Jonathan E. Page

A functional genomics screen identifies diverse transcription factors that regulate alkaloid biosynthesis in Nicotiana benthamiana

Biosynthesis of the alkaloid nicotine in Nicotiana species is induced by insect damage and jasmonate application. To probe the transcriptional regulation of the nicotine pathway, we constructed two subtracted cDNA libraries from methyl jasmonate (MeJA)-treated Nicotiana benthamiana roots directly in a viral vector suitable for virus-induced gene silencing (VIGS). Sequencing of cDNA inserts produced a data set of 3271 expressed sequence tags (ESTs; 1898 unigenes), which were enriched in jasmonate-responsive genes, and included 69 putative transcription factors (TFs). After a VIGS screen to determine their effect on nicotine metabolism, six TFs from three different TF families altered constitutive and MeJA-induced leaf nicotine levels. VIGS of a basic helix-loop-helix (bHLH) TF, NbbHLH3, and an auxin response factor TF, NbARF1, increased nicotine content compared with control plants; silencing the bHLH family members, NbbHLH1 and NbbHLH2, an ethylene response factor TF, NbERF1, and a homeobox domain-like TF, NbHB1, reduced nicotine levels. Transgenic N. benthamiana plants overexpressing NbbHLH1 or NbbHLH2 showed increased leaf nicotine levels compared with vector controls. RNAi silencing led to both reduced nicotine and decreased levels of transcript encoding of enzymes of the nicotine pathway. Electrophoretic mobility shift assays showed that recombinant NbbHLH1 and NbbHLH2 directly bind G-box elements identified from the putrescine N-methyltransferase promoter. We conclude that NbbHLH1 and NbbHLH2 function as positive regulators in the jasmonate activation of nicotine biosynthesis.

Functional Analysis of the Final Steps of the 1-Deoxy-d-xylulose 5-phosphate (DXP) Pathway to Isoprenoids in Plants Using Virus-Induced Gene Silencing

Isoprenoid biosynthesis in plant plastids occurs via the 1-deoxy-d-xylulose 5-phosphate (DXP) pathway. We used tobacco rattle virus (TRV) to posttranscriptionally silence the expression of the last two enzymes of this pathway, the IspG-encoded (E)-4-hydroxy-3-methylbut-2-enyl diphosphate synthase (HDS) and the IspH-encoded isopentenyl/dimethylallyl diphosphate synthase (IDDS), as well as isopentenyl/dimethylallyl diphosphate isomerase (IDI), the enzyme that interconverts IPP and DMAPP. TRV-IspG and TRV-IspH infected Nicotiana benthamiana plants had albino leaves that contained less than 4% of the chlorophyll and carotenoid pigments of control leaves. We applied [13C]DXP and [14C]DXP to silenced leaves and found that 2-C-methyl-d-erythritol 2,4-cyclodiphosphate accumulated in plants blocked at HDS while DXP, (E)-4-hydroxy-3-methylbut-2-enyl phosphate and (E)-2-methylbut-2-ene-1,4-diol accumulated in IDDS-blocked plants. Albino leaves from IspG- and IspH-silenced plants displayed a disorganized palisade mesophyll, reduced cuticle, fewer plastids, and disrupted thylakoid membranes. These findings demonstrate the participation of HDS and IDDS in the DXP pathway in plants, and support the view that plastid isoprenoid biosynthesis is metabolically and physically segregated from the mevalonate pathway. IDI-silenced plants had mottled white-pale green leaves with disrupted tissue and plastid structure, and showed an 80% reduction in pigments compared to controls. IPP pyrophosphatase activity was higher in chloroplasts isolated from IDI-silenced plants than in control plant chloroplasts. We suggest that a low level of isoprenoid biosynthesis via the DXP pathway can occur without IDI but that this enzyme is required for full function of the DXP pathway.

A polyketide synthase of Plumbago indica that catalyzes the formation of hexaketide pyrones

Plumbago indica L. contains naphthoquinones that are derived from six acetate units. To characterize the enzyme catalyzing the first step in the biosynthesis of these metabolites, a cDNA encoding a type III polyketide synthase (PKS) was isolated from roots of P. indica. The translated polypeptide shared 47–60% identical residues with PKSs from other plant species. Recombinant P. indica PKS expressed in Escherichia coli accepted acetyl‐CoA as starter and carried out five decarboxylative condensations with malonyl coenzyme A (‐CoA). The resulting hexaketide was not folded into a naphthalene derivative. Instead, an α‐pyrone, 6‐(2′,4′‐dihydroxy‐6′‐methylphenyl)‐4‐hydroxy‐2‐pyrone, was produced. In addition, formation of α‐pyrones with linear keto side chains derived from three to six acetate units was observed. As phenylpyrones could not be detected in P. indica roots, we propose that the novel PKS is involved in the biosynthesis of naphthoquinones, and additional cofactors are probably required for the biosynthesis of these secondary metabolites in vivo.

cDNA libraries for virus-induced gene silencing.

Virus-induced gene silencing (VIGS) exploits endogenous plant antiviral defense mechanisms to posttranscriptionally silence the expression of targeted plant genes. VIGS is quick and relatively easy to perform and therefore serves as a powerful tool for high-throughput functional genomics in plants. Combined with the use of subtractive cDNA libraries for generating a collection of VIGS-ready cDNA inserts, VIGS can be utilized to screen a large number of genes to determine phenotypes resulting from the knockdown/knockout of gene function. Taking into account the optimal insert design for VIGS, we describe a methodology for producing VIGS-ready cDNA libraries enriched for inserts relevant to the biological process of interest.