Arabidopsisplants grown in the field and climate chambers significantly differ in leaf morphology and photosystem components
© Mishra et al; licensee BioMed Central Ltd. 2011
Received: 15 April 2011
Accepted: 11 January 2012
Published: 11 January 2012
Plants exhibit phenotypic plasticity and respond to differences in environmental conditions by acclimation. We have systematically compared leaves of Arabidopsis thaliana plants grown in the field and under controlled low, normal and high light conditions in the laboratory to determine their most prominent phenotypic differences.
Compared to plants grown under field conditions, the "indoor plants" had larger leaves, modified leaf shapes and longer petioles. Their pigment composition also significantly differed; indoor plants had reduced levels of xanthophyll pigments. In addition, Lhcb1 and Lhcb2 levels were up to three times higher in the indoor plants, but differences in the PSI antenna were much smaller, with only the low-abundance Lhca5 protein showing altered levels. Both isoforms of early-light-induced protein (ELIP) were absent in the indoor plants, and they had less non-photochemical quenching (NPQ). The field-grown plants had a high capacity to perform state transitions. Plants lacking ELIPs did not have reduced growth or seed set rates, but their mortality rates were sometimes higher. NPQ levels between natural accessions grown under different conditions were not correlated.
Our results indicate that comparative analysis of field-grown plants with those grown under artificial conditions is important for a full understanding of plant plasticity and adaptation.
KeywordsArabidopsis thaliana Carotenoids Chlorophyll fluorescence Early light inducible proteins (ELIPs) Field Plants Indoor Plants Light harvesting proteins (LHCs)
Much of our understanding of plant growth, development and metabolism has come from studies--often using Arabidopsis thaliana as a model system--based on laboratory-grown specimens. Nevertheless, plants exhibit huge phenotypic plasticity and respond to differences in environmental conditions by acclimation [see for example [1, 2]], hence environmental conditions greatly influence the outcome of studies. Field studies are generally rare because (inter alia) the photoperiod, temperature and light intensity are not controlled and growth conditions are difficult to reproduce. However, in a few studies Arabidopsis grown in natural environments has been used to study, for example, reproductive timing, fitness-related quantitative traits and flowering time [e g [3–6]]. The main rationale for performing experiments under controlled conditions in growth cabinets or climate chambers is to minimize variations in measured traits apart from those due to applied treatments. However, even in the laboratory conditions are likely to vary to some extent, thus experimental results obtained using different brands of climate chambers, different standard procedures and different equipment in different laboratories are also likely to vary to some degree. The variations in the field are much greater, but few authors acknowledge that acquired results are strongly influenced by the growth conditions employed, and even fewer consider how the results may have differed had the experiments been performed under conditions that plants are actually adapted to, i.e. variable field conditions. Thus, the emphasis on controlling growth parameters to allow comparative investigation of plant physiology can provide valuable information, but it also constrains our understanding of how plants adapt to field conditions.
Due to the limitations outlined above, there is a need for comprehensive investigations of field-grown specimens to evaluate phenotypic characteristics expressed in plants grown under natural conditions, in which conditions are not controlled. We have therefore developed procedures and tools for analyzing field-grown Arabidopsis plants, including mutants and transgenics, in "semi-natural" conditions . We have also shown that mutants exhibiting no obvious phenotypic variation under laboratory conditions can suffer significant loss of fitness . For these reasons, studies on field-grown Arabidopsis (e.g. using high-throughput DNA microarray and metabolomics techniques) may be more informative for assessing plants' responses in real environments than those performed under controlled conditions . This raises complex problems, since a key characteristic of field conditions is that they vary in unpredictable ways, resulting in phenotypic variations among field-grown plants in each experiment, even at the same site. Responses to different plant ecotypes adapted to different environments are also likely to vary significantly at field sites. Nevertheless, failure to address these problems will inevitably constrain our understanding of plant responses.
