Trees

, Volume 19, Issue 4, pp 415–421

Is the bark of shining gum (Eucalyptus nitens) a sun or a shade leaf?

Authors

    • School of Forest and Ecosystem ScienceUniversity of Melbourne
  • Charles R. Warren
    • School of Forest and Ecosystem ScienceUniversity of Melbourne
  • Mark A. Adams
    • Centre for Excellence in Natural Resource Management, Faculty of Natural and Agricultural SciencesUniversity of Western Australia
Original Article

DOI: 10.1007/s00468-004-0400-5

Cite this article as:
Tausz, M., Warren, C.R. & Adams, M.A. Trees (2005) 19: 415. doi:10.1007/s00468-004-0400-5

Abstract

Photoinhibition and pigment composition of green stem tissues of field-grown adult Eucalyptus nitens were measured on clear spring days with low morning temperatures—conditions that cause photoinhibition in leaves of many plant species. The sun-exposed (north-facing) bark contained less chlorophyll a+b (531 vs 748 μmol m−2), neoxanthin (29 vs 41), and β-carotene (54 vs 73), more xanthophyll cycle pigments per unit surface area and per unit chlorophyll (71 vs 53 μmol m−2 and 141 vs 66 mmol mol−1 chlorophyll), and less lutein per unit chlorophyll (239 vs 190) than the shaded (southern) side. Maximum electron flow rates were 60 μmol m−2 s−1 on the sun-exposed side, and about 10 μmol m−2 s−1 on the shaded side. Fv/Fm was always lower than 0.8 on the sun-exposed side and the de-epoxidation state (DEPS) of the xanthophyll cycle was dominated by zeaxanthin in midday samples. Fv/Fm increased quickly after darkening, but DEPS recovered more slowly to 40% overnight. This suggested that rapidly reversible pH-dependent quenching was responsible for the bulk of changes in PS II efficiency. Fv/Fm remained below 0.8 overnight, which may well be indicative of photo-damage to PSII. In contrast, DEPS of the shaded side was lower, and Fv/Fm was higher, than for the sun-exposed side. We conclude that E. nitens chlorenchyma on the sun-exposed stem side has a photosynthetic pigment composition similar to sun leaves and it experiences significant photoinhibition in the field.

Keywords

PhotoinhibitionXanthophyll cyclePigmentsCarotenoidsBark

Introduction

Many, if not most, trees contain greenish, photosynthetically active tissues below the outer periderm or rhytidome of twigs, branches and even stems (Pfanz et al. 2002). Species that do not form a thick rhytidomal layer (e.g. Fagus), or that shed thicker bark layers regularly (e.g. Platanus) tend to keep these tissues on older branches and stems. Photosynthesis in these tissues contributes to the overall carbon budget of the tree. For several tree species, it has been estimated that young twigs refix up to 90% of the internal respiratory CO2 loss (Populus, Fagus, Wittmann et al. 2001). This proportion declines with the age of the tissue, but 10-year-old branches still refix up to 45% of respiratory CO2 (reported for Alnus in Pfanz et al. 2002).

The main stem and major limbs of smooth-barked eucalypts (“gum trees”) are typically green owing to the presence of chlorenchyma underneath a deciduous periderm (Chattaway 1953). Refixation of respiratory CO2 is likely to be especially important in smooth-barked eucalypts, because the leaf area index of eucalypt forests and plantations is low compared to, for example, European beech forests, and eucalypt leaves are pendulous. Hence, a greater proportion of incident light penetrates the canopy and reaches the stem and limbs. For aspen, it has been calculated that stems contribute up to 15% of the photosynthetic surface of trees (Schaedle et al. 1968 cited in Wittmann et al. 2001), a percentage that may be considerably higher in eucalypts because of their low leaf area index. This is supported by preliminary measurements in shining gum (Eucalyptus nitens), which indicated that the main stem was refixing between 70% and 75% of respiratory CO2 loss (K. Whittaker and M. Tausz, unpublished data).

