carbohydrate hemicellulose

CARBOHYDRATE METABOLISM OF VITIS VINIFERA:HEMICELLULOSE

A. J. WINKLER AND W. 0. WILLIAMS

LiteratureA review of plant science textbooks reveals the general opinion that hemi-

cellulose often functions as a reserve food. This viewpoint has probablyarisen from consideration of the early findings of SACHS (36), REISS (34),and SCHULZE (37) that hemicellulose appears to be a major food in certainseeds.

The more recent studies on the composition of hemicellulose have led toother theories as to its physiological function besides those of reserve foodand structural reinforcement. O 'DWYER (31) has suggested that since hemi-cellulose is closely related to pectins it serves as an intermediary form in asynthesis of lignin from pectic compounds. The support for this theory isfounded on the supposed decrease of pectin during the period of tissue matu-ration and on the apparently successful attempts to convert pectins to hemi-celluloses by mild alkaline hydrolysis (5), by hydrolysis with water underpressure (21), and by mild oxidation (28). The last reaction is presumablyanalogous to conversion in situ in the plant. Although it has been possible todemonstrate the transformation of pectin into hemicellulose, there is nosupport for the further transformation of hemicellulose into lignin (32).Since the structure of lignin has not been established, the theory of the trans-formation of hemicellulose into lignin must be considered as speculative.KERR and BAILEY (17) in their microchemical investigations of the cell wall,moreover, found no reason to believe that pectins disappear with cell matura-tion. A number of investigators have isolated a small amount of pectinsubstances from mature wood. The apparent percentage decrease in pectinaccompanying maturation appears to be the result of an increasing propor-tion of other constituents. Difficulties of extraction engendered by mechani-cal protection or perhaps by a physico-chemical union afforded by the laterdeposits may possibly have decreased the analytical yield of pectin. Hencepositive evidence that the absolute amount of pectin decreases is lacking.

Large variations of any of the constituents, notably that of the labilecarbohydrate reserves, cause an inverse change in the percentage of othercomponents. This is illustrated by the hemicellulose content of the fruit ofthe apple (13, 24), the peach (27), and the pear (22). In each case withmaturation a decreasing hemicellulose content was found on a percentagebasis which led to the opinion that hemicellulose is utilized as a reserve foodin the development of the fruit. Where the hemicellulose content has beenexpressed on a single fruit basis, however, there is a steady increase in the

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absolute amount of hemicellulose in the fruit during growth and ripening, asWIDDOWSON (42) has demonstrated for the apple, and SMITH (38) for theKieffer pear. In other words, hemicellulose deposition has only slowed downin respect to the rate of deposition of other substances in the fruit. Workingwith flowers and young fruits of the apple, HOWLETT'S (14) data show nosuch rapid reversible changes of hemicellulose to sugars as MURNEEK (24)found. WIDDOWSON'S data seem to show a slight decrease in the absoluteamount of hemicellulose as the fruit becomes overripe, but this might be theresult of tissue disintegration which in all probability has no connection withthe utilization of hemicellulose as a reserve. BUSTON (4), studying the podsof ripening peas and beans, found a decrease in the hemicellulose content ona percentage basis but the data for hemicellulose in grams per hundred podsshowed a steady increase.

Sufficient care has not always been taken to formulate an adequate con-ception of a reserve food. GARDNER, BRADFORD, and HOOKER (12) state,"The seasonal variation in the carbohydrate of plants gives evidence ofstorage." It seems necessary to make the further limitation that fluctuationswhich do not clearly result from conversion to other forms (such as starch tosugar in early winter) should correlate with conditions of vegetative activitywhich lead to utilization or storage of reserves. A large percentage utiliza-tion under extreme carbohydrate depletion should also be observable. Inthis latter connection the variability in composition of the hemicelluloseextract may raise the question as to whether or not we are dealing with theextraction of a homogeneous fraction or with the simultaneous extraction ofseveral fractions of hemicellulose. Recent studies of hemicellulose byO'DWYER (30), NORRIs and PREECE (29), and EHRLICH (11) have indicatedits varying composition-the hydrolysis products yielding hexose and pentosesugars together with a quantity of uronic acid. One such constituent orgroup of constituents may serve a structural function; the other, perhapsa minor fraction, may serve as a reserve food. If this were the case theutilization of a large proportion of the hemicellulose present would not beobservable.

