Identification of chloroplast membrane-localized proteins potentially contributing to biological yield in B. napus
We previously detected SNPs significantly associated with biological yield during a GWAS of 520 B. napus accessions (Lu et al., 2016). We selected 6,627 candidate genes contained within the intervals surrounding 88 significant quantitative trait loci (QTLs) associated with biological yield trait. Of those, 29 encoded proteins with potential localization to the chloroplast membrane, as determined by GO analysis (Fig. 1A, Table S1, Table S2). We further narrowed down the list of candidates to potential transporters that may be involved in the export of photosynthetic products. Notably, we identified two genes, BnaA04g02480D and BnaA07g17240D, that were closely linked with the significant SNP Bn-A07-p12412116 (Lu et al., 2016) and encoded orthologous to the Arabidopsis membrane protein FAX1, known to mediate plastid fatty acid export.
We focused on the characterization of these two FAX1 orthologous genes. The FAX protein family consists of seven members in Arabidopsis, named AtFAX1–7 (Li et al., 2015). To identify potential FAX orthologues in field mustard (Brassica rapa), wild cabbage (Brassica oleracea) and B. napus, we performed BLAST searches, using the 7 Arabidopsis FAX protein sequences as queries, leading to the identification of 9 putative orthologs each in B. rapa (BraFAX) and B. oleracea (BolFAX), and 21 in B. napus (BnaFAX). The physicochemical characteristics (amino acid number, theoretical isoelectric point (pI) values, relative molecular weight and number of transmembrane domains) for BnaFAX proteins are listed in Table S3.
We generated an unrooted neighbor-joining phylogenetic tree based on the 46 protein sequences of FAX family members (Fig. 1B) and discovered that AtFAX1 and six putative BnaFAX1 members (BnaFAX1-1 to BnaFAX1-6) clustered into one branch. To further characterize the B. napus FAX family, we analyzed the chromosomal locations and gene structures of the encoding genes (Figure S1A, S1B) and predicted the conserved motifs of BnaFAX proteins using the MEME program (Figure S1C). Of the six B. napus FAX1 members within the same branch as AtFAX1, BnaFAX1-1 (BnaA07g17240D) and BnaFAX1-2 (BnaCnng07490D) were closest to AtFAX1, as evidenced by their very similar gene structures and conserved protein motifs, suggesting that BnaFAX1-1 and BnaFAX1-2 may share the same functions as AtFAX1.
To determine what effect, if any, the six AtFAX1-like genes had on seed oil content, we analyzed their transcript levels across various tissues in one cultivar with high seed oil contents (H, cultivar name: ZS11) and one with low seed oil content (L, cultivar name: ZY821). BnaFAX1-1 was more highly expressed in the H cultivar relative to the L cultivar in all tissues tested (Fig. 1C). By contrast, BnaFAX1-3 (BnaA04g02480D) was barely detectable in either B. napus cultivar. Besides, we also observed the expression levels of 6 members of BnaFAX1 in 6 tissues of a pair of high- and low- seed oil content accessions grown in Chongqing (CQ24, CQ45) and Yunnan (YN24, YN45), among which CQ24 (seed oil content about 43%) and YN24 (seed oil content about 45%) are high- seed oil content (H-SOC) accessions, and CQ45(seed oil content about35%) and YN45 (seed oil content about 37%) are low- seed oil content (L-SOC) accessions. The result is shown in Figure S2, the expression level of BnaFAX1-1 in H-SOC accessions (CQ24, YN24) is higher than that of L-SOC accessions (CQ45, YN45) in the stem (St), leaf (Le), silique pericarps and seeds on the main inflorescence of 30 days after flowering (30ZP and 30ZS, respectively) and on the primary branch (30CP and 30CS, respectively) (Figure S2A, S2C). This further confirms the conjecture that BnaFAX1-1 may contribute to the formation of seed high oil content in B. napus. Furthermore, to further determine whether BnaFAX1-1, BnaFAX1-2 are conducive to the formation of high biological yield (Fig. 1A), the seedling leaves of four pairs with extremely high- (P281, P542, P125, P257-HBY) and low- biological yield accessions (P319, P276, P131, P81-LBY) were selected for qRT-PCR analysis, and biological yield dry weight per plant for each accession is shown in Figure S2G. The qRT-PCR results showed that the expression levels of BnaFAX1-1 and BnaFAX1-2 in high- biological yield accessions were higher than those in low- biological yield accessions (Figure S2E, S2F), which is consistent with the GWAS analysis result (Fig. 1A). Overall, to further determine whether BnaFAX1-1 can increase both seed oil content and biological yield, we further characterized the function of BnaFAX1-1.
