The role of auxin in the removal of dormancy callose and resumption of phloem activity in Vitis vinifera

RONI ALONI AND AYALA RAVIV Department of Botany, The George S . Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv 69978, Israel

AND

CAROL A. PETERSON' Department of Biology, University of Waterloo, Waterloo, Ont., Canada N2L 3Gl

Received January 25, 199 1

ALONI, R., RAVIV, A, , and PETERSON, C. A. 1991. The role of auxin in the removal of dormancy callose and resumption of phloem activity in Vitis vinifera. Can. J. Bot. 69: 1825-1832.

During winter the phloem of Vitis vinifera L. is dormant and is characterized by heavy deposits of callose on the sieve plates and lateral sieve areas of the sieve tubes. After bud break, the maturing leaves stimulate a nonpolar breakdown of this dormancy callose along the branch axis in the internodes located both above and below the leaves. However, the pattern of callose degradation in the radial direction is polar. It proceeds in a centrifugal direction so that the sieve tubes near the cambium become free of callose first and those adjacent to the periderm last. The effect of the leaves on the removal of dormancy callose can be replaced by auxin. Application of naphthaleneacetic acid to either the top or basal ends of excised dormant branches resulted in the removal of callose from sieve tubes, usually in less than a week. 'The fluorescent dye fluorescein was used to test phloem reactivation. Both acropetal and basipetal fluorescein movement occurred in sieve tubes in branches that were pretreated for 1 week with auxin, while much less movement of fluorescein occurred in the control branches, which remained dormant. Fluorescein translocation was observed in sieve tubes that had a reduced amount of callose and were wider than 20 prn, but was not detected in the narrow sieve tubes (diameters less than 15 pm) located next to the cambium. The possible roles of auxin, ethylene, and cytokinin in controlling callose 1evels.h the sieve tubes are discussed.

Key words: auxin, callose, fluorescein, Vitis vinifera, phloem, dormancy.

ALONI, R., RAVIV, A., et PETERSON, C. A. 1991. The role of auxin in the removal of dormancy callose and resumption of phloem activity in Vitis vinifera. Can. J. Bot. 69 : 1825-1832.

Au cours de l'hiver, le phlobme du Vitis vinifera L. est dormant et se caractCrise par une forte deposition de callose sur les plaques criblees et sur les plages criblees laterales des ClCments de vaisseaux criblCs. Aprbs le dkbourrement, les feuilles en maturation stimulent une rupture de cette dormance like a la callose le long de I'axe de la ramification, dans les entrenoeuds situCs au-dessus aussi bien qu'au-dessous des feuilles. Cependant, le patron de dCgradation de la callose dans la direction radiale est polaire. I1 procbde de f a ~ o n centrifuge, de sorte que les tubes criblCs pres du cambium deviennent les premiers libres de callose et ceux adjacents au piriderme les demiers. L'effet des feuilles Climiner la dormance liee i la callose peut Ctre remplack par l'auxine. L'application d'acide naphtalene acCtique, soit i la base, soit au sommet d'une branche dormante exciste, entraine llClimination de la callose des tubes criblCs, en moins d'une semaine gCnCralement. La fluoresceine, un colorant fluorescent, a CtC utilisC pour tester la rkactivation du phlobme. On retrouve un mouvement basipete aussi bien qu'acropbte de la fluorescCine chez les branches traitCes par l'auxine 1 semaine plus tat, alors qu'il y a beaucoup moins de mouvement de fluoresciine chez les branches temoins qui sont demeuries dormantes. Les auteurs ont pu observer un mou- vement de la fluoresckine dans les tubes criblCs montrant une quantite rCduite de callose et ayant un lumen plus grand que 20 pm, alors que ce mouvement Ctait absent des tubes criblCs Ctroits (diamktres infkrieurs i 15 km) 1ocalisCs prbs du cambium. Les auteurs discutent les r8les possibles de l'auxine, de 1'Cthylbne et de la cytokinine dans le contr8le des teneurs en callose dans les tubes criblCs.

