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Effect of temperature and water stresses on gas exchange, fluorescence kinetics, and solute levels of jojoba
INDUSTRIAL CROPS ANDPRODUCTS AN INTERNATIONAL
JOURNAL
Industrial Crops and Products 5 (19%) 279-290
Effect of temperature and water stresses on gas exchange, fluorescence kinetics, and solute levels of jojoba Toni L. Ceccardi a**,Irwin F! Ting b a University of Florida, IFAS, CitrusResearchand EducationCenter;L&e Alfred, FL, USA b Department of Botany and Plant Sciences, University of California, Riverside, CA, USA Recieved 17 January 1996; accepted 29 May 1996
Abstract
Seven year old jojoba, Simmondsia chinensis (Link) Schneid., plants grown under three irrigation treatments, wellwatered, intermediate, and dry, were exposed to controlled temperature treatments of +5, -5, and -10°C for six hours during February 1991. Following the temperature treatments, gas exchange and fluorescence kinetics were used to evaluate
the degree of injury to the plants. Following subfreezing temperatures, gas exchange was depressed, and fluorescence kinetics indicated greater damage to well-watered plants. Amino acid and carbohydrate levels, and osmotic potentials were measured before and after cold treatments to observe the effect of low temperatures on solutes. Freezing temperatures resulted in increased solute concentrations attributed to increases in total carbohydrates and total amino acids. Proline, arginine, aspartate family amino acids, glucose, and fructose increased while phenylalanine, tyrosine/ornithine, glutamate and sucrose decreased in response to freezing temperatures. Lathhouse plants exposed to -5°C natural frost for seven hours in December 1990 set seed, but, with the exception of dry treatment plants exposed to -5”C, plants exposed to either -5 or - 10°C in the laboratory in February 1991 did not set seed. Overall, the intermediate irrigation treatment had the least disruption of fluorescence kinetics and photosynthesis, and set the most seed following the controlled low temperature treatments. Keywords: Jojoba; Simmondsia chinensis; Freezing resistance; Drought; Fluorescence; Stress
1. Introduction Drought induced cold-hardening of jojoba is a common cultural practice in areas subject to subfreezing temperatures. Drought increases the supercooling capacity of jojoba by mechanisms which are not completely understood (Naqvi et al., 1988). Previous laboratory studies with jojoba showed increases in supercooling capacity of detached leaves and buds in response to decreased irrigation (Gold*Corresponding author.Fax:(941)956-4631.
stein et al., unpubl.). Since detached organs can supercool to much lower temperatures than intact plants (Sakai and Larcher, 1987) it was of interest to measure the frost-resistance and recovery of intact plants preconditioned with water stress. Physiological factors related to water status influence the capacity of a plant to supercool. A decrease in relative water content and the accumulation of solutes depress the freezing point of the cell sap (Alberdi and Corcuera, 1991; Sakai and Larcher, 1987), which in turn lowers the supercooling point. Furthermore, cold acclimation, as well as water stress,
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may induce an increase in solute concentration and a decrease in water content which may account for up to 50% of the decrease in freezing point (Yelenosky, 1979; Yelenosky and Guy, 1989; Young and Peynado, 1964). Drought and cold acclimation may have an additive influence on the supercooling point depression (Cox and Levitt, 1976), or they may work independently resulting in the same end (Chen et al., 1977). In the present study, the frost-hardiness of intact jojoba plants under three irrigation treatments was measured. Gas exchange and variable fluorescence were used as indicators of injury following cold treatments. In addition, low-temperatureinduced fluctuations of free amino acids and carbohydrates were measured in plants of the three irrigation treatments and compared to those levels found in plants under ambient winter temperatures. These parameters were compared before and after cold treatments and frost resistance was compared between irrigation treatments. 2. Materials and methods 2.1. Plant material
In the fall of 1990, six jojoba plants of the selection ‘Mirov’ from each of three irrigation treatments were transferred from a greenhouse to a latbhouse for acclimation to cooling nighttime temperatures. The seven year old plants had been transplanted from a field plot to pots (7.6 1 in UC&mix soil, a sandy loam) in a greenhouse in 1988 and maintained on three irrigation treatments. Prior to their transfer to the lathhouse, they were repotted into 19-1 pots of UC&mix soil. After the two years on the various irrigation treatments their sizes, measured as the volume of a sphere, ranged from dry treatment plants of approximately 0.30 m3 to wet treatment plants of 1.0 m3 (Ceccardi et al., 1990). The inigation treatments were maintained by hand-watering until water flowed from the bottom of the pots as follows: wet - watered every three days; intermediate - watered every four days; dry - watered every five days. On a monthly basis, the plants were fertilized by irrigation with a complete nutrient solution.
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2.2. Controlled freezing study
Between February 4-6, 1991, three plants from each irrigation treatment were randomly selected to undergo one of three freezing treatments. Beginning at 10 pm, whole plants were placed into a chest freezer (Kenmore 23, Sears Roebuck and Co., Chicago, IL) with an adjustable thermostat and subjected to temperatures of either 5°C (control), -5°C or -10°C for six hours. Cooling of the roots was avoided by wrapping the pots with a heating pad set on low and by covering the soil with Styrofoam pieces. Thirty-six gauge copper-constantan thermocouples were attached to two leaves with tape and to a cold junction compensator (Omega Engineering Inc., Stamford, CT) and a chart recorder, by which the mV outputs were recorded. The system was too insensitive to detect exotberms, but it monitored the freezer temperature. The plants were placed into the freezer at room temperature, and the thermostat was turned down to the target temperature. Cooling at a rate of 15°C h-l, the freezer decreased from room temperature to -10°C in about 2 h. A fan was used to circulate the air inside the freezer. After approximately eight hours (two hours cooling, six at set temperature), the thermostat was set to room temperature and the lid was propped open slightly. Two hours later, the plants were removed from the freezer. In addition to the controlled freezes, all lathhouse plants were exposed to air temperatures of -5°C on December 23, 1990 for a duration of approximately seven hours. Two weeks later, necrosis and chlorosis of plant tissues were compared between the irrigation treatments. 2.3. Physiological analyses 2.3.1. Gas exchange
Following the temperature treatments, gas exchange was measured using a portable photosynthesis system (Analytical Development Co., Ltd., Hoddesdon, UK) approximately every two hours until dusk (8 am to 6 pm) and once the next day at midday. The first fully expanded leaves (‘mature’) were chosen for measurements. Undamaged leaves were selected unless it was not possible, such as with the well-watered treatment following -WC, in which case all leaves appeared to have undergone a freeze-thaw.
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2.3.2. Fluorescence
Young (first or second node, not fully expanded) and mature (third or fourth node, fully expanded) leaf samples were collected approximately every 2 h after the plants were removed from cold for measurement of fluorescence kinetics. The leaves were dark adapted at room temperature for thirty minutes before modulated fluorescence was measured. The measurement system was described by Ogren and Baker (1985). The instrument (Hansatech Ltd., NuNorfolk, UK) consisted of a leaf chamber, detection probe, LED probe with a low level yellow light (1.5 pm01 me2 s -’ ) to detect background fluorescence, higher level light from a projector (150 pmol mm2 s-i) and highest level light for saturating pulses. The highest level light (1200 pmol me2 s-‘) was connected to a camera shutter and timer to give 0.2 or 0.3 s pulses of saturating light every 30 s. The outputs were recorded on a chart recorder. Photochemical quenching (qQ), maximum fluorescence (F,), variable fluorescence (F,), and F,l F, (ratio of variable fluorescence to maximum) were measured and calculated based on the modulated fluorescence kinetics according to Schreiber and Bilger (1987). 2.3.3. HPLC sample preparation Young and mature leaf samples were collected immediately before the plants were placed into the freezer, immediately after, and 12 h after the cold treatments. The samples were placed on dry ice in plastic bags and later stored at -80°C until they were analyzed. Statistical analyses of carbohydrates and amino acids were performed using Minitab analysis of variance (Minitab Inc., State College, PA: function, glm). Samples of approximately 10 mg leaf tissue from 2 leaves were used to extract carbohydrates and amino acids. The fresh weight was recorded and the samples were extracted in 80% ethanol until bleached. The leaf extracts were dried by vacuum centrifugation (Savant Instruments, Farmington, NY) and resuspended in 1 ml distilled water. The aqueous samples were partitioned into three fractions with a series of l-ml ion exchange columns (Bio-Rad Laboratories, Richmond, CA). Each sample was washed through the cohmms with a total of 15 ml of distilled water as described by Mitchell and Madore (1992). The first column retained amino acids using
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cation exchange resin (Bio-Rad AG 5Ow-X8. 50100 mesh, Hydrogen form). A second column of anion exchange resin (Bio-Rad AG l-X8 200-400 mesh, Formate form) retained organic acids, and the final, neutral fraction contained carbohydrates. 2.3.4. Amino acid determinations The amino acid fractions were eluted with 5 ml 4N ammonium hydroxide (NHaOH). Amino acid samples were dried and prepared for HPLC analysis as described by Madore (1990). The procedure included converting the amino acids to their phenylisothiocyanate (PITC) derivatives and resuspending in buffer (15 mM sodium acetate, 3% (v/ v) acetonitrile, and 0.025% (v/v) triethylamine, adjusted to pH 7.4 with phosphoric acid). The sample was filtered on a syringe filter and 20 ~1 were injected onto a Rainin Dynamax ODS column (4.6 x 25 mm) held at 48°C. Separation of the PITCamino acids was performed as described by Mitchell and Madore (1992). Samples were compared for differences due to irrigation treatment, temperature treatment, leaf age, and time of collection. 2.3.5. Carbohydrate determinations The neutral carbohydrate fractions were dried by vacuum centrifugation (Savant Instruments, Farmington, NY), resuspended in 100 ~1 water, and 20-~1 samples were analyzed by HPLC as described by Madore (1990), using a Waters SugarPak 1 column held at 90°C. Comparisons of the carbohydrate compositions of the three irrigation treatments before and after cold treatments were made. 2.3.6. Osmotic potentials Osmotic potentials were measured using the same leaf samples frozen for HPLC analyses. The leaf sap osmotic potentials were measured with a thermocouple psychrometer (Model 51OOC Wescor, Inc., Logan, UT). 2.4. Harvest In the summer of 1991, seeds were harvested to compare quantities of seed produced between irrigation treatments exposed either to just the December natural freeze or the additional February controlled freezes. Numbers of fully developed seeds
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and aborted seeds per plant were counted and weighed. No designation was made between unfertilized aborted seed or aborted seed due to frost injury, with the assumption that the ratio of unfertilized seed would be equivalent between the irrigation and temperature treatments. 3. Results 3.1. Gas exchange Midday peak values of photosynthesis and stomatal conductance following each of the three controlled cold treatments are shown in Table 1. Values shown in the first row in each irrigation treatment are from photosynthetic measurements taken on a day in January, not following any cold stress, for comparison. Compared to the control, only the wet treatment plant showed any photosynthetic depression following the 5°C treatment at 66% of that at ambient temperatures. With one exception, following the -5°C and - 10°C cold treatments, photosynthesis declined so that the plants were evolving CO,. The exception was the intermediate irrigation treatment which maintained low levels of photosynthesis following the -5°C treatment. After 24 h of recovery,
all of the irrigation treatments had higher photosynthesis levels, except that of the wet and intermediate irrigation treatments following the - 10°C treatment. 3.2. Fluorescence Table 2 shows fluorescence from midday on the day following the cold treatments. The intermediate and dry treatments showed practically no maximum fluorescence (F,,,) decline while the wet treatment had a 45% and 75% decrease following the -5°C and -10°C treatments, respectively (Table 2). Photocemical quenching, qQ, decreased by 58% and 75% in the wet treatment; by 16% and 35% in the intermediate; and 33% and 43% in the dry treatment after -5 and -10°C temperatures, respectively. The ratio of variable fluorescence to maximum fluorescence, F,IF,, decreased after -5°C in the wet treatment. It increased or remained approximately unchanged in the other treatments. 3.3. Amino acids Amino acid levels fluctuated in response to temperature and irrigation treatments, the time of sample collection, and the age of the tissue (Table 3). The
Table 1 Gas exchange aof jojoba under three irrigation treatments following low temperature treatments Treatment
Photosynthesis (pm01 COzmS2 s-t)
Conductance (mol mm2 s-l)
day 1
day 1
day 2
Wet Control +5”c -5°C -10°C
15.15 f 7.20 f -0.20 f -1.45 f
8.38 0.53 0.45 0.48
1.14 -2.05 f 0.18
Intermediate Control +5”c -5°C -10°C
4.41 4.34 2.0 -0.35
2.21 0.59 1.36 0.408
na 2.4 f 1.82 ‘deceased’
DrY Control +5”C -5°C -10°C
6.14 6.53 -0.18 -0.607
f2.74 f 1.20 f 0.85 f 0.108
na 1.20 f 1.55 -0.36 f 0.027
f f f f
&Sk
a Gas exchange measured with an ADC portable photosynthesis system. Values are the means and standard deviations of 4 measurements.
