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First published online March 30, 2007; 10.1104/pp.106.095182 Plant Physiology 144:468-478 (2007) © 2007 American Society of Plant Biologists OPEN ACCESS ARTICLE
Light and Metabolic Signals Control the Selective Degradation of Sucrose Synthase in Maize Leaves during Deetiolation1,[OA]Department of Plant Biology, University of Illinois, Urbana, Illinois 61801 (Q.-S.Q., S.C. Hardin, S.C. Huber); United States Department of Agriculture, Agricultural Research Service, Photosynthesis Research Unit, Urbana, Illinois 61801 (S.C. Huber); and Boyce Thompson Institute, Cornell University, Ithaca, New York 14853 (J.M., T.P.B.)
The content and activity of Suc (Suc) synthase (SUS) protein is high in sink organs but low in source organs. In this report, we examined light and metabolic signals regulating SUS protein degradation in maize (Zea mays) leaves during deetiolation. We found that SUS protein accumulated in etiolated leaves of the dark-grown seedlings but was rapidly degraded upon exposure to white, blue, or red light. This occurred concurrent with the accumulation of photosynthetic enzymes, such as Rubisco and Rubisco activase, and enzymes of Suc biosynthesis such as Suc-phosphate synthase. Deetiolation-induced SUS degradation was not inhibited by the proteasome inhibitor MG132. Moreover, neither full-length nor truncated SUS phosphorylated at the serine-170 site was found in the crude 26S proteasome fraction (150,000g postmicrosomal pellet) isolated in the presence of MG132. However, SUS degradation was strongly inhibited by feeding cycloheximide or amino acids to detached leaves, while Suc feeding had no effect. Of the amino acids tested, exogenous glutamate had the greatest effect. Collectively, these results demonstrate that SUS protein degradation during deetiolation: (1) is selective; (2) can be triggered by either blue- or red light-mediated signaling pathways; (3) does not involve the 26S proteasome; and (4) is inhibited by free amino acids. These findings suggest that SUS degradation is important to supply residues for the synthesis of other proteins required for autotrophic metabolism.
Suc is one of the primary end products of photosynthesis in plants. It is synthesized from UDP-Glc and Fru-6-P under the catalysis of Suc-P synthase (SPS) and Suc-6-P phosphatase. Suc is mainly produced in mature leaves, the predominant source organs of plants, and translocated via the phloem to photosynthetically inactive sink organs or tissues such as roots, reproductive structures, developing organs, and storage tissues. Suc is then metabolized in sink organs for energy production and carbon-skeleton formation (Winter and Huber, 2000  ; Fernie et al., 2002; Koch, 2004). The control of Suc transport and metabolism plays a critical role in plant growth and development, particularly during the source-to-sink transition (Lalonde et al., 1999; Winter and Huber, 2000; Truernit, 2001; Koch, 2004).
Suc synthase (SUS) converts Suc and UDP into UDP-Glc and Fru in plant cells (Tsai, 1974
SUS gene expression, protein level, and activity are tightly regulated during the sink-to-source transition in several organs. For example, there is a sharp decrease in SUS cleavage activity in the leaf-like cladodes of the Opuntia ficus-indica when they shift from sink to source (Wang et al., 1998
Deetiolation is an important developmental process that occurs when the dark-grown etiolated seedling is exposed to light (Quail, 2002
As a C4 plant, maize utilizes two morphologically and biochemically distinct cell types, the bundle sheath and mesophyll, to first fix carbon into C4 acids in the mesophyll and then decarboxylate them in the bundle sheath to provide Rubisco with a CO2-enriched environment (Sheen, 1999 In this study, we utilized maize seedlings undergoing deetiolation to study the degradation of SUS protein during the sink-to-source transition. We first established that emerged, etiolated maize leaf blades maintained high levels of SUS protein, and that exposure to light triggered SUS degradation (even in detached leaves). We report that SUS is degraded in response to white, red, or blue light. Amino acid and Suc feeding experiments were conducted to investigate the effects of metabolites on SUS degradation and the role of the 26S proteasome pathway was interrogated through the use of inhibitors. Collectively, the results suggest that SUS degradation was mediated through both red and blue light signals, but that SUS degradation was not mediated by the 26S proteasome. Inhibition of SUS degradation by free amino acids is a new level of control and suggests that SUS degradation in vivo may partially supply amino acids for synthesis of light-induced proteins.
