Photosynthetic metabolism during phosphate limitation in a legume from the Mediterranean-type Fynbos ecosystem

Journal Pre-proof Photosynthetic metabolism during phosphate limitation in a legume from the Mediterranean-type Fynbos ecosystem ´ Munoz, ˜ Stian Griebenow, Alejandra Zuniga-Feest, Gaston Pablo Cornejo, Aleysia Kleinert, Alex Valentine

PII:

S0176-1617(19)30174-9

DOI:

https://doi.org/10.1016/j.jplph.2019.153051

Reference:

JPLPH 153051

To appear in:

Journal of Plant Physiology

Received Date:

17 May 2019

Revised Date:

12 September 2019

Accepted Date:

12 September 2019

˜ G, Cornejo P, Kleinert A, Please cite this article as: Griebenow S, Zuniga-Feest A, Munoz Valentine A, Photosynthetic metabolism during phosphate limitation in a legume from the Mediterranean-type Fynbos ecosystem, Journal of Plant Physiology (2019), doi: https://doi.org/10.1016/j.jplph.2019.153051

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Photosynthetic metabolism during phosphate limitation in a legume from the Mediterranean-type Fynbos ecosystem.

Stian Griebenow1, Alejandra Zuniga-Feest2, Gastón Muñoz3, Pablo Cornejo4, Aleysia Kleinert1 and Alex Valentine1* 1

Botany and Zoology Department, University of Stellenbosch, Private Bag X1, Matieland, 7602, South Africa. 2

Laboratorio de Biología Vegetal, Instituto de Ciencias Ambientales y Evolutivas, Facultad de Ciencias, Universidad Austral de Chile, Campus Isla Teja s/n, Valdivia, Chile 3

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Facultad de Medicina Ciencia, Universidad San Sebastián, General Lagos 1163, Valdivia, Chile. 4

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Centro de Investigación en Micorrizas y Sustentabilidad Agroambiental (CIMYSA), Universidad de La Frontera, P.O. Box 54-D, Temuco, Chile.

*Corresponding

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author: e-mail: [email protected] Tel: (+27+21) 808-3067 Fax: (+27+21) 808-2405 Botany and Zoology Department, University of Stellenbosch, Private Bag X1, Matieland, 7602, South Africa.

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Abstract

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Phosphorus (P) is an essential mineral, required for crucial plant genetic, metabolic and signaling functions. Under P deficiency, normal physiological function can be disrupted, especially photosynthetic metabolism. The majority of photosynthetic studies of P stress has been on model organisms, and very little is known about plants that evolved on P deficient soils. Aspalathus linearis (Burm.f.) R.Dahlgren, a native to the Mediterranean ecosystem of South Africa was used to study the photosynthetic responses during short-term P limitation. A. linearis seedlings were cultured under glasshouse conditions and exposed to short-term P stress. Leaf photosynthetic gas exchange was coupled with metabolic analyses. In spite of the decline in leaf cellular Pi, the photosynthetic rates remained unchanged. These leaves also maintained their levels of light harvesting and reaction center pigments. The efficiency of the light reactions' utilization of ATP and NADPH increased during P-stress. Leaf glucose levels decreased during P-stress, while sucrose concentrations remained unaffected. These results show that during short-term P-stress, A. linearis can maintain its photosynthetic rates by altering the structural and functional components of the light reactions.

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Key words: Phosphorus, Photosynthesis, Asphalathus linearis, Nutrient-poor soils

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Introduction:

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Phosphorus (P) is an essential mineral which is required for a wide range of structural and metabolic functions, including essential processes such as photosynthesis and respiration (Ohlrogge et al., 2007; Raghothama, 1999; White and Hammond, 2008). Although P may be available in large quantities in soils, it is hower not always biologically available for plant uptake. Plants are only able to take up P in the forms of H2PO4– and HPO4 2– (Pi or orthophosphate) from the soil. (Hinsinger, 2001; Péret et al., 2011). Each of form of orthophosphate is only accessible at certain pH levels. The optimal pH for Pi uptake is between 4.5 and 5.8 (Raghothama, 1999). There is free Pi in the soil medium, but as Pi moves slowly through the medium there is localised deficiency around roots of plants (Hinsinger, 2001). Plants that have evolved on P deprived soils such as those in Australia, Chile and South Africa have modified their physiology to cope with P-stress (Lambers et al., 2008). Plants under P limitation have a suite of integrated mechanisms to adapt to P-stress, by either increasing P acquisition and recycling or increasing the efficiency of P utilisation in metabolism.

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Therefore, plants have various morphological and physiological responses to P deficiency which enable their survival in P-stressed conditions. 1) Root acidification of the rhizosphere, allows plants to mobilise Pi from organic and inorganic sources (White and Hammond, 2008). 2) Biomass allocation to alter the root architecture, for increased absorptive area of the roots (Vance et al., 2003). 3) Enhanced expression of Pi transporters to increase Pi uptake (White and Hammond, 2008). Nutrient deficiency can impact on many essential processes, including the photosynthetic capacity of plants (Longstreth and Nobel, 1980). Limited levels of Pi can reduce photosynthesis due to the energy demand required for the formation of phosphorylated intermediates in the Calvin cycle (Fredeen et al., 1990). The photosynthetic rate is regulated by the enzymes of the Calvin cycle, ATP and NADPH concentration and the pool of phosphorylated sugars, which act as a system (Geiger and Servaites, 1994). Importantly several variables interact with each other, thus if one is affected will result in the alteration to the other variables (Servaites et al., 1991). Photosynthesis is the primary source of plant productivity and consists of two distinct phases, the light and dark reaction (van Amerongen and Croce, 2013). The light reaction is based on the utilisation of light energy to produce nicotinamide adenine dinucleotide phosphate (NADPH), adenosine triphosphate (ATP) and oxygen (O2). The dark reaction consumes the products of the light reaction to fix CO2 via the Calvin cycle (Taiz et al., 2014). To obtain light energy, chlorophyll b and carotenoids pigments act as light absorbing pigments (Lichtenthaler, 1987). Chlorophyll a is the reaction centre pigment that transfers the harvested light energy to the different electron carriers (Lichtenthaler, 1987). The formation of light reaction products enables the fixation of CO2 through the Calvin cycle, with Triose-P being exported (Taiz et al., 2014). The Triose-P is then further used for the synthesis of essential sugars and the formation of starch (Zeeman, 2007). Reduction in Pi concentration can result in alterations to more than just to the photosynthetic mechanism, but also to respiration and metabolism, which can all lead to a reduction in maximum photosynthetic rate (Rychter et al., 2016). Under P-stressed conditions the efficiency of the Calvin cycle is reduced through the limitation of ATP, thus causing a reduction in the regeneration of ATP (Jacob and Lawlor, 1993). Chlorophyll pigments are also reduced to prevent damage to photosystems 3

