Plant Physiol. (1998) 117: 1281-1291
Induction of a Carbon-Starvation-Related Proteolysis in Whole
Maize Plants Submitted to Light/Dark Cycles and to Extended
Darkness1
Renaud Brouquisse*,
Jean-Pierre Gaudillère, and
Philippe Raymond
Station de Physiologie Végétale (R.B., P.R.), and
Station d'Agronomie (J.-P.G.), Institut National de la Recherche
Agronomique, Centre de Recherche de Bordeaux, BP 81, 33883 Villenave
d'Ornon cedex, France
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ABSTRACT |
Three-week-old maize (Zea
mays L.) plants were submitted to light/dark
cycles and to prolonged darkness to investigate the occurrence of
sugar-limitation effects in different parts of the whole plant. Soluble
sugars fluctuated with light/dark cycles and dropped sharply during
extended darkness. Significant decreases in protein level were observed
after prolonged darkness in mature roots, root tips, and young leaves.
Glutamine and asparagine (Asn) changed in opposite ways, with Asn
increasing in the dark. After prolonged darkness the increase in Asn
accounted for most of the nitrogen released by protein breakdown. Using
polyclonal antibodies against a vacuolar root protease previously
described (F. James, R. Brouquisse, C. Suire, A. Pradet, P. Raymond
[1996] Biochem J 320: 283-292) or the 20S proteasome, we showed that
the increase in proteolytic activities was related to an enrichment of
roots in the vacuolar protease, with no change in the amount of 20S proteasome in either roots or leaves. Our results show that no significant net proteolysis is induced in any part of the plant during
normal light/dark cycles, although changes in metabolism and growth
appear soon after the beginning of the dark period, and
starvation-related proteolysis probably appears in prolonged darkness
earlier in sink than in mature tissues.
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INTRODUCTION |
Carbohydrate deprivation is a fact of life for most higher plants.
When the emergence of young seedlings and their transition to
autotrophy is delayed (Elamrani et al., 1994b
), when competition occurs
between sink tissues such as roots, flowers, or fruits (Dejong and
Grossman, 1995; Ho, 1996), or when under environmental constraints, the
photosynthesis rate or the translocation of nutrients to sink tissues
decreases (Amthor and McCree, 1990; Setter, 1990) and higher plants may
experience carbon starvation. Similarly, in some types of senescence
(Noodén, 1988) or in postharvest situations (King et al., 1990),
the degradation of some tissue or cell structures is clearly related to
the appearance of carbon-starvation symptoms, such as a decrease in
sugar content, net lipid and protein breakdown, degradation of
plastids, or loss of membrane selective permeability. The metabolic
consequences of carbohydrate starvation have been studied in a number
of plant models, and the situation may be summarized as follows: to
survive, plant cells have to adapt to the lack of carbohydrates by
substituting protein and lipid metabolism for sugar metabolism through
autophagic processes (James, 1953; Thomas, 1978; Saglio and Pradet,
1980; Journet et al., 1986; Roby et al., 1987; Baysdorfer et al., 1988;
Peoples and Dalling, 1988; King et al., 1990; Brouquisse et al., 1991; Tassi et al., 1992; Elamrani et al., 1994b).
There are many reports of degradation and synthesis of
proteins during carbon deprivation. Thus, enzymic activities related to
sugar metabolism and respiration (Journet et al., 1986; Brouquisse et
al., 1991; Irving and Hurst, 1993), nitrogen reduction and assimilation
(Thomas, 1978; Peeters and Van Laere, 1992; Brouquisse et al., 1992),
or protein synthesis (Webster and Van't Hof, 1973; Tassi et al., 1992)
decrease during starvation. In contrast, the activity of enzymes
related to the catabolism of proteins (Thomas, 1978; Tassi et al.,
1992; James et al., 1993, 1996; Moriyasu and Ohsumi, 1996), amino acids
(Brouquisse et al., 1992), or fatty acids (Dieuaide et al., 1992, 1993;
Ismail et al., 1997) increases. Some genes encoding enzymes involved in
protein and lipid catabolism have been shown to be induced by sugar
depletion (Koch, 1996), and it is clear that changes in selective
synthesis or degradation of individual proteins could be important
components in the coordinated response to carbon starvation (Graham,
1996; Koch, 1996).
