Regeneration and Differentiation of Sieve Tube Elements

Regeneration and Differentiation of Sieve Tube Elements WILLIAM P.

JACOBS

Biology Department, Princeton University, Princeton, N e w Jersey

I. Introduction

.......................................

11. Regeneration of Sieve Tubes

.........................

What Factors Control Sieve Tube Regeneration? .... Excision of Organs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Substitution of Chemicals for Effective Organs ...... D. Generality of Results ............................ E. Specificity of IAA for Control of Sieve Tube Regeneration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . F. Are Sieve Tubes the Normal Path of IAA Movement? 111. Differentiation of Sieve Tubes in Organ or Tissue Culture IV. Normal Differentiation of Sieve Tubes . . . . . . . . . . . . . . . . V. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. B.

239 241 246 251 252 256 258 262 264 266 270 271

I. Introduction When one realizes that sieve tubes1 have been considered for 100 years to be the main pathways for the movement of organic food materials throughout the vascular plant, the relative paucity of critical developmental work should be questioned. The differentiation of sieve tubes has been studied much less than that of tracheary cells. The regeneration of sieve tubes has been studied even less than their differentiation. This is undoubtedly because sieve elements lack the easily distinguishable wall properties of tracheary cells and are typically tiny in diameter. Despite these difficulties, the absolute number of papers on the normal differentiation of sieve elements is sizeable, and even the regeneration of sieve tubes has been studied by several workers in recent years. The classic literature on the anatomy of normal differentiation of sieve tubes is summarized well in the reviews and books of Esau (1953, 1960, 1965), and there is no need to go over the material in detail. Figure 1 (Esau, 1953) shows stages in the development of sieve tube elements and associated companion cells. Nuclear disintegration and the development of slime and sieve plates are distinguishing features of sieve element development in the dicotyledons. Earlier work presents the pattern of sieve element differentiation in the angiosperm shoot apex as being very uniform, as well as strikingly different from that of tracheary differentiation. The first sieve tubes are reported to differentiate “continuously and acropetally” out into the young leaves, whereas the first tracheary 1 “Sieve tube” refers to a longitudinal chain of cells. The individual cells composing the sieve tube are called “sieve tube elements” (Esau, 1953, p. 2 6 8 ) .

239

FIG. 1. Differentiation of sieve tube members in Cucurbita. ( A ) Transection of primary phloem with the different stages numbered as follows: (1) meristematic phloem cell just before division; ( 2 ) after division into sieve tube member and companion cell; (3) slime bodies have just begun to develop in the sieve element protoplast: ( 4 ) slime bodies are of maximal size and the thick (nacrk) wall is present in the sieve element; ( 5 ) slime bodies have dispersed; (6) sieve element is partly obliterated. Similar stages are depicted in the longitudinal sections in B-G. ( B ) Meristernatic phloem cells in division (above) and just after division (below) into a sieve tube member and precursor of companioii cells. (C) Young sieve element and precursor of companion cells. ( D ) Sieve tube member with slime bodies beginning to develop; precursor of companion cells has divided illto three companion cells. ( E ) Slime bodies of maximal size, nucleus highly vacuolated, thick walls in the sieve element. ( F ) Slime bodies partly fused into amorphous masses and nu. cleus absent. (G) Mature sieve element with thin parietal cytoplasm, large vacuole containing vacuolar sap and slime (mostly in the lower end of element). The protoplast is connected with the lower sieve plate but is partly withdrawn from the upper. Note the depressions (sieve areas) in the sieve element walls facing the companion cells in E-G. (From Esau, 1953, Fig. 12.5.)

SIEVE TUBE ELEMENTS

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elements differentiate at an isolated locus near the base of the young leaf. The timing is different also; sieve tubes typically have been reported to differentiate in the young leaves before tracheary cells. The much sketchier information from studies of vascular regeneration in shoots suggests that the timing is different there, too, but in the opposite direction; sieve elements have been said to regenerate later than tracheary cells (von Kaan Albest, 1934; Simon, 1908). These differences in pattern and timing suggest that different chemicals control the differentiation and regeneration of these two vascular cell types. In this article, we shall emphasize recent studies of sieve tube regeneration and of the endogenous factors controlling sieve tube development.

11. Regeneration of Sieve Tubes Although tracheary regeneration had been investigated extensively as early as 1908 (Simon, 1908), it was not until 1934 that sieve tube regeneration was similarly investigated (von Kaan Albest, 1934). Eschrich’s elegant work in 1953 confirmed and extended von Kaan Albest’s. Both authors based most of their results on hand sections of regenerating vascular strands in elongating internodes of Impatiens. Impatiens was selected because Simon (1908) had used it for his studies of tracheary regeneration. Elongating internodes were used to avoid the problems of wound cambium formation that would be expected to occur if older, nonelongating internodes were used. von Kaan Albest reported that when the vascular strand was cut with a razor blade the first wound sieve tubes were not seen until 5 days later ( 1 day later than the first wound tracheary cells), when some were regenerated in direct connection with the phloem of the severed vascular strand. This regeneration was polar; it appeared first at the upper regeneration area (at the basal end of the severed vascular bundle). The wound sieve tubes differentiated in the unwounded cells of the procambial zone, running roughly parallel to the wound surface. After 5 days, the regenerating sieve tube strand bent around the edge of the wound and, still running more or less parallel to the wound, by 7-8 days had joined the apical end of the severed strand below the wound. (Except that the wound tracheids differentiated 2-3 days earlier than the wound sieve tubes, von Kaan Albest noted that the general course of regeneration was similar for the two types of vascular cells.) During the second week, more wound sieve tube cells regenerated, thickening the original regenerated strands. More cells differentiated above the wound than below it. A meristem finally developed in the callus 4-6 weeks after wounding and proceeded to cut off cells which differentiated into tracheids to the inside and sieve tubes to the outside. Seasonal effects were strong, the foregoing description applying to spring and summer. Eschrich (1953) confirmed this general pattern, adding that the rate of regen-

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WILLIAM P. JACOBS

eration varied with temperature and that Impatiens and Colezls regenerated sieve tubes faster than several other genera tested. The path of regeneration of the wound sieve tubes is shown in Fig. 2. They differentiate in the procambial zone, always interior to the starch sheath and

FIG. 2. Transverse section of cambial area of an Impatiens stem, showing cambial cells ( K ) , wound sieve tubes (a, b), recently laid down cell walls in early stage of sieve tube regeneration (c, d), and starch sheath ( S t ) . Several thick-walled wound tracheary cells are at the right of the cambial zone and two longitudinally running phloem strands have been cut through at A and B. (From von Kaan Albest, 1934, Fig. 12.)

SIEVE TUBE ELEMENTS

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often just interior to, but not joining, the xylem-less phloem strands (Eschrich, 1953). There are only one to three undifferentiated cells of the procambial zone between the wound sieve tubes and the wound tracheids (Fig. 2 ) . The earliest reported sign of the differentiation of wound sieve tube elements is the occurrence of numerous nuclear divisions in the cells of the procambial zone (Thompson, 1967, Fig. 1). von Kaan Albest stated, without illustrations to confirm it, that whole cells may differentiate directly into wound sieve tube elements, particularly during the period immediately after wounding when cell divisions are still scanty. This has not been confirmed by later investigators.

