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Gradients of seed photosynthesis and its role for oxygen balancing
BioSystems 103 (2011) 302–308
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Gradients of seed photosynthesis and its role for oxygen balancing Henning Tschiersch, Ljudmilla Borisjuk, Twan Rutten, Hardy Rolletschek ∗ Leibniz-Institut für Pflanzengenetik und Kulturpflanzenforschung (IPK), Corrensstr. 3, 06466 Gatersleben, Germany
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Article history: Received 24 June 2010 Received in revised form 17 August 2010 Accepted 18 August 2010 Keywords: Hypoxia Microsensor PAM fluorescence Seed photosynthesis
a b s t r a c t Seeds are generally viewed in the context of plant reproduction and the supply of food and feed, but only seldom as a site of photosynthesis. However, the seeds of many plant species are green, at least during their early development, which raises the issue of the significance of this greening for seed development. Here we describe the two contrasting modes of photosynthesis in the developing seed. The dicotyledonous pea seed has a green embryo, while the monocotyledonous barley caryopsis has a chlorenchymatic layer surrounding its non-green endosperm (storage organ). We have employed pulseamplitude-modulated fluorescence and oxygen-sensitive microsensors to localize and describe gradient distributions of photosynthetic activity across the seed/caryopsis, and have discussed its role in maintaining the endogenous O2 balance. We also report the lack of photosynthetic activity in the stay-green embryo axis of the sacred lotus (Nelumbo nucifera) seed following imbibition. The observations are discussed with respect to in vivo light supply and contrasted with the characteristics of leaf photosynthesis. © 2010 Elsevier Ireland Ltd. All rights reserved.
1. Introduction The seed of almost all our major crops as well as those of their wild relatives are green at some stage during their development. While seed development and associated storage processes has been studied in great detail, processes underlying the light response, seed’s own photosynthesis, diurnal adaptations and finally adjustment of seed metabolism to environmental changes remain largely unknown. So it is reasonable to ask whether or not the photosynthesis occurring within the immature seed is of any physiological relevance, and whether it makes any contribution to seed yield. The greening of the seed is initiated very early in development. In the legume seed, greening depends on the availability of light, and develops initially in the outer regions of embryo (Borisjuk et al., 2005). In the model plant Arabidopsis thaliana, greening is already visible when the embryo reaches the heart stage, and the expression of certain photosynthesis-related genes has been shown to be well correlated with that of key genes associated with oil storage (Ruuska et al., 2002). In the barley caryopsis, the expression of photosynthesis-related genes peaks before the assimilate storage phase (Sreenivasulu et al., 2004). Monitoring of global changes of gene expression identified 2091 differentially expressed genes involved in diurnally affected processes and light responses during development of barley caryopses (Mangelsen et al., 2010). Tight linkage between day/night cycles, changes in light supply, and
∗ Corresponding author. Tel.: +49 39482 5686; fax: +49 39482 5500. E-mail address: [email protected] (H. Rolletschek). 0303-2647/$ – see front matter © 2010 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.biosystems.2010.08.007
assimilate uptake was demonstrated. More than 350 genes were specifically expressed in distinct organs (pericarp, endosperm, embryo). An important event appears to be the differentiation of the plastids. Mechanisms initiating and governing this process are complex and involve integration of light and plastid signals (Larkin and Ruckle, 2008). The development of plastids has been mostly studied in plant leaves but remains largely on a descriptive level in seeds (Pyke, 2007; Rolletschek and Borisjuk, 2005). Embryonic plastids are of photoheterotrophic type, which differs both morphologically and physiologically from leaf chloroplasts (Rolletschek and Borisjuk, 2005). These plastids import carbon via specific translocators (Borisjuk et al., 2005; Rolletschek et al., 2007; Weber et al., 2005), and can utilize low light levels to drive energy production reactions (Browse and Slack, 1985), to activate certain enzymes (Ruuska et al., 2004) and to evolve oxygen (Borisjuk and Rolletschek, 2009). Seed photosynthesis may affect metabolism in a number of distinct ways. The photoheterotrophic plastids are capable of fixing carbon dioxide, although in the cereal caryopsis, this process is rather inefficient (Watson and Duffus, 1988). Plastidial photosynthesis can also contribute to the overall provision of energy, and help to counter the energy deficit generated by the high biosynthetic activity of the seed (Ruuska et al., 2004; Goffman et al., 2005; Allen et al., 2009). In addition, redox signals mediated via the ferredoxin/thioredoxin cascade can modulate the activity of certain biosynthetic enzymes (Buchanan and Balmer, 2005), allowing the plastidial redox pool to be involved in signal transduction between the plastid and the nucleus. Finally, the interior of the develop-
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ing as well as germinating seed is typically hypoxic (Borisjuk and Rolletschek, 2009), and given that low oxygen levels restrict respiration, any endogenous photosynthetic oxygen release could act to relieve shortages in the supply of respiratory energy, and thereby affect biosynthetic activity (Rolletschek et al., 2003). The impact of these various processes depends strongly on the structural organization of the seed. In dicotyledonous species, seed photosynthesis and assimilate storage activity within the embryo tend to spatially coincide with one another. However, in the monocotyledonous species, the chlorophyll-containing pericarp surrounds the nongreen endosperm (the major assimilate storage organ) and embryo, and these structures are physically separated from one another by layers of cutin/suberin, which act to inhibit solute exchange and, with the exception of the non-green crease area, also gas diffusion (Freeman and Palmer, 1984; Rolletschek et al., 2004). This spatial separation implies that the endosperm cannot be directly provided with either energy or redox signals derived from seed photosynthesis. While both oxygen and carbon dioxide are able to move across the cutin/suberin barrier, the significance (if any) of such movement to the regulation of storage metabolism in cereals remains undetermined. Another aspect is the regulation of photosynthetic activity within seed tissues. Photosynthetic function of the seed plastids has to be embedded in cellular metabolism and thus coupled to tissue differentiation. The latter occurs gradiently and is coupled with substantial changes in metabolite levels (Borisjuk et al., 2005; Rolletschek et al., 2007). Sucrose levels affect photosynthesisrelated gene expression (Koch, 2004) and sucrose is by itself gradiently distributed across the seed (Borisjuk et al., 2002). Here, we provide a functional characterization of the photosynthesis occurring within the developing pea seed and barley caryopsis. Spatial variation in photosynthetic activity within these structures has been assessed by a pulse-amplitude-modulated (PAM) fluorescence technique. Our focus has been on the contribution of photosynthetic oxygen release to the overall oxygen budget, which is of especial interest given that the interior of the seed is typically hypoxic (Borisjuk and Rolletschek, 2009). For comparative purposes, we have also included a study of the seed of the sacred lotus (Nelumbo nucifera), whose embryo axes remain green throughout development. The presence of (partially) green embryos in mature seeds is quite uncommon, and is thought to represent an evolutionary relict. However, the trait does give the opportunity to investigate whether this rather unique feature has any role to play during (hypoxic) seed germination.
