Jane M. Longland, Stephen C. Fry*, and Anthony J. Trewavas. Department of Botany, University of Edinburgh, The King's Buildings, Mayfield Road,. Edinburgh ...
Received for publication August 23, 1988 and in revised form January 25, 1989
Plant Physiol. (1989) 90, 972-976
0032-0889/89/90/0972/05/$01 .00/0
Developmental Control of Apiogalacturonan Biosynthesis and UDP-Apiose Production in a Duckweed1 Jane M. Longland, Stephen C. Fry*, and Anthony J. Trewavas Department of Botany, University of Edinburgh, The King's Buildings, Mayfield Road, Edinburgh, EH9 3JH, United Kingdom in wall composition that occur during the frond -- turion transition. Duckweed frond cell walls are rich in D-apiose residues (a branched-chain pentose), which occur in apiogalacturonan, a pectic polysaccharide (17). High concentrations of apiogalacturonan are restricted to certain fresh- and salt-water hydrophytic monocotyledons. In the cell walls of most other plants, apiose is only known to occur as a small proportion of the minor pectic polysaccharide, rhamnogalacturonan-II (9). The backbone of apiogalacturonan is an a-( 1->4)-linked polymer of D-galacturonic acid, to which apiose and apiobiose (=Dapiofuranosyl-,B-( l-13)-D-apiose) sidechains are linked via 02 or 0-3 (1, 6); D-xylose has also been detected (5, 6). Radioisotopic studies with cell-free extracts from Lemna minor ( 17) showed that D-glucuronic acid is a precursor of apiose. The step from UDP-glucuronic acid to UDP-apiose is catalyzed by the bifunctional enzyme UDP-apiose/UDP-xylose synthase. This enzyme can catalyze two reactions, UDP-glucuronic acid -- UDP-apiose and UDP-glucuronic acid -- UDP-xylose, whereas the more abundant UDP-glucuronic acid decarboxylase can only catalyze the single reaction, UDP-glucuronic acid - UDP-xylose (17). It was therefore possible for us to investigate changes in the synthesis of UDP-apiose and polysaccharide-bound apiose residues in vivo by feeding exogenous [3H]glucuronic acid as well as ['4C]glucose.
ABSTRACT Vegetative fronds of Spirodela polyrrhiza were induced to form dormant turions by the addition of 1 micromolar abscisic acid or by shading. The cell wall polymers of fronds contained a high proportion of the branched-chain pentose, D-apiose (about 20% of total noncellulosic wall sugar residues), whereas turion cell walls contained only trace amounts (about 0.2%). When the fronds were fed D-[3H]glucuronic acid for 30 minutes, the accumulated UDP-[3H]apiose pool accounted for about 27% of the total phosphorylated [3H]pentose derivatives; in turions, the UDP[3H]apiose pool accounted for only about 4% of the total phosphorylated [3H]pentose derivatives. We conclude that the developmentally regulated decrease in the biosynthesis of a wall polysaccharide during turion formation involves a reduction in the supply of the relevant sugar nucleotide. One controlling enzyme activity is suggested to be UDP-apiose/UDP-xylose synthase. However, since there was a 100-fold decrease in the rate of polysaccharide synthesis and only a 9-fold decrease in UDPapiose accumulation, there is probably also control of the activity of the relevant polysaccharide synthase.
Spirodela polyrrhiza is a temperate duckweed which forms resting buds (turions) at the onset of adverse environmental conditions. There are a number of structural and functional differences between fronds and turions (15). Turions are densely cytoplasmic, nongreen structures, containing large quantities of starch, whereas fronds are highly vacuolated, green, and contain little starch. One of the most striking differences is that turions have much thicker cell walls than fronds, but there are no reports of the chemical composition of turion cell walls ( 14). Spirodela can be induced to form turions by addition of ABA to the growth medium (1 1). Only small fronds of less than 1 mm diameter, which are still undergoing cell division, can be induced to develop into turions in this way; the developmental stage of the meristematic tissue is critical ( 15). During the frond -- turion transition, one of the earliest changes is a deceleration of cell expansion; turion-inducing concentrations of ABA reduce the plastic extensibility of the cell walls of small, fast-growing fronds within a few hours (8). This observation, coupled with the fact that turions have much thicker walls than fronds, led us to investigate changes
MATERIALS AND METHODS Radiochemicals D-[U-'4C]Glucose (250 mCi/mmol) was from Amersham International. D-[ 1-3H]Glucuronic acid, sodium salt, was prepared at Amersham by catalytic exchange of the pure sugar with 3H2 gas (method TL7). It was purified by paper chromatography in system 6 (see later) to yield 105 mCi at approximately 5 Ci/mmol and was repurified immediately prior to each experiment by paper electrophoresis at pH 3.5. Culture Conditions
Spirodela polyrrhiza fronds were grown on Hutner's medium diluted 1:1 with water (H/2) as described previously (15). Turions were produced from the parent fronds either by
'
J. M. L. and A. J. T. thank the Agricultural and Food Research Council, and S. C. F. thanks the Nuffield Foundation, for research grants.
