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First published online December 5, 2008; 10.1104/pp.108.131417 Plant Physiology 149:1179-1195 (2009) © 2009 American Society of Plant Biologists De Novo Synthesis and Degradation of Lx and V Cycle Pigments during Shade and Sun Acclimation in Avocado Leaves1School of Biochemistry and Molecular Biology, Australian National University, Canberra, Australian Capital Territory 0200, Australia
The photoprotective role of the universal violaxanthin cycle that interconverts violaxanthin (V), antheraxanthin (A), and zeaxanthin (Z) is well established, but functions of the analogous conversions of lutein-5,6-epoxide (Lx) and lutein (L) in the selectively occurring Lx cycle are still unclear. We investigated carotenoid pools in Lx-rich leaves of avocado (Persea americana) during sun or shade acclimation at different developmental stages. During sun exposure of mature shade leaves, an unusual decrease in L preceded the deepoxidation of Lx to L and of V to A+Z. In addition to deepoxidation, de novo synthesis increased the L and A+Z pools. Epoxidation of L was exceptionally slow, requiring about 40 d in the shade to restore the Lx pool, and residual A+Z usually persisted overnight. In young shade leaves, the Lx cycle was reversed initially, with Lx accumulating in the sun and declining in the shade. De novo synthesis of xanthophylls did not affect  - and β-carotene pools on the first day, but during long-term acclimation -carotene pools changed noticeably. Nonetheless, the total change in - and β-branch carotenoid pools was equal. We discuss the implications for regulation of metabolic flux through the - and β-branches of carotenoid biosynthesis and potential roles for L in photoprotection and Lx in energy transfer to photosystem II and explore physiological roles of both xanthophyll cycles as determinants of photosystem II efficiency.
It has long been recognized that photosynthesis in plants must resolve two conflicting requirements, the need to ramp up maximum light-harvesting efficiency in dim light and to wind back to lower efficiency when light is in excess, in order to maintain high rates of growth and productivity in varying light environments (Björkman, 1981  ; Pearcy, 1990). A wealth of research has established that plants adjust through an array of morphological and molecular events that confer photoprotection, mitigate and repair photoinactivation of PSII, and facilitate acclimation of the photosynthetic apparatus over different time scales in response to variable light regimes in wild plants, crops, and algae (Osmond et al., 1999; Demmig-Adams et al., 2006). In the context of the light reactions, low light acclimation optimizes light harvesting and energy transfer to the photosystems, particularly PSII, via enlarged functional antennae, accumulation of accessory light-harvesting pigments, and down-regulation of unnecessary competing photoprotective processes. High light acclimation involves increased photoprotection and photorepair, downsized antennae, fewer photosystems, and sometimes changes in the PSI to PSII stoichiometry (Osmond et al., 1999; Förster et al., 2005). Along with their function in energy transfer to the photosynthetic reaction centers as accessory pigments to chlorophylls, the xanthophyll pigments violaxanthin (V), antheraxanthin (A), and zeaxanthin (Z) play a central role in these transformations of the photosynthetic apparatus, especially in thermal energy dissipation and detoxification of reactive oxygen species.
Two xanthophyll cycles are now known in terrestrial plants, the lutein epoxide cycle (Lx cycle) based on interconversions of lutein-5,6-epoxide (Lx) and lutein (L) synthesized from
The functions attributed to the Lx cycle were initially based on structural analogies between Lx and A and between L and Z (Bungard et al., 1999
Broader issues, such as the roles of short-term dynamics of the two cycles in relation to long-term processes of shade and sun acclimation and in relation to leaf development and age, are poorly understood. Nonfruiting shoots of avocado trees constitute a very suitable model system in which to address these issues. Long-lived leaves of shade-grown avocado contain some of the highest levels of Lx thus far recorded (Esteban et al., 2007 Since there have been very few studies of these complex responses, we carried out a series of short- and long-term light treatments that are likely to reflect what leaves may experience in natural environments, with the aim to gain further insight into the physiological relevance of the Lx and V cycles under those circumstances. Four types of acclimation experiments were undertaken in this study. First, short-term acclimation from shade to sun addressed fast responses to a drastic increase in the light environment, simulating a prolonged sun fleck in shaded mature leaves or exposure to a bright sunny day in young leaves that had emerged during a prolonged overcast (shaded) growth period. These experiments revealed an unexpected loss of L prior to deepoxidation of Lx and V and a reverse Lx cycle in young leaves. Second, long-term acclimation of sun leaves to prolonged shade simulated normal processes of shading by further growth of outer canopy leaves. These treatments established the very slow accumulation of Lx in avocado leaves. Third, sequential sun exposures of mature leaves over several days, followed by continuous shade, were applied to simulate successive prolonged sun flecks, mimicking stochastic canopy disturbance during severe weather events, which confirmed many responses in the above experiments, particularly the very slow epoxidation of L to Lx in prolonged shade. Fourth, long-term acclimation of young and mature leaves to sun was examined. These experiments simulated sudden changes to canopy architecture as experienced during pruning and extended our understanding of the comparative rates and magnitude of Lx and V cycle engagement. We discuss the short-and long-term kinetics of both cycles in avocado leaves of different ages during acclimation, with particular attention to the stoichiometric relationships between xanthophyll and carotenoid pools and changing PSII efficiency.
