Maturation in Larch - NCBI

Plant Physiol. (1990) 94, 1308-131 5 0032-0889/90/94/1 308/08/$01 .00/0

Received for publication March 28, 1990 Accepted July 17, 1990

Maturation in Larch' II.

Effects of Age on Photosynthesis and Gene Expression in Developing Foliage

Keith W. Hutchison*, Christopher D. Sherman2, Jill Weber, Sandra Schiller Smith, Patricia B. Singer, and Michael S. Greenwood Department of Biochemistry (K.W.H., S.S.S., P.B.S) and Department of Forest Biology (C.D.S., J.W., M.S.G.), University of Maine, Orono, Maine 04469 ABSTRACT The effect of maturation on the morphological and photosynthetic characteristics, as well as the expression of two genes involved in photosynthesis in the developing, current year foliage of Eastern larch (Larix laricina [Du Roi]) is described. These effects were observed on foliage during the third growing season after grafting of scions from trees of different ages onto 2 year old rootstock. Specific leaf weight (gram dry weight per square meter), leaf cross-sectional area (per square millimeter), and chlorophyll content (milligram per gram dry weight) all increase with increasing age in long shoot foliage from both indoor- and outdoor-grown trees. Net photosynthesis (NPS) (mole of CO2 per square millimeter per second) increases with age on indoor- but not outdoor-grown trees. NPS also increases with increased chlorophyll content, but outdoor-grown scions of all ages had higher chlorophyll content, and chlorophyll does not appear to be limiting for NPS outdoors. To extend these studies of maturationrelated differences in foliar morphology and physiology to the molecular genetic level, sequences were cloned from the cab and rbcS gene families of larch. Both cab and rbcS gene families are expressed in foliage but not in roots, and they are expressed in light-grown seedlings of larch but only at very low levels in dark-grown seedlings (-2% of light-grown seedlings). Steadystate cab mRNA levels are relatively higher (-40%) in newly expanding short shoot foliage from juvenile plants compared to mature plants. Unlike cab, the expression of the rbcS gene family did not seem to vary with age. These data show that the maturation-related changes in morphological and physiological phenotypes are associated with changes in gene expression. No causal relationship has been established, however. Indeed, we conclude that the faster growth of juvenile scions reported previously (MS Greenwood, CA Hopper, KW Hutchison [1989] Plant Physiol 90: 406-412) is not due to increased NPS or cab expression. Long shoot foliage is the dominant foliar type on young trees and its lower specific leaf weight will permit production of more photosynthetic surface area per unit of leaf biomass.

generally associated with changes in vegetative growth characteristics and the onset of flowering. In a wide variety of conifers and other woody plants, foliar characteristics also change in association with the onset of maturation (18). Changes in needle form, dimensions, pigment (Chl and anthocyanin) content, surface waxes, and CO2 fixation have all been reported in the conifers Cupressus, Pinus, Picea, and Larix, and in English ivy (Hedera helix) (2, 16, 17, 28). Following germination, conifers generally exhibit indeterminate growth resulting in acicular needles throughout most of the first growing season. In many cases these simple needles are considerably smaller and distinctly different from the foliage produced after the first bud is set. However, the postjuvenile foliage that forms thereafter also exhibits gradual change with increasing age. For example, the fascicular needles of loblolly pine are thicker and slightly longer with increasing age ( 16). We (17) previously reported on a number of changes associated with increasing age in Eastern larch, including changes in needle dimensions, a reduction in total foliar surface area, and an increase in Chl content. Chl content on a needle dryweight basis increases almost 50% between 1 and 74 years ( 17). The time course for the increase in Chl content with age is inversely correlated with the decline in height and diameter growth. In contrast to larch, the analysis of the foliage of Hedera indicates that there is more Chl present in the juvenile form of the plant than in the mature form (2). This paper reports our further study of the foliar changes associated with maturation in larch. These changes occur at the morphological, physiological, and molecular genetic level. To approach the problem at the level of gene expression we reasoned that the changes in Chl content may be reflected in more general changes in the photosynthetic apparatus. This would suggest that genes encoding elements of the photosynthetic apparatus would be differentially expressed between juvenile and mature plants. Two of the most highly studied gene families in plants are those comprising the rbcS3 and cab sequences. Genomic and/or cDNA clones for cab and rbcS sequences have been obtained from a variety of angiosperms, including maize (7, 32), wheat (6, 29), Arabidopsis (26, 30),