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470 nm) for 6 min at an irradiance of 172 mol quanta m−2 s−1 (pea and lotus) or 232 mol quanta m−2 s−1 (barley) to achieve a steady state rate of photosynthesis. Thereafter, actinic irradiances in the range of 12–172 mol quanta m−2 s−1 (6–232 mol quanta m−2 s−1 for barley) were imposed for 30 s, starting with the highest light intensity. Measurements of the steady state chlorophyll fluorescence (Ft ) were acquired under actinic illumination, and the maximum fluorescence yield ) was measured during a 800 ms exposure to saturating light intensity. The result(Fm ing rapid light–response curves were used to derive light saturation properties, following White and Critchley (1999). Estimates of the effective quantum yield of by a pixel-by-pixel PSII (˚II ) were obtained from the digitized images of Ft and Fm calculation of [(Fm − Ft )/Fm ]. The ˚II images were interpreted to reveal a map of the relative photosynthetic electron transport rate (ETR), since the value of ˚II lies close to the overall quantum yield of photosynthesis (Genty et al., 1989). ETR represented 0.5 times the product ˚II × PPFD × abs (PPFD: photosynthetic photon flux density, abs: PAR absorptivity). A non-rectangular hyperbola was fitted to estimate the maximum ETR. Abs estimates were obtained from the expression (1 − R/NIR), where R was the re-emission value of diffuse red, and NIR that of infra-red radiation. ˚NPQ was calculated from the expression [(Ft /F m ) − (Ft /Fm )] and ˚NO from (Ft /Fm ). The fluorescence parameter images were displayed using false colour for clarity. The size of the selected sample areas was 0.07–1.1 mm2 , depending on the identity of the test tissue. An estimate for the photosynthetically active surface area of the seed was used to enable the calculation of gross O2 , NADPH and ATP evolution. For pea, a spherical shape was assumed, while for barley, an ellipsoid was chosen. This assumption led to estimates of 113 mm2 and 63 mm2 for the surface areas of, respectively, the pea seed and the barley caryopsis. 2.3. Determination of Respiratory Activity In order to measure respiration in the barley caryopsis, the material was placed inside a 10 ml measuring chamber (equipped with a magnetic stirrer), in which an oxygen-sensitive microsensor (Presens GmbH, Regensburg, Germany) was inserted. The incubation buffer comprised 150 mM sucrose, 30 mM glutamine, 30 mM asparagine, 20 mM KCl, 4 mM CaCl2 , 2 mM K2 SO4 , 2 mM KH2 PO4 , 3 mM MgSO4 and 10 mM MES, pH 5.9. After a 5 min pre-incubation, respiration was measured as O2 uptake in darkness over a 15 min period. The respiration rate in pea was taken from an earlier study (Rolletschek et al., 2003). 2.4. Confocal Laser Scanning Microscopy (CLSM) CLSM was applied to analyse the distribution of chlorophyll in 50 m vibratomeproduced sections of the developing barley caryopsis. The sections were illuminated with 488 nm laser light, and the resulting chlorophyll auto-fluorescence measured by applying a band-pass of 600–650 nm. 2.5. Determination of O2 Concentration in Seeds using Microsensors The O2 concentration inside the N. nucifera seed was determined using a microsensor (Presens, Neuburg, Germany) as detailed by Rolletschek et al. (2004). A tunnel (80 m diameter, 500 m length) was first drilled into the seed, and then the microsensor (30–50 m tip diameter) was mounted in a micro-manipulator and inserted into the tunnel. The insertion point was sealed with rubber to prevent diffusive gas exchange. Once a stable sensor signal had been established, the seed was imbibed inside the germination box. The O2 concentration within the seed was recorded at 10 min intervals.
2. Methods 2.6. Transmission Electron Microscopy (TEM) 2.1. Plant Growth Pea (cv. Erbi) plants were grown in a growth chamber set to provide a lit period of 16 h/19 ◦ C and a dark period of 8 h/16 ◦ C. The seeds were harvested 25 days after pollination (DAP). Barley plants (cv. Barke) were grown in a glasshouse under a 16 h/8 h light/dark regime, and caryopses at various developmental stages were harvested. The pea and barley materials were subjected to PAM analysis immediately after harvest. Seeds of the sacred lotus were soaked in distilled water for 24 h in the dark.
Tissues of pericarp and endosperm of barley caryopses (12 DAP) were chemically fixed with 2% glutaraldehyde and 2% formaldehyde in cacodylate buffer (50 mM, pH 7.0) for 16 h. After three 20 min washes with the same buffer, the tissues were postfixed with 1% OsO4 for 2 h. At the end of this procedure embryos were washed again with buffer and aqua dest followed by dehydration in a graded ethanol series and subsequent embedding in Spurr’s low viscosity resin. After thin sectioning, samples were stained with 4% uranyl acetate and lead citrate. Digital recordings were made on a Zeiss 902 electron microscope at 80 kV.
2.2. Chlorophyll Fluorescence Imaging and Calculation of PAR Absorptivity Estimates of key chlorophyll fluorescence parameters were obtained using an IMAGING-PAM chlorophyll fluorometer (Heinz Walz GmbH, Effeltrich, Germany). This device measures the partition of photosystem II excitation energy into three distinct pathways: photochemical utilization, regulated heat dissipation and nonregulated heat dissipation. These three fluxes are quantified by the quantum yield parameters Y(II) (hereafter referred to as ˚II ), Y(NPQ) [˚NPQ ] and Y(NO) [˚NO ], respectively, which together sum to unity (Kramer et al., 2004). ˚II corresponds to the photo-chemically utilized energy, while ˚NPQ and ˚NO reflect the energy lost as heat and fluorescence. Thus high values of ˚NPQ indicate high photo-protective capacity, whereas high values of ˚NO reflect a lack of protection against damage inflicted by excessive illumination. The imaged area was 17 mm × 22 mm for both pea and lotus, and 7 mm × 9 mm for barley. Seeds were sliced through their centres, covered with a glass plate and pre-illuminated with blue light (peak at
3. Results 3.1. Photosynthesis in Pea is Distributed Across the Embryo and is Adapted to Low Light Conditions The entire embryo of the developing pea seed is green without showing pronounced gradients (Fig. 1A). However, the level of ˚II varied remarkably across the pea embryo (Fig. 1B), showing a steep gradient from the outside to the inside. The abaxial (outer) regions of the embryo had a higher PAR absorptivity than the adaxial (inner) ones (Fig. 1C). Thus the differences
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Fig. 1. Gradient distribution of seed photosynthesis in pea measured by PAM fluorescence. The scale shows the relationship between the colour and the relevant fluorescence parameter. (A) Images of a representative pea seed (21 DAP). (B) Images of effective quantum yield of PSII (˚II ) measured at a light intensity of 135 mol quanta m−2 s−1 . (C) Image of PAR absorbtivity (abs) captured in the dark adapted state. (D) Electron transport rate (ETR in mol electrons m−2 s−1 ) measured at a light intensity of 135 mol quanta m−2 s−1 .
in ETR between the abaxial and adaxial regions were also amplified by differences in PAR absorptivity (Fig. 1D). The ETR reached a peak of 21.3 ± 0.8 mol electrons m−2 s−1 in the seed coat (Fig. 2A), while in the embryo abaxial region it was less (14.0 ± 0.6 mol electrons m−2 s−1 ) but nevertheless greater than in the adaxial region (6.2 ± 1.0 mol electrons m−2 s−1 ). Light saturation in the abaxial region occurred at a photon flux density of ∼75 mol quanta m−2 s−1 (Fig. 2B), whereas in the adaxial region this was only ∼40 mol quanta m−2 s−1 . The leaf ETR was much higher, and no light saturation was observed up to 180 mol quanta m−2 s−1 . Fig. 2C shows a typical dark-light fluorescence induction curve, and indicates that the time course of fluorescence induction for the seed resembles that for a leaf. The maximum fluorescence (Fm ) is
induced by a saturation pulse (SP). From the onset of the actinic light treatment, fluorescence emission peaked rapidly before declining to a nearly steady state level. During this period, repeated satu , which is less than F ration pulses were applied to evaluate Fm m as a result of non-photochemical fluorescence quenching. At the end of the actinic light treatment, the recovery process started and non-photochemical quenching became relaxed within a few minutes. The steady state fluorescence level in the pea seed sections was surprisingly high. The IMAGING-PAM chlorophyll fluorometer allows assessment of excitation energy flux at PS II into photochemical utilization, regulated heat dissipation and non-regulated heat dissipation. These three fluxes are described by the quantum yields Y(II), Y(NPQ) and Y(NO), respectively (for details see Section 2). As shown in Fig. 2D, unlike in the leaf, the pea seed showed an ele-
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Fig. 2. Photosynthetic parameters of pea seeds. (A) Comparison of maximum photosynthetic electron transport activity in various regions of pea seeds. The data represent the means ± S.E. of 6 replicate measurements. (B) Rapid light response curves of ETR in leaves and embryos. Actinic light was applied during consecutive 30 s periods with stepwise decreasing intensity. The data are the means ± S.E. of 5–6 replicate measurements. (C) Typical recording of a dark–light induction curve with repetitive application of saturation pulses measured in abaxial region of the embryo. Intensity of actinic irradiance was 172 mol quanta m−2 s−1 . (D) Excitation flux at PSII: differences between leaves and various seed regions. Complementary changes in quantum yields Y(II) (black), Y(NPQ) (light grey) and Y(NO) (dark grey) were calculated as described in Section 2. Samples were light adapted for 6 min at a light intensity of 172 mol quanta m−2 s−1 . The data are displayed as means of 7 replicate measurements.
