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First published online July 14, 2006; 10.1104/pp.106.084483 Plant Physiology 142:88-97 (2006) © 2006 American Society of Plant Biologists OPEN ACCESS ARTICLE
The Role of Pheophorbide a Oxygenase Expression and Activity in the Canola Green Seed Problem1,[W],[OA] inskáDepartment of Plant Biology, University of Illinois, Urbana, Illinois 61801 (D.W.C., D.R.O.); Department of Biology, University of Bern, CH–3013 Bern, Switzerland (A.P., S.H.); and Photosynthesis Research Unit, United States Department of Agriculture/Agricultural Research Service, Urbana, Illinois 61801 (D.R.O.)
Under normal field growth conditions, canola (Brassica napus) seeds produce chloroplasts during early seed development and then catabolize the photosynthetic machinery during seed maturation, producing mature seeds at harvest that are essentially free of chlorophyll (Chl). However, frost exposure early in canola seed development disrupts the normal programming of Chl degradation, resulting in green seed at harvest and thereby significantly devaluing the crop. Pheophorbide a oxygenase (PaO), a key control point in the overall regulation of Chl degradation, was affected by freezing. Pheophorbide a, the substrate of PaO, accumulated during late stages of maturation in seeds that had been exposed to freezing during early seed development. Freezing interfered with the induction of PaO activity that normally occurs in the later phases of canola seed development when Chl should be cleared from the seed. Moreover, we found that the induction of PaO activity in canola seed was largely posttranslationally controlled and it was at this level that freezing interfered with PaO activation. The increased accumulation of PaO transcript and protein levels during seed development was not altered by the freezing episode, and the increase in PaO protein was small compared to the increase in PaO activity. We found that PaO could be phosphorylated and that phosphorylation decreased with increasing activity, implicating PaO dephosphorylation as an important posttranslational control mechanism for this enzyme. Two PaO genes, BnPaO1 and BnPaO2, were identified in senescing canola leaves and during early seed development, but only BnPaO2 was expressed in maturing, degreening seeds.
Canola (Brassica napus) is an important oil seed crop grown extensively in North America and northern Europe with annual yields exceeding 7 million metric tons. Canola is the world's third-most important vegetable oil crop, in significant part due to the low levels of erucic acid and glucosinolates in canola oil (Levadoux et al., 1987  ; Zhang et al., 2004). However, the chlorophyll (Chl) content is significantly higher than that found in other major vegetable oils and is the biggest quality impediment in the canola oil industry.
During the early stages of seed development, photosynthetically produced carbohydrate is transferred from the leaves and silique walls to the seeds for the synthesis of oil and other storage products. In canola seeds, the conversion of sugars to fatty acids is the primary metabolic flux with more than 60% of carbon stored as oil (Schwender et al., 2004b
The key reactions in Chl degradation are catalyzed by chloroplast localized enzymes (Matile et al., 1999
Magnesium-dechelatase, for which the gene and protein are yet to be identified, is responsible for removing the magnesium ion from the tetrapyrrole producing the chlorin molecule pheophorbide (Pheide) a (Shioi et al., 1996
PaO, a nonheme iron monooxygenase localized to the inner envelope of maturing gerontoplasts, opens the porphyrin macrocycle by adding two oxygen atoms (Matile and Schellenberg., 1996
The conversion of Pheide a to the colorless primary fluorescent Chl catabolite (pFCC) is a complex step that involves not only PaO but also red Chl catabolite reductase (RCCR), a stromal protein, and is the causal gene in acd2 mutant of Arabidopsis. The opening of the Pheide a macrocycle by PaO produces a red colored catabolite (RCC), an intermediary product, which in turn is reduced by RCCR in a reaction requiring ferredoxin to form pFCC, a colorless compound that is detected by its distinctive blue fluorescence (Wüthrich et al., 2000 The major objective of this study was to identify those steps in Chl degradation in maturing canola seeds that are disrupted by exposure to freezing temperatures early in seed development. The results show that freezing interfered with the induction of PaO activity that normally occurs in the later phases of canola seed development. Moreover, we found that the regulation of PaO activity was largely posttranslational and it was at this level that freezing interfered with PaO activation in canola seeds.
