Acta Physiol Plant (2012) 34:2155–2164 DOI 10.1007/s11738-012-1016-9
ORIGINAL PAPER
Does shading explain variation in morphophysiological traits of tropical epiphytic orchids grown in artificial conditions? Marcel Viana Pires • Alex-Alan Furtado de Almeida Priscilla Patrocı´nio Abreu • Delmira da Costa Silva
•
Received: 28 June 2011 / Revised: 26 April 2012 / Accepted: 7 May 2012 / Published online: 24 May 2012 Ó Franciszek Go´rski Institute of Plant Physiology, Polish Academy of Sciences, Krako´w 2012
Abstract Light is one of the main factors of physical environment and it controls plant growth and development by interfering with photosynthesis, especially concerning CO2 assimilation. Photosynthetic characteristics and growth of C3 epiphytic orchids Miltonia flavescens and Miltonia spectabilis var. moreliana were analyzed under four radiation regimens (25, 50 and 75 % of global radiation and full sunlight). Anatomical characterizations were performed on plants grown at 25 % shade. Artificial shading was obtained using different shading nylon nets. The highest values of lightsaturated photosynthetic, dark respiration, net photosynthetic and leaf transpiration rates, stomatal conductance and intercellular to atmospheric CO2 concentration ratio were observed at full sunlight and 25 % shade. Moreover, both species allocated greater amount of leaf dry weight in those treatments. On the other hand, it was observed a greater investment in pseudobulb biomass in more shaded conditions (50 and 75 %), corroborating with the highest values of intrinsic water-use efficiency observed in those treatments. It was found a significant effect of shading on leaf area and specific leaf area. The anatomical features reflected strategies to save water. The phenotypic plasticity and principal component analysis suggested that the physiological traits were more responsive to light levels than the morphological traits. The results indicate that those species appear to be adapted to
Communicated by B. Zheng. M. V. Pires A.-A. F. de Almeida (&) P. P. Abreu D. da Costa Silva Departamento de Cieˆncias Biolo´gicas, Universidade Estadual de Santa Cruz, Rod. Ilhe´us-Itabuna, km 16, Ilhe´us, BA 45650-000, Brazil e-mail:
[email protected]
high irradiances conditions and are capable of adjusting, via morphophysiological changes, to light availability. Keywords Anatomy Biomass allocation Epiphytic orchids Irradiance Leaf gas exchanges
Introduction In the Southern region of Bahia state, Brazil, the Atlantic Rain Forest is being affected by deforestation caused by agricultural expansion, pasture and wood extraction. Those land-use practices have been harming, in an irreversible way, the local fauna and flora, and the fragmentation of natural habitats has been causing an increase in incident irradiance over shaded plants, such as the epiphytic orchids. The increase in irradiance leads to several physiological and anatomical effects on C3 orchids Miltonia flavescens (Lindl.) Lindl., and Miltonia spectabilis var. moreliana Henfr. Mart., as well as on their growth and development patterns. About 72 % of orchid species are estimated to be epiphytic (Gravendeel et al. 2004), with the majority of these being restricted to tropical regions. Thus, an intimate knowledge of the abiotic conditions, such as light intensity, is important for successful cultivation and preservation of orchid wild species (Lin and Hsu 2004). Changes in light exposure may cause damages on the photosynthetic apparatus of orchids. Low light can cause stress due to photosynthesis limitations, leading to lower carbon assimilation and growth. On the other hand, high light may likewise damage the photosynthetic apparatus, sometimes irreversibly (Pastenes et al. 2003). It has been commonly observed that the capacity of plants to grow and develop in regimens differing from their original habitats depends on their ability for photosynthetic acclimation to
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changing environment (Zhang et al. 2007). In this way, plants have developed light acclimation strategies, such as increasing photosynthetic capacity and thermal dissipation. Photosynthetic acclimation to changing irradiance is usually associated with anatomical and physiological modifications at the leaf (Evans and Poorter 2001; Lin and Hsu 2004) or whole plant levels, resulting from changes in growth patterns and biomass allocation. Several authors have reported that changes in light intensity may induce phenotypic variations in plants (Valladares et al. 2000; Aranda et al. 2008). This photosynthetic acclimation is important in order to optimize carbon allocation and to search for light in heterogeneous environments. Epiphytic orchids are able to survive in dry habitats of canopies based on morphophysiological mechanisms, such as maximum photosynthetic capacity, stomatal functioning, nitrogen investment, and leaf anatomy. Those adaptations serve to increase CO2 uptake, to reduce water and nutrient loss as well as to optimize their storage (Lorenzo et al. 2010). Photosynthetic parameters are frequently used as tools to measure environmental stress and to determine optimal growth conditions for different plant species (Pastenes et al. 2003; Lin and Hsu 2004). However, there are currently no known studies on artificial cultivation of species of the genus Miltonia. For this reason, a better understanding of the photosynthetic and anatomical features of those species under different environmental conditions should help researchers grow them artificially and introduce them into optimum natural habitats. By analyzing photosynthetic and growth characteristics of M. flavescens and M. spectabilis var. moreliana under four radiation regimens (three shading levels and full sunlight) and carrying out anatomical characterization (root, pseudobulb and leaf) from plants grown at 25 % shade, this study aimed at (i) testing photosynthetic acclimation capacity under different shading levels, (ii) determining the anatomical characteristics, (iii) finding optimal light required for the growth of both orchid species, then providing support to further programs for the conservation and reintroduction of orchids into natural habitats.
