The growth potential generated in citrus fruit under
water stress and its relevant mechanisms
Xu-Ming Huang
*, Hui-Bai Huang, Fei-Fei Gao
Department of Horticulture, South China Agricultural University Guangzhou, 510642, Peoples Republic of China
Accepted 1 June 1999
Abstract
A mild water stress was imposed upon potted tangerine trees (Citrus sinensisBlanco. cv. Zhuju) by water withholding during early juice sac expansion stage. While fruit growth was inhibited by water stress a growth potential was built up inside the fruit, which was not expressed until re-watering. The more powerful water uptake force of the stressed fruit was caused by its more negative fruit water potential. The mechanisms involved were both passive and active in nature: more water loss from fruit to transpiring leaves during water stress and some active adaptive physiological responses of fruit to water stress. The physiological responses involved both osmotic adjustment and cell wall adjustment. The former was re¯ected in higher soluble solute contents in both fruit juice and fruit skin (on a dry weight basis) resulting in the drop of osmotic potential (cs).
The latter was re¯ected in the cell wall loosening of fruit skin in response to water stress causing a further fruit turgor (cs) drop. These two responses further reduced fruit water potential, which
promoted post-stress fruit expansion growth.#2000 Elsevier Science B.V. All rights reserved.
Keywords: Citrus sinensis; Fruit expansion growth; Water stress; Water potential; Cell wall adjustment; Osmotic adjustment
1. Introduction
It has been well established that fruits on trees once subjected to a certain period of water stress can grow faster after re-watering than those on regularly watered trees (Chalmers and Wilson, 1978; Goell et al., 1981; Chalmers et al.,
*
Corresponding author.
E-mail address: [email protected] (X.-M. Huang).
1986; Mitchell et al., 1986; Huang et al., 1986; Cohen and Goell, 1988; Li et al., 1989). Huang et al. (1986, 1994) reported that orange fruits on trees once subjected to water stress exhibited an abrupt expansion growth much greater than those on non-stressed trees during the typhoon weather when atmospheric vapour pressure de®cit (VPD) drastically dropped, albeit under a plastic roof. Cohen and Goell (1988) suggested that dry matter accumulated in grapefruit during water stress accounted for a higher growth rate after re-watering. The phenomenon of post-stress faster growth of plant parts was termed as `stored growth' or `compensatory growth' by Kramer (1983), its mechanisms yet being under-explored. Nuemann (1995) considered that cell wall hardening in response to water stress was a major cause of growth inhibition during stress, and post-stress resumption of growth was a result of cell wall loosening. Our studies aimed at revealing the mechanisms underlying the after-effect of water stress on fruit growth.
2. Materials and methods
2.1. Materials
Two experiments were carried out in late autumn of 1995 and 1996, when sunny and dry weather was prevalent in Guangzhou. Two-year-old tangerine (Citrus reticulata Blanco cv. Zhuju) potted test trees on rough lemon rootstocks were used in both the years but with different sets of trees. The trees were planted in 5 l pots ®lled with loam medium. They blossomed in the middle of June after a period of water withholding to synchronise ¯ower initiation.
2.2. Treatments
In 1995, normal watering (NW) and water stress/re-watering (WS-RW) treatments were carried out under a plastic roof on 10 potted trees for each treatment with uniform tree size and fruit load (26±30 fruits per tree). Water withholding for the WS-RW trees was started on 30 October when the fruits were in the early stage of juice sac expansion. The pots were watered twice daily at dawn and dusk. For the NW trees, about 500 ml water per pot was supplied each time, while for the WS-RW trees, water supply was only 50 ml each time during the `stress-on' period. A soil tensiometer was installed in each of the pots to monitor soil water status. Soil in the WS-RW pots was kept at a tension between 85±100 kPa being read at dusk before daily watering, while in the NW pots it was kept within 10 kPa. Leaf water potential (cs) of the WS trees was about 0.2 MPa lower than that of the NW trees. The water stress in the WS-RW treatment during the `stress-on' period was mild according to Hsiao's scale (Hsiao, 1973). The
potted trees in the WS-RW treatment were re-watered on 22 November, thus entering a `stress-off' period. In 1996, water stress was started from 29 October in the same way as in 1995. On 15 November, the trees (10 pots per treatment) were moved into a phytotron where diurnal temperature and humidity changes were programmed to simulate `sunny weather' for three consecutive days. After that, `typhoon weather' was simulated starting on 18th and ending on 21st November. During this time, a steep and continuous depression of atmospheric VPD was programmed. The re-watering of the WS-RW pots commenced at the advent of the simulated `typhoon weather'. After the ending of the simulated `typhoon weather', the trees were again subjected to a 4-day period of simulated `sunny weather' for observations. The temperature and humidity changes programmed in the phytotron are shown in Fig. 1.
