Cloning and Characterization of Protein Prenyltransferase Alpha Subunit in Rice

Wang Tao, Lou Lijuan, Li Zeyu, Shang Lianguang, Wang Quan

Research Paper

Cloning and Characterization of Protein Prenyltransferase Alpha Subunit in Rice

Wang Tao, Lou Lijuan, Li Zeyu, Shang Lianguang, Wang Quan

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Protein prenylation plays a crucial role in plant development and stress response. We report the function of prenyltransferase α-subunit in rice. Protein-protein interactions showed that the farnesyl- transferase (OsPFT)/geranylgeranyltransferase-I (OsPGGT I-α) protein interacted together with OsPFT-β and OsPGGT I-β. The α- and β-subunits of OsPFT formed a heterodimer for the transfer of a farnesyl group from farnesyl pyrophosphate to the CaaX-box-containing peptide N-dansyl-GCVLS. Furthermore, the tissue expression patterns of the OsPFT and OsPGGT I subunits were similar, and these subunits were localized in the cytoplasm and nucleus. Moreover,-deletion homozygous rice mutants had a lethal phenotype, and the heterozygous mutants exhibited reduced pollen viability. These results indicated that prenylation plays an important role in rice development.

protein prenylation; plant development; rice; pollen viability

Protein prenylation is a posttranslational modification (PTM), in which isoprenoid lipid chains are added to target proteins to regulate plant development processes. Prenylation facilitates the targeting of lipid proteins to various cellular membranes and mediates protein- protein interactions through hydrophobic chains (Running,2014). There are three heterodimeric enzyme complexes capable of performing prenylation: protein farnesyl- transferase (PFT), protein geranylgeranyltransferase I (PGGT I) and Rab geranylgeranyltransferase (PGGT II) complexes. PFT and PGGT I share a common α- subunit (PFT/PGGT I-α) but have a distinct β-subunit (PFT-β or PGGT I-β) that determines substrate specificity. Compared with PFT and PGGT I, PGGT II contains different α- and β-subunits and also requires Rab escort protein (REP) for its activity (Leung et al, 2006). PFT recognizes the CaaX-box (‘C’ stands for cysteine; ‘a(chǎn)’ is usually an aliphatic amino acid; and ‘X’ represents alanine, serine, methionine, glutamine or cysteine) at the C-terminus of its target proteins. PGGT I modifies the same protein sequence as PFT does, except that X is usually leucine. PGGT II does not recognize the specific C-terminus box and is less characterized in plants. It has been demonstrated that a more complex C-terminal protein sequence is required for PGGT II, including the presence of one or two cysteines in the target protein (Hala et al, 2005).

Prenylation facilitates the addition of C15 isoprene farnesyl or C20 isoprene geranylgeranyl groups to various proteins, which is beneficial for their adhesion to cell membranes (Zhang and Casey, 1996; Maurer-Stroh et al, 2003; Turnbull and Hemsley, 2017). Protein prenylation was discovered almost four decades ago in fungi, and it involves modification of peptide pheromonesused during mating (Kamiya et al, 1978; Sakagami et al, 1981). To date, most researches on prenylation focus on mammals and yeast. Thegene in mammals is a substrate of farnesyltransferase and is one of the most easily activated oncogenes (Rowinsky et al, 1999). The biological activity of Ras proteins is closely related to farnesylation. It is known that interfering with the farnesylation of Ras inhibits tumor growth. However, the complete loss of farnesyltransferase in yeast or animals is lethal (Defeojones et al, 1985; Kataokaet al, 1985). Accordingly, the farnesylation modification pathway has received widespread attention, especially in terms of human cancer therapies.

A number of genetic studies have revealed the important roles of PFT and PGGT I in plant development and in response to environmental stress. For instance, the() gene encodes PFT/PGGT I-α;mutants exhibit severe developmental defect phenotypes, including delayed flowering time, a substantially larger shoot meristem, the presence of extraflower organs or decreased pollen fertilization. The relatively large shoot meristems ofmutants may result from defects in primordial differentiation initiated from the shoot apical meristem (Running et al, 2004). Furthermore,mutants are more sensitive to abscisic acid (ABA) and therefore, more drought tolerance compared withwild type (WT) plants (Wang et al, 2009).

