Cloning and characterization of a cDNA encoding hexokinase from tomato

Area of Science:

• Plant Molecular Biology
• Enzymology
• Biochemistry

Background:

• Hexokinases (HXK) are crucial enzymes in carbohydrate metabolism, catalyzing the initial phosphorylation of hexoses.
• Understanding HXK gene families in plants like tomato (Lycopersicon esculentum) is vital for elucidating sugar signaling and metabolic pathways.
• Previous studies indicated the presence of multiple HXK genes in various plant species, but their specific roles remain to be fully elucidated.

Purpose of the Study:

• To isolate and characterize novel hexokinase genes from tomato (Lycopersicon esculentum).
• To determine the functional role and substrate specificity of the identified tomato hexokinase.
• To analyze the expression patterns of the characterized hexokinase gene across different tomato tissues and developmental stages.

Main Methods:

• Isolation of partial HXK sequences from tomato using RT-PCR with degenerate primers.
• Screening of a tomato cDNA library to obtain a full-length LeHXK2 clone.
• Functional expression of LeHXK2 in a glucose/fructose phosphorylation-deficient yeast mutant.
• Kinetic analysis of purified LeHXK2 protein.
• RT-PCR analysis to determine LeHXK2 mRNA expression levels in various tomato tissues.

Main Results:

• Two distinct partial HXK sequences were identified, leading to the isolation of the full-length LeHXK2 cDNA.
• LeHXK2 encodes a 496 amino acid protein with significant homology to known plant HXKs, particularly potato StHXK2.
• Expression of LeHXK2 in a mutant yeast strain restored its ability to grow on glucose and fructose, with a preference for glucose.
• Kinetic studies confirmed LeHXK2's hexokinase activity and higher affinity for glucose.
• LeHXK2 mRNA was ubiquitously detected in all tomato organs, with the highest expression observed in flowers.

Conclusions:

• LeHXK2 represents a functional hexokinase in tomato (Lycopersicon esculentum) involved in glucose and fructose phosphorylation.
• The enzyme exhibits kinetic properties suggesting a primary role in glucose metabolism.
• LeHXK2's high expression in flowers indicates a potential specialized role in floral development or function.