FIG. 3. Diagrammatic reconstruction of a longitudinal radial section through cambial cell b of Fig. 2, showing the new curved walls. A transverse section cut through level I of this cell group would look like ‘c’ or ‘d in Fig. 2. A transverse section through level I1 would look like ‘a’ or ‘b’ in Fig. 2. (From von Kaan Albest, 1934, Fig. 12a.)

According to Eschrich, up to four nuclei may be present before new cell walls are formed (Eschrich, 1953, Fig. 5 ) . The first new cell wall is somewhat curved (Fig. 3), and the smaller subdivision of the original cell may then differentiate into a wound sieve tube element, or further cell divisions may occur within the smaller subdivision (Eschrich, 1953, Fig. 6 ) . Because the long axis of the wound sieve tube usually runs tangentially, its cross walls are typically part of the radial (longitudinal) wall of the original brick-shaped procambial cell. This portion of the radial wall that serves as the cross wall for the wound sieve tube usually includes several pits, and “it is probable” that the sieve plate differentiates from several of these neighboring pits (Eschrich, 1953, Fig. 10). Two wound sieve tube elements often differentiate from such subdivisions of a single brick-shaped cell of the procambial zone. von Kaan Albest thought it unlikely that the other daughter cells could be considered companion cells because they were too wide, were not particularly rich in cytoplasm, and did not have as many cross walls relative to the sieve tube elements as might be expected of normal companion cells (cf. Esau, 1953, pp. 281-284). Eschrich (1953), however, who used a variety of stains to investigate the cytoplasm and nuclei more closely, concluded that at least some of the other daughter cells were com-

2 44

WILLIAM P. JACOBS

panion cells, judging by cell shape, as well as by the shape and staining properties of the nucleus. Before the pits on the radial wall of the procambial cells were changed into recognizable sieve plates for the new wound sieve tube element, they already showed a thin layer of callose, judging by resorcin blue stains (Eschrich, 1953). Even after the sieve plate was first differentiated, plasmolysis of wound sieve

FIG.4. Tangential section through the area in which wound sieve tubes are regenerating below a stem wound in Impatiens. (From Eschrich, 1953, Fig. 15.) tube elements with weakly developed sieve plates caused the protoplasm to pull away from the cell wall all around the cell, as well as from the thin sieve plate. The callose stayed on the wall. Only when the sieve plate had differentiated even thicker strands, judging by resorcin blue stain, did the cytoplasm fail to pull away from the sieve plates upon plasinolysis (Eschrich, 1953, Fig. 12). After sieve plate development there was a long pause before the nucleus of the wound sieve tube eleinent was resorbed. It did not begin until 10 days after wounding.

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Not all wound sieve tube elements are shaped like these narrow tubes. Some have three to four sieve plate areas instead of two (Eschrich, 1953, Figs. 7 and 8). Eschrich noted that nucleated wound sieve tubes that regenerated around the wound always connected to sieve tubes running longitudinally that still contained nuclei and, by that criterion, were probably differentiated after the

FIG. 5 . Diagrams of fluorescence observed in Inzputiens steins when fluorescein was added at different times before or after wounding. 1, Results obtained when fluorescein was added 2 hours before wounding; 2, fluorescein added at wounding time; 3, fluorescein added 2 days after wounding; 4, 4 days after wounding; 5 , 6 days after wounding; 6 , fluorescein added 8 days after wounding. (From Eschrich, 1953, Fig. 27.)

wounding (Fig. 4 ) . (When these longitudinally running sieve tube elements lacked nuclei, so did the wound sieve tubes connecting to them.) These nucleated “long sieve tubes” did not connect by sieve plates with the eiiucleate sieve tubes of the old vascular strand; they maintained an independent course in the strand, with the only histological sign of 3 relation being a slight development of callose on pits of the common longitudinal walls. Do the wound sieve tubes function in transport? von Kaan Albest (1934, pp. 34-35) presented some very indirect evidence that they do. She cut all the vascular strands of the five-sided Impatiens stem, by making cuts at five different levels. (Each large corner strand was thereby cut at two levels.) Seven days later all 10 plants looked fresh, and sections of two revealed that wound sieve tubes and wound tracheids had regenerated completely around the wounds, The other eight experimental plants continued their development, differing from the normal plants in no way noticeable to von Kaan Albest. Eschrich’s evidence was more direct and more convincing ( 1 9 5 3 ) . At various intervals after wounding, he added potassium fluorescein above the severed vascular strands (Fig. 5 ; note

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WILLIAM P. JACOBS

that these experiments were run in the winter, so regeneration was somewhat slower). His unwounded control plants gave results as expected from the work of Schumacher (1933) ; fluorescence was restricted to the sieve tubes and companion cells. With wounded plants, fluorescein added immediately ( 2 in Fig. 5 ) gave fluorescence in the sieve tubes down to about five to seven sieve tube elements above the wound edge (1.5 mm) . Four to six hours later, some fluorescence also appeared in phloem parenchyma cells adjacent to the most basal 1 mm or so of fluorescing sieve tube elements. Fluorescein added 2 days after wounding gave a pattern not much changed; as the callus formed over the wound, the fluorescence moved down into the basal 1.5 mm of sieve tubes and parenchyma ( 3 in Fig. 5 ) . If regeneration is allowed to proceed for 4 days before fluorescein is added, a striking and unexpected difference is found; the fluorescence is now strong in the cells of the starch sheath just above the wound, spreading from the cut middle strand out to the intact side strands but not entering their vascular elements. Transverse sections were said to show that the fluorescence was restricted to the cells of the one-layer-thick starch sheath, and was not visible in the procambial tissue (4 in Fig. 5 ) . Six days after wounding, the fluorescence in the starch sheath had spread down around the sides of the wound, and some wound sieve tubes were in their first stages of differentiation ( 5 in Fig. 5 ) . Sections of the new wound sieve tubes, comprised of two to five elements, showed fluorescence that was weak when the sieve plate was not fully differentiated and strong when it was. Because these fluorescing sections were not necessarily continuous with the cut middle strand, Eschrich presumed that their fluorescein came from the cells of the overlying starch sheath. Eight days after wounding, all the fluorescence was in the sieve tubes and companion cells, as in intact tissue (6 in Fig. 5 ) . Eschrich emphasized that the wound sieve tubes were apparently functioning to move fluorescein not only long before their nuclei were resorbed but to a noticeable extent even before their sieve plates were fully differentiated. This was a big change in viewpoint from that common in the earlier sieve tube literature in which theories (more than data) led investigators to expect that the only sieve tube elements functioning in transport were those in the enucleate, “late” stage.