Subcellular localization and transcript levels of BnaFAX1-1 in B. napus
To determine the subcellular localization of BnaFAX1-1 in plant cells, we tagged BnaFAX1-1 with green fluorescent protein (GFP) and expressed the construct under the control of the constitutive Cauliflower mosaic virus (CaMV) 35S promoter (Fig. 2A). We transiently transfected Arabidopsis protoplasts with the BnaFAX1-1-GFP construct, using AtFAX1-GFP as a marker for chloroplast envelopes. We observed a ring of fluorescence at the periphery of chloroplasts, which is consistent with a plastid envelope localization for BnFAX1-1, as seen previously with AtFAX1 (Fig. 2B).
We next measured BnaFAX1-1 transcript levels of in seven tissues across five stages of development (roots, stems, leaves, flowers, buds, seeds, and silique pericarp after flowering 7, 14, 21, 30, 40 d). We observed the highest expression level for BnaFAX1-1 in leaves and seeds after 40 days of flowering (Fig. 2C). We had previously determined that AtFAX1 was mainly expressed in leaves, but not in seeds (Li et al., 2015). This result suggested that BnaFAX1-1 function may differ from that of AtFAX1, which did not contribute to seed oil accumulation in Arabidopsis.
Overexpression of BnaFAX1-1 in Arabidopsis and B. napus
Next, we generated BnaFAX1-1 overexpression constructs and transformed both Arabidopsis (Col-0 accession) and B. napus (Westar cultivar). We validated overexpression lines (OE) by RT-qPCR using total RNA extracted from B. napus and Arabidopsis transgenic individuals with BnaFAX1-1-specific primers (Table S4). We selected four homozygous BnaFAX1-1 overexpressing lines in Arabidopsis, named OE/At#1, OE/At#2, OE/At#3, and OE/At#4 (Fig. 3A). Similarly, we obtained four BnaFAX1-1 overexpressing lines in B. napus, named OE#17, OE#19, OE#20 and OE#21 (Fig. 4A).
Increased biological yield and seed oil production by BnaFAX1-1 overexpression in Arabidopsis
Next, we phenotyped Arabidopsis lines overexpressing BnaFAX1-1 and compared their growth to wild-type (WT) plants. We noticed that all Arabidopsis BnaFAX1-1 overexpressing lines were slightly larger and produced more biomass than WT plants (Fig. 3B, Table 1). After reaching reproductive maturity, overexpression lines were significantly bigger than the WT, with thicker inflorescence stalks and more siliques (Fig. 3C, Table 1). A detailed analysis of different tissues and organs in transgenic and WT plants grown for 7 weeks revealed that plant height, rosette fresh and dry weight, fresh and the dry weight of biological yield was significantly higher in overexpressing lines relative to WT plants (Table 1). Likewise, stem fresh weight and stem diameter in the transgenic lines were significantly increased compared to WT plants. Furthermore, we observed an increase in seed yield per plant in overexpression lines, largely due to an increase in silique number per plant (Table 1). We also determined the total lipid contentof mature seeds, which indicated that overexpression lines accumulated more total lipid content relative to the WT (Fig. 3D). Collectively, these results indicate that the heterologous overexpression of BnaFAX1-1 in Arabidopsis promoted plant growth and development, and led to an increase in seed oil production.
Table 1
Plant biomass and seed yield in Arabidopsis Col-0 plants and Arabidopsis lines overexpressing BnaFAX1-1.
