maturation in larch1

Plant Physiol. (1989) 90, 406-4120032-0889/89/90/0406/07/$01 .00/0

Received for publication May 23, 1988and in revised form January 11, 1989

Maturation in Larch1

1. Effect of Age on Shoot Growth, Foliar Characteristics, and DNA Methylation

Michael S. Greenwood*, Catherine A. Hopper, and Keith W. HutchisonDepartment of Forest Biology (M.S.G.) and Department of Biochemistry, University of Maine,

Orono, Maine 04469 (C.A.H., K.W.H.)

ABSTRACT

The time course of maturation in eastern larch (Larix laricina[Du Roil K. Koch) was examined by grafting scions from trees ofdifferent ages onto 2-year-old root stock and following sciondevelopment for several years. Height, diameter, foliar chlorophyllcontent, and rooting ability of scion-derived cuttings all variedlinearly as a function of log10 age. Chlorophyll content (milligramsper gram of dry weight) increased while height, diameter, andability to root decreased with age (P < 0.01). The tendency towardorthotropic growth and branch formation per centimeter of mainstem decreased abruptly between age 1 and 5 years (P < 0.01).Total chlorophyll content of both long and short shoot foliageincreased by 30 to 50% with increasing age, but the chlorophylla/b ratio did not change. Also, juvenile long shoot needles weresignificantly longer than mature (P < 0.01). Surprisingly, thejuvenile scions produced more total strobili over two successiveyears, but the mature scions produced a significantly higherproportion of male strobili (P < 0.001 year 1; P < 0.02 year 2).The age-related changes in foliar traits were not associated withchanges in DNA methylation between juvenile and mature scions.Using HPLC, we found that 20% of foliar DNA cytosine residueswere methylated in both scion types.

Maturation, or phase change, in woody plants refers to anongoing process which results in relatively permanent devel-opmental changes involving decreased growth rate, increasedfrequency of reproductive differentiation, and changes in anumber of morphological characteristics. The phenomenonofmaturation in woody plants, with emphasis on plant growthregulators in the process, has been recently and thoroughlyreviewed (4, 15, 30). Most of the work on maturation hasbeen descriptive, and comprehensive experimental studies onwoody species are rare. Nonetheless, maturation is not onlyan interesting developmental phenomenon, but a major con-cern to those wishing to use tissue culture for propagation or

application of biotechnological methods to forest trees. Thereis an almost total lack of methods to regenerate plants fromtissues of coniferous trees once they have passed the embry-onic or seedling stage (13, 18). Besides the difficulty in regen-erating mature individuals by tissue culture, several recent

' Supported by U.S. Department of Agriculture Competitive grantNo. 87-FSTY-9-0237 to M.S.G. and K.W.H. Maine AgriculturalExperiment Station Miscellaneous Publication No. 1338.

observations that tissue culture plantlets derived from embry-onic tissue of pines behave like mature trees have beenreported (13, 19). Furthermore, without reversal of matura-tion, clonal propagules (if obtainable) from mature trees willnot exhibit the period of rapid growth associated with thejuvenile phase. Thus, juvenile behavior of seedling explantsmay be easily lost, but mature characteristics appear quitepersistent. An ability to manipulate the mature phase willrequire new knowledge about its developmental basis. Hackett(15) evaluates past evidence as to whether the mature phaseis determined at the level of the individual cell or the entireapical meristem, or is due to correlative effects involving thewhole plant, and concludes that there is equivocal evidencein support of all three hypotheses.Mature characteristics in woody plants can be observed

directly on plants of different ages (2) and usually persist ongrafted scions or rooted cuttings (12, 14, 25). Some changesin developmental behavior associated with age are a functionof the increased size and complexity of the tree, and are lostfollowing grafting. For example, the growth rate of scionsfrom older trees grafted onto young rootstock appears to bereinvigorated to some extent, and flowering is suppressed.Wareing (27) refers to such reversible changes as 'aging,' incontrast to the relatively more permanent changes due tomaturation. Unfortunately, experimental demonstrations ofthe time course of maturational changes in woody plants arealmost totally lacking (13). We report herein results from twoexperiments designed to: (a) demonstrate the juvenile andmature characteristics of eastern larch, and (b) determine thetime course ofsome ofthese maturational changes. This workis part of an ongoing study to determine the role, if any, ofgene expression in the maturation process.

