Identification and phylogenetic analysis of 4CL from Fraxinus mandschurica
We identified 12 4CL genes from the Fraxinus mandshurica transcriptome databases using the BioEdit local Blast method with 5 4CL genes of Arabidopsis thaliana and 3 4CL genes of Populus trichocarpa nucleotide sequences retrieved from NCBI, named Fm4CL1- Fm4CL12, which have all been uploaded to GenBank and assigned accession numbers (KJ531400- KJ531404, KF994781, KJ531405-KJ531410), characteristics of 12 Fm4CL gene sequences are in Additional file 1: Table S1. To understand the evolutionary relationship of 4CL proteins between Fraxinus mandshurica and other species, we collected the sequences of 53 4CL proteins from Populus, Oryza sativa, Arabidopsis, and other species and constructed phylogenetic trees (Fig. 1). The 53 4CL proteins could be divided into five broad categories. Fm4CL3, Fm4CL7, Fm4CL8, and Fm4CL10 had higher homologies with 4CL sequences from Vitis vinifera, Populus, and Arabidopsis. Fm4CL2, Fm4CL6, and Fm4CL9 had high homology with sequences from Cucumis sativus, Zea mays, Vitis vinifera, and Populus. Fm4CL5, Fm4CL11, and Fm4CL12 had high homology with sequences from Betula luminifera, vitis vinfera, and Salvia miltiorrhiza. The phylogenetic analysis demonstrate that Fm4CL4 and its orthologs in Arabidopsis, Nicotiana sylvestris, Nicotiana tabacum, Oryza sativa, Salvia miltiorrhiza belong to the 4CL-like gene subfamily, so we named it Fm4CL-like 1. This indicates that Fm4CLs is closely related to these species.
Expression analysis of the Fm4CL/4CL-like gene family
To assess the differential expression pattern of the Fm4CL genes family in different tissues of Fraxinus mandshurica, samples of leaves, xylem, bark, flowers, bud, stem, petiole and seeds were collected in May. The qRT-PCR results showed that 12 Fm4CL transcripts could be detected in different tissues of Fraxinus mandshurica and has obvious tissue specific expression (Fig. 2a). Fm4CL2, Fm4CL6, Fm4CL9 and Fm4CL11 were expressed at low levels in all the tissues and organs of Fraxinus mandshurica. Among 12 4CL/4CL-like genes, Fm4CL-like 1 expression was the highest in bark, while Fm4CL-like 1 and Fm4CL8 were expressed at the highest level in xylem, indicating that Fm4CL8 and Fm4CL-like 1 may be involved in the biosynthesis of xylem.
Fraxinus mandshurica was subjected to tension treatment. Control check (CK), Tension Wood (TW), and Opposite Wood (OW) indicate untreated samples, tension stress surface samples, and corresponding surface samples, respectively (Fig. 2b). The expression of Fm4CL/4CL-like genes in 24 h-treated Fraxinus mandshurica tension wood (TW) was almost unchanged, but the expression in OW samples was generally higher than that in TW. After 3 d treatment, the expression level of Fm4CL genes in OW was significantly higher than TW. The Fm4CL genes were almost undetectable in Fraxinus mandshurica tension treated wood at 7 d. The expression pattern of the Fm4CL genes in tension wood differed, but all genes responded to the tension treatment. Among them, Fm4CL-like 1 had the highest expression level in OW after 3 d treatment, it was 3~20 times more than other genes, which indicated that Fm4CL-like 1 might be involved in the synthesis of cell wall and lignin.