In plant cells protein breakdown is mediated by different proteolytic
systems: (a) vacuolar proteolysis, (b) selective nuclear and cytosolic
proteolysis, and (c) organellar proteolysis (for review, see Vierstra,
1993). Because each cellular compartment is affected by sugar
starvation, these different proteolytic systems might be involved in
starvation-induced proteolysis. Vacuolar macroautophagy has been
reported for tobacco and sycamore cells submitted to Suc deprivation
(Aubert et al., 1996; Moriyasu and Ohsumi, 1996) and may occur in maize
(Zea mays L.) root tips, where James et al. (1996)
demonstrated the induction of a vacuolar Ser endopeptidase in response
to carbon starvation. The ubiquitin-dependent pathway and 20S/26S
proteasome, present in eukaryotic cells, mediate a selective cytosolic
and nuclear proteolysis (Hershko and Ciechanover, 1992; Coux et al.,
1996). Ubiquitin and components of the ubiquitin-dependent proteolysis
have been shown to be induced in response to various stresses
(Vierstra, 1993; Callis, 1995) but, to date, their involvment in
response to carbon starvation has not been studied in plants. Very
little is known about the changes in proteolytic activities in
organelles under carbon deprivation. One study reports the induction of
plastidial aminopeptidase and endopeptidase activities in cotyledons of
sugar beet seedlings during prolonged dark growth (Elamrani et al.,
1994a).
In all of these studies, relatively long-term carbohydrate starvation
was investigated and, most of the time, the effects were analyzed in
one type of organ (leaf, root, cotyledon, spear tip, etc.) or in model
systems such as callus or cell suspensions. However, depending on the
protocol used to induce carbon starvation (shading, darkening, leaf
pruning, excision, girdling, etc.), the response to starvation might
vary significantly (Feller and Fischer, 1994). Thus, in spite of the
interest in such models and protocols, there are few data available on
the changes in biochemical parameters at the level of the whole plant
during carbon starvation.
In the present study we used 3-week-old maize plants submitted to
light/dark cycles and to prolonged darkness to investigate the effects
of moderate or strong sugar deprivation on protein breakdown in
different parts of the plant. At various times during the experiment,
the following plant parts were harvested: mature, senescent, and
youngest leaves; mature roots; root tips; stems and sheaths; and green
and yellow remainders (intermediate parts of the leaves). For each
plant part, soluble sugar, protein, amino acid, and
NH4+ content were analyzed,
endopeptidase activities were measured, and the amount of the vacuolar
RSIP, a protease previously shown to be induced in carbon-starved maize
roots (James et al., 1996), and of the 20S proteasome (G. Basset, R. Brouquisse, L. Malek, and P. Raymond, unpublished data) were
investigated by immunodetection. The growth rate of roots was also
measured. The occurrence of carbon starvation during light/dark cycles
and under prolonged darkness is discussed on the basis of changes in
protein and free-amino acid content and proteolytic activities, used as
carbon-starvation symptoms.
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MATERIALS AND METHODS |
Plant Culture and Harvest
Maize (Zea mays L. cv DEA, Pioneer France Maïs,
Toulouse, France) seeds were germinated at 25°C in the dark
between sheets of Whatman 3MM chromatography paper soaked in mineral
nutrient solution (solution A) as described by Saglio and Pradet
(1980). After 5 d the seedlings were transferred to a hydroponic
culture system in a growth room for another 16 d. The culture medium
consisted of solution A concentrated 2-fold and supplemented with 1 mM
Fe-ethylenediamine-N,N-di(2-hydroxy-5-methylphenyl) acetic acid. Plants were grown under a 15-h photoperiod with a photosynthetic photon flux density of 200 to 250 µmol photons m
2 s1. The light/dark
temperature was 25/20°C and the RH was maintained close to 70%.
Nitrate and phosphate concentrations were checked and readjusted daily.
After 3 weeks maize plants were submitted to two additional light/dark
cycles and then to a 48-h dark period. Samples of two to three plants
were harvested independently after 15 h of light, 9 h of
darkness, 15 h of light, and 9, 17, 24, and 48 h of darkness. In each plant the following tissues were distinguished: root tips (the
last 3 mm of primary and secondary roots), mature roots (whole roots
minus the root tips), stems and sheaths, senescent leaves (the first 2 leaves), mature leaves (the apical 30 cm of the 4th, 5th, and 6th
leaves), and the youngest leaves (i.e. the two most recently formed
leaves; these are leaves 11 and 12 and are yellow). The remaining leaf
tissues were pooled into two parts on the basis of their color and
called the "green remainder" (the 3rd leaf and the remaining green
part of the 4th, 5th, and 6th leaves) and "yellow remainder" (the
remaining yellow part of the 4th, 5th, and 6th leaves, and the
7th-10th leaves). Immediately after harvest, the different tissues
were weighed, rapidly frozen in liquid nitrogen, and either directly
stored at 
80°C (root tips, senescing leaves, and youngest
leaves), or ground to a fine powder in a liquid nitrogen prechilled
mortar and stored at 80°C. For sugar, protein, and amino acid
analysis, tissue samples were freeze-dried for 24 h (Tray Drying
Chamber, FTS Systems, Stone Ridge, NY) before metabolite extraction.
Fresh tissues were used for enzymatic activity and
NH4+ measurements and for
immunoblotting experiments.
Extraction and Determination of Soluble Sugars and Amino Acids
Soluble sugars were extracted by boiling ethanol/water and
analyzed according to the method of Moing et al. (1992). Free amino acids were extracted according to the method of Stitt and ap Rees (1978) and analyzed as described in Brouquisse et al. (1992).
