The chlorophyll content and structure can be regulated by long-chain acyl-CoA synthetase, BnLACS9, by stimulating triacylglycerol metabolism in Brassica napus

doi:10.21203/rs.3.rs-1759226/v1

Download PDF

Research Article

The chlorophyll content and structure can be regulated by long-chain acyl-CoA synthetase, BnLACS9, by stimulating triacylglycerol metabolism in Brassica napus

https://doi.org/10.21203/rs.3.rs-1759226/v1

This work is licensed under a CC BY 4.0 License

Journal Publication

published 06 Jan, 2023

Read the published version in Biotechnology for Biofuels and Bioproducts →

You are reading this latest preprint version

Background

Chlorophyll is a very important pigment involved in photosynthesis, while plant Acyl-CoA biosynthesis is derived from plastid-localized fatty acids (FAs). Until now, the regulation of Acyl-CoA pathway for chlorophyll biosynthesis is still unknown.

Results

Here, we identified a Long-chain Acyl-CoA Synthetase (LACS) gene BnLACS9 from Brassica napus. BnLACS9 complemented a LACS-deficient yeast strain YB525, which indicated BnLACS9 have the LACS function. BnLACS9 was localized in chloroplast outer envelope (OE), while mainly expressed in young leaves and flowers. Overexpression of BnLACS9 in Nicotiana benthamiana lead to the contents of total CoA and MGDG increased significantly. In B. napus with overexpression of BnLACS9, the number of the chloroplast grana lamellae and the content of chlorophyll, as well as the MGDG and DGDG contents, were all increased compared to wide-type. The net photosynthetic rate, dry weight of the whole plant and oil content of seeds were significantly increased accompanied by the increased of chlorophyll content. Transcriptome analysis revealed that overexpression of BnLACS9 upgraded the pathway of acyl-CoA biosynthesis and further upgraded the enzymes in glycolipids synthesis pathway, while acyl-CoA was the substrate for glycolipids synthesis. The increased glycolipids, especially MGDG and DGDG, accelerated the formation of the chloroplast grana lamellae, which increased the number of chloroplast thylakoid grana lamella and further leading to increased chlorophyll content.

Conclusion

In the present study, we demonstrated that BnLACS9 played a crutial role in glycolipids and chlorophyll biosynthesis in B. napus. The result also provides a new direction and basis theoretical for the improvement of agronomic traits of plants.

BnLACS9

chloroplast

chlorophyll

galactolipid

glycolipid

Long-chain acyl-coA synthetases (LACSs) play a significant role in lipids formation and are associated with oil content accumulation in plants [1–5]. In plant, the LACSs gene family encodes long-chain acyl-coenzyme, which involved in the fatty acids (FAs) metabolism, activates free FAs and esterifies free FAs to acyl-CoAs [6, 7].

In Arabidopsis, nine genes (AtLACS1, AtLACS2, AtLACS3, AtLACS4, AtLACS5, AtLACS6, AtLACS7, AtLACS8, AtLACS9) that encode LACSs have been identified [6, 8]. Amino acid sequence analysis find they all have one very highly conserved motif, called AMP-binding protein (AMPBP) superfamily [9], which LACS belongs. Seven of the nine can complement a LACS-deficient strain of yeast (Saccharomyces cerevisiae) and successfully restored the growth of LACS-deficient yeast except AtLACS6, AtLACS7 [6]. AtLACS1 and AtLACS2 was located in ER (Endoplasmic Reticulum) [10], and the AtLACS1 have been found that has overlapping functions with AtLACS2 in plant wax and cutin synthesis in Arabidopsis thaliana [11]. Dirk Jessen found that AtLACS1 and AtLACS4 play an important roles in proper pollen coat formation in Arabidopsis [12]. Expression of AtLACS1, AtLACS2, and AtLACS3 enzyme in yeast also resulted in uptake of the long-chain fatty acid [13], whereas AtLACS6 and AtLACS7 play an important roles in activating FAs for β-oxidation in the peroxisome [14]. The AtLACS9 is known to reside in the chloroplast, a major contributor to chloroplastic LACS activity, involved in plastidial FA export for TAG formation [15]. Direct assays demonstrated that AtLACS1 enzyme functionally overlaps with AtLACS9 in Arabidopsis seed oil biosynthesis [16].

The chloroplast, the site for chlorophyll biosynthesis was formed with membranes which was filled with lipids. It completely development plays a crucial roles in the biosynthesis of chlorophyll [17]. The chloroplast membrane was mainly comprised by monogalactosyldiacylglycerol (MGDG), digalactosyldiacylglycerol (DGDG), phospholipid phosphatidylglycerol (PG), and sulfolipid sulfoquinovosyldiacylglycerol (SQDG), which are crucial to the development of chloroplasts [18]. On one hand, most of the different functional membrane lipids of the chloroplast are assembled in the chloroplast envelope membranes. On the other hand, some enzymes in the pathway of chlorophyll biogenesis are chloroplast membrane-related proteins. In particular, the galactolipids, accounting for 84% of the lipids of thylakoid membrane and the inner chloroplast membrane which have a major impact to the development of the chloroplast and the synthesis of chlorophyll [19–22]. The thylakoid membrane is more or less exclusively composed of these lipids. The galactolipids provides a membrane scaffold for photosynthetic pigment-protein complexes [19, 20]. In addition, galactolipids is also directly involved in photosynthesis. MGDG which was belonged to galactolipids is required for energy coupling between light-harvesting chlorophyll complex II (LHCII) and photosystem II (PSII) core complexes [23]. In Arabidopsis thaliana, loss galactolipids mutant had no visible green tissues under Pi-sufficient conditions and accumulated chlorophyll to barely detectable levels [24].

In plants, there are two kinds of galactolipids, the prokaryotic galactolipids and the eukaryotic galactolipids, which hold each half of the total galactolipids of the chloroplast membranes [18]. The prokaryotic galactolipids used the acyl-ACP as the initial substrate, the acyl was transfer to the G-3-P under the acyltransferase ATS1 and ATS2 to form the PA and DAG [18]. In Arabidopsis, ATS1 and ATS2 knockout mutants showed lack of the prokaryotic galactolipids and the plant undergrown [25, 26]. Final structure of PA formed in the chloroplast envelope is then C18:1- sn-1, C16:0- sn-2. This is typically a prokaryotic structure because of the exclusive C16:0 at sn-2 position of glycerol. The eukaryotic galactolipids used the acyl-CoA as the initial substrate which was catalyzed by LACS. The acyl was transfer to the G-3-P to form the PA and DAG with C18 FAs at the sn-2 position of glycerol, which about half of the chloroplast galactolipids in Arabidopsis. This structure is the consequence of the ER acyltransferase specificity for acyl-CoA during formation of PA and DAG. Two acyltransferase LPAT2 and LPAT3 have been identified in the endoplasmic reticulum [27]. The PA and DAG then was transferred to chloroplast and was involved in the synthesis of the MGDG, DGDG, PG and SQDG.

The synthesis of the MGDG was catalyzed by MGD1, MGD2 and MGD3 [28]. The final step of MGDG biosynthesis is catalyzed by MGDG synthase, which was occurred on the chloroplast. DGDG is subsequently synthesized by DGDG synthase used MGDG as the substrate [29]. Thus, MGDG synthase is the key enzyme for the biosynthesis of MGDG and DGDG, hence, for the biogenesis of chloroplast membranes [30]. Analysis of mutant caused by lack of MGDG and DGDG indicated that they are crucial for chloroplast biogenesis. Previous study have identified that the mgd1-2 mutant that is impaired in MGDG synthesis has a very low MGDG content and exhibits a complete loss of chlorophyll and photosynthetic activity [31]. Most chloroplast membrane glycerolipids are glycolipids which the major constituent is galactolipids. Mutations of galactolipids-deficient lead to severely hypoplasia of the chloroplast in Arabidopsis [32–34]. Therefore, the total content of the galactolipids in a plant tissue reflects the expansion of chloroplast membranes [35]. PG was synthesized by CDP-DAGS and PGP1 or PGP2 using the prokaryotic PA synthesized in the chloroplast envelope as the substrate. Many studies showed that lack of PG severely affected the chloroplast development and photosystem assembly [36, 37]. In MGD1 completely missing mutants of Arabidopsis thaliana, the content of MGDG and DGDG was severely reduced, the communication between chloroplast and thylakoid was hindered, the chlorophyll content was also reduced [31]. In Arabidopsis thaliana, DGD1 and DGD2 are involved in the formation of the DGDG [21, 38]. Double dgd1 and dgd2 mutants also have DGDG synthesis, because a new gene SFR2 was identified to be involved in the synthesis of DGDG [22]. PG was used as the chloroplast membrane lipid materials have the typical prokaryotic structure indicated that the substrate of the PG is the chloroplast PA. The PA from chloroplast and ER was catalyzed to form the CDP-DAG under the CDP-DAGS [27]. The CDP-DAG was then catalyzed by PGP1 and PGP2 to form PG [39, 40]. The synthesis of SQDG is under the action of genes SQD1 and SQD2 [41, 42].

