Planta (1992)187 : 242-246

P l a n t a 9 Springer-Verlag1992

Inter- and intracellular distribution of amino acids and other metabolites in maize (Zea mays L.) leaves Hendrik Weiner* and Hans W. Heldt**

Institut fiir Biochemieder Pflanze, Universit/it G6ttingen, Untere Karspfile 2, W-3400 G6ttingen, Federal Republic of Germany Received 21 October 1991; accepted 13 January 1992 Abstract. In illuminated maize ( Z e a m a y s L.) leaves, the distribution of triose phosphates, 3-phosphoglycerate, malate and various amino acids between the chloroplastic and the extrachloroplastic compartments of mesophyll and bundle-sheath cells, and the total vacuolar fraction of the leaves, was determined by a combination of previously published methods, for separating mesophyll from bundle-sheath material, and for nonaqueous subcellular fractionation. The results show that the triose phosphate/3-phosphoglycerate ratio in the extrachloroplastic fraction of the mesophyll cells is about 20-fold higher than in the bundle-sheath cells, which is in accordance with a triose phosphate/phosphoglycerate shuttle postulated previously. Whereas the vacuolar compartment was shown to contain most of the cellular malate, amino acids were found to be almost absent from this compartment. The amino-acid pattern in the extrachloroplastic fraction of the bundle-sheath cells largely resembled the pattern in whole leaves. These results show that for future studies the analysis of amino-acid contents in whole maize leaves can be used as a measure for the amino-acid levels in the cytosol of bundle-sheath cells. Key words: Amino acid (inter-, intracellular distribution) - Leaf (metabolite distribution) - Photosynthesis (metabolites) - Z e a (metabolite distribution)

* Present address: BotanischesInstitut, Universit~itHeidelberg, Im Neuenheimer Feld 360, W-6900 Heidelberg, Federal Republic of Germany ** To whom correspondence should be addressed Abbreviations: BS= bundle sheath; Chl = chlorophyll; ctMan= ctmannosidase; ME = malicenzyme;MDH = malatedehydrogenase; MS=mesophyll; PEPCase=phosphoenolpyruvate carboxylase; PGA = 3-phosphoglycerate; trioseP= triose phosphate

Introduction

Besides sucrose, amino acids are the major metabolites exported from the leaves to other parts of the plant via the phloem system. The specificity of uptake into the phloem, as well as phloem transport, can be evaluated by a comparison of the amino-acid content of the phloem sap, as measured using the aphid technique (Barlow and McCully 1972), with that in the cytosol of the source leaf (Riens et al. 1991; Winter et al. 1992). In spinach and barley leaves, the amino-acid content of the cytosol has been determined by nonaqueous fractionation of frozen leaf material (Gerhardt and Heldt 1984). With maize leaves, where the export into the phloem system proceeds from the cytosol of the bundle sheath (BS) cells, a reliable measurement of the metabolite composition in that compartment requires not only a subfractionation of each cell type into vacuolar, chloroplastic and cytosolic fractions, but also a separation of the BS and mesophyll (MS) material of the leaves, under conditions where the metabolites are not altered during the separation procedure. Nonaqueous fractionation, allowing the subfractionation of lyophilized leaf material (Heber 1957; Stocking 1959; Slack et al. 1969) has been employed by Gerhardt and Heldt (1984) to separate MS and BS chloroplasts from each other, and this method has been further refined by Usuda (1988). Leegood (1985) has determined the total metabolite content in the MS and BS fraction by crushing leaves by means of a roller device to yield a sap of almost pure mesophyll which was then quenched in perchloric acid within 1 s. In an alternative approach by Stitt and Heldt (1985a), maize leaves were frozen rapidly in liquid N2 and then partially homogenized while still suspended in liquid N2. Subsequently, particles enriched in MS cells, because of their smaller size, were separated from the larger particles of BS strands by filtering the homogenate (kept under liquid N2) through a series of nylon nets. This technique allowed a quantitative determination of the metabolite contents of the MS and BS cells of maize leaves (Stitt and Heldt 1985b).

