Increased Nicotiana tabacum fitness through positive regulation of carotenoid, gibberellin and chlorophyll pathways promoted by Daucus carota lycopene β-cyclase (Dclcyb1) expression

Abstract

Carotenoids, chlorophylls and gibberellins are derived from the common precursor geranylgeranyl diphosphate (GGPP). One of the enzymes in carotenoid biosynthesis is lycopene β-cyclase (LCYB) that catalyzes the conversion of lycopene into β-carotene. In carrot, Dclcyb1 is essential for carotenoid synthesis in the whole plant. Here we show that when expressed in tobacco, increments in total carotenoids, β-carotene and chlorophyll levels occur. Furthermore, photosynthetic efficiency is enhanced in transgenic lines. Interestingly, and contrary to previous observations where overexpression of a carotenogenic gene resulted in the inhibition of the synthesis of gibberellins, we found raised levels of active GA4 and the concommitant increases in plant height, leaf size and whole plant biomass, as well as an early flowering phenotype. Moreover, a significant increase in the expression of the key carotenogenic genes, Ntpsy1, Ntpsy2 and Ntlcyb, as well as those involved in the synthesis of chlorophyll (Ntchl), gibberellin (Ntga20ox, Ntcps and Ntks) and isoprenoid precursors (Ntdxs2 and Ntggpps) was observed. These results indicate that the expression of Dclcyb1 induces a positive feedback affecting the expression of isoprenoid gene precursors and genes involved in carotenoid, gibberellin and chlorophyll pathways leading to an enhancement in fitness measured as biomass, photosynthetic efficiency and carotenoid/chlorophyll composition.

Keywords: Biomass; carotenoids; chlorophyll; gene expression; gibberellins; lycopene β-cyclase; tobacco..

Fig. 1.

Carotenoid and directly-related biosynthesis pathways. Enzymatic conversions are shown by arrows with the enzymes involved in each reaction. The carotenoid pathway is in the center of the figure enclosed in grey, flanked by the chlorophyll and gibberellin (GA) pathways. Genes analyzed in this work are underlined. The non-mevalonate pathway (MEP) takes place in plastids to produce geranylgeranyl pyrophosphate (GGPP) that is required for carotenoid, chlorophyll and gibberellin synthesis, among others. DXS, deoxyxylulose synthase; DXR, 1-deoxyxylulose 5-phosphate reductoisomerase; CMS, 2C-methyl-D-erythritol 4-phosphate cytidyltransferase, CMK, 4-(cytidine 5-diphospho)-2-C-methyl-D-erythritol kinase, MCS, 2C-methyl-D-erythritol 2,4-cyclodiphosphate synthase, HDS, 4-hydroxy-3-methylbut-2-enyl diphosphate synthase; HDR, 4-hydroxy-3-methylbut-2-enyl diphosphate reductase; IPI, isopentenyl diphosphate isomerase; GGPPS, geranylgeranyl pyrophosphate synthase; PSY, phytoene synthase; PDS, phytoene desaturase; Z-ISO, z-carotene isomerase; ZDS, Z-carotene desaturase; CRT-ISO, carotenoid isomerase; LCYB, lycopene β-cyclase; LCYE, lycopene ε-cyclase; CHXB, β-carotene hydroxylase; CHXE, ε-carotene hydroxylase; GGRS, geranylgeranyl reductase; CHL, chlorophyll synthase; CPS, copalyl diphosphate synthase; KS, ent-kaurene synthase; KO, ent-kaurene oxidase; KAO, ent-kaurenoic acid oxidase, GA20ox, GA 20-oxidase, GA3ox, GA 3-oxidase; NCED, 9-cis-epoxycarotenoid dioxygenase. The LCYB1 enzyme from Daucus carota which was overexpressed in this work is boldfaced in the respective reaction of the pathway. The enzymes for which the expression of their corresponding coding sequences was quantitatively analyzed by qRT-PCR are underlined. Dashed lines represent multiple reaction steps.

Fig. 2.

