Phosphate Starvation Inducible Metabolism in

Plant Physiol. (1989) 91, 175-182 0032-0889/89/91/01 75/08/$01 .00/0

Received for publication January 12, 1989 and in revised form April 17, 1989

Phosphate Starvation Inducible Metabolism in Lycopersicon esculentum' Ill. Changes in Protein Secretion under Nutrient Stress Alan H. Goldstein*, Stephen P. Mayfield, Avihai Danon, and B. K. Tibbot Department of Plant Sciences, University of Arizona, Tucson, Arizona 85721 (A.H.G.); and Department of Molecular Biology, Research Foundation of the Scripps Clinic, La Jolla, California 92037 (S.P.M., A.D., B.K.T.) ABSTRACT

are secreted from the cell. When these molecules are proteins, biosynthesis and extracellular secretion processes will presumably play a role in the psi response. Much progress has been made on elucidating the pathway for secretion of proteins from plant cells (cf 1). However, little is known about the mechanisms whereby plant cells may target proteins for secretion out of the symplast/apoplast into the media. We have shown that, in suspension cultured tomato cells, no significant amount of the epsi acid phosphatase is retained in the apoplast (10). In the present study we examined the effect of phosphate starvation on the number of types of proteins secreted into the media by suspension cultured tomato cells. Phosphate-starved cells increased the secretion of proteins into the media. This increase occurred in a biphasic manner. In the first phase, shortly after the cells had been transferred to a Pi-depleted media, biomass accumulation continued at a rate equal to that of the unstressed control. Enhanced secretion of three proteins was seen during this phase. We considered these secreted proteins to be psi-specific. In the second phase, Pi-starved cells were no longer capable of growth as measured by biomass accumulation. We observed increased levels of three additional media proteins during this phase. During this type of severe Pi starvation, it was not possible to discriminate between psi phenomena and a more global stress response (or both). Severely stressed cells had a respiration rate twice the unstressed control and produced 4.4 times the amount of media proteins per unit biomass. By immunoblotting these media proteins with antibodies directed specifically against N-linked oligosaccharides that contain xylose, it was possible to identify those proteins processed in the Golgi apparatus. Results from these studies showed that increases in the levels of media proteins during both phases were the result of Golgi-mediated secretion processes. We also present data to demonstrate that phosphate starvation induced an increase in the rate of Pi uptake into suspension-cultured cells. Finally, we examined starvation for other nutrients and showed that depletion of exogenous N or Fe resulted in increased levels of specific media proteins.

Phosphate starvation increased the secretion of at least six proteins by suspension cultured tomato (Lycopersicon esculentum L. and L. pennellii) cells. Cells exhibited a biphasic response to phosphate (Pi) starvation. The early phase involved enhanced secretion of three proteins in response to transfer to a Pi-depleted media, while biomass accumulation continued at the same rate as in the Pi-sufficient cells. Severe starvation, defined as inhibition of biomass accumulation, induced enhanced secretion of three additional proteins. After sodium dodecyl sulfate-polyacrylamide gel electrophoresis, media proteins were immunoblotted with antibodies reacting specifically to oligosaccharides processed by the Golgi apparatus. Binding pattems showed that the enhancement in secretion during both phases of starvation was Golgi-mediated. Cells undergoing severe starvation had a respiration rate approximately twice that of unstressed cells and secreted 4.4 times more protein into the media per unit biomass. These data suggest overlapping Pi starvation-specific and global stress responses in plant cells. Under these conditions, Golgimediated protein secretion is enhanced. We present evidence for phosphate starvation inducible enhancement of Pi uptake. Secreted proteins specific for N and Fe starvation are also identified.

