The F-box protein COI1 functions upstream of MYB305 to regulate primary carbohydrate metabolism in tobacco (Nicotiana tabacum L. cv. TN90)

Abstract

Jasmonate (JA) plays an important role in regulating plant male fertility and secondary metabolism, but its role in regulating primary metabolism remains unclear. The F-box protein CORONATINE INSENSITIVE 1 (COI1) is a critical component of the JA receptor, and mediates JA-signalling by targeting JASMONATE ZIM-domain (JAZ) proteins for proteasomal degradation in response to JA perception. Here, we found that RNA interference-mediated knockdown of NtCOI1 in tobacco (Nicotiana tabacum L. cv. TN90) recapitulated many previously observed phenotypes in coi1 mutants, including male sterility, JA insensitivity, and loss of floral anthocyanin production. It also affected starch metabolism in the pollen, anther wall, and floral nectary, leading to pollen abortion and loss of floral nectar. Transcript levels of genes encoding starch metabolism enzymes were significantly altered in the pollen, anther wall, and floral nectary of NtCOI1-silenced tobacco. Changes in leaf primary metabolism were also observed in the NtCOI1-silenced tobacco. The expression of NtMYB305, an orthologue of MYB305 previously identified as a flavonoid metabolic regulator in Antirrhinum majus flowers and as a floral-nectar regulator mediating starch synthesis in ornamental tobacco, was extremely downregulated in NtCOI1-silenced tobacco. These findings suggest that NtCOI1 functions upstream of NtMYB305 and plays a fundamental role in coordinating plant primary carbohydrate metabolism and correlative physiological processes.

Keywords: COI1; jasmonate; primary metabolism; starch; tobacco..

Fig. 1.

Identification of NtCOI1-silenced tobacco lines. (A) Relative NtCOI1 transcript levels in NtCOI1-silenced (RI) and control (WT, wild type; VC, VC transformed) plants. The expression value of NtCOI1 in WT leaves is set as 1. Results are shown as means ±standard error (SE). (B) Detection of transiently expressed HA-tagged NtCOI1 protein by western blotting. Anti-HA and anti-Act indicate blotting with antibodies against HA tag and β-actin, respectively. VC_N and RI_N indicate different independent transgenic lines. Significant differences from the data of control plants are indicated: **P<0.005 (Student’s t-test).

Fig. 2.

Male fertile phenotypes of NtCOI1-silenced (RI) and control (Ctrl) plants. (A) Floral phenotypes at indicated various floral development stages. (B) Fertilization phenotypes. Top panel, capsules; middle panel, seeds (Ctrl) and abortive seeds (RI) released from capsules; the inset shows an enlargement of the region marked by a red square; bottom panel, pollen germination assay: the percentage of normally germinated pollen grains is given. (C) Pollen staining using I₂/KI solution. Arrows indicate round pollen grains of NtCOI1-silenced tobacco. The percentage of shrunk pollen grains is given in the left corner of each image. Insets in the S6 panel show enlargements of typical control and RI pollen grains. The representative control pictures were taken from VC-transformed plants.

Fig. 3.

Carotenoid accumulation in floral nectaries and floral-nectar production of NtCOI1-silenced (RI) and control (Ctrl) plants. (A) Visualization of carotenoid in the nectaries at the indicated floral development stages. (B) TLC assay of carotenoids in nectaries at S12. The red arrowhead indicates β-carotene spots and the black arrowhead indicates an unidentified pigment. The black bracket indicates chlorophylls. WT, wild type; VC, VC transformed. RI_N indicates independent transgenic lines. (C) Quantification of carotenoid in nectaries at the indicated stages. Results are shown as means ±SE. (D) Relative transcript levels of genes involved in carotenoid biosynthesis in nectaries at the indicated stages. GGPS, geranylgeranyl diphosphate synthase. The expression value of each gene in S9 WT nectaries is set as 1. Results are shown as means ±standard deviation (SD). (E) Floral-nectar content at indicated stages. Nectar volumes are based on measurement of five flowers from each plant line. Results are shown as means ±SD. (F) Visualization of floral nectar at S12. (G) The relative transcript levels of the nectarin gene Nec1 in nectaries at the indicated floral development stages. The expression value of Nec1 in S9 WT nectary is set as 1. The representative Ctrl pictures were taken from the VC plant. Significant differences from the data of control plants are indicated: **P<0.005 (Student’s t-test). Results are shown as means ±SD.

