AJCS 5(6):695-701 (2011) ISSN:1835-2707

Structural, physiological and metabolic integrated responses of two tomato

(Solanum lycopersicum L.) cultivars during leaf rehydration
A.M. Calcagno3, M. Rivas2, M. Castrillo*1
1
Dept. Biología de Organismos, Universidad Simón Bolívar, Apdo. postal 89000, Caracas 1080, Venezuela
2
Postgrado Cs. Biológicas,
3
Present address: Imperial College London, South Kensington Campus, London SW7 2AZ, UK
*Corresponding author: [email protected]

Abstract

The aim of this work was to characterize the recovery responses of two tomato cultivars, Rio Grande (RG) and Pera Quibor (PQ), to
rehydration after water deficit. Recovery responses were measured in terms of timing and magnitude, and analysed as indicative of
tolerance. The parameters evaluated were: soil water potential (soil Ψw) and leaf water potential (leaf Ψw); leaf osmotic potential
(leaf Ψs); leaf relative water content (LRWC); stomatal conductance (gs); Rubisco activity; protein content; fresh mass (FM) and dry
mass (DM); leaf area (LA); specific leaf area (SLA) and leaf density (D). Different types of responses were observed: as common
responses of both cultivars, full recovery of soil Ψw, Ψs, and LRWC were found regardless of stress level and length of rehydration
time. Recovery of protein was full in PQ and partial in RG. Full recovery of gs and fresh mass and partial recovery of Ψw and leaf
area was observed in both cultivars regardless of the level of stress but regarding rehydration time. Full recovery of Rubisco activity
was found regarding level of stress and rehydration time. As different responses between cultivars, full recovery of DM was found in
PQ and of SLA and leaf D in RG regardless of the level of stress but regarding rehydration time. Partial recovery of SLA and leaf D
was found in PQ regarding both level of stress and rehydration time. The delayed recovery of leaf structural changes promoted full
recovery of DM and protein in PQ. The correlations between physiological and metabolic parameters and observed sclerophylly
indices, reveal integrated tolerance mechanisms intended to protect plant cultivars from intermittent changes in water conditions.

Keywords: leaf density; leaf osmotic potential; leaf relative water content; leaf water potential; protein content ; Rubisco; soil water
potential; specific leaf area; stomatal conductance.
Abbreviations: D, leaf density ; DM, dry biomass; FM, fresh biomass; gs, stomatal conductance; LRWC, leaf relative water content;
PQ, Pera Quibor cultivar; RG, Rio Grande cultivar; SLA, specific leaf area; Ψw, leaf water potential; sΨw, soil water potential; Ψs,
leaf osmotic potential.

Introduction

Lack of water is the most unfavorable environmental stress availability of internal CO2, suggesting some non-stomatal
factor affecting cultivars’ growth and productivity, its effects limitation of photosynthesis (Souza et al, 2004). Under
being the most harmful among those of other environmental normal growing conditions field plants are subjected to
stress agents (Kramer, 1980). Crops grown under rain-fed environmental fluctuations, such as water deficit periods
conditions are usually affected by drought stress at different followed by rainy periods. Reports have shown the recovery
stages resulting in negative effect on yield (Amjad Ali et of measured parameters following a water deficit period,
al.2011). Water deficit induces changes in leaf anatomical particularly at the beginning of the rehydration period.
parameters, altering the CO2 conductance diffusion However, recovery from water stress on rehydration has not
components, and thus collaborating with the maintenance of been studied sufficiently, even though intermittent water
photosynthetic rates even at low stomatal conductance deficit, on a daily and seasonal basis and for short and long
(Chartzoulakis et al. 1999). Lowered photosynthetic rate periods of time, is commonly experienced by plants in natural
under water deficit has been attributed to both stomatal and environments. Furthermore, the study of recovery responses
non-stomatal limitations (Galle et al. 2010; Souza et al., on rehydration as an indication of tolerance has not been fully
2004) involving programmed stomatal closure and reduced considered. Therefore, the objective of the present work was
photosynthetic enzyme activity (Tabaeizadeh, 1998). to analyse the recovery responses to rehydration, in terms of
Working with chickpea cultivars under water deficit magnitude and timing as a signal of tolerance, of two tomato
Mafakheri et al (2010) reported that mesophyll resistance is cultivars, Rio Grande and Pera Quibor, after being subjected
the basic determinate of rate of phototosynthesis under to moderate and severe water deficit. Responses are observed
drought stress conditions. A reduction in Rubisco activity at two points: after day one of the water deficit process (both
during water deficit has also been reported (Castrillo et al. severe and moderate) and after day eight of the water deficit
2001; Vu and Allen, 2009). Water deficit effects in tomato process (both severe and moderate). The parameters
(Torrecillas et al. 1995; Castrillo and Calcagno, 1989) have evaluated after rehydration at day 1 and day 8 of the water
been reported. Studies show that re-watering after a water deficit process were soil water potential, leaf water potential,
deficit period, induces a recovery in assimilation rates. leaf osmotic potential, leaf relative water content, stomatal
Assimilation rates recovered only partially, despite the

