Seasonal Variations in Major Nitrogenous Buds, Leaves, Bark and Wood of Mandarin Treesl

J. Japan. Soc. Hort. Sci. 53(1) : 17-22. 1984. Seasonal Variations in Major Nitrogenous Buds, Leaves, Bark and Wood of Mandarin Treesl Components Satsuma in Tadashi KATO2, Makoto YAMAGATA and Sadao TSUKAHARA Shikoku National Agricultural Experiment Station, Zentsu ji, Kagawa 765 Summary Buds, leaves and 2 to 3-year-old bark and wood were sampled from 21-year-old satsuma mandarin trees (Citrus unshiu Marc.) throughout the year and analysed for total nitrogen, 70% ethyl alcohol soluble and insoluble N, amino acids, mainly arginine and proline, and amides. Many nitrogenous components showed seasonal variations in their content. In old parts, it was the highest in April, the sprouting season, and the lowest in July, the final stage of new shoot development, while it was lowest in September in new leaves. Bark and wood showed temporary increases in the content of total and soluble N, free proline, and especially free arginine during the sprouting period from late March to mid April, and then declines in insoluble N. Buds, on the other hand, showed a marked decrease in proline together with a drastic increase in arginine during the sprouting period. These findings suggest that the soluble N components, such as proline and arginine, are used first for new shoot development, followed by the insoluble N. In old leaves, most of these components began to decrease in early May and the percentage decline of total N from early May to July was about 16% on a dry weight basis, about 40% of which was accounted for by the loss of free proline N. On the whole, all of these components increased in all parts of the tree from autumn to winter. The increases of proline in bark and leaves were particularly marked. Introduction Fruit trees have 2 sources of N for the formation of new organs in the spring season : reserve N and fertilizer N currently absorbed. In a citrus tree, more than 75% of the N used for new shoot development in spring was found to come from the reserve (9). Thus, reserve N plays an important role in the formation of new organs. The citrus tree accumulates significant amounts of N in all its parts, leaves, shoots, trunk and roots(6). However, less is known of the pattern of utilization of reserve N. The nitrogenous constituents of plant tissues are known to show seasonal changes in their content(2, 10, 12). In citrus trees, 1 2 Recived for publication 22 December, Present address : National Grassland Institute, Nishinasuno, Tochigi 768 1983 Research Kubota et al. (8) found seasonal changes in some amino acids such as proline and arginine, and amides in various parts of youngg trees. They failed, however, to obtain information on the seasonal variation of protein. Kato et al. (6) reported that protein content decreased in various parts of a 21- year-old tree during the new shoot development. This suggests that protein also hass a seasonal variation. To learn more of the utilization of reserve N for the formation of new shoots, the present study was undertaken to elucidate seasonal variations in the nitrogenous components of dormant and burst buds, leaves, and 2-3-year-old bark and wood. Materials and Methods Experiments were conducted from February 1981 until February 1982 using six 21- year-old satsuma mandarin trees. These 17

18 KATO, T., M. YAMAGATA AND S. TSUKAHARA trees were grown individually in concrete frames(2 m x2 m), and nitrogenous fertilizers of 306 g N per tree for several years until autumn 1980 and thereafter 460 g N per tree were split.applied annually in 3 times; spring N was given in mid March, summer N in mid June and autumn N in mid November. Thus 53% of the annual fertilizer N was applied as spring N, 18% as summer N, and 29% as autumn N. These trees were divided into 2 groups of 3 trees. Six 2 to 3-year-old stems were collected from each group (2 stems/tree) at about 1 P. M. on all sampling dates throughout the year from February 1981 to February 1982. From them, dormant buds, burst buds, new leaves, old leaves(10 to 15 months old) and 2 to 3-yearold stem sections were collected. The stem sections were further separated into bark and wood. All of the plant parts were assayed for nitrogenous constituents. Samples of freshly harvested plant parts were homogenized and extracted in 70% (V/V) ethyl alcohol (containing 100 mm KC1). Total and 70% alcohol-soluble N were determined by steam distillation after Kjeldahl digestion. Protein N(70% alcohol-insoluble components) was estimated from the difference between them. Nonprotein amino acids and amides in the extracts were determined as follows. A portion of extract was dried under vacuum, dissolved in a lithiumbased buffer(ph 2.20) and filtered. Amino compounds in the filtrate were determined in an autoanalyzer with a series of lithiumbased buffers. April. shoot Thereafter, development, in mid July, and winter. Protein N it declined rapidly with reached the minimum again increased until in old and new leaves Fig. l Seasonal variations in total (T) and protein (P, 70% alcohol-insoluble). N contents in old (OL) and new (NL) leaves, bark and wood. Fig.2. contents and Seasonal wood. in variations in 70% old (OL) and nee; alcohol-soluble N (N L) leaves, bark Results Total, soluble and protein N Seasonal variations of total and protein N content in leaves, bark and wood are shown in Fig. 1. Total N in leaves decreased from early May until late July. The percentage decrease was about 16% on a dry weight basis. In new leaves, the content of total N was minimal in early September and increased thereafter until winter. In bark and wood, the total N contents began to increase in late March to early April, reaching the maximum in early to late Fig.3. Seasonal contents in variations in major free old (OL) and new (NL) amino leaves. acid