MATERIALS AND METHODS

Plant Material

Experiment 1

Scions were taken in March 1985 from vigorously growingbranches in the upper one-third of the crown of four sexuallymature larch trees (from a local natural stand) ranging 13 to25 years in age on a site in Medford, ME. Simultaneously,scions were taken from the main stem of 1-year-old seedlingsgrown from open pollinated progeny of the first four trees. Inorder to standardize scion diameter and minimize possible

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MATURATION IN LARCH

topophytic effects, the scions were pruned down to 20 cmstem segments, and terminal buds were always removed.Thus, all shoots emerging after grafting arose from lateralbuds. All scions were side grafted onto uniform 2-year-oldlarch potted rootstock (grown from open pollinated seed in2:1:1 peat:vermiculite:sand, fertilized with 9 month Osmocote18-7-10, a timed release fertilizer) in March 1985. Twelvegrafts were made from each mature tree as were 12 from itsseedlings, for a total of 96 grafts. All surviving grafted scionswere measured (see below) after 1 year. Five grafts from eachmature tree, as well as five from its seedling progeny, wererandomly chosen for longer term experiments. These 40grafted scions have been kept in a temperature-controlledglasshouse, without supplemental lighting, with temperaturesmaintained at 4°C or above October through March, andgenerally below 25°C the rest of the year. Male and femalestrobilus buds were counted after the 1987 and 1988 growingseasons and the means were compared using ANOVA.

Experiment 2

Scions were taken in March 1986 (as described above) fromtrees ranging from 1 to 74 years in age. Except for 1-year-oldmaterial, all scions were taken from a small natural stand inBingham, ME, which was probably seeded in from several ofthe oldest trees in the stand. Scions were taken from 27 treesranging 3 to 74 years in age, but there were three distinct ageclasses represented in the stand, 3 to 7 years (X = 5 years),16 to 19 years (X = 17 years), and 33 to 74 years (X = 45years). Since no 1-year-old seedlings could be found in thefield, scions of this age were taken from a container-grownseedling population of five half-sibling families from openpollinated mature trees. Five to six grafts were made fromeach of the 27 trees. The surviving grafts were divided intotwo replications of 40 trees each, with 10 trees in each of fourage classes per replication. Within each age class, geneticrelatedness was minimized, with five to seven different clonesor families represented.

Measurements

Height (from the graft union) and diameter (just above thegraft union) measurements were taken at the end of eachgrowing season. In addition, the primary branches werecounted on the main stem. All grafts were visually scored fororthotropic versus plagiotropic growth after the first growingseason. Scions whose leaders were growing close to verticalwere considered orthotropic, while scions growing horizon-tally or about 300 or more from vertical were calledplagiotropic.

DNA Methylation

We compared the amount of methylcytosine in the DNAofjuvenile and mature foliage since methylation ofDNA mayaffect gene expression (21) and changes in DNA methylationhave been proposed as a basis for woody plant maturation(3). DNA was extracted from 5 to 10 g of fully expandedneedles (collected in September) by a modification of themethod described by Murray and Thompson (20). Ten g of

fresh needles were frozen in liquid N2 and ground in a Brauncoffee grinder for 5 s. The ground tissue was then added to200 mL of ice-cold 50 mM Tris (pH 8.0), 5 mM EDTA, 350mM sorbitol, 0.1% BSA, 0.1% ,B-mercaptoethanol, and 10%polyethylene glycol 8000. It was then homogenized in aBrinkman Polytron for 5 to 10 s at a setting of 4. Thehomogenate was filtered through several layers of cheeseclothand one layer of Miracloth and pelleted by centrifugation at2,500g, for 5 min, at 4°C. The pellet ofnuclei was resuspendedin 20 mL of 50 mm Tris (pH 8.0), 5 mM EDTA, 350 mMsorbitol, and 0.1% f-mercaptoethanol. Sodium sarkosyl wasadded to a final concentration of 1% and the nuclei incubatedat least 30 min at room temperature. The solution was thenmade to 710 mm NaCl, 0.1I% CTAB and incubated for 10min at 60°C. It was then extracted with 10 mL of chloroform-isoamyl alcohol (24:1), and the layers were separated bycentrifugation at 1 3,000g), for 15 min, at 1 5°C. The aqueouslayer was removed and the DNA precipitated by the additionof two-thirds volume of ice-cold isopropanol and placing at-20C for 10 min. The DNA was recovered with a glass hook,washed with 20 mL of 76% ethanol and 10 mM ammoniumacetate at room temperature, and redissolved in 10 mM Tris(pH 8.0), 1 mm EDTA. Any remaining RNA contaminationwas removed by digestion with RNase (20 ,g/mL) for 30min, at 37°C, followed by phenol extraction and precipitationwith ethanol.