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Table 1. Correlations among leaf relative water content (LRWC), water potential (Ψw) and osmotic potential (Ψs) and stomatal
conductance (gs), soluble protein content (Prot) and Rubisco activity after moderate (WDI) and severe (WDII) water deficit levels,
and two rehydration levels (RI and RII) after eight days rehydration; RI 8d and RII 8d, for two tomato cultivars, Rio Grande (RG)
and Pera Quibor (PQ),*Spearman coefficient significantly different (P=0.05)

LRWC Ψw Ψs
RI 8d 0.2453 0.1599 0.6439*
RG
RII 8d 0.2657 0.0262 0.6645*
gs
RI 8d 0.1425 0.0517 0.4676*
PQ
RII 8d 0.6755* 0.7834* 0.7463*

RI 8d 0.6897* 0.0625 0.4134

RG
RII 8d 0.9733* 0.8757* 0.9563*
Prot
RI 8d 0.2652 0.0348 0.0047
PQ
RII 8d 0.4993* 0.6765* 0.7989*

RI 8d 0.7289* 0.2721 0.5811*

RG
RII 8d 0.8923* 0.7929* 0.9633*
Rubisco
RI 8d 0.3398 0.0787 0.4795*
PQ
RII 8d 0.6554* 0.6988* 0.7589*

conductance, Rubisco activity, protein content, leaf fresh and additional group of plants was watered daily and maintained
dry biomass and leaf schlerophylly indices. as control. Measurements were made on young, fully
expanded leaves of 6 plants (i.e. n = 6) per group.
Materials and methods
Water status measurements and stomatal conductance (gs).
Plant material
To obtain predawn water potential measurements, leaves
Tomato seeds of Río Grande cultivar and Pera Quibor were sampled in the morning (05:30 - 06:30h) and their water
cultivar (nationally produced cultivar in Venezuela) were potential was measured on leaf discs in C-52 chambers
germinated on wet paper in plastic trays. The seedlings were attached to a HR-33T Dew Point Microvoltmeter (Wescor,
then transferred to 5L plastic pots (one seedling per pot) Logan, USA). Soil water potential was determined
containing a mixture of clay and sand (3:2); watered daily; simultaneously using the same technique. After measuring
and maintained in a greenhouse at Simón Bolívar University water potential, the leaves were frozen in liquid nitrogen and
in Caracas, Venezuela. Fifty mL of Hoagland solution plus their leaf osmotic potential was measured using the
0.25mM CaCl2 and commercial fertilizer (POKON, Bendien, Hygrometric (dew point) method on sap obtained from
Naarden; Netherlands) were added to each pot twice a week. samples after thawing. Leaf relative water content was
determined according to Turner’s method (1981). Stomatal
Treatments conductance was measured between 07:00 and 08:00h using
a porometer (LI-65 with sensor LI-25, Lambda, Lincoln,
Plants were watered daily for 28 days. After this period, USA). Abaxial and adaxial conductances were measured and
watering was withheld in 80 plants (water deficit plants). total stomatal conductance was calculated.
When a significant number of plants of both cultivars reached
a leaf water potential of between -1.60 ± 0.5 to -1.70 ± 0.5 Growth and sclerophylly indices
(which occurred after 8 days without watering), they were re-
watered. This moderately stressed group of plants was Leaf area was measured by planimetry (Lasiko-Est 1929, Los
labeled Rehydration I (RI). Another group was kept under Angeles Scientific Instrument Co., Inc., Los Angeles,
water deficit until its leaf water potential reached a value of California, USA). Fresh biomass (FM) was determined
between -2.40 ± 0.65 to -2.55 ± 0.70MPa (occurred after 11 immediately after sampling, and dry biomass (DM) was
days without watering), after which the plants were obtained through oven drying at 80ºC for 48h. Specific leaf
rehydrated. This severely stressed group of plants was named area of individual leaves was calculated using the ratio leaf
Rehydration II (RII). The rehydration period lasted 8 days, area to dry biomass, and leaf density (D) was obtained using
during which the plants were watered daily. Plant responses the following formula: D = (DM/FM) x 1000 (Guerfel et al.
to rehydration were evaluated on day 1 (RI 1 and RII 1) and 2009.
on day 8 (RI 8 and RII 8) after the start of re-watering. An

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Leaf extraction, Rubisco activity and protein content were performed using SPSS 12.0 (SPSS Inc., Chicago, IL,
estimation USA).

