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Hormone transport and action in the green shoot:long-term studies of a clonal stock of Coleus blumei(Labiatae)¹

^a Biology Department,Princeton University, Princeton, New Jersey 08544

	ABSTRACT

TOP ABSTRACT INTRODUCTION METHODS OF ESTIMATING NATURE... HORMONE MOVEMENT AND ACTION... DISCUSSION REFERENCES

 
A decades-long study of hormone production, transport, developmentalactions, and hormonal interactions in the green shoots of mature plantshas exploited a clone of Coleus blumei. To obtain data bothquantitative and reproducible, we greatly increased sample size over theclassical anatomical models, initiated round-the-clock collections, andcountered that increased workload by clearing and staining organs ratherthan by embedding and serially sectioning them. Major developmentalevents occurred at night. The control of the normal differentiation andregeneration of tracheary cells and sieve-tube members byindole-3-acetic acid (IAA) and cytokinins and of fibers by IAA andgibberellic acid have been major findings from this approach. IAA fromthe leaf blade controls the timing of leaf abscission. As the leafages, the ability of the petiole to transport IAA from the blade to theabscission zone declines, with abscisic acid (ABA) decreasing IAAtransport down the petiole and concomitantly increasing the conjugationof IAA with aspartic acid. Evidence for transport barriers was found atnodes and abscission zones.

Key Words: abscisicacid • abscission • Coleusblumei; • cytokinins • fibers • gibberellicacid • indole-3-aceticacid • Labiatae • transport • vascularcells

	INTRODUCTION

TOP ABSTRACT INTRODUCTION METHODS OF ESTIMATING NATURE... HORMONE MOVEMENT AND ACTION... DISCUSSION REFERENCES

 
This article is designed to provide an overview of major researchcontributions from studies of a clonal stock of Coleus. Thequestion posed was what roles do hormones and their movements play inthe development of the green shoot. The approach is described below. Early research on plant hormones was mostly based on studies ofseedlings grown in the dark, the coleoptiles of grasses beingparticularly favored. The use of seedlings made economic sense: theplants were usable a few days after sowing, occupied little space,needed no lights, and could grow for several days on the nutrientsstored in the seeds. However, as research on such dark-grown seedlingsaccumulated through the 1940s, I wondered how typical such results wereof most of the plants on the earth's surface--namely, green plantsabsorbing nutrients from the soil, photosynthesizing, undergoing dailylight–dark cycles, and coordinating much more complex interactionsof tissues and organs than etiolated seedlings possessed. My approachto studying hormone actions in the mature green plant was stronglyinfluenced by the penchant of my mentor, Ralph Wetmore, for training hisstudents in both anatomy and physiology. As an anatomist, I wanted toextend the usual interest of physiologists in length, mass, and organdifferentiation to problems of cell differentiation. As a physiologist,I wanted to use larger sample sizes than anatomists were then typicallyusing—sample sizes that would be large enough to providequantitative data and to validate the use of statistical analyses. Also, as a physiologist I expected that the light–darkalternations that mature green plants experienced every 24 h wouldresult in some events of cell differentiation occurring only in thenight: the daytime samplings typical of the anatomists of the1930–1940s would miss them.

The labor involved in processing the larger,"physiological-size" anatomical samples, much less thoseneeded for the round-the-clock collections, would be almost impossiblyonerous if the conventional histological serial sectioning was used. Wedevised the following stratagems for decreasing that workload. First,we focussed on a clonal stock of Coleus blumei Benth. (the"Princeton clone"). By using a clone we eliminated geneticvariation and could obtain quantitative results from smaller samplesthan would otherwise be required. Plants from the clone were made evenmore uniform for critical experiments by growing them under controlledconditions of light and temperature in growth chambers. To obviate thelabor of intensive histological sectioning, whenever possible we clearedand stained whole organs instead. These preparations allowed us to lookthrough the transparent outer layers of an organ to count the stainedvascular cells inside.

I was fortunate to have the long-term and generous support of A. K.Parpart and J. T. Bonner, two successive chairmen of PrincetonUniversity's former Biology Department. In the greenhouses thatthey provided, the Coleus plants were grown to a uniformsize, with 6–8 pairs of unfolded leaves on the main stem and withthe youngest four unfolded leaf pairs showing increasing blade length. Each week a new leaf pair unfolded from the apical bud. Leaf pairs werenumbered from the apical bud down, leaf pair 1 being just below theapical bud. For a given experiment, plants were selected from a largerpopulation of the same age and were then matched by the blade length ofthe fastest growing leaf pair (No. 2). Internode 2 was just below leafpair 2. Treatments were assigned by mathematically random methods,results were routinely analyzed statistically, and all major experimentswere repeated at least 2–3 times to demonstrate reproducibility. Axillary buds were usually removed to increase growth of the main shoot,and supplementary lights were added in the greenhouse to maintain fastgrowth during winter months.

Starting with such uniform plant material in 1948 and with the helpover the decades of research assistants, students, postdoctoralassociates, and visiting professors, we have been able to develop adetailed picture of how several plant hormones function inColeus shoots. That picture, based on the Princeton clone ofC. blumei, will be described here, along with less detailed references to related work on other stocks of C. blumei andother species and genera. Because the uniformity of the Princeton clonalstock has been paralleled by a uniformity of our transport techniquesover the decades of our research on hormone physiology, the resultsfrom our studies of one hormone can be more validly compared with ourstudies of the other hormones than is true for transport studiesselected randomly from the literature.

Because coverage of the hormones' actions in the Princetonclone already makes this paper much longer than the usual invited paper, only a few of the later confirmations of our results will be mentioned.Experiments with active compounds that are not known to be endogenous(such as kinetin or the weed-killer 2,4-D) will not be discussed indetail, except when such experiments fill in gaps in our knowledge ofthe corresponding endogenous hormones (e.g., zeatin orIAA).