A. WHATFACTORSCONTROLSIEVETUBEREGENERATION ? Although 44 pages of von Kaan Albest’s article are on physiological experiments, by modern standards this is an unsatisfactory section. The experiments were qualitative when they need to be quantitative. They were poorly designed; effects of developmental age were inextricably confused with effects of treatment; controls were not routinely used; and there was no sign of randomization. With these flaws it would be pointless to discuss the results in detail. Suffice it to say that von Kaan Albest followed up the conclusion of Kabus (1912) which

SIEVE TUBE ELEMENTS

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stated that the presence of leaves or buds increased the chances of a graft being successful because of some “correlative influence” that came from them. von Kaan Albest excised leaves or buds above wounded internodes and found less regeneration of both sieve tubes and tracheary cells from the excisions. Tracheary regeneration was delayed by several excision treatments that had no detectable effect on the rate of regeneration of sieve tubes. A differential effect on the two types of vascular cells was also reported for the effect of leaves and buds below the wound; von Kaan Albest interpreted her experiments as showing sieve tubes to be unaffected by these proximal organs but tracheary regeneration to be inhibited by their presence (von Kaan Albest, 1934, p. 88). Decades later, von Kaan Albest’s pioneering study was extended by a group that had found it useful to apply a quantitative physiological approach to the study of tracheary regeneration. They had collected evidence that the normal limiting factor for tracheary regeneration was the auxin indole-+acetic acid (IAA) formed in young leaves (Jacobs, 1952, 1954, 1956). The amount of auxin coming from the leaves could be measured, this amount of synthetic IAA substituted for the leaves, and tracheary regeneration normal in amount and appearance obtained. To put the studies on a quantitative basis, existing techniques had been adapted so that whole mounts could be made of regenerating areas that had been made transparent after the tracheary cells were selectively stained. This allowed the entire tracheary regeneration to be seen at once and made possible the routine use of physiologically adequate sample sizes. Statistical techniques were used to obtain more information from the same amount of work, and genotypic variance was reduced by the use of a clonal stock. Because this approach had worked well for tracheary regeneration, it was natural to try to apply it to sieve tube regeneration also (LaMotte and Jacobs, 1962, 1963). At the time LaMotte started his work, there was no clue in the literature as to what the normal limiting factor for sieve tube differentiation might be. From the differences cited in the literature between tracheary cells and sieve tubes in timing and pattern, both in normal and regenerative development, one could only expect that sieve tubes would be controlled by a substance different from the IAA that controlled tracheary cells. Accordingly, his primary interest was in developing a technique capable of providing a quantitative and reproducible measure of the number of sieve tube elements regenerated. Coleus was used as experimental material because earlier workers had used it for studies of vascular regeneration and because its square stem facilitated the preparation of a flat “phloem strip” from one side of the stem. Regenerated sieve elements in Coleus were too smalI to be seen in whole segments of internode cleared by the same method employed in the studies on tracheary regeneration (Jacobs, 1952), so the anatomical technique of clearing

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WILLIAM P. JACOBS

was combined with the physiological one of making a “phloem strip” (LaMotte and Jacobs, 1962). The phloem and the tissues external to it were separated from the xylem-pith cylinder on one side of the stem (Pig. 6). This strip, removed from an internode previously killed, fixed, and toughened, as well as

Phloem strip

Sieve t u b e s

Wound

-

FIG. 6. Diagram of the LaMotte technique for removing a phloem strip f r o m one side of the “square” Coleus stem.

cleared, in lactic acid, was then stained with aniline blue. The stain brought out the sieve elements strongly enough so that the strip could be accurately mxde thinner and more transparent by dissecting off the epidermis, cortex, and scar tissue. The thinned strip was then restained in aniline blue before being made into permanent mounts with the originally inner surface of the strip facing the cover-slip. [Aniline blue is the stain most frequently used for sic\ e elements (Johansen, 1940) .] Although fixing the internode before dissectiiig off the strip improved the regularity of the separation, the youngest internode to give easy separation was no. 5 (Fig. 7). This is also the youngest one to cease elongntioii, and from both criteria is presumably the youngest internode with well-developed cambium. For this purely technical reason, our first studies of thc regeneration of sieve elements were performed on internode no. 5. To improve the precision of counting the regenerated cells, we switched froin a V-shaped wound in the large corner strand (as used for our early xylem studies) to a slit wound in the strand running midway along one of the flat sides of the Coleus internode. This change to a “side wound” not only iiiakes the stripping easier, but causes all the sieve elements to regenerate in essentially one plane rather than in the three-dinaensional pattern of the “corner w o d . ”

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SIEVE TUBE ELEMENTS

A microphotograph of such a preparation illustrates how easily the individual

sieve elements can be seen through the cleared and stained strip (Fig. 8). The wound sieve tubes are seen through only one to three radially thin, undifferentiated cells of the cambial region. Apical bud Apical internode Internode no. I Internode no.

2

FIG. 7. Diagram of Colem plant to show the method of designating Ieaves and internodes. Internode no. 2, which is still elongating, was the internode used i n early studies on xylem regeneration and auxin transport. Internode no. 5 is the youngest internode with cambium. (The leaves, which are actually decussate, are here represented as being all in one plane.) (From Jacobs, 1954, Fig. 2 with permission of the editors of The Ameiicnti Naturalist and the University of Chicago Press.

The number of strands of regenerated sieve tubes was counted, using a counting convention designed to measure the number of actual sieve elements regenerated (LaMotte and Jacobs, 1962). A diagram of a typical strip, illustrating the counting convention, is shown in Fig. 9. Once the counting convention was established and demonstrated to give reproducible counts, it was determined that there was an average of 10 sieve elements per strand [9.9 i0.7 (mean standard error) (LaMotte and Jacobs, 1962, Table II)]. As usual, all slides were coded, to eliminate subconscious bias, before counting was done, and blind repeats were routinely run to check on the reproducibility of the counts. The plants were selected from the clonal stock for age and developmental stage, as in the earlier tracheary studies. To reduce the variation still further, the plants were grown in growth chambers with controlled light cycles, light intensities, and temperatures. Long-day conditions (16-8) gave more and faster

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WILLIAM P. JACOBS

FIG. 8. Regenerated sieve tubes in Coleus stems, photographed in a phloem strip. A cross-strand with a branch strand joining it in the interfascicular region is shown. Note that many of the sieve tube members were formed diagonal to the long axis of preexisting cells. (From LaMotte and Jacobs, 1962, Fig. 3. @ 1962 The Williains & Wilkins Cn.)

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regeneration than short-day cycles (8-26), so long-day conditions were usually used (LaMotte and Jacobs, 1963, Table 1). B. EXCISIONOF ORGANS

In repeating von Kaan Albest’s experiments, but in this quantitative way, primary leaves and branches were cut off the plants in varying patterns both

apical

basal end 2

4 4.5

4.5

4

0

total 19.0 FIG. 9. Diagram of a phloem strip preparation (left) and its enlarged regeneration area (right) which show the original wound site and the various types of regenerated phtoem strands found in wounded CaZem internode no. 5. Assessment of the phloem strands in counting is shown by numerals. The letters a-g designate types of strands described in the original paper. The circles on the extreme left represent low power fields of view. (From Lahfotte and Jacobs, 1762, Fig. 1 . @ 1762 The Williams & Wilkins Co.) above and below the regeneration area, in the hope that lateral shoot organs of some age or position would be controlling the regeneration of sieve elements. When all leaves and buds were excised, a striking and statistically significant decrease occurred in the number of strands of sieve elements that regenerated (LaMotte and Jacobs, 1963, Fig. lo). The control group, with all leaves and buds intact, regenerated an average of 22.2 sieve tube strands in 5 days. The plants with only the root system and the main stem remaining regenerated only 4.7 strands. Thompson confirmed these findings (Thompson, 1966, Table I).

