Enhancement of Arabidopsis growth characteristics using genome interrogation with artificial transcription factors

Abstract

The rapidly growing world population has a greatly increasing demand for plant biomass, thus creating a great interest in the development of methods to enhance the growth and biomass accumulation of crop species. In this study, we used zinc finger artificial transcription factor (ZF-ATF)-mediated genome interrogation to manipulate the growth characteristics and biomass of Arabidopsis plants. We describe the construction of two collections of Arabidopsis lines expressing fusions of three zinc fingers (3F) to the transcriptional repressor motif EAR (3F-EAR) or the transcriptional activator VP16 (3F-VP16), and the characterization of their growth characteristics. In total, six different 3F-ATF lines with a consistent increase in rosette surface area (RSA) of up to 55% were isolated. For two lines we demonstrated that 3F-ATF constructs function as dominant in trans acting causative agents for an increase in RSA and biomass, and for five larger plant lines we have investigated 3F-ATF induced transcriptomic changes. Our results indicate that genome interrogation can be used as a powerful tool for the manipulation of plant growth and biomass and that it might supply novel cues for the discovery of genes and pathways involved in these properties.

Fig 1. Variation in rosette phenotypes and growth characteristics among a population of primary transformants (T1) harboring 3F-EAR encoding T-DNA constructs.

The presented individuals are representative of the extent of variation, but not in a quantitative manner. The plants were first grown on selection medium containing kanamycin, and were transferred to soil after approximately 2 weeks. The presented individuals are approximately 1 month old. The size of the individual in the right top corner is representative of a wild-type Col-0 plant at this stage of development. A truly wild-type Col-0 plant would not have survived the in vitro selection procedure used to obtain the transgenic plants, and was therefore not included in this Figure.

Fig 2. Growth curves of the T2 progeny (segregating) of the 100 largest 3F-EAR primary transformants in terms of RSA (n = 100 for Col-0; n = 7 for the transgenic lines).

The growth curve of the Col-0 is presented in black.

Fig 3

A) Quantification of the relative RSA of the selected 3F-EAR lines (T2; segregating) and 3F-VP16 lines (T3; only VP16-05-014 segregating) compared to Col-0 at 25 dpg. The RSA of each plant was calculated in terms of percentage of the average of Col-0. Error bars represent SEM values (n = 182 for Col-0, n = 16–18 for the transgenic lines). Significant differences with the Col-0 are indicated by an * (p < 0.05). B) Number of true leaves with discernable petioles at 25 dpg. Error bars represent SEM values (n = 36 for Col-0, n = 16–18 for the transgenic lines). Significant increases compared to Col-0 indicated by asterisks (*) (p < 0.05). C) Overview of the rosette phenotypes of the selected 3F-ATF lines (25 dpg), and of the 9 bp DNA recognition sequences of the 3Fs that were isolated from them. The presented individuals had RSA values closest to the average value found for their genotypes.

Fig 4. Growth curves of the wild-type Col-0, VP16-02-003 (T3) and retransformants reconstituted from VP16-02-003 (T2; segregating) (n = 48 for Col-0, n = 15–18 for the transgenic lines).

Significant differences with Col-0 at 28 dpg are indicated by an * (p < 0.05). For each line the average normalized expression values of the 3F-VP16 construct as determined by RT-qPCR analysis at 15 dpg are provided. For the lines with significantly larger RSA than Col-0 the average relative growth rate between 10 and 28 dpg and the total number of with discernable petioles at 28 dpg are provided. Significant differences with Col-0 are indicated by an * (p < 0.05).

Fig 5. Quantification of the relative fresh weight (A) and the relative dry weight (B) of VP16-02-003 plants (T3, non-segregating) and retransformant plants reconstituted from VP16-02-003 (T2; segregating) compared to the Col-0 (28 dpg).

The fresh and dry weights of each plant were calculated in terms of percentage of the average of Col-0. Error bars represent SEM values (n = 48 for Col-0, n = 15–18 for the transgenic lines). Significant increases compared to Col-0 are indicated by the letter ‘a’ (p < 0.05), and significant decreases are indicated by the letter ‘b’ (p < 0.05).

Fig 6. Venn diagrams of Differentially Expressed Genes (DEGs) compared to the Col-0 in the RNA sequencing data sets of the indicated 3F-EAR transgenic lines (p < 0.0001).

‘Background’ refers to RNA expression data derived from the pool of lines expressing 3F-EAR fusions similar to the specific 3F-EAR fusion expressed in the selected lines, but without a noticeable increase in RSA. A) Upregulated DEGs. B) Downregulated DEGs.

Fig 7. Venn diagrams of Differentially Expressed Genes (DEGs) compared to the wild-type Col-0 in the RNA sequencing data sets of the indicated 3F-EAR transgenic lines (p < 0.0001).

‘Background’ refers to RNA expression data derived from the pool of lines expressing 3F-VP16 fusions similar to the specific 3F-VP16 fusion expressed in the selected lines, but without a noticeable increase in RSA. A) Upregulated DEGs. B) Downregulated DEGs.