The first piece of evidence concerning the involvement of prenylation in plant development, as well as the stress response, was found by screening mutants that presented enhanced sensitivity to ABA (Cutler et al, 1996; Pei et al, 1998). Specifically,plants with a mutation in the farnesyl- transferase β-subunit gene, which is also called(), present increased sensitivity to ABA (Cutler et al, 1996; Pei et al, 1998; Allen et al, 2002; Brady et al, 2003). Owing to its involvement in the negative regulation of ABA signaling, farnesylation can coordinate physiological processes to regulate responses to heat and drought stress. Briefly, mutations in thegene confer drought and heat tolerance phenotypes in, canola, wheat, barley and soybean (Wang et al, 2005; Manmathan et al, 2013; Ogata et al, 2017; Daszkowska-Golec et al, 2018; Wu et al, 2019). In addition,mutants also exhibit developmental defects, such as increased shoot apical meristem and floral meristem sizes, resulting in abnormal phyllotaxy and extra sepals and petals (Bonetta et al, 2000; Yalovsky et al, 2000a). In subsequent research, the farnesylation target transcription factor APETALA1 is responsible for floral meristem organization (Cutler et al, 1996; Bonetta et al, 2000; Yalovsky et al, 2000b). However, it took a long time to explain the phenotype of the enhanced response to ABA and stomatal closure, because there is no obvious farnesyltransferase recognition motif present among components of the ABA signal transduction pathway. Finally, the HSP40 protein was identified as a target of farnesylation, and mutations in the gene encoding this protein result in phenotypic defects. Another farnesylated protein, CYP85A2, a cytochrome P450 protein, is also proposed to participate in the development of the pleiotropicphenotype (Northey et al, 2016; Barghetti et al, 2017).

To date, most of the studies on prenylation in plants focus on, while little is known about its function in rice. It is essential to characterize this corresponding type of modification in rice to reveal the mechanism underlying prenylation as well as its facilitation of stress resistance mechanisms in crop plants, such as the responses to heat and drought stress. Protein-protein interaction analysis indicated that OsPFT/OsPGGT I-α interacts together with OsPFT-β and OsPGGT I-β. Continuous fluorescence assays revealed that OsPFT/OsPGGT I-α and OsPFT-β have biochemical activity, and confocal fluorescence microscopy revealed that these subunits are localized in the cytoplasm and nucleus. In addition, the expression patterns of OsPFT/OsPGGT I-α, OsPFT-β and OsPGGT I-β were similar across different rice tissues. Rice-deletion homozygousmutants had a lethal phenotype, and heterozygous mutants showed some developmental defects and decreased pollen viability. All of these results indicated that prenylation plays an important role in rice.

Results

Sequence alignment and phylogenetic analysis of OsPFT/OsPGGT I-α, OsPFT-β and OsPGGT I-β

To identify the rice prenyltransferase subunit, relatedgenes(),(),() were used to query the rice genome database via BLAST (Rice Genome Annotation Project, http://rice.plantbiology. msu.edu). The results showed that(),() and() were most similar to,andsubunit genes, respectively. All the three genes were subsequently cloned from rice. Thecoding DNA sequence (CDS) comprises 1 020 nucleotide (nt) and encodes 339 amino acid, while theCDS comprises 1 530 nt and encodes 509 amino acid. When querying the sequence ofin the different databases via BLAST usingas a template, we found three isoforms with lengths of 535, 666 and 1 044 nt.

