J. Env. Bio-Sci., 2015: Vol.

29 (2):419-422
(419) ISSN 0973-6913 (Print), ISSN 0976-3384 (On Line)

BIOCHEMICAL RESPONSES OF COMMON BEAN (PHASEOLUS VULGARIS L.)

GENOTYPES TO WATER STRESS UNDER MID HILL CONDITIONS
Usha Rana* and Meena Kumari
Department of Biology & Environmental Sciences, College of Basic Sciences, CSKHPKV,Palampur–1760624-230311
[Corresponding author E-mail*: rana.usha@rediffmail.com]
Received: 31-08-2015 Accepted: 06-10-2015
Experiment was conducted to assess drought-tolerance in seven common bean genotypes by inducing stress at flower initiation
stage on the basis of inherent stress tolerance characters viz.total chlorophyll content, chlorophyll stability index, nitrate
reductase activity, total free proline content, total free amino acids and drought susceptibility index. Data of various biochemical
parameters was recorded and revealed significant differences among the various common bean genotypes for chlorophyll
content and proline accumulation. KR 15 and KR 186-I, showed minimum reduction in chlorophyll content, chlorophyll stability
index and nitrate reductase activity and maximum decline in these parameters was observed in KR 84 and KR 257. Total free
proline content and total free amino acids were minimum in the genotypes KR 15 and KR 186-I, whereas, KR 84 and KR 257
showed marked increase. KR 186-I, KR 15 and KRC 5 were found to be most drought tolerant genotypes with lower drought
susceptibility index, while KR 257 and KR 84 were most drought susceptible genotypes with higher drought susceptibility index.
It was concluded that genotypes KR 186-I, KR 15 and KRC 5 proved to be tolerant to water stress and genotypes KR 60 and KR 142
were moderately drought sensitive, whereas genotypes KR 84 and KR 257 were highly susceptible to water stress.