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WILLIAM P. JACOBS

Leaves and buds above the regenerating internode were more important than those below; excision of proximal leaves and branches caused no change in the number of regenerated sieve tube strands, but excision of distal leaves and buds gave a statistically significant decrease (LaMotte and Jacobs, 1963, p. 88, foot-

C

CONTROL

ALL SHOOT

D

A L L BUDS OFF

ALL PRIMARY

ORGANS OFF

22.2 k 4 . 4

4.7?2

** I

LEAVES OFF

21 2

t 3.5

E

*

F l

Wound

ALL P R O X I M A L ORGANS OFF

I 5 6 _ f 30

ALL DISTAL ORGANS OFF

25.2 k 2 . 7

11.5 & 2.6

*

FIG. 10. The effects of excising various shoot organs on the number of sieve tube? regenerated in Coleus internode no. 5 (the mean and standard eiror for each treatment is shown). All plants were grown in long-day (16 hours) conditions; regeneration was for a period of 5 days; n = 4. Asterisks show significant differences from the control mean (A) (i.e., * = .05 and ** = .Ol). (From LaMotte and Jacobs, 1963, Fig. I.)

note 3). With this clue as to the role of the distal organs, one could expect signs of polarity of regeneration in time course experiments. When all leaves and buds were left on, more sieve tubes regenerated above the wound thaii below it during the first 3-5 days. By 7 days, this difference had disappeared (Fig. 11).

c.

SUBSTITUTION OF CHEMICALS FOR

EFFECTIVEORCANS

To our surprise, IAA replaced completely the striking effect of the distal shoot organs on sieve tube regeneration. The addition through the stem stumj;

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FIG. 11. The polarity of phloem regeneration above and below the wound rls shown by the time course. (Data from LaMotte and Jacobs, 1963, Table 2.)

of IAA at 2 ppm in aqueous solution, the concentration shown to replace exactly leaves 1 and 2 in their effect on tracheary regeneration in internode no. 2 (Jacobs, 1956), restored exactly the level of sieve tube regeneration to that found in intact control plants (Table I ) . When added mixed with lanolin, IAA at 0.1% and 1.0% evoked significantly more regeneration of sieve tubes than the attached organs themselves. To further isolate the reacting system, and specifically to make certain that the root system was not necessary for the restoration of the normal level of sieve tube regeneration by added IAA, internodes no. 5 were excised from the plant and treated at their apical ends with IAA in lanolin. A highly significant inEFFECTSOF IAA

TABLE I SUCROSE ON SIEVE TUBE REGENERATION IN Coleus PLANTS STANDING 5 DAYSWITHOUT SHOOTORGANS~

AND

Treatment Intact control plants All shoot organs excised Water Sucrose ( 2 0 gm/liter) IAA ( 2 mg/liter) TAA sucrose

+

5

I,

Number of regenerated strands, mean & S.E. 23.2 t 2.2 7.2 t 3.4b 7.6 2.5h 23.9 & 4.5 20.9 t 3.9

From Table 3 of LaMotte and Jacobs (1963); u = 4. Significantly different (at 5% level) from the values from the intact contmls.

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WILLTAM P. JACOBS

crease in the number of sieve tubes was found after 7 days (LaMotte and Jacobs, 1963, Table 4 ) . The fact that isolated internode no. 5 did not give fewer regenerated sieve tubes than did the main stem with an intact root system remaining supports the view that the root system suppIied no significant amount of sieve tube-forming material.

I A A CONCENTRATION (%) FIG. 12. The effect of apically applied IAA at various “concentrations” in lanolin on xylem and sieve-tube regeneration around a wound in isolated no. 5 internodes of Coielts (Princeton clone). (From Thompson and Jacobs, 1966, Fig. 1.)

After this unexpected finding, it was obviously necessary to check, in the same internode, the effects of IAA on the regeneration of both sieve elements and tracheary cells. (Our earlier studies on tracheary regeneration had used elongating internode no. 2.) The LaMotte technique was used for sieve tubes, and a similar flat strip, stained for tracheary cell walls, was saved from the xylem side of the cambium. When excised internodes no. 5 were treated on their apical ends with different “concentrations” of IAA in lanolin, given the standard slit wound midway down the internode, then checked after 7 days for the number of regenerated tracheary cells and sieve element strands, the p o l e d results of all experiments were as shown in Fig. 1 2 (Thompson, 1965; Thompson and Jacobs,

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1966). When no IAA was added externally, no tracheary cells and only a few strands of sieve elements regenerated. As the concentration of IAA was increased to 0.01% IAA, there was a slow linear rise in the number of sieve elements; 0.1% IAA gave a sharp increase with still more sieve elements being regenerated when 1.0% IAA was added. When the number of sieve strands that had regenerated in an otherwise intact plant was determined and that value interpolated on the concentration curve for isolated internodes, the “intact” value was equivalent to an IAA concentration of 0.05% (asterisk in Fig. 1 2 ) . The number of tracheary cells that had regenerated in this same period, just across the cambial layer in the same internodes, is also shown in Fig. 1 2 . (The units on the two Y axes were selected so that the units represent the same iiumber of cells.) No xylem cells regenerated in untreated internodes, nor did they start to regenerate in noticeable amounts until 0.01 % IAA was added apically. There was the same striking increase in regeneration between 0.01 and 0.17; IAA that was noticed for the sieve elements. The values for tracheary regeneration in the intact plant fell at the same equivalent IAA concentration of 0.05% as did those for sieve strands. The regeneration of both sieve elements and tracheary cells were unaffected by IAA added at the original base of the isolated internode (Thompson and Jacobs, 1966, Table 11). Internode no. 5 , in other words, showed absohte polarity of IAA movement, as measured by vascular cell regeneration. A direct test of IAA-I4C movement in corresponding internode cylinders confirmed the strong basipetal polarity of IAA movement (Fig. 1 3 ; also see Jacobs and McCready, 1967). The fact that the curve for tracheary cells parallels so closely that for sieve elements in Fig. 12, but at a lower level, suggests that prospective sieve elements are either more sensitive than prospective tracheary cells to a given concentration of IAA, or that they have greater access to auxin in the stein-or perhaps both. If sieve tubes are the major path of normal transport for auxin, then the sieve tube side of the cambial layer would obviously have greater access to auxin after severance of the vascular continuity. The results with the untreated, excised internodes fit this hypothesis; the 60 or so sieve elements regenerated would be attributable to the small amounts of endogenous auxin available in the phloeni, while the lack of regenerated tracheary cells would reflect the lack of an auxin surplus to move radially and internally across the cambium. The time course of vascular regeneration was found to fit either hypothesis; regenerated sieve elements could be detected (with the stains used) a full day before tracheary cells in internode no. 5 (Thompson, 1967). The one fact that seemed to indicate that auxin first reaches the phloem side is that the first tracheary cells to regenerate are radially exactly opposite an already regenerated strand of sieve elements. The individual regenerated tracheary cell is so much larger than the individual cell of the regenerated sieve strand that it is startling

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WILLIAM P, JACOBS

to see, in preparation after preparation, how exactly the path of differentiation of the tracheary strand follows the course already laid down on the other side of the cambial layer by the sieve tube (Fig. 1 4 ) .

I

I

FIG. 13. The polarity of movement through pith or vascular (“cornc‘r”) cylinders of Coleus internode no. 5 of 14C from IAA and 2,4-D, each supplied at an initial concentration of 5 pM. Radioactivity in receivers is shown for basipetal (solid lines) and for acropetal (dashed lines) receivers. The diagram in the upper left corner represents the transport set-up, with a 3-mm-long agar cylinder on each end of a horizontal, 5-mm-long cylinder of tissue. (From Jacobs and McCready, 1967, Fig. 1 with permission fi-om the editors of American Journal o! Botany.)