Methylation was estimated by both restriction enzymeanalysis (results not shown) and HPLC after Singer et al. (23).Prior to HPLC, 15 ug ofDNA was hydrolyzed for 20 min at180°C in 88% formic acid, evaporated to dryness (under anN2 stream), and the residue redissolved in 25 ,uL of 0.1 MHCl. Ten uL of hydrolysate was injected into a Waters U6Kinjection port and isocratically eluted from a 25 cm Partisil10 SCX column (Whatman) using 0.1 M ammonium phos-phate buffer adjusted to pH 2.4 with 85% phosphoric acid ata pressure of 750 p.s.i., using a Waters 6000A solvent deliverysystem at a flow rate of 1 mL min-'. Bases were detectedusing a Lambda-Max Model 480LC spectrophotometer (at280 nm) combined with a Hewlett Packard 3392A integrator.

Chi Content

Total Chl was extracted either by grinding tissue in 80%acetone (1) or placing needle segments in DMSO at 65°C for5 h (16). Extinction coefficients were calculated for bothsolvents using the equations of Amon (1), based on spectraobtained (using a Milton Roy Spectronic 1201 spectropho-tometer) from known amounts of Chl a and b from spinach(Fisher Scientific). When tissues with widely varying Chlcontents were extracted using both methods, the results wereclosely correlated (r = 0.93), but DMSO consistently extractedmore Chl, with less variation between samples. In addition,the DMSO method is much less labor intensive, and was thusused for comparisons of relative Chl contents between indi-viduals in different age classes when large sample sizes wererequired. However, in experiment 1, Chl was extracted usingthe acetone method from mature long shoot foliage taken inJuly. In experiment 2, foliar samples were taken in April,May, and September, using the DMSO method.

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Plant Physiol. Vol. 90,1989

Statistical Analysis

The effect of age on graft development was evaluated byANOVA. Age class was considered a fixed effect, while clone,family, or replication was considered a random effect. Theerror mean square was used to test all main effects unlessgenetic or replication effects were significant, in which casethe interaction term was used to test the effect of age. Allstatistical analyses were performed using SAS (SAS InstituteInc., Box 8000, Cary, NC 27511-8000). The effect of age onfrequency of orthotropism by age class was tested using a x2test for unequal sample sizes (24).

RESULTS

Sources of Variation

The experiments described here are designed to determinethe effect of developmental state of the donor tree (whichshould be mainly a function of age) on the developmentalbehavior of their scions after grafting onto seedling rootstock.Potential sources of variation that could have obscured age-related differences are the effect ofage on grafting success, thegenetic variability of both the donor trees and the rootstock,and the vigor and quality of the scions collected. Graftingsuccess in these experiments has been uniformly high, withfailure probably due to poor scion quality. In the first exper-iment, 75% of the grafts made from 1-year-old donors sur-vived, compared with 94% for 13- to 25-year-old donors. Inthe second experiment, graft survival ranged from 91 to 100%across the four age classes. Despite all sources of variation,ANOVA indicated that age was the only main effect in bothexperiments with a significant impact on the traits measured.Genetic effects were insignificant in experiment 1 becausereplication within family or clone was small (n = 5) and thereis considerable between-tree variation within families andclones probably due in part to genetic variation in the root-stock, which came from a wide variety of genotypes.