	METHODS OF ESTIMATING NATURE OR MOVEMENT OF THEHORMONES

TOP ABSTRACT INTRODUCTION METHODS OF ESTIMATING NATURE... HORMONE MOVEMENT AND ACTION... DISCUSSION REFERENCES

 
Evaluating reports of hormone movement is difficult for botanists whoare not actively working on such topics, because of the many differenttechniques that have developed over the years—techniques rangingfrom those that give a slight indication that a hormone might be presentto those that actually definitively identify the hormone. The followingsummary of the most used techniques is basically chronological, althoughall the techniques are still used. (The summary is intended for thosebotanists who specialize in areas far removed from hormonal physiologyand biochemistry. Physiologists, much less experts in hormonalphysiology, will find this simplified discussion of techniquessuperfluous.)

Went (1928) created the Avena curvature test, the first bioassay for determining the amount of auxinin receiver blocks of agar. (For a full description of the assay, seeChapter 1 of Jacobs, 1979.) Theendogenous material moving across a cut surface into the agar wasinitially, if tacitly, assumed to be auxin and only auxin (so-called"diffusible auxin"). Much later, gibberellins (GAs) andabscisic acid (ABA) were also reported in some diffusates. Researcherslater separated bioactive compounds in extracts of receiver agar or oftissue sections from some of the other material in the extracts bychromatography, first on paper, then with thin-layer chromatography(TLC), and more recently by high-pressure liquid chromatography (HPLC). Increased resolution of compounds resulted from each new chromatographictechnique. The location of the active compounds on the paper andthin-layer chromatograms is denoted by Rf (the ratio of the active spotto the solvent front) and on HPLC by Rt (the retention time). Thus,when investigators conclude after TLC that IAA is present in theirextract, they typically mean that at the Rf typical of synthetic IAAsome IAA-like property was found (e.g., auxin-like bioassay activity,positive color test, appropriate fluorescence, or a peak of countedradioactivity if IAA with a radioactive label had been added to theplant). Obviously, the more such properties are demonstrated the morelikely it is that IAA is actually there. A convenient shorthand forsuch chromatographic evidence is to say "IAA wasfound in the extract" (Jacobs,1979).

Gas chromatography (GC) can be used directly for analysis of the gasethylene, but IAA, gibberellins, abscisic acid, and cytokinins mustfirst be derivatized into more volatile compounds. The Rt of thederivatized sample is then compared to that of derivatized standards fortentative identification. The detectors used vary in specificity fromthe relatively unspecific flame ionization detector to the more specificelectron capture detector. Capillary GC provides sharper resolution ofpeaks than does column GC.

The most convincing, practicable identification method currently inuse for plant growth substances is GC combined with mass spectrometry(GC-MS). The partially purified sample is typically purified further byHPLC, then derivatized for GC . Material separated by the GC is fedinto the mass spectrometer, bombarded with electrons to break up themolecules, and the initial molecule and all its fragments are comparedwith the spectrum of a standard. Such a "full massspectrum" provides the current state of the art in proving that agiven growth substance is present in the plant extract. If instead ofthe initial molecule and all its fragments only one or two fragments aremeasured (referred to as SIM, for selected ions mass spectrum), obviously the identification is much less secure. Quantitativeestimates of endogenous IAA can be made by adding to the initial samplea known amount of an internal standard. IAA labelled in all thecarbons of the benzene ring with the nonradioactive C isthe current state-of-the-art internal standard (Cohen, Baldi, and Slovin, 1986). Thespectroscopist then uses SIM to compare a few peaks from the endogenousIAA with a few from the C-IAA. But even with a fullspectrum MS to demonstrate the presence of IAA in the sample, followedby SIM to estimate the amounts, it is rare for both types of MS to beused throughout an experiment. The few ion fragments followed in SIMare assumed to come solely from the IAA and not to be supplemented oreven replaced by ions from other compounds that were formed by thetreatments or changing age of the tissue used in the experiment.

After IAA with radioactive labels became available commercially inthe 1960s, most studies of hormone movement have used such tracer IAA. Despite the sensitivity of the radioisotope counters (particularly ofthe liquid scintillation counters), some problems with the use of tracerhormones have not been always considered. For instance, although tracerIAA added to transport sections is typically found aschromatographically pure IAA in receiver agar so long as transport timesare 6 h (Fig. 1), yetwithin the green transport sections IAA is quickly metabolizedto other compounds (e.g., Veen and Jacobs,1969a, where more than half the tracer added toColeus transport sections was at Rfs other than that typicalof IAA after only 5 h of transport). Hence, it is clearly not valid tomeasure the total counts from slices of a transport section as being atrue measure of C-IAA movement per se. In addition, thosewho study the movement of tracer hormones by extracting successiveslices of transport sections should, but often do not, demonstrate thatthey are extracting all the radioactivity in the slices. More seriousis the addition of tracer hormone that has not been checked regularlyfor purity: a very small amount of impurity in a C-IAAstock (too small to be detected by a Geiger-tube paper scanner) willprovide acropetal movement of tracer through transport sections thatshow no acropetal movement after the IAA stock is purified (Jacobs, 1979, p. 246).

View larger version (22K): [in this window] [in a new window]   Figs. 1–4. IAA transport through shoot sections. 1. Distribution of radioactivity on a paper chromatogram on which an extract of basal receiver blocks of agar had been spotted and run after C-IAA transport through Coleus internode tissue (Jacobs and McCready, 1967 ). The black Rf zones represent the zones to which the pure IAA standard ran in parallel chromatograms. Such "chromatographic purity" of basal receiver-agar after auxin transport is typical. 2. Time course of C-IAA accumulation in basal receiver agar as measured by radioisotope counting after transport through green Phaseolus petiole sections (McCready and Jacobs, 1963 ). The lines drawn for the data from two different donor concentrations are the best-fitting straight lines by the least-squares technique. 3. The time course of basipetal movement of C-IAA through sections cut from Coleus petioles of different ages (Fig. 8-5 of Jacobs, 1979 ). Petioles of each higher number shown are ~2 wk older than petioles of the nearest lower number. The same amount of IAA was added in each donor block. 4. Pretreating young Coleus petioles with an extract of senescent petioles decreased the radioactivity at IAA and increased the amount at indoleacetylaspartate when transport sections that had transported C-IAA for 4 h were extracted and the extracts run on TLC (Chang and Jacobs, 1973 ).

