Extracellular invertase is an essential component of cytokinin-mediated delay of senescence

Abstract

Leaf senescence is the final stage of leaf development in which the nutrients invested in the leaf are remobilized to other parts of the plant. Whereas senescence is accompanied by a decline in leaf cytokinin content, exogenous application of cytokinins or an increase of the endogenous concentration delays senescence and causes nutrient mobilization. The finding that extracellular invertase and hexose transporters, as the functionally linked enzymes of an apolasmic phloem unloading pathway, are coinduced by cytokinins suggested that delay of senescence is mediated via an effect on source-sink relations. This hypothesis was further substantiated in this study by the finding that delay of senescence in transgenic tobacco (Nicotiana tabacum) plants with autoregulated cytokinin production correlates with an elevated extracellular invertase activity. The finding that the expression of an extracellular invertase under control of the senescence-induced SAG12 promoter results in a delay of senescence demonstrates that effect of cytokinins may be substituted by these metabolic enzymes. The observation that an increase in extracellular invertase is sufficient to delay leaf senescence was further verified by a complementing functional approach. Localized induction of an extracellular invertase under control of a chemically inducible promoter resulted in ectopic delay of senescence, resembling the naturally occurring green islands in autumn leaves. To establish a causal relationship between cytokinins and extracellular invertase for the delay of senescence, transgenic plants were generated that allowed inhibition of extracellular invertase in the presence of cytokinins. For this purpose, an invertase inhibitor was expressed under control of a cytokinin-inducible promoter. It has been shown that senescence is not any more delayed by cytokinin when the expression of the invertase inhibitor is elevated. This finding demonstrates that extracellular invertase is required for the delay of senescence by cytokinins and that it is a key element of the underlying molecular mechanism.

Figure 1.

Cytokinin-Mediated Delay in Senescence Correlates with an Increase in the Activity of Extracellular Invertase. Extracellular invertase activity has been determined in top, middle, and bottom leaves of tobacco plants expressing the ipt gene under control of the senescence-activated promoter SAG12 (SAG12:ipt; Gan and Amasino, 1995) and of wild-type plants (W38). Bars represent the mean value of three independent replications ± se. DW, dry weight.

Figure 2.

Senescence-Induced Expression of Extracellular Invertase Cin1 Results in a Delay of Senescence. (A) Phenotype of the transgenic SAG12:Cin1 and of the wild-type (W38) tobacco plants 17 weeks after sawing. The transgenic plants show a delay in senescence of mature leaves in respect to the loss of mature leaves in the wild-type plants. (B) Delay in senescence of detached young leaves from the transgenic SAG12:Cin1 and wild-type (W38) tobacco plants incubated in the light for 4 weeks. The results have been reproduced in five independent experiments with three independent transgenic lines, and representative results obtained with line NT58-5 are shown.

Figure 3.

The Increase in Extracellular Invertase Activity in Transgenic Tobacco Plants Expressing Extracellular Invertase under Control of the Senescence-Activated Promoter SAG12 Is Specific and Does Not Result in an Increased Glucose Concentration. (A) Extracellular invertase activity measured in bottom and top leaves of SAG12:Cin1 and wild-type (W38) plants 17 weeks after sawing. Bars represent the mean value of three independent replications ± se. (B) Vacuolar invertase activity measured in bottom and top leaves of SAG12:Cin1 and wild-type (W38) plants 17 weeks after sawing. Bars represent the mean value of three independent replications ± se. (C) Glucose contents of bottom and top leaves of SAG12:Cin1 and wild-type (W38) plants 17 weeks after sawing. Bars represent the mean of three independent replication leaves ± se. The results have been reproduced with three independent transgenic lines, and representative results obtained with line NT58-5 are shown.

Figure 4.

Chemical Induction of Extracellular Invertase Cin1 Results in Delay of Senescence. (A) Effect of the infiltration of tetracycline into detached leaves of a transgenic line expressing the extracellular invertase Cin1 under control of the tetracycline-inducible TetR promoter. The left leaf was infiltrated with an MS control solution, whereas the right one was infiltrated with the same solution containing tetracycline at a final concentration of 10 μg/L. (B) Chlorophyll fluorescence image of the leaves shown in (A). (C) RNA gel blot showing the accumulation of the Cin1 transcripts in leaves after 2 h of treatment with tetracycline in comparison to control leaves. (D) Effect of the localized induction of Cin1 by spotting chlorotetracycline onto transgenic leaves. An MS solution containing either 0.02% Silwet (left control leaf) or the detergent plus chlorotetracycline (10 μg/L) (right leaf) was spotted onto the marked zones. The results have been reproduced in five independent experiments with three independent transgenic lines, and representative results obtained with line NT35-7 are shown.

Figure 5.

Effect of the Inhibition of Extracellular Invertase Activity on the Delay of Senescence by Kinetin. (A) Detached leaves of a transgenic tobacco line expressing the tobacco invertase inhibitor P17A under control of the cytokinin-inducible promoter Lin6 (Lin6:P17A) and wild-type plants (W38) were infiltrated with water containing kinetin at a final concentration of 30 μg/L. (B) RNA gel blot analysis showing the accumulation of the transcripts for extracellular invertase Ntβfruct1 and the invertase inhibitor P17A in wild-type (W38) and transgenic (Lin6:P17A) plants after 1 and 3 d of treatment with kinetin (K), respectively. The results have been reproduced in five independent experiments with three independent transgenic lines, and representative results obtained with line NT71-29 are shown.