Selection and functional identification of Dof genes expressed in response to nitrogen in Populus simonii × Populus nigra

2.1 Plant material

Tissue culture-generated seedlings of Populus simonii × Populus nigra were grown in a growth chamber at 23°C, under 16 h of light and 8 h of darkness, and under a light intensity of 100 μmol m⁻² s⁻¹. Hydroponic cultivation was performed at 25°C, under 16 h of light and 8 h of darkness, and under a light intensity of 120 μmol m⁻² s⁻¹. Populus simonii × Populus nigra seedlings were cultured in lactate aqueous solution supplemented with 1 mM ammonium-nitrate for 1 week. After 3 days of N being withheld, the N supply was restored for 2 or 48 h, and then the N was withheld again for another 2 or 48 h. The solutions were replaced every 3 days. Samples were collected at each time point and frozen in liquid N for further study.

Seeds of Arabidopsis thaliana plants were disinfected in 75% ethanol comprising 0.05% Triton X-100 for 15 min, washed with absolute ethanol, dried, and germinated on 1/2-strength Murashige and Skoog (MS) solid media. An additional 25 mg L⁻¹ kanamycin was used for screening the transgenic lines. When the seedlings had developed their first pair of true leaves, they were transplanted into the soil for the eventual harvesting of their seeds. To carry out the hydroponic experiment, a special device was first constructed. The two ends of a 1.5 mL centrifuge tube were removed, and the middle part was filled with 6 g L⁻¹ agar and placed on a rectangular plastic plate. An Arabidopsis thaliana seed was placed in the center of the agar to germinate, and the device was placed in the liquid nutrient solution, which was an improved version of Hoagland solution (pH = 5.8). Each hydroponic container was placed in 4 L of nutrient solution, which was changed every 3 days. The N concentrations of the nutrient solutions were 0.15, 0.3, and 3 mM (NH₄NO₃ and KNO₃ were at the same molar ratio). Images were collected, and the growth data were statistically significant after 25 and 45 days. Whole plants were then frozen in liquid N for further study.

2.2 Gene cloning and vector construction

On the basis of the sequence of Populus trichocarpa, we designed the following gene-specific primers for cloning: for PnDof19, 5′-AACCAATACTCACTCCTCCAACA-3′ and 5′-AGGGCACATAAAGTAACCAAATC-3′; for PnDof20, 5′-AAAGATGATTCAAGAACTCTTAGGA-3′ and 5′-AATTGTTCTTAAGGATATGCACC-3′; and for PnDof30, 5′-ACCTGGTCTTTGTCTGTTTACTCTT-3′ and 5′-CTTCCACACCTGTCTTATACCCTTG-3′. Fragment amplification and vector construction involved the use of a KOD Plus Neo high-fidelity DNA polymerase (Toyobo), a pEASY cloning vector (TransGen Biotech), and Escherichia coli Trans1-T1 sensitive cells (TransGen Biotech).

The plant transient expression vector pBS-GFP was used for subcellular localization. The primers used were as follows: for pBS-PnDof19, 5′-agggtaccATGCCGGCAGAATTA-3′ and 5′-tgactagtTTTAAGACCATTCCC-3′; for pBS-PnDof20, 5′-agggtaccATGATTCAAGAACTC-3′ and 5′- tgactagtAGGATATGCACCATT-3′; and for pBS-PnDof30, 5′-agggtaccATGATTCCTTCGAGA-3′ and 5′-tgactagtAAGAAGTACTGAAGA-3′. The 5′ end of each pair of primers was added to the KpnI and SpeI restriction sites (New England Biolab). Arabidopsis plants were genetically transformed using the plant expression vector pROK2, and the primers used for pROK2-PnDof30 included 5′-actctagaATGATTCCTTCGAGA-3′ and 5′-tgggtaccTCAAAGAAGTACTGA-3′. The 5′ end of the primers was added to the KpnI and SpeI restriction sites (New England Biolab). After restriction enzyme digestion, T4 DNA ligase (TransGen Biotech) was used for fragment ligation. The primers were synthesized and the sequencing was performed by Harbin Boshi Biotechnology.