Protein Analysis
Proteins were extracted with the following extraction buffer: 50 mM Hepes-Na, pH 7.3, 0.2% sodium deoxycholate, 0.1%
Triton X-100, and 5 mM sodium ascorbate. They were measured
according to the method of Bradford (1976) using BSA as a standard.
NH4+ Analysis
NH4+ was measured, after
extraction from frozen powdered material with 0.1 M HCl, by
the phenol-hypochlorite method, as described in King et al. (1990).
Proteolytic Activity Measurements
Forty to 500 mg of fresh tissue was crushed in a glass potter or
in a mortar at 4°C with 200 µL to 1 mL of extraction medium (50 mM Tris-HCl, pH 7.5, 5 mM 
-mercaptoethanol,
and 0.5% [v/v] polyvinylpolypyrrolidone). Crushed extracts
were then centrifuged (15,000g, 15 min) and the supernatants
were used for endopeptidase and chymotrypsin-like activity
measurements. Endopeptidase activities (against azocasein) were
measured as described in James et al. (1993). The extinction
coefficient E1% azocaseine in 1 M
NaOH = 37 L cm1 g1 was used
to calculate the azocaseinase activity. Chymotrypsin-like activities
were measured using Succ-Leu-Leu-Val-Tyr-AMC as a substrate. Sixty
microliters of enzymic extract, 20 µL of water, and 20 µL of
substrate solution (2 mM stock in dimethyl formamide) were incubated for 1 h at 37°C. The reaction was stopped by the
addition of 100 µL of 10% SDS (w/v) and 2 mL of 100 mM
Tris-HCl, pH 9.0. The released 7-amino-4-methyl coumarin radicals were
measured fluorometrically (excitation at 380 nm, emission at 460 nm) in an F-2000 spectrofluorimeter (Hitachi, Tokyo, Japan).
Electrophoresis and Immunoblots
Native- and SDS-PAGE were performed with 5% (w/v) and 12.5%
(w/v) polyacrylamide gels, respectively, by the procedure of Laemmli (1970). Protein from native- and SDS-PAGE was transferred to a nitrocellulose membrane (BA 85, Schleicher & Schuell) for 1 h at 3 mA/cm2 in a Fast-Blot B33 semidry system
(Biometra, Göttingen, Germany). Blots were blocked with TBS
containing 0.2% Tween 20 and 5% (w/v) nonfat milk powder. RSIP and
20S proteasome were detected with polyclonal anti-RSIP (James et al.,
1996) and anti-20S proteasome (G. Basset, R. Brouquisse, L. Malek, and
P. Raymond, unpublished data) antibodies plus goat anti-rabbit
IgG-alkaline phosphatase conjugate (Sigma).
Immunoprecipitation Method
One milliliter of each crude extract supernatant was desalted by
centrifugation through a G-25 Sephadex column (Pharmacia) equilibrated
with 50 mM Mes-Na, pH 6.1, and 20 mM NaCl.
Equal azocaseinase activities (400 nmol azocasein
min1) were incubated for 2 h at 25°C
with an increasing volume of immune or preimmune anti-RSIP serum.
Immune complexes were incubated for 1 h at 6°C with a 2-fold
(IgG-binding) excess of Protein A-Agarose (Bio-Rad) and then
centrifuged for 10 min at 10,000g. The azocaseinase activity
was then measured in each supernatant fraction.
Root-Growth Measurement
Attached maize roots were put on a sloping gutter in the growth
chamber. Recycled nutrient solution was supplied continuously at the
top of the gutter, creating a wet film around the bundle. A camera
(Minolta, Ramsey, NJ) was fitted above the gutter to take pictures of
the root tips every 3 h. The root system was kept in darkness
except for short xenon flashes needed to take pictures. A grid was
drawn at the bottom of the gutter and root length was measured manually
on pictures with a 5-fold magnification of the actual root size.
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RESULTS |
To investigate changes in metabolite content and enzymic
activities during light/dark cycles and during an extended dark period, 3-week-old maize plants were harvested at the end of the light (15 h)
and the dark (9 h) periods of two successive light/dark cycles and then
after 17, 24, and 48 h of darkness.
Changes in Soluble Sugar and Protein Contents
Soluble sugars were measured in whole roots (including root tips),
stems and sheaths, senescent leaves, mature leaves, yellow remainder
(including the youngest leaves), and green remainder (Fig.
1). Depending on the tissue, the total
soluble sugar content varied from 85 to 140 µg mg1 dry
weight in whole roots and mature leaves, respectively, with Suc
accounting for 50% to 80% of the soluble sugars. After measurement of
the fresh and dry weights of the different tissues, dry weight was
shown to represent about 15 ± 4% of the fresh weight (data not
shown). Thus, the soluble sugar content was estimated to be between 70 and 120 µmol Glc equivalents g1 fresh weight.
