American Journal of Plant Sciences, 2011, 2, 318-324
doi:10.4236/ajps.2011.23036 Published Online September 2011 (http://www.SciRP.org/journal/ajps)
Responses of Transgenic Tobacco Plants with
Increased Proline Content to Drought and/or
Heat Stress
Jana Pospisilova1*, Daniel Haisel1, Radomira Vankova2
1Institute of Experimental Botany, Academy of Sciences of the Czech Republic, Na Karlovce, Prague, Czech Republic; 2Institute of
Experimental Botany, Academy of Sciences of the Czech Republic, Rozvojová, Prague, Czech Republic.
Received March 28th, 2011; revised May 3rd, 2011; accepted May 11th, 2011.
ABSTRACT
Transgenic tobacco plants (M51-1) constitutively over-expressing a modified gene for the proline biosynthetic enzyme
∆2-pyrroline-5-carboxylate synthetase (P5CSF129A) and the corresponding wild-type plants (WT) were compared
during drought or heat stress and under combination of both stresses. The proline content in M51-1 was several times
higher than in WT plants. Under optimal conditions, the transpiration rate and stomatal conductance of M51-1 plants
were lower than those in WT plants. The differences in net photosynthetic rate were not significant and water use effi-
ciency and contents of chlorophyll and xanthophyll cycle pigments were higher in M51-1 than in WT plants. Drought
induced by cessation of watering for 7 d resulted in decrease of all gas exchange parameters and chlorophyll content,
but in an increase of the content of xanthophyll cycle pigments and degree of their de-epoxidation. After application of
heat stress (40˚C/60 min) to control or water-stressed plants the gas exchange parameters decreased considerably.
Short-term heat stress alone, however, did not affect pigment contents. The responses of M51-1 and WT plants to the
tested stresses did not differ significantly. Therefore, a decisive contribution of elevated proline content to drought or
heat stress tolerance of tobacco was not proved.
Keywords: Carotenoids, Chlorophyll, Net Photosynthetic Rate, Stomatal Conductance, Transpiration Rate, Xanthophyll
Cycle Pigments
1. Introduction
In many plant species, osmotic adjustment (lowering of
the osmotic potential in order to maintain the pressure
potential) occurs in response to water stress induced by
drought, salinity or low temperature. The composition of
solutes contributing to osmotic adjustment differs ac-
cording to the plant species or genotype, duration as well
as severity of water stress 1,2. One of the well-known
osmotically active compounds is proline (for review see
e.g. 3). It may serve not only as osmoprotectant, but
also as a molecular chaperone, antioxidant, regulator of
redox homeostasis or source of carbon and nitrogen (for
review see 4). Therefore, its accumulation occurs not
only under water stress but also under other abiotic and
biotic stresses.
In higher plants, proline is synthesized mainly from
glutamate. The oxidation of glutamate to glutamic-γ-se-
mialdehyde (GSA) is catalysed by a ∆1-pyrroline-5-car-
boxylate synthetase (P5CS). GSA undergoes spontaneous
cycling to ∆1-pyrroline 5-carboxylate (P5C). The next step
involves the reduction of P5C to proline by ∆1-pyrroline-
5-carboxylate reductase (P5CR). Proline biosynthesis ste-
adily occurs in the cytosol, while it is augmented to the
chloroplasts during stress conditions 4. As proline bio-
synthesis requires NADPH, the enhanced rate of its bio-
synthesis in chloroplasts maintains the low NADPH:
NADP+ ratio, resulting in reduction of photoinhibition
under high irradiance 5. Thippeswamy et al. 6 sug-
gested that the proline synthesis from glutamate has been
limited by P5CS activity. Proline degradation by proline
dehydrogenase (PHD) occurs in mitochondria 4. Intra-
cellular proline content thus depends on its biosynthesis,
degradation and transport from other plant parts 7. For
example, P5CS activity in Arabis stelleri was increased
by mannitol and sorbitol, but not by NaCl, while activity
of PHD was decreased by mannitol and NaCl, which
resulted in an increase of free proline amount under all,

Responses of Transgenic Tobacco Plants with Increased Proline Content to Drought and/or Heat Stress319
above mentioned, treatments 8. During stress condi-
tions, elevation of proline content coincided with modu-
lation of the enzyme activities (increase in case of P5CR
and decrease in PDH, respectively), as well as with cha-
nges in expression of the corresponding genes 6,9,10.