Nonlethal Freezing Exposure Prevented the Complete Clearing of Chl from Mature Canola Seeds
At 20 d after pollination (DAP), when seeds had attained about 45% of final dry weight (Fig. 1A
) and 60% of maximum Chl content (Fig. 2
), canola plants were cooled in the dark at 5°C/h until reaching –4°C, where the temperature was held for 6 h followed by rewarming at 5°C/h back to the growth temperature. The Chl content of seeds collected at intervals from 13 to 46 DAP was measured spectrophotometrically in N, N-dimethylformamide using the specific absorption coefficients of Porra and Grimme (1974)
The Effects of Freeze Exposure on Chl Loss Was Direct and Not Mediated by Differential Effects of Freezing on Seed Moisture Content
Although the interference with developmentally programmed Chl degradation in maturing canola seeds by freeze exposure is well established (Johnson-Flanagan and Thiagarajah, 1990
To determine candidate steps in the Chl degradation of canola seed that may be sensitive to freeze exposure, we investigated the effects of freeze exposure on pools of Chl degradation catabolites during canola seed development (Fig. 3
). The catabolites were separated by HPLC based on their polarity in organic solvent and were quantified by fluorescence spectroscopy, which has the sensitivity required to detect Chl catabolites in the trace amounts normally present. The concentration of each catabolite was determined from the fluorescence intensity data using equations developed for the quantification of tetrapyrrole moieties (Rebeiz, 2002
Freeze exposure on 20 DAP had little effect on catabolite pool sizes until 8 d later. By 28 DAP, there was a 3- to 4-fold freeze-induced increase in Pheide a levels. The accumulation of Pheide a in the freeze-treated seeds became more exaggerated as seed development progressed, showing a nearly 10-fold increase compared to control by 46 DAP (Fig. 3). During the later stages of seed development, 35 DAP and after, freeze treatment induced chlorophyllide (Chlide) a accumulation and, at seed maturity (i.e. 46 DAP), the percent increase of Chlide a exceeded that of Pheide a. The increased levels of Pheide a, and eventually Chlide a, in freeze-exposed seeds suggested that freezing interferes in some fashion with PaO function. That Pheide a accumulation preceded Chlide a accumulation suggested that a progressive feedback within the degradation pathway had developed.
In principle, a decrease in the products of PaO would also be anticipated if the increase in Pheide a in freeze-treated seeds is due to a decrease in PaO activity. However, in senescing leaves, pFCC, the product of the PaO/RCCR reaction, is present in exceedingly low and difficult-to-quantify amounts, and FCCs and RCCs do not accumulate to detectable levels. Noncolored catabolites are the only products downstream of PaO that accumulate in senescing leaves in canola (Pru
The gene sequence of PaO from Arabidopsis was used to clone and identify the orthologous genes in canola. Two different cDNA clones of PaO were isolated from senescing canola leaves (Supplemental Fig. 1), which will be referred to as BnPaO1 and BnPaO2. The codon-derived amino acid sequences had 92% identity with AtPaO. In comparison to each other, the derived amino acid sequences of the two canola clones were 98% identical. Like PaO from maize and Arabidopsis (Gray et al., 2004 There are two notable differences in protein sequence at the N-terminal region of BnPaO1, BnPaO2, and AtPaO, possibly due to an insertion/deletion event postdating the divergence of Arabidopsis and canola (Supplemental Fig. 2). Alternatively, as canola is an allotetraploid, BnPaO1 and BnPaO2 could be derived from the two ancestral genomes of canola. BnPaO2 has an additional Ser residue (Ser-29) when compared to BnPaO1 and AtPaO. In the same region, AtPaO has two Thr residues (Thr-27 and Thr-28) positioned where both BnPaO clones have an Asn and a Ser residue (Asn-27 and Ser-28). Both BnPaO clones have an extra Ala residue (Ala-30) not found in AtPaO. Furthermore, in the 75 to 78 region, BnPaO2 is completely missing a sequence of G-D-K-E found in both AtPaO and BnPaO1. Because BnPaO2 has an additional Ser residue (Ser-29) and is missing Gly-75, Asp-76, Lys-77, and Glu-78, collectively this protein has three fewer amino acids than BnPaO1. AtPaO has one less amino acid than BnPaO1 due to the missing Ala residue (Ala-30). While the expression of BnPaO2 was measurable in seeds throughout seed development, BnPaO1 transcripts were detectable only during early seed development. At 8 to 10 DAP, BnPaO2 transcripts showed nearly 5.5-fold higher levels of expression when compared to BnPaO1 transcripts, and BnPaO2 transcripts at 8 to 10 DAP were expressed at similar levels to 21 DAP canola seeds (data not shown). BnPaO2 transcripts accumulated as seed development progressed with >10-fold increase from 21 to 41 DAP, and the accumulation was not affected by the 6-h freezing exposure on 20 DAP (Fig. 4A ). The expression of both BnPaO transcripts was readily detectable in senescing canola leaves (data not shown).