CZAPEK (7) and KOSTYTSCHEW (18) and others have emphasized theimportance of particular hemicellulose constituents as reserves, indicating aless prevalent belief that only certain fractions of the hemicellulose groupmay function primarily as reserves.

Still another type of finding that has led to an apparently erroneousconclusion is illustrated by the data of NELSON (25) and of LEUKEL (20)which show small decreases in hemicellulose in alfalfa roots that had under-gone repeated top cutting-treatments, and of NIGHTINGALE (26) which showa significant percentage decrease of hemicellulose in tomato stems grown indarkness for an extended period. These decreases most likely represent the

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WINKLER AND WILLIAMS: HEMICELLULOSE METABOLISM

net effect of a lessened hemicellulose deposit in the newly formed tissue onthe percentage of the hemicellulose in the entire plant part utilized. This isconfirmed by ALBERT'S (1) data on alfalfa which show that new top growthproduced in the dark room had a hemicellulose percentage much lower thannormal. The difference would have been larger had the hemicellulose con-tent of the normal material been expressed as percentage on a carbohydrate-(starch and sugar) free basis. The evidence seems to indicate that a largeror smaller quantity of hemicellulose may be laid down according to the nutri-tional conditions within the plant.A number of investigations show no particularly significant variations in

the hemicellulose content. Only a few of these need be mentioned, chieflyto indicate some of the plants that have been studied. DENNY (10) seems tohave found no consistent variation in the hemicellulose content of leaves oftobacco, sunflower, cotton, grape, hawthorn, peach, bean, and lilac, usingtwin-leaf samples collected at night and in the morning. On the average hisresults indicate a slight gain. JONES and BRADLEE (16) found very smallseasonal variations in the maple tree. BENNETT (2) in a seasonal study ofthe apricot found no reason to believe that hemicellulose functioned as areserve. WILLIAMS (43) working with the dewberry found that the acidhydrolyzable material showed minor seasonal changes. L. D. Davis (unpub-lished data), working with bearing and non-bearing trees of the sugar prune,found no significant seasonal variation in hemicellulose although large fluctua-tions occurred in the starch content (8) of the same material.

MURNEEK (24), LEONARD (19), and CLEMENTS (6) support the theorythat hemicellulose serves as a reserve food in the plant. In their investiga-tions, the observed variations of the hemicellulose content were irregular, andfurthermore, are not correlated with the vegetative condition of the plant.Their variations certainly do not point to hemicellulose as a reserve, but mayhave arisen largely from sampling and analytical errors. Arbitrary chemicaldeterminations of hemicellulose may show comparatively large deviations.

Several investigators have employed treatments leading to carbohydrateutilization and have studied the effect on the hemicellulose content. DELEANO(9) divided carefully selected leaves of Vitis vinifera along the midrib. Oneportion served as a check and the other was placed in a light-excludingrespiration chamber for varying periods of time from 22 to 288 hours.Under these conditions of decreasing carbohydrate reserves, the percentageof hemicellulose seemed to increase slightly. This increase might be ascribedto continued hemicellulose deposition in the leaf, but it more probably wascaused by the effect of the decrease of other constituents on the percentageof hemicellulose. A slight increase of total nitrogen was also shown. If thehemicellulose data are corrected, using the nitrogen increase as a basis, theresultant figures for hemicellulose are remarkably constant. Althougb

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BUSTON (4) found a decrease in the hemicellulose content in grape leaves heldin darkness, however, his findings are limited. PROEBSTING (33) in experi-ments on the apple found that continued defoliation did not decrease thehemicellulose content. The work of ALBERT (1), in which he endeavored toexhaust the reserve from alfalfa roots by growth under conditions of light ex-clusion, resulted in a consistent increase in the percentage by weight of hemi-cellulose, doubtless an effect of the carbohydrate utilization on the percentagecomposition of the residue. It is apparent that hemicellulose was not utilizedto any appreciable extent as a reserve under these conditions.

