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Rapid Dark Repression of 5-Aminolevulinic Acid Synthesis in Green Barley Leaves

Rapid Dark Repression of 5-Aminolevulinic Acid Synthesis in Green Barley Leaves Abstract In photosynthetic organisms chlorophyll and heme biosynthesis is tightly regulated at various levels in response to environmental adaptation and plant development. The formation of 5-aminolevulinic acid (ALA) is the key regulatory step and provides adequate amounts of the common precursor molecule for the Mg and Fe branches of tetrapyrrole biosynthesis. Pathway control prevents accumulation of metabolic intermediates and avoids photo-oxidative damage. In angiosperms reduction of protochlorophyllide (Pchlide) to chlorophyllide is catalyzed by the light-dependent NADPH:Pchlide oxidoreductase (POR). Although a correlation between down-regulated ALA synthesis and accumulation of Pchlide in the dark was proposed a long time ago, the time-resolved mutual dependency has never been analyzed. Taking advantage of the high metabolic activity of young barley (Hordeum vulgare L.) seedlings, in planta ALA synthesis could be determined with high time-resolution. ALA formation declined immediately after transition from light to dark and correlated with an immediate accumulation of POR-bound Pchlide within the first 60 min in darkness. The flu homologous barley mutant tigrina d12 uncouples ALA synthesis from dark-suppression and continued to form ALA in darkness without a significant change in synthesis rate in this time interval. Similarly, inhibition of protoporphyrinogen IX oxidase by acifluorfen resulted in a delayed accumulation of Pchlide during the entire dark period and a weak repression of ALA synthesis in darkness. Moreover, it is demonstrated that dark repression of ALA formation relies rather on rapid post-translational regulation in response to accumulating Pchlide than on changes in nuclear gene expression. Introduction In higher plants the formation of 5-aminolevulinic acid (ALA) is the first committed step in tetrapyrrole biosynthesis leading to the end products chlorophyll, heme, siroheme and phytochromobiline (Beale 1999, Papenbrock and Grimm 2001, Tanaka and Tanaka 2007). The synthesis of tetrapyrroles is adjusted in response to changes in environmental conditions (e.g. light, temperature) and development. The regulation of cellular tetrapyrrole accumulation is inextricably linked to the formation of ALA, which is considered to be a rate-limiting step controlling influx into the entire pathway. ALA is formed in a three-step reaction including the ligation of glutamate to tRNAGlu catalyzed by glutamyl-tRNA synthetase, the reduction of glutamate to glutamate-1-semialdehyde by glutamyl-tRNA reductase (GluTR) and a final transamination step mediated by glutamate-1-semialdehyde aminotransferase (GSAT; Kannangara et al. 1988). Light-dependent induction of ALA synthesis during de- etiolation and growth under dark/light conditions is well studied (Ilag et al. 1994, McCormack and Terry 2002). Within the metabolic pathway of tetrapyrrole biosynthesis ALA production is controlled by several feedback loops regulating gene expression or post-translational modifications. Signals are proposed to originate from heme and the Mg branch of the pathway. It is presently suggested that GluTR is the main target of regulatory mechanisms modulating ALA formation. The expression of HEMA1 encoding GluTR is regulated by a wide range of stimuli (e.g. cytokinin, the circadian clock, plastid-derived signals and light), whereas GSA encoding GSAT responds only weakly (Cornah et al. 2003, Gough et al. 2003, Eckhardt et al. 2004, Tanaka and Tanaka 2007). In in vitro experiments the activity of barley GluTR was inhibited by heme (Pontoppidan and Kannangara 1994), which exerted the inhibiting action through the first 30 N- terminal amino acids of the enzyme (Vothknecht et al. 1998). In Chlamydomonas an additional factor was required to mediate inhibition of GluTR by heme (Srivastava et al. 2005). Additionally, enzyme activies of the Mg branch feedback- control ALA synthesis. Tobacco mutants affected in the synthesis of CHLI or CHLH subunits of Mg chelatase not only showed decreased Mg chelatase activity, but also reduced ALA synthesizing activity. This metabolic feedback control acts at the transcriptional level (Papenbrock et al. 2000a, Papenbrock et al. 2000b) and prevents accumulation of tetrapyrrole intermediates generating reactive oxygen species upon excitation by light (Vavilin and Vermaas 2002). ALA synthesis is drastically reduced in darkness. Previous observations revealed accumulation of the chlorophyll precursor protochlorophyllide (Pchlide) in etiolated seedlings of angiosperms, since Pchlide can be converted to chlorophyllide by the light-dependent NADPH:Pchlide oxidoreductase (POR) only (Griffiths et al. 1996). Several experiments with etiolated seedlings and green plants unveiled a strong inverse correlation between Pchlide accumulation and ALA synthesis in darkness (Fluhr et al. 1975, Gough 1978, Ford and Kasemir 1980, Huang and Castelfranco 1989, Stobart and Ameen-Bukhari 1984, Stobart and Ameen-Bukhari 1986). Thus, it was assumed that a high Pchlide level induces down-regulation of chlorophyll biosynthesis at the level of ALA synthesis (Beale 2006, Tanaka and Tanaka 2007). The Arabidopsis flu mutant with a deficient negative regulator for ALA synthesis fails to down-regulate ALA synthesis in darkness and accumulates massive amounts of Pchlide (Meskauskiene et al. 2001). It was shown that FLU directly interacts with the Arabidopsis GluTR encoded by HEMA1, but not with the HEMA2 product. Furthermore, the FLU-dependent inactivation of GluTR works independently of the above- mentioned GluTR inhibition by heme (Goslings et al. 2004). Both the Arabidopsis flu mutant and the homologous barley tigrina d12 mutant (Lee et al. 2003) show a necrotic phenotype during photoperiodic growth emphasizing the importance of rapid and efficient down-regulation of ALA synthesis in darkness. To date, very little is known about the nature of the rapid control resulting in repression of ALA formation. Conclusive evidence for the role of Pchlide in the dark repression of ALA synthesis is still missing. The present study shows in a time course analysis rapid changes in the ALA synthesis rate and Pchlide accumulation after light–dark transition in green barley leaves and provides evidence for an almost instantaneous repression of ALA synthesis in darkness in correlation with an accumulation of POR-bound Pchlide. Results ALA synthesis in green barley leaves is immediately repressed in darkness Incubation of green barley leaves with levulinic acid, a competitive inhibitor of ALA dehydratase, facilitated time-resolved determination of ALA accumulation in both light and darkness. Several pre-experiments were performed to optimize our analysis of the in vivo metabolic activities. Using increasing amounts of levulinic acid we found that application of 40 mM levulinic acid was sufficient for rapid, reproducible accumulation of ALA within the experimental time period which correlates with the ALA-synthesis capacity (Supplementary Fig. S1a). Despite the detectable amounts of accumulated ALA due to inhibition by levulinic acid we found that Pchlide accumulation after 2 h of dark incubation was almost similar in leaves incubated with and without levulinic acid. These findings are explained by incomplete inhibition of ALA dehydratase (Supplementary Fig. S1b) allowing simultaneous analysis of ALA and Pchlide accumulation in the barley leaves. Leaves exposed to light intensity of 100 μmol photons m−2 s−1 accumulated ALA at a constant rate of 260 pmol ALA mg FW−1 h−1 (Fig. 1a). After transfer to darkness ALA accumulation decreased instantaneously and continued at a very low rate (30 pmol ALA mg FW−1 h−1). When leaves were retransferred to light after 90 min dark incubation, enzyme activity was restored rapidly and ALA synthesis proceeded at the rate observed before dark incubation. Northern (Fig. 1b) and Western (Fig. 1c) blot analyses revealed no differences in expression of analyzed enzymatic steps of tetrapyrrole biosynthesis between light-exposed and dark-incubated samples with the exception of HEMA1 RNA and POR protein. The POR level was elevated in darkness and was diminished during long exposure to light following diurnal oscillation. In green tissue the anti-POR antibody mainly recognizes PORB (Holtorf and Apel 1996). Unfortunately, the GluTR antibody available in the laboratory did not recognize the homologous barley protein, so we cannot prove influence of reduced HEMA1 transcript level on GluTR amounts. However, a rapid increase in ALA synthesis after re-illumination reflects constant or elevated GluTR content. Fig. 1 View largeDownload slide (a) ALA accumulation in barley leaf discs incubated in a buffer containing 40 mM levulinic acid for indicated time at light intensities of 100 μmol photons m−2 s−1 and darkness. Linear accumulation of ALA was observed in light (open squares). When samples were transferred to darkness after 30 min preincubation in light further accumulation of ALA was prevented (filled squares). When dark-incubated samples were re-transferred to light ALA accumulation started again (open triangles). Data are given as means ± SD. The calculated ALA synthesis rates were 260 pmol ALA mg FW−1 h−1 in continuous light, 30 pmol ALA mg FW−1 h−1 in darkness and 225 pmol ALA mg FW−1 h−1 after re-illumination. (b) Northern blot analyses of RNA extracted from leaf samples after different light and dark (L, D) incubations for up to 120 min. Transcript levels of HEMA1, PORB and LHCB were determined. As additional control RNA was analyzed from green light-exposed intact barley leaves. (c) Western blot analyses of the protein levels of GSAT, POR and the FLU homologous barley TIGRINA D protein (FLU) in light- and dark-incubated leaf samples. Fig. 1 View largeDownload slide (a) ALA accumulation in barley leaf discs incubated in a buffer containing 40 mM levulinic acid for indicated time at light intensities of 100 μmol photons m−2 s−1 and darkness. Linear accumulation of ALA was observed in light (open squares). When samples were transferred to darkness after 30 min preincubation in light further accumulation of ALA was prevented (filled squares). When dark-incubated samples were re-transferred to light ALA accumulation started again (open triangles). Data are given as means ± SD. The calculated ALA synthesis rates were 260 pmol ALA mg FW−1 h−1 in continuous light, 30 pmol ALA mg FW−1 h−1 in darkness and 225 pmol ALA mg FW−1 h−1 after re-illumination. (b) Northern blot analyses of RNA extracted from leaf samples after different light and dark (L, D) incubations for up to 120 min. Transcript levels of HEMA1, PORB and LHCB were determined. As additional control RNA was analyzed from green light-exposed intact barley leaves. (c) Western blot analyses of the protein levels of GSAT, POR and the FLU homologous barley TIGRINA D protein (FLU) in light- and dark-incubated leaf samples. Pchlide accumulation in green barley leaves is stopped within one hour of dark incubation In darkness Pchlide is supposed to influence ALA formation by feedback inhibition (Reinbothe and Reinbothe 1996). Time-resolved Pchlide accumulation was monitored after light–dark transition of green barley leaves (Fig. 2). Within the first 30 min of dark incubation leaves accumulated up to 6-fold more Pchlide compared with Pchlide levels during illumination. Prolonged dark incubation (e.g. up to a 20 h period) did not significantly increase levels of accumulated Pchlide compared with short-term dark incubation during the experiments. When green leaves were re-exposed to light their Pchlide pool decreased and reached the steady-state level of illuminated leaves within minutes (Fig. 