Maturation is a developmental process described in a wide variety of plants, both woody and herbaceous (20). It is This work was supported by U.S. Department of Agriculture grant 87-FSTY-9-0237, and funds administered through the Maine Agricultural Experiment Station. Maine Agricultural Experiment Station publication No. 1492. 2 Some of this work was in partial fulfillment of the requirements for a M.S. degree (C. D. S.).

3 Abbreviations: rbcS, the gene for small subunit of ribulose 1,5bisphosphate carboxylase/oxygenase; cab, the gene for light harvesting Chl-a/b-binding protein of PSII; SLW, specific leaf weight; NPS, net photosynthesis.

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MATURATION IN LARCH

and pea (3, 9). The identification of cDNA clones for both sequences has also been reported in pine (24, 39, 40). rbcS and cab sequences have been shown to be expressed in both a tissue-specific and light-regulated manner. Expression is generally associated with the foliage, stem, and fruit of the plant and is absent from the roots (10, 12, 35). Light regulation of the expression of the rbcS sequences has been recently reviewed by Tobin and Silverthorne (37) and Fluhr

et al. (15). Using heterologous probes for both cab and rbcS sequences we have isolated both cDNA and genomic clones from larch. The rbcS cDNA clones have been sequenced and the results are published elsewhere (23). The sequence for the cab cDNA clones is currently in preparation. The larch clones have, in turn, been used to determine the expression of these sequences in the needles of juvenile and mature plants, in roots, and in light- and dark-grown seedlings. MATERIALS AND METHODS Plant Material

The material, (Larix laricina [DuRoi]) used for these studies has been previously described (17; experiments 1 and 2). Briefly, experiment 1 trees represent a familial juvenile-mature pairing with the mature trees being the family progenitor and the juvenile trees being open-pollinated progeny. Four families are represented, with the mature trees ranging in age from 13 to 25 years. In experiment 2, 40 trees are separated into 4 distinct age classes of 10 trees each. The average ages were 1 year (age class 1), 5 years (age class 2), 17 years (age class 3), and 45 years (age class 4). In October 1987, after two seasons of growth, one of the replicates of experiment 2 was transplanted to the field. The trees remaining in the greenhouse were repotted into 57 L containers (containing 2:1:1 peat:vermiculite:sand, fertilized with Osmocote 18-7-10, a timed release fertilizer). Long and short shoot foliage from the following year's shoots were used to assess the effects of age on net photosynthetic capacity, needle morphology, and gene expression during needle development. For analysis of gene expression in experiment 1 trees, actively expanding short shoot foliage from the indoor-grown plants was harvested, quick-frozen in liquid N2, and stored at -70°C until used for RNA extraction. Tissue samples for each juvenile-mature pairing were harvested coincidentally to eliminate variability in expression due to diurnal rhythms. For analysis of gene expression in experiment 2 trees, newly expanding short shoot needles were harvested from each of the 10 trees in each of the four age classes. The samples within an age class were pooled, quick frozen in liquid N2, and stored at -70°C. For the analysis of gene expression in light- and dark-grown seedlings, seeds were germinated in either light or dark and incubated for 2 weeks. The samples were harvested under green light and quick-frozen in liquid N2 as above. Two-month-old, light-grown seedlings were also harvested and the plantlets divided into aerial (shoot) and root portions. Each fraction was quick-frozen in liquid N2 and stored as above until the RNA was extracted.