The PA from chloroplast and ER was used to the synthesis of MGDG, DGDG, PG and SQDG. Under the condition of lack of DAG, the MGD1 showed no activity. But the MGD1 activity was restored by PA indicated the PA can regulate the activity of MGD1 [43]. It is a very effectively way to control the development of the chloroplast by regulate the contents of the MGDG which was catalyzed by the MGD1. As the precursor of MGDG synthesis, the content of PA was known as the regulatory factor of the activity of MGD1 [18]. In rice, overexpression of the PHD1 increased the content of UDP-Gal, the substrates of galactolipids, upgraded the formation of the galactolipids and led to increasing the number of the thylakoids and the contents of the chlorophyll [30]. The formation of MGDG was regulated by the substrate of UDP-Gal. The LACS gene family provided the initial substrates acyl-CoA for the synthesis of MGDG, have been shown taking contribution to the pathway of the lipid biogenesis, may play the function of regulating the synthesis of MGDG.

Chlorophyll is an important photosynthetic pigment in the chloroplast of plant, because photosynthesis is the most important event on the Earth, providing energy for all animals and humans surviving. Chlorophyll’s central role in photosynthesis is to harvest light energy and converse it to chemical energy [44]. Disruption of the pathway of chlorophyll synthesis will lead to the chloroplast dysfunction, photosynthetic inefficiency, the plant growth retardation reduced and even lead to plant death [45–47]. So, the pathway of chlorophyll biosynthesis attracted much attention. All land plants, green algae, and prochlorophytes possess chlorophyll a and chlorophyll b [48]. The biosynthesis of chlorophyll is complex and the whole process (L-glutamyl-tRNA→chlorophyll a→chlorophyll b) need 15-step reaction. In the model plant Arabidopsis thaliana, 27 genes encoding the enzymes involved in the pathway of Chl biosynthesis was isolated and cloned [49–51]. HEMA, CHLD, PROB and CAO play a key role in regulating the chlorophyll synthesis. Acyl-CoA, one of the initial substrate of MGDG, was formed by LACS. We hypothesized that LACS might be a positive regulatory factor for the synthesis of MGDG and chlorophyll.

In the present study, a comprehensive genome wide analysis of this gene family in Brassica napus was performed and 34 B. napus LACS genes (BnLACSs) were identified. Comparative expression analysis between extremely high-oil-content and low-oil-content B. napus cultivars revealed that BnLACS9 may be involved in FA synthesis in chloroplast. The BnLACS9 was isolated from developing rape embryos, its cDNA encoding a novel acyl-CoA synthase. Our results showed that over-expression of BnLACS9 in Nicotiana benthamiana can increase the content of galactolipids of the leaf accompanied by the content of the chlorophyll increased significantly. Overexpression of BnLACS9 in B. napus also caused the chlorophyll content was upgraded significantly compared with the wild-type. So, our data provide a new insight about the pathway of chlorophyll biosynthesis. The BnLACS9 can regulate the content of the chlorophyll by influence the chloroplast biosynthesis through regulated the chloroplast functional lipid biogenesis. The photosynthetic rate is directly related to the chlorophyll content of the plant, so we take a new way to increase the biomass of B. napus by enhance the expression of the BnLACS9 gene in some way.

BnLACS9 is highly homologous with AtLACS9

In Arabidopsis thaliana, nine long-chain acyl-CoA synthetases (LACSs) belong to a large superfamily of acyl-activating enzymes [6, 8], and are involved in FA transport. In order to search the homologous proteins in B. napus L., the amino acid sequences of AtLACS1-9 (AT2G47240, AT1G49430, AT1G64400, AT4G23850, AT4G11030, AT3G05970, AT5G27600, AT2G04350, AT1G77590) were used as the query probe to search in Brassica genome sequences databases (http://www.genoscope.cns.fr/blat-server/cgi-bin/colza/webBlat). In total, 29 significant homologous proteins, BnaA01g13470D, BnaA01g23830D, BnaA02g21860D, BnaA03g29320D, BnaA03g57930D, BnaA05g00630D, BnaA05g00640D, BnaA05g07920D, BnaA05g16170D, BnaA05g31340D, BnaA06g28680D, BnaA07g20920D, BnaA09g22150D, BnaA10g24190D, BnaC01g15670D, BnaC02g28920D, BnaC03g25940D, BnaC03g34500D, BnaC03g44430D, BnaC04g00320D, BnaC04g26780D, BnaC04g51420D, BnaC05g45860D, BnaC05g51350D, BnaC06g20910D, BnaC07g28060D, BnaC09g11030D, BnaC09g26090D, were gained. The phylogenetic tree analysis was performed based on the similarities of the conserved domain sequences among these proteins. As shown in Fig. 1A, except AtLACS3, other AtLACSs have 2 or 4 homologous proteins in B napus, because the B. napus is allopolyploidy, and it has a duplicate genome [52]. All LACS proteins could be divided into three branches, LACS1 to LACS5 formed the first branch, LACS6 and LACS7 formed the second branch, and LACS8 and LACS9 formed the third branch (Fig. 1A), showed that these LACS proteins might have different function. The phylogenetic tree analysis showed that AtLACS9, BnaA07g20920D (BnLACS9-A07) and BnaC06g20910D (BnLACS9-C06) were in one clade (Fig. 1A), and they share 91.6% and 91.9% similarity in amino acid sequence, respectively (Fig. 1B). AtLACS9, BnLACS9-A07 and BnLACS9-C06 are closely related in terms of amino acid sequence and evolutionary relationship, which imply they likely have similar functions. The similarity between BnLACS9-A07 and BnLACS9-C06 was as high as 99.7%, so we selected BnLACS9-C06 as the target gene in this study.

The Expression pattern of BnLACS9

To better understand the function of BnLACS9, we examined the expression pattern of BnLACS9 in various organs, including the root, stem, young leaf, old leaf, flower and silique of Ningyou 12 (NY12). We performed Quantitative real-time PCR (qPCR) analysis to estimate the level of the BnLACS9 transcript. As shown in Fig. 2, BnLACS9 is mainly expressed in young leaf and flower. We could detect the expression of the BnLACS9 gene in other organs, but lowest expression in old leaf. These results show that the BnLACS9 gene is temporally and spatially expressed.

Subcellular localization of BnLACS9

Localization studies were further performed on BnLACS9 to understand its mechanism of action. To ascertain the subcellular localization of BnLACS9, GFP was fused to the C terminus of BnLACS9 under the control of 35S promoter, and the fusion gene was transformed into N. benthamiana leaves and the cotyledons of B. napus, respectively. We found that the expression pattern of BnLACS9-GFP overlapped with the chlorophyll autofluorescence in N. benthamiana leaves and B. napus cotyledons (Fig. 3A-F), suggesting that BnLACS9 may be targeted to the chloroplasts. In order to further study the location of BnLACS9-GFP in chloroplasts, we expressed 35S:BnLACS9-GFP in N. benthamiana leaves for subsequent protoplast isolation and observed the N. benthamiana protoplasts by three-dimensional modeling. From the Fig. 3G-I, we observed circular structures around the chloroplast, and formed loop structures and thin tubules form the chloroplast. All these observations we could conclude that BnLACS9 may located on the chloroplast envelope.

BnLACS9 can complement LACS function in deficient yeast YB525

To determine whether BnLACS9 has LACS activity, a yeast vector pYES2-BnLACS9 was constructed and analyzed by yeast complementary expression system. Yeast strain YB525 is a strain with affected LACS activity [53], and this defective yeast can’t grow in the medium only using FA as the sole carbon source. Yeast complementarity test was used to determine the growth of yeast cells transferred into different vectors in drop-out medium with different FAs as the sole carbon source for 72 hours. The YB525 yeast cells transferred into pYES2 empty vector could not grow in drop-out medium with different FAs as the sole carbon source. While the YB525 yeast cells transferred into pYES2-BnLACS9 vector grow better and has different growth rates in drop-out medium with different FAs as the sole carbon source (Fig. 4). The OD value of YB525 yeast cells transferred into pYES2-BnLACS9 vector could reach to 0.38 in drop-out medium with only fatty acid C18:0 as the sole carbon source, while the OD value of yeast 0.05 in drop-out medium with only fatty acid C12:0 as the sole carbon source (Fig. 4). These results indicate that the heterologous expression of BnLACS9 gene can complement the deficient yeast and has the activity of LACS protease.