In all of the tissues, soluble sugar content exhibited significant
variations during light/dark cycles, showing a 50% drop at the end of
the dark period. Under prolonged darkness soluble sugars continued to
decrease and reached 10% of their initial value after 48 h of
darkness. In roots soluble sugars decreased to 2.5 µg
g1 dry weight (around 2 µmol Glc equivalents
g1 fresh weight).
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| Figure 1.
Changes in soluble sugars, Suc, Glc, and Fru, in
different maize plant parts (whole roots, mature leaves, senescent
leaves, stems and sheaths, green remainder, and yellow remainder)
during light/dark cycles and 48 h of darkness. Each point
represents the mean (±SD) of four independent experiments.
DW, Dry weight.
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Such a decrease in soluble sugar content during more or less extended
dark periods has been described for many plant species and may be
related to cessation of photosynthetic activity during the dark period,
whereas respiration and biosynthetic processes continue, although at a
lower rate (Wittenbach, 1978; Frossard, 1985; Kerr et al.,
1985). This situation, if naturally or artificially prolonged, may lead
to a state of carbon starvation and to the beginning of controlled
autophagic processes, particularly in sink organs with active
metabolism (Thomas, 1978; Baysdorfer et al., 1988). The changes in
proteins were thus investigated in the different tissues of the plant
during light/dark cycles and extended darkness. As shown in Figure
2, the protein contents did not change
significantly in any tissue during the light/dark cycle. However, the
decrease in protein occurred after 48 h of darkness, particularly
in growing tissues such as mature roots, root tips, and youngest
leaves. In these tissues the protein decrease was 21% to 30% after
48 h of dark treatment. In the other tissues, the decrease in
protein was significant only after 72 h of darkness (data not
shown). Previous studies on sugar deprivation in excised maize root
tips showed that protein degradation was linked to the release of the
protein nitrogen as amino acids, particularly Asn, and
NH4+ (Brouquisse et al., 1992)
and to an increase in proteolytic activities (James et al., 1993,
1996). Thus, we investigated the fate of these various parameters in
the different parts of the maize plant, and particularly in sink
tissues (mature roots, root tips, and youngest leaves), in the course
of the light/dark cycle and extended darkness.
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| Figure 2.
Changes in protein content in different maize
plant tissues (root tips, mature roots, senescent leaves, mature
leaves, youngest leaves, stems and sheaths, green remainder, and yellow
remainder) during light/dark cycles and 48 h of extended darkness.
Each point represents the mean (±SD) of five different
experiments. DW, Dry weight.
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Changes in Free Amino Acids and NH4+
As shown in Figure 3, Gln and Asn
changed in opposite ways under varying light conditions. Under a normal
light/dark cycle, in mature roots and root tips, Gln content was higher
than that of Asn, increasing during the day and decreasing during the
night, whereas Asn content increased during the night and decreased
during the day. Although smoothed by the variability between the
samples of the four (root tips) or five (mature roots) experiment
series, such changes between the end of the day and the end of the
night were significant in a same series. These changes were not clearly observed in the youngest leaves. After 48 h of darkness, Gln
dropped while Asn dramatically increased by a factor of 5, 16, and 18 in mature roots, root tips, and youngest leaves, respectively. It may
be noted that Ser, which has been shown to accumulate in carbon-starved, excised root tips (Brouquisse et al., 1992), also increased in the different tissues with a pattern similar to that of
Asn. In the other plant tissues Asn content (3-10 nmol
g1 fresh weight) increased by a factor of 1.2 to 9 after 48 h of darkness (data not shown). The increase in Asn
has been related to the transient storage of the nitrogen released by
the degradation of protein amino acids under carbohydrate starvation
(James, 1953; Sieciechowicz et al., 1988; Genix et al., 1990;
Brouquisse et al., 1992), since
NH4+ is toxic for the cell and
cannot be accumulated at a high concentration (Givan, 1979).
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| Figure 3.
Changes in free amino acids, Asn, Gln, and Ser, in
youngest leaves (top), mature roots (middle), and root tips (bottom) of
maize plants submitted to light/dark cycles and to 48 h of
darkness. Each point represents the mean (±SD) of three
(youngest leaves), four (root tips), or five (mature roots) independent
experiments. DW, Dry weight.
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NH4+ content appeared to be
quite different between the youngest leaves and the root tissues, at
0.5 and 5 µmol g1 fresh weight, respectively
(Fig. 4). In these tissues its content did not change during the light/dark cycle, but increased 2- to 3-fold
between 17 and 48 h of darkness to reach 1.4, 14, and 15.4 µmol
g1 fresh weight in youngest leaves, mature
roots, and root tips, respectively. In the other tissues the
NH4+ content ranged between 0.6 and 2.3 µmol g1 fresh weight and did not vary
significantly during the experiment (data not shown). Considering that
85% of the fresh weight corresponds to cellular water and assuming an
even distribution in cellular water, the
[NH4+] in youngest leaves,
roots, and root tips was estimated to be 0.7, 5.9, and 6.1 mM, respectively, at the end of the light period (as a
comparison, the [NH4+] in the
liquid medium was 0.75 mM) and 1.6, 16.5, and 18 mM after 48 h of darkness. However, it has been shown
that most of the cell NH4+ is
located in the vacuole (Lee and Ratcliffe, 1991; Roberts and Pang,
1992). In the present work, on the basis of certain physiological parameters (i.e.