Canola Seed PaO Shown to Be Regulated by a Freezing-Sensitive, Posttranslational Mechanism The amount of PaO protein from the membrane fraction of developing canola seeds was measured by immunoblot analysis and quantified by infrared imaging. PaO protein levels increased only about 2-fold (Fig. 4B) over the period of seed development in which BnPaO2 transcripts increased >10-fold (Fig. 4A). Freezing exposure given on 20 DAP did not affect PaO protein content at any subsequent point during seed development. The immunoreactive complexes of PaO protein from canola seed resolved into a doublet on miniblots using 10% or lower acrylamide, differing in apparent molecular mass by approximately 0.5 kD (Fig. 4C). Only the lighter, bottom band showed increasing intensity as the seeds matured. No effect of freezing was evident for either PaO band. Whereas PaO protein levels only doubled between 21 and 41 DAP and were unaffected by freezing exposure, PaO activity increased more than 10-fold over this period, and this induction was suppressed >20% by freezing (Fig. 4D). That the increase in PaO activity was at least 5 times greater than the increase in PaO protein implies strong posttranslational regulation of PaO during seed maturation.
Analysis of the BnPaO2 codon-derived amino acid sequence revealed two potential Ser/Thr calcium-dependent protein kinase (CDPK) phosphorylation recognition sites at Ser-18 and Thr-402 (Supplemental Fig. 1). These candidate phosphorylation sites are fully conserved in BnPaO1 and AtPaO. To investigate if there were changes in PaO phosphorylation corresponding to the changes we had observed in PaO activity, we used immobilized metal affinity chromatography (IMAC), which separates phosphorylated and nonphosphorylated proteins by binding the phosphorylated form, followed by immunoblot analysis. PaO phosphorylation was measured at 21 DAP when significant PaO protein was present (Fig. 4B) but activity was low (Fig. 4D) and compared with 41 DAP when PaO protein and activity levels were greatest. At 21 DAP, PaO was detected in both phosphorylated and nonphosphorylated fractions; however, the phosphorylated fraction contained nearly 5-fold higher amounts of PaO protein (Fig. 5A ). At 41 DAP, phosphorylated PaO proteins did not show a significant increase from 21 DAP; however, the nonphosphorylated fraction increased by 3-fold, showing that the increase in PaO content between 21 and 41 DAP could be nearly accounted for by the nonphosphorylated form. Neither antiphos-Thr nor antiphos-Ser antibodies (Zymed) reacted with PaO from either fraction (data not shown).
As validation of this approach to investigate PaO phosphorylation, we used IMAC columns with sucrose synthase (SUS), which has two well-known phosphorylation sites at Ser-15 and Ser-170 (Huber and Huber, 1996 ; Winter et al., 1997; Hardin et al., 2004). Phosphorylated SUS was isolated from basal elongating maize leaf tissue and purified by anion-exchange chromatography. After separation on the phosphoprotein affinity column, a SUS phosphospecific antibody detected phosphorylated SUS in both the fraction initially immobilized and subsequently eluted from the column with phosphate as well as in the flow-through fraction that did not bind to the column (Fig. 5C). The proportion of phosphorylated SUS in the bound versus unbound fraction was 5.6 to 1 when 0.5 mg of SUS was loaded on the column but only 2.45 to 1 when loading was increased to 1 mg. This result is indicative of exceeding the phosphoprotein binding capacity of the column, which is reported to have a maximum phosphoprotein binding capacity of 0.5 mg. Nevertheless, because the PaO-containing lysate loaded on the IMAC column contained only 10% to 20% of the total phosphoprotein of the SUS samples, overloading, and thus contamination, of the unbound fraction with phospho-PaO was unlikely. To further verify the phosphorylation of PaO, Pro-Q Diamond-blot staining (Invitrogen) was used to scrutinize the phosphorylated and nonphosphorylated IMAC fractions collected at 21 DAP (Fig. 6 ). As expected, Pro-Q analysis showed no bands representing phosphorylated proteins, including PaO, in the IMAC flow-through fraction, which should contain only nonphosphorylated proteins. However, a 52-kD band corresponding to PaO (western blot) was shown to align with a Pro-Q staining protein of a similar running molecular weight in the phosphorylated fraction eluted from the IMAC column.
