Possible Control Sites of Polysaccharide Synthesis during Cell Growth and Wall Expansion of Pea Seedlings (Pisum sativum L.) G. Dalessandro* and D.H. Northcote Department of Biochemistry, Universityof Cambridge, Cambridge CB2 1QW, U.K.
Abstract. The activities of the enzymes of uridine
diphosphate sugar interconversions (UDP-D-glucose 4-epimerase, UDP-D-glucuronate 4-epimerase, UDPD-xylose 4-epimerase, UDP-D-glucose dehydrogenase and UDP-D-glucuronate decarboxylase) were measured by using enzymic preparations (protein precipitated between 40-65% (NH4)2SO 4 saturation) isolated from segments at different stages of elongation of the third internode of pea seedlings. All enzymic activities increased from dividing and non-elongated cells to fully elongated cells. At all stages of growth, the specific activity or the activity per cell of UDP-D-glucose dehydrogenase was much lower than that of UDP-D-glucuronate decarboxylase and this may represent a controlling step in the formation of UDP-D-xylose. During elongation, changes were also found in the activities of the epimerases. These could be correlated with the corresponding variations which occur in the chemical structure and physical properties of pectins during cell wall extension. However, the high levels of the epimerases present in cells which have completed elongation growth suggest that pectin synthesis is mainly controlled at the sites of the synthetase reactions. Key words: Cell-growth -
Cell wall - Control En-
The increase in cell wall area during cell elongation involves movement, rearrangement and change in the physical state of the wall polysaccharides already present (Wilson, 1964; Lockhart, 1965, 1967; Cleland, 1971) as well as synthesis of new wall materials (Bonnet, 1934; Ray, 1962; Srivastava et al., 1975). Pectin, hemicellulose and cellulose are deposited in the wall * Present address." G. Dalessandro, Istituto di Botanica, Universit~ di Bari, Via Amendola 175, 1-70126Bari, Italy
as a part of the process of continued cell elongation in higher plants. Soluble precursors of these polysaccharides are mainly uridine diphosphate sugars which are interconverted in the cell by the action of specific enzymes (Hassid, 1967, 1969; Northcote, 1969). Higher plants, contain a UDP-D-glucose dehydrogenase (E.C. 220.127.116.11) (Strominger and Mapson, 1957; Davies and Dickinson, 1972) that oxidizes UDP-D-glucose to UDP-D-glucuronic acid, the latter can be decarboxylated to UDP-D-xylose by the action of a UDP-D-glucuronic acid decarboxylase (E.C. 18.104.22.168) (Feingold et al., 1960; Ankel and Feingold, 1965). These two enzymes catalyse irreversible reactions. Enzymic epimerizations of UDP-D-glucose UDP-D-galactose, UDP-D-glucuronic acid ~UDP-D-galacturonic acid, and UDP-D-xylose ~UDP-L-arabinose are catalysed respectively by a UDP-D-galactose 4-epimerase (E.C. 22.214.171.124) (Fan and Feingold, 1969), UDP-D-glucuronate 4-epimerase (E.C. 126.96.36.199) (Feingold et al., 1960) and UDP-Larabinose 4-epimerase (E.C. 188.8.131.52) (Fan and Feingold, 1970). UDP-D-galactose, UDP-D-galacturonic acid and UDP-L-arabinose (galactose series) are precursors of pectin synthesis whereas UDP-D-glucose, UDP-D-glucuronic acid and UDP-D-xylose (glucose series) are precursors of hemicellulose synthesis in angiosperms (Northcote, 1969). The present investigation is concerned with the changes in the activities of the enzymes related to nucleoside diphosphate sugar interconversions (UDPD-galactose 4-epimerase, UDP-D-glucuronate 4-epimerase, UDP-L-arabinose 4-epimerase, UDP-D-glucose dehydrogenase and UDP-D-glucuronate decarboxylase) isolated from stem segments of the third internode of etiolated pea seedlings at different stages of cell elongation. Materials and Methods