1 309

Net Photosynthetic Capacity NPS (,4mol CO2 m-2 S-') and stomatal conductance (,umol * m-2. S-) were measured using a Li-Cor 6200 Portable Photosynthesis System (Li-Cor, Inc., Lincoln, NE 68504) by clamping a segment of long shoot with fully expanded foliage into a 0.25 L cuvette. All measurements were made between July 27 and September 26, 1988 inside a well ventilated building under a 90-W halogen lamp (G.E. Wattmiser II) between 9 AM and 5 PM, at an average temperature of 220C (range 18-260C). Light intensity was varied using neutral density filters (made from Chartpak No. PF06 grey film applied to 3.2 mm thick plexiglass), and IR light was filtered out by a water bath placed between the light source and the filters. An air-tight seal was created by removing some needles and applying plastic putty between the stem and the wall of the cuvette. The needles that had been removed were saved for Chl analysis according to Greenwood et al. (17). Following NPS measurement, the needles on the stem segment inside the chamber were removed and placed between two glass plates, photocopied, and dry weight determined after oven drying at 65°C for 24 h. Projected surface area was measured from the photocopied images using a Zeiss digitizing pad. Projected surface area will therefore slightly underestimate the true surface area of the needle, since the photocopied image does not take into account the threedimensional cross-section of the needle. Needle Morphology

Two long shoot needles from each tree were collected for thin sectioning in early October 1988, before needle senescence had begun. The middle thirds of the needles were tied into bundles of five each with thread, divided into two groups, one fixed in CRAF III and the other in FPA; both fixatives yielded similar results so the results were pooled. All the needles were dehydrated, embedded in paraffin, sectioned transversely at 10 u, and stained with safranin-fast green (4). Needle thickness, width, perimeter, and cross-sectional area were measured at x100 using a Zeiss microscope equipped with a drawing tube and digitizing pad. The product of perimeter/width x 2 and the projected surface area (measured from the photocopied needles) provided an estimate of needle surface area adjusted for the three-dimensional shape of the needles, which was used to calculate NPS. Specific leafweight was calculated from the projected surface area of one side of the leaf after Oren et al. (33).

Statistical Analysis One way analysis of variance was performed to determine the significance of age class on needle morphological and photosynthetic characteristics. Regression analysis was used to establish relationships between Chl content, SLW, and NPS. If the main effect (age class) was significant at P < 0.05, a Scheffe multiple comparison was performed for mean separation. Overall comparisons between indoor and outdoor replications were done using a t test. All analyses were performed using SAS (SAS Institute, Inc., Cary, NC 27511-

8000).

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HUTCHISON ET AL.