Recently, two closely related genes, P5CS1 and P5CS2,
were identified in Arabidopsis 11. The former one is
involved in regulation of development, while the latter
one is stress-responsible 4. Similarly, two genes for
proline degradation, MsPHD1 and MsPHD2 were dis-
tinguished in Medicago sativa 9.
The correlation between proline accumulation and
plant stress tolerance is not always clear (for review see
4,12). For example, with increased NaCl concentration,
salt resistant rice cultivars accumulated less proline than
the salt sensitive ones 13. With the aim to contribute to
the elucidation of the role of proline in response to drought
and heat stress alone or in combination, transgenic tobacco
plants (M51-1) constitutively over-expressing a modified
gene (P5CSF129A) for the proline biosynthetic enzyme
∆2-pyrroline-5-carboxylate synthetase and the correspond-
ing wild-type plants (WT) were compared. While previ-
ous papers focused on M51-1 plants described predomi-
nantly proline and phytohormone contents 14,15, in the
present paper net photosynthetic rate, transpiration rate,
stomatal conductance and pigment contents were followed.
2. Materials and Methods
2.1. Plants and Cultivation
The seeds of transgenic tobacco plants were kindly do-
nated by Dr. Jozef Gubis. The transformation was de-
scribed in detail by Gubis et al. 14. Wild type (WT) and
transgenic (M51-1) tobacco (Nicotiana tabacum L.)
seedlings were grown in Perlite with nutrient solution in
a growth chamber at a 16-h photoperiod, an irradiance of
250 mol (photon) m–2·s–1 (400 - 700 nm), day/night tem-
perature of 25˚C/20˚C, and relative humidity of about
50%. The below mentioned parameters were followed in
control plants sufficiently supplied with water, after ces-
sation of watering for 7 days and again after rehydration
for 7 days. Heat shock (40˚C, 60 min) was applied either
to control plants or to water-stressed plants and meas-
urements were done immediately after treatment.
2.2. Measurements of Gas Exchange and
Photosynthetic Pigments
Net photosynthetic rate (PN), transpiration rate (E) and
stomatal conductance (gs) were measured on attached
leaves using the commercial gas exchange system LCA-4
(ADC Bio Scientific, Hoddesdon, UK). All measurement
were done at a temperature of 25˚C, saturating irradiance
of 750 mol m–2·s–1, CO2 concentration of 350 mol·mol–1,
and relative humidity of about 30%. Contents of photosy-
nthetic pigments were determined in acetone extracts of
leaf discs by HPLC (ECOM, Prague, Czech Republic) us-
ing a reverse phase column (Watrex Nucleosil 120-5-C18,
5 m particle size, 125  4 mm). The solvent system was
acetonitrile:methanol:water (80:12:10) followed by me-
thanol:ethylacetate (95:5), the gradient was run from 2 to
6 min. The flow rate was 1 cm3·min–1, the detection
wavelength 445 nm. The pigment analyses were per-
formed using software Clarity (DataApex, Prague, Czech
Republic). Contents of proline and abscisic acid were
measured as described previously by Dobra et al. 14.
2.3. Data Analysis
Presented results are means  standard error of 9 (gas
exchange) or 3 (pigments) independent samples. Signifi-
cance of differences between WT and M51-1 as well as
between control and treated plants was evaluated by
t-test (Tables 1 and 2). The experiments were repeated
twice with similar results.
3. Results
The characteristic feature of transgenic tobacco plants
M51-1 was several times higher proline content in com-
parison with WT 14,15. In both genotypes, prolonged
water stress resulted in a highly significant increase in
proline content (Figure 1), while no significant changes
in proline content were observed after heat stress 15.
0
300
600
900
1200
1500
WT
M51-1
Proline content [ pmol g-1(d.m.)]
0
20
40
60
ABA content [ mol g-1(d.m.)]
control stress rehydration
Figure 1. Contents of proline and abscisic acid in wild type
(WT) and transgenic (M51-1) tobacco plants during water
stress and rehydration (both lasted 7 days).