There have been numerous demonstrations that the inhibition of PaO activity during leaf senescence leads to the accumulation of Pheide a and the inhibition of Chl degradation. In pao1, the insertional knockout mutant of Arabidopsis, approximately 80% of the Chl is retained during dark-induced leaf senescence; Chl that is degraded is largely accounted for by Pheide a accumulation in the leaf because no further downstream products can be detected (Pru inská et al., 2005). Similarly, senescing leaves of stay-green mutants of Festuca pratensis and Lolium temulentum accumulate Chlide and Pheide a, and reduced PaO activity has been shown to be the biochemical defect in these mutants (Vicentini et al., 1995; Roca et al., 2004). In freeze-exposed canola seeds, the induction of PaO activity later in seed development is impaired but not obliterated (Fig. 4D), resulting in a leaky phenotype where Pheide a accumulates (Fig. 3) and Chl degradation is slowed (Fig. 2) but not stopped. It might be expected that the 20% lower maximum induction of PaO activity in freeze-exposed maturing canola seeds (Fig. 4D) would prolong but not prevent full clearing of Chl from the seeds. However, another factor that can ultimately limit Chl clearing from the maturing seed is seed moisture content, and we believe that the intersection of these two control mechanisms is the cause of the green seed problem. When the moisture content of canola seeds dips below approximately 40%, many aspects of seed metabolism, including Chl degradation (Green et al., 1998), come to a halt. Under field conditions, freeze exposure may enhance the rate of seed desiccation (Green et al., 1998), further exacerbating the effects of the impaired induction of PaO activity on Chl degradation that are evident even when accelerated desiccation is prevented (Figs. 1B and 4D).
In this work, as with all cases in which PaO activity has been shown to be impaired (Hilditch et al., 1989 The profiles of PaO transcript level, protein content, and activity all qualitatively correlated with the progression of Chl degradation during seed development. However, PaO protein increased only 2-fold over the measured period of seed development, while transcript levels indicated >10-fold increase in expression. Some of the apparent discrepancy could be explained if PaO protein were highly stable compared to PaO transcript. If so, the rate of newly synthesized PaO protein could track the transcript level, yet the amount of new protein would be small in comparison to the accumulated stable pool. Indeed, PaO protein levels were one-half their maximum level at 21 DAP (Fig. 4B), prior to any measurable losses of Chl (Fig. 2) and when BnPaO transcript levels (Fig. 4A) were low. Beyond the dependence of PaO expression on transcript abundance, it is evident from our results that posttranslational control of the induction of PaO activity accompanies the degreening of canola seed. Our data showed that PaO protein content and activity have starkly different profiles during canola seed development. Whereas freeze exposure caused a statistically significant >20% reduction in the induction of PaO activity during 36 to 46 DAP, freezing was without any significant effect on PaO transcript or protein amounts. Although the posttranslational regulatory mechanism is not yet known, it appears likely that reversible protein phosphorylation is involved. Using both IMAC and Pro-Q Diamond-blot staining, we demonstrated a correlation between PaO dephosphorylation and increasing PaO activity during seed maturation. The stoichiometry of phosphorylated/dephosphorylated PaO decreased from 5 to 1 on 21 DAP to 2 to 1 on 41 DAP, with the dephosphorylated form of PaO increasing more than 3-fold over this interval. The 10-fold increase in PaO activity (Fig. 4D) illustrates that the observed change in PaO phosphorylation is large enough to have played a significant, although perhaps not exclusive, role in the posttranslational activation of this enzyme.
