We treaded the Jinhui 10 seeds (JH10) (Oryza sativa L. ssp. indica) with 1% ethylmethane sulfonate. In T1 generation, the dvb1 was found and was inherited stably after more than three years of observation. JH10, a restorer, was used as the wild-type (WT) in all experiments, all plants were bred in Southwest University, Chongqing, China, under natural conditions.
Characterization of Mutant Phenotypes
The phenotypes of the dvb1 mutant and the wild type were compared over the entire growth period. Agronomic traits, including root length, plant height, and length and width of the panicle and internodes, were measured. The wild type and dvb1 mutant plants were photographed using a Canon 5DIII digital camera (Tokyo, Japan). The inner surface of the sheath was observed using a SU3500 scanning electron microscope (Hitachi, Japan) at an accelerating voltage of 5.0 kV at − 4 °C.
Paraffin Sectioning, Cryosectioning, and Histological Analysis
Leaves, panicle base, and culms were collected at the tillering stage from wild type and dvb1 mutant plants, and fixed in 50% ethanol, 0.9 M acetic acid, and 3.7% formaldehyde overnight at 4 °C. The fixed samples were dehydrated with a graded series of ethanol (50%, 70%, 85%, 95%, 100%, 100%, and 100%), infiltrated with xylene, and embedded in paraffin (Sigma, USA). A rotary microtome RM2245 (Leica, Germany) was used to cut 8-μm-thick sections, which were transferred onto poly-L-lysine coated glass slides, deparaffinized in xylene, dehydrated through an ethanol series, and stained with safranin-O and fast green. For cryosectioning, wild-type and dvb1 mutant leaves were cut into 0.5 cm pieces, placed in an embedding agent ENG50 (Thermo, USA), and rapidly frozen. The frozen samples were sliced to about 10 μm thickness, stained in hematoxylin and eosin solution, then dehydrated through a gradient ethanol series (30%, 50%, 70%, 85%, 95% and 100%). The sections were observed using an Eclipse E600 light microscope (Nikon, Japan).
Genetic Analysis and Fine-Mapping of DVB1
The dvb1 mutant was crossed with rice ‘Xinong 1A’ (bred by the Southwest University Rice Research Institute) to generate the F1 population; the F2 population was derived by self-fertilization of the F1 population. The F2 individuals that exhibited the mutant phenotype were selected and used to map DVB1. Gene fine-mapping was conducted using simple sequence repeat markers obtained from the publicly available rice databases Gramene (http://www.gramene.org) and the Rice Genomic Research Program (http://rgp.dna.affrc.go.jp/E/publicdata/caps/index.html). Insertion/deletion markers were developed from comparison of the genomic sequences of ‘Xinong 1A’ and ‘Jinhui 10’.
Phylogenetic Tree Construction
Sequences of DVB1 homologous proteins from other plant species were acquired by conducting a BLAST search of the Phytozome portal (v12.1.6) (http://Phytozome.jgi.doe.gov/pz/portal.html#!Search?show=BLAST). Vector NTI Advance 10 was used to align the amino acid sequences. The alignment was saved as a FASTA file and used for phylogenetic analysis with MEGA (v5.2). A phylogenetic tree was constructed using the neighbour-joining method based on the evolutionary model with the lowest Bayesian information criterion score. Bootstrap support values from 1000 replicates are presented for each node. The peptide sequences used for phylogenetic tree construction are listed in Supplementary Table S1.
Complementary Vector Construction of DVB1
To complement the mutant phenotype of dvb1, the 4215 bp genomic fragment that contained the DVB1 coding sequence, coupled with 1874 bp upstream (including the transcription promoter) and 450 bp downstream sequences, was amplified from the wild-type genomic DNA and cloned into the binary vector pCAMBIA1301. The recombinant complementation plasmids were introduced into the dvb1 mutant using the Agrobacterium tumefaciens-mediated method. The primer sequences used for vector construction are listed in Supplementary Table S2.
RNA Extraction and Quantitative Real Time PCR Analysis
Total rice RNA was extracted from dissected tissues of the root, culm, leaf sheath, leaf, and panicle, and purified using the RNAprep Pure Plant Kit (Tiangen, China) in accordance with the manufacturer’s instructions. The concentration, purity, and integrity of extracted RNA were determined using a NanoDrop 2000 spectrophotometer (Bio-Rad, China) and 1% agarose gel electrophoresis. All reverse transcriptions were performed using 1 μg total RNA with SuperScript® III Reverse Transcriptase (Invitrogen, China) employing the oligo(dT) 18 primer in accordance with the manufacturer’s instructions. After cDNA synthesis, all samples were diluted tenfold with sterilized water, and qRT-PCR analysis was performed with the SYBR Supermix Kit (Bio-Rad, China) and the ABI 7500 Sequence Detection System (Thermo Fisher, USA) in accordance with the manufacturer’s instructions. Three replicates were performed and normalized relative expression levels were calculated by the ∆∆Ct method using ACTIN1 as an endogenous control (Livak and Schmittgen 2001). The primers used are listed in Supplementary Table S2.
In Situ Hybridization
The 204 bp specific probe for DVB1 was amplified and labelled using the DIG RNA Labelling Kit (Roche, Switzerland) in accordance with the manufacturer’s recommendations. Pretreatment of sections, hybridization, and immunological detection were performed as described previously (Ma et al. 2017). The primer sequences used are listed in Supplementary Table S2.
