Indian Journal of Biochemistry & Biophysics Vol. 45, February 2008, pp. 37-43

Application of fast chlorophyll a fluorescence transient (OJIP) analysis to monitor functional integrity of pea (Pisum sativum) mesophyll protoplasts during isolation

B Sunil1, K Riazunnisa1, T Sai Krishna1, Gert Schansker2, Reto J Strasser2, Agepati S Raghavendra1 and Prasanna Mohanty1*#

1Department of Plant Sciences, School of Life Sciences, University of Hyderabad, Hyderabad 500 046, India 2Bioenergetics Laboratory, University of Geneva, CH-1254, Jussy-Geneva, Switzerland

Received 10 May 2007; revised 28 November 2007

Intact and metabolically very active mesophyll protoplasts were isolated rapidly from pea (Pisum sativum) leaves. The functional performance of protoplasts at various stages of their isolation was analyzed by using fast Chl a fluorescence OJIP transients and compared with that of intact leaves. The results demonstrated that the OJIP transients could successfully be used to monitor the quality of mesophyll protoplasts at different isolation steps. The protoplasts maintained their integrity and photosynthetic status very well, and their performance was very similar to that of the intact leaves.

Keywords: Chl a fluorescence, Functional integrity, Mesophyll protoplasts, O-J-I-P Transients, Pisum sativum, Protoplast isolation

Plant protoplasts form unique, single cell and versatile system for use in a variety of biochemical, biophysical and physiological experiments1. They find use in tissue culture, plant transformation and in several other fields of modern biotechnology. The protoplasts, which represent cell wall less mesophyll cells have been isolated from a variety of tissues both from monocotyledonous and dicotyledonous plants. The yield and efficiency besides the intactness are important for protoplast isolation. Application of chlorophyll (Chl a) fluorescence fast-transient analysis is a simple and useful non-invasive tool to monitor chloroplast function in vivo as well as in vitro2. The fast OJIP transient and its quantification by the JIP test provide a sensitive and reliable test for the functionality of photosynthetic system and vitality of green tissues3-6. The Chl a fluorescence transients, both fast and slow are used extensively in studies on various aspects of plant biology from physiology, biotechnology to eco-physiology1,4,6. These Chl a fluorescence transients

have the potential to be exploited in studies on plant protoplasts. Isolation of intact mesophyll protoplasts from leaves involves short, but physiologically stressful steps, such as enzymatic digestion at acidic pH and alternate periods of light and darkness7. During such isolation procedure, the bioenergetic organelles of chloroplasts and mitochondria could experience metabolic disturbances. Earlier, we have developed an extremely efficient procedure for isolating intact mesophyll protoplasts from pea (Pisum sativum) leaves for studies on photosynthesis and respiration7,8. In addition, a fast and reliable procedure for isolation of metabolically active mesophyll protoplasts from model plant Arabidopsis thaliana has recently been developed9. In this communication, we have used for the first time the fast Chl a fluorescence transients for monitoring and assessing the functional photochemical performance of mesophyll protoplasts at different stages of isolation from pea (Pisum sativum) leaves. Our results demonstrate the similar photosynthetic performance of isolated mesophyll protoplasts to that of intact leaves.

___________ *Author for correspondence E-mail: prasanna37@hotmail.com, photosis@rediffmail.comFax: 0674 -2550274 # Mailing address: INSA Honorary Scientist, C\o Regional Plant Resource Centre, Nayapalli, Bhubaneswar 751015, Orissa, India Abbreviations: Chl, chlorophyll; FDA, fluoresceine diacetate; PQ, plastoquinone; PS, photosystem; QA, primary quinone electron acceptor of PS II; QB, secondary quinone electron acceptor of PSII

Materials and Methods Plant material Pea (Pisum sativum L. cv. Arkel) plants were raised from seeds (procured from Pocha Seeds Co. Ltd, Pune, India) in the green house with average

