OPEN ACCESS ARTICLE

This Article Free via Open Access: OA OA Abstract Full Text (PDF) OA All Versions of this Article: 144/1/468    most recent pp.106.095182v1 Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in ISI Web of Science Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via CrossRef Citing Articles via ISI Web of Science (1) Citing Articles via Google Scholar Google Scholar Articles by Qiu, Q.-S. Articles by Huber, S. C. Search for Related Content PubMed PubMed Citation Articles by Qiu, Q.-S. Articles by Huber, S. C. Agricola Articles by Qiu, Q.-S. Articles by Huber, S. C.

BIOCHEMICAL PROCESSES AND MACROMOLECULAR STRUCTURES

Light and Metabolic Signals Control the Selective Degradation of Sucrose Synthase in Maize Leaves during Deetiolation^1,[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.)

	ABSTRACT

TOP ABSTRACT RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

 
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 synthase (SUS) converts Suc and UDP into UDP-Glc and Fru in plant cells (Tsai, 1974; Winter and Huber, 2000; Koch, 2004), providing substrates for carbohydrate synthesis. SUS is highly expressed in storage organs such as seeds, fruits, and tap roots and is often positively correlated with starch content and fruit size (Herbers and Sonnewald, 1998; Fernie et al., 2002). In the analysis of transgenic potato (Solanum tuberosum) tubers with reduced SUS expression, starch content and tuber yield were markedly decreased (Herbers and Sonnewald, 1998). Similarly, recent studies have demonstrated that SUS is the most actively expressed Suc-metabolism enzyme in the storage roots of sweet potato (Ipomoea batatas) and its expression pattern is very similar to ADP-Glc pyrophosphorylase, an essential enzyme in starch synthesis (Li and Zhang, 2003). Therefore, SUS is an important determinant of sink strength in plants (Herbers and Sonnewald, 1998; Barratt et al., 2001; Fernie et al., 2002).

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). SUS protein levels also vary along a developmental gradient within monocot leaves. In rice (Oryza sativa), SUS accumulates to higher levels in the lower part of the sheath (sink) relative to the upper portion of the sheath (source; Ishimaru et al., 2004). Similarly, SUS protein accumulation varies in a position-dependent manner in the nonchlorophyllous maize (Zea mays) leaf elongation zone that is enclosed within the leaf sheath; SUS protein is abundant within the leaf base (sink) but is reduced to the low level characteristic of green leaves as the leaf emerges from the sheath (Hardin et al., 2003). The decrease in SUS protein from base to tip is associated with increased phosphorylation at Ser-170, and it was postulated that phosphorylation at this site might be a trigger for degradation via the proteasome during the sink-to-source transition in developing maize leaves (Hardin et al., 2003).

Deetiolation is an important developmental process that occurs when the dark-grown etiolated seedling is exposed to light (Quail, 2002). During deetiolation, seedlings are redirected from skotomorphogenic to photomorphogenic development through blue and red/far-red sensing photoreceptors that mediate rapid changes in gene expression (Tepperman et al., 2001, 2004; Folta et al., 2003) and cell physiology (Folta and Spalding, 2001a, 2001b). In maize, a shift from dark to light growth induces an inhibition of mesocotyl elongation, a rapid greening of emerged leaves, and an increase in photosynthetic gene expression. At least three classes of photoreceptors likely mediate this transition in maize including the red/far-red sensing phytochromes and the blue light sensing phototropins and cryptochromes (Sawers et al., 2005).

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). This metabolic cooperation involves cell-specific accumulation of photosynthetic gene transcripts and enzymes (Sheen, 1999; Majeran et al., 2005). Transcript profiling has confirmed that genes involved in photosynthesis and carbohydrate synthesis, such as SPS, are highly up-regulated by light while genes that encode enzymes responsible for carbohydrate catabolism, such as SUS, are down-regulated by light (Ma et al., 2001). Phytochrome appears to play an important role in modulating the accumulation of photosynthetic transcripts in response to light, but may not be required for maintaining the pattern of photosynthetic gene expression in the leaf (Markelz et al., 2003).