0.208 0.13 0.105 -0.94 0.097 0.085 0.08 0.090
day 2 f 0.057 f0.0047 f 0.015 f 0.019
:a75 f0.37 0.034 f 0.0033
f f f f
ia11 f0.015 ‘deceased’
0.0508 f 0.090 f 0.12 f 0.066 f
0.016 0.0086 0.015 0.028 0.016 0.010 0.0073 0.012
BY05 f 0.010 0.048 f 0.0068
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Table 2 Fluoresence parameters calculated following temperature treatments Irrigation
Temperature (“c)
ElIa (% Control)
9Qa (% Control)
&IF,
Wet
+5 -5 -10
100 f41.0 58.6 f 44.6 23.2 f 26.4
100 f0.8 60.3 f 22.1 24.9 f 1.9
0.095 f 0.038 0.034 f 0.048 1.57 f 1.31
Intermediate
+5 -5 -10
100 f0.7 120.4 f 51.9 81.5 f 10.7
100 f 1.6 74.6 f 23.5 66.7 f 16.3
0.082 f 0.070 0.091 f 0.13 0.18 f 0.079
Dry
+5 -5 -10
100 f65.8 71.6 k 67.9 112.3 f 24.2
100 f2.0 66.0 f 28.5 57.8 f 24.8
0.14 f0.051 0.18 f 0.074 0.20 f0.17
a Fluorescence values for 5°C were used as controls. Values are the means and standard deviations of 2 measurements.
pretreatment values differed between the mature and young leaves for most of the amino acids (totals, Table 3). Total amino acids were highest in the dry and wet irrigation treatments with a high degree of significance (P = 0.005). Also, total amino acids were generally higher in the young leaves (P = 0.02, Table 3). Total amino acids tended to increase after the cold treatments (P c 0.001, Table 3). Proline was significantly higher in the young leaves than mature (all treatments combined: young: 10.8f7.0; mature: 8.5f7.0 pmol/gfw, P = 0.025). Levels of proline made up the majority of total amino acids and were highest in the dry treatment, followed by the wet treatment (P = 0.01). Proline also increased in the wet and intermediate treatments after cold treatments, and decreased in the dry plants (Fig. 1). Similarly to proline, arginine levels were significantly higher in the young leaves (young: 0.51 f 0.04; mature: 0.21 f 0.04 pmol/gfw, P -c 0.001). Levels of arginine were highest in the wet treatment, followed by dry (P = 0.03). In the dry treatment, arginine decreased after the cold treatments, but increased in the wet and intermediate treatments (Fig. 2a). The pretreatment levels of the aspartate family (selected members: lysine, threonine, valine, isoleucine, leucine) were higher in the young leaves (young: 0.88 f 0.08; mature: 0.77 f 0.08 pmol/gfw, n.s.) with the exception of isoleucine. On average for all samples, aspartate family amino acids were highest in the wet treatment, descending to the lowest
Proline
II
181
I
II+ I Wet
2’l
+5
I -5
B
-10
Temperature ( C)
p=.oo Fig. 1. Proline levels following three temperature treatments in pmollgfw. Values are averages of young and mature leaves directly after cold treatments and 12 h later. The P-value shows the significance of the interaction of irrigation and temperature treatments on proline levels.
levels in the dry treatment (not significant). The increased concentration seen in this amino acid family after the subfreezing treatments, compared to the 5°C levels was highly significant (P < 0.001, Fig. 2b). The concentration of glutamate was highest in the mature leaves (young: 1.4 f 0.1, mature: 2.0 f 0.1 hmol/gfw, P < O.OOl),and declined after cold treatments (P < 0.001) . Glutamate was highest in the dry treatment and lowest in the intermediate (Fig. 2~). Levels of phenylalanine, tyrosine/omithine followed the same pattern as that of glutamate, being
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Table 3 Total amino acids and total carbohydrates for young and mature leaves of each irrigation treatment before (pre), directly after (post), and 12 h (+12 h) following three low temperature treatments Total carbohydrates (mg gfw-‘)
Total amino acids (pmol gfw-‘) Weta
Young a +5”c a -5°C a -lo”ca Mature a +5”c -5°C -10°C
Intermediate
Young +5”c -5°C -10°C Mature +5”c -5°C -10°C
Dry
Young +5”c -5°C -10°C Mature +5”c -5°C -10°C
Prea Post a +12 ha Post +12 h Post +12 h Pre Post +12 h Post +12 h Post +12 h
34.6f 7.0 33.9f 11 19.7 f 2.6 33.8 f 1.4 15.5 f 1.4 10.9 f 4.3 17.8 f 0.9 13.0f 5.7 19.1 f 1.4 15.9 f 0.9 30.0 f 4.4
Pre Post +12 h Post +12 h Post +12 h Pre Post +12 h Post +12 h Post +12 h
14.0 f 12.9 f 6.0f 15.9 f 28.4f 13.9f 20.7 f 12.4f 12.5 f 12.5 f 15.9 f 17.4 f 15.5 f 32.9 It
Pre Post +12 h Post +12 h Post +12 h Pre Post +12 h Post +12 h Post +12 h
22.4 f 1.7 23.4 f 5.6 31.8f 9.0 30.2f 5.5 26.5 f 1.5 14.4f 4.2 21.4f 11 17.6f 2.5 21.2f 0.9 27.7 f 2.4 22.9f 5.3 16.4f 4.6 13.6f 2.1 25.1 f 6.0
21.1 f 11.8f 18.43~
2.4 1.6 4.6
3.7 4.7 0.3 6.1 0.8 3.1 8.3 2.0 4.5 1.9 0.8 7.2 4.6 16
Weta
Young b +5”C a -5°C a -1o”ca Mature b +5”C -5°C -10°C
Intermediate
Young +5”C -5°C -10°C Mature +5”C -5°C -10°C
DrY
Young +5”C -5°C -10°C Mature +5”C -5°C -10°C
Preb
Postb +I2 hb Post +12 h Post +12 h Pre Post +12 h Post +12 h Post +12 h
207.9 f 188.9 f 220.6f 186.9f 256.7 f 196.9 f 191.5 f 175.9 f 186.7 f 151.5 f 188.7 f 154.1 f 184.4 f 373.6 f
24.2 18.7 3.6 3.8 28.2 10.7 11.9 24.5 0.2 23.4 44.2 55.8 10.3 137
Pre Post +12 h Post +12 h Post +12 h Pre Post +12 h Post +12 h Post +12 h
151.1 f 36.7 187.1 f 26.9 101.2f 6.5 191.7 f 24.8 159.1 f 2.7 206.6 f 22.2 207.7 f 15.4 95.5 f 5.3 74.9 f 22.7 122.3 f 1.10 112.2f0.19 114.1 f 15.4 198.5 f 0.90 310.7 f 67.0
Pre Post +12 h Post +12 h Post +12 h Pre Post +12 h Post +12 h Post +12 h
164.0 f 6.26 147.2 f 35.1 244.0 f 88.6 133.1 f 34.3 136.3 f 16.1 173.6 f 66.0 157.6 f 55.0 131.0f4.99 112.6 f 26.8 228.8 f 2.09 163.6 f 16.9 143.4 f 11.7 98.4 f 18.8 145.8 f 3.03
ab Levels of significance due to various treatments are shown in the wet treatment rows and apply to all treatments in that column. Significance was calculated by Anova (glm, Minitab). a Significance 5 0.05. b Significance 5 0.10. Significant interactions include irrigation x temperatureBVb; irrigation x age a, ns.; temperature x ageBsb, for amino acids and carbohydrates respectively. Each value represents the mean and standard deviation of two measurements.