Leaf traits, including those relating to photosynthesis, have particularly plastic responses to the growth environment. Various leaf acclimation responses have been recorded at many levels, from whole-plant morphology down to the stoichiometry of the photosynthetic apparatus [10, 11], for example, adjustments in reaction center stoichiometry and Rubisco levels [12, 13]. Pronounced changes in response to environmental variations have been well documented in levels of photosynthetic antenna, i e pigments and pigment-binding light-harvesting chlorophyll-binding (LHC) antenna proteins, Lhca protein and Lhcb proteins associated with photosystems I (PSI) and II (PSII), respectively, and in other members of the light-harvesting chlorophyll-binding (LHC) "superfamily", notably PsbS  and early light induced proteins (ELIPs) [15, 16]. Changes in light-harvesting pigments and proteins influence several photosynthetic parameters, e.g. the capacity for qE energy-dependent non-photochemical quenching (NPQ) or feedback de-excitation, which harmlessly dissipates excess absorbed light energy as heat, and the xanthophyll cycle (XC) pool size differs both between species [17, 18] and during acclimation [19–21]. Much less is known about how the light regime influences so-called state transitions in which the excitation energy inputs into the two photosystems  are balanced by reversible phosphorylation of the LHC proteins catalyzed by Stn7 kinase  and Pph1 phosphatase . However, this process too may be profoundly affected by environmental variations in the field.
A comparative analysis of Arabidopsis plants grown under various light intensities has been published , but the "high light" conditions used in this and other laboratory studies, typically 600-800 μmol quanta m-2 s-1, are equivalent to rather "low light" in the field, where light intensities on sunny days can exceed 2000 μmol quanta m-2 s-1. A systematic comparison between Arabidopsis plants grown in the laboratory with those grown under field conditions could therefore be informative for optimizing field-growth and reproducibility in future experiments. Hence, in the study presented here we examined Arabidopsis plants grown under three different light intensities in climate chamber conditions and related the magnitude of differences among them to those observed in field-grown plants. We also examined in more detail than previously changes in a number of regulatory processes whose importance our previous data suggest could be over- or under-estimated when analyzed plants are grown under "unnatural laboratory conditions" in terms of light intensity and lack of fluctuations in light and temperature. We used field-grown plants as references, as we believe that they best reflect the status of plants under the growth conditions to which Arabidopsis is adapted. Our comparison shows that Arabidopsis plants in climate chambers are similar in many respects to those grown in the field, but we also pinpoint some parameters for which extrapolating results from analyses of plants--in particular those grown under short day (SD) photoperiods--in controlled conditions to plants grown under natural conditions could be misleading.
Plants grown indoors have enlarged leaves, different leaf shapes and longer petioles
Indoor plants have much less xanthophyll cycle pigments
Carotenoid contents of Arabidopsis grown indoors under low light (LL), normal light (NL) and high light (HL) conditions, and in the field
1.4 ± 0.3b
1.0 ± 0.2a
1.1 ± 0.1a
1.5 ± 0.1b
1.4 ± 0.2a
1.6 ± 0.1b
2.5 ± 0.1c
8.5 ± 0.2d
0 ± 0
0 ± 0
0.24 ± 0.1a
1.4 ± 0.1b
0 ± 0
0 ± 0
0 ± 0
1.3 ± 0.1a
1.4 ± 0.1a
1.6 ± 0.1b
2.74 ± 0.1c
11.2 ± 0.1d
13.5 ± 0.9b
12.4 ± 0.4a
15.3 ± 0.1c
28.1 ± 0.2d
2.3 ± 0.2c
2.4 ± 0.1b
1.4 ± 0.1a
1.4 ± 0.1a
Lhca5, a component of PSI antenna complex, is significantly reduced in field-grown plants
Indoor plants accumulate high levels of Lhcb1 and Lhcb2
Early light-induced proteins (ELIPs) are absent from indoor-grown plants
Indoor plants have a much-reduced NPQ (non-photochemical quenching) capacity
Field-grown plants containing small amounts of Lhcb1 and Lhcb2 still perform state transitions