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.

	RESULTS

TOP ABSTRACT RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

 

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; Nguyen-Quoc et al., 1990; Winter and Huber, 2000; Carlson et al., 2002), but it was not known whether etiolated seedling leaves contained significant amounts of SUS protein (emerged green leaves do not). To monitor SUS accumulation, maize seeds were germinated and grown in the dark for 7 d. On day 8 one set of plants was transferred to the light and the other remained in darkness. Leaf tissue was harvested for immunoblot analysis of SUS protein using the pan-isoform SUS antibodies, anti-SUS-PH (Fig. 1, A and B ). The anti-SUS-PH antibodies (Hardin et al., 2004) were produced against a conserved peptide sequence and react with all three maize isoforms (Duncan et al., 2006). As shown in Figure 1A (top section), there was abundant SUS in the etiolated leaf blade tissues that was maintained and, in fact, increased slightly in continued darkness. Upon illumination, the etiolated leaves began greening and at the same time SUS protein levels declined to reach a minimum after 4 d of light exposure (Fig. 1B). Thus, SUS protein level is controlled by the light-regulated sink-to-source transition. These results also suggest that SUS degradation is not a consequence of leaf development per se (e.g. leaf emergence), but requires a light signal.

View larger version (28K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 1. Reciprocal changes in SUS and SPS protein during deetiolation. Maize (‘Pioneer 3183’) seeds were germinated and grown in the dark for 7 d and then maintained in the dark or transferred to white light for another 6 d. Emerged leaves were harvested and proteins were extracted for analysis. A, Immunoblot analysis of SUS, SUS phosphorylation at the Ser-15 and Ser-170 sites, and SPS during deetiolation using the antibodies listed on the right side of each section. B to D, Densitometry of immunoblots in A showing changes in SUS protein (B), SPS protein (C), relative phosphorylation status of SUS at the Ser-170 (D) and Ser-15 (E) sites during deetiolation. In B to E, values are expressed on a relative basis with dark day 0 set as 100%; data are the average of three (with error bars) or two independent experiments. F, Peptide kinase activity assay in extracts from emerged leaves of dark-grown seedlings, or after 1 d of deetiolation. Proteins were fractionated by anion-exchange chromatography, and peptide kinase activities were assayed as described in "Materials and Methods."

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 M_r 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). Thus, the upper band represents the SPS isoforms present in both dark- and light-grown tissues (see also Hardin et al., 2004). In the dark, SPS protein levels remained low and constant, and were increased greatly after transfer to the light (Fig. 1, A and C).

View larger version (66K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 4. Analysis of SUS association with the microsome and proteasome fraction by cosedimentation. Maize (‘B73’) seeds were germinated and grown in the dark for a week and then transferred to light for another 3 d. The emerged leaves were harvested and microsomes and proteasomes were isolated by differential centrifugation as described in "Materials and Methods." A, Immunoblot analysis of SUS protein, SUS phosphorylation, and SPS protein using the antibodies listed on the right side of each section. 100 KP, 100,000g pellet (microsomes); 150 KS, 150,000g supernatant (soluble proteins); 150 KP, 150,000g postmicrosomal pellet (proteasome fraction). B and C, Densitometry of immunoblots in A showing changes in SUS protein (B) and relative phosphorylation of SUS at the Ser-15 site (C) in the proteasome, membrane, and soluble fractions. Values in B and C are expressed on a relative basis with values on dark day 0 set as 100%. D, Chymotrypsin-like activity of the 150 KP proteasome fraction assayed in the presence and absence of 10 µM MG132. Proteasome activity is expressed as nmol AMC mg protein–1 h–1. Mean values from two independent experiments are presented.

