Using intracellular pH to evaluate growth inhibition of strawberry plants

Pkmt Physiol. Biochem., 1999.37

(2). 155- 160

Using intracellular plants

pH to evaluate growth inhibition

of strawberry

Fabien Robert*, Micbel Gendraud, Gilles Pete1 Physiologie

inte’gre’e de l’arbre fruitier, Universite’ Blaise-Pascal,

* Author to whom correspondence

should be addressed

(Received August 6, 1998; accepted November

24, avenue des Landais, 63177 Aubi&e cedex, France.

(fax +33 4 73 40 79 16; e-mail [email protected])

24, 1998)

Abstract - At present, many strawberry

plants (Frugaria x anana~~u Duch.) are cold-stored in autumn and replanted the next summer to provide good crops the followin g spring. These plants should be dormant (low growth potential) with abundant nutrient reserves (e.g. carbohydrates) to withstand storage. Because no rapid test was available to assess the induction of autumnal dormancy of strawberry plants, we have evaluated the level of relative nutrient deficiency of growth organs (correlative inhibition, one cause of dormancy) by intracellular pH measurements on Elsanta strawberry plants. To control the validity of this evaluation, carbohydrate accumulation in the storage parenchyma was measured and the dormancy level was estimated by measuring the petiole length of Elsanta plants under controlled conditions. The results showed that petiole length and intracellular pH of buds were low at the beginning of autumn, implying that the measurements of intracellular pH can be a marker of dormancy induction in strawberry plants. Then, in the middle of autumn, when the first changes occurred, growth inhibition

remained even as the intracellular pH measurements implied a break of correlative inhibition. This should suggest that another growth inhibition remains after the first chilling. 0 Elsevier, Paris Correlative inhibition / [14C]-DMO / growth inhibition / intracellular pH / strawberry DMO, $5dimethyloxazolidine-2,4 dione / pHc, cytoplasmic pH / pHi, intracellular pH / pHv, vacuolar pH / NMR, nuclear magnetic resonance I s.a.p., sub-apical parenchyma I st.p., storage parenchyma

relative inhibition 151). In a ‘trophic’ theory can explain, at

1. INTRODUCTION of summer, of strawberry 201. Then, these plants recover capability under the effect of

in autumn

vegetative

[ 12, and floral

In order to pro-

of dormancy in temperate

do not of their

by bud [ 10,241. These dormant states are classified according to of growth to climatic to exterto internal of the of the

Plant Physiol. Biochem.,

of bud

0981-9428/99/2/O

Elsevier, Paris

[ 131 showed that the intracellular pH (pHi) of bud cells in dormant tubers is lower than that of underlying parenchyma. Alkalinization of the latter was the result of an active plasmalemma H+-ATPase which generate a transmembrane pH gradient via proton extrusion out of the cell [27-29, 341. A cytoplasmic change was the main cause of pHi changes [7, 351. The transmembrane pH gradient lead to an active nutrient uptake involving co-transporters [6, 11, 141. The underlying parenchyma thus exhibited an important accumulation of nutrients. Therefore, it acts as a ‘nutrient sink’, inhibiting bud growth, at least partly, through a relative carbohydrate deficiency [ 151. Under

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F. Robert et al.