NH4+/NH3
pKa = 9.2, pH vacuole = 5.8, and pH cytoplasm = 7.3) and assuming that the cytoplasm and the vacuole each occupy 45% of the
total tissue volume in the root tips (Patel et al., 1990), and 10% and
80%, respectively, in the mature roots, the cytoplasmic [NH4+] after 48 h of
darkness was calculated to be 0.28 mM in the root tips and
0.37 mM in the mature roots.
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| Figure 4.
Changes in NH4+ content in
youngest leaves, mature roots, and root tips of maize plants submitted
to the light/dark cycle and to 48 h of darkness. Each point
represents the mean of three independent experiments. For clarity, only
one side of the SD is indicated for root-tip and mature
root data. FW, Fresh weight.
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Based on changes in proteins, amino acids, and
NH4+, it was possible to
calculate partial nitrogen balances before and after 48 h of
darkness in the different tissues and in the whole plant (Table
I, see 
N values). In sink
tissues (root tips, mature roots, youngest leaves, stems and sheaths,
and yellow remainder), 38% to 94% of the nitrogen released by net
protein breakdown was retrieved as Asn, for only 2% to 9% in mature
or senescent tissues (mature and senescent leaves, green remainder).
However, in these latter tissues, 54% (green remainder) to 82%
(mature and senescent leaves) of the nitrogen released by proteins was
lost after 48 h of darkness for only 30% to 49% in root tips,
mature roots, and stems and sheaths. In contrast to this, the amount of
nitrogen accumulated in NH4+,
Asn, and other free amino acids in the youngest leaves and yellow remainder after 48 h of darkness was 19% and 53% higher than the amount of nitrogen released by protein breakdown (Table I). Thus, it
appeared that the fate of nitrogen differed according to the tissue.
Considered at the level of the whole plant, 53% of the nitrogen
released by net protein breakdown was retrieved as free amino acids
(two-thirds of which was Asn), 4% as
NH4+, and the remaining 42% was
lost, presumably as NH4+, in the
hydroponic culture medium via the root system (which contained 76% of
the NH4+ accumulated
in the whole plant, Table I).
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Table I.
Distribution of nitrogen between protein, Asn, other
free amino acids (other AA), and NH4+ in the
different plant tissues and in the whole plant after 15-h light (15h L)
and 48-h dark (48h D) periods
The amount of nitrogen in protein was calculated assuming that the
nitrogen content of protein is 16.7%. The conversion of protein, Asn,
and other amino acid contents from micromoles per gram dry weight to
micromoles per gram fresh weight was calculated on a basis that dry
weight represents 15% of fresh weight, as mentioned in ``''.
 N represents the sum of (protein plus Asn plus other AA plus
NH4+) nitrogen amounts present in each tissue.
N represents the difference in nitrogen amount present in either
protein, Asn, other amino acids, NH4+, or N
between 48-h dark- and 15-h light-treated tissues.
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Changes in Endoproteolytic Activities
In previous work we showed that endoproteolytic activities
increase in excised maize root tips submitted to carbon starvation (James et al., 1993), and suggested that both the vacuolar and the
specific nuclear and cytosolic proteolytic systems were potentially involved in the plant response to starvation (James et al., 1996). Thus, we followed first the changes in global endopeptidase activities in youngest leaves, roots, and root tips, and second, by western-blot experiments, the changes in the amounts of vacuolar RSIP and in the
nuclear and cytosolic 20S proteasome.
Total endopeptidase activities, measured with azocasein as a substrate,
were found to be higher in mature roots than in the youngest leaves
(25-30 and 5-7 µg azocasein min1
g1 fresh weight, respectively), and to remain
essentially unchanged during light/dark cycles (Fig.
5A). However, after 48 h of darkness a significant and reproducible 1.5-fold increase in endopeptidase activity was observed in mature roots, and to a lesser extent in the
youngest leaves. Western-blot data showed that in both tissues the
amount of 20S proteasome remained unchanged throughout the experiment
(Fig. 5B). RSIP was found to be present in the mature roots and to
slightly increase under permanent darkness, but it was not detectable
in the youngest leaves (Fig. 5B). These last results are in agreement
with a previous study (James et al., 1996) that showed that RSIP is
absent in leaves and only present in roots of young maize seedlings.