More detailed treatment of these and other analytical techniques isprovided by various experts in Rivier and Crozier(1987). Limitations of bioassays and problems encounteredwith identifying native auxin are discussed in Jacobs (1979, pp.14–23, and Chapter3).

Basic to the original definition of "hormone" is theconcept of movement from the site of hormone production to the site ofhormone action. The usual method of studying IAA movement has been toadd IAA in donor agar to one cut end of a 3–10 mm transportsection and to measure what comes out through the other cut end intoreceiver agar during the next 3–6 h. As illustrated in Fig. 2, the typical results forbasipetal IAA movement through such sections show a linear accumulationof IAA in the receiver agar. Calculation of the best-fitting straightline to such data by least squares regressions, as used in Fig. 2, has been routinely used byJacobs and coworkers since 1961. Comparison of the results usingbioassay (as in Fig. 1 of Jacobs, 1961)with radioisotope counting (Fig.2) makes clear the advantage of the latter: pooling thebioassays of three successive days was required to reveal the linearrelation in the gynophore that a single experiment revealed usingC-IAA in the petiole. The reproducibility of theradioisotope data is also strikingly apparent, since the 2–3 datapoints plotted at any one hour were from experiments run on differentdays. (Each data point from the bean petiole figure is an average from16 transport sections, making these calculated straight lines probablythe most reliable in the hormone transport literature so far as samplesize is concerned.) Extrapolation of this calculated best-fitting lineback to the time axis gives a less subjective estimate than "fitting by eye" of the time at which the main front of theIAA starts to come out of the transport section into the receiver agar. From this time intercept workers can calculate the velocity ofmovement. The slope of the line is usually referred to as the"transport intensity." The significance of the velocity ofauxin movement was emphasized by the early research on etiolatedcoleoptiles. Both the phototropic curve in intact coleoptiles and thezone of increased elongation when auxin was added to the outer surfaceof an intact coleoptile moved basipetally at essentially the same 10mm/h velocity as auxin moved through excised coleoptile sections(summary in Chapter 1 of Jacobs, 1979). The velocity is markedly less than the rate of 30–135 mm/h atwhich metabolites have been reported to move in the phloem (e.g., Table 14.1 in Canny, 1973), supporting theview that two quite different transport systems are involved. Incomparing time courses (as for those in Fig. 2 showing the amount of IAAtransported from donor blocks initially containing 5 or 50 µmol/LIAA), we used statistical tests to assess the probability that the timeintercepts—and thus the velocities—were different. (For thePhaseolus data of Fig.2 they were not significantly different.)

Hormonal effects are typically in the one micromolar (µmol/L)range of concentration. Adding 1000 µmol/L IAA, as someinvestigators have done, will produce pharmacological results (such asthe production of ethylene), not physiological ones. (Before thegeneral use of micromolar designations, many papers reportedconcentrations as "mg/L." For IAA 10 µmol/L isequivalent to 1.75 mg/L).

By uniformly applying the techniques described above to our clonalstock, we can directly and validly compare our results with differentplant growth regulators despite the decades separating the earliest fromthe most recent experiments. The results follow.

	HORMONE MOVEMENT AND ACTION IN GREENSHOOTS

TOP ABSTRACT INTRODUCTION METHODS OF ESTIMATING NATURE... HORMONE MOVEMENT AND ACTION... DISCUSSION REFERENCES

 
Leaf longevity andabscission
Indoleacetic acid (IAA) is the endogenous controller of leaflongevity in the Princeton clone of Coleus. The young,fast-growing blades of leaf pair 2 produce the maximal amount ofdiffusible auxin (calculated as IAA). The amounts collected fromprogressively older leaves decline steadily (Fig. 5 of Jacobs, 1952). The auxin is IAA, as firstindicated by the location on chromatograms of auxin activity and colortests (Scott and Jacobs, 1964) and asconfirmed recently by full spectrum GC-MS of purified extracts of youngshoots (LaMotte, Li, and Jacobs, inpress). The number of days before abscission of leaves ofdifferent ages is significantly correlated with the amount of diffusibleauxin the leaves produce (Wetmore and Jacobs,1953). Cutting off the blade of one member of each leaf paircauses fast and uniform abscission of the debladed leaf at allpositions. The effect of the blades in preventing abscission can bereplaced completely at all nodes by substituting synthetic IAA for theblades.

The amount of auxin that the leaf blades produce is not the solecontrol of leaf longevity, however: the ability of progressively olderpetioles to transport IAA from their blades toward the basal abscissionlayer declines in parallel to the normal decline in amounts ofendogenous auxin from the ageing blades (Werblinand Jacobs, 1967; Veen and Jacobs,1969a; Brennan and Jacobs,1983). The declining ability to move IAA basipetally,therefore, can act as a safety valve: if too much more auxin thannormal is produced by the blade, the limiting transport capacity willprevent the excess from moving into the stem and disrupting thecoordinated development of the plant. The decreasing amount of IAAmoved in older leaves is a function of declining intensity, rather thandecreasing velocity (which averaged 5 mm/h as calculated from theextrapolated time intercept) (Fig.3).

The decrease with leaf age in basipetal IAA movement through thepetiole sections correlates with increasing immobilization in thesections of the IAA in the form of the conjugateIAAspartate (Veen and Jacobs,1969a). Inactivation of IAA by increasing decarboxylation isnot the basis of the decrease in basipetal transport (Brennan and Jacobs, 1983). Despite themetabolism of IAA within the petioles, only IAA is found in thereceiver agar on the base of the transport sections (Werblin and Jacobs, 1967; Veen and Jacobs, 1969a).

IAA substituted for the leaf blades stimulates petiolar elongationbut does not replace fully the effect of the younger blades inmaintaining elongation (Jacobs, Kaushik, andRochmis, 1964). IAA's full replacement of theblades' effect on abscission is due to accumulation in theabscission zone of much of the IAA moving down the petioles (Kaldewey and Jacobs, 1974).

