cDNA isolation and sequence analysis of CbFAD7 and CbFAD8from C. bungeana
Based on the nucleotide sequences of known plastidial ω-3 FADgenes, two pairs of degenerate primers were designed and used for getting conserved sequences. After RACE PCR reactions and full length verification, two full length cDNA of 1805 and 1563 bp were obtained and designated as CbFAD7 (KY069282) and CbFAD8 (KY069283), respectively. CbFAD7 contains an ORF encoding a predicted protein (CbFAD7) of 439 aa, which corresponds to a calculated molecular mass of 50.2 kDa and a pI of 7.89. The deduced CbFAD7 sequence displayed significant similarity to other known plant FAD7 sequences, exhibiting the highest identity (85%) to Brassica napus BnFAD7 (FJ985690). CbFAD8 contains an ORF encoding a predicted protein (CbFAD8) of 397 aa, which corresponds to a calculated molecular mass of 45.6 kDa and a pI of 8.92. The deduced CbFAD8 sequenceshowed remarkable similarity to other known plant FAD8 sequences and the highest identity (84%) to the Arabidopsis AtFAD8(NM120640). These data suggest that CbFAD7 and CbFAD8 cloned from C. bungeana encode the plastidial ω-3 FADs.
Analysis of the deduced CbFAD7 and CbFAD8 sequences with targeting prediction tools, showed the 50 and 60 aa N-terminal transit peptides with the characteristic features of chloroplast targeting peptide (Arrowed in Fig. 1a), respectively, predicting a subcellular localization in chloroplasts. Alignment of the two deduced protein sequences found there were three conserved histidine clusters (HDGCH, HXXXXXHRTHH and HHXXXXHVIHH, asterisked in Fig. 1a) and four transmembrane domains (TMD, underlined in Fig. 1a), suggesting that the predicted proteins of CbFAD7 and CbFAD8 are chloroplast membrane-bound ω-3 FADs.
To elucidate the phylogenetic relationship, the predicted proteins of CbFAD7, CbFAD8 and CbFAD3 were included in a dendrogram representing the known plant ω-3 FADs for comparison. CbFAD7 (ARL62096) and CbFAD8 (ARL62097) were positioned in the group corresponding to plastidial ω-3 FADs, whereas CbFAD3 (AKN35208) was grouped with those of microsomal location (Fig. 1b).
The functionality of CbFAD7 and CbFAD8 were verified in Arabidopsis mutant
To determine the functionality of CbFAD7 and CbFAD8, the ORF of the two genes were expressed in double fad7fad8 mutants under the CaMV 35S promoter of pBI121 vector, respectively. The fatty acids of leaf lipids showed that though the C18:3 contents in the complemented mutants F7 and F8 were still lower than that in WT plants, they were markedly higher than that in fad7fad8 mutants; moreover, the C18:3 content in F7 lines was much higher than that in F8 lines, which may indicate a stronger conversion effect of CbFAD7 on C18:3 (Fig. 2a). Being exposed to 15 ℃, the germination rates of F7 and F8 seeds were significantly higher than that of fad7fad8 mutant seeds, and close to that of WT seeds (Fig. 2b). These data confirmed that both CbFAD7 and CbFAD8 were functional plastical ω-3 FAD genes.
The tissue-specific and cold-responsive expressions of ω-3 CbFAD genes in C. bungeana
The expression profiles of plastical and microsomal ω-3 CbFAD genes were analyzed in the cell suspensions and the regenerated plants of C. bungeana. Both CbFAD7 and CbFAD8 were highly expressed in leaves and lowly expressed in roots (Fig. 3a), showing the tissue-specific expression parttern with the characteristic feature of plastidial ω-3 FAD genes. However, the expression level of CbFAD7 in suspension-cultured cells was just lower than that in leaves, while the expression level of CbFAD8 in cultured cells was as low as that in roots. CbFAD3 were highly expressed in non-green tissues, e.g. suspension-cultured cells and roots, and lowly expressed in stems and leaves (Fig 3a), showing the tissue-specific expression pattern with the characteristic feature of microsomal ω-3 FAD genes.