Maize SUS has been shown to be phosphorylated in vivo at two sites: Ser-15 and Ser-170 (Huber et al., 1996; Hardin et al., 2003). Serine-15 is a major phosphorylation site that affects cleavage activity and membrane association, whereas Ser-170 is a minor phosphorylation site that may be a trigger for degradation via the ubiquitin/26S proteasome. The changes of pS170- and pS15-SUS levels during deetiolation were determined using previously characterized pS170- and pS15-specific antibodies (Hardin et al., 2003, 2004). These antibodies specifically detect the phosphorylated forms of the corresponding peptides and do not recognize the unphosphorylated sequences. We found that the absolute pS170- and pS15-SUS levels decreased after exposure to light, generally in parallel with the decrease in SUS protein level (Fig. 1A). However, when expressed as a ratio with full-length SUS protein, the apparent phosphorylation stoichiometries at the Ser-170 and Ser-15 sites increased severalfold during deetiolation (Fig. 1, D and E). The anti-pS170 antibodies detected full-length SUS, as well as a smaller protein with a M_r of approximately 75 kD, and in some cases, a smaller fragment (Fig. 1A). However, the proteins smaller than approximately 90 kD were not detected by other SUS antibodies and thus may not be authentic degradation products of SUS.

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). However, in etiolated maize shoots, the SUS1 isoform accounts for >92% of the total SUS protein (Duncan et al., 2006) and thus the changes in phosphorylation status and protein level reported in this study can be almost completely attributed to the SUS1 isoform.

Calcium-Dependent Protein Kinase Activity in Maize Leaves Is Increased during Deetiolation

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.

Both Red and Blue Light Induced SUS Degradation in Maize Seedlings

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.

View larger version (48K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 2. Both red and blue light mediate SUS degradation and SPS synthesis during deetiolation. Maize (‘B73’) seeds were germinated and grown in the dark for 7 d and then maintained in the dark or transferred to light for another 4 d. The emerged leaves were harvested and proteins were extracted for analysis. A, Immunoblot analysis of SUS and SPS protein during deetiolation under white, red, and blue light. B to E, Densitometry of immunoblots challenged with anti-SUS (B), SPS (C), Rubisco (D; RbcL protein reported), and activase antibodies (E). Values are expressed on a relative basis with the corresponding dark control values at each day set as 100%.

MG132 Does Not Prevent SUS Degradation during Deetiolation

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.

View larger version (47K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 3. Effect of proteasome and protein synthesis inhibitors on SUS degradation during deetiolation. Etiolated maize (‘B73’) leaves were cut at the base and transferred to incubation medium (5 mM MES-KOH, pH 6.0) containing CHX (50 µM) or MG132 (100 µM), as indicated. Seedlings were harvested after 1 or 2 d of treatment in the light or dark as indicated. A, Immunoblot analysis of SUS, SUS phosphorylation at the Ser-15 and Ser-170 sites, and SPS during deetiolation. B to E, Densitometry of immunoblots in A showing changes in SUS (B), and relative phosphorylation status of SUS at the Ser-170 (C) and Ser-15 (D) sites after 2 d of deetiolation. In B to D, values of light day 2 samples are expressed on a relative basis with values on dark day 0 set as 100%. E, Immunoblot showing ubiquitinated proteins in seedlings provided water (control) or MG132 after 1 and 2 d of deetiolation (D1 and D2, respectively). F, densitometry analysis of immunoblot in E. Representative immunoblots are shown and densitometry values are averages from two independent experiments.

Full-Length SUS Cosediments with 26S Proteasomes

We isolated proteasomes by differential centrifugation (Hardin et al., 2003) from etiolated and deetiolated leaves and found that a substantial amount of the 90-kD SUS protein cosedimented with the proteasomes (150 KP fraction; Fig. 4A , top section). There were several lower molecular bands (75–50 kD) that were picked up by the anti-SUS-PH antibodies in the 150 KP fraction; however, these putative SUS fragments were not related to deetiolation since they were found in both day 0 and day 1 samples (Fig. 4A). The fractionation procedure also showed that, as expected, full-length SUS protein was associated with the microsomal membrane and soluble protein fractions (100 KP and 150 KS fractions, respectively, in Fig. 4A). SUS protein in all three fractions was progressively reduced upon exposure to light, and surprisingly, loss of SUS was slowest from the 150 KP fractions (Fig. 4B).

