Impact of altered gibberellin metabolism on biomass accumulation, lignin biosynthesis, and photosynthesis in transgenic tobacco plants

Abstract

Gibberellins (GAs) are involved in regulation of many aspects during plant development. To investigate the impact of altered GA levels on plant growth and metabolism, transgenic tobacco (Nicotiana tabacum) plants have been engineered to express either a GA20-oxidase (AtGA20-ox) or a GA2-oxidase (AtGA2-ox) gene from Arabidopsis under control of the cauliflower mosaic virus 35S promoter. Resulting plants were characterized by elongated or stunted shoot growth, respectively, indicating changes in the content of bioactive GAs. In accordance with the effect on plant growth, biomass production was increased or decreased in AtGA20-ox or AtGA2-ox plants, respectively, and was found to be positively correlated with the rate of photosynthesis as determined at the whole plant level. Differences in dry matter accumulation were most likely due to changes in lignin deposition as indicated by histochemical staining and quantitative measurements. Altered lignification of transgenic plants was paralleled by up- or down-regulation of the expression of lignin biosynthetic genes. Short-term GA3 feeding of excised petioles induced lignin formation in the absence of a transcriptional activation of pathway-specific genes. Thus, short-term GA treatment mediates lignin deposition most likely by polymerization of preformed monomers, whereas long-term effects on lignification involve elevated production of precursors by transcriptional stimulation of the biosynthetic pathway. Interestingly, analysis of stem cross sections revealed a differential effect of GA on the formation of xylem and pith cells. The number of lignified vessels was increased in AtGA20-ox plants pointing to a stimulation of xylem formation while the number of pith cells declined indicating a negative regulation.

Figure 1.

Expression of GA20-oxidase and GA2-oxidase from Arabidopsis in transgenic tobacco plants. a, Schematic structure of AtGA20-ox and AtGA2-ox constructs used to transform tobacco plants. b, RT-PCR of selected transgenic lines using gene-specific primers for either AtGA20-ox or AtGA2-ox as given in “Materials and Methods.” Size of amplified products corresponds to the size of the respective cDNA clones (1,133 bp for AtGA20-ox, 990 bp for AtGA2-ox). Lane 1 to 2, wild type (SNN); lane 3 to 5, independent transgenic lines of AtGA20-ox expressing plants (nos. 15, 10, and 7); and lanes 6 to 8, independent transgenic lines of AtGA2-ox expressing plants (nos. 41, 32, and 2). c, Phenotypic alteration of transgenic tobacco plants caused by the expression of AtGA20-ox or AtGA2-ox after 8 weeks in the greenhouse. From left to right: AtGA20-ox line numbers 15 and 7; wild type; AtGA2-ox line numbers 74 (low expression), 2, 32, and 41. The inset schematically illustrates the GA biosynthetic pathway. GA20-ox catalyzes the conversion from GA_12/53 to GA_9/20. GA2-oxidase inactivates the bioactive GA_4/1 and their precursors GA_9/20.

Figure 2.

Histochemical detection of lignin in stem cross sections of transgenic tobacco plants altered in their GA biosynthesis. Stem material from different parts of the axis of wild type, AtGA20-ox number 15, and AtGA2-ox number 41 was dehydrated by increasing ethanol concentrations (plants at 16-leaf stage). Subsequently, cross sections were stained for lignin using phloroglycinol-HCl reagent. Similar results were obtained in independent experiments. a–c, Cross sections through the upper stem part corresponding to eighth internode (counted from bottom to top) of wild type, AtGA20-ox, and AtGA2-ox, respectively. d–f, Cross sections through the middle stem part corresponding to fifth internode of wild type, AtGA20-ox, and AtGA2-ox, respectively. g–i, Cross sections through the lower stem part corresponding to second internode of wild type, AtGA20-ox, and AtGA2-ox, respectively. Bar is equivalent to 200 μm.

Figure 3.

Effect of altered GA metabolisms on expression of lignin biosynthesis genes. Northern-blot analysis of PAL and transcripts specific for lignin biosynthesis in stems of wild type (lanes 4–6), AtGA20-ox number 15 (lanes 1–3), and AtGA2-ox number 41 (lanes 7–9). Samples were taken from upper (eighth internode, lanes 1, 4, and 7), middle (fifth internode, lanes 2, 5, and 8), and lower stem parts (second internode, lanes 3, 6, and 9) of plants at 16-leaf stage. Twenty micrograms of total RNA were loaded per lane and probed with PAL, COMT, 4CL, CCR, and CAD, respectively. An actin probe was used as a loading control.

Figure 4.

Effect of GA₃ treatment on lignin content. Petioles of mature tobacco plants were infiltrated with water, 10 μm GA₃, or 50 μm GA₃, respectively, and kept for 24 h in darkness at room temperature. a–c, Histochemical detection of lignin in cross sections using thioglycol-HCl staining: (a) SNN + water, (b) SNN+ 10 μm GA₃, and (c) SNN + 50 μm GA₃. Bar corresponds to 200 μm. d, Lignin content in petioles treated with water, 10 μm GA₃, or 50 μm GA₃. Values represent the mean ± sd of three different experiments (n = 9).

Figure 5.

Single leaf measurements of photosynthesis. A, Light response curves of net photosynthetic rate were monitored in fully mature leaves of wild-type (SNN, black circles) and transgenic plants with altered GA biosynthesis (AtGA2-ox no. 41 [GA2], white circles; AtGA20-ox no. 15 [GA20], gray circles). Each data point is the mean ± se of 18 to 20 independent measurements. B, Photosynthetic activity of whole plants was calculated from 4 to 5 leaves of different age at 200 μmol quanta m⁻² s⁻¹ light intensity. Columns represent the difference (in percent) to the mean value of SNN plants (black circle). The mean gas exchange rate of wild-type plants was 0.762 ± 0.044 μmol CO₂ s⁻¹/plant. Data shown are the mean ± se of 24 to 27 independent values.

Figure 6.

Photosynthetic activities of whole plants measured in the canopy chamber PMK1. A, CO₂ uptake rates of AtGA2-ox (GA2) and AtGA20-ox (GA20 expressing tobacco plants are shown as difference, in percent, to the mean value of wild-type plants (SNN). Mean CO₂ uptake of wild-type plants was 0.722 ± 0.033 μmol CO₂ s⁻¹/plant. Light intensity was 200 μmol quanta m⁻² s⁻¹. Each bar is the mean ± se of 24 to 27 independent measurements. B, Schematic illustration of the continuous flow canopy chamber system PMK1 for measuring whole plant gas exchange.