J.

Agronomy & Crop Science 190, 256—261 (2004)

2004 Blackwell Verlag, Berlin
ISSN 0931-2250

Instituto Tecnológico de Tuxtla Gutie´rrez, Chiapas, Me´xico

Effects of Partial Defoliation on Sucrose Accumulation, Enzyme Activity and

Agronomic Parameters in Sugar cane (Saccharum spp.)
Dr F. A. Gutiérrez-Miceli, R. Morales-Torres, Y. de Jesús Espinosa-Castañeda, R. Rincón-Rosales, J. Montes-
Molina, M. A. Oliva-Llaven, and Dr L. Dendooven

AuthorsÕ addresses: F. A. Gutiérrez-Miceli, R. Morales-Torres, Y. de Jesús Espinosa-Castañeda, R. Rincón-Rosales, J. Montes-

Molina, M. A. Oliva-Llaven and L. Dendooven (corresponding author; e-mail: dendoove@mail.cinvestav.mx or
lucdendo@prodigy.net.mx), Instituto Tecnológico de Tuxtla Gutiérrez, Carr. Panam km 1080, Tuxtla Gutiérrez, Chiapas CP
29000, México
With 3 figures
Received November 21, 2003; accepted December 15, 2003

Abstract resulting in yields which may exceed 150 mt ha )1

Mechanical defoliation of sugar cane plants (Saccharum
spp.) will provide leaves that can be used as fodder. The
effect of partial mechanical defoliation on sucrose content,
enzyme activities and agronomic parameters of sugar cane
is still unknown. We investigated how sucrose accumula-
tion, activities of sucrose phosphate synthase, soluble acid
invertase, sucrose synthase, neutral invertase, brix grades,
purity grade, moisture content, fibre content, maturity
index and reduced sugars of two commercial sugar cane
plants (Mex 69-290 and Mex 57-473) were affected in a field
experiment. The concentration of sucrose in stems of
partial defoliated plants was not significantly different from
that found in intact plants. Agronomic parameters and
enzyme activities were not different in defoliated plants
compared with intact plants except for the moisture content
which was higher in defoliated plants than in intact ones.
These results indicated that sugar cane plants could be
partially defoliated without changing sucrose production
and agronomic parameters while providing leaves that
could be used as fodder.
Key words: neutral invertase — partial defoliation
— soluble acid invertase — sucrose accumulation
— sucrose phosphate synthase — sucrose synthase
Introduction
Sugar cane is a commercially important crop that
accounts for approximately 65 % of the global sugar
production (Carson and Botha 2002). The factors
that contribute to high yields of sugar are its
perennial growth habit and the continuous accumu-
lation of sucrose in the vegetative plant structure
(Moore 1995). Sugar cane is a C4 plant with carbon
fixation rates as high as 2.8 mg CO2 m)2 s)1 and a
mean total dry matter production of 40 g m)2 day)1
year)1 (Moore and Maretzki 1996). In the USA, the
sugar industry uses glyphosate (N-phosphonomet-
hilglycine), a desiccant, to increase yields of sucrose,
but the response differs between sugar cane varieties
(Martin et al. 1981). Su et al. (1992) reported that
glyphosate significantly reduces the activity of acid
invertase and the concentration of sucrose is thus
higher in the stem. Although glyphosate has been
granted approval as a chemical ripener in sugar cane
by the United States Environmental Protection
Agency since 1975, the defoliated sugar cane leaves
are not used as fodder due to low palatability for
animals (Martin et al. 1981) and possible risks to
animals (Linz et al. 1996) as glyphosate is a non-
selective herbicide with half-life of 47 days (Ahrens
1994).
Sugar cane leaves with an average production
15 mt ha )1 year)1, however, are normally highly
palatable with good intake characteristics for
ruminants so they have a great potential as fodder
(Naseeven 1986). Integrating production of sugar
and using the leaves as fodder will be economically
(Naseeven 1986, Namer 1991) and ecologically
beneficial (Stuart 1988) especially in areas of
Mexico where cost of fodder are high and manual
labour cheap. Pammenter and Allison (2002) found
that sucrose yield per plant was slightly, although
not significantly, greater in half-defoliated sugar
cane plants than in intact ones. Partial defoliation
has resulted in increases in sucrose contents in
Cherry trees (Prunus cereasus) (Layne and Flore
1995), but Neefs et al. (2002) reported that
mechanical partial defoliation of witloof chicory

