Lotus Newsletter (2009) Volume 39 (1), 21-27.
Incidence of drought stress and rewatering on Lotus tenuis.
Effects on cell membrane stability
ARIEL CLUA1*, MATIAS PAEZ1, HIPOLITO ORSINI1 and JOSE BELTRANO12
1
INFIVE. Instituto de Fisiología Vegetal. Facultad de Ciencias Agrarias y Forestales.
Universidad Nacional de La Plata. CC 327 La Plata 1900. Argentina.
2
CIC BA.
*Corresponding author
Abstract
Plants subjected to water stress are affected in its morphology, anatomy and physiological
processes. Morphological and physiological changes can be a survival strategy of Lotus
tenuis (Wald. et Kit) to tolerate water stress variable conditions. The purpose of this
experiment was to investigate Lotus tenuis response to variable drought stress conditions
and rewatering, on biomass production and cell membrane stability. L. tenuis seeds obtained
from a naturalized population of Saladillo (Buenos Aires Province, Argentina) were
subjected to the following treatments: Control: plants were watered daily to maintain soil
water potential (Ψs) close to –0.03 MPa. (C); Water stress: plants were stressed by
withholding watering (Ψs ≈ –0.9 MPa) for 15 days and rewatering close to -0.03 Mpa, until
the end of the experiment (ME1); plants were stressed by withholding watering (Ψs ≈ –0.9
MPa) for 30 days and rewatering close to -0.03 Mpa until the end of the experiment (ME2);
plants were stressed by withholding watering (Ψs ≈ –0.9 MPa) during all experiment (ME).
At 14 days intervals were determined aerial and roots dry weight (ADW and RDW
respectively), roots volume (RV), leaf area (LA) and roots and leaves cell membrane
stability (RCMS and LCMS respectively). Water stress treatments reduced ADW, RDW,
RV, LA and enhanced cell membrane damage although was recovered by rewatering. This
study showed a L. tenuis drought tolerance and its adaptation to extreme water stress
condition (75 days), manifested by persistence and continuous aerial and root growth, its
response to rewatering and cell membrane reparation.
Key words
Lotus tenuis, drought tolerance, rewatering, cell membrane stability.
Introduction
Drought and water stress is considered one of most important environmental factor, limiting
crop yields in the world (Panozzo and Eagles, 1999). During water stress, soil water is
strongly retained, interfering water and mineral nutrients absorption by plants. Plants
responses to water stress depend of several factors such as developmental stage, stress
21
22
A. Clua, M Paez, H. Orsini, J. Beltrano
severity and duration, and plant tolerance capacity. Common plant symptoms to water
deficit are stunted growth, limited CO2 diffusion to chloroplasts by stomatal closure,
reduced photosynthesis rate, and accelerated leaf senescence. Moreover, water stress can
increase reactive oxygen species synthesis (ROS), increasing proteins, membrane lipids and
photosynthetic pigments degradation and cell membrane damages (Anderson et al., 1990;
Navari-Izzo et al., 1997; Beltrano et al., 1997).
Lotus tenuis is important forage in the Argentinean Salado river basin, where flood and
drought alternation periods often reduce plant growth and yields (Durán, 2002). There are
experimental evidence about drought and flood tolerance conditions of L. tenuis (Mazzanti
et al., 1988), nevertheless physiological responses of this specie to water stress and
rewatering is incomplete and represent an important knowledge about L. tenuis stress
adaptability.
The purpose of this experiment was to investigate Lotus tenuis response to variable drought
stress conditions and rewatering on biomass production and cell membrane stability.
Materials and Methods
Seeds of L. tenuis obtained from a naturalized population of Saladillo, (Buenos Aires
Province, Argentina) were washed with 0.04% (w/v) NaHClO3, rinsed with deionized water
and sown in 3000 cc pots, filled with a representative Salado river basin soil. The soil was an
argiudol vertic (Soil Survey Staff, 1999; pH 5.5; ECe, 6.1 dS/m; P total 12 mg.kg-1; OM 3.5
%; C total 2.0 % and N total 0.24 %). Pots were placed under natural conditions in the field
and were watered daily, until the beginning of the experiment.
Twenty five plants were used for each treatment in a completely randomized block design.
The trial was initiated 7 months after sowing, when plants have a well developed crown.
Treatments were: Control (C): plants were watered daily; to maintain soil water potential
(Ψs) close to –0.03 MPa.; Water stress: plants were stressed by withholding watering (Ψs ≈
–0.9 MPa for 15 days and rewatering close to -0.03 Mpa, until the end of the experiment
(ME1); plants were stressed by withholding watering (Ψs ≈ –0.9 MPa for 30 days and
rewatering close to -0.03 Mpa until the end of the experiment (ME2); plants were stressed by
withholding watering (Ψs ≈ –0.9 MPa during all experiment (ME).