SEASONAL VARIATIONS IN MAJOR NITROGENOUS COMPONENTS IN BUDS, LEAVES 19 Fig.4 in Seasonal bark. variations in mayor free amino acid and amide contents ivlonth Fig.5. Seasonal in wood. variations showed the same seasonal variation as total N, while these in the bark and wood remained constant until late April, and then decreased from early May, reaching the minimum in mid June. Soluble N remained almost constant from February to late May in major free ammo acid and amide contents and then began to decrease in old leaves (Fig. 2). In new leaves, it was minimal in September, and thereafter increased until winter. In the bark and wood, soluble N showed roughly the same seasonal variation as total N.

20 KATO, T., M. YAMAGATA AND S. TSUKAHARA Free amino compounds In old leaves(fig. 3), proline was the main amino acid, amounting to 0.7-1.3 mg NJ g fresh weight in winter. It began to decrease in early May, when new shoots showed active development, and continued to decrease until abscission. In new leaves, proline remained constant at a low level until October and then rapidly increased to mid December. Other amino acids were also found, but most of them were present only in small quantities. In the bark(fig.4), the major free amino acids were proline and arginine. Proline showed an increase in early April, then a sharp decrease as new shoots developed and again an increase after September. Arginine increased markedly from late March through early April, decreased sharply as new shoots developed, and then increased again from October. Asparagine, the third main component, showed a maximum in mid May and a minimum in winter. In the wood (Fig. 5), proline showed a seasonal variation similar to that in the bark. Arginine also showed a similar variation to that in the bark except a peak in October. Asparagine had 2 peaks, one in May and the other in September. Dormant buds contained much proline(fig. 6). With sprouting proline declined sharply, accompanied by a drastic increase in arginine. Fig.6. Variations in contents in burst mayor buds free amino acid and amide during sprouting. When the buds elongated to about 3 mnx in April, arginine decreased drastically, accompanied by a gradual increase in asparagine. Other amino acids were present in small quantities during this period. Discussion Variations in leaves As new shoots developed, the total N content in old leaves decreased mostly due to decrease in protein and proline(figs.1, 2' and 3). The percentage decrease amounted to about 16% during the senescence in spring and summer. In deciduous trees, much of the leaf N is transported to the woody tissues during the senescence of leaves in autumn(3, 11). In citrus trees, however, the amount of N transported to the woody tissues seems. to be considerably smaller than in diciduous. trees. The drastic decline of leaf proline during the stage of new shoot development seems to be explained by its translocationn to the newly developing organs(1). During November and December, proline accumulated markedly in leaves, while other amino acids showed no accumulation. This is explained by the fact that proline is synthesized more actively than the other amino acids in leaves in autumn(5). Variations in bark and wood In the bark and wood tissues, total and soluble N began to increase simultaneously in late March, while protein N began to decrease from early May (Figs. 1 and 2). These facts mean that prior to protein breakdown there appears a significant increase in soluble N in both bark and wood. This increase seems also not to be due to the spring N, which was applied in mid March, because, as shown in the separate paper(7), it required about 2 months to contribute to the increase of soluble N in the bark and wood. On the other hand, Kato et al. (4) showed that most of N taken up in the winter season was kept in the roots, and a large portion of it began to be translocated upwards after late February to early March. Therefore, the increase of soluble N in the bark and wood in spring seems to be caused by the translocation of soluble reserve N