Transient expression of BnLACS9 in N. benthamiana leaves could enhance the content of FA, MGDG and total chlorophyll

In order to further study the function of BnLACS9 gene, BnLACS9 was transient expressed in N. benthamiana leaves. In plants, FAs are uniquely synthesized in plastids [54]. FA compositions from N. benthamiana leaves of both accessions were determined by gas chromatography-mass spectrometry (GC-MS). From Table 1, we could find that the transient expression of BnLACS9 gene in N. benthamiana leaves can increase the content of FAs, especially the C16:0 and C18:2, their contents were 221.3034 ± 14.0447 avfmol/mg and 59.2165 ± 9.703042 avfmol/mg, respectively. While the C16:0 and C18:2 are 134.9407 ± 7.264136 avfmol/mg and 23.56197 ± 3.008565 avfmol/mg in the control, respectively (Table 1). As a result, the total FA content in the transient expression of BnLACS9 gene in N. benthamiana leaves was 574.5768 ± 35.79813 avfmol/mg, while that of control was only 395.4662 ± 13.63986 avfmol/mg. These results indicated that BnLACS9 could increase the FA content in N. benthamiana leaves.

Table 1

The CoA content in *N. benthamiana* leaves
Acyl-CoA	P19 CoA content (avfmol/mg)	±SD		±SD
C2:0	6.61083	1.39379	7.1831	1.358259
C4:0	7.387154	6.789183	14.32233	0.97188
C6:0	8.322458	2.74276	6.547055	1.406957
C8:0	36.06325	6.266329	42.23341	5.334781
C10:0	0.647754	0.567659	0.869375	0.580957
C12:0	1.564231	0.299744	0.770367	0.516654
C14:0	10.88591	0.777906	12.78282	0.763995
C18:3	47.59071	6.161552	41.7954	6.390999
C16:1	4.173502	1.654766	7.576914	0.711642
C18:2	23.56197	3.008565	59.2165	9.703042
C16:0	134.9407	7.264136	221.6034	14.0447
C18:1	6.365882	0.315986	7.941865	1.109978
C20:2	0.967406	0.186929	1.215209	0.579532
C18:0	30.6479	0.65538	36.49893	1.843828
C20:1	24.87717	1.749918	45.81828	3.616426
C20:0	12.76891	0.180914	16.41325	0.587022
C22:1	26.27505	0.531116	38.57146	1.448839
C22:0	11.81541	0.246871	13.21715	0.53581
TOTAL	395.4662	13.63986	574.5768	35.79813

We also detected the chlorophyll content in N. benthamian leaves of transient expression of BnLACS9 and its control. We found that within 5 days after BnLACS9 gene was transferred into N. benthamiana leaves, there was no significant difference in chlorophyll content between the two groups. From the sixth day, the chlorophyll content of N. benthamiana leaves transfected with BnLACS9 gene was significantly higher than that of wild type, and reached the maximum on eighth day (Fig. 5A). MGDG is one of major component of chloroplast photosynthetic membrane [54]. We also detected the monogalactosyl-diaclyglycerol (MGDG) content in N. benthamian leaves between transient expression of BnLACS9 and its control. The content of MGDG in N. benthamiana leaves transfected with BnLACS9 gene was significantly higher than that of the control (Fig. 5B). All these results indicated that BnLACS9 could enhance the content of FA, MGDG and total chlorophyll.

Overexpression of BnLACS9 in B. napus could enhance the content of total chlorophyll

We found that the transient overexpression of BnLACS9 in N. benthamiana enhanced the content of FAs, MGDG and total chlorophyll (Fig. 5 and Table 1). To further address the function of BnLACS9 in rapeseed plant, transgenic rapeseed plants overexpressing BnLACS9, as well as those suppressing BnLACS9 (Bnlacs9), were generated (Fig. S1). Homozygous BnLACS9-overexpressed lines (#6, #12 and #18) were selected from the positive transgenic plants to evaluate the function of BnLACS9.

It was found that the cotyledons and leaves of BnLACS9-overexpressed plants were significantly greener than those of the wild type (Fig. 6A and B). Chlorophyll content of BnLACS9-overexpressed plants was also higher than that of wild type in 7-day-old cotyledons and 30-day-old leaves (Fig. 6C and D). We also found that the siliques of BnLACS9-overexpressed plants could delay senescence compared with control (Fig. 6E and F). So, we measured the chlorophyll content of the silique at 30 DAF and 50 DAF, and showed that the chlorophyll content in overexpressed plants was significantly higher than that in wild type plants (Fig. 6G and H). These results indicated that overexpression of BnLACS9 gene can increase chlorophyll content and delay senescence in B. napus.

Overexpression of BnLACS9 in B. napus could increase the number of thylakoid layer structure in chloroplast

The BnLACS9-overexpressed plants had higher chlorophyll content (Fig. 6), so we observed the chloroplast structure by transmission electron microscopy (TEM). Ultrastructural observation showed that the number of thylakoid layer structure in BnLACS9-overexpressed plants was more than that in wild type plants (Fig. 7). We also measured the photosynthetic efficiency of leaves of overexpressed plants and wild-type plants. We found that the photosynthetic efficiency of leaves of BnLACS9-overexpressed plants was significantly higher than that of wild-type plants (Fig. S2). These results indicated that overexpression of BnLACS9 gene could increase the number of chloroplast thylakoid layer structure, make leaves greener and improve photosynthetic efficiency.

In order to characterize the mechanism that BnLACS9 influenced the content of chlorophyll through regulating the genes in the pathway of the chlorophyll synthesis, we sequenced the transcriptome of the leaves of the overexpression of BnLACS9 plants and wide type. Transcriptome analysis showed 9 genes in chloroplast synthesis pathway (comp613754_c0, comp879850_c0, CHLD comp44567_c0, comp956830_c0, comp55669_c1, comp58218_c0, POR comp57696_c0, comp38830_c0, comp57073_c0) were up-regulated in BnLACS9-overexpressed plants (Fig. 8). We confirmed the transcript levels of genes HEMA, CHLD, PORB, CAO by RT-PCR (Fig. 8B), and showed that these genes were all up-regulated. The result suggested the BnLACS9 upgraded the key genes of chlorophyll synthesis pathway and further increased the content of the chlorophyll.

Overexpression of BnLACS9 in B. napus increased the content of the galactolipids

Our studies showed that the BnLACS9-overexpressed plants had more thylakoid layer structure than wild type plants (Fig. 7). It’s known that the main lipid component of the thylakoid layer structure is MGDG, DGDG, PG and SQDG. MGDG and DGDG account for approximately 84% of thylakoid membrane lipids [24]. PG is the main chloroplast phospholipid, represents about 8% of the chloroplast lipids. Another glycolipid, sulfoquinovosyldiacyl glycerol (SQDG), is present from 3–10% of the chloroplast membrane lipids [55]. Transient expression of BnLACS9 in N. benthamiana increased the synthesis of MGDG and chlorophyll. Overexpression of BnLACS9 also increased the content of the chlorophyll in B. napus (Fig. 9). In addition, the number of the thylakoid of the chloroplast was also increased.

Previous studies have shown that overexpression of BnLACS9 could improve the chlorophyll content and photosynthetic efficiency of leaves in rapeseed (Fig. 6 and S2). And overexpression of BnLACS9 in N. benthamiana leaves could increase TAG content (Table 1). We also found the transcript sequencing results of over-expressed BnLACS9 plants showed a significant increase in the expression of TAG synthesis pathway-related genes (Fig. 8). So, we measured the dry weight of the BnLACS9-overexpressed lines at 20 and 40 days after germination, and detected the seeds oil content of the transgenic plants by Nuclear Magnetic Resonance (NMR) and Near Infrared (NIR). At 20 days, we could see that the dry weights have been increased. At 40th day after germination, the dry weight of BnLACS9-overexpressed lines was particularly increased (Fig. 3S). As shown in Table 2, the oil content of BnLACS9-12 lines increased most obviously, reaching 45%, BnLACS9-6, BnLACS9-13 and BnLACS9-18 lines increased about 3%. In addition, the seed oil content of overexpressed BnLACS9 plants was determined by NIR, and the results also showed the same with NMR (Table 2). From the above, overexpression of BnLACS9 in plants could increase the biomass of the plant and increase the seed oil content in rapeseed.