Extraction of RNA and Construction of cDNA Clones

RNA was extracted from larch needles, roots, and seedlings by the method of Whitmore and Kreibel (38), with the exception that 0.1% Triton X-100 (Boehringer Mannheim) was substituted for the SDS in all phases of the procedure. Poly(A+) RNA was purified using poly-U mAP filters (Amersham) as described by the supplier, except that the RNA was preincubated with the mAP filters for 5 min at room temperature before being blotted through the filter. Ten grams wetweight of tissue gave approximately 5 mg of total RNA of which 0.3 to 0.5% was purified as poly(A+) RNA. Poly(A+) RNA used as a template for cDNA synthesis came from short shoot needles. The cDNA was synthesized via a modification of the method of Gubler and Hoffman (19). The reaction mixture (50 ,uL) contained: Tris-HCl, 50 mM (pH 8.3); KCI, 75 mM; DTT, 10 mM; MgCl2, 3 mM; dATP, dCTP, dGTP, TTP, 1 mM each; random hexamer primers (Pharmacia), 250 ug/mL; RNA, 5 ,ug; and cloned Moloney MLV reverse transcriptase (BRL), 1000 units. The reaction was incubated at 37°C for 1 h to synthesize the first strand. For second strand synthesis the entire reaction was diluted to 250 ,uL with buffer and dH20 to maintain the concentrations of the Tris, KCI, DTT, and MgCl2. RNase H, 4 units (Amersham) and Esherichia coli DNA polymerase I, 115 units (New England Biolabs) were added and the reaction incubated at I2°C for 1 h, and then 22°C for 1 h. The reaction was stopped by heating to 70°C for 10 min. The ends of the cDNA were 'polished' by the addition of T4 DNA polymerase, 2 units/ mg (Promega) and incubation for 10 min at 37°C. The cDNA was treated with EcoRI methylase (Promega) and EcoRI linkers (New England Biolabs) were ligated onto the ends. Complementary DNA of greater than 500 base pair was size-selected on agarose gels and cloned into the vector X-gtlO (21). Typical libraries contained 1 to 4 x 105 plaques. Identification of cab and rbcS cDNA Clones Two thousand plaques were plated at a density of 300 plaques in a 15 x 100 mm Petri dish using E. coli C600 hfl as a host. Plaque lifts were performed as described by Maniatis et al. (31), except that Hybond nylon filters (Amersham) were used. The DNA was fixed to the filter using short wavelength UV light. The filters were hybridized with either a probe for rbcS or for cab. The rbcS clone used to identify the larch homolog was from maize and provided by W. C. Taylor. The cab clone used in these experiments was from pea (9) and provided by N.-H. Chua. Purified inserts from the clones were labeled using a modification of the random primer labeling method of Feinberg and Vogelstein (13). The specific activity was approximately 2 x 108 cpm/mg. Hybridization conditions for the plaque lifts were a modification ofthose described by Church and Gilbert (8). The filters were prehybridized in 30 mL of 0.5 M NaPO4 (pH 7.2), 1 mM EDTA, and 7% SDS, at 65C, for at least 10 min. The filters were then probed with 32P-labeled DNA (5 x 106 cpm/mL) in 10 mL of the same buffer, at 65°C, overnight. The filters were washed in 0.5 M NaPO4 (pH 7.2), 1 mM EDTA, 5% SDS at 65°C for the first wash, and then the same buffer but with 1% SDS for all

Plant Physiol. Vol. 94, 1990

subsequent washes. The filters were then briefly rinsed with 2 x SSC at room temperature, and exposed to Kodak X-Omat AR5 x-ray film overnight at -70°C, using a DuPont Lightning Plus Intensifying Screen. Northem and Slot Blots For Northern blots 2 ,ug of total RNA were loaded per lane and electrophoresed in an agarose-formaldehyde gel as described by Maniatis et al. (31). The gels were blotted overnight onto Zetaprobe nylon membranes (Bio-Rad) using 20 x SSC. For slot blots serial dilutions of RNA were denatured by heating at 65°C for 10 min in 50% deionized formamide, 20 mm Mops buffer (pH 7.0). The samples were cooled on ice and loaded onto Zetaprobe nylon filters using a BRL Slot Blot apparatus. For both Northern and slot blots the RNA was fixed to the filter using UV light. Northern and slot blots were prehybridized at 42°C for 3 to 4 h in 50% formamide, 5 x SSC, 50 mM NaPO4 (pH 7.2), 1 x Denhardt's solution (11), 1% SDS, 50 ,ug/mL each of poly(A) and poly(C) (Pharmacia), and sonicated salmon sperm DNA (50 gg/mL). The filters were then hybridized overnight at 42°C in fresh hybridization buffer and labeled probes (1 x 106 cpm/mL). After hybridization the filters were washed four times in 0.2 x SSC, 1% SDS at 50°C. The filters were then blotted dry and exposed to either X-Omat AR5 or X-Omat K x-ray film as described above. After exposure to the film the blots were erased by putting them in 500 mL of 0.2 x SSC, 0.2% SDS at 95°C and shaking while the solution cooled to room temperature. This procedure was repeated a second time after which the filters were blotted dry and stored until used in the next hybridization.