SEASONAL VARIATIONS IN MAJOR NIT ROGEN OUS COMPONENTS IN BUDS, LEAVES 21 from lower parts. Arginine increased markedly in both tissues in early April(Figs.4 and 5). The increase in arginine N was quantitatively equal to that in the soluble N and also to that in the total N. Therefore, the increases in total and soluble N seemed to be mostly due to the increase in arginine having been translocated from the lower parts. Proline in the bark and wood began to 'decrease half a month or more earlier than that in old leaves. This suggests that proline in the bark and wood is used for the new shoot development more rapidly than that in old leaves. Variations in burst buds The changing patterns of proline and arginine in dormant and burst buds(fig.6) :suggest the possibility of proline being converted to arginine. However, according to our unpublished data on the metabolism of 14C-proline, burst buds could scarcely convert proline to arginine, so proline and arginine seem to be metabolised and utilized separately. The decrease of proline and the concomitant increase of arginine during the early stage of new shoot development suggest that proline is used first for shoot development, followed by arginine. Burst buds, bark and wood displayed similar arginine patterns. Thus the arginine in burst buds probably originates in the bark and wood. By contrast, proline in dormant buds probably is transported from leaves in autumn, because it is actively synthesized in leaves in autumn(5), and transported to the bark (1). High levels of proline in dormant buds seem to be related to cold tolerance. Literature Cited 1. AKAO, S. and S. KUBOTA. 1976. Studies on the nitrogen metabolism in satsuma mandarin trees. IV. Movements of proline in the leaves concurrent with the sprouting of spring shoots and their early growth. Bull. Shikoku Agric. Exp. Stn. 29:67-77. 2. HILL-COTTINGHAM, D. J. and E. G. BOLLARD. 1965. Chemical changes in apple tree tissues following applicatidn of fertilizer nitrogen. N. Z. J. Agric. Res. 8:778-787. 3. KANG, S. M., H. MATSUI and J. S. TITUS. 1982. Characteristics and activity changes of proteolytic enzymes in apple leaves during autumnal senescence. Plant Physiol. 70: 1367-1372. 4. KATO, T., S. KUBOTA and S. BAMBANG. 1982. Uptake of 15N-nitrate by citrus trees in winter and repartitioning in spring. J. Japan. Soc. Hort. Sci. 50:421-426. 5. KATO, T. and S. KUBOTA. 1982. Effects of low temperature in autumn on the uptake, assimilation and partitioning of nitrogen in citrus trees. J. Japan. Soc. Hort. Sci. 51: 1--8. 6. KATO, T., M. YAMAGATA and S. TSUKAHARA. 1984. Storage forms and reservoirs of nitrogen used for new shoot development in a satsuma mandarin tree. J. Japan. Soc. Hort. Sci. 52 : 393-398 (In Japanese with English summary) 7. KATO, T., M. YAMAGATA and S. TSUKAHARA. 1984. Seasonal variation in nitrogenous compounds in xylem sap of satsuma mandarin trees. J. Japan. Soc. Hort. Sci. 53 13-16. 8. KUBOTA, S., H. FUKUI and S. AKAO. 1974. Studies on nitrogen metabolism in satsuma mandarin trees. Part 3. Seasonal changes with composition of amino acids under Jeff erent nitrogen nutrition supplies. Bull. Shikoku Agric. Exp. Stn. 28:133-150. 9. KUBOTA, S., T. KATO, S. AKAO and C. BUNYA. 1976. 15N absorption and traslocation by satsuma mandarin trees. III. Behavior of nitrogen supplied in early spring. Bull. Shikoku Agric. Exp. Stn. 29 : 49-53. 10. O'KENNEDY, B. T., M. J. HENNERTY and J. S. TITUS. 1975. Changes in the nitrogen reserves of apple shoots during the dormant season. J. hort. Sci. 50:321-329. 11. SPENCER, P. W. and J. S. TITUS. 1972. Biochemical and enzymatic changes in apple leaf during autumnal senescence. Plant Physiol. 49 : 746-750. 12. TROMP, J. 1970. Storage and mobilization of nitrogenous compounds in apple trees with special reference to arginine. p. 143-159. In : L. C. Luckwill and C. V. Cutting(eds) Physiology of tree crops. Academic Press, London and New York.