The bouquet of grapevine (Vitis vinifera L. cv. Cabernet Sauvignon) flowers arises from the biosynthesis of sesquiterpene volatiles in pollen grains

Abstract

Terpenoid volatiles are important information molecules that enable pollinators to locate flowers and may protect reproductive tissues against pathogens or herbivores. Inflorescences of grapevine (Vitis vinifera L.) are composed of tiny green flowers that produce an abundance of sesquiterpenoid volatiles. We demonstrate that male flower parts of grapevines are responsible for sesquiterpenoid floral scent formation. We describe temporal and spatial patterns of biosynthesis and release of floral volatiles throughout the blooming of V. vinifera L. cv. Cabernet Sauvignon. The biosynthesis of sesquiterpene volatiles, which are emitted with a light-dependent diurnal pattern early in the morning at prebloom and bloom, is localized to anthers and, more specifically, within the developing pollen grains. Valencene synthase (VvValCS) enzyme activity, which produces the major sesquiterpene volatiles of grapevine flowers, is present in anthers. VvValCS transcripts are most abundant in flowers at prebloom stages. Western blot analysis identified VvValCS protein in anthers, and in situ immunolabeling located VvValCS protein in pollen grains during bloom. Histochemical staining, as well as immunolabeling analysis by fluorescent microscopy and transmission electron microscopy, indicated that VvValCS localizes close to lipid bodies within the maturing microspore.

Fig. 1.

Flower development on rooted cuttings of grapevine (V. vinifera L. cv. Cabernet Sauvignon) used for headspace volatile collection. (A) Flower panicle (top node) of a rooted cane inside the volatile collection chamber. The lateral vine (lower node) is below the chamber (not shown). (B) Individual flowers clockwise from top right at stage VI, just opening, and in bloom. A penny is used as a scale reference. (C) Stage IV flowers; the flowers are separated from one another. (D) Stage V flowers; the flowers are lengthened. (E) Stage VI flowers; just before bloom, when the flowers are lengthened. (F) Flowers at bloom (b) and at fruit set (fs) several days after bloom are identified by a swelling of the pistil.

Fig. 2.

Time course of volatile emissions from grapevine flowers at bloom. Data represent the amounts of volatiles detected by GCMS. Gray bars illustrate the period of darkness for each experiment. (A) Time course of volatile emissions divided into hourly segments between 0600 and 1100 hours. Bars represent 2–6 replicates + SEM. (B) Volatile emissions during constant darkness after the first day of blooming. Bars represent 5 replicates + SEM. (C) Volatile emissions during constant light conditions. Bars represent 3 replicates + SEM.

Fig. 3.

GCMS analysis of volatile compounds extracted from grapevine flower parts. (A) Total pentane-extractable compounds present in anthers, pistils, and caps. Bars represent 4 independent replicate extractions + SEM, with each replicate comprising 10 individual flowers per stage. (B) Pentane extracts from anthers analyzed by type of compound present: sesquiterpenes, diterpenes, or aliphatics. Bars represent 4 replicates + SEM. (C) Quantitative analysis of the pollen pentane and chloroform/methanol extracts from stage V and VI flowers. Bars represent 4 replicates + SEM. (D) Light microscopy image of a stage VI anther stained with Oil Red O. (Scale bar = 100 μm.) (E) High-magnification image of a pollen grain showing abundant lipid bodies stained with Oil Red O inside the pollen. (Scale bar = 5 μm.) (F) GCMS total ion chromatogram of the products from VvValCS enzyme (solid black line) compared with the anther extracts (solid gray line) and the flower volatile emissions collected on a day of peak release (dashed line). Compounds are as follows: Ε-β-caryophyllene (1), spirolepechinene (2)*, α-humulene (3), unknown sesquiterpene (4), Ε-β-farnesene (5), selina-4,11-diene* (6), (+)-valencene (7), tridecanone/cis-α-bergomatene (8), Ε,Ε-α-farnesene (9), and (–)-7-epi-α-selinene (10). Tentative designations are labeled with an asterisk. Structures of major compounds shown from left to right include valencene, Ε,Ε-α-farnesene, and 7-epi-α-selinene.

Fig. 4.

Stage-specific expression of VvValCS gene and VvValCS protein. (A) Quantitative RT-PCR analysis of VvValCS mRNA during flower development. VvValCS transcript abundance is shown at stages I–VI relative to transcript abundance at bloom. Each bar represents measurements from 3 technical replicates for each of 2 biological replicates ± SEM. GAPDH was used as an endogenous control. ΔCt for stages I–III was <2 cycles different from no template controls = no expression. (B) Protein expression of VvValCS in flower (stages I—VI and bloom) and berry stages (fs, fruit set; ps, pea size). SDS/PAGE gels were loaded with protein from ≈3 mg of tissue. Actin was measured as a loading control. (C) Protein expression of VvValCS in anthers, pistils, and caps at stages V and VI and bloom. Gels were loaded with protein for each flower organ pooled from 15 flowers for each stage of development. Actin was measured as a loading control.

Fig. 5.

Immunofluorescence localization of VvValCS protein to the pollen grains of stage V flowers. (A) Longitudinal section of a grape flower (f) stained with toluidine blue. The box highlights the portion of the anther (a) imaged in (B–M). The pistil (p) and the cap (c) are indicated in white. (Scale bar = 200 μm.) (B–E) The first column of images shows fluorescence from VvVal primary antibody with Alexa-488 secondary, the second column shows the same section with 332 Hoechst staining of nuclei, and the third column shows the 2 channels overlaid. VvVal is seen in the images with discrete green clusters. Diffuse autofluoresence is present for the blue and green channels for all treatments. (B and C) Images of a section of an anther locule containing pollen grains. VvVal is localized primary inside the stage V pollen grains. Some label is found on the edges of the pollen grains. Little label is found on the outside of the pollen grains or in other parts of the anther locule. (Scale bar = 50 μm.) (D and E) High-magnification (63×) images of representative pollen grains from stages V. Most of the label is present within the pollen grain, although some label is seen on the inner edges of the pollen grains as well. (Scale bar = 10 μm.)

Fig. 6.

TEM immunogold localization of VvVal within pollen grains. (A) Pollen grain with a single nucleus (n). (Scale bar = 2 μm.) (B–F) Pollen grain sections with lipid vesicles (v). Arrows indicate fusions of vesicles. Immunogold labeling of α-VvVal primary is highlighted in red after imaging; the original image is provided for reference as a supplementary figure. Most of the label is present on the outer edges of these lipid vesicles. No label is seen in the preimmune control. [Scale bar = 500 nm for (B), (C), (E), and (F) and 100 nm for (D).].