Annals of Applied Biology ISSN 0003-4746

RESEARCH ARTICLE

Antioxidant system is essential to increase drought tolerance

of sugarcane
R.D. Vilela1 , B.K.L. Bezerra1 , A. Froehlich2 & L. Endres1
1 Universidade Federal de Alagoas, Centro de Ciencias Agraria, Maceio, Alagoas, Brazil
2 Instituto Federal de Educação, Ciência e Tecnologia de Alagoas, Campus Satuba, Alagoas, Brazil

Keywords Abstract
Antioxidant activity; photoinhibition; ROS;
Saccharum; water stress. Drought is one of the main factors affecting the productivity of agricultural
crops, and plants respond to such stress by activating various physiological and
Correspondence
biochemical mechanisms against dehydration. The present study investigated
L. Endres, Universidade Federal de Alagoas,
Centro de Ciências Agraria, Maceio 57072-900,
two varieties of sugarcane (Saccharum spp.) with contrasting responses to
Alagoas, Brazil. Email: drought (RB867515, more tolerant; and RB855536, less tolerant) and subjected
lauricioendres@hotmail.com them to progressive drought conditions (2, 4, 6 and 8 days) followed by
rehydration. Drought caused a decrease in water potential (𝜓 w ) and osmotic
Received: 21 June 2016; revised version
potential (𝜓 os ) in the leaves, which recovered to normal levels after rehydration
accepted: 24 July 2017.
only up to the fourth day of drought. Water stress changed the carbon
doi:10.1111/aab.12387 metabolism of leaves by reducing starch and sucrose contents and increasing
glucose and fructose contents in both varieties. Water deficit caused a significant
reduction in the maximum quantum efficiency of photosystem II (Fv /Fm )
and effective quantum yield (ΦPSII ) in both varieties; however, RB867515
recovered faster after rehydration. Under water stress, the more tolerant
variety RB867515 exhibited increased activity of the antioxidant enzymes
catalase, ascorbate peroxidase and superoxide dismutase compared with the
RB855536 variety. The results suggest that RB867515 is more tolerant to
drought conditions because of a more efficient antioxidant system, which
results in reduced photosynthesis photoinhibition during water stress, thus
revealing itself as a potential physiological marker for drought tolerance studies.

Introduction and 2500 mm during their growth cycle (Doorenbos &

Sugarcane (Saccharum spp.) is one of the crops of greater
economic importance in the world. Brazil is the largest
cane producer with a planted area of 8.9 million hectares
and estimate production for the harvest (2015/2016) of
650 million tons, mainly for sugar and ethanol produc-
tion (CONAB, 2015). Sugarcane production continuously
increased and has gained featured as a feedstock for
second generation ethanol, considered a cleaner energy
source, compared to fossil fuels (Aguiar et al., 2016).
Among the features of culture, there is the fact that
sugarcane has C4 metabolism, it is well adapted to
tropical regions and has high efficiency of light energy
conversion into biomass (Endres et al., 2010). Neverthe-
less, to demonstrate its maximum production potential,
sugarcane requires favourable environmental conditions,
among which, the availability of water between 1200
Kassam, 1979; Singh et al., 2007). Ensuring this water
demand under field conditions has been a problem since
most farming areas suffer from seasonal droughts (Chaves
et al., 2002).
Water stress is one of the limiting factors for the devel-
opment and productivity of agricultural crops. Productiv-
ity losses caused by water scarcity exceed 60% for several
crops (Boyer, 1982; Ortolani & Camargo, 1987). In sug-
arcane, the damage is even more significant, especially
in the early developmental stages (Machado et al., 2009),
and it causes a reduction in biomass, disrupts the growth
of stems and causes a drastic reduction in productivity
(Rodrigues, 1995).
To perceive drought, the plants trigger a num-
ber of molecular, biochemical and physiological
responses, which develop under such condition, adaptive

