JXB Advance Access originally published online on February 13, 2004
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Journal of Experimental Botany, Vol. 55, No. 397, pp. 719-729, March 1, 2004
© 2004 Oxford University Press
Regulation of Growth, Development and Whole Organism Physiology |
Carbon and water relations for developing fruits of Opuntia ficus-indica (L.) Miller, including effects of drought and gibberellic acid
Received 23 May 2003; Accepted 16 December 2003
Department of Organismic Biology, Ecology, and Evolution, University of California, Los Angeles, CA 90095-1606, USA
* To whom correspondence should be addressed. Fax: +1 310 825 9433. E-mail: psnobel{at}biology.ucla.edu
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Abstract |
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Growth, gas exchange rates, and carbohydrate content were studied for developing fruits of the cultivated cactus Opuntia ficus-indica (L.) Miller, including effects of drought and exogenous gibberellic acid (GA3). Fruit development required 110 d from the time of bud differentiation to ripening at 80 d after anthesis, when the fruit mass averaged 67 g. Stomatal conductance and net CO2 uptake rates for fruits were higher during the night; they were maximal at 7 d before anthesis and decreased as development progressed. Fruits undergoing drought, imposed by detaching terminal stems bearing fruits, were 50% smaller than the control at 80 d after anthesis and did not ripen. Fruits injected with 2 ml of 500 ppm GA3 were 30% smaller than the control at 80 d after anthesis; they contained a large proportion of aborted seeds that produced a weak sink signal for dry mass accumulation. Gas exchange was higher at 21 d after anthesis for fruits treated with GA3. Total soluble sugars represented 40% of the fruit’s dry mass until 45 d after anthesis, when the sugar content rapidly increased, reaching 90% at 73 d after anthesis. Such an increase was not observed for fruits treated with GA3, and the sugar content for fruits undergoing drought remained low throughout development. Starch content increased for developing fruits of O. ficus-indica until 14 d after anthesis and, except for the fruits undergoing drought, decreased thereafter. Fruit development for O. ficus-indica is apparently regulated by water availability as well as hormonal signals originating both within and outside the fruit.
Key words: Abscisic acid, cactus pear, fruit development, fruit photosynthesis.
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Introduction |
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Fruit development requires a major investment of carbon and water (Heim et al., 1979; Galen et al., 1999; Taiz and Zeiger, 2002). Photosynthetic contributions by young green fruits to their daily carbon budget are considerable for various species, such as apple (Malus pumila; Jones, 1981), orange (Citrus sinensis; Moreshet and Green, 1980), peach (Prunus persica; Pavel and De Jong, 1993), and cactus pear (Opuntia ficus-indica; Nobel and De la Barrera, 2000), but the rest of the plant still supplies over 90% of the fruit daily carbon gain via the phloem (Ho et al., 1987; Nobel and De la Barrera, 2000). In addition, most, if not all, of the water can be supplied by the phloem, as suggested by the higher (less negative) water potentials observed for developing fruits compared with the adjacent stems of apple (Lang, 1990; Mills et al., 1997), avocado (Persea americana; Blanke and Whiley, 1995), tomato (Lycopersicon esculentum; Mingo et al., 2003), some tropical trees (Chapotin et al., 2003), and cactus pear (Nobel et al., 1994; Nobel and De la Barrera, 2000; De la Barrera and Nobel, 2004). Also, the hydraulic resistance for the xylem of various developing fruits is high (Lovisolo and Schubert, 1998; Nijsse et al., 2001; van Ieperen et al., 2003).
Opuntia ficus-indica is widely cultivated in arid and semi-arid regions worldwide, mainly for forage, but with increasing importance as a fruit crop (Barbera, 1995; Inglese et al., 2002). Its young fruits take up CO2 primarily at night for this CAM species at a rate equivalent to 35% of that for the underlying cladode, with the fruit contribution decreasing as development progresses (Inglese et al., 1994; Nobel and De la Barrera, 2000). Similarly, the water vapour conductance for young fruits of O. ficus-indica decreases as they ripen (Nerd and Nobel, 2000). The massive succulent stems of O. ficus-indica can buffer the plants against prolonged periods of drought by providing considerable quantities of water for fruit development, which, in turn, allows fruit production to occur without irrigation (Pimienta Barrios, 1990; Nerd and Mizrahi, 1995).