2). Fig. 2 View largeDownload slide Accumulation of Pchlide in barley leaf discs incubated in levulinic acid-containing buffer. Samples that were transferred to darkness (filled squares) accumulated Pchlide to a level six times higher than light-exposed plants (open squares). The dotted line indicates the Pchlide level reached after 20 h dark incubation of intact barley seedlings. Dark Pchlide content was diminished within <30 min of re-illumination (after 90 min). Data are given as means ± SD. Fig. 2 View largeDownload slide Accumulation of Pchlide in barley leaf discs incubated in levulinic acid-containing buffer. Samples that were transferred to darkness (filled squares) accumulated Pchlide to a level six times higher than light-exposed plants (open squares). The dotted line indicates the Pchlide level reached after 20 h dark incubation of intact barley seedlings. Dark Pchlide content was diminished within <30 min of re-illumination (after 90 min). Data are given as means ± SD. These data correlate very well with the observation that ALA synthesis is rapidly down-regulated in darkness to avoid massive flux into the tetrapyrrole biosynthesis pathway. To address the question of whether Pchlide accumulates only in the photo-convertible POR-bound or also in the free non-photo-convertible form within the first 2 h of darkness, we performed 77K fluorescence analyses of crude barley leaf extracts followed by Gaussian deconvolution of fluorescence data to detect potential hidden maxima. Non-photo- and photo-convertible Pchlide (Ex 433 nm) show a fluorescence emission maximum at 635 nm and 650 nm, respectively (Böddi et al. 1992, Franck and Strzalka 1992). During dark incubation only an emission maximum at 650 nm was detectable, indicating the photo-convertible POR-bound form of Pchlide. After 20 h dark incubation of young green barley leaves the fluorescence emission maximum at 635 nm can be assigned to free Pchlide (Fig. 3). The barley tigrina d12 mutant accumulated both the non-photo- and the photo-convertible Pchlide (Fig. 3). Fig. 3 View largeDownload slide 77K fluorescence spectra of intact barley wild-type Lomerit (WT) leaves at different incubation times in light and darkness. Within 1 h of darkness only the POR-bound photoactive form of Pchlide accumulated having a fluorescence emission maximum at 650 nm (excitation at 433 nm). After 20 h of darkness initial accumulation of free non-photo-convertible Pchlide (emission at 635 nm) was observed. The fluorescence spectrum of tigrina d12 seedlings was obtained after 20 h of darkness. Spectra were normalized to chlorophyll fluorescence emission maximum at 680 nm. Using the Gaussian deconvolution method emission bands in the complex spectra were assessed. Fig. 3 View largeDownload slide 77K fluorescence spectra of intact barley wild-type Lomerit (WT) leaves at different incubation times in light and darkness. Within 1 h of darkness only the POR-bound photoactive form of Pchlide accumulated having a fluorescence emission maximum at 650 nm (excitation at 433 nm). After 20 h of darkness initial accumulation of free non-photo-convertible Pchlide (emission at 635 nm) was observed. The fluorescence spectrum of tigrina d12 seedlings was obtained after 20 h of darkness. Spectra were normalized to chlorophyll fluorescence emission maximum at 680 nm. Using the Gaussian deconvolution method emission bands in the complex spectra were assessed. ALA synthesis is not suppressed in darkness when Pchlide levels do not increase As ALA synthesis in green barley leaves immediately declined after dark transition and Pchlide rapidly accumulated, it was important to perform additional experiments to confirm a Pchlide accumulation-dependent short-term feedback inactivation of ALA formation in darkness. Therefore, Pchlide accumulation in darkness was minimized by inhibiting the tetrapyrrole biosynthesis pathway upstream of Pchlide formation. The photosensitizing herbicide acifluorfen inhibits protoporphyrinogen IX oxidase (PPOX) resulting in a massive accumulation of protoporphyrinogen IX (Protogen). Fig. 4a displays the strongly reduced Pchlide formation in acifluorfen-treated and dark-incubated leaves compared with untreated leaves. It is worth mentioning that acifluorfen-treated leaves synthesized slightly more ALA than control leaves in light (Fig. 4b). Fig. 4 View largeDownload slide Acifluorfen treatment impedes accumulation of Pchlide and causes stimulated ALA synthesis in darkness. (a) Barley leaves that were transferred to darkness accumulate Pchlide to a level almost five times higher than that of illuminated leaves within <1 h of dark incubation (black squares). Leaves that were treated with acifluorfen accumulated Pchlide to a lower extent in darkness (grey squares). (b) Acifluorfen-treated leaves showed a slightly increased ALA accumulation in the light, but failed to repress ALA synthesis in darkness (30 min light + 60 min dark). The dashed line indicates the average pool of ALA formed within 30 min light. (c) The treatment of barley leaves with acifluorfen caused a massive accumulation of Proto. Data are given as means ± SD. The bar on the top indicates the light (white) and dark (black) incubation periods. Fig. 4 View largeDownload slide Acifluorfen treatment impedes accumulation of Pchlide and causes stimulated ALA synthesis in darkness. (a) Barley leaves that were transferred to darkness accumulate Pchlide to a level almost five times higher than that of illuminated leaves within <1 h of dark incubation (black squares). Leaves that were treated with acifluorfen accumulated Pchlide to a lower extent in darkness (grey squares). (b) Acifluorfen-treated leaves showed a slightly increased ALA accumulation in the light, but failed to repress ALA synthesis in darkness (30 min light + 60 min dark). The dashed line indicates the average pool of ALA formed within 30 min light. (c) The treatment of barley leaves with acifluorfen caused a massive accumulation of Proto. Data are given as means ± SD. The bar on the top indicates the light (white) and dark (black) incubation periods. However, it is more important that ALA synthesis of acifluorfen-treated leaves was not reduced in darkness, while ALA synthesis of untreated leaves showed the expected dark repression (Fig. 4b). This indicates that ALA formation in darkness depends on the level of Pchlide in plastids and ALA synthesis can be uncoupled from dark-repression by acifluorfen-mediated prevention of Pchlide accumulation. Heme is proposed to act as feedback inhibitor of ALA synthesis (Cornah et al. 2003, Goslings et al. 2004). To exclude possible heme effects, the heme content was determined in acifluorfen-treated and control leaves. The level of non- covalently bound heme was not reduced in acifluorfen-treated barley leaves, during neither light nor dark exposure (Table 1). Table 1 Content of non-covalently bound heme in barley leaves Sample  Treatment  Non-covalently bound heme (pmol mg FW−1)  30 min light + 60 min light  Control  8.0 ± 0.5    Acifluorfen  7.6 ± 1.0  30 min light + 60 min dark  Control  8.0 ± 0.1    Acifluorfen  9.0 ± 1.4  Sample  Treatment  Non-covalently bound heme (pmol mg FW−1)  30 min light + 60 min light  Control  8.0 ± 0.5    Acifluorfen  7.6 ± 1.0  30 min light + 60 min dark  Control  8.0 ± 0.1    Acifluorfen  9.0 ± 1.4  Leaf discs of acifluorfen-treated and control barley plants were incubated in levulinic acid-containing buffer and exposed to light, or 30 min light followed by 60 min darkness. The amount of non-covalently bound heme was determined. Data are given as means ± SD. View Large The acifluorfen treatment caused the expected massive accumulation of Protogen, which is rapidly oxidized to Proto (Witkowski and Halling 1988, Witkowski and Halling 1989). HPLC analysis revealed similar contents of Proto(gen) in dark and light-incubated acifluorfen-treated barley leaves, because ALA synthesis was not inactivated in darkness and resembled that of light-incubated leaf samples (Fig. 4c). Eventually, treatment with acifluorfen leads to photodamage of plants (Lermontova and Grimm 2006). To exclude any adverse effects of acifluorfen on plastid integrity and physiology within the time frame of ALA synthesis analysis, photosynthetic parameters and membrane lipid peroxidation were determined. Tables 2, 3 point out that acifluorfen treatment of barley plants did not perturb photosynthetic processes within the first 6 h of illumination. The pigment content did not differ significantly between treated and untreated leaves (Table 2). Maximum quantum yield of PSII photochemistry, actual quantum yield of photochemical energy conversion in PSII and non-photochemical quenching of variable chlorophyll fluorescence in acifluorfen-treated leaves resembled control leaves (Table 2). The content of thiobarbituric acid reactive substances (TBARS) did not differ significantly between control and acifluorfen-treated plants within the first 2 h of illumination indicating integrity of plastid membranes (Table 3). But continued photoperiodic growth for 2 days caused substantial phototoxic effects of acifluorfen as indicated by elevated amounts of TBARS in barley leaves compared with control values and leaf necrosis, which are explained by massive accumulation of Proto(gen) in herbicide-treated leaves (Table 3, Supplementary Fig. S2). Table 2 Photosynthetic parameters and pigment contents of barley leaves treated with acifluorfen for 20 h in darkness and subsequently transferred to light Parameter  % of control  Fv/Fma  99.7 ± 1.0  ΦPSIIb  102.5 ± 10.0  qNc  104.2 ± 19.0  Chlorophyll content  90.7 ± 7.3  Carotenoid content  90.4 ± 6.7  Parameter  % of control  Fv/Fma  99.7 ± 1.0  ΦPSIIb  102.5 ± 10.0  qNc  104.2 ± 19.0  Chlorophyll content  90.7 ± 7.3  Carotenoid content  90.4 ± 6.7  Acifluorfen-treated plants did not show any reduction in photosynthetic capacity compared with untreated controls within the first 6 h of light treatment. The pigment contents of untreated barley leaves were 287 ± 33 ng mg FW−1 total chlorophyll and 125 ± 15 ng mg FW−1 carotenoids. The chlorophyll a/b ratio was 4.7. aMaximum quantum yield of PSII photochemistry. bActual quantum yield of photochemical energy conversion in PSII. cNon-photochemical quenching of chlorophyll fluorescence. View Large Table 3 Membrane integrity of barley plants treated with acifluorfen for 20 h in darkness and subsequently transferred to light Sample  Treatment  TBARS (μmol mg FW−1)  2 h light  Control  9.1 ± 1.2    Acifluorfen  10.1 ± 3.2  2 d lighta  Control  11.4 ± 4.0    Acifluorfen  18.1 ± 1.5  Sample  Treatment  TBARS (μmol mg FW−1)  2 h light  Control  9.1 ± 1.2    Acifluorfen  10.1 ± 3.2  2 d lighta  Control  11.4 ± 4.0    Acifluorfen  18.1 ± 1.5  Accumulation of TBARS corresponds to membrane integrity. Acifluorfen-treated leaves did not show any membrane disintegration after 2 h of light exposure, while membrane damage was indicated after 2 d of light/dark incubation. aPhotoperiodic growth. View Large All other attempts to inhibit Pchlide accumulation during dark incubation of barley leaves and to reproduce the results obtained in the presence of acifluorfen by independent experimental approaches were unsuccessful. Inhibitors, such as the iron-chelators dipyridyl (Duggan and Gassman 1974) and thujaplicin (Oster et al. 1996) were applied similarly to acifluorfen, but prevented Pchlide accumulation in darkness less efficiently (data not shown). This may be attributed to limitations of inhibitor uptake through the leaf surface. Non-repressed ALA synthesis of the tigrina d12 mutant in darkness resembles response to acifluorfen treatment While dark-grown wild-type angiosperms immediately down-regulate ALA biosynthesis, the Arabidopsis flu and the homologous barley tigrina d12 mutants accumulate excessive amounts of Pchlide as a consequence of elevated ALA formation during the