Parenchyma Cell Respiration and Survival in Secondary Xylem: Does Metabolic Activity Decline with Cell Age?

Plant, Cell and Environment (2007) 30, 934–943 doi: 10.1111/j.1365-3040.2007.01677.x

Parenchyma cell respiration and survival in secondary xylem: does metabolic activity decline with cell age?

R. SPICER1 & N. M. HOLBROOK2

1Rowland Institute at Harvard University, 100 Edwin H. Land Boulevard, Cambridge, MA 02142, USA and 2Organismic and Evolutionary Biology, Harvard University, 16 Divinity Avenue, Cambridge, MA 02138, USA

ABSTRACT defines (and arguably drives) heartwood formation, a form of tissue senescence during which the oldest, non- Sapwood respiration often declines towards the sapwood/ functional xylem is compartmentalized in the centre of the heartwood boundary, but it is not known if parenchyma stem. The cause of parenchyma cell death is not known, metabolic activity declines with cell age. We measured but evidence for decreased metabolic activity in the inner- sapwood respiration in five temperate species (sapwood age most sapwood has led to a view of parenchyma ageing as range of 5–64 years) and expressed respiration on a live cell a gradual, passive decline in metabolism that terminates in basis by quantifying living parenchyma. We found no effect cell death. of parenchyma age on respiration in two conifers (Pinus Multiple reports suggest that sapwood respiration strobus, Tsuga canadensis), both of which had signifi- declines towards the sapwood/heartwood boundary cant amounts of dead parenchyma in the sapwood. In (Goodwin & Goddard 1940; Higuchi, Shimada & Watanabe angiosperms (Acer rubrum, Fraxinus americana, Quercus 1967; Pruyn, Gartner & Harmon 2002a,b; Pruyn, Harmon & rubra), both bulk tissue and live cell respiration were Gartner 2003; Pruyn, Gartner & Harmon 2005), although reduced by about one-half in the oldest relative to the there have been reports of no change (Bowman et al. 2005) youngest sapwood, and all sapwood parenchyma remained or even an increase in respiration in the ‘transition zone’, a alive. Conifers and angiosperms had similar bulk tissue res- narrow region between the sapwood and heartwood (Shain piration despite a smaller proportion of parenchyma in & MacKay 1973; Bowman et al. 2005). The vast majority conifers (5% versus 15–25% in angiosperms), such that of these studies have been in conifers, and reports on conifer parenchyma respired at rates about three times angiosperms are mixed as to whether respiration is reduced those of angiosperms. The fact that 5-year-old parenchyma in inner sapwood (Goodwin et al. 1940; Higuchi et al. 1967; cells respired at the same rate as 25-year-old cells in conifers Pruyn et al. 2003). At the cellular level, parenchyma nuclei suggests that there is no inherent or intrinsic decline in shrink (Yang 1993) and the number of mitochondria may respiration as a result of cellular ageing. In contrast, it is not decline with tissue age (Frey-Wyssling & Bosshard 1959), known whether differences observed in cellular respiration both of which suggest a reduction in metabolic activity. rates of angiosperms are a function of age per se, or whether In contrast, the ability of xylem parenchyma to active regulation of metabolic rate or positional effects (e.g. de-differentiate and form callus tissue is unaffected by age proximity to resources and/or hormones) could be the in some Pinus species (Allen & Hiatt 1994), and there is cause of reduced respiration in older sapwood. evidence that certain Krebs cycle enzymes (malic and suc- ccinate dehydrogenase) and glucose-6-phosphate dehydro- Key-words: ageing; heartwood; parenchyma; respiration; genase are more active in the innermost sapwood of Pinus sapwood; senescence. radiata (Shain et al. 1973; Hillis 1987; Hauch & Magel 1998). This latter enzyme catalyzes the first reaction of the oxida- INTRODUCTION tive pentose phosphate pathway, which supplies intermedi- ates for phenolic synthesis, an activity that characterizes Parenchyma cells within secondary xylem can be heartwood formation in many species. extremely long-lived. Individual cells may live for 2–200 Although most evidence points towards a reduction in years with longevities that are specific to a species but are parenchyma cell metabolism with age, there are logical also shaped by the environment. Surrounded by a matrix reasons to expect that this may not be true. First, given that of non-living cells that function in water transport and (1) the majority of work demonstrating a reduction in res- mechanical support, parenchyma cells form an important piration with tissue age is in conifers, and (2) that conifers point of exchange between