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 M_r (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).

Deetiolation-Induced SUS Degradation Is Inhibited by Feeding Amino Acids But Not by Suc

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.

View larger version (44K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 5. Amino acids inhibit SUS degradation during deetiolation. Etiolated leaves were detached and cut ends were placed in incubation medium (5 mM MES-KOH, pH 6.0) containing 20 mM Suc or 10 mM amino acid as indicated. Seedlings were harvested after 1 and 2 d in the light or dark. A, Immunoblot analysis of SUS and SPS protein. B, Densitometry of immunoblots in A showing loss of SUS (B and D) and accumulation of SPS (C) protein after 2 d of exposure to light. B and D, Values of light day 2 (D2) samples are expressed on a relative basis with values on dark day 0 set as 100%. C, Values of day 2 samples are expressed on a relative basis with light day 2 control (water) set as 100%. All values are means from three independent experiments. Asterisks denote significant differences (P < 0.05).

View larger version (31K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 6. Effect of CHX on free amino acid levels during deetiolation. A, Immunoblot analysis of SUS during deetiolation. B, Densitometry of immunoblots in A showing changes in SUS protein level. C, Total free amino acid pools. Amino acids were extracted and analyzed as described in "Materials and Methods." Dark day 0 leaves contained 27.8 µmol amino acids g–1 fresh weight of leaves. In B and C, values of day 1 (D1) and day 2 (D2) samples are expressed on a relative basis with values on dark day 0 set as 100%.

	DISCUSSION

TOP ABSTRACT RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

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; Hardin et al., 2004). However, when maize seedlings are grown in the dark, the large seed reserves allow substantial growth and interestingly, the emerged etiolated leaves contain substantial SUS protein. Upon exposure to light, SUS pools are degraded during the deetiolation process. The turnover of SUS appears to be a selective process as many photosynthetic enzymes (e.g. RbcL and activase; Fig. 2) show a concomitant accumulation (also see Majeran et al., 2005). Light quality experiments demonstrated that both red and blue light can mediate the degradation of SUS, suggesting that the trigger for degradation is not strictly dependent on the phytochrome signaling pathway. Future studies utilizing red and blue light signal transduction mutants in maize will help better define the role of phytochromes, cryptochromes, and possibly phototropins in mediating SUS protein turnover.

Deetiolation-Induced SUS Degradation Is Not Mediated by Proteasomes

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; Hellmann and Estelle, 2002; Thompson and Vierstra, 2005; Thompson et al., 2005). The proteasome pathway is one of the major mechanisms by which proteins are selectively degraded in eukaryotes (Coux et al., 1996; Ingvardsen and Veierskov, 2001; Hellmann and Estelle, 2002; Thompson and Vierstra, 2005). In this pathway, proteins that are targeted for turnover are tagged by multiple ubiquitin molecules, and the polyubiquitinated proteins are then recognized by the 26S proteasome and degraded. In yeast (Saccharomyces cerevisiae) and animal systems, metabolic enzymes such as Glc-6-P dehydrogenase, Gln synthetase, and Fru-1,6-bisphosphatase, are degraded by the proteasome pathway (Coux et al., 1996). As previously shown, SUS appears to be regulated by the proteasome pathway during the sink-source transition in developing maize leaves and phosphorylation of SUS at Ser-170 site may trigger turnover (Hardin et al., 2003, 2004). Phosphorylation at Ser-170 occurred frequently on truncated fragments of the SUS protein that were enriched within the base of maize leaves and the transition zone between heterotrophic (enclosed) and photosynthetic (emerged) leaf regions. Moreover, these pS170-SUS fragments were spatially coincident with proteasome activity within developing leaves and cosedimented with proteasomes (Hardin and Huber, 2004). The pS170-SUS was unstable in cultured leaf segments and was significantly stabilized by inclusion of the proteasome inhibitor MG132. Collectively, these results suggested that the proteasome pathway might be a common mechanism for the degradation of carbohydrate metabolism-related enzymes in yeast, animal, and plant systems.