H. Weiner and H.W. Heldt: Amino-acid distribution in maize leaves

243

In the w o r k described in the present report, the contents o f m a j o r a m i n o acids in the cytosolic a n d chloroplastic fraction o f MS a n d BS cells, a n d also in the total v a c u o l a r fraction, were estimated by a c o m b i n a t i o n o f the n o n a q u e o u s fractionation o f lyophilized leaf material and the differential filtration o f frozen leaf material mentioned above.

a marker enzyme for whole cytosolic leaf material on the assumption that the BS and MS cytosolic leaf materials have a very similar density. From the distribution of metabolites and marker enzymes in the four fractions, the content of each compartment, represented by the marker enzymes, was calculated according to Riens et al. (1991). For the assay of 3-phosphoglycerate (PGA), triosephosphate (trioseP), malate and Chl see Stitt and Hetdt (1985a). Amino acids were determined by high-performance liquid chromatography as described by Riens et al. (1991).

Material and methods

Results and discussion

Four- to five-week-old maize (Zea rnays L., cv. Balda; Fa, KrSbel, Grttingen, FRG) plants, grown in a climatized growth chamber under a 12 h light/12 h dark cycle were used for the measurements. From plants with about ten leaves, samples were taken from the upper one-third of the sixth to eighth leaves (counted from below) and immediately frozen in liquid N 2. The leaf samples were collected 30 min before the end of the illumination period. The light level at these leaves was 600-800 ~mol photons- m -2 9s -2. The midrib was discarded from the frozen leaves and the deribbed frozen leaf material was kept in liquid N2 until further fractionation. The method of Stitt and Heldt (1985a) was employed for the homogenization and filtration of maize leaves kept under liquid N2, and for the preparation of extracts from the leaf fractions. The distribution of metabolites in the two cell types was evaluated by linear regression analysis after assay of metabolites and markerenzyme activities [phosphoenolpyruvate carboxylase (PEPCase: MS), NADP-malic enzyme (NADP-ME: BS)] in the four different fractions (Stitt and Heldt 1985a, b). The nonaqueous fractionation of lyophilized maize leaves was carried out essentially as described for the fractionation of spinach leaves by Gerhardt and Heldt (1984). Maize leaves were snap-frozen in liquid N2, deribbed and lyophilized. Samples [100-150 g material containing 1.2-1.8 mg chlorophyll (Chl)] were suspended in 20 ml of a mixture of tetrachlorethylene/heptane (density = 1.25 g- era-3), and sonicated for 3 min (5-s periods followed by pauses of 5 s) using a Branson Sonifier Cell disruptor B 15 (Fa. Schfitt, G6ttingen, FRG). During the whole procedure the sample was kept in a mixture of dry ice and heptane at --40 ~ C. The homogenate was filtered through a 20-lain nylon net to separate coarse material. The filtrate was distributed to four centrifugation tubes, and to these pure heptane was added to yield a volume of 10 ml in each tube. The samples were centrifuged for 2 rain at 3000 rpm with a swing-out rotor in a centrifuge (Digifuge; Christ, Osterode, FRG). The clear supernatant, devoid of metabolites, was discarded and the sediment resuspended in 2 ml tetrachlorethylene/ heptane, (density= 1.25 g. crn-3), and filtered once more through a 20-lam nylon net. Aliquots were taken from the filtrate for assay of marker enzymes, chlorophyll and metabolites. A 1-ml aliquot of the filtrate, containing 200-600 lag Chl was layered on top of a logarithmic density gradient of a mixture of tetrachlorethylene and heptane with a density between 1.25 and 1.59 g. cm -3. The formation of the gradient and the centrifugation were carried out as described earlier (Gerhardt and Heldt 1984). After centrifugation the gradient was separated into four fractions, each mixed with a threefold volume of pure heptane, and centrifuged at 3000 rpm for 3 rain as above. The clear supernatant was discarded and the resultant sediment was suspended in 1.5 1111heptane and divided into two samples (one for assay of marker enzymes and one for metabolites). After the addition of extraction medium the resulting suspensions were homogenized in an ice bath using a homogenizer (PotterElvehjem, Zurich, Switzerland) and further processed according to Gerhardt and Heldt (1984). The distribution of metabolites among the different leaf compartments was evaluated from the metabolite content and the activities of the marker enzymes NADP-malate dehydrogenase (NADP-MDH: MS chloroplasts), NADP-ME (BS chloroplasts), PEPCase (MS cytosol), a-mannosidase 0xMan: vacuoles). As no marker enzyme is available for BS cytosol, PEPCase was used as