the symplast and apoplast in appear to have a gradual loss of living cells with tissue age woody tissue, functioning in carbohydrate storage, trans- (i.e. there is evidence for a gradual increase in the number port and wound response. It is the death of these cells that of dead parenchyma towards the sapwood/heartwood Correspondence: R. Spicer. Fax: 617 497 4627; e-mail: boundary; Nobuchi & Harada 1983; Yang 1993; Gartner, [email protected] Baker & Spicer 2000; Nakaba et al. 2006), it may be © 2007 The Authors 934 Journal compilation © 2007 Blackwell Publishing Ltd Parenchyma respiration in secondary xylem 935 that this metabolic decline is eliminated when respiration is was sampled with a 12 mm increment borer at 1.4 m above expressed on a per live cell volume basis.Although attempts ground, deep enough to reach the darker-coloured heart- have been made to relate respiration rate to parenchyma wood. Five trees per species were sampled in mid-July 2003 volume (Ryan 1990; Stockfors & Linder 1998; Pruyn et al. and again in mid-June 2004 for a total of 10 trees per 2005), there has never been an explicit accounting of the species. Stem diameter and sapwood age and depth were live cell fraction. Secondly, parenchyma cells play an active recorded and did not differ between sampling dates role in the transition from sapwood to heartwood: they (Table 1). synthesize complex polyphenolic compounds (Necˇesaný The phloem and cambium were removed from each 1973; Magel 2000), produce cellulosic material to form core with a razor blade immediately following extrac- tyloses and tylosoids (blocking angiosperm vessels and tion. Increment cores were then wrapped in moist paper conifer resin canals, respectively; Chattaway 1949; Chafe towel and stored in a cooler (~5 °C) for transport to the 1974; Saitoh, Ohtani & Fukazawa 1993), secrete gums laboratory. (Chattaway 1949) and, in some cases, deposit lignin in their cell walls (Balatinecz & Kennedy 1967; Yamamoto 1982). Respiration measurements An irreversible decline in metabolic rate with cell age would run counter to the acquisition of new physiological Fresh tissue was sampled from the outermost (youngest) roles at this late stage in development. and innermost (oldest) sapwood by removing two approxi- Our main objective in this study was to test the hypoth- mately 1 cm3 cylinders from each increment core. The out- esis that parenchyma in secondary xylem respires at the ermost position was adjacent to but did not include the same rate, regardless of age, by expressing respiration on a cambial zone (i.e. the cambium and developing xylem were live cell volume basis. Secondary objectives were to ask removed with a razor blade). The innermost position was whether species that differ widely in sapwood quantity and adjacent to but did not include any heartwood. The ‘white physiology also differ in sapwood respiration rate, and ring’ (i.e. transitional ring between sapwood and heartwood whether tissue-level respiration is simply a function of the in conifers; Nobuchi et al. 1983) in Pinus strobus and Tsuga volume proportion of parenchyma. By expressing respira- canadensis, when present, was also excluded from the inner- tion on a live cell volume basis, we were also able to most sample. Distance and ring number from the cambium compare respiration rates of individual parenchyma cells to were recorded for the outer and inner ends of each sample, those of other plant cell types. Finally,by using mature trees, and the mean of these two ends was used to define each normalizing sample position relative to the cambium and sample’s age and position (Table 2). sapwood/heartwood boundary,and equilibrating samples to Sapwood samples were equilibrated to 10% O2 (v/v) a uniform internal gas composition before measurement, before respiration measurements to eliminate potential we were able to eliminate several developmental and envi- effects of different native, internal gas compositions ronmental variables that might have confounded previous between inner and outer sapwood. Ten per cent O2 was results. chosen as a realistic midrange for the species studied (Spicer & Holbrook 2005) and to minimize equilibration times. Each sample was weighed, then wrapped in a MATERIALS AND METHODS 2 ¥ 5 cm strip of dampened cheesecloth and placed in an Plant material open 10 mL glass vial. The open vials were enclosed in a gas-impermeable glove bag and flushed repeatedly with

For sapwood respiration measurements, mature trees of 10% O2 (balance N2). Glove bags were then stored at 4 °C two conifer [Tsuga canadensis (L.) Carr. and Pinus strobus for a total of 36 h with additional repeated ﬂushing of 10%

