Ectopic expression of WRINKLED1 in rice improves lipid biosynthesis but retards plant growth and development.

WRINKLED1 (WRI1) is a transcription factor which is key to the regulation of seed oil biosynthesis in Arabidopsis. In the study, we identified two WRI1 genes in rice, named OsWRI1a and OsWRI1b, which share over 98% nucleotide similarity and are expressed only at very low levels in leaves and endosperms. The subcellular localization of Arabidopsis protoplasts showed that OsWRI1a encoded a nuclear localized protein. Overexpression of OsWRI1a under the control of the CaMV 35S promoter severely retarded plant growth and development in rice. Expressing the OsWRI1a gene under the control of the P1 promoter of Brittle2 (highly expressed in endosperm but low in leaves and roots) increased the oil content of both leaves and endosperms and upregulated the expression of several genes related to late glycolysis and fatty acid biosynthesis. However, the growth and development of the transgenic plants were also affected, with phenotypes including smaller plant size, later heading time, and fewer and lighter grains. The laminae (especially those of flag leaves) did not turn green and could not unroll normally. Thus, ectopic expression of OsWRI1a in rice enhances oil biosynthesis, but also leads to abnormal plant growth and development.

Introduction

Angiosperm seeds accumulate storage protein, oil (mainly triacylglycerols, TAG) and carbohydrate (mainly starches) during seed filling and degrade these compounds upon germination in order to support early seedling growth. The relative abundance of storage reserves varies among seeds of different species. Many oilseeds produce 50–70% oil, some legumes contain 40% protein, whereas the seeds of most cereals have starch as 70–85% of the dry weight [1]. The accumulation of storage components requires the coordinated expression of many genes that encode the enzymes of the corresponding pathways [2]. Several transcription factors have been reported to play roles in regulating these pathways. One of the most important of these genes is WRINKLED1 (WRI1) [3].

The WRI1 gene products belong to the AINTEGUMENTA (ANT) subfamily in AP2/ERF transcription factor family, members of which contain two plant-specific (AP2/EREB) DNA-binding domains [4]. The function of the WRI1 gene in plants is first reported in Arabidopsis. Arabidopsis wri1 mutant seedlings have no obvious phenotype during vegetative development, but they produce wrinkled seeds and the ability of developing seeds to convert sucrose and glucose into precursors of fatty acid (FA) biosynthesis is impaired, subsequently causing an 80% reduction in seed oil content [3]. As a transcription factor, the WRI1 protein can target, and promote the expression of, many genes encoding enzymes involved in late glycolysis and FA biosynthetic pathways [5, 6]. In Arabidopsis [4, 7], Brassica napus [8, 9], Nicotiana benthamiana [10], and Solanum tuberosum [11], overexpression or ectopic expression of WRI1 genes increases the oil content of seeds, leaves, or tubers.

In maize, overexpression of ZmWri1a increases the fatty acid content of the mature grain. The higher seed oil content in ZmWri1a overexpressing lines is not accompanied by any reduction in starch. The transgenic kernels do not show any macroscopically visible defects such as wrinkled or plump phenotypes, and there is no significant difference in kernel weight between transgenic and wild-type kernels [12, 13]. The ectopic expression of BdWRI1 in the grass Brachypodium distachyon induces the transcription of genes predicted to be involved in glycolysis and FA biosynthesis, and TAG content is increased up to 32.5-fold in 8-week-old leaf blades. However, ectopic expression of BdWRI1 also cause cell death in leaves [14].

Rice (Oryza sativa L.) has two genes, LOC_Os11g03540 and LOC_Os12g03290, which are close to the Arabidopsis WRI1 gene on a phylogenetic tree [15]. The LOC_Os11g03540 gene (AJ575217) is found to be mainly expressed in embryos, however, the expression of this gene is hardly detected in leaves and endosperms [16]. Sun et al. [17] expresses the CoWRI1 gene of Coconut (Cocos nucifera L.) using an endosperm-specific promoter EnP2 from rice changes the seeds oil content in transgenic rice. In order to explore whether the expression of OsWRI1 could increase oil accumulation in non-oil storage organs such as endosperms and leaves of rice plants, we overexpressed the LOC_Os11g03540 gene under the control of the CaMV 35S promoter. We observed that the resulting transgenic plants were severely retarded with respect to growth and development. Expressing this gene under the control of the P1 promoter of Brittle2 (small subunit of ADP–glucose pyrophosphorylase) [18] increased the oil content of leaves and endosperm but affected plant growth and development in rice.

Materials and methods

Plant materials

Using the japonica rice (Oryza sativa L.) cv. Zhonghua 11 (ZH11) as the wild type materials. After the seeds of wild-type and transgenic plants germinated, they were planted in a glass greenhouse under natural light in the South China Botanic Garden, China. Roots, leaves, embryos and endosperms from seeds of plants 10 days after flowering were sampled, and stored at −80°C until required for expression profiles analysis.