Etiolated Emerged Maize Leaves Have Abundant SUS Protein That Is Degraded in Deetiolation
SUS is an abundant protein in many heterotrophic organs including maize endosperm, roots, stems, and leaf elongation zone (Chourey et al., 1986
SPS Is Very Low in Emerged Etiolated Leaves and Increased in Deetiolation
To follow the changes in Suc biosynthetic capacity during deetiolation, we monitored SPS protein levels using SPS-specific antibodies (Fig. 1, A and C). The antibodies detected two bands: an upper band that had a Mr of approximately 105 kD and a lower band of around 90 kD (Fig. 1A). The 90-kD lower band is likely an antigenically related protein, as it was found exclusively in the microsomal fraction whereas SPS is strictly soluble (see Fig. 4). Maize has five isoforms of SPS and their sizes range from 108 to 119 kD (Castleden et al., 2004
SUS Is Phosphorylated at Ser-15 and Ser-170 in Deetiolation
Maize SUS has been shown to be phosphorylated in vivo at two sites: Ser-15 and Ser-170 (Huber et al., 1996
It should be noted that the anti-pS15 and anti-pS170 antibodies cross-react to different extents with the three maize SUS isoforms (Duncan et al., 2006
To understand whether the increased phosphorylation of SUS protein during deetiolation was due to increased protein kinase activity, we assayed calcium-dependent protein kinase (CDPK) and calcium-independent kinase (SnRK1-like) activities before and after exposure to light. Soluble leaf proteins were extracted from etiolated and deetiolated leaves and fractionated by FPLC anion exchange chromatography. Peptide kinase activities of CDPK and SnRK1 were determined using peptides SS4 (based on Ser-15 of SUS1) and SP49 (based on Ser-158 of SPS) as substrates, respectively. As shown in Figure 1F, maize seedlings generally had a higher level of CDPK activity compared to SnRK1-like activity. Importantly, CDPK activity was increased during deetiolation, whereas SnRK1-like activity was unchanged (Fig. 1F). Increased activity of CDPKs might contribute to the increased phosphorylation of SUS protein at the Ser-15 and Ser-170 sites during deetiolation.
To begin to characterize the light signals that induce SUS degradation during deetiolation, maize seedlings were grown in the dark, then given white, red, or blue light treatments. As shown in Fig. 2A , under both red and blue light conditions, SUS protein levels decreased with light exposure up to 4 d but at a slightly slower rate compared to white light (Fig. 2B). These results indicated that SUS degradation during deetiolation could be mediated by both red and blue light signaling pathways. The difference in rates may reflect the higher fluence rate of white light (100 µmol m–2 s–1) compared to red (3 µmol m–2 s–1) and blue (12 µmol m–2 s–1) light used in these experiments. However, it is also possible that SUS degradation requires a synergism of red- and blue light-mediated signals.
Exposure of etiolated leaves to white, red, or blue light also induced a 2- to 4-fold accumulation of SPS, involved in Suc biosynthesis, and two enzymes of CO2 fixation (Rubisco large subunit [RbcL] and Rubisco activase). Interestingly, light quality affected the proteins somewhat differently. With SPS and RbcL, the increase in protein level paralleled the fluence rates of light used, with white > blue > red light (Fig. 2, C and D). In contrast, Rubisco activase was induced equally under the three light regimes (Fig. 2E). In addition, Rubisco activase and SPS began to accumulate without an apparent lag following the onset of illumination, whereas RbcL protein only began to accumulate at a high rate following 1 d of exposure to light. Thus, the three enzymes differed somewhat, but the important result is that all three were induced by either red or blue light photoreceptors, concurrent with the loss of SUS protein.
To elucidate the mechanism of SUS degradation, etiolated leaves were detached and placed in water (control) or solutions containing MG132, an inhibitor of the 26S proteasome, or cycloheximide (CHX), an inhibitor of cytoplasmic protein synthesis. As shown in Figure 3A , illumination of detached leaves resulted in loss of SUS protein concurrent with accumulation of SPS. Importantly, the detached leaves provided an experimental system to feed inhibitors to leaves during deetiolation. Feeding leaves with the proteasome inhibitor, MG132, had only a slight effect on SUS degradation (Fig. 3B) or SPS accumulation (Fig. 3A), but did increase ubiquitinated proteins (Fig. 3, E and F), indicating that the inhibitor was effective in blocking their degradation via the 26S proteasome. These results suggest that: (1) the 26S proteasome is not essential for SUS degradation; and (2) SPS accumulation in the light is a result of increased protein synthesis rather than a reduction in the rate of protein degradation.