vated ˚NO value. The highest values of ˚II and ˚NPQ occurred in the seed coat and in the abaxial regions of the embryo. Both parameters decreased towards the centre of the seed, generally paralleled by an increase in ˚NO . The large ˚NO component reflects the limited photo-protective capacity of the pea seed. In addition to its low light saturation with respect to photosynthetic electron transport (Fig. 2B), this observation underlines the adaptation of seed photosynthesis to low light conditions. 3.2. Photosynthetic Activity in the Barley Caryopsis is Localized within Non-storage Tissues and is Adapted to a High Light Supply In the barley caryopsis, CLSM analysis showed that chlorophyll is only present in a distinct layer of the pericarp surrounding the entire endosperm, except for the region containing the vein and the nucellar projection (Fig. 3A). A characteristic fluorescence image of the effective quantum yield of PS II (measured at 160 mol quanta m−2 s−1 ) during mid-storage stage (12 DAP) is shown in Fig. 3B. As expected, photosynthetic activity was restricted to the chlorenchymatic regions of the pericarp. Thus, the photosynthesizing region envelops the starchy endosperm (major storage organ). The ETR followed a clear stage specific time course during grain development (Fig. 3C): in its early stages (6 DAP), the peak ETR averaged 12.3 ± 1.6 mol electrons m−2 s−1 , while the highest levels (from 27 to 32 mol electrons m−2 s−1 ) were reached during the mid-storage stage (12–22 DAP). Light saturation of
photosynthetic electron transport was not fully reached at a photon flux density of 232 mol quanta m−2 s−1 (mid storage stage, see Fig. 3F). In both the early and late storage stages, photosynthetic electron transport was saturated at a photon flux density of ∼150 mol quanta m−2 s−1 . Thus the extent of photosynthetically active tissue is much smaller in barley than in pea, but its photosynthetic activity is almost double, and is adapted to a high light supply. The respiratory activity of the developing barley caryopsis was assessed by evaluating the rate of O2 uptake under in vitro conditions. This reached its maximum during the early stages of development (5 DAP: 369 ± 22 nmol O2 g−1 FW min−1 ), then declined until 9 DAP to 113 ± 13 nmol O2 g−1 FW min−1 , a level which remained stable until 16 DAP. The barley caryopsis is a complex reproductive organ and actually comprises two generations: the maternal and the (new) filial ones. This raises the questions about the identity of plastids in maternal and filial tissues. Despite the maternal inheritance of the chloroplast genome, the functional identity can be strictly different in the distinct seed organs. As demonstrated in Fig. 3D and E, plastids within the chlorenchyma layer of pericarp (maternal tissue) appear on the ultrastructural level quite similar to those in leaves. This is in contrast to plastids localised in the endosperm (filial tissues), lacking grana and consequently photosynthetic activity. Both types of plastids differ in gene expression and metabolism (Radchuk et al., 2009). The complete lack of photosynthetic abili-
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Fig. 3. Photosynthesis in the barley caryopsis. (A) Chlorophyll auto-fluorescence in a seed cross-section performed by CLSM. (B) Image of effective quantum yield of PSII of a cross-section of caryopsis (12 DAP) detected at a light intensity of 160 mol quanta m−2 s−1 . The scale shows the relationship between the colour and ˚II . (C) Changes in photosynthetic electron transport activity during seed development. ETR was calculated in cross-sections of caryopsis illuminated with a light intensity of 232 mol quanta m−2 s−1 . The data are the means ± S.E. of 4–5 replicate measurements. (D and E) Transmission electron micrographs showing the two contrasting types of plastids within the seed. (D) Plastids in the chlorenchyma of pericarp reveals well developed, regularly distributed grana and small starch grains located between the grana. (E) Plastids in starchy endosperm possess features of amyloplasts with large densely packed starch grains. Abbreviations: g – grana, st – starch, cw – cell wall, m – mitochondria. (F) Rapid light–response curves of ETR of distinct developmental stages of seeds. ETR in response to light intensity was measured in the selected area as indicated in B. Actinic light was applied during consecutive 30 s periods with stepwise decreasing intensity. The data are the means ± S.E. of 4–5 replicate measurements.
ties is a characteristic feature of plastids from starchy endosperm in monocots and embodies their extreme specialisation to starch storage. 3.3. The Stay-green Embryo Axis in the Sacred Lotus is not Photosynthetically Active During Early Germination The sacred lotus embryo axis is green in the mature seed (Fig. 4A). To test whether this region is photosynthetically active upon early germination, we imbibed the seeds, and applied PAM imaging. The PAR absorptivity (Fig. 4B) and steady state fluorescence (Ft ; Fig. 4C) images of the imbibed seed indicated some absorption of photosynthetic active radiation, particularly in the embryo axis itself. However, an examination of illuminated seed cross-sections revealed no differences between the steady state (Fig. 4D). Consequently the estimated effecfluorescence and Fm tive quantum yield of PSII and of ETR were both zero (images not shown). Neither extending the duration of illumination, nor increasing the light intensity varied this result. The maximum quantum yield of PSII in dark-adapted seeds was also undetectable (data not shown). Thus, PAM fluorescence analysis suggested that the imbibed green embryo axis showed no photosynthetic activity. In a confirmatory experiment, oxygen-sensitive microsensors were used to detect photosynthetic O2 release. The microsensor was inserted into the dry seed near the embryo axis, and its O2 status was monitored during imbibition. In the dry seed, the internal O2 levels were ∼250 M, which corresponds to almost saturation, reflecting the absence of (or at least a very low rate of) respiration. Upon imbibition, O2 levels declined gradually over time, reaching strongly hypoxic levels of <10 M (Fig. 5), a time course
very similar to that observed in imbibed pea seeds (Benamar et al., 2008). Although the embryo axis swelled visibly with imbibition, the endogenous O2 level remained rather low, and there was no evidence for any light-dependent O2 evolution. Thus, the green embryo axis appears not to contribute towards O2 evolution within the seed, nor play any role in controlling endogenous hypoxia during germination. 4. Discussion We have sought to apply chlorophyll fluorescence imaging as a platform to clarify gradients of photosynthetic activity within the pea seed and the barley caryopsis. The major advantage of this method is its spatial resolution, which is sufficient to allow for resolution at the tissue level. Thus it has been possible to determine the specific contribution of seed photosynthesis to both developmental events and assimilate storage activity. In barley, photosynthetic activity was restricted to the chlorenchymatic regions of the pericarp, whereas photosynthetic ETR was present throughout the pea seed, with a substantial gradient of activity between the abaxial and adaxial regions. This distribution resembled that in the soybean seed (Borisjuk et al., 2005). The highest ETR occurred in the seed coat, and photosynthetic activity was much reduced in the centre of the seed. Such a gradient derives from (1) the start of degreening in the interior, and (2) the poor penetration of PAR through the seed, where probably <3% is transmitted to the adaxial region (Rolletschek et al., 2005). We have also been able to document the adaptation of seed photosynthesis to low light conditions. Light saturation of the ETR in the seed was typically reached at the comparatively low light intensity of 75–150 mol photons m−2 s−1 .
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Fig. 4. Fluorescence parameters in seeds of sacred lotus. (A) Cross-section of a seed showing the green embryo axis. (B) Image of PAR absorptivity captured in the dark adapted state. (C–D) Images of steady state fluorescence level (C) and of maximum fluorescence yield (D) were measured at a light intensity of 172 mol quanta m−2 s−1 . The scale shows the relationship between the colour and the relevant fluorescence parameter.
