Plant Science, 67 (19901253- 257 Elsevier Scientific Publishers Ireland Ltd.

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COMPARATIVE ULTRASTRUCTURAL AND FLUORESCENCE STUDIES OF GRAPEVINE (VIZ’IS VINIFERA L.) CHLOROPLASTS ISOLATED FROM STEM AND LEAF TISSUES

ALEXANDER G. IVANOW*, NADEZHDA S. IGNATOVAb and ALEXANDER M. CHRISTOV

‘Central Laboratory of Biophysics, Bulgarian Academy of Sciences, Sofia 1113, bInatitute of Viticulture and Enology, Pleven 5800andcInstitute ofPlant Physiology, Bulgarian Academy of Sciences, Sofia 1113 (Bulgarial

(Received July llth, 19891 (Revision received November 21st, 19891 (Accepted November 30th, 19891

Ultrastructural observations of chlorophyll-containing stem tissues (woody shoots) of Vitis vinifera L. show that the chloro- plasts in mature sieve elements (phloem and xylem) contain preferentially a large number of appressed (granall thylakoids. This finding is in close relation with the extremely low chlorophyll CJb ratios in the stem tissues, which characterizes the pho- tosystem II (PSIIkontaining granal membranes. These structural differences are accompanied also by differences in the prop- erties of the low temperature (77Kl chlorophyll fluorescence emission. In both phloem and xylem chloroplasts the peak at 735 nm originating from photosystem I (PSI1 is strongly reduced and the F,,,/F,, ratio is much more lower than in leaf chloroplasts. Furthermore, the value of the F,,/Fws ratio in stem tissues depends to a lower extent on the presence of Mgl’, i.e., the cation control of excitation energy distribution between PSI1 and PSI (spillover) is considerably restricted in both phloem and xylem thylakoid membranes.

Key words: pitis vinifera L.; stem chloroplasts; ultrastructure; low temperature (77K) fluorescence; photosystems; energy transfer

Introduction

Since the first study of Pearson and Lawr- ence [l] on the bark photosynthesis, the photo- synthetic activity of chlorophyllous woody stem tissues had been established in a variety of species [2-41. Recently, the primary pro- cesses of photosynthesis in chloroplast-contain- ing stem tissues have been investigated by means of induction phenomena of chlorophyll fluorescence [5] and delayed fluorescence emis-

*To whom correspondence should be sent at: Central Labo- ratory of Biophysics, Bulgarian Academy of Sciences, Acad. G. Bonchev str. bl.21,1113 Sofia, Bulgaria. Abbreviations: EDTA, ethylenediamine tetraacetic acid; HEPES. N-2-hydroxyethylpiperazine-N’-ethane-sulfonic- acid; LHCdb, light-harvesting chlorophyll o/h-protein complex of PSII; MES, 2-W-morpbolinolethanesulfonic acid; PS, photosystem; Tricine, N-(2-hydroxyl,l-bis/hydroxyme- thyl/etbyllethyllglicine

sion [6]. These studies have shown strong evi- dence that there is an effective electron transport capacity, oxygen evolving activity [S] and a high reduction state of the primary elec- tron acceptor Q, [5], implying high photochemi- cal efficiency of photosysetm II (PSI11 in the stem plastids.

Although the photosynthetic function, ultrastructure and pigment composition are relatively well studied [2,3,5], there is still no information concerning the functional chloro- phyll-protein complexes of PSI1 and PSI as well as the light-harvesting chlorophyll a/b II (LHCII) within the chloroplast membranes of the green stem tissues. Our work further explores the chlorophyll fluorescence phenom- ena (7’7K fluorescence, chlorophyll emission under F, conditions1 aiming to additional char- acterization of the photosynthetic apparatus in isolated chloroplasts from grapevine (Vitis mini-

0168-9452190/$03.50 0 1990 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland

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fera L.) woody shoots tissues (phloem and xylem) in comparison with leaf chloroplasts.

