W. CHENG and R. WU
Section of Biochemistry. Molecular and Cell Biology, Cornell University, lthaca. New York 14853, USA
Potato- and tomato-derived proteinase inhibitors are powerful inhibitors of serine proteinase, which help defend plants against insect predators by reducing the digestibility and nutritional quality of the leaves (Ryan 1978). In vivo studies have shown that the genes encoding proteinase inhibitors are activated at the transcriptional level following wounding (Graham et al. 1986). Transgenic tobacco plants resulting from direct transformation with proteinase inhibitor I and II genes also demonstrated significant retardation of the growth of Manduca sexta larvae (Johnson et al. 1989). We have transformed Japonica rice plants with the potato proteinase inhibitor II gene driven by its own promoter together with the first intron of the rice actin I gene and found that the wolind-inducible expression of PinII gene resulted in high-level accumulation of the PINII protein in the transgenic rice plants. These plants showed resistance to a major rice insect pest, pink stem borer (Sesamia inferens) (Duan et al. 1996).
It has been reported that abscisic acid (ABA), jasmonic acid (JA), and systemin, as well as mechanical injury, activate PinII gene expression (Pena-Cortes et al. 1995). By using the bacterial β-glucuronidase gene (GUS) as a reporter, we also demonstrated that the expression of the PinII-Actin 1 intron-GUS fusion gene displays a systemic wound response in rice (Xu et al. 1993). However, the kinetics of induction of PinII gene and the stability of the PINII protein produced in transgenic rice plants remain unknown.
In this report, we show that both wounding site and wounding intensity have apparent effects on the expression level of the PinII gene. Different leaves of the same plant, and different regions of the same leaf, respond to wounding with different sensitivities. Both the Pinll mRNA and PINII protein levels correlate to wounding intensity and distance from the wounding site.
After initial wounding, both the mRNA and PINII protein are highly stable in transgenic plants. The level of PinII mRNA reached a peak one week after wounding and the level was still detectable for at least one more week. The PINII protein level reached a peak two weeks after wounding and the level was still detectable for at least two more weeks.
(1) Effects of location of wounding sites on PinII gene expression and systemic induction
Transgenic rice plants at the 6-leaf-stage were used to test the effect of wounding (by small cuts with scissors) different regions of leaves and different leaves of a plant on the induction of local and systemic expression of the induction of the PinII gene. After collecting leaf samples, the activity of PINII protein was measured based on proteinase inhibitory activity in leaf extracts against commercial bovine pancreatic trypsin (Duan et al. 1996). We found that the highest expression of PinII gene always occurred at the wounded regions. In the same leaf, wounding different regions gave different maximum PINII activities. Wounding the top region (tip) gave the highest activity in the entire leaf, the bottom region was second, and the middle region gave the lowest activity. In the plant wounding of the bottom leaf gave the highest systemic PINII activity in other leaves of the plant, wounding the top leaf was second, and wounding the middle leaf gave the lowest activity.
(2) Effects of wounding intensity on local and systemic induction of PinII gene expression
The wounding intensity (the number of cuts per leaf) has positive effects on both local and systemic induction of PinII gene expression in transgenic rice plants. Apparently, with increase of the number of cuts per leaf of transgenic plants, the PINII protein levels increase accordingly (Fig. 1 ). It appears that the systemic transduction of wounding signal spreads efficiently to a long distance from leaf#l to leaf#6 (Fig. 2).
(3) Kinetics of accumulation and stability of PINII protein synthesized in transgenic rice plants upon wounding
To determine the kinetics of PinII gene expression in transgenic rice plants upon wounding, only the top leaf was wounded and these leaves were collected at different time intervals. The level of PINII protein, upon wounding, increased continuously and reached the highest level two weeks after wounding, and then decreased slowly in thirty days (Fig. 3). After wounding, the kinetics of accumulation and the high degree of stability of PINII protein produced in transgenic rice plants suggest that these plants can protect themselves starting 12 hours after wounding and continuing for a long period of time after the initial attack by insects.
(4) Inhibitory potential and efficiency of the two reactive sites in the PINII protein
The PINI protein contains two separate reactive sites (Plunkett el al. 1982) with Arg-Glu in the trypsin inhibitory site and Leu-Asn in the chymotrypsin inhibitory site (Thornburg et al. 1987). We tested the inhibitory potentials and efficiencies of inhibition of proteases in the two reactive sites of PINII protein produced in transgenic rice plants. The results in Fig. 4 show that leaf extracts from PinII gene-transformed rice plants strongly inhibited the activities of both chymotrypsin and trypsin. Leaf extracts from PinII transgenic rice line #6-8-6-3 showed 84% and 45% of chymotrypsin activity remaining, and 80% and 45% of trypsin activity remaining from non wounded and wounded plants, respectively. Moreover, the relationship between the amount of leaf proteins and the chymotrypsin or trypsin inhibitory percentages shows linear relationships up to 75 μg of extract protein. At higher concentrations of protein, the slope of the line is less steep. Therefore, the PINII protein produced in PinII-transgenic rice plants remains intact in molecular structure and it is equally active in inhibiting both chymotrypsin and trypsin.
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