Overexpression of MdFRK2 increased cellulose level in transgenic apple
We generated MdFRK2-OE transgenic apple lines (OE-4 and OE-9) using the cauliflower mosaic virus 35S (CaMV 35S) promoter (Fig. 1a). Compared to wild-type (WT), MdFRK2 expression in the stems increased by 12.55- and 13.79-fold in lines OE-4 and OE-9, respectively (Fig. 1b). The activity of FRK was markedly increased in the OE lines (Fig. 1c). Cellulose contents of MdFRK2-OE transgenic apple stems were increased 0.12- and 0.15-fold, respectively, over WT (Fig. 1d). Light microscopy revealed that the MdFRK2-OE lines had significantly thicker cambium and primary phloem compared to WT (Fig. 1e). These results showed that MdFRK2 is a major player in cellulose synthesis in apple plants.
Alteration of soluble carbohydrate concentration in MdFRK2 transgenic apple
To decipher how changes in the level of soluble carbohydrates affects cellulose synthesis with MdFRK2 overexpression, the Fru concentrations in OE-4 and OE-9 transgenic lines were measured. Fru levels decreased by 15.00% in OE-4 and 18.28% in OE-9 (Fig. 2). It is worth mentioning here that the Suc concentration in the OE-4 transgenic line decreased significantly, by 7.50%, while Glc concentrations significantly decreased by 18.68% and 15.26%, respectively (Fig. 2). These results indicated that the links between MdFRK2 and cellulose synthesis might be related to sugar metabolism in the sink.
Heterologous expression of MdFRK2 in poplar plants
To further understand the cellulose synthesis-related functions of MdFRK2 in stems, we heterologously expressed it in poplar (Populus clone 717). Poplar is rich in cellulose and is an important source material for everyday products such as cloth, paper and biofuels [25]. Analysis of the DNA and mRNA levels revealed that three heterologous lines (OE#1, OE#4 and OE#9) (Fig. 3a) exhibited increases in MdFRK2 transcript levels in stems relative to the levels in WT controls (Fig. 3b-c). All three lines also exhibited significantly increased enzyme activity. Interestingly, the FRK activity of mature leaves did not differ between the transgenic and wild-type plants (Additional file 1: Fig. S1). Lines OE#1, OE#4 and OE#9 displayed 0.70, 0.57 and 0.49-fold increases, respectively, in FRK activity in the stems, relative to the levels of the untransformed WT controls (Fig. 3d). Regardless of these observed differences, there were no significant changes in stem height and diameter in transgenic lines (Additional file 3: Table S1). These data showed that apple FRK2 functioned very specifically in sugar metabolism in poplar.
Heterologous expression of MdFRK2 altered soluble sugar concentration of poplar plants
To examine the affect of overexpression of MdFRK2 on carbohydrate metabolism in poplar, we determined sugar concentrations in transgenic lines using gas chromatography mass spectrometry GC/MS (Fig. 4). The high MdFRK2 transcript levels in stems resulted in lower concentrations of Fru in the three transgenic lines, with levels reduced to 13.87% of the control level for OE#1, 15.76% for OE#4 and 15.80% for OE#9. The concentrations of Suc in these three transgenic lines were decreased by 17.65%, 15.16% and 15.84%, respectively. In the transgenic lines, the concentration of Glc was also decreased. These data demonstrated that MdFRK2 modulate sucrose and hexose metabolism in a heterologous species.
Carbohydrate metabolism pathway in stems of transgenic poplar
To determine why the sugar concentrations changed in the transgenic poplar lines expressing MdFRK2, enzyme activity and expression of genes related to sugar metabolism were assessed (Fig. 5a). The activity of cell wall invertase (CWINV) was not statistically different between the wild-type and transgenic lines. However, the activities of neutral invertase (NINV) and sucrose synthase (SUSY), both of which are related to Suc breakdown, were significantly increased (Additional file 2: Figure S2). A similar pattern was observed for HxK and FRK activities. These results showed that the breakdown of Suc and Fru in sink cells is through the activities of FRK and SUSY, respectively. The changes in Suc and Fru were similar to the pattern of enzyme activities involved in Suc and Fru metabolism.
Additionally, the transcript abundance of genes encoding these enzymes were investigated (Fig. 5b). Gene involved in Suc degradation (PtrSUSY1) was significantly upregulated in the transgenic poplar. In accord with the reduced Glc concentrations (Fig. 4), Transcripts for PtrHxK1, were increased. However, the expression level of PtrCWINV2 was unchanged relative to control levels in transgenic lines. Taken together, these findings further suggested that the decreased Suc and Fru concentrations in MdFRK2-transgenic Populus were due to increased cleavage of Suc and Fru into hexose phosphates via the increased SUSY and FRK activities and transcript levels of PtrSUSY1 and MdFRK2, respectively.
Heterologous expression of MdFRK2 accelerated UDPG accumulation of poplar plants
SUSY directly produces UDPG, which is the substrate for cellulose synthesis in sink organs. In order to confirm that alteration of UDPG in the transgenic lines was caused by increased SUSY or FRK activity, the UDPG concentration was detected (Fig. 6). In poplar overexpressing MdFRK2, the concentrations of UDPG were significantly increased, by 0.95-, 0.70- and 0.69-fold, respectively. Furthermore, the levels of fructose 6-phosphate (F6P), glucose 6-phsophate (G6P) and glucose 1-phsophate (G1P) were increased greatly in all three OE lines relative to the levels in WT. These results suggested that MdFRK2 overexpression could accelerate UDPG accumulation and that the capacity for cellulose synthesis via UDPG would be increased.
Heterologous expression of MdFRK2 accelerated cellulose accumulation in Populus plants
After seeing increased UDPG levels in stems of these three transgenic lines with up-regulated MdFRK2 expression, the cellulose content of all transgenic lines were measured (Fig. 7a). The contents of cellulose increased 0.49-, 0.21- and 0.24-fold in the three transgenic lines as compared with WT, respectively. Accordingly, the three transgenic poplar lines contained increased hemicellulose contents, by 6.90% in OE#1, 15.64% in OE#4 and 14.84% in OE#9 (Fig. 7b). The pectin contents in the transgenic poplar stems decreased 0.22-, 0.19- and 0.15-fold compared to WT (Fig. 7c), whereas the lignin content showed no significant changes (Fig. 7d).
To further identify key genes contributing to changes in cellulose levels in the transgenic lines, the expression levels of genes related to these traits were measured using qRT-PCR. The results suggested that the expression levels of genes involved in the biosynthesis of cellulose (PtrCesA3 and PtrCesA6), were highly up-regulated in OE#1, OE#4 and OE#9 transgenic lines compared to in wild-type (Fig. 7e). However, the expression levels of hemicellulose (PtrFRA8 and PtrIRX9) and lignin biosynthetic genes (PtrPAL1 and PtrC4H1) did not obviously differ. These results demonstrated that cellulose is a major sink for FRK2-metabolized carbon.
Heterologous expression of MdFRK2 changed primary phloem in Populus plants
As reported previously [26], the cambium is a meristem between xylem and phloem, which will further differentiate outward to form primary phloem. To test whether the increased cellulose level altered the primary phloem of transgenic lines, double staining with safranin and fast green were used to stain cellulose and lignin, respectively. Examination by light microscopy revealed that OE#1, OE#4 and OE#9 transgenic lines had significantly thicker primary phloem (Fig. 8a-d), which was increased by 8.33%, 10.96% and 12.94% compared to wild-type, respectively (Fig. 8e). These results indicated that cellulose might be major contributors to phloem development.