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temperatures of 25-30°C (day) and 20-25°C (night). The first and second fully expanded leaves were picked from 8-10 days old plants and used for isolating mesophyll protoplasts7,8. Isolation of mesophyll protoplasts The abaxial epidermis of pea leaves was stripped-off with forceps and floated on pre-plasmolysis medium (0.3 M sorbitol, 1 mM CaCl2, 10 mM 2-(N-morpholino ethanesulfonic acid (MES)-KOH, pH 6.0). After 15 min, preplasmolysis medium was removed and digestion medium containing 2% (w/v) cellulase Onozuka R-10, 0.2% (w/v) Macerozyme R-10, 0.25% BSA, 10 mM sodium ascorbate, 0.25 mM EDTA, 0.4 M sorbitol, 1 mM CaCl2, 10 mM (MES-KOH, pH 5.5) was added. The leaf pieces were digested at 30°C for 30 min under an illumination of 300 µmol photons m-2 s-1. Thereafter, the protoplasts were collected in washing medium (0.4 M sorbitol, 1 mM CaCl2, 10 mM N-2-hydroxyethylpiperazine-N’-2-ethanesulfonic acid (HEPES-KOH, pH 6.0), and centrifuged at 50 × g for 3 min. This process was repeated once. The supernatant was discarded and protoplasts were suspended in suspension medium (0.4 M sorbitol, 1 mM CaCl2, 0.5 mM MgCl2, 10 mM HEPES-KOH, pH 7.0) and centrifuged for 2 min. The protoplast pellet was suspended in suspension medium. Further details of protoplast isolation have been described elsewere8,10. The different steps for isolation of mesophyll protoplasts from pea leaves are shown in Fig. 1. The

three centrifugation steps in our isolation procedures (see Fig. 1) were shown to be crucial for obtaining intact protoplasts in high yield and good physiological state7-9. The intactness of protoplasts was assessed by either staining with fluoresceine diacetate (FDA) or by assaying glycolate oxidase11 and ranged from 90-95%. Photomicrographs of protoplasts (Fig. 2) were taken using a light microscope (DMR, Leica, Germany). Estimation of chlorophyll Chlorophyll (Chl) was estimated in protoplast preparations by extraction with 80% (v/v) acetone12. An aliquot of 12.5 µl of protoplast suspension was added to 5 ml of 80% (v/v) acetone and mixed on a cyclo-mixer. The absorbance of acetone extract was measured at 652 nm (A652, to determine chl) and 710 nm (A710 to correct for turbidity) using a spectrophotometer (Shimadzu UV-160A). An aliquot of 12.5 µl of suspension medium mixed with 80% acetone served as the blank. The Chl concentration was calculated using the following formula12.

Chl (mg ml-1 of protoplast suspension) = (A652 – A710) × 11.11

The room temperature absorption spectrum of isolated protoplasts was recorded using a Shimadzu UV 1801 spectrophotometer. Measurement of fast Chl a fluorescence induction kinetics A plant efficiency analyzer (Handy PEA, Hansatech Ltd., UK) was used. Protoplast samples at selected stages of isolation (see Fig. 1) were transferred into a 1.5 ml eppendorf tube, kept in darkness for 5 min and then examined for Chl a fluorescence transient pattern using the procedure of Strasser et al13 OJIP-transients. A typical sets of original transient traces is presented in Fig. 3. Table 1

Fig. 1—Protoplast isolation showing different steps and treatments [The photographs of typical protoplast preparation are presented in Fig. 2]

Table 1—List of symbols used to describe technical fluorescence parameters calculated from OJIP measurements

Fo = Minimal Chl a fluorescence (at ~ 20 µs) FJ = Fluorescence intensity at j-step (at ~ 2 ms) FI = Fluorescence intensity at i-step (at ~ 30 ms) FP = Fluorescence intensity at p-step (at ~ 1000 ms) FM = Maximal fluorescence intensity Ft = Fluorescence at a given time V, Relative variable fluorescence

= (Ft-Fo)/(FM-Fo)

(See Appendix I-II in Ref. 5)