L.] and three angiosperm (Acer rubrum L., Fraxinus ameri- O2 at 12 and 24 h. Prior tests had shown that 36 h was cana L., Quercus rubra L.) species were randomly selected sufﬁcient to fully equilibrate the samples so that their inter- within a 5 ha tract of natural forest in Harvard Forest, Peter- nal gas compositions matched that of treatment gas, while sham, MA, USA (42.5°N, 72°W, 220 m elevation). Sapwood cold storage suppressed respiration and extended the

Table 1. Outer diametera, depth and age of SW age (rings) sapwood (mean Ϯ SE, n = 10) of trees sampled from Harvard Forest, Petersham Species Diameter (cm) SW depth (cm) Mean Range MA Acer rubrum 33.1 Ϯ 2.1 8.3 Ϯ 1.1 43 Ϯ 429–60 Fraxinus americana 34.4 Ϯ 2.0 7.5 Ϯ 1.2 48 Ϯ 334–64 Pinus strobus 35.4 Ϯ 1.5 3.5 Ϯ 0.5 21 Ϯ 212–29 Quercus rubra 36.0 Ϯ 1.2 1.5 Ϯ 0.2 8 Ϯ 15–13 Tsuga canadensis 34.7 Ϯ 1.7 4.1 Ϯ 0.4 28 Ϯ 417–47 aDiameter measured over-bark at 1.4 m above ground. SW, sapwood. © 2007 The Authors Journal compilation © 2007 Blackwell Publishing Ltd, Plant, Cell and Environment, 30, 934–943 936 R. Spicer & N. M. Holbrook

Table 2. Age (ring number) and distance Outer sapwood Inner sapwood (mm) from the cambium for inner and outer sapwood positions (mean Ϯ SE, n = 9 Species Age Distance Age Distance or 10) Acer rubrum 4.7 Ϯ 0.3 0.5 Ϯ 0.2 40.7 Ϯ 3.3 7.7 Ϯ 1.1 Fraxinus americana 4.8 Ϯ 0.7 0.5 Ϯ 0.3 45.2 Ϯ 3.4 7.0 Ϯ 1.2 Pinus strobus 4.1 Ϯ 0.7 0.5 Ϯ 0.3 17.6 Ϯ 1.4 3.0 Ϯ 1.5 Quercus rubra 1.7 Ϯ 0.2 0.3 Ϯ 0.4 5.9 Ϯ 0.6 1.2 Ϯ 0.2 Tsuga canadensis 3.1 Ϯ 0.4 0.5 Ϯ 0.3 24.0 Ϯ 3.4 3.6 Ϯ 0.4

length of time respiration rates (after return to 20 °C) from vials, measured for fresh volume by displacement, and remained stable. Although high levels of CO2 may exist vacuum infiltrated with a cold, phosphate-buffered (pH 7.2) within the stem, CO2 was not included in the measure- 3.5% paraformaldehyde fixative. ment gas because good estimates of realistic levels in situ were not available, and because different-aged sapwood responded similarly to CO2 (Spicer & Holbrook 2007). Parenchyma volume estimates Following equilibration and a final flush of 10% O2, the vials were sealed with crimp-top lids each containing a butyl The proportion of volume occupied by living parenchyma rubber septum. Sealed vials were then transferred to a was determined for each sample through image analysis of water bath where they were incubated at 20 °C throughout light and fluorescence microscopy sections. Tangential sec- the measurement period. Oxygen was measured in each vial tions (20 mm) were cut from both ends of each sample with every 4–6 h for 24–36 h by penetrating the septum with a a sliding microtome, stained in 1% aqueous safranin-o, needle-tipped fiber optic O2 probe (USB2000 spectrometer mounted and viewed with a 10¥ objective. Ten randomly and FOXY probe, Ocean Optics, Inc., Dunedin, FL, USA). located images (0.66 mm2 area) were captured for each tan- The probe, which operates using principles of fluorescence gential section for a total of 20 images per sample (10 quenching in the presence of O2, was calibrated with images from each end) and processed in ImageJ (http:// humidified O2 standards at 20 °C. The slope of the linear rsb.info.nih.gov/ij/). Rays were manually highlighted with a portion of the curve (typically the first 12–24 h) was taken paint tool, and the proportion of tangential surface occu- -1 as the rate of O2 consumption and converted to mol hr by pied by ray was measured (Fig. 1a,b). This proportion of calculating the total volume of gas in the vial. At the end of area was assumed to equal the proportion of volume, as rays this period, O2 levels were between 3 and 7% depending on were often continuous radially through the sample, and the rate of O2 consumption. Samples were then removed both ends were measured.The mean value for all 20 images