Two other hormones speed abscission in Coleus byinterfering with this transport of IAA to the abscission zone. Gibberellic acid (GA-3) increases both elongation and the speed ofabscission of younger petioles (Jacobs and Kirk,1966) by keeping more IAA that is in transit in the petioleand away from the abscission zone (Kaldewey andJacobs, 1975). Abscisic acid also speeds abscission bydecreasing the intensity of IAA transport down the petiole (Chang and Jacobs, 1973), but does so presumablyby the increasing formation of IAAspartate that is observed accompanying increasing doses of abscisic acid or of diffusate fromsenescent leaves (Fig. 4).Abscisic acid has recently been conclusively identified by fullspectrum GC-MS in young shoots of the Princeton clone (LaMotte, Li, and Jacobs, in press).

Although a clearly defined histological abscission layer develops atthe base of the petioles, it is not a prime causative factor inabscission, because it is detectable 3–5 wk before the leavesnormally abscise (Wetmore and Jacobs,1953). Only after IAA movement from the old blade hasessentially ceased is IAA available from the younger parts of the shoot able to force abscission of the old leaf (Rossetter and Jacobs, 1953; Jacobs, 1955, 1958).

The movement of IAA differs from that of the other hormones whosemovement through petiolar sections has been tested: its velocity isgreater [5 mm/h vs. 1–2 mm/h for gibberellic acid,cytokinins, or abscisic acid (Jacobs and Pruett,1972; Veen and Jacobs, 1969b;and Veen, 1975, respectively)], andIAA's movement through petioles of various ages is strictly polar,moving only from the blade end to the abscission zone end of thepetioles (Werblin and Jacobs, 1967;Veen and Jacobs, 1969a), whereas themovement of GA-1 and GA-5 is nonpolar (Jacobs,Beall, and Pharis, 1988), as is that of radiolabelled GA-3 (W.P. Jacobs and M. Jacobs, unpublished data). [Our earlier reports ofpolar movement of GA-3 (Jacobs and Kaldewey,1970; Jacobs and Pruett,1972) were based solely on bioassays in which more inhibitionof the assay in the apical receivers would be seen as less acropetalGA-3 movement.] Various cytokinins, whether endo- or exogenous,also move without polarity through petiole sections of the Princetonclone (Veen and Jacobs, 1969b; Koevenig, 1973; Jacobs,1976), confirming the work of Fox and Weis(1965) for several genera including Coleus. Thecytokinin kinetin increases the basipetal movement of IAA throughsections cut from the petioles of isolated, rooting leaves (Fig. 5). The development of roots onthe cuttings, which was expected to act like kinetin through itsputative production of cytokinin, had no such effect (Chang, 1971).

View larger version (25K): [in this window] [in a new window]   Fig. 5. The effects of kinetin and root regeneration on C-IAA basipetal transport through petiolar sections from Coleus leaf cuttings on various days after excision of the leaves. The arrow indicates when roots appeared on the leaf cuttings (figure modified from Chang, 1971 ).

The regeneration of tracheary cells in the young internode iscontrolled by IAA. Quantitative studies, using cleared and stainedwhole mounts, reveal that IAA at the exact replacement level of 2 mg/Lrestores completely the decrease in number of regenerated trachearystrands caused by excision of distal leaves (Jacobs, 1952, 1956). If the IAA is mixed with lanolin (a moreconvenient method of long-term application), 1% IAA in lanolinprovides full replacement of the auxin from the distal shoot (Jacobs et al., 1959) and slightly more thanfull restoration of normal amount of tracheary regeneration (Thompson and Jacobs, 1966). The more precisedata of Thompson and Jacobs, based on counts of individual regeneratedtracheary cells rather than of complete strands, reveal that0.05% IAA in lanolin exactly replaces the distal leaves, while0.1 and 1.0% IAA are on the transport saturation plateau andcause somewhat more tracheary regeneration (Thompson and Jacobs, 1966).

These same young internodes differ from the strictly polar petiolesin allowing sizeable acropetal movement through transport sections(Jacobs, 1952). One-third as much IAAmoves acropetally as basipetally when the same amount is added at eachend (Jacobs, 1954). [Naqvi and Gordon (1965) confirmed the 3:1polarity in another clone of Coleus blumei.] Theacropetal movement is physiologically meaningful since a similar 3:1ratio is found when tracheary regeneration is counted with all distalauxin-sources present compared to regeneration when only leaves belowthe wounded internode are present (Jacobs,1954).

Vascular regeneration in the older stem (internode 5, which is 3 wkolder than the young internode described above and just starting cambialactivity) was examined with similar cleared and stained material(LaMotte and Jacobs, 1962, 1963; Thompson andJacobs, 1966). IAA at an exact replacement level for theendogenous auxin also exactly replaces the whole distal shoot in thenumber of both tracheary cells and sieve tubes that regenerate in the collateral vascular strands. As increasingconcentrations of IAA are added at the distal end of the sections,increasing numbers of tracheary and sieve-tube cells regenerate, with 2mg IAA/L in solution or 0.05% IAA in lanolin providing the samenumbers of regenerated cells as the intact controls. At all levels ofadded IAA, more sieve-tube cells than tracheary cells regenerate(Fig. 6). [A similardose-response curve was reported for callus cultures ofGlycine and Syringa (Aloni,1980).] Linear regression analysis of regeneration datafrom 1 to 4 d shows exact duplication of the time course of sieve-tuberegeneration in the intact plant by distally applied IAA at 0.05%in lanolin (Thompson, 1967; Fig.4–18 in Jacobs, 1979). Thompsonnoted that the regenerated tracheary strands, are exactly opposite thepreviously regenerated sieve-tube strands, and pointed out how similarhis observations on vascular regeneration in this nonelongating, olderinternode were to the descriptions by others of normal vasculardevelopment in leaf primordia of a shoot apex: in both cases sievetubes develop first, followed by tracheary cells in the other side ofthe vascular bundle and with the tracheary cells initially not connectedto previously differentiated xylem.

