Suppression of LX ribonuclease in tomato results in a delay of leaf senescence and abscission

Abstract

Although present in different organisms and conserved in their protein sequence, the biological functions of T2 ribonucleases (RNase) are generally unknown. Tomato (Lycopersicon esculentum) LX is a T2/S-like RNase and its expression is known to be associated with phosphate starvation, ethylene responses, and senescence and programmed cell death. In this study, LX function was investigated using antisense tomato plants in which the LX protein level was reduced. LX protein levels normally become elevated when leaves senesce and antisense inhibition of LX retarded the progression of senescence. Moreover, we observed a marked delay of leaf abscission in LX-deficient plants. This correlated with specific induction of LX protein in the tomato mature abscission zone tissue. LX RNase gene regulation and the consequences of antisense inhibition indicate that LX has an important functional role in both abscission and senescence.

Figure 1.

Induction of the LX protein level during senescence and in response to ethylene and its inhibition in LX antisense lines. A, Regulation of the LX protein level during leaf senescence. Proteins extracted from leaves at different stages of natural senescence from very young leaf (L5) up to fully yellowed leaf (L1). The arrow indicates the progress of senescence. Proteins extracted from the same fresh weight of leaf tissue were used in western-blot analysis for measuring the LX protein level. B, Changes in LX protein level in young leaves following ethylene treatment. Plants at 6 weeks of age with six developed leaves were exposed to ethylene in air at 2 μL L⁻¹ for 24 h, followed by air streaming. Proteins extracted from leaves at different positions on the stem and at different times were used in western-blot analysis for measuring the LX protein level. L1 to L4, Leaves at position 1 (lowest) to position 4 on the stem, respectively. C, Comparison of LX protein level between wild-type and antisense lines in young leaves following ethylene treatment. Intact 6-week-old plants were subjected to ethylene (10 μL L⁻¹) treatment for 18 h. Protein samples (10 μg) extracted from young leaves at two different positions were subjected to western-blot analysis. SL, Senescing leaf; VF36, wild-type control; A2, H9, and T2, independent LX antisense transgenic lines.

Figure 2.

Comparison between wild-type and antisense lines for their anthocyanin and chlorophyll content. Tomato plants were grown under Pi-deficient conditions. Anthocyanin and chlorophyll were extracted from the first leaf in all plants and measured, as described in “Materials and Methods.” Error bars correspond to ±sd. VF36, Wild-type control; A2, H9, and T2, independent LX antisense transgenic lines.

Figure 3.

Quantitation of chlorophyll, protein, and LX protein levels in wild-type and LX antisense lines. A, Chlorophyll, protein, and LX levels in cotyledons of plants that were grown in small-volume soil containers for 4 weeks until four leaves had developed. All three measurements were performed with the same extracts. Error bars correspond to ±sd. VF36, Wild-type control; A2, H9, and T2, independent LX antisense transgenic lines. B, Chlorophyll, protein, and LX levels in the first true leaves of plants that were grown in small-volume soil containers for 7 weeks. Labels are the same as in A. C, Chlorophyll and protein levels in the first true leaves of plants grown on perlite watered with full nutrient solution for 5 weeks, followed by Pi starvation for an additional 3 weeks. Labels are the same as in A.

Figure 4.

Abscission of cotyledons and leaf petioles in wild-type and LX antisense plants following growth under nutrient limitation (A) or following deblading plus ethylene treatment (B and C). A, Control and LX antisense plants grown on perlite and continuously watered with tap water. VF36, Wild-type control; A2 and T2, independent LX antisense transgenic lines. B, Abscission of cotyledons in ethylene-treated 6-week-old wild-type and LX antisense lines. Plants grown on soil were subjected to ethylene at 2 μL L⁻¹ for 36 h after which they were moved back to the greenhouse. The numbers of abscising cotyledons were recorded just after the ethylene treatment (day 0) and on the following 2 d. The percentages of abscising cotyledons out of the total number (40) are presented. Error bars correspond to ±sd. VF36, Wild-type control; A2, H9, and T2, independent LX antisense transgenic lines. C, Abscission of debladed petioles in ethylene-treated 6-week-old wild-type and LX antisense lines. In each plant, the three lowest leaves were debladed and the plants were ethylene treated as in B. The numbers of abscising petioles were recorded and the percentages of abscising leaves out of the total number (60) are presented. Experiment details and labeling as in B.

Figure 5.

Abscission of debladed leaf petioles in wild-type and LX antisense plants grown under optimal conditions. Control and LX antisense plants were grown on soil for 3 months until flowering was initiated and the plants developed about 10 leaves. Deblading was performed for the leaves in the four indicated positions on the stem and the number of abscising petioles was recorded for each position separately. The percentages of abscising petioles out of the total number (20) are presented for each petiole position in the four graphs. VF36, Wild-type control; A2, H9, and T2, independent LX antisense transgenic lines.

Figure 6.

Expression of LX protein in tomato AZ. A, Increased LX protein level in the petiole AZ tissue. Proteins were extracted from tissue slices a few millimeters in thickness that included the AZ or from control tissue slices of the same size from a few millimeters away on the petiole (C). The slices were taken from either a young leaf or a senescing leaf that approached abscission. Following separation of 10 μg protein on SDS-PAGE, the LX protein was immunodetected with LX antibodies. S, Senescing leaf protein. B, Comparison of LX protein levels in the AZs of wild-type and antisense plants. AZ, AZ tissue; C, control tissue; VF36, control wild-type plant; A2, H9, independent LX antisense plants; S, senescing leaf protein. C, Increased LX protein level in AZ of ripe fruits. Proteins were extracted from tissue slices a few millimeters in thickness that included the AZ or from control tissue slices of the same size taken from a few millimeters away on the fruit pedicle petiole (C). The slices were cut from either wild-type (VF36) or antisense plants (A2, H9, T2). Following separation of 10 μg of proteins on SDS-PAGE, immunodetection was performed with LX antibodies. S, Senescing leaf protein.

Figure 7.

Expression of LX-related RNase in tobacco. A, Immunodetection of RNase in senescing (TS) or young (TY) tobacco leaves with LX antibodies. S, Tomato senescing leaf protein. B, Immunodetection of the LX-related RNase in tobacco leaf AZ. Proteins were extracted from tissue slices, a few millimeters in thickness, which included the AZ, or from control tissue slices of the same size cut from a few millimeters away on the leaf petiole (C). Following separation of 10 μg protein on SDS-PAGE, immunodetection was performed with LX antibodies.