Pea seeds (Pisum sativum L. var. Feltham First) were soaked in running tap water (8 h), planted in vermiculite and grown for
G. Dalessandro and D.H. Northcote: Control of Wall Synthesis
8 days in darkness at 25 ~ C. By using a dim green light, etiolated seedIings having the third internode approximately 40 mm long were chosen and used for all experiments. The third internode was cut into four segments (5 mm each) as illustrated in Figure 1. These segments corresponded to different stages of elongation growth and differentiation. Segments I (apical hook) had dividing and non-elongated cells whereas segments II, had slightly elongating and expanding cells. Segments III and IV had elongating and fully elongated cells. Segments I, II, III and IV (6-7 g) were collected in ice-cold beakers and rapidly washed three times with ice-cold phosphate buffer 0.2 M, pH 7.0, and used for enzyme extraction. 40
}II E 20
tion growth and differentiation catalysed the formation of UDP-D-[UJ4C]galactose, UDP-D-[U14C]glucuronic acid, UDP-D-[U-l~C]galacturonic acid, UDP-D-[UJ4C]xylose and UDP-L-[U-I~C]ara binose in the presence of UDP-D-[UJ4C]glucose as substrate plus NAD § By using substrates such as UDP-D-[U-14C]glucose or UDP-D-[UJ4C]galactose, UDP-D-[UJ4C]glucuronic acid and UDP-D-[U14C]xylose it was ascertained that the enzymic preparations contained the enzymes UDP-D-galactose 4epimerase, UDP-D-glucuronate 4-epimerase, UDPD-xylose 4-epimerase, UDP-D-glucose dehydrogenase and UDP-D-glucuronate decarboxylase which were responsible for the nucleoside diphosphate sugar interconversions. After dialysis the enzymic preparations had the same activities as before dialysis. The enzymic preparations (protein precipitated between 40-65% (NH4)2SO4 saturation) were used for all ex-
Fig. 1. Diagram showing the segments cut fiom the third internode of the pea seedlings
Enzyme Preparation Preparation of crude extract, MnC1 z treatment and fractions of the extracts prepared from segments I, II, III and IV were obtained as previously described (Dalessandro and Northcote, 1977a). The protein fraction precipitated between 40-65% ammonium sulfate saturation was used for all enzymic assays.
Analytical Methods Paper chromatography, electrophoresis, radioactivity counting procedure, detection methods, protein estimation and procedures for counting of cells were similar to those previously described (Dalessandro and Northcote, 1977a).
Enzyme Assays UDP-D-galactose 4-epimerase (E.C. 184.108.40.206), UDP-D-glucuronate 4-epimerase (E.C. 220.127.116.11), UDP-D-xylose 4-epimerase (E.C. 18.104.22.168), UDP-D-glucose dehydrogenase (E.C. 22.214.171.124), UDP-D-glucuronate decarboxylase (E.C. 126.96.36.199) were assayed radiochemically as previously described (Dalessandro and Northcote, 1977a, b).
Chemicals and Radiochemieals All chemicals and radiochemicals used have been described by Dalessandro and Northcote (1977a, b).
Results Preliminary experiments showed that the enzymic preparations (protein precipitated between 40-65% (NH4)2SO4 saturation) obtained from the third internode of pea stem segments at various stages of elonga-