View larger version (22K): [in this window] [in a new window]   Fig. 6. The dose-response curve of regeneration of sieve-tube strands (open circles) and tracheary cells (closed circles) in isolated Coleus internodes 5 to IAA in lanolin that was added at the apical end of the sections (Thompson and Jacobs, 1966 ). Because each sieve-tube strand consists of an average of ten cells (LaMotte and Jacobs, 1962 ), the axis coordinates are selected to show the number of cells of the two vascular tissues. The two asterisks interpolated on the curves show the average values for control plants that were intact except for a slit to initiate regeneration.

Between the large corner strands and the collateral strands runningvertically up the middle of the flat sides of Coleus are stillsmaller strands that have differentiated sieve tubes but not xylem (cf.diagram of Fig. 6 in Jacobs, 1970). Wounding these strands causes regeneration around the wound of sievetubes, but no xylem forms. If the distal organs are removed, sieve-tuberegeneration decreases, but can be fully restored by substituting IAAfor the distal organs. However, if the stem and root systembelow the regenerating internode are also excised, IAA fails torestore >20% of the full amount, unless zeatin or zeatinriboside is also added (Houck and LaMotte,1977). They suggested that both IAA from young leaves andcytokinins from the roots are required for sieve-tube regeneration, butthat in the the larger bundles of internode 5 (such as those thatThompson studied) there is sufficient cytokinin available so that onlyIAA is limiting. They also applied this interpretation to Thompson andJacobs' results on excised internode 2, where IAA fully replacedthe rest of the plant for xylem regeneration, but could only partiallyreplace it for sieve-tube regeneration (Fig. 5 in Thompson and Jacobs, 1966).

Evidence that IAA is directly involved in tracheary celldifferentiation rather than directly affecting only sieve tubes camefrom radioautographic studies of isolated internode 5 (Sabnis, Hirshberg, and Jacobs, 1969). Theyimproved resolution over earlier studies by using ultrathin sections andIAA labeled with tritium (rather than with C). The IAA wasof such high specific activity that hormonal (as contrasted topharmacological) concentrations of IAA could be added. Theradioautographs collected after 3 h show very specific labeling of thesecondary walls of young tracheary cells that are in the process ofdifferentiating. Already differentiated tracheary cells (i.e., thoseradially farther from the cambium), as well as young tracheary cellslongitudinally farther from the site of IAA application, are notlabeled. Similar results were later reported from other Coleus stocks (Wangermann, 1970; Gee, 1972).

The final stages of differentiation of fiber cells in the primaryphloem, which typically occur in internode 6, involve changes in thesecondary walls (Aloni, 1976b). Excision of distal but not proximal leaves prevents this finaldifferentiation, suggesting that IAA might be limiting (Aloni, 1978). However, IAA substituted for thewhole shoot distal to internode 5 causes only 20% of the~500 fibers to fully differentiate. GA-3, which has no effect whenadded alone as a substitute for the distal shoot, causes the full numberof fibers to differentiate when it is added with IAA (Aloni, 1979).

Auxin and normal vascular differentiation
The normal differentiation of tracheary cells is an obvious extensionof the studies on tracheary regeneration. Based on a large sample ofleaves 1–4, the number of tracheary cells is linearly related tothe length of the leaves (Fig. 15 in Jacobs andMorrow, 1957). Converting this relation to the rate at whichthe tracheary cells are formed, we found that the relative rate ofproduction of tracheary cells and diffusible auxin is essentially thesame for a given leaf age (Fig. 16 of Jacobs andMorrow, 1957). Comparison of the number of tracheary cellsformed by a given amount of auxin in petiole 2 during its normaldevelopment with the number regenerated in internode 2 during the sametime reveals that 14 times more tracheary cells differentiate frompetiolar procambium than regenerate from parenchyma cells in theinternode below. Jacobs and Morrow interpreted the results as anexplanation of why parenchyma does not differentiate into trachearycells unless the vascular strand is cut—only then does thetransporting vascular tissue pump IAA out "into the adjoiningtissues faster than it can be transported away," thus raising thelocal IAA concentration to the 14 times higher level that initiatesregeneration.

Pursuing questions about normal tracheary cell differentiation, theynoted that if the relations between IAA level and normal tracheary celldifferentiation that were seen in unfolded leaves 1–4 also held inthe five smaller leaf pairs still in the apical bud, the first trachearycell should be differentiating in leaves ~1.3 mm long. Large-sample, round-the-clock collections confirmed that this is so: the shortest leaf with walled xylem was 1296 µm long, the longestwithout it was 1386 µm.

With such evidence of the close relation between leaf length andvascular differentiation, Jacobs and Morrow focused on the full traces of leaves 1000–1500 µm long, exploiting the linear relationthey found between the lengths of the measurable leaf 1 and the tinyleaf III (their Fig. 11) to select plants for sectioning that would haveleaf III, the third leaf pair down from the apical meristem dome, in thedesired size range. They found a previously unreported and earlierforming locus of tracheary cell differentiation in the traces to leafIII. These isolated loci transfix the node below leaf III and were seenexclusively in nighttime collections. Emphasizing the value of thelarge sample size, in addition to nighttime collections, is the factthat only five of the 18 "night" plants were caught with thenode-transfixing locus prior to its connection with the well-knownseparate locus out in the leaf.

Applying these methods to early differentiation of sieve tubes in theapical bud, they uncovered a close relation between leaf length andfirst sieve tube, with leaf primordia 400–450 µm longdifferentiating the first sieve tubes out in the primordia (Jacobs and Morrow, 1958). They pointed out thatit makes physiological sense for sieve tubes to develop as specializedtransport channels for the movement of food into the tiny leaf beforethe primordium grows too large for mere diffusion to suffice. Theyalso found near the base of the young leaf a new locus of sieve-tubedifferentiation that is isolated initially from the acropetallydifferentiating sieve tubes in the leaf trace below the node. Theleaves with the isolated locus grow only a tiny amount more before thislocus connects with the sieve tubes in the trace below (Jacobs and Morrow, 1967). Hence, even with thenighttime collections, in which 80% of the isolated sieve-tubeloci were found, and their large samples, they did not find the lociuntil quantitative relations from a linear regression of leaf lengthagainst level of sieve-tube differentiation pointed them to theparticular leaf lengths in which the separate loci were found. It isnot surprising, therefore, that the small-sample, daytime collections ofclassical anatomists would have led them to think that the pattern offirst sieve-tube differentiation in angiosperms is continuous andacropetal into young leaves, in contrast to the isolated locus of firsttracheary cell differentiation that they had observed out in the leaf(Esau, 1953). The evidence from thePrinceton clone is much more unifying: for both differentiation in theyoung leaves of the apical bud and for regeneration in the olderinternode, the first sieve tube forms on the outer side of theprocambial/cambial strand, then a few days later and opposite thepreviously formed sieve tubes the first tracheary cell develops(Jacobs and Morrow, 1967).