972
CONTROL OF APIOGALACTURONAN BIOSYNTHESIS
treatment with 1 gM ABA (Sigma Chemical Co.) or by incubation in subdued light (50 ,umol photons s-' m-2 [=25% of normal intensity]) for 7 d after confluent growth had been
attained. Radioactive Labeling of Fronds and Turions
Radioactive sugars were supplied to the plant material without the addition of nonradioactive carriers so as to maximize specific radioactivity and minimize interference with normal photosynthetic metabolism. ['4C]Glucose and [3H] glucuronic acid were used at 40 ,gCi and 1 mCi, respectively, per 25 mL of H/2 incubation medium. For analysis of 14Cincorporation, the cultures were grown under standard conditions, aseptically, for 7 d in the presence of 40 ,uCi of [14C] glucose. For analysis of 3H-incorporation into wall polysaccharides, the plants were incubated with 1 mCi of [3H]glucuronic acid for 24 h. For analyis of short-term incorporation into nucleoside diphosphate-sugars, uptake had to be facilitated by use ofvacuum-infiltration: the plants were held below the surface with a wire grid, in a crystallizing dish containing 25 mL of medium and 1 mCi of [3H]glucuronic acid. The dish was placed in a glass chamber, which was then evacuated for 2 min. The vacuum was released, the grid removed, and the plants were maintained under normal growth conditions for 30 min.
Sampling and Extraction of Plant Material The following procedures were carried out at 0 to 4°C except where otherwise stated. Plants were rinsed briefly in H20, blotted, and weighed. They were ground in a mortar with 1 mL of an emulsion produced by mixing equal volumes of 90% (w/v) aqueous phenol and 10 mm potassium phosphate buffer (pH 6.5). The homogenate was centrifuged for 2 min at 6000g. The aqueous (top) phase was analyzed for free [3H]glucuronic acid and phosphorylated derivatives. The organic phase was shaken at 85 to 95°C for 1 h to complete the solubilization of proteins and then centrifuged as before to pellet the cell wall material. The pellet was washed with 5 x 1 mL of ethanol and air-dried. Destarching of Turion Cell Wall Material To avoid confusion with wall glucans and to prevent overloading of chromatograms, we removed starch by the method of Ring and Selvendran (12). About 3 mL of 90% (v/v) aqueous DMS0 was added per 1 g fresh weight of tissue and the mixture stirred magnetically for 1 h at 25°C. The cell wall material was pelleted again (2 min at 6000g) and the DMSO treatment repeated until there was no iodine-detectable starch in subsequent extracts. Analysis of Wall Polysaccharides The wall pellets were resuspended in 2 M trifluoroacetic acid (0.1 mL per mg dry material) and hydrolyzed at 1 20°C for 1 h. Insoluble material was pelleted again, and the soluble sugars were chromatographed on Whatman No. 1 paper as described later.