Given that microsomal and plastical ω-3 CbFAD genes mostly expressed in suspension-cultured cells and plant leaves, respectively, the cold-responsive expression of these genes were detected in the two tissues. In cell suspensions, the expression of CbFAD3 rapidly increased at 4 (6.3-fold) and 0 ℃ (5.7-fold), and decreased at -4 ℃ (Fig. 3b). Conversely, the expression of CbFAD8 was almost no change at 4 ℃, but sharply increased at 0 (13.1-fold) and -4 ℃ (27.7-fold) (Fig. 3b). In fact, the increases in CbFAD3 and CbFAD8 mRNA all peaked at being treated for 3 h, and were accompanied by a decrease in CbFAD7 mRNA at different low temperatures (Fig. 3b). Being compared with cell suspensions, the expression pattern of CbFAD3 and CbFAD7 in plant leaves interchanged, the former decreased at all tested low temperatures, the latter rapidly increased at 4 (4.1-fold) and 0 ℃ (2.6-fold) (Fig. 3b). Like that observed in cultured cells, the up-regulation of leaf CbFAD8 was only induced by severe low temperatures, with the increase of 3.4-fold at 0 ℃ and 6.7-fold at -4 ℃ (Fig. 3b). In this case, the cold-induced increase in CbFAD7 and CbFAD8 mRNA peaked at being treated for 3 and 6 h, respectively. On the whole, the expression of ω-3 CbFAD genes presented a non-redundant pattern in respond to different low temperatures.
The hormone- and inhibitor-responsive expressions of ω-3 CbFAD genes in C. bungeana
The expression of ω-3 CbFAD genes respond to various exogenous hormones were studied in the cell suspensions and the plant leaves of C. bungeana. In suspension-cultured cells, the expression of CbFAD3 was up-regulated by ABA, SA and BRs, but down-regulated by JA and GA3; among these regulations, the BRs-induced up-regulation (peaked at being treated for 3 h, 6.2-fold) was similar to those induced by chilling temperatures, and the JA-resulted down-regulation was in accord with that reduced by subzero temperatures (Fig. 3a and 4a). Although the expression of CbFAD7 was increased by ABA, SA and GA3, the decreased expression regulated by BRs and JA were consistent with the cold-reduced ones (Fig. 3a and 4a). The increase in CbFAD8 mRNA was induced by all tested hormones except GA3, however, only the JA- and the BRs-induced increases (9.3-fold and 8.1-fold, respectively) peaked at being treated for 3 h and resembled those induced by severe low temperatures (Fig. 3a and 4a). Unlike that be found in cell suspensions, the expression of CbFAD3 in leaves was down-regulated by all tested hormones with different dynamics, each of which approached the cold-reduced ones in varied degrees (Fig. 3b and 4b). Meanwhile, the expression of leaf CbFAD7 was increased by BRs, JA and GA3, but reduced by ABA; among these changes, the increases induced by BRs and GA3 (1.6-fold and 1.9-fold, respectively) peaked at being treated for 3 h and were like those induced by chilling temperatures (Fig. 3b and 4b). Similarly, the expression of leaf CbFAD8 was also induced by three hormones (ABA, BRs and JA) and reduced by one (GA3), however, only the increases induced by ABA and JA (4.3-fold and 3.6-fold, respectively) peaked at being treated for 6 h and resembled those induced by severe low temperatures (Fig. 3b and 4b). The Pearson correlations between the cold- and the hormone-responsive expressions of ω-3 CbFADgenes (Table 1 and 2) indecated that JA and BRs may participate in the cold-responsive expresssions of thesegenes in both cultured cells and plant leaves, while ABA and GA3 may only participate in the cold-responsive gene expressions in leaves.
To further confirm the participations of these hormones, the corresponding inhibitors were used. Data showed that in cell suspensions (Fig. 5a), the cold-induced expression of CbFAD3 was completely inhibited by Pcz (a synthetic inhibitor of BRs), but the inhibition could be partly eliminated by DIECA (a synthetic inhibitor of JA, 45.1%); the cold-reduced expression of CbFAD7 was mainly eliminated by DIECA (65.8%) and partially eliminated by Pcz (32.9%), but completely eliminated by the synergism of them; in contrast, the cold-induced expression of CbFAD8 gene was mostly inhibited by either DIECA (79.9%) or Pcz (78.9%), but completely inhibited by the cooperation of them. In plant leaves (Fig. 5b), the cold-reduced expression of CbFAD3 was completely eliminated by the coordination of Pcz, DIECA, Pac (a synthetic inhibitor of GA3) and Flu (a synthetic inhibitor of ABA), and the synergistic effect of Pcz and DIECA play a major role (79.0%); the cold-induced expression of CbFAD7 can be mainly inhibited by Pcz (66.0%) and partially inhibited by Pac (33.9%), but completely inhibited by the collaboration of them; likewise, the cold-induced expression of CbFAD8 can be mostly inhibited by DIECA (73.1%) and partially inhibited by Flu (29.9%), but completely inhibited by the combined effect of them.
All these results suggested that in C. bungeana cultured cells, the cold-responsive expression of CbFAD3 may be induced by BRs and reduced by JA; the synergism of JA and BRs may decrease CbFAD7 mRNA and increase CbFAD8 mRNA in response to low temperatures, however, the over-high concentration of BRs can in turn inhibit the expression of CbFAD8 (Fig. 3b, 6b and S2). In C. bungeana leaves, the cold-reduced expression of CbFAD3 was regulated by the cooperation of BRs, JA, GA3 and ABA; while the cold-induced expressions of CbFAD7 and CbFAD8 were regulated by the coordination of BRs and GA3 as well as JA and ABA, respectively.