Figure 1. Light microscopy images illustrating the estimation of parenchyma proportion in woody tissue. Tangential sections in (a) Fraxinus americana showing multiseriate rays and a background composed predominantly of fibers with some axial parenchyma (a) (b) (arrows), and (b) Acer rubrum showing both uniseriate and multiseriate rays painted black following area measurement in ImageJ (http://rsb.info.nih.gov/ij/). Axial parenchyma painted black in transverse sections of (c) F. americana, showing an annual ring border with a large earlywood vessel in the lower left-hand corner, smaller latewood vessels above and (d) Quercus rubra latewood; single star (*) indicates a ray; double star (**) (c) (d) indicates a large, multiseriate ray. Scale bar = 50 mm. © 2007 The Authors Journal compilation © 2007 Blackwell Publishing Ltd, Plant, Cell and Environment, 30, 934–943 Parenchyma respiration in secondary xylem 937 was taken to characterize the proportion of ray parenchyma Insoluble for each sample. amyloplasts formazan from (starchAmyloplasts storage) insoluble ‘ In addition to ray parenchyma, both Fraxinus americana fromreduction reduction of of and Quercus rubra contain significant volumes of axial tetrazoliumtetrazolium salt parenchyma (axial parenchyma is absent from Acer rubrum Ray tracheid and T. canadensis). Axial parenchyma was quantified for transverse sections (20 mm) taken from both the upper and lower surfaces of F.americana and Q. rubra samples, stained for starch (indicative of parenchyma) with 1% aqueous Ray parenchyma potassium iodide, and viewed with a 20¥ objective. Addi- tional transverse sections were stained with safranin-o. Ten randomly located images (0.17 mm2 area) were captured per section for a total of 20 images per sample. As described Ray tracheid above, parenchyma was manually highlighted with a paint Nuclei tool (Fig. 1c,d); the proportion of surface area occupied by 50 µm parenchyma was measured, and the mean of all 20 images was used to characterize the sample. Resin canal epithelial cell volume in P. strobus was estimated by measuring the Figure 2. Radial tissue section from the outer sapwood of average transverse area of individual resin canals (cell area Pinus strobus. A single ray is shown, composed of four radial files only,10 resin canals) and counting resin canals on two trans- (stacked) of ray parenchyma and two files (at top and bottom) of ray tracheids. Ray parenchyma contain insoluble red formazans verse surfaces per sample.Proportion volume was calculated produced by reduction of the vital stain tetrazolium chloride in assuming that canals ran through the length of the sample. oxidative respiration, and DAPI-stained nuclei. Tests with vital staining showed that the presence/absence of nuclei reliably Live parenchyma estimates indicated live/dead cells. Ray tracheids die during differentiation in the cambial zone, and so lack a protoplast (and nucleus), and Radial tissue sections were used to determine the presence are easily distinguished by the presence of circular bordered pits. Fluorescence (365ex l/450em l) overlaid onto brightfield image; and absence of nuclei to quantify the proportion of paren- scale bar = 50 mm. chyma that was alive. Preliminary work using vital stains (triphenyl-tetrazolium chloride, which when reduced pro- duces coloured, insoluble formazans) and a fluorescent species as a ‘between subject’ factor, and radial position as nucleic acid stain 4′,6-diamidino-2-phenylindole (DAPI) a ‘within subject’ factor (i.e. radial position was spatially had shown that the presence of a nucleus is a sufficient repeated within each tree, the subject). Differences between indicator of cell vitality (Fig. 2). Radial sections (20 mm) of radial positions within each species were tested with paired all five species were rinsed in phosphate-buffered saline t-tests (two-tailed). Orthogonal contrasts were used to test (PBS) (pH 7.5), incubated in 1% (w/v) Triton-X in PBS a priori comparisons where possible. Multiple a priori overnight, and stained with 0.001% DAPI in PBS. Sections comparisons (e.g. those among interaction terms) were were mounted in 4:1 glycerol : PBS and viewed with a 4¥ tested with Bonferroni-adjusted P-values to minimize (P. strobus and T. canadensis)or10¥ (A. rubrum, F. ameri- the experiment-wise error rate (Sokal & Rohlf 1995). cana, Q. rubra) objective on an epifluorescent microscope Unplanned multiple comparisons were made with Fisher’s (365ex l/450em l). least significant difference (LSD) corrected P-values. For each annual ring within a sample, the total number of rays and the number of rays containing nuclei were tallied. RESULTS Rays of angiosperms (A. rubrum, F. americana, Q. rubra) retained nuclei until their abrupt disappearance at the The effect of radial position was highly significant (Table 3) sapwood/heartwood boundary. In contrast, conifer species such that the youngest, outermost sapwood respired at a (P. strobus and T. canadensis) showed dead rays (those higher rate than the oldest, innermost sapwood in all completely lacking nuclei) in the outer sapwood, with species except P. strobus when expressed on a per tissue increasing frequency towards the inner sapwood. The pro- volume basis (Fig. 3a, Table 4). Sampling date was a signifi- portion of live rays was calculated for each annual ring, and cant blocking factor (Table 3) such that respiration rates a mean value (proportion live ray) was determined for each were slightly higher, and the magnitude of the difference section by weighting the contribution of each ring according between inner and outer sapwood slightly greater in 2003 to its radial width. relative to 2004. Most pairwise species comparisons were significant for the outer sapwood (Pinus versus Tsuga and Statistical analyses Fraxinus versus Acer were exceptions), whereas only Fraxi- nus and Quercus differed significantly for the inner The effects of species and radial position on sapwood res- sapwood (P = 0.02, Fisher’s LSD). piration were tested in a repeated measures analysis of There were large species differences in parenchyma variance (anova) that included sampling year as a block, content and vitality (Fig. 4, Tables 3 & 5). Conifers had less © 2007 The Authors Journal compilation © 2007 Blackwell Publishing Ltd, Plant, Cell and Environment, 30, 934–943 938 R. Spicer & N. M. Holbrook