Fig. 2. Specific activities and units of enzyme activities per cell of the enzymes bringing about UDP-D-sugar interconversions during cell elongation in the third internode of pea seedlings (Fig. 1). Spec. act., specific activity (nmol min ~ mg-1 protein). Units per cell, units of enzyme activity per cell (nmol min -~ cell 1). Ea ' UDP-D-galactose 4-epimerase; E2, UDPD-glucuronate 4-epimerase; E3, UDP-D-xylose 4-epimerase; @, UDP-D-glucose dehydrogenase; (~), UDP-D-glucuronate decarboxylase. Segment I, apical hook; segment II, slightly elongating and expanding cells; segment III, elongating cells; segment IV, fully elongated cells. Reaction mixtures. UDP-D-galactose 4epimerase (El) ; 20 nmol UDP-D-galactose, 0.408 nmol UDP-D[U-t4C]galactose (105,700 c.p.m.) and 30 ~tg enzyme (protein precipitated between 40-65% (NH4)~SO4 saturation) in 0.2 M glycinesodium hydroxide, pH 9.0, in a total volume of 20 gl. Reaction time at 30~ C was 5 min. One nmole substrate was equivalent to 5,179 cpm. UDP-D-glucuronate 4-epimerase (Ez); 50 nmol UDPD-glucuronic acid, 0,864nmol UDP-D-[UJ4C]glucuronic acid (270,000 cpm) and 50 lag enzyme (protein precipitated between 40-65% (NH~)zSO 4 saturation) in 0.2 M sodium phosphate buffer, pH 7.0, in a total volume of 20 lal. Reaction time at 37 ~ C was 10 rain. One nmole substrate was equivalent to 5,308 cpm. UDPD-xylose 4-epimerase (E3); 10 nmol UDP-D-xylose 0.511 nmol UDP-D-[UJ4C]xylose (98,270 cpm.) and 25 pg enzyme (protein precipitated between 40-65% (NH4)zSO4 saturation) in 0.2 M sodium phosphate buffer, pH 8.0, in a total volume of 20 ~1. Reaction time at 30 ~ C was 5 rain. One nmole substrate was equivalent to 9,349 cpm. UDP-D-glucose dehydrogenase @ ; 5 nmol UDP-Dglucose, 0.4 nmol UDP-D-[UJ4C]glucose (125,000 cpm.), 20 nmol NAD + and 25 lag enzyme (protein precipitated between 40-65% (NH4)2SO4 saturation) in 0.2 M sodium phosphate buffer, pH 8.0, in a total volume of 20 lal. Reaction time at 30 ~ C was 10 min. One nmole substrate was equivalent to 23,148 cpm. UDP-D-glucuronate decarboxylase (~); 50 nmol UDP-D-glucuronic acid, 0.432 nmol UDP-D-[U-14C]glucuronic acid (135,000cpm.) and 25 gg enzyme (protein precipitated between 40 65% (NH4)zSO4 saturation) in 0.2 M sodimn phosphate buffer, pH 7.0, in a total volume of 20 lal. Reaction time at 37~ was 5 min. One nmole substrate was equivalent to 2,677 cpm. The amount of UDP-D-[Ut4C]xylose and UDP-L-[U-t4C]arabinose formed in 5 min was determined. UDP-D-[UJ*C]xylose and UDP-L-[UJ4C]arabinose were corrected for the loss of the carbon C-6 from the [UJ*Clglu curonic acid
G. Dalessandro and D,H. Northcote: Control of Wall Synthesis
Specific Activities and Units of Enzyme Activities per Cell of the Enzymes of UDP-sugar Interconversions during Elongation Growth in Pea Stem Segments
periments. They retained full activities for at least a m o n t h when stored frozen at - 1 5 ~ C. Linearity of enzyme reactions with time and protein concentrations were observed over a period of 15 rain. The a m o u n t of enzymic preparation which was able to convert less than 15% of the substrate, was added to the incubation mixture for all assays. All reactions were carried out at optimal p H and temperature.
Figure2 shows the specific activities (nmol m i n - 1 mg 1 protein) and the units of enzyme activities per cell (nmol min = 1 cell- 1) of UDP-D-galactose 4-epimerase, UDP-D-glucuronate 4-epimerase, U D P -
I I "13.2 I I I : 3o.6 IV:35.2 El
"~" U D P - D - G a l a c t o s e
I : 1.3x10-6
I 1:0.3 act
(~) : Spec,
El: U n i t s per
/ I I :0.5
II : 2 . 3 x 1 0 - 6 I I I : 5.OxlO-8 I V " 7.7 x 10-6
Q I : 3.3 x 10-8 (~:Units cell
I I :8.5 xlO -8
I I :1.3
III:8.8 • -8 IV: 8.9 • - 8
I l l :1.3 EZ: Spec. a c t . / i i i :2'6 /
U D P - D - G l u c u r o n i c acid ~ .