In general, photosynthesis in stem chlorenchyma is strongly light limited. This has been confirmed for several tree species inasmuch as chlorophyll concentrations and chlorophyll a/b ratios were typical of shade leaves (Aschan et al. 2001). Unlike leaves, photosynthesis of stem chlorenchyma is unlikely to be limited by CO2 or inhibited by O2 because respiration by non-photosynthetic stem tissues leads to high internal CO2 and low O2 concentrations. Reported minimum O2 concentrations in stems and branches of different tree species are of the order of 0–10 vol%, while estimated CO2 concentrations are 5–800 times ambient (Pfanz et al. 2002).

Photosynthesis in forest trees is limited by photoinhibition under a wide array of environmental conditions (Alves et al. 2002). Photoinhibition—the decline in photochemical efficiency induced by high levels of irradiance—is caused by an imbalance between absorbed light energy and its use in assimilation. Light energy in excess of the assimilatory need is potentially harmful and is either dissipated by photoprotective systems or leads to photodegradation of photosystems. Photoprotective systems include flexible conversion of the xanthophyll cycle (Adams et al. 2002), pH-dependent fluorescence quenching associated with photosynthetic electron transport (Niyogi et al. 2001), and scavenging of harmful active oxygen species. Photodegradation mainly affects the D1 protein in PS II and can be observed as a slowly recovering decrease in PS II quantum efficiency (Maxwell and Johnson 2000). While we might expect a low probability of photo-oxidative stress and photoinhibition in the high CO2 and low light environment of stem chlorenchyma, there have been few confirmatory studies. One study using chlorophyll fluorescence techniques found—in contrast to expectations—a photoinhibitory effect of high internal CO2 on corticular photosynthesis in young twigs of several tree species (Manetas 2004). This suggests that in species where stem photosynthesis is a significant phenomenon, susceptibility to photoinhibition might also be greater than expected.

In the present study we addressed this question and analysed photosynthetic pigments and photoinhibition in bark chlorenchyma of Eucalyptus nitens stems. This species was chosen because of the availability of comparative data on photochemistry, photoinhibition, and photosynthetic pigments in leaves measured with comparable methods under comparable environmental conditions (Close et al. 2001; Hovenden and Warren 1998; Warren et al. 1998). Measurements were made in the field on clear spring days with low morning temperatures—conditions that are known to cause photoinhibition in leaves of this (Hovenden and Warren 1998; Warren et al. 1998) and other species (Adams et al. 2002). We tested two hypotheses: (1) stem chlorenchyma has photoprotective pigments and photochemistry typical of shade leaves, and (2) stem chlorenchyma does not experience significant photoinhibition.

Materials and methods

Research site

This study was conducted at an experimental plantation of Eucalyptus nitens (Deane and Maiden) Maiden located near the School of Forest and Ecosystem Science in Creswick, Victoria, Australia. The trees were 8 years old, about 12-m high, and had diameters at breast height of 24±7 cm. Three individual trees at the northern (sun-exposed) border of the plot were selected.

Measurements were made on four completely clear days in the first 2 weeks of September 2003 (early spring). Morning temperatures were between 1 and 4°C, and afternoon temperatures between 14 and 16°C. Photosynthetic photon flux density (PPFD) was measured with the PAR (photosynthetic active radiation) sensor of the LiCor 6400.

Plant material

Sun-exposed (north-facing) and shaded (south-facing) surfaces of the stem of three trees were selected. Measurements were made at breast height (1.2±0.3 m) of the stems.

Pigment analyses

Pigments were analysed as described in Tausz et al. (2003). In short, bark disks were punched out of the tree, put in pre-labelled vials, and immersed in liquid nitrogen. The material was freeze-dried, ground in a dismembrator under liquid nitrogen (Mixer Mill, Retsch), and extracted in ice-cold acetone. Separation was carried out on a gradient HPLC system (Waters 600E with WISP 701 Autosampler, C18 Spherisorb column, solvent A: acetonitrile:water:methanol 100:10:5 (v/v/v), solvent B: acetone:ethylacetate 2:1 (v/v), gradient 10% B–70% B in 18 min, 4 min on 80% B, return to 10% B in 5 min, 90% A for 5 min) with pigments detected at 440 nm (Waters 486 detector).