Materials and methods Plant material and experimental conditions The experiment was conducted at the Universidade Estadual de Santa Cruz (UESC), in the city of Ilhe´us, Bahia, Brazil, (14°450 S, 39°130 W). Mature plants of Miltonia flavescens and Miltonia spectabilis var. moreliana were collected in the city of Itapebi, Bahia (15°570 S, 39°320 W). The plants were transplanted to perforated adobe pots filled with dry seeds of Spondias mombin L. (substratum). Each
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pot had four to five stumps and represented one sample unit. To study the effect of different radiation regimens, plants grown in pots were transferred to full and intermediate sunlight. Artificial shading was obtained with different shading nylon nets, fixed in iron frames of 4 9 1 9 2 m, under field conditions, which allowed the reduction of 25 % (*160 lmol m-2 s-1), 50 % (*380 lmol m-2 s-1), and 75 % (* 600 lmol m-2 s-1) of global photosynthetic active radiation, along with a control treatment under full sunlight (*800 lmol m-2 s-1). The photosynthetic photon flux density (PPFD) values, measured between 8:00 a.m. and 6:00 p.m. (n = 5 days), were obtained using a LI250 light meter with a linear quantum sensor LI-191SA (Li-Cor Biosciences Inc., Lincoln, NE, USA). The plants were irrigated daily, fertilized through the leaves (N:P:K ratio of 1:1:1), and sprayed with fungicide once every 15 days. Gas exchange and PPFD response curves Leaf gas exchanges were evaluated 90 days after the plants were exposed to the shade treatments, between 8:00 and 12:00 a.m., on five individual plants per species (a fully expanded and not self-shaded leaf per plant), using a Portable Photosynthesis System LI-6400 (Li-Cor Biosciences Inc., Lincoln, NE, USA) equipped with an artificial irradiance source 6400-02B RedBlue. Light response curves were created with ten levels of PPFD (0, 5, 10, 25, 50, 100, 200, 400, 600, and 800 lmol m-2 s-1). Readings were taken in decreasing order, with 1–2 min intervals between each reading (CV \ 0.3 %). The CO2 flux was adjusted to maintain a concentration of 380 lmol mol-1 inside the chamber; the leaf chamber temperature was maintained at 26 °C. The net photosynthetic rate (PN), leaf transpiration rate (E), stomatal conductance to water vapor (gs), intercellular to atmospheric CO2 concentration ratio (Ci/Ca), and intrinsic water-use efficiency (PN/gs) were calculated using the values of CO2 and humidity variation inside the chamber, both measured by the infrared gas analyzer of the portable photosynthesis system when PPFD C 600 lmol photon m-2 s-1 (saturating, but not inhibitory irradiance observed for both species studied, in the four levels of shading). Non-linear regression for exponential equations was used to estimate the photosynthetic parameters. The following equation was used for the construction of PN versus PPFD curves (Webb et al. 1974): PN = Pmax (1 exp (-a 9 PPFD/Pmax)) - Rd, where Pmax is the lightsaturated rate of gross photosynthesis, a is the apparent quantum yield, and Rd is the dark respiration rate. Based on the adjusted values, the light compensation point was calculated (LCP).