Traits | WT | OE/At#1 | OE/At#2 | OE/At#3 | OE/At#4 |
Stem fresh weight (mg/cm) | 12.57 ± 0.79 | 12.63 ± 2.03 | 15.31 ± 2.00** | 15.9 ± 1.28** | 15.86 ± 2.66** |
Stem diameter (mm) | 0.97 ± 0.11 | 1.04 ± 0.08 | 1.16 ± 0.99** | 1.23 ± 0.14** | 1.21 ± 0.08** |
Plant height (cm) | 32.6 ± 3.37 | 39.74 ± 2.21** | 38.9 ± 1.13** | 37.75 ± 2.5** | 37.49 ± 2.37** |
Rosette fresh weight (g) | 1.09 ± 0.14 | 1.28 ± 0.17* | 1.43 ± 0.3* | 1.58 ± 0.36** | 1.44 ± 0.32* |
Rosette dry weight (g) | 0.17 ± 0.02 | 0.18 ± 0.02 | 0.22 ± 0.02** | 0.23 ± 0.05** | 0.23 ± 0.04** |
Fresh weight of BYAG (g) | 2.91 ± 0.24 | 4.44 ± 0.48** | 4.20 ± 0.45** | 4.85 ± 0.57** | 4.31 ± 0.43** |
Dry weight of BYAG (g) | 0.45 ± 0.05 | 0.65 ± 0.07** | 0.68 ± 0.04** | 0.76 ± 0.10** | 0.65 ± 0.14** |
Length of siliques (cm) | 1.6 ± 0.09 | 1.57 ± 0.04 | 1.62 ± 0.09 | 1.67 ± 0.07 | 1.59 ± 0.1 |
Number of siliques per plant | 349.7 ± 24.18 | 408.7 ± 32.1** | 416.4 ± 39.05** | 501.9 ± 45.95** | 412.75 ± 57.54* |
Number of seeds per silique | 61.06 ± 6.24 | 64.33 ± 3.56 | 60.78 ± 4.93 | 63.33 ± 5.69 | 61.94 ± 6.63 |
Weight of per 1000 seeds (mg) | 19.15 ± 0.28 | 18.37 ± 0.68 | 18.58 ± 0.11 | 19.12 ± 0.12 | 19.36 ± 0.58 |
Seed yield per plant (mg) | 118.5 ± 34 | 179.8 ± 45** | 174.9 ± 54* | 241.7 ± 51** | 172.0 ± 42* |
*P < 0.05, **P < 0.01, Student’s t-test (n = 6–10 ± SD). BYAG: biological yield of above ground organs.
Increased biological yield, gibberellin and leaf lipid contents in B. napus plants overexpressing BnaFAX1-1
To test the effect of BnaFAX1-1 overexpression in B. napus on biomass accumulation, we analyzed the growth kinetics of three independent BnaFAX1-1 overexpression lines selected at random (OE#17, OE#19 and OE#21) (Fig. 4A). We grew all plants hydroponically in Hoagland nutrient solution for 32 d. All BnaFAX1-1 overexpression lines were larger and produced more biomass than their non-transgenic WT control (Fig. 4B). This increase in leaf biomass was reflected in all phenotypes measured: leaf fresh /dry weight and leaf size (including leaf length, leaf width and leaf area; Fig. 4C, 4D). Compared to the WT, overexpression of BnFAX1-1 also resulted in a significant increase in total root length, root area, root volume, root fresh and dry weight (Fig. 4D). Overexpression of BnaFAX1-1 in B. napus therefore promoted plant growth.
Toward the identification of the potential mechanism linking FAX1 and B. napus growth and biomass improvements, we quantified phytohormone contents in the leaves of two transgenic lines (OE#19 and OE#21) and their WT using liquid chromatography followed by tandem mass spectrometry (LC-MS/MS). We observed that gibberellic acid A4 (GA4) accumulated to significantly higher levels in transgenic leaves relative to the WT (Fig. 5A). By contrast, the contents of indole-3-acetic acid (IAA), salicylic acid (SA), and jasmonic acid (JA) were similar in the overexpression lines and the WT (Figure S3). GA4 is a bioactive gibberellin that plays critical roles in plant growth and development (Eriksson et al., 2000). To explore the reason behind the increase in GA4 content in the transgenic lines, we performed transcriptome sequencing from leaves of the transgenic lines (OE#19 and OE#21) and WT. We discovered that the GA4 biosynthetic genes COPALYL DIPHOSPHATE SYNTHASE (CPS), KAURENOIC ACID OXIDASES (KAOs) and GA20 OXIDASE (GA20OX) were more highly expressed in the transgenic lines relative to the WT (Fig. 5B). We validated these results by RT-qPCR (Fig. 5C). These results indicate that overexpression of BnaFAX1-1 led to up-regulated GA4 biosynthesis, which may in turn contribute to biological yield increase in B. napus.