Furthermore, the proportion of light energy lost in form of heat and fluorescence (˚NO ) under saturating light conditions was much greater than that experienced in the leaf. The interior of a developing seed is characterized by a state of O2 deficiency (Borisjuk and Rolletschek, 2009). When the internal O2 level is low enough to limit respiration (i.e., hypoxia), raising the supply of O2 through seed photosynthesis could in principle alleviate this stress, by promoting the supply of respiratory energy
Fig. 5. Changes of internal oxygen concentration during imbibition of sacred lotus seeds. Measurements were done by inserting a microsensor near the embryo axis.
and limiting fermentation. Such an improved energy status should result in a higher rate of storage product synthesis with an impact on seed yield. The increase in internal O2 level achieved by photosynthetic O2 release in the pea seed and the barley caryopsis has already been highlighted in the context of internal O2 balance (Rolletschek et al., 2003, 2004). The present data allow a direct calculation of both potential photosynthetic O2 production and the coupled NADPH formation/ATP synthesis. The intensity of full summer sunlight can reach 2000 mol photons m−2 s−1 , but only a fraction of this reaches the photosynthetic active part of the seed. In the case of pea, some 75% of it is absorbed or reflected by the pod, and of the remainder, only 32% penetrates the seed coat to reach the embryo surface. Consequently a maximum intensity of 160 mol photons m−2 s−1 is not only available but also sufficient to drive photosynthesis in the pea embryo. In barley, the situation is somewhat different, since ∼18% of PAR reaches the caryopsis, and thus as much as 350 mol photons m−2 s−1 can be used for photosynthetic purposes. This difference in exploitable PAR probably explains the major differences in the architecture and physiology of photosynthesis between the barley caryopsis and the pea seed. Nevertheless, under nearly saturating light conditions, the calculated gross rates of O2 evolution were comparable: the pea embryo can produce up to 1.3 mol O2 h−1 and the barley caryopsis 1.8 mol O2 h−1 . At the equivalent developmental stages, respiratory O2 demand is 2.5 mol O2 h−1 for pea (Rolletschek et al., 2003) and 0.4 mol O2 h−1 for barley. Thus, under light saturated conditions, local photosynthesis in the pea can contribute about 50% of its total oxygen demand, and the seed has a net O2 uptake rate.
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In contrast, in barley, the evolution of photosynthetic O2 exceeds the caryopsis’ respiratory requirement. As reported for other cereal grains (Nutbeam and Duffus, 1978), therefore, the immature barley caryopsis is a net producer of O2 in the presence of PAR. Using the method proposed by Ruuska et al. (2004), we have calculated that each pea embryo is responsible for the production of 2.5 mol NADPH h−1 through its localized photosynthetic activity, while the equivalent level for the barley caryopsis is 3.5 mol h−1 . Photosynthetic electron transport may generate an ATP synthesis rate of ∼1.7 mol h−1 in the pea embryo and 2.3 mol h−1 in the barley caryopsis. The size of these rates demonstrates the potential impact of seed photosynthesis on assimilate storage. Most of this energy will be provided to the outer regions of seed where most (in the case of barley, all) of the photosynthetic activity occurs. Its contribution to assimilate storage and growth (both of which are also heterogeneously distributed across the seed – see Borisjuk et al., 2004) therefore depends heavily on the seed’s topography. The photosynthetic activity in the barley caryopsis declines during maturation, and by the time of grain maturity, photosynthetic ETR has been completely abolished (data not shown). The pea (and other dicotyledonous species) seed behaves in a similar fashion (own unpublished data), except some varieties with stay-green phenotype. Thus it was not unexpected to find that photosynthetic activity was lacking in the stay-green embryo within the mature sacred lotus seed. Although embryo axis of mature seeds contains chlorophyll, chlorophyll-binding proteins (Ushimaru et al., 2003) and probably Rubisco (Maeda et al., 1996), we have no evidence for photosynthetic activity of the embryo at this early stage of germination. However, as part of an adaptation strategy to extreme environments, it has been suggested that the dry seed of certain halophytic and xerophytic species do trigger photosynthetic activity upon imbibition and germination (Zhang et al., 2010). Thus, the ability to reactivate, rather than to degrade, the plastids can be advantageous under certain conditions. Mechanisms of redifferentiation of embryonic plastids are largely unknown (Tuquet and Newman, 1980; Solymosi et al., 2007). Although a good deal is known about the role of plastids in embryo, the identification of molecular components involved in plastid differentiation during embryogenesis is at the very beginning. Recent studies on albino and embryo lethal mutants of Arabidopsis (sco1-1, sco1-4, wco1, sig6) have proven fundamental differences between plastid development in embryo-derived cells and cells derived from the apical meristem (Albrecht et al., 2006; Ruppel and Hangarter, 2007). Acknowledgement We thank J. Shen-Miller (University of California) for providing seed material for Nelumbo nucifera. References Albrecht, V., Ingenfeld, A., Apel, K., 2006. Characterization of the snowy cotyledon 1 mutant of Arabidopsis thaliana: the impact of chloroplast elongation factor G on chloroplast development and plant vitality. Plant Mol. Biol. 60, 507– 518. Allen, D.K., Ohlrogge, J.B., Shachar-Hill, Y., 2009. The role of light in soybean seed filling metabolism. Plant J. 58, 220–234. Benamar, A., Rolletschek, H., Borisjuk, L., Avelange-Macherel, M.-H., Curien, G., Mostefai, A., Andriantsitohaina, R., Macherel, D., 2008. Nitrite–nitric oxide control of mitochondrial respiration at the frontier of anoxia. Biochim. Biophys. Acta 1777, 1268–1275. Borisjuk, L., Rolletschek, H., 2009. The oxygen status in the developing seeds. New Phytol. 182, 17–30. Borisjuk, L., Walenta, S., Rolletschek, H., Mueller-Klieser, W., Wobus, U., Weber, H., 2002. Spatial analysis of plant metabolism: sucrose imaging within Vicia faba cotyledons reveals specific developmental patterns. Plant J. 29, 521–530.
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Biophys. Acta 990, 87–92. Goffman, F.D., Alonso, A.P., Schwender, J., Shachar-Hill, Y., Ohlrogge, J.B., 2005. Light enables a very high efficiency of carbon storage in developing embryos of rapeseed. Plant Physiol. 137, 1115–1129. Koch, K., 2004. Sucrose metabolism: regulatory mechanisms and pivotal roles in sugar sensing and plant development. Curr. Opin. Plant Biol. 7, 235–246. Kramer, D.M., Johnson, G., Kiirats, O., Edwards, G.E., 2004. New fluorescence parameters for the determination of QA redox state and excitation energy fluxes. Photosynth. Res. 79, 209–218. Larkin, R.M., Ruckle, M.E., 2008. Integration of light and plastid signals. Curr. Opin. Plant Biol. 11, 593–599. Maeda, Y., Minamikawa, T., Xu, B.M., 1996. Metabolic activities in germinated ancient lotus seeds. J. Exp. Bot. 47, 577–582. Mangelsen, E., Wanke, D., Kilian, J., Sundberg, E., Harter, K., Jansson, C., 2010. Significance of light, sugar, and amino acid supply for diurnal gene regulation in developing barley caryopses. Plant Physiol. 153, 14–33. Nutbeam, A.R., Duffus, C.M., 1978. Oxygen exchange in the pericarp green layer of immature cereal grains. Plant Physiol. 62, 360–362. Pyke, K., 2007. Plastid structure: development and differentiation. In: Hoober, J.K. (Ed.), Chloroplast: The Organelle that Sustains Us, The Biomedical & Life Sciences Collection. Henry Stewart Talks Ltd., London (online at http://hstalks.com/bio). Radchuk, V.V., Borisjuk, L., Sreenivasulu, N., Merx, K., Mock, H.P., Rolletschek, H., Wobus, U., Weschke, W., 2009. Spatiotemporal profiling of starch biosynthesis and degradation in the developing barley grain. Plant Physiol. 150, 190–204. Rolletschek, H., Borisjuk, L., 2005. Photosynthesis in seeds: localisation, features and role in storage. Recent Res. Dev. Plant Sci. 3, 25–45. Rolletschek, H., Weber, H., Borisjuk, L., 2003. Energy status and its control on embryogenesis of legumes. Embryo photosynthesis contributes to oxygen supply and is coupled to biosynthetic fluxes. Plant Physiol. 132, 1196–1206. Rolletschek, H., Weschke, W., Weber, H., Wobus, U., Borisjuk, L., 2004. Energy state and its control on seed development: starch accumulation is associated with high ATP and steep oxygen gradients within barley grains. J. Exp. Bot. 55, 1351–1359. Rolletschek, H., Radchuk, R., Klukas, C., Schreiber, F., Wobus, U., Borisjuk, L., 2005. Evidence of a key role for photosynthetic oxygen release in oil storage in developing soybean seeds. New Phytol. 167, 777–786. Rolletschek, H., Nguyen, T.H., Häusler, R.E., Rutten, T., Göbel, C., Feussner, I., Radchuk, R., Tewes, A., Claus, B., Klukas, C., Linemann, U., Weber, H., Wobus, U., Borisjuk, L., 2007. Antisense inhibition of the plastidial glucose-6-phosphate/phosphate translocator in Vicia seeds shifts cellular differentiation and promotes protein storage. Plant J. 51, 468–484. Ruppel, N.J., Hangarter, R.P., 2007. Mutations in a plastid-localized elongation factor G alter early stages of plastid development in Arabidopsis thaliana. BMC Plant Biol. 7, 37. Ruuska, S.A., Girke, T., Benning, C., Ohlrogge, J.B., 2002. Contrapuntal networks of gene expression during Arabidopsis seed filling. Plant Cell 14, 1191–1206. Ruuska, S.A., Schwender, J., Ohlrogge, J.B., 2004. The capacity of green oilseeds to utilize photosynthesis to drive biosynthetic processes. Plant Physiol. 