Yeast two-hybrid (Y2H) interaction assays were performed between OsPFT/OsPGGT I-α and the three isoforms, and the final results showed that the 1 044 nt isoform () can interact with OsPFT/OsPGGT I-α, implying that this isoform was functional in rice (Fig. S1). As the prenyltransferase subunit usually contains a prenyltrans domain that is conserved in different species, multiple sequence alignments of deduced amino acid sequences of the prenyltransferase subunit in rice,and humans were performed (Fig. 1). The conserved prenyltransferase α-subunit repeat domains of PFT/ PGGT I-α were shown in the red rectangles. The similarities in protein sequence between OsPFT/ OsPGGT I-α with AtPFT/AtPGGT I-α and HsaPFT/ HsaPGGT I-α were 63.69% and 54.59%, respectively (Fig. 1-A). In Fig. 1, squalene oxidase repeat domains in PFT-β and PGGT I-β are marked by solid and dotted blue rectangles. The similarities in protein sequence between OsPFT-β with AtPFT-β and HsaPFT-β were 53.23% and 47.03%, respectively (Fig. 1-B), and the similarities in protein sequence between OsPGGT-β with AtPGGT-β and HsaPGGT-β were 65.87% and 83.00%, respectively (Fig. 1-C). Taken together, the results indicated that these subunits of prenyltransferase were conserved among rice,and humans.

A phylogenetic tree was constructed based on data from different plant species, including,,,,,,,,,and.The protein sequences of PFT/PGGT I-α, PFT-β and PGGT I-β from these species obtained from the Plaza Plant Comparative Genomics Database (http:// bioinformatics.psb.ugent.be/plaza/) were queried via BLAST (Fig. S2). The three subunits involved in protein prenylation in rice clustered closely with those in other monocots, such asand, but clustered far from those inand(Fig. S2).

Fig. 1. Multiple sequence alignment of deduced amino acid sequences of prenyltransferase subunit and molecular.

A, Amino acid sequence alignment of OsPFT/OsPGGT I-α subunit with HsaPFT/HsaPGGT I-α and AtPFT/AtPGGT I-α subunits. Letters in the black boxes indicate highly conserved amino acid residues. The red solid frames represent the protein prenyltransferase alpha subunit repeat domain.

B, Amino acid sequence alignment of OsPFT-β subunit with HsaPFT-β and AtPFT-β subunits. The blue solid frames represent the prenyltransferase and squalene oxidase repeat domain.

C, Amino acid sequence alignment of OsPGGT I-β subunit with HsaPGGT I-β and AtPGGT I-β subunits. The blue dotted frames represent the prenyltransferase and squalene oxidase repeat domain.

OsPFT/OsPGGT I-α interacts together with OsPFT-β and OsPGGT I-β

Since prenyltransferase subunits usually form heterodimers to function, we investigated the interactions between the OsPFT/OsPGGT I-α subunit with the OsPFT-β subunit or OsPGGT I-β subunit. We initially performed Y2H assays. As shown in Fig. 2-A, all clones grew well in SD-L-W media, and when on selective SD-L-W-H-A media, only cells coexpressing OsPFT/OsPGGT I-α and OsPFT-β could grow. Moreover, luciferase complementation imaging (LCI) analysis revealed that OsPFT/OsPGGT I-α/OsPFT-β coexpression displayed strong luminescence signals that were not detected in the control (Fig. 2-C). Furthermore, co-immunoprecipitation (Co-IP) experiments indicated that OsPFT/OsPGGT I-α and OsPFT-β interacted with each other (Fig. 2-E). Similarly, interactions between OsPFT/OsPGGT I-α and OsPGGT I-β were also confirmed by Y2H, LCI and Co-IP assays (Fig. 2-B, -D and -F). In summary, these results indicated that the rice OsPFT/OsPGGT I-α subunit interacts together with OsPGGT I-β and OsPFT-β to form heterodimers.

Fig. 2. OsPFT/OsPGGT I-α interacts with OsPFT-β and OsPGGT I-β.

A and B, Yeast-two hybrid analysis of OsPFT/OsPGGT I-α and OsPFT-β interaction (A) and OsPFT/PGGT I-α and OsPGGT I-β interaction (B). Bait of OsPFT-β or OsPGGT I-β was fused to the GAL4 DNA-binding domain, and prey of OsPFT/OsPGGT I-α was fused to the GAL4 activation domain. Colonies growth on the SD-L-W-H-A medium represent positive interaction.