(Jacob and Lawlor, 1991). This leads to decreased ATP and NADPH concentrations, due to the decreased efficiency of the light reaction (Zhang et al., 2009). By limiting the production of light reaction products, P-stress can also indirectly affect the synthesis of products via the Calvin cycle (Campbell and Sage, 2006). Under P-stress, the reduced triose-P export from the chloroplasts can limit sugar synthesis in the cytosol, and may rather be used for the starch formation instead (Kang et al., 2014; Zhang et al., 2014).

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Most studies that on plant P stress, have focussed on known model species, such as Arabidopsis, wheat, maize and the common bean. However there is a lack of understanding how species that have evolved on the P-limited soils in nutrient-poor ecosystems of the world, adapt their photosynthetic processes to P limitation. Recent studies on indigenous plants to naturally P-poor soils have looked at below-ground P acquisition strategies and alterations in root metabolism (Vardien et al., 2016). As such it is important to obtain information regarding the above-ground photosynthetic adjustments of plants, which are native to nutrient-poor ecosystems. Aspalathus is a genus of Fabaceae (legumes) that is endemic to the P-poor soils of the nutrient-poor Fynbos ecosystem (Dahlgren, 1968). A. linearis, or more commonly known as “Rooibos”, is an erect shrub that grows up to 2m in height, with needle like leaves and bright yellow flowers. It can be found in mountainous fynbos areas at an elevation between 100m to 1300m (Manning and Goldblatt, 2012). The distribution of the species ranges from the Cape Peninsula to the western and south eastern sections of the Western Cape and infiltrates sections of the south western sections of the Northern Cape (Manning and Goldblatt, 2012). A. linearis grows in sandstone derived soils that are well drained, nutrient poor and highly acidic (pH 3 – 5.3) (Muofhe and Dakora, 1999). Although these acidic soils are generally poor in biologically available P, the levels of P can be quite variable in the micro-molar range or even lower (Maseko and Dakora, 2013). Moreover, it is well-established that Fynbos plants are able to experience P-stress under extremely low P supply (Magadlela et al., 2016).

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The aim of this work was to assess the photosynthetic capacity of A. linearis under P-stress conditions. Since the plant has evolved in nutrient poor conditions, it should be more resistant to disruption in soil P supply. Therefore, we hypothesise that during short-term P deficiency, this species would be able to prevent excessive declines in photosynthetic metabolism. The hypothesis for this study was that photosynthetic adjustment in A. linearis during P-stress is more flexible to prevent excessive decline. This was addressed by determining the leaf photosynthetic gas exchange and the associated metabolic physiology of P-stressed A. linearis. Materials and Methods Experimental setup A. linearis seeds were obtained from the standard stock of commercially available seeds, used in the cultivation of rooibos tea. Plants were germinated and grown in a north-facing glasshouse at the Department of Botany and Zoology (Stellenbosch University, Stellenbosch) with average midday irradiances ranging between 600 and 800 mol.m-2.s-1 and day/night time temperatures 4

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in the range of 25°C and 15°C. Plants were watered twice weekly and were provided 100 mL of quarter-strength Long Ashton Nutrient Solution once a week. The nutrient solution contained 500 μM of Phosphorus (P) as Na2HPO4 and 250 μM of Nitrogen (N) as NaNO3. This levels of nutrients applied were appropriate for Fynbos soils as established by previous studies (Magadlela et al., 2016). Plants remained unnodulated, to prevent the metabolic costs of nodule Nitrogen assimilation to affect the root carbon (C) costs of P-stress adaptations (Kleinert et al., 2014). Nodulation was prevented by not supplying any bacterial inoculum to the seedlings. Furthermore, the seeds and the growth medium were sterilized to prevent contamination. During the cultivation, nutrient solutions were also supplied using sterilized distilled water. Fifty plants were grown for a subsequent 6 months and translocated twice a week as to limit the effect of local environmental variation. Once a suitable size of 30cm was obtained plants were assigned a random category via randomised selection. Plants were allocated as either control or P-stressed plants. P-stress was induced for 3 weeks on 25 plants by altering the relative concentration of P in the provided Long Ashton nutrient solution. Control and P starved plants were provided twice a week with 250 mL of reverse osmotic water and 100 mL of Long Ashton nutrient solution. Control plants were grown under adequate P concentration of 500 μM while P starved plants were grown under reduced P concentration of 5 μM. This concentration of P has been shown in model legumes (Olivera et al., 2004; Zhang et al., 2014) and legumes from nutrient-poor ecosystems (Magadlela et al., 2016; Magadlela et al. 2017) to produce the effects of P-stress. The experiment was terminated after three weeks as the onset of P starved morphological stress. Plants were subsequently harvested and flushed with liquid nitrogen and stored at -80°C till further analysis.

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Biomass determination

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Orthophosphate

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For each treatment 13 plants were harvested and wet weight of roots and shoots were obtained using a mass balance. Weights were recorded for both roots and shoots. Plants were subsequently dried at 50°C for 7 days in a drying oven (Labcon). Once plants were dried, root and shoot biomass of both treatments were recorded. Biomass allocations were calculated for each treatment as the dried root to dried shoot weight ratio.