Effect of Age on Scion Development

The results ofexperiment 1, comparing the growth ofscionsfrom four trees averaging 19 years old (mature) with scionsof 1-year-old open pollinated progeny of the same trees (ju-venile), are shown in Table I and Figure 1. Although thejuvenile scions exhibited more height growth during the firstgrowing season (60 versus 48 cm, difference significant at P< 0.01), the mature scions caught up after the second growingseason. However, the juvenile scions produced more thantwice as many lateral branches during the second growingseason (Table I; Fig. 1). Therefore, the juvenile scions haveproduced more total shoot growth (combined terminal andlateral long shoots) in addition to more diameter growth.Most (79%) of the mature scions grew plagiotropically, com-pared with only 7% for the juvenile scions. The mature foliagewas noticeably greener than the juvenile foliage in late Julyand exhibited more total Chl (Table I). In addition, long shootneedles on juvenile grafts were longer than those on maturegrafts (4.3 versus 3.5 cm, significant at P < 0.0001), but theirwidth and thickness were similar. The total estimated foliarsurface area (surface area = total branch ln (cm) x No.

needles-cm-' x surface area-needle-') for the juvenile graftsaveraged more than twice that for the mature grafts. A com-parison of rooting by cuttings taken from the grafted scionsin July of the first growing season shows that the cuttingsderived from the mature scions rooted only about half as wellas the juvenile. The methylcytosine content of foliar DNAdid not differ at all betweenjuvenile and mature foliage overallor by family (see Table I). In addition, we did not observeany differences in overall DNA fragment size following diges-tion with methylation sensitive and insensitive restrictionenzymes (data not shown).The scions from juvenile and mature trees in experiment 1

produced very few strobili until 3 years after grafting. Butafter the 1987 and 1988 growing seasons, 60 and 100% of thescions flowered; results by family are shown in Table II. Thejuvenile scions produced more strobili of both sexes in bothyears, but the difference was only significant the first year (P< 0.01 for year 1, P < 0.58 for year 2). But in both years thejuvenile scions produced a significantly higher proportion offemale strobili, whereas the mature scions produced about 4times as many males as females in both years. The effect ofage on sex ratio is highly significant (P < 0.001 for year 1, P< 0.02 for year 2).The purpose of experiment 2 was to demonstrate the time

course of the maturational changes demonstrated by experi-ment 1. The time courses, as a function of the log1o of age,are shown in Figure 2. Height, diameter, Chl content androoting vary linearly with the log10 of age, while orthotropismand branch frequency decrease abruptly, then stabilize. Theeffect ofage on height, diameter, branches. cm-', rooting, andChl content are significant at P< 0.001 according to ANOVA.The grafted scions from 1-year-old trees exhibited significantlymore orthotropic growth than the older scions (x2, p< 0.001).The effect of age on Chl content is not apparent until well

after the needles are fully expanded (Table III). In September,age-related differences in the Chl content of both long andshort shoot foliage are similar. Short shoot foliar clustersemerge first, since the needles are preformed the previousyear, and are borne on lateral shoots without internode elon-gation. Long shoot foliage is borne along the elongating axisof the terminal long shoots and are separated by internodes.Most long shoot needles are not preformed the previous year.Overall, long shoot needles are longer than those of shortshoots (see table III), but unlike short shoots, their averagelength appears to decrease with increasing age.

DISCUSSION

We have assessed maturational changes in shoot activity inEastern larch by grafting similar sized scions from trees ofdifferent ages onto 2-year-old rootstock. Similar results canbe obtained with rooted cuttings, but grafted scions are pref-erable because they generally grow relatively faster than rootedcuttings or seedlings (7), but most importantly grafting successdoes not decline with increasing age of the scion donor. Weobserved no evidence of graft incompatibility, nor evidenceof overgrowth of the rootstock by the scion. While expressionof plagiotropism by mature Douglas fir scions can be avoidedby grafting onto relatively large rootstock (4 year old), theplagiotropic response by all but the 1 year scions in this study

408 GREENWOOD ET AL.



MATURATION IN LARCH

Table I. Shoot Growth and Foliar Characteristics of Grafted Juvenile (1y) and Mature (16-23 year old) Scions after Two Growing SeasonsMature scions were taken from four different trees and juvenile scions were taken from the open pollinated families of these trees and grafted

onto seedling rootstock. Heights, diameters, and branch counts are means of five trees from each family (juv, mat) combination. Ten to 12cuttings were taken from each family (juv, mat) combination for evaluation of rooting. Foliar samples (long shoots) from several trees in eachfamily (juv, mat) combination were taken and pooled for Chl (acetone method) and % methylcytosine determinations.