C and D, Firefly luciferase complementation imaging (LCI) assay in. The N-terminal half of luciferase (Nluc) was fused into OsPFT/OsPGGT I-α or OsPGGT I-β and the C-terminal half of luciferase (Cluc) was fuse into OsPFT/OsPGGT I-α or OsPFT-β.

E and F, Co-immunoprecipitation (Co-IP) assay for OsPFT/OsPGGT I-α and OsPFT-β(E) or OsPFT/OsPGGT I-α and OsPGGT I-β (F). OsPFT/OsPGGT I-α was fused to green fluorescent protein (GFP), OsPFT-β and OsPGGT I-β were fused to Nluc, respectively. These constructions and GFP control vector were transformed intoGV3101 strain and infiltrated into. OsPFT/OsPGGT I-α-GFP or GFP were immobilized on GFP-trap beads and incubated with OsPFT-β-Nluc or OsPGGT I-β-Nluc. Input represents equal amounts of purified OsPFT/OsPGGT I-α-GFP or GFP fusions and OsPFT-β-Nluc or OsPGGT I-β-Nluc before Co-IP.

Substrate assays of OsPFT/OsPGGT I-α and OsPFT-β heterodimers

To further characterize the function of OsPFT/ OsPGGT I-α/OsPFT-β heterodimers, we performed ancontinuous fluorescence assays. Briefly, OsPFT/OsPGGT I-α and OsPFT-β were purified fromthat contained pET28a::OsPFT/ OsPGGT I-α and pET28a::OsPFT-β recombinant plasmids, respectively. DANSYL-Gly-Cys-Val-Leu- Ser-OH (Dansyl-GCVLS) acts as a farnesyltransferase substrate peptide that contains the specific CaaX motif recognized by farnesyltransferase. As farnesylated Dansyl-GCVLS has an emission fluorescence at 505 nm when excited at a wavelength of 340 nm, the 505 nm fluorescence intensity signal was monitored during the assays. The fluorescence intensity did not increase in the mixture containing only OsPFT/OsPGGT I-α or OsPFT-β, implying that each individual subunit cannot transfer a farnesyl unit from farnesyl pyrophosphate (FPP) or a geranylgeranyl unit from geranyl geranyl pyrophosphate (GGPP) to Dansyl- GCVLS (Fig. 3). However, the fluorescence intensity increased rapidly when OsPFT/OsPGGT I-α, OsPFT-β and FPP but not GGPP were mixed together (Fig. 3). The results showed that OsPFT/OsPGGT I-α and OsPFT-β heterodimers can transfer a farnesyl unit from FPP but not a geranylgeranyl group from GGPP to the corresponding proteins that can be farnesylated.

Tissue expression pattern and subcellular location of rice prenyltransferase subunits

Next, we checked the expression levels of,andin different rice tissues, including the panicle, leaf blade, sheath, root and floral organs (anther, stigma and ovary) (Fig. 4), via real-time PCR analysis. The results showed that OsPFT/OsPGGT I-α, OsPFT-β and OsPGGT I-β exhibited higher expression levels in leaf blade. Furthermore, we measured their expression levels in floral organs and found thatexpressed higher level compared withandin floral organs. To identify the subcellular localization of prenyltransferase subunits, the CDSs of,andwere fused to a green fluorescent protein (GFP) tag, the constructs of which were transiently coexpressed together with the nuclear marker mCherry-NLS inleaves, and an empty GFP vector was used as a negative control. As shown in Fig. 5, all the three subunits were expressed mainly in the cytoplasm and nucleus.