2.3 Genome-wide analysis of the Dof transcription factor family members in Populus trichocarpa

The Phytozome (https://phytozome.jgi.doe.gov) [44] and Plant Transcription Factor Database (PlantTFDB) (http://planttfdb.cbi.pku.edu.cn) websites were queried to obtain genome-wide information concerning the Dof transcription factor family in Populus trichocarpa [45]. The website of the ProtParam tool (http://web.expasy.org/protparam/) was used to analyze the protein physicochemical properties [46], and the gene structure was analyzed via the Gene Structure Display Server 2.0 website (http://gsds.cbi.pku.edu.cn/) [47]. The WoLF PSORT website was used for subcellular localization predictions (http://www.genscript.com/psort/wolf_psort.html). The chromosome localization of the Dof genes was performed based on data from the Phytozome database and from the complete genome sequence of Populus trichocarpa, which was obtained in 2006 [48]. ClustalX software was used for multiple comparisons of protein sequences [49], and MEGA 5 software was used to construct phylogenetic trees [50]. Selecton software was used to analyze the evolutionary selection pressure (http://selecton.tau.ac.il) [51], and the mechanistic–empirical combination model was used for the analysis [52]. The AspenDB (http://aspendb.uga.edu/), poplar EFP browser (http://bar.utoronto.ca/efppop/cgi-bin/efpWeb.cgi), and Phytozome websites were used to analyze gene expression patterns, and the MeV 4.7.4 website was used to construct heat maps. The protein sequences of the cloned genes were analyzed with BioEdit multiple comparison software.

2.4 PCR, RNA extraction, and qRT-PCR

The RNA extraction reagent used in this experiment was pBIOZOL (Beijing Biomars-Technology). In addition, a PrimeScript^TM RT Reagent Kit (TaKaRa) was used for reverse transcription, an SYBR Green Real-time Quantitative Kit (CWBIO) was used to quantify the reagents, and the quantitative PCR instrument used was an ABI 7500 system. The reactions and steps were performed according to the manufacturers’ instructions.

2.5 Subcellular localization

PDS-1000 was used for subcellular localization. Microcarriers were bombarded into lower onion epidermal cells. One day after dark culture, the cells were examined via confocal laser microscopy. The nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI) reagent and imaged under fluorescent light, and combined fields.

2.6 Genetic transformation of Arabidopsis thaliana

After transforming the pROK2-PnDof30 vector into Agrobacterium tumefaciens GV3101, the gene was transformed into the genotype of Arabidopsis thaliana ecotype Col-0 plants by the floral-dip method [53]. The seeds of Arabidopsis thaliana homozygous lines were screened on 1/2-strength MS plates supplemented with 25 mg L⁻¹ kanamycin for 3 continuous generations.

2.7 Measurements of physiological parameters

The chlorophyll, soluble protein, and soluble sugar contents and the activities of PEPC, PK, GS, and NR were determined via standard kits (Suzhou Comin Biotechnology) in accordance with the product instructions.

3.1 Identification and bioinformatic analysis of the Dof transcription factor family members in Populus trichocarpa

Dof members usually have a conserved Dof domain. To identify all the Dof members in Populus trichocarpa, we searched the Populus trichocarpa V3.0 database for the conserved protein sequence from the Phytozome website and ultimately obtained 45 candidate sequences. After comparing the candidate sequences with the Populus trichocarpa Dof members in the PlantTFDB, the redundant sequences were removed, and 44 genes that might encode Dof transcription factors were ultimately identified. According to their chromosomal location information, these members were named PtDof01–44 (Table 1). Among these members, 20 had no introns, 21 had 1 intron, and 3 had 2 introns. The proteins encoded by these genes were 159–506 AAs in length, had a molecular weight ranging from 17.73 to 55.26 kDa, and had an isoelectric point ranging from 4.46 to 10.86. Subcellular localization prediction showed that all the members were localized in the nucleus, except PtDof19 which was localized in the mitochondria.