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Growth parameters For standard procedures, three rhizomes of about 10 cm were collected from each sample (n = 5). Subsequently, the greatest possible amount of roots inserted in each rhizome was collected, in addition to the selection of 10 pseudobulbs and 20 leaves from each sample. Leaf area (LA) was estimated using an automatic Leaf Area Meter LI-3100 (Li-Cor Biosciences Inc., Lincoln, NE, USA). Root (RDW), rhizome (RzDW), pseudobulb (PbDW), leaf (LDW), and total (TDW) dry weight were obtained after drying at 70 °C until reaching a constant mass. The specific leaf area (SLA) was estimated by the quotients between individual mean values of LA and LDW, according to Hunt et al. (2002). Light microscopy The anatomical characterization was carried out from plants grown at 25 % shade. Roots, pseudobulbs and leaves of three plants of each species were collected for light microscopy analysis 90 days after the plants were exposed to the shade treatment. Small pieces (2 mm2) of roots, pseudobulbs and leaves were fixed in a solution of 70 % FAA. The samples were dehydrated in graded ethanol series (30, 50, 70, 85, 90 and 100 %). Free-hand sections for light microscopy were stained with Astra blue and safranin (Kraus and Arduin 1997) and photographed with an OLYMPUS BX50 light microscope (Center Valley, PA, USA). Furthermore, histochemical tests were performed with Lugol and Sudan III on samples of pseudobulbs and leaves. Statistical analysis The experiment was conducted in a completely randomized design, using a 4 9 2 factorial arrangement, with four light availability levels (full sunlight and 25, 50, and 75 % shade) and two Miltonia species, with five replicates. The results were submitted to analysis of variance (ANOVA) followed by Tukey’s mean comparison test (P \ 0.05). To compare levels of phenotypic plasticity, a phenotypic plasticity index (PIV) for each trait was calculated as the difference between the minimum and maximum mean response along a standardized gradient of light. To obtain an index between 0 and 1, the difference was divided by the maximum mean value for each trait (Valladares et al. 2000). The phenotypic plasticity data were transformed by the Kolmogorov–Smirnov test, to present normal distribution. Furthermore, a relative distance plasticity index (RDPI) was also obtained for each genotype as RDPI = R[dij ? i0 j0 /(xd ? xd)]/n, where n is the total number of distances. For a simple trait x, phenotypic plasticity is
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considered as a random variable, in which each realization is described by the absolute distance between two randomly selected replicates (j and j0 ) of the same genotype belonging to different treatments (i and i0 , where i is always different from i0 , as replicates were grown in different treatments). Therefore, relative distances rdd?d0 d0 are defined as dd?d0 d0 /(xd0 d0 ? xd) for all pairs of replicates of a given genotype grown in different treatments (Valladares et al. 2006). Differences in the plasticity index were analyzed using one-way ANOVA followed by Scott-Knott test (P \ 0.05). Those statistical procedures were performed using the software Statistica 6.0 (StatSoft Inc., Tulsa, OK, USA). Multivariate principal components analysis (PCA) was used in order to evaluate the combination of physiological and morphological characteristics of the species studied in both environments. PCA is a mathematical manipulation of the data matrix designed to reduce its original dimension and is based on the correlation among the variables, in which the highly correlated ones are grouped into a new variable, called the principal component (PC) (Morgano et al. 1999). From a mathematical point of view, the matrix of data decomposed in two matrices, the first consisting of scores and the second of loadings. The PCs with eigenvalues C1 were assumed and used for further analysis. The score matrix was used to create the diagrams between the PCs. PCA was performed using the software Stata 11.0 (StataCorp LP, College Station, TX, USA).
Results The artificial shading significantly (P \ 0.05) influenced the values of the light-saturated rate of gross photosynthesis (Pmax) and dark respiration rate (Rd) for both species, especially under full sunlight and 25 % shade. M. flavescens presented significant differences for the light compensation point (LCP) showing the highest mean value under full sunlight (Table 1). No differences were observed between the apparent quantum yield (a) mean values for both species. Furthermore, there were no significant differences between both species in relation to the light response curves parameters (Table 1). Plants of M. flavescens and M. spectabilis var. moreliana showed typical C3 photosynthetic behavior, with the net photosynthetic rate (PN) increasing as the PPFD increased, until reaching saturation at approximately 500–600 lmol m-2 s-1, corresponding to photosystem saturation. The highest values of PN for M. flavescens and M. spectabilis var. moreliana were 4.26 and 4.47 lmol CO2 m-2 s-1, respectively, and were observed at full sunlight (Table 2). There was an increase of leaf transpiration rates (E), stomatal conductance (gs) and intercellular to atmospheric CO2 concentration ratio (Ci/Ca)