Relative expression levels of Dclcyb1 and key endogenous carotenoid genes in transgenic tobacco lines. (A) Relative expression levels of Dclcyb1 in leaves of empty vector (e.v.) and Dclcyb1 transgenic (L14, L15 and L16) 8-week-old T₁ tobacco plants. (B) Relative expression of endogenous Ntpsy1, Ntpsy2 and Ntlcyb. Actin was used as normalizer in qRT-PCR measurements. Columns and bars represent the mean and SE (n=6). Asterisks indicate significant differences between Dclcyb1 transgenic lines and the e.v. transformants. Non-paired two-tailed Student’s t-tests were performed for all transgenic lines: *, P<0.05; **, P<0.01; *** P<0.001.

Fig. 3.

Phenotype of transgenic Dclcyb1 tobacco lines. (A) Left: representative 12-week-old T₁ empty vector (e.v.), centre: representative 12-week-old T₁ Dclcyb1 transgenic (L14, L15 and L16) tobacco lines, right: representative 16-week-old T₁ e.v. line. (B) Detached leaves correspond to the 4th node (from the base of the plant) of plants shown in panel A. (C) Representative 8-week-old T₁ e.v. and Dclcyb1 transgenic tobacco lines are shown. Scale bar, 10cm.

Fig. 4.

Relative expression of key genes involved in isoprenoid precursors, chlorophyll, gibberellins and ABA synthesis pathways. Relative expression of Ntdxs1, Ntdxs2 and Ntggpps genes that codify for key enzymes acting in the synthesis for carotenoid, gibberellin and chlorophyll precursors, as well as Ntchl (chlorophyll synthesis), Ntcps and Ntks (gibberellin synthesis) and Ntnced (ABA synthesis) was measured in leaves of 2-month-old T₁ empty vector (e.v.) and Dclcyb1 transgenic (L14, L15 and L16) tobacco lines. Actin was used as normalizer in qRT-PCR measurements. Columns and bars represent the means and SE (n=6). Asterisks indicate significant differences between Dclcyb1 transgenic lines and e.v. transformants, as determined by non-paired two-tailed Student’s t-tests. *, P<0.05; **, P<0.01; ***, P<0.001.

Fig. 5.

Gibberellin levels in transgenic tobacco plants. (A) Phentotype of 1-month-old empty vector (e.v.) and Dclcyb1 transgenic (L14, L15 and L16) T₁ tobacco lines. (B) Fresh weight and (C) leaf surface area for 1-month-old e.v., L14, L15 and L16 transgenic lines. Columns and bars represent the means and SE (n=16). Letters indicate significant differences between transgenic lines and the e.v. plants as determined by ANOVA analysis with Tukey’s post-test, P<0.0001. Scale bar, 2cm. (D) The bioactive gibberellin (GA1 and GA4ng/g FW) contents in e.v. and Dclcyb1 transgenic T₁ tobacco lines are shown. Three biological replicates and two technical replicates from leaves of 1-month-old tobacco plants were used. Asterisks indicate significant differences between Dclcyb1 transgenic lines and the e.v. transformants, as determined by non-paired two-tailed Student’s t-tests, *, P<0.05; ***, P<0.001. (This figure is available in colour at JXB online.)

Fig. 6.

Scheme of a proposed model for boosting carotenoid, gibberellin and chlorophyll synthesis in Dclcyb1 transgenic tobacco. Carotenoid, chlorophyll and gibberellin biosynthesis pathways are presented. The over expression of Dclcyb1 causes a greater production of β-carotene (1) that may be cleaved by singlet oxygen to produce apocarotenoids such as β-cyclocitral or dihydroactinidiolide that may act as retrograde signaling molecule(s) to activate the expression of endogenous psy genes (2), or of dxs and ggpps genes involved in early isoprenoid production (2), raising probably the synthesis of the common precursor, GGPP, resulting in higher accumulation of gibberellins, chlorophylls and carotenoids (3). (This figure is available in colour at JXB online.)