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Mechanical Defoliation of Saccharum
(Cichorium intybus) affected plant growth. They
found that the total fresh weight of defoliated
plants stayed markedly lower compared with intact
plants. Little is known how partial mechanical
defoliation might affect enzyme activity and thus
sucrose concentration in sugar cane nor how it
might affect plant characteristics and development
so we investigated this in a field experiment with
two commercial 10-month-old sugar cane varieties.
Materials and Methods
Plant material
The sugar cane cultivars Mex 57-473 and Mex 69-290 were
cultivated under field conditions at ÔIngenio La FeÕ located
in Venustiano Carranza, Chiapas, México. These cultivars
were selected as they showed differences in sugar accumu-
lation and sugar yield. Mex 57-473 is characterized by a
large production (140 t ha)1) and is considered of medium
maturity and Mex 69-290 by a medium production
(110 t ha)1) and early maturity. Plants were grown from
vegetative cuttings using standard practices for water and
fertilizer applications (Zhu et al. 1997). Five hundred
kilograms of fertilizer (180, 45, 30 for N, P, K, respectively)
was added 45 days after sugar cane was planted and
another 500 kg of the same fertilizer was added 30 days
after the first application. Sugar cane was planted on
2 December and irrigation trough furrows was applied each
21–25 days. Irrigation frequency increased to one irrigation
each 15 days in March and April, but was suspended
1 month before harvest. Sugar cane plants were grown on
calcareous sandy soil. Plant density was 15 stalks m2 for an
experimental plot of 10 m2. After 10 months, stalk height
of Mex 57-473 was 2.7 ± 0.3 m and internode number
17 ± 4, while stalk height of Mex 69-290 was 3.0 ± 0.2 m
and internode number 15 ± 5. All leaves from 10-month-
old sugar cane plants were removed with a knife except for
the four uppermost leaves.
The experimental design used complete randomized
blocks of 10 · 10 m with 44 plots with defoliated plants
and 44 with undefoliated plants serving as control. After 0,
3, 6, 9, 12, 15, 18, 21, 24, 27 and 30 days, all plants of four
plots were sampled, pooled and analysed for sucrose
content, enzyme activity and agronomic parameters.
Sample preparation
More details of the sample preparation can be found in
Gutiérrez-Miceli et al. (2002a). Briefly, stems were cut into
individual internodes and the three internodes that made
up the middle of the stem were selected. The internode rind
and epidermis were removed and the remaining internode
was weighed and cut lengthwise into four equal pieces. Two
pieces were chopped into small pieces and frozen in liquid
nitrogen before being stored in a freezer until used for sugar
and enzyme assays. The data presented are mean values of
at least three independent extractions.
257
The remaining two pieces were amended with 1 l distilled
water, placed in an industrial blender (Waring Co., Walnut
Creek, CA), and mixed for 5 min at 200 rpm. The sample
was passed through a Whatman no. 4 filter paper and brix
grades, purity grade, reduced sugar and sucrose content
were determined. The fibre retained on the filter was dried
in an oven at 80 C until constant weight and measured.
Enzyme extraction and enzyme assays
Frozen tissue of an internode was weighed and ground to a
fine powder in liquid nitrogen in a chilled mortar. The
extraction and enzyme assays were carried out as described
in Gutiérrez-Miceli et al. (2002b).
Analytical determinations
A 20-ll aliquot of the filtrate was applied to an amino-silica
column with a Varian HPLC (Marian LA, CA) fitted with
an R1-4 refraction index detector and a 4400 integrator to
quantify sucrose and reduced sugars. Acetonitrile : water
(80 : 20) was used as the mobile phase with a flow rate of
1.0 ml min)1.
Protein concentration was determined in dialysed extract
using bovine serum albumin as standard as described by
Bradford (1976). Enzyme activity was calculated and
expressed as micromoles of product formed per gram of
total protein per minute. The data presented are the mean
values of at least four replicates.
Agronomic parameters
Brix grades, fibre content, moisture content, purity grade
and maturity index were determined using methods des-
cribed in ICUMSA (1979). The maturity index was defined
as: [100 · sucrose in sugar cane/(moisture content ·
reduced sugars)].
Statistical analysis
The enzyme activities for soluble acid invertase (SAI),
sucrose synthase (SUSY), neutral invertase (NI) and
sucrose phosphate synthase (SPS), sucrose content, Brix
grades, purity grade, moisture content, fibre content,
maturity index and reduced sugars in defoliated plants
were subjected to one-way analysis of variance to test for
significant differences between defoliated plants and non-
defoliated plants of the two varieties. All analyses were
performed using SAS (SAS Institute 1989).
Results
Sucrose content
The sucrose concentrations in defoliated and intact
plants for both varieties ranged between 11.9
and 15.1 lmol g)1 fresh weight and showed no
258 Gutiérrez-Miceli et al.