Fig. 4.

Starch synthesis and transcription of NtMYB305 in the floral nectaries of NtCOI1-silenced (RI) and control (Ctrl) plants. (A) Visualization of nectary starch at S12 by I₂/KI staining. The representative Ctrl picture was taken from VC-transformed plants. (B) Starch content in nectaries at the indicated floral development stages. WT, wild type. Error bar, mean ± SE. (C) Relative expression levels of starch metabolic genes in nectaries at indicated floral development stages. Results are shown as means ±SD. The expression value of each gene in the S9 WT nectaries is set as 1. (D) Relative NtMYB305 transcript levels in nectaries at indicated floral development stages. Results are shown as means ±SD. The expression value of NtMYB305 in S9 WT nectary is set as 1. Significant differences from the data of control plants are indicated: *P<0.05; **P<0.005 (Student’s t-test). (This figure is available in colour at JXB online.)

Fig. 5.

Starch accumulation in the anther wall, pollen, and corolla of NtCOI1-silenced (RI) and control (Ctrl) plants. (A) I₂/KI staining of the anther wall and pollen. The inset in each panel is an enlargement of the region marked by the smaller red square. The red arrow in the S9 RI anther wall indicates a black-stained pollen. (B) I₂/KI staining of RI (right) and control (left) corollas at the indicated floral development stages. Red arrows indicate the stamen filaments. (C) Starch content in the anther wall, corolla, and pollen. Coloured curves indicate the change of the absolute values of starch content differences in the control and RI organs at indicated floral development stages. Error bar, mean ± SE. The representative control pictures and data were taken from the VC plant. Significant differences from the data of control plants are indicated: *P<0.05; **P<0.005 (Student’s t-test).

Fig. 6.

Analyses of total soluble sugar and invertase gene expression in the anther wall. (A) The amount of total soluble sugars in the anther wall at the indicated floral development stages. WT, wild type; VC, VC-transformed; RI, NtCOI1 silenced. Trendlines indicate change curves in VC and RI anthers over the indicated floral development stages. (B) The expression patterns of invertase genes in the wild-type anther wall at the indicated floral development stages. INV1–5 indicates cell-wall invertase genes 1–5, respectively. The expression value of INV1 in the S4 anther wall was set as 1. (C) Relative transcript levels of invertase genes in the anther wall at the indicated floral development stages. Significant difference from the data of control plants are indicated: *P<0.05 (Student’s t-test).

Fig. 7.

Transcriptional analyses of NtMYB305 and starch metabolic genes in the anther wall, corolla, and pollen. WT, wild type; VC, VC transformed; RI, NtCOI1 silenced. The transcription of each gene in S4 wild-type organs was set as 1. Results are shown as means ±SD. Significant differences from the data of control plants are indicated: *P<0.05; **P<0.005 (Student’s t-test).

Fig. 8.

Leaf morphology of NtCOI1-silenced (RI) tobacco. (A) I₂/KI staining of leaves. Insets show leaves stained after ethanol decolorization. The representative control (Ctrl) pictures were taken from VC-transformed plant. (B). Starch content in leaves. Results are shown as means ±SE. The representative Ctrl data were taken from the VC plant. (C) Relative expression levels of NtMYB305 and starch metabolic genes in leaves. WT, wild type. The transcription of each gene in WT leaves was set as 1. Results are shown as means ±SD. Significant differences from the data of control plants are indicated: *P<0.05; **P<0.005 (Student’s t-test).