The increase in endopeptidase activity in the whole roots was linked to
the increase in RSIP amount, since in both 15-h light and 48-h dark
tissue extracts, RSIP was found to represent about 70% to 75% of the
total activity after immunoprecipitation experiments (Table
II).
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| Figure 5.
Changes in total endopeptidase (EP) activity and
immunodetection by western-blot analysis of RSIP (SDS-PAGE) and 20S
proteasome (native-PAGE) in mature roots (Root) and youngest leaves
(Y.L.) of maize plants submitted to light/dark cycles and 48 h of
darkness. A, Azocasein was used as a substrate. Each point represents
the mean (±SD) of three (youngest leaves) or six (mature
roots) independent experiments. B, The equivalent to 0.5 mg dry weight
of mature roots (bottom, 100-70 µg of protein) and youngest leaves
(top, 90-60 µg of protein) was loaded onto each lane. FW, Fresh
weight.
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Table II.
Percentage of endopeptidase activity brought about
by RSIP in mature roots and root tips after 15-h light and 48-h dark
periods
Equal amounts of azocaseinolytic activity (400 nmol azocasein
min1) were analyzed by an immunoprecipitation test in the
presence of immune or preimmune anti-RSIP serum, as described in
``Materials and Methods''. The data are the results of two
independent experiments.
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In root tips global endopeptidase activities (6.1 µg azocasein
min1 g1 fresh weight)
were found to be lower than in mature roots (Fig. 6A). However, these activities showed
significant and reproducible variations during light/dark cycles,
increasing and decreasing during the dark and the light periods,
respectively, and then increased 5-fold after 48 h of darkness
(Fig. 6A). This increase in endopeptidase activities was linked to a
significant enrichment of the root tips in RSIP, as shown after
immunodetection on western-blot (Fig. 6B) and immunoprecipitation
experiments (Table II). Using a synthetic substrate,
Succ-Leu-Leu-Val-Tyr-AMC, we also measured the
chymotrypsinlike activity, which was found to be a good
marker for the activity of the 20S proteasome in maize root tips (G. Basset, R. Brouquisse, L. Malek, and P. Raymond, unpublished data). Neither the chymotrypsin-like activity nor the amount of 20S proteasome protein showed any change during light/dark cycles or prolonged darkness (Fig. 6) in spite of the decrease in the bulk of cell proteins.
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| Figure 6.
Changes in total endopeptidase and
chymotrypsin-like activities and immunodetection by western-blot
analysis of RSIP (SDS-PAGE) and 20S proteasome (native-PAGE) in root
tips of maize plants submitted to light/dark cycles and 48 h of
darkness. A, Azocasein was used as a substrate for total endopeptidase
activity measurement, and Succ-Leu-Leu-Val-Tyr-MCA was used
for chymotrypsin-like activity measurement. Each point represents
the mean (±SD) of four (azocasein) or two
(chymotrypsin-like) independent experiments. B, The equivalent to 0.25 mg dry weight of root tips (100-75 µg of protein) was loaded onto
each lane. FW, Fresh weight; Chymo. Act., chymotrypisin activity;
Azoca. Act., azocaseinase activity.
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In senescent leaves, mature leaves, and stems and sheaths,
endoproteolytic activities ranged from 3 to 8 µg azocasein
min1 g1 fresh weight
and did not change significantly during the experiment (data not
shown).
Root-Growth Measurement
To assess the effect of carbon deprivation on root growth, we
followed root elongation during the light/dark cycle and under extended
darkness. As reported in Figure 7,
primary and secondary roots exhibited changing elongation rates during
light/dark cycles, speeding up in the light and slowing down in the
dark. Such an effect has been reported in various studies and was
attributed to the decrease of carbohydrate supply to the root system
(Williams and Farrar, 1990; Bingham and Stevenson, 1994; Merlo et al.,
1994). In the present experiment the pattern of the root elongation
rate (Fig. 7) closely resembled that of root sugar content (Fig. 1). It
is striking that the mean elongation rate dropped within the first
3 h of the dark period. Under prolonged darkness the roots ceased
to elongate after 15 to 24 h, when the decrease in protein and the
increase in Asn, NH4+, and in
endopeptidase activities became significant in the root tips (Figs. 2,
3, 4, and 6). In addition, secondary root elongation stopped after 15 to 18 h of darkness, whereas primary roots continued to grow up to
21 to 24 h (Fig. 7, arrows). This suggests that sugar limitation
was effective in the secondary roots first and only 6 h later in
the primary roots. On the basis of root elongation, it was found that
roots survived at least to 96 h in darkness, since they were able
to reinitiate their growth after return to initial light/dark
conditions (data not shown).
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| Figure 7.