through nutrient distribution, can be determined by the measurement of pHi in involved tissues [21, 351. Today, few studies are made on the biochemistry of strawberry plant dormancy. Previous works showed seasonal change in metabolite contents in reserve organs (e.g. roots, storage parenchyma) [5, 251. Some morphological observations showed that petiole elongation depends on the physiological state of strawberry plants [3, 191. Under controlled conditions, the petioles of non-dormant strawberry plants are longer than those of dormant strawberry plants [33]. This observation provides a tool to assess the growth potential of strawberry plants. Because its result can only be known three months after the beginning of the observations (at the end of petiole growth), other possible markers should be studied to permit a rapid evaluation of the growth potential and particularly the induction of growth inhibition. So, in this study, we have, during two years, first measured the pHi in different parts of Elsanta strawberry plants to assess the changes in correlative inhibition (one cause of dormancy). To complete these observations, we have evaluated the carbohydrate content in the storage parenchyma. Finally, we measured the petiole length of Elsanta plants under controlled conditions to evaluate their dormancy level. 2. RESULTS 2.1. Intracellular pH measurements For 1992, the results of measurements showed a relatively constant pHi of the sub-apical parenchyma (s.a.p.) whereas the pHi of the buds were more variable @gure I A). (a) A decrease in bud pHi was observed at the end of summer; from August 14 to October 23, the buds’ pHi values were different from s.a.p. pHi (r = 0.78, P > 0.05). (b) After October 23, a significant increase in bud pHi indicated a physiological change when the first cold temperatures in autumn occur. Three hundred hours of temperatures below 8 “C were measured on November 13. For 1993 (figure I B), pHi of buds, sub-apical parenchyma (s.a.p.) and storage parenchyma (st.p.) showed a similar pHi evolution to the first year. (a) The pHi of buds was more acid than that of the two kinds of parenchyma from August 20 to October 26 (t = 6.36, P < 0.05). After this period, an increase of bud pHi was observed (250 h of chilling were measured on November 5). (b) A difference was also observed between the pHi of sub-apical parenchyma and those of storage parenchyma. Sub-apical parenchyma was more acid than storage parenchyma from September 8 Plant Physiol. Biochem.

7.0 6.7 6.4 600 6.1

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Days Figure 1. Intracellular pH (k SE) in buds, sub-apical parenchyma (s.a.p.). storage parenchyma (st.p.), and sum of hours below 8 “C. Measurements (five repetitions for each date) were carried out on samples of field-grown Elsanta strawbetry plants. Results are reported against calendar days of measurements. Intracellular pH was determined, for 1992 (A) and 1993 (B) measurements, using an adapted [14C]-DMO method (see Methods). Local temperatures were provided by M&Co-France.

to October 8 (t = 4.34, P < 0.05). Then, a slight increase of sub-apical parenchyma pHi and a concomitant more important decrease of’storage parenchyma pHi were observed on October 26 (150 h of chilling). 2.2. Carbohydrate measurements Relative changes in carbohydrate between the two years of measurements were similar, even if absolute values were different (due to different age of plants). Starch in storage parenchyma increased during autumn (figure 2) to a maximum at 300 h of chilling. Then, starch quantities decreased. Soluble sugars (sucrose, glucose and fructose) increased after 300 h of chilling (figure 2). 2.3. Morphological measurements Table I shows short petioles during autumn 1992 from October 16 to November 25 and the beginning of petiole length increase on December 24 (852 h of chilling). For 1993, petioles were short from September 23 to November 25 and the petiole length increased on December 20 (1 020 h of chilling).

Cellular pH of dormant strawberry

1400

200

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160

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Days Figure 2. Starch and soluble sugars (sucrose, glucose and fructose) accumulation (k SE) in storage parenchyma and sum of hours below 8 “C. Soluble sugars were extracted with hot ethanol/water (80/20, v/v) from storage parenchyma powder (four repetitions for each date; four plants per repetition); D.W. = dry weight. Starch and soluble sugars were evaluated by an enzymatic method (kit TC starch and kit TC sucrose, D-fructose and D-glucose from Boehringer Mannheim). Results are reported against calendar days of measurements. Local temperatures were provided by M&o-France. Table I. Petiole lengths (in mm) of Elsanta strawberry plants transferred from outdoors to a growth chamber. These plants were left under natural climatic conditions before the transfers. For each date, growing petioles were measured (four plants per group in the first year of observations and six in the second year) for three months after the transfer. Values followed by different letters differ significantly at 0.05 probability level. Calendar days of transfers into growth chamber