Water stress usually reduces fruit yield (Nerd and Mizrahi, 1995; Pessarakli, 1995; van Iersel et al., 1994), leading, in some cases, to the abscission of immature fruit (Pessarakli, 1995). By contrast, drought hastens ripening and seed maturation for tomato (García-Martínez and Hedden, 1997) and reduces fruit abscission for lychee trees (Litchi chinensis; Batten et al., 1994). Responses to drought are often hormonally mediated by the roots rather than by a change in the shoot water status (Davies and Zhang, 1991; Nobel and De la Barrera, 2002; Mingo et al., 2003), which can complicate the analysis of water limitations. Detached cladodes of O. ficus-indica provide a model system for studying drought responses without the chemical signalling from the roots; their net CO2 uptake rates are similar to those for cladodes on droughted plants (Raveh and Nobel, 1999) and their succulence allows them to remain alive for many months (Nobel, 1996; Nobel and Castañeda, 1998).
Gibberellins control fruit development in various ways and at different developmental stages. For O. ficus-indica, the endogenous level of gibberellins (including GA3) increases during flower development and becomes maximal during anthesis (Inglese et al., 1998). At this stage, GA3 inhibits the differentiation of new flower buds, as also does its injection into detached cladodes (Nobel, 1996). For many species, GA3 produced by developing seeds stimulates the growth and maturation of that fruit (Srivastava, 2002; Taiz and Zeiger, 2002). Moreover, treating emasculated flowers of O. ficus-indica or seedless grapes with GA3 leads to the development of normal-sized, though seedless, fruits (Pharis and King, 1985; García-Martínez and Hedden, 1997). However, treatment of flowers at or before anthesis with GA3 induces seed abortion for pea (Pisum sativum) and O. ficus-indica (Gil and Espinoza, 1980; García-Martínez and Hedden, 1997). The fruit pulp of O. ficus-indica originates from the funicle, which connects the seed to the ovary, indicating that fruit development depends on the presence of seeds (Pimienta Barrios, 1990). Yet the carbon and the water relations of fruits whose seed abortion is induced by gibberellin, compared with those having fertilized seeds, are not known during fruit development for O. ficus-indica.
The 24 h patterns of net CO2 uptake and water vapour conductance at various stages throughout fruit development were therefore studied in the field for O. ficus-indica to evaluate the effects of drought and GA3 on fruit development. The following two hypotheses were tested: (1) drought will negatively affect fruit development for this cactus—in particular, fruits will have a lower stomatal conductance, which will lead to lower rates of CO2 uptake and reduced growth; and (2) treatment of flowers with GA3 will induce seed abortion, arresting normal fruit development—in particular, fruits will be smaller and will ripen at a slower pace than the control, and rates of gas exchange will also be reduced by GA3. In addition, the contents of soluble sugars and starch during fruit development were determined to gain insight into carbohydrate polymer and sugar patterns for O. ficus-indica.
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Materials and methods |
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Plant material and treatments
Fruit development was studied for nine-year-old plants of Opuntia ficus-indica (L.) Miller (accession number 1279 of Texas A&M University, Kingsville, TX) approximately 2 m in height at the Agricultural Research Station, University of California, Riverside, CA, from April to August 2000 with supplemental data on fruit size from 2001 and 2002. Daily mean air temperature averaged 26.3±1.2 °C, ranging from 20.6 °C at 14 d before anthesis to 31.0 °C at 73 d after anthesis. The total daily photosynthetic photon flux (PPF, wavelengths of 400–700 nm; measured with a LI-188S integrating quantum sensor, Li-Cor, Lincoln, NE) averaged 57±2 mol m–2 d–1 on a horizontal surface. Fruits of O. ficus-indica are unilocular, polyspermic, fleshy berries (Pimienta Barrios, 1990; Nerd and Mizrahi, 1995). At the time of anthesis, 80 flowers growing on 16 terminal cladodes (five flowers per cladode, with all additional flowers removed) were marked for growth measurements (the ‘control’).
Gas exchange for detached cladodes of O. ficus-indica is similar to that of cladodes growing on droughted plants (Raveh and Nobel, 1999). Therefore, a drought treatment was imposed by detaching eight terminal cladodes, each one with five remaining flowers; the cladodes were placed on a table approximately 1 m above the ground to avoid herbivory by rodents. Also at the time of anthesis, five flowers on each of eight (attached) terminal cladodes were injected with 2 ml of a 500 ppm (w/w) gibberellic acid solution (GA3; Sigma, St Louis, MO). In all three cases (control, drought, and GA3-treated), cladodes were selected whose floral buds had differentiated at approximately the same time (within 2–3 d); anthesis occurred within 3–4 d of each other. On each sampling date, five fruits of the control and one of the experimental treatments were randomly selected from different cladodes for destructive analysis.