Table I profiles baseline control measurements (at 6 AM) of carotenoid pool sizes on a chlorophyll basis in shade- and sun-acclimated avocado leaves in relation to age (size) that provide a foundation for our investigations of light responses in the Lx and V cycles in these plants. Young leaves had 50% lower chlorophyll content than mature leaves (Table I) but total carotenoid pools on a chlorophyll basis were similar in both leaf categories, so that total carotenoids per leaf area were substantially larger in mature leaves. The chlorophyll-based concentration of neoxanthin (N), an important structural component of antenna Lhcs, was remarkably stable in both sun and shade leaves and about one-third lower in sun leaves (Tables I and II ). The V+A+Z pool was noticeably larger in sun leaves that also contained higher residual levels of A and Z with age. In contrast, the Lx pool increased with age in shade leaves and to a lesser extent in sun leaves, whereas the L pool declined in mature shade leaves but was more stable in sun leaves. Pool sizes of -C increased markedly with age in shade leaves, β-C pools increased in sun leaves, and chlorophyll a/b ratios were consistently lower in shade leaves, as observed in other species (Thayer and Björkman, 1990; Krause et al., 2001). Photosynthetic efficiency estimated from maximum photochemical efficiency of PSII in the dark-adapted state (Fv/Fm) was slightly lower in sun leaves than in shade leaves, and generally PSII efficiency was highest in mature leaves. Mature shade leaves sampled at the start and at the conclusion of the 3-month experimental period showed stable chlorophyll concentration on a leaf area basis and a trend toward increased total carotenoid concentration on a chlorophyll basis (Table II). The fact that there was no loss of chlorophyll confirmed that mature leaves did not senesce in long-term experiments.
Mature Shade Leaves Exposed Short Term to Sunlight Showed Distinct Effects on the Lx and V Cycle Pigment Pools Typical daily photosynthetic photon flux densities (PPFDs) experienced by leaves of avocado plants during growth in the shade enclosure, and on exposure to sun in the open greenhouse, are shown in Figure 1A . The maximum light intensity in the sun (1,400 µmol photons m–2 s–1) was about 10 times higher than the maximum in the shade (150 µmol photons m–2 s–1). Insignificant or no diurnal change in pigment composition and PSII efficiency in leaves that were kept in the shade indicated that neither of the xanthophyll cycles was active in this growth environment (Table III ). In fact, neither A nor Z were detectable at any time, and Fv/Fm remained constant at its maximum level. When a shade-grown plant was transferred to sunlight in the unshaded glass house at 8 AM (experiment 1), the increase in light intensity from 80 to 290 µmol photons m–2 s–1 by 9 AM was insufficient to activate either xanthophyll cycle in mature leaves (Fig. 1, B, C, E, and F). Interestingly, the first apparent effect on the xanthophylls was a decrease in L during this time (Fig. 1C). With further increases in light intensity, xanthophyll deepoxidation was induced, decreasing both Lx and V at similar rates (Fig. 1, B and E). However, the concomitant increase in L and A+Z exceeded the decline in Lx and V up to 2-fold (Fig. 1, B, C, E, and F). Lutein epoxide levels did not recover after the plant was returned to shade, indicating that epoxidation of L in the Lx cycle was either not occurring or was to slow to be detected. Indeed, L continued to increase for another 2 h in the shade, rising to a steady level throughout the following night (Fig. 1, B and C). The Lx+L and V+A+Z pools were each augmented by 15 to 20 mmol mol–1 chlorophyll (Fig. 1, C and F), indicating that substantial de novo xanthophyll synthesis occurred in addition to the deepoxidation reactions.