Sequence retrieval and analysis

TAIR database (http://www.) and Phytozome database (http://www.phytozome.net/) were used to download AINTEGUMENTA (ANT) amino acid sequences of Arabidopsis and rice, respectively. However, the ANT sequences of maize and Jatropha curcas were derived from GenBank (http://www.ncbi.nlm.nih.gov/). ClustalW software was used to analyze the full-length ANT amino acid’ bioinformatics characteristics. And then the NJ (neighbor-joining) method was used to construct a rootless phylogenetic tree based on the Mega software version 5 [19].

Subcellular localization of OsWRI1a protein

Using rice leaves as a template, we cloned the OsWRI1a gene (without stop codon) by RT-PCR technology. Subsequently, we ligated the sequenced correct OsWRI1a gene into the pBWA(V)HS-GLosgfp vector to construct the OsWRI1a-GFP fusion protein vector. We further co-transformed the constructed OsWRI1a-GFP fusion expression vector and empty vector into Arabidopsis protoplast cells by a PEG-mediated method [20], and observed under a confocal electron microscope.

Plasmid constructs and plant transformation

Using rice leaf cDNA as template, specific primers were designed (primers given in S1 File), and the coding sequence of full-length OsWRI1 gene was cloned by RT-PCR. The cloned and purified DNA sequence was connected to the pMD18-T vector (Takara, http://www.takara.com.cn/). The qualified recombinant cloning vector pMD18-T -OsWRI1 plasmid was digested with BamH I and Sal I. Then, the DNA fragment cut by BamH I and Sal I was connected to pCAMBIA1301 vector under the control of the CAMV 35S promoter (35S) by T4 DNA ligase (EcoR I ‒ 35S ‒ Sac I, BamH I ‒ OsWRI1a ‒ Sal I, Pst I ‒ ocs ‒ Hind III).

To construct an expression vector containing OsWRI1a under the control of the promoter of Brittle2 (Bt2, endosperm ADP–glucose pyrophosphorylase small subunit; LOC_Os08g25734), the P1 promoter of the Brittle2 gene was amplified by PCR from rice genomic DNA extracted from leaves (primers listed in S1 File) [18]. The correctly sequenced PCR product was connected to the pMD18-T vector, and the pMD18-T-OsWRI1 plasmid was digested by Hinc II and BamH I. Then we linked the target gene to the pCAMBIA1301 vector through T4 DNA ligase (containing OsWRI1a and ocs) at the Sma I/BamH I site (Sma I/Hinc II ‒ Bt2P1 ‒ BamH I ‒ OsWRI1a ‒ Sal I, Pst I ‒ ocs ‒ Hind III). Next, we transformed the constructed OsWRI1 plant expression vector into Agrobacterium (strain EHA105) by the freeze–thaw procedure. Finally, Agrobacterium containing OsWRI1 plasmid was used to transform rice callus as described by Li and Li [21, 22], and obtained OsWRI1 transgenic plants. In addition, the screening of homozygotes was based on a segregation ratio of about 3:1 observed in T2 plants. The segregation ratios of four transgenic plants, were confirmed by GUS staining, were 237:78, 197:64, 169:55 and 206:67, respectively. Then we randomly selected 30 T3 plants for GUS staining. We speculated that these plants were homozygous plants for all GUS-stained plants.

RNA isolation and qRT-PCR

According to the instructions of the kit, plant total RNA was extracted by TRIzol reagent (Invitrogen, http://www.thermofisher.com). Then we took 2 μg total RNA, and reversed transcribe the RNA into cDNA through M-MLV reverse transcriptase (Promega, http://www.promega.com). An OsRUB (rice ubiquitin) gene was used as a reference. The LightCycler 480 (a Mini Option real-time PCR system) was used to perform qRT-PCR. Cycling conditions were as follows: 95°C for 30 s, 95°C for 5 s, 60°C for 20 s, and 72°C for 20 s. The reaction was carried out for 40 cycles. Three biological replicates were performed in this study, and average values were used to calculate the relative expression of genes [23, 24]. The primers were shown in S1 File.

Chemical assays

Chlorophyll concentrations of samples were determined according to Arnon [25]. The chloroplast pigments were extracted from fully-expanded 2nd leaves with 80% acetone.

TAG and free FA analyses were performed as described by previously Yang et al. [14]. Total lipid samples were separated by TLC on silica plates (Si250PA; 20*20cm; Mallinckrodt Baker) developed with ether: ethyl ether:acetic acid (80:20:1, v/v). After development, TAG and free FA bands were sprayed with 0.01% (v/v) Primuline in 80% (v/v) acetone and visualized under UV light. TAG or free FA bands were isolated from the TLC plate. FA methyl esters were prepared and quantified as described previously [26].

For fatty acid composition analysis. To determine fatty acid composition, fatty acid methyl esters were prepared and quantitatively analyzed by gas chromatography according to Wu et al. [27]. An Agilent 7890A GC system apparatus with a flame ionization detector on an HP-88 column (30 m × 0.25 mm I.D., 0.20 μm film thickness) was used. Results are expressed as mol percentage [26]. Statistical analysis of the data was performed using Student’s test.