Table 2

The content of seed oil of *BnLACS9* overexpressed lines is measured by NMR and NIR
line	measured by NMR (%)	measured by NIR (%)
NY12	39.70	38.99
BnLACS9-6	43.34	41.93
BnLACS9-12	45.64	44.97
BnLACS9-13	41.18	41.81
BnLACS9-17	41.42	39.25
BnLACS9-18	43.86	41.59
NMR: Nuclear magnetic resonance
NIR: Near-infrared

The BnLACS9 affected the biogenesis of the chlorophyll by regulating the chloroplast membrane lipid biosynthesis. In order to make a thorough inquiry about how the content of the glycolipid was increased in the overexpression of BnLACS9 plants and to elucidate the gene regulatory network of the pathway. Transcriptomes in the overexpression of BnLACS9 line and wild type were analyzed. The unigenes related to the glycolipid (Fig. 10A) synthesis were picked out from the transcriptomes data of the B. napus. The unigene was shown as ‘comp…_c0’. The expression level of the unigenes was represented by RPKM [56]. Nine unigenes for glycolipid biosynthesis were chosen for RT-PCR to confirm the expression level of the unigenes with RPKM (Fig. 10B). These unigenes were ATS1 (comp_775367c0), LPAAT (comp50451_c0), PP (comp30817_c0), PGPS (comp41948_c0), PGPP (comp56019_c0), MGDGS (comp55779_c0), DGDGS (comp56744_c0), SLS (comp52127_c0), CDP-DAGS (comp42015_c0), which the RPKM is the highest (Fig. 10). The diacylglycerol backbones LPA is assembled by the ATS1 [57], LPA in turn is then acylated by the lysophosphatidic acid acyltransferase (LPAAT) to phosphatidic acid (PA), PA is dephosphorylated to DAG by an inner-envelope localized enzyme PP [25]. CDP-DAGS, PGPS and PGPP are the genes involved in the PG synthesis [58, 59]. MGDGS and DGDGS are the enzymes responsible for the synthesis of MGDG and DGDG [29, 60]. The SLS takes charge of synthesis of SQDG [41, 61]. The result showed that ATS1, LPAAT, PP, PGPS, PGPP, PP, MGDGS, DGDGS, SLS, CDP-DAGS had higher expression in the overexpression of BnLACS9 plant than the wild type. In particular, the expression level of the ATS1 and SLS is four times higher more than the CK. Other genes in overexpression of BnLACS9 plant is exceeded double than the CK. Taken together, the results suggest that BnLACS9 regulated the expression of genes in the pathway of glycolipid biosynthesis. The higher expression level of these genes increased the content of the glycolipid.

In this study, we characterized the function of BnLACS9 in B. napus. As shown previously, most of the LACS genes have the ability to complement a LACS-deficient strain of yeast [6]. The BnLACS9 can complement the LACS-deficient yeast mutant YB525 and recover the growth rate of the YB525 (Fig. 4). Subcellular localization analysis indicated BnLACS9 might be located in chloroplast outer membrane of B. napus (Fig. 3). This result was similar to previous studies [62]. RT-PCR analysis showed that the expression of BnLACS9 was very low in the old leaves, but high in the flowers and young leaves (Fig. 2). The metabolism of FAs in flowers and young leaves is more active, the results suggest that BnLACS9 plays a certain role in the metabolism of FAs and the synthesis of glycolipids. Overexpression of BnLACS9 increased the content of the acyl-CoA, the content of the TAG and glycolipid in N. benthamiana leaves (Table 1). Interestingly, the content of the chlorophyll was also increased in the leaves of the transient expression in N. benthamiana leaves (Fig. 5). Overexpression of BnLACS9 in B. napus, the content of chlorophyll was also increased in the leaves (Fig. 6). Transcriptome analysis found that the BnLACS9 increasing the content of chlorophyll through regulating the pathway of glycolipid synthesis which is the main component of the chloroplast membrane and the thylakoid membrane (Fig. 8). BnLACS9 increased the expression level of the key enzymes in the chlorophyll synthesis.

In plant, activation of free FAs to acyl-CoA derivatives is necessary to provide the substrates for glycolipid biogenesis. The activation of CoA is catalyzed by LACSs. Chloroplast LACS activity is essential to glycolipid synthesis since chloroplasts are the main site of de novo FA synthesis [63]. The AtLACS9, which has been shown to reside in the plastid [39], is considered to be the major LACS isoform involved in the production of acyl-CoA for biosynthesis of membrane glycerolipids and storage TAGs [16]. The BnLACS9 was also shown to reside in the chloroplast outer membranes which transform the FA into acyl-CoA (Fig. 3). Overexpression of BnLACS9 stimulated much more FAs from chloroplast, which are further used for the synthesis of glycerolipids. In plants, the synthesis of glycolipid can be divide in chloroplast and endoplasmic reticulum: the ER-localized eukaryotic pathway and the inner envelope-localized prokaryotic pathway [64]. Plastid-derived DAG contains 16-carbon FAs in the sn-2 position, whereas ER-derived DAG contains 18-carbon FAs in the sn-2 position [19]. In Arabidopsis chloroplast, about one of three newly synthesized FAs staying in the chloroplast and are used to the synthesis of lipids via the prokaryotic pathway but the remaining two of three are exported to the eukaryotic pathway in the ER. During the process of the synthesis of glycolipids, the acyl-CoA as the principal substrate is regulated by the acyl-CoA synthase. The BnLACS9 is belonged to acyl-CoA synthase. Overexpression of BnLACS9 in N. benthamiana and B. napus increased the content of the acyl-CoA. Then the acyl-CoA was a positive regulatory factor as the substrate of lipids synthesis to upgrade the following steps of glycolipids biogenesis.

The main ingredient of the chloroplast membrane is lipids which are mostly composed of MGDG, DGDG, PG, and SQDG, especially the galactolipids MGDG and DGDG. The biosynthesis of MGDG and DGDG play an important role in the synthesis of thylakoid. A lipid bilayer for manufacture membranes enriched in galactolipids and PG is necessary for the extension of the envelope membranes and for the development of thylakoids. In the overexpression of BnLACS9 plants, the content of the galactolipids were increased which was used to participate in the synthesis of chloroplast membrane lipid. The expression level of MGD, CDP-DAGS, PGP1 and PGP2 was upgrade under the function of the BnLACS9. The increased galactolipids and PG result in increasing of the number of the thylakoid grana and chloroplast leading to the increased of the chlorophyll content.

The content of chlorophyll make a great difference with the gene of the chlorophyll synthesis, which the synthesis is mediated by more than 17 enzymes in higher plants [65]. In the overexpression BnLACS9 plants, the key genes in the pathway of chlorophyll biogenesis were significantly upgraded. The synthesis of ALA is the key step of the chlorophyll biogenesis which was regulated by the HEMA gene [65]. The steps of forming Mg-protoporphyrin IX catalyzed by protoporphyrin IX Mg-chelatase, which is coded by CHLD, is the second key regulation gene [66, 67]. The third regulation gene of the pathway is POR, encoding NADPH-protochlorophyllide oxidoreductase which is thylakoid membrane-associated protein. The POR makes it function in transforming the protochlorophyllide to chlorophyllide [43]. In addition, the enzymes of chlorophyllide a oxygenase was encoded by the CAO gene which catalyze the formation of chlorophyll, is the fourth key enzymes of chlorophyll biogenesis [68]. In the overexpression BnLACS9 plants, the expression level of the four key genes was also upgraded, which indicated the BnLACS9 may regulate them by means of signal molecule.

In general, BnLACS9 was located on the chloroplast envelope, which could promote the transport of Fatty Acyl-CoA from chloroplast to ER. In turn, the increased Fatty Acyl-CoA in ER can promote the content of galactolipids in chloroplasts, increase the number of thylakoid layer structure and improve the photosynthetic efficiency in chloroplast, and finally increase the synthesis of TAG (Fig. 11). Therefore, BnLACS9 could increase the oil content of rape seeds.

Plant materials and growth conditions

The seeds of WT rapeseed cultivar ‘Ningyou 12 (NY12)’ and ‘Zhongshuang 11 (ZS11)’ as well as N. benthamiana storing in our lab were sown in a soil mix (peat moss/perlite/vermiculite, 5/3/2, v/v/v) in flower pots (4–5 seedlings/pot) and grown in a plant growth room under the following growth conditions: 22 ± 2°C with a 16-h light: 8-h dark photoperiod at a light intensity of 5000 LX and 60% relative humidity.