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Antioxidant system is essential to increase drought tolerance of sugarcane
mechanisms that vary with the intensity, stress time,
interaction with other types of stress, development stage
and genotype (Bohnert & Jensen, 1996; Bray, 1997).
There are several studies reporting the physiological
and biochemical differences between sugarcane varieties
when subjected to drought (Inman-Bamber & Smith,
2005; Smit & Singels, 2006) including decrease in the
maximum quantum efficiency of photosystem II (Silva
et al., 2007; Silva et al., 2011; Medeiros et al., 2013),
reducing the relative water content (Graça et al., 2010;
Zhao et al., 2010; Silva et al., 2013), drop in photosyn-
thesis and changes in gas exchange (Medeiros et al.,
2013; Santos et al., 2014), reducing the osmotic potential
(Molinari et al., 2007) and increased activity of antioxi-
dant enzymes (Begcy et al., 2012; Cia et al., 2012). Such
parameters have been sensitive in order to characterise
the response of sugarcane to drought, and has helped to
understand how the cane adapts and tolerates drought,
but the accumulation of information is still small when
compared to other economically important crops as soy-
bean, rice and maize (Bruce et al., 2002; Sinclair et al.,
2007; Farooq et al., 2009).
Although there is a great demand for the development
of varieties that are more tolerant to dehydration, sugar-
cane breeding programmes have not yet produced geno-
types with good productive performance under moderate
drought conditions, a very common condition in Brazil-
ian sugarcane fields and also sugarcane fields around the
world. Thus, a comparative analysis of sugarcane varieties
that have varying degrees of tolerance to water deficit
may reveal differences in physiological and biochemical
responses to help determine the mechanisms of drought
tolerance in plants.
The present study aimed to physiologically characterise
the responses to drought of two sugarcane varieties with
different degrees of water deficit tolerance, with the goal
of contributing information related to the drought toler-
ance of sugarcane.
Materials and methods
Growing conditions
Two varieties of sugarcane (Saccharum spp.) with contrast-
ing responses to drought were used, with the RB867515
variety considered more tolerant and RB855536 variety
considered less tolerant to drought. The tolerance of these
varieties to drought was based on producer reports.
Pots containing 22.0 kg broken-up and sieved soil in
a greenhouse under natural light were prepared. Four
single-bud billets were planted in each pot. At 22 days
after planting, thinning was performed, and the two
plants that exhibited the best development were selected.
2 Ann Appl Biol (2017)
R.D. Vilela et al.
Until the beginning of treatment, the soil was maintained
at field capacity.
The moisture in the pots was measured using an
SM200 sensor coupled to an HH2 moisture meter (Delta-T
Devices, Cambridge, England). The maximum, mean and
minimum temperatures, photosynthetically active radi-
ation (PAR) and vapour pressure deficit (VPD) were
recorded by an automatic WS GP1 AT weather station
(Delta-T Devices) installed inside the greenhouse (Fig. 1).
Experimental design and statistical analysis
At 109 days after planting, two varieties (factor 1) were
submitted to the following stress situations (factor 2):
1 Control plants, well-watered;
2 2 days of drought;
3 4 days of drought;
4 6 days of drought;
5 8 days of drought;
6 2 days of drought, followed by 2 days of rewatering
recovery;
7 4 days of drought, followed by 2 days of rewatering
recovery;
8 6 days of drought, followed by 2 days of rewatering
recovery;
9 8 days of drought, followed by 2 days of rewatering
recovery.
Experimental design was completely randomised, with
four replications and the experimental unit was a pot.
Physiological analyses were performed for each repli-
cation (pot). All data were subjected to analysis of
variance (ANOVA) with the factor variety (2: RB867515,
RB855536) and factor stress condition (9: treatments
1–9) to examine significant main effects or interac-
tions between factors (Table 1). Before the analysis,
ANOVA assumptions were tested with Levene’s test for
homoscedasticity and the Shapiro–Wilk’s test for nor-
mality. The test indicated that ANOVA assumptions were
not violated.
Physiological analyses
The age of the leaves chosen for physiological measure-
ments was standardised after counting the leaves starting
at the apex according to the classification described by
Moore (1987). Thus, the +1 leaf was considered the first
leaf with visible ligule, etc.
Assessment of photochemical efficiency
Photoinhibition was evaluated at between 4 and 5 h
and 11 and 12 h by measuring the maximum quantum
© 2017 Association of Applied Biologists
R.D. Vilela et al. Antioxidant system is essential to increase drought tolerance of sugarcane

A 45 Maximum B
Average
Minimum
Air Temperature °C 40

35

30

25

20

C D
12

10

8
MJ/m2/day

E F
4.0
3.5
3.0
DPV (KPa)

2.5
2.0
1.5
1.0
0.5
0.0
0 20 40 60 80 100 120 0 2 4 6 8 10
Time (days) Time (days)

Figure 1 Minimum, average and maximum temperature, photosynthetically active radiation and vapour pressure deficit during normal plant growth and during
drought registered inside the greenhouse during the experimental period.