In contrast, epoxidation of A+Z was initiated in the shade without delay. The decrease in A+Z was equal to the overnight increase in V, but 50% of the maximum A+Z levels were still present after one night, reflecting de novo synthesis. However, the respective -C and β-C pools that are the substrates for L and A+Z synthesis and the N pool that is synthesized from V did not change significantly (Fig. 1D). Overall, the ratio of total change in -C branch carotenoids and xanthophylls to total change in β-C branch carotenoids and xanthophylls was close to 1 (Table IV
). The deepoxidation state (DPS) of the V cycle and PSII efficiency changed in opposite directions. Increased DPS in the sun was accompanied by more than 50% decrease in Fv/Fm, and neither fully recovered during the following 21 h of shade (Fig. 1G). The two parameters were strongly and linearly correlated (r2 = 0.98; data not shown).
Short-Term Sunlight Exposure of Young Shade Leaves Induced Unexpected Synthesis of Lx and L The substantially lower Lx and higher L concentrations in very young avocado leaves that had emerged in the shade (Table I) led us to examine responses of the Lx and V cycle to strong light in these tissues (experiment 2; Fig. 2 ). In marked contrast to mature leaves, the Lx cycle was initially reversed in young leaves. Levels of Lx increased during sunlight exposure at a rate of 0.6 mmol mol–1 chlorophyll h–1, which was 15 times faster than usually observed in the shade (see below), and Lx actually decreased in the shade overnight, almost returning to initial levels (Fig. 2A). Interestingly, shade-acclimated young leaves contained less than 10% of the Lx found in mature leaves. The L pools increased at a rate of 2 mmol mol–1 chlorophyll h–1 in the first 24 h, both during the sun and the shade, but gradually declined to initial levels over several shade days (Fig. 2B).
Similar to mature leaves, V decreased in sunlight, and deepoxidation of V accounted for half of the increases in both A and Z (Fig. 2, E and F). Epoxidation of A+Z overnight increased the V pool accordingly, and again a small residual amount of A+Z persisted. The Fv/Fm was linearly and inversely correlated with the V cycle DPS (r2 = 0.79; data not shown).
The increase of the Lx+L pool (45 mmol mol–1 chlorophyll) and the V+A+Z pool (30 mmol mol–1 chlorophyll) by de novo synthesis in young leaves (Fig. 2, C and F) was twice as much as in mature leaves. The young leaves had less than half the amount of
Long-term shade acclimation was examined to discover whether sun leaves could accumulate Lx in the shade and reestablish xanthophyll pigment pools characteristic of shade leaves (experiment 3). A shade-grown plant was acclimated to sun for 30 d and then returned to shade for 43 d (Fig. 3
). In spite of the long-term sun exposure, mature and young leaves initially contained amounts of Lx similar to those in shade-grown leaves in experiments 1 and 2. The Lx pools increased in the shade by another 15 to 20 mmol mol–1 chlorophyll in both mature and young leaves (Fig. 3A). However, Lx accumulation (0.041 mmol mol–1 chlorophyll h–1) occurred in mature leaves about 60 times slower than the decrease of Lx by deepoxidation observed during short-term sun exposure (Fig. 1B). In the young sun leaves, Lx accumulation (0.021 mmol mol–1 chlorophyll h–1) was 30 times slower in the shade than in young shade leaves during short-term sun exposure (Fig. 2A). The L pool in mature leaves increased transiently for 1 d after transfer to shade and then decreased by 50 mmol mol–1 chlorophyll (about 2-fold more than the increase in Lx) to stable levels (Fig. 3B). Young leaves showed a similar, transient increase (compare with Fig. 2, B and C), followed by a stronger decline in L pool size (about 90 mmol mol–1 chlorophyll within 7 d), before recovering about 50% of this loss over the next 20 d. In contrast to mature leaves that showed no changes in
V cycle pigment pools generally showed less variation during shade acclimation, and V+A+Z pools were not changed significantly after 43 d (Fig. 3, E–G). The transient increase and decrease of V during the first shade day was more prominent in young leaves and coincided with similar increases in β-C (Fig. 3H). Only traces of A (and no Z) were detectable in both leaf types during the first 4 d, when the V cycle was fully epoxidized (Fig. 3F). The transient increases in V during the first shade day coincided with similar increases in β-C (Fig. 3H), and pools of V or A showed no significant changes after long-term shade acclimation (Fig. 3, E and F). The (C+X) and β(C+X) pools declined by the same amounts in leaves of both age categories, and the ratio of change was about 1 (Table IV). Thus, long-term shade acclimation of sun leaves primarily affected the pools of Lx cycle pigments in both mature and young leaves and -C specifically in young leaves. On the whole, the acclimation process seems to be associated with larger fluctuations of pigment pool sizes in the young leaves.