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Table 1 Light-saturated net photosynthetic rate (Pmax) [lmol(CO2) m-2 s-1], dark respiration rate (Rd) [lmol m-2 s-1], light compensation point (LCP) [lmol m-2 s-1], and apparent quantum yield (a) Species
Shading
Pmax
M. flavescens
Full Sun
4.78 ± 0.1Aa
25 % 50 % M. spectabilis var. moreliana
[mol mol-1] estimated for M. flavescens and M. spectabilis var. moreliana grown under different light intensities Rd
4.59 ± 0.08
Aa
4.13 ± 0.07
Ab Ac
a
LCP
0.74 ± 0.01Aa
14.28 ± 1.9Aa
0.06 ± 0.004Aa
Aa
Ab
0.05 ± 0.008Aa
Ab
0.05 ± 0.006Aa
0.69 ± 0.03
13.27 ± 2.2
Ab
0.56 ± 0.03
11.46 ± 1.1
75 % Full Sun
3.86 ± 0.06 5.12 ± 0.2Aa
0.42 ± 0.04 0.87 ± 0.06Aa
13.56 ± 2.3 12.46 ± 2.8Aa
0.03 ± 0.004Aa 0.07 ± 0.006Aa
25 %
4.97 ± 0.1Aa
0.76 ± 0.05Aa
9.48 ± 1.9Aa
0.08 ± 0.009Aa
50 %
4.35 ± 0.1
Ab
Ab
Aa
0.04 ± 0.007Aa
4.16 ± 0.1
Ab
Aa
0.04 ± 0.010Aa
75 %
Ab
Ab
0.45 ± 0.04
11.31 ± 2.6
Ab
0.41 ± 0.05
9.27 ± 2.2
Mean ± SE values from five replicates. Mean comparisons were done using Tukey’s test (P \ 0.05). For each variable lowercase letters indicate comparisons between treatments and uppercase ones comparisons between species. Mean followed by the same letter is not significantly different Table 2 Net photosynthetic rate (PN) [lmol(CO2) m-2 s-1], stomatal conductance (gs) [mol m-2 s-1], leaf transpiration rate (E) [lmol (H2O) m-2 s-1], intercellular to atmospheric CO2 concentration ratio
(Ci/Ca), and intrinsic water-use efficiency (PN/gs) [lmol(CO2) mol(H2O) m-2 s-1] from M. flavescens and M. spectabilis var. moreliana grown under different light intensities
gs
E
Species
Shading
PN
M. flavescens
Full Sun
4.26 ± 0.22Aa
0.09 ± 0.004Aa
Aa
Aa
25 % 50 % M. spectabilis var. moreliana
4.02 ± 0.27
Ab
3.56 ± 0.16
0.08 ± 0.005
Ab
0.04 ± 0.003
Ci/Ca
1.21 ± 0.12Aa Aab
1.23 ± 0.16
Ab
0.88 ± 0.09
0.88 ± 0.06Aa
51.49 ± 5.9Ac
Ab
55.07 ± 6.2Ac
Ab
94.50 ± 5.7Aa
Ab
0.73 ± 0.08 0.63 ± 0.08
75 % Full Sun
Ab
3.44 ± 0.11 4.47 ± 0.21Aa
Ab
0.05 ± 0.002 0.11 ± 0.007Aa
0.72 ± 0.07 1.47 ± 0.16Aa
0.68 ± 0.07 0.87 ± 0.11Aa
77.06 ± 6.6Ab 42.46 ± 1.7Bd
25 %
4.38 ± 0.25Aa
0.09 ± 0.006Aa
1.55 ± 0.13Aa
0.85 ± 0.09Aa
51.32 ± 2.5Ac
Ab
Ab
Aa
66.07 ± 5.8Bb
Aa
74.79 ± 4.7Aa
50 % 75 %
Aab
4.11 ± 0.14
Ab
3.82 ± 0.12
0.06 ± 0.006
Ab
0.05 ± 0.007
Abc
PN/gs
1.16 ± 0.11
Ab
0.94 ± 0.09
0.84 ± 0.12 0.82 ± 0.08
Mean ± SE values for five replicates. Mean comparisons were done using Tukey’s test (P \ 0.05). For each variable lowercase letters indicate comparisons between treatments and uppercase ones comparisons between species. Mean followed by the same letter is not significantly different
as PN increased, reaching the highest values at full sunlight. On the other hand, it was observed an increase of the intrinsic water-use efficiency (PN/gs) as light intensities decreased (Table 2). Both species allocated the highest amount of leaf dry weight (LDW) under full sunlight and 25 % shade. On the other hand, greater investment in pseudobulb dry weight (PbDW) was observed in the intermediate treatments (50 and 75 % shade). Proportional allocation of root (RDW) and rhizome (RzDW) dry weight did not differ significantly (P \ 0.05) among the different shading levels (Fig. 1). There were significant effects (P \ 0.05) of shading on leaf area (LA) and specific leaf area (SLA) in both species, with the highest mean values found at 75 % shade (Fig. 2). It was also observed a significant increase on total dry weight (TDW) of M. flavescens at 50 % shade (Fig. 2), probably due to the large investment in pseudobulbs in this environment. The phenotypic plasticity indexes (PIV) of the physiological traits were 11.8 and 16.7 % larger than those of morphological traits for M. flavescens and M. spectabilis
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var. moreliana, respectively (Table 3). Among the physiological traits, Pmax (*0.19 for both species) and PN (*0.19 and 0.14 for M. flavescens and M. spectabilis var. moreliana, respectively) showed the lowest phenotypic plasticity in the different shading levels. Some physiological traits revealed to be more plastic in response to shading, such as Rd (*0.43 and 0.54 in the above order) and gs (*0.55 and 0.54). Furthermore, LA (*0.51 and 0.52) and SLA (*0.47 and 0.49) were the morphological traits that showed the highest phenotypic plasticity. Similar results were found for the relative distance plasticity index (RDPI). Mean values of RDPI of the physiological features were 17.8 and 20 % larger than those of morphological features for M. flavescens and M. spectabilis var. moreliana, respectively (Table 3). As RDPI gives an unbiased estimation of the levels of phenotypic variation and allows the exploration of plasticity with strong statistical power to test for differences in plasticity between species (Valladares et al. 2006), a one-way ANOVA was performed to evaluate differences between both species. However, no significant differences were observed (Table 3), indicating