efficiency of PSII (Fv /Fm ), which was obtained with
a portable modulated light fluorometer (Opti-Sciences,
OS1-FL, Hudson, NH, USA) according to Maxwell &
Johnson (2000).
The effective quantum yield of PSII (Yield) was deter-
mined at 10:00 h under ambient light. Two readings of the
middle third of the +1 leaf per plant were performed.
Leaf water potential
The leaf water potential (𝜓 w ) was determined at 04:30 h
in the +2 leaf using a Scholander pressure pump
(Soilmoisture Equipment Corporation, Santa Barbara,
CA, USA). The leaves were cut at the ligule and analysed
immediately after collection to measure the 𝜓 w .
Osmotic potential
After quantifying the 𝜓 w , the same leaf was used to
quantify the osmotic potential 𝜓 os . The tissue of the
middle third of the leaf, without the midrib, was pressed
to extract the cell sap, which was stored in 1.5 mL tubes at
−20∘ C. An aliquot of 10 μL was used to quantify the 𝜓 os in
a VAPRO 5520 vapour pressure osmometer (Wescor, Inc.
Logan, UT, USA).

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Antioxidant system is essential to increase drought tolerance of sugarcane R.D. Vilela et al.

Table 1 Analysis of variance, mean square (MS) and P value for physiological characteristics of sugarcane predawn and midday maximum quantum efficiency
of photosystem II (Fv /Fm ); effective quantum yield of PSII (ΦPSII ); leaf sucrose, glucose, fructose and starch; enzymatic activity of catalase (CAT), and superoxide
dismutase (SOD), and ascorbate peroxidase (APX)

Variety (V) Treatment (T) V×T

Source of
variation df MS F Prob. > F df MS F Prob. > F df MS F Prob. > F Error

Predawn Fv /Fm 1 0.000 0.091 0.764 8 0.047 15.487 <0.001 8 0.001 0.282 0.969 0.003
Midday Fv /Fm 1 0.003 0.670 0.417 8 0.051 11.845 <0.001 8 0.004 0.846 0.567 0.004
ΦPSII 1 0.033 3.976 0.051 8 0.183 22.330 <0.001 8 0.035 4.326 <0.001 0.008
Sucrose 1 11,195.757 200.044 <0.001 8 5,840.235 104.352 <0.001 8 1,488.401 26.595 <0.001 55.966
Glucose 1 583.617 57.773 <0.001 8 744.955 73.745 <0.001 8 178.872 17.707 <0.001 10.102
Fructose 1 7.871 1.019 0.317 8 1,269.701 164.358 <0.001 8 124.403 16.104 <0.001 7.725
Starch 1 2,183.259 68.824 <0.001 8 512.660 16.161 <0.001 8 550.729 17.361 <0.001 31.722
Enzyme CAT 1 0.001 4.743 0.034 8 0.002 8.221 <0.001 8 0.000 0.730 0.664 <0.001
Enzyme SOD 1 0.681 0.519 0.474 8 28.756 21.908 <0.001 8 11.674 8.894 <0.001 1.313
Enzyme APX 1 0.628 58.686 <0.001 8 0.138 12.859 <0.001 8 0.076 7.138 <0.001 0.011