To gain insight into the effect of intermittent sun exposure on long-term shade acclimation, a shade-grown plant was exposed to sunlight from 6 AM to 2 PM on days 0, 2, and 9, followed by uninterrupted shade until day 44 (experiment 4). Frequent sampling for pigments and measurements of chlorophyll fluorescence during the first diel (data not shown) confirmed the response pattern of mature shade leaves to short-term sun exposure shown in Figure 1. Changes in pigment composition showed two phases that distinguished days 0 to 10 (intermittent sun exposure) from days 10 to 44 (unperturbed shade). In agreement with experiment 1, sun exposure on days 0 and 2 initiated substantial deepoxidation of Lx and V and de novo pigment synthesis in mature shade leaves, but sun on day 9 had no additive effect (Fig. 4 ). The increase in L (100 mmol mol–1 chlorophyll), initiated by two separate days of sun, doubled the L pool and was much larger than the decrease in Lx (30 mmol mol–1 chlorophyll) that reduced the Lx pool to 25% of its original size (Fig. 4, A and B). The net increase of the Lx+L pool indicated that de novo synthesis of L was strongly induced in this first phase. During the following unperturbed 35 d in the shade, Lx and L pools readjusted to their initial levels. The 3-fold greater decline in L than increase in Lx during this phase suggested that L epoxidation was accompanied by other turnover processes. Moreover, the extent of truncation of the Lx cycle was apparent from the approximately 130 times faster rate of Lx deepoxidation on day 0 (3.5 mmol mol–1 chlorophyll h–1) compared with L epoxidation from days 10 to 44 (0.027 mmol mol–1 chlorophyll h–1).
Changes in the V cycle pigment pools were less pronounced than in the Lx cycle (Fig. 4, D and E), and although A+Z increased (from 25 to 37 mmol mol–1 chlorophyll) in the sun as expected (2 PM data points not shown), 5 to 15 mmol mol–1 chlorophyll A+Z was detected after each night at 6 AM for many days (Fig. 4E). Part of the overnight epoxidation of the A+Z pool during days 0 and 2 contributed to the substantial increase in V (24 mmol mol–1 chlorophyll) that persisted almost throughout the experiment, but the doubling of the V+A+Z pool indicated de novo synthesis of V cycle pigments in addition. The residual A+Z was reflected in sustained DPS (Fig. 4F) and was highly correlated with the Fv/Fm (r2 = 0.88; Fig. 7C) that declined following sun exposure on days 0 to 2 from 0.74 to 0.41 and that recovered to about 80% during long-term shade. As observed previously, there were no significant changes in the -C or β-C pool sizes during sun exposure on days 0 and 2. However, the decline in -C (35 mmol mol–1 chlorophyll) over 9 d was matched by an increase in the pool of β-C (Fig. 4C), and the -C to β-C ratio declined from 1.9 to 0.6.
Long-Term Sun Acclimation of Mature and Young Shade Leaves Showed Large Increases in Both Lx and V Cycle Pigments
Long-term changes in the pigment composition of mature and young shade leaves during sun acclimation were examined in experiment 5 (Figs. 5
and 6
). Mature leaves showed the expected 60% deepoxidation of Lx (about 10 mmol mol–1 chlorophyll) when first exposed to sunlight on day 0 (Fig. 5A) but, uncharacteristically, the Lx pool increased slightly from day 10 onward. Young leaves had much lower Lx levels initially (15% of mature leaves), showed the same light stimulation of Lx synthesis and lack of Lx deepoxidation as in the short-term sun exposure (Fig. 2), and accumulated Lx (0.009 mmol mol–1 chlorophyll h–1) to the same level as in mature leaves in the long term (Fig. 5A). The L pool in mature leaves rapidly increased over the first 10 d of sun exposure (Fig. 5B), doubling the pool size, and by day 39 there was still 50% more L than at day 0. The decrease in Lx (10 mmol mol–1 chlorophyll) and
The V pool in mature leaves increased much more during the first 18 d (55 mmol mol–1 chlorophyll) than in young leaves over 39 d (10 mmol mol–1 chlorophyll) in the sun (Fig. 5E). Overnight, both leaf types retained similar A and Z pools (up to 26 mmol mol–1 chlorophyll A+Z), so that the V cycle was never fully epoxidized throughout the sun acclimation period (Fig. 5, E and F). De novo synthesis increased the total V+A+Z pool by about the same amount as the β-C pool (Fig. 5, G and H). Interestingly, the increase in (C+X) during long-term sun acclimation was only about half that of β(C+X) in leaves of both age classes (Table IV).