dark period (Hansson et al. 1997, Meskauskiene et al. 2001, Lee et al. 2003). To substantiate the regulatory relationship between immediate dark-repression of ALA synthesis and rapid Pchlide accumulation, we included examinations of the kinetics of ALA synthesis and Pchlide accumulation of the tigrina d12 mutant and its wild-type variety Bonus in darkness. Bonus wild-type leaves followed the same pattern of light-stimulated and dark-repressed ALA synthesis as the wild-type variety Lomerit, but show a lower ALA synthesis rate in light (Figs. 1, 5). The tigrina d12 leaves (200 pmol ALA mg FW−1 h−1) showed a 2-fold increased ALA synthesis in light compared with Bonus wild type (100 pmol ALA mg FW−1 h−1). Due to the lack of the functional repressor protein tigrina d12 did not decelerate ALA formation in darkness (Fig. 5). As a consequence of non-repressed ALA synthesis Pchlide content increased during darkness to a level that exceeds the amount of available POR protein resulting in the occurrence of free non-photo- convertible Pchlide (Fig. 3; Gough and Kannangara 1979, Hansson et al. 1997). Fig. 5 View largeDownload slide ALA accumulation in wild-type and tigrina d12 leaves during light and dark incubation. When exposed to light, the tigrina d12 (open circles) leaves exhibited an elevated ALA synthesis rate as opposed to the corresponding wild type (open squares). After dark transition wild type (filled squares) rapidly repressed ALA synthesis while the mutant (filled circles) continued without decreasing enzyme activity. Data are given as means ± SD. Fig. 5 View largeDownload slide ALA accumulation in wild-type and tigrina d12 leaves during light and dark incubation. When exposed to light, the tigrina d12 (open circles) leaves exhibited an elevated ALA synthesis rate as opposed to the corresponding wild type (open squares). After dark transition wild type (filled squares) rapidly repressed ALA synthesis while the mutant (filled circles) continued without decreasing enzyme activity. Data are given as means ± SD. Diminished ALA synthesis in low light also correlates with Pchlide accumulation ALA synthesis rate can be modulated over a range of increasing light intensities. It remains open whether ALA synthesis is attenuated at very low light intensities by the same regulatory mechanism as in darkness. ALA synthesis in green leaves of barley wild-type Lomerit was determined at light intensities <30 μmol photons m−2 s−1 to examine enzymatic activities at the transition between dark-repression and light-stimulation of ALA formation. Below 10 μmol photons m−2 s−1 ALA synthesis was reduced. In parallel the Pchlide steady-state level increased with decreasing light quantities (Fig. 6a). It is suggested that this elevated Pchlide level contributed to the diminished ALA synthesis rate at very low light intensities. Fig. 6 View largeDownload slide ALA (filled squares) and Pchlide (filled circles) accumulation in barley leaf discs of (a) wild-type Lomerit, (b) wild-type Bonus and (c) tigrina d12 incubated for 2 h in a buffer containing 40 mM levulinic acid at different light intensities (white light). In wild type but not in tigrina d12 ALA synthesis rate was decreased at light intensities <10 μmol photons m−2 s−1 down to a very low level in darkness. Data are given as means ± SD. Fig. 6 View largeDownload slide ALA (filled squares) and Pchlide (filled circles) accumulation in barley leaf discs of (a) wild-type Lomerit, (b) wild-type Bonus and (c) tigrina d12 incubated for 2 h in a buffer containing 40 mM levulinic acid at different light intensities (white light). In wild type but not in tigrina d12 ALA synthesis rate was decreased at light intensities <10 μmol photons m−2 s−1 down to a very low level in darkness. Data are given as means ± SD. ALA synthesis and Pchlide levels at different light intensities were also determined in Bonus wild-type and tigrina d12 seedlings (Fig. 6b,c). The light-intensity dependency of ALA synthesis differed between tigrina d12 seedlings and its wild type. Consistent with the results of tigrina d12 ALA synthesis in darkness (Fig. 5), ALA synthesis was not reduced at low light intensity (3 μmol photons m−2 s−1), although the Pchlide levels (3 pmol mg FW−1) were already elevated compared with that of wild type (0.55 pmol mg FW−1). Discussion Rapid post-translational control represses dark ALA synthesis in green leaves Light-dependent Pchlide reduction in angiosperms enables synthesis of new chlorophyll molecules exclusively upon light exposure. Pchlide is the only tetrapyrrole intermediate accumulating in darkness. It was previously demonstrated that Pchlide accumulation is inversely correlated with down- regulation of ALA synthesis in dark-grown green leaves and etiolated seedlings and therefore a regulatory role for Pchlide was proposed (Nadler and Granick 1970, Castelfranco et al. 1974, Fluhr et al. 1975, Gough 1978, Ford and Kasemir 1980, Stobart and Ameen-Bukhari 1984, Stobart and Ameen-Bukhari 1986, Huang and Castelfranco 1989). This regulatory mechanism is reasonable as down-regulated ALA synthesis in darkness prevents excessive accumulation of Pchlide. Its photoreactivity is harmful for light-exposed plants. However, due to experimental limitations a time course analysis of the interdependence between Pchlide accumulation and activities of ALA synthesis has ultimately never been provided. Moreover, the proposed feedback control from Pchlide and POR could not be substantiated by protein–protein interactions between enzymes involved in Pchlide reduction and ALA synthesis. The first substantial information about the mode of action was provided by the analysis of the negative regulator FLU, which interacts with GluTR and was shown to be required for dark-repression of ALA synthesis (Meskauskiene et al. 2001). Inhibition of ALA formation was previously studied at light–dark transitions in leaves of different plant species or isolated chloroplasts. A decline in ALA synthesis was demonstrated after the transfer of the samples to darkness. But the elucidation of rapid turn-off mechanisms of ALA synthesis was hindered by technical limitations. ALA synthesis was determined in leaves after levulinic acid incubation for several hours (Fluhr et al. 1975, Huang and Castelfranco 1989, Beator and Kloppstech 1993, Kruse et al. 1997, Papenbrock et al. 1999, Goslings et al. 2004). Taking advantage of both high metabolic activities of green primary barley leaves and improved methodology ALA synthesis could be analyzed within 30 min. In our experimental setup the synthesis of ALA was constant during light exposure and stopped almost instantaneously when leaf samples were transferred to darkness (Fig. 1). During the subsequent dark period a slow but continuing synthesis of additional ALA molecules was detected (Figs. 1, 4b). Observed differences at the level of transcripts and proteins involved in ALA formation may not explain the rapid dark-repression of ALA synthesis; an effective and rapid mechanism most likely acts at the post-translational level. In analogy, ALA formation recovers almost immediately and completely after dark to light transition (Fig. 1). Under these assay conditions Pchlide accumulation in dark-incubated barley leaf samples reached a maximum within 30–60 min that did not further increase during prolonged dark periods. The underlying mechanism repressing ALA synthesis in darkness avoids Pchlide levels exceeding the binding capacity of POR and efficiently prevents accumulation of photosensitizing free Pchlide (Fig. 3). However, the molecular mechanisms of the light–dark switch of ALA formation are still not entirely elucidated. Inhibition of Pchlide accumulation hinders repression of ALA synthesis Our results corroborate the hypothesis of a negative feedback regulation of ALA formation in green tissues originating from the Mg branch of the tetrapyrrole biosynthesis pathway. When Pchlide accumulation was prevented, inactivation of ALA synthesis did not occur. This was achieved by inhibition of PPOX with acifluorfen that effectively stopped the metabolic flow in the tetrapyrrole biosynthetic pathway (Fig. 4; Witkowski and Halling 1988) without affecting the integrity of plastid physiology within the experimental time period (Tables 2, 3). However, Pchlide accumulation was inhibited (Fig. 4a) and, in turn, ALA formation was not repressed indicating a deficit in the repression signal in dark-incubated acifluorfen-treated leaves. This deregulation of ALA synthesis is rather attributed to deficient Pchlide accumulation than to increased levels of Proto(gen). Protogen and Proto are intermediates that do not accumulate in significant amounts, when green leaves are transferred from light to darkness. Moreover, transgenic tobacco plants with reduced amounts of the PPOX do not show deregulation of ALA formation in darkness (Lermontova and Grimm 2006). Becerril et al. (1992) reported that acifluorfen-treated duckweed accumulated Proto(gen) in darkness over a period of 5 h, before ALA synthesis was stopped and the metabolite flow was negatively affected. It was suggested that acifluorfen treatment did not completely block PPOX leading to delayed Pchlide accumulation and Pchlide-mediated feedback inhibition of ALA synthesis. However, Pchlide accumulation is mandatory for efficient post-translational inactivation of ALA synthesis in darkness. It was previously proposed that elevated levels of free heme control ALA synthesis in darkness (Masuda et al. 1990, Cornah et al. 2003, Goslings et al. 2004, Beale 2006) and GluTR activity was directly inhibited either by heme (Pontoppidan and Kannangara 1994) or by an unknown plastid-localized factor (Srivastava et al. 2005). In our experiments the contents of non-covalently bound heme in light-exposed barley leaves did not differ from dark-incubated samples. Furthermore acifluorfen treatment did not result in significant reduction in the heme content (Table 1). Since we determined the heme content of green tissue but not of isolated plastids we cannot fully exclude changes in the so-called free heme pool within plastids. As a light-dependent enzymatic step involved in heme metabolism has not been reported, yet, it is implausible that free heme accumulates rapidly after light to dark transition and contributes to the observed rapid dark-repression of ALA synthesis. It is likely that the remaining ALA synthesis in dark-incubated green leaves is directed into the Fe branch of tetrapyrrole biosynthesis, because maximum Pchlide accumulation was observed after 1 h of dark incubation. It is not excluded that heme-dependent inhibition of ALA synthesis or GluTR activity reported previously, plays a role in an efficient long-term tuning of ALA synthesis in planta. It was shown that in light–dark grown tobacco plants heme accumulated and Fe chelatase activity increased in the dark periods (Papenbrock et al. 1999). POR and FLU are involved in rapid repression of ALA synthesis ALA synthesis was shown to be repressed at low light intensities with simultaneous accumulation of Pchlide (Fig. 6). Keeping in mind that ALA synthesis is almost immediately repressed in darkness and Pchlide accumulation occurs rapidly, it is conclusive that elevated Pchlide contents are almost instantaneously communicated to repress ALA synthesis by a post-translational mechanism. The regulatory mechanism of Pchlide-mediated dark repression of ALA synthesis involves possibly the ternary complex of NADPH:Pchlide POR (Oliver and Griffiths 1982). It is suggested that accumulating Pchlide bound to POR is responsible for the slow-down of ALA synthesis under low light conditions or in darkness (Fig. 6). Stobart and Ameen-Bukhari (1986) used ALA feeding experiments and titration with different amounts of ALA to investigate the light-induced ALA synthesis in etiolated barley seedlings. Both authors emphasized that