State transition parameters in LL, NL, HL and field grown plants
0.69 ± 0.015a
0.79 ± 0.016c
0.73 ± 0.05b
0.72 ± 0.08b
0.017 ± 0.005a
0.045 ± 0.001a, b
0.049 ± 0.014b
0.058 ± 0.011b
t 1/2 (s)
146.61 ± 7.41a
197.69 ± 0.85b
158.46 ± 1.30c
115.0 ± 4.35d
Photoperiod is the main determinant of leaf size and shape, other factors are more important for photosynthetic traits
Variation in chlorophyll, leaf traits, Lhca 5, ELIP I, ELIP II, non-photochemical quenching (NPQ) and state transition parameters
HL (short day)
Leaf area (mm2)
531.28 ± 123.73
175.608 ± 40.40
61.75 ± 30.01
Leaf length (mm)
37.96 ± 5.05
20.76 ± 2.65
11.10 ± 3.04
Leaf width (mm)
18.39 ± 2.59
11.55 ± 1.24
7.0 ± 2.1
Leaf width: Leaf length ratio
0.48 ± 0.017
0.56 ± 0.03
0.63 ± 0.15
Chlorophyll content μg cm-2
28.23 ± 2.96
15.23 ± 0.57
18.76 ± 0.23
2.96 ± 0.18
3.56 ± 0.18
3.70 ± 0.10
38.49 ± 2.20
33.93 ± 1.30
9.36 ± 0.65
0.0 ± 0.0
0.0 ± 0.0
63.14 ± 3.34
0.0 ± 0.0
0.0 ± 0.0
22.77 ± 2.45
0.84 ± 0.005
0.84 ± 0.007
0.81 ± 0.009
2.15 ± 0.13
2.53 ± 0.29
3.10 ± 0.25
1.97 ± 0.11
2.35 ± 0.12
2.92 ± 0.23
0.73 ± 0.05
0.69 ± 0.02
0.72 ± 0.08
0.049 ± 0.014
0.044 ± 0.004
0.058 ± 0.011
158.46 ± 1.30
166.05 ± 1.91
115.0 ± 4.35
ELIPs are dispensable under field conditions
Arabidopsis mutants affected in xanthophyll synthesis and metabolism have been extensively studied by our research group  and others , but mutants lacking ELIPs have been less thoroughly characterized. However, in a recent study a double knock out (KO) mutant lacking both ELIP proteins, ELIP1 and ELIP2, was generated. This mutant did not exhibit obvious phenotypic deviations from wild-type in growth traits when grown under photoinhibitory conditions . In the light of findings by us and others  that ELIPs can accumulate to high levels in field-grown plants, we analyzed growth and silique production of the ELIP double mutant in the field in two different years. In terms of both growth and visible phenotype, double ELIP mutants were indistinguishable from wild-type plants. In 2008 the number of siliques produced by wild type-plants and the ELIP double mutant were not significantly different (201 ± 19 and 178 ± 14, respectively), but the ELIP double mutants had a lower survival rate in the experiment (57%) compared to wild-type plants (83%). In 2009, neither survival (100 vs. 93%) nor the number of siliques (11.6 ± 1.4 vs. 14.3 ± 1.7) differed significantly between wild-type and ELIP double mutants.
NPQ levels in Arabidopsisaccessions grown in the field and indoors are not correlated
Discussion and Conclusions
In recent decades our understanding of the molecular basis of photosynthesis has increased impressively. It is increasingly evident that the fundamental structure of the photosynthetic apparatus is an example of the capacity for complex, highly sophisticated systems to evolve, since cyanobacteria, green algae and higher plants (which apparently diverged hundreds of millions of years ago) have very similar photosynthetic machineries [39, 40]. The main differences in the photosynthetic apparatus of these taxa are in the "peripheral" parts, such as the antenna systems. For example, the phycobilisomes of cyanobacteria have been replaced with the LHC proteins in green algae and higher plants, and there are wide variations in their photosynthetic pigments, many of which were previously used as key taxonomic descriptors. Much regulation is exerted by the antenna systems, where the qE type of NPQ (feedback de-excitation) and state transitions occur in conjunction with dynamic changes in antenna size in acclimation responses to, inter alia, changes in light conditions . More recently, the less abundant LHCI proteins Lhca5 and Lhca6 have been implicated in the regulation of cyclic electron transport , which is known to be subject to both evolutionary adaptation and environmental acclimation [see e.g. ]. Comparative studies of plants from diverse taxa, ecological niches or habitats (field or laboratory) show that the regulatory properties of the antenna systems typically vary more than the properties of the "core engine" of the system .