Interestingly, SUS degradation appears to be mediated by a light-dependent process that requires cytoplasmic protein synthesis. As shown in Figure 3, A and B, SUS degradation during deetiolation was almost completely inhibited by CHX, which is an inhibitor of cytoplasmic protein synthesis. As would be expected, SPS protein accumulation was also completely blocked by feeding CHX (Fig. 3A). Finally, it is also noteworthy that in the presence of CHX, the stoichiometry of SUS phosphorylation at the Ser-15 and Ser-170 sites was greatly reduced relative to the controls (Fig. 3, C and D), perhaps indicating that phosphorylation was carried out by a process in which some component(s) was light sensitive and de novo synthesis was required.
We isolated proteasomes by differential centrifugation (Hardin et al., 2003 The pS170-SUS content was very low in the 150 KP proteasome fractions whereas there was significant pS170-SUS in the soluble fraction (150 KS; Fig. 4A). However, the full-length pS170-SUS signal was very weak in the soluble fraction and the major form of pS170-SUS was a slightly truncated fragment (Fig. 4A). The membrane-associated SUS (100 KP; Fig. 4A) also contained relatively little pS170-SUS. In contrast, pS15-SUS protein was very high in the proteasome, membrane, and soluble fractions (Fig. 4A) and relative phosphorylation state increased with time after exposure to light (Fig. 4C). SPS protein only existed in the 150 KS soluble fraction and increased with light exposure (Fig. 4A, bottom section). The approximately 90 kD protein detected by the anti-SP68 antibodies was primarily found in the microsomal membrane fraction and thus was judged not to be an authentic form of SPS. It is also worth noting that the authentic, higher Mr (approximately 105 kD subunit) form of soluble SPS was never found in the 150 KP proteasome fraction. Thus, the presence of full-length SUS in the proteasome fraction cannot be ascribed to contamination or carry over of soluble proteins. We also assayed proteasome activity in the 150 KP fraction isolated from leaves and found that the MG132-inhibited 26S proteasome activity in the preparations was essentially unchanged after 1 d of light exposure (Fig. 4D).
In preliminary experiments, we found that detached leaves lost SUS protein more rapidly than attached leaves during deetiolation (data not shown). This prompted us to conduct sugar and amino acid feeding experiments to determine whether SUS degradation during deetiolation is controlled by metabolite levels. It was found that supply of Gln to detached leaves decreased the rate of SUS degradation relative to control leaves (provided only water) during deetiolation as evidenced by 2.5-fold higher levels of SUS protein after 2 d of deetiolation (Fig. 5, A and B ). Supply of Suc alone to detached leaves had no effect on SUS degradation and the presence of Suc did not affect the stabilizing action of Gln (Fig. 5B). Accumulation of SPS protein after 2 d of deetiolation was relatively unaffected by exogenous Gln and Suc (Fig. 5C), suggesting that amino acids and energy reserves required for protein synthesis during deetiolation are not normally rate limiting. Other amino acids were also effective in reducing SUS degradation. As shown in Figure 5D, exogenous supply of Asn, Asp, Glu, or Gln all resulted in significant stabilization of SUS protein with Glu being the most effective. Thus, Glu and/or Glu-derived metabolites function in stabilizing SUS protein during deetiolation, suggesting that the degradation of SUS could be coordinated with concurrent protein synthesis by changes in the pool of free amino acids.
To further test the coupling between SUS degradation and concurrent protein synthesis, we measured free amino acid pools in detached leaves undergoing deetiolation in the presence and absence of CHX. In the control leaves (supplied with water), there was a progressive loss of SUS protein over the course of the experiment, and degradation was almost completely inhibited by CHX (Fig. 6, A and B ), consistent with the results presented in Figure 3. The total free amino acid pool decreased about 20% in control leaves during deetiolation compared to the amino acid pool in etiolated leaves at the start of the experiment. In the presence of CHX, the free amino acid pool was consistently slightly higher than in the control leaves (Fig. 6C). These results are consistent with the notion that there is a close coupling between free amino acid pools and the degradation of heterotrophic proteins, such as SUS.