Intercellular distribution of metabolites. T h e distribution o f metabolites between the BS a n d MS cells o f frozen maize leaves was determined using the m e t h o d o f Stitt and Heldt (1985a). T h e results are s h o w n in Table 1. The distribution o f P G A , occurring primarily in the BS cells, and that o f trioseP, located m a i n l y in the M S cells, was very similar to earlier results o f L e e g o o d (198 5) a n d Stitt and Heldt (1985a, b). The distribution o f aspartate, glutamate a n d alanine resembled earlier results o f Leeg o o d (1985). H o w e v e r , with regard to malate, which was mainly f o u n d in the BS, o u r results differ m a r k e d l y f r o m earlier w o r k in which the BS cells were f o u n d to contain only 5% ( L e e g o o d 1985) or 33% (Stitt a n d Heldt 1985a, b) o f the total malate. T h e cause o f this variation presumably lies in the subcellular localization o f malate, as will be discussed later.

Subcellular distribution of metabolites. F o r the determ i n a t i o n o f subcellular metabolite levels in maize leaves, the frozen, lyophilized a n d n o n a q u e o u s l y h o m o g e n i z e d leaf material was subjected to centrifugation in a density gradient c o m p o s e d o f a mixture o f tetrachlorethylene and heptane. After centrifugation, the material c o n t a i n e d in the gradient was divided into f o u r fractions, n u m b e r e d f r o m the t o p to the b o t t o m o f the gradient. As s h o w n in Fig. 1, the material deriving f r o m vacuoles, as represented by the m a r k e r e n z y m e ~tMan, was c o n c e n t r a t e d in the heaviest fraction, 4, which also contained a large p a r t o f the M S cytosol material designated by P E P C a s e activity. A s s u m i n g t h a t the densities o f the cytosolic material o f the M S a n d BS cells are similar, we e m p l o y e d P E P Case as a m a r k e r for the total cytosolic material. The BS chloroplasts (marker N A D P - M E ) were f o u n d mainly in the middle o f the gradient, whereas the M S chloroplasts represented by N A D P - M D H were f o u n d to be concentrated m o s t l y in the lightest fraction. A similar separation o f the m a r k e r enzymes N A D P - M D H , N A D P - M E a n d P E P C a s e was f o u n d by n o n a q u e o u s density gradient centrifugation o f lyophilized leaf m a t e rial by U s u d a (1988). The contents o f trioseP, P G A , m a l a t e a n d various a m i n o acids were determined in aliquot samples o f the f o u r fractions s h o w n in Fig. 1. U s i n g a calculation p r o g r a m described previously by Riens et al. (1991), the percentage distribution o f the various metabolites a m o n g the f o u r c o m p a r t m e n t s represented b y the f o u r m a r k e r enzymes was evaluated f r o m the distribution o f m a r k e r enzymes and metabolites in the four different fractions o f the gradient. T h e results o f this evaluation are s h o w n in Table 2. The metabolite contents o f M S and BS