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Analysis of Sugar Nucleotides and Sugar Phosphates
UDP-Apiose is extremely unstable, and to minimize breakdown all the following procedures were carried out swiftly. The aqueous phase from the phenol extraction (cooled to 0°C) was concentrated to near dryness in vacuo with a 'SpeedVac' (Savant Instruments Inc., Hicksville, NY); during this process the solution froze. The frozen, concentrated solution was thawed to 4°C, loaded on to Whatman No. 1 chromatography paper, dried under a stream of cold air in a cold room (4°C), and immediately electrophoresed at pH 6.5. UDP-apiose is not available commercially but is known to coelectrophorese with UDP-xylose (7), which was hence used as a marker for UDP-apiose. Material comigrating with UDPxylose was eluted into 2 M trifluoroacetic acid by the method of Eshdat and Mirelman (2). To distinguish 3H associated with UDP-apiose from that associated with UDP-xylose and other coelectrophoresing sugar nucleotides, the eluate from the electrophoretogram was hydrolyzed at 100°C for 20 min and the 3H-sugars produced were resolved by paper chromatography as described below. Chromatography Chromatography was performed on Whatman No. 1 paper by the descending method in the following solvents (all compositions by volume unless otherwise stated): 1. Ethyl acetate/pyridine/H20 (8:2:1) (20 h). 2. Ethyl acetate/pyridine/H20 (8:2:1) (60 h). 3. Ethyl acetate/acetic acid/H20 (9:2:2) + 0.25% (w/v) phenylboronic acid (9.5 h). 4. Butan-l-ol/ethyl acetate/H20 (40:11:19) (60 h). 5. Butan-l-ol/acetic acid/H20 (12:3:5) (9 h), followed by ethyl acetate/pyridine/H20 (8:2:1) (7 h) in the same dimension. 6. Ethyl acetate/acetic acid/H20 (3:3:1) (12 h). Radioactive wall hydrolyzates were chromatographed in system 5. The chromatograms were cut into strips, which were assayed for radioactivity by scintillation counting in 0.5% (w/ v) ppO2 in toluene, and then selected strips (where there was any doubt as to the identity of radioactive peaks) were washed in toluene, dried, eluted with water by the method of Eshdat and Mirelman (2), and reanalyzed in one of the following systems: system 4 to separate xylose from fucose; system 2 to resolve glucose, galactose, and mannose; system 3 to separate apiose from rhamnose (apiose migrates considerably faster than any other common sugar in this system [13]); and electrophoresis at pH 3.5 to separate galacturonic acid from glucuronic acid. RF values are given elsewhere (3). Electrophoresis
Electrophoresis was performed on Whatman No. 1 paper in pH 6.5 buffer (acetic acid/pyridine/H20, 3:100:897 by volume) at 1.5 kV for 30 min or in pH 3.5 buffer (acetic acid/ pyridine/H20, 10:1:189 by volume) at 3 kV for 30 min. Electrophoresis was carried out in tanks filled with toluene (for the pH 6.5 buffer) or white spirit (for pH 3.5), which were 2 Abbreviations: PPO, 2,5-diphenyloxazole; Mbromophenol blue, electrophoretic mobility relative to that of bromophenol blue.
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Plant Physiol. Vol. 90,1989
LONGLAND ET AL.
continuously water-cooled to 20 to 30°C. The cathode was located at the top of the tank. Bromophenol blue was used as a tracker dye at pH 6.5, and picric acid at pH 3.5. While electrophoresis at pH 6.5 gives poorer resolution of uronic acids, sugar-phosphates and UDP-sugars than at pH 3.5 (4), pH 6.5 was chosen for analysis of the UDP-sugars because at pH 3.5 UDP-apiose is acid-hydrolyzed to UDP + free apiose, even at room temperature (7, 17). The pH 6.5 system was used for the initial separation of UDP-apiose (mbromophenol blue of UDP-xylose = 1.2) from any free apiose (mbromophenol blue = 0.0) before proceeding to the chromatographic separation systems. Coelectrophoresis of an apiose-derivative with UDPxylose does not prove definitively that it is UDP-apiose; however, it does prove that it carries a strong negative charge, and UDP-apiose is the only known naturally occurring phosphorylated derivative of apiose, so this is taken as the most likely identity. Some of the material could have been a-Dapiosyl 1,2-phosphate, the product of base-catalyzed degradation of UDP-apiose (7). Detection of Marker Compounds on Paper Papers developed in solvent systems containing pyridine were dried, dipped in butan-l-ol, and redried to remove residual pyridine before staining. Monosaccharides were detected with aniline hydrogen-phthalate (3); apiose stained lightly but exhibited yellow fluorescence under UV (13). UDP-Sugars and sugar phosphates were stained with perchloric acid/molybdate (3). Cellulose Estimation The cellulose content of the destarched cell walls was measured by the method of Updegraff (16). Microdissection of Young Fronds of Turion-Inducible Size from Parental Tissue Fronds were killed by immersion in boiling methanol for 5 min. The small fronds (less than 1 mm in diameter) were dissected out from inside the parental pockets with a scalpel and a binocular microscope.