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lists the symbols used to describe different components of fluorescence transients. Further details on OJIP measurements and the use of different formulae are described elsewhere4-6,14. At each step, the Chl content and protoplast numbers were different. Therefore, the transients in selected figures were normalized, so as to present comparative changes in the kinetics of OJIP phase, if any, during isolation procedures or treatment. Further details are given in the figure legends. Monitoring photosynthesis: CO2-dependent oxygen evolution and benzoquinone-dependent Hill activity Photosynthetic oxygen evolution by mesophyll protoplasts was monitored using a Clark-type O2 electrode (DW2, Hansatech Ltd., King’s Lynn, UK). The reaction medium of 1 ml for monitoring photosynthesis by mesophyll protoplasts contained 0.65 M sorbitol, 1 mM CaCl2, 1 mM MgCl2, 10 mM HEPES-KOH, pH 7.5, protoplasts equivalent to 10 µg Chl, and 1 mM sodium bicarbonate (unless otherwise specified) and the saturating light intensity of 600 µmol photons m-2 s-1 was used9. When required, the oxygen evolution was assayed in the presence or absence of 1 mM benzoquinone (for PSII activity) and/or 5 mM glycolaldehyde (an inhibitor of Calvin-Benson cycle). The water (25°C) was circulated through the outer jacket of reaction chamber. Illumination was provided by a 35 mm slide projector, equipped with a 24V/150W halogen lamp (Philips Focusline, Projection Lamp Type 7158XHP). Calibration of oxygen content in electrode chamber was done with

air-saturated water, assumed to contain 253 nmol O2

ml-1 at 25°C15. The rates were corrected for dark respiration5. The data presented were the average values (± SE) from three to four experiments conducted on different days. Results The protoplast preparation required about an hour and involved dark-light adaptations (Fig. 1). The intact protoplasts were separated by centrifugation. The micrograph in Fig. 2A demonstrates that before centrifugation, there were some broken fragments. The centrifugation step did not cause any further breakage of the protoplasts (Fig. 2B). The room temperature absorption spectrum (Fig. 2C) of protoplasts exhibited typical peaks corresponding to Chl a, Chl b and carotenoids. A comparison of Chl a fluorescence transient curves measured during the isolation procedure with the transients of intact leaves provided a good test for the functional integrity of the photosynthetic electron transport chain. Because of the need for quick monitoring of Chl a fluorescence transients at frequent intervals, we measured only OJIP transient in this study. Table 2 shows the oxygen evolution ability of same set of protoplasts preparation used for the measurement of the OJIP-transients. A high rate of photosynthetic oxygen evolution, even without any added bicarbonate, is a consistent feature of our protoplast preparations. The rate of benzoquinone (BQ)-dependent O2 evolution which reflects PSII activity was significantly higher than that with 1 mM

Fig. 2—Appearance and absorption spectrum of mesophyll protoplasts of pea (Pisum sativum) [(A): Micrograph of mesophyll protoplasts in washing medium before centrifugation step; (B): Micrograph of mesophyll protoplasts after final centrifugation and kept in suspension medium; and (C): Absorption spectrum of mesophyll protoplasts in suspension medium]

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Table 2—Photosynthetic oxygen evolution (µmol mg-1 Chl h-1) by mesophyll protoplasts of pea before and after centrifugation of protoplast suspension in presence or absence of benzoquinone (BQ) or glycolaldehyde or bicarbonate.

[Values represent mean of three independent experiments ± SD]

No BQ or Glycolaldehyde 1 mM BQ + 5 mM Glycolaldehyde Step Without HCO3

- 1 mM HCO3- Without HCO3

- 1 mM HCO3-

Before centrifugation 59 ± 1.5 113 ± 2.0 190 ± 2.5 233 ± 1.6 After centrifugation 64 ± 2.6 121 ± 2.2 236 ± 3.4 248 ± 2.5

NaHCO3, which represents a measure of PSI + PSII + Calvin cycle (Table 2). Fig. 3 shows the typical Chl a fluorescence fast OJIP-transients, recorded from 5 samples starting from pea leaves (control), peeled leaf segments, and enzyme-digested preparations including washing and suspension steps during the isolation procedure. The typical curves of samples at different intervals revealed no major changes during isolation procedure. The basic level of fluorescence would vary with the

amounts of Chl in the same sample as well as the duration of exposure to light or darkness14,16. These differences in Chl fluorescence could be overcome by normalizations to Fo, Fi or Fm of the fast transient. These normalizations make a comparison of the fluorescence transients within the same figure easy. Fig 4A shows the Chl fluorescence transients of peeled leaf pieces (without epidermis), while these measurements made after 0, 10, 20 and 30 min of digestion are shown in Fig. 4B. The Chl fluorescence transients measured after 30 min of digestion (Fig. 3B) showed a rise in J peak. Fig. 5A shows a comparison between transients measured at the end of digestion (after 30 min) and soon after the suspension of protoplasts in washing medium (pH 6) (see Fig. 1). The results showed that the washing of protoplasts affected the kinetics of Chl fluorescence, while slowing down of the initial rise. The OJIP-transients measured after the subsequent centrifugation step were similar to the ones measured before centrifugation, except that the initial fluorescence rise was fast (Fig. 5B). Thus,