244

H. Weiner and H.W. Heldt: Amino-acid distribution in maize leaves

Table 1. Metabolite content of maize

leaves (11.5 h illumination, ambient CO2) and the distribution of the metabolites between the MS and BS cells of these leaves. For details see Material and methods. Average values from four different experiments 4- SD

Metabolite a

Total leaf content (nmol 9(mg Chl)-1)

Proportion Content of total content (nmol " (mg total Chl)-1) in BS (%) BS MS

Asp Glu Gln Ala Gly Ser Mal PGA TrioseP

1100 + 370 1200 4- 360 4104- 260 32004- 1700 2304- 15 2604- 15 39004- 290 810 4- 140 410 4- 84

48 + 2 42 4- 4 504- 5 57+ 3 614-5 594-7 774- 7 73 4- 8 18 4- 7

530 505 205 1820 140 150 3000 590 70

570 695 205 1380 90 110 900 220 340

a Ala, alanine; Asp, aspartic acid; Gin, glutamine; Glu, glutamic acid; Gly, glycine; Mal, malate; Ser, serine

Table 2. Distribution of metabolites in fractions of maize leaf material as characterized by marker enzymes. Average values of four experiments _+SD. The leaves, same as used in Table 1, were quenched in liquid N2 after 11.5 h illumination at ambient CO2. For details of fractionation and evaluation see Methods and text

Metabolite

Asp Glu Gln Ala Gly Ser Mal PGA TrioseP

Distribution (%) NADP-MDH

ME

PEPCase

ctMan

28_+6 30_+5 27-t-9 15+5 19_+1 20_+2 2-+1 14_+3 21_+2

28 _+ 3 27 _+ 3 30 +19 28 __+ 4 26 +_ 7 31 _+ 1 0.8__ 1.3 43 _+ 3 11 _+ 4

39_+ 6 40_+ 6 10_+ 8 55+ 8 54_+10 48___ 1 3_+ 3 42_+ 5 64_+ 8

5 _+5 2 _-!-3 34 _+9 2 _+2 2 _+2 0 93 _+3 0.6_+0.5 3 _+5

100 [ ] NADP - MDH

8O

7

S / 6O N

[] NADP-ME

[ ] PEPCase 9 a Man

/

r

4O

r

20

r

1 2 3 4 Fig. 1. Distribution of marker enzymes in fractions after centrifugation of lyophilized maize leaf material in a nonaqueous density gradient. The fractions are numbered from top to bottom of the centrifugation tube. Mean values from four experiments. For details see Material and methods and text. The leaf material was collected 30 min before the end of a 12-h illumination period

chloroplasts, a n d o f the c o m b i n e d fraction o f BS and MS vacuoles, can be calculated f r o m the d a t a o f Tables 1 a n d 2. By subtracting the c o n t e n t o f the c o r r e s p o n d i n g chloroplast fractions f r o m the metabolites f o u n d in the total BS a n d M S fraction (Table 1) the extrachloroplastic p o r t i o n o f BS and MS cells, consisting o f cytosolic and v a c u o l a r material, can be evaluated. Distribution ofmalate. The results o f Table 3 show clearly that the bulk o f the malate is c o n c e n t r a t e d in the vac-

uoles. Since a b o u t three-quarters o f the total malate is f o u n d in the BS cells, the vacuoles containing malate m u s t be primarily located in the BS cells. N o n a q u e o u s fractionation o f spinach leaves had shown earlier that during the light period malate is accumulated in the vacuoles ( G e r h a r d t and Heldt 1984). A p p a r e n t l y this also holds for maize leaves. The extent o f malate accum u l a t i o n in maize leaves appears to be extremely variable. In earlier experiments carried out in our l a b o r a t o r y the majority o f the malate was f o u n d in the MS p o r t i o n o f the maize leaf material (Stitt and Heldt 1985a, b). It should be noted that in these leaves the total content o f malate was only one-third o f that f o u n d in Table 3, reflecting variations in the leaf material. In earlier studies the existence o f a diffusion gradient o f malate between the M S and BS cells was postulated f r o m the difference in total content o f malate in the MS and BS fractions o f maize leaves (Leegood 1985; Stitt and Heldt 1985b). Because o f the high malate content in the vacuoles, a definite conclusion a b o u t c o n c e n t r a t i o n differences in the cytosol o f MS and BS cells c a n n o t be reached for the maize leaves studied in the present work. Distribution o f trioseP and PGA. A l m o s t no P G A and trioseP were f o u n d in the vacuolar fraction. As it is k n o w n that these substances do not occur in the vacuoles, the above result demonstrates the reliability o f o u r method. Owing to the absence o f trioseP and P G A f r o m the vacuoles, it is possible to determine the t r i o s e P / P G A ratios in the chloroplast and cytosol fractions o f b o t h MS