~' U D P - D - G a l a c t u r o n i c I
I ,'28.9 I I : 34.3 ( ~ :Spec. act. I I I : 34.3
E 2 : U n i t s per
I I :2.1x10-7 I I I : 4 . 2 x l O -7 I V:2.6~10 -7
| II (~):cellUnitS per [ i i i
: 4,1 xl0 -6 : 5,7x10 -6
C02 i[[ :1.8 : 3.7 E3:Spec. a c t ' / I I [ : 5.2 / /IV:6.o
"~ UDP L - A r a b i n o s e ~[ : 1.5~10 -7 E3:Units per cell
: 4"4• /III
: 9.o~1o -7
[ I V : 12.3x10 -7
42 D-xylose 4-epimerase, UDP-D-glucose dehydrogenase and UDP-D-glucuronate decarboxylase during elongation growth of the third internode of pea seedlings. Both the specific activity and the units of enzyme activity per cell of UDP-D-galactose 4-epimerase and UDP-D-xylose 4-epimerase increased from the non-elongated cells of the apical hook (segment I) to the fully elongated and differentiated cells (segment IV). In general the activity of UDP-D-galactose 4-epimerase was about six times greater than that of UDP-D-xylose 4-epimerase. The activity of UDPD-glucuronate 4-epimerase was constant in segment I and segment II whereas it increased in elongating cells (segment III) and decreased in elongated cells (segment IV). UDP-D-glucose dehydrogenase activity rose from the apical hook to the slightly elongating cells (segment II) and then remained nearly constant. However the specific activity of UDP-D-glucuronate decarboxylase remained nearly constant from the apical hook through elongation whereas it increased in the fully elongated cells (segment IV), when expressed as units of enzyme activity per cell there was a sharp increase from segment [ to segment IV. The specific activity and the units of enzyme activity per cell of UDP-D-glucuronate decarboxylase were approximately 100 times greater than that of UDP-D-glucose dehydrogenase at all stages of growth. UDP-D-galactose 4-epimerase, UDP-D-glucuronate 4-epimerase, UDP-D-xylose 4-epimerase and UDP-D-glucuronate decarboxylase activities were not affected by NAD § , NADH, Ca 2§ Mg 2+ and IAA (auxin) at concentrations between 0.001 m M - 1 mM.
The composition of the cell wall changes during growth and differentiation and in particular the pectic substances are only synthesised during the period of active division and expansion of cell wall area (Thornber and Northcote, 1961a, b; Northcote, 1963). During secondary thickening, a large amount o f hemicellulose and cellulose are deposited but no pectin. In addition to these overall changes in deposition of pectin and hemicellulose the nature of the pectin deposited alters during primary growth (Stoddart and Northcote, 1967; Bowles and Northcote, 1972; Wright and Northcote, 1974) and it can also be influence~l by the application of plant growth hormones (Rubery and Northcote, 1970). Since the pectin polysaccharides hold water in the form of gels and their chemical composition can vary so that their gel properties change, these polysaccharides are extremely important in bringing about changes in the physical
G. Dalessandro and D.H. Northcote: Control of Wall Synthesis nature of the wall which precedes expansion in area and allows it to take place during growth (Northcote, 1972). The pectin is made up of chains of polygalacturonorhamnans with attached blocks of arabinogalactans and separate neutral polysaccharides composed of arabinose and galactose (Barrett and Northcote, 1965). The presence of the arabinogalactans either attached to the negatively charged uronic acid chains or as separate neutral polysaccharides contributes to the characteristic physical nature of the pectin and especially its relationship to bound water. They must therefore considerably influence the physical nature of the wall in which they occur and control of the type of pectin material that is deposited in the wall at any time during growth is important, especially during the early stages of wall formation and extension (Boffey and Northcote, 1975; Hanke and Northcote, 1974). There are therefore at least three stages of development at which control of polysaccharide synthesis that produces changes in the type or amount of polysaccharide deposited in the wall must be exerted. These are: 1)variation in the ratio of polygalacturonan to the arabinogalactan of the pectin formed during the initial stages of growth, 2) the large increase in hemicellulose (xylan) production during wall thickening, 3) the cessation