Results

The WRI1 genes of rice

WRINKLED1 (WRI1) encodes a transcription factor of the AP2/ERF class. The protein has two plant-specific (AP2/ERF) DNA-binding domains, and in Arabidopsis it is involved in the control of storage compound biosynthesis [3, 4]. According to a phylogenetic tree based on AP2/ERF family proteins from selected plant species, seven AP2 proteins from rice fall into the ANTb clade which contains the AtWRI1 protein [15] (Fig 1). LOC_Os11g03540 and LOC_Os12g03290 proteins share the highest amino acid similarities with Arabidopsis WRI1 and maize WRI1 proteins, so we named them OsWRI1a and OsWRI1b respectively. The coding domain sequences of the two OsWRI1 genes share very high (over 98%) nucleotide similarity. Their levels of expression could not be determined separately by RT-PCR. The expression levels measured by qRT-PCR in this study therefore represent the combined expression of the OsWRI1a and OsWRI1b genes, which were expressed mainly in roots and embryos (Fig 2). Of the two OsWRI1 genes, we could only obtain cDNA clones for OsWRI1a by RT-PCR from total RNA of young seeds. Therefore, the OsWRI1a gene was selected for further functional verification.

Fig 1

Neighbor-joining unrooted tree.

Bootstrap values were calculated for 100 replicates, and values are indicated at the corresponding nodes. The database accession numbers of sequences are indicated in brackets.

Fig 2

Expression analysis of the OsWRI1a/OsWRI1b genes.

qRT-PCR applied to total RNA samples was used to measure OsWRI1a/OsWRI1b gene expression. Relative expression was normalized to that of the reference gene ubiquitin (internal control). The 10 d represent endosperms and embryos were sampled from seeds at 10 days after flowering. Bars show means ± SD of three biological replicates.

OsWRI1a localizes to the nucleus

In order to clarify the protein properties of OsWRI1a, we constructed an OsWRI1a-GFP fusion protein expression vector. Then we transferred the constructed vector and empty control vector into Arabidopsis thaliana protoplast cells by PEG-mediated method, and placed them under a fluorescence confocal microscope to observe the localization. The subcellular localization results showed that green fluorescence signal was detected in all cells with the empty control vector, while the fluorescence signal was only observed in the nucleus of the OsWRI1a-GFP fusion expression vector (Fig 3). The results show that the OsWRI1a protein is localized in the nucleus.

Fig 3

Subcellular localization of OsWRI1a.

Transiently expressed the GFP (upper) and OsWRI1a-GFP fusion protein (lower) in Arabidopsis protoplasts which observed under a laser scanning confocal microscope. Bars = 10 μm.

Overexpression of OsWRI1a in rice seriously retarded plant growth and development

Ectopic expression of the WRI1 gene in several plant species results in an increase in oil accumulation in leaves and other organs. To investigate whether ectopic expression of this gene exerted the same effect in rice, we overexpressed the WRI1a gene under the control of the CaMV 35S promoter. More than twenty transgenic lines were obtained. The transgenic seedlings exhibited multiple morphological defects, including dwarfism, chlorotic leaves, and failure to produce seeds. The upper parts of the laminae of most leaves were chlorotic and curly, and the chlorotic parts died quickly (S1 Fig). Because a high level of expression of the OsWRI1a gene seriously inhibited rice growth, and since expression of ZmWRI1 under the control of the 19 KD ZEIN promoter (expressed at the later stage of endosperm development) do not lead to an increase in seed oil content in maize [12], we next expressed the OsWRI1a gene under the control of the P1 promoter of the Brittle2 (Bt2) (LOC_Os08g25734) gene. The Bt2 gene encodes two proteins, a cytosolic and a plastidial small subunit of ADP–glucose pyrophosphorylase. The GUS gene under the control of the Bt2 P1 (P) is highly expressed in endosperm but lower in leaves and roots of rice plants, whereas under the control of the P2 promoter, it is expressed strongly in roots and leaves but at a low level in endosperms [18]. Four independent P::OsWRI1a transgenic lines (P1-6, -7, -8, and -10) were established for use in our experiments.

