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SUMMARY Nectar volume and sugar composition are key determinants of the strength of plant–pollinator mutualisms. The main nectar sugars are sucrose, glucose and fructose, which can vary widely in ratio and concentration across species.Brassica spp. produce a hexose‐dominant nectar (high in the monosaccharides glucose and fructose) with very low levels of the disaccharide sucrose. Cell wall invertases (CWINVs) catalyze the irreversible hydrolysis of sucrose into glucose and fructose in the apoplast. We found thatBrCWINV4Ais highly expressed in the nectaries ofBrassica rapa. Moreover, abrcwinv4anull mutant: (i) has greatly reduced CWINV activity in the nectaries; (ii) produces a sucrose‐rich nectar; but (iii) with significantly less volume. These results definitively demonstrate that CWINV activity is not only essential for the production of a hexose‐rich nectar, but also support a hypothetical model of nectar secretion in which its hydrolase activity is required for maintaining a high intracellular‐to‐extracellular sucrose ratio that facilitates the continuous export of sucrose into the nectary apoplast. The extracellular hydrolysis of each sucrose into two hexoses by BrCWINV4A also likely creates the osmotic potential required for nectar droplet formation. These results cumulatively indicate that modulation of CWINV activity can at least partially account for naturally occurring differences in nectar volume and sugar composition. Finally, honeybees prefer nectars with some sucrose, but wild‐typeB. rapaflowers were much more heavily visited than flowers ofbrcwinv4a, suggesting that the potentially attractive sucrose‐rich nectar ofbrcwinv4acould not compensate for its low volume.
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Functional annotation of haloacid dehalogenase superfamily structural genomics proteins
Haloacid dehalogenases (HAD) are members of a large superfamily that includes many Structural Genomics proteins with poorly characterized functionality. This superfamily consists of multiple types of enzymes that can act as sugar phosphatases, haloacid dehalogenases, phosphonoacetaldehyde hydrolases, ATPases, or phosphate monoesterases. Here, we report on predicted functional annotations and experimental testing by direct biochemical assay for Structural Genomics proteins from the HAD superfamily. To characterize the functions of HAD superfamily members, nine representative HAD proteins and 21 structural genomics proteins are analyzed. Using techniques based on computed chemical and electrostatic properties of individual amino acids, the functions of five structural genomics proteins from the HAD superfamily are predicted and validated by biochemical assays. A dehalogenase-like hydrolase, RSc1362 (Uniprot Q8XZN3, PDB 3UMB) is predicted to be a dehalogenase and dehalogenase activity is confirmed experimentally. Four proteins predicted to be sugar phosphatases are characterized as follows: a sugar phosphatase from Thermophilus volcanium (Uniprot Q978Y6) with trehalose-6-phosphate phosphatase and fructose-6-phosphate phosphatase activity; haloacid dehalogenase-like hydrolase from Bacteroides thetaiotaomicron (Uniprot Q8A2F3; PDB 3NIW) with fructose-6-phosphate phosphatase and sucrose-6-phosphate phosphatase activity; putative phosphatase from Eubacterium rectale (Uniprot D0VWU2; PDB 3DAO) as a sucrose-6-phosphate phosphatase; and hypothetical protein from Geobacillus kaustophilus (Uniprot Q5L139; PDB 2PQ0) as a fructose-6-phosphate phosphatase. Most of these sugar phosphatases showed some substrate promiscuity.
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- PAR ID:
- 10468505
- Publisher / Repository:
- Portland Press
- Date Published:
- Journal Name:
- Biochemical Journal
- Volume:
- 480
- Issue:
- 19
- ISSN:
- 0264-6021
- Page Range / eLocation ID:
- 1553 to 1569
- Subject(s) / Keyword(s):
- Haloacid dehalogenase Hypothetical protein Structural genomics Function prediction POOL SALSA Substrate specificity Sugar phosphate phosphatase
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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- Free Publicly Accessible Full Text
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Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.1042/BCJ20230057
