Overexpression of a pine Dof transcription factor in hybrid poplars: A comparative study in trees growing under controlled and natural conditions

Abstract

In this work, the role of the pine transcriptional regulator Dof 5 in carbon and nitrogen metabolism has been examined in poplar trees. The overexpression of the gene and potential effects on growth and biomass production were compared between trees growing in a growth chamber under controlled conditions and trees growing in a field trial during two growth seasons. Ten-week-old transgenic poplars exhibited higher growth than untransformed controls and exhibited enhanced capacity for inorganic nitrogen uptake in the form of nitrate. Furthermore, the transgenic trees accumulated significantly more carbohydrates such as glucose, fructose, sucrose and starch. Lignin content increased in the basal part of the stem likely due to the thicker stem of the transformed plants. The enhanced levels of lignin were correlated with higher expression of the PAL1 and GS1.3 genes, which encode key enzymes involved in the phenylalanine deamination required for lignin biosynthesis. However, the results in the field trial experiment diverged from those observed in the chamber system. The lines overexpressing PpDof5 showed attenuated growth during the two growing seasons and no modification of carbon or nitrogen metabolism. These results were not associated with a decrease in the expression of the transgene, but they can be ascribed to the nitrogen available in the field soil compared to that available for growth under controlled conditions. This work highlights the paramount importance of testing transgenic lines in field trials.

Fig 1. Analysis of the transgenic poplar lines.

(A) Diagram of the construct used to transform hybrid poplar clone INRA 7171-B (P. tremula x P. alba): the pBI121 vector containing the PpDof5 sequence under the control of a 35S gene promoter (CaMV35S) was used for the transformation. (B) Determination of copy number in the L2 and L6 transgenic lines using quantitative PCR. The poplar single copy gene FAD-dependent oxidoreductase (Potri.018G112600) was used as the standard. The ratio of the amplicons derived from the integrated PpDof5 and the internal control is indicated at the bottom of each line. (C) Expression analysis of PpDof5 by real-time qPCR in controls (white bar) and two transgenic lines: L2 (gray bar) and L6 (black bar). The expression data were normalized using ubiquitin as the reference gene. (D) Representative picture of 10- week-old untransformed control and L2 and L6 transgenics. (E) Root volume of control and transgenic poplar plants. (F) Length of the stem in cm of the plants (five plants were used for each determination).

Fig 2. ¹⁵N uptake in hydroponic culture.

(A) Plants acclimatized in transparent plastic pots as described in Materials and Methods and grown in KNO₃ as the sole N source were incubated for 2 h in the same solution but containing ¹⁵N-label as 1 mM K¹⁵NO₃. δ¹⁵N (‰) values were determined in roots using an elemental analyzer coupled to a Delta V Advantage isotope ratio mass spectrometer. Values are in triplicate, and asterisks indicate that the difference between the control and transgenic poplars is significant by the t-test (P<0.05).(B) The same as in (A), except that plants were grown under NH₄Cl as the sole N source.

Fig 3. Field trial of transgenic poplar overexpressing PpDof5.

(A) Diagram of the ground for the field trial experiment. (B) General overview of ground-field trees in the field trial. (C) Confirmation of the presence of the transgene in field-growing trees. PCR analysis was performed using primers specific for PpDof5 on genomic DNA from poplar. As a control, the internal poplar gene PtGS2 was amplified. Two kinds of samples from years 2012 and 2013, the two growing periods considered, were analyzed. (D) The mRNA level of PpDof5 was determined by qPCR in leaves of field-growing trees in 2012 and 2013. Determinations were made in triplicate. (E) Content of chlorophyll a and b in controls and transgenic poplar was determined in the upper (UL) and basal (BL) leaves of trees during the growing period of 2012 and in leaves and branches during the growing period of 2013. White bars correspond to control samples, gray bars are L2 and black bars are L6 samples.

Fig 4. Carbohydrate content in PpDof5 transgenic poplar.

(A) Analysis of carbohydrate content in 10-week-old control and transgenic poplar lines grown in the growth-chamber. Values are expressed as nmol mg^-1 of fresh weight (FW). The determination is the mean of three independent biological samples measured in triplicate in upper leaves (UL), basal leaves (BL), stems and roots. Asterisks indicate that the difference between the control and transgenic poplars is significant by the t-test (P<0.05). (B) Analysis of carbohydrate content in control and transgenic poplar lines grown in the field during the two-growing seasons of the experiment. Values are expressed as nmol mg^-1 FW and are the means of three independent biological samples measured in triplicate. The samples were upper leaves (UL) and basal leaves (BL) for 2012 and leaves and branches for 2013. Asterisks indicate that the difference between the control and transgenic poplars is significant by the t-test (P<0.05) for starch content in samples of 2012.

Fig 5. Cellulose and lignin content in PpDof5 transgenic poplar.

(A) Determination of cellulose and lignin content in 10-week-old poplar plants grown in a growth-chamber. Values are expressed as mg/g FW. The determination was performed in triplicate in three independent biological samples. Asterisks indicate significant differences between controls and lines by the t-test (P<0.05). (B) Determination of cellulose and lignin content in poplar trees grown in the field. Values are expressed as mg/g FW. The determination is the mean of three independent biological samples measured in triplicate.

Fig 6. Expression analysis of PAL1 and GS1.3.

(A) Diagram of the location in the pathway of the two enzymes involved in the synthesis of phenylalanine for lignin synthesis. The ammonium flux involving the two enzymatic reactions is highlighted in black. (B) Expression analysis by qPCR of PAL1 and GS1.3 genes in the basal section of the stem in control and transgenic poplar trees. Measurements were performed in triplicate in three independent biological replicas. Asterisks indicate that the difference between the control and transgenic poplars is significant by the t-test (P<0.05).