Carbohydrates assessment reaction medium contained 50 mM TFK buffer (pH 7.5),

Leaf samples from the middle third of the +1 leaf were
collected at midday, immediately frozen in liquid nitro-
gen and stored at −70∘ C. The extraction of sugar and
starch was performed according to Lisec et al. (2006) using
approximately 10 mg of lyophilized leaf tissue. The lev-
els of glucose, fructose and sucrose were determined
enzymatically according to Stitt et al. (1989) using an
enzyme immunoassay reader (ELISA VersaMax, Molec-
ular Devices, Sunnyvale, California, USA) at 340 nm. The
starch levels were measured first by incubating an aliquot
of the starch extract at 55∘ C for 60 min with 30 μL of
300 mM citrate buffer (pH 4.6) and 2.0 U amyloglucosi-
dase (EC 3.2.1.3) in a final volume of 300 μL. The starch
was measured enzymatically using the glucose released
from the starch (Antunes et al., 2012).
Antioxidant enzymes
Catalase and ascorbate peroxidase. Samples of the middle
third of the +1 leaf were collected at midday, immedi-
ately frozen in liquid nitrogen and stored at −70∘ C. Cata-
lase (CAT) and ascorbate peroxidase (APX) extraction fol-
lowed the method described by Havir & McHale (1987)
with modifications. Briefly, approximately 30 mg fresh
leaf material was manually macerated in 2 mL 50 mM
potassium phosphate buffer (pH 7.5), 2 mM ethylenedi-
aminetetraacetic acid (EDTA), 20 mM ascorbate, 0.1%
Triton X-100, 1% polyvinylpolypyrrolidone (w/v) and
deionised H2 O, and the sample was then centrifuged for
15 min at 14 000 g at 4∘ C.
To determine the CAT activity, the samples were main-
tained at 4∘ C. The reaction was quantified using an
ultraviolet (UV) spectrophotometer (Genesys 10, Thermo
Scientific, Madison, WI, USA) for 1 min at 240 nm. The
12.5 mM H2 O2 and 20 μL of the extract.
The APX activity was determined according to Nakano
& Asada (1981). The reaction medium used in the analysis
consisted of 50 mM TFK buffer (pH 7.5), 0.1 mM H2 O2 ,
0.5 mM sodium ascorbate and 25 μL extract. The reaction
was quantified using a UV spectrophotometer for 1 min at
290 nm.
Superoxide dismutase. The method described by Gian-
nopolitis & Ries (1977) with modifications was used
for the extraction and determination of superoxide
dismutase (SOD) activity. Initially, 50 mg of the sam-
ple were extracted in buffer [100 mM TFK (pH 6.8),
0.1 mM EDTA, 1.0 mM dithiothreitol (DTT), 0.1%
Triton X-100, 1.0 mM phenylmethylsulfonyl fluoride
(PMSF), 1% polyvinylpolypyrrolidone (PVPP) (w/v)
and deionised H2 O] and subsequently centrifuged
for 15 min at 14 000 g at 4∘ C. The SOD activity was
determined using reaction medium [50 mM TFK
(pH 7.8), 13.0 mM methionine, 75.0 𝜇M nitro blue
tetrazolium (NBT), 0.1 mM EDTA, 2.0 𝜇M riboflavin,
and deionised H2 O] and 10 μL enzyme extract. The
reaction was measured at 560 nm using an enzyme
immunoassay reader (ELISA VersaMax, Molecular
Devices).
Results
Environmental conditions
During the experimental period, the mean temperature
inside the greenhouse was approximately 28∘ C; how-
ever, there was a slight increase in the mean temperature

4 Ann Appl Biol (2017)

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R.D. Vilela et al. Antioxidant system is essential to increase drought tolerance of sugarcane

0.18 RB867515 Leaf water potential (𝜓 w ) and osmotic potential (𝜓 os )

0.16 RB855536
0.14
0.12
The suspension of irrigation caused a similar reduction in
m-3.m-3

0.10 Well-watered the 𝜓 w in both varieties on the second and fourth day
0.08 Drought-stressed
0.06 compared with that of the control (Fig. 3). Starting at
0.04 the fourth day of drought, the 𝜓 w could not be quan-
0.02
0.00 tified in the dehydrated plants due to severe drought.
2 4 6 8 10 2 4 6 8 10
Time (days) Time (days) After rehydration, variety RB867515 recovered its 𝜓 w
compared with plants hydrated up to the fourth day of
stress, whereas this behaviour was not observed for vari-
Figure 2 Volumetric soil moisture of soil in vases during sugarcane growth to
109 days old and then during irrigation suspension in the sugarcane varieties ety RB855536. Neither variety presented a recovery of
RB855536 and RB867515 under drought-stressed. The bars represent the 𝜓 w relative to the values found for the control when sub-
standard error of the mean (n = 6). jected to more than 4 days of stress.
The leaf 𝜓 os of the more tolerant variety RB867515
decreased and remained more negative than the control
when subjected to water deficit for 4 days or more, how-
(±30∘ C) and strong increase in the maximum tempera- ever, significant changes were not observed in the more
ture (±43∘ C) during sample collection (Fig. 1). The inter- sensitive variety (Fig. 3C and Fig. 3D).
cepted PAR was high throughout the period of vegetative
growth (Fig. 1C and Fig. 1D). The VPD was also high,
especially during the sample collection period (Fig. 1E
Photochemical efficiency
and Fig. 1F). As an effect of this, soil moisture decreased
rapidly after irrigation was suspended, and decreases were The photoinhibition was observed after 2 days of drought
observed until the sixth day of drought in both varieties in the RB867515 variety and 4 days of drought in the
and then followed by a small change until the 10th day of RB855536 variety (Fig. 4A–D). Although RB867515
water restriction (Fig. 2). underwent a larger drop in Fv /Fm under stress, the plants

Time (days) Time (days)

A 0 2 4 6 8 10 B 0 2 4 6 8 10
0.0
-0.1
-0.2
-0.3
ψw MPa

-0.4
-0.5
-0.6
Well-watered
-0.7
Drought-stressed
-0.8 Afer 2 days of recovery

C D
0 2 4 6 8 10 0 2 4 6 8 10
-0.4
-0.5
-0.6
ψos MPa

-0.7
-0.8
-0.9
RB855536 RB867515
-1.0

Figure 3 Leaf water potential (𝜓 w ) (A and B) and leaf osmotic potential (𝜓 os ) (C and D) at predawn of sugarcane varieties RB855536 (A and C) and RB867515
(B and D) of 109 days old, submitted to drought-stressed and recovery.