136, 1–10. Solymosi, K., Vitányi, B., Hideg, E., Böddi, B., 2007. Etiolation symptoms in sunflower (Helianthus annuus) cotyledons partially covered by the pericarp of the achene. Ann. Bot. 99, 857–867. Sreenivasulu, N., Altschmied, L., Radchuk, V., Gubatz, S., Wobus, U., Weschke, W., 2004. Transcript profiles and deduced changes of metabolic pathways in maternal and filial tissues of developing barley grains. Plant J. 37, 539–553. Tuquet, C., Newman, D.W., 1980. Aging and regreening in soybean cotyledons. 1. Ultrastructural changes in plastids and plastoglobuli. Cytobios 29, 43–59. Ushimaru, T., Hasegawa, T., Amano, T., Katayama, M., Tanaka, S., Tsuji, H., 2003. Chloroplasts in seeds and dark-grown seedlings of lotus. J. Plant Physiol. 160, 312–324. Watson, P.A., Duffus, C.M., 1988. Carbon dioxide fixation by detached cereal caryopses. Plant Physiol. 87, 504–509. Weber, H., Borisjuk, L., Wobus, U., 2005. Molecular physiology of legume seed development. Ann. Rev. Plant Biol. 56, 253–279. White, A.J., Critchley, C., 1999. Rapid light curves: a new fluorescence method to assess the state of the photosynthetic apparatus. Photosynth. Res. 59, 63–72. Zhang, S., Song, J., Wang, H., Feng, G., 2010. Effect of salinity on seed germination, ion content and photosynthesis of cotyledons in halophytes or xerophyte growing in central Asia. J. Plant Ecol., doi:10.1093/jpe/rtq005.
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Gradients of seed photosynthesis and its role for oxygen balancing Henning Tschiersch, Ljudmilla Borisjuk, Twan Rutten, Hardy Rolletschek ∗ Leibniz-Institut für Pflanzengenetik und Kulturpflanzenforschung (IPK), Corrensstr. 3, 06466 Gatersleben, Germany
a r t i c l e
i n f o
Article history: Received 24 June 2010 Received in revised form 17 August 2010 Accepted 18 August 2010 Keywords: Hypoxia Microsensor PAM fluorescence Seed photosynthesis
a b s t r a c t Seeds are generally viewed in the context of plant reproduction and the supply of food and feed, but only seldom as a site of photosynthesis. However, the seeds of many plant species are green, at least during their early development, which raises the issue of the significance of this greening for seed development. Here we describe the two contrasting modes of photosynthesis in the developing seed. The dicotyledonous pea seed has a green embryo, while the monocotyledonous barley caryopsis has a chlorenchymatic layer surrounding its non-green endosperm (storage organ). We have employed pulseamplitude-modulated fluorescence and oxygen-sensitive microsensors to localize and describe gradient distributions of photosynthetic activity across the seed/caryopsis, and have discussed its role in maintaining the endogenous O2 balance. We also report the lack of photosynthetic activity in the stay-green embryo axis of the sacred lotus (Nelumbo nucifera) seed following imbibition. The observations are discussed with respect to in vivo light supply and contrasted with the characteristics of leaf photosynthesis. © 2010 Elsevier Ireland Ltd. All rights reserved.
1. Introduction The seed of almost all our major crops as well as those of their wild relatives are green at some stage during their development. While seed development and associated storage processes has been studied in great detail, processes underlying the light response, seed’s own photosynthesis, diurnal adaptations and finally adjustment of seed metabolism to environmental changes remain largely unknown. So it is reasonable to ask whether or not the photosynthesis occurring within the immature seed is of any physiological relevance, and whether it makes any contribution to seed yield. The greening of the seed is initiated very early in development. In the legume seed, greening depends on the availability of light, and develops initially in the outer regions of embryo (Borisjuk et al., 2005). In the model plant Arabidopsis thaliana, greening is already visible when the embryo reaches the heart stage, and the expression of certain photosynthesis-related genes has been shown to be well correlated with that of key genes associated with oil storage (Ruuska et al., 2002). In the barley caryopsis, the expression of photosynthesis-related genes peaks before the assimilate storage phase (Sreenivasulu et al., 2004). Monitoring of global changes of gene expression identified 2091 differentially expressed genes involved in diurnally affected processes and light responses during development of barley caryopses (Mangelsen et al., 2010). Tight linkage between day/night cycles, changes in light supply, and
∗ Corresponding author. Tel.: +49 39482 5686; fax: +49 39482 5500. E-mail address: [email protected] (H. Rolletschek). 0303-2647/$ – see front matter © 2010 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.biosystems.2010.08.007
assimilate uptake was demonstrated. More than 350 genes were specifically expressed in distinct organs (pericarp, endosperm, embryo). An important event appears to be the differentiation of the plastids. Mechanisms initiating and governing this process are complex and involve integration of light and plastid signals (Larkin and Ruckle, 2008). The development of plastids has been mostly studied in plant leaves but remains largely on a descriptive level in seeds (Pyke, 2007; Rolletschek and Borisjuk, 2005). Embryonic plastids are of photoheterotrophic type, which differs both morphologically and physiologically from leaf chloroplasts (Rolletschek and Borisjuk, 2005). These plastids import carbon via specific translocators (Borisjuk et al., 2005; Rolletschek et al., 2007; Weber et al., 2005), and can utilize low light levels to drive energy production reactions (Browse and Slack, 1985), to activate certain enzymes (Ruuska et al., 2004) and to evolve oxygen (Borisjuk and Rolletschek, 2009). Seed photosynthesis may affect metabolism in a number of distinct ways. The photoheterotrophic plastids are capable of fixing carbon dioxide, although in the cereal caryopsis, this process is rather inefficient (Watson and Duffus, 1988). Plastidial photosynthesis can also contribute to the overall provision of energy, and help to counter the energy deficit generated by the high biosynthetic activity of the seed (Ruuska et al., 2004; Goffman et al., 2005; Allen et al., 2009). In addition, redox signals mediated via the ferredoxin/thioredoxin cascade can modulate the activity of certain biosynthetic enzymes (Buchanan and Balmer, 2005), allowing the plastidial redox pool to be involved in signal transduction between the plastid and the nucleus. Finally, the interior of the develop-
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ing as well as germinating seed is typically hypoxic (Borisjuk and Rolletschek, 2009), and given that low oxygen levels restrict respiration, any endogenous photosynthetic oxygen release could act to relieve shortages in the supply of respiratory energy, and thereby affect biosynthetic activity (Rolletschek et al., 2003). The impact of these various processes depends strongly on the structural organization of the seed. In dicotyledonous species, seed photosynthesis and assimilate storage activity within the embryo tend to spatially coincide with one another. However, in the monocotyledonous species, the chlorophyll-containing pericarp surrounds the nongreen endosperm (the major assimilate storage organ) and embryo, and these structures are physically separated from one another by layers of cutin/suberin, which act to inhibit solute exchange and, with the exception of the non-green crease area, also gas diffusion (Freeman and Palmer, 1984; Rolletschek et al., 2004). This spatial separation implies that the endosperm cannot be directly provided with either energy or redox signals derived from seed photosynthesis. While both oxygen and carbon dioxide are able to move across the cutin/suberin barrier, the significance (if any) of such movement to the regulation of storage metabolism in cereals remains undetermined. Another aspect is the regulation of photosynthetic activity within seed tissues. Photosynthetic function of the seed plastids has to be embedded in cellular metabolism and thus coupled to tissue differentiation. The latter occurs gradiently and is coupled with substantial changes in metabolite levels (Borisjuk et al., 2005; Rolletschek et al., 2007). Sucrose levels affect photosynthesisrelated gene expression (Koch, 2004) and sucrose is by itself gradiently distributed across the seed (Borisjuk et al., 2002). Here, we provide a functional characterization of the photosynthesis occurring within the developing pea seed and barley caryopsis. Spatial variation in photosynthetic activity within these structures has been assessed by a pulse-amplitude-modulated (PAM) fluorescence technique. Our focus has been on the contribution of photosynthetic oxygen release to the overall oxygen budget, which is of especial interest given that the interior of the seed is typically hypoxic (Borisjuk and Rolletschek, 2009). For comparative purposes, we have also included a study of the seed of the sacred lotus (Nelumbo nucifera), whose embryo axes remain green throughout development. The presence of (partially) green embryos in mature seeds is quite uncommon, and is thought to represent an evolutionary relict. However, the trait does give the opportunity to investigate whether this rather unique feature has any role to play during (hypoxic) seed germination.