D. GENERALITY OF RESUJ-TS IAA stimulates the regeneration of both sieve tubes and tracheary cells in plants other than the Princeton clone of Coleus, as shown for the “Golden Bedder” variety of Coleus blumei and for the “Yellow Plum” variety of tomato (Lyropersicon esculentum Mill.) (Thompson and Jacobs, 1966, Table 111). In excised tomato internodes, 0.1% IAA in lanolin fully restored the numbers of vascular cells to those found in intact controls. Both tomato and “Golden Bedder” were like the Princeton clone in showing a strong basipetal polarity when vascular regeneration resulting from apical application of added IAA was compared to that from basal application.

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WILLIAM P. JACOBS

E. SPECIFICITY OF IAA

FOR

CONTROL OF SIEVETUBE REGENERATION

Because the activity of IAA would be expected to be a function of its activity as an auxin, other synthetic auxins were tested for activity in isolated internode no. 5 (Fig. 15; data taken from Thompson, 1965). Each of the auxins was

0 0

10

I

I

NUMBER

OF SIEVE TUBE 30

I

I

STRANDS

70

50 I

I

I

Tryptophan Lanolin Control

0 tracheary cells

0

100

300 NUMBER OF TRACHEARY

500

3300

3400

CELLS

FIG. 15. Activity of various auxins in causing regeneration of sieve tubes and trachea0

ceiis in isolated Coletls internode no. 5. All compounds added as 0.1%) mixtures in lanolin (except for tryptophan, which was at 1.0%). Asterisks next to 2,4-D and 2,4,5-T indicate such proliferation of sieve tubes that counts were impossible to make accurately. (Data from Thompson, 1965, Table IX.)

active in causing the regeneration of both sieve tubes and tracheary cells. The two weed killers with auxin activity, 2,4-dichlorophenoxyaceticacid (2,4-D) and 2,4,5-trichlorophenoxyaceticacid (2,4,5-T), were at least as active as IAA in causing tracheary regeneration, and were so much more active in causing sieve tube regeneration that their counts for 7 days are minimum estimates only. We already knew that 2,4-D and IAA showed striking similarities in their polar movement through sections of whole internode or whole petiole (Jacobs, 1967). The unusually high vasculogenic activity of 2,4-D could be explained, however, if 2,4-D showed preferential movement through vascular tissue. By comparing movement through cylinders of tissue including only pith parenchyma with cyiinders including vascular tissue, we could show that 2,4-D did move preferentially through the latter-although basipetally polar movement occurred in both. On

SIEVE TUBE ELEMENTS

259

the contrary, more IAA moved through pith cylinders (Fig. 13; see Jacobs and McCready, 1967). This striking effect on sieve tube regeneration of 2,4-D added at hormonal levels seems to be a manifestation of the same action that causes “distortion of phloem” reported earlier as a typical result of using more nearly herbicidal levels of 2,4-D (Eames, 1950). Tryptophan, a presumed precursor of IAA in many plants including Coleus (Valdovinos and Perley, 1966) can also cause sieve tubes and tracheary cells to regenerate. Its lesser activity and more erratic effectiveness (Thompson, 1965) fit the idea that its activity depends on prior transformation to IAA. Several non-auxin compounds tested similarly on isolated internode no. 5 did not affect the number of sieve tubes or tracheary cells (Thompson, 1965). Thiamin and ascorbic acid, investigated because of an earlier report that they stimulated cambial activity in other genera (Kunning, 1950), were without effect at 1% in lanolin. So was nicotinic acid. Gibberellic acid (GA,), added to the top of internode no. 5 alone or in varying concentrations with 0.1% IAA, had no statistically significant effect on sieve tube or tracheary regeneration (Thompson, 1965). (When added with IAA, it decreased the average number of sieve tube strands regenerated in all four experiments, but never to the 5% level. In three of the four experiments, it increased the average number of tracheary cells regenerated, but again not to the 5% level by the “t” test.) Thinking that the stimulating effects of GA, on other actions of IAA-so often reported in the literature-might be the specific result of GA, affecting transported IAA (as had been shown in two legumes by Jacobs and Case, 1965; Pilet, 1965), Thompson added IAA-I4C alone or with GA, to internode no. 2 and checked the effect on vascular regeneration in internode no. 5 (Thompson, 1966). The GA, did not increase the amount of either vascular regeneration or of radioactivity in internode no. 5. (In all these experiments with GA,, IAA was added at a level low enough so that internode no. 5 would give more vascular cells if more IAA reached it.) When substituted for the distal organs, sucrose at 2 % had no effect on the number of sieve tubes regenerated in internode no. 5 , whether added alone or with IAA (LaMotte and Jacobs, 1963, Table 3). With such evidence from the older, nonelongating internode no. 5 that the regeneration of both sieve tubes and tracheary cells was controlled by the endogenous hormone IAA, some similar experiments were run on the younger, elongating internode no. 2. [This means that, in contrast to the earlier studies on tracheary regeneration (Jacobs, 1952, 1754, 1956), the internodes were wounded in one of their flat sides, rather than cutting a V-shaped hole from one of the large corner strands.] Excised wounded internodes no. 2 treated apically with various “concentrations” of IAA in lanolin during their week of regeneration gave the results shown in Fig. 16 (Thompson and Jacobs, 1966). The tracheary

2 60

WILLIAM P. JACOBS

cells responded much as they did in the older nonclongating internode, even to the interpolated values for intact plants falling at the same equivalent IAA concentration (vit., 0.05%). One of the few differences is that internode no. 2 showed no further increase as the IAA concentration was raised from 0.1 to l.OO/o.r This result fits expectations, however, from data on the transport of auxin added apically to sections cut froin the two internodes; internode no. 2 was found to show a plateau in the amount of basipetal transport as IAA coil-

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Intact plant

(Its sieve tubes= 175)

z

0 0 7

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0 5 : 8 I

0001 IA A

001

CONCENTRATION

m r

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10

0

(OX)

FIG. 16. The effect of apically applied IAA at various concentrations on xylem and sieve tube regeneration around a wound in isolated no. 2 internodes of Coleus (Princeton clone). Each point is the average of two experiments. (Corrected data from Thompson and Jacobs, 1966, Fig. 5 . )

centration was increased, while internode no. 5 did not (Fig. 17; see Scott and Jacobs, 1963; Fig. 18; see Naqvi, 1963). Internode no. 2 showed a quite different response from internode no. 5 in its sieve tube regeneration. Although IAA added apically did cause an increase in the number of regenerated sieve tube elements, no IAA concentration tried fully replaced the rest of the plant in its effect on sieve tube regeneration. Presumably some other substance or substances in addition to IAA are needed to provide the normal number of sieve tube elements in this elongating internode. The hormonal auxin of Coleus is IAA and only IAA by a iiumber of criteria. In the work on tracheary regeneration cited earlier, Jacobs had shown that the young leaves of Coleus were the main endogenous source of auxin. This auxin was entirely IAA, judging by Rf in two solvent systems (Fig. 19), by activity in two bioassays, and by color tests and ultraviolet fluorescence (Scott and Jacobs, 1964). In addition, exact substitution of synthetic IAA for the endogenous 2 The original figure was in error in showing a larger decrease from 0.1 to 1.0% than does the present figure. (The error resulted from not using weightitig factors in averaging experiments of different sample size.)

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PIG. 18. Basipetal transport through 7.O-mm sections cut from Coleus internodes nos. 2 and 5 (from Naqvi, 1963). Effect of different donor concentrations on the amount of IAA-14C transported through internodes nos. 2 and 5 in 8 hours. (From Jacobs, 1967, Fig. 8.)