of pectin synthesis after celt expansion has ended. Control can take place by a metabolic modulation of the various enzymic steps that occur during the formation of the polysaccharides. This will regulate the flux of carbohydrate through the various paths from the nucleoside diphosphate sugars to the polymerised product by variations in the activity of the enzyme and represents a rapid biochemical regulatory mechanism. The regulation may however, at the different stages of the differentiation process, be brought about by the repression or expression of different enzymes by a control of the genome and protein synthesis. The enzymic reactions to be considered for hemicellulose synthesis in the pea seedling are the UDPD-glucose dehydrogenase, UDP-D-glucuronate decarboxylase and the polysaccharide synthetases. It can be seen from our results during the formation of UDP-D-xylose (the precursor of xylans) that the activity of the UDP-D-glucose dehydrogenase was considerably lower than that of the UDP-D-glucuronate decarboxylase and that this probably therefore represented a limiting enzymic step. That it could act in metabolic control is shown by the modulating effect of UDP-D-xylose on the activity of the enzyme (Neufeld and Halt, 1965; Ankel et al., 1966; Davies and Dickinson, 1972; Dalessandro and Northcote, 1977b). Nevertheless this possible control mechanism
G. Dalessandro and D.H. Northcote: Control of Wall Synthesis
can be overcome in the plant tissue since there is another route to produce UDP-D-glucuronic acid from glucose via myo-inositol (Loewus et al., 1973). Our results also show that there was an additional overall control of xylan synthesis, brought about during differentiation of the cell which resulted in an increase in the activity of the decarboxylase. As the cell walls ceased to increase in area and became thickened (in segment IV) the activity of the decarboxylase increased so that there was an abundant supply of UDP-D-xylose. This increase was probably due to an increase in the amount of enzyme synthesized during the differentiation. Similar results to these have been found during the differentiation of cambium cells to xylem cells in sycamore and poplar (Dalessandro and Northcote, 1977a). Pectin synthesis depends in additon to the enzymes already discussed, on the three epimerases and the polysaccharide synthetases. It is clear from our results that during the period of rapid cell elongation (segment III) there was a large increase in the activities of the UDP-D-glucose and UDP-D-xylose 4-epimerases compared to the UDP-D-glucuronate 4-epimerase. These changes in the amounts of the activities of the three epimerases with respect to one another will undoubtedly result in the alteration in the ratio of the amounts of the nucleoside diphosphate sugars of the galactose series and probably produce corresponding changes in the composition of the pectic substances which result in the physical changes of the wall that occur during cell wall extension. However even in segment IV where little or no extension of the cells was occurring the levels of the activities remained high to produce precursors of the pectin polysaccharides which were no longer being formed. It seems likely therefore that one of the main control points of pectin synthesis must occur at the synthetase steps and that the cell maintains the epimerases necessary for the formation of the whole complement of nucleoside diphosphate sugars even though some of these are no longer used in wall synthesis. G.D. thanks The British Council for a European Fellowship during the tenure of which this work was carried out.
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43 Barrett, A.J., Northcote, D.H. : Apple fruit pectic substances. Biochem. J. 94, 617-627 (1965) Boffey, S.A, Northcote, D.H.: Pectin synthesis during the wall regeneration of plasmolysed tobacco leaf cells. Biochem. J. 150, 433-440 (1975) Bonner, J. : Studies on the growth hormone of plants. V. The relation of cell elongation to cell wall formation. Proc. Nat. Acad. Sci. U.S.A. 20, 393-397 (1934) Bowles, D.J., Northcote, D.H. : The sites of synthesis and transport of extracellular polysaccharides in the root tissues of maize. Biochem. J. 130, 1133-1145 (1972) Cleland, R. : Cell wall extension. Ann. Rev. Plant Physiol. 