H. Weiner and H.W. Heldt: Amino-acid distribution in maize leaves Table 3. Metabolite contents in subcellular fractions of maize leaves, as evaluated from the data of Tables 1 and 2. For details, see Methods and text. CC, chloroplastic compartment; EC, extrachloroplastic compartment

Metabolite

Asp Glu Gln Ala Gly Ser Mal PGA TrioseP TrioseP/PGA

and BS cells (Table 3). In comparison with spinach leaves, where in the steady state o f photosynthesis the trioseP/PGA ratio in the chloroplasts was found to be 0.05 (Gerhardt et al. 1987), this ratio in the BS chloroplasts differs only by a factor of 2, but is 15-fold higher in the MS chloroplasts. As in spinach leaves, in maize MS cells the trioseP/PGA ratio in the cytosol is about three times higher than in the chloroplasts. With isolated spinach chloroplasts a similar difference in stromal and cytosolic trioseP/PGA ratios was found to be the result of the effect of light on the transport of trioseP and P G A by the chloroplast phosphate translocator (Flfigge and Heldt 1986). Our results show that in maize MS chloroplasts the phosphate translocator functions similarly in this respect. On the other hand, there appears to be no difference between the trioseP/PGA ratios in the cytosol and chloroplast stroma of BS cells. The different quotients between the trioseP/PGA ratios in the chloroplastic and extrachloroplastic compartments of MS and BS cells reflect the different functions of the phosphate translocators in the chloroplasts of MS and BS cells. During C4 photosynthesis of maize the trioseP-PGA exchange across the inner-envelope membrane o f BS chloroplasts has to proceed in the opposite direction from that of mesophyll chloroplasts o f C4 or C3 plants. It has been recently shown that in the C4 plant Panicum miliaceum the phosphate translocators of MS and BS chloroplasts were distinctly different in their properties (Ohnishi et al. 1989). The existence of high diffusion gradients of trioseP and P G A suitable for driving a trioseP/PGA shuttle between the MS and BS cells has been postulated in earlier studies from measurements o f trioseP/PGA ratios in the total MS and BS fractions of maize leaves (Leegood 1985; Stitt and Heldt 1985b). Our measurements of trioseP and P G A contents in the cytosol of MS and BS cells verify this postulate. Although, because o f uncertainties about the sizes of the subcellular compartments in maize leaves, the concentrations o f trioseP and P G A in the cytosolic compartments of the MS and BS cells cannot be evaluated with high reliability, the 20-fold difference between the trioseP-PGA ratios in the cytosol of the MS and BS cells clearly indicates that a gradient exists between the two compartments, facilitating the

245 Content (nmol 9(mg total Chl)-1) Total BS MS CC EC CC 1 100 1200 410 3 200 230 260 3900 810 410

308 329 123 896 60 86 31 348 45 0.13

220 180 82 928 80 67 2970 243 29 0.12

308 360 110 480 44 50 78 113 86 0.76

EC 264 336 95 896 46 57 821 106 250 2.36

BS + MS vacuoles 55 24 140 64 3 0 3600 5 12

Table 4. Distribution of amino acids in whole maize leaves and in the BS cytosolic fraction (extrachloroplastic compartment) of these leaves. Data from Table 3 Amino acid Asp Glu Gin Ala Gly Ser Y.