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Pi concentrations on 10 P-stressed and control plants were determined according to Nanamori et al. (2004), modified to 500 mg of frozen plant material homogenised in 1 mL of pre-chilled 10% (v/v) Perchloric acid, and diluted 4.6x with pre-chilled 5% (v/v) Perchloric acid.Following the addition of reaction mixtures and incubation periods, the sample absorbance was measured at a wavelength of 820 nm on a spectrophotometer (Biotek, PowerWave HT). Pi concentration was calculated using a standard curve of KH2PO4. Pi values were expressed as μmol/g fresh weight, for standardisation between treatments.. Photosynthetic measurements Photosynthetic responses were determined for four replicates for each treatment. Using the youngest, fully expanded leaf, the photosynthetic rate was determined using a portable infra5

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red gas analyser (Li-6400, LI-COR, IRGA, Lincoln, NE, USA). Plant replicates were measured under the same conditions inside the greenhouse they were grown in at midday. Light response curves were derived by using the built-in function on the Li-6400; by altering the irradiance level in the leaf chamber. Photosynthetic readings were done at 11 irradiance levels (PAR 0, 50, 100, 150, 200, 300, 500, 800, 1100, 1300, 1600, 1800 μmol m-2 s-1) with 5 minute intervals at each irradiance level before value was recoded. Air was regulated at a flow rate of 200 (μmol/s) to maintain chamber temperature at 25°C; CO2 was set at 410 μmol mol-1 to match ambient levels. Leaves used for photosynthetic measurements were removed and surface area calculated and photosynthetic data was adjusted. All photosynthetic values were adjusted and expressed on a leaf mass (g) basis. Light–response curves were used to derive the lightsaturated rate of photosynthesis (Pmax), dark respiration and apparent quantum yield (φ). Light response curves were constructed on a model basis. Model selection was done as described in (Lobo et al., 2013). The model that was used was described by Prioul and Chartier (1977). The model is as follows: PN = ((φ (Io) x I + Pgmax – (φ (Io) x I + Pgmax)2 - 4θ x φ (Io) x I x Pgmax)0.5)/2θ) - RD

Chlorophyll extractions

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The model parameters is as follows, where PN = net photosynthesis rate [μmol (CO2) m-2.s-1], φ (Io) = quantum yield at I = 0 [μmol (CO2) μmol-1 (photons)], I = photosynthetic photon flux density [μmol (photons) m-2.s-1], Pgmax = maximum gross photosynthesis rate [μmol (CO2) m2 -1 .s ], θ = convexity (dimensionless), RD = dark respiration rate [μmol (CO2) m-2.s-1]. The model parameters were adjusted accordingly by altering the flow rate and the leaf surface area in the camber to match which was used during the experiment.

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Chlorophyll and carotenoid pigments were extracted using a modified version of Pocock et al., 2004, using 100% acetone instead of the recommended 70%. This was changed according to (Stian to add this). In the modified protocol, 0.5g of fresh weight was extracted in 5 mL of 100% acetone and centrifuged at 10°C for 5 minutes at 3100g. Supernatant was collected and chilled for 3h at 4°C. Once cooled 0.05 mL of extract was added to 0.95 mL of 100% acetone. The mixture was centrifuged again at 4°C for 7 minutes at 2000g. The supernatant was collected and absorbance was measured using a spectrophotometer (Biotek, PowerWave HT)] at, 663.5, 646.5 and 470. To calculate the total chlorophyll a and b, carotenoids and total chlorophyll concentration a modified equation of Porra (2002) was used. The calculations are as follows: Chlorophyll a (μg/ml) = 12.25 (A663.5) - 2.55 (A646.5) Chlorophyll b (μg/ml) = 20.31 (A646.5) - 4.91 (A663.5) Carotenoids (μg/ml) = (1000 A470 - 3.27 [chl a] – 104 [chl b])/227 Total chl (μg/ml) = 17.76 (A646.5) + 7.34 (A663.5) ATP assay 6

ATP quantification was done using the ATP colorimetric kit from Sigma-Aldrich (MAK190). 10 mg of leaf material of each plant was used. The procedure that was followed was the prescribed method as stated by the manufactures. To quantify the amount of ATP in each sample a standard curve was constructed using the supplied ATP standard solution. From the line of best fit equation sample ATP concentration was calculated. ATP was calculated according to the equation prescribed in the kit. C= Sa/Sv

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Where C is the concentration of ATP in sample, Sa is the amount of ATP calculated from standard curve and Sv is the sample volume (50 μL) added to each well. ATP concentration was normalized using the amount of the sample fresh weight, added extraction buffer and reaction mixture for internal standardization of volume variations to obtain normalized responses (per gram FW) and response ratios. Values were expressed as μmol/g fresh weight, for comparison. NADPH assay

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NADPH was quantified using the total NADPH/NADP colorimetric kit from Abcam (ab186033), the protocol is as follows. Leaf material of 10 replicates was taken for both control and treatment plants. Frozen 100mg of plant material were ground in liquid nitrogen; the provided lysis buffer was added in a 3:1 ratio as an extract. Extracted samples were centrifuged for 5 minutes at 2.0 CPR at 20°C. Supernatant was collected and 50 μL was added to individual wells, samples were done in triplicate to compensate for pipetting errors. 50 μL of the provided reaction mixture was added to each sample in each well and incubated for 1 and a half hours in the dark at room temperatures. Subsequent incubation, absorbance of samples was measured at 460 nm on a spectrophotometer (Biotek, PowerWave HT). NADPH concentration was obtained from a calculated standard curve with the provided standard, with PBS as a blank NADPH concentration was normalized using the amount of the sample fresh weight, added extraction buffer and reaction mixture for internal standardization of volume variations to obtain normalized responses (per gram FW) and response ratios. Values were expressed as μmol/g fresh weight, for comparison. Sugar profiles

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Sugar analysis was done according to the method in Magadlela et al. (2017). The quantification of carbohydrates was done using GC-MS unit at the Central Analytical Facility Stellenbosch. Analyses were done for three plants per treatment for roots and shoots with Pentaerythitol as internal standard. Sugars were screened for by adding increasing concentration of the added substance in factors of 2.5 ppm. Samples that were detected below a concentration of 2.5 ppm were removed from analysis. Sugar peak height representing arbitrary mass spectral ion currents of each fragment mass was normalized using the amount of the sample FW and Pentaerythitol for internal standardization of volume variations to obtain normalized responses (per gram FW) and response ratios.