The functional state of the V cycle was tracked throughout the experiment by comparing the differences in V, A, and Z levels (
Correlations between PSII efficiency (Fv/Fm), DPS, and differential Lx and V cycle pigment accumulation of potential physiological relevance were investigated during sun and shade acclimation (Fig. 7 ). Strong inverse linear correlations were found between Fv/Fm and DPS of the V cycle throughout each diel during long-term sun acclimation (experiment 5), in accordance with the general notion that accumulation of A+Z reduces PSII efficiency. Interestingly, the slope of the regression line was much steeper during the first diel after transfer from shade to sun than during the diels after 10, 19, and 39 d in the sun (Fig. 7A). Likewise, decrease in Fv/Fm correlated well with increased DPS during long-term shade acclimation interrupted by intermittent sun exposure (experiment 4). The slope of declining Fv/Fm against increasing DPS in sunlight during the initial diels (days 0 and 2) was half the slope found between increasing Fv/Fm and decreasing DPS (due to residual A+Z at 6 AM) during the slow recovery of PSII efficiency in the following 34 unperturbed shade days (Fig. 7B). In addition to the strong inverse correlation between decline in Fv/Fm and increase in A+Z pool size throughout long-term shade acclimation in this experiment (Fig. 7C), there was a direct, but weaker, correlation between the decline in Fv/Fm and that in Lx (Fig. 7D). Clearly, there was a link between Fv/Fm, DPS, or Lx, but many factors, such as duration of light treatments and/or the age of the leaves, modified these relationships.
Loss of Lutein Precedes Engagement of the Lx and V Cycles and De Novo Synthesis of Xanthophylls
Our experiments on shade-to-sun and sun-to-shade acclimation in avocado, a species in which shade leaves accumulate high concentrations of Lx, revealed a variety of novel adjustments in carotenoid pigment pools that may have roles in optimizing photosynthetic efficiency in changing light environments. An interesting observation was that exposure of a shade-grown plant to the natural, gradual increase in irradiance to 1,400 µmol photons m–2 s–1 over 7 h in the sun caused an initial, small decline in L (5–15 mmol mol–1 chlorophyll) prior to measurable deepoxidation of Lx or V (Figs. 1 and 8
). A similar but more rapid loss of L over the first 15 to 120 min was also observed when avocado plants were transferred abruptly from shade to full sunlight (Esteban et al., 2008
Implications for the Concurrent Operation of VDE and ZE in the Lx and V Cycles in Avocado Leaves
Restoration of the Lx pool after fast deepoxidation in just hours of sun exposure is exceptionally slow in mature avocado leaves (Figs. 1, 4, and 8). A month or more in deep shade is required, which is 1 to 2 orders of magnitude slower than in other Lx-accumulating species and represents the slowest recovery of Lx so far reported (García-Plazaola et al., 2007
Other processes may also contribute to the slow recovery of Lx pools in avocado. For example, residual VDE activity in the shade could delay Lx accumulation. However, this seems less likely because VDE requires a low lumen pH of about 5 for association with the antenna, which is a prerequisite for activation (Hieber et al., 2000
Our comparative analysis of mature and young leaves of shade-grown plants opens up even more interesting features of Lx cycle regulation in avocado. Whereas mature leaves showed the typical truncated Lx cycle, the Lx cycle in young leaves was fundamentally different and appeared to function in reverse. During short-term sun exposure, Lx accumulated in the sun and decreased in the shade (Figs. 2 and 8). In fact, this Lx accumulation in the sun was 1 order of magnitude more rapid in young leaves than the slow recovery of Lx pools in mature leaves in the shade. This unusual response is clearly related to the developmental stage of the leaf, since it disappeared between 10 and 20 d as young leaves grew older in sunlight (experiment 5; data not shown). The Lx accumulation in sunlight may be related to the fact that young leaves are sink rather than source leaves (Schaffer et al., 1991