T.L. Ceccanii, 1.P lining/Industrial Crops and Products 5 (1996) 279-290 Arginine
285
Aspartate family
0.7
1.4
1
r
Wet 4 Int + Dry *
0.6 -
0.2 -.-__.--------
0.1
1A +5
-5 Temperature
0.21’ B +5 ( C)
p=.o5
-5 Temperature ( C)
p=.13
Phenylalanine, Tyrosine/Ornithine 0.3
3. Wet *
2.5
Int
Int
2+___ AL
CZy *
------I
1.5 -
1 /c +5
p=.of3
0.2
;y *
aI 0.15 0.1 -
1 0.5
Wet *
0.25 -
-5 Temperature ( C)
-10
I-I
0.05 +5
-5 Temperature ( C)
-10
p=.oz
Fig. 2. Amino acid levels following three temperature treatments of (a) arginine, (b) aspartate family selected members (lysine, threonine, valine, isoleucin, leucine), (c) glutamate, and (d) phenylalanine, tyrosinelomithine, in Kmollgfw. Values are averages of young and mature leaves at two times following- temperature treatments. The P-values show the significance of the interaction of irrigation and _ temperature treatments on amino acid levels.
highest in mature leaves (young: 0.15 f 0.01, mature: 0.19f0.01 ,xmollgfw, P = 0.02) and declining after cold treatments (P <: 0.001). The decline was greater following -5 than -10°C (Fig. 2d). Levels were highest in the intermediate treatment and lowest in the wet treatment. The amino acid group consisting of glycine and serine showed no significant response to either irrigation or cold treatment (data not shown). In general, they were highest in the young leaves and in the wet irrigation treatment, lowest in intermediate. 3.4. Carbohydrates Total carbohydrates were highest in the wet and lowest in the dry plants (P = 0.001, Table 3). Young leaves had higher levels than mature leaves (Ta-
ble 3). Carbohydrates increased after cold treatments in the wet and intermediate treatments, while they decreased in the dry treatment (Table 3). Following the -10°C treatment the total carbohydrate levels were higher in the mature leaves, in those of the wet and intermediate irrigation treatments (Table 3). Glucose levels were significantly higher in young leaves (all treatments combined: young: 155 f 7, mature: 132 k 7 mg/gfw, P = 0.02). Overall, the levels of glucose were highest in wet and lowest in dry plants (P = 0.001). Glucose levels increased after the cold treatments in wet and intermediate plants, but decreased in dry plants, similarly to total carbohydrates (Fig. 3a). The levels of glucose were much higher than any other sugar, and were probably responsible for changes seen in total carbohydrates (up to 90%).
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Glucose 220
significant. Sucrose increased after the -5°C treatment, but there was no change in the level following -10°C compared to +5”C. The overall trend was toward increased levels after cold treatments, except in young leaves treated with -10°C of the wet and intermediate treatments in which there was a 2-fold decrease (not shown). The dry plants following - 10°C had levels approximately equal to or less than +S’C (Fig. 3b) with no difference in mature and young leaves (not shown). Fructose levels were significantly higher in the wet irrigation treatment than intermediate and dry, prior to cold treatments (not shown). After cold treatments, levels were highest in the dry plants (P=O.O3, Fig. 3~). Additionally, the levels were higher in the young leaves (young: 3.7 f 0.4, mature: 1.5 f 0.4 mg/gfw, P c 0.001). Fructose tended to increase after -5 and - lO“C, except in wet treatment, in which it stayed approximately equal to the 5°C treatment (Fig. 3c).
I
sot4 A +5
-5 Temperature (“c)
p=.oo Sucrose
3.5. Osmotic potential 12” B +5
I -5 Temperature (%)
pz.72
1 Wet 4 Int + Dry *
-5
-10
Temperature (“c) p=.o3
Fig. 3. Carbohydrate levels following three temperature treatments of (a) glucose, (b) sucrose, and (c) fructose, in mglgfw. Values are averages of young and mature leaves at two times following temperature treatments. The P-values show the significance of the interaction of irrigation and temperature treatments on carbohydrate levels.
Sucrose levels were higher in mature leaves (young: 14 f 1.3, mature: 18 f 1.3 mg/gfw, P = 0.02), and the differences due to irrigation were not
The pretreatment values of osmotic potential were consistently lowest in the dry plants, and in general, highest in the wet plants. The osmotic potential tended to decrease after low temperature treatments, with the lowest water potentials following -10°C (Table 4). 3.6. Harvest 1991 llvo weeks after a natural frost in December, 1990, the wet plants had necrotic, yellow leaf tips, and brown flower buds. The dry treatment plants had little visible damage to either leaves or buds. The intermediate plants had less injury than the wet plants. The results of the 1991 summer harvest are shown in Table 5. None of the plants exposed to -10°C in the laboratory in February produced seed, neither did plants of the intermediate and wet treatments following the -5°C cold treatment. The dry irrigation treatment exposed to -5°C in the controlled study did set seed. In contrast, all of the plants under ambient conditions were exposed to a low temperature of -5°C for approximately seven hours in December, but still set seed.
LL. Ceccanii, I.R Lting/Industrial Crops and Products 5 (1996) 279-290 Table 4 Osmotic potentials of jojoba under three irrigation treatments before and after low temperature treatments a Pretreatment $x (MPa) Wet irrigation -3.3 f 1.0
Intermediate irrigation -3.7 f 0.25
Dry irrigation -4.2 f 0.3
Temperature (“C)
Posttreatment & (MPa)
+w -5°C -10°C
-3.9 f 0.01
+YC -5°C -10°C
-5.5 f 0.01 -3.6 f 0.80 -4.6 f 0.01
+5”c -5°C -10°C
-2.2 f 0.39 -3.6 f 0.53 -5.0 f 0.66
-Y9*0.6
aValuesare the means and standard deviations of 9 (pretreatment) or 3 osmometer readings. Overall, the intermediate treatment set the greatest mass of seed, followed by dry and then wet (Table 5). 4. Discussion The decreased photosynthesis and stoma&l conductance seen after the cold treatments in jojoba is an indication of injury. The negative values of photosynthesis could be due to photorespiration or photoinhibition. Loreto and Bongi (1989) found in jojoba, that following low temperatures, in the light, recovery to the initial rates of photosynthesis took 24 h to one week and they concluded that the delay was due to photoinhibition. In the present study, the dry and intermediate treatment plants showed some recovery of their gas exchange rates 24 h after removal from the -5°C treatment. Recovery from freezing injury requires energy either from stored reserves or from photosynthesis (Steffen and Palta, 1987), and therefore total recovery of the plants depends on the early recovery of photosynthetic capacity. Overall, the intermediate plants had the least photosynthetic depression after the subfreezing treatments which may indicate that their photosynthetic apparatus was more cold resistant than the wet and dry treatments. Following the - 10°C treatment, photosynthesis did not recover even after 2 days, but respiration still occurred, as seen by the output of CO2 (Ta-
287
ble 1). This may be evidence that photosynthesis is more sensitive to freeze-thaw injury than respiration (Steffen and Palta, 1987) in jojoba. Chlorophyll fluorescence is a good indicator of photosynthetic activity and can be used to monitor damage to the photosynthetic apparatus (Baker and Horton, 1987). Fluorescence parameters indicate plant injury with declining F,,,, qQ and FJF, such as in the case of photoinhibition (Franklin et al., 1992). Fluorescence measurements of jojoba leaves decreased more in the wet treatment than in either of the low-irrigation treatments (Table 2). F, showed practically no decline in the intermediate and dry treatments, possibly due to drought-induced acclimation causing structural changes in lipid and membrane compositions which may protect the photosynthetic apparatus (Guy, 1990). Photochemical quenching (qQ) also showed less depression following cold in the dry and intermediate treatments. The intermediate treatment had the highest qQ activities after both the subfreezing treatments, which correlates with the greater capacity for gas exchange recovery seen in that treatment. Likewise, in the dry and intermediate treatments, the negligible change in FJF,,, again indicates hardiness of the photosynthetic apparatus. In the wet treatment, the F,IF, value decreased following the -5°C treatment; then in a result which may appear surprising, it increased about 600% after the -10°C treatment. The maximum fluorescence peak decreased below the height of variable fluorescence peaks, which remained high due to an increase in Fo, baseline fluorescence. High F. indicates inactivation of PSI1 (Baker and Horton, 1987), and in this case the resulting high FJF,,, indicated tissue death. The total amino acid levels were higher in the dry treatment due to osmotic adjustment, measured by the accumulation of compatible solutes. Interestingly, the intermediate treatment had less solute accumulation than the wet treatment. Because jojoba is adapted for growth in dry environments, and does not increase biomass production as much as other Cs crops when given excess water (Wardlaw et al., 1983), the well-watered plants may have been stressed due to over-watering. Therefore, total amino acids were found in higher levels than in the intermediate plants, which appeared to be the least stressed (Ceccardi et al., 1990).
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Table 5 Harvest data from plants of three irrigation treatments and various cold treatments Irrigation
Temperature (“C)
Wet
ambient ambient ambient +5 -5 -10 total
Intermediate
Fruit (number)
Seed weight (8)
Aborts a (number)
Fruit set (%)
46 31 60 54 0 0
36.64 21.38 38.85 33.7 0 0 130.57
63 37 62 98 121 21
36.5 45.6 49.2 35.5 0 0
ambient ambient ambient +5 -5 -10 total
42 73 115 205 0 0
31.97 51.78 73.15 77.46 0 0 234.36
233 110 136 300 125 24
15 40 46 40 0 0
ambient ambient ambient +5 -5 -10 total
45 74 91 2 81 0
27.2 44.07 61.84 0.915 47.07 0 181.10
70 104 167 18 152 28
39 42 35 10 35 0
a ‘Aborts refers to undeveloped seeds noted by an empty seed coat, which may include seeds damaged by frost or unpollinated flowers (see text). Plants under ‘ambient’ conditions underwent a natural frost of -5°C for 7 h on December 23, 1990. All other treatments were additional controlled treatments,
Proline, which was highest in the dry treatment and increased in the other treatments after cold temperatures, often accumulates during water stress and may protect thylakoid membranes against electrolyte damage occurring during freezing stress (Stewart and Larher, 1980). In cabbage, free proline levels correlated with freezing tolerance of different tissues in plants which were either acclimated to low temperatures or nonacclimated (Stewart and Larher, 1980). During poststress periods proline can be used as a source of carbon, nitrogen, or reductant, and may have for this reason accumulated in the wet and intermediate plants following the freezing treatments. Alternatively, proline may have been a desiccation product (Stewart and Larher, 1980). Because of the high initial levels of proline in the dry treatment, there was no additional increase following the cold treatments. The average proline levels for the three irrigation levels were: wet, 10.0; intermediate, 7.1; dry, 12.2 pmoll gfw. Proline was a major component of total amino acid levels, especially in the young leaves.