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) in a yeast two-hybrid screen as interacting with SUS. Whether this association occurs in vivo and has any functional consequence is not known.

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; Thompson et al., 2005). However, autophagy is not thought to function in selective protein degradation, and thus the mechanism involved in the degradation of SUS during deetiolation needs to be explained further in future studies.

SUS Degradation during Deetiolation May Provide Amino Acids for the Synthesis of Other Proteins

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). Thus, SUS degradation could contribute in a significant way to the amino acid pool required for synthesis of photosynthetic enzymes. Our working model is supported by the observation that SUS degradation was blocked by CHX (Figs. 3 and 6). One interpretation of this result is that an essential component involved in SUS degradation turns over quickly and must be continually synthesized, and as a result protein degradation is blocked by CHX. However, we favor the view that CHX reduces amino acid utilization and thereby indirectly (but effectively) inhibits SUS degradation. Consistent with this view is the observation that exogenous amino acids inhibited SUS degradation. Also consistent with this notion is the finding that free amino acid pools decreased somewhat in detached leaves undergoing deetiolation, and that pools were increased, but only slightly, in the presence of CHX (Fig. 6). Collectively, the results suggest that the degradation of heterotrophic enzymes, such as SUS, is a major source of amino acids for photosynthetic protein biosynthesis, and that there is a close coupling between protein degradation and free amino acid pools. In intact seedlings, the free amino acids are potentially supplied both by breakdown of seed reserves as well as selective degradation of heterotrophic enzymes such as SUS. Thus, SUS degradation would be predicted to be more rapid in detached leaves versus attached leaves, and this has been observed (data not shown). The observation that Glu was the most effective amino acid for inhibition of SUS turnover (Fig. 5D) suggests that Glu signaling may be involved. Indeed, plants possess Glu-gated nonselective ion channels (Kang and Turano, 2003) that may mediate ligand-induced changes in cytosolic [Ca²⁺] (Dubos et al., 2003). Future studies will focus on the possible roles of amino acid signaling and nutrient sensors in the regulation of SUS and on the mechanism of SUS degradation, with special attention to the autophagy pathway.

	MATERIALS AND METHODS

TOP ABSTRACT RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

 

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). The temperature was constant at 28°C for the duration of the experiments.

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.

Protein Extraction and Immunological Blots

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, 2004). Both of the modification specific antibodies were highly phosphorylation and sequence specific. The rabbit anti-Rubisco and anti-Rubisco activase antibodies were kindly provided by Dr. Archie Portis (U.S. Department of Agriculture-Agricultural Research Service, University of Illinois at Urbana-Champaign). Anti-ubiquitin antibodies were obtained from Sigma.

Proteasome Isolation and Activity (Chymotrypsin-Like) Assay

For measurement of enzyme activity, proteasomes were isolated as previously described (Hardin et al., 2003). Briefly, extracts were prepared in 100 mM MOPS (pH 7.5), 10 mM dithiothreitol (DTT), 1 mM EGTA, 8 mM MgCl₂, 4 mM ATP, 0.5 M Suc, 0.5 mM phenylmethylsulfonyl fluoride, 2 µM N-(trans-epoxysuccinyl)-Leu-4-guanidinobutylamide (E64), 2 µM leupeptin, 1 mM caproic acid, and 1% (w/v) polyvinylpolypyrrolidone (PVPP), and centrifuged at 100,000g and 4°C for 1 h. The supernatant was centrifuged at 150,000g at 4°C for 5 h to produce a proteasome pellet, which was resuspended in 20 mM MOPS (pH 7.5), 2 mM DTT, 2 mM ATP, 4 mM MgCl₂, and 0.25 M Suc. Chymotrypsin-like activity was determined using cleavage of the fluorogenic peptide Suc-Leu-Leu-Val-Tyr-amido-methyl coumarin (AMC; Calbiochem) to monitor 26S-proteasome activity in the presence of ATP (Coux et al., 1996). Assays were performed in 20 mM Tris (pH 8.0), 5 mM MgCl₂, 1 mM DTT, 0.1 mM peptide, 1 mM ATP, and 10 µM MG132 as indicated at 37°C for 30 min, and stopped with 1 mL of 1% (w/v) SDS. The released AMC was excited at 380 nm, and fluorescence intensity was measured at 440 nm. Activity was calculated using an AMC standard curve made under the same conditions.