Scion Scion Number of Long Shoot Methylcytosine Rooting GrongFamily Height Diameter' Branches" Chlorophyll (foliar DNA) Abiliy Plagiotropicalln

Plagiotropicallya

Juv Mat Juv Mat Juv Mat Juv Mat Juv Mat Juv Mat Juv Matcm min cm-' mg-g dry wt-h % % %

1 178 164 21 14 0.34 0.17 4.0 8.3 21 21 100 82 0 642 160 153 22 15 0.35 0.21 4.7 7.4 18 17 100 70 8 803 164 172 20 15 0.35 0.13 5.3 6.5 25 23 100 18 20 754 161 158 23 17 0.42 0.18 6.0 6.9 16 20 100 50 0 91X 166 162 22 15 0.37 0.17 5.0 7.3 20 20 100 55 7 78

±SE ±4 ±4 ±0.5 ±0.5 ±0.02 ±0.02 ±0.5 ±0.8 ±1.8 ±1.3 ±0 ±14 ±10 ±11a Juvenile means differ from mature at P 6 0.05.

J \:XM

AA I/

Figure 1. Two-year-old grafted scions from a mature larch tree (right)and from a juvenile (1 -year-old) tree (left) grown from seed collectedfrom the same tree. Pole graduated in 10 cm increments. Graft unionis about 40 cm above pot level. Note high branch density of juvenilegraft, plagiotropic growth of mature graft.

were very similar to that reported by Copes (8) using 1- to 2-year-old rootstock. Since the scions from mature trees weretaken (by a professional tree climber) from the upper 25% ofthe live crown of large trees, direct measurement of thedevelopment of similar branches in situ was not practical.Based on observation, the grafted scions grew much morethan their counterparts on the original ortet, which is usuallythe case following grafting onto vigorous, well fertilized green-house-grown rootstock (7). This apparent revitalization oc-curred on all age classes and cannot be considered rejuvena-tion, since age-related developmental differences persistedlong after grafting. Instead, the reinvigoration (of the olderscions) reverses the effects of limited nutrients and/or theincreased size and complexity of the tree. Wareing (27) refersto the latter effect as aging. The seedling scions also grewmore than their intact counterparts, since the rootstock pro-vided a root system considerably larger than the one theyoriginated from (see also ref. 7).The mature characteristics of eastern larch are very similar

to those described for many other woody species (12, 15). Inparticular, the maturation patterns for vegetative shoot growthcharacteristics appear to be closely similar for larch, loblollypine, and radiata pine (12, 14, 25). Reduced height anddiameter growth, reduced rooting, decreased branch fre-quency, a tendency toward plagiotropic growth, changes inbranch habit and foliar morphology are all associated withmaturation in all three species. However, to our knowledge,this is the first report that Chl content increases during mat-uration, although the juvenile foliage of both loblolly andradiata pine appear more yellow than mature (unpublishedKodachrome slides, MS Greenwood).The maturation-related increase in Chl content is substan-

tial (between 33 and 50% between age classes 1 and 4, TableIII). If Chl content is expressed per unit of surface area, asimilar trend across age classes is evident. Also, the Chl a/bratio (about 4) does not vary with scion age or throughout theseason. The external dimensions of both juvenile and maturefoliage appear to be similar, although long shoot needle lengthdecreases with age. The foliar surface area of the juvenile

409



Plant Physiol. Vol. 90, 1989

Table II. Male, Female Flowering per Scion and Sex Ratio (Male/Female) on Two Successive Years,for Each of the Four Families Used in Experiment 1

There were a total of 3 grafted scions in each family juvenile-mature combination, for a total of 24trees. The between family differences for total strobilus production between years were not significantat P < 0.05 in either year. The juvenile scions produced significantly (P < 0.01) more total strobili inyear 1, but not in year 2 (P < 0.58). The sex ratios between juvenile and mature scions were differentin both years (P < 0.001 year 1, P < 0.02 year 2).