2.3. Fruit growth behaviour
Two fruits on each tree were tagged and numbered. Two paint dots were marked at opposite sides of fruit cheek to assure reproducible precise diameter measurements. Fruit diameters were measured with a vernier calliper (precision 0.02 mm) at dawn (0600±0700 h) and at dusk (1800±1900 h). Growth rate (diameter increase per day, mm dayÿ1
) for each traced fruit, was taken as the slope obtained from the linear regression of fruit diameter against days from the start of water stress.
2.4. Water potential of leaf and of fruit
Water potential of leaf (c1) and water potential of fruit (cf) were measured with a PMS pressure chamber at dawn, with at least 5 replicates (fruits or leaves) for each time.
2.5. Rapid determination of fruit turgor
The following device (Fig. 2) was used for the rapid determination of fruit turgor. The probe was inserted into a sampled fruit until the rubber stopper was pressed tightly against the fruit peel so that there was no juice escaping along the edge of the probe. The juice was then drained out of the fruit by the internal fruit turgor, escaping through the opening on the head side of the probe, which was connected to a manometer. Two minutes were allowed for balancing before the fruit turgor carried by the fruit skin was read from the manometer. At least ®ve fruits (replicates) were used for each measurement.
2.6. Measurement of TSS in juice
For each treatment, juice from eight fruits (eight replicates) was individually extracted for the determination of TSS (total soluble solids) content with an Abbe refractometer.
2.7. Measurement of skin mechanical properties
Skin mechanical properties were measured with a Swedish-made L and W6-2 wet strength tester. The tester stretched the skin strip (5 mm in width and 15 mm in length) at a speed of 1.5 mm sÿ1
and automatically plotted a strain-stress curve by an auto-recorder until the sample strip was broken. Skin compliance was calculated as strain/stress (mm kgÿ1
). Ten replicates (skin strips from 10 fruits) were used.
Fig. 2. A simple device for the rapid determination of turgor of citrus fruit. 1. Fruit sample 2. Probe 3. Manometer (®lled with mercury) 4. Opening on the head of the probe 5. Juice passage 6. Rubber stopper.
2.8. Skin soluble sugars, wall pectin and calcium analysis
Extraction of cold water-soluble sugar, hot water-soluble pectin and insoluble pectin from the skin was conducted following a procedure described by Bouranis and Niavis (1992). Soluble and insoluble pectin extracts were hydrolysed, respectively with 3 mol lÿ1
sulphate in boiling water bath for 2 h. A solution containing 0.1% potassium polygalacturonate (Sigma product) was taken as pectin standard. Content of soluble sugars in the hydrolysate was measured with anthrone method (Zhang, 1990) to represent the pectin content. Wall calcium was extracted together with insoluble pectin and then determined with an atomic spectrometer. The above mentioned measurements were conducted with four replicates.
2.9. X-ray micro-analysis (electron probe) of skin cell wall calcium
Fruit skin was cut into 5 mm wide stripes, which were embedded in warm agar and quickly frozen in liquid nitrogen. The brittle frozen embedded skin stripes were split into halves and mounted on a sample support with the fresh fracture surface facing upwards, immediately dried in a high vacuum evaporator and then coated with carbon. The fracture surfaces were then observed under a Hitachi S-550 scanning electron microscope (20 kev, 10±9 A) and wall Ca was analysed with an IMIX 2C mode X-ray energy dispersive spectrometer equipped with a SUN 3/8 data analysing computer system. Spot analysis was made on the middle lamella of wall fracture surfaces under a magni®cation of 2000. X-ray spectra were obtained in 50 s.