Material and Methods

Chloroplast isolation Grapevine (Vitis vinifera L., cv. Merlot)

leaves (100 g) were homogenized three times for 15 s at high-speed in ice-cold medium containing 0.33 M sucrose, 5 mM MgC12, 1 mM MnC12, 2 mM EDTA, 20 mM NaCl, 2mM ascor- bic acid, 0.5 mM K~IPO 4 and 50 mM MES buffer (pH 6.1). All subsequent operations were carried out at 4 °C. The resulting slurry was fil- tered through eight layers of cheese cloth and centrifuged at 600 × g for 20 s. The supernatant was recentrifuged at 2000 × g for 2 rain. The resultant pellet was resuspended in medium containing 0.33 M sucrose, 5 mM MgCI~, 1 mM MnC12, 2 mM EDTA, 20 mM NaCI and 50 mM HEPES buffer (pH 6.7) and the cen- trifugation steps were repeated. During the isolation procedure 5°/0 (w/v) polyviny]-pyrroli- done (Polyclar AT, Serva) was also present in the buffers. The harvested chloroplasts were resuspended in solution containing 0.33 M suc- rose, 5 mM MgCI~ and 10 mM Trieine buffer (pH 8.0) at a concentration of 3 mg Chl/ml.

Chloroplasts from the chlorophyllous stems were obtained after mechanical separation of phloem and xylem tissues from either developing green shoots or 1-year~ld shoots. Thin slices of phloem and xylem were first hand-ground with quartz sand in MES buffer (pH 6.1) as described above except that sucrose concentration was 0.5 M. All other subsequent isolation steps were the same as in the case of isolation of leaf chloroplasts. Chlorophyll a and b concentrations were determined in 800/0 ace- tone extract [7].

Electron microscopy Thin slices of both leaves and stems were

fixed with 50/0 glutaraldehyde in 10 mM Tricine buffer (pH 8.0), 5 mM MgCI 2 and 0.5 M sucrose. After fixing for 6 h at 4°C the slices were thoroughly washed. Postfixation of the samples in 1% OsO 4 buffered with phosphate buffer (pH

7.2) containing 0.137 M NaC1, 2.7 mM KC1 and 0.6 M sucrose was carried out overnight at 4 °C. The fixed material was dehydrated by treating with increasing concentrations of ethanol and propylene oxide and embedded in Durkupan ACM (Fluka). Ultrathin sections were stained with uranyl acetate and lead citrate and exam- ined on a JEM 100B electron microscope.

The chloroplast ultrastructure was analyzed from the electron micrographs by measuring the average number of thylakoids per grana stack (AT) and adjacent stromal thylakoids (N), respectively, the ratio of the total length of appressed to non-appressed thylakoids (~ LfL L) and the average ratio of the length of grana stack to adjacent stroma GLg/L [8].

Fluorescence measurements Low temperature (77K) fuorescent measure-

ments were performed in quartz cuvette in medium containing 10 mM Tricine buffer (pH 8.0), 0.33 M sucrose, 10 mM KC1 -+ 5 mM MgCI 2 and 10 pM Chl/ml in the probe. Fluorescent spectra were recorded on a Jobin Yvon JY3 spectrofluorimeter equipped with a red sensi- tive photomultiplier (Hamamatsu R928) and a liquid nitrogen device. Chlorophyll fluores- cence was excited at 436 nm. Exciting and mea- suring slits were 4 nm.

Room temperature chlorophyll fluorescence was monitored at wavelength longer than 660 nm (Schoot RG 11 cut~)ff filter). The weak exci- tation light beam (lO/o of the saturating light intensity) at 436 nm was provided by monochrc~ mator SPM2 (Carl Zeiss, Jena). Chlorophyll concentration in the probe was 5 ~g/ml.