RESULTS Needle Morphology Long shoot foliage, which occurs on the current year's shoot, exhibits distinct morphological changes with increasing age which include increases in thickness, cross-sectional area, and SLW (as defined by Oren et al. [33]), which are shown in Table I. These changes are evident on leaves that developed either indoors or outdoors, and the effect of age is highly significant (P < 0.001). Needle width also tended to increase slightly with age, but this effect was only significant in the outdoor-grown needles (P < 0.01). The ratio of needle perimeter to width did not differ significantly between the four age classes (P < 0.1), and no trend with increasing age was evident. Therefore, projected surface area was used to calculate both NPS and SLW. Net Photosynthesis

NPS measurements were made both on intact (the entire tree was moved to the measurement site) and clipped long shoots. Clipped shoots were recut under water, and the cut end inserted through a rubber stopper into a water-filled test tube. As long as NPS was measured within 20 min of clipping, both clipped and intact twigs yielded similar results. Significant variation in NPS was observed as a function of

MATURATION IN LARCH

Table I. Morphological and Physiological Characteristics of Long Shoot Larch Foliage on Grafted Scions from Trees of Four Different Age Classes Morphological characteristics include needle thickness, width, cross-sectional area (X-S area) and specific leaf weight (SLW). Physiological characteristics include Chl content (Chl) stomatal conductance (conductance) and net photosynthesis (NPS). Different letters in rows across indicate that means differ at P < 0.05 using Scheffe's multiple comparison test, and that ANOVA showed that effect of trait shown is significant at P < 0.05. Morphological or Physiological Characteristic

Indoor Thickness (mm) Width (mm) X-S area (mm2) SLW (g m-2) Chl (mg-g dry wt-1) Conductance (,umolm-2. S-1) NPS (umol C02 m2_

s

1 (1 y)

Age Class 2 (5 y) 3 (17 y)

4 (45 y)

0.48a 0.95a 0.27a 84a 6.2a 0.06a

0.51 a 0.94a 0.28a 86a 7.2ab 0.06a

0.56b 1.08b 0.35b 89a 6.7ab 0.06a

0.60b 1.04ab 0.36b 101 b 7.4b 0.06a

3.3a

3.7ab

3.8ab

4.2b

0.46a 0.94a 0.23a 78a 7.Oa 0.06a

0.47a 0.90a 0.24a 88ab 7.9ab 0.07a

0.52b 0.92a 0.27a 97b 7.5ab 0.06a

0.59c 0.97a 0.33b 99b 8.3b 0.06a

4.3a

4.6a

4.5a

4.2a

1)

Outdoor Thickness (mm) Width (mm) X-S area (mm2) SLW (g . m2) Chl (mg.g dry wt-1) Conductance (,umol-

NPS (gmol C02m-2M

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Clearly, older needles can carry out more NPS at high light intensities. Gene Expression

The phenotypic differences between juvenile and mature plants in foliar morphology, Chl content, and net photosynthesis suggested that there may also be a differential response in the expression of genes involved in photosynthesis. Larch cDNA clones were constructed from foliar poly(A+) RNA isolated from short shoots of experiment 1 trees, and were probed with rbcS or cab sequences from maize and pea, respectively. Expression of cab and rbcS sequences have been shown to be tissue-specific and light-regulated in a number of angiosperms. To determine if these gene families were similarly regulated in larch, seeds were germinated and grown in either the light or dark. After 2 weeks the seedlings were 2 to 3 cm in length. Dark-grown seedlings were etiolated. RNA isolated from the light- and dark-grown seedlings were Northern blotted and probed with either a cab probe (clone pCLLH 1.1) or a rbcS probe (clone pCLRu3.3). Cab and rbcS probes hybridized to transcripts of 1100 and 1000 nt, respectively (Fig. 3). Both sequences can be detected in light- and in dark-grown seedlings (Fig. 3). However, the level of cab and rbcS rnRNAs are much lower in the dark-grown seedlings than in the lightgrown seedlings. Based on densitometric tracings of the Northern blots, the sequences are expressed at 25- to 50-fold higher levels in the light-grown seedlings.