Overexpression of Fm4CL-like 1 gene in transgenic tobacco
According to the expression analysis of the Fm4CL gene family in Fraxinus mandshurica, Fm4CL-like 1 was chosen for overexpression in transgenic tobacco. Agrobacterium strain LBA4404 containing the pBI121- Fm4CL-like 1 recombinant plasmid was used to mediate the genetic transformation of tobacco leaves. The transformed tobacco was placed directly in differentiation-selective MS medium (with 50 mg/L NAA and 500 mg/L 6-BA) containing 50 mg/L kanamycin and 300 mg/L cefotaxime. The above-mentioned tobacco leaves were differentiated into shoots on differentiation media to obtain kanamycin-resistant tobacco plants. Finally, the tobacco plants were rooted in MS medium containing 200 mg/L NAA (Fig. 3a). Then, the expression of Fm4CL-like 1 in transgenic tobacco was detected by real-time RT-PCR. Ten independent transgenic events were obtained. Fm4CL-like 1-overexpressed transgenic line 1 was selected for further study based on the expression level.
GUS staining of transgenic plants showed that the GUS activity in the young leaves and stems was the highest among tissues in the three-week-old transgenic tobacco seedlings, while the activities in the root and mature petiole were lower. GUS expression was also detected in transgenic tobacco flower tissues. Among these, the GUS activity was highest in the petals, anthers, receptacle, calyx, and stigma. However, the filaments and pedicel hardly exhibit GUS activity. The above results are consistent with the results of real-time RT-PCR, demonstrating that Fm4CL-like 1 is highly expressed in transgenic tobacco (Fig. 3c).
Fm4CL-like 1 plays an important role in lignin biosynthesis
To understand the function of Fm4CL-like 1, the contents of lignin, cellulose, and hemicellulose were measured in transgenic tobacco. The lignin content in overexpression lines (OE) was approximately 39.5% higher than that in WT (P < 0.05) (Fig. 4b), and the S/G ratio of transgenic tobacco increased by 19.7% compared with WT (P < 0.05) (Fig. 4c). The cellulose content in WT was approximately 14.78% higher than that in OE lines (P < 0.05) (Fig. 4d). The hemicellulose content was very similar in both conditions (Fig. 4e). In addition, the stems and petioles of OE line were sectioned and stained with phloroglucinol-HCl (Fig. 4a). Based on xylem staining, the lignin content of OE plants was higher than that of WT, indicating that Fm4CL-like 1 promoted lignin deposition. These results demonstrate that overexpression of Fm4CL-like 1 contributes to the accumulation of lignin in tobacco.
Fm4CL-like 1 altered the xylem of transgenic tobacco
The third section of OE and WT was paraffin-embedded (Fig. 5a) and we found that the number of xylem cells in OE lines increased by 40% compared with WT plants (Fig. 5b). Xylem cell walls of OE and WT were observed by scanning electron microscopy (Fig. 5c). The cell wall thickness of WT and OE samples was respectively about 1.91 ± 0.13 μm and 2.43 ± 0.08 μm, indicating that the cell wall thickness of OE was increased by 21.6% compared to WT (Fig. 5d). This is the further evidence that Fm4CL-like 1 is involved in the synthesis of plant xylem.
Fm4CL-like 1 confers mannitol tolerance
Transgenic and WT tobacco plants were treated with 200 mM Mannitol to study their tolerance to osmotic stress. Under the control conditions, no significant difference in the phenotype, growth rate, fresh weight, or root length of WT and OE samples was noted (Fig. 6a), indicating that Fm4CL-like 1 did not affect the phenotype or growth rate of the plant.
After two and four weeks of mannitol treatment, OE showed a higher growth rate, greener leaves compared to WT plants. After four weeks treatment, root length of OE is 64% longer than WT (Fig. 6a and c). Treatment of WT and OE seedlings with 200 mM Mannitol, the result was similar, OE seedlings have a longer root and greener leaves than WT, while WT plants were grown slowly and the leaves turned yellow (Fig. 6b and d).
The seed germination rate of WT and OE lines grown in normal MS medium were similar. However, with 200 mM mannitol treatment, the seed germination rate of OE is 47% higher than that of WT (Fig. 6e). Moreover, OE seedlings began to germinate on the 4th day after sowing, while WT seedlings began to germinate on the 6th day. These results suggest that overexpression of Fm4CL-like 1 significantly increased osmotic stress tolerance in tobacco.