Pchlide bound to POR affects light-stimulated ALA synthesis and free POR is mandatory to synthesize ALA. We proposed that due to its low content relative to etiolated tissue, POR is rapidly saturated with Pchlide in dark-incubated green tissue and this correlates with the fast post-translational inhibition of ALA formation. The negative regulator FLU is probably a substantial part of the above-mentioned feedback control. FLU deficiency in the barley mutant tigrina d12 prevents repression of ALA synthesis under low light intensities and in darkness and causes higher ALA synthesis during illumination (Figs. 5, 6). Further details of the feedback regulation sensing Pchlide accumulation in dark-incubated and low-light-exposed green leaves (Fig. 6) need to be elucidated. ALA formation is likely inhibited at the post-translational level. Future experiments are required to demonstrate that FLU also functions in response to light intensity. The current knowledge comprises Pchlide accumulation in darkness as the starting point of the feedback mechanism controlling ALA synthesis in green leaves. It is suggested that under low light intensities ALA synthesis experiences a similar Pchlide-induced repression. It is very likely that the FLU protein represents a signaling component that interacts directly with GluTR. The potential of FLU to inhibit GluTR activity in darkness and to modulate ALA synthesis at different light intensities in green tissues remains a subject of further investigation. Materials and Methods Plant growth conditions Barley seedlings [Hordeum vulgare L. var. Lomerit and Bonus (wild type and tigrina d12)] were grown on vermiculite for 6 days in a 14 h light/10 h dark rhythm or continuous light, respectively, in a growth chamber at 22–23°C with normal light intensity of 80–100 μmol photons m−2 s−1. Experimental design Equal-sized primary leaves were harvested 2 h after onset of light and cut 3–4 cm below the tip into pieces of 1 cm length. Unless otherwise stated, 100 mg leaf material (corresponding to six leaf segments) was used for each measurement. For herbicide treatment 5-day-old barley seedlings were sprayed four times with 200 μM acifluorfen [in 10 mM Tris–HCl, pH 8.0, 0.05% (v/v) Tween 80] during a 20 h dark incubation. For analysis of the ALA synthesis rate, RNA and protein expression, and levels of Pchlide, porphyrins and heme, leaf material was incubated in 50 mM Tris–HCl, pH 7.2 and 40 mM levulinate for 30 min at 22–23°C. After preincubation the samples were transferred to darkness for a time period indicated in the figure legends or remained under the same light condition. Standard light intensity was 100 μmol photons m−2 s−1. Different light intensities were applied as indicated. Samples were taken at different time points during the incubation time, dried on paper towels, weighed and frozen in liquid nitrogen. Leaf samples of 20 h dark-incubated acifluorfen-treated or control plants were subjected to measurements of TBARS and photosynthetic capacity at different time points after beginning of illumination. Determination of ALA ALA content was measured using the method of Mauzerall and Granick (1956). Frozen samples were homogenized, resuspended in 20 mM potassium phosphate buffer pH 6.8 and centrifuged for 10 min at 16 000 × g; 400 μl of the supernatant was mixed with 100 μl of ethyl acetoacetate and boiled for 10 min at 100°C. Samples were mixed with 500 μl of modified Ehrlich’s reagent [373 ml of acetic acid, 90 ml of 70% (v/v) perchloric acid, 1.55 g of HgCl2, 9.10 g of 4-dimethylaminobenzaldehyde and 500 ml of H2O] and centrifuged for 5 min at 16 000 × g. Absorption was measured at 526, 553 and 720 nm and the ALA content of the samples was calculated using a standard curve generated by commercial ALA (Sigma-Aldrich Inc.). Determination of the Pchlide content Extraction of Pchlide followed the protocol of Koski and Smith (1948). Leaf samples were fixed with steam for 2 min, frozen, ground in liquid nitrogen and extracted three times in alkaline acetone (9:1, 100% acetone: 0.1 N NH4OH). After centrifugation at 16 000 × g for 10 min the supernatants were collected and chlorophyll was removed by a stepwise extraction with 1 vol, 1 vol and 0.3 vol of 100% n-hexane. The Pchlide content of the samples was quantified using HPLC according to Langmeier et al. (1993). Samples were injected and separated by a reverse phase column (Waters RP-18 ODS Hypersil 3 μM, 12.5 × 4 cm). Solvents A (20% 1 M ammonium acetate pH 7.0, 80% methanol, v/v) and B (20% acetone, 80% methanol, v/v) were used with the following program: 15 min linear gradient from 100% A to 100% B, continued with a 10 min isocratic run with B, returned to 100% A with a 3 min linear gradient and followed by a 2 min isocratic run with solvent A. The flow rate was set to 1.0 ml min−1. Eluates were analyzed by a fluorescence detector using an excitation wavelength of 435 nm and recording at 644 nm. For calibration a Pchlide standard was extracted from 7-day-old etiolated barley leaves (Koski and Smith 1948) and quantified using the extinction coefficient ε(Pchlide in diethyl ether, 623 nm) of 35 600 M−1 cm−1 (Dawson et al. 1986). 77K fluorescence spectroscopy Leaf material (10 mg) was ground in a buffer containing 50 mM Tricine and 0.4 M Sorbitol, pH 7.8 (Krupa et al. 1987). A 77K spectroscopy capillary tube was filled with a mixture of 150 μl of the homogenate and 350 μl of glycerol and frozen in liquid nitrogen. 77K fluorescence emission (600–700 nm) was recorded on a FP-6500 fluorescence spectrometer (JASCO Inc.) using an excitation wavelength of 433 nm. Spectra were fitted by multiple Gaussian deconvolution using the nonlinear least squares fitting of the R software package (R Development Core Team 2008). Initial estimates of means and standard deviation were derived by analyzing the first and second derivatives of the spectra. Determination of heme The content of non-covalently bound heme was determined as described in Weinstein and Beale (1984) and Yaronskaya et al. (2003). Frozen leaf material (300 mg) was homogenized and chlorophyll was removed by washing with cold acetone: 0.1 N NH4OH (9:1, v/v) followed by centrifugation at 16 000 × g for 10 min. Non-covalently bound heme was extracted from the pellets twice with 5 ml of extraction solution [2 ml of dimethyl sulfoxide, 10 ml of acetone and 0.5 ml of 37% (v/v) HCl], followed by centrifugation at 16 000 × g for 10 min. Heme was transferred to ether by adding 3 ml of diethyl ether, 2 ml of saturated NaCl and 10 ml of water. The ether phase was transferred to a new tube. Washing with 3 ml of ether was repeated and both ether phases were combined and concentrated to a final volume of 2 ml. A 3 ml DEAE–Sepharose CL-6B column was equilibrated with 1 column volume of diethyl ether: ethanol (3:1, v/v). Heme extracts were mixed with 0.7 ml of ethanol and applied to the column. The column was washed with two volumes of diethyl ether:ethanol (3:1, v/v), 1 vol of diethyl ether: ethanol (1:1, v/v) and finally with 1 vol of ethanol. Heme was eluted with a mixture of ethanol:acetic acid: water (81:9:10, v/v/v) and quantified spectrophotometrically using the extinction coefficient ε(398 nm) of 144 mM−1 cm−1 (Weinstein and Beale 1983). Determination of pigments and other porphyrins Chlorophyll and carotenoids were extracted using 80% (v/v) acetone with 10 μM KOH and determined spectrophotometrically according to Lichtenthaler (1987). Protoporphyrin IX (Proto) and Mg-porphyrins were extracted with acetone: methanol: 0.1 N NH4OH (10:9:1, v/v/v) and analyzed via HPLC as described in Papenbrock et al. (1999). RNA analysis Plant total RNA was extracted following the TRIsure protocol (Bioline GmbH, Luckenwalde, Germany). Ten micrograms of each sample was separated electrophoretically and transferred to Hybond N+ membrane (GE Healthcare) using standard protocols (Sambrook and Russel 2001). Probes were amplified by PCR from a barley cDNA using gene-specific primers for HvHEMA1 (5′-GCAGCCGCGGGCGCATTCGCCGCCGCCA AG-3′, 5′-TGCCAATTTCTGACAACTTGTTTGACT-3′), HvPORB (5′-GCGGCCACTTCCTTCCTCCCGTCGG-3′, 5′-AGCTGGCGC ACGTTCTTGACGAACTGG-3′), and HvCAB (chlorophyll a/b binding protein 5′-TCTGTCTTCCTCCACCTTCGCCGGG-3′, 5′- ACATGGAGAACATGGCCAGCGTCC-3′) and labelled with [α-32P]dCTP using random oligonucleotide priming (HexaLabel DNA labelling Kit; Fermentas GmbH, Germany). Prehybridization and hybridization were performed in Church buffer (Church and Gilbert 1984) at 70°C. Final washings were carried out to a stringency of 0.1 × SSC and 0.1% (w/v) SDS at 65°C. Protein analysis Plant total protein was extracted by grinding frozen leaf material in a buffer containing 2% (w/v) SDS, 56 mM NaCO3, 12% (w/v) sucrose, 56 mM DTT and 2 mM EDTA, pH 8.0, followed by heating at 70°C for 20 min and centrifuging at 16 000 × g for 10 min. The protein concentration in the supernatant was measured using BCA protein assay reagent (Perbio Science) after trichloracetic acid precipitation. Proteins were separated on 12% or 15% polyacrylamide gels, transferred to Hybond-C membranes (GE Healthcare) and probed with specific antibodies using standard protocols (Sambrook and Russel 2001). Determination of membrane lipid peroxidation Approximately 200 mg leaf material was homogenized in 0.25% (w/v) thiobarbituric acid in 10% (w/v) TCA and heated at 95°C in a glass tube in a water bath for 30 min to form malondialdehyde according to De Vos et al. (1989). The amount of TBARS was calculated from the difference in absorbance at 532 nm and 600 nm using an extinction coefficient ε(532 nm – 600 nm) of 155 mM−1 cm−1. Measuring the photosynthetic capacity Chlorophyll fluorescence parameters were measured using a PAM 2000 (Walz, Effeltrich, Germany). Conventional fluorescence nomenclature was used (van Kooten and Snel 1990, Rohácek 2002). Attached leaves were dark adapted for 30 min for determination of the maximum quantum yield of PSII photochemistry (calculated as ratio Fv/Fm = (Fm – Fo)/Fm) After determining the basic fluorescence (Fo) an 800 ms saturating pulse was applied to ascertain the maximum fluorescence (Fm). ΦPSII, the actual quantum yield of photochemical energy conversion in PSII, was determined according to Genty et al. (1989) on growth light-adapted leaves. The non-photochemical quenching of variable chlorophyll fluorescence, qN, was calculated by the formula qN = 1 – (Fm′ – Fo′)/(Fm – Fo). Fm and Fo were determined as described above following the determination of Fm′ and Fo′ upon 15 min adaptation to actinic light at an intensity appropriate to growth light intensity. Miscellaneous All experiments were performed with three to six independent experiments. In order to test significant differences between calculated values equality of variances was tested by a F-test followed by Student’s t-test using a P-value of <0.05. Acknowledgments The authors thank B. Hickel for help with the HPLC analyses and acknowledge funding by Deutsche Forschungsgemeinschaft (DFG – Research Unit 804 and Sonderforschungsbereich 429) to B.G. and Studienstiftung des deutschen Volkes to E.P. Abbreviations Abbreviations ALA 5-aminolevulinic acid GluTR glutamyl t-RNA reductase GSAT glutamate 1-semialdehyde aminotransferase Pchlide protochlorophyllide POR NADPH:protochlorophyllide oxidoreductase PPOX protoporphyrinogen IX oxidase Proto protoporphyrin IX Protogen protoporphyrinogen IX Proto(gen) accumulated protoporphyrinogen IX and protoporphyrin IX PSII photosystem II TBARS thiobarbituric acid reactive substances. 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Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oxfordjournals.org http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Plant and Cell Physiology Oxford University Press

Rapid Dark Repression of 5-Aminolevulinic Acid Synthesis in Green Barley Leaves

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Oxford University Press
