Directory UMM :Data Elmu:jurnal:S:Scientia Horticulturae:Vol83.Issue3-4.March2000:
Scientia Horticulturae 83 (2000) 227±240
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 sinensis Blanco. 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 rewatering. 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).
0304-4238/00/$ ± see front matter # 2000 Elsevier Science B.V. All rights reserved.
PII: S 0 3 0 4 - 4 2 3 8 ( 9 9 ) 0 0 0 8 3 - 7
228
X.-M. Huang et al. / Scientia Horticulturae 83 (2000) 227±240
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 underexplored. 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
X.-M. Huang et al. / Scientia Horticulturae 83 (2000) 227±240
229
Fig. 1. Diurnal humidity (A) and temperature (B) changes of simulated `sunny weather' and
`typhoon weather' in a phytotron (in 1996) Continuous line: `sunny weather'; Broken line: `typhoon
weather'.
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.
230
X.-M. Huang et al. / Scientia Horticulturae 83 (2000) 227±240
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.
2.5. 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 AbbeÂ
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 kg ÿ1). Ten replicates (skin strips from 10 fruits)
were used.
X.-M. Huang et al. / Scientia Horticulturae 83 (2000) 227±240
231
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 8000 g
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
The extraction of crude enzyme followed the procedure of peroxidase
extraction, except the extraction medium used was acetate buffer (0.2 mol lÿ1
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X.-M. Huang et al. / Scientia Horticulturae 83 (2000) 227±240
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
NW
WS-RW(stress-on)
WS-RW(stress-off)
a
Growth rate (mm dayÿ1)
1995
1996
0.10b
0.059a
0.181c
0.11b
0.06a
0.172c
% of diameter increase from
stress until harvest (1996)
27.3a
31.4b
Growth 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.
X.-M. Huang et al. / Scientia Horticulturae 83 (2000) 227±240
233
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.
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,
c1 recovered 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 of cf between the two treatments was narrowed to
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
1
7
ÿ0.63a
ÿ0.76a
ÿ0.66a
ÿ0.83b
ÿ0.79a
ÿ0.65a
ÿ.079a
ÿ0.94a
ÿ0.77a
ÿ1.05b
ÿ1.15b
ÿ0.84a
1996
ÿ2
1a
7
ÿ0.58a
ÿ0.34a
ÿ0.58a
ÿ0.75b
ÿ0.37a
ÿ0.59a
ÿ0.57a
ÿ0.54a
ÿ0.53a
ÿ0.95b
ÿ0.79b
ÿ0.59a
a
VPD dropped re-watering (stress-off period).
234
X.-M. Huang et al. / Scientia Horticulturae 83 (2000) 227±240
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)
ÿ4
ÿ1
1
4
9
NW
WS-RW
5.78a
4.97a
5.50a
4.42a
3.86a
3.34b
3.08b
4.18b
3.55b
3.31a
a non-signi®cant level a week after the water stress was removed, cf of WS-RW
was still about 10% lower than that of NW. Thus, c1 seemed to be more sensitive
towards the alteration of environmental water status than cf. 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 4
Solute contents in juice and skin cells (Results of 1996)
Days after
re-watering
ÿ3
1
7
TSS of juice (%)
Soluble sugar in
skin (mg gÿ1 FW)
Dray matter of skin (%)
NW
WS-RW
NW
WS-RW
NW
WS-RW
6.3a
6.7a
6.5a
7.6b
7.4b
7.0a
59.6a
54.3a
53.8a
81.1b
63.9b
60.0b
24.0a
24.5a
24.5a
29.9b
25.7b
24.8a
X.-M. Huang et al. / Scientia Horticulturae 83 (2000) 227±240
235
Fig. 4. Stress-strain curves of citrus skin strips. Continuous line: NW; broken line: WS-RW.
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
Table 5
Skin Compliance on stretch and skin cell wall componentsa (Results of 1996)
Treatment
ÿ1
Skin compliance (mm kg )
Hot water soluble pectin (mg gÿ1DW)
Hot water insoluble pectin (mg gÿ1DW)
Wall structural Ca (mg gÿ1 DW)
Wall Ca signal in X-ray probe analysis (counts in 50 s)
NW
WS-RW
9.07a
2.79a
306.1a
0.34a
16989a
10.4b
4.92b
277.59a
0.20b
12156a
a
All analyses were carried out with samples 3 days before re-watering (stress-on period) in WSRW treatment.
236
X.-M. Huang et al. / Scientia Horticulturae 83 (2000) 227±240
Fig. 5. Representative X-ray spectra obtained at 2000 magni®cation in spot analysis on skin cell
walls. Continuous line: NW, broken line: WS-RW.