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Statistical analysis Statistical inferences was done on the amount of the specific enzyme (ATP, n = 6, NADPH, n = 10, Pi, n = 10), chlorophyll concentration and the rate of photosynthesis (n = 4). Statistical inference was completed in R studio (RStudio Team, 2018). For all analysis results, data were tested for normality. When data was not normally distributed, data points were log transformed and re-tested for normality. All parameters that were conducted were tested using students Ttest between treatments. Significant differences were noted for P-value < 0.05. Results Pi assay

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Plants exposed to low P supply, experienced P-stress as evidenced by the decline in metabolically available cytosolic orthophosphate (Pi). Between the treatments there was a significant decrease (T 16.018= 4.55, P-value < 0.05) in leaf Pi concentrations in P-stressed plants (Figure 1). During the 3 weeks of P-stress there was a 16.3% decrease in overall Pi.

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Biomass data

ATP and NADPH

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P-stress had a significant enhancing effect on the total plant biomass of A. linearis (Table 1) (T13.181= -6.52, P-value < 0.05). As such, A. linearis plants are able to adapt to P-stress by altering their biomass partitioning to different plant organs. Under P-stressed conditions there was a significant alteration to the weight allocated to roots and shoots (Table 1). Shoot weight did not alter amongst the two treatments (T23.383 = -1.37, P-value = 0.184), while root biomass significantly did (T12.42= -7.28, P-value < 0.05). Under P-stress there was a significantly higher root to shoot ratio obtained (T15.72 = -11.48 and p-value < 0.05), indicating that under P-stress A. linearis increases root biomass as shoot biomass remained the same (Table 1). This means that in 3 weeks P-stressed plants were able to increase root biomass 5 fold.

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Leaf ATP concentration increased by 8.04% under P-stressed conditions in A. linearis, however this was not large enough for statistical significance as can be seen in Figure 2 (T9.963 = -1.04, P-value = 0.321). Under P-stressed conditions there was a significant increase in the total NADPH concentration per g fresh weight in the leaves (Figure 2) (T17.644= -11.46, Pvalue < 0.05). This corresponds to an 84.5% increase in the concentration of NADPH in leaves over the 3 weeks of P-stress.

Photosynthetic Measurements Maximum photosynthetic rate did not differ between the control and P-stressed group (T3.108 = 0.62, P-value = 0.578). There was no significant difference between the two treatments but, there was a 3.71% increase in photosynthetic capacity under P-stressed conditions (Figure 2). 8

Respiration rate did not significantly differ (Table 2) (T5.59 = 0.28, P-value = 0.786), however there was an 87.81% increase in dark respiration rate under P-stress. Apparent quantum yield also did not show any significant differences between treatments (Table 2) (T3.12= 1.06, P-value = 0.362). These parameters show that photosynthetic capacity was unaltered under P-stressed conditions in A. linearis. Chlorophyll measurements Under P-stressed conditions there was no significant effect on the concentration of chlorophyll a (T13.5 = 0.66, P-value = 0.5203), chlorophyll b (T13.892 = 1.62, P-value = 0.127), total chlorophyll concentration (T11.412= 1.0408, P-value = 0.3195) and total carotenoid concentration (T12.521= 0.51, P-value = 0.616) under P deprivation. A. linearis did not shift its concentration of chlorophylls and carotenoids under P deprivation conditions (Table 2).

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Sugar analysis

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In A. linearis seedlings glucose concentration in the roots and shoots show different results under P-stressed conditions (Table 2). In the shoots there was a significant difference in glucose concentration between the control and P-stressed treatments (T4 = 2.87, P-value = 0.046), which corresponds to a 40.85% decrease under P-stress. In the roots there was no significant difference between P-stressed and control treatments (T3.316 = -0.428, P-value = 0.695). Sucrose concentrations in leaves (Table 2) showed no change during P stress (T3.896 = 1.26, P-value = 0.278). Root sucrose concentration declined by 36.44% in P stressed treatments (T2.741 = 9.07, P-value = 0.004).

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Metabolite ratios

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Normalized concentrations of the different parameters were used for ratio comparisons. Ratio comparisons enabled the determination efficiency usage under P-stressed conditions. Ratios were also tested for significance using Student’s t-tests. The ratios that were tested can be seen in Table 2. These ratios enabled the efficiency of production in exported products, enzymes catalysing metabolic reactions and the use efficiency of photosynthesis under P-stress. In Table 2, the results of comparing different measured parameters are shown. Under P-stress there was a significant alteration of ATP to light harvesting pigments, while ATP/Total chlorophyll concentration decreased. NADPH alterations had the most differing results between treatments. NADPH to Pi and ATP showed significant increases in their ratios under P-stress. This indicates that under P-stress NADPH use efficiency increase per unit Pi, and that the production of NADPH was increased over ATP. The production of NADPH to chlorophyll and carotenoid concentration were all enhanced under P-stress. None of the photosynthetic parameter ratios were significant between the different treatments. For sugar analysis, glucose shows a higher efficiency of ATP and NADPH usage under control conditions; while there was no significant production of glucose per unit Pi. Discussion

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Plants such as A. linearis, which evolved on P-poor soils, are able to maintain their photosynthetic rates during short-term P-stress. This is due to alterations in the structure and function of light reaction components.

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The reduction of metabolic P (Pi) in leaves of A. linearis plants under P-stressed conditions, serves as an as an indicator for P-stress, as found in previous studies. Plants are able to adapt to P-stress by changing the allocation of carbon and mineral resources to growth and metabolism. By altering biomass allocation to their roots, the plants are thereby able to acquire the deficient P more effectively (Vance et al., 2003). Our finding of increased root biomass under P-stress concurs with our data of lower sucrose concentration roots, which indicates higher utilisation to fuel this growth (Lambers et al., 2015). This altered root growth with Pstress, is congruent with responses in clover plants, which was attributed to lower respiration rates in roots due to assimilates being partitioned to increased root growth, rather than metabolism (Fan et al., 2016). Under P-stressed conditions, efficient P. vulgaris plants are able to lower the root respiration level, which allowed the plants to maintain high biomass allocation without increasing the total carbon costs (Chaudhary et al., 2008). This allocation difference can be seen in the root: shoot ratio of plants (Hammond and White, 2008), but this may not always be the case for all Aspalathus species.