The finding that successive leaf pairs are so closely related inlength that one can measure leaves 20–60 mm long and from theselengths calculate the length of leaf primordia still in the apical bud(Jacobs and Morrow, 1957) was extended to show that the lengths of leaf primordia are in turn quantitativelyrelated to the height of the apical meristem dome. They used theserelations to measure unfolded leaves in order to collect apicalmeristems of specific heights. Round-the-clock collections uncovered adiurnal rhythm of leaf initiation, with leaves most likely to beinitiated near the middle of the dark period (Figs. 3 and 4 in Jacobs and Morrow,1961).

Auxin andcompensatory growth
We have analyzed main-shoot development in terms of compensatorygrowth. Excising all axillary buds and branches causes much greatergrowth of the leaves on the main stem, an effect which could not benullified by substituting IAA for all the excised axillary buds(Jacobs and Bullwinkel, 1953). Accompanying the compensatory growth is a speeding of floraldevelopment, which by contrast can be slowed again when IAA issubstituted for the axillary buds (Jacobs,Davis, and Bullwinkel, 1959). By substituting IAA for leafblade 2, Aloni (1976a) confirmed thatIAA could inhibit flowering. Students of flowering physiology havetypically found that true long days and short days with light given inthe middle of the night affect flowering similarly. However, that isnot true for the compensatory growth caused by excising the axillarybranches of the Princeton clone: interrupted nights cause less growththan true long days. Jacobs, Davis, and Bullwinkel correlated thisunexpected difference with the discovery that leaves are most apt to beinitiated between 2300 and 0100 (Jacobs andMorrow, 1957, 1961) andsuggested that the light given then inhibited the compensatorygrowth.

In many species excising the apical bud of the main shoot increasesthe growth of some or all axillary shoots. Botanists have traditionallycalled such compensatory growth "release of the axillary branchesfrom apical dominance." IAA substituted for the apical budrestores some or all of the apical dominance in a number of species, butonly rarely has exact replacement of IAA from the shoot tipbeen attempted.

Because such quantitatively exact agreements had been found in thePrinceton clone between the effects of added IAA and endogenous"IAA" from leaf blades, whether on vascular regeneration orleaf abscission, the Princeton clone seemed an ideal organism forinvestigating IAA and apical dominance. To obtain a more completepicture of the responses to decapitation and IAA substitution, thelengths of all the axillary buds were measured [as contrasted tojust the two lowermost ones that Thimann andSkoog followed in Vicia (1934)].

Excision of the tip of the main shoot releases the younger axillarybuds of the Princeton clone from apical dominance (Fig. 4 in Jacobs et al., 1959). The extent of therelease is essentially the same as in Thimann and Skoog'sVicia, as measured by the ratio of lengths of the axillary budshowing the greatest response in the decapitated plants to controls. Substitution of 1% or 5% IAA in lanolin for the excisedapex consistently restores none of the apical dominance. Our earlierexperiments had demonstrated that 1% IAA in lanolin completelyand exactly replaces the effect of the shoot tip in speeding abscissionof debladed leaves down to at least node 4 (Figs. 7 and 8 in Jacobs, 1955). Direct assays with theAvena bioassay and a xylem regeneration assay of the amount ofauxin activity collected below node 2 confirm that IAA substituted forthe top of the shoot actually gets down the stem to the level of thereleased axillary buds (Fig. 6 and Table 3 in Jacobs et al., 1959). (A surprising findingwas that IAA substituted for the top of the shoot regularlystimulates the growth of the axillary bud that was at node 3 atthe start of the experiment. Clearing and staining the tracheary cellsof the shoot revealed that the tracheary cells in the trace to bud 3 hadalmost but not quite completely differentiated at the start of theexperiments. The authors hypothesized that IAA stimulated the growth ofthat particular axillary bud by speeding the short, final stretch of itsvascular differentiation.) So these healthy, vigorously growingColeus plants show clear apical dominance, but IAA from theshoot tip is obviously not the controlling agent, in contrast to theclassic theory based originally on legumes such asVicia.

Other factorsaffecting IAA movement in shoots
Irradiation of sections cut from internode 2 increases basipetalC-IAA transport by 176% (Koevenig and Jacobs, 1972). Blue, red, orfar-red (each monochromatic and of equal energy) gave the same increasein basipetal counts.

Most transport studies cited so far used sections cut from the middleof petioles or internodes. Such sections are anatomically uniform alongtheir length. However, other locations in the shoot support thehypothesis that barriers to IAA transport exist. The accumulation ofIAA in the abscission zone at the base of the petiole has already beenmentioned (Kaldewey and Jacobs, 1974,1975). Jacobsand Morrow (1957) hypothesized the first differentiation oftracheary cells at the two isolated loci in young leaf traces,transfixing the node and forming near the base of the young leaf, toresult because "auxin increases locally in concentration due to apiling up at a physiological barrier." They pointed out that thenode provides an obvious anatomical break from the smooth continuity ofinternodal tissue and that the first tracheary cells out in the leafform in the main vein in the region where the two larger lateral veinsempty into the main vein. Both sites they proposed as transportbarriers, where local concentrations of IAA would be expected toincrease to a level that would initiate tracheary celldifferentiation.

