrc plants suggest that disturbances in chloroplast metabolism interfere with the circadian clock-mediated regulation of plant growth. This interpretation is consistent with a recent report by Hassidim et al. (2007)
Light intensity and CO2 partial pressure are well-known environmental factors that control the number of stomata in plants. Shading and elevated CO2 partial pressure both decrease the stomatal density in leaves (Lake et al., 2001
The stomatal density is significantly higher in short-day ntrc leaves than in wild-type plants (Table II). This is accompanied by the repression of two genes encoding important negative regulators of stomatal development, SDD1 and EPF1, in ntrc plants (Table IV), which speaks for disturbed stomatal development in the absence of NTRC. Stomatal development starts with an asymmetric division of the epidermal cell that creates a meristemoid cell (reviewed by Bergmann and Sack, 2007
Interestingly, in the regulatory model of stomatal development, the membrane proteins SDD1 and EPF1 control the first steps of the signaling cascade immediately after an unknown environment-dependent initiatory factor (Casson and Gray, 2008
The plastid-localized shikimic acid pathway serves as a biosynthetic route for aromatic amino acids (Herrmann, 1995
Attempts to identify thiol-redox-regulated enzymes have revealed putative targets for the thioredoxin systems both in the shikimate pathway and in the biosynthesis of aromatic amino acids, including 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase and Trp synthase β (Entus et al., 2002
Materials and Growth Conditions
Homozygous T-DNA insertion mutants deficient in NTRC (At2g41680; SALK_096776 and SALK_114293) were identified from the SALK Institute's collection by PCR analysis of genomic DNA according to the institute's protocols (http://signal.salk.edu/cgi-bin/tdnaexpress; Alonso et al., 2003
Seeds were sterilized with 50% (v/v) ethanol + 0.5% (v/v) Triton X-100 for 2 min, subsequently rinsed with 95% (v/v) ethanol, and dried. Sterilized seeds were sown on half-strength Murashige and Skoog salts in 1% (w/v) agar. After incubation at 4°C for 2 d, the plates were transferred to growth chambers under different spectral qualities of light: white, 100 µmol photons m–2 s–1; red, 30 µmol photons m–2 s–1; high-fluence-rate blue, 30 µmol photons m–2 s–1; and low-fluence-rate blue, 3 µmol photons m–2 s–1. For far-red light, germination was first induced by a 2-h white light treatment under 100 µmol photons m–2 s–1, after which the plates were kept in darkness at 20°C for 70 h and finally placed under far-red light. Hypocotyl length was measured after 7 d.
Sterilized seeds were sown on half-strength Murashige and Skoog salts in 1% agar containing 40 µM Trp, 40 µM Phe, 40 µM Ile, 40 µM Ala, or 1 µM IAA. After incubation at 4°C for 2 d, the plates were transferred to the growth chamber, where the seedlings were grown under short-day conditions for 2 weeks.
Foliar chlorophyll content was determined by punching two leaf discs, 3 mm in diameter, into 1 mL of dimethylformamide. The leaf discs were incubated overnight at 4°C in darkness, and the chlorophyll content was measured spectrophotometrically according to Inskeep and Bloom (1985)
Gas exchange of intact ntrc and wild-type plants was measured with the CIRAS-1 combined infrared gas analysis system (PP Systems) equipped with an Arabidopsis pot chamber (PP Systems). The response of net photosynthesis to the reference CO2 was measured at 20°C under a photosynthetically active photon flux density of 500 µmol m–2 s–1, which was saturating for net photosynthesis. The parameters for maximal carboxylation rate of Rubisco (Vcmax; µmol CO2 m–2 s–1), maximal electron transport rate (Jmax; µmol m–2 s–1), and rate of mitochondrial respiration in light (Rd; µmol m–2 s–1) were obtained by modeling the response of net CO2 assimilation to increasing extracellular CO2 concentration according to Farquhar et al. (1980) The photoinhibition state of PSII in intact leaves was recorded as the ratio of variable to maximal fluorescence (Fv/Fm, where Fv is the difference between maximal fluorescence [Fm] and initial fluorescence [Fo]), measured with a Hansatech PEA fluorometer after a 30-min dark incubation.