Multiple alignments of the Dof domain sequences of the Dof transcription factor members showed that 43 Dof domains were conserved; these domains comprised 54–55 AAs and especially included cysteines at sites 3, 6, 28, and 31. These four cysteines are essential for the formation of zinc-finger structures (Figure 1). Notably, the Dof domain of the PtDof29 protein is incomplete and does not have a typical C2–C2 zinc-finger structure.

Figure 1

Multiple alignment of 44 conserved PtDof protein sequences. Two sets of four cysteines highlighted in yellow form a zinc-finger structure, and the underlined area is the Dof domain.

To study the evolutionary relationships among members of the Dof family, we constructed a neighbor-joining (NJ) phylogenetic tree of the Dof protein sequences via MEGA 5 software (Figure 2). The results showed that the Dof members of poplar could be divided into 4 subfamilies (I, II, III, and IV) that contained 15, 11, 8, and 9 Dof members, respectively. Although the two branches of the fourth subfamily were not on the same trunk, the evolutionary relationships were very similar between each other, and the gene structures were similar; thus, they were combined into one subfamily. PtDof29 is relatively independent and does not belong to any subfamily. The gene exon and intron structure maps effectively support the classification results of the subfamily.

Figure 2

Phylogenetic relationships and gene structure of Dof family members in Populus trichocarpa. The NJ phylogenetic tree on the left was constructed via MEGA 5; the tree comprises the aligned protein sequences of 44 PtDof members, and the 4 subfamilies are named I, II, III, and IV. The gene exon/intron structure is shown on the right. The blue lines represent untranslated regions, the yellow lines represent coding areas, and the thin lines represent introns.

The coding DNA sequence (CDS) of the Populus trichocarpa Dof gene was input into the Selecton server, and a selection pressure map of each site was obtained (Figure 3). In the maps, yellow represents a positive selection site with less distribution; there was no positive selection site distributed within the Dof domain. White to purple represents negative selection sites; almost all AAs in the Dof domain are associated with a negative selection site, and most of them are dark purple on the map, representing substantial purifying selection. These results indicate that the Dof domain is under a substantial amount of purifying selection, maintaining a high degree of evolutionary rigor; most of the non-Dof domains are under neutral selection. Compared with the other sites, these sites are less conserved and have a higher probability of mutation. This conclusion also explains why the sequences of the Dof family members are quite different.

Figure 3

Evolution pressure analysis of PtDofs in Populus trichocarpa. The black underlined area represents the Dof domain.

Based on the chromosomal location information of the 44 Dof genes whose sequence is on the Phytozome website and the homologous recombination map published in 2006, we mapped the chromosomal location map of the Populus trichocarpa Dof genes (Figure 4); notably, PtDof29 was unable to be mapped because it belonged to the scaffold structure. The results showed that the Dof genes were distributed throughout the chromosomes, indicating that Dof genes might be ancient. In general, genes within the homologous recombination region of a chromosome may originate from the same ancestor gene. After combining these results with the results of the phylogenetic tree, we identified nine pairs of genes that may have been generated by homologous recombination events of chromosomes in recent evolutionary years: PtDof1/PtDof7, PtDof2/PtDof24, PtDof4/PtDof36, PtDof5/PtDof37, PtDof11/PtDof31, PtDof22/PtDof26, PtDof23/PtDof25, PtDof32/PtDof38, and PtDof34/PtDof40. These nine gene pairs were present not only in the homologous recombination region of the same chromosome but also in the same branch of the evolutionary tree. Among the Dof genes in Populus trichocarpa, these nine pairs are most homologous. Therefore, we speculate that these nine pair of genes may have originated from homologous recombination events throughout the evolution.

Figure 4

Chromosome mapping of Dof family members in Populus trichocarpa. The same colored regions represent chromosomal homologous recombination regions.