Chlorophyll fluorescence

Fluorescence measurements were made using the leaf chamber fluorometer of a Li-Cor 6400 gas exchange system (Li-Cor, USA). To access the stem, the chamber was detached from the Li-Cor head and strapped to the stem surface avoiding intrusion of ambient light. Optimal quantum efficiency of PS II (Fv/Fm) was determined after dark adaptation (using aluminium foil to exclude light) for 10 and 30 min, and overnight. Using the built in software function of the Li-Cor we made sure that the light intensity of the flash used for Fm and Fm′ measurements was saturating. Effective quantum yield of PS II (ΦPSII, calculated as (Fm′−Fs)/Fm′) and heat dissipation rate (calculated as 1−Fv′/Fm′) were measured under actinic light intensity set to the measured ambient conditions at the stem surface. Actinic light in the chamber was switched on before the chamber was mounted to avoid darkening of the measured spot on the bark. Measurements were taken once the fluorescence signal was stable, which took a maximum of 5 min. Definition and interpretation of fluorescence parameters followed Maxwell and Johnson (2000). Electron transport rates (ETR) were calculated as \(\Phi _{\text{PSII}} \times \text{PPFD} \times 0.5 \times \text{trans}_{\text{out}} \times \text{abs}_{\text{green}}\). PPFD is the photon flux density in the PAR range on the stem surface, transout is the light transmission through the bark layers outside of the chlorenchyma, absgreen is the light absorbance in the green tissue, and the factor 0.5 assumes that captured light is distributed evenly between PS I and PS II. Analogous to ETR, the heat dissipation rate (HDR) was calculated as \((1 - F_\text{v} ^\prime /F_\text{m} ^\prime ) \times \text{PPFD} \times 0.5 \times \text{trans}_{\text{out}} \times \text{abs}_{\text{green}}\) (Hovenden and Warren 1998).

Light transmission

Light transmission of the outer layer and light absorption of the green tissue was measured using a LiCor PAR sensor (LiCor, USA) according to Aschan et al. (2001). The chlorenchyma was separated from the layers underneath and mounted on a black paper frame. Light transmission through chlorenchyma + outer layer was determined using the light sensor. Thereafter, the green layer was removed using a scalpel blade, and the transmission through the remaining outer periderm layers was also determined and thus the contribution of chlorenchyma could be determined.

Statistics

Differences between sun- and shade-exposed stem surfaces were evaluated by Mann-Whitney’s two-sample test. Significance of differences between sampling times was calculated by Kruskal-Wallis test followed by cross-comparisons according to Conover (Bortz et al. 1990). P<0.05 is regarded as significant. Reported values are medians and median deviations, which are recommended for relatively small sample sizes (Sachs 1992).

Results

Light reception of the green cortex tissue

The bark (periderm) layers outside the green tissues transmitted 56.5% (5.8) of incident PAR (median and median deviation of n=6, sun-exposed and shaded sides of three trees), while the chlorenchyma absorbed 88.5% (1.0). There were no significant differences in these properties between sun-exposed and shaded sides of the stem. When making measurements under full sunlight (1,500 μmol m−2 s−1 incident on the vertical stem surface) the bark chlorenchyma cells received about 750 μmol m−2 s−1 photons in the PAR range.

Pigment contents

There were clear differences in pigmentation between the sun-exposed and shaded side of the stems. On an area basis, the bark of the sun-exposed stem surface contained less chlorophyll a and b (Fig. 1), neoxanthin, and β-carotene, and more \(V + A + Z\) (the sum of violaxanthin V, antheraxanthin A, and zeaxanthin Z) than the shaded surface (Fig. 2a). On a total chlorophyll basis, there were no differences in neoxanthin and β-carotene, and the sun-exposed side contained more \(V + A + Z\) and lutein (Fig. 2b).
Fig. 1

Chlorophyll contents of bark chlorenchyma in Eucalyptus nitens stems. Shaded south-facing side (black), sun-exposed north-facing side (clear). Medians and median deviations of 2–3 replicate bark disks taken from each of three trees (n=8–9). Numbers report the significance (P) of differences between sun and shade samples estimated by Mann-Whitney U-test

Fig. 2

Carotenoid contents of bark chlorenchyma in Eucalyptus nitens stems. a Based on stem surface area. b Based on total chlorophyll. Shaded south-facing side (black), sun-exposed north-facing side (clear). \(V + A + Z\) sum of violaxanthin, antheraxanthin, and zeaxanthin. Medians and median deviations of 2–3 replicate bark disks taken from each of three trees (n=8 or 9). Numbers report the significance (P) of differences between sun and shade samples estimated by Mann-Whitney U-test