Mots elks : auxine, callose, fluoresckine, Vitis vinifera, phloeme, dormance. [Traduit par la redaction]

Introduction

The phenomenon of phloem reactivation in spring was well documented in Vitis vinifera L. (Esau 1948, 1969) and has also been followed in some other species (see Evert 1990). Normally, the sieve tubes of the grapevine function for two growing seasons. In the intervening winter, the sieve tubes are plugged with heavy dormancy callose. In the beginning of the second growing season these callose masses are gradually digested; the callose diminishes in thickness and the sieve tubes

'Author to whom correspondence should be addressed until April 15, 1992: Dr. Carol A. Peterson, c/o Prof. Dr. Emst Steudle, Lehr- stuhl fiir Pflanzenokologie, Universitat Bayreuth, Universitatsstrasse 30, Postfach 12 1251, 8580 Bayreuth, Germany.

assume the appearance of fully active tubes. Phloem reacti- vation does not show any close relationship to bud growth (Esau 1948). The mechanism that controls the removal of dor- mancy callose and resumption of phloem activity in spring is unknown.

The amount of callose in a sieve tube results from the activ- ities of two enzymes, p-1,3-glucan synthetase that synthesizes this carbohydrate and P-1,3-glucanase that digests it. The !at- ter enzyme is under developmental regulation in the intact plant. There is an increasing gradient of P-1,3-glucanase activ- ity in leaves in the basipetal direction. Its activity is low or absent in young leaves near the apical bud and is high in mature leaves of Phaseolus vulgaris (Abeles and Forrence 1970), Nicotiana glutinosa (Moore and Stone 1972), and Nicotiana tabacum (Felix and Meins 1986). This enzyme also plays an

Rinred in Canada / Imprim6 au Canada

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1826 CAN. J. BOT. VOL. 69, 1991

important role in the defense reaction of plants against path- ogens (Boller 1985; Mauch and Staehelin 1989). Opposite effects of auxin on callose deposits and on the enzymes that influence its level are apparent under different conditions. Application of l o p s M NAA to Phaseolus vulgaris signifi- cantly reduced callose deposits in the sieve tubes, while 5 - l o p 4 M NAA caused a greater amount of callose than in the controls (Scott et al. 1967). Auxin application to decapitated seedlings of Pisum sativum increased P-I ,3-glucanase activity, thus causing callose degradation (Datko and Maclachlan 1968). Conversely, in another study also on P. sativum, auxin increased P-1,3-glucan synthetase activity that stimulated cal- lose deposition (Ray 1973). These results indicate that auxin is likely involved in regulating callose levels in sieve tubes, but that the regulating mechanism is probably not a simple one since such disparate results have been obtained.

The auxin that will affect callose levels in the phloem of the plant axis originates in the leaves. The auxin produced by young leaves moves in a polar fashion through the procam- bium and cambium (Moms and Kadir 1972; Moms and Thomas 1978), while the auxin produced by mature leaves moves nonpolarly through the sieve tubes themselves (Eschrich 1968; Goldsmith et al. 1974; Moms and Kadir 1972).

The present study was designed to elucidate the role of auxin in controlling the reactivation of sieve tubes in dormant branches of V. vinifera in spring. We studied the effects of leaf development and auxin application on callose breakdown and analyzed the resumption of phloem activity in the auxin- treated branches by fluorescein translocation in their sieve tubes. This dye has been used to detect functioning sieve tubes in other plants (Aloni and Peterson 1990; Eschrich 1953; Schulz 1987).

Materials and methods Plant material and growth conditions

Tel Aviv Dormant branches from 5- to 10-year-old V. vinifera plants grown

in a garden at Tel Aviv University were used in experiments run during February and March of 1985 and 1986. For each experiment, 1-year-old branches were collected from one grapevine only.

Waterloo Dormant branches, approximately 0.7 m long, of V. vinifera cv.

Optma were collected at the Vineland station of the Horticultural Research Institute of Ontario on April 11, 1988, for translocation experiments. Their basal ends were kept in water and they were trans- ferred to the University of Waterloo in a cool darkened container. These dormant branches were kept in water in the laboratory (tem- perature approximately 21°C) near a window (light intensity approx- imately 1.5 pmol m-2 s-I).