After searching the AspenDB website for sequences of gene probes for the Populus trichocarpa Dof family members, we searched the expression data of each gene in the EFP database. In total, 34 Dof gene expression data points were ultimately identified and used to construct an expression map (Figure 5). The results showed that the expression of the Dof genes was mostly downregulated in the mature leaves. However, in the young leaves, the expressions of 9 genes was downregulated, and the expressions of the other 25 genes were upregulated. The expressions of the PtDof10, PtDof18, PtDof20, and PtDof41 genes were relatively high, and the expression of the PtDof19 gene was the highest. In the roots, the expressions of 7 genes was downregulated, and that of 27 genes were upregulated, of which the expressions of PtDof5, PtDof14, and PtDof20 were the highest. In the young leaves of plants growing in the darkness, the expressions of 15 genes was upregulated, and that of 19 genes were downregulated. In the young leaves of plants growing in darkness but then exposed to light for 3 h, the expressions of 12 genes was upregulated, and that of 22 genes were downregulated. In seedlings subjected to continuous light, the expressions of 17 genes were upregulated, and that of 17 genes were downregulated. In the female flowers, the expressions of 13 genes were upregulated, and that of 21 genes were downregulated, and in the male flowers, the expressions of 20 genes were upregulated, and that of 14 genes were downregulated. In the xylem, the expressions of 11 genes was upregulated, and that of 33 genes were downregulated. In conclusion, most of the members of the Populus trichocarpa Dof gene family were expressed in young leaves and roots, and the expression patterns in other plant parts were more complex, which indicated that the function of Dof genes might be substantially different.

Figure 5

Expression patterns of 34 Populus trichocarpa Dof members based on EFP data. ML: Mature leaves; YL: Young leaves; Rt: Roots; Ds: Dark-grown seedlings; Ds3h: Dark-grown seedlings exposed to light for 3 h; Cls: Continuous light-grown seedlings; fc: Female catkins; mc: Male catkins; xy: Xylem.

The fragments per kilobase of transcript per million mapped reads (FPKM) data of the Populus trichocarpa Dof genes were obtained from the Phytozome website, and the expression data were used to construct a gene expression heat map (Figure 6). The results showed that the expressions of PtDof10 and PtDof30 were higher in the leaves (early stage of female floral buds) than in the other organs. In the leaves (immature ones), the most highly expressed genes were PtDof30 and PtDof19. In the young leaves, PtDof10 and PtDof30 were highly expressed; in the roots, the PtDof5 gene was expressed the most. The most highly expressed genes in the root tips were PtDof10 and PtDof30. In the stems (internodes), the most highly expressed genes were PtDof32 and PtDof39; in the stem nodes, the most highly expressed genes were PtDof19, PtDof10, PtDof38, and PtDof39. The expression of PtDof05, PtDof10, PtDof19, PtDof20, PtDof23, PtDof30, and PtDof39 in various tissues was significantly higher than that of the other studied genes.

Figure 6

Expression patterns of 44 Populus trichocarpa Dof members based on FPKM data obtained from the Phytozome website. Lf: Leaves (early-stage female floral buds); Li: Leaves (immature); Ly: Leaves (young); Rt: Roots; RtT: Root tips; Si: Stems (internodes); Sn: Stems (nodes).