Responses of Transgenic Tobacco Plants with Increased Proline Content to Drought and/or Heat Stress
320
3.1. Comparison of Transgenic and Wild Type
Plants under Sufficient Water Supply
0
3
6
9
WT
M51-1
PN [ mol m-2 s-1]
E [ mmol m-2 s-1]
gs [ mol m-2 s-1]
0
1
2
0.04
0.08
contro l stress rehyd ra tion
In non-stressed plants, transpiration rate (E) and stomatal
conductance (gs) of M51-1 were lower than those of WT,
which was in agreement with higher abscisic acid content
in M51-1 (Figure 1). Net photosynthetic rate (PN) was
not significantly different between the plant types (Fig-
ure 2). Water use efficiency (WUE = PN/E) in M51-1
and WT plants was 5.95 and 5.08 mmol (CO2) mol–1
(H2O), respectively. Contents of chlorophyll (Chl a + b)
or xanthophyll cycle pigments (Xan = zeaxanthin + an-
teraxanthin + violaxanthin) were significantly higher in
M51-1 than WT (Figure 3).
3.2. Response of Transgenic and Wild Type
Plants to Drought and Rehydration
Cessation of watering for 7 days induced water stress and
decreased PN, E and gs, the response being similar in
M51-1 and WT plants. All gas exchange parameters were
partially recovered after rehydration in both plant types
(Figure 2). The water stress slightly decreased Chl a + b
content, similarly in both plant types. The content of Car
was slightly increased under drought, this increase was
higher in M51-1 than in wild type. The Xan content and
the degree of their deepoxidation DEPS = zeaxanthin +
0.5 antheraxanthin)/(antheraxanthin + violaxanthin + ze-
axanthin) were markedly increased under drought in both
plant types. These changes were partially reversed after
rehydration (Figure 3).
Figure 2. Net photosynthetic rate (PN), transpiration rate (E)
and stomatal conductance (gs) in wild type (WT) and trans-
genic (M51-1) tobacco plants during water stress and rehy-
dration (both lasted 7 days).
0
3
6
Chl a+b [ g cm-2]
0
1
2
Car [ g cm-2]
WT
M51-1
Xan [ g cm-2]
0.5
1.0
DEPS
0.2
0.4
control stress rehydrationcontrol stress rehydration
Figure 3. Contents of chlorophylls (Chl a + b), carotenoids (Car) and xanthophyll cycle pigments (Xan) and Xan degree of
deepoxidation (DEPS) in wild type (WT) and transgenic (M51-1) tobacco plants during water stress and rehydration (both
lasted 7 days).

Responses of Transgenic Tobacco Plants with Increased Proline Content to Drought and/or Heat Stress321
Table 1. Results of statistical evaluation (P values) of the effects of material (WT  M51-1) under control conditions, 7-d wa-
ter stress and 7-d rehydration and effect of treatments (control  stress or rehydration) measured in different materials.
Parameter Materials Treatments
Control Stress Rehydration Stress Rehydration
WT M51-1 WT M51-1
PN 0.355 0.194 0.926 0.040 0.003 0.010 0.086
E 0.007 0.317 0.329 0.003 0.021 0.881 0.656
gs 0.001 0.014 0.765 0.016 0.197 0.043 0.262
Chl a + b 0.017 0.003 0.019 0.002 0.002 0.061 0.044
Car 0.530 0.001 0.252 0.007 0.001 0.041 0.137
Xan 0.001 0.001 0.007 0.001 0.001 0.023 0.387
DEPS 0.097 0.681 0.130 0.001 0.001 0.770 0.971
Table 2. Results of statistical evaluation (P values) of the effects of material (WT  M51-1) under control conditions, heat and
water stress + heat and effect of treatments (control  heat or stress + heat) measured in different materials.
Parameter Materials Treatments
Control Heat Stress + Heat Heat Stress + Heat
WT M51-1 WT M51-1
PN 0.831 0.380 0.008 0.012 0.012 0.001 0.001
E 0.389 0.137 0.270 0.001 0.001 0.001 0.001
gs 0.145 0.061 0.033 0.001 0.001 0.001 0.001
Chl a + b 0.017 0.001 0.002 0.148 0.786 0.001 0.002
Car 0.530 0.336 0.001 0.530 0.336 0.009 0.001
Xan 0.001 0.020 0.001 0.012 0.005 0.001 0.001
DEPS 0.097 0.240 0.026 0.002 0.031 0.001 0.001
3.3. Response of Transgenic and Wild Type
Plants to Heat Stress and Combined
Heat and Water Stress
After heat stress (40˚C/60 min) applied to control or wa-
ter-stressed plants PN, E and gs decreased considerably in
both M51-1 and WT plants (Figure 4). These effects
were much more pronounced at the combined drought
and heat stress than at drought or heat alone. The lowest
values of all gas exchange parameters were found in
M51-5 plants under combined drought and heat stress.