In addition, 2-year-old dormant plants of V. vinifera cv. Sovereign Coronation were planted on April 15, 1988, in Pro-mix BX (Premier Bands, New Rochelle, NY) in plastic pots (200 mm diameter, 200 mm high). These plants were kept in a greenhouse and were also used for translocation experiments. Greenhouse temperatures ranged from 20 to 30°C.

Hormone application and experimental conditions Tel Aviv Correlations between leaf development and the level of callose in

the sieve tubes were studied in intact grapevine plants grown at Tel Aviv University garden as well as in dormant branches, six internodes long, which were transferred from the garden to a growth room. All the buds on these branches were excised except one in the middle of each branch. Control branches that had been completely debudded

were also prepared. The growth room temperature was approximately 24"C, with 16 h light (approximately 10 pmol mP2 s-') and 8 h dark. Light and dark relative humidities were 70 and 85%, respectively.

For the auxin experiments, debudded branch segments two inter- nodes long were used. The lengths of these segments ranged from 150 to 180 mm. Both ends were carefully recut with a razor blade just prior to treatment. Three different auxin experiments were run with 1-naphthaleneacetic acid (NAA, Sigma No. N-0640) as follows: (i) Auxin was applied in lanolin paste at three concentrations (0.1%, 1%, and 2% NAA, wlw) to the upper end of the youngest internode; the basal end was kept in a tube with tap water. The auxin-containing lanolin pastes were prepared as described by Aloni (1979). (ii) Auxin was applied in a 0.02 M K-Na phosphate buffer, pH 5.8 (Goldsmith et al. 1974), at two concentrations M and lo-' M NAA) that have been reported to decrease callose levels (Scott et al. 1967). The auxin solutions were applied to the upper or lower ends of the dormant branches by immersing the end to be treated in the appropriate solu- tion in a test tube and sealing the remaining opening with Parafilm (American Can. Co. Greenwich, CT). The branch was laid on its side on wet filter paper in a chamber with a relative humidity of 96%. (iii) Auxin was applied in 0.02 M K-Na phosphate buffer, pH 5.8, to either the upper or the lower ends of the branches at the two con- centrations given in (ii) above. In this case, the branches were kept vertically between two test tubes, so that both their ends were in liquid. Both ends of the branches were sealed into the tubes by Para- film as in (ii) above.

The auxin-containing lanolin pastes and the auxin solutions were renewed every 3 d. Preliminary experiments run during February extended over 5, 7, 10, and 14 d. Final experiments run during March 1985 and 1986 were completed after 5-7 d. Each experiment was repeated three times with five branches per treatment.

Waterloo The branches for the translocation experiments started to show bud-

break on April 21. On April 24, auxin (1% NAA in lanolin, wlw) was applied to the apical ends of five branches that showed early bud development. Additionally, two groups of five branches, which started to show bud development 2 and 3 d later, were treated sim- ilarly with the auxin-containing lanolin. Control branches at the same stages of bud development were treated with a lanolin paste without auxin. The auxin-containing lanolin was renewed after 3 and 6 d.

The fluorescent dye fluorescein (0.1% disodium fluorescein, C.I. 45350; Baker, Phillipsburg, NJ) in 0.07 M K-Na phosphate buffer, pH 5.3, was fed to the phloem of branches pretreated for 1 week with auxin, as well as to the control branches, by a method described earlier (Aloni and Peterson 1990; Peterson and Cumer 1969). Using a new razor blade, the peridem was carefully shaved away from a 10-mm zone in the middle of an internode between two actively grow- ing buds (young lateral branches). This location was 250 to 350 rnrn from the end of the branch where the lanolin was applied. A well that encircled the abraded area of the branch was fashioned from modelling clay, filled with about 20 mL fluorescein, and covered with Parafilm to prevent evaporation of the dye solution. After 24 h, the translocation of the dye was examined in sections taken 20 and 40 mm both above and below the fluorescein application site. The fluorescein experiment was repeated three times with excised branches in the laboratory near the window and twice on intact plants in the green- house, with five branches per treatment.

In all experiments, the levels of callose were evaluated as detailed in the following section.

