Planta

Planta 148,28-34(1980)

9 by Springer-Verlag 1980

Osmoregulation and the Control of Phloem-Sap Composition in Ricinus communis L. J. Andrew C. Smith* and John A. Milburn Department of Botany, The University, Glasgow G12 8QQ, U.K.

Abstract. Phloem-sap composition was studied in plants of Ricinus communis L. grown on a waterculture medium. The sap possessed a relatively high K + : Na + ratio and low levels of Ca 2+ and free H +. Sucrose and K § (together with its associated anions) accounted for 75% of the phloem-sap solute potential (~s). In plants kept in continuous darkness, a decrease in phloem-sap sucrose levels over 24h was accompanied by an increase in K § levels. Measurements of phloem-sap ~s and xylem water potential (~) indicated that this resulted in a partial maintenance of phloem turgor pressure (~p). In darkness there was also a marked decrease in the malate content of the leaf tissue, and it is possible that organic carbon from this source was mobilized for export in the phloem. The results support the concept of the phloem sap as a symplastic phase. We interpret the increase in K § levels in the phloem in darkness as an osmoregulatory response to conditions of restricted solute availability. This response can be explained on the basis of the sucrose-H § co-transport mechanism of phloem loading.

Key words: Osmoregulation - Phloem sap - Potassium Ricinus - Solute loading/unloading - Sucrose.

Introduction

Phloem transport in vascular plants provides developing tissues with a supply of organic and inorganic * Present address': Institut ffir Botanik, Technische Hochschule Darmstadt, Schnittspahnstrage 3 5, D-6100 Darmstadt, Federal Republic of Germany

(/-'=water potential; tP~=solate potential; ~Up= pressure potential Abbreviations:

solutes. Maintenance of longitudinal transport through the conducting tissue depends ultimately on the processes by which the solutes are transferred into and out of the phloem. The system is therefore delimited by these 'loading' and 'unloading' fluxes, and their properties must be understood before the transport process as a whole can be adequately described. The contents of the phloem can be regarded in essence as a symplastic phase (Miinch 1930; Crafts 1961). Consequently, some of the osmoregulatory characteristics of solute uptake and accumulation at the cellular level (e.g. Cram 1976; Raven 1977a) may also apply to phloem loading. Phloem sap composition has been documented fl'om a range of species (see Ziegler 1975; Pate 1976), but there is little information on the way in which the solute content of the sap is controlled. An examination of the effects of changes in solute availability on sap composition would help to elucidate the properties of the loading process. These properties must in turn have an important bearing on the control of long-distance transport in the phloem. In this paper we describe changes in phloem-sap composition observed in plants of Ricinus communis subjected to the stress of continuous darkness. Sap can be obtained directly from incisions into the bark (Milburn 1971), and there is evidence that this sap represents a true sample of the sieve-tube contents (Hall et al. 1971; Hall and Baker 1972; Mengel and Haeder 1977). The experiments were prompted by the observation that sucrose and K § levels in phloem sap from Salix tend to vary reciprocally under appropriate conditions (Hoad and Peel 1965). We show that a similar response in Ricinus has osmoregulatory characteristics, and discuss how this is related to the mechanism of phloem Ioading.

0032-0935/80/0148/0028/$01.40

J.A.C. Smith and J.A. Milburn: Osmoregulation and Phloem-Sap Composition

29

Materials and Methods

Analytical Methods

Plant Material and Growth Conditions

(a) Determination of Plant Water Status: In the description of plant water relations we shall use the following relation (Milburn 1979) :

Seeds of Ricinus communis L. var. gibsonii Nichols. (Daggs Ltd., Glasgow, U.K.) were sown in moist vermiculite. Before sowing, the vermiculite was washed through with three volumes of tap water followed by two volumes of nutrient solution (see below). The seedlings were then raised in a 'propagating tray' at 25 _+2~ C, relative humidity (RH) 95 100%. Nine to 10 d after sowing, the seedlings were transferred to nutrient solution. This solution was based on Long Ashton formula (Hewitt and Smith 1975), and the composition was as follows. Macronutrients (in mol m-3): KNO3, 4.0; Ca(NO3)2 anhydrous, 4.0; MgSO4.7 H20, 1.5; Na2HPOr H20, 1.3. Micronutrients (in mmolm-3): Fe citrate-5H20, 100; MnSO4.4H20, 10.0; ZnSO4.7H20, 1.00; C o S O 4 * 5 H 2 0 , 1.00; H3BO> 50.0; NazMoO4.2HiO, 0.50; NaCI, 100; CoSO~.7H20, 0.20. Each seedling was transferred to a plastic pot of volume 10-3m 3 containing two-thirds strength nutrient solution. To minimize the effects of irradiation on the temperature of the solution, the pots were covered with an undercoat of matt-black and a topcoat of whitegloss paint. Plant stems were support in holes of diameter 18 mm in the lids of the pots by split rubber tubing and cotton-wool padding. The solution in each pot was aerated for 10 rain in every hour. After 2 weeks the pots were refilled with full-strength solution; they were subsequently topped-up with fresh solution as required and rinsed out with hot water at least once a week. Plants were raised in a heated glasshouse with solar irradiation supplemented by artificial lighting on a photoperiod of 16 h provided by Atlas 400 W MBFR/U Kolorlux high-pressure mercuryvapour lamps. The temperature range was 12~ C (night minimum) to 36~ C (day maximum) and the RH 60-100%.