Phosphate (Pi) starvation induces a wide array of metabolic effects that modify plant growth at the organ, tissue, and cellular levels (1 1). We have recently presented evidence for the existence of a psi2 starvation rescue system in higher plants (10, 1 1). One functional aspect of such a system must involve the secretion of molecules into the media (or rhizosphere in vivo) that can act to enhance the availability of phosphate in the external environment. One member of this system, the epsi (pronounced ee') acid phosphatase has been identified and characterized (1 1). It is reasonable to propose that a phosphate starvation rescue system that acts to enhance the availability of exogenous phosphates must include molecular components that 'Financial support: This work was conducted while A. H. G. was a Visiting Scientist at The Scripps Research Foundation and was

MATERIALS AND METHODS Cell Culture and Isolation Of Extracellular Proteins All experiments were carried out with either Lycopersicon esculentum (cv VF36) or L. pennellii (ATICO 716). Phosphate

supported in part by PPG Inc. 2 Abbreviations: psi, phosphate starvation inducible; epsi, excreted phosphate starvation inducible. 175

176

GOLDSTEIN ET AL.

starvation experiments were conducted with suspension-cultured cells as previously described (10) except that the cells used in these experiments had been in continuous culture for 1 year. Cells were removed from the media by filtration and centrifugation at 2, 4, 6, and 8 d after transfer to media +Pi or -Pi. Proteins were isolated from the media via acetone precipitation also as previously described (1 1). Immunology

The epsi acid phosphatase (fraction Mll) was partially purified from other extracellular proteins on a Sephadex G150 column as previously described (1 1). This fraction was concentrated via ultrafiltration using the Amicon Centricon 30 and further purified by DEAE anion exchange column chromatography using the conditions described by Ninomiya et al. (16). SDS/PAGE followed by silver staining showed that the fractions with acid phosphatase activity still contained several bands. The electrophoresis sample buffer in all cases contained 2.5% SDS only, so that enzyme activity could be restored by washing the gel in sodium acetate buffer. The post-DEAE fraction was separated via chromatofocusing on a 20 x 1 cm column using PBE 94 and polybuffer 74 (Pharmacia). Two closely eluting peaks of acid phosphatase activity were obtained from a linear gradient (pH 7 to 4, 10 mL h-'). The two peaks of activity, probably corresponding to the isozymes seen on native PAGE ( 11) had apparent pIs of 5.2 and 5.3. The fractions containing these two peaks were pooled and reconcentrated with the Amicon 30. SDS/PAGE followed by silver staining showed only a few protein bands, none of which migrated near the band which showed acid phosphatase activity. The epsi acid phosphatase band was cut out of an acrylamide gel which still contained SDS, mixed 1:1 with Freund's adjuvant and injected into white New Zealand rabbits three times over an 8 week period. The rabbits were checked for production of antisera after the 6th week. A polyclonal antisera (designated AP3) was obtained that recognized the epsi acid phosphatase as well as other secreted proteins on immunoblots.

Plant Physiol. Vol. 91, 1989

electroblotted onto CN-Br activated Whatman 52 paper (14) and antibody-protein binding carried out after Tobin et al. (19) with slight modifications. AP3 antiserum was used at 1:1000 and 949 at 1:500 dilution. Antibody-protein complexes were visualized via autoradiography after further binding to [1251]protein A (Amersham).

Scanning Laser Densitometer Analysis of Protein Gels

Protein bands were visualized by silver staining using the method of Sammons et al. (17). These gels were analyzed directly with the LKB Ultroscan XL Scanning Laser Densitometer. The peak areas were calculated by signal integration and mol wt estimated by a parabolic polynomial method using the GelScan software (LKB version 1.2). Pi Uptake

Three d old cells were used. At this growth stage, the Pi concentration of the +Pi cells was approximately 0.7 mm (10). A sterile K2HPO4 solution (pH 5.6) was used to bring the -Pi cells to the same exogenous Pi concentration. The cells were then returned to the shaker for one-half h. One mL aliquots of cells grown +Pi or -Pi were transferred to shallow bottom sterile 2 mL microfuge tubes (Sarstadt, Inc.) and then 20 ,uCi of carrier-free [32P]orthophosphate was added to all tubes. The tubes were shaken horizontally at 300 rpm. Cells were pelleted in a microfuge for 2 min at 0.5, 1, 2, and 4 h. The supernatant was withdrawn and residual liquid drained by inverting the tubes for 1 h. The pellet was resuspended in scintillation cocktail by bath sonication for 5 min and counted directly. Dry weights were determined on duplicate samples.