2.10. Extraction and enzyme assay of peroxidase in the skin
Skin of known weight was ground with 3 ml of cold 1 mol lÿ1
NaCl phosphate buffer solution (0.02 mol lÿ1
BPS, pH 6.8) in a mortar and centrifuged at 8000g for 10 min. The precipitate was further extracted with 3 ml of 1 mol lÿ1
NaCl BPS and centrifuged twice again. The supernatant liquid was the crude enzyme. Protein content in the crude enzyme solution was determined colorimetrically with Coommassie brilliant blue G250 (Zhang, 1990). The rate of guaiacol oxidation was measured likewise to indicate peroxidase activity, which was represented as unit enzyme activity per mg protein. One unit of enzyme activity was taken as 0.01 increase of OD490 nm in 1 min. Four replicates were used.
2.11. Cellulase and pectinase in fruit skin
pH 5.0) containing 1 mol lÿ1
NaCl. Cellulase and pectinase were assayed with a viscosimeteric method described by Fry et al. (1992). Reaction media for the assays of cellulase and pectinase were respectively 0.1% sodium carboxymethyl cellulose and 0.1% citrus pectin. Enzyme activity that caused the ¯ow rate of the meniscus to decrease by 1% in an hour was taken as one enzyme activity unit. Since citrus contained a gelling enzyme and the pectinase was not further puri®ed from the crude extract, the activity of pectinase thus measured was actually the net effect of gelling enzyme and pectin degrading enzyme. Four replicates were used for the enzyme assays.
2.12. Statistics
Signi®cance of difference of the above-mentioned parameters between NW and WS-RW treatments was analysed with one-way ANOVA.
3. Results
3.1. Growth behaviour of fruits
Water stress signi®cantly reduced growth rate (Table 1) while causing greater range of diurnal expansion and contraction of fruit diameter (Fig. 3). A peculiar `growth jump' was clearly observed for the stressed fruits with the advent of simulated `typhoon weather' (Table 1and Fig. 3). It was re¯ected in an abruptly accelerated growth rate of the WS-RW fruits in sharp contrast to the gradual increase in growth rate of the non-stressed fruits (NW). Also interestingly, the absolute fruit diameter eventually acquired by the WS-RW treatment was remarkably greater than by the NW treatment (Fig. 3). This suggests that a greater growth potential was built-up during the `stress-on' period and was expressed on re-watering.
Table 1
Growth rates of citrus fruits under different treatmentsa
Treatment Growth rate (mm dayÿ1
) % of diameter increase from stress until harvest (1996)
1995 1996
NW 0.10b 0.11b 27.3a
WS-RW(stress-on) 0.059a 0.06a
WS-RW(stress-off) 0.181c 0.172c 31.4b
aGrowth rate during `stress-off' period was the slope of diameter-day linear regression in the
duration of 3 weeks after stress. The lower 0case letters indicate differences between treatments are signi®cant at 0.05 level.
3.2. Leaf and fruit water potentials
The leaf water potential (c1) of the WS-RW trees in the `stress-on' period was about 0.2MPa (30%) lower than that of the NW trees (Table 2). On re-watering, c1recovered to the same level as that of NW trees just a day after the stress was removed. The restoration of fruit water potential (cf), however, was apparently lagging. Though the difference ofcfbetween the two treatments was narrowed to
Fig. 3. Growth behaviour of citrus fruits under NW and WS-RW treatments Arrow: a `growth jump' (caused by VPD drop plus re-watering) occurred with the advent of simulated `typhoon weather'. The inset shows the details of the growth curves of the NW fruit and WS-RW fruit during the `stress-on' and `stress-off' periods. The horizontal bar denotes the duration of simulated `typhoon weather'. Each column represents a single day. Dots falling on the vertical bars represent diameter measured at dawn and those falling in between the bars were measured at dusk. Note the greater magnitude of daily expansion and contraction of WS-RW fruit during the `stress-on' period.