Expression of OsWRI1a in rice under the control of the Bt2P1 promoter affected plant growth and development

The phenotypes of P::OsWRI1a transgenic rice lines were observed in the T4 plants. It was found that P::OsWRI1a transgenic rice plants were smaller and their heading date was about 7 days later than the wild type (ZH11) (Fig 4A‒4C). The number of tillers in P::OsWRI1a plants (from 7.6±1.6 to 8.8±1.7) was less than that in the wild type (14.6±1.5) at the heading stage under present pot-culture conditions. The root lengths of P::OsWRI1a seedlings were about 5% less than the wild type (S2 Fig). The P::OsWRI1a plants had shorter internodes, shorter panicles and fewer grains than wild type plants (Fig 4E and 4F). The middle part of the lamina of all flag leaves and a small proportion of the second leaves did not turn green and failed to unroll properly during the period from emergence to death (Fig 5A‒5D). Laminae of the second leaves were shorter, looked yellowish-green (Fig 5E) and contained less chlorophyll compared to the leaves of the same part of the wild-type plants (Fig 5F). Changes in levels of OsWRI1a transcripts in leaves were observed by qRT-PCR analysis (Fig 4D). The levels of OsWRI1a transcripts in the first leaf (flag leaf) was higher than that in the second and third leaves at the flag leaf stage of about 10 cm in length in P::OsWRI1a transgenic plants (S3A Fig).

Fig 4

The phenotypes of PBt2P1::OsWRI1a plants.

(A) Plants at the jointing-booting stage. (B) Plants at heading stage. (C) Plants at the mature stage. (D) Expression analysis of the OsWRI1a gene in leaves of plants at heading stage. The experiment included three biological replicates, each with two technical replicates. Values represent means of n = 6 ± SD (Duncan test: **, P < 0.01). (E) and (F) Lengths of internodes and panicles of the main stems. Values in (F) represent means of n = 30 ± SD. P1-6, -7, -8, and -10, P::OsWRI1a transgenic lines.

Fig 5

The abnormal phenotypes of leaves in PBt2P1:: OsWRI1a plants.

(A) to (D) The developing course of abnormal lamina of flag leaves in P:: OsWRI1a plants. P1-6, -7, -8, and -10, P:: OsWRI1a transgenic lines. (E) The lamina of the second leaf of heading stage plants. Bar = 10 cm. (F) The chlorophyll content in the second leaves of heading stage plants. Values represent means of n = 6 ± SD (Duncan test: **, P < 0.01). (G) The FA and TAG content in leaves of heading stage plants. Values represent means of n = 3 ± SD (Duncan test: **, P < 0.01). DW = dry weight. (H) and (I) FA composition in free FA (H) and TAG (I) in leaf blades of heading stage plants. Values represent means of n = 3 ± SD (Duncan test: **, P < 0.01). C16:0, palmitic acid; C16:1 palmitoleic acid; C18:0, stearic acid; C18:1, oleic acid; C18:2, linoleic acid; C18:3, linolenic acid. DW = dry weight. P1-6 and -7, P::OsWRI1a transgenic lines.

The crude lipid content of the leaves of P::OsWRI1a plants was increased by 40% to 65% (S3B Fig), and the expression of several genes involved in later glycolysis and fatty acid biosynthetic pathway was upregulated (S3C Fig). To explore whether the levels of leaf TAG was also increased, we examined the content of TAG in the leaf blades of two P::OsWRI1a lines. As a result, the content of TAG in P::OsWRI1a leaf blades was increased by more than 40% (Fig 5G). After analyzing the fatty acid composition, we observed that the TAG of the PBt2P1::OsWRI1a lines contained more C18:1 and C18:2, but less C18:0 and C18:3 compared to wild-type lines (Fig 5I). Similar to the ectopic expression of BdWRI1 in B. distachyon [14], the total free fatty acid content and total unsaturated fatty acid content was also increased in P::OsWRI1a leaf blades (Fig 5G and 5H). These results indicate that the expression of OsWRI1a influences fatty acid biosynthesis in leaves and plant growth and development in rice.

The PBt2P1::OsWRI1a lines had increased TAG content and altered fatty acid composition in endosperms

The 1000-seed weight of P::OsWRI1a transgenic rice plants was approximately 15% lower than those of wild type (S4A Fig). The total crude lipid content of the endosperm of the mature seeds from P::OsWRI1a lines was about 75% more than that of the wild type, while the net crude lipid content was no more than 1% (S4B Fig). We also analyzed the TAG content of endosperm and embryo of two P::OsWRI1a lines. The TAG content in the endosperm of mature seeds from P::OsWRI1a lines was over 30% more than that of the wild type (Fig 6A). The C16:0, C18:0, C18:1, and C18:2 fatty acids were the main components of TAG of rice endosperm. The TAG of endosperm from P::OsWRI1a lines contained more C16:0, but less C18:1, compared to wild type (Fig 6B).

Fig 6

Changes in TAG accumulation in endosperm and embryo.

(A) The TAG content in endosperm of mature seeds. (B) FA composition in TAG of endosperm. Values represent means of n = 3 ± SD (Duncan test: **, P < 0.01; *, P < 0.05). DW = dry weight. P1-6, and -7, PSh2P1::OsWRI1a transgenic lines. C16:0, palmitic acid; C18:0, stearic acid; C18:1, oleic acid; C18:2, linoleic acid; C18:3 arachidic acid.