Mutations of OsPFT/OsPGGT I-α via CRISPR/ Cas9 system

To investigate the biological function ofin rice development and/or stress responses, we generatedknockout mutants via the CRISPR/Cas9 strategy. Two guide RNA targets corresponding specifically to the first exon ofwere designed (Fig. 6-A). A total of six positive transgenic T0lines with mutations in the targets were obtained. Two lines with 2 bp and 182 bp deletions in thegene were selected for further analysis, and both of these mutations resulted in in-frame mutations in(Fig. 6-B). Notably, we failed to obtain any homozygous mutants, and only WT or heterozygous plants were obtained in each generation, suggesting that mutations inmay be lethal. Further observation of heterozygous plants revealed that, unlike WT plants, the CRISPR mutants had curled anthers (Fig. 6-C). Moreover, an I2-KI staining assay was performed to test the pollen viability. The results showed that the pollen viability in the mutants obviously decreased, with a concurrent reduction in the number of starch grains (Fig. 6-D). This was further verified by counting and calculating the ratio of nonviable pollen, which revealed that approximately 50% of the pollen was nonviable. The statistics analysis showed a significant difference between the CRISPR mutants and WT (Fig. 6-E). In summary,plays an important role in rice development.

Fig. 3.continuous ?uorescence assay for OsPFT/OsPGGT I-α and OsPFT-β activity measurement.

A,OsPFT/OsPGGT I-α and OsPFT-β activity assay using farnesyl pyrophosphate (FPP) as substrate. OsPFT/OsPGGT I-α + FPP and OsPFT-β + FPP were used as the negative controls.

B,OsPFT/OsPGGTI-α and OsPFT-β activity assay using geranyl geranyl pyrophosphate (GGPP) as substrate. OsPFT/OsPGGT I-α + GGPP and OsPFT-β + GGPP were used as the negative controls.

The fluorescence assays were performed in the Tecan Infinite Pro Microplate Reader and monitored using an excitation wavelength of 340 nm and emission wavelength of 505 nm.

Fig. 4. Gene expression pattern in different orgens and tissues.

Gene expression levels of,andin different rice organs or tissues. Ricewas used as the reference. Data are Mean ± SD (= 3).

Fig. 5. Subcellular localization assay of GFP-tagging OsPFT/OsPGGT I-α, OsPFT-β and OsPGGT I-β in.

The assays were performed using mCherry-NLS as a nucleus-location signal by the confocal laser microscope scanning. The scale bar is 10 μm in the first column, and 25 μm in the next three columns.

Discussion

Protein prenylation was first discovered in animals and is important for cell signaling and membrane trafficking, with great potential in biomedicine and biotechnology applications (Defeojones et al, 1985; Kataoka et al, 1985; Rowinsky et al, 1999). In plants, an increasing number of attempts have been carried out to modify prenylation levels in plants to enhance drought tolerance. The first application of prenylation was conducted in canola (), in which the expression of the α- and β-subunits of farnesyl- transferase was downregulated under drought-inducible conditions based on an antisense hairpin (Wang et al, 2009). Similar studies have also shown that, compared with WT plants, transgenic plants produce substantially more seeds under drought conditions (Wang et al, 2005). Subsequently studies showed that the loss of function of the β-subunit involved in farnesylation also confer drought tolerance to soybean and barley plants compared with WT plants (Ogata et al, 2017; Daszkowska-Golec et al, 2018).

Fig. 6. Mutation analysis inby CRISPR/Cas9-mediated genome editing system.

A, Schematic diagram of the designed targeted sites in. 3′/5′ UTRs, exons and introns are indicated by green rectangles, blue rectangles and black lines, respectively. The target sequences are underlined, and the protospacer adjacent motif (PAM) is emphasized in red letters. Sequence analyses of the targeted regions inare in T3transgenic plants. The deletion sequence is shown by short solid line. CR-1 and CR-2 refer to the two OsPFT/OsPGGT I-α mutants; WT refers to the wild type.

B, PCR assay of T3transgenic lines in CR-2. Each lane represents an independent CRISPR/Cas-mediated editing line.

