Inorganic Markers of Stress in Natural and Genetically Modified Plants After Chemical and Physical Stress

 

A. Giacomino1*, M. Malandrino2, I. Zelano2, O. Abollino2

1 University of Torino; Department of Drug Science and Technology, Via Giuria 9, 10125 Torino2 University of Torino, Department of Chemistry, Via Giuria 5,10125 Torino

(agnese.giacomino@unito.it) 

The action of biotic and abiotic stresses on plants can induce within the plant the production of compounds able to contrast the effects of the attack. The knowledge of the response of the plants to unfavourable conditions can have useful effects in many fields (biological, environmental, agronomic), taking into account the climatic changes which occur in these last years.[1,2]

The study of the behavior of the plants and the effect of genic modifications in different environmental conditions can be very useful to predict the variation induced in the normal development of the plants caused by environ-mental pollution and climatic variations, taking into account the increasing water scarcity and temperature warning that are taking place on Earth. The availability of genetically modified forms of the investigated plant allowed us to evaluate the role played by the modifications for the improvement or the reduction of the resistance of such plants to the different environmental stresses.

Samples: Nicotiana langsdorfii (NL ) Wild Type (WT) without stress (WT) and stressed by Cr (WT Cr) or by water stress (WT W) or by termal strrss (WT T) NL modified with the rat gluco-corticoid receptor gene (GR) without stress (GR) and stressed by Cr (GR Cr) or by water stress (GR W) or by termal strrss (GR T)NL modified with rolC gene from Agrobacterium rhizogenes (rolC) without stress (rolC) and stressed by Cr (rolC Cr) or by water stress (rolC W) or by termal strrss (rolC T)Cultures were incubated in a growth chamber at 24±1°C with a photoperiod of 16 h of light (1500 lux) and 80% relative humidity. After germination, seedlingswere grown on LS0 medium for one month. Plant Incubation and growth were carried out at the Department of Evolutionary Biology ‘‘Leo Pardi”, University ofFlorence, by Prof. M. Buiatti and Dr. P. Bogani.Inorganic Markers[3]: Cl-, NO3

-,Na+, K+

Sample pretreatment: the samples were frozen with liquid nitrogen and freeze-dried. Extraction with water: 0.5 g of each sample were treated with 25 ml of high purity water. The suspension was shaken for 2 h at room temperature; then it was centrifuged and the supernatant was filtered through a 0.45 mm cellulose acetate filter and analysed for anions and cations content. Total concentrations: 0.2 g of each sample were treated with a mixture of 3 ml HNO3/3ml H2O2 in a microwave oven.

References(1) C. Poschenrieder, R. Tolra, J. Barcelò, Trends in Plant Science 11 (2006).(2) N. Candan, L. Tarhan. Plant Science 165 (2003).(3) M. Patra, N. Bhowmikb, B. Bandopadhyay, A. Sharma. Environmental and Experimental Botany 52 (2004).

From the analysis:The genetically modified plants contain different concentrations of cations and anions in comparison to the wild type form, in particular all the modified forms (GR, rolC and rolD) contain lower amount of the markers-ions.Also the results obtained from the water extracts analysis confirmed that the considered stresses cause a decrease of the markers concentration, with the exception of rolC samples in which [Na] increases in the presence of stresses, and [K] in rolC-WS.

From the chemometric treatment of the results: The plot of the scores obtained by the PCA considering only the investigated inorganic markers shows a symilar behaviour of the samples after the same stress, in particular for the plants after termal stress. None separation is observed for the different plant forms.The symilarity among the plants seems to be influenced more by the type of the stress than by the genetic modification.

Sample Cl- NO3- Na+ K+

NL-WT 16,3 63,8 1,0 36,9

NL-WT Cr8,5

(-47,7)

36,0

(-43,6)

0,5

(-50,0)

23,7

(-35,8)

NL-WT W2,4

(-85,1)

11,6

(-81,9)

0,5

(-50,0)

32,9

(-10,8)

NL-WT T5,4

(-67,1)

55,3

(-13,5)

0,3

(-70,0)

0,7

(-98,1)

NL-GR 13,9 53,7 1,0 32,2

NL-GR Cr10,8

(-22,2)

30,9

(-42,5)

0,7

(-30,0)

21,7

(-32,6)

NL-GR W12,0

(-13,8)

45,3

(-15,7)

0,6

(-40,0)

29,2

(-9,3)

NL-GR + T6,1

(-56,0)

43,0

(-20,0)

0,5

(-50,0)

4,8

(-85,1)

NL-RolC 15,8 55,8 0,3 22,4

NL-RolC Cr8,8

(-44,4)

30,3

(-45,6)

0,4

(33,3)

17,8

(-20,5)

NL-RolC W10,1

(-36,2)

36,2

(-35,1)

0,5

(66,7)

30,9

(38,0)

NL-RolC T7,2

(-54,4)

55,3

(-0,9)

0,4

(33,3)

4,8

(-78,6)

NL-RolD 4,2 37,2 0,2 3,5

Sample Ca Mg Cr Fe Mn Zn P

NL-WT 1,46 0,96 ≤ 0,05 0,039 0,18 0,08 8,64

NL-WT + Cr1,22

(-16,4)