There are two CDPK recognition sites in BnPaO1, BnPaO2, and AtPaO protein sequences (Supplemental Fig. 1). The first CDPK site is located within the putative chloroplast target sequence, most likely cleaved once the protein is translocated; thus, this CDPK site is not likely to be involved in regulation of the enzyme in the chloroplast. The common CDPK consensus phosphorylation site is
That the freezing episode and any decrease in the observed rate of Chl degradation or PaO activity can be separated by more than a week indicates that freezing does not interfere directly with the PaO protein but with the program controlling Chl clearing from the seed. Since freezing appears to interfere with the activation of PaO by dephosphorylation, the delayed effect may be mediated at the level of PaO phosphorylation/dephosphorylation. Cold stress may indirectly lead to changes in protein phosphorylation and significant changes in CDPK activity by affecting the fluctuations in cytosolic Ca2+ levels (Martin and Busconi, 2001 The doublet seen in the immunoblots of PaO protein from canola seeds (Fig. 4C) is most likely due to the presence of both BnPaO1 and BnPaO2 proteins in the sample. The two distinct clones of PaO were isolated and identified from leaves of canola, differing by 526 D, which would account for the difference between the two bands. Although BnPaO1 transcript was expressed only early in seed development, it seems likely that its protein product persisted after BnPaO1 transcript disappeared. Only BnPaO2 transcript was detected at later stages in seeds. Interestingly, the doublet is not seen when the protein has been isolated on IMAC columns (Figs. 5 and 6). The protein retained on the IMAC column (i.e. phosphorylated form) corresponds to the lighter band of the doublet and is the band that increases during seed development (Fig. 4C). That the doublet is not seen when the sample is run over the IMAC column (Figs. 5 and 6) could support the notion that phosphorylation of BnPaO2 plays a role in the regulation of PaO activity in canola seed. Another possibility is that the doublet is due to differential posttranslational modification, including phosphorylation. However, because a single phosphorylation will add only 80 D, it seems unlikely that even multiple sites of reversible phosphorylation could alone account for the approximately 500 D difference estimated from electrophoretic mobility on SDS-PAGE.
The extent to which posttranslational control of PaO operates in leaf senescence is uncertain. Pru
Freezing exposure of developing canola seeds hinders the programmed degreening of the seed by interfering with the posttranslationally controlled induction of PaO activity. Although the rate of Chl degradation is slowed by only approximately 20%, the inhibition is sufficient to prevent Chl from fully clearing from the seed before seed moisture content dips below the threshold at which seed metabolism is suspended. The mechanism of the posttranslational control is unknown, but the increase in PaO activity during seed maturation corresponds to a decrease in the phosphorylation of the PaO enzyme. Canola has two highly homologous PaO genes that contain two candidate CDPK phosphorylation sites.
Plant Material Canola (Brassica napus L. cv Westar) seeds were germinated in moist vermiculite. Two-week-old seedlings were transplanted to 12-inch pots containing Sunshine Mix LC1 soil (SunGro Horticulture) and grown in growth chambers at 12-h photoperiod of 450 µmol photons m–2 s–1 and 22°C-day/16°C-night thermoperiod with a relative humidity of 70%. Canola plants were fertilized weekly with 20-20-20 Peters Professional fertilizer (United Industries). A set of 10 to 12 canola plants was grown for each of the control and freeze condition experiments. After bolting and prior to flowering, inflorescences with similar maturity were chosen from each canola plant for hand pollination. The tip of each flowering bud was cut open and hand pollinated. Each pollination was marked on the stems of the flower bud. When collecting samples, siliques were randomly chosen from different inflorescences of each plant and pooled. Freeze treatment was on whole plants in pots at 20 DAP in a darkened, controlled-environment chamber initially set at 22°C with high humidity. The temperature was decreased 5°C/h until reaching –4°C, where the temperature was held for 6 h and then increased 5°C/h until reaching the initial temperature of 22°C. Chamber conditions were then reset to normal growth conditions. Seeds were collected at preset intervals throughout the completion of seed development.