C, Comparison of WT and Hetero CR-1/CR-2 in floret at the mature pollen stage. Hetero CR-1 and Hetero CR-2 represent the heterozygous OsPFT/OsPGGT I-αCR-1/2. Scale bars are 1 mm.

D, Pollen was stained with I2-KI. The red arrows marked the sterile pollen. Scale bars are 30 μm.

E, Statistic of sterile pollen. The error bars at each data point indicate the standard error obtained from three biological replicates. **, Significant difference at the 0.01 level.

In this study, we isolated prenylation-related enzymes in rice and found that these enzyme subunits can form heterodimers, while PFTase α- and β-subunitscan farnesylate CaaX motif peptides. Importantly, the deletion of the α-subunit constituent of both farnesyltransferase and geranylgeranyltransferase resulted in a lethal phenotype, indicating that prenylation plays a significant role in rice development. Interestingly, a previous report showed thatα-subunit mutants exhibit no lethal phenotypes (Running et al, 2004), suggesting that prenylation is specific in different plant species. In line with this prenylation specificity, the loss of the geranylgeranyltransferase β-subunit causes more severe phenotypic defects than the loss of the constituent geranylgeranyltransferase α-subunit in the moss(Thole et al, 2014). In addition, all the three components are expressed at different levels in different tissues, suggesting the potential role of different substrate levels in the output of protein prenylation. For example, in, AP1 and CAL have specific CaaX motifs (Yalovsky et al, 1999, 2000b), but their orthologs in rice do not have this motif. Similarly, CYP85A2 proteins that can be farnesylated arespecific (Northey et al, 2016). Taken together, given the regulatory effects of prenylation on the growth and stress tolerance of plants, our results are beneficial for understanding the prenylation of functional proteins in plants, especially crop plants.

methods

Sequence analysis and phylogenetic tree construction

The protein sequences of PFT/PGGT I-α, PFT-β and PGGT I-β in different species were searched against Plaza Plant Comparative Genomics database (https://bioinformatics.psb. ugent.be/plaza/). Multiple amino acid sequence alignment was performed using the MEGA7.0 and GeneDoc softwares and the protein domain was analyzed by SMART (http://smart.embl. de/). The phylogenetic tree was constructed based on the full-length amino acid sequence by the MEGA7.0 software using the maximum likelihood method and 1000 bootstrap replicates.

Yeast strain and yeast two-hybrid assay

The yeast strain Y2H gold (,,,,,,,and) was used for Y2H assays. The Y2H interaction assay was performed using Frozen-EZ Yeast TransformationIIKit (ZYMO Research, Orang, USA). Briefly, the appropriate bait and prey constructions were co-transformed into the Y2H gold competent yeast cells following the standard transformation protocol of the kit. A total of 2 μL liquid culture was dropped on the SD-L-W double dropout medium and SD-L-W-H-A quadruple dropout medium respectively and incubated at 30 oC for 72 h.

Luciferase complementation imaging (LCI) assay

LCI assays were analyzed as described previously (Chen et al, 2008). The full-length CDS ofandwere ligated into pCAMBIA-Cluc and pCAMBIA-Nluc vectors, respectively to generate Cluc-OsPFT-β and OsPFT/ OsPGGT I-α-Nluc. The Cluc-/Nluc-derivative constructs were transformed intostrainGV3101 and cultured for 48 h. After overnight cultivation, thestrain were resuspended and mixed with equal volume. Then, the mixture was infiltrated intoleaves. The luciferase activity was detected with a Tanon 6600 Luminescent Imaging Workstation (Tanon, China).

Co-immunoprecipitation assay

Theandfull length CDS were ligated into the pCAMBIA-1300-221-GFP.1 and pCAMBIA- Nluc vector, respectively. The GFP and Nluc-derivative constructs were transformed intostrain GV3101 and cultured for 48 h, then injected intoleaves after cultivated at 28 oC. Total proteins of OsPFT/OsPGGT I-α-GFP or GFP alone were extracted from leaves with native buffer, incubated with anti-GFP beads for 2 h, then added the OsPFT-β-Nluc fusion protein into the protein bound beads for another 2 h. The binding proteins on the beads were resuspended using 30 μL protein loading buffer (1×) and boiled for 5 min, and the interacting proteins were then detected through Western Blotting using GFP and luciferase antibody. The Co-IP interaction of OsPGGT I-β-Nluc and OsPFT/OsPGGT I-α-GFP was conducted as above mentioned.