Changes in the elongation rate of primary and
secondary roots of maize plants during light/dark cycles and extended
darkness. These data are from a representative experiment that has been
repeated two times. The first and second points of each curve represent
the mean growth rates over the last 3 h of the light period and
the first 3 h of the dark period, respectively. Each point
represents the mean (±SD) of two (primary roots) or four
(secondary roots) measurements. For clarity, only one side of the
SD was indicated. Arrows indicate the moment when secondary
(SR) or primary (PR) roots stopped growing.
|
|
| |
DISCUSSION |
In 3-week-old maize plants grown under medium light intensity, the
soluble sugar content strongly changed during the light/dark cycle in
all of the plant tissues, and dropped dramatically during extended
darkness (Fig. 1). Numerous studies have shown that following carbohydrate depletion, the growth and respiration of plant tissues decrease, and net protein and lipid breakdown starts (James, 1953; Thomas, 1978; Wittenbach, 1978; Baysdorfer et al., 1988; King et
al., 1990; Williams and Farrar, 1990; Brouquisse et al., 1991; Bingham
and Stevenson, 1994). In the present experiments no net protein
breakdown was observed during the light/dark cycle, but when the dark
period was extended, proteins significantly decreased in growing
tissues such as roots or youngest leaves, whereas they moderately
decreased in mature tissues (Fig. 2). Actively metabolizing and growing
tissues are known to be very sensitive to carbohydrate depletion, and
the early decrease in protein may be related to the cessation of growth
and to the beginning of degradative processes (Journet et al., 1986;
Baysdorfer et al., 1988; Brouquisse et al., 1991; Moriyasu and Ohsumi,
1996). Carbon deprivation-related proteolysis is generally
accompanied by associated symptoms such as increased Asn and
NH4+ contents and increased
endopeptidase activities (Thomas, 1978; King et al., 1990; Brouquisse
et al., 1992; Peeters and van Laere, 1992; James et al., 1993).
During light/dark cycles Gln and Asn exhibited opposite trends in root
tissues (Fig. 3). Both amino acids play a crucial role in plant growth
and development, since they are the primary nitrogen-transport compounds within most plants (Urquhart and Joy, 1981) and their synthesis is regulated by sugar supply and other regulatory signals (for review, see Lam et al., 1996). Thus, whereas in light-grown plants
Gln is preferentially synthesized, in dark-grown plants in which
photosynthetic carbon is limiting, Asn is synthesized preferentially to
transport nitrogen (Lam et al., 1996). In the present experiment, as
neither NH4+ nor proteins
changed significantly during the light/dark cycle (Figs. 2 and 4), the
increase in root Asn during the dark period cannot be ascribed to an
increase in net protein breakdown, but may rather be explained by the
regulation of the nitrogen assimilation pathways.
Under prolonged darkness the situation is different: the dramatic
increase in Asn is related to the breakdown of proteins and to an
increase in Ser and NH4+contents
(Figs. 2, 3 and 4). As already suggested (Brouquisse et al., 1992), in
carbon-starved tissues Asn plays the role of a nitrogen storage
compound and avoids a toxic accumulation of NH4+ inside the cell (Givan,
1979; Siechiechowicz et al., 1988). Considering the compartmentation of
NH4+ between cytoplasm (<0.4
mM) and vacuole (19-36 mM) in the root tissues, NH4+ should not affect
cytoplasmic metabolism. As reported in Table I, the nitrogen released
by protein degradation is differently metabolized according to the
plant tissue. In actively growing sink tissues (root tips, youngest
leaves, and yellow remainder), it is significantly retrieved as Asn
(more than 70%) and other free amino acids, whereas it is essentially
lost (more than 80%) in mature or senescent source tissues (mature and
senescent leaves), the other tissues (mature roots, stems and sheaths,
and green remainder) showing intermediate behavior. Taken together,
these observations suggest that in whole maize plants submitted to
transient starvation, the nitrogen issued from net proteolysis is
metabolized or lost. In aerial source tissues (mature and senescent
leaves, and to some extent, green remainder), it is massively exported to be either released in the incubation medium via the root system or
partially reallocated to aerial sink tissues (youngest leaves and
yellow remainder). In contrast, in the sink organs (root tips, youngest
leaves, yellow remainder, and to a lesser extent mature roots and stems
and sheaths), the nitrogen issued from proteolysis is primarily stored
as Asn or free amino acids and, to a lesser extent, partly lost into
the incubation medium. Thus, in these latter tissues, dividing and
elongating cells will be able to use a "cheap" (in terms of energy
cost) and readily available source of nitrogen as soon as the carbon
supply to growing tissues increases again (Brouquisse et al., 1992).
The synthesis of Asn is probably catalyzed by Asn synthetase, the main
route for Asn synthesis in plants (Siechiechowicz et al., 1988; Lam et
al., 1996), because its activity and mRNA level were found to increase in maize root tips submitted to carbon starvation (Brouquisse et al.,
1992; Chevalier et al., 1996).