Microscopy Tel Aviv To assess callose levels in the grape phloem, thick, transverse sec-

tions were prepared from the middle of all internodes above and below the remaining bud on long branches or branches on intact plants, and from the middle of each internode of the shorter branch pieces used in the auxin experiments. The sections were stained with 2% lacmoid (National Aniline Division, Allied Chemical and Dye Corp., NY), in

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ALONI ET AL.

TABLE I. Effect of auxin (NAA) in lanolin on the breakdown of dormancy callose deposits in Vitis branches (N = 5)

Control Expt.

NAA

No. Day 0 Untreated Lanolin 0.1% 1 % 2%

1 5.0?0 5.0?0 3.8k0.4" 3.8?0.4* 3.2?0.6* 2.6?0.5* 2 5.0?0 5 . 0 5 0 5.0?0 2.6k0.4" I .6?0.5* 2.0?0.6* 3 5.0?0 5 . 0 5 0 5.0?0 1.8?0.4* 1.620.5" 1.2?0.4*

NOTE: Results are expressed as rated callose deposits that could vary from 0 (absent) to 5 (heavy). Day 0 control, branch segments sampled at the beginning of the experiment; untreated control, branch segments without lanolin; lanolin control, branch segments with lanolin without NAA. *, difference from the day 0 control is highly significant ( P < 0.01).

TABLE 2. Effect of auxin (NAA) in phosphate buffer on the breakdown of dormancy callose deposits in Vitis branches kept horizontal with one tube at the treated end (N = 5)

Auxin from Auxin from Control above below Expt.

No. Day 0 Buffer Water M M M

- -

NOTE: Results expressed as in Table I. Day 0 control, branch segments sampled at the beginning of the experiment; buffer control, tube contained buffer; water control, tube contained water. Auxin from above, auxin solution at the distal end of the segment; auxin from below, auxin solution at the proximal end of the segment; *, difference from the day 0 control is highly significant ( P < 0.01).

TABLE 3. Effect of auxin (NAA) in phosphate buffer on the breakdown of dormancy callose deposits in Vitis branches kept horizontal with a tube at each end (N = 5)

10-4 M lo-5 M Water D Expt . Day 0 Buffer D Water D NAA D NAA D + 1 0 - j M No. control + water P + water P + water P + water P NAA P

NOTE: Results expressed as in Table I . Day 0 control, branch segments sampled at the beginning of the experiment; D , applied to the distal ends of the segments; P, applied to the proximal ends of the segments; *, difference from the day 0 control is highly significant ( P < 0.01).

96% ethanol for a few seconds, washed in tap water, mounted in 60% sodium lactate (Aloni 1980), and viewed with an Olympus BH light microscope. The callose in the sieve tubes was identified by its pos- itive reaction with lacmoid, which produced a sky blue color. Quan- tification of callose was accomplished by scoring its level on the sieve plates from 0 (callose free) to 5 (heavy deposits). The level of callose for each section was calculated from five fields of view (at magni- fication x 400).

Waterloo The effect of auxin on callose levels was studied with a Zeiss Axio-

phot epifluorescence microscope with a violet filter assembly (excit- ing wavelengths 361435 nm). The callose was stained with 0.01% aniline blue (C.I. 42755; Polysciences, Wamngton, PA) in 0.05 M K-Na phosphate buffer, pH 7.2, for 5 min, and mounted in buffer of the same pH (Prazak and Peterson 1987). A positive reaction for callose was a yellow fluorescence under violet excitation light.

Fluorescein translocation was analyzed after 24 h in fresh, free- hand, thick, transverse sections taken 20 and 40 mm above and below the site of fluorescein application. The sections were mounted in 0.07 M K-Na phosphate buffer, pH 5.3, and were viewed immedi- ately with the epifluorescence microscope described above.

Photographs of either aniline blue-stained or fluorescein-containing sections were taken with 200 ASA Ektachrome slide film; black and white photomicrographs were prepared from internegatives of the color slides.

Statistics Statistical methods followed Zar (1984) with the analysis of vari-

ance (ANOVA) test being used routinely.