Length and diameter (cm) of 10 floral buds or the ensuing fruits (hereafter collectively referred to as fruits) were measured weekly with a vernier caliper (gradations of 0.02 mm) for the control and both experimental treatments (10 fruits under each condition); assuming that the fruits were prolate spheroids (Nobel and De la Barrera, 2000), their surface areas were calculated from 0.5
d2+0.5dl[sin–1(1–d2/l2)0.5]/(1–d2/l2)0.5, where d (cm) is the diameter at mid-fruit and l (cm) is the fruit length. Five fruits from the control condition and five from one of the two experimental treatments were collected every 2 weeks. Fruit fresh mass (g) was recorded before removing a sample for carbohydrate determination. Fruits were then dried in a forced-draught oven at 80 °C until no further mass changes occurred, usually within 48 h, to determine dry mass (g). Regression curves for length versus fresh mass and versus dry mass were constructed based on all fruits harvested every 2 weeks; these curves were used to determine fresh and dry mass non-destructively for fruits developing in the field. Gas exchange was measured in the field during the intervening weeks.
Gas exchange
Fruit transpiration (mmol m–2 s–1), fruit surface temperature (°C), air temperature (°C), and relative humidity (%) were measured every 2 h over 24 h periods on alternate weeks with a Li-Cor LI-1600 steady-state porometer for which the acrylic top had been removed to allow a tight seal with the fruits using the existing closed-pore foam gasket; water vapour conductance (mmol m–2 s–1) was calculated by dividing the transpiration rate by the difference in water vapour mole fraction between the fruit, assumed to be at water vapour saturation, and the air (Nobel, 1999). Concomitantly, net CO2 uptake (µmol m–2 s–1) was measured with a Li-Cor LI-6200 portable photosynthesis system using a 0.25 l cuvette whose lid had been replaced with an acrylic extension with a square opening 1 cm2 in area that was fitted with closed-pore foam gasket to ensure an air-tight seal when placed in contact with the fruits. Such gas exchange measurements, which were performed on the same fruits throughout their development, were done during clear days, on east-facing unshaded fruits, and avoiding the areoles.
Carbohydrate determination
Tissue samples were obtained with a cork borer 14 mm in diameter, weighed, and ground in 8 ml of boiling ethanol (80% w/w) in a mortar. The fine slurry was transferred to a centrifuge tube together with an additional 6 ml of boiling ethanol used to rinse the mortar and pestle. After centrifuging for 15 min at 1500 g, the content of total soluble sugars in the supernatant fluid was analysed with a colorimetric phenol method (Sturgeon, 1990). The pellet obtained after centrifugation was dried at 40 °C for 24 h and then washed three times with methanol:chloroform:water 12:5:3 by vol. and twice with distilled water. Starch from this pellet was hydrolysed with amyloglucosidase followed by an enzymatic glucose determination (Wang and Nobel, 1995).
Statistical analyses
Statistical analyses were performed with SigmaStat (SPSS Science, Chicago, IL, USA). Unless otherwise indicated, P values are from pairwise Tukey tests following a Friedman repeated measures ANOVA on ranks. Data are presented as means ±SE.
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Results |
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Length/diameter changes
Fruit growth for Opuntia ficus-indica occurred over approximately 110 d (Fig. 1). Three growth phases were observed: (1) relatively rapid growth for small flower buds until anthesis 28 d later, when the experimental treatments of drought and GA3 were imposed; (2) slow growth for 7 d; and (3) a second period of growth during which the fruits ripened and asymptotically approached their final size at about 80 d after anthesis (Fig. 1). Drought reduced fruit size, and GA3 induced substantial seed abortion. In particular, the latter seeds were smaller, deformed, and less lignified than viable seeds. At 73 d after anthesis, seed mass was 52% lower and fruit mass was 65% lower following GA3 treatment compared to the control (Table 1).