For immunoblot analysis, the preparation of proteasomes was as described (Hardin et al., 2003). One gram of tissue was ground in 1 mL extract buffer containing 50 mM MOPS, pH 7.5, 5 mM DTT, 1 mM EGTA, 10 mM ATP, 10 mM MgCl₂, 10% glycerol, 1 mM AEBSF, 5 µM E64, 2 µM leupeptin, 1% PVPP, 100 µM MG132, and 0.25 M Suc. The extract was centrifuged at 25,000g for 10 min at 4°C. The supernatant was transferred to a new tube and centrifuged at 100,000g for 1 h. The pellet was resuspended in 1x SDS buffer and the supernatant was transferred to a new tube and centrifuged at 150,000g for 5 h. The resulting supernatant was mixed with 3x SDS buffer and the pellet was resuspended in 1x SDS buffer.

Protein Purification and Peptide Kinase Assays

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) with 0.1 mg mL^–1 peptide (SS4: VLARLHSVRERIKK; SP49: KGRMRRISSVEMMK). The SS4 and SP49 peptides correspond to the residues flanking the Ser-15 site in maize SUS1 and the Ser-158 site in SPS, respectively. Reactions were initiated with 0.1 mM [-³²P] ATP (150 cpm pmol^–1) and 10 mM MgCl₂, and stopped after 10 min at room temperature.

Amino Acid Analysis

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), and analyzed by gas chromatography in the Metabolomics Center at the University of Illinois at Urbana-Champaign. All 20 protein amino acids were individually resolved and quantitated, but only the total free amino acid pool is presented in Figure 6.

	ACKNOWLEDGMENTS

 
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.

	FOOTNOTES

 
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.

	LITERATURE CITED

TOP ABSTRACT RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

 
Barratt DH, Barber L, Kruger NJ, Smith AM, Wang TL, Martin C (2001) Multiple, distinct isoforms of sucrose synthase in pea. Plant Physiol 127: 655–664[Abstract/Free Full Text]

Carlson SJ, Chourey PS, Helentjaris T, Datta R (2002) Gene expression studies on developing kernels of maize sucrose synthase (SUS) mutants show evidence for a third SUS gene. Plant Mol Biol 49: 15–29[CrossRef][ISI][Medline]

Castleden CK, Aoki N, Gillespie VJ, MacRae EA, Quick WP, Buchner P, Foyer CH, Furbank RT, Lunn JE (2004) Evolution and function of the sucrose-phosphate synthase gene families in wheat and other grasses. Plant Physiol 135: 1753–1764[Abstract/Free Full Text]

Chourey PS, Latham MD, Still PE (1986) Expression of two sucrose synthase genes in endosperm and seedling cells of maize: evidence of tissue specific polymerization of protomers. Mol Gen Genet 203: 251–255[CrossRef][ISI]

Coux O, Tanaka K, Goldberg A (1996) Structure and functions of the 20S and 26S proteasomes. Annu Rev Biochem 65: 801–847[CrossRef][ISI][Medline]

Dubos C, Huggins D, Grant GH, Knight MR, Campbell MM (2003) A role for glycine in the gating of plant NMDA-like receptors. Plant J 35: 800–810[CrossRef][ISI][Medline]

Duncan KA, Hardin SC, Huber SC (2006) The three maize sucrose synthase isoforms differ in distribution, localization and phosphorylation. Plant Cell Physiol 47: 959–971[Abstract/Free Full Text]

Fernie AR, Willmitzer L, Trethewey RN (2002) Sucrose to starch: a transition in molecular plant physiology. Trends Plant Sci 7: 35–41[CrossRef][ISI][Medline]