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Antioxidant system is essential to increase drought tolerance of sugarcane R.D. Vilela et al.

A B
0.85
RB855536 RB867515
0.80
0.75
Fv/Fm - Predawn
0.70
0.65
0.60 Well-watered
0.55 Drought-stressed
After 2 days of recovery
0.50
0 2 4 6 8 10 0 2 4 6 8 10

C D
0.90
0.80
0.70
Fv/Fm - Midday

0.60
0.50
0.40
0.30
0.20
0 2 4 6 8 10 0 2 4 6 8 10
E F
0.80
0.70
0.60
Φ PSII - 10h

0.50
0.40
0.30
0.20
0.10
0 2 4 6 8 10 0 2 4 6 8 10
Time (days) Time (days)

Figure 4 Maximum quantum efficiency of PSII (Fv /Fm ) at predawn (A and B) and midday (C and D) and effective quantum yield of PSII (ΦPSII ) (E and F) of sugarcane
varieties RB855536 (A, C and E) and RB867515 (B, D and F) of 109 days old, submitted to drought-stressed and recovery.

recovered better in the investigated recovery period of
2 days, especially at midday (Fig. 4C and Fig. 4D).
The effective yield (ΦPSII ) of PSII was also severely
affected by the water deficit, reaching an average reduc-
tion of 61% in both varieties on the eighth-day dry
(Fig. 4E and Fig. 4F). However, after rehydration, only
variety RB867515 was capable of full recovery, even
when subjected to severe stress.
Carbohydrate evaluation
The more drought-tolerant variety RB867515 exhibited a
24% greater starch content in the leaves compared with
the less tolerant variety under normal growing condi-
tions. Under water deficit, this variety underwent a major
change in starch composition at the beginning of stress,
and this change was less intense in RB855536 (Fig. 5A
and Fig. 5B). The water deficit drastically reduced the
concentration of sucrose in the leaves of the two varieties,
with a trend towards recovery of the original content after
rehydration (Fig. 5C and Fig. 5D). This recovery was less
intense in plants under severe stress, and the more toler-
ant variety displayed a 55% decline in the concentration
of sucrose with 4 days of water restriction (Fig. 5D). Over-
all, the decrease in this carbohydrate was higher in the
more tolerant variety RB867515 compared with the less
tolerant variety RB855536.
Compared with the decrease in sucrose levels, increases
in the glucose and fructose contents were observed in
both varieties in response to dehydration (Fig. 5E–H).
The content of leaf glucose reached 39.52 mmol kg−1 in
the less tolerant variety and 32.94 mmol kg−1 dry weight
(DW) in the more tolerant variety with 6 days of water
restriction (Fig. 5E and Fig. 5F). With 4 days of drought,
greater fructose accumulation at 37.99 and 47.74 mmol
kg−1 DW in the less tolerant and more tolerant vari-
eties, respectively (Fig. 5G and Fig. 5H). After rehydra-
tion of the plants, the leaf glucose and fructose contents

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R.D. Vilela et al. Antioxidant system is essential to increase drought tolerance of sugarcane

A RB855536 B RB867515
Starch mg g-1 DW 50.0 50.0

40.0 40.0

Starch mg g-1
30.0 30.0

20.0 20.0

10.0 10.0

0.0 0.0
0 2 4 6 8 10 0 2 4 6 8 10
Time (days) Time (days)
C RB855536 D Well-watered
RB867515
Drought-stressed
140 140 After 2 days of recovery
120 120
mmol Kg-1 DW

mmol Kg-1 DW
Sucrose level

100

Sucrose level
100
80 80
60 60
40 40
20 20
0 0
0 2 4 6 8 10 0 2 4 6 8 10
Time (days)
E RB855536 F RB867515
50 50

40 40
mmol Kg-1 DW

mmol Kg-1 DW
Glucose level

Glucose level

30 30

20 20

10 10

0 0
0 2 4 6 8 10 0 2 4 6 8 10
Time (days) Time (days)
G RB855536 H RB867515
60 60
50 50
mmol Kg-1 DW

mmol Kg-1 DW
Fructose level

Fructose level

40 40
30 30
20 20
10 10
0 0
0 2 4 6 8 10 0 2 4 6 8 10
Time (days) Time (days)