Sequence analysis

The amino acid sequences of B. napus were retrieved through a database search using the amino acid sequences of Arabidopsis thaliana LACS proteins: AtLACS1 (AT2G47240), AtLACS2 (AT1G49430), AtLACS3 (AT1G64400), AtLACS4 (AT4G23850), AtLACS5 (AT4G11030), AtLACS6 (AT3G05970), AtLACS7 (AT5G27600), AtLACS8 (AT2G04350) and AtLACS9 (AT1G77590). These sequences were aligned by Clustal-W, and a neighbor-joining tree was built using MEGA version 4.0 [69]. The LACS protein sequences were analyzed using the MEME software [70]. Domains analyzes were performed in Pfam domains database (http://pfam.sanger.ac.uk/search/sequence).

RNA extraction, cDNA synthesis and Real-time PCR (RT-PCR)

Total RNA extractions from various tissues and reverse transcription were performed according to Wang et al. with some modifications [71]. For RT-PCR, 25 ng of cDNA was applied using corresponding gene-specific primer pairs. BnACTIN was amplified as the control. RT-PCR was performed on a cycler apparatus (Bio-Rad, USA) using the SYBR Green Master Mix (Vazyme, China) according to the manufacturer’s instructions. Amplification was conducted in 96-well optical reaction plates with the following protocol: 94℃ for 4 min, 40 cycles of 94℃ for 15 s, 58℃ for 15 s, and 72℃ for 15 s. The relative expression levels were estimated using the 2^−∆∆CT method of Livak and Schmittgen [72]. The RT-PCR was repeated with three biological replications. Primers used in the study are listed in Table S1.

For transcriptome analysis, one hundred milligrams of the leaf were used for RNA extraction. The purified RNA was detected and qualified using OneDrop OD-1000 + spectrophotometer (RockGene, Shanghai, China). Library construction was performed according to the standard protocol. The sequencing of the library was performed using the BGISeq-500 platform. The experiment was repeated three times with independent samples. The raw data were filtered to trim adaptor sequences and to remove low-quality sequences (Q < 20) with > 10% uncertain (N) bases. The NCBI database was used to annotate gene function. Differentially expressed genes (DEGs) were screened according to the NOIseq method [73]. Transcripts reach the criterion of log₂ fold change ≥ 1 (or ≤-1) and probability ≥ 0.8 were selected as DEGs.

Subcellular localization of BnLACS9 by transient expression in N. benthamiana leaves and B. napus cotyledons

To determine the expression location of the BnLACS9 protein, the full-length BnLACS9 cDNA was PCR amplified using the primers (BnLACS9-LF/BnLACS9-LR) with the cDNA as a template. The PCR product was cloned into the pENTR™ TOPO® vector (Invitrogen, USA), and the clone with correct sequencing was recombined into pK7WG2.0 by Gateway cloning (Invitrogen, USA) to produce GFP fusion proteins. The recombinant plasmid was transformed into the Agrobacterium tumefaciens strain GV3101. Overnight cultured A. tumefaciens strain GV3101 harboring the recombinant vectors were washed with sterile 10 mM MMA (10 mM MES, 10 mM MgCl2, and 100 mM acetosyringone, pH 5.6) and suspended with injection buffer MMA to the OD600 = 1.6. The GV3101 containing the construct 35S::p19 was also cultured and suspended with MMA to the OD600 = 1.6, which expression the Tomato bushy stunt virus p19 protein to suppress the gene silencing [74]. The two cultures were mixed 1:1 to the OD600 = 0.8, and followed by incubation at 28°C with shaking for 3 h. Agrobacterium infiltration into three- to four-week-old N. benthamiana leaves was performed as described previously [75, 76]. For B. napus, the culture mixture was injected into cotyledons at the fourth day after germination. Plant culture conditions is the same with the N. benthamiana culture conditions as follow: illumination weekly 12 h (light intensity 5000 LX, temperature 24°C, humidity 60%) and dark 12 h (temperature 22°C, humidity 60%). An important step is that the injected B. napus cotyledons must be maintained humidity in a film bag. The protoplasts from N. benthamiana leaves were performed as described previously [62]. Four to seven leaf disks with a diameter of 0.8 cm of transfected were cut with a cork borer and transferred into a 10 ml syringe containing 2 ml of cell wall digestion solution (CWDS: 1.5% cellulase R-10, 0.4% macerozyme R-10, 0.4 M mannitol, 20 mM KCl, 20 mM MES (pH 5.6), 10 mM CaCl2, 0.1% BSA). Then, Protoplasts and plant tissue were monitored by confocal microscopy (Leica TCS SP5, Wetzlar, Germany). The fluorescence emissions were at 510–540 nm for eGFP and 658–665 nm for chloroplast.

Expression of BnLACS9 cDNAs in Yeast

To generate the recombinant plasmids of pYES2-BnLACS9, the primers (BnLACS9-YF/BnLACS9-YR) were used to amplify the BnLACS9, then insert the sequenced BnLACS9 gene into the multiple cloning sites (BamH I/Kpn I) of pYES2 to obtained the recombinant plasmids of pYES2-BnLACS9. The recombinant plasmids were transformed into the competent E. coli. Then the recombinant plasmid DNA from the identified bacterial colonies was transformed into yeast YB525 cells (http://www.atcc.org/). The YB525 containing the recombinant plasmids of pYES2-BnLACS9 was identified and representative colonies were chosen and grown until mid- to late-log phase in dropout base and using long-chain FAs 12:0 lauric acid, 14:0 myristic acid, 16:0 palmitic acid, 18:0 stearic acid, 18:1 oleic acid and 22:1 erucic acid as a sole carbon source liquid medium. After 84 h at 30°C, the growth ratio was determined by Spectrophotometer at OD600.

Plant expression vector constructs and A. tumefaciens-mediated plant transformations

To construct the expression vector contained the BnLACS9, the full-length BnLACS9 cDNA was PCR amplified using the primers (BnLACS9-LF and BnLACS9-NR) with the cDNA as a template. The PCR product was cloned into the pENTR™ TOPO® vector (Invitrogen, USA), and the clone with correct sequencing was recombined into the vector pB2WG7.0 by gateway (Invitrogen, USA) to create the vector under the control of the constitutive Cauliflower mosaic virus (CaMV) 35S promoter. The expression vector contained the BnLACS9 was transformed into the A. tumefaciens strain GV3101. For transient expression in N. benthamiana leaves, Agrobacterium infiltration into three- to four-week-old N. benthamiana leaves was performed as described previously [75, 76].

To construct a vector for the constitutive expression of BnLACS9, a full-length BnLACS9 (BnLACS9-OF/BnLACS9-OR) cDNA was PCR amplified from its cDNA clone, and the CaMV 35S promoter (CaMV 35S-F/CaMV 35S-R) and CaMV Nos terminator (CaMV Nos-F/CaMV Nos-R) was generated from the vector pEGAD. Then, all three products were inserted into the EcoR I/Hind III sites of pCAMBIA1300, creating the pCAMBIA1300-35S-BnLACS9-NOS vector, which was then transformed into GV3101. To construct the vector to suppress BnLACS9 expression, a highly conserved 164-bp cDNA fragment of the 5′-open reading frame was amplified with primers Bnlacs9-F and Bnlacs9-R. This cDNA fragment was subcloned into the pENTR/D-TOPO vector, creating pENTR::Bnlacs9. Then, the pENTR::Bnlacs9 plasmid was transferred into the destination vector pHellsgate 12 to generate pHellsgate 12::Bnlacs9 using the Gateway LR recombinase (Invitrogen, USA). The plasmid pHellsgate 12::Bnlacs9 was transformed into GV3101. And the two vectors were transformed using an Agrobacterium-mediated transformation method as described by Wang et al. [77]. At least three independent transformants were examined as described in the results section. Hygromycin-resistant T2 generation plants were identified by PCR from the progeny of the primary transformants.

Ultrastructural observation of chloroplasts

The leaves of wild type and BnLACS9-overexpressed lines were cut into 2 mm segments and fixed in 2.5% glutaraldehyde solution. Then they were put into refrigerator at 4°C for 24 hours. The sample was replaced every 3 days during storage. Fixed samples were sent to the Electron Microscope Center of Nanjing Academy of Agricultural Sciences for sample preparation, and the ultrastructure of chloroplasts was observed by transmission electron microscopy (TEM). TEM sample preparation methods: samples were fixed in 3% glutaraldehyde and 0.1 mol/L phosphate buffer (pH 7.2) for 4 hours after collection, then fixed in 1% osmium acid (pH 7.2) for 4 hours after washing, and then embedded and polymerized with SPURR after dehydration of acetone step by step. After dehydration of ethanol step by step, the embedded samples were sliced and then dried at the critical point by isoamyl acetate. The samples were observed and photographed by JEM-1230 transmission electron microscopy (JEOL LTD, Japan).