Experimental Conditions Three d before experimentation, 9-week-old plants were transferred from the glasshouse to a thermostatically controlled growth-room with forced ventilation. A bank of five fluorescent tubes (Omega 65/80 W White) provided a photoperiod of 16 h. The irradiance at upper-leaf height was 22 28 W m -2 in the range 400 1000 mm, measured with a Model 40X Opto-meter with foot-candle diffuser (United Detector Technology Inc., Santa Monica, California, USA). The temperature during the light-period was 23.0+_ 1.5~ C and the RH 65-100%. Plants were subjected to periods of darkness by transferring them to a growth-room of identical design but without lighting. Temperature and RH were kept within the range of values in the control growth-room.

Experimental Techniques Phloem sap was obtained from incisions into the bark with a clean, previously unused razor-blade as described by Hall et al. (1971). The exuding sap was collected in glass microcapillaries ('Microcap': Drummond Scientific Co., USA) of volume 50 or 100.10 9m 3. In the continuous darkness experiment, analyses were carried out on the first 10-10-9m 3 of sap exuding from each incision. Leaf-cell sap was extracted by freeze-disruption of the leaf tissue. The leaves were frozen at - 17~ C for 2.0 h and subsequently allowed to thaw at room temperature. Samples of interveinal tissue were centrifuged and the sap filtered through a layer of filter paper. There is evidence that the dilution error caused by mixing of the cell contents with apoplastic water is relatively small in Ricinus leaves, and at most about 12% (Milburn and Weatherley 1971; Dodoo 1978). For comparative purposes, the relative magnitude of this error was assumed to be the same for all the leaves used.

I/.t = I/] s __ I/Jp

where ~v is the total water potential, 7is is the solute potential and 7~p is the (turgor) pressure potential. We implicitly accept that 7J~ and tpp have thermodynamic equivalents in energetic and entropic terms, and that there are ambiguities involved in their definition (see Weatherley 1970 ; Spanner 1973 ; Zimmermann and Steudle 1978). The matric component of t/, was assumed to be numerically small and at least partially accounted for in the determination of ~e. Sap ~ was measured cryoscopically using a freezing-point osmometer (Knauer and Co. GmbH, Berlin, F.R.G.). Xylem 7~ was estimated for a fully expanded alternate leaf that had been sealed in a polyethylene bag on the plant for 2.0 h: this greatly reduced transpiration, thereby allowing bulk leaf 7~ to approach equilibrium with xylem 7*. The leaf was then excised and a value for xylem t/'p obtained using a pressure bomb (Scholander et al. 1964). To this was then added the value of xylem-sap 7~ determined on the extruded sap. (b) Chemical Analysis." Sucrose and reducing sugars were estimated by the method of Nelson and Somogyi (see Hall and Baker 1972). The cations K § Na +, Mg 2+ and Ca 2+ were quantified by atomic absorption spectrophotometry for samples diluted x 1000 by volume with distilled water using an EEL Atomic Absorption Spectrophotometer Mark 2 (Evans Electroselenium Ltd., Essex, U.K.). Chloride activities were determined using a chloride-sensitive electrode (Electronic Instruments Ltd., Surrey, U.K.) for samples diluted x 10 by volume with distilled water. Each sample was further diluted with an 'ionic-strength and pH-adjustment buffer' (EIL), 1 part buffer: 10 parts sample by volume; the buffer was ammonium acetate-acetic acid. Phosphate was measured colorimetrically by the method of Murphy and Riley (1962), and nitrate by the method of Wood et al. (1967), both on diluted samples. Organic acids in sap samples were separated by ion-exchange chromatography following the procedure of ap Rees and Beevers (1960). The samples were titrated to neutrality with NaOH to give a value for total titratable acidity. Malic acid was quantified by enzymic assay using the method of M611ering (1974), except that the buffer was 600 tool m-3 glycylglycine. Amino acids were quantified using a Jeol JLC-5AH autoanalyzer (Japan Electronics Optics Laboratory Co. Ltd., Japan) with a sodium-citrate buffer system. This analysis also gave a figure for the level of free NH~~. Serine and the amides glutamine and asparagine were separated using a Locarte system (Locarte, London, U.K.) with lithium-citrate buffer. The amino acids and amides are referred to only on the basis of their net charge at pH 7.0 (pI values taken from Mahler and Cordes 1971). The pH of the phloem sap was measured during collection from the plant in a microcapillary fitted to a glass bulb around the tip of an Activion pH combination microelectrode (Activion Glass Ltd., Fife, U.K.).

Results A summary of the solute content of the phloem sap from plants grown on NO3--N

Long Ashton solution

is p r e s e n t e d i n T a b l e 1. T h e s o l u t e s a r e g r o u p e d o n

30

J.A.C. Smith and J.A. Milburn: Osmoregulation and Phloem Sap Composition

Table 1. S u m m a r y of solute content of phloem sap from 9-week-old Rieinus plants grown on N O s - - N Long A s h t o n solution. Figures are means • for 6 plants or values for pooled samples. For calculation of the approximate theoretical contribution of the solutes to sap 7~ it was assumed that the monovalent and divalent cations were balanced by monovalent and divalent anions, respectively Concentration/ Theoretical tool m - s contribution to sap 7~s/MPa

Neutral compounds sucrose reducing sugars amino acids + amides bearing no net charge Cations K+ Na + amino acids ( + v e ) + N H 2 Mg 2 § Ca z+

Anions CIHzPO~/HPO~

NO3 organic acids amino acids ( - v e )

200 _,__o

I'llllllllll,[,

I'lllllllllld

I

~_~T

SUCROSE

150 z

100

o Y / ~ - - - - - ~~

"r

T

K+

I---

z

LLI L-J

50

z CD L..A

259 • 21 trace 86.5

-0.71 0

o2O0

[~1/111111/1~1111111111111111111111111111111111111111/[

0.22

15o 68.1 +4.1 t 6.7+ l . g f 2.7J 3.9 _+0.3 1.3+_0.4J

-0.36

c~