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Fruit length for O. ficus-indica increased by an average of 1.3 mm d–1 for the 28 d before anthesis, when it was 4.8 cm. Length for the control then increased by 0.4 mm d–1 for 7 d and by 0.9 mm d–1 for the next 14 d; thereafter the rate decreased until the final length of 7.7 cm was reached (Fig. 1A). At 80 d after anthesis, fruits undergoing drought were 25% shorter than the control (Fig. 1A; P <0.05) and those treated with GA3 were 12% shorter (P <0.05). Fruit diameter increased by 0.6 mm d–1 for the 28 d just before anthesis, when it was 2.6 cm. Diameter for the control then increased by 0.2 mm d–1 for 7 d and by 0.5 mm d–1 for the next 14 d; thereafter the rate decreased to zero at 80 d after anthesis (Fig. 1B). At 80 d after anthesis, the diameter was 4.6 cm for ripe control fruits, 29% less for fruits undergoing drought (P <0.05), and 14% less for those treated with GA3 (P <0.05). Based on the length and diameter, the surface area of fruits of O. ficus-indica increased by 0.63 cm2 d–1 for the 28 d before anthesis, when it was 21.3 cm2 (Fig. 1C). At 80 d after anthesis, the surface area was 52.5 cm2 for control fruits (Fig. 1C), 44% less for fruits undergoing drought (P <0.05), and 22% less for those treated with GA3 (P <0.05).
Rate of growth
Based on cubic regressions of fresh mass (fm) on fruit length (l) [fm=0.352l+0.475l2+0.0752l3 (R2=0.92, P <0.0001)], the fresh mass for control fruits of O. ficus-indica was 1.19 g for small buds at 28 d before anthesis, 21.1 g at anthesis, and 67.4 g at 80 d after anthesis (Fig. 2A). At 80 d after anthesis, the fresh mass for fruits undergoing drought was 51% less than the control (P <0.05) and was 34% less for those treated with GA3 (P <0.05). Similarly, the dry mass (dm) for control fruits of O. ficus-indica, obtained from cubic regressions on fruit length [dm= –0.106+1.41l–0.459l2+0.0488l3 (R2=0.87, P <0.0001)], was 0.94 g for small buds, 1.52 g at anthesis, and 6.46 g at 80 d after anthesis (Fig. 2B). At 80 d after anthesis, dry mass for fruits undergoing drought was 66% less than the control (P <0.05) and was 42% less (P <0.05) for those treated with GA3 (Fig. 2B). Although initially the dry mass/fresh mass ratio was higher, beginning at anthesis when it was 0.072, it increased by 3.0x10–4 d–1 for the control and by 1.5x10–4 d–1 for fruits treated with GA3, but decreased by 7.2x10–5 d–1 for fruits undergoing drought.
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The daily rate of water gain (calculated as the daily change in fresh mass minus the daily change in dry mass) averaged 0.38 g d–1 per fruit at 25 d before anthesis, increasing 3-fold at 11 d before anthesis (P <0.05), and then decreasing back to its initial value at 4 d after anthesis (P <0.05; Fig. 3A). A second period of relatively rapid water gain occurred for fruits 7–21 d after anthesis, averaging 0.97 g d–1 per fruit, after which it steadily decreased essentially to zero as the fruits ripened (P <0.001 from an ANOVA). For fruits undergoing drought, the rate of water gain was only 0.23 g d–1 per fruit from 7–80 d after anthesis. For fruits treated with GA3, the rate of water gain was maximal at 7 d after anthesis at 0.95 g d–1 per fruit and then steadily decreased essentially to zero at 80 d after anthesis (P <0.001 from an ANOVA).
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The rate of dry mass gain averaged 45 mg d–1 per fruit at 25 d before anthesis, decreasing to 14 mg fruit–1 d–1 (P <0.05) from 14 d before anthesis to anthesis (Fig. 3B). The rate increased to a maximum of 127 mg d–1 per fruit at 31 d after anthesis (P <0.05) and then decreased essentially to zero as the fruits ripened. For fruits undergoing drought, the rate of dry mass gain at 7 d after anthesis was 86% less than for the control (P <0.05) and averaged 10 mg d–1 per fruit throughout fruit development. For fruits treated with GA3, the maximum rate of dry mass gain was 61 mg d–1 per fruit at 31 d after anthesis, decreasing to essentially zero at 80 d after anthesis (P <0.01 from an ANOVA).