Table 3. Repeated measures analysis of P-values for effects on respiration variance (ANOVA) testing effects of species and radial position on respiration Model term d.f. Rtiss Rlive Vpar per tissue volume (Rtiss), respiration per live Intercept 1 <0.0001 <0.0001 <0.0001 parenchyma volume (Rlive) and proportion Sampling date 1 0.005 0.005 0.6 of tissue volume occupied by parenchyma Species 4 0.002 <0.0001 <0.0001 (Vpar) Error 41 Radial position 1 <0.0001 0.7 0.5 Radial position ¥ sampling date 1 0.01 0.5 0.2 Radial position ¥ species 4 <0.0001 0.07 0.4 Error 41

Sampling date is included as a blocking factor, and radial position is treated as a spatially repeated measure within each stem. d.f., degrees of freedom. than one-third the parenchyma volume of angiosperms smallest total proportion of parenchyma (Fig. 4). Fraxinus (P < 0.0001, orthogonal contrast). Angiosperms differed in had the highest proportions of both of axial and total paren- both the total amount and composition of parenchyma such chyma volume. Angiosperms showed no evidence of paren- that A. rubrum, which lacks axial parenchyma, had the chyma death, either ray or axial, throughout the sapwood (Fig. 5). In contrast, both conifers showed measurable proportions of dead ray parenchyma starting in the outer sapwood and increasing towards the sapwood/heartwood (a) Inner boundary (Fig. 6). Both species had a greater proportion of Outer living parenchyma in the outer sapwood (Table 5; paired t-test P-values < 0.01). Tsuga had a smaller proportion of living parenchyma in the inner but not the outer sapwood relative to Pinus (P = 0.0003 and 0.5, respectively, orthogonal contrasts). In no case was there any difference in total (live plus dead) parenchyma volume between outer and inner sapwood (Table 3). When respiration was expressed on a per live cell volume basis, there were large species differences (Table 3) such that conifers had almost three times the respiration rate of angiosperms (Fig. 3b, contrast P < 0.001). The effect of radial position on live cell respiration depended on species (Table 3, interaction term marginally signiﬁcant with (b) P = 0.07). Parenchyma in the outermost sapwood respired