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© The Author 2010. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oxfordjournals.org
ISSN
0032-0781
eISSN
1471-9053
DOI
10.1093/pcp/pcq047
pmid
20375109
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Abstract

Abstract In photosynthetic organisms chlorophyll and heme biosynthesis is tightly regulated at various levels in response to environmental adaptation and plant development. The formation of 5-aminolevulinic acid (ALA) is the key regulatory step and provides adequate amounts of the common precursor molecule for the Mg and Fe branches of tetrapyrrole biosynthesis. Pathway control prevents accumulation of metabolic intermediates and avoids photo-oxidative damage. In angiosperms reduction of protochlorophyllide (Pchlide) to chlorophyllide is catalyzed by the light-dependent NADPH:Pchlide oxidoreductase (POR). Although a correlation between down-regulated ALA synthesis and accumulation of Pchlide in the dark was proposed a long time ago, the time-resolved mutual dependency has never been analyzed. Taking advantage of the high metabolic activity of young barley (Hordeum vulgare L.) seedlings, in planta ALA synthesis could be determined with high time-resolution. ALA formation declined immediately after transition from light to dark and correlated with an immediate accumulation of POR-bound Pchlide within the first 60 min in darkness. The flu homologous barley mutant tigrina d12 uncouples ALA synthesis from dark-suppression and continued to form ALA in darkness without a significant change in synthesis rate in this time interval. Similarly, inhibition of protoporphyrinogen IX oxidase by acifluorfen resulted in a delayed accumulation of Pchlide during the entire dark period and a weak repression of ALA synthesis in darkness. Moreover, it is demonstrated that dark repression of ALA formation relies rather on rapid post-translational regulation in response to accumulating Pchlide than on changes in nuclear gene expression. Introduction In higher plants the formation of 5-aminolevulinic acid (ALA) is the first committed step in tetrapyrrole biosynthesis leading to the end products chlorophyll, heme, siroheme and phytochromobiline (Beale 1999, Papenbrock and Grimm 2001, Tanaka and Tanaka 2007). The synthesis of tetrapyrroles is adjusted in response to changes in environmental conditions (e.g. light, temperature) and development. The regulation of cellular tetrapyrrole accumulation is inextricably linked to the formation of ALA, which is considered to be a rate-limiting step controlling influx into the entire pathway. ALA is formed in a three-step reaction including the ligation of glutamate to tRNAGlu catalyzed by glutamyl-tRNA synthetase, the reduction of glutamate to glutamate-1-semialdehyde by glutamyl-tRNA reductase (GluTR) and a final transamination step mediated by glutamate-1-semialdehyde aminotransferase (GSAT; Kannangara et al. 1988). Light-dependent induction of ALA synthesis during de- etiolation and growth under dark/light conditions is well studied (Ilag et al. 1994, McCormack and Terry 2002). Within the metabolic pathway of tetrapyrrole biosynthesis ALA production is controlled by several feedback loops regulating gene expression or post-translational modifications. Signals are proposed to originate from heme and the Mg branch of the pathway. It is presently suggested that GluTR is the main target of regulatory mechanisms modulating ALA formation. The expression of HEMA1 encoding GluTR is regulated by a wide range of stimuli (e.g. cytokinin, the circadian clock, plastid-derived signals and light), whereas GSA encoding GSAT responds only weakly (Cornah et al. 2003, Gough et al. 2003, Eckhardt et al. 2004, Tanaka and Tanaka 2007). In in vitro experiments the activity of barley GluTR was inhibited by heme (Pontoppidan and Kannangara 1994), which exerted the inhibiting action through the first 30 N- terminal amino acids of the enzyme (Vothknecht et al. 1998). In Chlamydomonas an additional factor was required to mediate inhibition of GluTR by heme (Srivastava et al. 2005). Additionally, enzyme activies of the Mg branch feedback- control ALA synthesis. Tobacco mutants affected in the synthesis of CHLI or CHLH subunits of Mg chelatase not only showed decreased Mg chelatase activity, but also reduced ALA synthesizing activity. This metabolic feedback control acts at the transcriptional level (Papenbrock et al. 2000a, Papenbrock et al. 2000b) and prevents accumulation of tetrapyrrole intermediates generating reactive oxygen species upon excitation by light (Vavilin and Vermaas 2002). ALA synthesis is drastically reduced in darkness. Previous observations revealed accumulation of the chlorophyll precursor protochlorophyllide (Pchlide) in etiolated seedlings of angiosperms, since Pchlide can be converted to chlorophyllide by the light-dependent NADPH:Pchlide oxidoreductase (POR) only (Griffiths et al. 1996). Several experiments with etiolated seedlings and green plants unveiled a strong inverse correlation between Pchlide accumulation and ALA synthesis in darkness (Fluhr et al. 1975, Gough 1978, Ford and Kasemir 1980, Huang and Castelfranco 1989, Stobart and Ameen-Bukhari 1984, Stobart and Ameen-Bukhari 1986). Thus, it was assumed that a high Pchlide level induces down-regulation of chlorophyll biosynthesis at the level of ALA synthesis (Beale 2006, Tanaka and Tanaka 2007). The Arabidopsis flu mutant with a deficient negative regulator for ALA synthesis fails to down-regulate ALA synthesis in darkness and accumulates massive amounts of Pchlide (Meskauskiene et al. 2001). It was shown that FLU directly interacts with the Arabidopsis GluTR encoded by HEMA1, but not with the HEMA2 product. Furthermore, the FLU-dependent inactivation of GluTR works independently of the above- mentioned GluTR inhibition by heme (Goslings et al. 2004). Both the Arabidopsis flu mutant and the homologous barley tigrina d12 mutant (Lee et al. 2003) show a necrotic phenotype during photoperiodic growth emphasizing the importance of rapid and efficient down-regulation of ALA synthesis in darkness. To date, very little is known about the nature of the rapid control resulting in repression of ALA formation. Conclusive evidence for the role of Pchlide in the dark repression of ALA synthesis is still missing. The present study shows in a time course analysis rapid changes in the ALA synthesis rate and Pchlide accumulation after light–dark transition in green barley leaves and provides evidence for an almost instantaneous repression of ALA synthesis in darkness in correlation with an accumulation of POR-bound Pchlide. Results ALA synthesis in green barley leaves is immediately repressed in darkness Incubation of green barley leaves with levulinic acid, a competitive inhibitor of ALA dehydratase, facilitated time-resolved determination of ALA accumulation in both light and darkness. Several pre-experiments were performed to optimize our analysis of the in vivo metabolic activities. Using increasing amounts of levulinic acid we found that application of 40 mM levulinic acid was sufficient for rapid, reproducible accumulation of ALA within the experimental time period which correlates with the ALA-synthesis capacity (Supplementary Fig. S1a). Despite the detectable amounts of accumulated ALA due to inhibition by levulinic acid we found that Pchlide accumulation after 2 h of dark incubation was almost similar in leaves incubated with and without levulinic acid. These findings are explained by incomplete inhibition of ALA dehydratase (Supplementary Fig. S1b) allowing simultaneous analysis of ALA and Pchlide accumulation in the barley leaves. Leaves exposed to light intensity of 100 μmol photons m−2 s−1 accumulated ALA at a constant rate of 260 pmol ALA mg FW−1 h−1 (Fig. 1a). After transfer to darkness ALA accumulation decreased instantaneously and continued at a very low rate (30 pmol ALA mg FW−1 h−1). When leaves were retransferred to light after 90 min dark incubation, enzyme activity was restored rapidly and ALA synthesis proceeded at the rate observed before dark incubation. Northern (Fig. 1b) and Western (Fig. 1c) blot analyses revealed no differences in expression of analyzed enzymatic steps of tetrapyrrole biosynthesis between light-exposed and dark-incubated samples with the exception of HEMA1 RNA and POR protein. The POR level was elevated in darkness and was diminished during long exposure to light following diurnal oscillation. In green tissue the anti-POR antibody mainly recognizes PORB (Holtorf and Apel 1996). Unfortunately, the GluTR antibody available in the laboratory did not recognize the homologous barley protein, so we cannot prove influence of reduced HEMA1 transcript level on GluTR amounts. However, a rapid increase in ALA synthesis after re-illumination reflects constant or elevated GluTR content. Fig. 1 View largeDownload slide (a) ALA accumulation in barley leaf discs incubated in a buffer containing 40 mM levulinic acid for indicated time at light intensities of 100 μmol photons m−2 s−1 and darkness. Linear accumulation of ALA was observed in light (open squares). When samples were transferred to darkness after 30 min preincubation in light further accumulation of ALA was prevented (filled squares). When dark-incubated samples were re-transferred to light ALA accumulation started again (open triangles). Data are given as means ± SD. The calculated ALA synthesis rates were 260 pmol ALA mg FW−1 h−1 in continuous light, 30 pmol ALA mg FW−1 h−1 in darkness and 225 pmol ALA mg FW−1 h−1 after re-illumination. (b) Northern blot analyses of RNA extracted from leaf samples after different light and dark (L, D) incubations for up to 120 min. Transcript levels of HEMA1, PORB and LHCB were determined. As additional control RNA was analyzed from green light-exposed intact barley leaves. (c) Western blot analyses of the protein levels of GSAT, POR and the FLU homologous barley TIGRINA D protein (FLU) in light- and dark-incubated leaf samples. Fig. 1 View largeDownload slide (a) ALA accumulation in barley leaf discs incubated in a buffer containing 40 mM levulinic acid for indicated time at light intensities of 100 μmol photons m−2 s−1 and darkness. Linear accumulation of ALA was observed in light (open squares). When samples were transferred to darkness after 30 min preincubation in light further accumulation of ALA was prevented (filled squares). When dark-incubated samples were re-transferred to light ALA accumulation started again (open triangles). Data are given as means ± SD. The calculated ALA synthesis rates were 260 pmol ALA mg FW−1 h−1 in continuous light, 30 pmol ALA mg FW−1 h−1 in darkness and 225 pmol ALA mg FW−1 h−1 after re-illumination. (b) Northern blot analyses of RNA extracted from leaf samples after different light and dark (L, D) incubations for up to 120 min. Transcript levels of HEMA1, PORB and LHCB were determined. As additional control RNA was analyzed from green light-exposed intact barley leaves. (c) Western blot analyses of the protein levels of GSAT, POR and the FLU homologous barley TIGRINA D protein (FLU) in light- and dark-incubated leaf samples. Pchlide accumulation in green barley leaves is stopped within one hour of dark incubation In darkness Pchlide is supposed to influence ALA formation by feedback inhibition (Reinbothe and Reinbothe 1996). Time-resolved Pchlide accumulation was monitored after light–dark transition of green barley leaves (Fig. 2). Within the first 30 min of dark incubation leaves accumulated up to 6-fold more Pchlide compared with Pchlide levels during illumination. Prolonged dark incubation (e.g. up to a 20 h period) did not significantly increase levels of accumulated Pchlide compared with short-term dark incubation during the experiments. When green leaves were re-exposed to light their Pchlide pool decreased and