0,82

(-14,6)0,19

0,02

(-46,2)

0,10

(-44,4)

0,05

(-37,5)

6,53

(-24,4)

NL-WT + W1,56

(6,8)

1,1

(17,7)≤ 0,05

0,00

(-82,0)

0,18

(0)

0,05

(-37,5)

9,89

(14,5)

NL-WT + T0,67

(-54,1)

0,6

(-31,2)≤ 0,05

0,00

(-84,6)

0,06

(-66,7)

0,03

(-62,5)

1,55

(-82,1)

NL-GR 0,94 0,76 ≤ 0,05 0,011 0,13 0,05 8,35

NL-GR+ Cr0,72

(-23,4)

0,5

(-34,2)0,15

0,01

(18,2)

0,07

(-46,1)

0,03

(-40,0)

5,71

(-31,6)

NL-GR + W0,62

(-34,0)

0,7

(-7,9)≤ 0,05

0,00

(-63,6)

0,09

(-30,8)

0,04

(-20,0)

12,72

(52,3)

NL-GR + T0,3

(-67,0)

0,4

(-42,1)≤ 0,05

0,00

(-54,5)

0,03

(-76,9)

0,02

(-60,0)

1,55

(-81,4)

NL-RolC 0,85 0,81 ≤ 0,05 0,006 0,11 0,06 8,59

NL-RolC+ Cr0,7

(-12,9)

0,5

(-35,8)0,11

0,01

(133)

0,06

(-45,5)

0,02

(-66,7)

7,32

(-14,8)

NL-RolC+ W0,9

(10,6)

1,0

(23,5)≤ 0,05

0,01

(133)

0,13

(18,2)

0,06

(0)

11,60

(35,0)

NL-RolC+ T0,

(-52,9)

0,

(-38,3)≤ 0,05

0,00

(16,7)

0,04

(-63,6)

0,02

(-66,7)

1,36

(-84,2)

NL-RolD 0,74 0,26 ≤ 0,05 0,048 0,10 0,05 1,33

WT

WT+Cr

WT+W

WT+T

GR

GR+Cr

GR+W

GR+T

RolC

RolC+Cr

RolC+W

RolC+T

RolD

0

2

4

6

8

10

12

14

16

18

Cl-

g/K

g D

W

WT

WT+Cr

WT+W

WT+T

GR

GR+Cr

GR+W

GR+T

RolC

RolC+Cr

RolC+W

RolC+T

RolD

0

10

20

30

40

50

60

70

NO3-

g/K

g D

W

WT

WT+Cr

WT+W

WT+T

GR

GR+Cr

GR+W

GR+T

RolC

RolC+Cr

RolC+W

RolC+T

RolD

0

0.2

0.4

0.6

0.8

1

1.2

Na+

g/K

g D

W

WT

WT+Cr

WT+W

WT+T

GR

GR+Cr

GR+W

GR+T

RolC

RolC+Cr

RolC+W

RolC+T

RolD

0

5

10

15

20

25

30

35

40

K+g

/Kg

DW

Concentration (g/kgDW = dried weight) of Cl-, NO3-, Na+ and K+ in WATER EXTRACTS

and percentage in comparison with the corresponding plant without stress

-3 -2 -1 0 1 2 3 4

-2

-1

0

1

2

3

WT

WT + Cr

WT + W

WT + T

GRGR+ Cr

GR + W

GR + T

RolC

RolC+ Cr

RolC+ W

RolC+ T

RolD

Observations (F1 e F2: 65,21 %)

F1 (40,55 %)

F2

(24,

66 %

)

PCAConsidering only the

markers (Cl-, NO3

-, Na+, K+)

ICP-OESPerkin Elmer Optima 7000

IC Dionex DX-500

(Column: ion pack AS9-HC)

Markers

Other analytes

=

Element

WT GR RolC WT Cr GR Cr RolC Cr WT W GR W RolC W WT T GR T RolC T Rol D

Mg 1.87 1.28 1.77 1.70 1.77 1.43 1.94 1.10 1.63 2.48 1.87 2.02 2.57Na 0.97 1.15 0.56 0.71 0.74 0.63 0.62 0.95 0.48 0.62 1.78 1.13 1.97Ca 6.87 5.94 2.34 5.93 6.20 5.09 7.39 5.49 5.64 6.00 4.48 4.90 6.51P 12.6 10.9 5.61 11.1 11.6 13.6 12.8 8.96 14.9 5.70 5.73 4.66 6.85Fe 0.40 0.22 14.9 0.31 0.32 0.25 0.27 0.17 0.21 0.16 0.16 0.18 0.14Al 0.01 0.00 0.28 0.02 0.02 0.02 0.01 0.00 0.01 0.01 0.00 0.15 0.01Mn 0.36 0.32 0.00 0.24 0.25 0.21 0.37 0.22 0.32 0.23 0.22 0.22 0.40Zn 0.15 0.13 0.29 0.10 0.10 0.07 0.15 0.07 0.12 0.08 0.10 0.08 0.12Si 0.09 0.06 0.05 0.31 0.33 0.18 0.05 0.07 0.06 0.35 0.11 0.10 0.25K 220 213 186 169 181 142 202 152 172 82.8 67.8 63.0 59.2

TOTAL[ ]