Rosettes of ntrc and wild-type plants were immediately frozen in liquid nitrogen. Total root and leaf extracts were collected after homogenization of the tissue in ice-cold isolation buffer (330 mM Suc, 25 mM HEPES-KOH, pH 7.4, 10 mM MgCl2, and 10 mm NaF) and filtration through Miracloth under dim light. For isolation of thylakoids, the filtrate was centrifuged at 6,000g for 5 min at 4°C. The thylakoid pellet was gently resuspended in 25 mM HEPES-KOH, pH 7.4, 10 mM MgCl2, and 10 mM NaF, centrifuged at 6,000g, for 5 min at 4°C, and finally suspended in the isolation buffer and stored at –80°C. The chlorophyll content of isolated thylakoids was determined according to Porra et al. (1989)
Blue-native PAGE was performed according to Kügler et al. (1997)
Soluble and total extracts corresponding to 5 to 15 µg of protein were solubilized and separated by SDS-PAGE (Laemmli, 1970
For enzymatic assays, 500 mg of leaves was ground to a fine powder in liquid nitrogen. Subsequently, initial and maximal activities of chloroplast NADP-MDH and FBPase were measured according to Scheibe and Stitt (1988)
Laser scanning confocal microscopy images were obtained with an inverted confocal laser scanning microscope (Zeiss LSM510 META; http://www.zeiss.com) with a 20x/0.50 water objective. Stomata were imaged by exciting autofluorescing compounds at 488 nm on the adaxial surface of 4-week-old leaves, followed by detection with a 420- to 480-nm passing emission filter. Stomatal density (number of stomata per square millimeter) and stomatal index (the ratio of the number of stomata to the total number of epidermal cells x 100) were calculated from the images. Chloroplasts and mesophyll cells were imaged by chlorophyll autofluorescence, which was excited at 488 nm with an argon diode laser, and detected with a 650- to 710-nm passing emission filter. Maximal projections of the sequential confocal images were created with the Zeiss LSM Image Browser version 3,5,0,376.
Plant hormones were analyzed using a modified vapor-phase extraction method described by Schmelz et al. (2003)
Approximately 100 mg of 10-d-old plant material was frozen in liquid nitrogen and ground with the Retsch Tissue Lyser (Qiagen). The plant metabolites were extracted twice with 500 µL of 50% (v/v) methanol by shaking vigorously for 30 min with the Tissue Lyser. The extract was centrifuged for 5 min at 33,000g (Jouan MR 23i; Thermo Electron Industries), and the supernatant was dried in vacuo for 2 h (SC250 Express SpeedVac Concentrator SDP121P; Thermo Electron Industries). The dried residue was redissolved in 100 µL of 10% methanol. Amino acids in the extracts were further extracted and derivatized with the Ez:faast liquid chromatography-mass spectrometry kit (Phenomenex) using the procedure described by Husek (1998)
Global changes in gene expression were explored with spotted Arabidopsis 24k oligonucleotide arrays (MWG Biotech; http://www.mwg-biotech.com; ArrayExpress database accession no. A-ATMX-2; http://www.ebi.ac.uk/arrayexpress). A total of 500 mg of 10-d-old seedlings or rosette leaves (28 d old, grown in short days, and 21 d old, grown in long days) of wild-type and ntrc mutant plants was collected 4 h after the onset of the light period, and total RNA was isolated with Trizol as described previously (Piippo et al., 2006 The arrays were scanned with an Agilent scanner (G2565BA; http://www.agilent.com), and spot intensities were quantified with the ScanArray Express Microarray Analysis System 2.0 (Perkin-Elmer Life Sciences; http://las.perkinelmer.com) using the adaptive circle method. Low-quality spots were flagged and not included in the analysis. The raw data were normalized using the Lowess method in GeneSpring GX 7.3 (Agilent; http://www.agilent.com). Normalized data from three biological replicates were used, and the genes with Student's t test P values below 0.05 were chosen for further analysis. The gene annotation used was derived from The Arabidopsis Information Resource (TAIR 7; http://www.arabidopsis.org).
The probe for GUN5 was amplified from cDNA by PCR with primers 5'-CTCACGGACTCCCATTTTGT-3' and 5'-AGGGACTGCAGCTTACCTCA-3'. Northern blotting and hybridization of the membranes with GUN5 and probe for 18S rRNA were performed according to Mulo et al. (2003) Array design and data from this article have been deposited at ArrayExpress under accession number E-MEXP-1697.
The following materials are available in the online version of this article.
We thank Jouko Sandholm, Colin Ruprecht, Briitta Ruokamo, and Kati Thiel for excellent technical assistance and Eva-Mari Aro and Paula Mulo for critical reading of the manuscript. The Cell Imaging Core of the Turku Center for Biotechnology of the University of Turku and Åbo Akademi University is acknowledged for providing laser scanning confocal microscopy. We are grateful to CSC–Scientific Computing Ltd. for providing the national license for GeneSpring. The Salk Institute Genomic Analysis Laboratory, funded by the National Science Foundation, is acknowledged for providing the sequence-indexed Arabidopsis T-DNA insertion mutants. Received December 5, 2008; accepted January 13, 2009; published January 16, 2009.
1 This work was supported by the Academy of Finland (project nos. 107039 and 204521) and the Finnish Graduate School in Plant Biology. 
2 These authors contributed equally to the article. The author responsible for 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: Eevi Rintamäki (evirin{at}utu.fi).
[W] The online version of this article contains Web-only data.
[OA] Open Access articles can be viewed online without a subscription. www.plantphysiol.org/cgi/doi/10.1104/pp.108.133777 * Corresponding author; e-mail evirin{at}utu.fi.
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-amino-n-caproic acid, 30% [w/v] Suc, and 50 mg mL–1 Serva Blue G) and run on a native gel with a 5% to 12% gradient of acrylamide on the separation gel. Electrophoresis was performed with the Hoefer Mighty Small system (Amersham Biosciences) at 3°C for 3.5 h by gradually increasing the voltage from 75 to 200 V. Photosynthetic thylakoid protein complexes were identified according to Aro et al. (2005)