In vitro continuous ?uorescence assay

Thecontinuous fluorescence assays were performed as described previously (Dozier et al, 2014). Briefly, the peptide N-dansyl-GCVLS (Meilunbio, Dalian, China) was incubated with dithiothreitol (DTT) at 4 oC for 30 min in a final 100 μL volum, prior to adding the assay buffer (100 μL) with a final concentration of 4 μmol/L of dansyl-GCVLS, 10 mmol/L of DTT, 50 mmol/L of Tris-HCl (pH 7.5), 10 mmol/L of MgCl2, 10 μmol/L of ZnCl2, 0.2% of-octyl-β-dglucoside, 25 μmol/L of FPP or 25 μmol/L of GGPP, individually. Nearly 20 μg of the enzyme (His-tag fused OsPFT-β and OsPFT/OsPGGT I-α proteins) were mixed with the assay buffer, and the fluorescence were monitored in the Tecan Infinite Pro Microplate Reader (Tecan, Austria) with an excitation wavelength of 340 nm and an emission wavelength of 505 nm.

Subcellular localization

Theandfull length CDS were ligated into the vector pCAMBIA-1300-221-GFP.1, mCherry- NLS was used as the nucleus location marker. The GV3101 harboringorwas mixed with mCherry-NLS in a 1:1 ratio. After transiently infiltrated inleaves, the green and red florescence signals were visualized with a confocal microscopy (Leica TCS SP8, Germany). The excitation wave lengths of GFP and mCherry were 488 and 580 nm, respectively, and the emission wave lengths were 506 and 610 nm, respectively.

I2-KI staining

The spikelet in the heading rice was taken into the 75% ethanol. The anthers were crushed with forceps to fully release the pollen grains and add 1 to 2 drops of 1% I2-KI solution in the slides. The glass was covered and pressed gently for 2–3 min and finally observed the pollen viability under a 10× microscope (OLYMPUS, Japan).

Real-time PCR

Total RNAs were isolated from rice tissues using a RNA extraction kit (QIAGEN Rneasy Mini Kit, Germany) according to the manual. The RNA samples were used as the template for cDNA synthesis according to the Quantscript RT Kit (Tiangen, Beijing, China) manual. Real-time PCR was analyzed using primer pairs in the ChamQ SYBR qPCR Master Mix (Vezyme, Nanjing, China) with the Bio-Rad CFX384 detection system. Firstly, we made the standard curves with the target gene and reference gene (). Then, samples, including panicle, leaf blade, sheath, root, anther, stigma and ovary were detected by real-time PCR. The gene expression pattern was calculated according to the standard curves.

SUPPLEMENTAL DATA

The following materials are available in the online version of this article at http://www.sciencedirect.com/journal/rice-science; http://www.ricescience.org.

Fig. S1. Interaction between OsPFT/OsPGGT I-α and its three isoforms in yeast-two-hybrid.

Fig. S2. Molecular phylogenetic tree analysis of selected prenyltransferase subunit amino acids among different species.

ACKNOWLEDGEMENTS

This study was supported by the Science Technology and Innovation Committee of Shenzhen Municipality of China (GrantNos. JCYJ20170303154319837 and JCYJ20170412155447658), and the Science Technology Innovation and Industrial Development of Dapeng New District, Shenzhen, China (Grant Nos. PT201901-18 and PT201901-20).

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Wang Quan (wangquan03@caas.cn)

15 August 2020;

14 May 2021

Copyright ? 2021, China National Rice Research Institute. Hosting by Elsevier B V

This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/)

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http://dx.doi.org/10.1016/j.rsci.2021.05.014

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