Fig. 4—Chl a fluorescence fast OJIP transients recorded during the first two crucial steps of protoplasts isolation [(A): Measurements with abaxial epidermis peeled leaf segments kept in dim light at room temperature (240C); samplings were examined at different time intervals in different peeled samples marked as 1 to 5. The curves were normalized at P level of the transients; and (B): Stripped leaf samples in digestion medium: samples were measured at 0 to 30 min, at 10 min intervals (loss in signal amplitude and change in the time course was noticeable due to enzymatic digestion)]

Fig. 3—Typical set of fast Chl a fluorescence OJIP transient measurements made during protoplast isolation (see, Fig. 1) [Raw OJIP transient traces as recorded by Handy PEA are shown. Green: Pea leaves from green house as well as their detached leaf segments; Blue: peeled leaves before digestion; Light turquoise blue: peeled leaf segments in digestion medium at different times of digestion in light; Red: samples incubated in washing medium; Dark blue green: final protoplast suspension. Large number of repetitive measurements were made to monitor, if any rapid occurrence of changes in OJIP kinetics and none were observed during isolation procedure. We further noted that only a few selected measurements of Chl a fluorescence would suffice]

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Fig. 5—Chl a fluorescence fast transient of protoplasts samples at washing and centrifugation steps. [(A): Protoplasts in the washing medium (curve 1) and after resuspension in the washing medium (curve 2); (B): protoplasts in washing medium, before centrifugation (curve 1) or after centrifugation (curve 2). Both these traces were normalized at Fo; and (C): OJIP transient curves of leaf and final protoplast suspension, normalized at Fo and at FM. The curves 1 and 2 represent averages of 3 to10 measurements.

centrifugation of the protoplasts at 50 × g had no harmful effect on fluorescence kinetics of the protoplasts (Fig. 5B). A strong recovery of the initial OJIP kinetics was observed on transfer of the protoplasts to an iso-osmotic suspension medium (pH 7) at the end of the washing procedure (Fig. 5C). This transient of protoplasts showed only minor differences with the OJIP-transient of the source leaves. Part of the differences between the OJIP-transient at the end of the digestion step and the transient of the sample suspended in washing medium (Fig. 6A) could be attributed to differences in the PQ-pool redox state. A brief (1s) pre-illumination given 10s before the fluorescence measurement can cause a reduction of the PQ-pool14,17. Such a ‘pre illumination’ pre-treatment given to protoplasts kept in washing medium made the OJIP-transients of protoplasts after washing (Fig. 6A) quite similar to those before washing. Fig. 6B, shows the effect of illumination and temperature on the OJIP-transients in leaves. There was a decrease in the extent of Chl a fluorescence in leaves kept at 0°C, compared to the leaves kept at 25°C. However, the OJIP transient curves of leaves became quite similar when a second light pulse was given 10 s after the first, the temperature effect was disappeared.

Discussion The fast Chl a fluorescence OJIP transient is a powerful tool for assessing the photochemical

electron transport activities as well as the overall vitality and physiological performance of oxygenic photosynthetic organisms and their tissues4,6,18-20. Although the intactness of protoplasts was assessed routinely during our studies, the functional integrity of the mesophyll protoplasts has to be ascertained, as the digestion involves exposure to acidic pH and high osmoticum. Rapid non-invasive spectroscopic method like Chl a fluorescence analysis can, therefore, be beneficial to monitor the photosynthetic performance of protoplasts during the isolation.