Short-term heat stress alone, however, did not affect sig-
nificantly pigment contents. The pigment contents, how-
ever, were affected by combined heat and water stress in
pattern similar to that of water stress alone (Figure 5).
Under combined heat and drought, the pigment contents
as well as DEPS were higher in M51-1 than in WT plants.
4. Discussion
Accumulation of proline during drought was repeatedly
reported in many species. The pathways of proline bio-
synthesis and degradation were recently described 4,
but its biological functions have not been fully elucidated
yet. The unsolved question is whether proline accumula-
tion is a sign of stress tolerance or only consequence of
the stress (for review see 12). The reply to this question
was searched by determination of changes in proline
content during different stresses, by application of pro-
line with the aim to ameliorate negative effects of stress
(for review see e.g. 3,12) and recently by using trans-
genic plants with increased endogenous proline content.
The last approach has been based either on increased of
proline biosynthesis achieved by over-expression of P5CS
gene 16-18 or on decrease of proline degradation by re-

Responses of Transgenic Tobacco Plants with Increased Proline Content to Drought and/or Heat Stress
322
pression of PDH genes 9. In addition, it has been found
that antisense P5CS transgenic Arabidopsis plants, show-
ing significantly lower proline accumulation than respe-
ctive wild type, were hypersensitive to osmotic stress 19.
General feature of transgenic plants over-expressing
P5CS gene was high accumulation of proline, especially
under stress conditions. In indica rice accumulation of
proline was accompanied by better biomass production
and growth performance under drought or salt stress
18,20. It was rather surprising that in sugarcane, Vigna
aconitifolia and tobacco the increase in proline accumu-
lation was not accompanied by markedly higher osmotic
adjustment in transgenic plants than in respective WT
plants 15,20,21.
In our experiments, transgenic tobacco plants M51-1
had a little lower E and gs that WT plants under sufficient
water supply, which might be caused by slightly higher
ABA content in M51-1 than in the WT plants. Lower E
might led to lower water consumption, more conserva-
tive water use and better growth as observed by Molinari
et al. 20, Vendruscolo et al. 21 and Dobra et al. 15.
Chl content was slightly higher in M51-1 than in WT
plants. Due to the higher Chl content but lower gs in M51-1
than in WT, PN was similar in both genotypes. Due to
similar PN and lower E, WUE was higher in M51-1 than
in WT plants. This can be a positive feature of M51-1
plants leading to better utilization of water sources. On
the other hand, transpiration efficiency (measured as ac-
cumulation of biomass per amount of water transpired) in
chickpea plants was similar in transgenic plants with
high proline content to that in WT plants 22.
0
3
6
WT
M51-1
PN [ mol m-2 s-1]
0
1
E [ mmol m-2 s-1]
gs [ mol m-2 s-1]
0.04
0.08
control stress
25 oC 40 oC 25 oC 40 oC
Figure 4. Net photosynthetic rate (PN), transpiration rate (E)
and stomatal conductance (gs) in wild type (WT) and trans-
genic (M51-1) tobacco plants after heat shock (40˚C/60 min)
imposed to plants sufficiently supplied with water or to
water-stressed plants.
Chl a+b [ g cm-2]
Car [ g cm-2]
Xan [ g cm-2]
0.5
1.0
DEPS
0.2
0.4
0
3
6
0
1
2
WT
M51-1
control stress
25 oC 40 oC 25 oC 40 oC control stress 25 oC 40 oC 25 oC 40 oC
Figure 5. Contents of chlorophylls (Chl a + b), carotenoids (Car) and xanthophyll cycle pigments (Xan) and Xan degree of
deepoxidation (DEPS) in wild type (WT) and transgenic (M51-1) tobacco plants after heat shock (40˚C/60 min) imposed to
plants sufficiently supplied with water or to water-stressed plants.