Table 2
Changes of leaf and fruit water potentials in NW and WS-RW treatments
Year Day after
re-watering
c1(Mpa) c1(Mpa)
NW WS-RW NW WS-RW
1995 ÿ3 ÿ0.63a ÿ0.83b ÿ.079a ÿ1.05b
1 ÿ0.76a ÿ0.79a ÿ0.94a ÿ1.15b
7 ÿ0.66a ÿ0.65a ÿ0.77a ÿ0.84a
1996 ÿ2 ÿ0.58a ÿ0.75b ÿ0.57a ÿ0.95b
1a ÿ0.34a ÿ0.37a ÿ0.54a ÿ0.79b
7 ÿ0.58a ÿ0.59a ÿ0.53a ÿ0.59a
a non-signi®cant level a week after the water stress was removed,cfof WS-RW was still about 10% lower than that of NW. Thus,c1seemed to be more sensitive towards the alteration of environmental water status thancf. The lasting effect of water stress made the stressed fruit a stronger competitor for water.
3.3. Changes of fruit turgor
Turgor pressure of the fruits on the WS-RW trees in the `stress-on' period was ca. 40% lower than that of the fruits on the NW trees (Table 3). Slow restoration of fruit turgor was seen after the removal of water stress.
3.4. Solute contents in fruit juice and fruit skin cells
The TSS content in juice and the soluble sugars content and % dry matter in the skin all increased under water stress as compared with the NW control (Table 4). However, the % increase of soluble sugar content was greater than the dry matter increase in skin. This suggests that soluble sugar concentration increase was not a sheer result of water loss and that osmotic adjustment in response to the stress existed at least in skin cells. Re-watering resulted in a gradual drop of the above three parameters.
Table 3
Fruit turgor of NW treatment and WSRW treatment prior to and after re-watering (result of 1995)
Days after re-watering Fruit turgor (kPa)
NW WS-RW
Solute contents in juice and skin cells (Results of 1996)
Days after re-watering
TSS of juice (%) Soluble sugar in skin (mg gÿ1
FW)
Dray matter of skin (%)
NW WS-RW NW WS-RW NW WS-RW
ÿ3 6.3a 7.6b 59.6a 81.1b 24.0a 29.9b
1 6.7a 7.4b 54.3a 63.9b 24.5a 25.7b
7 6.5a 7.0a 53.8a 60.0b 24.5a 24.8a
3.5. Mechanical properties of cell wall and metabolism in skin
Stress-strain curves of fruit skin strips on the NW and WS-RW trees (Fig. 4) showed a drop of skin mechanical strength under water stress. This was also re¯ected by a skin compliance in the WS-RW treatment, which was higher than that in NW treatment (Table 5). Hot water soluble pectin content in the skin was increased, while cell wall calcium and insoluble pectin contents decreased under water stress. The decrease of wall calcium was also evidenced by X-ray electron probe analysis shown in Fig. 5. The wall Ca signal in the skin of the WS-RW fruits was weaker than that of the NW fruits. It was very likely that water stress in our study led to pectin hydrolysis and to the release of calcium from the skin cell walls as well, and resulted in cell wall loosening, causing a drop in skin mechanical strength. Analyses of cell wall metabolism-related enzymes (Table 6) illustrate that water stress did activate pectinase and cellulase in the fruit skin. Peroxidase has been claimed to irreversibly rigidify cell walls by forming diphenolic covalent bonds among wall polymers (Lamport, 1980). The cell wall
Fig. 4. Stress-strain curves of citrus skin strips. Continuous line: NW; broken line: WS-RW.
Table 5
Skin Compliance on stretch and skin cell wall componentsa(Results of 1996)
Treatment
NW WS-RW
Skin compliance (mm kgÿ1) 9.07a 10.4b
Hot water soluble pectin (mg gÿ1
DW) 2.79a 4.92b
Hot water insoluble pectin (mg gÿ1DW) 306.1a 277.59a
Wall structural Ca (mg gÿ1
DW) 0.34a 0.20b
Wall Ca signal in X-ray probe analysis (counts in 50 s) 16989a 12156a
a
rigidi®cation in fruit skin induced by water stress, however, was not seen in our study. It is thus clear that wall loosening characterised the wall adjustment in citrus fruit skin in response to water stress.