2 62

WILLIAM P. JACOBS

auxin estimated to be IAA gave exact replacement of the effect of the young leaves on tracheary regeneration (Jacobs, 1956, Fig. 4 ) . Exact substitution by synthetic IAA also gave exact replacement of endogenous auxin in the abscission-speeding effect of the apical bud (Jacobs, 1955; Jacobs et al., 1957, Fig. 6). c

I

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b w

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lsopropanol -ammonia -water Synthetic I A A R f = 0.38

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IAA (mg/liter)

FIG. 19. Evidence that diffusible auxin is I A A and only IAA. All the auxin activity of the agar diffusion extract runs to the R, typical of synthetic IAA (right side), Shading indicates significant difference from 2% sucrose control. Calibration curve of 0, 0.01, 1.0 mg/liter synthetic IAA is shown on left. Star indicates activity of elution controls of s and 50 pg synthetic IAA chromatographed like the unknown. Triangle indicates activ. ity of agar extract at Rf zone corresponding to synthetic IAA. (From Scott and Jacobs, 1964, Fig. 2.)

F. ARE SIEVE TUBES THE NORMAL PATHOF IAA MOVEMENT? As cited above, several pieces of evidence concerning timing and sensitivity of vascular regeneration in Coleas fit the view that sieve tubes are the major path of IAA movement in the intact plant. Although it is possible for IAA to move in polar fashion through isolated cylinders of tissue comprised solely of pith parenchyma from internode no. 5 (Fig. 13; see Jacobs and McCready, 1967), this is probably not the normal path. [We know that these pith cylinders act differently when isolated than in the intact plant; for example, they elongate considerably when isolated, a characteristic correlated with polar transport of auxin (McCready and Jacobs, 1963) .] Fischnich (1935) presented indirect evidence that IAA added to the intact Coleus leaf moved mostly in the vascular strand; root initiation on internodes below occurred only opposite the vascular strands. Camus' (1747) work on regeneration in cultured pieces of root (see Section 111) also supports the view that the cambial area is the preferred

SIEVE TUBE ELEMENTS

263

path of auxin movement (cf. Camus, 1949, Figs. 61 and 74). A particularly high content of endogenous auxin was found in scrapings from the general cambial zone of several trees (Soding, 1937). It was only recently, with the help of aphids, that the presence and movement of auxin in sieve tubes was demonstrated. Maxwell and Painter (1962) found auxins in extract of aphids that fed exclusively on sieve tube sap, and Eschrich (1968) found no labeled material in the aphids’ honey dew except the IAA-14C he had added to an intact leaf. At present, therefore, we know that IAA is present in large amounts in the cambial area (including young xylem, cambium, and young phloem), and that it can move in sieve tubes (although evidence is lacking so far as to the exclusiveness of this path). It takes 14 times as much IAA for a tracheary cell to differentiate in regeneration as in normal development (Jacobs and Morrow, 1957). To raise the endogenous level 14 times is apparently the reason why the vascular strand must be cut before regeneration occurs. Merely making a wound of about the same size will not suffice (von Kaan Albest, 1934). If sieve tubes are the normal path of IAA movement, then severing them should be the stimulus for vascular regeneration. von Kaan Albest cited 10 cases in which she had severed strands small enough to consist entirely of phloem. In three cases, no vascular cells regenerated; in the other seven, wound sieve tubes regenerated but no tracheary cells formed. [She interpreted these results as an indication that one must sever phloem strands to obtain phloem regeneration, and xylem strands to obtain tracheary regeneration. I think it more likely that the lack of regenerated tracheary cells was a result of the fact that much more auxin is required to regenerate tracheary cells (cf. Fig. 1 2 ) and that a small strand-such as would consist entirely of phloem cells-would not carry enough auxin to make anything but sieve tube elements.] Further evidence that it is the non-xylem part of the vascular strand that must be severed came from Roberts and Fosket (1962) ; pith punctures that damaged the xylem led to no xylem regeneration, but deep cortical punctures did. Also confirming the hypothesis that auxin movement is in the sieve tubes or cambium or both were the results of Thompson (1967). He physically separated the xylem from the phloem region of a severed vascular strand and inserted a very thin glass coverslip. Tracheary cells regenerated only on the phloeni side of the cover-slip and in strands that matched the path of the sieve tubes that had regenerated a few cells external to them. Thompson also noted a case among his treatments in which a strand containing no differentiated tracheary cells had been accidentally severed; a substantial number of tracheary cells regenerated, nonetheless (Thompson, 1965, p. 8 2 ) .

2 64

WILLIAM P. JACOBS

111. Differentiation of Sieve Tubes i n Organ or Tissue Culture There are many papers describing the growth and differentiation observed in sterile cultures of organs or tissues. In addition to their other points of interest, such cultures may be thought of by the developmental physiologist as representing a step beyond, for instance, isolated internodes in the direction of further isolating the reaction system that controls cell differentiation (Jacobs, 1959). Cultures do have a serious limitation, however. By cutting away most of the plant or animal from the cultured tissue, we are likely to have cut away sources of chemicals that are normally present in nonlimiting amounts. W e will have made some chemicals “artificially limiting.” For instance, it is reasonable to expect that in an intact organism of the many chemicals presumed to be necessary for the differentiation of a sieve tube, only one or two of these will actually be controlling sieve tube differentiation by their limiting availability. In the artificially deprived culture situation, however, the limiting factor may be one of the compounds always present in superfluity in the intact organism. To correct for this limitation of the culture technique it is necessary to relate quantitatively the results of cultures back to the limiting factor in the intact organism. Such a “correction for the intact organism” was not needed in one of the early papers on sterile culture, because the author grew an entire sterile seedliiig in a test-tube. Molliard (1907) found that adding sugars to the clearly noaoptimal medium increased the number of sieve tubes that differentiated. His Figures 45 and 46 of transverse sections of Ipoinuea show this in striking fashion. Most of the later workers who studied vascular differentiation in sterile cultures were, to the contrary, growing only small pieces of the organisin and, therefore, the relevance of their results to the limiting factors in the intact plant needs to be demonstrated. The elegant and extensive work of Camus (1949) showed that a bud grafted onto the “shoot-end” of a cultured piece of Cichorizm root induced a vascular strand that eventually joined with the cambial zone of the root tissue. Various synthetic auxins could substitute to a sizeable extent for the vasculogenic effect of the grafted bud, with IAA and indolebutyric acid giving results most like the bud itself. Soon after IAA had been shown to be able to control the regeneration of titcheary cells in young Colezls stems, it was shown to be able to speed the regeneration of tracheary cells in Syringd callus using the techniques of Camus (Wetmore and Sorokin, 1955), and in cultured Piszlm roots (Torrey, 1953). Both papers reported that there was no discernible effect of auxin on sieve tube differentiation. Further investigation of the cultured Syringu callus, however, colifirmed and extended Molliard’s results; Wetmore and Rier ( 1 9 6 3 ) reported that in the presence of auxin higher sucrose concentrations (4-5%) favored the de.