Glutamate is central to ammonium metabolism and is the immediate precursor of the amino group of most protein amino acids (Thompson, 1980). Not only does glutamate contribute the nitrogen atom, but also most of the carbon atoms, to proline and arginine synthesis (Thompson, 1980). Arginine may be another compatible solute produced in response to freezing-induced dehydration strain, and it can also be converted to proline without prior conversion to glutamate (Stewart and Larher, 1980). Therefore, glutamate probably decreased following the cold stress by conversion to proline and arginine. Arginine accumulation is consistent with its role as an osmoticum and its easy conversion to proline. Total carbohydrates and total amino acids in the stressed leaves underwent the same changes following cold treatments. First, levels of both constituents were generally higher in young leaves. Levels increased following cold treatments of -5 and - lO“C, using +5“C as the control, in wet and intermediate
T.L. Ceccardi, 1.P. ling/lndustrial
plants. In contrast, the levels decreased in dry plants following subfreezing treatments. Unlike amino acids, total carbohydrates did not accumulate in the dry plants, which had the lowest levels of carbohydrates. Well-watered plants had the highest rates of photosynthesis under ambient conditions and therefore had higher levels of phloem sugars. Following cold treatments, carbohydrate levels (based on fresh weight) may have accumulated in response to freezing dehydration or due to water loss through leaky cell membranes. Mitchell and Madore (1992) reported that, in muskmelon, chilling caused an increase in phloem sugars, including sucrose, fructose, and glucose. Sucrose transport, or production, was inhibited following -lO“C, indicated by a decrease in young, sink leaves (not shown). Glucose and fructose levels most likely began to increase as vacuolated sucrose was hydrolyzed into its hexose components. Mitchell and Madore (1992) found that sugar translocation was inhibited by low temperatures in muskmelon, causing accumulation of hexose sugars. Similarly, low temperatures imposed a sink limitation on sunflower and rape, resulting in the accumulation of sucrose (Paul et al., 1991). The timing of the freeze may be crucial in jojoba. Plants subjected to a -5°C cold night for seven hours in December, considered a harsh freeze in Riverside, still set seed. Those subjected to additional controlled freezes of -5°C in early February set no seed except in dry plants. It is possible that by February most of the plants had deacclimated. According to the National Weather Service (Ann. report of fruit-frost activities in the Corona-Riverside District, 1990-91 winter season), December was followed by a unseasonably mild January and a warm February. The freezing temperature of jojoba buds increases at later dates (Ceccardi, 1993). The rate of freezing may also have been a deciding factor in the survival of the cold-treated plants. A cooling rate of 15”C/h is much faster than in nature (usually no more than 3“C/h, from UC Riverside weather station data). Rapid rates of cooling may result in injury before metabolism adjusts. Osmotic potentials decreased, on average, following low temperature treatments, possibly due to the loss of water from the leaves following the lethal treatments of -5 and -10°C.
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The dry plant subjected to -5°C in the laboratory was better protected against freezing than the more irrigated treatments, probably due to its lower water content, resulting in seed production. Drought does not increase the length of time to break dormancy, so a longer dormancy would not be the reason for the added protection (Nelson and Palzkill, 1990). The intermediate treatment, which appeared to recover photosynthetic capacity more rapidly than either the dry or wet plants, set the most seed. An intermediate level of water stress seems to give the best level of frost protection without sacrificing yield. The low yield of the dry treatment demonstrates that there is a cut-off point where increased drought does not improve frost resistance and decreases yield. The wet treatment itself may be a stress imposed upon these plants which are better adapted to surviving in harsh, dry conditions, and are not well suited for high production under what would be ample water for a more typical Cs crop (Wardlaw et al., 1983). 5. Conclusion Photosynthesis measurements, including gas exchange and fluorescence kinetics are good indicators of freezing injury and recovery. In this study with jojoba, if photosynthesis had not recovered by the second day following the low temperature treatments, the above ground tissue did not survive. Using photochemical quenching as an indicator, levels below approximately 60% of ambient levels indicated an inability to recover. The differences in solute patterns and photosynthesis levels explain the differences in freezing resistance and productivity between the irrigation treatments. The dry treatment had the lowest osmotic potentials, the highest proline levels, and was the only plant to survive and set seed after -5°C but it resulted in slower growth and fewer seeds set than the intermediate or wet treatments. The intermediate treatment resulted in lower levels of proline than the wet or dry treatments, and intermediate plants had lower photosynthesis rates and lower levels of carbohydrates than the wet treatment plants. The moderate levels of growth and water stress of the intermediate plants resulted in a balance between drought-induced frost resistance and metabolism, leading to higher yields than in the more extreme treatments. The na-
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ture of the intermediate treatment’s frost resistance contradicts Khalafalla’s (1987) report that more frost resistant lines of jojoba accumulated more soluble sugars, sucrose and proline. The well-watered plants had the highest osmotic potentials, levels of sugars, and rates of growth and photosynthesis. The more severe damage caused by low temperatures resulted in the lowest yields in the well-watered plants. Acknowledgements The authors thank Dr. Monica Madore and Michelle Gadus for their assistance in performing the HPLC analyses of carbohydrate and amino acid levels. References Alberdi, M. and Corcuera, L.J., 1991. Cold acclimation in plants. Phytochemistry, 30: 3117-3148. Baker, N.R. and Horton, P., 1987. Chlorophyll fluorescence during photoinhibition. In: D.J. Kyle, C.B. Osmond and C.J. Amtzen (Editors), Photoinhibition. Elsevier Science Publishers, Amsterdam, pp. 145-168. Ceccardi, T.L., 1993. The effects of drought on the frost resistance of Simmondsia chinensis. Ph.D. dissertation, University of California, Riverside, CA. Ceccardi, T.L., Naqvi, H.H. and Ting, I.P., 1990. Physiological responses of jojoba to drought and frost stress. In: Estilai, Naqvi and Ting (Editors), Proc. 1st Ann. Meeting Assoc. Adv. Ind. Crops. October 1990, Riverside, CA, 47-52. Chen. PM., Li, PH. and Burke, M.J., 1977. Induction of frost hardiness in stem cortical tissues of Comus stolonifera Michx. by water stress. I. Unfrozen water in cortical tissues and water status in plants and soil. Plant Phys., 59: 236-239. Cox, W. and Levitt, J., 1976. Interrelations between environmental factors and freezing resistance of cabbage leaves. Plant Physiol., 57: 553-555. Franklin, L.A., Levavasseur, G., Osmond, C.B., Henley, W.J. and Ramus, J., 1992. Two components of onset and recovery during photoinhibition of Ulva mtundata. Planta, 186: 399408. Guy, C.L., 1990. Cold acclimation and freezing stress tolerance: role of protein metabolism. Ann. Rev. Plant Physiol. Plant Mol. Biol., 41: 187-223. Khalafalla, MS., 1987. Frost tolerance studies on jojobaSimmondsia chinensis (Link) Schneider: Clonal variation, compositional relationship and effect of cultural practices. Ph.D.dissertation. University of Arizona, Tucson, AZ. Loreto, F. and Bongi, G., 1989. Combined low temperature-high light effects on gas exchange properties of jojoba leaves. Plant
Physiol., 91: 1580-1585. Madore, M.A., 1990. Carbohydrate metabolism in photosynthetic and nonphotosynthetic tissues of variegated leaves of Coleus blumei Benth. Plant Physiol., 93: 617-622. Mitchell, D.E. and Madore, M.A., 1992. Patterns of assimilate production and translocation in muskmelon (Cucunds melo L.). II. Low temperature effects. Plant Physiol., 99: 966-971. Naqvi, H.H., Goldstein, G., Ratanyaka, C., Ceccardi, T. and Ting, II?, 1988. Jojoba breeding and agronomic investigations at UC Riverside. Proc. 7th Int. Conf. on Jojoba and its Uses. Jan. 17-22, 1988, Phoenix, AZ, pp. 395-409. Nelson, J.M. and Palzkill, D.A., 1990. Water stress for preventing jojoba yield-loss due to frost damage. Jojoba Happenings, 19: l-2. Ogren, E. and Baker, N.R., 1985. Evaluation of a technique for the measurement of chlorophyll fluorescence from leaves exposed to continuous white light. Plant Cell Environ., 8: 539-547. Paul, M.J., Driscoll, S.I? and Lawlor, D.W., 1991. Sinkregulation of photosynthesis in relation to temperature in sunflower and rape. J. Exp. Bot., 43: 147-153. Sakai, A. and Larcher, W., 1987. Frost Survival of Plants. Responses and Adaptation to Freezing Stress. Springer-Verlag. Berlin Heidelberg. Schreiber. U. and Bilger, W., 1987. Rapid assessment of stress effects on plant leaves by chlorophyll fluorescence measurements. In: J.D. Tenhunen (Editor), Plant Response to Stress, Vol. G15. Springer-Verlag, Berlin Heidelberg, pp. 27-53. Steffen, K.L. and Palta, J.P., 1987. Photosynthesis as a key process in plant response to low temperature: alteration during low temperature acclimation and impairment during incipient freeze-thaw injury. In: PH. Li (Editor), Plant Cold Hardiness. Alan R. Liss, Inc., NY, pp. 67-99. Stewart, G.R. and Larher, F., 1980. Accumulation of amino acids and related compounds in relation to environmental stress. In: B.J. Miflin (Editor), The Biochemistry of Plants, Vol 5. Acad. Press, NY pp. 609-635. Thompson, J.F., 1980. Arginine synthesis, proline synthesis, and related processes. In: B.J. Miflin (Editor), The Biochemistry of Plants, Vol. 5. Acad. Press, NY, pp. 375-402. Wardlaw, I.F., Begg, J.E., Bagnall, D. and Dunstone, R.L., 1983. Jojoba: temperature adaptation as expressed in growth and leaf function. Aust. J. Plant Physiol., 10: 299-312. Yelenosky, G., 1979. Accumulation of free proline in citrus leaves during cold hardening of young trees in controlled temperature regimes. Plant Physiol., 64: 425427. Yelenosky, G. and Guy, C.L., 1989. Freezing tolerance of citrus, spinach, and petunia leaf tissue. Osmotic adjustment and sensitivity to freeze induced cellular dehydration. Plant Physiol., 89: 444-451. Young, R. and Peynado, A., 1964. Changes in cold hardiness and certain physiological factors of red blush grapefruit seedlings as affected by exposure to artificial hardening temperatures. Proc. Am. Sot. Hortic. Sci., 86: 244-252.