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
Table 6
Activities of wall metabolism-related enzymes ( Results of 1996)
Enzymes
Days after re-watering
ÿ1
Pectinase (unit mg
protein)
Cellulase (unit mgÿ1 protein)
Peroxidase (unit mgÿ1 protein)
ÿ3
7
ÿ3
7
ÿ3
7
Treatment
NW
WS-RW
134.1a
230.2a
5460a
2670a
18864a
11901a
152.6a
266.8b
7340b
4230a
17882a
10836a
X.-M. Huang et al. / Scientia Horticulturae 83 (2000) 227±240
237
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 `stressoff' 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.
Results obtained from the present studies indicate that passive water loss was
not the sole contribution to the more negative cf. Therefore, the role of metabolic
changes within the stressed fruit must not be neglected. Since more TSS in juice
and soluble sugars in skin cells (on a dry weight basis) were found in the WS-RW
fruits (Table 4), these changes might include soluble solute accumulating
mechanisms related to osmotic adjustment (cs dropped). Our results have also
substantiated a wall adjustment mechanism in the fruit, which involved wallloosening metabolism (Table 5). This was different from the wall-hardening
adjustment observed in leaves, stems and roots under water stress (Nuemann,
1995). Wall-hardening adjustment in conjunction with osmotic adjustment in
these organs is in favour of cell turgor maintenance. That is undoubtedly
necessary for keeping the stomata open to maintain photosynthetic activity in
leaves, and necessary for the extension of roots in search of more soil water. For
fruit, however, the maintenance of turgor seems of no special bene®t for its
adaptation to water stress. Actually, in our experiments, the WS-RW fruits lost
turgor considerably when the stress was on (Table 3). Wall-loosening adjustment
in fruit might be interrelated with solute accumulation in the stressed fruits. Wall
loosening involved hydrolysing enzymes (Table 6), which decomposed wall
polymers into soluble solutes and thus reduced cs in fruit cells. The possibility
that more assimilates were synthesized in leaves and then transported
continuously into the stressed fruits, to contribute to osmotic adjustment, should
not be excluded here. Wall-loosening adjustment (causing cp to decrease) in
conjunction with osmotic adjustment (causing cs to decrease) could further
238
X.-M. Huang et al. / Scientia Horticulturae 83 (2000) 227±240
Fig. 6. Diagrammatic scheme of suggested mechanisms for fruit growth potential built up by water
stress (arrow with a broken line: relation uncertain).
reduce the cf of 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
X.-M. Huang et al. / Scientia Horticulturae 83 (2000) 227±240
239
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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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 sinensis Blanco. 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 rewatering. 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).
0304-4238/00/$ ± see front matter # 2000 Elsevier Science B.V. All rights reserved.
PII: S 0 3 0 4 - 4 2 3 8 ( 9 9 ) 0 0 0 8 3 - 7
228
X.-M. Huang et al. / Scientia Horticulturae 83 (2000) 227±240
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 underexplored. 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
X.-M. Huang et al. / Scientia Horticulturae 83 (2000) 227±240
229
Fig. 1. Diurnal humidity (A) and temperature (B) changes of simulated `sunny weather' and
`typhoon weather' in a phytotron (in 1996) Continuous line: `sunny weather'; Broken line: `typhoon
weather'.
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.
230
X.-M. Huang et al. / Scientia Horticulturae 83 (2000) 227±240
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.
2.5. 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 AbbeÂ
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 kg ÿ1). Ten replicates (skin strips from 10 fruits)
were used.
X.-M. Huang et al. / Scientia Horticulturae 83 (2000) 227±240
231
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 8000 g
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
The extraction of crude enzyme followed the procedure of peroxidase
extraction, except the extraction medium used was acetate buffer (0.2 mol lÿ1
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X.-M. Huang et al. / Scientia Horticulturae 83 (2000) 227±240
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
NW
WS-RW(stress-on)
WS-RW(stress-off)
a
Growth rate (mm dayÿ1)
1995
1996
0.10b
0.059a
0.181c
0.11b
0.06a
0.172c
% of diameter increase from
stress until harvest (1996)
27.3a
31.4b
Growth 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.
X.-M. Huang et al. / Scientia Horticulturae 83 (2000) 227±240
233
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.
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,
c1 recovered 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 of cf between the two treatments was narrowed to
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
1
7
ÿ0.63a
ÿ0.76a
ÿ0.66a
ÿ0.83b
ÿ0.79a
ÿ0.65a
ÿ.079a
ÿ0.94a
ÿ0.77a
ÿ1.05b
ÿ1.15b
ÿ0.84a
1996
ÿ2
1a
7
ÿ0.58a
ÿ0.34a
ÿ0.58a
ÿ0.75b
ÿ0.37a
ÿ0.59a
ÿ0.57a
ÿ0.54a
ÿ0.53a
ÿ0.95b
ÿ0.79b
ÿ0.59a
a
VPD dropped re-watering (stress-off period).