RESULTS Composition of Cell Walls from Fronds and Turions The cellulose content of the cell walls of fronds and turions was 25.5 and 37.0% of the dry weight, respectively. To compare the sugar composition of the noncellulosic wall polysaccharides of fronds and tdrions, we carried out longterm in vivo feeding of D-[U-14C]glucose. Since the first product of glucose metabolism, glucose 6-phosphate, is metabolically central, the radioactivity was expected to be incorporated into all organic components of the cell with approximately equal efficiency. The partitioning of "'C between the various sugar residues of the wall after 7 d exposure to ["'C]glucose (Table I) is therefore expected to correspond closely to the total sugar composition (radioactive + nonradioactive) of the cell walls. The high degree of labeling of glucose in turions (63.3% of
Table I. Long-Term Partitioning of 14C from D-[14C]glucose between the Sugar Residues of Noncellulosic Wall Polysaccharides of Spirodela polyrrhiza Values represent mean of three determinations ± SE. Included are data for turions induced both by ABA-treatment and by shading. No significant difference was noted in the labeling of turions produced by these two methods. Similarly, there was no effect of ABA-treatment on mature fronds (data not shown). Total Noncellulosic Poly-
Sugar Residue
sacchande-Bound Sugar Residues Fronds
Apiose Arabinose Fucose Galactose Galacturonic acid Glucose Glucuronic acid Mannose Rhamnose Xylose
21.4 ± 2.3 11.3 ± 1.0 1.3 ± 0.3 8.7 ±0.1 25.6 ± 0.5 13.0 ± 2.0 5.5 ± 2.2
Undetectable 4.6 ± 1.0 8.6 ± 2.9
Turions
0.13 ± 0.06 5.4 ± 2.6 0.63 ± 0.32 4.8±0.13 15.0 ± 4.9 63.3 ± 7.4 5.3 ± 3.5 Undetectable 0.40 ± 0.20 5.2 ± 1.6
total noncellulosic "'C-monosaccharides) contrasts sharply with the value for fronds (13.0%). The ["'CJglucan of the turions is unidentified and will be the subject of a future investigation. Since the walls had been destarched by an extremely effective method (12), the polymer is unlikely to be ["'C]starch. Since the high ["'C]glucose was not accompanied by high ["'C]xylose, the polymer cannot have been ["'C]xyloglucan. It cannot have been ["'C]cellulose, as this polysaccharide is not significantly hydrolyzed under the acidic conditions employed (3). It is most likely to be ["'C]callose or ,#-( 1 >3),(1--4)-['4C]glucan. The ['4C]arabinose:"'4C]xylose ratio was 1:0.76 in fronds and 1:0.96 in turions. ['4C]Rhamnose was 11.5-fold more abundant in frond walls than in turion walls. However, the most striking difference was that, in the fronds, ["'C]apiose constituted 18 to 26% of the total wall-bound noncellulosic ['4C]polysaccharide, whereas ['4C]apiose was virtually absent in turions (0-0.2%). The high level of ['4C]galacturonic acid found in fronds (25.6%) suggests that the apiose could be present in the form of apiogalacturonan, although the turions were found also to contain a relatively high proportion of ['4C]galacturonic acid (15.0%) while lacking ['4C]apiose. Incorporation of Apiose Residues into Wall Polysaccharides of Fronds of Various Ages In growing fronds, an experiment was conducted to monitor the medium-term partitioning of material between the various pentoses from their common precursor, [3H]glucuronic acid. Spirodela incorporated 3H from exogenous [3H]glucuronic acid into polysaccharide-bound [3H]arabinose, [3H]xylose, [3H]apiose, [3H]galacturonic acid, and [3H]glucuronic acid residues. Negligible radioactivity was incorporated into the major neutral hexose residues, glucose and galactose. The ratio [3H]xylose:[3H]arabinose:[3H]apiose in the noncellulosic wall polysaccharides varied with age (Fig. 1). As the frond
CONTROL OF APIOGALACTURONAN BIOSYNTHESIS
diameter increased from 2.2 to 7.0 mm, partitioning into [3H] apiose residues doubled, mainly at the expense of [3H]arabinose; however, at all stages examined, [3H]apiose was a major product in contrast to the situation in turions (Table I). In a single analysis of the cell walls of roots of mature fronds, [3H] apiose was a major constituent, the [3H]pentoses being 41% [3H]apiose, 35% [3H]xylose, and 24% [3H]arabinose. Investigation of UDP-apiose Pools
The inability of turions to make polysaccharide-bound apiose resiudes could be due to (a) failure to synthesize UDPapiose or (b) failure to incorporate the apiose residue from UDP-apiose into nascent polysaccharides. To determine which of these alternatives was the case, we investigated the presence, or absence, of UDP-apiose within turions. The presence of normal or elevated levels of UDP-apiose would prove that option (b) was true, whereas its absence would indicate that (a) was true. To investigate the ability of fronds and turions to make UDP-apiose, we fed [3H]glucuronic acid to the plants for 30 min. During this period only a small proportion of the [3H]glucuronic acid was taken up, so all obligatory intermediates en route to 3H-polysaccharides
40 .