Means of final petiole lengths

1992

1993

1992

289 303 329 358 48 72

266 291 329 354 58 -

23.8 19.8 22.6 27 b 52 c 57.7

Sum of hours at temperature below 8 “C

1993 a a a

c

49.4 49.1 39.8 60.7 78.1 -

b b a c d

1992

1993

20 30 432 852 1 780 2 258

0 30 700 1 020 2 100 _

3. DISCUSSION Many changes were observed in the intracellular pH (pHi) of buds, sub-apical parenchyma and storage parenchyma. The bud pHi decreased at the end of summer yigure I) and then remained low during the beginning of autumn. Also, the pHi of the storage

plant

157

parenchyma measured in 1993 (figure I B) was more alkaline than that of buds, whereas pHi of the sub-apical parenchyma was also alkaline but less than that of storage parenchyma. Consequently, underlying parenchyma should be a ‘sink’ for buds, as has been observed in Jerusalem artichoke [ 131, in Japanese artichoke 1351,in ash-tree [S], and in oak [ 11. In Jerusalem artichoke, pHc calculated from pHi and pHv values of bud and underlying parenchyma of dormant and nondormant tubers were confirmed by NMR measurement of pHc [7]. Tort et al. [3.5] also showed a correlation between pHi and pHc values in Japanese artichoke tubers. In Jerusalem artichoke, the underlying parenchyma of dormant tubers, which have a pHi more alkaline than that of buds, exhibits an important accumulation of nutrients [ 14, 151, and also a very active plasmalemma H+-ATPase [27-291. This H+ATPase generates a transmembrane pH gradient via proton extrusion out of the cell [27-29,341 which leads to an alkaline pHc and an active nutrient uptake, involving co-transporters [6, 11, 141. In non-dormant tubers, non active H+-ATPase and decrease of pHi in underlying parenchyma permit the growth of the bud [ 15, 291. At the end of summer, the change of pHi should be the main cause of the decrease of strawberry growth potential, by trophic inhibition of storage parenchyma on buds. Carbohydrate measurements showed starch accumulation in storage parenchyma during the beginning of autumn figure 2), supporting the potential role of trophic inhibition of storage parenchyma on buds. Similar carbohydrate changes were observed in strawberry plants by BCdard and Beaumont [4] and Paquin et al. [26]. This correlative inhibition was also confirmed by morphological measurements on Elsanta plants under controlled conditions. These measurements, which provide a tool to detect growth inhibition [33], showed short petiole lengths during autumn (table I), revealing that these strawberry plants did not grow actively. No increase in petiole length was observed when the pHi measurements revealed correlative inhibition. Therefore, the measurements of pHi provided a good tool to assess the first growth inhibition at the beginning of autumn. They allow the determination of the potential direction of trophic accumulation and thus the cause of growth inhibition. The measurement of starch accumulation in the storage parenchyma support these observations @gure 2) but can not be used alone to determine the cause of inhibition. Then, our results showed similar pHi in both subapical parenchyma and storage parenchyma after 150 h of chilling @gure I B), and an increase of bud vol. 37 (2) 1999

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F. Robert et al.

pHi after 250-300 h of chilling figure 1 A, B). At these chilling values, starch accumulation stopped (figure 2). The underlying parenchyma should thus not be considered as a trophic inhibitor for buds. A decrease in starch content was then observed after 350-400 h of chilling, probably permitting cryo-protection of cells @gure 2). Although these results revealed the breaking of the correlative inhibition, the morphological observation of plants under controlled conditions continued to show low petiole lengths. This fact indicates that, at the end of autumn, another growth inhibition exists in strawberry plants after the first chilling. Previous work showed, at the end of autumn, that the low capability to synthesize nucleotides is related to growth inhibition of strawberry plants [33]. Therefore, at the beginning of autumn, the correlative inhibition should permit the induction of this growth inhibition which continued beyond 300 h of chilling. The results reported in this paper suggest that pHi measurements of the studied parts of Elsanta plants could be a good marker to assess the first growth inhibition. The growth inhibition which remained after the first chilling is not due to correlative inhibition, but should be due to energetic inhibitions [33]. Moreover, because morphological results can only be known three months after the beginning of the observations, pHi measurements provide a better means for evaluating the decrease in growth potential. 4. METHODS 4.1. Plant material. For pH and carbohydrate measurements, strawberry (Fragan’a x trnanassa Duch., Elsanta cultivar) plants from cold storage (first year of measurement), or obtained from runner production were replanted in July in the field in Clermont-Ferrand (45”47’ N, 3”7’ E) and left exposed to the natural climate. For 1992, plants had been planted during the previous year. Thus, these plants were one year older than those used for 1993. For morphological measurements, the same plants were replanted at the same period in pots (1.7810-*.m3, a sufficient volume for root development in autumn), and left outside, as for plants used for pH measurements, until they were transferred into a growth chamber. 4.2. Intracellular pH measurements. An adapted [14C]DMO ([2-‘4C] $5dimethyloxazolidine-2,4 dione) method was used to determine the pHi of the strawberry parts studied. This method, first used for the determination of intracellular pH in giant cells of algae [37], has been used by Kurkdjian et al. [22] in isolated Acer pseudoplutunus cells or in tissues from Jerusalem artichoke (Heliunthus tuberosus L. [13]) and chestnut-tree (Custunea saliva Miller [31]). The