Folta KM, Pontin MA, Karlin-Neumann G, Bottini R, Spalding EP (2003) Genomic and physiological studies of early cryptochrome 1 action demonstrate roles for auxin and gibberellin in the control of hypocotyl growth by blue light. Plant J 36: 203–214[CrossRef][ISI][Medline]

Folta KM, Spalding EP (2001a) Opposing roles of phytochrome A and phytochrome B in early cryptochrome-mediated growth inhibition. Plant J 28: 333–340[CrossRef][ISI][Medline]

Folta KM, Spalding EP (2001b) Unexpected roles for cryptochrome 2 and phototropin revealed by high-resolution analysis of blue light-mediated hypocotyl growth inhibition. Plant J 26: 471–478[CrossRef][ISI][Medline]

Hardin SC, Huber SC (2004) Proteasome activity and the post-translational control of sucrose synthase stability in maize leaves. Plant Physiol Biochem 42: 197–208[CrossRef][ISI][Medline]

Hardin SC, Tang GQ, Scholz A, Holtgraewe D, Winter H, Huber SC (2003) Phosphorylation of sucrose synthase at serine 170: occurrence and possible role as a signal for proteolysis. Plant J 35: 588–603[CrossRef][ISI][Medline]

Hardin SC, Winter H, Huber SC (2004) Phosphorylation of the amino terminus of maize sucrose synthase in relation to membrane association and enzyme activity. Plant Physiol 134: 1427–1438[Abstract/Free Full Text]

Hellmann H, Estelle M (2002) Plant development: regulation by protein degradation. Science 297: 793–797[Abstract/Free Full Text]

Herbers K, Sonnewald U (1998) Molecular determinants of sink strength. Curr Opin Plant Biol 1: 207–216[CrossRef][ISI][Medline]

Holtgräwe D, Scholz A, Altmann B, Scheibe R (2005) Cytoskeleton-associated, carbohydrate-metabolizing enzymes in maize identified by yeas two-hybrid screening. Physiol Plant 125: 141–156[CrossRef]

Huang JZ, Huber SC (2001) Phosphorylation of synthetic peptides by a CDPK and plant SNF1-related protein kinase: influence of proline and basic amino acid residues at selected positions. Plant Cell Physiol 42: 1079–1087[Abstract/Free Full Text]

Huber SC, Huber JL, Liao PC, Gage DA, McMichael RW, Chourey PS, Hannah CL, Koch K (1996) Phosphorylation of serine-15 of maize leaf sucrose synthase: occurrence in vivo and possible regulatory significance. Plant Physiol 112: 793–802[Abstract]

Ingvardsen C, Veierskov B (2001) Ubiquitin- and proteasome-dependent proteolysis in plants. Physiol Plant 112: 451–459[CrossRef][Medline]

Ishimaru K, Kosone M, Sasaki H, Kashiwagi T (2004) Leaf contents differ depending on the position in a rice leaf sheath during sink-source transition. Plant Physiol Biochem 42: 855–860[CrossRef][ISI][Medline]

Kang J, Turano FJ (2003) The putative glutamate receptor 1.1 (AtGLR1.1) functions as a regulator of carbon and nitrogen metabolism in Arabidopsis thaliana. Proc Natl Acad Sci USA 100: 6872–6877[Abstract/Free Full Text]

Koch K (2004) Sucrose metabolism: regulatory mechanisms and pivotal roles in sugar sensing and plant development. Curr Opin Plant Biol 7: 235–246[CrossRef][ISI][Medline]

Lalonde S, Boles E, Hellmann H, Barker L, Patrick JW, Frommer WB, Ward JM (1999) The dual function of sugar carriers: transport and sugar sensing. Plant Cell 11: 707–726[Free Full Text]

Li XQ, Zhang DP (2003) Gene expression activity and pathway selection for sucrose metabolism in developing storage root of sweet potato. Plant Cell Physiol 44: 630–636[Abstract/Free Full Text]