Transpiration
The water vapour conductance for developing fruits of O. ficus-indica was greatest at night, reaching a maximum near 04.00 h, and decreased as ripening progressed (Fig. 4). For flower buds at 7 d before anthesis, the maximal conductance was 25 mmol m–2 s–1 and the minimum was 13 mmol m–2 s–1 (Fig. 4A). At 7 d after anthesis, the maximum was 32% lower (P <0.05) and the minimum was 74% lower (P <0.05) and drought had no apparent effect (Fig. 4A). At 38 d after anthesis, the maximal conductance was 3.8 mmol m–2 s–1 for the control and 30% lower for fruits undergoing drought (P <0.05; Fig. 4B). At 66 d, the maximal conductance for the control was also 3.8 mmol m–2 s–1 and drought had no apparent effect (Fig. 4C). At 21 d after anthesis, the maximal water vapour conductance for fruits treated with GA3 was 14 mmol m–2 s–1, 56% greater than for the control (P <0.05), and the minimum was 3.0 mmol m–2 s–1 in both cases (Fig. 4D). At 52 d after anthesis, the maximal conductance was 4.9 mmol m–2 s–1 and no effect of GA3 was apparent (Fig. 4E). At 80 d after anthesis, the maximal water vapour conductance was 4.5 mmol m–2 s–1 for the control and 28% lower for those treated with GA3 (P <0.05; Fig. 4F).
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Daily transpiration was 24 mol m–2 d–1 for flower buds 7 d before anthesis and then steadily decreased for fruits, becoming 73% lower at 38 d after anthesis (P <0.05) and averaging 80% lower thereafter (Fig. 5A). Drought had little effect on daily transpiration for developing fruits of O. ficus-indica (Fig. 5A). For fruits treated with GA3, daily transpiration was 28% higher than for the control at 21 d after anthesis (P <0.05), but 38% lower at 80 d (P <0.05; Fig. 5A). Taking into consideration the increase in surface area during fruit development (Fig. 1C), daily transpiration for buds at 7 d before anthesis was 0.77 g d–1 per fruit, decreasing by only 33% for fruits at 38 d after anthesis (Fig. 5B) and by 30% thereafter (Fig. 5B). The smaller surface areas for fruits undergoing drought (Fig. 1C) led to a daily transpiration per fruit that averaged 48% lower than for the control at 38 d and 66 d after anthesis (P <0.05; Fig. 5B). For fruits treated with GA3, daily transpiration per fruit was 22% greater than for the control at 21 d (P <0.05; Fig. 5B), but their smaller surface area (Fig. 1C) and lower transpiration per unit area (Fig. 5A) led to a daily transpiration per fruit that was 57% lower than for the control at 80 d after anthesis (P <0.05; Fig. 5B).
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Net CO2 uptake
The net CO2 uptake rate for developing fruits of O. ficus-indica was greatest at night, reaching a maximum near 00.00 h, and decreased as ripening progressed (Fig. 6). A maximal rate of 6.1 µmol m–2 s–1 and a minimal rate of –1.3 µmol m–2 s–1 were observed for flower buds at 7 d before anthesis (Fig. 6A). At 7 d after anthesis, the maximal rate of net CO2 uptake decreased by 39% for control fruits (P <0.05) and by 56% for those undergoing drought (P <0.05), while the minimum did not change for either case compared with 7 d before anthesis (Fig. 6A). At 38 d after anthesis, the maximal rate was 1.4 µmol m–2 s–1 for control fruits and 51% lower for those undergoing drought (P <0.05; Fig. 6B). At 66 d after anthesis, the maximal rate of net CO2 uptake averaged 0.51 µmol m–2 s–1 and no effect of drought was apparent (Fig. 6C). At 21 d after anthesis, the maximal rate of net CO2 uptake was 37% lower than at 7 d before anthesis (P <0.05) and no effect of GA3 was apparent (Fig. 6D). At 52 d after anthesis, the maximal rate of net CO2 uptake was 0.78 µmol m–2 s–1 and also no effect of GA3 was apparent (Fig. 6E). At 80 d after anthesis, the maximal rate of net CO2 uptake was 0.78 µmol m–2 s–1 for the control fruits and 31% lower for those treated with GA3 (P <0.05; Fig. 6F).