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In Aspalathus nivea and Aspalathus subtingens root to shoot ratio did not alter under P-stressed conditions (Power et al., 2010), suggesting that the standard P-stress adaptations for model plants, may not always hold true for species which are native to P-poor soils. In the current study, the P-stressed plants were under the initial stages of P-stress, based on the increased root to shoot ratios and the decline in cellular Pi concentration (Hammond and White, 2008). Moreover, the P-stressed A. linearis plants were able to maintain their photosynthetic rates, which is largely in contrast with published literature of model plants (Jacob and Lawlor, 1991; Lima et al., 1999). Adaptations to maintain photosynthetic rates during P limitations have been reported in Pinus massoniana (Fan et al., 2016), Phaseolus vulgaris (Lima et al., 1999) and sunflower (Rodríguez et al., 1998). The maintenance of photosynthetic rates during P stress in A. linearis plants was underpinned by the functional and structural alterations to photosynthesis in A. linearis.

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Although the absence of proportional investment of the pigments in light harvesting and reaction centre components of the photosystems during P-stress, concurs with previous studies (Jacob and Lawlor, 1991; Lin et al., 2009; Olivera et al., 2004; Zhang et al., 2014), there were unexpected modifications in the efficiencies with which light reaction products were synthesised and utilised. The increased efficiency of the synthesis of light reaction products, ATP and NADPH per unit of pigments, indicates that there was a preferential alteration in light reaction efficiency under P-stressed conditions. This enhanced efficiency may have enabled the P stressed plants to maintain their photosynthetic rates, at similar levels to the control plants. Since the apparent photon yield is an indication of how efficiently ATP and NADPH are produced and utilised by the dark reactions in the stroma, the unchanged levels ATP and 10

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NADPH during P-stress, therefore suggests that this may be one of the underlying alterations which allows P-stress plants to match the photosynthetic rates of control plants (Björkman and Demmig, 1987). Typical responses to P-stressed conditions are the reduction in ATP production (Zhang et al., 2009, 2014). Plants regulate their ATP concentration to avoid damaging photochemical apparatus, by up-regulating the processes which are not being affected (Sage, 1990). However, the unchanged ATP concentrations during P-stress, may be a consequence of the pigment concentrations, which were unaltered by the low level of P supply. The increase in NADPH under P-stressed conditions concurs with previous studies (Jacob and Lawlor, 1993; Juszczuk and Rychter, 1997). These changes to pyridine nucleotides are possibly the result of P-stress induced modifications to the equilibrium of the oxidation-reduction reactions towards storage of redox equivalents (Juszczuk and Rychter, 1997; Maciejewska and Kacperska, 1987). Furthermore, the efficiency of synthesises for both these light reaction products were also greatly enhanced per unit of metabolic Pi. The increase in the leaf ATP to Pi ratios of the P-stressed plants, indicate that plants under P limitation can reduce the use of P-requiring metabolic reactions, by circumventing the ATP dependant pathways (Hammond and White, 2008; Plaxton and Carswell, 1999), but may also suggest that there is an alteration in the efficiency of light reaction product synthesis per unit Pi. This increased efficiency can arise from increased transfer of electrons or excitation energy between light harvesting and reaction centre components (Geiger and Servaites, 1994). The utilisation of light reactionsgenerated ATP and NADPH in the dark reactions, indicate that there may be further alterations during P deficiency.

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During the Calvin cycle of photosynthesis, the export of triose-P from the chloroplasts can be limited by P-stress, but this triose-P accumulation can also limit photosynthesis by end-product feedback inhibition (Huber and Huber, 1992). Owing to the unchanged rates of photosynthesis in P-stressed leaves, it likely that triose-P accumulation was prevent by starch synthesis (Kang et al., 2014; Walker and Sivak, 1986; Zhang et al., 2014). This concurs with the lower glucose concentrations in the P-stressed leaves, and resonates with other studies on legume leaves (Olivera et al., 2004). Triose-P partitioning between sucrose or starch production appears to be regulated at both the biochemical and genetic level of a plant (Cakmak et al., 1994). In contrast to the glucose levels in P stressed leaves, the unaffected sucrose concentrations during P limitation may be related to the constant ATP levels, which can enable the continued metabolism of sucrose. Under P-stressed conditions it is generally thought that the hyperaccumulation of sucrose in P stressed plants, is likely due to the reduction in ATP and P dependant metabolism of sucrose (Kondracka and Rychter, 1997; Rychter and Randall, 1994). Owing to effects of P supply on the light and dark reactions, the lower ratios of glucose per unit of ATP and NADPH in P-stressed plants, may be indicative of a shift in the balance participation of the light and dark reactions. Thus in conclusion, A. linearis, a native to P -poor soils, is able to adapt to P-stress via alterations in growth allocations and photosynthetic metabolism. The biomass allocation is congruent with previous work. However, the alterations in the light reactions of photosynthesis, present new perspectives to P-stress adaptions. Moreover, this indicates that model plants are 11

limited for inferences of P-stress adaptation and that plants which evolved in P poor soils may provide more valuable insights. Conflicts of Interest: No conflict of interest is noted. Acknowledgements:

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We would also like to thank Amoré Malan, Derek Rossouw, Elbé du Toit, Lida-Mari Groenewald and Darryn Whisgary for their help with laboratory work. Guy Midgley and Marius Rossouw for giving guidance with the gas exchange. We want to thank Lucky Mokwena for helping in analysing sugar profiles at CAFs’ GC/MS unit. The study was funded by the National Research Foundation (NRF) of South Africa.