The Development of a Deetiolation Assay to Study SUS Metabolism
In this study, we used deetiolation of maize leaves as a system to study the degradation of SUS protein. During normal light development, maize leaves undergo the sink-to-source transition as the blade emerges from the surrounding sheath. Correspondingly, SUS protein is reduced to low levels just prior to emergence of the green leaf blade. The pattern of leaf SPS protein content is essentially the mirror image, in concert with the role of SUS in Suc degradation and SPS in Suc biosynthesis (Nguyen-Quoc et al., 1990
Selective protein turnover or degradation is essential for normal plant growth and development, and functions in many aspects of physiological and cellular processes such as the precise removal of short-lived regulatory proteins, the elimination of abnormal proteins, the maintenance of amino acid pools for continual protein synthesis, and the recycling of carbon and nitrogen during senescence and apoptosis (Vierstra, 1996
In this study, however, no significant inhibition of SUS degradation during deetiolation was observed in the presence of MG132 despite the fact that MG132 substantially increased the level of ubiquitinated proteins (Fig. 3, E and F). In addition, although SUS cosedimented with proteasomes, the polypeptide patterns were not suggestive of SUS degradation: (1) full-length rather than truncated SUS was the most abundant component that cosedimented with the proteasome fraction (Fig. 4A); (2) full-length SUS was associated with the proteasome even in the etiolated seedlings (Fig. 4A); and (3) there was little pS170-SUS in the proteasome fraction (Fig. 4A). Therefore, SUS degradation during deetiolation might not involve the proteasome pathway. It is possible that SUS cosediments with proteasomes as a result of binding to the proteasome ATPase RPT3, which was detected by Holtgräwe et al. (2005)
The autophagic pathway is another major protein degradation process in plants. In this pathway, proteins are engulfed in membrane vesicles and delivered into vacuoles for degradation by a wide range of proteases, peptidases, lipases, and other hydrolytic enzymes (Thompson and Vierstra, 2005
We showed in this report that amino acid feeding inhibited SUS degradation during deetiolation, while Suc feeding had no effect (Fig. 5). Thus, the degradation of SUS protein during deetiolation may be triggered by utilization of free amino acids for the synthesis of other proteins such as SPS and the array of photosynthetic enzymes that are required for autotrophic growth and development. Indeed, SUS is an abundant protein in heterotrophic maize tissues. The SUS1 protein is the predominant isoform expressed in etiolated maize shoots and has been estimated to constitute about 15% of the total soluble protein (Duncan et al., 2006
Plant Material and Growth Conditions
Maize (Zea mays; inbred B73 or Pioneer 3183) seeds were soaked in water overnight and germinated and grown in a soil mixture in the dark for a week. Seedlings were then transferred to white light (100 µmol m–2 s–1) and harvested daily for 6 d; seedlings kept in the dark served as controls. Leaves were harvested in liquid nitrogen and stored at –80°C prior to analysis. For light quality experiments, the same protocol was followed, except that the seedlings were moved to growth chambers and illuminated for up to 4 d with white (100 µmol m–2 s–1), red (3 µmol m–2 s–1), or blue (12 µmol m–2 s–1) light as indicated. Light chambers were as described previously (Markelz et al., 2003 For Suc and Gln feeding experiments, maize seeds were germinated and grown in the dark for a week. Then the seedlings were cut off from the base and placed in incubation medium (5 mM MES-KOH, pH 6.0) containing 20 mM Suc, 10 mM Gln, or 20 mM Suc + 10 mM Gln, in white light. Detached seedlings were harvested at days 1 and 2, frozen in liquid nitrogen, and stored at –80°C for immunoblot analysis. For inhibitor experiments, the same protocol was followed, except that detached shoots were placed in incubation medium (5 mM MES-KOH, pH 6.0) containing CHX (50 µM) or MG132 (100 µM) as indicated. Seedlings were harvested at days 1 and 2, frozen in liquid nitrogen, and stored at –80°C for immunoblot analysis.