Responses of Transgenic Tobacco Plants with Increased Proline Content to Drought and/or Heat Stress323
However, no marked difference in the responses to
water stress, including decrease in E, gs, PN and Chl con-
tent and increase in Xan content and DEPS, was ob-
served between the genotypes. The same holds for the
recovery after water stress. When P5CS gene was intro-
duced under stress-inducible promotor, Chl content and
variable to maximum Chl fluorescence ratio (Fv/Fm) in
transgenic and WT sugarcane plants did not differ under
sufficient water supply but, in contrast to our results,
these characteristics remained higher in transgenic plants
that in WT plants under water stress 20. During simul-
taneous drought and heat stress, dissociation of the oxy-
gen-evolving complex was bypassed by proline feeding
electrons into photosystem 2, maintaining the acceptable
NADPH level in transgenic soybean plants 23. Under
drought stress, the cumulative daily transpiration was
higher only in some lines of transgenic chickpea plants
than that in WT plants. Also gs in these transgenic
chickpea plants was slightly higher than of WT at both
control and stress conditions 22.
Short-term heat stress affected only gas exchange pa-
rameters but not pigment contents. Application of heat
stress to previously water-stressed plants led to further
decrease in E, gs and PN. Similar results were recently
mentioned in two pepper cultivars 24 and Ceratonia
siliqua 25. Our data indicate only relatively minor dif-
ferences between M51-1 and WT in responses to heat or
combined stresses.
All the above-mentioned results did not prove clear
correlation between proline accumulation and stress tol-
erance. However, in our experiments, the induced stre-
sses were rather mild than severe. Maybe, that under se-
vere stress, the proline plays more important role, espe-
cially as protectant against oxidative stress as suggested,
e.g., Molinari et al. 20) and Vendruscolo et al. 21.
5. Conclusions
The increased content of proline in transgenic tobacco
plants M51-1 was accompanied by slightly lower E and
gs and slightly higher Chl and Xan content under suffi-
cient water supply, which might suggest more conserva-
tive water regime in these transgenic plants than in re-
spective WT plants. The changes of all parameters in-
duced by mild water stress or/and heat shock reflect more
similarities than differences in response of M51-1 and
WT tobacco plants. Therefore, the clear correlation be-
tween proline content and stress tolerance was not
proved in our experiments.
6. Acknowledgements
The authors are grateful to Dr. Jozef Gubis, Res. Inst.
Plant Prod., Piestany, Slovakia, for providing plant mate-
rial. This work was supported by the Grant Agency of the
Czech Republic, project No. 522/09/2058.
REFERENCES
[1] A. Ogawa and A. Yamauchi, “Root Osmotic Adjustment
under Osmotic Stress in Maize Seedlings. 2. Mode of
Accumulation of Several Solutes for Osmotic Adustment
in the Root,” Plant Production Sciences, Vol. 9, No. 1,
2006, pp. 39-46. doi:10.1626/pps.9.39
[2] D. J. Walker, P. Romero and E. Correal, “Cold Tolerance,
Water Relations and Accumulation of Osmolytes in
Bituminaria bituminosa,” Biologia Plantarum, Vol. 54,
No. 2, 2010, pp. 293-298.
doi:10.1007/s10535-010-0051-x
[3] B. Heuer, “Osmoregulatory Role of Proline in Plants Ex-
posed to Environmental Stresses,” In: M. Pessarakli, Ed.,
Handbook of Plant and Crop Stress, 2nd Edition, Marcel
Dekker, New York, 1999, pp. 675-695.
[4] L. Szabados and A, Savouré, “Proline: A Multifunctional
Amino Acid,” Trends in Plant Science, Vol. 15, No. 2,
2010, pp. 89-97. doi:10.1016/j.tplants.2009.11.009
[5] P. D. Hare, W. A. Cress, “Metabolic Implications of Stress-
Induced Proline Accumulation in Plants,” Plant Growth
Regulation, Vol. 21, No. 2, 1997, pp. 79-102.
doi:10.1023/A:1005703923347
[6] M. Thippeswamy, P. Chandraobulreddy, B. Sinilal, M.
