Fragaria nilgerrensis, Schltdl. ex J.Gay

Mehmood, Nasir, Yuan, Yuan, Ali, Mohammed, Ali, Muhammad, Iftikhar, Junaid, Cheng, Chunzhen, Lyu, Meiling & Wu, Binghua, 2021, Early transcriptional response of terpenoid metabolism to Colletotrichum gloeosporioides in a resistant wild strawberry Fragaria nilgerrensis, Phytochemistry (112590) 181, pp. 1-12 : 3-4

2.3. Isoprenoid biosynthesis genes from F. nilgerrensis leaf transcriptome

Fruits of wild and cultivated varieties of strawberry contains numerous types of terpenoids as the major components of the aroma essential oils, represented mainly by myrtenol, α- pinene, β- pinene, myrtenyl acetate, sabinene, β- myrcene, α- phellandrene, β- phellandrene, dihydromyrcenol, α- terpinolene, α- terpineol, linalool, nerolidol, ()-limonene and (+)-limonene ( Aharoni et al., 2004). However, the composition of terpenoids in leave are rarely reported. Our GC/MS profile unveils that the leaf contained low amount (less than 2% of total extracts) of terpenoids in total. However, upon challenged with C. gloeosporioides , rapid induction in terpenoid biosynthesis is quite impressed. Thus, we played particular attention to the fungus-elicited transcriptional regulation of terpenoid biosynthesis network in the leaf based on our transcriptome data.

Plant terpenoids biosynthesis are initiated from the plastidial MEP (methylerythritol phosphate) and the cytosolic/peroxisomal MVA (mevalonic acid) pathways that generate the building blocks isopentyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) for diverse downstream conversions ( Fig. 4 ). Therefore, we first identified unigenes coding for enzymes in various steps of terpenoid biosynthesis pathways in the transcriptome, such as isopentyl diphosphate synthases (IPPS), dimethylallyl diphosphate synthases (DMADPS), geranyl diphosphate synthases (GPPS), farnesyl diphosphate synthases (FPPS) and geranylgeranyl diphosphate synthases (GGPPS). In addition, most unigenes associated with biosynthesis of isoprenoid in both MEP and MVA pathways were found, including FnDXR (1-deoxy-D-xylulose-5- phosphate reductoisomerase), FnISPF (2-C-methyl-D-erythritol-2,4- cyclodiphosphate synthase), FnHDS1 ((E)-4-hydroxy-3-methylbut- 2- enyl-diphosphate synthase 1), FnHDR (4-hydroxy-3-methylbut-2-enyl diphosphate reductase), FnIDI1 (isopentenyl-diphosphate deltaisomerase 1), FnAACT1 (acetyl-CoA C-acetyltransferase 1), FnHMGS (hydroxylmethylglutaryl-CoA synthase), FnHMGR4 and 5 (hydroxymethylglutaryl-CoA reductase (NADPH) 4 and 5), FnMDC (diphosphomevalonate decarboxylase). In total 57 unigenes related to MEP, MVA and other terpenoid backbone biosynthesis pathway were identified and assigned gene names based on homology (Table S4 and Fig. 4 ).

Terpene synthase (TPS) catalysis the final step for various terpenoid molecules. In total 59 homologous TPS unigenes from our dataset were identified and determined on the basis of TPS sequence resemblances that were discovered in the reference of canonical annotation database (Table S5 and Fig. 4 ). Fifteen of these unigenes were annotated as monoterpenes, limonene and pinene degradation biosynthesis, such as (3 S)-linalool synthase, (+)-neomenthol dehydrogenase, ()-alphaterpineol synthase, ()-camphene/tricyclene synthase and aldehyde dehydrogenase (NAD +). Twenty-three unigenes were annotated as sesquiterpene biosynthesis, such as farnesyl-diphosphate farnesyltransferase, NAD + -dependent farnesol dehydrogenase, alpha-farnesene synthase, ()-germacrene D synthase and beta-amyrin synthase. Also, twelve unigenes were annotated as diterpene biosynthesis, such as ent-copalyl diphosphate synthase, ent-kaurene synthase, ent-kaurene oxidase, ent-kaurenoic acid monooxygenase, gibberellin-44 dioxygenase, gibberellin 3 beta-dioxygenase and gibberellin 2 beta-dioxygenase. Finally, nine unigenes related to squalene monooxygenase were annotated as triterpene biosynthesis (Table S5 and Fig. 4 ).