To investigate the consequence of BnaFAX1-1 accumulation in the two selected overexpression lines above on membrane lipid contents, we analyzed lipids from 32-d-old leaves using LC-MS/MS (Fig. 6). We observed a higher lipid content for phosphatidylcholine (PC) and phosphatidylethanolamine (PE) in the leaves of OE#19 and OE#21 plants when compared to WT (Fig. 6). These results revealed that BnaFAX1-1 share the same role as AtFAX1 when ectopically expressed in leaves for the regulation of leaf lipid and biomass accumulation.
BnaFAX1-1 enhances biological yield and seed yield of B. napus plants grown in the field
To determine if the phenotypes seen in BnaFAX1-1 overexpression lines extended to the field, we sowed seeds for WT and OE lines in a randomized field plot design using four plots for each OE line and WT. We investigated growth characteristics in plants at flowering stage (grown for 175 d). OE plants were clearly bigger and taller compared to WT, and had produced more leaves on the main stem in the same growth period (Fig. 7A, 7C). In addition, OE plants showed larger leaves at the same position relative to WT plants, which was reflected in increased leaf length, leaf width and leaf area (Fig. 7B, 7C). Lastly, OE lines produced thicker main stems than WT plants; only chlorophyll content and the photosynthetic rate of OE lines were similar to those of the WT (Fig. 7C).
We next harvested mature plants from the field to carry out additional measurements. The BnaFAX1-1 OE lines were significantly taller than WT plants and bore more effective branches (i.e. branches bearing seeds) per plant (Fig. 8A, 8D-c). Although we observed no differences in the length of the main inflorescence between the WT and transgenic plants, the OE lines did exhibit more siliques per main inflorescence than in WT plants (Fig. 8B). Similarly, total silique number was significantly greater in all OE lines relative to WT (Fig. 8D-f), as were silique length (Fig. 8C) and number of seeds per silique. Together, these results revealed the greater seed yield per plant and biological yield in all OE plants (Fig. 8D), possibly by increasing the number of effective branches and siliques per plant. We did not observe such phenotypes when we overexpressed AtFAX1 in Arabidopsis in our previous work (Li et al., 2015). Therefore, BnaFAX1-1, unlike AtFAX1, may play a vital role in improving seed yield and biological yield in B. napus.
BnaFAX1-1 enhances B. napus seed oil production and improves oil quality
We examined total lipid content in seeds from OE lines and WT at 30 d and 45 d after flowering, as well as in dry seeds following harvest. Overexpression of BnFAX1-1 in B. napus resulted in an increase in total seed lipid contents at all development stages tested relative to WT (Fig. 9A). We also determined the range of TAG molecular species and total TAG content in BnaFAX1-1 OE lines and WT mature dry seeds. We saw a significant rise in the content of many TAG molecular species (TAG 50:2, 50:3, 52:2, 54:2, 54:3, 54:4, 54:5, 56:2, 56:4) and total TAG content in BnaFAX1-1 OE transgenic plants compared to WT (Fig. 9B, C). In addition, an analysis of fatty acid composition of mature dry seeds grown in the field revealed that oleic acid (C18:1) was significantly increased in the transgenic lines, whereas palmitic acid (C16:0), arachidic acid (C20:0) and eicosenic cis (C20:1) were significantly reduced. Stearic acid (C18:0), linoleic acid (C18:2) and linolenic acid (C18:3) contents were similar in the OE lines and WT (Fig. 9D). These results demonstrated that overexpression of BnaFAX1-1 effectively increased seed oil production and oleic acid content. These results therefore revealed that BnaFAX1-1 may have important application value in B. napus molecular breeding to improve seed oil content, oil quality, seed yield and biological yield.