Results The nonpolar axial effect of maturing leaves and auxin on

removal of dormancy callose Dormant V. vinifera branches as well as branches with

young, breaking buds possessed large deposits of dormancy callose on their sieve plates and along their longitudinal walls. Callose levels diminished considerably, at least in the sieve tubes near the cambium, at the time the first leaves reached 70-100% of their final size. Callose breakdown in the branch occurred simultaneously both above and below the site of insertion of the young lateral branch. This nonpolar effect of maturing leaves on callose degradation was observed in intact plants as well as in excised branches kept in a growth room. Leaf development was prevented in control branches by removing all their buds while they were still dormant. In these branches, callose levels remained consistently high for the duration of the experiment.

In experiments where auxin was applied to branches, there was no difference between the amounts of callose in samples taken from the two internodes. Therefore the results from these two locations were combined (Tables 1, 2, and 3). In the

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TABLE 4. Fluorescein translocation in branches pretreated with auxin (N = 5)

Distance below Distance above fluorescein fluorescein

Expt. application (mm) application (mm)

No. 20 40 20 40

NOTE: Numbers are the percentage of stems with visible fluorescein in the phloem. Numbers in parentheses are the controls, i.e., branches treated with plain lanolin. Experiments 10, 11, and 12 were done with excised branches in the laboratory, experiments 13 and 14 were done with intact plants in the greenhouse.

*Calculated from four branches.

experiment where the upper cut ends of debudded stem seg- ments were treated with lanolin Dastes. callose levels were maximum at the outset; they rekained high in debudded branches that were untreated or were treated with plain lanolin (Table I). All three concentrations of auxin significantly reduced the levels of callose on the sieve plates. The lowest concentration gave the least reduction but not significantly less than caused by the higher concentrations ( ~ a b k 1). In a sub- sequent experiment, auxin was applied in solution to either the proximal or distal ends of the branch segments. This auxin treatment dramatically reduced the amounts of callose on the sieve plates compared to the controls; in one of the replicates, the water control also had reduced callose levels (Table 2). The auxin effect on callose removal was the same regardless of whether the hormone was applied to the proximal or distal end of the segment, indicating that the effect is nonpolar (Table 2). When both ends of the branch were kept in water or buffer and auxin was added to either the proximal or basal end, callose levels were also reduced (Table 3). As in the pre- vious experiment, this effect was nonpolar. This auxin effect on callose deposits were relatively slow during February and was detected only after 10 or 14 d, but was much faster during March or April when only 5 or 7 d were needed to obtain obvious callose degradation.

The radial polar effect of maturing leaves and auxin on removal of dormancy callose

Sections taken from the day 0 controls from the experiments at Waterloo showed high levels of dormancy callose (Fig. 1). It occurred uniformly on all the sieve tubes regardless of their age (distance from the cambium). Application of auxin to the apical end of the branch induced a breakdown of this callose beginning with the last-formed sieve tubes located nearest to

the cambium (Figs. 2 and 3). The same pattern of callose removal was observed at the time of the development of the first mature leaves in branches kept in water. With time, cal- lose reduction gradually extended to the earlier formed sieve tubes located toward the periphery of the stem. The oldest sieve tubes located near the periderm were the last ones to show a decrease in callose levels. This radial polar pattern of callose breakdown was not observed in experiments performed in Tel Aviv.

Trunslocation of fluorescein in auxin pretreated branches The effect of auxin on translocation in the sieve tubes was - -

tested in excised branches kept in the laboratory as well as in intact branches grown in the greenhouse. The buds of both groups had broken dormancy but had not yet produced any mature leaves. The phloem in branches pretreated for 1 week with 1% NAA always showed more fluorescein translocation both above and below the dye application site than the control (Table 4). More fluorescein was translocated acropetally towards the more developed and fastest growing lateral branch (Table 4). Fluorescein translocation occurred through groups of sieve tubes (Figs. 4 , 5 , and 6) and was very rarely observed in a single sieve tube. During dormancy and before the pro- duction of new sieve tubes in the spring, the diameters of the sieve tubes of V. vinifera increased in the centrifugal direction. The sieve tubes of the first group located near the cambium (No. 1 in Fig. 1) were the narrowest and their diameters ranged from 10 to 16 pm. The diameters of the second group (No. 2 in Fig. 1) ranged from 21 to 27 pm, while the diameters of the third group (No. 3 in Fig. 1, arrow in Fig. 2) ranged from 26 to 32 pm. In the other groups located farther away from - -