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Daily net CO2 uptake was 168 mmol m–2 d–1 for flower buds at 7 d before anthesis and then steadily decreased for fruits, becoming 85% lower at 38 d after anthesis (P <0.05) and averaging 88% lower thereafter (Fig. 7A). Daily net CO2 uptake was lower than for the control for fruits undergoing drought, for example, at 7 d after anthesis it was 37% lower (P <0.05; Fig. 7A). For fruits treated with GA3, daily net CO2 uptake was 20% higher than for the control at 21 d after anthesis (P <0.05) and similar thereafter (Fig. 7A). The daily dry mass gain per fruit, based on net CO2 uptake and including the increasing surface area during development (Fig. 1C), was 13.7 mg d–1 per fruit from 7 d before anthesis to 21 d after anthesis; it then decreased by 44% at 38 d after anthesis (P <0.05) and by an average of 68% thereafter (P <0.05; Fig. 7B). Daily dry mass gain for fruits undergoing drought was 47% lower than for the control at 7 d after anthesis (P <0.05) and averaged 67% lower thereafter (P<0.05; Fig. 7B). Treatment with GA3 had no apparent effect on fruit daily dry mass gain based on net CO2 uptake (Fig. 7B).
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Carbohydrates
The content of soluble sugars steadily increased in developing fruits of O. ficus-indica, the rate averaging 43 mg d–1 per fruit from 14 d before anthesis to 31 d after anthesis; the rate increased to 264 mg d–1 per fruit from 45–59 d after anthesis, leading to 7.2 g of soluble sugars per fruit at 73 d after anthesis (Fig. 8A). For fruits undergoing drought, the content of soluble sugars was 63% lower than the control at 31 d after anthesis (P <0.05) and was 89% lower at 59 d (P <0.01). For fruits treated with GA3, an effect on soluble sugar content was apparent only at 73 d after anthesis, when it was 59% lower than for ripe control fruits (Fig. 8A).
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The content of starch for young flower buds of O. ficus-indica at 28 d before anthesis was 3.1 mg per fruit, steadily increased until a maximum of 9.4 mg fruit–1 at 14 d after anthesis, and then decreased to 4.4 mg per fruit at 73 d (Fig. 8B). Decreases in starch content were not observed for fruits undergoing drought, whose starch content at 59 d after anthesis was 2-fold higher than for the control (P <0.05; Fig. 8B). For fruits treated with GA3, the starch content was similar to that for the control (Fig. 8B).
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Discussion |
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Increases in fruit mass of Opuntia ficus-indica during development followed a double sigmoid pattern, consistent with previous observations (Nerd and Mizrahi, 1995). Changes in the growth rate apparently reflected discrete developmental stages. For instance, the first period of rapid growth is presumably due to cell elongation, as suggested by a rapid water gain and a slow dry mass gain that preceded anthesis. During the subsequent period of slow growth near anthesis, dry matter and water supply from the plant was reduced. Following pollination, a third developmental stage was characterized by a relatively rapid growth rate due to a sink signal, such as GA3 (Grange, 1993; Srivastava, 2002; Taiz and Zeiger, 2002), originated from fertilized ovules. The growth rate then decreased as fruits approached their final size and ripened at 80 d after anthesis. During this latter period, the proportion of the fruit’s mass represented by the locule (pulp) increases, while the receptacle (peel) softens and becomes thinner during colour break (Pimienta Barrios, 1990; Nerd and Mizrahi, 1995; Barbera et al., 1994).
The water vapour conductance for developing fruits of O. ficus-indica was greatest at 7 d before anthesis, when the greatest transpiration and highest net CO2 uptake rates were also observed. Indeed, at this time, transpiration by fruits was 50% greater than their daily water gain, and CO2 uptake by fruits contributed about 60% of their daily dry mass gain. After anthesis, fertilization can trigger various physiological processes (Grange, 1993; Srivastava, 2002) that, in this case, led to a decrease in water vapour conductance and, consequently, to lower gas exchange rates as development progressed, reflecting the senescence of maternal tissue and the development of embryonic tissue (Haig and Westoby, 1988; Srivastava, 2002). At 20 d after anthesis, transpiration was equivalent to only 50% of the daily water gain, and fruit CO2 uptake supplied only 10% of the daily dry mass gain. The senescence of maternal tissues became more evident by 70 d after anthesis, as fruits had then essentially ripened and transpiration had increased and was about equivalent to the daily water gain; at this time, CO2 uptake by fruits had decreased to 5% of the daily dry mass gain.