Twenty-five years ago, Arabidopsis emerged as the prime model organism for plant biology research . Its small size and rapid growth cycle has enabled photosynthesis researchers to move from experiments with "synthetic" (e.g. algal cultures) or imprecise (e.g. spinach) systems to more reproducible experiments with plants grown under highly controlled and reproducible conditions in climate chambers, growth rooms and cabinets. Although this has been important for scientific development, we believe that studies performed with plants grown under natural conditions can provide valuable complementary information. This study is a contribution to the growing body of literature describing experiments in which Arabidopsis has been exploited as a natural species rather than a "laboratory rat". In the wild, Arabidopsis grows in open, typically highly-disturbed, habitats and has significant capacity for photosynthetic acclimation . Therefore, whether or not Arabidopsis plants grown in climate chambers are like those grown in the field, which may seem trivial, is highly relevant for scientists addressing many aspects of plant biology. One aspect not covered in this work is the natural variation of the species; it is possible that our results may have been substantially different had we chosen to study a different accession rather than Colombia-0. We chose this accession because it has been used in most studies published to date; however this accession is not specifically adapted to our local study site environment in Umeå. Furthermore, as we have not compared plants grown at different sites, at different times of the year or with different photoperiods we cannot draw general conclusions about the plasticity of Arabidopsis in all environments. Additional phenotypic variations may be encountered in future experiments, and we make no claim that other field-grown Arabidopsis plants will necessarily be similar to those analyzed here. Nevertheless, we believe that the trends we have recorded are likely to represent some of the most prominent differences between indoor- and field-grown Arabidopsis plants. It is also obvious that plants grown in climate chamber under LD are better substitutes for field-grown plants than plants grown under SD--which is typically used for photosynthetic studies--although plants grown indoors under LD were still more similar in terms of photosynthetic characteristics to SD plants than to field-grown specimens (Table 3). Further studies are needed to determine if variations in LD conditions in climate chambers (e.g. 14, 16, 18 or 20 h photoperiods) significantly influence photosynthetic characteristics.
Our data show that the indoor-grown SD plants had, for example, different leaf morphology, higher levels of Lhca5, much higher levels of Lhcb1 and Lhcb2, less PsbS (and no ELIPs), and different pigment contents compared to the field-grown plants. In particular they were strongly depleted in xanthophyll cycle pigments. The differences in leaf morphology and plant stature are striking, and it is intriguing that some of the observed changes, for example in leaf size, did not follow simple patterns, notably both LL- and field-grown plants had smaller leaves than NL- and HL-grown plants. This indicates leaf developmental patterns are influenced by more than one factor. For example, several "typical photoreceptors" may respond both to differences in photoperiod and light intensity, and photosynthetic signals may influence leaf morphology. Accordingly, anatomical differences between typical sun and shade leaves seem to depend on photosynthetic signals . We also found that Lhca5, expression of which correlates well with light intensity in indoor plants, was almost undetectable in the field-grown plants. It appears, therefore, that (at least in Arabidopsis) Lhca5 is not simply a "light stress LHC", as exemplified by LI818 in Chlamydomonas , since it was down-regulated under our field conditions. It has been suggested that both the Lhca6 protein, which is present at very low levels in plants grown under most conditions, and Lhca5 regulate cyclic electron transport around PSI . However, we are not aware of any published analyses of the cyclic electron transport capacity of field-grown plants.