(a) (b)
30 30
Enzyme activity (µmol min–1 g–1 protein)

24 24

18 18

12 12

6 6

0 0
0 6 12 18 24 30 0 6 12 18 24 30
(c) (d)
30 30 Fig. 1: (a) Activity of
SUSY (h), SPS (n),
24 24
SAI (s) and NI ()
18 18 (lmol min)1 g)1 protein) in
defoliated plants of the
12 12 variety Mex 69-290 and
6 6
(b) control plants and in
(c) defoliated plants of the
0 0 variety Mex 57-473 and (d)
0 6 12 18 24 30 0 6 12 18 24 30 and control plants. Bars are
plus and minus standard
Time (days) deviation of four replicates

significant changes over time (no data shown). (a)

30
Sucrose concentration was not significantly differ-
ent between the defoliated and the intact plants, 20
nor was there a significant difference between the 10
two varieties.
0

–10
Enzyme activity
Enzyme activity (µmol min–1g–1 protein)

–20
On average, SAI showed the largest activity in –30
defoliated and intact plants of both varieties and
–40
SPS the lowest (Fig. 1a–d). The enzyme activities
for SAI, SUSY, NI and SPS in defoliated plants and 0 3 6 9 12 15 18 21 24 27 30
non-defoliated plants in each of the variety were not
(b)
significantly different (Fig. 1a–d). The activity of 30
SPS ) SAI was not affected by defoliation in both
20
varieties (Fig. 2a), but there was a difference in
patterns for both varieties. From day 3 onwards 10
until day 12, SPS ) SAI was larger in variety Mex 0
57-473 than in variety Mex 69-290, but from day
–10
21 the reverse was observed. The same was found
for (SPS ) SUSY) ) (SAI + NI) (Fig. 2b). –20

–30

Agronomic parameters –40

The maturity index, fibre content, purity grade and 0 3 6 9 12 15 18 21 24 27 30

the Brix grade all increased significantly in time, Time (days)

while reduced sugars and moisture content
decreased significantly over time, except for the Fig. 2: (a) Activity of (SPS ) SAI) and (b) [(SPS +
SUSY) ) (SAI + NI)] (lmol min)1 g)1 protein) in
moisture content in the intact plants of variety Mex
defoliated plants of the variety Mex 69-290 (n) and
57-473 in which it stayed constant (P < 0.05) control ones (h), and in defoliated plants of the variety
(Fig. 3a–f). No significant differences in Brix, Mex 57-473 () and control ones (s). Bars are plus and
purity, fibre content, maturity, were found between minus standard deviation of four replicates
Mechanical Defoliation of Saccharum 259

(a) (b)
18 90

88
17

Percentage
86

Percentage
16
84
15
82

14 80

0 6 12 18 24 30 0 6 12 18 24 30

(c) (d)
84 12.0

82

Percentage
80 11.5
Percentage
78
76 11.0
74
72 10.5

0 6 12 18 24 30 0 6 12 18 24 30

(e) (f)
Fig. 3: (a) Brix grades, (b) 4.5 0.8
purity grade, (c) moisture
content, (d) fibre content, 4.0
0.7
(e) maturity index (%) and
Percentage