Figure 5 Starch levels (A and B), sucrose (C and D), glucose (E and F) and fructose (G and H) of sugarcane varieties RB855536 (A, C, E and G) and RB867515
(B, D, F and H) of 109 days old, submitted to drought-stressed and recovery.

exhibited a downward trend that was more irregular in
the less tolerant variety RB855536 (Fig. 5E and Fig. 5G).
Activity of antioxidant enzymes
The activity of all antioxidant enzymes increased after
the start of water deficit conditions in both varieties,
and the activity tended towards stabilisation after rehy-
dration (Fig. 6). However, the activity of these enzymes
appeared to be more efficient in the tolerant variety
RB867515. For example, CAT activity was 15% higher
in RB867515, compared to the less tolerant variety
RB855536 under the same conditions (Fig. 6A and
Fig. 6B). Even after 8 days of drought, CAT activity

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Antioxidant system is essential to increase drought tolerance of sugarcane R.D. Vilela et al.

A RB 855536 B RB867515
0.14

u CAT min-1mg-1 protein

0.12
0.10
0.08
0.06
Well-watered
0.04
Drought-stressed
0.02 After 2 days of recovery
0.00
0 2 4 6 8 10 0 2 4 6 8 10

C D
1.2
u APX min-1 mg-1 protein

1.0
0.8
0.6
0.4
0.2
0.0
0 2 4 6 8 10 0 2 4 6 8 10

E F
10
u SOD min-1 mg-1 protein

0
0 2 4 6 8 10 0 2 4 6 8 10
Time (days) Time (days)

Figure 6 Catalase (A and B), ascorbate peroxidase (C and D) and superoxide dismutase (E and F) of sugarcane varieties RB855536 (A, C and E) and RB867515
(B, D and F) of 109 days old, submitted to drought-stressed and recovery.

returned to normal after rehydration in both analysed
varieties.
An improved APX activity response was observed in
the tolerant variety relative to that of the less tolerant
variety in response to drought (Fig. 6C and Fig. 6D). With
2 days of water deficit, the APX activity in the RB867515
variety was four times higher than that of the control.
Even after rehydration, the APX activity remained slightly
higher compared with the control plants in this variety,
whereas the same behaviour was not observed in the
more sensitive variety (Fig. 6C and Fig. 6D).
The SOD activity was stimulated by the water deficit
(Fig. 6E and Fig. 6F). In the most tolerant variety
RB867515, the increase in activity was gradual with
increased of drought, with the maximum activity (73%
increase) observed at 8 days of stress, whereas a peak in
enzyme activity (66% increase) occurred at 4 days with
the more sensitive variety after suspension of irrigation,
with values decreasing thereafter. Overall, the enzyme
activity decreased after rehydration.
General linear model is presented in Table 1. Analysis is
presented comparing time of drought, recovery and their
interactions on Chlorophyll fluorescence of PSII, carbo-
hydrate levels and antioxidant activity of both varieties.
Discussion
The reduction of 𝜓 w is an early event of plants in response
to drought (Silva et al., 2007). Two sugarcane varieties
exhibited a decline in predawn leaf 𝜓 w and 𝜓 o starting
at the beginning of water stress and recovery until the
fourth day of drought. Such behaviour of sugarcane, has