reached the steady-state level of illuminated leaves within minutes (Fig. 2). Fig. 2 View largeDownload slide Accumulation of Pchlide in barley leaf discs incubated in levulinic acid-containing buffer. Samples that were transferred to darkness (filled squares) accumulated Pchlide to a level six times higher than light-exposed plants (open squares). The dotted line indicates the Pchlide level reached after 20 h dark incubation of intact barley seedlings. Dark Pchlide content was diminished within <30 min of re-illumination (after 90 min). Data are given as means ± SD. Fig. 2 View largeDownload slide Accumulation of Pchlide in barley leaf discs incubated in levulinic acid-containing buffer. Samples that were transferred to darkness (filled squares) accumulated Pchlide to a level six times higher than light-exposed plants (open squares). The dotted line indicates the Pchlide level reached after 20 h dark incubation of intact barley seedlings. Dark Pchlide content was diminished within <30 min of re-illumination (after 90 min). Data are given as means ± SD. These data correlate very well with the observation that ALA synthesis is rapidly down-regulated in darkness to avoid massive flux into the tetrapyrrole biosynthesis pathway. To address the question of whether Pchlide accumulates only in the photo-convertible POR-bound or also in the free non-photo-convertible form within the first 2 h of darkness, we performed 77K fluorescence analyses of crude barley leaf extracts followed by Gaussian deconvolution of fluorescence data to detect potential hidden maxima. Non-photo- and photo-convertible Pchlide (Ex 433 nm) show a fluorescence emission maximum at 635 nm and 650 nm, respectively (Böddi et al. 1992, Franck and Strzalka 1992). During dark incubation only an emission maximum at 650 nm was detectable, indicating the photo-convertible POR-bound form of Pchlide. After 20 h dark incubation of young green barley leaves the fluorescence emission maximum at 635 nm can be assigned to free Pchlide (Fig. 3). The barley tigrina d12 mutant accumulated both the non-photo- and the photo-convertible Pchlide (Fig. 3). Fig. 3 View largeDownload slide 77K fluorescence spectra of intact barley wild-type Lomerit (WT) leaves at different incubation times in light and darkness. Within 1 h of darkness only the POR-bound photoactive form of Pchlide accumulated having a fluorescence emission maximum at 650 nm (excitation at 433 nm). After 20 h of darkness initial accumulation of free non-photo-convertible Pchlide (emission at 635 nm) was observed. The fluorescence spectrum of tigrina d12 seedlings was obtained after 20 h of darkness. Spectra were normalized to chlorophyll fluorescence emission maximum at 680 nm. Using the Gaussian deconvolution method emission bands in the complex spectra were assessed. Fig. 3 View largeDownload slide 77K fluorescence spectra of intact barley wild-type Lomerit (WT) leaves at different incubation times in light and darkness. Within 1 h of darkness only the POR-bound photoactive form of Pchlide accumulated having a fluorescence emission maximum at 650 nm (excitation at 433 nm). After 20 h of darkness initial accumulation of free non-photo-convertible Pchlide (emission at 635 nm) was observed. The fluorescence spectrum of tigrina d12 seedlings was obtained after 20 h of darkness. Spectra were normalized to chlorophyll fluorescence emission maximum at 680 nm. Using the Gaussian deconvolution method emission bands in the complex spectra were assessed. ALA synthesis is not suppressed in darkness when Pchlide levels do not increase As ALA synthesis in green barley leaves immediately declined after dark transition and Pchlide rapidly accumulated, it was important to perform additional experiments to confirm a Pchlide accumulation-dependent short-term feedback inactivation of ALA formation in darkness. Therefore, Pchlide accumulation in darkness was minimized by inhibiting the tetrapyrrole biosynthesis pathway upstream of Pchlide formation. The photosensitizing herbicide acifluorfen inhibits protoporphyrinogen IX oxidase (PPOX) resulting in a massive accumulation of protoporphyrinogen IX (Protogen). Fig. 4a displays the strongly reduced Pchlide formation in acifluorfen-treated and dark-incubated leaves compared with untreated leaves. It is worth mentioning that acifluorfen-treated leaves synthesized slightly more ALA than control leaves in light (Fig. 4b). Fig. 4 View largeDownload slide Acifluorfen treatment impedes accumulation of Pchlide and causes stimulated ALA synthesis in darkness. (a) Barley leaves that were transferred to darkness accumulate Pchlide to a level almost five times higher than that of illuminated leaves within <1 h of dark incubation (black squares). Leaves that were treated with acifluorfen accumulated Pchlide to a lower extent in darkness (grey squares). (b) Acifluorfen-treated leaves showed a slightly increased ALA accumulation in the light, but failed to repress ALA synthesis in darkness (30 min light + 60 min dark). The dashed line indicates the average pool of ALA formed within 30 min light. (c) The treatment of barley leaves with acifluorfen caused a massive accumulation of Proto. Data are given as means ± SD. The bar on the top indicates the light (white) and dark (black) incubation periods. Fig. 4 View largeDownload slide Acifluorfen treatment impedes accumulation of Pchlide and causes stimulated ALA synthesis in darkness. (a) Barley leaves that were transferred to darkness accumulate Pchlide to a level almost five times higher than that of illuminated leaves within <1 h of dark incubation (black squares). Leaves that were treated with acifluorfen accumulated Pchlide to a lower extent in darkness (grey squares). (b) Acifluorfen-treated leaves showed a slightly increased ALA accumulation in the light, but failed to repress ALA synthesis in darkness (30 min light + 60 min dark). The dashed line indicates the average pool of ALA formed within 30 min light. (c) The treatment of barley leaves with acifluorfen caused a massive accumulation of Proto. Data are given as means ± SD. The bar on the top indicates the light (white) and dark (black) incubation periods. However, it is more important that ALA synthesis of acifluorfen-treated leaves was not reduced in darkness, while ALA synthesis of untreated leaves showed the expected dark repression (Fig. 4b). This indicates that ALA formation in darkness depends on the level of Pchlide in plastids and ALA synthesis can be uncoupled from dark-repression by acifluorfen-mediated prevention of Pchlide accumulation. Heme is proposed to act as feedback inhibitor of ALA synthesis (Cornah et al. 2003, Goslings et al. 2004). To exclude possible heme effects, the heme content was determined in acifluorfen-treated and control leaves. The level of non- covalently bound heme was not reduced in acifluorfen-treated barley leaves, during neither light nor dark exposure (Table 1). Table 1 Content of non-covalently bound heme in barley leaves Sample  Treatment  Non-covalently bound heme (pmol mg FW−1)  30 min light + 60 min light  Control  8.0 ± 0.5    Acifluorfen  7.6 ± 1.0  30 min light + 60 min dark  Control  8.0 ± 0.1    Acifluorfen  9.0 ± 1.4  Sample  Treatment  Non-covalently bound heme (pmol mg FW−1)  30 min light + 60 min light  Control  8.0 ± 0.5    Acifluorfen  7.6 ± 1.0  30 min light + 60 min dark  Control  8.0 ± 0.1    Acifluorfen  9.0 ± 1.4  Leaf discs of acifluorfen-treated and control barley plants were incubated in levulinic acid-containing buffer and exposed to light, or 30 min light followed by 60 min darkness. The amount of non-covalently bound heme was determined. Data are given as means ± SD. View Large The acifluorfen treatment caused the expected massive accumulation of Protogen, which is rapidly oxidized to Proto (Witkowski and Halling 1988, Witkowski and Halling 1989). HPLC analysis revealed similar contents of Proto(gen) in dark and light-incubated acifluorfen-treated barley leaves, because ALA synthesis was not inactivated in darkness and resembled that of light-incubated leaf samples (Fig. 4c). Eventually, treatment with acifluorfen leads to photodamage of plants (Lermontova and Grimm 2006). To exclude any adverse effects of acifluorfen on plastid integrity and physiology within the time frame of ALA synthesis analysis, photosynthetic parameters and membrane lipid peroxidation were determined. Tables 2, 3 point out that acifluorfen treatment of barley plants did not perturb photosynthetic processes within the first 6 h of illumination. The pigment content did not differ significantly between treated and untreated leaves (Table 2). Maximum quantum yield of PSII photochemistry, actual quantum yield of photochemical energy conversion in PSII and non-photochemical quenching of variable chlorophyll fluorescence in acifluorfen-treated leaves resembled control leaves (Table 2). The content of thiobarbituric acid reactive substances (TBARS) did not differ significantly between control and acifluorfen-treated plants within the first 2 h of illumination indicating integrity of plastid membranes (Table 3). But continued photoperiodic growth for 2 days caused substantial phototoxic effects of acifluorfen as indicated by elevated amounts of TBARS in barley leaves compared with control values and leaf necrosis, which are explained by massive accumulation of Proto(gen) in herbicide-treated leaves (Table 3, Supplementary Fig. S2). Table 2 Photosynthetic parameters and pigment contents of barley leaves treated with acifluorfen for 20 h in darkness and subsequently transferred to light Parameter  % of control  Fv/Fma  99.7 ± 1.0  ΦPSIIb  102.5 ± 10.0  qNc  104.2 ± 19.0  Chlorophyll content  90.7 ± 7.3  Carotenoid content  90.4 ± 6.7  Parameter  % of control  Fv/Fma  99.7 ± 1.0  ΦPSIIb  102.5 ± 10.0  qNc  104.2 ± 19.0  Chlorophyll content  90.7 ± 7.3  Carotenoid content  90.4 ± 6.7  Acifluorfen-treated plants did not show any reduction in photosynthetic capacity compared with untreated controls within the first 6 h of light treatment. The pigment contents of untreated barley leaves were 287 ± 33 ng mg FW−1 total chlorophyll and 125 ± 15 ng mg FW−1 carotenoids. The chlorophyll a/b ratio was 4.7. aMaximum quantum yield of PSII photochemistry. bActual quantum yield of photochemical energy conversion in PSII. cNon-photochemical quenching of chlorophyll fluorescence. View Large Table 3 Membrane integrity of barley plants treated with acifluorfen for 20 h in darkness and subsequently transferred to light Sample  Treatment  TBARS (μmol mg FW−1)  2 h light  Control  9.1 ± 1.2    Acifluorfen  10.1 ± 3.2  2 d lighta  Control  11.4 ± 4.0    Acifluorfen  18.1 ± 1.5  Sample  Treatment  TBARS (μmol mg FW−1)  2 h light  Control  9.1 ± 1.2    Acifluorfen  10.1 ± 3.2  2 d lighta  Control  11.4 ± 4.0    Acifluorfen  18.1 ± 1.5  Accumulation of TBARS corresponds to membrane integrity. Acifluorfen-treated leaves did not show any membrane disintegration after 2 h of light exposure, while membrane damage was indicated after 2 d of light/dark incubation. aPhotoperiodic growth. View Large All other attempts to inhibit Pchlide accumulation during dark incubation of barley leaves and to reproduce the results obtained in the presence of acifluorfen by independent experimental approaches were unsuccessful. Inhibitors, such as the iron-chelators dipyridyl (Duggan and Gassman 1974) and thujaplicin (Oster et al. 1996) were applied similarly to acifluorfen, but prevented Pchlide accumulation in darkness less efficiently (data not shown). This may be attributed to limitations of inhibitor uptake through the leaf surface. Non-repressed ALA synthesis of the tigrina d12 mutant in darkness resembles response to acifluorfen treatment While dark-grown wild-type angiosperms immediately down-regulate ALA biosynthesis, the Arabidopsis flu and the homologous barley tigrina d12 mutants accumulate excessive