Content (%) Leaf 17 19 6 50 4 4 ~ 100

BS cytosol 16 20 6 53 3 3 ~ 100

diffusion of P G A from the BS to the MS cells and that of trioseP in the opposite direction. Distribution of amino acids. The vacuolar content of amino acids amounted to about one-third of the total, but in contrast to malate, and to a lesser extent glutamine, only a minor proportion of the other amino acids analyzed (representing the main amino acids of maize leaves) were found to be present in the vacuoles. As mentioned before, the sizes of the various subcellular compartments in maize leaves are not known exactly. However, as an extreme case, if all vacuolar amino acids (BS + MS) were located in the BS cells and the ratio of the sizes of the vacuolar and the cytosolic compartments in the BS cells were only 5, the data of Table 3 would yield a maximal estimate of the vacuolar concentrations of Asp, Glu, Ala, Gly and Ser of only 0-4% o f the corresponding concentrations in the cytosol. This result demonstrates that in maize, as in spinach (Riens et al. 1991) and barley (Winter et al. 1992), the amino acids mentioned above are virtually excluded from entering the vacuoles. As shown in Table 3, the amino acids are distributed quite evenly among the various compartments except the vacuolar one. The percentage distributions o f amino acids in whole maize leaves and in the extrachloroplastic compartment o f BS cells are almost identical (Table 4). Thus for all the amino acids listed the measured leaf contents are, in relative terms, very similar to the contents in the BS cytosol.

246 Assuming that the unknown volume of the cytosolic c o m p a r t m e n t in BS cells is 20 gl 9 (mg total Chl)-1 and allocating all the amino acids of the extrachloroplast fraction to the cytosol, the data of Table 3 yield the following concentration estimates: Asp 11 m M , Glu 9 m M and Ala 46 m M . Analogously, high amino-acid concentrations can be evaluated for the other maize leaf c o m p a r t m e n t s except the vacuoles. Very high contents of amino acids have been found in illuminated spinach leaves, with an estimated total concentration in the cytosol of 120 m M (Riens et al. 1991). Our results indicate that, as in spinach leaves, amino acids represent major solutes of the chloroplastic and cytosolic compartments of maize leaves. The aphid technique, by which phloem sap is collected from leaves by severing the stylet of an aphid feeding from these leaves (Barlow and McCully 1972; Fisher and F r a m e 1984), makes it possible to monitor the export of assimilates f r o m the leaves under various metabolic conditions. In order to investigate the factors governing amino-acid export f r o m maize leaves, the concentrations o f the amino acids in the cytosol o f the BS cells and in the phloem sap have to be known. The results of the present publication show that for routine measurements o f amino-acid export o f leaves in relation to leaf metabolism, e.g. in the course of a diurnal cycle, instead o f assaying cytosolic amino-acid levels by the complex fractionation techniques described in this paper, it appears sufficient for m o s t amino acids, and within a reasonable error, to analyze the total leaf contents and use them as a measure o f the content in the BS cytosol. Similarly in spinach, the amino-acid content of whole leaves was shown to reflect the amino-acid pattern in the cytosolic c o m p a r t m e n t (Riens et al. 1991). In a recent report from our laboratory, in which the percentage distribution of amino acids in illuminated maize leaves was compared with that in the phloem sap collected from these leaves, it was postulated that, under the conditions of the experiment, glutamine was extracted preferentially from the source cells to the sieve tubes whereas the export of glycine and aspartate was restricted by comparison with that o f the other amino acids (Weiner et al. 1991). The validity of the conclusions of this experimental approach is demonstrated in the present publication.

H. Weiner and H.W. Heldt: Amino-acid distribution in maize leaves This work was supported by the Bundesminister fiir Forschung und Technologie.

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