Leaf extraction and total Rubisco activity assay was Results

performed following the method reported by Castrillo et al. The values of soil water potential and leaf water potential,
(2001). Protein content estimation was carried out following leaf osmotic potential and leaf relative water obtained during
the Bradford (1976) method. recovery from water deficit, in all tested plants, are shown in
Figures 1, a, b, c and d respectively. In both moderate and
Statistical analysis severely stressed plants, soil water potential (sΨw), leaf water
potential (Ψw), leaf osmotic potential (Ψs) and leaf relative
Pearson and Spearman rank test was used for correlations;
water content(LRWC) obtained values were significantly
Friedman and Mann-Whitney u-test was used for comparison
lower than those obtained from the control group.During
of treatments (P = 0.05) (Sokal and Rohlf, 1995). Analyses
rehydration, soil water potential reached values similar to
those of control

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Table 2. Recovery responses types. PQ: Pera Quibor cultivar; RG Rio Grande cultivar; WD: Water deficit ; FR: Full
recovery; PR: Partial recovery; sΨw: soil water potential; Ψw: leaf water potencial; Ψs: leaf osmotic potential; LRWC: leaf
relative water content; Prot: protein content; Rubisco:Ribulose 1,5-Bis-P- carboxylase activity; gs: stomatal conductance; FM:
fresh mass; DM: dry mass; D: Leaf density; SLA: Specific leaf area
Types Commons No commons
PQ RG
WD independent-time independent sΨw; Ψs;LRWC (FR) Prot(FR) Prot(PR)
WD independent-time dependent gs; FM (FR) DM(FR) SLA&D(FR)
Ψw; LA (PR)
WD dependent-time dependent Rubisco act. (FR) SLA&D(PR) DM(PR)