JOURNAL
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Effect of temperature and water stresses on gas exchange, fluorescence kinetics, and solute levels of jojoba Toni L. Ceccardi a**,Irwin F! Ting b a University of Florida, IFAS, CitrusResearchand EducationCenter;L&e Alfred, FL, USA b Department of Botany and Plant Sciences, University of California, Riverside, CA, USA Recieved 17 January 1996; accepted 29 May 1996
Abstract
Seven year old jojoba, Simmondsia chinensis (Link) Schneid., plants grown under three irrigation treatments, wellwatered, intermediate, and dry, were exposed to controlled temperature treatments of +5, -5, and -10°C for six hours during February 1991. Following the temperature treatments, gas exchange and fluorescence kinetics were used to evaluate
the degree of injury to the plants. Following subfreezing temperatures, gas exchange was depressed, and fluorescence kinetics indicated greater damage to well-watered plants. Amino acid and carbohydrate levels, and osmotic potentials were measured before and after cold treatments to observe the effect of low temperatures on solutes. Freezing temperatures resulted in increased solute concentrations attributed to increases in total carbohydrates and total amino acids. Proline, arginine, aspartate family amino acids, glucose, and fructose increased while phenylalanine, tyrosine/ornithine, glutamate and sucrose decreased in response to freezing temperatures. Lathhouse plants exposed to -5°C natural frost for seven hours in December 1990 set seed, but, with the exception of dry treatment plants exposed to -5”C, plants exposed to either -5 or - 10°C in the laboratory in February 1991 did not set seed. Overall, the intermediate irrigation treatment had the least disruption of fluorescence kinetics and photosynthesis, and set the most seed following the controlled low temperature treatments. Keywords: Jojoba; Simmondsia chinensis; Freezing resistance; Drought; Fluorescence; Stress
1. Introduction Drought induced cold-hardening of jojoba is a common cultural practice in areas subject to subfreezing temperatures. Drought increases the supercooling capacity of jojoba by mechanisms which are not completely understood (Naqvi et al., 1988). Previous laboratory studies with jojoba showed increases in supercooling capacity of detached leaves and buds in response to decreased irrigation (Gold*Corresponding author.Fax:(941)956-4631.
stein et al., unpubl.). Since detached organs can supercool to much lower temperatures than intact plants (Sakai and Larcher, 1987) it was of interest to measure the frost-resistance and recovery of intact plants preconditioned with water stress. Physiological factors related to water status influence the capacity of a plant to supercool. A decrease in relative water content and the accumulation of solutes depress the freezing point of the cell sap (Alberdi and Corcuera, 1991; Sakai and Larcher, 1987), which in turn lowers the supercooling point. Furthermore, cold acclimation, as well as water stress,
0926-6690/96/$15.00 Copyright 0 19% Elsexier Science B.V. All rights reserved. PIISO926-6690(96)000026-X
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may induce an increase in solute concentration and a decrease in water content which may account for up to 50% of the decrease in freezing point (Yelenosky, 1979; Yelenosky and Guy, 1989; Young and Peynado, 1964). Drought and cold acclimation may have an additive influence on the supercooling point depression (Cox and Levitt, 1976), or they may work independently resulting in the same end (Chen et al., 1977). In the present study, the frost-hardiness of intact jojoba plants under three irrigation treatments was measured. Gas exchange and variable fluorescence were used as indicators of injury following cold treatments. In addition, low-temperatureinduced fluctuations of free amino acids and carbohydrates were measured in plants of the three irrigation treatments and compared to those levels found in plants under ambient winter temperatures. These parameters were compared before and after cold treatments and frost resistance was compared between irrigation treatments. 2. Materials and methods 2.1. Plant material
In the fall of 1990, six jojoba plants of the selection ‘Mirov’ from each of three irrigation treatments were transferred from a greenhouse to a latbhouse for acclimation to cooling nighttime temperatures. The seven year old plants had been transplanted from a field plot to pots (7.6 1 in UC&mix soil, a sandy loam) in a greenhouse in 1988 and maintained on three irrigation treatments. Prior to their transfer to the lathhouse, they were repotted into 19-1 pots of UC&mix soil. After the two years on the various irrigation treatments their sizes, measured as the volume of a sphere, ranged from dry treatment plants of approximately 0.30 m3 to wet treatment plants of 1.0 m3 (Ceccardi et al., 1990). The inigation treatments were maintained by hand-watering until water flowed from the bottom of the pots as follows: wet - watered every three days; intermediate - watered every four days; dry - watered every five days. On a monthly basis, the plants were fertilized by irrigation with a complete nutrient solution.
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2.2. Controlled freezing study
Between February 4-6, 1991, three plants from each irrigation treatment were randomly selected to undergo one of three freezing treatments. Beginning at 10 pm, whole plants were placed into a chest freezer (Kenmore 23, Sears Roebuck and Co., Chicago, IL) with an adjustable thermostat and subjected to temperatures of either 5°C (control), -5°C or -10°C for six hours. Cooling of the roots was avoided by wrapping the pots with a heating pad set on low and by covering the soil with Styrofoam pieces. Thirty-six gauge copper-constantan thermocouples were attached to two leaves with tape and to a cold junction compensator (Omega Engineering Inc., Stamford, CT) and a chart recorder, by which the mV outputs were recorded. The system was too insensitive to detect exotberms, but it monitored the freezer temperature. The plants were placed into the freezer at room temperature, and the thermostat was turned down to the target temperature. Cooling at a rate of 15°C h-l, the freezer decreased from room temperature to -10°C in about 2 h. A fan was used to circulate the air inside the freezer. After approximately eight hours (two hours cooling, six at set temperature), the thermostat was set to room temperature and the lid was propped open slightly. Two hours later, the plants were removed from the freezer. In addition to the controlled freezes, all lathhouse plants were exposed to air temperatures of -5°C on December 23, 1990 for a duration of approximately seven hours. Two weeks later, necrosis and chlorosis of plant tissues were compared between the irrigation treatments. 2.3. Physiological analyses 2.3.1. Gas exchange
Following the temperature treatments, gas exchange was measured using a portable photosynthesis system (Analytical Development Co., Ltd., Hoddesdon, UK) approximately every two hours until dusk (8 am to 6 pm) and once the next day at midday. The first fully expanded leaves (‘mature’) were chosen for measurements. Undamaged leaves were selected unless it was not possible, such as with the well-watered treatment following -WC, in which case all leaves appeared to have undergone a freeze-thaw.
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2.3.2. Fluorescence
Young (first or second node, not fully expanded) and mature (third or fourth node, fully expanded) leaf samples were collected approximately every 2 h after the plants were removed from cold for measurement of fluorescence kinetics. The leaves were dark adapted at room temperature for thirty minutes before modulated fluorescence was measured. The measurement system was described by Ogren and Baker (1985). The instrument (Hansatech Ltd., NuNorfolk, UK) consisted of a leaf chamber, detection probe, LED probe with a low level yellow light (1.5 pm01 me2 s -’ ) to detect background fluorescence, higher level light from a projector (150 pmol mm2 s-i) and highest level light for saturating pulses. The highest level light (1200 pmol me2 s-‘) was connected to a camera shutter and timer to give 0.2 or 0.3 s pulses of saturating light every 30 s. The outputs were recorded on a chart recorder. Photochemical quenching (qQ), maximum fluorescence (F,), variable fluorescence (F,), and F,l F, (ratio of variable fluorescence to maximum) were measured and calculated based on the modulated fluorescence kinetics according to Schreiber and Bilger (1987). 2.3.3. HPLC sample preparation Young and mature leaf samples were collected immediately before the plants were placed into the freezer, immediately after, and 12 h after the cold treatments. The samples were placed on dry ice in plastic bags and later stored at -80°C until they were analyzed. Statistical analyses of carbohydrates and amino acids were performed using Minitab analysis of variance (Minitab Inc., State College, PA: function, glm). Samples of approximately 10 mg leaf tissue from 2 leaves were used to extract carbohydrates and amino acids. The fresh weight was recorded and the samples were extracted in 80% ethanol until bleached. The leaf extracts were dried by vacuum centrifugation (Savant Instruments, Farmington, NY) and resuspended in 1 ml distilled water. The aqueous samples were partitioned into three fractions with a series of l-ml ion exchange columns (Bio-Rad Laboratories, Richmond, CA). Each sample was washed through the cohmms with a total of 15 ml of distilled water as described by Mitchell and Madore (1992). The first column retained amino acids using
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cation exchange resin (Bio-Rad AG 5Ow-X8. 50100 mesh, Hydrogen form). A second column of anion exchange resin (Bio-Rad AG l-X8 200-400 mesh, Formate form) retained organic acids, and the final, neutral fraction contained carbohydrates. 2.3.4. Amino acid determinations The amino acid fractions were eluted with 5 ml 4N ammonium hydroxide (NHaOH). Amino acid samples were dried and prepared for HPLC analysis as described by Madore (1990). The procedure included converting the amino acids to their phenylisothiocyanate (PITC) derivatives and resuspending in buffer (15 mM sodium acetate, 3% (v/ v) acetonitrile, and 0.025% (v/v) triethylamine, adjusted to pH 7.4 with phosphoric acid). The sample was filtered on a syringe filter and 20 ~1 were injected onto a Rainin Dynamax ODS column (4.6 x 25 mm) held at 48°C. Separation of the PITCamino acids was performed as described by Mitchell and Madore (1992). Samples were compared for differences due to irrigation treatment, temperature treatment, leaf age, and time of collection. 2.3.5. Carbohydrate determinations The neutral carbohydrate fractions were dried by vacuum centrifugation (Savant Instruments, Farmington, NY), resuspended in 100 ~1 water, and 20-~1 samples were analyzed by HPLC as described by Madore (1990), using a Waters SugarPak 1 column held at 90°C. Comparisons of the carbohydrate compositions of the three irrigation treatments before and after cold treatments were made. 2.3.6. Osmotic potentials Osmotic potentials were measured using the same leaf samples frozen for HPLC analyses. The leaf sap osmotic potentials were measured with a thermocouple psychrometer (Model 51OOC Wescor, Inc., Logan, UT). 2.4. Harvest In the summer of 1991, seeds were harvested to compare quantities of seed produced between irrigation treatments exposed either to just the December natural freeze or the additional February controlled freezes. Numbers of fully developed seeds
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and aborted seeds per plant were counted and weighed. No designation was made between unfertilized aborted seed or aborted seed due to frost injury, with the assumption that the ratio of unfertilized seed would be equivalent between the irrigation and temperature treatments. 3. Results 3.1. Gas exchange Midday peak values of photosynthesis and stomatal conductance following each of the three controlled cold treatments are shown in Table 1. Values shown in the first row in each irrigation treatment are from photosynthetic measurements taken on a day in January, not following any cold stress, for comparison. Compared to the control, only the wet treatment plant showed any photosynthetic depression following the 5°C treatment at 66% of that at ambient temperatures. With one exception, following the -5°C and - 10°C cold treatments, photosynthesis declined so that the plants were evolving CO,. The exception was the intermediate irrigation treatment which maintained low levels of photosynthesis following the -5°C treatment. After 24 h of recovery,
all of the irrigation treatments had higher photosynthesis levels, except that of the wet and intermediate irrigation treatments following the - 10°C treatment. 3.2. Fluorescence Table 2 shows fluorescence from midday on the day following the cold treatments. The intermediate and dry treatments showed practically no maximum fluorescence (F,,,) decline while the wet treatment had a 45% and 75% decrease following the -5°C and -10°C treatments, respectively (Table 2). Photocemical quenching, qQ, decreased by 58% and 75% in the wet treatment; by 16% and 35% in the intermediate; and 33% and 43% in the dry treatment after -5 and -10°C temperatures, respectively. The ratio of variable fluorescence to maximum fluorescence, F,IF,, decreased after -5°C in the wet treatment. It increased or remained approximately unchanged in the other treatments. 3.3. Amino acids Amino acid levels fluctuated in response to temperature and irrigation treatments, the time of sample collection, and the age of the tissue (Table 3). The
Table 1 Gas exchange aof jojoba under three irrigation treatments following low temperature treatments Treatment
Photosynthesis (pm01 COzmS2 s-t)
Conductance (mol mm2 s-l)
day 1
day 1
day 2
Wet Control +5”c -5°C -10°C
15.15 f 7.20 f -0.20 f -1.45 f
8.38 0.53 0.45 0.48
1.14 -2.05 f 0.18
Intermediate Control +5”c -5°C -10°C
4.41 4.34 2.0 -0.35
2.21 0.59 1.36 0.408
na 2.4 f 1.82 ‘deceased’
DrY Control +5”C -5°C -10°C
6.14 6.53 -0.18 -0.607
f2.74 f 1.20 f 0.85 f 0.108
na 1.20 f 1.55 -0.36 f 0.027
f f f f
&Sk
a Gas exchange measured with an ADC portable photosynthesis system. Values are the means and standard deviations of 4 measurements.
