Heterologous expression of chloroplast-localized geranylgeranyl pyrophosphate synthase confers fast plant growth, early flowering and increased seed yield

Abstract

Geranylgeranyl pyrophosphate synthase (GGPS) is a key enzyme for a structurally diverse class of isoprenoid biosynthetic metabolites including gibberellins, carotenoids, chlorophylls and rubber. We expressed a chloroplast-targeted GGPS isolated from sunflower (Helianthus annuus) under control of the cauliflower mosaic virus 35S promoter in tobacco (Nicotiana tabacum). The resulting transgenic tobacco plants expressing heterologous GGPS showed remarkably enhanced growth (an increase in shoot and root biomass and height), early flowering, increased number of seed pods and greater seed yield compared with that of GUS-transgenic lines (control) or wild-type plants. The gibberellin levels in HaGGPS-transgenic plants were higher than those in control plants, indicating that the observed phenotype may result from increased gibberellin content. However, in HaGGPS-transformant tobacco plants, we did not observe the phenotypic defects such as reduced chlorophyll content and greater petiole and stalk length, which were previously reported for transgenic plants expressing gibberellin biosynthetic genes. Fast plant growth was also observed in HaGGPS-expressing Arabidopsis and dandelion plants. The results of this study suggest that GGPS expression in crop plants may yield desirable agronomic traits, including enhanced growth of shoots and roots, early flowering, greater numbers of seed pods and/or higher seed yield. This research has potential applications for fast production of plant biomass that provides commercially valuable biomaterials or bioenergy.

Keywords: chloroplast; fast growth; geranylgeranyl pyrophosphate synthase; gibberellins; higher yield.

Figure 1

Transgenic plants carrying p35S::HaGGPS construct. (a) Schematic diagram showing the structure of the pBI121 transformation vector carrying HaGGPS. (b, d, e) Expression levels of HaGGPS were quantified using real‐time qPCR with the reference genes (expression level = 1), ubiquitin in T₃ tobacco lines (b), At1G13320 in T₃ Arabidopsis lines (d) and actin in T₂ dandelion lines (e). (c) Enzymatic activity of GGPS in control and HaGGPS‐transgenic tobacco and Arabidopsis plants. Error bars in (b–e) represent standard deviations from one representative control GUS‐ (n = 3) and two HaGGPS‐transgenic lines (n = 6), except in (c) where Arabidopsis control was wild type, **P < 0.01.

Figure 2

Subcellular localization of HaGGPS‐GFP‐fused proteins in tobacco leaves. (a) Green fluorescence showing the presence of HaGGPS‐GFP proteins observed through a confocal microscope. (b) Red autofluorescence of the chloroplasts of the leaf tissues. (c) A bright‐field photograph of the transiently transformed tobacco plant leaf. (d) Merged images of chlorophyll (red) fluorescence and HaGGPS‐GFP (green) fluorescence. Scale bars = 20 μm.

Figure 3

Enhanced growth and increased numbers of seed pods in T₃ HaGGPS ‐transgenic tobacco plants. (a and b) Photograph (a) and graph (b) showing accelerated shoot growth in 60 days old HaGGPS ‐transgenic tobacco lines compared with that of the control GUS ‐transgenic plants. (c and d) Photograph (c) and graph (d) showing HaGGPS ‐transgenic tobacco plants flowering earlier than control GUS ‐transgenic plants. (e and f) Photograph (e) and graph (f) showing flowers/seed pods produced in 120 days old GUS ‐ and HaGGPS ‐transgenic tobacco plants. Error bars in (b, d and f) represent standard deviations obtained from one representative control GUS ‐ (n = 5) and two HaGGPS ‐transgenic lines (n = 10), **P < 0.01. Wild‐type plants were examined as another control, and the results are described in the text.

Figure 4

Effect of HaGGPS overexpression on root biomass and morphology. (a) Images showing seedling growth of T₃ HaGGPS ‐ and GUS (control)‐transgenic tobacco plants on MS medium at 7 (upper) and 15 (below) days after germination, respectively. (b) Graphs depicting accelerated growth of the root systems in HaGGPS ‐transgenic tobacco plants compared with those of GUS ‐transgenic plants at 7 (upper) and 15 days (below), respectively, on MS medium. (c and d) Photograph (c) and graph (d) showing increased elongation of the primary root and increased lateral root formation in 60 days old tobacco plants expressing HaGGPS under greenhouse conditions compared to the control GUS ‐transgenic plants. Error bars in (b and d) represent standard deviations obtained from one control GUS ‐ (n = 4) and two GGPS ‐transgenic lines (n = 8), **P < 0.01.

Figure 5

Gibberellin content in the control GUS ‐ and HaGGPS ‐transgenic tobacco plants. Content (ng/g dry weight) of GA ₁ and GA ₄ and their precursors GA ₉ and GA ₂₀, respectively, in apical shoots from GUS ‐ and HaGGPS ‐transgenic plants. Error bars represent standard deviations, n = 4, *P < 0.05, **P < 0.01.

Figure 6

Enhanced growth in T₃ HaGGPS ‐transgenic Arabidopsis plants. (a and b) Photograph (a) and graph (b) showing accelerated seedling growth of HaGGPS ‐transgenic Arabidopsis plants compared with control GUS ‐transgenic plants on MS medium at 11 days after germination. (c and d) Photograph (c) and graph (d) showing enhanced growth of shoot and root tissues in HaGGPS ‐transgenic Arabidopsis plants compared with the control GUS ‐transgenic plants in soil pots at 18 days after germination. Error bars in (b and d) represent standard deviations obtained from three independent transgenic lines, n = 9 and 3, respectively, **P < 0.01.

Figure 7

Enhanced growth in T₂ HaGGPS ‐transgenic dandelion plants. (a and b) Photograph (a) and graph (b) showing enhanced seedling growth of HaGGPS ‐transgenic dandelion plants compared with control GUS‐transgenic dandelion plants on MS medium at 6 days after germination. (c and d) Photograph (c) and graph (d) showing accelerated increase in total fresh biomass of HaGGPS ‐transgenic dandelion plants compared with control GUS‐transgenic plants in soil pots at 36 days after germination. Error bars in (b, d) represent standard deviations obtained from three independent transgenic lines, n = 3, *P < 0.05, **P < 0.01.

Figure 8

HaGGPS expression influences neither petiole length nor chlorophyll content in tobacco plants. (a) Photograph showing the petioles in young leaves of 60 days old GUS (control)‐ and HaGGPS ‐transgenic tobacco plants. (b) A graph depicting identical petiole lengths in mature leaves of GUS ‐ and HaGGPS ‐transgenic tobacco plants. (c and d) Contents of chlorophyll a and chlorophyll b (c) and total chlorophyll (d) in young leaves of GUS ‐ and HaGGPS ‐transgenic tobacco plants. Error bars in (b, c and d) represent standard deviations obtained from one representative control GUS ‐ (n = 3) and two independent HaGGPS ‐transgenic lines (n = 6).