amounts of Pchlide as a consequence of elevated ALA formation during the dark period (Hansson et al. 1997, Meskauskiene et al. 2001, Lee et al. 2003). To substantiate the regulatory relationship between immediate dark-repression of ALA synthesis and rapid Pchlide accumulation, we included examinations of the kinetics of ALA synthesis and Pchlide accumulation of the tigrina d12 mutant and its wild-type variety Bonus in darkness. Bonus wild-type leaves followed the same pattern of light-stimulated and dark-repressed ALA synthesis as the wild-type variety Lomerit, but show a lower ALA synthesis rate in light (Figs. 1, 5). The tigrina d12 leaves (200 pmol ALA mg FW−1 h−1) showed a 2-fold increased ALA synthesis in light compared with Bonus wild type (100 pmol ALA mg FW−1 h−1). Due to the lack of the functional repressor protein tigrina d12 did not decelerate ALA formation in darkness (Fig. 5). As a consequence of non-repressed ALA synthesis Pchlide content increased during darkness to a level that exceeds the amount of available POR protein resulting in the occurrence of free non-photo- convertible Pchlide (Fig. 3; Gough and Kannangara 1979, Hansson et al. 1997). Fig. 5 View largeDownload slide ALA accumulation in wild-type and tigrina d12 leaves during light and dark incubation. When exposed to light, the tigrina d12 (open circles) leaves exhibited an elevated ALA synthesis rate as opposed to the corresponding wild type (open squares). After dark transition wild type (filled squares) rapidly repressed ALA synthesis while the mutant (filled circles) continued without decreasing enzyme activity. Data are given as means ± SD. Fig. 5 View largeDownload slide ALA accumulation in wild-type and tigrina d12 leaves during light and dark incubation. When exposed to light, the tigrina d12 (open circles) leaves exhibited an elevated ALA synthesis rate as opposed to the corresponding wild type (open squares). After dark transition wild type (filled squares) rapidly repressed ALA synthesis while the mutant (filled circles) continued without decreasing enzyme activity. Data are given as means ± SD. Diminished ALA synthesis in low light also correlates with Pchlide accumulation ALA synthesis rate can be modulated over a range of increasing light intensities. It remains open whether ALA synthesis is attenuated at very low light intensities by the same regulatory mechanism as in darkness. ALA synthesis in green leaves of barley wild-type Lomerit was determined at light intensities <30 μmol photons m−2 s−1 to examine enzymatic activities at the transition between dark-repression and light-stimulation of ALA formation. Below 10 μmol photons m−2 s−1 ALA synthesis was reduced. In parallel the Pchlide steady-state level increased with decreasing light quantities (Fig. 6a). It is suggested that this elevated Pchlide level contributed to the diminished ALA synthesis rate at very low light intensities. Fig. 6 View largeDownload slide ALA (filled squares) and Pchlide (filled circles) accumulation in barley leaf discs of (a) wild-type Lomerit, (b) wild-type Bonus and (c) tigrina d12 incubated for 2 h in a buffer containing 40 mM levulinic acid at different light intensities (white light). In wild type but not in tigrina d12 ALA synthesis rate was decreased at light intensities <10 μmol photons m−2 s−1 down to a very low level in darkness. Data are given as means ± SD. Fig. 6 View largeDownload slide ALA (filled squares) and Pchlide (filled circles) accumulation in barley leaf discs of (a) wild-type Lomerit, (b) wild-type Bonus and (c) tigrina d12 incubated for 2 h in a buffer containing 40 mM levulinic acid at different light intensities (white light). In wild type but not in tigrina d12 ALA synthesis rate was decreased at light intensities <10 μmol photons m−2 s−1 down to a very low level in darkness. Data are given as means ± SD. ALA synthesis and Pchlide levels at different light intensities were also determined in Bonus wild-type and tigrina d12 seedlings (Fig. 6b,c). The light-intensity dependency of ALA synthesis differed between tigrina d12 seedlings and its wild type. Consistent with the results of tigrina d12 ALA synthesis in darkness (Fig. 5), ALA synthesis was not reduced at low light intensity (3 μmol photons m−2 s−1), although the Pchlide levels (3 pmol mg FW−1) were already elevated compared with that of wild type (0.55 pmol mg FW−1). Discussion Rapid post-translational control represses dark ALA synthesis in green leaves Light-dependent Pchlide reduction in angiosperms enables synthesis of new chlorophyll molecules exclusively upon light exposure. Pchlide is the only tetrapyrrole intermediate accumulating in darkness. It was previously demonstrated that Pchlide accumulation is inversely correlated with down- regulation of ALA synthesis in dark-grown green leaves and etiolated seedlings and therefore a regulatory role for Pchlide was proposed (Nadler and Granick 1970, Castelfranco et al. 1974, Fluhr et al. 1975, Gough 1978, Ford and Kasemir 1980, Stobart and Ameen-Bukhari 1984, Stobart and Ameen-Bukhari 1986, Huang and Castelfranco 1989). This regulatory mechanism is reasonable as down-regulated ALA synthesis in darkness prevents excessive accumulation of Pchlide. Its photoreactivity is harmful for light-exposed plants. However, due to experimental limitations a time course analysis of the interdependence between Pchlide accumulation and activities of ALA synthesis has ultimately never been provided. Moreover, the proposed feedback control from Pchlide and POR could not be substantiated by protein–protein interactions between enzymes involved in Pchlide reduction and ALA synthesis. The first substantial information about the mode of action was provided by the analysis of the negative regulator FLU, which interacts with GluTR and was shown to be required for dark-repression of ALA synthesis (Meskauskiene et al. 2001). Inhibition of ALA formation was previously studied at light–dark transitions in leaves of different plant species or isolated chloroplasts. A decline in ALA synthesis was demonstrated after the transfer of the samples to darkness. But the elucidation of rapid turn-off mechanisms of ALA synthesis was hindered by technical limitations. ALA synthesis was determined in leaves after levulinic acid incubation for several hours (Fluhr et al. 1975, Huang and Castelfranco 1989, Beator and Kloppstech 1993, Kruse et al. 1997, Papenbrock et al. 1999, Goslings et al. 2004). Taking advantage of both high metabolic activities of green primary barley leaves and improved methodology ALA synthesis could be analyzed within 30 min. In our experimental setup the synthesis of ALA was constant during light exposure and stopped almost instantaneously when leaf samples were transferred to darkness (Fig. 1). During the subsequent dark period a slow but continuing synthesis of additional ALA molecules was detected (Figs. 1, 4b). Observed differences at the level of transcripts and proteins involved in ALA formation may not explain the rapid dark-repression of ALA synthesis; an effective and rapid mechanism most likely acts at the post-translational level. In analogy, ALA formation recovers almost immediately and completely after dark to light transition (Fig. 1). Under these assay conditions Pchlide accumulation in dark-incubated barley leaf samples reached a maximum within 30–60 min that did not further increase during prolonged dark periods. The underlying mechanism repressing ALA synthesis in darkness avoids Pchlide levels exceeding the binding capacity of POR and efficiently prevents accumulation of photosensitizing free Pchlide (Fig. 3). However, the molecular mechanisms of the light–dark switch of ALA formation are still not entirely elucidated. Inhibition of Pchlide accumulation hinders repression of ALA synthesis Our results corroborate the hypothesis of a negative feedback regulation of ALA formation in green tissues originating from the Mg branch of the tetrapyrrole biosynthesis pathway. When Pchlide accumulation was prevented, inactivation of ALA synthesis did not occur. This was achieved by inhibition of PPOX with acifluorfen that effectively stopped the metabolic flow in the tetrapyrrole biosynthetic pathway (Fig. 4; Witkowski and Halling 1988) without affecting the integrity of plastid physiology within the experimental time period (Tables 2, 3). However, Pchlide accumulation was inhibited (Fig. 4a) and, in turn, ALA formation was not repressed indicating a deficit in the repression signal in dark-incubated acifluorfen-treated leaves. This deregulation of ALA synthesis is rather attributed to deficient Pchlide accumulation than to increased levels of Proto(gen). Protogen and Proto are intermediates that do not accumulate in significant amounts, when green leaves are transferred from light to darkness. Moreover, transgenic tobacco plants with reduced amounts of the PPOX do not show deregulation of ALA formation in darkness (Lermontova and Grimm 2006). Becerril et al. (1992) reported that acifluorfen-treated duckweed accumulated Proto(gen) in darkness over a period of 5 h, before ALA synthesis was stopped and the metabolite flow was negatively affected. It was suggested that acifluorfen treatment did not completely block PPOX leading to delayed Pchlide accumulation and Pchlide-mediated feedback inhibition of ALA synthesis. However, Pchlide accumulation is mandatory for efficient post-translational inactivation of ALA synthesis in darkness. It was previously proposed that elevated levels of free heme control ALA synthesis in darkness (Masuda et al. 1990, Cornah et al. 2003, Goslings et al. 2004, Beale 2006) and GluTR activity was directly inhibited either by heme (Pontoppidan and Kannangara 1994) or by an unknown plastid-localized factor (Srivastava et al. 2005). In our experiments the contents of non-covalently bound heme in light-exposed barley leaves did not differ from dark-incubated samples. Furthermore acifluorfen treatment did not result in significant reduction in the heme content (Table 1). Since we determined the heme content of green tissue but not of isolated plastids we cannot fully exclude changes in the so-called free heme pool within plastids. As a light-dependent enzymatic step involved in heme metabolism has not been reported, yet, it is implausible that free heme accumulates rapidly after light to dark transition and contributes to the observed rapid dark-repression of ALA synthesis. It is likely that the remaining ALA synthesis in dark-incubated green leaves is directed into the Fe branch of tetrapyrrole biosynthesis, because maximum Pchlide accumulation was observed after 1 h of dark incubation. It is not excluded that heme-dependent inhibition of ALA synthesis or GluTR activity reported previously, plays a role in an efficient long-term tuning of ALA synthesis in planta. It was shown that in light–dark grown tobacco plants heme accumulated and Fe chelatase activity increased in the dark periods (Papenbrock et al. 1999). POR and FLU are involved in rapid repression of ALA synthesis ALA synthesis was shown to be repressed at low light intensities with simultaneous accumulation of Pchlide (Fig. 6). Keeping in mind that ALA synthesis is almost immediately repressed in darkness and Pchlide accumulation occurs rapidly, it is conclusive that elevated Pchlide contents are almost instantaneously communicated to repress ALA synthesis by a post-translational mechanism. The regulatory mechanism of Pchlide-mediated dark repression of ALA synthesis involves possibly the ternary complex of NADPH:Pchlide POR (Oliver and Griffiths 1982). It is suggested that accumulating Pchlide bound to POR is responsible for the slow-down of ALA synthesis under low light conditions or in darkness (Fig. 6). Stobart and Ameen-Bukhari (1986) used ALA feeding experiments and titration with different amounts of ALA to investigate the light-induced ALA