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470 nm) for 6 min at an irradiance of 172 mol quanta m−2 s−1 (pea and lotus) or 232 mol quanta m−2 s−1 (barley) to achieve a steady state rate of photosynthesis. Thereafter, actinic irradiances in the range of 12–172 mol quanta m−2 s−1 (6–232 mol quanta m−2 s−1 for barley) were imposed for 30 s, starting with the highest light intensity. Measurements of the steady state chlorophyll fluorescence (Ft ) were acquired under actinic illumination, and the maximum fluorescence yield ) was measured during a 800 ms exposure to saturating light intensity. The result(Fm ing rapid light–response curves were used to derive light saturation properties, following White and Critchley (1999). Estimates of the effective quantum yield of by a pixel-by-pixel PSII (˚II ) were obtained from the digitized images of Ft and Fm calculation of [(Fm − Ft )/Fm ]. The ˚II images were interpreted to reveal a map of the relative photosynthetic electron transport rate (ETR), since the value of ˚II lies close to the overall quantum yield of photosynthesis (Genty et al., 1989). ETR represented 0.5 times the product ˚II × PPFD × abs (PPFD: photosynthetic photon flux density, abs: PAR absorptivity). A non-rectangular hyperbola was fitted to estimate the maximum ETR. Abs estimates were obtained from the expression (1 − R/NIR), where R was the re-emission value of diffuse red, and NIR that of infra-red radiation. ˚NPQ was calculated from the expression [(Ft /F m ) − (Ft /Fm )] and ˚NO from (Ft /Fm ). The fluorescence parameter images were displayed using false colour for clarity. The size of the selected sample areas was 0.07–1.1 mm2 , depending on the identity of the test tissue. An estimate for the photosynthetically active surface area of the seed was used to enable the calculation of gross O2 , NADPH and ATP evolution. For pea, a spherical shape was assumed, while for barley, an ellipsoid was chosen. This assumption led to estimates of 113 mm2 and 63 mm2 for the surface areas of, respectively, the pea seed and the barley caryopsis. 2.3. Determination of Respiratory Activity In order to measure respiration in the barley caryopsis, the material was placed inside a 10 ml measuring chamber (equipped with a magnetic stirrer), in which an oxygen-sensitive microsensor (Presens GmbH, Regensburg, Germany) was inserted. The incubation buffer comprised 150 mM sucrose, 30 mM glutamine, 30 mM asparagine, 20 mM KCl, 4 mM CaCl2 , 2 mM K2 SO4 , 2 mM KH2 PO4 , 3 mM MgSO4 and 10 mM MES, pH 5.9. After a 5 min pre-incubation, respiration was measured as O2 uptake in darkness over a 15 min period. The respiration rate in pea was taken from an earlier study (Rolletschek et al., 2003). 2.4. Confocal Laser Scanning Microscopy (CLSM) CLSM was applied to analyse the distribution of chlorophyll in 50 m vibratomeproduced sections of the developing barley caryopsis. The sections were illuminated with 488 nm laser light, and the resulting chlorophyll auto-fluorescence measured by applying a band-pass of 600–650 nm. 2.5. Determination of O2 Concentration in Seeds using Microsensors The O2 concentration inside the N. nucifera seed was determined using a microsensor (Presens, Neuburg, Germany) as detailed by Rolletschek et al. (2004). A tunnel (80 m diameter, 500 m length) was first drilled into the seed, and then the microsensor (30–50 m tip diameter) was mounted in a micro-manipulator and inserted into the tunnel. The insertion point was sealed with rubber to prevent diffusive gas exchange. Once a stable sensor signal had been established, the seed was imbibed inside the germination box. The O2 concentration within the seed was recorded at 10 min intervals.
2. Methods 2.6. Transmission Electron Microscopy (TEM) 2.1. Plant Growth Pea (cv. Erbi) plants were grown in a growth chamber set to provide a lit period of 16 h/19 ◦ C and a dark period of 8 h/16 ◦ C. The seeds were harvested 25 days after pollination (DAP). Barley plants (cv. Barke) were grown in a glasshouse under a 16 h/8 h light/dark regime, and caryopses at various developmental stages were harvested. The pea and barley materials were subjected to PAM analysis immediately after harvest. Seeds of the sacred lotus were soaked in distilled water for 24 h in the dark.
Tissues of pericarp and endosperm of barley caryopses (12 DAP) were chemically fixed with 2% glutaraldehyde and 2% formaldehyde in cacodylate buffer (50 mM, pH 7.0) for 16 h. After three 20 min washes with the same buffer, the tissues were postfixed with 1% OsO4 for 2 h. At the end of this procedure embryos were washed again with buffer and aqua dest followed by dehydration in a graded ethanol series and subsequent embedding in Spurr’s low viscosity resin. After thin sectioning, samples were stained with 4% uranyl acetate and lead citrate. Digital recordings were made on a Zeiss 902 electron microscope at 80 kV.
2.2. Chlorophyll Fluorescence Imaging and Calculation of PAR Absorptivity Estimates of key chlorophyll fluorescence parameters were obtained using an IMAGING-PAM chlorophyll fluorometer (Heinz Walz GmbH, Effeltrich, Germany). This device measures the partition of photosystem II excitation energy into three distinct pathways: photochemical utilization, regulated heat dissipation and nonregulated heat dissipation. These three fluxes are quantified by the quantum yield parameters Y(II) (hereafter referred to as ˚II ), Y(NPQ) [˚NPQ ] and Y(NO) [˚NO ], respectively, which together sum to unity (Kramer et al., 2004). ˚II corresponds to the photo-chemically utilized energy, while ˚NPQ and ˚NO reflect the energy lost as heat and fluorescence. Thus high values of ˚NPQ indicate high photo-protective capacity, whereas high values of ˚NO reflect a lack of protection against damage inflicted by excessive illumination. The imaged area was 17 mm × 22 mm for both pea and lotus, and 7 mm × 9 mm for barley. Seeds were sliced through their centres, covered with a glass plate and pre-illuminated with blue light (peak at
3. Results 3.1. Photosynthesis in Pea is Distributed Across the Embryo and is Adapted to Low Light Conditions The entire embryo of the developing pea seed is green without showing pronounced gradients (Fig. 1A). However, the level of ˚II varied remarkably across the pea embryo (Fig. 1B), showing a steep gradient from the outside to the inside. The abaxial (outer) regions of the embryo had a higher PAR absorptivity than the adaxial (inner) ones (Fig. 1C). Thus the differences
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Fig. 1. Gradient distribution of seed photosynthesis in pea measured by PAM fluorescence. The scale shows the relationship between the colour and the relevant fluorescence parameter. (A) Images of a representative pea seed (21 DAP). (B) Images of effective quantum yield of PSII (˚II ) measured at a light intensity of 135 mol quanta m−2 s−1 . (C) Image of PAR absorbtivity (abs) captured in the dark adapted state. (D) Electron transport rate (ETR in mol electrons m−2 s−1 ) measured at a light intensity of 135 mol quanta m−2 s−1 .