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References

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Björkman, O., Demmig, B., 1987. Photon yield of 02 evolution and chlorophyll fluorescence characteristics at 77 K among vascular plants of diverse origins. Planta 170, 489–504. Cakmak, I., Hengeler, C., Marschner, H., 1994. Partitioning of shoot and root dry matter and carbohydrates in bean plants suffering from phosphorus, potassium and magnesium deficiency. J. Exp. Bot. 45, 1245–1250. https://doi.org/10.1093/jxb/45.9.1245 Campbell, C.D., Sage, R.F., 2006. Interactions between the effects of atmospheric CO2 content and P nutrition on photosynthesis in white lupin (Lupinus albus L.). Plant, Cell Environ. 29, 844–853. https://doi.org/10.1111/j.1365-3040.2005.01464.x Chaudhary, M.I., Adu-Gyamfi, J.J., Saneoka, H., Nguyen, N.T., Suwa, R., Kanai, S., ElShemy, H.A., Lightfoot, D.A., Fujita, K., 2008. The effect of phosphorus deficiency on nutrient uptake, nitrogen fixation and photosynthetic rate in mashbean, mungbean and soybean. Acta Physiol. Plant. 30, 537–544. https://doi.org/10.1007/s11738-008-0152-8 Dahlgren, R., 1968. Revision of the genus Aspalathus II: the species with ericoid and pinoid leaves. 7 subgenus Nortiera. With remarks on rooibos cultivation. 121, 165–208. Fan, F., Ding, G., Wen, X., 2016. Proteomic analyses provide new insights into the responses of Pinus massoniana seedlings to phosphorus deficiency. Proteomics 16, 504–515. https://doi.org/10.1002/pmic.201500140 Fredeen, A.L., Raab, T.K., Rao, I.M., Terry, N., 1990. Effects of phosphorus nutrition on photosynthesis in Glycine max (L.) Merr. Planta 181, 399–405. https://doi.org/10.1007/sl0869-007-9037-x Geiger, D.R., Servaites, J.C., 1994. Diurnal Regulation of Photosynthetic Carbon Metabolism in C3 Plants. Annu. Rev. Plant Physiol. Plant Mol. Biol. 45, 235–256. Hammond, J.P., White, P.J., 2008. Sucrose transport in the phloem: Integrating root responses to phosphorus starvation. J. Exp. Bot. 59, 93–109. https://doi.org/10.1093/jxb/erm221 Hinsinger, P., 2001. Bioavailability of soil inorganic P in the rhizosphere as affected by rootinduced chemical changes: a review. Plant Soil 237, 173–195. https://doi.org/10.1023/A:1013351617532 Huber, S., Huber, J., 1992. Role of Sucrose-Phosphate Synthase in Sucrose Metabolism in Leaves. Plant Physiol. 99, 1275–1278. Jacob, J., Lawlor, D.W., 1993. In vivo photosynthetic electron transport does not limit photosynthetic capacity in phosphate deficient sunflower and maize leaves. Plant Cell Environ. 16, 785–795. Jacob, J., Lawlor, D.W., 1991. Stomatal and mesophyll limitations of photosynthesis in phosphate deficient sunflower, maize and wheat plants. J. Exp. Bot. 42, 1003–1011. https://doi.org/10.1093/jxb/42.8.1003 Juszczuk, I.M., Rychter, A.M., 1997. Changes in pyridine nucleotide levels in leaves and roots of bean plants (Phaseolus vulgaris L.) during phosphate deficiency. J. Plant Physiol. 151, 399–404. https://doi.org/10.1016/S0176-1617(97)80003-X Kang, J., Yu, H., Tian, C., Zhou, W., Li, C., Jiao, Y., Liu, D., 2014. Suppression of Photosynthetic Gene Expression in Roots Is Required for Sustained Root Growth under Phosphate Deficiency. Plant Physiol. 165, 1156–1170. https://doi.org/10.1104/pp.114.238725 Kleinert, A., Venter, M., Kossmann, J., Valentine, A., 2014. The reallocation of carbon in P 13

Jo

ur

na

lP

re

-p

ro of

deficient lupins affects biological nitrogen fixation. J. Plant Physiol. 171, 1619–1624. https://doi.org/10.1016/j.jplph.2014.07.017 Kondracka, A., Rychter, A.M., 1997. The role of Pi recycling processes during photosynthesis in phosphate-deficient bean plants. J. Exp. Bot. 48, 1461–1468. https://doi.org/10.1093/jxb/48.7.1461 Lambers, H., Martinoia, E., Renton, M., 2015. Plant adaptations to severely phosphorusimpoverished soils. Curr. Opin. Plant Biol. 25, 23–31. https://doi.org/10.1016/j.pbi.2015.04.002 Lambers, H., Raven, J.A., Shaver, G.R., Smith, S.E., 2008. Plant nutrient-acquisition strategies change with soil age. Trends Ecol. Evol. 23, 95–103. https://doi.org/10.1016/j.tree.2007.10.008 Lichtenthaler, H.K., 1987. Chlorophylls and Carotenoids: Pigments of Photosynthetic Biomembranes. Methods Enzymol. 148, 350–382. https://doi.org/10.1016/00766879(87)48036-1 Lima, J., Mosquim, P., Da Matta, F., 1999. Leaf gas exchange and chlorophyll fluorescence parameters in Phaseolus vulgaris as affected by nitrogen and phosphorus deficiency. Photosynthetica 37, 131–121. https://doi.org/10.1023/A:1007079215683 Lin, Z.H., Chen, L.S., Chen, R.B., Zhang, F.-Z., Jiang, H.-X., Tang, N., 2009. CO2 assimilation, ribulose-1,5-bisphosphate carboxylase/ oxygenase, carbohydrates and photosynthetic electron transport probed by the JIP-test, of tea leaves in response to phosphorus supply. BMC Plant Biol. 9, 1–12. https://doi.org/10.1186/1471-2229-9-43 Lobo, F. de A., de Barros, M.P., Dalmagro, H.J., Dalmolin, Â.C., Pereira, W.E., de Souza, É.C., Vourlitis, G.L., Rodríguez Ortíz, C.E., 2013. Fitting net photosynthetic lightresponse curves with Microsoft Excel - a critical look at the models. Photosynthetica 51, 445–456. https://doi.org/10.1007/s11099-013-0045-y Longstreth, D.J., Nobel, P.S., 1980. Nutrient Influences on Leaf Photosynthesis: Effects of Nitrogen, Phosphorus and Potassium for Gossypium Hirsutum L. Plant Physiol. 65, 541–543. https://doi.org/http://dx.doi.org/10.1104/pp.65.3.541 Maciejewska, U., Kacperska, A., 1987. Changes in the level of oxidized and reduced pyridine nucleotides during cold acclimation of winter rape plants. Physiol. Plant. 69, 687–691. Magadlela, A., Beukes, C., Venter, F., Steenkamp, E., Valentine, A., 2017. Does P deficiency affect nodule bacterial composition and N source utilization in a legume from nutrientpoor Mediterranean-type ecosystems? Soil Biol. Biochem. 104, 164–174. https://doi.org/10.1016/j.soilbio.2016.10.021 Magadlela, A., Vardien, W., Kleinert, A., Steenkamp, E.T., Valentine, A.J., 2016. Variable P supply affects N metabolism in a legume tree , Virgilia divaricata , from nutrient-poor Mediterranean-type ecosystems. Funct. Ecol. 43, 287–297. Manning, J., Goldblatt, P., 2012. Plants of the Greater Cape Floristic Region, 1: The Core Cape Flora, Strelitzia 29., 1st ed. South African National Biodiversity Institute, Pretoria. Maseko, S., Dakora, F., 2013. Plant enzymes, root exudates, cluster roots and mycorrhizal symbiosis are the drivers of P nutrition in native legumes growing in P deficient soils of the Cape Fynbos in South Africa. J. Agric. Sci. Technol. A 3, 331–340. Muofhe, M.L., Dakora, F.D., 1999. Nitrogen nutrition in nodulated field plants of the shrub tea legume Aspalathus linearis assessed using 15N natural abundance. Plant Soil 209, 181–186. https://doi.org/10.1023/A:1004514303787 Nanamori, M., Shinano, T., Wasaki, J., Yamamura, T., Rao, I.M., Osaki, M., 2004. Low 14