30
':u 20 20
C-
*-4In 10
Cq 40
C30 -404-
0
o
40
._
o
30
0. C-
20 2
5 6 4 3 Frond diameter (mm)
7
Figure 1. Medium-term partitioning of 3H from D-[3H]glucuronic acid between pentose residues of the noncellulosic wall polysaccharides of S. polyrrhiza fronds: effect of frond size.
975
should still have been present in radioactive form. Turions incorporated much less 3H into UDP-apiose than did fronds (Table II). In contrast, turions accumulated somewhat more UDP-[3H]arabinose and UDP-[3H]xylose than fronds. In turions, UDP-[3H]apiose constituted 1.7 to 5.5% of the total UDP-[3H]pentoses + [3H]pentose 1-phosphates, whereas in fronds it constituted 18 to 41% (Table II)-an average of ninefold higher than in turions. DISCUSSION While interest has previously been extended toward the apiose content of Lemna minor (1, 5, 6, 17), which does not form turions, there are no previous analyses of turion cell walls. Our results show that in Spirodela polyrrhiza the frond and turion cell walls are chemically very different: in particular, the large proportion of apiose found in frond walls is virtually absent in turion walls, despite the level of galacturonan (the apiosyl acceptor) remaining high in turions. Spirodela incorporated 3H from added [3H]glucuronic acid into polysaccharide-bound [3H]pentose and [3H]uronic acid residues. This indicates that exogenous [3H]glucuronic acid was taken up, phosphorylated, and nucleotidylated to form UDP-[3H]glucuronic acid, the immediate precursor of UDP[3H]galacturonic acid, UDP-[3H]xylose and UDP-[3H]apiose (Fig. 2). These UDP-3H-sugars, and the UDP-[3H]arabinose to which UDP-[3H]xylose epimerizes, are sugar donors for polysaccharide synthesis (4). While turions incorporated detectable levels of 3H from [3H]glucuronic acid into UDP-[3H]apiose, the extent of this incorporation was much lower than in fronds, despite the fact that turions had a slightly greater ability to accumulate UDP[3H]arabinose and UDP-[3H]xylose in the same experiments. This suggests that the reaction UDP-glucuronic acid -+ UDPapiose, catalyzed by UDP-apiose/UDP-xylose synthase, is minimal in turions. In most cases where there is developmental regulation of polysaccharide biosynthesis, control is believed to be imposed at the final step, catalyzed by the polysaccharide synthase(s) (10). We suggest an exception to this rule, since it would seem that, whether or not the activity of the relevant polysaccharide synthase (UDP-apiose: galacturonan apiosyltransferase) falls during turion formation, the approximately ninefold relative decrease in UDP-apiose accumulation will be epistatic and will probably of itself reduce the rate of apiogalacturonan synthesis (Fig. 2). We have measured steady state levels of UDP-[3H]apiose rather than the rate of its synthesis. It is possible that changes in the observed levels of UDP-[3H]apiose reflect changes in the rate of its utilization rather than changes in the rate of its synthesis. However, if any change in UDP-[3H]apiose utilization does accompany turion development, it is likely to be negative (to account for the cessation of apiogalacturonan synthesis), and this would be predicted to spare the substrate and cause an increase in UDP-[3H]apiose accumulation, not the observed decrease. Nevertheless, the 9-fold decrease in relative UDP-[3H] apiose accumulation (Table II) contrasts with the approximately 100-fold decrease in rate of deposition of ['4C]apiose residues in the cell wall during the frond -- turion transition
Plant Physiol. Vol. 90,1989
LONGLAND ET AL.