3.2 Screening of Dof genes in response to N changes in Populus simonii × Populus nigra

Two groups of tissue culture-generated seedlings were subjected to N-treatment experiments under LA hydroponic solution. In the first group, 1 mM ammonium-nitrate was used as the sole N source (Figure 7). After 1 week of cultivation, the seedlings were subjected to an N-deficient solution for 3 days. Afterward, they were subjected to an N-sufficient solution for 2 or 48 h, after which the N was withheld again for 2 or 48 h. Samples were taken at each of these time points. Populus simonii × Populus nigra seedlings were grown under N-deficient conditions in vitro for 3 days as controls and were provided different forms of N in vivo only to maintain biological activity. After the N was absorbed under in vivo conditions, the N treatments were carried out in vitro to obtain information on Dof genes induced in response to N in Populus simonii × Populus nigra. qRT-PCR was used to measure the expression of the Dof genes (the primers used are listed in Table 1). The internal reference gene used was cell division control protein 2 (CDC2), and the 2^−ΔΔCt method was used to calculate the relative expression. After several rounds of designing primers and performing quantitative experiments, the expression data of 38 genes were ultimately obtained. The results showed that the expression patterns of these genes in the leaves, stems, and roots were very different under different treatments, showing different degrees of tissue specificity. Compared with that in the control group, the expressions of 31 Dof genes in the leaves of the treatment group increased 2 h after the N supply was restored. After the N supply was restored for 48 h, the expression levels were lower than that after the N supply was restored for 2 h, and the expression levels of most of the Dof genes were lower after N was withheld than when the N was supplied for 2 and 48 h. It could be concluded that a short N supply induces the expressions of most Dof genes in the leaves but that a prolonged N supply inhibits the expressions of some of these genes. In the stems, 32 Dof genes presented higher expression levels when N was withheld for 48 h but lower expression levels when N was resupplied for 2 and 48 h. These results suggest that low N contents in vivo induced the expressions of Dof genes in the absence of an N supply in vitro. In the roots, the expressions of 34 Dof genes decreased 2 h after the N was resupplied, while the expressions of 17 Dof genes increased slightly within the 48 h during which the N was resupplied and withheld again. This may indicate that the expressions of some Dof genes was induced under low N levels in the roots.

Figure 7

Relative expression levels of the Populus simonii × Populus nigra PnDof genes under sufficient and deficient N supplies, as revealed by qRT-PCR (L: leaves; S: stems; R: roots).

In the second N treatment experiment, seedlings were treated with ammonium and nitrate N for 2 weeks at concentrations of 0.1, 1, or 10 mM (Figure 8). The results showed that the expressions of ten genes (Dof4, Dof10, Dof11, Dof13, Dof21, Dof28, Dof30, Dof32, Dof42, and Dof43) were induced in the leaves under low ammonium concentrations, that of eight genes (Dof4, Dof9, Dof19, Dof26, Dof30, Dof36, Dof37, and Dof40) were induced in the stems, and that of seven genes (Dof7, Dof9, Dof10, Dof21, Dof25, Dof36, and Dof42) were induced in the roots. Under low-nitrate conditions, the expressions of nine genes were induced in leaves, including Dof4, Dof10, Dof13, Dof21, Dof23, Dof30, Dof32, Dof33, and Dof36; the expressions of four genes were induced in stems, including Dof26, Dof28, Dof36, and Dof43; and the expressions of 19 genes were induced in the roots. In conclusion, the expressions of the following genes was induced under both low levels of N at the same time: Dof4, Dof10, Dof13, Dof21, Dof30, and Dof32 in leaves; Dof36 in the stems; and Dof25, Dof36, and Dof42 in the roots.

Figure 8

Relative expression levels of Populus simonii × Populus nigra PnDof genes under two kinds of N supplied at three different concentrations, as revealed by qRT-PCR (L: Leaves; S: Stems; R: Roots).

3.3 Cloning of the PnDof19, PnDof20, and PnDof30 genes from Populus simonii × Populus nigra

We cloned the CDSs of the PnDof19 (MK796000), PnDof20 (MK7960001), and PnDof30 (MK789595) genes from Populus simonii × Populus nigra cDNA (Figure 9), which were 747, 972, and 1,074 bp long, respectively, and encoded 248, 323, and 357 AAs, respectively.

Figure 9

Gene coding sequences of PnDof19, PnDof20, and PnDof30 alongside their translated protein sequences in Populus simonii × Populus nigra.

To explore whether the transcription factors encoded by the PnDof19, PnDof20, and PnDof30 genes cloned from Populus simonii × Populus nigra are involved in regulating C and N metabolism, we compared the sequences of these three proteins with the sequences of three functional proteins that specifically regulate C and N metabolism (Figure 10). The results showed that all six proteins had a complete Dof domain and four highly conserved cysteines, and all of them had a nuclear localization signal specific to Dof transcription factor family members (B1 and B2 regions).