Fruits growing on detached cladodes of O. ficus-indica were considerably smaller than for the control and had not ripened at 80 d after anthesis. Nevertheless, their stomatal conductance and net CO2 uptake rates were similar to the control throughout development, indicating that the drought treatment did not limit gas exchange. For many plants, ABA from the roots limits transpiration and net CO2 uptake through stomatal closure in response to water stress (Srivastava, 2002; Taiz and Zeiger, 2002), such as for fruits of O. ficus-indica growing on rooted cladodes (Nerd and Nobel, 2000), stems of the hemiepiphytic cactus Hylocereus undatus (Nobel and De la Barrera, 2002), and developing fruits of tomato (Mingo et al., 2003). Therefore, the use of detached cladodes of O. ficus-indica is a model for studying drought processes, such as fruit development, without the confounding effects of root chemical signalling. Considerable amounts of water were transpired by the control before anthesis, when the stomatal conductance of such fruits was highest, and presumably also by their petals during flower opening (Nobel, 1988; Galen et al., 1999). In addition, total transpiration for the control was 48 g of water per fruit, calculated by integrating the daily transpiration from anthesis to ripening at 80 d after anthesis, when the fruit water content was 61 g. Therefore, the amount of water required for the development of the five fruits per cladode considered here was greater than 45% of the fresh mass for a 1-year-old cladode (Nobel, 1996). Because about 9–15 fruits are allowed to develop out of the 30 or more flower buds that could set fruit for a given cladode in commercial plantations of O. ficus-indica (Acevedo et al., 1983; Inglese, 1995), the water supplied by the plant for fruit development must come from parts of the plant other than the cladode underlying the developing fruits.
Detached cladodes of O. ficus-indica can sustain net CO2 uptake for a period of weeks, the daily amount halving at about 30 d after detachment (Raveh and Nobel, 1999), as it also does for cladodes growing on droughted plants (Acevedo et al., 1983; Nobel, 1988). Thus detached cladodes fix limited, but probably sufficient, amounts of carbohydrate to sustain fruit development. However, because the translocation of photosynthates to developing fruits requires considerable amounts of water (Ho, 1988; Nobel and De la Barrera, 2000; Srivastava, 2002), development did not progress for the fruits undergoing drought.
Treatment of fruits of O. ficus-indica with GA3 at anthesis resulted in fewer and smaller (aborted) seeds, and the fruits at 80 d after anthesis were smaller than for the control, consistent with previous studies (Gil and Espinoza, 1980; Pimienta Barrios, 1990). Exogenous GA3 also inhibits seed development for grape (Agüero et al., 2000) and pea (García-Martínez and Hedden, 1997). Moreover, by contrast with many species for which treatment with GA3 leads to normal-sized or larger fruits (García-Martínez and Hedden, 1997; Taiz and Zeiger, 2002), for some grape cultivars treatment with GA3 leads to smaller fruits (Agüero et al., 2000). In apparent contradiction to the effects of exogenous GA3, endogenous levels of GA3 are maximal near anthesis for O. ficus-indica (Inglese et al., 1998), and this phytohormone is essential for embryo and seed development of grape (Agüero et al., 2000) and pea (Swain et al., 1997), and for pollen tube elongation of Arabidopsis thaliana (Singh et al., 2002). Shortly after anthesis, however, endogenous levels of GA3 are greatly decreased in O. ficus-indica (Inglese et al., 1998), grape (Agüero et al., 2000), and tomato (Srivastava, 2002). Such dual effects of GA3 have led to fruit development being described using ecological models of kin competition (Haig and Westoby, 1988) in which each developing, or potentially developing, fruit competes for maternal resources with its siblings. The mechanisms for control of fruit development by gibberellins are not fully understood, although phenylalanine ammonia-lyase (PAL; Agüero et al., 2000), an enzyme involved in cell-wall development and plant defence against pathogens (Taiz and Zeiger, 2002), and cytokinins have been implicated (Ho, 1988).
For O. ficus-indica (Inglese et al., 1998), tomato (Srivastava, 2002), and other species (Pharis and King, 1985), a second period of increased levels of GA3 occurs about half-way between fertilization and ripening. Concurrently, increased levels of ABA occur for tomato (Srivastava, 2002); this phytohormone is involved in seed maturation and in the acquisition of dormancy and acts antagonistically to GA3 (Srivastava, 2002; Taiz and Zeiger, 2002). As discussed above, ABA leads to stomatal closure in photosynthetic organs, including young fruits (Nerd and Nobel, 2000; Mingo et al., 2003). The considerably higher water vapour conductance for fruits treated with GA3 than for the control at 21 d after anthesis suggests that this phytohormone delayed fruit ripening; whether biosynthesis of ABA was inhibited by GA3 should be investigated. If such were the case, the biosynthesis of ABA in developing fruits of O. ficus-indica probably occurs initially in fertilized seeds. The decrease in water vapour conductance observed at 52 d after anthesis may, in turn, be caused by ABA of funicular origin (Srivastava, 2002). At 80 d after anthesis, the lower water vapour conductance for fruits treated with GA3 than for the control can reflect the low levels of GA3 in fruits with aborted seeds and hence a less antagonistic effect with the funicular ABA. Other factors contributing to reduced water vapour conductance at the later stages of fruit development for O. ficus-indica are that stomatal frequency decreases as fruit size increases (Nerd and Nobel, 2000) and that stomates become non-functional as ripening approaches (Srivastava, 2002).