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(a)
(a)
(b)
(b)
(c)
Fig. 1 Proportional allocation (%) of pseudobulb (PbDW), rhizome (RzDW), root (RDW), and leaf dry weight (LDW) for M. flavescens (a) and M. spectabilis var. moreliana (b) grown under different light intensities (full sun and 25, 50 and 75 % shade). Mean values from five replicates
that both orchid species show the same level of phenotypic plasticity in such experimental conditions. The principal component analysis (PCA) reduced the 11 input variables to three principal components (PCs), explaining 83.7 % of the total variance in the original data (Table 4) and validating the phenotypic plasticity analysis previously described. The relationship of the original variables with the PCs is indicated by PC loadings, which are similar to correlation coefficients between original characters and the PC. Loadings in PC1 were positively related to E, Pmax, gs, Rd and PN, explaining 62.4 % of the total variance. Loadings in PC2 were positively related to RzDW and RDW, explaining 11.6 % of the total variance. Loadings in PC3 were negatively related to SLA but positively related to LDW, PbDW and PN/gs, explaining 9.7 % of the total variance (Table 4). These results confirm that the physiological plasticity was more important for light acclimation than the morphological plasticity. The score diagrams obtained from PCA (Fig. 3) showed, based on the
Fig. 2 Leaf area, LA (a), specific leaf area, SLA (b), and total dry weight, TDW (c) for M. flavescens and M. spectabilis var. moreliana grown under different light intensities (full sun and 25, 50 and 75 % shade). Bars represent mean (n = 5) and mean comparison was done using Tukey’s test (P \ 0.05). Vertical bars denote the SE. For each variable lowercase letters indicate comparisons between treatments and uppercase ones comparisons between species
morphophysiological characteristics presented, the distinction between high light (full sunlight and 25 % shade) and intermediate light treatments (50 and 75 % shade). In relation to anatomical characterization, roots of M. flavescens and M. spectabilis var. moreliana showed thick velamen, lignified exodermis, single-layered endoderm and pericycle; and cortex formed by alternating xylem by phloem with many thick lignin wall fiber sheaths (Fig. 4a).
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Table 3 Plasticity of physiological and morphological traits of M. flavescens and M. spectabilis var. moreliana grown under different light intensities M. flavescens
M. spectabilis var. moreliana
RDPI
PIV
PIV
Variables
RDPI
Pmax Rd LCP
0.19
c
0.43
d
0.39
h
0.12
Ad
0.39
Bb
0.33
Ab Ac
PC1
PC2
PC3
Ea
0.35
–
–
Pmax
0.34
–
–
0.13
Ae
gs
0.33
–
–
0.49
Aa
Rd
0.31
–
–
0.21
Bd
PN
0.31
–
–
0.14
0.10
Ae
RzDW
–
0.72
–
Physiological traits h
Table 4 Principal components (PC) obtained from the matrix of correlation for morphophysiological characteristics of M. flavescens and M. spectabilis var. moreliana grown under different light intensities
f
0.19
a
0.54
e
0.26
g
PN
0.19
0.15
gs
0.55a
0.48Aa
0.54a
0.48Aa
RDW
–
0.42
–
E
c
0.37
Ab
c
0.32
Ac
LDW
–
–
0.50
0.41
Aa
0.29
Bc
PbDW
–
–
0.40
0.39
Ab
Ac
SLA
–
–
-0.38
PN/gs
–
–
0.36 1.07
Ci/Ca PN/gs
0.41
b
0.47
b
c
0.37
b
0.43
0.34
Mean 0.38a Morphological traits
0.33Aa
0.36a
0.30Aa
6.86
1.27
f
0.21
Ac
f
0.15
Be
Explained variance (%)
62.40
11.59
9.75
Ac
Bf
Cumulative variance (%)
62.40
73.99
83.74
PbDW
0.45
0.39
0.28
g
Eigenvalue 0.18
h
RzDW
0.23
0.17
0.09
0.04
RDW
0.22g
0.17Bc
0.26e
0.21Ad
Data are PC loadings
LDW
d
0.38
0.34
Ab
e
0.29
0.23Bd
a
TDW
0.31e
0.24Ac
0.33d
0.27Ac
LA
0.51a
0.44Aa
0.52a
0.46Aa
b
0.39
Ab
b
0.42Ab
0.28
Ab
b
0.25Ab
SLA Mean
0.47
b
0.34
0.49 0.30
Plasticity index values (PIV) for traits followed by the same lowercase letter are not significantly different. For relative distance plasticity index (RDPI), lowercase letters indicate comparisons between treatments and uppercase ones comparisons between species (Scott-Knott test, P \ 0.05). RDPI mean followed by the same letter is not significantly different Pmax light-saturated rate of gross photosynthesis, Rd dark respiration rate, LCP light compensation point, a apparent quantum yield, PN net photosynthetic rate, gs stomatal conductance to water vapor, E leaf transpiration rate, Ci/Ca intercellular to ambient CO2 concentration ratio, PbDW pseudobulb dry weight, RzDW rhizome dry weight, RDW root dry weight, LDW leaf dry weight, TDW total dry weight, LA leaf area, SLA specific leaf area, PN/gs intrinsic water-use efficiency