Most or all PSI and PSII core proteins are present in unit stoichiometry and this also probably applies to the PSI antenna proteins Lhca1-4 and the minor Lhcb antenna proteins Lhcb3-6. Our data show that the PSI antenna in the plants grown indoors was similar to that of the plants grown under field conditions but--as we have noted before--the PSII antenna may be more flexible. On a PSII basis, the levels of Lhcb5 (CP26) and, in particular, Lhcb6 (CP24) were lower in indoor plants, raising questions whether PSII centers lacked these proteins in the indoor plants, or a fraction of the proteins was present, but they were not bound in their "normal positions" in PSII in the field-grown plants (or both). Our results relating to the major LHCII proteins (Lhcb1, Lhcb2 and Lhcb3) are particularly intriguing. Taking known pigment and protein stoichiometries into account, there may have been three to four LHCII trimers per PSII monomer in the LL plants. The supermolecular structure of PSII has been studied extensively, and it is known that up to three LHCII trimers, denoted S, M and L, can associate with each PSII complex in a dimer . S, M and L refer to strongly, medium and loosely bound trimers, respectively. It is possible that the M trimer is composed of Lhcb3 and two Lhcb1 subunits . It is not known if there is any specificity for Lhcb1 and Lhcb2 at any position in the S and L trimers. It is conceivable that other LHCII trimers may aggregate in "LHCII-only domains", which must be attached to the photosystems, since energy transfer from all parts of the LHCII antenna into the photosystems is very efficient. Naïvely, the S, M and L trimers plus trimers found in LHCII-only domains may account for three to four trimers/PSII in LL plants. However, the field-grown plants contained only ca. a third of this amount of LHCII, i.e. one or at most two trimers/PSII. Lhcb3 was present in approximately equal amounts in field-grown and indoor plants, suggesting that M trimers were present in most or all of their PSII centers. Our data show that plants with only small amounts of LHCII trimers are perfectly capable of performing state transitions, consistent with the finding that the fitness of the Stn7 mutant grown under field conditions deviates from that of wild-type counterparts . However, since the M trimer--at least Lhcb3--is not believed to participate in state transition , Lhcb1 and Lhcb2 in S trimers are likely to be efficiently phosphorylated and participate in state transitions in field-grown plants. Alternatively, M trimers may become phosphorylated and detach from PSII. There are insufficient data from our study to enable us to confirm this possibility, but a more detailed study of PSII in Arabidopsis grown under field conditions may show which PSII supercomplexes are most abundant when Arabidopsis is exposed to its naturally-adapted light regimes. Taken together, although the LHCII content is much lower in field grown plants, antenna function is not much affected.
ELIPs, most likely involved in pigment metabolism in plastids, were originally identified as proteins that transiently accumulate during early plastid development, but subsequent studies have shown that they also accumulate under diverse stress conditions . ELIPs play an important protective role under light-saturated conditions, such as may occur in the field and, except in some artificially-controlled growth conditions in climate chambers; they are likely to be abundant thylakoid proteins. Nevertheless, our results indicate that the plants lacking ELIPs were well adapted to their growth conditions and had high levels of fitness; our 2-year study of double ELIP mutants suggests that ELIP functions in mature leaves may be redundant or of low importance. However, ELIPs may be more important in early developmental stages and it is also possible that they play crucial roles under conditions that the plants did not encounter during these 2 years.
Xanthophyll cycle pigments and PsbS are typically involved in photoprotective processes. In our experiments these factors were found at very low levels in indoor plants compared with field-grown samples. This is consistent with the view that under natural conditions photoprotection by NPQ and other mechanisms is of vital importance for the fitness of the plant . We have also shown that the level of NPQ is balanced and there is some evidence that selective forces act to reduce the level of photoprotection . Finally, our comparison of NPQ levels in a set of Arabidopsis accessions grown in the lab and the field illustrates how conclusions drawn from studies in the lab may be invalid for field-grown plants, due to phenotypic plasticity.
Plants have evolved many mechanisms that are involved in responses to changes in their growth conditions, ranging from long-term developmental processes that affect the morphology or physiology of the whole plant or individual leaves [25, 49], to adjustments in the functioning of individual proteins within the photosynthetic apparatus, operating on timescales ranging from seconds to hours . We have studied some of these adjustments, in particular relating to the functions of the photosynthetic light harvesting apparatus. In addition, adjustments to PSI/PSII ratios, variations in components of the inter-photosystem energy flow apparatus, and rates of cyclic electron transport, ATP generation and the photosynthetic dark reactions may be as important as those investigated here. We anticipate that other studies will focus on comparisons of photosynthetic properties that vary between and within species, or in single genotypes, as a result of phenotypic plasticity.