0.208 0.13 0.105 -0.94 0.097 0.085 0.08 0.090
day 2 f 0.057 f0.0047 f 0.015 f 0.019
:a75 f0.37 0.034 f 0.0033
f f f f
ia11 f0.015 ‘deceased’
0.0508 f 0.090 f 0.12 f 0.066 f
0.016 0.0086 0.015 0.028 0.016 0.010 0.0073 0.012
BY05 f 0.010 0.048 f 0.0068
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Table 2 Fluoresence parameters calculated following temperature treatments Irrigation
Temperature (“c)
ElIa (% Control)
9Qa (% Control)
&IF,
Wet
+5 -5 -10
100 f41.0 58.6 f 44.6 23.2 f 26.4
100 f0.8 60.3 f 22.1 24.9 f 1.9
0.095 f 0.038 0.034 f 0.048 1.57 f 1.31
Intermediate
+5 -5 -10
100 f0.7 120.4 f 51.9 81.5 f 10.7
100 f 1.6 74.6 f 23.5 66.7 f 16.3
0.082 f 0.070 0.091 f 0.13 0.18 f 0.079
Dry
+5 -5 -10
100 f65.8 71.6 k 67.9 112.3 f 24.2
100 f2.0 66.0 f 28.5 57.8 f 24.8
0.14 f0.051 0.18 f 0.074 0.20 f0.17
a Fluorescence values for 5°C were used as controls. Values are the means and standard deviations of 2 measurements.
pretreatment values differed between the mature and young leaves for most of the amino acids (totals, Table 3). Total amino acids were highest in the dry and wet irrigation treatments with a high degree of significance (P = 0.005). Also, total amino acids were generally higher in the young leaves (P = 0.02, Table 3). Total amino acids tended to increase after the cold treatments (P c 0.001, Table 3). Proline was significantly higher in the young leaves than mature (all treatments combined: young: 10.8f7.0; mature: 8.5f7.0 pmol/gfw, P = 0.025). Levels of proline made up the majority of total amino acids and were highest in the dry treatment, followed by the wet treatment (P = 0.01). Proline also increased in the wet and intermediate treatments after cold treatments, and decreased in the dry plants (Fig. 1). Similarly to proline, arginine levels were significantly higher in the young leaves (young: 0.51 f 0.04; mature: 0.21 f 0.04 pmol/gfw, P -c 0.001). Levels of arginine were highest in the wet treatment, followed by dry (P = 0.03). In the dry treatment, arginine decreased after the cold treatments, but increased in the wet and intermediate treatments (Fig. 2a). The pretreatment levels of the aspartate family (selected members: lysine, threonine, valine, isoleucine, leucine) were higher in the young leaves (young: 0.88 f 0.08; mature: 0.77 f 0.08 pmol/gfw, n.s.) with the exception of isoleucine. On average for all samples, aspartate family amino acids were highest in the wet treatment, descending to the lowest
Proline
II
181
I
II+ I Wet
2’l
+5
I -5
B
-10
Temperature ( C)
p=.oo Fig. 1. Proline levels following three temperature treatments in pmollgfw. Values are averages of young and mature leaves directly after cold treatments and 12 h later. The P-value shows the significance of the interaction of irrigation and temperature treatments on proline levels.
levels in the dry treatment (not significant). The increased concentration seen in this amino acid family after the subfreezing treatments, compared to the 5°C levels was highly significant (P < 0.001, Fig. 2b). The concentration of glutamate was highest in the mature leaves (young: 1.4 f 0.1, mature: 2.0 f 0.1 hmol/gfw, P < O.OOl),and declined after cold treatments (P < 0.001) . Glutamate was highest in the dry treatment and lowest in the intermediate (Fig. 2~). Levels of phenylalanine, tyrosine/omithine followed the same pattern as that of glutamate, being
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Table 3 Total amino acids and total carbohydrates for young and mature leaves of each irrigation treatment before (pre), directly after (post), and 12 h (+12 h) following three low temperature treatments Total carbohydrates (mg gfw-‘)
Total amino acids (pmol gfw-‘) Weta
Young a +5”c a -5°C a -lo”ca Mature a +5”c -5°C -10°C
Intermediate
Young +5”c -5°C -10°C Mature +5”c -5°C -10°C
Dry
Young +5”c -5°C -10°C Mature +5”c -5°C -10°C
Prea Post a +12 ha Post +12 h Post +12 h Pre Post +12 h Post +12 h Post +12 h
34.6f 7.0 33.9f 11 19.7 f 2.6 33.8 f 1.4 15.5 f 1.4 10.9 f 4.3 17.8 f 0.9 13.0f 5.7 19.1 f 1.4 15.9 f 0.9 30.0 f 4.4
Pre Post +12 h Post +12 h Post +12 h Pre Post +12 h Post +12 h Post +12 h
14.0 f 12.9 f 6.0f 15.9 f 28.4f 13.9f 20.7 f 12.4f 12.5 f 12.5 f 15.9 f 17.4 f 15.5 f 32.9 It
Pre Post +12 h Post +12 h Post +12 h Pre Post +12 h Post +12 h Post +12 h
22.4 f 1.7 23.4 f 5.6 31.8f 9.0 30.2f 5.5 26.5 f 1.5 14.4f 4.2 21.4f 11 17.6f 2.5 21.2f 0.9 27.7 f 2.4 22.9f 5.3 16.4f 4.6 13.6f 2.1 25.1 f 6.0
21.1 f 11.8f 18.43~
2.4 1.6 4.6
3.7 4.7 0.3 6.1 0.8 3.1 8.3 2.0 4.5 1.9 0.8 7.2 4.6 16
Weta
Young b +5”C a -5°C a -1o”ca Mature b +5”C -5°C -10°C
Intermediate
Young +5”C -5°C -10°C Mature +5”C -5°C -10°C
DrY
Young +5”C -5°C -10°C Mature +5”C -5°C -10°C
Preb
Postb +I2 hb Post +12 h Post +12 h Pre Post +12 h Post +12 h Post +12 h
207.9 f 188.9 f 220.6f 186.9f 256.7 f 196.9 f 191.5 f 175.9 f 186.7 f 151.5 f 188.7 f 154.1 f 184.4 f 373.6 f
24.2 18.7 3.6 3.8 28.2 10.7 11.9 24.5 0.2 23.4 44.2 55.8 10.3 137
Pre Post +12 h Post +12 h Post +12 h Pre Post +12 h Post +12 h Post +12 h
151.1 f 36.7 187.1 f 26.9 101.2f 6.5 191.7 f 24.8 159.1 f 2.7 206.6 f 22.2 207.7 f 15.4 95.5 f 5.3 74.9 f 22.7 122.3 f 1.10 112.2f0.19 114.1 f 15.4 198.5 f 0.90 310.7 f 67.0
Pre Post +12 h Post +12 h Post +12 h Pre Post +12 h Post +12 h Post +12 h
164.0 f 6.26 147.2 f 35.1 244.0 f 88.6 133.1 f 34.3 136.3 f 16.1 173.6 f 66.0 157.6 f 55.0 131.0f4.99 112.6 f 26.8 228.8 f 2.09 163.6 f 16.9 143.4 f 11.7 98.4 f 18.8 145.8 f 3.03
ab Levels of significance due to various treatments are shown in the wet treatment rows and apply to all treatments in that column. Significance was calculated by Anova (glm, Minitab). a Significance 5 0.05. b Significance 5 0.10. Significant interactions include irrigation x temperatureBVb; irrigation x age a, ns.; temperature x ageBsb, for amino acids and carbohydrates respectively. Each value represents the mean and standard deviation of two measurements.