Fatty Acyl-CoAs, Fatty acid, TAG and Galactolipids Analyses

The Fatty Acyl-CoAs fatty acid, TAG and galactolipids were measured using the method of Larson and Graham (2001) and Larson et al. (2002) [78, 79].

Chlorophyll determination

Leaves were crushed into powder in liquid nitrogen and then was homogenized in 80% acetone, and the debris was removed by centrifugation at 12,000 rpm for 5 min. The absorbance of the supernatant at 663 and 645 nm was measured using Spectrophotometer (GE Healthcare, USA). The chlorophyll A and chlorophyll B concentration of the samples was determined as described previously [80].

Photosynthetic activity

Photosynthetic parameters of the plants were measured using a portable photosynthesis system (LI-6400XT, LI-COR, http://www.licor.com/) in the field. Measurements were performed in the morning (9:00, 11:00 AM and 15:00 PM). All data represent the means obtained from five plants in one line.

Seed oil content

Seed oil content was determined using a Foss NIR Systems 5000 near-infrared reflectance spectroscope (Foss NIR Systems Inc., http://www.fossnirsystems.com) and Nuclear Magnetic Resonance (CNMR-1000, China).

Thin layer chromatography (TLC) analysis

Total lipid extracts were analyzed by thin layer chromatography (TLC) on silica gel 60A plates (Merck, 20 × 10 cm, layer thickness 0.2 mm). The plates were washed twice with chloroform/methanol (1:1, by volume) and activated at 180°C before use. Polar lipids were eluted with Solvent A (chloroform/methanol/acetic acid/water 85:15:10:3.5, by volume); the neutral lipids were separated by TLC in Solvent B (hexane/diethyl ether/acetic acid, 70:30:1, by volume).

Lipid detection was carried out by spraying the plate with 5% sulfuric acid in water, followed by charring at 180°C for 5 min, or exposing the TLC plate to iodine vapor, for staining all classes of lipids. The estimation of the content of individual polar and neutral lipids of the total lipid extracts was performed by video densitometry analysis of spots on TLC, obtained after averaging three replicates of C, NI, and NII (ImageJ software).

Acknowledgements

The authors would like to thank Liang Guo (Hangzhou University) for Fatty Acyl-CoAs, Fatty acid, TAG and Galactolipids analysis assistance.

Author contributions

KMZ and XFZ carried out the experiments, performed the data analysis and drafted the manuscript. NNL drafted the manuscript. YG, PD, RS and YLL analysed data. XLT conceived of the study, and drafted the manuscript. All the authors read and approved the manuscript.

Funding

This work was supported by the Jiangsu Provincial Agricultural Science and Technology Independent Innovation Fund Project (6181330000), the National Natural Science Foundation of China (31671720) and the National Key R & D Program of China (2016YFD0101900 and 2016YFD0100300).

Availability of data and materials

The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.

Declarations Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

The authors declare no conflicts of interest.