Shiva Kumar and Chinta Sudhakar, “Proline Accumula-
tion and the Expression of ∆1-Pyrroline-5-Carboxylate
Synthetase in Two Safflower Cultivars,” Biologia Plan-
tarum, Vol. 54, No. 2, 2010, pp. 386-390.
doi:10.1007/s10535-010-0070-7
[7] P. B. Kavi Kishor, S. Sangam, R. N. Amrutha, P. S.
Laxmi, K. R. Naidu, K. R. S. S. Rao, S. Rao, K. J. Reddy,
P. Theriappan and N. Sreenivasulu, “Regulation of Pro-
line Biosynthesis, Degradation, Uptake and Transport in
Higher Plants: Its Implications in Plant Growth and
Abiotic Stress Tolerance,” Current Science, Vol. 88, No.
3, 2005, pp. 424-438.
[8] Y. Jung, J. Park, Y. Choi, J.-G. Yang, D. Kim, B.-G. Kim,
K. Roh, D.-H. Lee, C.-K. Auh and S. Lee, “Expression
Analysis of Proline-Metabolism Genes from Halophyte
Arabis stelleri under Osmotic Stress Conditions,” Journal
of Integrative Plant Biology, Vol. 52, No. 10, 2010, pp.
891-903. doi:10.1111/j.1744-7909.2010.00990.x
[9] G. Miller, H. Stein, A. Honig, Y. Kapulnik and A. Zilber-
stein, “Responsive Modes of Medicago sativa Proline
Dehydrogenase Genes during Salt Stress and Recovery
Dictate Free Proline Accumulation,” Planta, Vol. 222, No.
1, 2005, pp. 70-79.
[10] C. O. Silva-Ortega, A. E. Ochoa-Alfaro, J. A. Reyes-
Agüero, G. A. Aguado-Santacruz and J. F. Jiménez-Bre-
mont, “Salt Stress Increases the Expression of P5CS Gene
and Induces Proline Accumulation in Cactus Pear,” Plant
Physiology and Biochemistry, Vol. 46, No. 1, 2008, pp.
82-92. doi:10.1016/j.plaphy.2007.10.011
[11] G. Szekely, E. Abraham, A. Cselo, G. Rigo, L. Zsigmond,
J. Csiszar, F. Ayaydin, N. Strizhov, J. Jasik, E. Schmelzer,
C. Koncz and L. Szabados, “Duplicated P5CS Genes of

Responses of Transgenic Tobacco Plants with Increased Proline Content to Drought and/or Heat Stress
324
Arabidopsis Play Distinct Roles in Stress Regulation and
Developmental Control of Proline Biosynthesis,” Plant
Journal, Vol. 53, No. 1, 2008, pp. 11-28.
doi:10.1111/j.1365-313X.2007.03318.x
[12] M. Ashraf and M. R. Foodlad, “Roles of Glycine Betaine
and Proline in Improving Plant Abiotic Stress Resis-
tance,” Environmental and Experimental Botany, Vol. 59,
2007, pp. 206-216. doi:10.1016/j.envexpbot.2005.12.006
[13] S. Lutts, J. M. Kinet and J. Bouharmont, “Effects of Salt
Stress on Growth, Mineral Nutrition and Proline Accu-
mulation in Relation to Osmotic Adjustment in Rice
(Oryza sativa L.) Cultivars Differing in Salinity Resis-
tance,” Plant Growth Regulation, Vol. 19, No. 2, 1996,
pp. 207-218. doi:10.1007/BF00037793
[14] J. Gubis, R. Vankova, V. Cervena, M. Dragounova, M.
Hudcovicova, H. Lichtnerovia, T. Dokoupil and Z. Jure-
kova, “Transformed Tobacco Plants with Increased Tol-
erance to Drought,” South African Journal of Botany, Vol.
73. No. 4, 2007, pp. 505-511.
[15] J. Dobra, V. Motyka, P. Dobrev, J. Malbeck, I. T. Prasil,
D. Haisel, A. Gaudinova, M. Havlova, J. Gubis and R.
Vankova, “Comparison of Hormonal Responses to Heat,
Drought and Combined Stress in Tobacco Plants with
Elevated Proline Content,” Journal of Plant Physiology,
Vol. 167, No. 16, 2010, pp. 1360-1370.
doi:10.1016/j.jplph.2010.05.013
[16] P. B. Kavi Kishor, Z. Hong, G.-H. Miao, C. A. Hu and D.