ExperimentationThe analytical data, presented in table I, were obtained from representa-

tive cross-sections of grape stems (Vitis vinifera var. Carignane). The label-ing of the sections selected, as indicated in table I, is self-explanatory, exceptthat the term shoot is used to indicate the herbaceous growth of the currentseason as opposed to the designation cane, used to indicate the mature stemsof the preceding season. The data on the annual hemicellulose level wereobtained by analysis of composite samples of each of the various sections,obtained by the removal of ten vines at each sampling, at intervals varyingfrom a few days up to one month during the course of the year period. Thesame sampling procedure was utilized in the severe defoliation treatment,except that only six vines were removed for the composite sample. Thedefoliation treatment consisted of leaf removal at intervals sufficiently shortthat the leaves never reached a diameter of more than 2 inches. The defolia-tion was repeated until the vines were unquestionably in a dying condition.Adjacent vines receiving the same treatment were dead at the time ofsampling. The composite samples were dried rapidly in a strong air blastat 550 to 600 C.

The sugars were removed by extraction for 6 hours with 95 per cent. ethylalcohol. 'The starch was liquefied by buffered taka-diastase (previously pre-cipitated from water solution by alcohol to remove most of the blank). Theresidue from the starch extraction was hydrolyzed with 2 per cent. HCI (byweight) for three hours in an enclosed steam bath. The resulting sugarswere determined by the MUNSON-WALKER-SHAFFER-HARTMAN method.

It is realized that this analytical procedure includes other polyuronideswhich are present (chiefly pectic substances) but there is no reason to believethat this affects the conclusions drawn. The data represent averages of theresults of duplicate analyses of each sample. All results are expressed interms of dextrose as percentage of the dry carbohydrate-free residue (starchand sugars) following the example of MASON and MASKELL (23). DENNY(10) found that this procedure gives results compatible with those calculatedon the unit organ basis. Large variations in the stored starch have intro-

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PLANT PHYSIOLOGY

duced apparent but erroneous inverse fluctuations in the data on the hemi-cellulose content.

DiscussionThe most striking feature of the data (table I) is the lack of significant

variation between the samples of each series except that for the base of theshoots. In the first sample of this series, taken when shoots were very suc-culent, the hemicellulose content was less than half that of the usual content(approximately 7 per cent. against the average of later values of 17.3 percent.) after the tissues had become more mature. At the second sampling,when the base of the shoot was becoming woody, there was still a significantlylow hemicellulose content in this section of the vine (12 per cent.). It isinteresting to note that these first samples present an inverse curve of thereducing sugar content which decreases rapidly between May 5 and May 26.Starch deposition was relatively very small until approximately a month afterthe hemicellulose had attained its normal magnitude. At this time the baseof the shoot was no longer succulent.

The appearance of a normal hemicellulose content during the period ofrapid growth and before starch storage actively begins, points to its functionas a structural material. This theory is supported by the lack of significantfluctuation in the hemicellulose content in the other sections. The starchcontent, which is the principal reserve in this case, fluctuated widely in eachpart of the vine represented by the samples analyzed. In certain cases (barksamples of canes and trunk) starch was absent during the period of thesummer minimum. The non-utilization of hemicellulose in periods when thestarch is deficient in certain areas of the vine or at the low summer minimumseems to show that it does not normally serve as a reserve food in the vine.

Although the above results indicate that hemicellulose does not functionnormally as a reserve food in the vine, it seemed desirable to determinewhether or not it could be utilized as a reserve of "last resort" as has beenproposed by NIGHTINGALE (26). In the vines dying because of repeateddefoliation treatments, the hemicellulose was found to be present in normalamounts. It is apparent (see table I) that hemicellulose was not utilized asa food reserve even under these conditions of abnormal depletion of carbo-hydrate reserves. There always remained a detectable amount of starch(with iodine test) in the woody stems. Perhaps this residue was unavailableor was being hydrolyzed very slowly to soluble carbohydrates.

The data for the average hemicellulose values of the various vine cross-sections in table I appear to support the theory of the structural function ofhemicellulose. There is a correlation with general observations on compara-tive thickness of all walls-structural rigidity-of the various tissues withtheir respective hemicellulose contents. The hemicellulose content wasgreatest in the xylem of the woody stem, successively decreasing in the

386




root-wood, bark, and leaves. A number of investigators, including THOMAS(40), PROEBSTING (33), and SMYTEH (39), have noted the difference inhemicellulose content between the wood and the bark of the stem. Oneyear MURNEEK (24) found less hemicellulose in leaves than in wood butnot consistently less in the following year. These differences are presum-ably caused by the greater amount of hemicellulose in the walls of xylemcells than in those of phloem and cortex cells. The decreased ratio ofcortex to xylem in older roots correlates with the increasing hemicellulosecontent (table I). BENNETT'S (2) data show distinctly less hemicellulose inlateral roots than in trunk wood for the apricot.