Expression of OsWRI1a upregulated the expression of glycolysis and fatty acid biosynthesis related genes in rice endosperm

In maize and many other plants, overexpression of the WRI1 gene upregulates the expression of many genes involved in glycolysis and in fatty acid biosynthetic pathways [6, 8, 12]. Therefore, we further examined the expression of several genes related to these pathways in the endosperm 5 and 10 days after flowering (DAF) by qRT-PCR. Compared to wild type, expression of 8 genes, PK, PDH-E1 α, PDH-E1β, PDH-E2, KASIII, ENR1, ACP, and FatA, was increased more than 2-fold at 5 DAF and/or 10 DAF (Fig 7).

Fig 7

Expression of genes predicted to encode proteins involved in the later stages of glycolysis and in fatty acid biosynthetic pathways in developing endosperms.

Total RNA was isolated from endosperms of the seeds of plants 5 and 10 days after flowering (5 d, 10 d). The experiment included three biological replicates, each with two technical replicates. Values represent means of n = 6 ± SD (Duncan test: **, P < 0.01; *, P < 0.05). P1-6, -7, -8, and -10, P::OsWRI1a transgenic lines. The gene accession numbers are: OsACP, LOC_Os08g43580; OsENR1, LOC_Os08g23810; OsFatA, LOC_Os09g32760; OsKASIII, LOC_Os04g55060; OsPDH-E1α, LOC_Os04g02900; OsPDH-E1β, LOC_Os12g42230; OsPDH-E2, LOC_Os12g08170; OsPK, LOC_Os10g42100; OsRUB1, LOC_Os06g46770.

Discussion

The WRI1 protein is a key regulator of oil biosynthesis first discovered in plants [3, 28, 29]. Mutation in the WRI1 gene causes a reduction in oil accumulation and wrinkled seeds. According to a phylogenetic analysis of AP2/ERF proteins, Arabidopsis has 3 AP2 proteins, while rice has 7 AP2 proteins that fall into the ANTb clade (Fig 1) [15]. As in the case of maize, the WRI1 gene is duplicated in rice. The OsWRI1 genes share very high nucleotide similarity, probably as a result of a recent segmental duplication between chromosomes 11 and 12 [30]. The OsWRI1 genes were barely expressed in leaves and endosperms (Fig 2) [16], but their expression was high in embryos, suggesting that they may play roles in the biosynthesis of storage fatty acids in rice embryos.

Partial Arabidopsis transgenic lines (106 and 107) overexpressing AtWRI1 show abnormal morphological phenotypes on agar plates. The seedlings do not turn green, lack expanded cotyledons, frequently do not open their apical hooks and often show elongated hypocotyls [4]. In the present study, we found that the upper parts of the laminae of most leaves in 35S::OsWRI1a expressing rice did not turn green and could not unroll. The growth and development of the transgenic seedlings were severely retarded (S1 Fig). In rice expressing P::OsWRI1a, growth and development were less strongly retarded. Laminae of the flag leaves did not turn green and failed to unroll (Fig 5A‒5D). These effects should base on the abundance of OsWRI1 transcripts in different leaves of P::OsWRI1a transgenic lines (S3A Fig). These results implied that a higher expression level of the OsWRI1 gene could inhibit chlorophyll biosynthesis and photomorphogenesis in transgenic rice leaves, as was suggested in the case of partial Arabidopsis overexpressing WRI1 lines under the control of the 35S promoter [4]. The development of rice leaves may be more sensitively to this inhibitory effect than Arabidopsis because it appears in all 35S::OsWRI1a expressing rice plants. Previous studies suggest that overexpression of AtWRI1 in rice shows a significant increase in vegetative tissue fatty acids, with some seed reserve changes, but no phenotypic off types observed [31, 32]. We speculate that the different phenotypes of AtWRI1-overexpressing rice plants and OsWRI1-overexpressing rice plants may be attributed to the fact that genes in different species may play different functions, even though these genes are capable of increasing fatty acid content and are highly homologous. Similarly, JcERF011 functions differently in Arabidopsis and rice [15]. In maize, overexpression of ZmWRI1a results in kernels that have elevated oil content, but do not show any visible defects with respect to seed yield and other agronomic traits [12, 13]. In Brassica napus, overexpression of BnWRI1 accelerates flowering and enhances oil accumulation in both seeds and leaves without leading to any visible inhibition of growth [9]. Ectopic expression of BdWRI1 in the grass Brachypodium distachyon induces the transcription of genes predicted to be involved in glycolysis and FA biosynthesis, and TAG content is increased in leaf blades. However, ectopic expression of BdWRI1 also cause cell death in the leaf blades; this may be due to the high content of free fatty acids [14]. Taking these findings together, it appears that the specific phenotypes resulting from ectopic expression of the WRI1 gene depend on plant species even within the same family.

