Cloning, functional characterization and evaluating potential in metabolic engineering for lavender ( +)-bornyl diphosphate synthase.

KEY MESSAGE: We isolated and functionally characterized a new ( +)-bornyl diphosphate synthase (( +)-LiBPPS) from Lavandula x intermedia. The in planta functions of ( +)-LiBPPS were evaluated in sense and antisense transgenic plants. The monoterpene ( +)-borneol contributes scent and medicinal properties to some plants. It also is the immediate precursor to camphor, another important determinant of aroma and medicinal properties in many plants. ( +)-Borneol is generated through the dephosphorylation of bornyl diphosphate (BPP), which is itself derived from geranyl diphosphate (GPP) by the enzyme ( +)-bornyl diphosphate synthase (( +)-BPPS). In this study we isolated and functionally characterized a novel ( +)-BPPS cDNA from Lavandula x intermedia. The cDNA excluding its transit peptide was expressed in E. coli, and the corresponding recombinant protein was purified with Ni-NTA agarose affinity chromatography. The recombinant ( +)-LiBPPS catalyzed the conversion of GPP to BPP as a major product, and a few minor products. We also investigated the in planta role of ( +)-LiBPPS in terpenoid metabolism through its overexpression in sense and antisense orientations in stably transformed Lavandula latifolia plants. The overexpression of ( +)-LiBPPS in antisense resulted in reduced production of ( +)-borneol and camphor without compromising plant growth and development. As anticipated, the overexpression of the gene led to enhanced production of borneol and camphor, although growth and development were severely impaired in most transgenic lines strongly and ectopically expressing the ( +)-LiBPPS transgene in sense. Our results demonstrate that LiBPPS would be useful in studies aimed at the production of recombinant borneol and camphor in vitro, and in metabolic engineering efforts aimed at lowering borneol and camphor production in plants. However, overexpression in sense may require a targeted expression of the gene in glandular trichomes using a trichome-specific promoter.

Cloning and functional characterization of a floral repressor gene from Lavandula angustifolia.

MAIN CONCLUSION: Using RNA-Seq, we identified genes involved in floral development in lavenders and functionally characterized the floral repressor LaSVP. The molecular aspects of flower initiation and development have not been adequately investigated in lavender (Lavandula). In order to identify genes that control these processes, we employed RNA-Seq to obtain sequence information for transcripts originating from the vegetative shoot apical meristem (SAM) and developing inflorescence tissues of Lavandula angustifolia and Lavandula × intermedia plants, and assemble a comprehensive transcriptome of 105,294 contigs. Homology-based annotation provided gene ontology terms for the majority of transcripts, including over 100 genes homologous to those that control flower initiation and organ identity in Arabidopsis thaliana. Expression analysis revealed that most of these genes are differentially expressed during flower development. For example, LaSVP, a homolog of the floral repressor SHORT VEGETATIVE PHASE (SVP), was strongly expressed in vegetative SAM compared to developing flowers, implicating its potential involvement in flowering repression in lavender. To investigate LaSVP further, we constitutively expressed the gene in transformed A. thaliana plants, evaluating its effects on flower initiation and morphology. Expression of the LaSVP in A. thaliana delayed flowering and affected flower organ identity in a dosage-dependent manner. Two of the highest expressing lines produced sepals instead of petals and were sterile as they failed to develop proper seed pods. This study provides the foundation for future investigations aimed at elucidating flower initiation and development in lavender.