the cambium the sieve tube diameters ranged from 25 to 39 " pm. Fluorescein did not move through narrow sieve tubes (less than 15 pm in diameter) even when they were callose-free. Dye translocation usually occurred through the relatively wide sieve tubes of the third group (marked by No. 3 Fig. 1 , arrow in Figs. 2, 4), although they showed some callose deposits on their sieve plates (Figs. 2 and 3). Fluorescein translocation was also observed in the second group from the cambium (Fig. 5) that was relatively callose-free (Fig. 2) and even in the fourth group (Fig. 6, arrow) that had thicker callose deposits.

Discussion

The results of our study demonstrate that the maturing leaves of V. vinifera stimulate the removal of dormancy callose and that an auxin source can mimic this effect. The actual pathway of movement of the auxin from the leaves to the phloem is not

FIGS. 1 4 . Radial polar effects of auxin (1% NAA) in removal of dormancy callose and resumption of sieve tube activity shown in transverse sections (Figs. 1, 2, and 4) and a longitudinal view (Fig. 3) in Vitis vinifera branches. Bars = 200 km. Fig. 1. A day 0 control branch with heavy deposits of dormancy callose showing a typical radial gradient of increasing sieve tube (s) diameter with increasing distance from the xylem (x ) . The numbers ( 1 to 5) mark groups, counted from xylem, of sieve tubes separated by fibers (f). No. 1, the latest formed group with very narrow sieve elements; No. 2, group of sieve tubes with medium diameters; Nos. 3, 4, and 5, wide sieve tubes that differentiated in the middle of the previous growing season. Fig. 2. Gradual radial gradient of callose degradation detected 7 d after auxin application. The two sieve tube groups closest to the xylem are almost callose-free. The arrow marks group No. 3 with low levels of callose; groups 4 and 5 have relatively high callose deposits. Fig. 3. Longitudinal structure and callose deposits 5 d after auxin application. A radial gradient of callose removal is apparent in the sieve tubes of the three groups (numbered) located near the xylem. Fig. 4. Pattern of functioning sieve tubes (arrow) 40 mm above the fluorescein application site, examined 24 h after fluorescein application to a branch pretreated for a week with auxin. Fluorescein was detected in all the sieve tubes of group No. 3, while no dye was seen in the other sieve tube groups. The protective tissue, the periderm @), is brightly autofluorescent.

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FIGS. 5 and 6. Patterns of functioning sieve tubes (bright patches) detected 40 rnrn above the fluorescein application site, examined 24 h after dye application to Vitis vinifera branches pretreated for a week with 1% NAA. Bars = 200 pm. Fig. 5. An almost continuous pattern of active sieve tubes around the xylem (x). r , ray; p , periderm. Fig. 6. A group of active sieve tubes (arrow) located further from the xylem. These sieve tubes are group No. 4 (according to Fig. I), while the three other active sieve tubes (left side) belong to group No. 3 (according to Fig. 1).

clear. On one hand, the radial gradient of callose disappear- ance (also noted by Esau 1948) suggests that the auxin is being transported in or near the vascular cambium of the branches and is diffusing from there to the surrounding tissues. With this pathway of hormone movement, one would expect an axial polar response, but this was not the case. The effects of both the maturing leaves and the applied auxin were clearly non- polar. The lack of polarity and coincidence of callose disap- pearance with the maturation of leaves, which would begin to export assimilates, suggests that auxin is also being supplied to the system via the assimilate stream in the sieve tubes. The fluorescein tracer experiments indicated that some of these tubes could function even with heavy deposits of callose. It is possible that dormancy callose removal may be brought about by auxin moving both in the cambium and in the sieve tubes.

The present study demonstrates that narrow sieve tubes located near the cambium, although almost callose-free, did not translocate fluorescein. On the other hand, wide sieve tubes with higher callose levels did show dye translocation. This difference may result from the relatively high resistance to mass flow through the narrow tubes, or it may be because these very young tubes do not yet connect sources and sinks. Further research is required to clarify this phenomenon.