Characterization of Cell Wall Proteins Cell wall proteins were extracted using both 2 M NaCl and EDTA treatments as described by Masuda et al. (13). SDSPAGE and silver staining were carried out as discussed above.

SDS-PAGE and Immunoblotting

mRNA Isolation and In Vitro Translation

The protein value for each time point +Pi or -Pi was normalized to the amount of cell biomass in that treatment so that all lanes on the gel contained an amount of secreted protein proportional to an equivalent amount of biomass (as measured by wet weight of cells). The absolute amount of protein in all samples was then adjusted to put them into an optimal range for silver staining. Protein values ranged from 0.15 ,ug protein for the 2 d +Pi cells to 0.45 ,tg protein for the 8 d -Pi cells. Lanes for immunoblots were loaded with 5 times this amount of protein. Proteins were resuspended to a final volume of 40 ,uL in sample buffer (0.4 M Tris [pH 8.0], 2.5% SDS, 2.5% f-mercaptoethanol, 15% sucrose), heated to 65°C for 5 min and then separated on a denaturing 7 to 15% gradient gel using the method of Chua (5). Gels were run at 8 mA constant current for 12 h at 8°C. Replicated samples were loaded on a single gel which was subsequently cut and used for both silver staining and western blots. Proteins were

Total RNA was isolated from 6 and 8 d cells growing -Pi and +Pi using the methods of Baltimore (2) and Glisin et al. (9) with slight modifications. Poly(A+) RNA was selected via column chromatography with oligo(dT) cellulose. Cell free translation with labeled [35S]methionine was carried out in the rabbit reticulocyte system according to the protocol supplied by the manufacturer (Promega, Inc.). Enhancement of incorporation of [35S]methionine into polypeptides (after deacylation) in the presence of poly(A+) RNA was usually 20 to 30 times that obtained in the -RNA control. One-dimensional SDS-PAGE was carried out according to Laemmli (12) using a 4% stacking gel and a 10 to 20% resolving gel. Yields of poly(A+) RNA per unit biomass were equivalent between treatments. Likewise, cell free translation levels were approximately equal so that loading each lane with equal cpm resulted in a profile of translation products representative of identical cell samples for both treatments at d 6 and 8.

psi PROTEIN SECRETION IN TOMATO CELLS

Starvation for N, K, and Fe. These studies were similar to the Pi starvation studies except that L. pennellii cells were used and the Murashige and Skoog salts modified in the following manner. The -N media contained no NH3NO3 or KNO3. The media was supplemented with 440 mg/L KS04. The -K media lacked KNO3 with P being supplied as (NH4)3PO4. The -Fe media was made up without Fe-EDTA.

2-

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Figure 1 gives an overview of the growth and media protein accumulation patterns of tomato cells grown +Pi or -Pi. Figure 1 A shows the total amount of protein secreted into the media at each time point for the two treatments. During the first 6 d of growth, there was little difference in total media proteins. Analysis of the normalized values in Figure B gave a different result. The two treatments remained equivalent in protein secretion only for the first 4 d. This corresponded to the period when lack of exogenous Pi had no effect on growth as measured by biomass accumulation (Fig. 1C). By d 6, the -Pi were no longer growing, but were secreting approximately 1.5 times the media proteins as the +Pi treatment on a unit biomass basis. This enhancement was the result of both a decline in the amount of proteins secreted per unit biomass by the +Pi cells and an increase by the -Pi cells. By d 8, the -Pi cells secreted approximately 4.4 times the protein as the +Pi controls. Once again, this enhancement resulted from a substantial decline in secretion by the +Pi cells accompanied by a large increase by the -Pi cells. The enhancement in epsi acid phosphatase activity is shown in Figure 1 D. This protein follows the general pattern seen in Figure lB. Figures 2 and 3 show equivalent silver-stained and AP3 immunoblotted gels of media proteins separated by SDSPAGE. Silver-stained total protein patterns for the various time points are seen in Figure 2. The scanning laser densitom-

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Figure 1. A, Effect of Pi starvation on total media proteins; B, effect of Pi starvation on the amount of media protein per g of cells; C, effect of Pi starvation on total biomass accumulation in 100 mL of suspension culture; D, effect of Pi starvation on the ratio of epsi acid phosphatase activity in the media per g of cells.