Chlorophyll fluorescence and xanthophyll cycle pigments

The diurnal course of the PS II quantum yield (ΦPSII) indicated photochemical activity in the bark tissues during illumination (Fig. 3). Using the transmittance of the outer layer and the absorbance of the green tissue, maximum ETR was 60 μmol m−2 s−1 on the sun-exposed side, and about 10 μmol m−2 s−1at the shaded side (Fig. 3c).
Fig. 3

Diurnal course of a photosynthetic photon flux density (PPFD) measured on the bark surface, b PS II quantum efficiency (black square, black circle ΦPSII and open square, open circle Fv/Fm), and c electron transport rates (ETR) at southern, shade-exposed (squares) and northern, sun-exposed (circles) bark chlorenchyma of Eucalyptus nitens on clear days. Variations in PPFD between 1000 and 1200 hours are caused by shading due to neighbouring trees. Astronomical sunrise at the measurement days was 0635–0643 hours, and sunset 1805–1811 hours. Fv/Fm was determined after 10 min dark adaptation. Medians and median deviations of 1–2 measurements done within 30 min on each of three different trees (n=3–4)

Optimal quantum yield (Fv/Fm measured after dark adaptation) was always lower than 0.8 on the sun-exposed side, even after overnight dark adaptation (Figs. 3b, 4). When patches of the bark were darkened at midday, the recovery of Fv/Fm was fast for the first 10 min, but changes thereafter were slow and not significant (Fig. 4). The shaded side had Fv/Fm values greater than 0.8 after 10 min of darkening (Fig. 3b).
Fig. 4

Time course of recovery of PS II quantum efficiency and xanthophyll cycle conversion states in north-facing (sun-exposed) bark chlorenchyma of Eucalyptus nitens. Time 0 corresponds to midday values (1330–1400 hours) at a PPFD of 1,300 μmol m−2 s−1 at the bark surface. Subsequent samples were taken from areas darkened by aluminium foil. Medians and median deviations of 1–2 measurements taken from each of three trees (n=4–5). Results from sampling times significantly different from each other have no letter in common. a PS II quantum efficiency (black square) and de-epoxidation status of the xanthophyll cycle (DEPS, open circle) calculated as \((Z + 0.5 \times A)/(V + A + Z) \times 100\). b Xanthophyll concentrations. V violaxanthin (black square), A antheraxanthin (open circle), Z zeaxanthin (black circle)

The de-epoxidation state (DEPS, calculated as \((0.5 \times A + Z)/(V + A + Z) \times 100)\) of the xanthophyll cycle was dominated by zeaxanthin in midday samples of the sun-exposed bark surface (Fig. 4). When the bark was subsequently darkened, the de-epoxidation state changed significantly within 30 min. This change did not correspond with the recovery of PS II quantum yield. Minimum de-epoxidation state after overnight darkening was about 40% on the sun-exposed surface, but further significant recovery of Fv/Fm was not observed (Fig. 4). In shade-exposed bark, midday DEPS was much lower (8±4%), and declined further after 30 min of darkening (3±2%).

Plotting ΦPSII and 1−Fv′/Fm′, respectively, against PPFD shows that the heat dissipation rate exceeded rates of electron transport at about 200 μmol m−2 s−1 at the sun-exposed stem surface (Fig. 5).
Fig. 5

a Light dependence of PS II quantum efficiency (ΦPSII, black circle) and heat dissipation rate (1−Fv′/Fm′, open circle) on northern, sun-exposed stem surfaces of Eucalyptus nitens recorded during one diurnal course. Curves shown employed exponential fit (R2=0.94 for 1−Fv′/Fm′ and 0.92 for ΦPSII). b Calculated electron transport rates (black circle) and heat dissipation rates (open circle). PPFD photosynthetic photon flux density

Discussion

The photosynthetic bark tissues of E. nitens are different to species investigated previously in at least three respects:
  1. 1.