No net protein degradation and no increase in endopeptidase activities
were observed during light/dark cycles. However, after 48 h of
darkness total endopeptidase activities increased in root tips and
mature roots (Figs. 2, 5, and 6). In the roots the increase in
proteolytic activity was associated with the induction of RSIP, a
vacuolar Ser endopeptidase shown to account for up to 80% of the
endopeptidase activity in roots of whole plants (Table II) and
sugar-starved, excised maize roots (James et al., 1996). As in yeast
(Baba et al., 1994) and animal cells (Seglen and Boley, 1992),
autophagy has been shown to occur in plant cells submitted to sugar
starvation (Aubert et al., 1996; Moriyasu and Ohsumi, 1996). In tobacco
cells deprived of Suc, the appearance of autophagic vacuoles has been
associated with an increase in endopeptidase activities (Moriyasu and
Ohsumi, 1996), and in Suc-starved sycamore cells, the development of
autophagic vacuoles was correlated with an increase in P-choline, a
marker of phospholipid degradation (Aubert et al., 1996). The presence
of an increasing amount of RSIP in maize roots during extended darkness
(Fig. 6B; Table II) suggests that vacuolar autophagy may be
involved in the degradative processes (e.g. proteolysis) that take
place in the root system. Although endopeptidase activities did not
increase significantly in the youngest leaves, net protein breakdown
and Asn increase clearly occurred after 48 h of darkness.
Preexisting proteolytic systems may be active enough to account for the
net degradation of the proteins in the youngest leaves or the delay in
endopeptidase induction in youngest leaves compared with root tissues.
Alternatively, azocasein could be a poor substrate for the newly
induced proteases in leaves, and the azocaseinolytic measurement would
not account for all of the endopeptidase induction during the dark
period.
Although vacuolar proteolysis and autophagy are known to be
nonselective processes (Marty, 1978; Seglen and Bohley, 1992), the
maintenance or increase of some enzymes, in a general context of
degradation, suggests that some degradative processes should be
selective. In addition, the observation that changes in metabolic state
(as shown by the change in Gln/Asn, Fig. 3) and in growth rate (Fig. 7)
occur before any significant decrease in the amount of total protein
(Fig. 2) suggests that the protein equipment is changed through
increased protein turnover, in which selective proteolysis may have a
major role. The proteasome-mediated, ubiquitin-dependent proteolysis,
which is present in the cytosol and the nucleus of eukaryotic cells, is
highly selective (Coux et al., 1996). Furthermore, ubiquitin and
proteasome have been shown to increase in skeletal rat muscle during
starvation (Medina et al., 1995). As we showed that 20S proteasome is
present in maize plants, and particularly in growing tissues (G. Basset, R. Brouquisse, L. Malek, and P. Raymond, unpublished data), it
was tempting to investigate its fate during the light/dark cycle and
extended darkness. On the basis of its immunosignal on western blots
and, in the case of the root tips, on its chymotrypsin-like activity
(Figs. 5 and 6), it can be seen that the proteasome remained steady for
up to 48 h of darkness, whereas the total protein decreased. This suggests that in the cytosol and the nucleus of carbon-deprived cells,
proteasome-mediated proteolysis could selectively hydrolyze some
proteins, thus allowing the synthesis of enzymes or proteic factors
necessary for the acclimation to starvation.
Finally, the time course of the appearance of starvation symptoms
varied according to the tissue; proteolysis first appears in sink
tissues and later in mature tissues. These observations raise the
problem of the regulation of proteolysis by sugar supply. The
expression of numerous plant genes is known to be regulated by
carbohydrate status (for review, see Koch, 1996) and it is clear that
global protein breakdown is also regulated by sugars (James et al.,
1993). The potential involvement of different proteolytic systems (i.e.
vacuolar- and ubiquitin-dependent proteolysis) raises questions
concerning their regulation and their respective roles in the response
to starvation. Whereas the involvement of the lysosomal/vacuolar
pathway in autophagy associated with nutrient deprivation is well
established, ubiquitin- and proteasome-dependent proteolysis has been
associated with actively growing tissues well supplied with nutrients
(Ichihara and Tanaka, 1995; Coux et al., 1996). The identification of
the natural substrates of the proteases and of the signals (and their
transduction pathways) that regulate the expression of the gene
encoding these proteases are two major objectives of our ongoing
research on proteolysis.
| |
FOOTNOTES |
1
This work was supported by the French Institut
National de la Recherche Agronomique.
*
Corresponding author; e-mail brouquis{at}bordeaux.inra.fr; fax
33-556-84-32-35.
Received February 19, 1998;
accepted April 29, 1998.
| |
ABBREVIATIONS |
Abbreviations:
RSIP, root starvation-induced protease.
Succ-Leu-Leu-Val-Tyr-AMC, N-succinyl-Leu-Leu-Val-Tyr-7-amino-4-methyl coumarin.
| |
ACKNOWLEDGMENTS |
We wish to warmly thank Soazig Ledeleter, Emmanuelle Constant,
Frédéric Madec, and Mikaël Laizet, the undergraduate
students who helped us to prepare and analyze the 2716 extracts
necessary for this study.
| |
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Abstract
Introduction