T.L. Ceccanii, 1.P lining/Industrial Crops and Products 5 (1996) 279-290 Arginine
285
Aspartate family
0.7
1.4
1
r
Wet 4 Int + Dry *
0.6 -
0.2 -.-__.--------
0.1
1A +5
-5 Temperature
0.21’ B +5 ( C)
p=.o5
-5 Temperature ( C)
p=.13
Phenylalanine, Tyrosine/Ornithine 0.3
3. Wet *
2.5
Int
Int
2+___ AL
CZy *
------I
1.5 -
1 /c +5
p=.of3
0.2
;y *
aI 0.15 0.1 -
1 0.5
Wet *
0.25 -
-5 Temperature ( C)
-10
I-I
0.05 +5
-5 Temperature ( C)
-10
p=.oz
Fig. 2. Amino acid levels following three temperature treatments of (a) arginine, (b) aspartate family selected members (lysine, threonine, valine, isoleucin, leucine), (c) glutamate, and (d) phenylalanine, tyrosinelomithine, in Kmollgfw. Values are averages of young and mature leaves at two times following- temperature treatments. The P-values show the significance of the interaction of irrigation and _ temperature treatments on amino acid levels.
highest in mature leaves (young: 0.15 f 0.01, mature: 0.19f0.01 ,xmollgfw, P = 0.02) and declining after cold treatments (P <: 0.001). The decline was greater following -5 than -10°C (Fig. 2d). Levels were highest in the intermediate treatment and lowest in the wet treatment. The amino acid group consisting of glycine and serine showed no significant response to either irrigation or cold treatment (data not shown). In general, they were highest in the young leaves and in the wet irrigation treatment, lowest in intermediate. 3.4. Carbohydrates Total carbohydrates were highest in the wet and lowest in the dry plants (P = 0.001, Table 3). Young leaves had higher levels than mature leaves (Ta-
ble 3). Carbohydrates increased after cold treatments in the wet and intermediate treatments, while they decreased in the dry treatment (Table 3). Following the -10°C treatment the total carbohydrate levels were higher in the mature leaves, in those of the wet and intermediate irrigation treatments (Table 3). Glucose levels were significantly higher in young leaves (all treatments combined: young: 155 f 7, mature: 132 k 7 mg/gfw, P = 0.02). Overall, the levels of glucose were highest in wet and lowest in dry plants (P = 0.001). Glucose levels increased after the cold treatments in wet and intermediate plants, but decreased in dry plants, similarly to total carbohydrates (Fig. 3a). The levels of glucose were much higher than any other sugar, and were probably responsible for changes seen in total carbohydrates (up to 90%).
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286
Crops and Products 5 (1996) 279-290
Glucose 220
significant. Sucrose increased after the -5°C treatment, but there was no change in the level following -10°C compared to +5”C. The overall trend was toward increased levels after cold treatments, except in young leaves treated with -10°C of the wet and intermediate treatments in which there was a 2-fold decrease (not shown). The dry plants following - 10°C had levels approximately equal to or less than +S’C (Fig. 3b) with no difference in mature and young leaves (not shown). Fructose levels were significantly higher in the wet irrigation treatment than intermediate and dry, prior to cold treatments (not shown). After cold treatments, levels were highest in the dry plants (P=O.O3, Fig. 3~). Additionally, the levels were higher in the young leaves (young: 3.7 f 0.4, mature: 1.5 f 0.4 mg/gfw, P c 0.001). Fructose tended to increase after -5 and - lO“C, except in wet treatment, in which it stayed approximately equal to the 5°C treatment (Fig. 3c).
I
sot4 A +5
-5 Temperature (“c)
p=.oo Sucrose
3.5. Osmotic potential 12” B +5
I -5 Temperature (%)
pz.72
1 Wet 4 Int + Dry *
-5
-10
Temperature (“c) p=.o3
Fig. 3. Carbohydrate levels following three temperature treatments of (a) glucose, (b) sucrose, and (c) fructose, in mglgfw. Values are averages of young and mature leaves at two times following temperature treatments. The P-values show the significance of the interaction of irrigation and temperature treatments on carbohydrate levels.
Sucrose levels were higher in mature leaves (young: 14 f 1.3, mature: 18 f 1.3 mg/gfw, P = 0.02), and the differences due to irrigation were not
The pretreatment values of osmotic potential were consistently lowest in the dry plants, and in general, highest in the wet plants. The osmotic potential tended to decrease after low temperature treatments, with the lowest water potentials following -10°C (Table 4). 3.6. Harvest 1991 llvo weeks after a natural frost in December, 1990, the wet plants had necrotic, yellow leaf tips, and brown flower buds. The dry treatment plants had little visible damage to either leaves or buds. The intermediate plants had less injury than the wet plants. The results of the 1991 summer harvest are shown in Table 5. None of the plants exposed to -10°C in the laboratory in February produced seed, neither did plants of the intermediate and wet treatments following the -5°C cold treatment. The dry irrigation treatment exposed to -5°C in the controlled study did set seed. In contrast, all of the plants under ambient conditions were exposed to a low temperature of -5°C for approximately seven hours in December, but still set seed.
LL. Ceccanii, I.R Lting/Industrial Crops and Products 5 (1996) 279-290 Table 4 Osmotic potentials of jojoba under three irrigation treatments before and after low temperature treatments a Pretreatment $x (MPa) Wet irrigation -3.3 f 1.0
Intermediate irrigation -3.7 f 0.25
Dry irrigation -4.2 f 0.3
Temperature (“C)
Posttreatment & (MPa)
+w -5°C -10°C
-3.9 f 0.01
+YC -5°C -10°C
-5.5 f 0.01 -3.6 f 0.80 -4.6 f 0.01
+5”c -5°C -10°C
-2.2 f 0.39 -3.6 f 0.53 -5.0 f 0.66
-Y9*0.6
aValuesare the means and standard deviations of 9 (pretreatment) or 3 osmometer readings. Overall, the intermediate treatment set the greatest mass of seed, followed by dry and then wet (Table 5). 4. Discussion The decreased photosynthesis and stoma&l conductance seen after the cold treatments in jojoba is an indication of injury. The negative values of photosynthesis could be due to photorespiration or photoinhibition. Loreto and Bongi (1989) found in jojoba, that following low temperatures, in the light, recovery to the initial rates of photosynthesis took 24 h to one week and they concluded that the delay was due to photoinhibition. In the present study, the dry and intermediate treatment plants showed some recovery of their gas exchange rates 24 h after removal from the -5°C treatment. Recovery from freezing injury requires energy either from stored reserves or from photosynthesis (Steffen and Palta, 1987), and therefore total recovery of the plants depends on the early recovery of photosynthetic capacity. Overall, the intermediate plants had the least photosynthetic depression after the subfreezing treatments which may indicate that their photosynthetic apparatus was more cold resistant than the wet and dry treatments. Following the - 10°C treatment, photosynthesis did not recover even after 2 days, but respiration still occurred, as seen by the output of CO2 (Ta-
287
ble 1). This may be evidence that photosynthesis is more sensitive to freeze-thaw injury than respiration (Steffen and Palta, 1987) in jojoba. Chlorophyll fluorescence is a good indicator of photosynthetic activity and can be used to monitor damage to the photosynthetic apparatus (Baker and Horton, 1987). Fluorescence parameters indicate plant injury with declining F,,,, qQ and FJF, such as in the case of photoinhibition (Franklin et al., 1992). Fluorescence measurements of jojoba leaves decreased more in the wet treatment than in either of the low-irrigation treatments (Table 2). F, showed practically no decline in the intermediate and dry treatments, possibly due to drought-induced acclimation causing structural changes in lipid and membrane compositions which may protect the photosynthetic apparatus (Guy, 1990). Photochemical quenching (qQ) also showed less depression following cold in the dry and intermediate treatments. The intermediate treatment had the highest qQ activities after both the subfreezing treatments, which correlates with the greater capacity for gas exchange recovery seen in that treatment. Likewise, in the dry and intermediate treatments, the negligible change in FJF,,, again indicates hardiness of the photosynthetic apparatus. In the wet treatment, the F,IF, value decreased following the -5°C treatment; then in a result which may appear surprising, it increased about 600% after the -10°C treatment. The maximum fluorescence peak decreased below the height of variable fluorescence peaks, which remained high due to an increase in Fo, baseline fluorescence. High F. indicates inactivation of PSI1 (Baker and Horton, 1987), and in this case the resulting high FJF,,, indicated tissue death. The total amino acid levels were higher in the dry treatment due to osmotic adjustment, measured by the accumulation of compatible solutes. Interestingly, the intermediate treatment had less solute accumulation than the wet treatment. Because jojoba is adapted for growth in dry environments, and does not increase biomass production as much as other Cs crops when given excess water (Wardlaw et al., 1983), the well-watered plants may have been stressed due to over-watering. Therefore, total amino acids were found in higher levels than in the intermediate plants, which appeared to be the least stressed (Ceccardi et al., 1990).
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Crops and Products 5 (1996) 279-290
Table 5 Harvest data from plants of three irrigation treatments and various cold treatments Irrigation
Temperature (“C)
Wet
ambient ambient ambient +5 -5 -10 total
Intermediate
Fruit (number)
Seed weight (8)
Aborts a (number)
Fruit set (%)
46 31 60 54 0 0
36.64 21.38 38.85 33.7 0 0 130.57
63 37 62 98 121 21
36.5 45.6 49.2 35.5 0 0
ambient ambient ambient +5 -5 -10 total
42 73 115 205 0 0
31.97 51.78 73.15 77.46 0 0 234.36
233 110 136 300 125 24
15 40 46 40 0 0
ambient ambient ambient +5 -5 -10 total
45 74 91 2 81 0
27.2 44.07 61.84 0.915 47.07 0 181.10
70 104 167 18 152 28
39 42 35 10 35 0
a ‘Aborts refers to undeveloped seeds noted by an empty seed coat, which may include seeds damaged by frost or unpollinated flowers (see text). Plants under ‘ambient’ conditions underwent a natural frost of -5°C for 7 h on December 23, 1990. All other treatments were additional controlled treatments,
Proline, which was highest in the dry treatment and increased in the other treatments after cold temperatures, often accumulates during water stress and may protect thylakoid membranes against electrolyte damage occurring during freezing stress (Stewart and Larher, 1980). In cabbage, free proline levels correlated with freezing tolerance of different tissues in plants which were either acclimated to low temperatures or nonacclimated (Stewart and Larher, 1980). During poststress periods proline can be used as a source of carbon, nitrogen, or reductant, and may have for this reason accumulated in the wet and intermediate plants following the freezing treatments. Alternatively, proline may have been a desiccation product (Stewart and Larher, 1980). Because of the high initial levels of proline in the dry treatment, there was no additional increase following the cold treatments. The average proline levels for the three irrigation levels were: wet, 10.0; intermediate, 7.1; dry, 12.2 pmoll gfw. Proline was a major component of total amino acid levels, especially in the young leaves.