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Figure 2. Pattern of media proteins per unit biomass present under conditions of increasing Pi starvation. Total media proteins were separated by SDS-PAGE and visualized by silver staining. Each lane represents the amount of media protein per g of cells grown -Pi or +Pi at a given time point. Seven proteins identified by immunoblotting with AP3 antiserum and two that did not bind this antiserum were selected for further analysis. These proteins were identified by apparent mol wt.

eter assigned between 37 and 47 bands of which about 16 were easily visible. When proteins were quantitated by integration of the scans, it was readily apparent that phosphate starvation increased the levels of several media proteins. Figure 3 is an autoradiogram of an immunoblot done on the other half of this gel using polyclonal antiserum AP3. This figure shows that increases in psi media proteins were accompanied by increased binding to AP3. Figure 4 shows an immunoblot of a gel run under conditions identical to those used for Figures 2 and 3. Both lanes contained proteins secreted by the 8 d -Pi cells. The absolute amount of protein per lane was twice that used in Figure 3. The paper was cut and immunoblotted with either AP3 or a polyclonal antiserum designated 949 (generously provided by M. J. Chrispeels, UC San Diego) that reacted specifically with N-linked oligosaccharides that contain xylose (8). This indicated processing through the Golgi-apparatus ( 18). It may be seen that the staining patterns for the two antisera were almost

identical. The autoradiogram of the immunoblot in Figure 3 could be matched with the silver-stained gel in Figure 2 to identify

178

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seven proteins that bound AP3.

Two additional proteins were also selected for quantitative analysis. The relative location of these nine proteins is shown in Figure 2. A quantitative comparison of the amounts of these proteins present at the different time points for both treatments, as determined by scanning laser densitometry, is shown in Figure 5. Since the values shown represent the areas obtained by scanning the gel in Figure 2, they are also normalized to show the amount of protein secreted per unit biomass. The ratios of these two areas for all treatments is shown in Table I. Figure 6 shows the distribution of cell wall proteins extracted sequentially by 2 M NaCl and EDTA according to the methods of Masuda et al. (13). The proteins were separated by SDS-PAGE and visualized by silver-staining. All lanes represented equal amounts of cell biomass. Lanes a and b show the cell wall proteins extracted by 2 M NaCl from 6 d -Pi and +Pi cells, respectively, while lanes c and d show the proteins extracted from 8 d -Pi and +Pi cells under the same conditions. Lanes e and f show the cell wall proteins extracted from 6 d cells by further treatment with 0.5% EDTA for 96 h at 320C. It may be seen in all cases that there was little similarity between the cell wall protein patterns and those of the media proteins shown in Figure 2. The 2 M NaCl-extracted proteins in the -Pi treatments had a darker background than the +Pi samples. It is interesting to note that the -Pi treatments turned dark brown immediately after initiation of both the cell wall and RNA extraction procedures. While there were some apparent differences between the cell wall proteins in the two treatments, they were small compared to the changes in the media proteins. There appeared to be some enhancement in the levels of several high mol wt proteins (85, 83, and 69 kD) extracted from the -Pi cell walls by 2 M NaCl at both 6 and 8 d. The psi media

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Figure 4. Comparison of immunoblots obtained with antisera AP3 and 949. Both antisera were reacted with proteins from the 8 d -Pi cells. The autoradiogram from the AP3 blot was superimposed on the silver stained protein pattern to identify bands that gave visible antibody binding. These seven proteins were selected for further analysis.