in ETR between the abaxial and adaxial regions were also amplified by differences in PAR absorptivity (Fig. 1D). The ETR reached a peak of 21.3 ± 0.8 mol electrons m−2 s−1 in the seed coat (Fig. 2A), while in the embryo abaxial region it was less (14.0 ± 0.6 mol electrons m−2 s−1 ) but nevertheless greater than in the adaxial region (6.2 ± 1.0 mol electrons m−2 s−1 ). Light saturation in the abaxial region occurred at a photon flux density of ∼75 mol quanta m−2 s−1 (Fig. 2B), whereas in the adaxial region this was only ∼40 mol quanta m−2 s−1 . The leaf ETR was much higher, and no light saturation was observed up to 180 mol quanta m−2 s−1 . Fig. 2C shows a typical dark-light fluorescence induction curve, and indicates that the time course of fluorescence induction for the seed resembles that for a leaf. The maximum fluorescence (Fm ) is
induced by a saturation pulse (SP). From the onset of the actinic light treatment, fluorescence emission peaked rapidly before declining to a nearly steady state level. During this period, repeated satu , which is less than F ration pulses were applied to evaluate Fm m as a result of non-photochemical fluorescence quenching. At the end of the actinic light treatment, the recovery process started and non-photochemical quenching became relaxed within a few minutes. The steady state fluorescence level in the pea seed sections was surprisingly high. The IMAGING-PAM chlorophyll fluorometer allows assessment of excitation energy flux at PS II into photochemical utilization, regulated heat dissipation and non-regulated heat dissipation. These three fluxes are described by the quantum yields Y(II), Y(NPQ) and Y(NO), respectively (for details see Section 2). As shown in Fig. 2D, unlike in the leaf, the pea seed showed an ele-
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Fig. 2. Photosynthetic parameters of pea seeds. (A) Comparison of maximum photosynthetic electron transport activity in various regions of pea seeds. The data represent the means ± S.E. of 6 replicate measurements. (B) Rapid light response curves of ETR in leaves and embryos. Actinic light was applied during consecutive 30 s periods with stepwise decreasing intensity. The data are the means ± S.E. of 5–6 replicate measurements. (C) Typical recording of a dark–light induction curve with repetitive application of saturation pulses measured in abaxial region of the embryo. Intensity of actinic irradiance was 172 mol quanta m−2 s−1 . (D) Excitation flux at PSII: differences between leaves and various seed regions. Complementary changes in quantum yields Y(II) (black), Y(NPQ) (light grey) and Y(NO) (dark grey) were calculated as described in Section 2. Samples were light adapted for 6 min at a light intensity of 172 mol quanta m−2 s−1 . The data are displayed as means of 7 replicate measurements.
vated ˚NO value. The highest values of ˚II and ˚NPQ occurred in the seed coat and in the abaxial regions of the embryo. Both parameters decreased towards the centre of the seed, generally paralleled by an increase in ˚NO . The large ˚NO component reflects the limited photo-protective capacity of the pea seed. In addition to its low light saturation with respect to photosynthetic electron transport (Fig. 2B), this observation underlines the adaptation of seed photosynthesis to low light conditions. 3.2. Photosynthetic Activity in the Barley Caryopsis is Localized within Non-storage Tissues and is Adapted to a High Light Supply In the barley caryopsis, CLSM analysis showed that chlorophyll is only present in a distinct layer of the pericarp surrounding the entire endosperm, except for the region containing the vein and the nucellar projection (Fig. 3A). A characteristic fluorescence image of the effective quantum yield of PS II (measured at 160 mol quanta m−2 s−1 ) during mid-storage stage (12 DAP) is shown in Fig. 3B. As expected, photosynthetic activity was restricted to the chlorenchymatic regions of the pericarp. Thus, the photosynthesizing region envelops the starchy endosperm (major storage organ). The ETR followed a clear stage specific time course during grain development (Fig. 3C): in its early stages (6 DAP), the peak ETR averaged 12.3 ± 1.6 mol electrons m−2 s−1 , while the highest levels (from 27 to 32 mol electrons m−2 s−1 ) were reached during the mid-storage stage (12–22 DAP). Light saturation of
photosynthetic electron transport was not fully reached at a photon flux density of 232 mol quanta m−2 s−1 (mid storage stage, see Fig. 3F). In both the early and late storage stages, photosynthetic electron transport was saturated at a photon flux density of ∼150 mol quanta m−2 s−1 . Thus the extent of photosynthetically active tissue is much smaller in barley than in pea, but its photosynthetic activity is almost double, and is adapted to a high light supply. The respiratory activity of the developing barley caryopsis was assessed by evaluating the rate of O2 uptake under in vitro conditions. This reached its maximum during the early stages of development (5 DAP: 369 ± 22 nmol O2 g−1 FW min−1 ), then declined until 9 DAP to 113 ± 13 nmol O2 g−1 FW min−1 , a level which remained stable until 16 DAP. The barley caryopsis is a complex reproductive organ and actually comprises two generations: the maternal and the (new) filial ones. This raises the questions about the identity of plastids in maternal and filial tissues. Despite the maternal inheritance of the chloroplast genome, the functional identity can be strictly different in the distinct seed organs. As demonstrated in Fig. 3D and E, plastids within the chlorenchyma layer of pericarp (maternal tissue) appear on the ultrastructural level quite similar to those in leaves. This is in contrast to plastids localised in the endosperm (filial tissues), lacking grana and consequently photosynthetic activity. Both types of plastids differ in gene expression and metabolism (Radchuk et al., 2009). The complete lack of photosynthetic abili-
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Fig. 3. Photosynthesis in the barley caryopsis. (A) Chlorophyll auto-fluorescence in a seed cross-section performed by CLSM. (B) Image of effective quantum yield of PSII of a cross-section of caryopsis (12 DAP) detected at a light intensity of 160 mol quanta m−2 s−1 . The scale shows the relationship between the colour and ˚II . (C) Changes in photosynthetic electron transport activity during seed development. ETR was calculated in cross-sections of caryopsis illuminated with a light intensity of 232 mol quanta m−2 s−1 . The data are the means ± S.E. of 4–5 replicate measurements. (D and E) Transmission electron micrographs showing the two contrasting types of plastids within the seed. (D) Plastids in the chlorenchyma of pericarp reveals well developed, regularly distributed grana and small starch grains located between the grana. (E) Plastids in starchy endosperm possess features of amyloplasts with large densely packed starch grains. Abbreviations: g – grana, st – starch, cw – cell wall, m – mitochondria. (F) Rapid light–response curves of ETR of distinct developmental stages of seeds. ETR in response to light intensity was measured in the selected area as indicated in B. Actinic light was applied during consecutive 30 s periods with stepwise decreasing intensity. The data are the means ± S.E. of 4–5 replicate measurements.