8 Ann Appl Biol (2017)

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R.D. Vilela et al.
been reported in other studies, Medeiros et al. (2013), for
example, noted a greater reduction in predawn leaf 𝜓 w
in RB867515 variety, followed by a recovery to normal
level after rehydration. Saliendra & Meinzer (1991) pro-
posed 𝜓 w values of −0.8 to −0.9 MPa as being critical
to the growth of leaves and stomatal aperture of sugar-
cane. Following this criterion, the 𝜓 w of both analysed
varieties recover after 8 days of drought, presenting −0.4
and −0.49 MPa in RB855536 and RB867515, respectively;
however, 𝜓 w did not reach the plants values under normal
conditions.
The decrease in 𝜓 w was also accompanied by a decline
in 𝜓 o in both varieties. This reaction of the plant before the
drought, is a strategy to keep the water absorption, and
minimise the effect of water deficit, and may be related to
increased tolerance to dehydration (Blum, 2005). After
rehydration, the more tolerant variety exhibited lower
𝜓 o than RB855536 (less tolerant variety) starting at the
sixth day of drought. According with Inman-Bamber &
Smith (2005) and Molinari et al. (2007), who noted the
same osmo-regulator effect on varieties of sugarcane in
response to dehydration, evidenced by a reduction in
osmotic potential and accompanied by a decrease in water
potential.
The drought also aggravates photoinhibition, especially
when accompanied by high irradiance (Galmés et al.,
2007). This photosynthesis process is slow and reversible
due to intense sun exposure (Dias & Marenco, 2007)
and can be dynamic or chronic, in the first case, pho-
tosynthesis recovers radiative stress in the late after-
noon with the decrease of the irradiance, already in
chronic photoinhibition, the PSII takes up to 12 h to
recover. In both varieties, the chronic photoinhibition
was evidenced by the reduction in Fv /Fm at predawn and
low values near midday. A leaf with intact photochem-
ical apparatus exhibits an Fv /Fm ratio of approximately
0.75–0.85, and a reduction in these values may indi-
cate photoinhibition (Bolhàr-Nordenkampf et al., 1989).
Already, second Björkman & Demmig (1987), the Fv /Fm
ratio of a plant without photoinhibition ranges from 0.8
to 0.83 𝜇mol m−2 s−1 .
The ratio Fv /Fm at midday was significantly affected
by drought, the more tolerant variety, RB867515, had
minor decrease in this parameter, around 0.48 compared
to more sensitive variety which showed 0.40 with 8 days
of drought. Other studies reported a greater reduction
in the ratio Fv /Fm of cane varieties with lower toler-
ance to water deficit (Gonçalves et al., 2010; Chen et al.,
2011; Silva et al., 2011; Silva et al., 2014) concluded that
this analysis was effective in the detection of photoin-
hibition in this culture. Santos et al. (2014), differently,
obtained higher values of Fv /Fm after the drought, around
0.6 in sugarcane varieties that showed greater reduction
Ann Appl Biol (2017)
© 2017 Association of Applied Biologists
Antioxidant system is essential to increase drought tolerance of sugarcane
parameter at 1000 h. The photochemical data shows that
the plants were tested in extremely limiting conditions,
and RB867515 variety had higher photochemical effi-
ciency, and probably best dissipation of excitation energy,
essential to avoid the formation of reactive oxygen species
and greater damage to PSII (Müller et al., 2001), associ-
ated with the antioxidant system. After 2 days of rehydra-
tion, the plants showed a recovery until the fourth day
of drought; after this period, the plants were unable to
recover from chronic photoinhibition.
In contrast, Santos et al. (2014) noted the recovery of
Fv /Fm at the control level in sugarcane after 12 days of
rehydration. This study, in contrast, demonstrates that
in a short time, 2 days of rehydration, sugarcane already
presents a considerable improvement in the photochem-
ical system. Already in eudicotyledonous species, such
as beans, Miyashita et al., 2005 found there is a rapid
decrease in rate Fv /Fm under drought and poor recovery
after rehydration.
The effective quantum yield of PSII (ΦPSII ) is well cor-
related with carbon assimilation and reflects the photo-
synthetic activity of the leaf (Maxwell & Johnson, 2000).
Both varieties exhibited a reduction in ΦPSII after drought.
However, only the more tolerant variety, RB867515,
showed a fully recovered ΦPSII after rehydration.
The ΦPSII represents the light harvesting efficiency and
excitation of the PSII reaction centre (Genty et al., 1990;
Maxwell & Johnson, 2000). In the present study, the rapid
response of the variety more tolerant to rehydration may
have been related to its greater drought tolerance relative
to the less tolerant variety. Zhao et al. (2010) reported a
decline in ΦPSII in sugarcane under drought conditions
and concluded that this parameter is useful in the early
detection of water stress in this crop. The same response
was obtained by Colom & Vazzana (2003), who observed
a reduction in ΦPSII and Fv /Fm in the grass Eragrostis curvula
after water deficit.
Drought conditions also dramatically affected the car-
bohydrate contents of both varieties. The sucrose concen-
tration in RB867515, the more tolerant variety, showed a
16% greater decrease compared with that of RB855536,
the less drought-tolerant variety. Invertase most likely
serves as a key factor in decreasing the sucrose content
(Castrillo, 1992; Lawlor & Cornic, 2002) because inver-
tase hydrolyzes sucrose into hexoses to provide energy to