For water levels control, Ψs was measured daily during the entire experiment using a
HR-33T dew-point psychrometer (Wescor Inc., Logan, UT, USA) with PST-55 probes,
placed 15 cm deep in the soil, at the beginning of the experiment. The amount of water lost
was added daily. Soil water availability was previously determined, and a curve of water
retention was made.
From the beginning of experiment and at 14 days intervals, 5 plants for treatment were
harvested. Roots were severed from shoots to determine aerial and roots dry weight (ADW
and RDW respectively), roots volume (RV), leaf area (LA) and roots and leaves cell
membrane stability (RCMS and LCMS respectively).
Response of Lotus tenuis to flooding
23
Data were analyzed statistically by ANOVA and differences between means were tested for
significance using the least significance difference test according to Snedecor and Cochran
(1980).
Results
RDW showed significant differences comparing ME1 and ME2 with control treatment, at 15
and 30 days respectively. ME showed significant differences since 45 days compared with
control, ME1 and ME2 (Figure 1).
12,00
ROOT DRY WEIGTH
AERIAL DRY WEIGHT
10,00
RADICAL VOLUME
70,00
60,00
50,00
40,00
6,00
30,00
cm3/root
g/pl
8,00
4,00
20,00
2,00
10,00
0,00
0,00
0 15 30 45 60 75
0 15 30 45 60 75
0 15 30 45 60 75
0 15 30 45 60 75
CONTROL
ME1
ME2
ME
Figure 1. Aerial and root dry weight (g/pl) and root volume (cm3) of Lotus tenuis plants
subjected to water stress treatments: without drought (C), 15 days drought period and
rewatering (ME1); 30 days drought period and rewatering (ME2); drought during all
experiment (ME).
ADW showed in ME1 and ME2 significant reduction compared with control, until 45 days.
Moreover, ME1 was significant different to ME2. Rewatering revert the effect and not
showed differences since 60 days, comparing ME1 and ME2 treatments. ME showed
significant differences with others treatments along the experiment. (Figure 1).
ME1 showed RV significant reduction, at 15 and 30 days, and ME2 at 30 days, compared
with control. Rewatering revert the effect and was not observed differences, since 45 days,
comparing ME1, ME2 and control treatments. ME showed significant differences with
others treatments along the experiment (Figure 1).
LA showed significant reduction for ME1 and ME2 treatment, during the experiment,
compared with control. In turn ME1 showed significant differences compared with ME2
24
A. Clua, M Paez, H. Orsini, J. Beltrano
until 60 days. ME1 and ME2 showed LA recovery by rewatering, since 15 and 30 days,
respectively. ME treatment showed significant differences with others treatments along the
experiment (Figure 2).
800
LEAF AREA
700
cm2/pl
600
500
400
300
200
100
0
0
15 30 45 60 75
CONTROL
0
15 30 45 60 75
0
15 30 45 60 75
ME1
0
15 30 45 60 75
ME2
ME
Figure 2. Leaf area (cm2) of Lotus tenuis plants subjected to water stress treatments: without
drought (C), 15 days drought period and rewatering (ME1); 30 days drought period and
rewatering (ME2); drought during all experiment (ME).
LCMS showed a significant reduction for ME1 and ME2 treatment, compared with control
at 15 and 30 days respectively. Rewatering revert the effect and was not observed
differences, since 45 days. ME treatment showed significant differences with others
treatments along the experiment. RCMS showed similar results (Figure 3).
120
LCMS
RCMS
100
CMS (%)
80
60
40
20
0
0
15 30 45 60 75
CONTROL
0
15 30 45 60 75
ME1
0
15 30 45 60 75
ME2
0
15 30 45 60 75
ME
Figure 3. Roots and leaves cell membrane stability (RCMS and LCMS respectively, %) of
Lotus tenuis plants subjected to water stress treatments: without drought (C), 15 days
drought period and rewatering (ME1); 30 days drought period and rewatering (ME2);
drought during all experiment (ME).
Response of Lotus tenuis to flooding
25
Discussion
Results observed in this work showed a detrimental effect of drought stress period in aerial
and roots Lotus tenuis biomass. Nevertheless was observed a Lotus tenuis drought tolerance,
according to absence of plants mortality even in extreme stress treatment (EM) and recovery
by rewatering.
Growth reduction by water stress is caused by changes in several physiological processes.
Drought stress induces stomatal closure, limiting CO2 diffusion rate, a photosynthetic rate
reduction (Augé et al., 1992). In addition water stress reduces leaf area as was observed by
Witkowski and Lamont (1991) and Parkhurst and Louks (1972).
Moreover, the aerial biomass reduction of Lotuis tenuis is coincident to others forage
legumes responses, as lucerne, where water stress caused a significant growth reduction
(Bolger, 1989; Halim et al., 1989).