ties is a characteristic feature of plastids from starchy endosperm in monocots and embodies their extreme specialisation to starch storage. 3.3. The Stay-green Embryo Axis in the Sacred Lotus is not Photosynthetically Active During Early Germination The sacred lotus embryo axis is green in the mature seed (Fig. 4A). To test whether this region is photosynthetically active upon early germination, we imbibed the seeds, and applied PAM imaging. The PAR absorptivity (Fig. 4B) and steady state fluorescence (Ft ; Fig. 4C) images of the imbibed seed indicated some absorption of photosynthetic active radiation, particularly in the embryo axis itself. However, an examination of illuminated seed cross-sections revealed no differences between the steady state (Fig. 4D). Consequently the estimated effecfluorescence and Fm tive quantum yield of PSII and of ETR were both zero (images not shown). Neither extending the duration of illumination, nor increasing the light intensity varied this result. The maximum quantum yield of PSII in dark-adapted seeds was also undetectable (data not shown). Thus, PAM fluorescence analysis suggested that the imbibed green embryo axis showed no photosynthetic activity. In a confirmatory experiment, oxygen-sensitive microsensors were used to detect photosynthetic O2 release. The microsensor was inserted into the dry seed near the embryo axis, and its O2 status was monitored during imbibition. In the dry seed, the internal O2 levels were ∼250 M, which corresponds to almost saturation, reflecting the absence of (or at least a very low rate of) respiration. Upon imbibition, O2 levels declined gradually over time, reaching strongly hypoxic levels of <10 M (Fig. 5), a time course
very similar to that observed in imbibed pea seeds (Benamar et al., 2008). Although the embryo axis swelled visibly with imbibition, the endogenous O2 level remained rather low, and there was no evidence for any light-dependent O2 evolution. Thus, the green embryo axis appears not to contribute towards O2 evolution within the seed, nor play any role in controlling endogenous hypoxia during germination. 4. Discussion We have sought to apply chlorophyll fluorescence imaging as a platform to clarify gradients of photosynthetic activity within the pea seed and the barley caryopsis. The major advantage of this method is its spatial resolution, which is sufficient to allow for resolution at the tissue level. Thus it has been possible to determine the specific contribution of seed photosynthesis to both developmental events and assimilate storage activity. In barley, photosynthetic activity was restricted to the chlorenchymatic regions of the pericarp, whereas photosynthetic ETR was present throughout the pea seed, with a substantial gradient of activity between the abaxial and adaxial regions. This distribution resembled that in the soybean seed (Borisjuk et al., 2005). The highest ETR occurred in the seed coat, and photosynthetic activity was much reduced in the centre of the seed. Such a gradient derives from (1) the start of degreening in the interior, and (2) the poor penetration of PAR through the seed, where probably <3% is transmitted to the adaxial region (Rolletschek et al., 2005). We have also been able to document the adaptation of seed photosynthesis to low light conditions. Light saturation of the ETR in the seed was typically reached at the comparatively low light intensity of 75–150 mol photons m−2 s−1 .
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Fig. 4. Fluorescence parameters in seeds of sacred lotus. (A) Cross-section of a seed showing the green embryo axis. (B) Image of PAR absorptivity captured in the dark adapted state. (C–D) Images of steady state fluorescence level (C) and of maximum fluorescence yield (D) were measured at a light intensity of 172 mol quanta m−2 s−1 . The scale shows the relationship between the colour and the relevant fluorescence parameter.
Furthermore, the proportion of light energy lost in form of heat and fluorescence (˚NO ) under saturating light conditions was much greater than that experienced in the leaf. The interior of a developing seed is characterized by a state of O2 deficiency (Borisjuk and Rolletschek, 2009). When the internal O2 level is low enough to limit respiration (i.e., hypoxia), raising the supply of O2 through seed photosynthesis could in principle alleviate this stress, by promoting the supply of respiratory energy
Fig. 5. Changes of internal oxygen concentration during imbibition of sacred lotus seeds. Measurements were done by inserting a microsensor near the embryo axis.
and limiting fermentation. Such an improved energy status should result in a higher rate of storage product synthesis with an impact on seed yield. The increase in internal O2 level achieved by photosynthetic O2 release in the pea seed and the barley caryopsis has already been highlighted in the context of internal O2 balance (Rolletschek et al., 2003, 2004). The present data allow a direct calculation of both potential photosynthetic O2 production and the coupled NADPH formation/ATP synthesis. The intensity of full summer sunlight can reach 2000 mol photons m−2 s−1 , but only a fraction of this reaches the photosynthetic active part of the seed. In the case of pea, some 75% of it is absorbed or reflected by the pod, and of the remainder, only 32% penetrates the seed coat to reach the embryo surface. Consequently a maximum intensity of 160 mol photons m−2 s−1 is not only available but also sufficient to drive photosynthesis in the pea embryo. In barley, the situation is somewhat different, since ∼18% of PAR reaches the caryopsis, and thus as much as 350 mol photons m−2 s−1 can be used for photosynthetic purposes. This difference in exploitable PAR probably explains the major differences in the architecture and physiology of photosynthesis between the barley caryopsis and the pea seed. Nevertheless, under nearly saturating light conditions, the calculated gross rates of O2 evolution were comparable: the pea embryo can produce up to 1.3 mol O2 h−1 and the barley caryopsis 1.8 mol O2 h−1 . At the equivalent developmental stages, respiratory O2 demand is 2.5 mol O2 h−1 for pea (Rolletschek et al., 2003) and 0.4 mol O2 h−1 for barley. Thus, under light saturated conditions, local photosynthesis in the pea can contribute about 50% of its total oxygen demand, and the seed has a net O2 uptake rate.
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In contrast, in barley, the evolution of photosynthetic O2 exceeds the caryopsis’ respiratory requirement. As reported for other cereal grains (Nutbeam and Duffus, 1978), therefore, the immature barley caryopsis is a net producer of O2 in the presence of PAR. Using the method proposed by Ruuska et al. (2004), we have calculated that each pea embryo is responsible for the production of 2.5 mol NADPH h−1 through its localized photosynthetic activity, while the equivalent level for the barley caryopsis is 3.5 mol h−1 . Photosynthetic electron transport may generate an ATP synthesis rate of ∼1.7 mol h−1 in the pea embryo and 2.3 mol h−1 in the barley caryopsis. The size of these rates demonstrates the potential impact of seed photosynthesis on assimilate storage. Most of this energy will be provided to the outer regions of seed where most (in the case of barley, all) of the photosynthetic activity occurs. Its contribution to assimilate storage and growth (both of which are also heterogeneously distributed across the seed – see Borisjuk et al., 2004) therefore depends heavily on the seed’s topography. The photosynthetic activity in the barley caryopsis declines during maturation, and by the time of grain maturity, photosynthetic ETR has been completely abolished (data not shown). The pea (and other dicotyledonous species) seed behaves in a similar fashion (own unpublished data), except some varieties with stay-green phenotype. Thus it was not unexpected to find that photosynthetic activity was lacking in the stay-green embryo within the mature sacred lotus seed. Although embryo axis of mature seeds contains chlorophyll, chlorophyll-binding proteins (Ushimaru et al., 2003) and probably Rubisco (Maeda et al., 1996), we have no evidence for photosynthetic activity of the embryo at this early stage of germination. However, as part of an adaptation strategy to extreme environments, it has been suggested that the dry seed of certain halophytic and xerophytic species do trigger photosynthetic activity upon imbibition and germination (Zhang et al., 2010). Thus, the ability to reactivate, rather than to degrade, the plastids can be advantageous under certain conditions. Mechanisms of redifferentiation of embryonic plastids are largely unknown (Tuquet and Newman, 1980; Solymosi et al., 2007). Although a good deal is known about the role of plastids in embryo, the identification of molecular components involved in plastid differentiation during embryogenesis is at the very beginning. Recent studies on albino and embryo lethal mutants of Arabidopsis (sco1-1, sco1-4, wco1, sig6) have proven fundamental differences between plastid development in embryo-derived cells and cells derived from the apical meristem (Albrecht et al., 2006; Ruppel and Hangarter, 2007). Acknowledgement We thank J. Shen-Miller (University of California) for providing seed material for Nelumbo nucifera. References Albrecht, V., Ingenfeld, A., Apel, K., 2006. Characterization of the snowy cotyledon 1 mutant of Arabidopsis thaliana: the impact of chloroplast elongation factor G on chloroplast development and plant vitality. Plant Mol. Biol. 60, 507– 518. Allen, D.K., Ohlrogge, J.B., Shachar-Hill, Y., 2009. The role of light in soybean seed filling metabolism. Plant J. 58, 220–234. Benamar, A., Rolletschek, H., Borisjuk, L., Avelange-Macherel, M.-H., Curien, G., Mostefai, A., Andriantsitohaina, R., Macherel, D., 2008. Nitrite–nitric oxide control of mitochondrial respiration at the frontier of anoxia. Biochim. Biophys. Acta 1777, 1268–1275. Borisjuk, L., Rolletschek, H., 2009. The oxygen status in the developing seeds. New Phytol. 182, 17–30. Borisjuk, L., Walenta, S., Rolletschek, H., Mueller-Klieser, W., Wobus, U., Weber, H., 2002. Spatial analysis of plant metabolism: sucrose imaging within Vicia faba cotyledons reveals specific developmental patterns. Plant J. 29, 521–530.
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