Plant material and growth conditions
Wild type Arabidopsis thaliana (Col-0) plants were grown from seeds under short photoperiods indoors under three growth irradiances: 30 (LL) and 300 (NL) μmol quanta m-2 s-1 in growth chambers equipped with metal halide lamps maintained at 8 h light, 16 h dark, 23/18°C and 75% relative humidity; and 600 (HL) μmol quanta m-2 s-1 in a chamber maintained at 9 h light, 15 h dark, 23/18°C and 75% relative humidity. LD indoor conditions were the same as HL conditions, except that the photoperiod was 16 h light, 8 h dark. In addition, another set were prepared and grown in the field as described by , as follows. After stratification, seeds were sown on June 29 2009, and seedlings were transferred to individual pots 10 days later on July 9 and pre-grown as above in a NL growth chamber. The resulting plants were transferred to our experimental garden in Umeå (N 63° 49' 9.96" E 20° 18') on July 22, when they had three to four leaves. The plants were shaded on the first day to allow for some acclimation. Photon flux density (PPFD) was monitored at the field site and ranged from very low levels up to 600 W/m2 (ca 2 300 μmol quanta m--2 s--1) during the photoperiods, which in the beginning of the experiment was ca. 20 h. The mid-day temperature varied between 16° and 28°C, and the relative humidity (RH) between 30 and 100%. A detailed description of the growing conditions is presented in Additional file 1: Figure S2 (A and B). Fluorescence data were recorded on individual plants in randomized order on August 8; measurements started around 10 am and finished around 6 pm. On August 10 at approximately noon all leaves from the plants used for fluorescence measurements were sampled for pigment and thylakoid protein analysis. Three sample pools, each consisting of leaves from 5 to 15 plants, were sampled and analyzed. The measurement and sampling schemes for LL, NL and HL plants were similar to those applied to plants grown under field conditions. Timings were adjusted to the growth rates under the different conditions, since the intention was to sample plants at similar developmental stages (before bolting), rather than those of the same age. Since the plants were small when transferred to the field but grew considerably before sampling, most of the leaf biomass analyzed consisted of leaves that had developed under field conditions.
We had performed a pilot experiment in the same garden in the summer (2008) prior to the study described above, in which we analyzed the plants' pigment and protein levels less comprehensively. The trends obtained were largely comparable to those found in the main study (data not shown). The plants used for measuring state transitions were grown in the summer of 2010. The chl levels of these plants were monitored to confirm that the size of the light-harvesting antenna was similar to that of the plants grown in 2009.
For the study of NPQ variation, we obtained 14 Arabidopsis accessions from the Nottingham Arabidopsis Stock Centre: Van-0, Can-0, Kas-1, Ws-2, Col-0, UK, Sf-2, Old-2, Mt-0, Br-0, Aa-0, Cvi-0, Mr- and Ron-0. We also included two Swedish accessions, and finally a mutant (npq4, Li et al., 2000) and a transgenic (oePsbS, Li et al., 2002) with varying levels of PsbS and, hence, NPQ. These lines were grown under two different conditions. First, plants were grown in a climate chamber (under NL) conditions as described above and NPQ was measured after 4 weeks of growth. A second batch of plants were grown under the same conditions for 6 weeks, then transferred to the field and measured 5 days later. In both experiments, the different genotypes were grown in a randomized pattern, to avoid misinterpretations of data due to local variations in (for example) light conditions; six plants of each genotype were analyzed.
Leaf size and shape
Leaf shape and size were quantified using the imaging software LAMINA .
Chlorophylls were extracted from leaf tissue with 80% (v/v) acetone and assayed spectrophotometrically using extinction coefficients according to .