Figure 3. Parenchyma respiration expressed as rate of O2 consumption (a) per unit fresh sapwood volume and (b) per unit live parenchyma volume. Parenchyma volume was quantiﬁed through image analysis of transverse and tangential tissue sections; live parenchyma fraction was determined by the Figure 4. Volume proportion of ray and axial parenchyma presence or absence of nuclei in radial sections. Means Ϯ SE are (proportion of fresh sapwood volume as estimated by image shown; n = 10 trees; ***indicates signiﬁcant differences between analysis). Axial parenchyma in Pinus includes only the epithelial inner and outer sapwood (P-values < 0.01, paired t-test). cells lining resin canals (Tsuga lacks resin canals). © 2007 The Authors Journal compilation © 2007 Blackwell Publishing Ltd, Plant, Cell and Environment, 30, 934–943 Parenchyma respiration in secondary xylem 939

Table 4. P-values for tests of signiﬁcance P-values for inner versus outer sapwood position (paired t-tests, two-tailed) comparing respiration rates expressed on a tissue Species Rtiss Rlive volume (Rtiss) and live cell volume (Rlive) Acer rubrum 0.005 0.003 basis between inner and outer sapwood Fraxinus americana <0.0001 <0.0001 positions within each species Pinus strobus 0.7 0.6 Quercus rubra 0.0003 0.0004 Tsuga canadensis 0.007 0.22

Note that given a total of ﬁve comparisons, the Bonferroni-adjusted P-value required for signiﬁcance is 0.01 (Sokal et al. 1995).

Table 5. Percent ray parenchyma living in outer versus inner DISCUSSION sapwood samples (mean proportion of rays retaining nuclei Ϯ SE, n = 9or10) We found little evidence for a decline in parenchyma cell respiration that is inherent to the process of cellular Percent live parenchyma ageing. First, there was no decline in parenchyma respiration with age in the conifers T. canadensis and P. strobus, Species Outer Inner despite maximum cell ages of about 30 and 20 years, Acer rubrum 100 100 respectively. Only Tsuga showed reduced respiration with Fraxinus americana 100 100 age on a per tissue volume basis, and this was eliminated Pinus strobus 93 Ϯ 385Ϯ 4 once the proportion of dead cells was taken into account. Quercus rubra 100 100 Second, although the angiosperms (A. rubrum, F. ameri- Tsuga canadensis 90 Ϯ 459Ϯ 7 cana and Q. rubra) had reduced respiration in older tissue – a difference that was unaffected by correcting for live Differences between inner and outer positions were significant for both Pinus and Tsuga (P = 0.01 and 0.001, respectively, two-tailed cell volume because all sapwood parenchyma remained paired t-test). alive – this does not necessarily reflect an inherent or intrinsic decline with age because age and position within the stem are confounded. In Quercus and Acer, paren- at almost twice the rate as that of inner sapwood for all chyma in the innermost sapwood respired at the same rate three angiosperms (Fig. 3b), whereas for conifers, there was despite a difference in age of 35 years. One would have no significant difference between the two radial positions to invoke dramatic species differences in cellular ageing (Fig. 3b, Table 4). trends to explain this result. Although species-specific

(a) (b) (c)

Figure 5. Radial tissue sections of the outer and inner sapwood (SW) of angiosperms showing ﬂuorescent DAPI-stained nuclei. The outermost SW (1-year-old annual ring) is shown for (a) Fraxinus americana,(b)Quercus rubra (d) (e) (f) and (c) Acer rubrum. The annual ring adjacent to the heartwood (HW) is shown for Fraxinus (d,e), Acer (f) and Quercus (g–i). The abrupt loss of nuclei at the SW/HW boundary is shown for Quercus (i). Tissue ages and depths within the stem for the innermost SW shown here are 54 years and 14 cm (Fraxinus), 40 years and 7 cm (Acer), 7 years and 3 cm (g) (h) (i) (Quercus). Scale bar = 50 mm.

(a) (b)