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age class, environment and Chl content. Significantly greater NPS with increasing age was observed on the indoor-grown replication (P 6 0.01), but not on the one grown outdoors (Table I). Increased Chl content was observed with increasing age (17), and similar, significant increases (P < 0.05) were observed among the four age classes in both the indoor and outdoor replications (Table I). Overall, the outdoor replication exhibited more NPS and total Chl than the indoor trees. In both replications, increased NPS was associated with increased Chl content, but the effect was most pronounced in the indoor replication, where NPS and Chl content show a significant positive correlation (cf Fig. 1, a and b). Although the range in Chl content is similar inside and out, the values are skewed toward the higher part of the range outside (Fig. lb). Although stomatal conductance is significantly correlated with NPS for both indoor- (r = 0.65, P < 0.001) and outdoorgrown trees (r = 0.52, P s 0.030), it did not exhibit significant variation among age classes or environment (Table I). Light saturation curves (done on intact long shoots) for age classes 1 and 4 are shown in Figure 2. The thicker needles of age class 4 exhibit progressively more NPS with increasing light intensity, and the differences are highly significant. Light saturation curves were also done on age classes 2 and 3, and, as would be expected, were intermediate between 1 and 4.

Outdoor Population

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Figure 1. a, Net maximum photosynthesis as a function of Chi content, outdoor population (n = 30), all age classes combined; b, photosynthesis as a function of Chl content, indoor population (n = 36), all age classes combined.

HUTCHISON ET AL.

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Plant Physiol. Vol. 94, 1990

5

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Figure 2. Light saturation curves of larch scions, indoorr population (trial 1). Age classes 2 and 3 have been omitted forr clarity; (*, significant difference at P = 0.05; **, significant differe nce at P < 0.01. Bar = SD).

To determine if the rbcS and cab sequences were expressed in a tissue-specific manner, RNA was isolated fronn the roots and from the aerial portion (shoots) of 2 month oldI seedlings. No RNA was detected with either probe in the rc)ots of the seedlings, but it was present in RNA extracted from shoots

(Fig. 3).

Total RNA from newly expanding long shoot foliage of grafted scions from the four tree families in experinnent 1 was Northern blotted and probed with a larch cab p)robe. The blots were then erased and reprobed with a larch r bcS probe. To control for the amount of RNA bound to the filters the blots were again erased and probed with a larch aictin probe (PB Singer, KW Hutchison, unpublished data) amd finally with a larch 185 rRNA probe (JL Crosby, KW IHlutchison, unpublished data). Figure 4 shows the results otf the four successive hybridizations to RNAs extracted frorrn juvenile and mature trees of family 4. Similar results were seen with the other families. In all four families the level of cab RNA ..

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Figure 3. Northern blots of RNA from light- and dark-grown seedlings, and from seedling roots and foliage hybridized with cab and rbcS cDNA probes. Two micrograms of total RNA for each sample were electrophoresed in formaldehyde agarose gels and blotted on to Zetaprobe nylon membranes. The membranes were then probed with either a larch cab cDNA probe (clone pCLLH1 .1) or a larch rbcS cDNA probe (clone pCLRu3.3). L, RNA from light-grown seedlings; D, RNA from dark-grown seedling; S, RNA from seedling shoots; R, RNA from seedling roots.