22, 197 222 (1971) Dalessandro, G., Northcote, D.H.: Change in enzymic activities of nucleoside diphosphate sugar interconversions during differentiation of cambium to xylem in sycamore and poplar. Biochem. J. 162 (1977a) Dalessandro, G., Northcote, D.H.: Changes in enzymic activities of nucleoside diphosphate sugar interconversions during differentiation of cambium to xylem in pine and fir. Biochem. J. 162 (1977b) Davies, M.D., Dickinson, D.B. : Properties of uridine diphosphoglucose dehydrogenase from pollen of Lilium longiJTorum. Arch. Biochem. Biophys. 152, 53-61 (1972) Fan, D.F., Feingold, D.S.: Nucleoside diphosphate-sugar 4-epimerases. I. Uridine diphosphate glucose 4-epimerase of wheat germ. Plant Physiol. 44, 599-604 (1969) Fan, D.F., Feingold, D.S.: Nucleoside diphosphate-sugar 4-epimerases. II. Uridine diphosphate arabinose 4-epimerase of wheat germ. Plant Physiol. 46, 592-595 (1970) Feingold, D.S., Neufeld, E.F., Hassid, W.Z.: The 4-epimerization and decarboxylation of uridine diphosphate D-glucuronic acid by extracts from Phaseotus aureus seedlings. J. Biol. Chem. 235, 910-913 (1960) Hanke, D.E., Northcote, D.H.: Cell wall formation by soybean callus protoplasts. J. Cell Sci. 14, 29-50 (1974) Hassid, W.Z. : Transformation of sugars in plants. Ann. Rev. Plant Physiol. 18, 253-280 (1967) Hassid, W.Z. : Biosynthesis of oligosaccharides and polysaccharides in plants. Science 165, 137-144 (1969) Lockhart, J.A.: Cell extension. In: J. Bonner and J.E. Varner, eds., Plant Biochemistry. Academic Press, New York 826 849 (1965) Lockhart, J.A.: The physical nature of irreversible deformation of growing plant cells. Plant Physiol. 42, 1545-1552 (1967) Loewus, F., Chen, M-S., Loewus, M.F. : The myo-inositol oxidation pathway to cell wall polysaccharides. In: Biogenesis of Plant cell wall polysaccharides, pp. 1-27, F. Loewus, ed. New York: Academic Press 1973 Neufeld, E.F., Hall, C.W. : Inhibition of UDP-D-glucose dehydrogenase by UDP-D-xylose: A possible regulatory mechanism. Biochem. Biophys. Res. Commun. 19, 456-46l (1965) Northcote, D.H. : Changes in the cell walls of plants during differentiation. Symp. Soc. exp. Biol. 17, 157-174 (1963) Northcote, D.H. : The synthesis and metabolic control of polysaccharides and lignin during the differentiation of plant cells. In: Essays in Biochemistry, Vol. 5, 89-137. Ed. Campbell, P.N., Greville, G.D. London: Academic Press 1969 Northcote, D.H.: Chemistry of the plant cell wall. Ann. Rev. Plant Physiol. 23, 113-132 (1972) Ray, P.M. : Celt wall synthesis and cell elongation in oat coleoptile tissue. Amer. J. Bot. 49, 928-939 (1962) Rubery, P.H., Northcote, D.H. : The effect of auxin (2,4-dichlorophenoxyacetic acid) on the synthesis of cell wall polysaccharides in cultured sycamore cells. Biochim. Biophys. Acta 222, 95 108 (t970) Srivastava, L.M., Sawhney, V.K., Taylor, I.E.P. : Gibberellic-acid-
44 induced cell elongation in lettuce hypocotyls. Proc. Nat. Acad. Sci. U.S.A. 72, 110%1111 (1975) Stoddart, R.W., Northcote, D.H. : Metabolic relationships of the isolated fractions of the pectic substances of actively growing sycamore cells. Biochem. J. 105, 45 59 (1967) Strominger, J.L., Mapson, L.W. : Uridine diphosphoglucose dehydrogenase of pea seedlings. Biochem. J. 66, 567-572 (1957) Thornber, J.P., Northcote, D.H. : Changes in the chemical composition of a cambial cell during its differentiation into xylem and phloem tissue in trees. 1. Main components. Biochem. J. 81, 449-455 (1961 a)
G. Dalessandro and D.H. Northcote: Control of Wall Synthesis Thornber, J.P., Northcote, D.H. : Changes in the chemical composition of a cambial cell during its differentiation into xylem and phloem tissue in trees. 2. Carbohydrate constituents of each main component. Biochem. J. 81,455-464 (1961 b) Wilson, K.: The growth of plant cell walls. Int. Rev. Cytol. 17, 1-50 (1964) Wright, K., Northcote, D.H.: The relationship of root-cap slimes to pectins. Biochem. J. 139, 525 534 (1974)
Received 17 September; accepted 11 October 1976
Possible control sites of polysaccharide synthesis during cell growth and wall expansion of pea seedlings (Pisum sativum L.).
The activities of the enzymes of uridine diphosphate sugar interconversions (UDP-D-glucose 4-epimerase, UDP-D-glucuronate 4-epimerase, UDP-D-xylose 4-...