Glutamate is central to ammonium metabolism and is the immediate precursor of the amino group of most protein amino acids (Thompson, 1980). Not only does glutamate contribute the nitrogen atom, but also most of the carbon atoms, to proline and arginine synthesis (Thompson, 1980). Arginine may be another compatible solute produced in response to freezing-induced dehydration strain, and it can also be converted to proline without prior conversion to glutamate (Stewart and Larher, 1980). Therefore, glutamate probably decreased following the cold stress by conversion to proline and arginine. Arginine accumulation is consistent with its role as an osmoticum and its easy conversion to proline. Total carbohydrates and total amino acids in the stressed leaves underwent the same changes following cold treatments. First, levels of both constituents were generally higher in young leaves. Levels increased following cold treatments of -5 and - lO“C, using +5“C as the control, in wet and intermediate
T.L. Ceccardi, 1.P. ling/lndustrial
plants. In contrast, the levels decreased in dry plants following subfreezing treatments. Unlike amino acids, total carbohydrates did not accumulate in the dry plants, which had the lowest levels of carbohydrates. Well-watered plants had the highest rates of photosynthesis under ambient conditions and therefore had higher levels of phloem sugars. Following cold treatments, carbohydrate levels (based on fresh weight) may have accumulated in response to freezing dehydration or due to water loss through leaky cell membranes. Mitchell and Madore (1992) reported that, in muskmelon, chilling caused an increase in phloem sugars, including sucrose, fructose, and glucose. Sucrose transport, or production, was inhibited following -lO“C, indicated by a decrease in young, sink leaves (not shown). Glucose and fructose levels most likely began to increase as vacuolated sucrose was hydrolyzed into its hexose components. Mitchell and Madore (1992) found that sugar translocation was inhibited by low temperatures in muskmelon, causing accumulation of hexose sugars. Similarly, low temperatures imposed a sink limitation on sunflower and rape, resulting in the accumulation of sucrose (Paul et al., 1991). The timing of the freeze may be crucial in jojoba. Plants subjected to a -5°C cold night for seven hours in December, considered a harsh freeze in Riverside, still set seed. Those subjected to additional controlled freezes of -5°C in early February set no seed except in dry plants. It is possible that by February most of the plants had deacclimated. According to the National Weather Service (Ann. report of fruit-frost activities in the Corona-Riverside District, 1990-91 winter season), December was followed by a unseasonably mild January and a warm February. The freezing temperature of jojoba buds increases at later dates (Ceccardi, 1993). The rate of freezing may also have been a deciding factor in the survival of the cold-treated plants. A cooling rate of 15”C/h is much faster than in nature (usually no more than 3“C/h, from UC Riverside weather station data). Rapid rates of cooling may result in injury before metabolism adjusts. Osmotic potentials decreased, on average, following low temperature treatments, possibly due to the loss of water from the leaves following the lethal treatments of -5 and -10°C.
Crops and Products 5 (1996) 279-290
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The dry plant subjected to -5°C in the laboratory was better protected against freezing than the more irrigated treatments, probably due to its lower water content, resulting in seed production. Drought does not increase the length of time to break dormancy, so a longer dormancy would not be the reason for the added protection (Nelson and Palzkill, 1990). The intermediate treatment, which appeared to recover photosynthetic capacity more rapidly than either the dry or wet plants, set the most seed. An intermediate level of water stress seems to give the best level of frost protection without sacrificing yield. The low yield of the dry treatment demonstrates that there is a cut-off point where increased drought does not improve frost resistance and decreases yield. The wet treatment itself may be a stress imposed upon these plants which are better adapted to surviving in harsh, dry conditions, and are not well suited for high production under what would be ample water for a more typical Cs crop (Wardlaw et al., 1983). 5. Conclusion Photosynthesis measurements, including gas exchange and fluorescence kinetics are good indicators of freezing injury and recovery. In this study with jojoba, if photosynthesis had not recovered by the second day following the low temperature treatments, the above ground tissue did not survive. Using photochemical quenching as an indicator, levels below approximately 60% of ambient levels indicated an inability to recover. The differences in solute patterns and photosynthesis levels explain the differences in freezing resistance and productivity between the irrigation treatments. The dry treatment had the lowest osmotic potentials, the highest proline levels, and was the only plant to survive and set seed after -5°C but it resulted in slower growth and fewer seeds set than the intermediate or wet treatments. The intermediate treatment resulted in lower levels of proline than the wet or dry treatments, and intermediate plants had lower photosynthesis rates and lower levels of carbohydrates than the wet treatment plants. The moderate levels of growth and water stress of the intermediate plants resulted in a balance between drought-induced frost resistance and metabolism, leading to higher yields than in the more extreme treatments. The na-
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ZL. Ceccardi, IJ? ling/Industrial Cmps and Products 5 (1996) 279-290
ture of the intermediate treatment’s frost resistance contradicts Khalafalla’s (1987) report that more frost resistant lines of jojoba accumulated more soluble sugars, sucrose and proline. The well-watered plants had the highest osmotic potentials, levels of sugars, and rates of growth and photosynthesis. The more severe damage caused by low temperatures resulted in the lowest yields in the well-watered plants. Acknowledgements The authors thank Dr. Monica Madore and Michelle Gadus for their assistance in performing the HPLC analyses of carbohydrate and amino acid levels. References Alberdi, M. and Corcuera, L.J., 1991. Cold acclimation in plants. Phytochemistry, 30: 3117-3148. Baker, N.R. and Horton, P., 1987. Chlorophyll fluorescence during photoinhibition. In: D.J. Kyle, C.B. Osmond and C.J. Amtzen (Editors), Photoinhibition. Elsevier Science Publishers, Amsterdam, pp. 145-168. Ceccardi, T.L., 1993. The effects of drought on the frost resistance of Simmondsia chinensis. Ph.D. dissertation, University of California, Riverside, CA. Ceccardi, T.L., Naqvi, H.H. and Ting, I.P., 1990. Physiological responses of jojoba to drought and frost stress. In: Estilai, Naqvi and Ting (Editors), Proc. 1st Ann. Meeting Assoc. Adv. Ind. Crops. October 1990, Riverside, CA, 47-52. Chen. PM., Li, PH. and Burke, M.J., 1977. Induction of frost hardiness in stem cortical tissues of Comus stolonifera Michx. by water stress. I. Unfrozen water in cortical tissues and water status in plants and soil. Plant Phys., 59: 236-239. Cox, W. and Levitt, J., 1976. Interrelations between environmental factors and freezing resistance of cabbage leaves. Plant Physiol., 57: 553-555. Franklin, L.A., Levavasseur, G., Osmond, C.B., Henley, W.J. and Ramus, J., 1992. Two components of onset and recovery during photoinhibition of Ulva mtundata. Planta, 186: 399408. Guy, C.L., 1990. Cold acclimation and freezing stress tolerance: role of protein metabolism. Ann. Rev. Plant Physiol. Plant Mol. Biol., 41: 187-223. Khalafalla, MS., 1987. Frost tolerance studies on jojobaSimmondsia chinensis (Link) Schneider: Clonal variation, compositional relationship and effect of cultural practices. Ph.D.dissertation. University of Arizona, Tucson, AZ. Loreto, F. and Bongi, G., 1989. Combined low temperature-high light effects on gas exchange properties of jojoba leaves. Plant
Physiol., 91: 1580-1585. Madore, M.A., 1990. Carbohydrate metabolism in photosynthetic and nonphotosynthetic tissues of variegated leaves of Coleus blumei Benth. Plant Physiol., 93: 617-622. Mitchell, D.E. and Madore, M.A., 1992. Patterns of assimilate production and translocation in muskmelon (Cucunds melo L.). II. Low temperature effects. Plant Physiol., 99: 966-971. Naqvi, H.H., Goldstein, G., Ratanyaka, C., Ceccardi, T. and Ting, II?, 1988. Jojoba breeding and agronomic investigations at UC Riverside. Proc. 7th Int. Conf. on Jojoba and its Uses. Jan. 17-22, 1988, Phoenix, AZ, pp. 395-409. Nelson, J.M. and Palzkill, D.A., 1990. Water stress for preventing jojoba yield-loss due to frost damage. Jojoba Happenings, 19: l-2. Ogren, E. and Baker, N.R., 1985. Evaluation of a technique for the measurement of chlorophyll fluorescence from leaves exposed to continuous white light. Plant Cell Environ., 8: 539-547. Paul, M.J., Driscoll, S.I? and Lawlor, D.W., 1991. Sinkregulation of photosynthesis in relation to temperature in sunflower and rape. J. Exp. Bot., 43: 147-153. Sakai, A. and Larcher, W., 1987. Frost Survival of Plants. Responses and Adaptation to Freezing Stress. Springer-Verlag. Berlin Heidelberg. Schreiber. U. and Bilger, W., 1987. Rapid assessment of stress effects on plant leaves by chlorophyll fluorescence measurements. In: J.D. Tenhunen (Editor), Plant Response to Stress, Vol. G15. Springer-Verlag, Berlin Heidelberg, pp. 27-53. Steffen, K.L. and Palta, J.P., 1987. Photosynthesis as a key process in plant response to low temperature: alteration during low temperature acclimation and impairment during incipient freeze-thaw injury. In: PH. Li (Editor), Plant Cold Hardiness. Alan R. Liss, Inc., NY, pp. 67-99. Stewart, G.R. and Larher, F., 1980. Accumulation of amino acids and related compounds in relation to environmental stress. In: B.J. Miflin (Editor), The Biochemistry of Plants, Vol 5. Acad. Press, NY pp. 609-635. Thompson, J.F., 1980. Arginine synthesis, proline synthesis, and related processes. In: B.J. Miflin (Editor), The Biochemistry of Plants, Vol. 5. Acad. Press, NY, pp. 375-402. Wardlaw, I.F., Begg, J.E., Bagnall, D. and Dunstone, R.L., 1983. Jojoba: temperature adaptation as expressed in growth and leaf function. Aust. J. Plant Physiol., 10: 299-312. Yelenosky, G., 1979. Accumulation of free proline in citrus leaves during cold hardening of young trees in controlled temperature regimes. Plant Physiol., 64: 425427. Yelenosky, G. and Guy, C.L., 1989. Freezing tolerance of citrus, spinach, and petunia leaf tissue. Osmotic adjustment and sensitivity to freeze induced cellular dehydration. Plant Physiol., 89: 444-451. Young, R. and Peynado, A., 1964. Changes in cold hardiness and certain physiological factors of red blush grapefruit seedlings as affected by exposure to artificial hardening temperatures. Proc. Am. Sot. Hortic. Sci., 86: 244-252.