In the fluorescein translocation experiments, we applied the dye to an internode located between two developing buds with fast-growing young leaves. At this developmental stage, the sieve tubes of the control branches still had large deposits of dormancy callose. This experimental design was planned to establish active sinks to enable fluorescein translocation. In fact, fluorescein movement in the sieve tubes was correlated

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with the size of the growing bud, always showing a higher rate Research Council of Canada. We thank Dr. K. Helen Fisher of acropetal translocation toward the upper bud that was grow- of the Horticultural Research Institute of Ontario for providing ing most rapidly. numerous grape branches.

The amount of callose in a sieve tube is controlled by the relative activities of P-1,3-glucan synthetase and P-1,3-glu- ABELES, F. B., and FORRENCE, L. E. 1970. Temporal and hormonal canase. To the best of our knowledge, the effects of plant hor- control of p-1,3-glucanase in Phaseolus vulgaris L. Plant Phys- mones on the activities of these enzymes have never been iol. 45: 395400.

investigated in dormant plants. Diverse and sometimes con- ALONI, R. 1979. Role of auxin and gibberellin in differentiation of

tradictory results have been obtained in other experimental sys- primary phloem fibers. Plant Physiol. 63: 609-6 14. 1980. The role of auxin and sucrose in the differentiation of

tems. The synthesis of P-1,3-glucanase was inhibited in sieve and tracheary elements in plant tissue cultures. Planta, 150: tobacco pith parenchyma tissue by both auxin and cytokinin 255-263. (Felix and Meins 19861, and this regulation was at the level ALONI, R., and PETERSON, C. A. 1990. The functional significance of mRNA (Mohnen et al. 1985). However, the activity of this of phloem anastomoses in stems of Dahlia pinnata Cav. Planta, enzyme was stimulated by auxin in decapitated Pisum stems 182: 583-590. (Datko and Maclachlan 1968), was unaffected by auxin in ALONI, R., BAUM, S. F., and PETERSON, C. A. 1990. The role of Nicotiana glutinosa (Moore and Stone 1972), or was inhibited cytokinin in sieve tube regeneration and callose production in by a combination of auxin, gibberellin, and cytokinin in Phas- wounded Coleus internodes. Plant Physiol. 93: 982-989.

eolus vulgaris (Abeles and Forrence 1970). ~ ~ h ~ l ~ ~ ~ may BOLLER, T. 1985. Induction of hydrolases as a defense reaction

mediate the process, since it is known to decrease callose against pathogens. In Cellular and molecular biology of plant stress. Edited by J. L. Key and T. Kosuge. Alan R. Liss, New

deposits during leaf abscission (Scott et al. 1967) and to stim- York. pp. 247-262. ulate callose degradation by increasing P-1,3-glucanase activ- DATKO, A. H., and MACLACHLAN, G. A. 1968. Indoleacetic acid ity in leaves of bean (Abeles and Forrence 1970), of tomato and the synthesis of glucanases and pectic enzymes. Plant Phys- (Pegg 1976), in excised leaf discs of N. glutinosa (Moore and iol. 43: 735-742. Stone 1972), and in pith parenchyma of N. tabacum (Felix and ESAU, K. 1948. Phloem structure in the grapevine, and its seasonal Meins 1986). Ethylene also increased P- 1,3-glucanase content changes. Hilgardia, 18: 217-296. in tobacco leaves (Felix and Meins 1987). Because ethylene - 1969. The Phloem. In Encyclopedia of plant anatomy. Vol. 5. is known to be induced by plant tissues exposed to high auxin Pt. 2. Edited by W. Zimmermann, P. Ozenda, and H. D. Wulff.

levels (Yang and Hoffman 1984), we suggest that in our exper- Gebriider Borntraeger, Berlin.

imental system, ethylene might have been involved in the cal- ESCHRICH, W. 1953. Beitrage zur Kenntnis der Wundsiebrohren- Entwicklung bei Impatiens holsti. Planta, 43: 37-74.