Plant Physiol. Biochem.

determination of pHi is based on the equilibrium distribution of the [‘4C]-DM0 (lipophilic and weak acid) between the cells of incubated samples and incubating solution. The relative proportion of the non-metabolized [‘4C]-DM0 in the incubated sample cells (Ci) and in the incubating solution (Ce), permits the computation of the pHi according to the equation of Waddel and Butler [36]:

gi Ce

1

+

10W-pW

1 + 10(~He-~Ka)

where pHe, the pH of the incubating solution, was measured with a Tacussel TSP pH glass electrode after the incubation of samples, and pKa is the pKa of 14C-DM0 with a value of 6.3. So, pHi

= log $(lOpKa

+ lOpHe )-lOPK”]

The measurements were made for 1992 (Aug. 2; Aug. 14; Aug. 27; Sept. 16; Oct. 10; Oct. 23; Nov. 13; Dec. 23) on five apical buds (5-7 young leaves and primordia) and on five subapical parenchyma pieces of Elsanta plants [33] harvested from a nursery in Clermont-Ferrand. They were repeated for 1993 (Aug. 20; Sept. 08; Sept. 24; Oct. 8; Oct. 26; Nov. 5; Nov. 19; Dec. 3) on the same parts, plus five storage parenchyma pieces. Buds and pieces of sub-apical and storage parenchyma (about 80 mg for each) of five plants were separately incubated in 1 mL [‘4C]-DM0 solution (2.1 mM KOHMES, pH 6.1; 4 yM [‘4C]-DM0 (1.84 MBq+‘mol-‘), Amersham) for 14 h in darkness at 15 f 1 “C. The results of preliminary tests showed that these were the best experimental conditions (data not shown). After quickly washing in distilled water, the [‘4C]-DM0 absorbed by incubated samples was extracted according to Jefford and Edelman [18]. [‘4C]-DM0 absorbed by samples (Ri) and that remaining in the incubating solution (Re) were measured with a Kontron scintillation counter, in 5 mL Beckman liquid scintillation cocktail. From Ri, we calculated Ci as Ci = Qi/Vi, where Qi = Ri - [(metabolized + intercellular) [‘4C]-DMO] and Vi = Total volume of sample - intercellular volume of sample. Metabolized [‘4C]-DM0 was previously determined by chromatography of the extracts of incubated samples loaded onto polyethylenimine-cellulose plates. After migration in isopropanol-ammonia-distilled water (8/1/l, v/v/v), the distribution of radioactivity on the plates was evaluated using a Bioscan radiochromatogram scanner (figure 3). From the value of the two peaks, which represent the metabolized (Rf = 0.74) and non-metabolized [‘4C]-DM0 (Rf = 0.85), it is possible to calculate the percentage of metabolized [14C]DMO in each type of organ: 29 % for buds and 2 1 % for subapical and storage parenchyma during the autumnal-winter season. The intercellular [‘4C]-DM0 was computed from intercellular volume and [‘4C]-DM0 concentration in the incubating solution. The intercellular volume was determined by incubating samples in 10 pM [‘4C]-inulin solution, a non-permeable metabolite for strawberry plant cells, as 12.9 % for buds and 12 % for sub-apical and storage paren-