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velopment of sieve tube elements, whereas lower sucrose concentrations ( 1-276) led to fewer sieve tube elements and more tracheary elements. This has been confirmed in Phaeolzls callus cultures by Jeffs and Northcote (1966), who also stated that kinetin added along with IAA and sucrose increased cell divisionsits classic effect-and “phloem differentiation.” Wetmore and Rier said, “It is striking that the majority of vascular elements, whether xylem or phloem, are formed in nodular centers and not as isolated elements.” Jeffs and Northcote noted this also. What this indicates is that the auxin-sucrose treatments are essentially causing the differentiation of a piece of complete vascular strand that contains sieve tube dements toward the outside of the callus,3 tracheary cells toward the interior, and a cambial zone in between the two. We should remember that there are special difficulties in trying to study sieve tube differentiation in callus cultures; not only are the cells apt to be very small, but they do not form in such predictable locations as in regenerating internodes, nor do they usually differentiate as a strand of sieve tube elements. The difficulty of searching through a whole callus for the few cells that might show sieve plates or slime plugs undoubtedly explains why the work published so far has not included actual counts of sieve elements, nor shown a photograph of recognizable sieve tube elements induced by chemicals. [Both Galavati ( 1 9 6 4 ) and Wetmore and Rier (1963, Fig. 3) have published convincing photographs of sieve tube elements induced in cultured tissue by developing buds.] One other report on cultures should be mentioned because of the possibilities it raises. Gautheret (1961) reported that GA,, if added to Jerusalem artichoke cultures along with IAA, caused broad areas of xylem and phloem to form from the cambial area (Gautheret, 1961, Figs. 1-3). GA3 added without the auxin had little effect-the auxin requirement for GA, activity agreeing with many though not all earlier reports on other processes. Higher concentrations of IAA added with the GA, differentially inhibited cell formation on the phloem side. Because no evidence was presented that the new phloem cells included an increased number of sieve tube elements, this seems like the equivalent in tissue culture of the GA-stimulation of cambial activity in trees that had been reported earlier (Bradley and Crane, 1957; Wareing, 1 9 5 5 ) . Similar effects on cambial activity resulted from GA,IAA additions to a number of noncultured, regenerating woody stems (Wareing et al., 1964; Digby and Wareing, 1966), although the authors apparently did not know of Gautheret’s paper. These later authors interpreted their results as an indication that GA, caused the d i f w e c tiation of sieve tube elements, but their actual evidence was that the cells induced by GA,IAA on the phloem side of the cambium are not differentiated 3 The legend for Fig. 1 2 of Wetmore and Rier (1963) describes the reverse of this, but Prof. Wetmore (personal communication) stated that the legend was incorrect and that the contrary statement on their p. 423 was correct.

2 66

WILLIAM P. JACOBS

into sieve tube elements nor into companion cells (Wareing et al., 1964). In their second paper, they were able to find some sieve plates in some cells on the phloem side of the cambium in one of the genera, but conspicuously refrained from claiming that the GA,-IAA treatment induced more of them. DeMaggio (1966) also reported more cells cut off on the phloem side of the cambium from GA, application to Pinus explants, but could not find the standard critical signs of sieve cell differentiation. (He could see more cells with walls that were birefringent in polarized light after GA, treatment, so some wall changes did occur.) To summarize the research on regeneration, at present it looks as if IAA is the hormone controlling the differentiation of sieve tubes in secondary tissues (i.e., those with a cambium, such as Coleas internode no. 5 ) , with GAS affecting the number of cells cut off by the cambium but not affecting the number that then differentiate into sieve tube elements. (Wareing et al., essentially counted cell number on the phloem side, whereas Thompson in Coleus counted sieve tube element differentiation.) In primary tissues (e.g., Coleus internode no. 2), IAA seems to be the sole and sufficient explanation for tracheary regeneration, but something in addition to IAA is required to fully replace the effects of the distal and proximal tissues on sieve tube regeneration. From the results with sterile culture, this additional factor may be sucrose. According to the work of Gautheret, Wareing et al., and DeMaggio, however, it might well be gibberellic acid. Although it was hypoth. esized many years ago that sugar reacted with auxin to determine the spatial and temporal pattern of normal vascular differentiation (Jacobs, 1752), sugar has not as yet been shown to actually limit vascular differentiation in intact healthy plants, Molliard’s sterile seedlings being semistarved according to his description. Favoring gibberellic acid as the second factor are the facts that the action of gibberellic acid is typically restricted to young tissue, and that GA, has been shown to be active in petioles no. 2-4 of this same clone of Coleus (Jacobs and Kirk, 1966). [A detailed and interesting argument for the importance of sugar in controlling vascular development is given in a review by Wetmore et ul. (1964).]

IV. Normal Differentiation of Sieve Tubes Our discussion of research on this topic will be brief and selective. Much of the recent work has been with the electron microscope, and I am not qualified to discuss that critically. Of the other papers on sieve tube differentiation published in the last decade, Esau has recently discussed most of them with her usual thoroughness and authority (1965). In a more recent paper, however, the

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quantitative methods used in the CoZeus regenerative studies were applied to the normal shoot tip. The results were illuminating and unifying. The rationale of the experiments was as follows. Our quantitative studies of sieve tube regeneration had shown that sieve tubes formed first, and that later and exactly opposite them tracheary strands regenerated. The earlier literature, qualitative as it was, had led us to expect quite a different spatiotemporal pattern. The literature had been similarly misleading as to the pattern of tracheary regeneration in relation to the polarity of auxin transport (Jacobs, 1952, 1954). Accordingly, it seemed reasonable to look with some scepticism on the accounts in the literature indicating that the pattern of differentiation of sieve tubes was “continuous and acropetal” and thus quite different from that of the first tracheary cells. W e investigated apical buds with quantitative methods and aroundthe-clock collecting. The latter was based on the fact that earlier anatomists had typically collected samples only during the daylight hours and on the belief that a sizeable percentage of the developmental activity would occur during the dark portion of the normal 24-hour cycle. Cell differentiation during the dark period would be particularly expected if auxin were the limiting factor, because auxin production had been reported to follow a diurnal cycle with a maximum at night (Yin, 1941). This expectation had already been confirmed for xylem differentiation (Jacobs and Morrow, 1 9 5 7 ) ; a new locus of tracheary differentiation was detectable only in collections made at night. The first applications of this method revealed that the first differentiation of sieve tubes in a leaf primordium was strictly correlated with the length of the leaf (Fig. 20; see Jacobs and Morrow, 1958), no leaf differentiating its first sieve tube element until it was 400-450 p long. All leaves longer than 470 p had a continuous strand of sieve elements extending up into them from the stem. It seemed appropriate that the leaves did not grow longer than about 0.5 inm before differentiating the cells specialized for the movement of organic food materials, because 0.5 mm is roughly the distance beyond which physical diffusion would inadequately provide for the growth of the primordium. In other respects, the first results were disappointing. There was no sign of a discontinuity in any one of the many “first sieve tube strands” differentiating in the young leaves (Fig. 2 0 ) ; the differentiation was apparently “continuous and acropetal” as the classic literature reported. For a leaf primordium too short to contain differentiated sieve tubes, the sieve tube strand destined for that leaf was presumably down in the stem differentiating acropetally toward the leaf base from the procambial cells of the leaf trace. This process of acropetal differentiation in the leaf trace occurred, at first glance, just as expected. As the young leaf grew progressively longer, the sieve tube (destined to connect it eventually with the mature part of the plant below) dif-

2 68

WILLIAM P. JACORS

ferentiated progressively closer to the leaf base (Fig. 2 1 ; see Jacobs and Morrow, 1967). Regression equations fitted to the data confirmed that there was a linear relation between the length of a young leaf and the distance from the base of the leaf to the top of the acropetally differentiating sieve tube in the leaf trace (Jacobs and Morrow, 1967, Table 1 ) . Scrutiny of the regression line 32r

-

0 2

1 Immature sieve tube

4

6

8

10

LENGTH

12

14 16

18

20 22 24 26 28

OF LEAF (microns x 100)

Longitudinal distribution of first sieve tubes within the main vein of young Coleus leaves. Each solid vertical line represents the sieve tube of a different leaf. (From Jacobs and Morrow, 1958, Fig. 1 with permission from the editors of Scieiice.)