    Light transmission through the outermost (periderm) layers of E. nitens stems is higher than normally reported (Pfanz et al. 2002; Wittmann et al. 2001). Hence chlorenchyma of E. nitens receives more than 50% of the incident photosynthetic active radiation, which at midday translates to maxima of about 750 μmol photons m−2 s−1 on the sun-exposed side.

     
  2. 2.

    Bark of E. nitens contains as much or more chlorophyll per unit area than the leaves of conspecifics grown under a similar irradiance (Fig. 1, compare to 386 μmol m−2 chlorophyll a+b in leaves, Close et al. 2001). These bark chlorophyll concentrations are greater than is normally reported. In other tree species, young twigs have chlorophyll contents that are 70% of those reported for leaves, with older twigs containing 2–3 times less chlorophyll (Pfanz et al. 2002).

     
  3. 3.

    E. nitens bark — even on the shaded stem side — has chlorophyll a/b ratios of about 4 which is surprisingly high and would be typical of sun leaves. In other tree species, chlorophyll a/b ratios from 1.8 to 2.7 are reported for bark chlorenchyma (Pfanz et al. 2002), a ratio that would be more typical of shade adapted tissues.

     

Differences in leaf area index, canopy architecture, and periderm anatomy are almost certainly responsible for the pigmentation of E. nitens bark being typical of sun leaves, while bark of other species investigated so far is more like shade leaves. The high transmittance of PAR through the outermost bark layer, high chlorophyll contents and low leaf area index of eucalypt forests support an assertion that bark of E. nitens will re-fix more CO2 than other species investigated so far. Preliminary investigations on main stems showed refixation rates as high as 0.7 μmol CO2 m−2 s−1 in sunlight (calculated as the difference between light and dark respiration rates, K. Whittaker and M. Tausz, unpublished). Values this high have been reported only in young twigs of aspen and beech (Aschan et al. 2001; Wittmann et al. 2001), and given the observed decrease of refixation with organ age (Aschan et al. 2001), suggest E. nitens may refix as much if not more CO2 than other species. Further research is clearly required.

The carotenoid composition of the green bark tissues has, to our knowledge, not been investigated before, but in the case of E. nitens these tissues seem remarkably similar to leaves. On a total chlorophyll basis, neoxanthin, the xanthophyll cycle pool \(V + A + Z\), and lutein in sun-exposed bark were within or close to the range reported for leaves of E. nitens seedlings (Close et al. 2001), and surface area based \(V + A + Z\) concentrations were even higher. Leaves of E. nitens grown under comparable conditions exhibited 129±26 mmol \(V + A + Z\), 54±18 mmol neoxanthin, and 219±67 mmol lutein per mol chlorophyll a+b during the same season as in our study. Leaf V+A+Z concentrations on an area basis were 50±10 μmol m−2 (Close et al. 2001; values for β-carotene are omitted in that paper). In several other tree species, sun foliage of adult trees contains from 80 to more than 100 mmol \(V + A + Z\) per mol chlorophyll a+b, whereas shade foliage exhibits values at or less than 50 (Niinemets et al. 1998, 2003; Tausz et al. 2005). These ranges are similar to sun- versus shade-exposed bark chlorenchyma in E. nitens and strongly suggest a higher photoprotective capacity of bark tissue on sun-exposed face of stems. The optimum PS II quantum efficiency (measured after overnight dark recovery) was lower, while the diurnal decreases in quantum efficiency and xanthophyll de-epoxidation state were greater, for the sun-exposed compared to the shade surface. These trends are consistent with differences in PPFD received and general observations for sunlit versus shaded foliage (Adams et al. 2002). In addition to differing in carotenoid composition, sun- versus shade-exposed bark also varied in chlorophyll concentrations. Shade-acclimated twigs contain greater concentrations of chlorophyll per unit surface area, with the upper parts of twigs having less chlorophyll than lower, more shaded parts (Pfanz et al. 2002). This is consistent with our study, in which the higher chlorophyll concentrations in bark on the shaded stem side probably represents a greater capacity for light capture. Leaves of E. nitens had 386±106 μmol m−2 chlorophyll a+b when grown under comparable environmental conditions in full sunlight (Close et al. 2001), which is only slightly less than we found in the sun-exposed bark.