While exogenous GA3 induced seed abortion for developing fruits of O. ficus-indica, resulting in smaller fruits and, presumably, in lower levels of endogenous ABA and GA3 throughout fruit development, the fruits still ripened. Thus fertilized seeds are not necessarily essential for the completion of fruit development in O. ficus-indica, similar to the case for various seedless varieties of commercially grown fruits (Pharis and King, 1985; Srivastava, 2002; Taiz and Zeiger, 2002). Moreover, some varieties of O. ficus-indica develop with high numbers of apomictic or sterile parthenocarpic seeds (Pimienta Barrios, 1990). Hence, the high level of endogenous GA3 that occurs near the time of anthesis probably acts as a physiological trigger for the maternal processes leading to fruit development. In such a case, the developmental control may be mediated by carbohydrate metabolism (Ho, 1988), perhaps with the involvement of PAL. Export of auxin from developing fruits has been proposed as a mechanism that inhibits the development of further fruits (‘primigenic dominance’; Bangerth, 1989, 1993). Indeed, the level of auxin increases for tomato shortly after pollination (Srivastava, 2002).
The content of total soluble sugars steadily increased for fruits of O. ficus-indica until 45 d after anthesis, but the contribution of sugars to the fruit’s dry mass remained near 40%. After 45 d after anthesis, however, the content of total soluble sugars increased dramatically, becoming 90% of the fruit dry mass at 73 d after anthesis. Indeed, during the final stages of ripening, the locule undergoes rapid growth, while the receptacle, chlorophyll and cell wall components degrade, leading to a thinner and softer fruit peel (Pimienta Barrios, 1990; Gutterman, 1995; Nerd and Mizrahi, 1995). Because the increase in fruit mass had slowed at this stage, an increase in locular biomass, especially soluble sugars, is mainly due to degradation of cell wall polysaccharides from the receptacle and their mobilization into the locule (Grange, 1993; Nerd and Mizrahi, 1995). This is accompanied by a decrease in starch content observed in the later stages of ripening. For fruits undergoing drought, the contribution of soluble sugars to the fruit’s dry mass did not change throughout development, averaging only 25%. At 59 d after anthesis, starch content for fruits undergoing drought was higher than at previous dates, representing 0.6% of the fruit’s dry mass, suggesting that the water limitation interrupted starch degradation in addition to limiting growth. For fruits treated with GA3, the amount of soluble sugars was similar to the control, except it did not increase at 73 d after anthesis, indicating that the aborted seeds induced by this phytohormone were weaker carbohydrate sinks than normal seeds.
In summary, the patterns of gas exchange for green fruits of O. ficus-indica were those typical for CAM plants, and the rates decreased as fruit development progressed. Fruits undergoing drought were smaller and did not ripen. This suggests that there are hydraulic constraints for fruit development, which are masked in the field because water for fruit development can be supplied from other parts of the plant during drought. Viable seeds are an important sink signal for driving fruit development. High levels of gibberellins produced by the fruits at the time of anthesis/fertilization (Inglese et al., 1998) induce physiological changes in the maternal tissue that ensure the supply of water and dry matter. By contrast, exogenous gibberellins induced seed abortion resulting in a weaker sink signal. During the final stages of fruit development for O. ficus-indica, a second pulse of gibberellins, first produced by the developing seeds and then by the funicle, apparently drives a second period of dry matter accumulation and the degradation of locular tissue and remobilization of its carbon compounds to produce the fruit pulp. Hence investments of water and carbon for fruit development are considerable and involve multifaceted interactions between the plant and the developing fruits.
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We thank Mindy Cherng, Craig Fiehler, and Mario Vallejo for their help during the gas exchange measurements. This research was financially supported by funds from the Council on Research of the University of California to PSN and a CONACYT Graduate Student Fellowship (No. 116385) and a ‘Stephen A Vavra’ Fellowship to EDlB.
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