As in other plant species, the WRI1 gene has a function in FA biosynthesis in Arabidopsis and rice [8, 12, 33]. The expression of OsWRI1a under the control of the P1 promoter from the Bt2 gene was sufficient to increase the lipid content of rice leaves and endosperms (Figs 5G and 6A). The underlying mechanism involves the transcriptional up-regulation of genes related to late glycolysis and fatty acid biosynthesis in leaves and endosperms (S3C Fig; Fig 7). An unresolved question was why, although the oil content was increased, the net content in mature endosperms was no more than 1% (Fig 6A and S4B Fig). The same phenomenon was also observed in maize seeds as a result of overexpression of ZmWRI1a [12, 13]. The first possible explanation is the cytosolic localization of ADP–glucose pyrophosphorylase in rice and maize endosperms, which is responsible for partitioning large amounts of carbon into starch [34]. Another possibility is that the WRI1 protein regulates mainly the expression of the genes involved in glycolysis and FA biosynthesis, not those of the TAG assembly pathway. The third possibility is that, in oilseeds, the accumulation of oils in endosperms or cotyledons accompanies the degradation of starch and the assembly of TAGs [2, 26]. In contrast, TAGs seem to be rapidly turned over in leaves in B. distachyon plants overexpressing BdWRI1 [14]. Thus, ectopically expressing solely the OsWRI1 gene in rice, another grass species, would not be expected to result in the large-scale accumulation of TAG in leaves or endosperms.

Conclusion

In this study, we cloned the OsWRIa gene and analyzed its function in rice. The OsWRI1a gene encoded a nuclear localization protein. OsWRI1a-overexpressing plants not only increased oil content in transgenic rice leaves and endosperm, but also resulted in abnormal growth and development of transgenic plants. This result adds to our understanding of the AP2 family gene WRI1 in plant growth and development.

Primers used in this study.

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Phenotype of transgenic plants carrying 35S::OsWRI1a constructs.

Putative transformants were selected by detecting GUS reporter gene expression by means of a GUS staining assay. The (three-month-old) seedlings exhibited dwarfism, the upper parts of the laminae of most leaves were chlorotic and curly, and these leaves died more rapidly than those of wild-type plants.

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Roots of 3-leaf stage in wild-type and transgenic plants carrying PBt2P1::OsWRI1a transgenic constructs.

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Lipid content and expression of genes predicted to encode proteins involved in the later stages of glycolysis and fatty acid biosynthetic pathways in leaves.

(A) The levels of OsWRI1 transcripts in flag leaf (1st leaf), the second (2nd leaf) and the third (3rd leaf) leaves at the stage of the flag leaf was about 10 cm in length in two P::OsWRI1a transgenic lines of P1-6 and P1-7. (B) The crude lipid content in leaves of heading stage plants. Values represent means of n = 6 ± SD (Duncan test: **, P < 0.01). DW = dry weight. (C) Expression of genes predicted to encode proteins involved in the later stages of glycolysis and fatty acid biosynthetic pathways in leaves. Total RNA was isolated from the second leaves of plants at heading time. The experiment included three biological replicates, each with two technical replicates. Values represent means of n = 6 ± SD (Duncan test: **, P < 0.01).

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Changes in grain weight and lipid accumulation in endosperm.

(A) The 1000-seed weight. Seed weights were calculated by randomly selected seeds. The mature seeds were dried under 37°C in an oven for three days. Values represent means of n = 3 ± SD (Duncan test: **, P < 0.01). (B) The crude lipid content in endosperm of mature seeds. Values represent means of n = 6 ± SD (Duncan test: **, P < 0.01; *, P < 0.05). DW = dry weight.

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7 Mar 2022

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Partly

Reviewer #2: Partly

**********

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

**********

3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: No

Reviewer #2: Yes

**********

4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: No

Reviewer #2: Yes

**********

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The manuscript lacks the novelty component and there are several technical points that makes this study not suitable for publication in the present form. The function of WRINKLED1 gene in context of lipid biosynthesis and their effect on plant growth has been already reported by various researcher previously in different plants. Author only reported the role of rice WRINKLED1 gene lipid biosynthesis and their effect on plant vegetative growth.

My specific comments are as under:

• Information on the confirmation of the integration and homozygosity of transgene in transgenic rice plants is missing.

• Line 189-190:- The author reported that the transcripts level of OsWRI1a in transgenic rice was around 25-35 fold high in leaves tissue (Fig 4D) as compared to control plant leaves. The author generated the OsWRI1a overexpression transgenic rice plant under endosperm specific Bt2 P1 (PBt2P1) promoter (highly expressed in endosperm and lower in leave; line:-174-175). It is not clear as to why OsWRI1a overexpression lines showed elevated OsWRI1a transcript levels in leaves tissue when it is an endosperm specific promoter.

• There is no insight into why OsWRI1a overexpression lines showed retarded plant growth phenotypes.