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at both rehydration levels (RI and RII) and measured times dependent/time dependent responses (recovery is more
(1d and 8d).On re-watering, leaf water potential increased in affected by severe water deficit and was observed only at day
both cultivars and reached a partial recovery during the 8 8 after rehydration). The observed responses that are common
days rehydration period. The values of leaf osmotic potential to both cultivars are: a) Water deficit independent/time
and leaf relative water content showed full recovery at both independent responses: full recovery of soil water potential,
rehydration levels. Moderate and severely stressed Río leaf osmotic potential and leaf relative water content in both
Grande and Pera Quibor plants showed a significantly lower cultivars and protein content in Pera Quibor; partial recovery
stomatal conductance (gs) at the early stage (Fig.1e) of re- of protein content in Río Grande; b) Water deficit
irrigation, however, after 8 days of rehydration they showed independent/time dependent responses: full recovery of
full recovery at both levels. Fresh biomass values were stomatal conductance and fresh biomass, and partial recovery
significantly lower than control in all stressed plants. Full of leaf water potential and leaf area and c) Water deficit
recovery was observed after 8 days of rehydration at dependent/time dependent responses: full recovery of
moderate and severely stressed plants (RI and RII) of both Rubisco activity. The different responses between both
cultivars (Fig. 2a). Dry biomass (Fig. 2c) values were cultivars were: a) Water deficit independent/time dependent
significantly lower than control values at both levels of water responses: full recovery of dry biomass in Pera Quibor;
deficit; recovery was observed in Pera Quibor after 8 days of specific leaf area and leaf density in Río Grande and b)Water
rehydration at both levels of water deficit. Leaf area values deficit dependent/time dependent responses: partial recovery
were significantly lower than control at both levels of water of specific leaf area and leaf density in Pera Quibor. The full
deficit and increased on rehydration of both cultivars; partial recovery responses in protein content and dry biomass in Pera
recovery was obtained after 1 day and 8 days of the Quibor, not seen in Río Grande, are the result of structural
rehydration period (Fig.2c). Leaf density values increased changes occurred during the water deficit period. These
during moderate and severe water deficit, showing recovery responses are based on delayed sclerophylly recovery through
at both levels in Río Grande on days 1 and 8 after leaf structural changes, which increase leaf thickness, and are
rehydration, and in Pera Quibor only on plants subjected to maintained after re-watering. The changes in leaf structural
severe stress and after 1 day (Fig. 2d). Specific leaf area parameters are due to reductions in cell expansion and
values decreased significantly during moderate and severe intercellular spacing, producing a compact leaf, with less leaf
water deficit in both cultivars; recovery was observed only in area and greater leaf thickness. In a C3 crop plant like
Río Grande on 1 day and 8 days of rehydration for both tomato, an increase in leaf thickness leads to a thicker
levels of stress (Fig. 2e). Protein content (Fig.3a) decreased palisade parenchyma, which could contain larger numbers of
significantly in comparison with control values in moderate CO2-fixation sites, while a thicker spongy parenchyma could
and severely stressed plants. Full recovery was observed at result in easier CO2 diffusion to these sites. The leaf structure
moderately stressed plants after 1 day in Pera Quibor, and changed to a more compact and thicker one, with a smaller
after 8 days at moderately and severely stressed plants in both area. This change could be considered as a tolerance
cultivars. Rubisco activity (Fig. 3b) decreased significantly structural leaf transition, which occurs during water deficit
with respect to control at both levels of water deficit. It periods and is maintained at rehydration in order to improve
showed full recovery in Pera Quibor cultivars under moderate tolerance and increase productivity during recovery events.
stress after 8 days, and in Río Grande in cultivars under The mesophyll conductance (gmes), that is, the conductance
severe stress after 8 days. Table 1 shows correlations for CO2 diffusion from the surface of mesophyll cell walls to
obtained at moderate stress (RI) and severe stress (RII) after the chloroplast stroma via plasma membranes, cytoplasm and
8 days of rehydration of both cultivars. Significant chloroplast envelopes under water deficit, could change
correlations in both cultivars were obtained for protein drastically. The partial recovery observed in leaf water
content and Rubisco activity with leaf relative water content, potential and leaf area (water deficit independent/time
leaf water potential and leaf osmotic potential at severe water dependent response) could be due to the fact that leaf water
deficit, and for stomatal conductance with leaf osmotic potential depends on its components: leaf osmotic potential
potential at moderate and severe water deficit. Similarly in and turgor potential, and on tissue rigidity. Leaf area is
Pera Quibor at severe stress, stomatal conductance correlated dependent on leaf cell expansion, which is also affected by
with leaf relative water content and leaf water potential, and turgor potential. With regards to cell expansion it has been
in Río Grande at moderate stress there was correlation of reported that in pea plants under water deficit, it could be the
Rubisco with leaf relative water content and soil water cause of changes in the area of leaves experiencing a delayed
potential. Table 2 shows recovery of the responses common development after stress (Lecour et al, 1995). It was also
to both cultivars, and the ones that are different between reported that leaf expansion and development were nearly
them. The observed responses include: water deficit halted during water deficit in advanced-stage leaves, being
independent/time independent responses; water deficit development solely due to cell expansion; this expansion is
independent/time dependent responses; and water deficit resumed after re-watering, but not sufficiently for cell size to
dependent/time dependent responses. equal that of controls at maturity (Alves and Setter, 2004). In
C3 plants leaf photosynthetic rate depends on photosynthetic
Discussion components contents, such as Rubisco, cytochrome f, H+-
ATPase and reaction centres, among others, but also on
During the experimentation period, different recovery structural parameters, such as leaf thickness and area per leaf
responses were observed (Table 2); the observed responses mass.The sclerophylly indices such as specific leaf area and
common to, and different in both cultivars include: water leaf density have been reported extensively in water deficit
deficit independent/time independent responses (recovery is studies and in drought tolerant plant varieties or species.
not influenced by water deficit intensity, moderate or severe, There have been many reports on reduced specific leaf area
and was observed at day 1 and day 8 after rehydration); water under drought conditions (Guerfel et al. 2009; Bacelar et al.
deficit independent/time dependent responses (recovery is not 2006 ; Nautiyal et al, 2002). Moreover, Ennajeh et al (2010)
affected by water deficit intensity, moderate or severe, and reported that under water deficit conditions, a drought-
was observed only at day 8 after rehydration; water deficit resistant olive cultivar maintained higher rates of photo-

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Fig 3 .Soluble protein content (a) (mg g-1DM) and Rubisco (carboxylase activity) (b) (μmol 14CO2mg-1DM) values in two tomato
cultivars, Rio Grande (RG) and Pera Quibor (PQ), after moderate (WDI) and severe (WDII) water deficit levels, and two rehydration
levels (RI and RI1): RI 1d and RI1 1d, one day after rehydration; RI 8d and RI1 8d, eight days after rehydration and control values
(C); values are average ± SE (n=6). *Mean is significantly lower from control (P=0.05)

synthetic assimilation and lower rates of transpiration due to intended to protect plant cultivars from intermittent changes
leaf morpho-anatomical adaptations to drought stress. Leaves in water conditions.
with high leaf density improve their survival rate during
severe drought because of a higher resistance to physical Acknowledgements
damage by desiccation (Mediavilla et al, 2001). The
significant correlations (Table 2) of leaf water potential, leaf Financial support was provided by Decanato de
relative water content, and leaf osmotic potential with protein Investigaciones y Desarrollo, Simón Bolívar University.
content and Rubisco at day 8 of rehydration, after severe Seeds were supplied by INIA (National Institute for
stress, revealed a close relationship of these parameters on Agricultural and Animal Husbandry Research, Maracay,
recovery. Results of response to rehydration after short Venezuela).
periods of time have been previously reported. The present
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