synthesis in etiolated barley seedlings. Both authors emphasized that Pchlide bound to POR affects light-stimulated ALA synthesis and free POR is mandatory to synthesize ALA. We proposed that due to its low content relative to etiolated tissue, POR is rapidly saturated with Pchlide in dark-incubated green tissue and this correlates with the fast post-translational inhibition of ALA formation. The negative regulator FLU is probably a substantial part of the above-mentioned feedback control. FLU deficiency in the barley mutant tigrina d12 prevents repression of ALA synthesis under low light intensities and in darkness and causes higher ALA synthesis during illumination (Figs. 5, 6). Further details of the feedback regulation sensing Pchlide accumulation in dark-incubated and low-light-exposed green leaves (Fig. 6) need to be elucidated. ALA formation is likely inhibited at the post-translational level. Future experiments are required to demonstrate that FLU also functions in response to light intensity. The current knowledge comprises Pchlide accumulation in darkness as the starting point of the feedback mechanism controlling ALA synthesis in green leaves. It is suggested that under low light intensities ALA synthesis experiences a similar Pchlide-induced repression. It is very likely that the FLU protein represents a signaling component that interacts directly with GluTR. The potential of FLU to inhibit GluTR activity in darkness and to modulate ALA synthesis at different light intensities in green tissues remains a subject of further investigation. Materials and Methods Plant growth conditions Barley seedlings [Hordeum vulgare L. var. Lomerit and Bonus (wild type and tigrina d12)] were grown on vermiculite for 6 days in a 14 h light/10 h dark rhythm or continuous light, respectively, in a growth chamber at 22–23°C with normal light intensity of 80–100 μmol photons m−2 s−1. Experimental design Equal-sized primary leaves were harvested 2 h after onset of light and cut 3–4 cm below the tip into pieces of 1 cm length. Unless otherwise stated, 100 mg leaf material (corresponding to six leaf segments) was used for each measurement. For herbicide treatment 5-day-old barley seedlings were sprayed four times with 200 μM acifluorfen [in 10 mM Tris–HCl, pH 8.0, 0.05% (v/v) Tween 80] during a 20 h dark incubation. For analysis of the ALA synthesis rate, RNA and protein expression, and levels of Pchlide, porphyrins and heme, leaf material was incubated in 50 mM Tris–HCl, pH 7.2 and 40 mM levulinate for 30 min at 22–23°C. After preincubation the samples were transferred to darkness for a time period indicated in the figure legends or remained under the same light condition. Standard light intensity was 100 μmol photons m−2 s−1. Different light intensities were applied as indicated. Samples were taken at different time points during the incubation time, dried on paper towels, weighed and frozen in liquid nitrogen. Leaf samples of 20 h dark-incubated acifluorfen-treated or control plants were subjected to measurements of TBARS and photosynthetic capacity at different time points after beginning of illumination. Determination of ALA ALA content was measured using the method of Mauzerall and Granick (1956). Frozen samples were homogenized, resuspended in 20 mM potassium phosphate buffer pH 6.8 and centrifuged for 10 min at 16 000 × g; 400 μl of the supernatant was mixed with 100 μl of ethyl acetoacetate and boiled for 10 min at 100°C. Samples were mixed with 500 μl of modified Ehrlich’s reagent [373 ml of acetic acid, 90 ml of 70% (v/v) perchloric acid, 1.55 g of HgCl2, 9.10 g of 4-dimethylaminobenzaldehyde and 500 ml of H2O] and centrifuged for 5 min at 16 000 × g. Absorption was measured at 526, 553 and 720 nm and the ALA content of the samples was calculated using a standard curve generated by commercial ALA (Sigma-Aldrich Inc.). Determination of the Pchlide content Extraction of Pchlide followed the protocol of Koski and Smith (1948). Leaf samples were fixed with steam for 2 min, frozen, ground in liquid nitrogen and extracted three times in alkaline acetone (9:1, 100% acetone: 0.1 N NH4OH). After centrifugation at 16 000 × g for 10 min the supernatants were collected and chlorophyll was removed by a stepwise extraction with 1 vol, 1 vol and 0.3 vol of 100% n-hexane. The Pchlide content of the samples was quantified using HPLC according to Langmeier et al. (1993). Samples were injected and separated by a reverse phase column (Waters RP-18 ODS Hypersil 3 μM, 12.5 × 4 cm). Solvents A (20% 1 M ammonium acetate pH 7.0, 80% methanol, v/v) and B (20% acetone, 80% methanol, v/v) were used with the following program: 15 min linear gradient from 100% A to 100% B, continued with a 10 min isocratic run with B, returned to 100% A with a 3 min linear gradient and followed by a 2 min isocratic run with solvent A. The flow rate was set to 1.0 ml min−1. Eluates were analyzed by a fluorescence detector using an excitation wavelength of 435 nm and recording at 644 nm. For calibration a Pchlide standard was extracted from 7-day-old etiolated barley leaves (Koski and Smith 1948) and quantified using the extinction coefficient ε(Pchlide in diethyl ether, 623 nm) of 35 600 M−1 cm−1 (Dawson et al. 1986). 77K fluorescence spectroscopy Leaf material (10 mg) was ground in a buffer containing 50 mM Tricine and 0.4 M Sorbitol, pH 7.8 (Krupa et al. 1987). A 77K spectroscopy capillary tube was filled with a mixture of 150 μl of the homogenate and 350 μl of glycerol and frozen in liquid nitrogen. 77K fluorescence emission (600–700 nm) was recorded on a FP-6500 fluorescence spectrometer (JASCO Inc.) using an excitation wavelength of 433 nm. Spectra were fitted by multiple Gaussian deconvolution using the nonlinear least squares fitting of the R software package (R Development Core Team 2008). Initial estimates of means and standard deviation were derived by analyzing the first and second derivatives of the spectra. Determination of heme The content of non-covalently bound heme was determined as described in Weinstein and Beale (1984) and Yaronskaya et al. (2003). Frozen leaf material (300 mg) was homogenized and chlorophyll was removed by washing with cold acetone: 0.1 N NH4OH (9:1, v/v) followed by centrifugation at 16 000 × g for 10 min. Non-covalently bound heme was extracted from the pellets twice with 5 ml of extraction solution [2 ml of dimethyl sulfoxide, 10 ml of acetone and 0.5 ml of 37% (v/v) HCl], followed by centrifugation at 16 000 × g for 10 min. Heme was transferred to ether by adding 3 ml of diethyl ether, 2 ml of saturated NaCl and 10 ml of water. The ether phase was transferred to a new tube. Washing with 3 ml of ether was repeated and both ether phases were combined and concentrated to a final volume of 2 ml. A 3 ml DEAE–Sepharose CL-6B column was equilibrated with 1 column volume of diethyl ether: ethanol (3:1, v/v). Heme extracts were mixed with 0.7 ml of ethanol and applied to the column. The column was washed with two volumes of diethyl ether:ethanol (3:1, v/v), 1 vol of diethyl ether: ethanol (1:1, v/v) and finally with 1 vol of ethanol. Heme was eluted with a mixture of ethanol:acetic acid: water (81:9:10, v/v/v) and quantified spectrophotometrically using the extinction coefficient ε(398 nm) of 144 mM−1 cm−1 (Weinstein and Beale 1983). Determination of pigments and other porphyrins Chlorophyll and carotenoids were extracted using 80% (v/v) acetone with 10 μM KOH and determined spectrophotometrically according to Lichtenthaler (1987). Protoporphyrin IX (Proto) and Mg-porphyrins were extracted with acetone: methanol: 0.1 N NH4OH (10:9:1, v/v/v) and analyzed via HPLC as described in Papenbrock et al. (1999). RNA analysis Plant total RNA was extracted following the TRIsure protocol (Bioline GmbH, Luckenwalde, Germany). Ten micrograms of each sample was separated electrophoretically and transferred to Hybond N+ membrane (GE Healthcare) using standard protocols (Sambrook and Russel 2001). Probes were amplified by PCR from a barley cDNA using gene-specific primers for HvHEMA1 (5′-GCAGCCGCGGGCGCATTCGCCGCCGCCA AG-3′, 5′-TGCCAATTTCTGACAACTTGTTTGACT-3′), HvPORB (5′-GCGGCCACTTCCTTCCTCCCGTCGG-3′, 5′-AGCTGGCGC ACGTTCTTGACGAACTGG-3′), and HvCAB (chlorophyll a/b binding protein 5′-TCTGTCTTCCTCCACCTTCGCCGGG-3′, 5′- ACATGGAGAACATGGCCAGCGTCC-3′) and labelled with [α-32P]dCTP using random oligonucleotide priming (HexaLabel DNA labelling Kit; Fermentas GmbH, Germany). Prehybridization and hybridization were performed in Church buffer (Church and Gilbert 1984) at 70°C. Final washings were carried out to a stringency of 0.1 × SSC and 0.1% (w/v) SDS at 65°C. Protein analysis Plant total protein was extracted by grinding frozen leaf material in a buffer containing 2% (w/v) SDS, 56 mM NaCO3, 12% (w/v) sucrose, 56 mM DTT and 2 mM EDTA, pH 8.0, followed by heating at 70°C for 20 min and centrifuging at 16 000 × g for 10 min. The protein concentration in the supernatant was measured using BCA protein assay reagent (Perbio Science) after trichloracetic acid precipitation. Proteins were separated on 12% or 15% polyacrylamide gels, transferred to Hybond-C membranes (GE Healthcare) and probed with specific antibodies using standard protocols (Sambrook and Russel 2001). Determination of membrane lipid peroxidation Approximately 200 mg leaf material was homogenized in 0.25% (w/v) thiobarbituric acid in 10% (w/v) TCA and heated at 95°C in a glass tube in a water bath for 30 min to form malondialdehyde according to De Vos et al. (1989). The amount of TBARS was calculated from the difference in absorbance at 532 nm and 600 nm using an extinction coefficient ε(532 nm – 600 nm) of 155 mM−1 cm−1. Measuring the photosynthetic capacity Chlorophyll fluorescence parameters were measured using a PAM 2000 (Walz, Effeltrich, Germany). Conventional fluorescence nomenclature was used (van Kooten and Snel 1990, Rohácek 2002). Attached leaves were dark adapted for 30 min for determination of the maximum quantum yield of PSII photochemistry (calculated as ratio Fv/Fm = (Fm – Fo)/Fm) After determining the basic fluorescence (Fo) an 800 ms saturating pulse was applied to ascertain the maximum fluorescence (Fm). ΦPSII, the actual quantum yield of photochemical energy conversion in PSII, was determined according to Genty et al. (1989) on growth light-adapted leaves. The non-photochemical quenching of variable chlorophyll fluorescence, qN, was calculated by the formula qN = 1 – (Fm′ – Fo′)/(Fm – Fo). Fm and Fo were determined as described above following the determination of Fm′ and Fo′ upon 15 min adaptation to actinic light at an intensity appropriate to growth light intensity. Miscellaneous All experiments were performed with three to six independent experiments. In order to test significant differences between calculated values equality of variances was tested by a F-test followed by Student’s t-test using a P-value of <0.05. Acknowledgments The authors thank B. Hickel for help with the HPLC analyses and acknowledge funding by Deutsche Forschungsgemeinschaft (DFG – Research Unit 804 and Sonderforschungsbereich 429) to B.G. and Studienstiftung des deutschen Volkes to E.P. Abbreviations Abbreviations ALA 5-aminolevulinic acid GluTR glutamyl t-RNA reductase GSAT glutamate 1-semialdehyde aminotransferase Pchlide protochlorophyllide POR NADPH:protochlorophyllide oxidoreductase PPOX protoporphyrinogen IX oxidase Proto protoporphyrin IX Protogen protoporphyrinogen IX Proto(gen) accumulated protoporphyrinogen IX and protoporphyrin IX PSII photosystem II TBARS thiobarbituric acid reactive substances. 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Journal

Plant and Cell PhysiologyOxford University Press

Published: Apr 7, 2010

Keywords: Acifluorfen 5-Aminolevulinic acid Chlorophyll Dark repression FLU Heme Protochlorophyllide tigrina d 12

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