Figure 10

Multiple alignment of protein sequences. The black box area is the conserved Dof domain, and B1 and B2 represent Dof-specific nuclear localization signals.

Moreover, we identified Dof genes with different functions (both AtDof1/NM_104048.4 and AtOBP1/OAP05220.1 in Arabidopsis thaliana and OsDof12/AAL84292.1 in rice) and constructed phylogenetic trees comprising the CDSs of both the three protein-coding genes cloned by us and the known Dof protein-coding genes (Figure 11). Because of the poor conservation of Dof protein sequences of non-Dof-domain regions, we used only conserved domain sequences to construct phylogenetic trees to determine their evolutionary relationships more accurately. The results showed that PnDof20 and PnDof30 branched together with the N metabolism regulatory genes AtDof1 and PpDof5, suggesting that PnDof20 and PnDof30 may also be N metabolism regulatory genes; PnDof19 and the cell cycle regulatory gene AtOBP1 were branched together on one branch, and thus, we speculated that PnDof19 might be related to cell cycle regulation. OsDof12 is a flowering regulatory gene that is independent of the other genes in the phylogenetic tree.

Figure 11

Phylogenetic tree comprising PnDof19, PnDof20, and PnDof30 in Populus simonii × Populus nigra and several specific functional Dof genes in other species.

3.4 Subcellular localization of the PnDof19, PnDof20, and PnDof30 proteins

To determine whether the three Populus simonii × Populus nigra Dof proteins have characteristics of general transcription factors, i.e., the localization of the protein in the nucleus, we fused the open reading frame of the PnDof19, PnDof20, and PnDof30 genes to the GFP gene within a PBS-GFP vector; onion subepidermal cells were subsequently transformed via gene gun bombardment. Cells displaying green fluorescence were observed via scanning laser confocal microscopy. The nuclei were stained with DAPI reagent and then observed and imaged under a microscope (Figure 12). The results showed that the PnDof19, PnDof20, and PnDof30 proteins localized to the nucleus, which is consistent with the localization of Dof proteins in other reported species [54,55].

Figure 12

Subcellular localization of the PnDof19, PnDof20, and PnDof30 proteins. a: Fluorescence field; b: Bright field; and c: Superimposition of the fluorescence and bright fields.

3.5 Functional analysis of PnDof30-overexpressing Arabidopsis thaliana lines

The PnDof30 gene was inserted into the genome of Arabidopsis thaliana ecotype Col-0 plants by the floral-dip method, and 24 independent transgenic lines were selected for extraction of their genomic DNA. The transgenic lines were identified via PCR (Figure 13). After identification, 11 transgenic lines were randomly selected to determine the expression level of their PnDof30 gene (Figure 14). The results showed that the expression level and stability in each line were substantially different. We chose three stable expression lines, L1, L2, and L15, for functional analysis and then screened and identified the homozygotes.

Figure 13

PCR-based identification of genomic DNA in transgenic Arabidopsis thaliana lines.

Figure 14

Relative expression of PnDof30 in transgenic Arabidopsis thaliana lines.

The seeds of the WT and homozygous lines were vernalized and planted in several unique hydroponic devices for germination and growth. The hydroponic solutions used were improved versions of Hoagland solution, consisting of 0.15, 0.3, or 3 mM N. The phenotypes were evaluated after 25 days of plant growth (Figure 15). The results showed that the phenotypes of the growth-related parameters of the PnDof30-overexpressing Arabidopsis thaliana lines, such as leaf size, lotus leaf diameter, and leaf number, were significantly better than those of WT plants under all three different N concentrations, especially under low-N conditions.

Figure 15

Phenotypic changes in PnDof30 transgenic Arabidopsis thaliana lines under N treatment for 25 days.