lose breakdown, at least near the site of the high auxin (1 and 1968. Translokation radioaktive markierter Indolyl-3-essig- 2% NAA) applications. Since we could not detect callose saure in Siebrohren von Vicia faba. Planta, 78: 144-157. breakdown in the control branches in which all buds were EVERT, R. F. 1990. Dicotyledons. In Sieve elements, comparative excised, we can conclude that wound-induced ethylene, which structure, induction and development. Edited by H. D. Behnke, would have been produced by cutting the branches and by bud and R. D. Sjolund. Springer-Verlag, New York. pp. 103-137. excision, was not sufficient to stimulate callose breakdown by FELIX, G., and MEINS, F., JR. 1986. Developmental and hormonal itself in our system. 'There was a requirement for either grow- regulation of P-1,3-glucanase in tobacco. Planta, 167: 206-21 l . ing leaves or a source of auxin for callose removal. However, - 1987. Ethylene regulation of P- 1,3-glucanase in tobacco. we cannot discount the possibility that the effect of this auxin Planta, 172: 386-392. is mediated by ethylene. GOLDSMITH, M. H. M., CATALDO, D. A., KARN J., BRENNEMAN, T.,

Recently, we found that high levels of cytokinin promote and TRIP, P. 1974. The rapid nonpolar transport of auxin in the

callose production on the sieve plates in Coleus internodes phloem of intact Coleus plants. Planta, 116: 301-317.

(Aloni et al. 1990) and proposed that in the intact plant, cyto- MAUCH, F., and STAEHELIN, L. A. 1989. Functional implications of the subcellular localization of ethylene-induced chitinase and kinin stimulates callose production toward the end of the grow- P-1,3-glucanase in bean leaves. Plant Cell, l: 447-457.

ing in plugging of the sieve tubes for the MOHNEN, D., SHINSHI, H, , FELIX, G,, and ME~NS, F., J R . 1985. winter. In the present study we found that the maturing leaves Hormonal regulation of P-1,3-glucanase messenger RNA levels show the opposite effect, namely that they stimulate the in tobacco tissues. EMBO J. 4: 163 1-1635. removal of dormancy callose in Vitis branches and that this MOORE, A. E., and STONE, B. A. 1972. Effect of senescence and effect can be mimicked by a source of auxin. Although the hormone treatment on the activity of P-1,3-glucan hydrolase in mechanism regulating the levels of callose in the sieve tubes Nicotiana glutinosa leaves. Planta, 104: 93-109. of the intact plant is as yet not clearly understood, we have MORRIS, D. A., and KADIR, G. 0. 1972. Pathways of auxin transport shown that auxin is a primary signal stimulating the breakdown in the intact pea seedling (Pisum sativum L.). Planta, 107: 171- of the dormancy callose and resumption of phloem activity in 182. spring. hi^ auxin effect might be partially mediated by eth- MORRIS, D. A., and THOMAS, A. G. 1978. A microautoradiographic

ylene, which is known to enhance P-1-3-glucanase activity and study of auxin transport in the stem of intact pea seedlings (Pisum

callose degradation (Abeles and Forrence 1970; Felix and sativum L.). J. Exp. Bot. 29: 147-158.

Meins 1987; Mauch and Staehelin 1989; Moore and Stone PEGG, G. F. 1976. The response of ethylene-treated tomato plants to

1972; Pegg 1976; Scott et al. 1967). infection by Verticillium albo-atrum. Physiol. Plant Pathol. 9: 215-226.

PETERSON, C. A., and CURRIER, H. B. 1969. An investigation of Acknowledgments bidirectional translocation in the phloem. Physiol. Plant. 22:

1238-1250. This research was supported by an International Scientific PRAZAK, J. A, , and PETERSON, C. A. 1987. A rapid fluorescence

Exchange award to R.A. and C.A.P. and an operating grant method for observing phloem regeneration in Coleus blumei. to C.A.P., both from the Natural Sciences and Engineering Stain Technol. 62: 276-278.

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SCHULZ, A. 1987. Sieve-element differentiation and fluorescein its regulation in higher plants. Annu. Rev. Plant Physiol. 35: translocation in wounded phloem of pea roots after complete sev- 155-189. erance of the stele. Planta, 170: 289-299. ZAR, J. H. 1984. Biostatistical analysis. Prentice-Hall, Englewood

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