The level of related phytohormones in C. bungeanaduring low-temperature treatments
To provide more evidence to the hormone-regulated cold response, the content changes of related phytohormones were detected in the cell suspensions and the plant leaves of C. bungeana at different low temperatures. Being treated at 0 ℃, the levels of all tested pyhtohormones presented a rapid and two-peaks increase in both tissues (Fig. 6a and 7a). In cultured cells, the level of BRs peaked at 1 (1.7-fold) and 3 h (1.5-fold), while that of JA peaked at 1.5 (2.6-fold) and 3 h (2.4-fold). In plant leaves, the content changes of BRs and JA were the same as or similar to those in cultured cells, except the peak times of JA increase (2 and 4 h) were a little later; at the same time, the level of GA3 peaked at 0.5 (1.8-fold) and 2.5 h (2.3-fold), and that of ABA peaked at 1 (1.6-fold) and 3 h (1.9-fold). Overall, the cold-induced hormone increases, especially the first peaks, were before the cold-responsive expressions of corresponding ω-3 CbFAD genes (Fig. 3b, 6a and 7a); furthermore, the content increases in synergistic pairs, such as JA and ABA, or BRs and GA3, presented a peak stagger trend to avoid redundant effect at the same temperature (Fig. 6a and 7a).
Actually, all tested low temperatures could induce the content increases in these phytohormones, but the increment of the same phytohormone was varied (Fig. 6b and 7b). In suspension-cultured cells (Fig. 6b), the level of BRs highly increased at 4 ℃ (2.6-fold) and slightly increased at 0 ℃ (1.7-fold); as for JA, the highest content increment was found at -4 ℃ (3.1-fold), and the increments at 4 and 0 ℃ were the same (2.6-fold). The cold-induced hormone increases in leaves were more regular than those in cultured cells (Fig. 7b): the content increment of BRs decreased as temperature decrease, conversely, that of JA increased; meanwhile, the level of GA3 showed the highest and the lowest increases at -4 (4.2-fold) and 0 ℃ (1.8-fold), respectively, while those of ABA presented in an opposite way (1.9-fold at 0 ℃ and 1.2-fold at -4 ℃). On the whole, the increment changes in these phytohormones, were consistent with the dynamic expression of corresponding ω-3 CbFAD genes at different low temperatures; furthermore, the content increment of antagonistic pairs, for example GA3 and ABA, showed a reverse tendency in response to temperature variation, which was due to the trade-offs between plant growth and cold stress response (Fig. 3b, 6b and 7b).
The level of TAs in C. bungeana during low-temperature and hormone-inhibitor treatments
Considering that the cold-responsive expressions of ω-3 CbFAD genes may affect the level of TAs, the C18:3 and the C16:3 contents of total lipids were detected in the cell suspensions and the plant leaves of C. bungeana under different low temperatures. Because of lacking chloroplast, no C16:3 was detected in suspension-cultured cells (Fig. 8a). Data showed that the level of C18:3 was obviously induced by all tested low temperatures and reached the maximum at being treated for 12 h in both tissues; the C18:3 content in cultured cells increased from about 20.6% to 46.2-55.0% of total fatty acids, and that in leaves increased from about 46.3% to 58.6-60.7% of total fatty acids (Fig. 8). Similarly, the C16:3 content of leaf lipids increased from about 2.8% to 4.3-5.2% of total fatty acids, and reached the maximum at being treated for 12 (4 and 0 ℃) or 24 h (-4 ℃) (Fig. 8b). In general, the marked increases in TAs were in accordance with but lagging behind the cold-responsive expressions of ω-3 CbFAD genes (Fig. 3 and 8).
To further confirm that the phytohormones affect the cold-induced level of TAs through regulating ω-3 CbFAD genes, the C18:3 and the C16:3 contents of total lipids were also detected in the cell suspensions and the plant leaves of C. bungeana under hormone-inhibitor treatments. In suspension-cultured cells, the cold-induced increase in C18:3 content was completely inhibited by the synergism of DIECA and Pcz, but partially inhibited by either of them (Fig. 8a); In plant leaves, the cold-induced increases in C18:3 and C16:3 contents were all completely inhibited by the synergistic effect of DIECA, Pcz, Pac and Flu, but partially inhibited by either of them (Fig. 8b). These results consisted with the inhibitor-responsive expressions of ω-3 CbFAD genes (Fig. 5 and 8), suggesting that with the help of ABA and GA3 in leaves, JA and BRs participated in maintaining appropriate level of TAs in C. bungeana through regulating these genes under low temperatures.