234
X.-M. Huang et al. / Scientia Horticulturae 83 (2000) 227±240
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)
ÿ4
ÿ1
1
4
9
NW
WS-RW
5.78a
4.97a
5.50a
4.42a
3.86a
3.34b
3.08b
4.18b
3.55b
3.31a
a non-signi®cant level a week after the water stress was removed, cf of WS-RW
was still about 10% lower than that of NW. Thus, c1 seemed to be more sensitive
towards the alteration of environmental water status than cf. 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 4
Solute contents in juice and skin cells (Results of 1996)
Days after
re-watering
ÿ3
1
7
TSS of juice (%)
Soluble sugar in
skin (mg gÿ1 FW)
Dray matter of skin (%)
NW
WS-RW
NW
WS-RW
NW
WS-RW
6.3a
6.7a
6.5a
7.6b
7.4b
7.0a
59.6a
54.3a
53.8a
81.1b
63.9b
60.0b
24.0a
24.5a
24.5a
29.9b
25.7b
24.8a
X.-M. Huang et al. / Scientia Horticulturae 83 (2000) 227±240
235
Fig. 4. Stress-strain curves of citrus skin strips. Continuous line: NW; broken line: WS-RW.
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
Table 5
Skin Compliance on stretch and skin cell wall componentsa (Results of 1996)
Treatment
ÿ1
Skin compliance (mm kg )
Hot water soluble pectin (mg gÿ1DW)
Hot water insoluble pectin (mg gÿ1DW)
Wall structural Ca (mg gÿ1 DW)
Wall Ca signal in X-ray probe analysis (counts in 50 s)
NW
WS-RW
9.07a
2.79a
306.1a
0.34a
16989a
10.4b
4.92b
277.59a
0.20b
12156a
a
All analyses were carried out with samples 3 days before re-watering (stress-on period) in WSRW treatment.
236
X.-M. Huang et al. / Scientia Horticulturae 83 (2000) 227±240
Fig. 5. Representative X-ray spectra obtained at 2000 magni®cation in spot analysis on skin cell
walls. Continuous line: NW, broken line: WS-RW.
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
Table 6
Activities of wall metabolism-related enzymes ( Results of 1996)
Enzymes
Days after re-watering
ÿ1
Pectinase (unit mg
protein)
Cellulase (unit mgÿ1 protein)
Peroxidase (unit mgÿ1 protein)
ÿ3
7
ÿ3
7
ÿ3
7
Treatment
NW
WS-RW
134.1a
230.2a
5460a
2670a
18864a
11901a
152.6a
266.8b
7340b
4230a
17882a
10836a
X.-M. Huang et al. / Scientia Horticulturae 83 (2000) 227±240
237
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 `stressoff' 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.
Results obtained from the present studies indicate that passive water loss was
not the sole contribution to the more negative cf. Therefore, the role of metabolic
changes within the stressed fruit must not be neglected. Since more TSS in juice
and soluble sugars in skin cells (on a dry weight basis) were found in the WS-RW
fruits (Table 4), these changes might include soluble solute accumulating
mechanisms related to osmotic adjustment (cs dropped). Our results have also
substantiated a wall adjustment mechanism in the fruit, which involved wallloosening metabolism (Table 5). This was different from the wall-hardening
adjustment observed in leaves, stems and roots under water stress (Nuemann,
1995). Wall-hardening adjustment in conjunction with osmotic adjustment in
these organs is in favour of cell turgor maintenance. That is undoubtedly
necessary for keeping the stomata open to maintain photosynthetic activity in
leaves, and necessary for the extension of roots in search of more soil water. For
fruit, however, the maintenance of turgor seems of no special bene®t for its
adaptation to water stress. Actually, in our experiments, the WS-RW fruits lost
turgor considerably when the stress was on (Table 3). Wall-loosening adjustment
in fruit might be interrelated with solute accumulation in the stressed fruits. Wall
loosening involved hydrolysing enzymes (Table 6), which decomposed wall
polymers into soluble solutes and thus reduced cs in fruit cells. The possibility
that more assimilates were synthesized in leaves and then transported
continuously into the stressed fruits, to contribute to osmotic adjustment, should
not be excluded here. Wall-loosening adjustment (causing cp to decrease) in
conjunction with osmotic adjustment (causing cs to decrease) could further
238
X.-M. Huang et al. / Scientia Horticulturae 83 (2000) 227±240
Fig. 6. Diagrammatic scheme of suggested mechanisms for fruit growth potential built up by water
stress (arrow with a broken line: relation uncertain).
reduce the cf of 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
X.-M. Huang et al. / Scientia Horticulturae 83 (2000) 227±240
239
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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