Long-distance transport of IAA down the stem of the Princeton clonewas demonstrated by Thompson (1966),who cut off the shoot above internode 2 and applied C-IAAto the cut surface. Some IAA moved down the stem at least as far asinternode 7 (the oldest internode examined), but progressively lessreached each successive internode (his Fig. 3). Movement to internode 7was judged by total radioactivity and to internode 5 by bothIAA and the statistically significant increase in thenumbers of sieve tubes and tracheary cells regenerated there from theaddition of IAA three internodes above. GA-3 added with the labelledIAA had no effect on either the number of vascular cells regenerated oron the amount of radioactivity reaching internode 5.

Thompson made the interesting observation that, although there was aclear-cut linear-logarithmic decrease in radioactivity recovered fromsuccessive internodes 2– 5, the decrease stopped or even reversed in internode 5, the internode where cambium normally begins to develop(his Fig. 1). In view of the extensive evidence linking IAA to cambialactivity, he suggested that "an internode shifting to secondarygrowth might attract a greater amount of basipetally movinghormone."

The first test of IAA movement through histologically defined tissuewas run by cutting cylinders with a cork borer from pith of internode 5of the Princeton clone and comparing the kinetics and polarity ofmovement with those through "corner cylinders," whichincluded all types of tissues in the internode transsection (Jacobs and McCready, 1967). The pithparenchyma moved labelled IAA with strong basipetal polarity, as did thecorner cylinders. The rate of movement, based on regressioncalculations of time intercepts and analysis of statisticalsignificance, was only slightly slower for pith cylinders. For bothcylinder types only C-IAA was in the receiveragar.

Flowering
The Princeton clone is day-neutral with respect to flowering(Jacobs, Davis, and Bullwinkel, 1959). Quantitative studies involving grafting leaves of the clone onto hostplants of C. frederici, a short-day plant, confirm that leavesof our day-neutral C. blumei clone provide ample stimulus(flowering hormone, or "florigen" in the parlance offlowering physiologists) in either short days or long days --thusconfirming for the Princeton clone the hypothesis of others that plantsare day neutral because they produce florigen irrespective ofphotoperiod and that florigen is not species specific (Jacobs, 1980). The normal inhibition offlowering in the clone by IAA from leaves was mentioned above undercompensatory growth.

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS OF ESTIMATING NATURE... HORMONE MOVEMENT AND ACTION... DISCUSSION REFERENCES

 
By using a clone, and especially by studying the IAA physiology ofadult plants growing under normal greenhouse conditions, rather than ofseedlings growing in the dark, we revealed a number of new phenomena. The main results with the Princeton clone emphasize the crucial role ofdiffusible IAA in the development of the green shoot. The amount of IAAmoving out of the leaf blades determines leaf longevity by its effecton the abscission zone at the base of the leaf. The full effect of theblades on longevity can be replaced by IAA alone that is substituted forthe blades. In the young internodes the regeneration of tracheary cellsis similarly controlled by IAA from the leaves: IAA quantitativelysubstituted for the leaves' normal supply of IAA fully replaces theleaves' controlling effect on the number of tracheary cellsregenerated (and partially replaces their effect on sieve-tuberegeneration). In older internodes, IAA fully replaces the leaves incontrolling regeneration of both sieve tubes and tracheary cells incollateral vascular strands. In strands small enough to have onlyphloem differentiated, excised internodes show full replacement of boththe distal shoot and proximal shoot and roots only when zeatin is addedalong with IAA. [Banko, Roberts, and Boe(1976) reported cytokinin-like activity in the exudate fromthe root system of a different stock of C. blumei, so in theintact plant zeatin presumably comes from the roots.] The finalstage of differentiation of phloem fibers, which occurs in olderinternodes normally under the control of distal leaves, is alsocontrolled by IAA—in this case requiring for full replacement ofthe leaves the addition of GA, which has no effect by itself.

The normal differentiation of vascular cells in young leaves, leafprimordia, and the apical bud is also controlled by IAA, from theresearch of Jacobs and Morrow (1957,1967) on the Princeton clone and ofWangermann (1967) and Bruck and Paolillo (1984a) on other stocks of C.blumei. Note that the maximal production of IAA by leaf 2, thefastest growing leaf on the plant, thus provides a second protectionagainst too much water loss to add to the well-known stomate-closingaction of ABA (e.g., Terry, Krizek, and Mirecki,1985). IAA stimulates the production of tracheary cells,which carry water to the leaves, and ABA decreases water loss if theleaf is reaching the wilting stage.

The movement of this potent plant regulatory hormone, IAA, is inturn regulated by the tissues through which it moves. Less and lessapplied IAA can move through the petioles as they age—even if anamount of IAA is applied that will replace the normal maximal amountthat moves out of young leaf 2. The excess IAA entering the olderpetiole sections is conjugated into IAAspartate within the sections. This increasing conjugation with age acts as the putative control valvefor IAA movement out of the leaves. In the young internode, basipetalmovement of IAA saturates when 5 mg/L are appplied: thisconcentration provides IAA through the internode at a level onlyslightly above the normal production by the distal leaves. Hence, adouble control exists on the amount of IAA moving through petioles andyoung internodes: first, the amount of diffusible IAA produced by theleaves and, secondly, the transport saturation levels. From the patternof vascular differentiation in the apical bud, nodes and vein junctionshave been proposed as IAA transport barriers (Jacobs and Morrow, 1957). Bruck and Paolillo (1984b) made a similarproposal about the nodes.

Two other hormones have major effects on the movement of IAA in thisclone. Abscisic acid speeds the abscission of petioles by keeping IAAin the petiole and away from the abscission zone, with concomitantconjugation of IAA with aspartic acid: increasing release of ABA inprogressively ageing petioles is the presumed basis for the decliningtransport of IAA through progressively older petioles. Pretreatmentwith GA also speeds petiolar abscission and increases petiolarelongation by keeping more IAA in the petiole and away from theabscission zone. (The GA effect is limited to young organs, likeseveral GA effects reported in other systems.) Kinetin, the onlycytokinin that has been tested for effects on IAA movement in thePrinceton clone, increases basipetal IAA movement through petioles.