Common bean (Phaseolus vulgaris L.) belongs to family
Fabaceae, is the world’s most important food legume. It is
cultivated globally on an area of 1.21 million ha with production
of 30.24 million tons1. Environmental abiotic stresses severely
affect plant growth and productivity worldwide and drought
being the most important environmental stress, severely
impairs plant growth and development, more than any other
factor2. About 60% of common bean production in developing
countries occurs under conditions of drought stress3. Drought
causes impaired mitosis, cell elongation and expansion,
resulting reduced growth and yield traits4. Drought stress
significantly reduced yield traits in common bean5. Water
stress at appropriate time of plant growth stages adversely
affect the metabolic processes like photosynthesis, protein
synthesis, accumulation of metabolites, chlorophyll content
as well as growth and development processes like leaf
area6.The response of plants to stresses depends on many
factors such as phenological stage, genotype, time and
strength of stresses7 .The assessment and quantification of
biochemical diversity for the traits contributing towards drought
tolerance at these stages is of vital importance. Biochemical
diversity is structured by genotypes and this diversity could
be utilized for cultivar breeding and germplasm conservation
programs aimed at improving drought tolerance.
Moisture deficit at the reproductive stage causes the principal
decline in yield as compared to stress happening at any other
NAAS Rating (2016)-4.20
growth stages. Fluctuations in water balance and soil available
water are critical to crop yields because they directly disturb
plant biochemical processes and responses8. As different crop
species have varying responses to water stress one finds a
spectrum of responses within same species and may be
classified as tolerant or sensitive to water stress9. However,
water stress tolerant plants have ability to maintain water
and ion homeostatic conditions by lowering water loss through
transpiration and accumulation of organic compatible
solutes10. Maintenance of osmoregulation is an important
mechanism of drought tolerance and tolerant plants usually
shows better osmo-regulation than the susceptible ones. In
view of water conservation strategy in plants to acclimatize
water stress, biochemical characterization is considered a
promising selection criterion for drought resistance. So, the
selection of stress tolerant/resistant species on the basis of
biochemical characterization is important in common bean
genotypes to improve productivity in drought condition due to
rainfed cultivation. Therefore, the present study is an attempt
to evaluate the genotypes of common bean for drought
tolerance on the basis of biochemical characterization which
shall be useful for the plant breeder to incorporate these
characters while selecting genotypes for crosses.
MATERIAL AND METHODS
The trial was carried out at Department of Biology and
Environmental Sciences in the College of Basic Sciences,
 BIOCHEMICAL RESPONSES OF COMMON BEAN
CSKHPKV, Palampur. The site is located at 32.60 N latitude,
76.30 S longitudes and an altitude of 1290 m above mean sea
level. Seeds were procured from MAREC, Sangla, HP and
planted on 26th July, 2012 in pots having 30 cm diameter in
completely randomized block design (CRD) with four
replications. The pots were prepared by mixing soil, vermi-
compost and sand in the 3: 2:1 ratio along with addition of N,
P and K in the ratio of 20:60:0. Nitrogen was applied in two
doses, one at time of sowing and other at the time of flowering.
Stress was created at flower initiation stage by withholding
water until the appearance of slight wilting symptoms and
control/unstressed plants were watered regularly in the net
house covered from the top and hand weeding was done
regularly. All the parameters were estimated after 30 days of
stress imposition. Biochemical parameters included were
chlorophyll content calculated by Dimethyl sulphoxide
(D.M.S.O.) method given by earlier workers11. The method of
chlorophyll stability index (CSI) was followed as suggested
by earlier workers12. Nitrate reductase activity was determined
by the standard method13. Total free proline was estimated
as per method14. The total free amino acids were determined
as per method and drought susceptibility index15-16.
RESULTS AND DISCUSSION
Total chlorophyll content was reduced in all common bean
genotypes under water stress (Table-1). KR 15 showed
minimum reduction (7.27%) under water deficit as compared
to unstressed condition. Maximum decline in chlorophyll
content was (35.03%) displayed by KR 84 in stressed plants
than unstressed plants. Total chlorophyll content significantly
reduced in all common bean genotypes under water stress.
Common bean genotypes KR 186-I, KR 15 and KRC 5 were
more drought tolerant genotypes exhibiting less negative
impact of drought stress on chlorophyll content while KR 84
and KR 257 were more sensitive to water deficit than other
genotypes. Water stress decrease the chlorophyll contents
and reduction in chlorophyll contents was very low in
genotypes Gazira-2 and Hassawi-2( tolerant genotype ) and
was maximum in the genotype Giza Blanka ( susceptible
genotype)17. Increase in chlorophyll content in tolerant
genotypes was due to higher leaf area duration, higher relative
water content and higher net assimilation rate18. The decrease
in chlorophyll content in drought stressed plant might possibly
(420)
due to changes in lipid protein ratio of pigment complex or
increased chlorophyllase activity19. Decrease in chlorophyll
under drought stress is mainly due to damage to chloroplasts
caused by active oxygen species20. Water stress decreases
the chlorophyll content under moderate water stress21. Water
stress significantly reduced chlorophyll stability index in all
common bean genotypes as compared to unstressed
condition. Phaseolus vulagris genotype KR 257 displayed
maximum reduction (46.76%) in stressed plants as compared
to unstressed plants, whereas KR 15 recorded minimum
reduction (4.43 %) under stress followed by KR 186-I.Data on
chlorophyll stability index showed that common bean
genotypes KR 15, KR 186-I, KRC 5 and KR 142 were relatively
more drought tolerant than KR 60 while KR 84, KR 257 were
drought susceptible genotypes. Chlorophyll stability index is
an indicator of stress tolerance capacity of plants and it
indicates how well chloroplast can perform under stress22. A
higher CSI helps plants to withstand stress through better
availability of chlorophyll by maintaining more dry matter
production and higher yield. Tolerant and moderately tolerant
cultivars and hybrids showed a lesser reduction in chlorophyll
stability index (6 and 12%) in response to irrigation at 50%
available soil moisture than control, while susceptible cultivars
and hybrids had higher reduction in CSI of upto 19% due to
water deficit than control23.
Nitrate reductase activity was significantly reduced due to
water deficit in all common bean genotypes (Table-1).
Minimum reduction (12.82%) was recorded in KR 186-I under
drought stress as compared to unstress, whereas, maximum
reduction (70.03 %) was observed in KR 84.Nitrate reductase
activity was greatly reduced in KR 257 and KR 84, indicating
their drought susceptible behavior, while KR 186-I, KR 15,
KRC 5 and KR 142 were relatively more drought tolerant
genotypes than KR 60 on the basis on nitrate reductase activity
reduction. Relative water content and leaf water content in
Phaseolus vulgaris decreased under water stress and nitrate
reductase activity declined in non watered plants as compared
to well watered plants24. Significant differences for nodulation
and nitrate reductase activity were observed among the
genotypes under various moisture regimes25. Decline in nitrate
reductase at low water potential is due to decrease in nitrate
flux which inturn regulates the synthesis of this enzyme26.
Water deficit inhibits the activities of many enzymes. Nitrate
(421) RANA AND KUMARI

Table-1. Effect of water stress on total chlorophyll content (µg/g FW), chlorophyll stability index (%) FW, nitrate
reductase activity (µM NO3- reduced per g fresh weight per hr), total free proline content (µg/g DW) ,total
free amino acids (µg/g) DW and drought susceptibility index in different genotypes of Phaseolus vulgaris
L. at flower initiation stage after 30 days of stress imposition
Genotypes Total chlorophyll content Chlorophyll stability index Nitrate reductase activity Total free proline (µg/g ) Total Free Amino acids (µg/g) Drought
(µg/g) (%) (µM NO 3 - reduced per g DW susceptibility
fresh weight per hr) index
S US RP(%) S S S S US RP(%) S US RP(%) S US RP(%)