Juvenile MatureYear Family No.

Male Female Sex Ratio Male Female Sex Ratio

1 1 5 80 0.06 0 0 0.002 4 47 0.09 7 1 7.003 8 152 0.05 61 23 2.704 0 35 0.00 11 2 5.50X 4 79 0.05 20 7 3.80

±SE 2 26 0.02 14 6 1.60

2 1 160 249 0.64 313 35 8.942 424 350 1.21 521 124 4.203 316 576 0.55 376 189 2.004 39 50 0.78 138 79 1.75

235 306 0.80 337 107 4.22±SE 85 109 0.15 79 33 1.67

120 _2

E1291001

86160 c .2S

40

20 -

'87 7.0

Figues. Tmcorefochneinttlsinegt,diaee

E

.c~~~~~:6.0 '87

lo' 986'(Nov) 0

E 6- 05.

20-

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1 17 45 1 17 45

Log10Age,Y LogloAge,Y

Figure 2. Time courses for changes in total scion height, diameter

growth, orthotropism (the fraction of scions that grew upright), total

number of branches cm-' of stem length, Chi content of long shoot

foliage, and rooting ability of cuttings derived from grafted scions as

a function of increasing age. The means of four age classes are

plotted versus the logio age.

grafts in experiment 1 was more than twice that for the maturegrafts, due mainly to significantly greater height growth andmore branches cm-'.The effect of maturation on the reproductive development

of larch scions grafted at different ages differs with that ofloblolly and radiata pine. Grafted scions of the latter speciesshow increased reproductive competence with increasing age(for both male and female strobili) soon after grafting, whilethe opposite results are reported here for larch. Fowler (1 1)notes that eastern larch flowers precociously relative to othersympatric conifers. Our results suggest that achievement ofminimum size may be more important than maturation statefor flowering by larch. Loblolly pine is not considered aprecocious species (9), and although juvenile scions of thisspecies also grow more and produce more branches per unitstem length, at first they flower significantly less than maturescions (12). However, after a number of years, grafts fromjuvenile scions flower more than those from more maturescions of loblolly pine (26). Mature scions of both larch andloblolly pine produce relatively more male strobili. As matu-ration progresses in conifers, female strobili usually appearfirst (22). In black spruce, the ratio of male to female strobiliincreases with advancing age (5).

Intact, full-grown trees may not produce 'mature' charac-teristics uniformly. Instead, maturation seems to increasetoward the top of the tree; for example, rooting ability bycuttings declines significantly with increasing height of thecrown in 29- to 65-year-old western hemlock (10). For thisreason we always took scions from the upper quarter of thelive crown. The maturational changes followed in this studyexhibited two distinct time courses with increasing scion agewhen plotted as the log1o of age. The tendency toward ortho-tropic growth and the number of branches-cm-' both de-creased abruptly between ages 1 and 5 years, but showed littlechange after that. In contrast, height, diameter, Chl content,




MATURATION IN LARCH

Table l1l. Chl Content (DMSO method), Needle Length for Short, Long Shoots of Larch from Four AgeClasses in April, May, and September, (Experiment 2)

Different letters in columns indicate means differ at P < 0.05. ANOVA showed the effect of age classon Chl content was significant at P < 0.001 in May and September. Only long shoot needle lengthvaries with age class (P < 0.01). Effects of replication were not significant (P > 0.05).

of Needles Needle LengthScion

(Age Class) Short shoots Long shoots Short shoots Long shoots

Apr May Sept Sept Sept Sept

mg-g9' dry wt cm

1 (1 year) 3.2 3.6a 5.2a 4.6a 2.8 6.5a2 (5 year) 3.2 4.3b 6.7b 5.6b 3.0 6.3a3 (17 year) 3.5 4.8bc 6.4b 6.Ob 2.8 5.3b4 (45 year) 3.1 5.1c 7.Oc 6.8c 2.8 5.Ob

and rooting changed steadily with increasing age. One possibleexplanation for the more gradual change exhibited by thosecharacteristics which varied linearly as the log10 age is thatpartial rejuvenation following grafting occurred for thesetraits, especially in age class two and three. However, nonlogplots of all these parameters show that the rate of change ismost rapid between ages 1 and 5 years. Thus, although thereappear to be two distinct types of time courses (Fig. 2), thequestion remains whether or not the maturational changesdescribed here vary independently of one another.

