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Efficient developmental mis-targeting by the sporamin NTPP vacuolar signal to plastids in young leaves of sugarcane and Arabidopsis

  • Cell Biology and Morphogenesis
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Abstract

Plant vacuoles are multi-functional, developmentally varied and can occupy up to 90% of plant cells. The N-terminal propeptide (NTPP) of sweet potato sporamin and the C-terminal propeptide (CTPP) of tobacco chitinase have been developed as models to target some heterologous proteins to vacuoles but so far tested on only a few plant species, vacuole types and “payload” proteins. Most studies have focused on lytic and protein-storage vacuoles, which may differ substantially from the sugar-storage vacuoles in crops like sugarcane. Our results extend the evidence that NTPP of sporamin can direct heterologous proteins to vacuoles in diverse plant species and indicate that sugarcane sucrose-storage vacuoles (like the lytic vacuoles in other plant species) are hostile to heterologous proteins. A low level of cytosolic NTPP-GFP (green fluorescent protein) was detectable in most cell types in sugarcane and Arabidopsis, but only Arabidopsis mature leaf mesophyll cells accumulated NTPP-GFP to detectable levels in vacuoles. Unexpectedly, efficient developmental mis-trafficking of NTPP-GFP to chloroplasts was found in young leaf mesophyll cells of both species. Vacuolar targeting by tobacco chitinase CTPP was inefficient in sugarcane, leaving substantial cytoplasmic activity of rat lysosomal β-glucuronidase (GUS) [ER (endoplasmic reticulum)-RGUS-CTPP]. Sporamin NTPP is a promising targeting signal for studies of vacuolar function and for metabolic engineering. Such applications must take account of the efficient developmental mis-targeting by the signal and the instability of most introduced proteins, even in “storage vacuoles”.

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Abbreviations

BGUS:

Bacterial β-glucuronidase

CCLR:

Cell culture lysis reagent

CTPP:

C-terminal propeptide

DW:

Dry weight

ER:

Endoplasmic reticulum

FW:

Fresh weight

GFP:

Green fluorescent protein

LAR:

Luciferase assay reagent

LUC:

Firefly luciferase

MGUS:

Modified glycosylation-resistant GUS

NR:

Neutral red

NTPP:

N-terminal propeptide

RGUS:

Rat lysosomal GUS

RLU:

Relative light units

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Acknowledgements

We thank Drs. Jean-Marc Neuhaus (University of Neuchatel, Switzerland) and Melvin G. Rosenfeld (New York School of Medicine, USA) for the RGUS constructs, Dr. J. Mylne (University of Queensland, Australia) for his help with Arabidopsis, and the University of Queensland for Postgraduate Research Scholarships (OPRS and UQPRS) to AG.

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  1. Department of Botany, The University of Queensland, Brisbane, QLD 4072, Australia

    Annathurai Gnanasambandam & Robert G. Birch

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  1. Annathurai Gnanasambandam
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  2. Robert G. Birch
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Correspondence to Robert G. Birch.

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Communicated by P. Lakshmanan

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Gnanasambandam, A., Birch, R.G. Efficient developmental mis-targeting by the sporamin NTPP vacuolar signal to plastids in young leaves of sugarcane and Arabidopsis. Plant Cell Rep 23, 435–447 (2004). https://doi.org/10.1007/s00299-004-0860-5

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