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Figure 5. Seven Golgi-processed proteins identified by immunoblotting and two proteins that did not give a visible AP3 binding signal were analyzed by scanning laser densitometry. The y-axis represents area units obtained from the laser scan for a given protein band. Each protein has two area values (-Pi or +Pi) at each time point along the x-axis.


179

Table I. Ratio of the Areas (-Pi/+Pi) of Selected Media Proteins from Tomato Cells Growing -Pi or +Pi The proteins are designated by mol wt and by apparent binding (B) or nonbinding (NB) to AP3 antiserum. Protein

Day

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42.9(NB)

1.14 0.91 0.92 1.12

2.89 2.01 5.90 7.56

0.00 0.84 2.03 3.69

1.40 0.56 1.60 3.11

0.46 0.73 0.43 0.50

2.40 2.30 4.15 6.45

Figure 7 is an autoradiogram of total cell free translation products from 6 and 8 d cells grown -Pi and +Pi. While there were some differences between treatments, the general pattern was similar. These experimental results are included as an internal control to demonstrate the physiological integrity of the phosphate starved cells. Currently, we are analyzing data from two-dimensional gels of cell free translation products from 4, 6, and 8 d cells grown -Pi and +Pi. These detailed quantitative analyses will be published elsewhere. Figure 8 shows Pi uptake by -Pi or +Pi cells at the various timepoints. It may be seen that the Pi uptake rate of the -Pi cells was approximately twice that of the +Pi cells fro the first 2 h (2.3-fold enhancement in cpm/g wet weight between 1

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proteins identified in Figure 2 fell between 67.2 and 42.9 kD. The pattern of cell wall proteins in this region was completely different and did not show the dramatic seen

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protein. The patterns of EDTAextractable proteins (lanes e and f) were almost identical for the two treatments. There was a higher level of an 83 kD protein in the -Pi treatment. It is interesting to note that a protein with identical electrophoretic mobility appeared at a higher level in the -Pi treatments extracted with 2 m NaCl. In addition, treatment with EDTA appeared to extract a poorly resolved protein at 32 kD that was more abundant in induction of

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the -Pi treatment.

cell wall

Figure 7. Autoradiogram of cell free translation products from mRNAs isolated from 6 d cells growing +Pi (a) or -Pi (b); 8 d cells growing +Pi (c) or -Pi (d); -mRNA control (e); mol wt standards (f); or BGMV control RNA (g).

180

GOLDSTEIN ET AL.

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Figure 9. Silver-stained SDS-PAGE of media proteins secreted from 4 d old suspension cultured cells of L. pennelli grown -Fe, -K, -N, -Pi, or in complete Murashige and Skoog salts. The amount of protein per lane is normalized to represent the same cell biomass across all samples.

and 2 h). Between 2 and 4 h the -Pi cells accumulated Pi at 1.7 times the rate of the +Pi cells. Figure 9 shows a SDS-PAGE silver stained gel of proteins secreted from 4 d old suspension cultured cells of L. pennellii grown either -Pi, -N, -K, or -Fe. It may be seen that, as with Pi, depletion of N or Fe from the media resulted in increased levels of specific media proteins. At 4 d, all treatments accumulated biomass at the rate of the unstressed control (data not shown).