P. S. Verma, “Overexpression of 1-Pyrroline-5-Carboxy-
lase Synthetase Increases Proline Production and Confers
Osmotolerance in Transgenic Plants,” Plant Physiology,
Vol. 108, No. 4, 1995, pp. 1387-1394.
[17] R. Dibax, C. Deschamps, J. C. Bespakhok Filho, L. G. E.
Vieira, H. B. C. Milinari, M. K. F. De Campos and M.
Quorin, “Organogenesis and Agrobacterium tumefaciens-
Mediated Transformation of Eucalyptus saligna with
P5CS Gene,” Biologia Plantarum, Vol. 54, No. 1, 2010,
pp. 6-12. doi:10.1007/s10535-010-0002-6
[18] V. Kumar, V. Shriram, P. B. Kavi Kishor, N. Jawali and
M. G. Shitole, “Enhanced Proline Accumulation and Salt
Stress Tolerance of Transgenic Indica Rice by over-Ex-
pressing P5CSF129A Gene,” Plant Biotechnology Re-
ports, Vol. 4, No. 1, 2010, pp. 37-48.
[19] T. Nanjo, M. Kobayashi, Y. Yoshiba, Y. Sanada, K.
Wada, H. Tsukaya, Y. Kakubari, K. Yamaguchi-Shino-
zaki and K. Shinozaki, “Biological Functions of Proline
in Morphogenesis and Osmotolerance Revealed in An-
tisense Transgenic Arabidopsis,” The Plant Journal, Vol.
18, No. 2, 1999, pp. 185-193.
doi:10.1046/j.1365-313X.1999.00438.x
[20] H. B. C. Molinari, C. J. Marur, E. Daros, M. K. Freitas de
Campos, J. F. R. Portela de Carvalho, J. C. B. Filho, L. F.
P. Pereira and E. L. G. Vieira, “Evaluation of the Stress-
Inducible Production of Proline in Transgenic Sugarcane
(Saccharum spp.): Osmotic Adjustment, Chlorophyll Fluo-
rescence and Oxidative Stress,” Physiologia Plantarum,
Vol. 130, No. 2, 2007, pp. 218-229.
doi:10.1111/j.1399-3054.2007.00909.x
[21] E. C. G. Vendruscolo, I. Schuster, M. Pileggi, C. A. Scapim,
H. B. C. Molinari, C. J. Marur and E. L. G. Vieira,
“Stress-Induced Synthesis of Proline Confers Tolerance
to Water deficit in Transgenic Wheat,” Journal of Plant
Physiology, Vol. 164, No. 10, 2007, 1367-1376.
doi:10.1016/j.jplph.2007.05.001
[22] P. Bhatnagar-Mathur, V. Vadez, M. J. Devi, M. Lavanya,
G. Vani and K. K. Sharma, “Genetic Engineering of
Chickpea (Cicer arietinum L.) with the P5CSF129A Gene
for Osmoregulation with Implications on Drought Toler-
ance,” Molecular Breeding, Vol. 23, No. 4, 2009, pp.
591-606. doi:10.1007/s11032-009-9258-y
[23] J. A. De Ronde, W. A. Cress, G. H. J. Kruger, R. J. Stre-
asser, J. Van Staden, “Photosynthetic Response of Trans-
genic Soybean Plants, Containing an Arabidopsis P5CR
Gene, during Heat and Drought Stress,” Journal of Plant
Physiology, Vol. 161, No. 11, 2004, pp. 1211-1224.
doi:10.1016/j.jplph.2004.01.014
[24] W. H. Hu, Y. A. Xiao, J. J. Zeng and X. H. Hu, “Photo-
synthesis, Respiration and Antioxidative Enzymes in
Pepper Leaves under Drought and Heat Stresses,” Biolo-
gia Plantarum, Vol. 54, No. 4, 2010, 761-765.
[25] M. L. Osório, J. Osório, A. C. Vieira, S. Gonçalves and A.
Romano, “Influence of Enhanced Temperature on Photo-
synthesis, Photooxidative Damage, and Antioxidant Stra-
tegies in Ceratonia siliqua L. Seedlings Subjected to
Water Deficit and Rewatering,” Photosynthetica, Vol. 49,
No. 1, 2011, 3-12.