Ma L, Li J, Qu L, Hager J, Chen Z, Zhao H, Deng XW (2001) Light control of Arabidopsis development entails coordinated regulation of genome expression and cellular pathways. Plant Cell 13: 2589–2607[Abstract/Free Full Text]

Majeran W, Cai Y, Sun Q, van Wijk KJ (2005) Functional differentiation of bundle sheath and mesophyll maize chloroplasts determined by comparative proteomics. Plant Cell 17: 3111–3140[Abstract/Free Full Text]

Markelz NH, Costich DE, Brutnell TP (2003) Photomorphogenic responses in maize seedling development. Plant Physiol 133: 1578–1591[Abstract/Free Full Text]

Nguyen-Quoc B, Krivitzky M, Huber SC, Lecharny A (1990) Sucrose synthase in developing maize leaves: regulation of activity by protein level during the import to export transition. Plant Physiol 94: 516–523[Abstract/Free Full Text]

Quail PH (2002) Phytochrome photosensory signaling networks. Nat Rev Mol Cell Biol 3: 85–93[CrossRef][ISI][Medline]

Sawers RJH, Sheehan MJ, Brutnell TP (2005) Cereal phytochromes: targets of selection, targets for manipulation? Trends Plant Sci 10: 138–143[ISI][Medline]

Sheen J (1999) C4 gene expression. Annu Rev Plant Physiol Plant Mol Biol 50: 187–217[CrossRef][ISI][Medline]

Silva BM, Casal S, Andrade PB, Seabra RM, Oliveira MB, Ferreira MA (2003) Development and evaluation of a GC/FID method for the analysis of free amino acids in quince fruit and jam. Anal Sci 19: 1285–1290[CrossRef][ISI][Medline]

Tepperman JM, Hudson ME, Khanna R, Zhu T, Chang SH, Wang X, Quail PH (2004) Expression profiling of phyB mutant demonstrates substantial contribution of other phytochromes to red-light-regulated gene expression during seedling de-etiolation. Plant J 38: 725–739[CrossRef][ISI][Medline]

Tepperman JM, Zhu T, Chang HS, Wang X, Quail PH (2001) Multiple transcription-factor genes are early targets of phytochrome A signaling. Proc Natl Acad Sci USA 98: 9437–9442[Abstract/Free Full Text]

Thompson AR, Doelling JH, Suttangkakul A, Vierstra RD (2005) Autophagic nutrient recycling in Arabidopsis directed by the ATG8 and ATG12 conjugation pathways. Plant Physiol 138: 2097–2110[Abstract/Free Full Text]

Thompson AR, Vierstra RD (2005) Autophagic recycling: lessons from yeast help define the process in plants. Curr Opin Plant Biol 8: 165–173[CrossRef][ISI][Medline]

Truernit E (2001) The importance of sucrose transporters. Curr Biol 11: R169–R171[CrossRef][ISI][Medline]

Tsai CY (1974) Sucrose-UDP glucosyltransferase of Zea mays endosperm. Phytochemistry 13: 885–891[CrossRef][ISI]

Vierstra RD (1996) Proteolysis in plants: mechanisms and functions. Plant Mol Biol 32: 275–302[CrossRef][ISI][Medline]

Wang N, Zhang H, Nobel PS (1998) Carbon flow and carbohydrate metabolism during sink-to-source transition for developing cladodes of Opuntia ficus-indica. J Exp Bot 49: 1835–1843[Abstract/Free Full Text]

Winter H, Huber SC (2000) Regulation of Suc metabolism in higher plants: localization and regulation of activity of key enzymes. CRC Crit Rev Plant Sci 19: 31–67[CrossRef]

This article has been cited by other articles:

				M. Patel and J. O. Berry Rubisco gene expression in C4 plants J. Exp. Bot., May 1, 2008; 59(7): 1625 - 1634. [Abstract] [Full Text] [PDF]

This Article Free via Open Access: OA OA Abstract Full Text (PDF) OA All Versions of this Article: 144/1/468    most recent pp.106.095182v1 Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in ISI Web of Science Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via CrossRef Citing Articles via ISI Web of Science (1)