Results and Discussion

The ultrastructural features derived from the analysis of the electron micrographs indi- cate that the ratio of appressed to non- appressed thylakoid membranes and the average number of partitions per grana stack are higher in the stem chloroplasts as com- pared to the values of • L/X L ratio and Ng observed in the chloroplasts from the meso- phyll leaf tissue (Table I). The L J L ratio which

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Table I. The ultrestruetural features of leaves and stem chloroplasts of Vitis t~tyera L. N. and N. represent the average number of appreseed thylakoick l~er grana stack and adjacent stromal thylakoids respectively. XL/XL. is the ratio of the stromal length of appressed to non-appressad thylakoid membranes. L~L is the average ratio of the lengths of grana stasks to adjacent stromal thylakoids and was calculated as in Ref. 8. Mean values ± S.E. were calcu- lated from 7 - 1 0 mierographs, which represent 10-- 14 typi- cal ehloroplasts. Significance levels were calculated from t- test on the differences between leaf and stem (phloem and xylem) means (*P < 0.05; * ' * P < 0.001).

Sample Leaf Stem

N 6.70 ± 0.80*** 14~0 ± 0.75 N~ 3.00 ± 1.00" 4.50 ± 0~4 LXL/~Y.L, 1.70 ± 0.25"'" 4.53±0.33 L~ o 0.72 ± 0.06*** 1.43 ± 0.11

is equivalent to the proportion of the projected area of granal to stromal lamellae is also about 2-fold higher (1.43) in the stem chloroplasts. These data are consistent with some earlier morphological studies [3] as well as with the recent observations of Larcher et al. [5] although no quantitative ultrastructural analy- sis has been presented.

The comparative analysis of the pigment composition indicates that the Chla/b ratio is much lower in the stem tissue than in the leaves (Table II). The values of Cha/b ratio decrease approximately linearly following the order leaf tissue > stem cortex (phloem) inner stem tissue (xylem). The total chlorophyll content decreases in the same order, the lowest value being observed in the green woody (xy-

lem) tissue. Similar extremely low Chla/b ratios have been reported recently by Lareher et al. [5] for green stem tissues of Fagus silvatica. Furthermore, the data presented in Table II show a slight increase of the chlorophyll con- tent and the Chla/b ratio in the stem tissues from January to June, indicating certain sea- sonal dynamics of both parameters. These data correspond to the seasonal patterns of the bark photosynthetic capability reported for Vitis vinifera grapevine woody shoots [3] as well as in some other species [2,4]. It should be noted that even increased in June, the values of Ch] content and the Chla/b ratios in the phloem and xylem remain far below those observed in the leaves.

The increased amount of PSII-associated Chl b and the proportion of appressed to non- appressed thylakoid membranes allow as to assume that the green plastids in grapevine stem tissues (phloem and xylem) could be referred to a low-light or shade type chloroplasts [8,9]. Such an assumption can be fully understood within the framework of the observations that ext remely small part of radiation light energy could penetrate the bark of unshaded grapevine woody shoots [3].

The 77K ratio of relative fluorescence intens- ities emitted from PSI (735 nm) to that emitted from PSII (695 nm) provides a reliable indication for the distribution of excitation energy to each of the photosystems [11,12]. It is generally accepted that Mg 2÷ may control the distribution of excitation light energy ('spillov- er') in isolated chloroplasts via a cation-depen- dent rearrangement of chlorophyll-protein

Table II. Distribution of chlorophylls per fresh weight in 80% acetone extracts from leaves of Vitis vini/era L. (cv. Merlot) and from phloem and xylem of 1-year~)ld vine shoots during January ('Winter') and June ('Summer'). Mean values ± S.E. were calculated from 7 independent experiments. Statistically significant differences are marked by: *P < 0.05; ***P < 0.001.