KR 186-I 10.08 11.30 10.79 183.98 183.98 183.98 1.36 1.56 12.82 183.98 164.62 -11.83 222.92 193.55 -17.58 0.43

KR 60 11.29 13.61 17.05 4.32 5.57 22.41 296.94 247.22 -20.11 1.36
163.82 163.82 163.82 163.82 125.82 -30.89

KR 142 9.36 11.63 19.52 146.58 146.58 146.58 1.59 2.14 25.70 146.58 116.92 -25.22 269.23 207.22 -29.93 1.13

KRC 5 9.38 11.19 16.18 148.41 148.41 148.41 2.67 3.47 23.05 148.41 127.45 -16.45 306.26 264.36 -15.85 0.63

KR 15 6.89 7.43 7.27 162.76 162.76 162.76 1.08 1.37 21.17 162.76 130.49 -24.73 187.09 159.29 -17.16 0.53

KR 84 7.79 11.99 35.03 178.61 178.61 178.61 0.77 2.57 70.03 178.61 134.99 -32.31 252.02 153.33 -64.37 1.48

KR 257 12.71 15.95 20.32 130.46 130.46 130.46 1.76 3.81 53.81 130.46 100.21 -30.19 295.23 150.54 -94.24 1.44
Mean 9.64 11.87 159.23 159.23 159.23 1.93 2.93 159.23 128.64 246.53 180.07
C.D. (5%)

Stress 0.29 3.89 3.89 3.89 0.06 3.89 6.17 0.036

Genotypes 0.56 7.28 7.28 7.28 0.11 7.28 11.54

Interaction 0.79 10.29 10.29 10.29 0.16 10.29 16.33

S- Stress, US- Unstressed, RP- Reduction percentage

reductase activity was highly sensitive to water stress and a
significant decrease in nitrate reductase activity was observed
in many plant species under drought conditions27. Common
bean genotype KR 84 showed maximum accumulation of
proline (32.31%) under drought stress than unstressed,
whereas, minimum proline accumulation (11.83%) was
recorded in KR 186-I due to drought stress (Table-1). Total
free proline increased in all seven genotypes under water
stress than unstress.In the present study drought tolerant
genotypes KR 186-I, KRC 5 and KR 15 showed lower
accumulation of proline followed by KR 142 and KR 60 as
compared to drought susceptible genotypes KR 257 and KR
84. Accumulation of proline in beans under drought stress
help the plant to survive and reproduce and this accumulation
of proline only occurred in drought susceptible cultivars28.
Genotype WA4502-1 showed lowest increase in proline
accumulation and had a relatively better performance under
drought when compared with the other whitebean genotypes29.
Higher tolerance of ILC482 genotype to drought stress was
observed which have lower accumulation of proline content30.
Accumulation of proline is an important indicator of drought
stress tolerance in bacteria, algae and higher plants31. This
imino acid has been reported to play multiple physiological
functions in plants subjected to drought, such as
osmoregulation, a sink for energy and nitrogen and a signal
of senescence. In present study, higher proline accumulation
in drought susceptible cultivars occurred as a symptom of
stress injury and not necessarily as a consequence of osmotic
adjustment. Significant accumulation of total free amino acids
was recorded under water stress than unstressed (Table-1).
Lowest increase (15.85%) in total free amino acids was
observed in KRC 5 under water deficit, while KR 186-I and KR
15 were at par with each other.KR 84 showed maximum
accumulation (93.34 %) of total free amino acids due to water
stress than unstressed. On the basis of present data genotype
KR 84 and KR 257 were proved to be more sensitive towards
 BIOCHEMICAL RESPONSES OF COMMON BEAN
drought stress than other genotypes. The findings are
supported by some earlier workers32.They found that genotype
A 320 was less sensitive to water stress with lower
accumulation of soluble sugars and free amino acids while
genotype Xodo was most sensitive with higher soluble sugars
and amino acids accumulation under water deficit. It has been
reported that free amino acids are amides, contribute to
osmotic adjustment but experimental results are inconsistent.
The increased accumulation of total free amino acids in
sensitive genotypes may be due to greater protein breakdown.
Amino acid content increased under drought stress apparently
due to hydrolysis of protein in chickpea (Cicer arietinum)
genotypes33. Generally, the environmental stresses especially
drought stress, give rise to accumulation of soluble
carbohydrates, proline and free amino acids as well as
antioxidants compounds. These solutes are low molecular
weight, highly soluble compounds that are non toxic at high
cellular concentration and protect cellular components from
dehydration injury, thus are referred to as osmoprotectants
and compatible solutes34. In the present study, Phaseolus
vulgaris L. genotypes KR 186-I,KR 15 and KRC 5 were most
drought tolerant genotypes with lower drought susceptibility
indexes, while KR 60 and KR 142 were moderately sensitive
to drought stress, whereas, KR 257 and KR 84 were most
drought susceptible genotypes with higher drought
susceptibility index. Yield obtained under drought condition
was 37 per cent less than that of under irrigated condition
and cultivars with drought susceptibility index less than 0.86
were considered as drought tolerant as given by earlier
workers35. DSI values 0.84 and 0.75 were observed in
response to continuous water stress and late water stress
respectively in tolerant variety36.
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