Pongdontri P, Hills M: Characterization of a novel plant acyl-CoA synthetase that is expressed in lipogenic tissues of Brassica napus L. Plant molecular biology 2001, 47(6):717-726.
Lu S, Song T, Kosma DK, Parsons EP, Rowland O, Jenks MA: Arabidopsis CER8 encodes LONG-CHAIN ACYL-COA SYNTHETASE 1 (LACS1) that has overlapping functions with LACS2 in plant wax and cutin synthesis. The Plant journal : for cell and molecular biology 2009, 59(4):553-564.
Pulsifer IP, Kluge S, Rowland O: Arabidopsis long-chain acyl-CoA synthetase 1 (LACS1), LACS2, and LACS3 facilitate fatty acid uptake in yeast. Plant physiology and biochemistry : PPB 2012, 51:31-39.
Zhao L, Katavic V, Li F, Haughn GW, Kunst L: Insertional mutant analysis reveals that long-chain acyl-CoA synthetase 1 (LACS1), but not LACS8, functionally overlaps with LACS9 in Arabidopsis seed oil biosynthesis. The Plant journal : for cell and molecular biology 2010, 64(6):1048-1058.
Singh AB, Dong B, Xu Y, Zhang Y, Liu J: Identification of a novel function of hepatic long-chain acyl-CoA synthetase-1 (ACSL1) in bile acid synthesis and its regulation by bile acid-activated farnesoid X receptor. Biochim Biophys Acta Mol Cell Biol Lipids 2019, 1864(3):358-371.
Shockey JM, Fulda MS, Browse JA: Arabidopsis contains nine long-chain acyl-coenzyme A synthetase genes that participate in fatty acid and glycerolipid metabolism. Plant physiology 2002, 129(4):1710-1722.
Graham IA: Seed storage oil mobilization. Annual review of plant biology 2008, 59:115-142.
Shockey JM, Fulda MS, Browse J: Arabidopsis contains a large superfamily of acyl-activating enzymes. Phylogenetic and biochemical analysis reveals a new class of acyl-coenzyme a synthetases. Plant physiology 2003, 132(2):1065-1076.
Babbitt PC, Kenyon GL, Martin BM, Charest H, Slyvestre M, Scholten JD, Chang KH, Liang PH, Dunaway-Mariano D: Ancestry of the 4-chlorobenzoate dehalogenase: analysis of amino acid sequence identities among families of acyl: adenyl ligases, enoyl-CoA hydratases/isomerases, and acyl-CoA thioesterases. Biochemistry 1992, 31(24):5594-5604.
Judy Schnurr, Jay shockey , Browse aJ: The Acyl-CoA Synthetase Encoded by LACS2 Is Essential for Normal Cuticle Development in Arabidopsis. The Plant Cell Online 2004, 16(3):629-642.
Lü S, Song T, Kosma DK, Parsons EP, Rowland O, Jenks MA: ArabidopsisCER8encodes LONG-CHAIN ACYL-COA SYNTHETASE 1 (LACS1) that has overlapping functions with LACS2 in plant wax and cutin synthesis. The Plant Journal 2009, 59(4):553-564.
Jessen D, Olbrich A, Knüfer J, Krüger A, Hoppert M, Polle A, Fulda M: Combined activity of LACS1 and LACS4 is required for proper pollen coat formation in Arabidopsis. The Plant Journal 2011, 68(4):715-726.
Pulsifer IP, Kluge S, Rowland O: Arabidopsis LONG-CHAIN ACYL-COA SYNTHETASE 1 (LACS1), LACS2, and LACS3 facilitate fatty acid uptake in yeast. Plant Physiology and Biochemistry 2012, 51:31-39.
Fulda M, Shockey J, Werber M, Wolter FP, Heinz E: Two long‐chain acyl‐CoA synthetases from Arabidopsis thaliana involved in peroxisomal fatty acid β‐oxidation. The Plant Journal 2002, 32(1):93-103.
Schnurr JA: Fatty Acid Export from the Chloroplast. Molecular Characterization of a Major Plastidial Acyl-Coenzyme A Synthetase from Arabidopsis. Plant Physiology 2002, 129(4):1700-1709.
Zhao L, Katavic V, Li F, Haughn GW, Kunst L: Insertional mutant analysis reveals that long-chain acyl-CoA synthetase 1 (LACS1), but not LACS8, functionally overlaps with LACS9 in Arabidopsis seed oil biosynthesis. The Plant Journal 2010, 64(6):1048-1058.
Block MA, Joyard J, Douce R: Site of synthesis of geranylgeraniol derivatives in intact spinach chloroplasts. Biochimica et Biophysica Acta (BBA)-General Subjects 1980, 631(1):210-219.
Block MA, Dubots E, Maréchal E: Glycerolipid Biosynthesis and Chloroplast Biogenesis. In: Plastid Development in Leaves during Growth and Senescence. Springer; 2013: 131-154.
Härtel H, Dörmann P, Benning C: DGD1-independent biosynthesis of extraplastidic galactolipids after phosphate deprivation in Arabidopsis. Proceedings of the National Academy of Sciences 2000, 97(19):10649-10654.
Mizusawa N, Wada H: The role of lipids in photosystem II. Biochimica et Biophysica Acta (BBA)-Bioenergetics 2012, 1817(1):194-208.
Dörmann P, Hoffmann-Benning S, Balbo I, Benning C: Isolation and characterization of an Arabidopsis mutant deficient in the thylakoid lipid digalactosyl diacylglycerol. The Plant Cell Online 1995, 7(11):1801-1810.
Kelly AA, Froehlich JE, Dörmann P: Disruption of the two digalactosyldiacylglycerol synthase genes DGD1 and DGD2 in Arabidopsis reveals the existence of an additional enzyme of galactolipid synthesis. The Plant Cell Online 2003, 15(11):2694-2706.
Zhou F, Liu S, Hu Z, Kuang T, Paulsen H, Yang C: Effect of monogalactosyldiacylglycerol on the interaction between photosystem II core complex and its antenna complexes in liposomes of thylakoid lipids. Photosynthesis Research 2009, 99(3):185-193.
Kobayashi K, Narise T, Sonoike K, Hashimoto H, Sato N, Kondo M, Nishimura M, Sato M, Toyooka K, Sugimoto K et al: Role of galactolipid biosynthesis in coordinated development of photosynthetic complexes and thylakoid membranes during chloroplast biogenesis in Arabidopsis. The Plant Journal 2013, 73(2):250-261.
Nakamura Y, Tsuchiya M, Ohta H: Plastidic phosphatidic acid phosphatases identified in a distinct subfamily of lipid phosphate phosphatases with prokaryotic origin. Journal of Biological Chemistry 2007, 282(39):29013-29021.
Yu B, Wakao S, Fan J, Benning C: Loss of plastidic lysophosphatidic acid acyltransferase causes embryo-lethality in Arabidopsis. Plant and cell physiology 2004, 45(5):503-510.
Joyard J, Ferro M, Masselon C, Seigneurin-Berny D, Salvi D, Garin J, Rolland N: Chloroplast proteomics highlights the subcellular compartmentation of lipid metabolism. Progress in lipid research 2010, 49(2):128-158.
Awai K, Maréchal E, Block MA, Brun D, Masuda T, Shimada H, Takamiya K-i, Ohta H, Joyard J: Two types of MGDG synthase genes, found widely in both 16: 3 and 18: 3 plants, differentially mediate galactolipid syntheses in photosynthetic and nonphotosynthetic tissues in Arabidopsis thaliana. Proceedings of the National Academy of Sciences 2001, 98(19):10960-10965.
Kelly AA, Dörmann P: Green light for galactolipid trafficking. Current opinion in plant biology 2004, 7(3):262-269.
Li C, Wang Y, Liu L, Hu Y, Zhang F, Mergen S, Wang G, Schläppi MR, Chu C: A rice plastidial nucleotide sugar epimerase is involved in galactolipid biosynthesis and improves photosynthetic efficiency. PLoS genetics 2011, 7(7):e1002196.
Kobayashi K, Kondo M, Fukuda H, Nishimura M, Ohta H: Galactolipid synthesis in chloroplast inner envelope is essential for proper thylakoid biogenesis, photosynthesis, and embryogenesis. Proceedings of the National Academy of Sciences 2007, 104(43):17216-17221.
Dormann P, Hoffmann-Benning S, Balbo I, Benning C: Isolation and characterization of an Arabidopsis mutant deficient in the thylakoid lipid digalactosyl diacylglycerol. The Plant cell 1995, 7(11):1801-1810.
Hartel H, Lokstein H, Dormann P, Grimm B, Benning C: Changes in the composition of the photosynthetic apparatus in the galactolipid-deficient dgd1 mutant of Arabidopsis thaliana. Plant physiology 1997, 115(3):1175-1184.
Jarvis P, Dormann P, Peto CA, Lutes J, Benning C, Chory J: Galactolipid deficiency and abnormal chloroplast development in the Arabidopsis MGD synthase 1 mutant. Proceedings of the National Academy of Sciences of the United States of America 2000, 97(14):8175-8179.
Joyard J, Maréchal E, Miège C, Block MA, Dorne A-J, Douce R: Structure, distribution and biosynthesis of glycerolipids from higher plant chloroplasts. In: Lipids in photosynthesis: Structure, function and genetics. Springer; 2004: 21-52.
Pineau B, Girard-Bascou J, Eberhard S, Choquet Y, Tremolieres A, Gerard-Hirne C, Bennardo-Connan A, Decottignies P, Gillet S, Wollman FA: A single mutation that causes phosphatidylglycerol deficiency impairs synthesis of photosystem II cores in Chlamydomonas reinhardtii. European journal of biochemistry 2004, 271(2):329-338.
Wu F, Yang ZL, Kuang TY: Impaired photosynthesis in phosphatidylglycerol-deficient mutant of cyanobacterium Anabaena sp PCC7120 with a disrupted gene encoding a putative phosphatidylglycerophosphatase. Plant physiology 2006, 141(4):1274-1283.
Dörmann P, Balbo I, Benning C: Arabidopsis galactolipid biosynthesis and lipid trafficking mediated by DGD1. Science 1999, 284(5423):2181-2184.
Frentzen M: Phosphatidylglycerol and sulfoquinovosyldiacylglycerol: anionic membrane lipids and phosphate regulation. Current opinion in plant biology 2004, 7(3):270-276.
Müller F, Frentzen M: Phosphatidylglycerophosphate synthases from< i> Arabidopsis thaliana</i>. FEBS letters 2001, 509(2):298-302.
Yu B, Xu C, Benning C: Arabidopsis disrupted in SQD2 encoding sulfolipid synthase is impaired in phosphate-limited growth. Proceedings of the National Academy of Sciences 2002, 99(8):5732-5737.
Sanda S, Leustek T, Theisen MJ, Garavito RM, Benning C: Recombinant Arabidopsis SQD1 converts UDP-glucose and sulfite to the sulfolipid head group precursor UDP-sulfoquinovose in vitro. Journal of Biological Chemistry 2001, 276(6):3941-3946.
Dubots E, Audry M, Yamaryo Y, Bastien O, Ohta H, Breton C, Maréchal E, Block MA: Activation of the chloroplast monogalactosyldiacylglycerol synthase MGD1 by phosphatidic acid and phosphatidylglycerol. Journal of Biological Chemistry 2010, 285(9):6003-6011.
Fromme P, Melkozernov A, Jordan P, Krauss N: Structure and function of photosystem I: interaction with its soluble electron carriers and external antenna systems. FEBS letters 2003, 555(1):40-44.
Jung YH, Kim SC, Kim M, Kim KH, Kwon HM, Oh MY: Chloroplast inheritance patterns in Actinidia hybrids determined by single stranded conformation polymorphism analysis. Molecules and cells 2003, 15(2):277-282.
Nakanishi H, Nozue H, Suzuki K, Kaneko Y, Taguchi G, Hayashida N: Characterization of the Arabidopsis thaliana mutant pcb2 which accumulates divinyl chlorophylls. Plant & cell physiology 2005, 46(3):467-473.
Zhang H, Li J, Yoo JH, Yoo SC, Cho SH, Koh HJ, Seo HS, Paek NC: Rice Chlorina-1 and Chlorina-9 encode ChlD and ChlI subunits of Mg-chelatase, a key enzyme for chlorophyll synthesis and chloroplast development. Plant molecular biology 2006, 62(3):325-337.
Tanaka A, Ito H, Tanaka R, Tanaka NK, Yoshida K, Okada K: Chlorophyll a oxygenase (CAO) is involved in chlorophyll b formation from chlorophyll a. Proceedings of the National Academy of Sciences 1998, 95(21):12719-12723.
Beale SI: Green genes gleaned. Trends in plant science 2005, 10(7):309-312.
Nonaka A, Tanaka M, Anguri H, Nagata H, Kita J, Shizukuishi S: Clinical assessment of oral malodor intensity expressed as absolute value using an electronic nose. Oral Dis 2005, 11 Suppl 1:35-36.
Nagata N, Tanaka R, Tanaka A: The major route for chlorophyll synthesis includes [3,8-divinyl]-chlorophyllide a reduction in Arabidopsis thaliana. Plant & cell physiology 2007, 48(12):1803-1808.
Chalhoub B, Denoeud F, Liu SY, Parkin IAP, Tang HB, Wang XY, Chiquet J, Belcram H, Tong CB, Samans B et al: Early allopolyploid evolution in the post-Neolithic Brassica napus oilseed genome. Science 2014, 345(6199):950-953.
Guo X, Jiang M, Wan X, Hu C, Gong Y: Identification and biochemical characterization of five long-chain acyl-coenzyme A synthetases from the diatom Phaeodactylum tricornutum. Plant physiology and biochemistry : PPB 2014, 74:33-41.
Li N, Xu C, Li-Beisson Y, Philippar K: Fatty Acid and Lipid Transport in Plant Cells. Trends in plant science 2016, 21(2):145-158.
Block MA, Maréchal E, Joyard J: Role of the plastid envelope in the biogenesis of chloroplast lipids. In: Regulation of photosynthesis. Springer; 2001: 195-218.
Mortazavi A, Williams BA, McCue K, Schaeffer L, Wold B: Mapping and quantifying mammalian transcriptomes by RNA-Seq. Nature methods 2008, 5(7):621-628.
Xu C, Yu B, Cornish AJ, Froehlich JE, Benning C: Phosphatidylglycerol biosynthesis in chloroplasts of Arabidopsis mutants deficient in acyl‐ACP glycerol‐3‐phosphate acyltransferase. The Plant Journal 2006, 47(2):296-309.
Andrews J, Mudd JB: Phosphatidylglycerol synthesis in pea chloroplasts pathway and localization. Plant physiology 1985, 79(1):259-265.
Babiychuk E, Müller F, Eubel H, Braun HP, Frentzen M, Kushnir S: Arabidopsis phosphatidylglycerophosphate synthase 1 is essential for chloroplast differentiation, but is dispensable for mitochondrial function. The Plant Journal 2003, 33(5):899-909.
Andersson MX, Dörmann P: Chloroplast membrane lipid biosynthesis and transport. In: The Chloroplast. Springer; 2009: 125-158.
Benning C: A role for lipid trafficking in chloroplast biogenesis. Progress in lipid research 2008, 47(5):381-389.
Breuers FK, Brautigam A, Geimer S, Welzel UY, Stefano G, Renna L, Brandizzi F, Weber AP: Dynamic Remodeling of the Plastid Envelope Membranes - A Tool for Chloroplast Envelope in vivo Localizations. Frontiers in plant science 2012, 3:7.
Browse J, Somerville C: Glycerolipid synthesis: biochemistry and regulation. Annual review of plant biology 1991, 42(1):467-506.
Joyard J, Maréchal E, Miège C, Block MA, Dorne A-J, Douce R: Structure, distribution and biosynthesis of glycerolipids from higher plant chloroplasts. In: Lipids in photosynthesis: Structure, function and genetics. Springer; 1998: 21-52.
Tripathy BC, Pattanayak GK: Chlorophyll Biosynthesis in Higher Plants. 2012, 34:63-94.
Beale SI: Enzymes of chlorophyll biosynthesis. Photosynthesis Research 1999, 60(1):43-73.
Zhang H, Li J, Yoo J-H, Yoo S-C, Cho S-H, Koh H-J, Seo HS, Paek N-C: Rice Chlorina-1 and Chlorina-9 encode ChlD and ChlI subunits of Mg-chelatase, a key enzyme for chlorophyll synthesis and chloroplast development. Plant molecular biology 2006, 62(3):325-337.
Oster U, Tanaka R, Tanaka A, Rüdiger W: Cloning and functional expression of the gene encoding the key enzyme for chlorophyll b biosynthesis (CAO) from Arabidopsis thaliana. The Plant Journal 2000, 21(3):305-310.
Tamura K, Dudley J, Nei M, Kumar S: MEGA4: molecular evolutionary genetics analysis (MEGA) software version 4.0. Molecular biology and evolution 2007, 24(8):1596-1599.
Bailey TL, Elkan C: Fitting a mixture model by expectation maximization to discover motifs in bipolymers. In.: Department of Computer Science and Engineering, University of California, San Diego; 1994.
Wang JL, Tang MQ, Chen S, Zheng XF, Mo HX, Li SJ, Wang Z, Zhu KM, Ding LN, Liu SY et al: Down-regulation of BnDA1, whose gene locus is associated with the seeds weight, improves the seeds weight and organ size in Brassica napus. Plant biotechnology journal 2017.
Livak KJ, Schmittgen TD: Analysis of relative gene expression data using real-time quantitative PCR and the 2(T)(-Delta Delta C) method. Methods 2001, 25(4):402-408.
Tarazona S, Garcia-Alcalde F, Dopazo J, Ferrer A, Conesa A: Differential expression in RNA-seq: a matter of depth. Genome Res 2011, 21(12):2213-2223.
Voinnet O, Rivas S, Mestre P, Baulcombe D: An enhanced transient expression system in plants based on suppression of gene silencing by the p19 protein of tomato bushy stunt virus. The Plant Journal 2003, 33(5):949-956.
Wood CC, Petrie JR, Shrestha P, Mansour MP, Nichols PD, Green AG, Singh SP: A leaf-based assay using interchangeable design principles to rapidly assemble multistep recombinant pathways. Plant biotechnology journal 2009, 7(9):914-924.
Sparkes IA, Runions J, Kearns A, Hawes C: Rapid, transient expression of fluorescent fusion proteins in tobacco plants and generation of stably transformed plants. Nature protocols 2006, 1(4):2019-2025.
Wang Z, Mao H, Dong CH, Ji RQ, Cai L, Fu H, Liu SY: Overexpression of Brassica napus MPK4 Enhances Resistance to Sclerotinia sclerotiorum in Oilseed Rape. Mol Plant Microbe In 2009, 22(3):235-244.
Larson TR, Graham IA: Technical advance: a novel technique for the sensitive quantification of acyl CoA esters from plant tissues. The Plant Journal 2001, 25(1):115-125.
Larson TR, Edgell T, Byrne J, Dehesh K, Graham IA: Acyl CoA profiles of transgenic plants that accumulate medium‐chain fatty acids indicate inefficient storage lipid synthesis in developing oilseeds. The Plant Journal 2002, 32(4):519-527.
MELIS A, Spangfort M, Andersson B: Light‐absorption and electron‐transport balance between photosystem II and photosystem I in spinach chloroplasts. Photochemistry and Photobiology 1987, 45(1):129-136.