Since ZE activity is evidently not confined to very low light and darkness (Frommolt et al., 2001
De novo synthesis of L and to a lesser extent of A and Z during sun acclimation may be a distinctive feature of avocado and a few other species (e.g. Anarcardium excelsum; Krause et al., 1999
During the first day after transfer to sunlight, most of the increase in L in mature and especially in young leaves can be attributed to de novo synthesis, most of which occurred in the light (Figs. 1 and 2; Table IV). Likewise, additional A and Z were synthesized in young leaves in these experiments, but synthesis of L exceeded that of total V+A+Z by up to 40%. In both cases, carotenoid pools remained unchanged. The flux ratios (de novo synthesis of xanthophyll-pool of carotene substrate) in mature leaves were about 0.5 (Fig. 1), implying that half the
Although flux regulation of de novo xanthophyll synthesis within the
Substrate-product relationships in the
Evidently, the activities of β,
There is little doubt that L has essential functions in environments of excess light. It has been proposed that L might augment photoprotection by the β-C pool in Chlamydomonas reinhardtii (Trebst and Depka, 1997
The best understood function of the V cycle is its relationship to photoprotection and PSII efficiency. The DPS of the V cycle has been viewed as an indicator of NPQ capacity (Demmig-Adams and Adams, 1992
The lower slope of the Fv/Fm versus DPS relationship during the first diel of sun exposure compared with that during the slow recovery of PSII efficiency during 44 d in the shade (Fig. 7B) may be due to the slowly reversible conversion of L to Lx. It has been proposed that photoconverted L "locks in" photoprotective energy dissipation in the shade (Matsubara et al., 2005
In addition to the putative protective function of photoconverted L in photoprotection, recent studies with Inga species suggested that the accumulation of Lx in the shade in long-lived inner canopy leaves of trees may enhance light-harvesting efficiency. In vitro experiments with thylakoid preparations and reconstitution experiments with recombinant Lhcbs, as well as analysis of fast chlorophyll fluorescence transients in vivo, indicated that less energy is lost by thermal dissipation in the presence of high Lx, possibly facilitating energy transfer between chlorophyll molecules (Matsubara et al., 2007
This study revealed an intriguing array of relationships among the pigments of the - and β-branches of the carotenoid pathways during short-term sun exposure and long-term shade and sun acclimation in shade-grown avocado leaves that are summarized schematically and semiquantitatively in Figure 8. These observations place the relationship of the Lx and V cycles in a new context, suggesting that L may initially confer protection from photooxidation and that subsequent de novo synthesis of L and A+Z may dominate the presumed functional roles of these pigments in the slowly and rapidly reversible down-regulation of light-harvesting efficiency in the antennae of photosystems. It becomes clear that there are significant ontogenetic differences in the light acclimation response, exemplified by the initial sun acclimation response of young leaves associated with reverse Lx cycle activity. In spite of large variations in the -C and β-C branch carotenoid and xanthophyll pigments, the balance between fluxes through the -C and β-C branches was equal irrespective of the acclimation processes. Understanding the extent to which these interactions of the two xanthophyll cycles sustain the performance of long-lived avocado leaves in the shade and their contribution to overall canopy photosynthesis, growth, and yield in avocado and other species becomes an exciting direction for future investigation.
