Physiological, cytological and genetic tools in assessing plant growth: case studies of exposure to biotic and abiotic stress
Conceição Santos & Glória Pinto
CESAM (Centre for Environmental and Marine
Studies), &
Department of Biology
University of Aveiro , Portugal
Physiological,
cytological and genetic tools must be combined for assessing the plant
performance under stress. Our laboratory performs studies mostly on abiotic
stresses (e.g. salt, Cd and Pb) and on biotic stresses such as dutch elm
disease and esca (Fig. 1). Within this scope we routinely follow, at the
Laboratory of Biotechnology and Cytomics, the performance of plants by
combining a battery of cytological, histo-anatomical, physiological and genetic
approaches:
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A) |
B) |
C) |
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Figure 1. Aspect of grapevine plants infected with Phaeomoniella chlamydospora: (A)
control at day 15; (B)
infected with Ph. chlamydospora
(1AS strain) at day 15; black arrow: chlorotic regions. (C)
infected with Ph. chlamydospora
(1AS strain) at day 30; black arrow: chlorotic regions. |
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a) Oxidative stress: oxidative stress often increases with stress and
ageing by increasing the levels of reactive oxygen species (ROS). Several
complementary parameters such as: a) lipid peroxidation (detected by e.g. MDA),
b) membrane permeability, c) reactive oxygen species contents (e.g. H2O2),
d) expression/activity of genes/enzymes involved in antioxidative stress (e.g.
CAT, POX, SOD), when used in combination, indicate the oxidative status of the
plant and its ability to efficiently scavenge ROS and prevent damaging effects
of free radicals (e.g. Santos et al 2001; 2006; Azevedo 2005a; Loureiro et al
2006; Oliveira et al 2008;). All these physiological changes
are also followed by functional cytometry (e.g. viability), histochemical (e.g.
H2O2 detection) and ultrastructural changes by SEM and
TEM (e.g. Silva et al 2007).
b) Photosynthesis and carbon and nitrogen metabolism - stresses
affect osmotic potential as well as chlorophyll metabolism and light harvesting
complex II of plants, affecting therefore, photosynthesis and carbohydrate
metabolism. Stresses often decrease both photophosphorilation process and/or
enzymes involved in Calvin cycle (e.g. rubisco) (e.g. Azevedo et al 2006b;
Santos et al 2001; 2005; Oliveira et al 2008). These stresses also affect
nitrogen metabolism (e.g. aminoacids such as proline contents and GS1 and GS2
transcripts and enzyme activities) (e.g. Santos et al 2004). All these physiological
changes are also followed by histochemical (e.g. for starch and protein
detection) and ultrastructural changes by SEM and TEM.
c) Genetic analyses: Plants
under stress often suffer genetic changes. We currently assess nDNA, ploidy and
in cell cycle (G0/G1, S and G2 phases) changes, as well as putative clastogenic
effects induced by several compounds such as metals (e.g. Silva et al 2007).
Also, also molecular markers, such as microsatellites and RAPDs may provide
valuable tools to assess genetic instability under metal stress (Monteiro et
al. 2007; Table 1). Finally, we have recently established DNA methylation and
COMEts applied to plants under stress.
Table 1. Description of
the lettuce SSRs variability under exposure to Cd used: locus, repeat structure,
allele size, number of alleles (and PIC value) and the PCR conditions and ABI
dyes used. Polymorphism Information Content, PIC = 1 - ∑ pi2,
where pi is the frequency of the ith allele (adapted from
Monteiro et al 2007).
|
SSR |
Repeat
Structure |
Allele
size (pb) |
No.
Alleles / PIC values |
PCR
Conditions |
ABI
dye |
|
|
1 |
LsA004a |
(GA)19(GT)7(GAGT)4(GA)10 |
200 |
2
/ 0.52 |
55ºC
/ 30 |
FAM |
|
2 |
LsB101 |
(GT)12(AT)5(GT)17 |
184 |
3
/ 0.56 |
55ºC
/ 30 |
NED |
|
3 |
LsB104 |
(GA)5(GT)7TATT(GT)12(T)4(GT)8(GA)11 |
164 |
4
/ 0.64 |
55ºC
/ 30 |
FAM |
|
4 |
LsD106G |
(TCT)17(T)5(TCT)2 |
190 |
3
/ 0.56 |
55ºC
/ 30 |
HEX |
|
5 |
LsD109 |
(TCT)22 |
155 |
4
/ 0.80 |
55ºC
/ 30 |
HEX |
|
6 |
LsE003a |
(TGT)24(TA)(TGT)10(TAT)2 |
208 |
2
/ 0.32 |
55ºC
/ 30 |
HEX |
|
7 |
LsB108a |
(GT)8(AT)7(GT)25(GA)2(GT)5 |
197 |
3
/ 0.56 |
55ºC
/ 30 |
FAM |
|
8 |
LsD110a |
(TCT)21(TCC)2(TCT)7 |
234 |
- |
55ºC
/ 30 |
JOE |
|
9 |
LsG001G |
(GATA)31(GA)17 |
299 |
3
/ 0.72 |
50ºC
/ 30 |
FAM |
Selected publications:
Monteiro M, Santos C, Mann R, Soares A, Lopes T (2007). Evaluation of cadmium
genotoxicity in Lactuca sativa L.
using nuclear microsatellites. Environmental
Experimental Botany 60, 421–427
Oliveira
H ; Barros A; Santos C (2008) Fourier transformation infrared
spectroscopy analysis of salt stressed and fungi infected in vitro grapevine
plants. Environmental Experimental Botany (in press).
Santos C, Campos A, Azevedo
H(2001). In situ and in vitro senescence induced by KCl
stress: nutritional imbalance, lipid peroxidation and antioxidant metabolism. Journal.
Experimental. Botany, 52 (355): 351-360
Santos C, Fragoeiro A, Oliveira H, Phillips A (2006). Response of Vitis vinifera L. plants inoculated with
Phaeoacremonium angustius and Phaeomoniella chlamydospora to
thiabendazole, resveratrol and sodium arsenite. Scientia Horticulturae, 107(2): 131-136
Silva S, Costa A, Rodriguez E, Santos C, Lopes
P, Pinto-Carnide O, Guedes-Pinto H (2007) Effect of aluminum short
term exposures on nutrient accumulation in Triticum
aestivum INCOMAM’07 International conference on microscopy and
microanalsis, LifeSci-IV5, pg 90: 6-7
Conceição Santos is a specialist of in vitro culture and plant physiology also working
on Xomics and molecular biology. She’s leader of the
Glória Pinto is a specialist of in vitro culture. In the past 6 years she has worked
(often in collaboration with Celbi Co.) in order to implement somatic
embryogenesis in their tree breeding programs. Presently she is Pos-Doc of the
Biotechnology & Cytomics Lab at UA with special interest in functional
genomics and proteomics studies in order to improve the efficiency of these
processes (or others) with which breeders can select plants with desirable
combination of genes in several conditions (wood quality, disease stress,
drought stress, climate changes, etc).