Figure 6. Radial tissue sections of the outer (a,b), middle (c,d) and inner (e,f) sapwood (SW) of Pinus strobus (a,c,e) and Tsuga canadensis (b,d,e) showing DAPI-stained nuclei. Parenchyma death (c) (d) occurs in Pinus in (c) the middle SW, where the upper file of cells in a short ray has died, and (e) the innermost SW, where parenchyma cells fill with polyphenolics at the SW/HW boundary. ThemiddleSWofTsuga contains both living and dead rays (d), but at the SW/HW boundary (f), all remaining rays die. Outer SW is 1 year old and adjacent to the cambium. Tissue ages/depths within the stem for the middle and inner SW, respectively, are 8 years/2 cm and 16 (e) (f) years/4 cm (Pinus); 22 years/5 cm and 56 years/6 cm (Tsuga). Scale bar = 50 mm. cellular ageing is certainly possible, it seems unlikely given heartwood (Shain et al. 1973). One obvious approach for the range of maximum parenchyma ages found both future work will be to determine radial profiles of metabolic within a species (30+ year range within this study alone) activity at a high spatial resolution across the sapwood (i.e. and within an individual (e.g. 15+ year differences in per annual ring), and at different times of the year. Simi- sapwood age between the stem base and top have been larly, one can begin to separate the effects of age and posi- observed; Spicer & Gartner 2001; Domec, Pruyn & tion by measuring respiration rates in tissue of the same age Gartner 2005). Similarly, substrate limitation is unlikely to sampled at different heights in the stem – the same annual cause the observed declines in respiration because paren- ring number will be closer to the sapwood/heartwood chyma cells often store large quantities of starch (some- boundary towards the top of the stem. times lipids), even in the innermost sapwood (Fischer & Species differences in respiration rate were much larger Höll 1992; Magel, Jay-Allemand & Ziegler 1994; Höll when expressed on a live cell basis than on a tissue volume 2000). Many of the innermost sapwood samples in this basis. These data suggest that bulk tissue respiration cannot study showed parenchyma filled with amyloplasts. be estimated by quantifying parenchyma volume because An alternative explanation is that parenchyma metabo- there may be large species differences in cellular respiration lism is actively regulated and effectively ‘down-regulated’, rates. Sapwood, rather than parenchyma, may behave uni- either with age or, more likely,with position in the stem.The formly across species: here, the large parenchyma volume in ability of parenchyma cells to de-differentiate and begin angiosperms (18–25%) combined with a low cellular respi- dividing at any age (Fisher 1981; Allen et al. 1994), as well as ration rate (about one-third that of conifers) produce tissue the role of parenchyma in synthesizing secondary com- respiration rates roughly similar to those of conifers. It will pounds and tyloses just prior to heartwood formation, be interesting to see if angiosperms and conifers with the suggest that communication routes exist for regulation. same volume fraction of parenchyma have similar cell-level There is also one account of an increase in metabolic activ- respiration rates [e.g. Salix and Tilia are reported to have ity in the narrow ‘transition zone’ between sapwood and proportions of parenchyma (6–8%) comparable to some © 2007 The Authors Journal compilation © 2007 Blackwell Publishing Ltd, Plant, Cell and Environment, 30, 934–943 Parenchyma respiration in secondary xylem 941 conifers]. These smaller differences in bulk tissue respira- That rates of parenchyma respiration vary among species tion may be normalized at the whole-tree level by varying may also suggest functional specialization of parenchyma volumes of sapwood.There is evidence among conifers that among taxonomic groups. Conifers have narrow (one to species with narrow sapwood have higher bulk tissue respi- two cells wide) rays with a single parenchyma cell type and ration rates than those with wide sapwood (Pruyn et al. almost no axial parenchyma.Angiosperms are more diverse 2003). Our data support this idea among angiosperms as in parenchyma composition, with wider rays (2–30 cells well, with the caveat that species differences in tissue res- wide) composed of several cell types, and large volumes of piration are not simply a function of parenchyma volume. axial parenchyma arranged in complex patterns, often sur- Quercus, which maintains a very narrow band of sapwood, rounding and linking vessels. There are almost certain to be has the highest rate of respiration (both for total sapwood functional/physiological differences between ray and axial and for outer sapwood alone) but an intermediate volume parenchyma in angiosperms, but it is not known to what of parenchyma. extent they differ in rates of respiration. If axial paren- Our results suggest that bulk sapwood respiration is quite chyma respires at a lower rate than ray parenchyma, for low, roughly an order of magnitude lower than cambial instance, it can explain the higher tissue respiration rate of tissue (Goodwin et al. 1940; Higuchi et al. 1967) and half