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Cloning and developmental expression of the sucrose-phosphate-synthase gene from spinach

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Abstract

A 561-base-pair (bp) polymerase-chain-reaction (PCR) product of sucrose-phosphate synthase (SPS) was amplified using degenerate oligonucleotide primers corresponding to tryptic peptides of SPS (EC 2.4.1.14) from spinach (Spinacia oleracea L). Crucial to the primer specificity and the synthesis of the 561-bp product was the use of primer pools in which the number of degenerate primer species was limited. A full-length cDNA was subsequently obtained by screening a cDNA bacteriophage library with the 561-bp product of SPS and 5′ PCR-RACE (Rapid Amplification of cDNA Ends). The 3530-bp cDNA of SPS encoded for a 1056-amino-acid polypeptide of predicted molecular mass of 117 kDa. The deduced amino-acid sequence of spinach SPS showed regions of strong homology with SPS from maize (A.C. Worrell et al., 1991, Plant Cell 3, 1121–1130); amino-acid identity was 54% over the entire protein. Western and Northern analyses of root, petiole and spinach leaf tissue showed that SPS was expressed in an organ-specific manner, being predominantly localized in the leaf. The accumulation of SPS protein and mRNA during leaf development coincided with the early rapid phase of leaf expansion and the apparent transition of the leaf from sink to source status. Levels of SPS mRNA and protein were reduced during the acclimation of leaves to low-irradiance conditions. Transfer of low-irradiance-adapted leaves to higher-irradiance conditions resulted in a gradual increase in SPS protein and mRNA. Diurnal changes in irradiance did not alter SPS protein or transcript levels, indicating that short-term regulation of SPS primarily involves a modulation of enzyme activity.

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Abbreviations

kbp:

kilobase-pair

kDa:

kilodalton

PCR:

polymerase chain reaction

PCR-RACE:

rapid amplification of cDNA ends

rbcS, SAc, sps1:

genes encoding the small subunit of Rubisco, actin, and SPS, respectively

Rubisco:

ribulose-1,5-bisphosphate carboxylase-oxygenase

SPS:

sucrose-phosphate synthase

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Authors and Affiliations

  1. USDA-ARS, 107 Animal Pathology Building, University of Kentucky, 40546-0076, Lexington, KY, USA

    Robert R. Klein, Steven J. Crafts-Brandner & Michael E. Salvucci

Authors
  1. Robert R. Klein
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  2. Steven J. Crafts-Brandner
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  3. Michael E. Salvucci
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Additional information

The investigation reported in this paper (No. 92-3-249) is in connection with a project of the Kentucky Agricultural Experiment Station

The authors wish to thank C.A. Patterson for his technical assistance in Western-blot and sequence analyses. The authors also wish to thank L.F. Staples for her technical assistance in conducting SPS enzyme assays. Special thanks to Dr. S.C. Huber (USDA-ARS, North Carolina State University, USA) for helpful discussion on conducting SPS enzyme assays.

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Klein, R.R., Crafts-Brandner, S.J. & Salvucci, M.E. Cloning and developmental expression of the sucrose-phosphate-synthase gene from spinach. Planta 190, 498–510 (1993). https://doi.org/10.1007/BF00224789

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