The pseudobulbs have single-layered epidermis, with no trichomes or stomata, and thin and lignified cuticle; cortex made up of aerenchymas, with abundant crystals, raphides and druses structures; parenchyma with large amounts of starch; and collateral bundles surrounded by fiber sheaths (Fig. 4b–e). The hypostomatic leaf showed a single-layered epidermis, coated by a thick cuticle; homogeneous mesophyll with only one layer of hypodermical bulliform cells; collateral bundles, with xylem in the direction of the adaxial face, and phloem towards the abaxial face, covered by fiber sheaths; and anomocytic and tetracytic stomata at the same level of the other epidermal cells or slightly elevated in comparison to them; we also observed large amount of crystals associated to parenchymatic cells (Fig. 4f–h).
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E leaf transpiration rate, Pmax light-saturated rate of gross photosynthesis, gs stomatal conductance to water vapor, Rd dark respiration rate, PN net photosynthetic rate, RzDW rhizome dry weight, RDW root dry weight, LDW leaf dry weight, PbDW pseudobulb dry weight, SLA specific leaf area, PN/gs intrinsic water-use efficiency
Discussion There are few studies regarding the photosynthetic behavior of tropical orchids based on light availability. Most of the researches are concentrated on Crassulacean Acid Metabolism (CAM), a widespread photosynthetic pathway that has evolved in plants of CO2- and waterlimited environments, due to the abundance of epiphytic orchids that present this metabolic pathway. Lin and Hsu (2004), in their studies on the Phalaenopsis amabilis, a worldwide cultivated CAM orchid, observed that such specie has considerable potential for light acclimation and presents shaded leaves with lower rates of CO2 fixation. According to Zhang et al. (2007), the photosynthesis of the orchid species Cypripedium guttatum is also influenced by irradiance levels. The same authors observed that a level of about 45 % sunlight appears to be optimal for photosynthesis of C. guttatum. Evidence from some studies indicates that many tropical epiphytes are subject to and often well adapted to frequent periods of drought stress between precipitation events (Hew and Yong 1997; Nowak and Martin 1997), in addition to stress from other environmental factors, such as light (He et al. 1998; Konow and Wang 2001). The highest mean values of PN observed under full sunlight and 25 % shade (Table 2), with decreases in intermediate shade levels, show the acclimation capacity of
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(a)
(b)
Fig. 3 PCA ordination diagrams displaying correlations between morphophysiological characteristics in plants of M. flavescens and M. spectabilis var. moreliana submitted to different light intensities, for first and second PCs (a) and for first and third PCs (b)
both species to environments with high irradiance, also revealing that significant light requirements are needed in order to reach Pmax (Table 1). While these results are consistent with those found for some orchid species (Boardman 1977), other studies have recorded photosynthetic rates in intermediate light conditions to be higher than those under low and high light conditions (Zhang et al. 2003, 2007). Thus, M. flavescens and M. spectabilis var. moreliana are adapted to full sunlight and highly capable of using light energy. However, the maintenance of high photosynthetic rates has high energy cost and is beneficial only under high irradiance conditions where the amount of Rubisco and the reaction center of photosystem II (PSII) increased with the expansion of the light harvest complex of PSII (Hikosaka and Terashima 1995). In this way, lower photosynthetic rate in shade-tolerant leaves has usually been attributed to a lower investment in enzymes and components of photosynthetic machinery (Niinemets 2007). Low values of LCP (Table 2) enable a positive carbon balance, likely related to a lower respiration rate (Table 1). The LCP phenotypic plasticity indexes (Table 3) seem to
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allow those species to adapt to different shading conditions, allowing acclimation at low irradiances as well as photoprotective capacity, then showing proper acclimation to high irradiances. The increase of gs usually causes the rise of PN, and its effect is more pronounced in C3 plants. Both light and air temperature influence stomatal responses. Moreover, the behavior of E is linked to gs, since gas exchanges conducted through the stomata are efflux of H2O and CO2 influx. Light-saturated photosynthesis is correlated to the electron transport chain saturation and decline in Rubisco activity in the Calvin Cycle. Thus, the stomatal opening is reduced because it does not need to absorb more CO2 in that situation, in addition to avoiding water loss through transpiration. The largest LDW accumulated under full sunlight and 25 % shade (Fig. 1) may be due