The mature wood had slightly more hemicellulose than the young shoots.This is an agreement with the results of TOTTINGHAM, ROBERTS, and LEPKOV-SKY (41) who found less hemicellulose in the new spur growth as comparedwith that of the old spurs or base wood. Neither of these findings seems toagree with the conclusions of BRADBURY AND ROBERTS (3) that acid hydrolysisattacks the young xylem, phloem, and cambium cells rather than the "re-serves" occurring as wall thickenings. Probably the increased hemicellulosecontent of older wood is caused by further hemicellulose deposition. It isreasonable, however, to assume that nutritive or other conditions mightreverse this result by influencing the amount of the hemicellulose depositwhich is laid down or at least might affect its percentage ratio.

Conclusions

A critical review of the data presented in the literature reveals lack ofdefinite evidence to support the assumption that hemicellulose functions as areserve food in the vegetative part of plants. On the contrary, a preponder-ance of evidence indicates that it does not function as a reserve. Definiteevidence of its utilization in the seeds of a few plants seems to have been ob-tained but it appears wise to avoid the reasoning that if hemicellulose is areserve food in the seed, it will necessarily perform this same function in theplant.

The data of this investigation obtained from Vitis vinifera stems fail toshow any indication of the metabolic utilization of hemicellulose and supportthe theory that hemicellulose does not function as a reserve material in thevine even as a reserve of "last resort. " The data further indicate that hemi-cellulose probably fanctions as a structural material.

UNIVERSITY OF CALIFORNIADAVIS, CALIFORNIA

LITERATURE CITED

1. ALBERT, W. B. Studies on the growth of alfalfa and some perennialgrasses. Jour. Amer. Soc. Agron. 19: 624-654. 1927.

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2. BENNETT, J. P. The distribution of carbohydrate foods in the apricottree. Proc. Amer. Soc. Hort. Sci. 21: 372-384. 1924.

3. BRADBURY, DOROTHY, and ROBERTS, R. H. The effect of acid hydrolysisupon a hemicellulose reserve in apple trees. Proc. Amer. Soc. Hort.Sci. 23: 298-299. 1926.

4. BUSTON, H. W. Observations on the nature, distribution, and develop-ment of certain cell-wall constituents of plants. Biochem. Jour. 29:196-218. 1935.

5. CANDLIN, E. J., and SCHRYVER, S. B. Investigations of the cell wallsubstance of plants with special reference to the chemical changestaking place during lignification. Proc. Roy, Soc. London B 103:365-376. 1928.

6. CLEMENTS, H. F. Hourly variations in carbohydrate content of leavesand petioles. Bot. Gaz. 89: 241-272. 1930.

7. CZAPEK, F. Biochemie der Pflanzen. Fisher. Jena. 1922.8. DAvIS, L. D. Some carbohydrate and nitrogen constituents of alternate-

bearing sugar prunes associated with fruit bud formation. Hil-gardia 5: 119-154. 1931.

9. DELEANO, N. T. Studien iiber den Atmungsstoffwechsel abgeschnittenerLaubbliitter. Jahrb. wiss. Bot. 51: 541-592. 1912.

10. DENNY, F. E. Changes in leaves during the night. Contrib. BoyceThompson Inst. 4: 65-83. 1932.

11. EHRLICE, F. Neue Untersuchung iuber Pectinstoffe. Zeitschr. angew.Chem. 40: 1305-1313. 1927.

12. GARDNER, V. R., BRADFORD, C. F., and HOOKER, H. D., JR. The funda-mentals of fruit production. McGraw-Hill. 1922.

13. GERHARDT, F. Some chemical changes incident to ripening and storagein the Grimes apple. Plant Physiol. 1: 251-264. 1926.

14. HOWLETT, FREEMAN S. The nitrogen and carbohydrate composition ofthe developing flowers and young fruits of the apple. Cornell Univ.Agr. Exp. Sta. Memoir 99. 1926.

15. JOHNSTON, EARL S., and DORE, W. H. The influence of boron on thechemical composition and growth of the tomato plant. PlantPhysiol. 4: 31-62. 1929.

16. JONES, C. H., and BRADLEE, JENNIE L. The carbohydrate contents of themaple tree. Vermont. Agr. Exp. Sta. Bull. 358. 1933.