Leaf tissue was extracted into 1x SDS buffer containing 62.5 mM Tris-HCl, pH 6.8, 0.7 M 2-mercaptoethanol, 2% (w/v) SDS, 1 M urea, 10% (v/v) glycerol, 0.005% (w/v) bromphenol blue, 5 mM NaF, 1 mM Na3VO4, 1 mM 4-(2-aminoethyl) benzenesulfonylfluoride hydrochloride (AEBSF), and 2 mM EDTA. SDS-PAGE (7% acrylamide) was conducted by loading 10 µg protein per lane. Proteins were electrophoretically transferred to polyvinylidene difluoride membranes (Immobilon-FL, Millipore) for immunoblot analysis. Membranes were blocked with 2% (w/v) fish gelatin (Sigma) in phosphate-buffered saline containing 5 mM NaH2PO4, pH 7.4, and 150 mM NaCl. The Alexa Fluor 680-conjungated secondary antibodies (Molecular Probes) were detected by an Odyssey infrared imager system (LI-COR), and densitometry was performed with the instrument's image processing software.
Production of rabbit polyclonal antibodies against the SUS1 peptides pS15 (CRVLSRLHpSVRERIGD), pS170 (CQFLNRHLpSSKLFHDK), and PH (CHILRVPFRTENGIVRKWISR), and the SPS peptide (KAQVDVGNLKFPAIRRRKC) have been described previously (Hardin et al., 2003
For measurement of enzyme activity, proteasomes were isolated as previously described (Hardin et al., 2003
For immunoblot analysis, the preparation of proteasomes was as described (Hardin et al., 2003
Frozen maize leaf samples were extracted into protein extraction buffer (100 mM MOPS, pH 7.5, 10 mM DTT, 5 mM EDTA, 1 mM EGTA, 20 mM NaF, 5 mM Na2MoO4, 1 mM Na3VO4, 0.5 µM microcystin-LR, 1 mM phenylmethylsulfonyl fluoride, 5 mM caproic acid, 1 mM benzamidine, 2 µM E64, 2 µM leupeptin, 10 µM MG132, 5 µg mL–1 soybean [Glycine max] trypsin inhibitor, 1% [w/v] PVPP, 0.25 M Suc, and 2% [w/v] polyethylene glycol [PEG-8000]). Clarified extracts were produced by filtration through Miracloth (CalBiochem) and centrifugation at 35,000g and 4°C. Proteins precipitated by addition of PEG-8000% to 20% were collected by centrifugation and solubilized in resuspension buffer (50 mM MOPS, pH 7.5, 5 mM DTT, 50 mM Suc, 1 mM EDTA, 10 mM NaF, 1 mM Na2MoO4, 0.1 mM Na3VO4, 0.5 mM AEBSF, 2.5 mM caproic acid, 0.5 mM benzamidine, and 1 µM E64). Proteins were applied to a 5-mL SOURCE 15Q (Amersham) anion-exchange column in buffer A (50 mM MOPS, pH 7.5, 2 mM DTT, 50 mM Suc) and eluted at 4°C with a 50-mL linear gradient of 0 to 500 mM NaCl in buffer A at a flow rate of 1 mL min–1.
Peptide kinase activities were as described by Huang and Huber (2001)
Etiolated maize leaves (‘Pioneer B73’) were cut at the base and transferred to incubation medium (5 mM MES-KOH, pH 6.0) containing 50 µM CHX as indicated. Leaves were harvested at the start of the experiment (day 0) and after 1 or 2 d of exposure to light. Leaf samples (about 0.7 g fresh weight) were extracted with 0.1 M HCl (5 x 2 mL), purified, and derivatized according to Silva et al. (2003)
The authors gratefully acknowledge the contribution of anti-Rubisco and anti-Rubisco activase polyclonal antibodies by Dr. Archie R. Portis (U.S. Department of Agriculture-Agricultural Research Service, University of Illinois at Urbana-Champaign). Mention of a trademark or proprietary product does not constitute a guarantee or warranty by the U.S. Department of Agriculture-Agricultural Research Service and does not imply its approval to the exclusion of other products that might also be suitable. Received December 21, 2006; accepted March 26, 2007; published March 30, 2007.
1 This work was supported by funds from the U.S. Department of Energy (grant no. DE–AI05–91ER20031 to S.C. Huber). 
2 Present address: BASF Plant Science, 26 Davis Drive, Research Triangle Park, NC 27709. 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: Steven C. Huber (schuber1{at}life.uiuc.edu).
[OA] Open Access articles can be viewed online without a subscription. www.plantphysiol.org/cgi/doi/10.1104/pp.106.095182 * Corresponding author; e-mail schuber1{at}life.uiuc.edu; fax 217–244–4419.
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ABSTRACT
RESULTS
-32P] ATP (150 cpm pmol–1) and 10 mM MgCl2, and stopped after 10 min at room temperature. 