to increased leaf thickness, which usually occurs in leaves under high light availability, and also to resource protection to the photosynthetic pigments (Scalon et al. 2001). Sun leaves presented a large amount of dry matter and mineral nutrients per unit area, while shade leaves show the opposite behavior (Popma et al. 1992). On the other hand, the increase in PbDW in more shaded treatments (Fig. 1) was probably due to storage of water, nutrients and carbohydrates to maintain cellular integrity and metabolic activity, whereas the photosynthetic activity decreased, corroborating with the highest values of PN/gs observed in these treatments (Table 2). This appears to be an unexpected result because, under shade, water consumption is usually reduced. However, the function of pseudobulbs is to supply plants during periods of rapid resource utilization (Zimmerman 1990). Thus, under shade stress conditions, those species can use the water, nutrients and carbohydrates reserves to maintain vegetative growth as well as flowering in low photosynthetic conditions. Increases in LA and SLA reflect changes in leaves dimensions and forms in response to irradiance (Fig. 2), thus compensating the lower photosynthetic rates, typical of shade leaves (Scalon et al. 2001). Such morphological changes are related to the efficiency of utilization of available sunlight to compensate for low irradiance conditions (Campos and Uchida 2002). Leaf thickness reduction is due to the consumption of assimilates during leaf area expansion (Cooper and Qualls 1967). On the other hand, there is an increase in leaf thickness, creating greater internal volume for CO2 diffusion and greater cell volume to accommodate the photosynthetic apparatus, with a concomitant reduction of leaf area in sun leaves (Bjo¨rkman 1981). The increase in SLA (Fig. 2b) under more shaded treatments may represent an adaptive mechanism, demonstrating the most efficient use of assimilates, since a greater photosynthetic area per accumulated biomass unit is produced (Cooper 1966). In contrast, low SLA benefits plants
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Fig. 4 a Transverse section of a root of M. flavescens showing the velamen (Ve), pericycle (Pe), endoderm (Ed), and exodermis (Ex); b Transverse section of a pseudobulb of M. spectabilis var. moreliana. Histochemical test with Lugol showing fibers associated with the vascular bundles (VB) and parenchymal cells (Pr) containing starch (St); c Aerenchyma cells containing crystal (Cr); d Transverse section of a pseudobulb of M. flavescens. Histochemical test with Sudan III showing the presence of suberin in the cuticle (Cu). Observe, too, the parenchyma (Pr) and aerenchyma (Ar) cells; e Aerenchyma cells containing raphide (Ra); f Transverse section of a leaf of M. flavescens showing the vascular bundle (VB), hypodermical bulliform cells (BC), cuticle (Cu) on the upper epidermis and fiber sheaths (FS) on the lower epidermis; g Parenchyma cells containing crystal (Cr); and h Paradermal section of lower epidermis of M. spectabilis var. moreliana showing the anomocytic (AS) and tetracytic (TS) stomatal. Scale bar 65 lm (a–d, f), 40 lm (e, g) and 10 lm (h)
under high irradiance because it reduces tissue exposure to the sun, then reducing water loss and self-shading (Claussen 1996). The efficiency of plants in using solar energy for growth and their ability to adapt to environmental conditions is affected by management practices used during their cultivation, being genetically controlled. The phenotypic plasticity (Table 3) and the principal component analysis (Table 4) suggest that the physiological traits were more responsive to light levels than the morphological traits. The greater plasticity for functional rather than morphological traits as displayed by the orchid leaves is consistent with a sun-tolerant status (Table 3). On the other hand, morphological plasticity is likely to play a secondary role in photoacclimation. High morphological and low physiological plasticity are often associated with shade-tolerant species (Valladares et al. 2000; Niinemets and Valladares 2004). Variation in all input variables was adequately explained by three PCs (Table 4). One of these components, PC1, which combined E, Pmax, gs, Rd and PN—physiological traits related to photosynthesis, leaf
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transpiration and gas exchange—was considered the most important component in explaining the variation in our data. The loading diagrams (Fig. 3) show a clear separation among the light environments. The plants grown in high light (full sunlight and 25 % shade) were grouped in a different area in contrast to plants grown in low light (50 and 75 % shade). However, some caution is needed in the interpretation of the results. Only a few features have been analyzed and the PCA left over 16 % of the phenotypic variation unexplained. Moreover, our phenotypic estimates are inevitably restricted and there might be a range that has not been accounted for. On the other hand, it is clear that the differences found for the morphophysiological characteristics are explained by the variation of light levels (Fig. 3). The organization of the orchid’s vegetative organs is notably diverse, thus contributing to increase the variety of growth patterns (Pabst and Dungs 1975) and favoring adaptation to different environments (Benzing et al. 1983). One of these adjustment mechanisms is the presence of