cells to conduct various metabolic activities (Leite et al.,
2009), including maintenance of the biological processes
involved in tolerance to dehydration. Vu & Allen (2009)
emphasise that increases in soluble carbohydrate levels is
a common response to drought.
Reducing the sucrose levels is an important osmotic
adjustment mechanism in response to water stress
(Castrillo, 1992; Lawlor & Cornic, 2002). The decrease
9
Antioxidant system is essential to increase drought tolerance of sugarcane
of this carbohydrate appears to be related to the biggest
drop shown in osmotic potential of the more tolerant
variety, RB867515, and it may have provided adaptive
advantages to it. The increased hexose levels were cor-
related with decreased sucrose and starch contents in
both varieties, which is consistent with the results of Vu
& Allen (2009), who observed an increase in glucose
and fructose levels associated with a marked decrease in
starch levels and subtle reduction in sucrose in the leaves
of sugarcane after 13 days of drought. Similarly, Keller
& Ludlow (1993) reported that water stress reduced the
levels of starch and sucrose in the leaves of Cajanus cajan
and increased the levels of glucose and fructose because
of an increase in the activity of the enzymes amylase
and invertase, which are responsible for the hydrolysis of
starch and sucrose, respectively. Liu et al. (2004) obtained
similar results for carbohydrates in the leaves of soybean;
however, they did not report changes in invertase activity.
In contrast, Whittaker et al. (2001) observed that hexose
levels in the leaves of Sporobolus stapfianus decreased and
sucrose levels increased with decreases in the relative
water content.
The increase in the activity of antioxidant enzymes
in response to water deficit has been largely used as an
indicator of drought tolerance (Apel & Hirt, 2004). It
was observed in this study, an increased activity of all
antioxidant enzymes in response to drought, followed by
reduction after rehydration. The activity of CAT and APX
was highest in the RB867515 variety. The SOD showed
similar behaviour in the two varieties.
The CAT is on the front line in the fight against free
radicals, even with lower affinity for H2 O2 . Compared to
APX, CAT activity is essential at the beginning of stress,
it helps the action of other enzymes of the antioxidant
system reducing the action of reactive oxygen species
(Mhamdi et al., 2010). Several studies have related higher
CAT activity in drought tolerant varieties, Osipova et al.
(2011) reported differences in the activities of CAT in
wheat genotypes subjected to water deficit and associ-
ated the increased activity of this enzyme with increased
drought tolerance. So, in sugarcane we can suggest that
the greater activity of CAT in RB867515 may be indicative
of increased drought-tolerance in this variety. Boaretto
et al. (2014) also observed differences in CAT activity in
sugarcane varieties with contrasting responses to drought.
For these authors, the integrated action of the enzymes
glutathione reductase, CAT and SOD in the more toler-
ant variety were an important system of drought stress
tolerance.
The antioxidant activity of APX was 34% higher in
the more tolerant variety compared with the less toler-
ant variety in response to dehydration. These results cor-
roborate those of Sairam et al. (1998), who reported that
10
R.D. Vilela et al.
the activity of CAT and APX was higher after drought
in the more tolerant wheat genotype compared with the
less tolerant variety. Cia et al. (2012) compared sugarcane
varieties that were contrasting in drought tolerance and
observed an increase in the activity of the enzymes SOD
and CAT in the tolerant variety with imposed drought
stress. This result was also observed in the present study.
The SOD is the first enzyme to act in detoxification
against superoxide radicals (Alscher et al., 2002). The
rapid response of this enzyme along with others of antiox-
idant systems are crucial in the development and pro-
ductivity of the crops (Gill & Tuteja, 2010), as is the
case of sugarcane, largely grown in areas susceptible to
drought. The SOD activity was slightly higher in the vari-
ety RB867515 during dehydration, and similar results
were found by Chugh et al. (2011), who observed that
SOD activity was slightly higher in tolerant maize geno-
types after water deficit, significant inhibition of SOD
activity was observed in the less tolerant variety. However,
Sairam & Srivastava (2001) reported that SOD activity
was similar between wheat genotypes with contrasting
drought tolerance. The SOD and APX are two enzymes of
action in chloroplasts, it is likely that both acted synergis-
tically in detoxification against ROS in RB867515 variety,
promoting greater recovery of Fv /Fm with only 2 days of
rehydration.
Conclusion
Carbohydrate metabolism is seriously affected by water
stress, and decreases in sucrose and increases in glu-
cose and fructose were observed in the leaves. How-
ever, the carbohydrate metabolism of the varieties under
study could not be differentiated. In contrast, antioxi-
dant activity, which was demonstrated by increases in the
enzymes CAT, APX and SOD, appears to be a more effi-
cient mechanism for maintaining photosynthetic activity
in the drought-tolerant variety RB867515, and it may
have caused a smaller reduction of photoinhibition and
effective quantum yield during the hottest hours of the
day in the drought-tolerant variety compared with that
of the drought-sensitive variety RB855536.
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