Fig. 6—Effect of pre-illumination on fluorescence fast transient of protoplasts and leaf segments. [(A): Fluorescence transients of protoplasts; and (B): Fluorescence transients of leaf segments kept at 25°C (curves 1 and 2) or at 0°C (curves 3 and 4). The characteristics of leaf segments without pre-illumination (curves 1 and 3) or with pre-illumination (curves 2 and 4) are shown separately]

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1

An important cause of variability in Chl fluorescence transients time course is due to change in the FJ-intensity, ascribed to differences in the chloroplast PQ-pool redox state16. Despite the fact that the abaxial peeling was done in dim room light (<10 µmol photons m-2 s-1), a partially reduced PQ-pool was observed in leaves, whose epidermis was stripped-off (Fig. 4A). Since the leaf pieces were illuminated during digestion, the fluorescence amplitude was suppressed (Fig 4B), possibly due to non-photochemical quenching. Such effect of light on the OJIP-transients was also reported in earlier study16. Room temperature Chl a fluorescence in higher plants gets emanated from chloroplasts and the Chl a fluorescence transient (Kautsky transient) reflects the photo reduction of electron transport carriers of two interactive photosystems and also the development of transthylakoid proton gradient which ensures the coupling of electron transport to ATP synthesis12,19,21. The intactness and integrity of chloroplasts ensures retention of both fast O-J-I-P phase as well as slow P-S-M-T phases of Kautsky transient. These phases are reliable criteria of the functional integrity of chloroplasts in vivo and in situ19,22. Any perturbation of chloroplast structure and function would alter these fluorescences4,5,18. Generally isolated intact chloroplasts do not retain their functional ability for a long time because of CO2 and Pi limitations15. However, intact protoplasts isolated from the pea mesophyll cells in the present study retained their CO2 fixation ability, respiratory and photosynthetic control and metabolic fluxes during their isolation and for several hours thereafter. Thus, the protoplasts may be quite useful for studies on chloroplast-mitochondrial interactions, metabolic fluxes and modulation of metabolites as well as on cellular signaling23. Earlier studies on Chl fluorescence properties of barley protoplasts demonstrated a slow decline in their redox state, transthylakoid pH gradient and phosphate metabolism24,25. The protoplast suspension exhibited a slight slow down of the kinetics up to the J-step and a much smaller contribution of the JI-phase to the whole transient (Fig. 5A). The smaller contribution of the JI-phase could be a side effect of the illumination during digestion. Light-induced changes in the amplitude of the JI-phase have been suggested to be related to changes in the LHCII-phosphorylation state16,22. The slower rise up to the J-step could be related to this

process. However, the reason for the slower IP-rise remains to be elucidated. The decrease in Chl a fluorescence at 0oC could be due to the fact that the second transient was measured in the presence of a reduced PQ-pool and therefore, the OJ-rise was not affected by temperature-dependent electron transport between QA and QB

13,18,19. However, some warming-up of the leaf during the measurement could also not be excluded. The transients in Fig. 6A indicated that slow rise kinetics up to the J-step largely disappeared, if the PQ-pool was pre-reduced by a pre-illumination flash. In the present study, we measured only the first OJIP phase fluorescence transient which only monitors light-driven electron flow involving two photosystems from H2O to FNR. Thus, the Chl fluorescence could be used as a quick non-invasive probe to assess if the isolation steps followed in our protocol impart any damage to protoplasts. The OJIP- transients monitored using Handy PEA showed clearly that our preparative steps were gentle and none of the steps seemed to alter the leaf tissues or protoplasts. Microscopic and electron transport assay (Fig. 1, Table 2) adequately supported these conclu-sions. However, the slow fluorescence phases of the transient would also be extremely useful to characterize the transthylakoid ∆pH development and state transitions and CO2 fixation19. These transient changes and changes in other spectroscopic features would be quite appropriate for further elucidating and probing the metabolic functions of protoplasts. Acknowledgements The visits of P M and R J S were possible through the visiting professorships under UPE Program of University of Hyderabad. This work was supported by grants (to ASR) from Council of Scientific and Industrial Research, New Delhi (No. 38(1063)/03/EMR) and DST JC Bose National Fellowship Grant (No. SR/S2/JCB-06/2006). K R and B S were recipients of Senior Research Fellowships from CSIR. We thank Dr. K K Sharma, ICRISAT, Hyderabad for sparing their Handy PEA instrument. We acknowledge the kind help of Drs. J S S Prakash and S Rajagopal of University of Hyderabad. We also thank the anonymous reviewers for their suggestions for the improvement of the manuscript. References

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