17. KERR, T., and BAILEY, I. W. The cambium and its derivative tissues.X. Structure, optical properties, and chemical composition ofthe so-called middle lamella. Jour. Arnold Arboretum 15: 327-349.1934.

18. KOSTYTSCHEW, S. Lehrbuch der Pflanzenphysiologie. Julius Springer,Berlin. 1926.

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19. LEONARD, 0. A. Seasonal study of tissue function and organic solutemovement in the sunflower. Plant Physiol. 11: 25-61. 1936.

/0. LEUKEL, W. A. Deposition and utilization of reserve foods in alfalfaplants. Jour. Amer. Soc. Agron. 19: 596-623. 1927.

21. LINGOOD, F. V. The decarboxylation of pectin. Biochem. Jour. 24:262-265. 1930.

22. MAGNESS, J. R. Investigations in the ripening and storage of Bartlettpears. Jour. Agr. Res. 19: 473-500. 1920.

23. MASON, T. G., and MASKELL, E. J. Studies on the transport of carbo-hydrates in the cotton plant. I. A study of diurnal variation inthe carbohydrates of leaf, bark, and wood and of the effects of ring-ing. Ann. Bot. 42: 190-252. 1928.

24. MURNEEK, A. E. Nitrogen and carbohydrate distribution in organs ofbearing apple spurs. Missouri Agr. Exp. Sta. Res. Bull. 119.1928.

25. NELSON, N. T. The effects of frequent cutting on the production, rootreserves, and behavior of alfalfa. Jour. Amer. Soc. Agron. 17: 100-113. 1925.

26. NIGHTINGALE, G. T. The chemical composition of plants in relation tophotoperiodic changes. Wisconsin Agr. Exp. Sta. Res. Bull. 74.1927.

27. NIGHTINGALE, G. T., ADDOMS, R. M., and BLAIE, M. A. Developmentand ripening of peaches as correlated with physical characteristics,chemical composition, and histological structure of the fruit flesh:III. Macrochemistry. New Jersey Agr. Exp. Sta. Bull. 494.1930.

28. NORMAN, A. G., and NORRIS, F. W. Studies on pectin. IV. The oxida-tion of pectin by Fenton's reagent and its bearing on the genesis ofthe hemicelluloses. Biochem. Jour. 24: 402-409. 1930.

29. NORRIS, F. W., and PREECE, I. A. Studies on hemicelluloses. I. Thehemicelluloses of wheat bran. Biochem. Jour. 24: 59-66. 1930.

30. O 'DWYER, MARGARET H. The hemicelluloses. Part IV. The hemi-celluloses of beech wood. Biochem. Jour. 20: 656-664. 1926.

31. . Preliminary investigations on the constitution of thehemicelluloses of timber. Biochem. Jour. 22: 381-390. 1928.

32. PHILLIPS, M., and Goss, M. J. Composition of the leaves and stalks ofbarley at successive stages of growth with special reference to theformation of lignin. Jour. Agr. Res. 51: 301-319. 1935.

33. PROEBSTING, E. L. The relation of stored food to cambial activity in theapple. Hilgardia 1: 81-106. 1925.

34. REIss, R. Ueber die Natur der Reservecellulose und fiber ihre

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Aufl6sungsweise bei der Keimung der Samen. Landw. Jahrb. 18:711-765. 1889.

35. DU SABLON, LECLERC. Recherches physiologiques sur les matrieres dereserves des arbres. Rev. gen. Bot. 18: 5-25. 1906.

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Lupinus luteus and Lupinus angustifolius and uber ihr Verhaltenwahrend des Keimungsvorganges. Zeitschr. physiol. Chem. 21:392-411. 1895-96.

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39. SMYTH, ELSIE S. The seasonal cycles of nitrogenous and carbohydratematerials in fruit trees. 2. The seasonal cycles of alcohol solublematerials and of carbohydrate fractions and lignin in the wood,bark, and leaves, portions of terminal shoots of apple trees under twocultural systems-grass plus annual spring nitrate and arable with-out nitrogenous fertilizer. Jour. Pom. and Hort. Sci. 12: 249-292.1934.

40. THOMAS, W. Composition of current and previous season's branchgrowth in relation to vegetative and reproductive responses in Pyrusmalus L. Plant Physiol. 7: 391-445. 1932.

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