Because the plant size was too small for further determination of growth-related parameters, we cultured Arabidopsis thaliana plants in liquid media for 45 days and observed their phenotype (Figure 16). The results showed that when the culture period was extended to 45 days, the overexpression plants under low-N conditions still grew better than WT plants, and the leaf color was greener than that of WT plants. Under 3 mM N, the difference in growth between the overexpression lines and WT narrowed; nonetheless, the L2 transgenic lines were significantly better than the WT, and L1 and L3 transgenic lines were slightly better than the WT.

Figure 16

Phenotypic changes in PnDof30 transgenic Arabidopsis thaliana lines under N treatment for 45 days.

The diameter of the leaves, the number of leaves, and fresh weight were further determined (Figure 17). Compared with that of the WT plants, the diameter of the lotus leaves of the L1 plants increased significantly at a 0.3 mM N concentration, and the diameter of the lotus leaves of the L2 and L3 plants increased significantly at all three N concentrations, which was consistent with the observed phenotypes. The number of leaves in the L2 and L3 plants was significantly different from that of WT plants at the 0.15 mM N concentration, and the L2 plants had significantly more leaves than the WT plants at the 0.3 and 3 mM N concentrations. Although the number of leaves was not significantly different between the L1 and L3 plants and the WT plants, we observed that the fresh weight of the three transformed lines was significantly higher than that of the WT plants at the three concentrations, and the difference between L2 and the WT was the most significant. Taken together, these results indicated that the PnDof30 gene could improve the growth index of Arabidopsis thaliana, especially under low-N levels.

Figure 17

Rosette leaf diameter, leaf number, and fresh weight of Arabidopsis transgenic lines and WT plants subjected to 3 different N concentrations for 45 days (p < 0.05).

To further evaluate the effect of overexpression of the PnDof30 gene on the growth of Arabidopsis thaliana, we measured the contents of soluble sugars, soluble protein, and chlorophyll (Figure 18). The results showed that the soluble protein contents in the three transgenic lines were higher than those in the WT at the 0.15, 0.3, and 3 mM N concentrations, while the contents in the L3 plants at the 0.15 mM N concentration and in the L1 and L3 plants at 0.3 mM N concentrations were not significantly different from those in the WT, but they were slightly higher. Under the 0.15 mM N concentration, the soluble sugar content in the three transgenic lines decreased significantly; under the 0.3 mM N concentration, the content in the L1 plants decreased slightly, whereas in the L2 and L3 plants, the contents decreased significantly. However, there was no significant difference between the transgenic and WT plants at the 3 mM N concentration. Under the three N concentrations, the chlorophyll content in the transgenic lines was significantly higher than that in the WT plants. The increase in soluble protein content and the decrease in soluble sugar content indicated that the efficiency of N utilization improved and that C skeletons were consumed in the transgenic Arabidopsis thaliana lines, which resulted in a decrease in the soluble sugar content and an increase in both the soluble protein content and the chlorophyll content, which effectively promoted photosynthesis and C/N metabolism. Taken together, these results indicate that the PnDof30 gene can increase the C/N metabolic level in transgenic Arabidopsis thaliana, especially under low N levels.

Figure 18

Contents of soluble sugars, soluble proteins, and chlorophyll in Arabidopsis transgenic lines and WT plants subjected to 3 different N concentrations for 45 days (p < 0.05).

The activities of the PEPC, PK, GS, and NR enzymes were subsequently determined (Figure 19). According to the PEPC enzyme activity results, the activities in the three transformed lines were significantly higher than that in the WT plants at 0.15 mM N. Under 0.3 mM N, the activity in the L1 plants was slightly higher than that in the WT plants, and that in the L2 and L3 plants was significantly higher than that in the WT plants. Under 3 mM N, the enzyme activity in the L2 plants was significantly higher than that in the WT plants, while the enzyme activity in the L1 and L3 plants did not significantly differ from that in the WT plants.

Figure 19

Enzyme activity of PEPC, PK, GS, and NR of Arabidopsis transgenic lines and WT under 3 concentrations of N treatment for 45 days (p < 0.05).