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Table II. Short-Term Partitioning of iH from r,3H]Glucuronic Acid between Phosphorylated Pentose-Derivatives in Spirodela polyrrhiza Values represent mean of eight determinations ± SE. Organ
UDP-arabinose + arabinose-phosphates (A)
Fronds Turions
13.2 ± 0.8 15.8 ± 0.7
Radioactivity Accumulated in UDP-xylose + xylose-phosphates (B) cpm/mg fresh wt 10.7 ± 1.5 16.8 ± 6.5
10Glcuronic acidi El D-Glucuronic acid 1-phosphate
E2
3. 4.
5.
UDP-D-Glucuronic acid
6. 7. 8.
'olysaccharide-bound pentose residuesIl
9.
Figure 2. Proposed pathways by which 3H from exogenous D-[3H] glucuronic acid is incorporated into phosphorylated pentose denvatives and polysaccharide-bound pentose residues. The enzymes involved are: El, D-glucuronokinase; E2, UDP-D-glucuronic acid pyrophosphorylase; E3, UDP-D-glucuronic acid decarboxylase; E4, UDP-D-apiose/UDP-D-xylose synthase; E5, UDP-D-xylose 4-epimerase; E6, E7, E8, polysaccharide synthases. UDP-L-Arabinose and UDP-D-xylose are likely to be in dynamic equilibrum with the corresponding pentose 1-phosphates (4).
10.
(Table I). It thus seems likely that turions have decreased levels of both the UDP-apiose generating system and also of the apiogalacturonan synthase system.
14.
11.
12.
13.
15.
LITERATURE CITED
16. 1. Duff RB (1965) The occurrence of apiose in Lemna (duckweed) and other Angiosperms. Biochem J 94: 768-772 2. Eshdat Y, Mirelman D (1972) An improved method for the
17.
UDP-apiose + apiose phosphates (C)
C/[A + B + C]
8.98 ± 1.53 1.12 ± 0.19
26.6 ± 2.4 3.7 ± 1.1
%
recovery of compounds from paper chromatograms. J Chromatogr 65: 458-459 Fry SC (1988) The growing plant cell wall: chemical and metabolic analysis. Wiley, New York Fry SC, Northcote DH (1983) Sugar-nucleotide precursors of the arabinofuranosyl, arabinopyranosyl, and xylopyranosyl residues of spinach polysaccharides. Plant Physiol 73: 1055-1061 Hart DA, Kindel PK (1970) Isolation and partial characterization of apiogalacturonans from the cell wall of Lemna minor. Biochem J 116: 569-579 Hart DA, Kindel PK (1970) A novel reaction involved in the degradation of apiogalacturonans from Lemna minor and the isolation of apiobiose as a product. Biochemistry 9: 2190-2196 Kindel PK, Watson RR (1973) Synthesis, characterization and properties of uridine 5'-(a-D-apio-D-furanosyl pyrophosphate). Biochem J 133: 227-241 Longland JM (1986) The molecular mode of action of abscisic acid in the induction of dormancy. PhD thesis, University of Edinburgh McNeil M, Darvill AG, Fry SC, Albersheim P (1984) Structure and function of the primary cell walls of plants. Annu Rev Biochem 53: 625-663 Northcote DH (1985) Control of cell wall formation during growth. In CT Brett, JR Hillman, eds, The Biochemistry of Plant Cell Walls. Cambridge University Press, Cambridge, pp 177-197 Perry TO, Byrne OR (1969) Turion induction in Spirodela polyrrhiza by abscisic acid. Plant Physiol 44: 784-785 Ring SR, Selvendran RR (1978) Purification and methylation analysis of cell wall material from Solanum tuberosum. Phytochemistry 17: 745-752 Sandermann H (1969) Specific and rapid determination of Dapiose. Phytochemistry 8: 1571-1575 Smart CC, Trewavas AJ (1983) Abscisic-acid-induced turion formation in Spirodela polyrrhiza L. II. Ultrastructure of the turion, a stereological analysis. Plant Cell Environ 6: 515-522 Smart CC, Trewavas AJ (1984) Abscisic-acid-induced turion formation in Spirodela polyrrhiza L. III. Specific changes in protein synthesis and translatable RNA during turion development. Plant Cell Environ 7: 121-132 Updegraff DM (1969) Semi-micro determination of cellulose in biological materials. Anal Biochem 32: 420-424 Watson RR, Orenstein NS (1975) Chemistry and biochemistry of apiose. Adv Carbohydr Chem Biochem 31: 135-184