Figure 4. Northern blot of total RNA from foliage a mature tree (family 4) and from its half-sibling progeny (juvenile) hybridized in succession with a larch cab probe, a larch rbcS probe, and larch actin probe and a larch 18S rRNA probe. Probes: cab, pCLLH1.1; rbcS, pCLRu3.3; actin, pCLAcl; 18S rRNA, pCLRi4.1. J, juvenile; M, mature.

in short shoots was higher in juvenile plants than it was in mature plants. The measured level of rbcS RNA was not significantly different between juvenile and mature trees (Fig 4; see below). Slot blots of the RNA samples from the four tree families were probed in the same sequential fashion except that the rbcS probe was used first, followed by the cab

probe, then the 185 rRNA probe. The autoradiograms were scanned with a densitometer to quantitate the amount of RNA. The average ratio of expression of cab RNA in juvenile versus mature trees was 1.34 ± 0.05 SE. The ratio of expression of the rbcS sequences in juvenile versus mature plants was 1.00 ± 0.04 SE. The difference in the ratio of expression ofcab and rbcS sequences in juvenile trees versus their expression in mature trees was highly significant (P 6 0.001). The population of trees from experiment 2 provided us with a second, and larger, number of trees with a much broader range of ages. Total RNA from pooled needle samples of short shoots from 10 trees in each age class were analyzed by probing either Northern or slot blots. Autoradiograms of the blots were scanned with a densitometer to quantitate the amount of RNA present. Figure 5 shows the relative level of expression for both cab and rbcS expression in the four age classes. The cab and rbcS data were normalized to the amount of rRNA detected by the 18S rRNA probe and are expressed relative to the level ofRNA in age class 1 (1 year). Cab mRNA levels were consistently lower in the older age classes than in the juvenile trees. Within experimental error, rbcS mRNA levels were the same in age classes 1, 2, and 4 (1, 5, and 45 years, respectively), but were lower in the age class 3 sample (17 years) (Fig. 5). DISCUSSION Maturation in larch results in an array of changes in leaf morphology, which in turn affect photosynthesis. In addition to an increase in Chl content, the length of long shoot foliage decreases with age (17). Furthermore, we report here that long shoot needles (on both indoor- and outdoor-grown trees) are significantly thicker, with increased cross-sectional area, which contributes to increased specific leaf weight. Thus, mature foliage appears to be more massive, which may be a

MATURATION IN LARCH

1.2

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rbcS

3

1.0

0.8Ca CA w

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0.4

0.2-

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2

3

4

Age Class

Figure 5. Expression of cab and rbcS sequences in newly expanding short shoots from four classes of larch. Slot blots of serial dilutions of total RNA extracted from pooled foliage samples of 10 trees in each age class were probed successively with a larch rbcS cDNA probe, a larch cab cDNA probe, and a larch 18S rRNA probe. The results are normalized to the amount of 18S rRNA on the filters and expressed relative to the level of mRNA in the 1 year age class. Age class 1, 1 year; age class 2, 5 years; age class 3, 17 years; age class 4, 45 years. Bars = standard error.

attribute of maturation in most woody species, since increased leaf thickness due to maturation has also been reported for such diverse species as European beech (Nordhausen, referenced in Skene [36]), English ivy (2), loblolly pine (16), and red spruce (22). Although the effects of maturation on leaf morphology appear to be consistent among several species, the effect of maturation on NPS is highly inconsistent. The juvenile foliage of English ivy has more Chl but less NPS than mature foliage, because the mature foliage has much higher stomatal conductance (2). The juvenile foliage of red spruce has the same amount of Chl as mature, but has higher NPS and stomatal conductance (22). In this study, Chl content increased with age in both indoorgrown trees and outdoor-grown trees, on both long and short shoot foliage. However, NPS exhibited a significant increase correlating positively with Chl content only on indoor-grown common