Sample June January

Cld a + b Cldo/b Chla + b Chlo/b

Leaves 132.00 ± 6.69 3.31 ± 0.14 - - Phloem 15.52 ± 0.33 2.05 ± 0.11"** 8.29 ± 0.20 1.69 ± 0.04 Xylem 4.33 ± 0.37 1.74 ± 0.06*** 4.97 ± 0.60 1.49 ± 0.05*

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complexes of PSII and PSI within the thylakoid membranes [12,13]. The F73s/Fs9 s emission ratio for leaf chloroplasts is higher (Table III) when the chloroplasts are suspended in low-salt (10 mM NaC1) medium as compared to the respec- tive value obtained in high-salt (5 mM MgC12) medium.

The values for the F735/F69 s ratio of both phloem and xylem chloroplasts are much more lower that in leaf chloroplasts (Table III) although remarkable enhancement is regis- tered in the 'Summer' stem plastids. It is of interest that the cation dependence of the F735/ F695 ratio is negligible in stem chloroplasts. As proposed earlier by Anderson and Andersson [14] spillover type changes could not be regis- tered if there are little or no PSI complexes in grana partitions.

The Mg 2÷ considerably increases the ratio of F695/Fs5 in the 77K chlorophyll emission spectra of both phloem and xylem chloroplasts (Table III). It seems very likely that the increased F69 s amplitude could be attributed to the Mg 2*- induced enhancement of the energy transfer from LHCa/b to the PSII reaction center com- plex [15]. In addition, the spectral shift of 2.5-- 5.0 nm towards the longer wavelengths of the secondary peak in stem chloroplasts in the presence of Mg 2÷ (spectra are not shown), imply the appearance of an aggregated form of the LHCa/b complex [10] which could be also at least partially responsible for the observed

enhancement of the Fs,/F~s ratio. Moreover, as pointed by Leong and Anderson [16] the main portion (31% of the total chlorophyll con- tent) of LHCa/b in low light intensities-adapted chloroplasts could be referred to its oligomeric form (LHCP1).

Data concerning the composition and/or alterations at the LHCa/b-PSII pigment level are delivered also from room temperature chlo- rophyll fluorescence measurements under F 0 conditions [11]. The F 0 registered in green stem (phloem and xylem) plastids are almost three fold higher than in leaf chloroplasts (Table III). The differences observed between 'winter' and 'summer' samples well correspond to the sea- sonal dynamics of the pigment composition (Chla/b ratio) of stem plastids. Since the level of F o in isolated chloroplasts does not depend on photochemical events when the first stable electron acceptor (Qa) of PSII is fully oxidized (as in our experiments), it is reasonable to assume that the observed higher E 0 values rep- resent the increased quantity of LHCa/b com- plex within the thylakoid membranes of the stem chloroplasts.

There are data that LHCa/b is involved in the molecular mechanisms responsible for the thylakoid stacking [17], therefore a higher degree of appressed thylakoids should be expected as a result of the increased LHCa/b amount since LHCa/b-PSII supramolecular complexes are localized mainly in granal mere-

Table III. Fluorescence properties at 77K of chloroplasts membranes isolated from Vitis vinifera leaves and shoots (phloem and xylem}. The values are averages from 3 separate experiments. F 0 values are normalized to the F o level registered in leaf chloroplasts.

Sample F 735/F e9 s F 695/ F~s F °

+ Mg - Mg + Mg - Mg

June Leaves 1.030 3.050 1.130 0.940 1.00 Phloem 0.370 0.410 1.113 0.784 2.65 Xylem 0,275 0.315 1.890 0.662 2.80

January Phloem 0,246 0.236 0.703 0.705 2.45 Xylem 0.310 0.353 0.743 0.667 2.30

branes [14]. Actually, we observed a higher degree of thylakoid stacking in the stem chloro- plasts as compared to the leaf chloroplasts (see Table I). The physiological significance of the increased amount of LHCa/b complex under low light intensity and/or shade conditions is proposed to ensure higher efficiency of light harvesting in PSII [9,16]. The well functioning PSII recently reported in stem chloroplasts [5,6] entirely confirms this suggestion.

Acknowledgement

This work was supported in part by the Bul- garian Ministry of Culture, Science and Educa- tion under Research contract N 519.

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