Carotenoid composition was determined by high-pressure liquid chromatography (HPLC)  with modifications described by . The de-epoxidation state of the xanthophyll pool was calculated as (Z + A/2)/(V+A+Z) where V = [Violaxanthin], A = [Antheraxanthin] and Z = [Zeaxanthin].
Chlorophyll fluorescence and state transition measurements
Chlorophyll fluorescence of the plants was measured, after dark-adaptation, with a Dual PAM 100 chlorophyll fluorescence photosynthesis analyzer (Heinz Walz) as previously described . For NPQ measurements, actinic illumination was 660 μmol photons m-2 s-1 for 20 min, followed by darkness. A saturating pulse of light (5000 μmol photons m-2 s-1) was given every 1-2 min.
Immunoblot analysis of thylakoid membrane proteins
Immunoblot analysis of thylakoid membrane proteins was performed as described by , with modifications. Five- to six-week-old leaves were homogenized and filtered using a nylon mesh with a 20 μm mesh size (Millipore). The filtered homogenate was pelleted and resuspended in hypotonic buffer to break the chloroplasts. The thylakoid membranes were pelleted then resuspended in 0.33 M sorbitol, 20 mM Tricin (pH 7.8) and 5 mM MgCl2. All of the preparation steps were performed on ice or in a cold room (4°C) under a green safe light. Thylakoid proteins were prepared for immunoblot analysis by addition of Laemmli denaturation buffer  and incubation at 90°C for 10 min . One microgram of chlorophyll was loaded per lane, and the proteins were separated in a 16% denaturing SDS-PAGE gel (with non-urea buffers) using the Bio-Rad Mini Protean III system. The proteins were blotted on nitrocellulose membranes (Bio-Rad; 0.2 μm), using a Bio-Rad wet blotting system with methanol-containing buffers, according to the manufacturer's instructions. The nitrocellulose membranes were blocked using 5% (w/v) non-fat dried milk in TBS-T buffer with 0.1% Tween 20 for 1 h (Sigma-Aldrich Sweden AB) and incubated using rabbit primary antibodies against photosynthetic proteins [54, 56, 57] (provided by Agrisera, Vännäs, Sweden) at 1:5000 dilution for all antibodies (except anti-Lhca5 antibody, which was diluted 1:2000), for 1 h in TBS-T buffer with 0.1% Tween 20 and 5% non-fat dried milk. The membranes were washed three times for 5 min in TBS-T buffer, 0.05% Tween 20 and incubated with anti-rabbit donkey antibody horseradish peroxidase (HRP) conjugate (GE Healthcare Bio- Sciences) for 1 h at 1:10,000 dilution in TBS-T buffer with 0.1% Tween 20 and 5% non-fat dried milk. Immunoblotted membranes were incubated for 2 min in ECL plus HRP substrate (GE Healthcare Bio-Sciences), and chemoluminescence was then detected using a LAS-3000 cooled CCD camera. Optimal exposure times ranged from 5 to 10 min, and identical exposure times were used to quantify signals for each antibody used. Images were recorded using Image Reader software with 1 min incremental recording and standard CCD sensitivity (Fujifilm Medical Systems). The images were processed and quantified by the Multi Gauge application (Fujifilm Medical Systems), using profile lane quantification with automatic background subtraction and band detection. Standard parameters for peak detection were used according to the manufacturer's instructions.
Results were statistically analyzed using one-way ANOVA implemented in SPSS18 software applying Duncan's new multiple range tests to analyze all possible differences between LL, NL, HL and field plants. In addition, an orthogonal contrast analysis was done to see the difference between indoor and field plants (contrast indoor vs. field plants). The number of independent variables for each experiment was three.
We thank Nathaniel Street for help with leaf size and shape measurements and comments on the ms, Carlo Soave for the gift of ELIP KO seeds, Pär Ingvarsson for seeds of the two Swedish Arabidopsis accessions and Alexander Ruban for useful inputs. This work was supported by the Kempe foundation, the Swedish Research Council and the Swedish Research Council for Environment, Agricultural Sciences and Spatial Planning.
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