Abstract
Sucrose-phosphate phosphatase (SPP) catalyses the final step in the sucrose biosynthesis pathway. Arabidopsis thaliana genome codifies four SPP isoforms. In this study, the four Arabidopsis thaliana genes coding for SPP isoforms have been cloned, expressed in Escherichia coli and the kinetic and regulatory properties of the purified enzymes analysed. SPP2 is the isoform showing the highest activity, with SPP3b and SPP3a showing lower activity levels. No activity was detected for SPP1. We propose that this lack of activity is probably due to the absence of an essential amino acid participating in catalysis and/or in the binding of the substrate, sucrose-6-phosphate (Suc6P). The expression patterns of Arabidopsis SPP genes indicate that SPP2 and SPP3b are the main isoforms expressed in different tissues and organs, although the non-catalytic SPP1 is the main isoform expressed in roots. Thus, SPP1 could have acquired new unknown functions. We also show that the three catalytically active SPPs from Arabidopsis are dimers. By generating a chimeric SPP composed of the monomeric cyanobacterial SPP fused to the higher plant non-catalytic S6PPc domain (from SPP2), we show that the S6PPc domain is responsible for SPP dimerization. This is the first experimental study on the functionality and gene expression pattern of all the SPPs from a single plant species.
Highlights
Sucrose is an essential carbohydrate for higher plants and other photosynthetic organisms and considered to be one of the main products of photosynthesis [1, 2]
SPP1 and SPP3a were PCR-amplified from total cDNA preparations from roots or rosette leaves, respectively. cDNA fragments were amplified by PCR using specific primers (Table 1) which were designed with the appropriate restriction sites and a start codon (Met)
When we tested the affinity for Suc6P of the three active Sucrose-phosphate phosphatase (SPP) (Table 3), SPP2 was shown to have the lowest Km for Suc6P (0.73 mM), SPP3a showed a similar Km value (0.87 mM), while SPP3b displayed a 5-times higher Km (3.46 mM) than SPP2. These results suggest that SPP2 is the main isoform responsible for sucrose synthesis in Arabidopsis with the highest activity and affinity for the substrate, while SPP3a is the isoform with the lowest activity and catalytic efficiency for the substrate (Table 3)
Summary
Sucrose is an essential carbohydrate for higher plants and other photosynthetic organisms and considered to be one of the main products of photosynthesis [1, 2]. Sucrose is primarily synthesized in photosynthetic cells and transported to the rest of the plant to provide carbon and energy for growth and for the accumulation of carbon reserves. Sucrose is involved in the regulation of different processes including transcriptional and post-transcriptional control and stress responses [2,3,4,5,6,7,8]. Arabidopsis thaliana Sucrose Phosphate Phosphatases role in study design, data collection and analysis, decision to publish, or preparation of the manuscript
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