DISCUSSION Phosphate starvation had pleiotropic effects on protein secretion into the medium. We have operationally defined as psi those effects which are specifically stimulated by removal of Pi from the medium. As shown in Figure IC and in our previous work (10), -Pi cells continued to accumulate biomass at a rate equivalent to +Pi cells for several days. For the specific cell line used in this experiment, unstressed growth was maintained for 4 d. During this time, protein secretion into the medium was approximately the same for both treatments (Fig. 1, A and B). However, during this period of unstressed growth, we observed increases in the levels of two psi media proteins at 53.6 and 42.9 kD. It is possible that the amount of the 59.5 kD protein also increased after removal of Pi from the media. Table I shows that the psi enhancement of the 53.6 kD protein was 2.89, 2.01, 5.90, and 7.56 at d 2, 4, 6, and 8, respectively. At these same time points, the psienhancement in acid phosphatase activity was 1.4, 1.9, 2.4, and 7.1 (Fig. ID). Based on these data, and our previous estimation of 57 kD for the molecular mass of the epsi acid phosphatase (using a different electrophoretic system, [1 1]), we assume the 53.6 kD protein to be the epsi acid phosphatase. Enhancement in enzyme activity was accompanied by an increase in total protein and antibody binding by the 53.6 kD protein (Figs. 3 and 5). Therefore, we conclude that psi enhancement in acid phosphatase activity observed by ourselves (10) and others (cf 16) was the result of increased protein secretion into the media. In addition to psi-specific enhancement of protein secretion, there appeared to be a more global effect which was not observed until nutrient starvation became severe enough to stop cell growth. This effect is summarized in Figure 1, A and B. Total media protein levels remained the same in the two treatments up to d 6. However, by d 8 the media from the -Pi treatment contained almost twice the protein as the +Pi treatment. Figure lB shows that, when normalized to a unit of biomass, this enhancement was the result of a gram of -Pi cells secreting 4.4 times the protein at 8 d as a gram of +Pi cells. It is interesting to note that at d 8 the respiration rate of the -Pi cells was approximately twice that of the +Pi treatment (4.83 ± 0.18 ,Umol 02 min-' mg dry weight-' versus 2.39 ± 0.07 ,Umol 02 min-' mg dry weight-'). Figures 2, 3, and 4 show that Pi starvation resulted in increases in the levels of seven media proteins that bind both AP3 and 949 antisera. It was not possible to discriminate between psi effects induced by severe Pi starvation and global starvation stress effects that were caused or affected by the cessation of cell growth. Figure 2 clearly shows that psi media protein levels increased as nutrient starvation mediated inhibition of cell growth became more severe. From the immunoblot shown in Figure 4, we concluded that the polyclonal antiserum AP3 was directed at N-linked oligosaccharides that contain xylose. The pattern of staining was almost identical to antiserum 949 from the laboratory of M. J. Chrispeels whose group has shown that this antiserum specifically reacted with this carbohydrate moiety (8). Both antisera were obtained by injecting rabbits with a purified secreted plant protein.

181


Proteins having this carbohydrate group have been processed through the Golgi apparatus (18). Figure 3 shows that phosphate starvation induced an increase in secretion of some Golgi-processed media proteins. In addition, we examined two media proteins that did not bind either AP3 or 949 antisera. In general, media proteins could be divided into three categories (a) proteins that were clearly psi and showed increased levels long before growth was reduced; (b) proteins whose levels increased simultaneously with the starvation-mediated cessation of growth; (c) proteins whose levels remained unaffected by nutrient starvation. The 53.6 kD (presumably the epsi acid phosphatase), the 42.9 kD, and possibly the 59.5 kD proteins fell into the first category. We have previously shown that transfer of cells to phosphate depleted media induced specific secretion of proteins into the media (11). Labeling with [35S]methionine demonstrated that the steady state levels of several media proteins increased dramatically while the general profile of media proteins remained constant. It is of interest to note that major induced proteins of approximately 44 and 60 kD were identified by steady state labeling. These proteins may be the 42.9 and 59.5 kD proteins identified in this study. An induced 56 kD protein was also visible in that study, although it was obscured by a larger uninducible band that ran at a slightly higher mol wt. That 56 kD protein was probably the epsi acid phosphatase, while the higher mol wt protein corresponded to the 54.5 kD protein identified in Figure 2. The 67.2, 48.2, and 45.3 kD proteins are examples of the second category, while secretion of the 60.2 and 54.5 kD proteins appeared to be unaffected by either Pi starvation or the cessation of cell growth. The 57.7 kD protein was either unaffected or repressed very early in starvation metabolism. At the present time, it is not possible to know if the two proteins that do not bind AP3 (57.7 and 42.9 kD) were secreted through the Golgi apparatus. The twofold enhancement in total media protein secreted by 8 d old cultures (Fig. IA) was completely accounted for by a twofold increase in the areas of the proteins identified by AP3 binding. The profile of cell wall proteins was similar in the -Pi and +Pi treatments (Fig. 6) indicating that phosphate starvation did not induce mistargeting or leaching of cell wall proteins into the media. The cell-free translation data (Fig. 7) further demonstrated that, in all probability, phosphate starvation did not induce gross disruptions in metabolism and/ or cell necrosis. The fact that we did not observe a large number of new proteins under severe starvation further strengthens the argument that nutrient stress does not result in nonspecific leakage of intracellular proteins. Finally, we have previously shown that there was no leakage of the intracellular pool of acid phosphatase into the media up to 16 d after transfer to -Pi media (10). These combined data support the hypothesis that phosphate starvation induces Golgi-mediated secretion of specific proteins into the media. While the effect of phosphate starvation on Golgi-mediated secretion has not been studied previously, Chrispeels and Greenwood (4) observed that heat stress inhibited transport of proteins out of the endoplasmic reticulum but also noted that certain Golgi functions appeared unaffected by heat