)
3.5
(f) reduced sugars (mg l)1) –1
(mg l 0.6
in defoliated plants of the 3.0
variety Mex 69-290 (n) and
control ones (h), and in 2.5 0.5
defoliated plants of the vari-
ety Mex 57-473 () and 2.0 0.4
control ones (s). Bars (|) 0 6 12 18 24 30 0 6 12 18 24 30
are plus and minus standard
deviation of four replicates Time (days)

defoliated plants and intact ones for both varieties.
Moisture content, however, was significantly larger
in defoliated plants than in intact ones (P < 0.05).
Discussion
In sugar cane, the regulation of sucrose storage is
complicated as it is produced in the leaf, translo-
cated in the phloem and stored in the stem
(Hawker and Hatch 1965) regulated both spatially
and temporally (Moore 1995). Several metabolic
routes and mechanisms of transport involved in
this regulation have been characterized (Glasziou
and Gayler 1972, Hawker et al. 1991, Komor
1994). In sugar cane cell suspension cultures,
Wendler et al. (1990) reported that sucrose
concentration in sugar cane is the result of a
simultaneous synthesis and degradation with SPS
and SAI as key enzymes.
Zhu et al. (1997) found while working with sugar
cane varieties, which differ in the accumulation of
sucrose, a strong correlation between the activities
of SPS and SAI and the sucrose accumulated as
found in this experiment. Lingle (1999) found
similar results while investigating enzyme activities
and concentrations of sucrose during plant growth.
He found that when sugar cane grows rapidly the
plant needs carbon sources for biochemical activ-
ities and therefore SAI activity is higher and
sucrose concentration is lower.
After an initial difference, partial defoliated sugar
cane plants had similar sucrose concentration as the
intact plants. Pammenter and Allison (2002) also
found that sucrose yield per plant was not affected
260
by sugar cane defoliation. The leaves that remain on
the stalks in the partial defoliated plants normalize
the source–sink relations in the sugar cane stem and
this could have been the consequence of either
increased illumination incident on, and/or a change
in the photosynthetic characteristics of, the remain-
ing leaves (Pammenter and Allison 2002). This
effect has been observed in Phaseolus vulgaris plants
(Alderfer and Eagles 1976). Leaves of partially
defoliated plants became thicker, dark green and
leathery. The same changes were also observed in
leaves of Prunus vulgaris plants partially defoliated
(Carmi and Koller 1979). In the experiment des-
cribed here, enzyme activities in defoliated and non-
defoliated plants were not significantly different and
neither the activity of SPS ) SAI.
Chemical defoliation with glyphosate leads to a
slowing of growth, alteration of the distribution of
carbohydrates and a decrease in acid invertase
(Maretzki et al. 1976), but an increase in transloca-
tion to the ripening internodes possibly mediated by
a decrease in auxin (Su et al. 1992) and consequently
the quantity of sucrose-accumulated increases. The
differences between chemical and partial mechanical
defoliation is that when glyphosate is applied all
leaves of sugar cane plants are affected, whereas with
partial defoliation in this experiment, the four
uppermost leaves were not removed. Additionally,
the defoliated plants were not inhibited in their
growth as 10-month sugar cane plants have reached
the final growth phase (Glasziou and Gayler 1972).
Although partial mechanical defoliation affected
growth of chicory plants and their root mass was up
to 20 % lower and the total fresh weight 50 % (Neefs
et al. 2002), in sugar cane, agronomic parameters
were not affected. It might be that 10-month-old
sugar cane plants had reached the final growth
phase, so defoliation did not affect agronomic
parameters negatively (Glasziou and Gayler 1972).
It was found that sucrose content, enzyme activ-
ities and agronomic parameters were similar in
defoliated and normal developing sugar cane plants,
while the obtained leaves might serve as fodder.
However, it might be important to investigate
whether this applies to other sugar cane varieties
as the response to glyphosate differed between them.
Acknowledgements
This research was funded by Sistema de Investigación
Benito Juarez (SIBEJ) del Consejo Nacional de Ciencia y
tecnologı́a (CONACyT) grant A-22 and Fundación Pro-
duce Chiapas A.C. (México).
Gutiérrez-Miceli et al.
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