Fig. S1 The identification of BnLACS9 overexpression and RNAi transgenic plants. (A) The BnLACS9 overexpression vector and identification of pCAMBIA1300-35S-BnLACS9-NOS transgenic Brassica napus plants by PCR. F and R are the primers used to identify the transgenic plants. Line 1: 5000 DNA Maker; line 2 to line 21: BnLACS9 overexpression transgenic lines; Line 22: PCR result using pCAMBIA1300-35S-BnLACS9-NOS plasmid as positive control; line23-24: the negative control. (B) The BnLACS9 RNAi vector and identification of pHellsgate 12::Bnlacs9 transgenic plants by PCR. RKAN-F and RKAN-R are the primers used to identify the transgenic plants. Line 1: 2000 DNA Marker; Line 2 to Line 9: BnLACS9 RNAi transgenic lines; Line 10: The negative control; Line 11: PCR result using pHellsgate 12::Bnlacs9 plasmid as positive control. (C) RT-PCR analysis of BnLACS9 overexpression and RNAi transgenic plants. NY12: Wide type; The BnLACS9 overexpression lines (Over-BnLACS9-6, Over-BnLACS9-12, Over-BnLACS9-13, Over-BnLACS9-17, Over-BnLACS9-18); The BnLACS9 RNAi lines (RNAi-Bnlacs9-2, RNAi-Bnlacs9-4, RNAi-Bnlacs9-6, RNAi-Bnlacs9-9). BnACTIN was used as an internal control. The data show means ± standard errors (N = 3). * p < 0.05, ** p < 0.01, *** p < 0.001. Student’s t-test was used to generate p-value.

Fig. S2 The net photosynthetic rate of the leaves from BnLACS9 overexpression transgenic lines

Fig. S3 The dry weight of the seedling from BnLACS9 overexpression transgenic lines.

No competing interests reported.

Download PDF

Journal Publication

published 06 Jan, 2023

Read the published version in Biotechnology for Biofuels and Bioproducts →

Editorial decision: Major revision
03 Sep, 2022
Reviews received at journal
10 Aug, 2022
Reviewers agreed at journal
26 Jul, 2022
Reviewers invited by journal
26 Jul, 2022
Editor assigned by journal
16 Jun, 2022
Submission checks completed at journal
15 Jun, 2022
First submitted to journal
14 Jun, 2022

You are reading this latest preprint version

The chlorophyll content and structure can be regulated by long-chain acyl-CoA synthetase, BnLACS9, by stimulating triacylglycerol metabolism in Brassica napus

Status:

Journal Publication

Version 1

Abstract

Figures

Background

Results

BnLACS9 is highly homologous with AtLACS9

The Expression pattern of BnLACS9

Subcellular localization of BnLACS9

Discussion

Experimental Procedures

Plant materials and growth conditions

Sequence analysis

RNA extraction, cDNA synthesis and Real-time PCR (RT-PCR)

Ultrastructural observation of chloroplasts

Fatty Acyl-CoAs, Fatty acid, TAG and Galactolipids Analyses

Chlorophyll determination

Photosynthetic activity

Seed oil content

Thin layer chromatography (TLC) analysis

Declarations

References

Supplemental Figure Legends

Additional Declarations

Supplementary Files

Status:

Journal Publication

Version 1