Fm4CL-like 1 affects the accumulation of reactive oxygen species (ROS), biosynthesis of MDA, and relative conductivity
ROS is a plant signaling molecule. Under drought stress, plant cells will produce a large amount of ROS resulting in inhibition of plant growth [39]. Therefore, we investigated whether Fm4CL-like 1 could affect ROS accumulation. DAB staining was used to assess the level of H2O2 (a major ROS) (Fig. 7a). No significant difference in DAB staining was observed between WT and OE lines under control conditions. However, OE showed reduced DAB staining compared to WT plants after 3rd d of treatment with 200 mM Mannitol, indicating that Fm4CL-like 1 overexpression reduced H2O2 accumulation in plants. We further measured the H2O2 level (Fig. 7d). Consistent with DAB staining, there was no difference between WT and OE under control conditions. However, with 200 mM mannitol treatment, H2O2 levels were 22% higher in WT than OE. These results show that Fm4CL-like 1 can affect ROS accumulation.
Due to the obvious differences in H2O2 levels in WT and OE lines after mannitol treatment, the activities of POD and SOD were measured in mannitol-treated tobacco. The results showed that the POD and SOD activity in the OE line was higher by 35 and 24% than that in WT (Fig. 7b, c).
The study measured leaf cell conductivity and MDA content of plants on the 3rd d of growth under normal conditions and 200 mM mannitol treatment, respectively. Under control conditions, the MDA and relative conductivity of WT and OE samples were similar. However, under the mannitol treatment, the MDA level of the WT was 25% higher than that of OE (Fig. 7e). The relative conductivity of the WT was 15% higher than that of OE (Fig. 7f). These results indicate that WT tobacco is more susceptible to damage than transgenic tobacco in a mannitol-simulated arid environment.
Fm4CL-like 1 reduces water loss by reducing the stomatal apertures
Since OE is more resistant to osmotic stress than WT, we suspect that it may be also related to changes in the stomata. The leaf stomata of WT and OE plants were observed by light microscopy. The stomatal apertures of OE were 25% smaller than those of WT under control conditions. Under mannitol stress, the stomatal apertures of both WT and OE were reduced in size, while the stomatal apertures of OE were 30% smaller than those of WT (Fig. 8a-b). The photosynthetic rate of OE was 22% higher than that of WT under the control conditions. However, the photosynthetic rate of OE was 48% higher than that of WT under mannitol stress conditions (Fig. 8c). These results indicate that photosynthesis of plants can be affected by changes in the stomatal structure of the leaves. Since transgenic tobacco plants had a stronger drought tolerance than WT, we measured the water loss rates of WT and transgenic tobacco plants in vitro. The water loss rate of WT plants was 16.7% higher than that of the OE group (Fig. 8d). The higher water loss was detected when the detached leaves exposed to air, indicating that Fm4CL-like 1 overexpression reduced the plant transpiration rate.
Fm4CL-like 1 affect the expression of stress-related genes
From the previous determination of physiological indicators (e.g., ROS), five stress-related response genes were selected for the quantification of expression levels, to analyze the role of Fm4CL-like 1 in anti-retrograde pathway at the molecular level (Fig. 9). The results showed that the transcriptional expression of NtABF2, NTZFP, NTCAT, NtHAK1, and NTAPX genes were significantly different between WT and OE samples. Under normal growth conditions, the expression levels of NtABF2, NtCAT, NtAPX, and NtHAK1 in WT and OE were not significantly different, while the expression level of NtZFP in OE was 7 times higher than in WT. After mannitol treatment, these five genes were up regulated in WT and OE. The expression of NTHAK1, NTABF2, NTZFP, and NTAPX was at the peak after 1 h treatment. The expression level of NtCAT peaked 6 h after mannitol treatment. The expression levels of these five genes in OE after mannitol treatment were higher than those in WT. These results indicate that Fm4CL-like 1 can affect stress response gene expression.