Seeds harvested at various developmental stages were ground in liquid nitrogen, and total RNA was extracted using Trizol reagent (Invitrogen) according to the manufacturer's protocol. RNA quality was checked on 1% Tris-acetate EDTA agarose gel and the A260 was determined. RNA (20 µg) was mixed with RQ1 DNase (Promega), buffer, RNasin, and water to a total volume of 50 µL and DNase treated following the manufacturer's protocol. One to two micrograms of DNase-treated total RNA was used as a template for cDNA synthesis using manufacturer's protocol (Invitrogen).
First-strand cDNA synthesis was performed using 1 to 2 µg DNase-treated RNA and Oligo(dT)18 primer according to manufacturer's instructions (Invitrogen). Quantitative real-time reverse transcription-PCR used QuantiTect SYBR Green PCR kit (Qiagen) with Cepheid SmartCycler according to the manufacturer's suggestions. Actin-3 was used as internal control. The primers used for amplifications were: BnPaO (i.e. BnPaO1 and BnPaO2), forward, 5'-GAAGCTCGCGCTGTTAAATC-3', reverse, 5'-CCCTTTGAATTGTCACCGTT-3'; BnPaO1, forward, 5'-ACGGCGGAGATAAGGAAGAA-3', reverse, 5'-CTCGACCCAGGAGCTGAA-3'; BnPaO2, forward, 5'-GACGGAAACTTCTCGACAGC-3', reverse, 5'-TTGAACTCAGACCCTTCTTCG-3'; actin-3, forward, 5'-ATGGTTAAGGCTGGTTTTGCT-3', reverse, 5'-ATCCTTCTGTCCCATTCCAAC-3'. All primers used were within the optimal amplicon range between 100 and 200 bp. For each gene, a range of six dilutions of genomic DNA of known concentration was amplified under the same conditions as the cDNA samples and then used as the standard curve to determine the number of cDNA molecules present in the experimental samples. At least four values were produced for each sample and repeated independently at least twice.
The primers for cloning PaO from canola leaves 5 d after darkening were designed based on the open reading frame sequence of At3g44880: forward, 5'-ATGTCAGTAGTTTTACTCTCTTCT-3', reverse, 5'-TCGATTTCAGAATGTACATAATCT-3'. PaO cDNA corresponding to the size of the open reading frame, approximately 1,600 bp, was cloned using a commercial cloning kit, pDrive Cloning Vector (Qiagen). Multiple colonies were sequenced from both directions with internal primers, M13 reverse and M13 forward (–20). The canola PaO open reading frame was completely sequenced in both directions using an automated DNA sequencing system, ABI 373A DNA sequencer (Applied Biosystems). Sequencher 4.5 (Genecodes) was used to align sequences, view chromatograms, and edit sequences at the Biotechnology Center of University of Illinois at Urbana-Champaign.
Canola seeds were homogenized in 5 mL/g fresh weight of a medium containing 400 mM sorbitol, 25 mM tricine-KOH, pH 8.0, 2 mM EDTA, 1 mM MgCl2, 0.1% bovine serum albumin (w/v), 5 mM polyethylene glycol 4,000, and 10 mM cysteamine-HCl using a chilled mortar and pestle. After filtration through a layer of nylon membrane, the homogenate was centrifuged at 10,000g for 4 min. The membrane pellet was resuspended with the above medium without EDTA, MgCl2, and bovine serum albumin, corresponding to 2 mL/g fresh weight leaf tissue and centrifuged at 10,000g for 4 min. The supernatant was removed and pellet frozen in liquid nitrogen and stored at –80°C.
Membrane fractions from chloroplasts were isolated as described above with the addition of the following: 1 µM E64 Cys protease inhibitor, 0.1 µM Microcystin-LR, 1 mM 4-(2-aminoethyl) benzenesulphonyl fluoride, 1 mM p-aminobenzophenone, 5 mM caproic acid, 10 µM leupeptin, 1 mM dithiothreitol, 1 mM NaF, 1 mM NaVO4, 1 mM EDTA, and 1 mM EGTA. The membrane pellet was resuspended in 2 µM E64 Cys protease inhibitor, 0.5 µM Microcystin-LR, 10 µM MG132 Mycoplasma genitalium proteasome inhibitor, 1 mM 4-(2-aminoethyl) benzenesulphonyl fluoride, 1 mM p-aminobenzophenone, 5 mM caproic acid, 5 µM leupeptin, 10 mM dithiothreitol, 20 mM NaF, 1 mM NaVO4, 5 mM EDTA, 1 mM EGTA, 10 mM NaMO4, and 5 µg/µL SBT1 subtilisin-like Ser protease.