Bochert (2) questions the existence of a 'uniform juvenilestate' in woody plants and suggests thatjuvenile characteristicsvary independently of one another. In our opinion, the simi-larity in curve shape for the maturational time courses ofsuchdiverse traits as rooting, Chl content, or diameter growth donot necessarily support this concept. An alternative hypothe-sis, that a single process may affect at least groups of traitsduring maturation, needs to be given serious consideration.The observation that the mature characteristics described

here persist on grafted scions as well as cuttings rooted fromthem indicates that continued expression of the mature phe-notype by the scion is unaffected by changed inputs, followinggrafting, of nutrients or plant growth regulators from theseedling root system. While Wareing (28) proposes that root-produced plant growth regulators affect maturation, their rolein the process is confusing. For example, BA has been impli-cated in both the promotion and reversal of maturation inconifers, and the GAs promote flowering in conifers butreverse the mature phase of English ivy (see reviews in refs.13, 30). Although the log-linear response to increasing agemimics the dose-response to most plant growth regulators,there is little evidence that the latter play a primary role incontrolling maturation. While gibberellin promotes floweringin many conifers, there is ample evidence that their applica-tion cannot offset a genetic indisposition of individual treesto flower (6, 29). The control mechanisms for flowering, andpossibly maturation as well, are probably the result of theproducts or regulatory activities of many different genes.Some of the genes may affect the levels of plant growthregulators; others may affect the ability ofthe plant to respondto them, while still others may affect flowering by mechanismsthat do not involve plant growth regulators at all. Thus,

hypotheses that either flowering or maturation are controlledby plant growth regulator levels alone overlooks the potentialrole of tissue responsiveness, in addition to other factors, asyet uncharacterized.

Bolstad and Libby (3) suggest that maturation in trees mightbe associated with varying levels of DNA methylation, sinceincreased levels of methylation are usually associated withdecreased gene expression at the transcriptional level (21). Weobserve here that there is no apparent difference in DNAmethylation between total DNA extracted from the foliage ofjuvenile and mature scions. The methods we used, however,would not detect methylation of only one or a few genes.Preliminary analysis of cDNA libraries made from poly(A+)RNA (mRNA) from expanding shoots ofjuvenile and matureplants suggest there may be some differences in gene expres-sion (17). Although gene expression differences, if any, be-tween the juvenile and mature states will be subtle, we dohave evidence that the Chl a/b binding protein gene is differ-entially expressed in juvenile and mature material. We arecurrently working to further define this observation.

ACKNOWLEDGMENTS

We thank Jill Weber, Mary Olien, Throstur Eysteinsson, and PattySinger for their technical help, and Mary Lou Hodge for typing andediting the manuscript.

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2. Bochert R (1976) The concept ofjuvenility in woody plants. ActaHortic 56: 21-33

3. Bolstad PV, Libby WJ (1982) Comparison of radiata pine cut-tings of hedge and tree-form origin after 7 growing seasons.Silvae Genet 31: 9-13

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Plant Physiol. Vol. 90,1989

7. Copes DL (1976) Comparative leader growth ofDouglas fir grafts,cuttings and seedlings. Tree Planter's Notes 27: 13-16

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13. Greenwood MS (1987) Rejuvenation offorest trees. Plant GrowthRegul 6: 1-12

14. Greenwood MS, Nussbaum ES (1981) Rooting ability and veg-etative growth performance of stem cuttings from one- andfive-year-old ortets of loblolly pine. Proceedings of the 16thSouthern Forest Tree Improvement Conference, Blacksburg,VA, May 26-29, pp 176-183

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