Over-expressing CuZnSOD gene for controlling oxidative burst in Hevea brasiliensis (Müll. Arg)

Design and production: CIRAD - 2007

Over-expressing CuZnSOD gene for controlling

oxidative burst in Hevea brasiliensis (Müll. Arg).

French Agricultural Research Centre for International Development

H

evea brasiliensis (Müll. Arg.) is the main source of natural

rubber which is biosynthesized in latex cells. Ethephon

application can enhance the biosynthesis activity required

for latex regeneration after each tapping (figure 1A),

optimizing the yield potential of rubber tree. However,

a good management of both tapping frequencies and

ethephon applications is required for avoiding excessive metabolic activation

which can lead to Tapping Panel Dryness (TPD) (figure 1B, 1C).

This physiological disorder is a consequence of an oxidative stress in the latex

cells, leading to membrane damages, flocculation of rubber particles and

plugging of the latex vessels (Figure 1C). Three genotypes (PB 260, PB 217

and RRIM 600) with contrasted metabolism activity and response to ethylene

stimulation have been selected for further studies.

Leclercq J., Martin F., Lardet L., Rio M., Gebelin V., Chabaud M., Ayar A. and Montoro P.

UMR DAP, Department BIOS, CIRAD, TA A-96 / 03, Avenue Agropolis, 34 398 Montpellier Cedex 5, France.

CIRAD

Expression analysis of CuZnSOD gene by Q-PCR

CuZnSOD over-expression by genetic transformation of rubber tree

Expression analyses of CuZnSOD gene involved in

detoxifying the reactive oxygen species (ROS, Figure 2) has been studied in healthy trees, TPD trees and also in young budded plants. During photosynthesis, ROS production is enhanced (Foyer and Noctor, 2003). CuZnSOD gene expression is stimulated slightly in PB 260 at 16h00 and strongly in PB 217 and RRIM 600 at 12h00 (figure 3A).

In response to ethylene, the expression of the CuZnSOD gene is highly enhanced in PB 260, remains constant in PB 217 but is down-regulated in RRIM 600 even though in this genotype the expression level of CuZnSOD is always higher than other ones (figure 3B). In response to wounding, CuZnSOD transcript level is reduced in PB 260 and PB 217 (figure 3C). CuZnSOD gene has also been shown to be down-regulated in TPD tree compared to its healthy

counterpart (unpublished data).

An Agrobacterium tumefaciens-mediated genetic transformation has been developed (Blanc et al., 2006) based on the successful plant regeneration procedure using

H. brasiliensis PB 260 somatic embryos (Lardet et al., 1999). The GFP was used as a

visual marker (Figure 4). The CuZnSOD gene placed under the control of CaMV35S promoter was over-expressed in transgenic calli (data not shown). Regeneration of transgenic plants harbouring a single copy of the T-DNA is on-going (Figure 5).

(B) Tapping panel of a healthy tree. Figure 1 : (A) Application of ethephon

to a rubber tree tapping panel.

K

us

w

anhad

i

(C) A sick tree showing tapping panel dryness (TPD).

O2 O2-+ O2-+ 2 H+ O2 H2O2 H2O M D H A D H A A s c o rb a te 2 G S H 2 G S S G N A D P+ _{N A D P H + H}+ N A D P+ N A D P H + H+ SOD APX GR DH AR MDHAR H2O + O2 CAT O2 O2-+ O2-+ 2 H+ O2 H2O2 H2O M D H A D H A A s c o rb a te 2 G S H 2 G S S G N A D P+ _{N A D P H + H}+ N A D P+ N A D P H + H+ SOD APX GR DH AR MDHAR H2O + O2 CAT GR APX CAT SOD DHAR MDHAR Figure 2: The

ascorbate-glutathione cycle with the major antioxidative enzymes (Noctor, 1998; Alscher et al., 2002) 0 0 ,0 5 0 ,1 0 ,1 5 0 ,2 0 ,2 5 0 ,3 0 ,3 5 0 ,4 0 ,4 5 8h0 0 12 h0 0 1 6 h0 0 8 h0 0 1 2 h0 0 16 h0 0 8h0 0 12 h0 0 1 6 h0 0 PB 2 6 0 PB 2 17 RRIM 6 0 0 A Normalized ratio 0 0 ,2 0 ,4 0 ,6 0 ,8 1 1 ,2 1 ,4 1 ,6 1 ,8 T0 T4 h T0 T4 h T0 T4h PB 2 6 0 PB 2 1 7 RRIM 6 0 0 B _{Normalized ratio} 0 0 ,0 1 0 ,0 2 0 ,0 3 0 ,0 4 0 ,0 5 0 ,0 6 0 ,0 7 0 ,0 8 0 ,0 9 0 ,1 T0 T15 min T4 h T0 T1 5 min T4 h PB 2 60 PB 21 7 c _{Normalized ratio}

Figure 3: (A) CuZnSOD gene expression analysis by Q-PCR during the day

in young budded plant.

(B) CuZnSOD gene expression analysis by Q-PCR in response to ethylene treatment (T0 = air, T4h = 1 ppm during 4 hours) in young budded plant.

(C) CuZnSOD gene expression analysis by Q-PCR in response to wounding in young budded plant (15 min and 4 hours after wounding).

Figure 5: Genetic transformation steps:

(A) GFP fluorescence observed in callus after 5 days of coculture with A. tumefaciens. (B) GFP fluorescence in callus after antibiotic selection.

(C) GFP fluorescence in a somatic embryo. (D) GFP fluorescence in a regenerated plantlet.

A B _C

D

Conclusion

The gene encoding CuZnSOD displays different expression profiles in the three

genotypes in response to day light, ethylene and wounding. Analysis of over-expressing

CuZnSOD gene transgenic plants will allow a better understanding of the implication

of CuZnSOD activity in oxidative burst tolerance in rubber tree.

. Alscher et al. (2002) Journal of Experimental Botany 53(372), 1331-1341

. Blanc et al. (2006) Plant Cell Report 24(12) 724-733

. Foyer and Noctor (2003) Physiologia Plantarum 119: 355-364

. Lardet et al. (1999) Canadian Journal of Botany 77(8): 1168-1177 . Noctor (1998) Annual Review of Plant Physiology and Plant Molecular Biology 49 : 249-279

References...

NptI 35S GFP 35S SOD 35S 35S Gus

Figure 4: Constructs used for genetic transformation