Reviewer #2: Comments on the manuscript entitled, “Ectopic expression of Wrinkled1 in rice improves lipid biosynthesis but retards plant growth and development”

The authors looked at two rice gene calls with functional annotation with the transcription factor that acts as a global regulator of fatty acid biosynthesis wrinkled1 (Wri1). They demonstrated that as expected the OsWri1a is post translationally targeted to the nuclear compartment using an Arabidopsis GFP fusion platform. The authors then assembled two cassettes to ectopically express OsWri1 constitutively and in a seed preferred fashion. The rationale for doing this is found on line 163 of the manuscript: “Ectopic expression of the WRI1 gene in several plant species results in an increase in oil accumulation in leaves and other organs. To investigate whether ectopic expression of this gene exerts the same effect in rice, we overexpressed the WRI1a gene under the control of the CaMV 35S promoter.”

This is where the problem lies in their communication. There are previous publications relating to expression of the AtWri1 in rice, that include additional alleles to pull carbon towards TAG biosynthesis (AtDGAT/AtPDAT) and protect TAG turnover (AtOle) see Izadi-Darbandi et al 2020 Mol Biol Rep 47:7917, and ectopically express AtWri1 alone in rice see Yang et al 2019 Biosci, Biotechnol, Biochem 83:1807. In the previous publications, significant increase in vegetative tissue fatty acids, with some seed reserve changes, but no phenotypic off types observed. The two key differences between this manuscript and the previous published ones, are ectopic expression of the OsWri1 and the rice genotype. The authors cannot ignore these publications, and will need to address, or at least attempt to explain why the different outcomes. In addition, Mano et al 2019 Plants8, 207; doi:10.3390/plants8070207 communicate on much deeper in silico analysis on rice Wri1 alleles. This publication is also not mentioned in this manuscript.

Secondly, the rationale for targeting lipid biosynthesis in a grain feedstock would be to see if this approach can impact harvest index, and thus grain yield. The outcome of merely stating see what happens, was expected.

**********

6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

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Reviewer #1: Yes: Arun Kumar

Reviewer #2: Yes: Tom Elmo Clemente

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19 Mar 2022

Dear Editor and Reviewers:

Thank you, Respected Editor and Reviewers, for your valuable comments, and thank you for your precious time dedicated to reviewing this paper. The manuscript has been carefully revised, and all the sections and parts were modified according to the comments. The authors hope that the manuscript in the revised form meets the expectations of the reviewer and can express the content of our research to the community with more clarity. Revised portions of the paper are marked in red. In addition, the paper has been edited by a native-English speaker with a PhD in a relevant discipline working for the professional editing company Sees-editing Ltd.

We hope you will consider this work for publication in PLos One. Thank you again for your consideration.

Sincerely,

Huawu Jiang

Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The manuscript lacks the novelty component and there are several technical points that makes this study not suitable for publication in the present form. The function of WRINKLED1 gene in context of lipid biosynthesis and their effect on plant growth has been already reported by various researcher previously in different plants. Author only reported the role of rice WRINKLED1 gene lipid biosynthesis and their effect on plant vegetative growth.

Response: Thank you for your comments concerning our manuscript. Professor, the purpose of this paper is to test whether WRINKLED1 gene can improve the endosperm oil content of starchy crops. The results showed that in the endosperm of starchy crops, increasing the expression of WRINKLED1 could only slightly increase the oil content. At the same time, we further found that increasing the expression of WRINKLED1 affected the growth of leaves of transgenic plants. Finally, thank you very much for your time and effort in reviewing our manuscript.

My specific comments are as under:

• Information on the confirmation of the integration and homozygosity of transgene in transgenic rice plants is missing.

Response: Thank you, professor, for your comments and valuable addition. This is an important point that we should have mentioned. Professor, we have added such important information forward to lines 114-118 in the manuscript as you suggested. The contents are as follows: The screening of homozygotes was based on a segregation ratio of about 3:1 observed in T2 plants. The segregation ratios of four transgenic plants, were confirmed by GUS staining, were 237:78, 197:64, 169:55 and 206:67, respectively. Then we randomly selected 30 T3 plants for GUS staining. We speculated that these plants were homozygous plants for all GUS-stained plants.

• Line 189-190: The author reported that the transcripts level of OsWRI1a in transgenic rice was around 25-35 fold high in leaves tissue (Fig 4D) as compared to control plant leaves. The author generated the OsWRI1a overexpression transgenic rice plant under endosperm specific Bt2 P1 (PBt2P1) promoter (highly expressed in endosperm and lower in leave; line:-174-175). It is not clear as to why OsWRI1a overexpression lines showed elevated OsWRI1a transcript levels in leaves tissue when it is an endosperm specific promoter.

Response: Thank you, Professor, for your comments. Although Bt2 gene was mainly expressed in endosperm, it was also weakly expressed in leaves. Therefore, we speculate that using the promoter of Bt2 gene to drive OsWRI1 gene expression will further increase the expression of OsWRI1 gene in the leaves of transgenic plants. In addition, in the endosperm, the blue product produced by GUS reaction appeared after 10 minutes of reaction and reached saturation in 30 min. However, in the leaves, it was difficult to detect GUS activity after 2 hours of reaction, and there was blue product after 4 hours of reaction (Pan X, Jiang H, Yan H, Li M, Wu G. Promoter analysis of the gene encoding ADP-glucose pyrophosphorylase small subunit I in rice. Journal of Tropical and Subtropical Botany. 2008; 16: 189–194 (in Chinese).).