Chl was extracted from ground canola seeds with 500 µL of N, N'-dimethylformamide in a microfuge tube using a mini plastic pestle. After three subsequent washings with 300 µL N, N'-dimethylformamide, the homogenate was centrifuged at 12,000g for 2 min at room temperature. The pellet was then extracted further with 300 µL N, N'-dimethylformamide and the pooled supernatants adjusted to a final volume of 2 mL. The Chl content of the seeds was determined spectrophotometrically using the specific absorption coefficients for Chl a and b of Porra and Grimme (1974)
Chl catabolites were separated by HPLC based on their polarity in organic solvent and were quantified by fluorescence spectroscopy according to published procedures (Rebeiz, 2002
The chloroplast membrane pellet (equivalent to 25 g fresh weight), isolated as described above, was resuspended in 1.25 mL of 25 mM Tris-MES, pH 8.0, and centrifuged twice at 12,000g for 5 min at 4°C to remove debris. The supernatant containing RCCR was transferred to a new tube and stored at –80°C until used for PaO assay. Following the removal of soluble proteins, the membrane pellet was washed three times in 5 mL 25 mM Tris-MES, pH 8.0, and centrifuged at 12,000g for 5 min followed by the removal of the supernatant. The washed membrane pellets were then resuspended in 750 µL Tris-MES, pH 8.0, and mixed with Triton X-100 to a final concentration of 1%. The membrane proteins were solubilized by shaking for 30 min at 4°C and centrifuged at 10,000g for 5 min. The supernatant containing the solubilized membrane proteins was used for PaO assay.
PaO activity was assessed by using a coupled PaO/RCCR assay according to established protocols (Hörtensteiner et al., 1995
Membrane proteins were extracted from seeds (Pru
Phosphoproteins were detected on polyvinylidene difluoride membranes using Pro-Q Diamond-blot staining protocol (Invitrogen). A Peppermint Stick phosphoprotein standard was used where 1 µL corresponded to 0.5 µg. Images were acquired on a Typhoon 8600 Variable Mode Imager (Amersham Pharmacia Biotech) following Pro-Q, with 532-nm laser, 580-nm bandpass filter at normal sensitivity, and a photomultiplier tube voltage of 300. IMAC separation of phosphorylated and nonphosphorylated PaO was accomplished using Qiagen PhosphoProtein Purification kit (Qiagen) according to manufacturer's instructions.
All data were analyzed by a mixed model ANOVA (PROC MIXED; SAS Institute, 1996) with treatment as a fixed factor, time as a repeated factor, and a compound symmetry covariance structure. Preplanned comparisons of means for each time point were analyzed with linear contrasts. Sequence data from this article have been deposited with the EMBL/GenBank data libraries under accession number DQ388373 for BnPaO1 and DQ388372 for BnPaO2.
We are grateful to Dr. John Gray for providing antibodies for PaO and Dr. Steven Huber for supplying SUS proteins and antibodies for SUS. We thank Dr. Adriana Ortiz-Lopez for her contributions to the initial stages of this research. We acknowledge Kateri Duncan, Dr. Shane Hardin, Dr. Aleel Grennan, and Qingiu Gong for their contributions to this research and Dr. Aleel Grennan for her expert help with the manuscript. Received June 2, 2006; accepted July 6, 2006.
1 This work was supported in part by the Integrative Photosynthesis Research Training Grant from the Department of Energy (grant no. DEFGO2–92ER20095), funded under the Program for Collaborative Research in Plant Biology, and by the Swiss National Science Foundation (grant no. 3100A0–105389).  The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Donald R. Ort (d-ort{at}uiuc.edu).
[W] The online version of this article contains Web-only data.
[OA] Open Access articles can be viewed online without a subscription. www.plantphysiol.org/cgi/doi/10.1104/pp.106.084483 * Corresponding author; e-mail d-ort{at}uiuc.edu; fax 217–244–0656.
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ABSTRACT
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-x-Basic-x-x-S/T, where the underlined Ser or Thr is phosphorylated, x is any residue, and 