Cellular pH of dormant strawberry

0.5

0.6

0.7

0.8

1

0.9

Rf

Figure 3. Example of radioactive peaks of chromatography

of extracts of samples. Extracts were loaded onto polyethylenimine-cellulose plates and migrated in isopropanol-ammonia-distilled water (8/1/l, v/v/v). Samples were pre-incubated in 1 mL [‘4C]-DM0 solution for 14 h in darkness at 15 + 1 “C. Percentage of metabolized [14C]DMO = area of metabolized [‘4C]-DM0 peak (A; Rf = 0.74) /area of metabolized [‘4C]-DM0 peak (A) + area of non-metabolized [14C]DMO peak (B; Rf = 0.85).

chyma. The volume of the samples was calculated after incubation, from the sample weights and the densities (previously measured as 1.1 for buds, 1.15 for sub-apical parenchyma and 1.21 for storage parenchyma). The chemical hydrolysis of [‘4C]-DM0 in the incubating solution was determined in the same way (5 %) as the metabolized [‘4C]-DM0 of samples and leads to Ce = Re x 0.95. 4.3. Carbohydrate measurements. For carbohydrate measurements, storage parenchyma of Elsanta plants were first dried. Soluble sugars (sucrose, fructose, glucose) were extracted with hot ethanol/water (80120, v/v) from storage parenchyma powder. Starch and soluble sugar were evaluated by an enzymatic method (kit TC starch and kit TC sucrose, D-fructose and D-glucose from Boehringer Mannheim): p fructosidase + H20 --------_)D-glucose

Sucrose

Starch

amyloglucosidase h

+ (n - l)H,O

D-glucose

+ D-fructose (1)

(D-fluctose)

n D-glucose

(2)

+ ATPS

(3)

G-6-P (F-6-P) + ADP phosphoglucoseisomemse F-6-P

)

G-6-P

(4)

)

+

+ glucosed-phosphate G-6-P + NADP NADPH + H+

_dehydrogenase

D-gluconate-6-P

(5)

NADPH was evaluated by spectrophotometry at 340 nm. Glucose was determined after reactions (3) and (5). Fructose was determined after reactions (3), (4) and (5), with the glu-

plant

159

cose value subtracted. Sucrose was determined after reactions (2), (3), (4) and (5), with the fructose and glucose values subtracted. Starch was determined after reactions (2), (3) and (5). The measurements were made for 1992 (Oct. 1, Oct. 16, Oct. 29, Nov. 25, Dec. 14, Jan. 13) and 1993 (Sept. 29, Oct. 19, Nov. 11, Nov. 26, Dec. 3 l), with four repetitions (four plants by repetition) by date. 4.4. Morphological measurements. Four Elsanta plants per date were transferred from outdoor to a growth chamber for 1992 (Oct. 16, Oct. 30, Nov. 25, Dec. 24, Feb. 17, Mar. 12) and six plants for 1993 (Sept. 23, Oct. 18, Nov. 25, Dec. 20, Feb. 27). The growth chamber conditions consisted of a 12 h/12 h photoperiod with day/night temperatures of 23/13 f 1 “C, 80 % relative humidity, and artificial metal halogen (Power Star HQIL Osram) lighting (180 + 15 pmo1.m-2.s-‘). To assess the growth potential of Elsanta plants, the morphological evolution was evaluated by measuring the full grown petioles that emerged during the three months after the transfer. The first two petioles growing in the growth chamber were not measured because the growth of these young petiales began in field conditions and could be modified by transfer to the growth chamber. The results were expressed in relation to the hourly sum of temperatures below 8 “C. 4.5. Temperature measurements. The sum of hours below 8 “C during autumn and winter were calculated from October 1 with local data provided by Mtteo-France. This parameter provided a good approximation of the chilling effect which promotes strawberry plant growth [32], as had been observed with other species [16]. 4.6. Statistical analysis. Regression and differences (t-test) between computed pHi were tested using the EViews software (Quantitative Micro Software, Irvine, California). Differences between the means of each transfer date were tested using a multiple range test (P < 0.05). Acknowledgements. The authors gratefully thank the ‘Centre interregional de recherche et d’experimentation de la fraise’ for its contribution of equipment and the ‘Centre technique interprofessionnel des fruits et legumes’ for tinancial support.

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