Based on the transcript sequences, we also detected simple sequence repeat (SSR) motifs in the identified unigenes associating with terpenoid biosynthesis, e.g. Hmgcr 4, FnDXS 3, FnDXR, FnMCT, FnIDI 1, FnFPPS 1, FnGGPPS 1, FnCMT, FnFNTB 2, FnSPS 1, and unigenes related to (3 S)- linalool synthase, aldehyde dehydrogenase (NAD +), 3, 4, 6, ent-copalyl diphosphate synthase, ent-kaurene synthase 1, ent-karene oxidase, ent-kaurenoic acid monooxygenase 2, gibberellin-3-beta dioxygenase, farnesyl-diphosphate, farnesyltransferase, squalene monooxygenase 2, and beta-amyrin synthase 3 (Table S6).

The identified homologous genes provide an overview for further deciphering terpenoid biosynthesis in leaves of the N. nilgerrensis and may be a reference for other strawberry cultivars as well ( Fig. 4 ). Next, we examined the expression profiles of these unigenes during the timecourse after fungal challenge. In the general terpenoid biosynthesis pathways, more unigenes from the MVA, MEP and those related to diphosphate synthases (PPS) showed up-regulation as early at 3 hpi with various fold-change levels, while only a few unigenes were repressed, especially c27310. graph_c0 (FnDHDDS4) and c15242. graph_c0 (FnDHDDS1) ( Fig. 5A ). For TPS -like unigenes, ten unigenes (c26859. graph_c0, c23001. graph_c0, c26382. graph_c2, c24450. graph_c0, c34218.

graph_c0, c34395. graph_c0, c39471. graph_c0, c31932. graph_c0, c24276. graph_c0 and c27886. graph_c0) were up-regulated remarkedly at 3–6 hpi, and further fourteen unigenes (c24788. graph_c1, c27601. graph_c0, c9433. graph_c0, c26113. graph_c2, c8446. graph_c0, c26141. graph_c0, c13323. graph_c0, c11811. graph_c0, c9937. graph_c0, 18669. graph_c0, c28365. graph_c0, c1733. graph_c0, c45499. graph_c0, c19725. graph_c0 and c19236. graph_c0) were induced significantly at 12–18 hpi ( Fig. 5B ). These results indicate that in response to C. gloeosporioides challenge, terpenoid biosynthesis genes are among the most active transcripts of the specialised metabolism during the early stage.

Figures

Fig. 4. Schematic illustration of general terpenoid biosynthesis pathways showing identified unigenes from F. nilgerrensis leaf transcriptome data. The interconvertible precursors IPP and DMAPP, two phosphorylated C5 unites, are produced by the MVA and MEP pathways which are exchangeable from both compartmentations. The chloroplast is a major site for synthesis of hemiterpene (C5), monoterpenoids (C10), diterpenoids (C20) carotenoids (C40) and chlorophyll, while the cytosol and other organelle are responsible for synthesis of monoterpenoids (C10), sesquiterpene (C15) and triterpene (C30). But that is not strictly conclusive. Arrow with lines indicate reactions catalyzed by enzymes and the encoding genes, with unigenes identified in this experiment boxed. The color highlights are for better visualization. Abbreviations: AACT, acetoacetyl-CoA thiolase; CMK, 4-diphosphocytidyl-2-C-methyl-D-erythritol kinase; DXR, 1-deoxy-D-xylulose 5-phosphate reductase; DXS, 1-deoxy-D-xylulose 5-phosphate synthase; HDR, (E)-4-hydroxy-3-methyl-but-2-enyl diphosphate reductase; HDS, (E)-4-hydroxy-3- methyl-but-2-enyl diphosphate synthase; HMGR, 3-hydroxy-3-methylglutaryl-CoA reductase; HMGS, 3-hydroxy-3-methylglutaryl-CoA synthase; IDI, isopentenyl diphosphate isomerase; MCT, MEP cytidyltransferase; MDC, mevalonate-5-diphosphate decarboxylase; MDS, 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase; MVK, mevalonate kinase. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 5. Heat maps depicting the transcript levels of terpenoid biosynthesis genes from F. nilgerrensis leaf transcriptome in response to C. gloeosporioides. (A) Fifty-six MVA and MEP pathway-associated genes. Annotation of the unigenes by homologous are listed in Table S4. (B) Fifty-nine terpene synthase-like or related genes. Annotation of the unigenes by homologous are listed in Table S5. Average FPKM (n = 2) were log2 transformed. The heatmap was generated by Prism GraphPad version 7.04.