With respect to PK enzyme activity, the results showed that at 0.15 mM N concentration, the activities in the L1 and L2 plants were slightly higher and that in the L3 plants was significantly higher than that in the WT plants. Under 0.3 mM N, compared with the WT plants, the three transformed lines presented significantly higher PK activity. Under 3 mM N, the activity in the L1 plants was significantly higher than that in the WT plants, and the activities in the L2 and L3 were not significantly different from that in the WT plants.

In terms of GS enzyme activity, the results showed that the three transformed lines did not significantly differ from the WT plants at the 0.15 mM N concentration but that the activity in the former was significantly higher than that in the WT plants at the 0.3 and 3 mM N concentrations.

In terms of NR enzyme activity, the results showed that the activities of the three transgenic lines were significantly higher than those in the WT plants at all three N concentrations.

PEPC and PK are important enzymes involved in the process of C metabolism. The activities of the PEPC and PK enzymes in the transgenic lines increased under low-N conditions, indicating that the metabolism of C increased. Reactions involving GS constitute the first step of ammonium assimilation, and reactions involving NR constitute the first step of nitrate assimilation. The enzyme activities in all transgenic lines improved under low-N conditions. The results of the enzyme activity assay showed that overexpression of PnDof30 in Arabidopsis thaliana could promote C/N assimilation efficiency under low-N conditions.

We selected 13 genes that play a major role in the C/N pathways and measured their relative expression (Figure 20). Under 0.15 mM N, the expression of the PEPC1 gene in the three transgenic lines was not significantly different from that in the WT plants, but the expression of PEPC2 significantly increased. Under 0.3 and 3 mM N, the expressions of PEPC1 and PEPC2 in the transgenic lines increased. The expressions of PK1 and PK2 in the three transgenic lines significantly increased under all three N concentrations. The expression changes of the PEPC and PK genes were consistent with the results of the PEPC and PK enzyme activities.

Figure 20

Relative expression levels of the PEPC and PK genes in Arabidopsis transgenic lines and WT plants subjected to 3 different concentrations of N for 45 days (p < 0.05).

The ammonium transporter (AMT) protein data showed that the expression of 3 genes in the 3 transgenic lines significantly increased under 3 mM N (Figure 21). Under low-N conditions, the expressions of AMT1.1 and AMT1.2 were downregulated, and that of AMT1.3 was upregulated. All three genes were major functional genes involved in the high-affinity ammonium transport system in Arabidopsis thaliana; however, the contribution of each gene was unknown, so it was uncertain whether ammonium uptake increased in general.

Figure 21

Relative expression levels of AMT genes in Arabidopsis transgenic lines and WT plants subjected to 3 different concentrations of N for 45 days (p < 0.05).

The nitrate transporter (NRT) data showed that the expression of two NRT genes increased in all three transgenic lines under 3 mM N, but the expression of NRT1.1 was downregulated and that of NRT2.1 was upregulated under low-N conditions (Figure 22). Like with the AMT gene, it was unclear whether the nitrate transport level improved.

Figure 22

Relative expression levels of NRT genes in Arabidopsis transgenic lines and WT plants subjected to 3 different concentrations of N for 45 days (p < 0.05).

The GS data showed that the expression of GS1.1 in the three transgenic lines was significantly higher than that in the WT plants at all three N concentrations (Figure 23). The expressions of GS1.2, GS1.3, and GS2 were upregulated under 0.3 and 3 mM N; however, the expression of GS1.2 was upregulated while the expressions of both GS1.3 and GS2 were downregulated at 0.15 mM N. Under 0.15 mM N, GS1.2 expression in the transgenic lines was not significantly different from that in the WT plants, and GS1.3 and GS2 expression was downregulated. We speculate that 0.15 mM N is the key concentration responsible for GS1.2, GS1.3, and GS2 gene expressions. The change in GS gene expression under low-N levels is consistent with the change in GS enzyme activity.

Figure 23

Relative expression levels of GS genes in Arabidopsis transgenic lines and WT plants subjected to 3 different concentrations of N for 45 days (p < 0.05).