Jo

ur

na

lP

re

-p

ro of

Phosphorus Tolerance Mechanisms : Phosphorus Recycling and Photosynthate Partitioning in the Tropical Forage Grass , Brachiaria Hybrid Cultivar Mulato Compared with Rice. Plant Cell Physiol. 45, 460–469. Ohlrogge, J., Browse, J., Jaworski, J., Somerville, C., 2007. Lipids, in: Buchanan, B.B., Gruissem, W., Jones, R. (Eds.), Biochemistry & Molecular Biology of Plants. John Wiley & Sons, Chichester, pp. 337–400. Olivera, M., Tejera, N., Iribarne, C., 2004. Growth, nitrogen fixation and ammonium assimilation in common bean (Phaseolus vulgaris): effect of phosphorus. Physiol. Plant. 121, 498–505. https://doi.org/10.1111/j.1399-3054.2004.00355.x Péret, B., Clément, M., Nussaume, L., Desnos, T., 2011. Root developmental adaptation to phosphate starvation: Better safe than sorry. Trends Plant Sci. 16, 442–450. https://doi.org/10.1016/j.tplants.2011.05.006 Plaxton, W., Carswell, M., 1999. Metabolic aspects of the phosphate starvation response in plants., in: Lerner, H. (Ed.), Plant Responces to Environmental Stresses: From Phytohormones to Genome Reorganization. Dekker, New York, pp. 349–372. Pocock, T., Król, M., Huner, N., 2004. The Determination and Quantification of Photosynthetic Pigments by Reverse Phase High Performance Liquid Chromatography, Thin-Layer Chromatography, and Spectrophotometry, in: Carpentier, R. (Ed.), Photosynthesis Research Protocols. Totowa, pp. 137–148. Power, S.C., Cramer, M.D., Verboom, G.A., Chimphango, S.B.M., 2010. Does phosphate acquisition constrain legume persistence in the fynbos of the Cape Floristic Region? Plant Soil 334, 33–46. https://doi.org/10.1007/s11104-010-0311-8 Prioul, J.L., Chartier, P., 1977. Partitioning of Transfer and Carboxylation Components of Intracellular Resistance to Photosynthetic CO₂ Fixation : A Critical Analysis of the Methods Used. Ann. Bot. 41, 789–800. R Core Team, 2018. R: A language and environment for statistical computing. R foundation for statistical computing. Vienna, Austria. https://www.R-project.org/ Raghothama, K.G., 1999. Phosphate Acquisition. Annu. Rev. Plant Physiol. Plant Mol. Biol. 50, 665–693. https://doi.org/10.1146/annurev.arplant.50.1.665 Rodríguez, D., Zubillaga, M.M., Ploschuk, E.L., Keltjens, W.G., Goudriaan, J., Lavado, R.S., 1998. Leaf area expansion and assimilate production in sunflower (Helianthus annuus L.) growing under low phosphorus conditions. Plant Soil 202, 133–147. https://doi.org/10.1023/A:1004348702697 Rychter, A.M., Randall, D., 1994. The effect of phosphate deficiency on carbohydrate metabolism in bean roots. Physiol. Plant. 91, 383–388. Rychter, A.M., Rao, I.M., Cardoso, J.A., 2016. Photosynthesis and its relationship with plant nutrient elements, in: Pessarakli, M. (Ed.), Handbook of Photosynthesis. Taylor & Francis Group, New York, pp. 603–625. https://doi.org/10.1016/S0261-2194(97)811878 Sage, R.F., 1990. A Model Describing the Regulation of Ribulose-1,5-Bisphosphate Carboxylase, Electron Transport, and Triose Phosphate Use in Response to Light Intensity and CO2 in C3 Plants. Plant Physiol. 94, 1728–1734. https://doi.org/10.1104/pp.94.4.1728 Servaites, J.C., Shieh, W.J., Geiger, D.R., 1991. Regulation of photosynthetic carbon reduction cycle by ribulose bisphosphate and phosphoglyceric Acid. Plant Physiol. 97, 1115–21. https://doi.org/10.1104/pp.97.3.1115 15

Jo

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-p

ro of

Taiz, L., Zeiger, E., Moller, I.., Murphy, A., 2014. Plant Physiology and Development, 6th ed. Sinauer Associates Inc., Sunderland, USA. https://doi.org/10.3119/0035-4902117.971.397 van Amerongen, H., Croce, R., 2013. Light harvesting in photosystem II. Photosynth. Res. 116, 251–263. https://doi.org/10.1007/s11120-013-9824-3 Vance, C.P., Uhde-Stone, C., Allan, D.L., 2003. Phosphorus aquisition and use: critical adaptations by plants for securing a nonrewable resource. New Phytol. 157, 423–447. https://doi.org/10.1046/j.1469-8137.2003.00695.x Vardien, W., Steenkamp, E.T., Valentine, A.J., 2016. Legume nodules from nutrient-poor soils exhibit high plasticity of cellular phosphorus recycling and conservation during variable phosphorus supply. J. Plant Physiol. 191, 73–81. https://doi.org/10.1016/j.jplph.2015.12.002 Walker, D.A., Sivak, M.N., 1986. Photosynthesis and phosphate : a cellular affair ? Trends Biochem. Sci. 11, 176–179. White, P.J., Hammond, J.P., 2008. Phosphorus nutrition of terrestrial plants, in: White, P.J., Hammond, J.P. (Eds.), The Ecophysiology of Plant-Phosphorus Interaction. Springer, New York, pp. 31–50. Zeeman, S., 2007. Carbohydrate Metabolism, in: Buchanan, B.B., Gruissem, W., Jones, R. (Eds.), Biochemistry & Molecular Biology of Plants. John Wiley & Sons, Chichester, pp. 567–609. Zhang, H., Huang, Y., Ye, X., Shi, L., Xu, F., 2009. Genotypic differences in phosphorus acquisition and the rhizosphere properties of Brassica napus in response to low phosphorus stress. Plant Soil 320, 91–102. https://doi.org/10.1007/s11104-008-9873-0 Zhang, K., Liu, H., Tao, P., Chen, H., 2014. Comparative proteomic analyses provide new insights into low phosphorus stress responses in maize leaves. PLoS One 9. https://doi.org/10.1371/journal.pone.0098215