trees, which overall had less Chl than those grown outdoors

(Table I; Fig. 1). Outdoors, no significant correlation between NPS and Chl content occurs because Chl levels are skewed toward the higher part of the range, and therefore do not appear to be limiting for NPS. SLW is commonly positively correlated with NPS (33), which is also true here, but, as with Chl, the correlation is much stronger indoors (r = 0.57, P s0.00l) than outdoors (r = 0.33, P s 0.07). While, overall, SLW was the same for both indoor and outdoor populations (P s 0.83), needles from indoor-grown trees were significantly (P s 0.01) thicker and wider, which was reflected in greater cross-sectional surface area. Thus, the outdoor-grown needles are more compact, have higher Chl content and NPS, and exhibit less variation in NPS with increasing age. Chl content and SLW are not correlated with one another at all, either indoors or out (r = 0.01). Therefore, even though both increase with age, the increases appear to occur independently of one another. Stomatal conductance does not vary with age, either indoors or out (Table I). Larch is unique among conifers in that it bears two distinct

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types of foliage. Short shoot needles are preformed in the resting bud, and emerge during bud burst in the spring, forming a rosette, since there is almost no internode elongation between them. Long shoot needles are borne on the expanding long shoot, and some are preformed in the resting bud, but in vigorous shoots most are the result of indeterminate growth. Long shoot needles are longer than short shoot needles, and are separated by internodes. Short shoot needles all emerge at the same time so that foliage develops synchronously. Short shoots are therefore ideal for gene expression studies. However, the rosette habit of short shoot foliage renders estimates of NPS very difficult because of difficulties in surface area determination. In contrast, long shoot foliage is well-adapted for NPS determinations (on fully expanded foliage) but since the needles expanded sequentially as the shoot elongates, harvest of needles at the same developmental stage is very difficult. In young, vigorous trees, the great majority of the foliage is made up of long shoot needles at the end of the growing season. On older trees with less vigorous long shoots, short shoot foliage is relatively more abundant, since short shoots may persist for several years. Although short shoot Chl content increases with age, SLW does not (P < 0.96). What, then, is the adaptive significance of producing juvenile long shoot foliage of lower specific leaf weight? One obvious answer to this question is that juvenile plants can produce more photosynthetic surface area per unit of biomass invested in foliage. In fact, juvenile scions may produce about twice as many needles as mature, which is a function primarily of production of a greater number of lateral branches, as well as more shoot growth (17). The number of needles per unit of long shoot stem length is the same for juvenile and mature scions (our unpublished data). Thus, juvenile plants produce much more photosynthetic surface area than mature, which would more than offset the slight decrease in NPS due to less Chl and juvenile and mature needles are approximately the same weight. Furthermore, outdoor-grown juvenile long shoot foliage of larch has reduced SLW, but not reduced NPS. Thus, we can certainly conclude that slower growth by mature plants is not due to reduced NPS, since mature foliage indoors exhibits more NPS. Therefore, differences in NPS do not explain the difference in growth rates between juvenile and mature scions. The tendency of mature needles to be more massive with higher Chl content would also permit them to carry out more NPS in bright light, as is observed in Figure 2. Mature shoots near the tops of tall trees will certainly be exposed to higher light intensities, and will be under more water stress due to the effect of gravity and exposure. Thicker leaves use water more efficiently, since they possess a higher ratio of photosynthetic mesophyll to transpiring leaf area ( 14). The developmental changes in foliar morphology and physiology that are associated with phase change in larch prompted us to initiate a study of the expression of genes involved in photosynthesis in juvenile and mature plants. We identified and isolated cDNA clones from the rbcS and cab multigene families. In a preliminary analysis of the expression of these sequences in larch we have determined that both the cab and the rbcS sequences are expressed in the foliage but not the roots of the larch tree (Fig. 4). The cab and rbcS transcripts

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are nearly identical in length at approximately 1000 nt. An additional band of low mol wt can be seen in the shoot RNA hybridized with the rbcS probe (Fig. 4). Whereas it is possible that this band represents one member of the rbcS multigene family (23), its small size (200 nt) would be unable to code for a complete protein. Quantitation of the band by densitometric scanning indicates that it represents