Plant Material and Growth Conditions
Avocado (Persea americana ‘Edranol’) seedlings (60 cm), purchased from Vallance's Nursery in September 2005, were kept in a deeply shaded part of a temperature-controlled (28°C day/18°C night) glasshouse in Canberra. Maximum PPFDs were 50 to 150 µmol photons m–2 s–1 at 12 PM. Seedlings were maintained in their original 8-L containers of potting soil with regular irrigation and additions of slow-release nutrients until preliminary experiments were conducted in November 2005 (García-Plazaola et al., 2007
Plants (1–1.5 m tall) with suitably displayed leaves (i.e. not subject to self-shading) and of requisite age and size were selected so that four similar leaves in each age category were sampled repeatedly by excising leaf discs from the same plant for pigment and fluorescence analysis throughout the experiments. Biological replication was achieved by repeating experiments on comparable leaves on different plants at different times but under similar conditions. Two leaf size categories were used to analyze the influence of age and developmental stage: mature leaves were fully expanded (18–25 cm length), whereas young leaves were rapidly expanding. Short-term responses (1–5 d) of the Lx and V cycle pigments upon transfer of shade-grown leaves to sun were examined in plants moved to the open glasshouse for exposure to natural sunlight on cloudless days. In experiment 1, the plant was transferred to sun at 8 AM and returned to shade at 3 PM after 7 h of exposure to light intensities that increased from 80 to 1,400 µmol photons m–2 s–1. Mature leaves were sampled frequently before, during, and after these transfers and at 6 AM the next day. Comparable mature leaves on another plant that remained in the shade enclosure were sampled throughout the day as controls. In experiment 2, young leaves in early (y1; 6–7 cm) and later (y2; 11–13 cm) stages of expansion were sampled before the plant was transferred to the open greenhouse at 6 AM (<10 µmol photons m–2 s–1), after 8 h of sun exposure directly before plants were returned to the shade enclosure at 2 PM, and at 6 AM the next morning (6-2-6 protocol). In the following shade growth period, samples were taken at 6 AM on days 2 and 5. Mature leaves were also sampled as additional controls. Two types of long-term shade acclimation experiments were performed to examine the truncated nature of the Lx cycle in avocado leaves. In experiment 3, well displayed young (y2, as above) and mature leaves of the current growth flush on a plant that had been acclimated to sunlight for 30 d were analyzed during the 6-2-6 diel on day 0 to establish a baseline for comparison with other experiments and then at 6 AM at intervals during the following 43 d in the shade. This experiment simulated the shading of outer canopy leaves that could be expected in rapidly growing shoots of avocado trees in the field. In experiment 4, a shade-grown plant was transferred to sun at 6 AM and returned to shade at 2 PM on days 0, 2, and 9. Samples were taken from mature leaves according to the 6-2-6 protocol during these transfers. Subsequently, unperturbed shade acclimation was followed in samples taken at 6 AM at intervals until day 44. This experiment may reasonably reflect natural events that temporarily disturb inner canopy light exposures. Long-term sun acclimation was examined to compare the engagement of the Lx and V cycles in young and mature leaves. This situation may arise when the outer canopy structure is permanently altered by natural factors (e.g. wind damage) or management procedures (e.g. canopy pruning to reduce water use). In experiment 5, a shade-grown plant was transferred to sunlight for 39 d. Leaf samples were collected using the 6-2-6 protocol on days 0, 9, 19, and 38.
Leaf discs (1 cm in diameter) were cut out from tip to base of attached leaves for pigment and fluorescence analyses. Chlorophyll fluorescence parameters were determined on leaf discs after 30 min of dark adaptation using the PEA fluorometer (Hansatech) according to the manufacturer's protocol. Maximum efficiency of PSII was derived as Fv/Fm = (Fm – Fo)/Fm (van Kooten and Snel, 1990
We are grateful to Matthew Gordon for technical support in HPLC measurements, to Steve Dempsey for maintenance of the avocado seedlings, and to Fred Chow for access to chlorophyll fluorescence measuring equipment. Received October 16, 2008; accepted December 1, 2008; published December 5, 2008.
1 This work was supported by the Australian Research Council (grant nos. DP0666289 to C.B.O. and CE056195 to B.J.P.). 
2 Present address: School of Biochemistry and Molecular Biology, Australian Research Council Centre of Excellence in Plant Energy Biology, Linnaeus Way, Building 41, Australian National University, Canberra, Australian Capital Territory 0200, Australia. The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Britta Förster (britta.forster{at}anu.edu.au). www.plantphysiol.org/cgi/doi/10.1104/pp.108.131417 * Corresponding author; e-mail britta.forster{at}anu.edu.au.
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ABSTRACT
RESULTS
Car; mmol mol–1 chlorophyll) and total chlorophylls (
) in pool size were assessed by comparing the amounts of pigments at the beginning and end of the respective sun and shade periods. Negative values indicate decreases in total pool size.
- and β,β-cyclase, which could determine overall flux from all-trans-lycopene to 
ka K (2004) The xanthophyll cycle: molecular mechanism and physiological significance. Acta Physiol Plant 26: 197–212