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FIGURE CAPTIONS

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Figure 1. Leaf Pi concentrations of 6-month old A. linearis seedlings, grown in sand culture under controlled P (500 μM P) and P-stressed (5 μM P) conditions. Values represent the means (n=10) with standard error bars. The * indicates significant differences between the two treatments, (P ≤ 0.05).

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Figure 2. Leaf ATP concentrations (a), NADPH concentrations (b) and maximum photosynthetic rates (c) of 6-month old A. linearis seedlings, grown in sand culture under controlled P (500 μM P) and P-stressed (5 μM P) conditions. Values represent the means (n= 6-10) with standard error bars. The * indicates significant differences between the two treatments, (P ≤ 0.05).

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Table 1. Biomass allocation of 6-month old A. linearis seedlings, grown in sand culture under controlled P (500 μM P) and P-stressed (5 μM P) conditions. Values represent the means (n=13) with standard errors. Statistical differences between the treatments are indicated by * (significant) and NS (non-significant) at 95% confidence (P ≤ 0.05). Parameter

P-stressed

Control

Shoot biomass (g dry weight)

0.123 ± 0.011 NS

0.102 ± 0.0097 NS

Root biomass (g dry weight)

0.576 ± 0.06 *

0.113 ± 0.008

Total biomass (g dry weight)

0.699 ± 0.07 *

0.215 ± 0.016

Root : Shoot ratio

4.708 ± 0.274 NS

1.186 ± 0.109 NS

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Values represent the means (n=13) with standard errors. Statistical differences between the treatments are indicated by * (significant) and NS (non-significant) at 95% confidence (P ≤ 0.05).

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Table 2. Leaf pigment concentrations, photosynthetic light response curve parameters and metabolite analyses of 6-month old A. linearis seedlings, grown in sand culture under controlled P (500 μM P) and P-stressed (5 μM P) conditions. Values represent the means (n = 3-13) with standard errors. Statistical differences between the treatments are indicated by * (significant) and NS (non-significant) at 95% confidence (P ≤ 0.05).

Parameter

P-stressed

Control

Chl a (μg/g fw)

703.625 ± 22.917 NS

679.785 ± 42.393 NS

Chl b (μg/g fw)

424.426 ± 37.536 NS

340.869 ± 30.341 NS

Total Chl (μg/g fw)

1548.947 ± 178.199 NS

1315.027 ± 104.295 NS

Carotenoids (μg/g fw)

234.736 ± 34.0627 NS

0.0037 ± 0.00024 NS

Dark respiration (μmol CO2/g fw)

0.1372 ± 0.107 NS

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Apparent photon yield (mmol (CO2) mmol-1 (photons)/g fw)

Plant sugar concentration

0.00329 ± 0.0013 NS

0.0556 ± 0.0267 NS

11.288 ± 1.171 *

19.083 ± 1.902

Root Glucose (mol/g fresh weight)

14.918 ± 1.823 NS

13.662 ± 2.236 NS

Shoot Sucrose (mol/g fresh weight)

923.070 ± 174.342 NS

1437.331 ± 142.021 NS

Root Sucrose (mol/g fresh weight)

11959.012 ± 431.381*

32814.919 ± 5804.797

ATP/Pi

0.000031 ± 1.136E-06 *

0.000024 ± 1.484E-06

ATP/Chl a (μmol/μg)

8.317E-08 ± 4.039E-09 NS

ATP/Chl b (μmol/μg)

0.00015 ± 7.30996E-06 *

0.00011 ± 5.370E-06

ATP/Total Chl (μmol/μg)

4.299E-08 ± 2.088E-09 *

3.379E-08 ± 1.621E-09

ATP/Carotenoids

2.728E-07 ± 1.325E-08 *

2.229E-07 ± 1.070E-08

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Shoot Glucose (mol/g fresh weight)

207.250 ± 16.842 NS

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Photosynthetic parameters

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Leaf Pigments

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Leaf metabolite ratios

7.437E-08 ± 3.569E-09 NS

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NADPH/Pi

0.316 ± 0.0121 *

0.143 ± 0.005

NADPH/ATP

10342.87 ± 395.60 *

6059.825 ± 200.38

NADPH/Chl a (μmol/μg)

0.00086 ± 0.0000323*

0.000451 ± 0.000051

NADHP/Chl b (μmol/μg)

1.557 ± 0.0595*

0.678 ± 0.102

NADPH/Total Chl (μmol/μg)

0.000445 ± 0.000017*

0.0002 ± 0.000028

NADPH/Carotenoids (μmol/μg)

0.00282 ± 0.000108*

0.00135 ± 0.000177

Glucose/ATP (mol/μmol)

199641.291± 20712.474*

Glucose/NADPH (mol/μmol)

19.302 ± 2.003*

60.176 ± 5.997

Glucose/Pi (mol/μmol)

6.099 ± 0.633 NS

8.631 ± 0.860

364657.514 ± 36340.760

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NS

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Values represent the ratios with mean values of each parameter with standard error bars . Values represent the means (Chl a, n = 10, Chl b, n = 8, Carotenoids, n = 8, Total chlorophyll, n = 7, Glucose Root and shoot and Sucrose Root and Shoot n = 3, Dark respiration, n = 4, Apparent Photon Yield, n = 4) with standard errors.

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