(as drawn through the data of Fig. 2 1 ) , however, revealed an unexpected problem; although the sieve tubes in the leaf traces were differentiating acropetally as the leaf grew, they were lagging progressively farther behind the leaf tip. At zero leaf length they averaged 442.9 p below the “tips” of the leaf; at 4SOp leaf length, they were 601 p below (450 f 101 p). This trend of iizcrediiiig distance of sieve tubes from the leaf tip somehow had to be reversed, because all leaves more than 470 p long had continuous sieve tubes out into the leaf (Fig. 20). To investigate how this increasing gap was closed we used our knowledge of the close relation between leaf length and sieve tube differentiation to select leaves just about to show sieve tubes out in the leaf. This technique uncovered a new phenomenon of sieve tube differentiation; in leaves 387-459 11 long a separate locus of sieve tube differentiation was found near the base of the young leaf (Fig. 2 2 ) . Within a very short span of leaf lengths, the sieve tube of the first locus connected basipetally with the acropetally differentiating sieve tubes in the stem-thus closing the gap. Why was this separate locus of sieve tube differentiation, found reproducibl) year after year in this clone of Coleus (Jacobs and Morrow, 1967), not discov-

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ered before among the angiosperms ? I A separate locus for sieve cells had been found in young leaves of Equisetzlm (Queva, 1907) and of Selaginella (Jacobs, 1947) but was thought to be correlated with the microphylls of those genera, in contrast to the megaphylls of the angiosperms.] Aside from the remote possi-

- 500‘

0

I

too

I

200

I

I

300

400

1 500

LENGTH OF LEAF (microns) FIG. 21. Data for leaf length plotted against the most distal position of sieve tubes in the corresponding leaf‘ traces, including linear regression line and its equation. (From Jacobs and Morrow, 1967, Fig. 3 with permission from the editors of Aineiicuii Jou17)d of Botany. )

bility that it occurs only in Coleus, there are three obvious reasons: (1) The separate loci were mostly found in nighttime collections; ( 2 ) even though our sample sizes were larger than those of most of our predecessors, we did not find the separate locus until the quantitative relations from the linear regression pointed out to us that the “differentiation gap” occurred and thereby directed us to the particular range of leaf lengths in which the locus was found; and ( 3 ) ColeuJ has a fairIy long leaf plastochron under our growth conditions-forming a new leaf pair once a week-and genera with plastochrons of 1 or 2 days, such as Xunthizlm, would presumably go through this developmental stage 3-7 times faster.

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WILLIAM P. .JACOBS

V. Conclusions The quantitative studies of Coleus provide a unifying picture of vascular differentiation and regeneration. The pattern of first differentiation of sieve tubes in the young leaf is not different from that of tracheary cells in being “continuous and acropetal” as the literature would lead us to expect, but rather remark-

0 387

387

100 p

H FIG. 2 2 . Isolated loci of sieve tube differentiation shown in stereodiagrammatic chart of three different shoot tips (F, I, J ) , with leaves 387-459 p long (leaf pair 11). Stiypling shows expected level of sieve tubes as derived from the regrcssion line of Pig. 21. (For comparable xylem differentiation, see F, I, J, Jacobs and Morrow, 1957, p. 830.) Nodes, which are represented as flat plates labeled with Roman numbers, are shown as transparent where traces would end in this area. The scale shown is valid for vertical measurement but not for horizontal. (From Jacobs and Morrow, 1967, Fig. 5 with per. mission from the editors of American Jolrrnal of Botany.)

ably similar to that of tracheary cells. The first sieve tube element in the leaf forms at a separate locus on the outer side of the procambial strand near the base of the leaf. This is the region where the first tracheary cell will form on the inner side of the procambial strand several days later. Vascular regeneration in internode no. 5 shows the same pattern (again, contrary to expectations from the literature) ; sieve tubes regenerate first, then 1 day later and exactly opposite them on the inner side of the cambium the tracheary cells differentiate. [In elongating internode no. 2, regenerated strands of tracheary cells are also exactly opposite the regenerated sieve tubes (Thompson, 1967, Fig. 5 ) , but the timing has not yet been investigated in Colez~s.] The regeneration of both cell types in internode no. 5 is controlled by the amount of basipetally moving endogenous auxin, IAA, coming from the leaves.

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The IAA moves in the sieve tubes (and probably in the procambium-cambium also, particularly in locations where no sieve tubes have yet differentiated, such as in the youngest leaf primordia). When the sieve tube strands are severed, the local IAA concentration in the cells adjoining the cut end builds up enough to initiate the differentiation of wound sieve tube elements. Subsequently, tracheary cells regenerate opposite the wound sieve tubes. Recent radioautographic evidence confirms that IAA itself (or a derivative of it) moves across from the sieve tube-cambial zone to the differentiating tracheary cells (Sabnis et ul., 1969). When IAA labeled with tritium was added in normal amounts to Coleus internodes, the “acetone-insoluble” label was found to be specifically localized in the secondary walls of those tracheary cells that were in a stage of early differentiation. In such radioautographs, there was no localization of tritium in the sieve tubes, the basipetally moving IAA-3H presumably having been extracted with the acetone used to prepare the sections for ultramicrotoming. With regeneration in internode no. 5 so fully explained by IAA, it is reasonable to hypothesize that the very similar patterns of normal vascular differentiation in the shoot tip are also explainable by IAA, particularly since reasonable correlations have already been shown between normal IAA production and normal tracheary differentiation (e.g., Jacobs and Morrow, 1957, Fig. 16). Regeneration of sieve tubes in young elongating internode no. 2 is not fully understood. Whether GA, or sugars are interacting with IAA to limit sieve tube regeneration in internode no. 2 , we can not say at present. The lack of a cambium in that developmental stage may well be critical, however, because von Kaan Albest reported that several species with isolated procambial strands did not regenerate sieve tubes when their vascular strands were severed (although some did regenerate tracheary cells). There is a great need for quantitative and critical studies of cambial activity in relation to sieve tube differentiation. From our present knowledge, the relation between leaf growth and vascular differentiation seems reasonable; as a leaf grows more quickly, it produces more IAA, which in turn allows more vascular tissue to differentiate in the traces supplying the leaf with the wherewithal to grow. A declining growth rate is associated with declining IAA production (Jacobs, 1952, Fig. 5 ) and, therefore, with declining differentiation of vascular tissue in the leaf traces. ACKNOWLEDGMENTS The cost of preparing the manuscript and figures was aided by an NSF research grant. I am also grateful to Mrs. Mary Leksa for deciphering and typing the manuscript, and to research assistants Mrs. Paula Edwards and Mrs. Hannah Suthers for their help in assembling figures and legends. (Mrs. Suthers also constructed Figure 6.)

REFERENCES Bradley, M. V., and Crane, J. C. (1957). Science 126, 972. Camus, G. (1349). Rev. Cytol. Bid. Vegetales 11, 1.

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