Likewise water stress reduced leaf area significantly, with a variable defoliation, depending
of stress severity and duration, as was observed by Halim et al. (1989). These responses
represent an adaptation plant strategy by diminish water loss and increasing water stress
tolerance.
Also water stress can increase reactive oxygen species synthesis (ROS) that produce
proteins, membrane lipids and photosynthetic pigments degradation with a loss of cell
membrane stability (Anderson et al., 1990; Navari-Izzo et al, 1997; Beltrano et al., 1997).
Plants can recovery its physiological functions when stress factor is reverted and the
recovery celerity is consequence of plant stress tolerance. In this study, Lotus tenuis showed
a rapid recovery by rewatering and increase RV, LA and CMS.
Leaf area recovery of Lotus tenuis after rewatering was observed by basal bud regrowth
(data not shown). This event could be attributed to ethylene-cytokins balance modifications,
as was observed in others species (Beltrano et al., 1994).
Tolerance drought strategy could be associated to integrity cell membrane preservation and
its rapid reparation (Oliver, 1991). These events represent an important factor of cellular
organization during rewatering period after drought stress, and could be Lotus tenuis
adaptation mechanisms to drought stress tolerance.
References
AUGÉ R.M., STODOLA A.J.W., BROWN M.S. and BETHLENFALVAY G.J. 1992. Stomatal
response of mycorrhizal cowpea and soybean to short term osmotic stress. New
Phytologyst, 120, 117-125.
26
A. Clua, M Paez, H. Orsini, J. Beltrano
ANDERSON, J.V., HESS J.L., and CHEIONE B.J. 1990. Purification characterization, and
immunological properties for two isoforms of glutathione reductase from eastern white
pine needles. Plant Physiology, 94, 1402-1409.
BELTRANO J., CARBONE A., MONTALDI E.R. and GUIAMÉT J.J. 1994. Ethylene as promoter
of wheat grain maturation and ear senescence. Plant Growth Regulation, 15, 107-112.
BELTRANO J., MONTALDI E.R., BÁRTOLI, C. and CARBONE A. 1997. Emission of water
stress ethylene in wheat (Triticum aestivum L.) ears: effects of rewatering. Plant Growth
Regulation, 21, 121-126.
BOLGER T.P. 1989. Water use, yield, quality and dinitrogen fixation of sainfoin and alfalfa
under gradient irrigation. Diseases Abstract International, 50, 376-381.
DURÁN D. 2002. Las sequías como riesgo natural. [Drought as natural hazard.] Lic en
Geografía de la Universidad del Salvador. Investigadora del CONICET en la Comisión
Nacional del Programa Hidrológico Internacional de la UNESCO. [In Spanish]
http://www.produccionbovina.com/inundacion/07-sequias_riesgo_natural.htm
HALIM R.A., BUXTON D.R., HATTENDORF M.J. and CARLSON R.E. 1989. Water estress
effect on alfalfa forage quality after adjustment for maturity differences. Agronomy
Journal, 81, 189-194.
MAZZANTI A., MONTES L., MIÑON D., SARLANGUE H. and CHEPPI C. 1988. Utilización de
Lotus tenuis en establecimientos ganaderos de la Pampa Deprimida: Resultados de una
encuesta. [Utilization of Lotus tenuis in ranches of the Pampa Deprimida: Results of the
survey] Revista Argentina de Producción Animal, 8(4), 301-305. [In Spanish]
NAVARI-IZZO F., MENEGUZZO S., LOGGINI B., VAZZANA C. and SGHERRI C.L.M. 1997.
The role of the glutathione system during dehydration of Boea hygroscopica.
Physiologia Plantarum, 99, 23-30.
OLIVER M.J. 1991. Influence of protoplasmic water loss on the control of protein synthesis
in the desiccation-tolerant moss Tortula ruralis: ramification for a repair-based
mechanism of desiccation – tolerance. Plant Physiology, 97, 1501-1511.
PANOZZO J. and EAGLES H. 1999. Rate and duration of grain filling and grain nitrogen
accumulation of wheat cultivars grown in different environments. Australian Journal of
Agricultural Research, 50, 1007-1015
PARKHURST D.F. and LOUKS O.L. 1972. Optimal leaf size in relation to environment.
Journal of Ecology, 60, 505-511.
Response of Lotus tenuis to flooding
27
SNEDECOR G.W. and COCHRAN W.G. 1980. Statistical methods. 7.ed. Iowa State
University, Iowa, 507p.
SOIL SURVEY STAFF. 1999. Soil taxonomy, a basic system of soil classification for making
and interpreting soil surveys, 2nd ed.: U.S. Department of Agriculture, Natural
Resources Conservation Service, Agriculture Handbook Number 436, 870 p.
WITKOWSKI E.T.F. and LAMONT B.B. 1991. Leaf specific mass confounds leaf density and
thickness. Oecology, 88, 486-492.