• There is no insight into why OsWRI1a overexpression lines showed retarded plant growth phenotypes.

Response: Thank you, Professor, for your comments and suggestions. Professor, actually during the revision process of the manuscript we can make changes to the manuscript. With due respect Sir, contribution to the field is an option while submitting the new article. Sir, your suggestion is valuable for us and we will carefully keep this valuable point in our mind for the next time we submissions to PLos One Journals. In addition, because a high level of expression of the OsWRI1a gene (under the control of the CaMV 35S promoter) seriously inhibited rice growth, the transgenic plants died and we couldn't get the seeds. Therefore, we have not carried out the research on the relevant mechanism.

Reviewer #2: Comments on the manuscript entitled, “Ectopic expression of Wrinkled1 in rice improves lipid biosynthesis but retards plant growth and development”

Response: Thank you, Respected Professor, for your valuable comments, and thank you for your precious time dedicated to reviewing this paper.

• The authors looked at two rice gene calls with functional annotation with the transcription factor that acts as a global regulator of fatty acid biosynthesis wrinkled1 (Wri1). They demonstrated that as expected the OsWri1a is post translationally targeted to the nuclear compartment using an Arabidopsis GFP fusion platform. The authors then assembled two cassettes to ectopically express OsWri1 constitutively and in a seed preferred fashion. The rationale for doing this is found on line 163 of the manuscript: “Ectopic expression of the WRI1 gene in several plant species results in an increase in oil accumulation in leaves and other organs. To investigate whether ectopic expression of this gene exerts the same effect in rice, we overexpressed the WRI1a gene under the control of the CaMV 35S promoter.” This is where the problem lies in their communication.

Response: Thank you, Professor, for your valuable comments. Professor, we have carefully revised the manuscript according to your suggestions. Thank you very much for your time and effort in reviewing our manuscript.

There are previous publications relating to expression of the AtWri1 in rice, that include additional alleles to pull carbon towards TAG biosynthesis (AtDGAT/AtPDAT) and protect TAG turnover (AtOle) see Izadi-Darbandi et al 2020 Mol Biol Rep 47:7917, and ectopically express AtWri1 alone in rice see Yang et al 2019 Biosci, Biotechnol, Biochem 83:1807. In the previous publications, significant increase in vegetative tissue fatty acids, with some seed reserve changes, but no phenotypic off types observed. The two key differences between this manuscript and the previous published ones, are ectopic expression of the OsWri1 and the rice genotype. The authors cannot ignore these publications, and will need to address, or at least attempt to explain why the different outcomes. In addition, Mano et al 2019 Plants8, 207; doi:10.3390/plants8070207 communicate on much deeper in silico analysis on rice Wri1 alleles. This publication is also not mentioned in this manuscript. Secondly, the rationale for targeting lipid biosynthesis in a grain feedstock would be to see if this approach can impact harvest index, and thus grain yield. The outcome of merely stating see what happens, was expected.

Response: Thank you, Professor, for your valuable comments. Professor, this is an important point that we should have mentioned in the discussion. According to your comments, we have added a discussion of why AtWRI1 and OsWRI1 function differently in rice in the revised MS, in lines 245-251.

In addition, professor, we have added the references about Mano et al 2019 Plants8, 207; doi:10.3390/plants8070207 in the revised MS.

Finally, we hope that the manuscript in the revised form meets the expectations of the reviewer and can express the content of our research to the community with more clarity. Thank you again for your consideration.

Submitted filename: Response to reviewers.doc

Click here for additional data file.

14 Apr 2022

Ectopic expression of WRINKLED1 in rice improves lipid biosynthesis but retards plant growth and development

PONE-D-22-01261R1

Dear Dr. Jiang,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

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Kind regards,

Tamar Juven-Gershon, Ph.D.

Academic Editor

PLOS ONE

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #2: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: Authors have addressed my comments satisfactorily. Just found one typo mistake in Figure 3 (Please change Brihgt to Bright).

Reviewer #2: The authors likely observed relatively high miss expression of the Brittle2 promoter given the use of pCAMBIA1301binary vector, for whatever orientation the Brittle2-OsWRI1 cassette was cloned in, the Brittle2 and a 35S CaMV promoter element would reside 5' to 5', which will impact specificity of Brittle2 expression

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: Yes: Dr. Arun Kumar

Reviewer #2: Yes: Tom Elmo Clemente

11 Aug 2022

PONE-D-22-01261R1

Ectopic expression of WRINKLED1 in rice improves lipid biosynthesis but retards plant growth and development

Dear Dr. Jiang:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

If we can help with anything else, please email us at plosone@plos.org.

Thank you for submitting your work to PLOS ONE and supporting open access.

Kind regards,

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on behalf of

Prof. Tamar Juven-Gershon

Academic Editor

PLOS ONE