Quercus relative to Fraxinus. that of secondary phloem and cambial tissue combined It remains to be seen if differences between conifers and (Pruyn et al. 2002a,b; Pruyn et al. 2003; Bowman et al. 2005; angiosperms hold with a wider sampling of species, but Pruyn et al. 2005). By expressing respiration on a live cell there may be fundamental differences in the manner in volume basis, we were able to ask whether this low tissue which sapwood senesces in these two taxonomic groups. respiration rate is simply a function of the small proportion Parenchyma cell death within the sapwood of conifers may of living cells, or whether parenchyma cells have a low be the result of a cambial process, and unrelated to sapwood metabolic rate as well. Quantifying live cell volume in plant ageing and death at the sapwood/heartwood boundary. Ray tissue is rarely done, and there are no published accounts of initials can be lost from the cambium, at least in conifers, respiration rates for live cells within a more complex tissue. and this occurs with increasing frequency as the cambium However, there are two extremes of tissues composed of ages or when growth is slowed (Bannan 1934; Barghoorn pure living cells against which we may compare our rates: 1940). Once a cambial ray initial is lost, that radial file tubers and meristems. ‘Resting’ (mature, non-growing) ceases to be continuous with the phloem, and this loss of -3 -1 potatoes respire at 0.5–1.5 mmol O2 cm h (per fresh access to carbohydrates could result in cell death. Ray ini- volume; Bidwell 1974; Hajirezaei et al. 2003), a rate on tials are typically lost from the top and bottom of rays, and par with our lowest angiosperm values (1.4–4.2 mmol short rays may be eliminated altogether (Bannan 1934; -3 -1 O2 cm h ), but an order of magnitude lower than our Barghoorn 1940; Larson 1994). We observed that ray death -3 -1 range for conifers (11.6–14.5 mmol O2 cm h ). In contrast, in conifer sapwood began with the top and bottom radial -3 -1 fine root meristems respire at 100–250 mmol O2 cm h cell files (Fig. 6c; see also Nakaba et al. 2006), and that short (Lambers 1985; Tang & Peters 1995; Bidel et al. 2000; rays were more likely to die than tall rays, particularly in Asplund & Curtis 2001), an order of magnitude higher than Tsuga. In addition to their outright loss from the cambium, our highest conifer values. Parenchyma cells are apparently ray initials at the top and bottom of a ray may switch from not in a ‘quiescent’ state, and instead have a metabolic rate producing ray parenchyma to producing ray tracheids slightly above simple storage tissue but well below that of (Fig. 2; Bannan 1934; Barghoorn 1940; Larson 1994), dividing, meristematic tissue. thereby severing a symplasmic connection to the phloem Within-stem variation in rates of cellular respiration may (ray tracheids die during differentiation). In contrast, paren- reflect changes in parenchyma function with age/position in chyma cell death caused by cambial dynamics is probably the stem, or more specifically, with the water-transport insignificant in angiosperms given their wide rays and/or capacity of the xylem. Both Quercus and Fraxinus are ring- abundant axial parenchyma, which can link radial files. porous species and only transport water in the outermost In summary, we found that long-lived xylem parenchyma annual ring(s) (Spicer et al. 2005). Axial parenchyma in cells respire at relatively low rates that vary across species, Fraxinus frequently surrounds vessels (more so than in and that respiration rates do not necessarily decline as cells Quercus; Fig. 1c,d) and may remain alive for 45+ years after age. Further work is needed to tease apart the effects of age its associated vessels have ceased to conduct water. This and position on respiration within tree stems. The fact that may represent a shift towards a resting state in which trees maintain a fairly species-specific sapwood depth (and primary functions are in wound response and carbohydrate thus volume), rather than number of years of sapwood, is storage, rather than in transpiration stream maintenance suggestive of positional and not age-based controls. This is and modification (Bucci et al. 2003; Salleo et al. 2004; Zwie- in keeping with the idea that tree size, and not age, accounts niecki et al. 2004). In support of a functional shift, sample for physiological limits to growth (Koch et al. 2004; Men- age was a far better predictor of parenchyma respiration in cuccini et al. 2005). Clearly, there is still much to learn about Fraxinus (linear regression P < 0.0001; R2 = 0.67, data not parenchyma metabolism and function in secondary xylem, shown) than it was in Acer (P = 0.03, R2 = 0.28, data not and with longevities ranging from 2 to 200 years, these cells shown), which maintains transpiration throughout its may make an interesting model system for cellular ageing sapwood (Spicer et al. 2005). and non-replicative senescence. © 2007 The Authors Journal compilation © 2007 Blackwell Publishing Ltd, Plant, Cell and Environment, 30, 934–943 942 R. Spicer & N. M. Holbrook

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