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velamen in roots (Fig. 4a). This structure is capable of absorbing water and mineral salts, reduce transpiration, and provide mechanical protection (Benzing 1987). This feature becomes very important for M. flavescens and M. spectabilis var. moreliana, when they are in epiphytic environments, due to water-use restrictions. Pseudobulbs can be seen as another adjustment mechanism in situations of limited water availability, since those can store water and help in the maintenance of water balance. In those cases, small parenchyma cells commonly store starch (Fig. 4b), which is used for the periodic growth of pseudobulbs and formation of new branches, whereas the large aerenchyma store water, druses (Fig. 4c) and raphides (Fig. 4e), such as described for other orchid species, including M. flavescens (Oliveira and Sajo 2001). Leaf succulence is another adaptive mechanism that improves water storage in epiphytes. Hypodermis tissue (Fig. 4f) is commonly found in epiphytic species of several families of mono- and dicots, particularly in Orchidaceae (Oliveira and Sajo 1999). That tissue is considered to be adjustable to different environments and is the most common structure for leaf water storage, besides performing a supporting function. The distribution of cuticle wax was visibly thicker on the adaxial surface of the leaves (Fig. 4f). Such aspect reflects the genetic control of plant and its needs in relation to the environment. Helbsing et al. (2000) reported that epiphytes and hemiepiphytes show, in general, a lower leaf cuticular permeance to water in comparison to terrestrial forms. Salatino et al. (1986) indicated that wax crystals (Fig. 4g) can be important features in adjusting to environmental conditions in which high light intensity and high transpiration rates are predominant. One of the most constant characteristics among monocots is the large amount of fibers in different vegetative organs (Fahn 1990). Those fibers play different roles, such as tissue support, protection against water loss and light intensity filter. We believe that these functions are performed by the fibers observed in the material under study, both in pseudobulbs and leaves (Fig. 4f). M. flavescens and M. spectabilis var. moreliana leaves are hypostomatic, with anomocytic and tetracytic stomata (Fig. 4h), a characteristic that is also observed in the Cattleya and Sophronitis genus (Bonates 1993). The presence of stomata only on the abaxial surface is a common trait among dicots (Fahn 1990) and is considered a photoprotective mechanism especially in high light intensity (Dickison 2000). In conclusion, variations in the photosynthetic and morphological characteristics of M. flavescens and M. spectabilis var. moreliana growing in different light environments reflect physiological adaptations to changing light environments. The photosynthetic acclimation of both species to different levels of irradiation was strongly
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correlated with changes in stomatal conductance, transpiration and leaf area. Moreover, the acclimation of both species was related to physiological rather than morphological traits. The results show that these species are capable of adjusting, via morphophysiological changes, to light availability. Furthermore, they reveal that the anatomical features represent strategies intended to save water and that these species should be grown in environments with high irradiance or full sunlight. Thus, these orchids present typical characteristics of epiphytic species adapted to high irradiances. Author contribution M.V. Pires and A-A.F. Almeida were responsible for experimental design, performance of experiments, data analysis and manuscript preparation. P.P. Abreu and D.C. Silva were involved in anatomical characterizations and also performed the experiments. Acknowledgments Thanks are due to FAPESB (Foundation for Research Support of the State of Bahia) for the scholarships granted to M.V. Pires and P.P. Abreu, and CNPq (Brazilian Council for Advancement of Science and Technology) for the scholarships awarded to A-A.F. Almeida and financial support. M.V. Pires thanks Prof. D.A. Cunha for the support on Principal Components Analysis. Conflict of interest interest.
The authors state that they have no conflict of
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