stress. Recent work with yeast (3, 7) provides strong evidence for a role for heat shock proteins in transmembrane translocation of newly synthesized secretory proteins. It appears that severe Pi starvation stress may act differently at the cellular level. The 4.4-fold enhancement in Golgi-mediated extracellular secretion demonstrates clearly that protein secretion levels may be uncoupled from cell growth under these conditions. The psi enhancement of Pi uptake is another example of the pleiotropic effects of starvation on cell metabolism. Preincubation with cold Pi for one-half h makes it unlikely that this enhancement is due to adsorption in the cell wall space. Since enhanced uptake is seen days in advance of a decreased growth, it is unlikely that the increased rate of Pi uptake is due to passive movement of Pi. Psi enhancement of whole plant Pi uptake has been reported by others (6), and it is probable that this phenotype is a component of a Pi starvation rescue mechanism. The data presented here, when viewed in conjunction with our previous work, clearly demonstrate the multiple effects of phosphate starvation on plant metabolism. It is reasonable to suppose that at least some of these metabolic changes result from changes in gene expression. Recently, we have identified several novel or enhanced, cell-free translation products synthesized from poly(A+) RNA isolated from -Pi cells (manuscript in preparation). The term stimulon is used to refer to the entire set of genes responding to a given environmental stimulus (15). Based on the accumulated physiological and molecular evidence, it is reasonable to propose the existence of a higher plant Pi stimulon. The secretion of unique media proteins under N and Fe starvation indicates that starvation rescue mechanisms for these mineral nutrients may also involve media proteins. It will be instructive to attempt to determine the degree of overlap between the molecular components of the various mineral nutrient starvation rescue systems in higher plants. ACKNOWLEDGMENTS The authors would like to thank M. J. Chrispeels, M. B. Hein, A. Hiatt, and J. B. Hicks for useful discussions and suggestions. LITERATURE CITED 1. Akazawa T, Hara-Nishimura I (1985) Topographic aspects of biosynthesis, extracellular secretion and intracellular storage of proteins in plant cells. Annu Rev Plant Physiol 36: 441-472 2. Baltimore D (1966) Purification and properties of polyvirus double-stranded ribonucleic acid. J Mol Biol 18: 421-428 3. Chiroco WJ, Walters MG, Blobel G (1988) 70K heat shock related proteins stimulate protein translocation into microsomes. Nature 332: 805-810 4. Chrispeels MJ, Greenwood JS (1987) Heat stress enhances phytohemagglutinin synthesis but inhibits its transport out of the endoplasmic reticulum. Plant Physiol 83: 778-784 5. Chua N-H (1980) Electrophoretic analysis of chloroplast proteins.

Methods Enzymol 69: 434-446 6. Clarkson DD, Scattergood CB (1982) Growth and phosphate transport in barley and tomato plants during the development of, and recovery from, phosphate stress. J Exp Bot 33: 865875 7. Deshaies RJ, Bloch BD, Werner-Washburne M, Craig EA, Shekman R (1988) A subfamily of stress proteins facilitates

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9. 10.

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12. 13.

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