Manetas (2004) observed consistently lower rates of photochemistry in bark chlorenchyma of five European tree species compared to the leaves. For example, Prunus cerasus twigs showed an ETR of 10 μmol m−2 s−1 under an irradiance of 500 μmol m−2 s−1 directed at the green tissues, compared to above 100 μmol m−2 s−1 in leaves, i.e. ETR in leaves was 10 times greater. In bark of E. nitens, however, the light dependence of photochemistry and energy dissipation was quite similar to E. nitens leaves (Hovenden and Warren 1998; Warren et al. 1998). Maximal ETR in sun-exposed bark (at a PPFD of about 750 μmol m−2 s−1 directed at the green tissue) was about 60 μmol m-2 s−1, which was more than half of that measured in leaves at comparably low temperatures (maximum of 100 μmol m-2 s−1 at 5°C; at 20°C leaves had an ETR of about 180 μmol m−2 s−1, Hovenden and Warren 1998). In sun-exposed bark, heat dissipation rates exceeded electron transport rates at an incident PPFD of about 200 μmol m−2 s−1, or about 100 μmol m−2 s−1 at the surface of the green tissue. In seedling leaves at 5°C, the corresponding value was 500 μmol m−2 s-1 (Hovenden and Warren 1998). That means that for a large part of each and every sunny day (Fig. 3a) bark on the sun-exposed side of E. nitens has a larger requirement for photoprotective mechanisms than leaves.

The recovery of PS II quantum efficiency and the xanthophyll de-epoxidation state were not in tune. This pattern suggests processes other than xanthophyll conversion were responsible for changes in Fv/Fm. Within the first 10 min of darkening, quantum efficiency recovered quickly, whereas changes in DEPS were not significant. The bulk of the change in quantum efficiency might be attributed to the rapidly reversible, pH-dependent quenching (Niyogi et al. 2001). It was reported recently that high CO2 concentrations in bark tissues would increase the risk of photoinhibition due to acidification processes that limit this energy-dependent quenching (Manetas 2004). This is unlikely the case in E. nitens because a fast-recovering component accounted for most of the light-related decrease in photochemical efficiency, and the persistent component of PS II efficiency decline was much less (pre-dawn Fv/Fm of more than 0.75 compared to values between 0.55 and 0.71 reported by Manetas 2004). It is not known why energy-dependent quenching dominates in bark of E. nitens but is apparently inhibited in other species. It may be speculated that differences in internal stem CO2 concentrations play a role.

Persistent declines in PS II efficiency in sun-exposed bark at midday may well be indicative of damage to PS II. After 30 min of darkness, xanthophylls were significantly more epoxidised (mainly due to conversion of zeaxanthin to antheraxanthin), but the concomitant further small changes in Fv/Fm were not significant. A persistent de-epoxidation of the xanthophyll cycle pool has been reported in foliage of cold-exposed plants and was ascribed to a low-temperature limitation of the enzyme-mediated epoxidation reaction (Öquist and Huner 2003). This might explain persistent reductions in Fv/Fm observed in sun-exposed bark. However, this is an unlikely explanation in the present study because xanthopylls returned to a DEPS of about 40% upon longer dark adaptation, whereas no further recovery in Fv/Fm was observed. This indicates that a persistent decrease in PS II efficiency was not directly related to xanthophyll conversion, but possibly due to photodegradation processes or structural acclimation in the thylakoids (Öquist and Huner 2003).

We conclude that of all bark chlorenchyma investigated so far, the photochemical properties and pigment composition of E. nitens bark tissues are closest to the corresponding traits in leaves reported for this (Hovenden and Warren 1998; Warren et al. 1998; Close et al. 2001) and other tree species. At the sun-exposed stem side, growth irradiance of bark chlorenchyma is high enough to induce photochemistry and pigmentation resembling light-adapted leaves, whereas chlorenchyma at the shaded side is more like shade-adapted tissues (except for a remarkably high chlorophyll a/b ratio). The present results lead us to reject the hypothesis that photoinhibition and photoprotection in stem chlorenchyma does not play an important role. Instead we suggest that bark on the sun-exposed side of E. nitens regularly experiences photoinhibitory conditions, and this can lead to small but persistent reductions in maximal photochemical efficiency.

Acknowledgements

The Australian Research Council is warmly thanked for providing financial support

Copyright information

© Springer-Verlag 2005