4. Discussion
The phenomenon of compensatory growth has been well-established in water stressed fruits. It has also been a commonplace observation that accelerated growth after the removal of water stress could last for weeks and resulted in a greater fruit volume at harvest relative to non-stressed fruits (Chalmers et al., 1986; Huang et al., 1986; Mitchell et al., 1986, and Fig. 3). This suggests that the post-stress growth was not solely `compensatory' but also a realisation of a
Fig. 5. Representative X-ray spectra obtained at2000 magni®cation in spot analysis on skin cell walls. Continuous line: NW, broken line: WS-RW.
Table 6
Activities of wall metabolism-related enzymes ( Results of 1996)
Enzymes Days after re-watering Treatment
NW WS-RW
Pectinase (unit mgÿ1protein) ÿ3 134.1a 152.6a
7 230.2a 266.8b
Cellulase (unit mgÿ1protein) ÿ3 5460a 7340b
7 2670a 4230a
Peroxidase (unit mgÿ1
protein) ÿ3 18864a 17882a
7 11901a 10836a
commonly greater growth potential built up inside the fruit by the stress. In our experiments, stressed fruits on the WS-RW trees were ready to expand to a greater magnitude during the night even in the `stress-on' period, and much greater throughout the simulated `typhoon weather' period in the phytotron (Fig. 3).
The greater growth potential of the WS-RW fruits stemmed from their more negative water potential. The more negative cf was conducive to preventing further water loss when the stress was becoming protracted, resulting in the creation of greater water uptake force within the fruit for the follow up `stress-off' period (Table 2). The more negative cf in the WS-RW fruits during the `stress-on' period resulted passively from a greater daytime water translocation from the fruits to the transpiring leaves. It was clearly shown by a greater shrinkage exhibited in the stressed fruits rather than in the NW fruits (Fig. 3). Research with intact orange trees has recon®rmed the `Midday Water Reservoir' theory by demonstrating tritiated water transport from fruit to leaf during daytime, and even more under water stress conditions (Ye et al., 1989). In our experiments, the water restoring capacity of stressed fruits in the night-time was greater than that of the NW fruits (Fig. 3), yet there was still a net daily water loss resulting in the inhibition of fruit growth during the `stress-on' period.
reduce thecfof stressed fruits. In this way, a greater water uptake force could be created and a greater growth potential could be built up. Mechanisms for creating more fruit growth potential by water stress are summarized and shown in Fig. 6. In what manner water stress affects the growth of a post-stressed fruit is de®nitely associated with the time of imposition relating to the developmental stage of fruit, the duration and the intensity of the stress per se. Water stress imposed upon a fruit during the rapid fruit expansion stage has been shown inhibitory to fruit growth and the adverse effect is hardly reversible (Hardie and Considine, 1976; Lotter et al., 1985; Huang et al., 1986; Li et al., 1989). The positive effect of water stress on fruit growth lasting through the post-stress period has been found when water stress was removed before the rapid expansion stage of fruit (Chalmers et al., 1986; Huang et al., 1986; Mitchell et al., 1986; Li et al., 1989). In the present study, the water stress imposed upon the test trees was mild and the duration was shorter than a month during the early stage of juice sac expansion well before the fruit expanded rapidly. This proved bene®cial to the sizing and internal quality of the fruits. Further studies are yet necessary to clarify the mechanisms of differential effects of water stress on fruit growth when it
Fig. 6. Diagrammatic scheme of suggested mechanisms for fruit growth potential built up by water stress (arrow with a broken line: relation uncertain).
occurs at different stages of development. Results gained from these studies will certainly serve as the base for developing an ef®cient restricted irrigation system in citrus groves.
Acknowledgements
This research was supported by the National Foundation for Natural Sciences of P.R. China. Thanks are due to Professor Jing-Ping Xiao who has offered constructive suggestions for our research.
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