In comparison with the other hormones that have been tested inColeus, the crucial role of IAA is correlated with both itsbasipetally polar transport and its greater velocity of movement. Bothcharacteristics are favorable attributes for a major developmentalcoordinator. (The slower movement of GA and ABA than of IAA is the mostlikely reason that pretreatment with them is more effective thansimultaneous treatment with IAA: the ABA or GA needs to get down intothe petiole before the faster moving IAA overtakes them.)

The combined anatomical-physiological approach has been especiallyuseful for some developmental problems, in our case for studies of theregeneration and differentiation of vascular tissues. We have foundhigh correlations between leaf size and the extent of vasculardifferentiation and between the latter and IAA levels. Researchers haveconfirmed in several genera the close relation between the size of leafprimordia and the intial stages of vascular differentiation (e.g.,Jacobs and Raghavan, 1962; Swift and O'Brien, 1971; Larson, 1975; Colemanand Greyson, 1976; Bruck and Paolillo,1984a).

Our use of large samples and round-the-clock collections uncoveredpreviously unknown phenomena of vascular differentiation and leafinitiation. The earliest differentiation of tracheary cells was foundonly in nighttime collections and most cases of isolated loci ofsieve-tube differentiation were also from night collections. Bruck and Paolillo (1984c) confirmed in theirstock of C. blumei that a majority of the cases of isolatedloci of tracheary cells at nodes are found in nighttime collections.Leaves are preferentially initiated near the middle of the night(Jacobs and Morrow, 1961), a findingconfirmed for Xanthium (Jacobs,1992). From such results it seems eminently sensible to urgemore widespread use of round-the-clock sampling—particularly sincethe production of several plant growth substances has been shown tofollow diurnal cycles with maxima at night (e.g., Yin, 1941; Krekule et al.,1985; Gocal et al., 1991;Lopez-Carbonell et al., 1992). Hence, there is every reason to expect other developmental phenomena tobe night related, too.

Our use of quantitative analysis, including testing of thestatistical significance of differences and linear regression analysis,has been fruitful, also. For instance, the linear regression of leaflength on number of tracheary cells in the petiole helped developevidence that the relative rate of production of IAA and tracheary cellswas the same for each leaf size from leaf 1 to leaf 4. A regression ofleaf length on the height to which sieve tubes differentiate in theleaf's procambial strand alerted us to the gap in normal sieve-tubedifferentiation in young leaf primordia—and thereby to thediscovery of the separate, isolated locus of sieve-tube differentiationin the base of the young leaf. Similarly, regression analysis ofhormone movement made clear that the velocity of IAA movement inpetioles of various ages was unchanging and was greater than that of GAor cytokinins: only the slope ("intensity") of the linearrelation between time and the amount of IAA moved through petiolesdeclined with age (Fig. 3). And when ABA decreased IAA movement through petioles, it also changedthe intensity not the velocity.

In attempting to unravel the contribution of a hormone or hormones toa given aspect of development we have striven to measure the normalamount of the hormone produced by an organ and then to substituteexactly that amount of the hormone for the excised organ. Ifsuch exact substitution replaces exactly the full effect of the excisedorgan, as IAA does for leaf abscission and vascular regeneration, onehas unusually strong evidence for the normal controlling role of thathormone. If one does not get full replacement of effect by such exactsubstitution of a single hormone, one can look for interactions withother hormones, such as the need for the addition of GA to IAA for fullrestoraton of phloem fiber differentiation (Aloni,1979) or of zeatin for full restoration of sieve tubes inphloem-only strands (Houck and LaMotte,1977). Exact substitution is particularly valuable in cases ofapparent inhibition, because an inhibitory effect is apt to be much lessspecific than a stimulatory one, as Jacobs etal. (1959) pointed out. In apical dominance, for example, wecould demonstrate by exact replacement that IAA was not the controllingagent in our plants. [Confirmation of our results on apicaldominance has come from studies of other clones and species (Thimann, Sachs, and Mathur, 1971; Cline, 1996), although exact replacement of thenormal IAA supply was not demonstrated in these cases.]

How generally do the conclusions based on our clone of Coleusblumei apply to a larger group of species? The critical role ofIAA in controlling tracheary cell differentiation and regeneration inColeus has been confirmed in a very wide range of plantspecies, including many angiosperms, some gymnosperms, and even someferns [cf. reviews in Roberts, Gahan, andAloni (1988) and Fukuda(1992); Steeves and Briggs,(1960) for fern experiments). Auxins added to plants inhibitabscission, as has been known since Laibach's (1933) report, and it is part ofthe standard practice in controlling the timing of "fruitdrop" in several tree crops (cf. Weaver,1972). However, with the partial exception ofPhaseolus and cotton, I know of no plants other thanColeus where the normal relation of IAA to ageing andabscission has been so thoroughly investigated. The recentdemonstration by GC-MS of IAA in Coleus shoots fits whatothers have reported from a wide range of genera and plant groups, evenincluding several algae (Jacobs,1986). An exception has been reported from legumes, where4Cl-IAA was identified in extracts (Marumo etal., 1968; Pless et al.,1984). The presence of the highly active 4Cl-IAA in legumesshows the need to re-examine apical dominance and other auxin effects inlegumes, where it has been silently assumed that IAA was the endogenousauxin. More recently, indole-butyric acid, long used as an exogenousstimulator of rooting on stem cuttings, has been identified by GC-MS asan endogenous auxin in several angiosperms (e.g., Schneider, Kazakoff, and Wightman, 1985;Epstein, Chen, and Cohen, 1989).

In summary, mature plants, growing in normal light–darkconditions, show complexities of hormonal transport and action onewould not suspect from studies of dark-grown coleoptiles of grassseedlings, valuable though such studies of seedlings have been, andcontinue to be, in understanding the movement of the "growthhormone" IAA.

	FOOTNOTES

 
¹ The author thanks Prof. Darleen A. DeMason for her kindness and generosity in offering to comment on an early draft of this paper, the anonymous reviewers, and Editor Karl Niklas for his patience and advice.

² Current address: 64 Maclean Circle, Princeton, NJ 08540.

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