Subcellular Localization in Rice Protoplasts
The full-length coding region of DVB1 without the stop codon was amplified and cloned into the Pan580 vector driven by double 35S promoter (2 × 35S promoter) to generate the 2 × 35S::DVB1-GFP (green fluorescent protein) fusion protein. Both Pan580-DVB1-GFP and empty Pan580-GFP plasmids were introduced into rice protoplasts as described previously (Ma et al. 2017). After incubation at 28 °C overnight, GFP fluorescence was observed using a LSM 780 confocal laser scanning microscope (Zeiss, Germany). Protoplasts were incubated with 0.5 mM MitoTracker™ Orange CM-H2TMRos and MitoTracker™ Orange CMTMRos (Invitrogen, China) for 30 min to stain the mitochondria (Wang et al. 2016). To explore whether subcellular distribution of DVB1 overlapped with a nuclear marker, DVB1-GFP and OsH2B-mCherry plasmids were co-introduced into rice protoplasts (Guo et al. 2018). To check DVB1G90E’s location, the full-length coding region of DVB1G90E without the stop codon was amplified and cloned into the Pan580 vector driven by double 35S promoter (2 × 35S promoter) to generate the 2 × 35S:: DVB1G90E-GFP (green fluorescent protein) fusion protein. The transformation method is as above. The primer sequences used are listed in Supplementary Table S2.
Subcellular Localization in Nicotiana benthamiana
The full-length coding sequence of DVB1 was cloned into the pCAMBIA1300 vector driven by single 35S promoter to generate the 35S::DVB1-GFP fusion protein. Plasmids were introduced into leaf cells of Nicotiana benthamiana using an Agrobacterium (strain GV3101)-mediated infiltration. The pCAMBIA1300 vector was infiltrated as a control. After infiltration for 36–56 h, leaf cells were incubated with 0.5 mM MitoTracker™ Orange CMTMRos (Invitrogen, China) for 30 min to stain the mitochondria (Tabatabaei et al. 2019). The GFP and MitoTracker signals were visualized using a LSM 780 confocal laser scanning microscope (Zeiss, Germany). The primer sequences used are listed in Supplementary Table S2.
Transmission Electron Microscopy
Tissue fixation and preparation for TEM followed a previously described method with minor modifications (Scafaro et al. 2011). Leaves at the tillering stage were collected and cut into 1 mm2 squares, fixed in fixative solution (3.5% glutaraldehyde, 2% paraformaldehyde, and 0.1 M phosphate buffer) for at least 48 h, and then post-fixed in 1% osmium tetroxide for 2 h after washing with 0.1 mol L− 1 phosphate saline buffer. Tissues were stained with uranyl acetate, dehydrated in ethanol, and embedded in Spurr’s medium before thin sectioning. Samples were stained again and mitochondria examined using a H-7500 transmission electron microscope (Hitachi, Japan).
Measurement of ATP Content
ATP extraction was performed as previously described (Zeng et al. 2013). Leaf tissue (0.1 g fresh weight [FW]) sampled at the tillering stage was homogenized in liquid nitrogen, mixed immediately with 400 μL of 5 M perchloric acid, and boiled for 10 min in a water bath. The solution was centrifuged at 16,000 g for 15 min at 4 °C. The clear supernatant was collected for ATP estimation by high-performance liquid chromatography (HPLC) using an ACQUITY™ UPLC™ BEH-C18 column (2.1*100 mm, 1.7 μm; Waters, USA) and H-Class UPLC™ Tunable UV Detector (Waters, USA).
Yeast Two-Hybrid Assay
The Matchmaker™ GAL4 Two-Hybrid System (Clontech, China) was used to determine whether the DVB1 and DVB1G90E proteins could form homotypic oligomers. The full-length DVB1 and DVB1G90E coding sequences were cloned into the pGAD-T7 and pGBK-T7 vectors, respectively. The 1–237 bp sequence of DVB1 (lacking the DVB1 mutation site) was cloned into the pGAD-T7 and pGBK-T7 vectors, and transformed into yeast cells. The DO-Leu-His-Ade and DO-Leu-Trp-His-Ade filter assays were performed in accordance with the manufacturer’s instructions to check self-activation of DVB1 BD and DVB1G90E BD and protein–protein interactions.
Gel Electrophoresis and Immunoblot Analysis
To produce a His-tagged protein, the full-length DVB1 and DVB1G90E coding sequences were cloned into the expression vector pET-32a (Novagen, China) using the Kpn1 and EcoR1 restriction enzymes, and introduced into E. coli strain BL 21 (DE3). Positive colonies were grown at 37 °C to an optical density at 600 nm (OD600). The fusion protein was induced by addition of isopropyl.
β-D-thiogalactopyranoside to a final concentration of 1 mM, and then was purified with Ni-NTA Sefinose™ Resin (BBI, China) following the manufacturer’s protocol. Immunoblot analysis by SDS-PAGE and Native PAGE using a His-tag antibody (Proteintech, USA) were performed as described previously (Fujita et al. 2003).
Leaves of wild-type and dvb1 mutant plants were sampled at the tillering stage for transcriptome sequencing experiments with three biological replicates (Allwegene Company, China). Total RNA was extracted from the samples using the TRIzol Reagent (Invitrogen, China) in accordance with the manufacturer’s instructions. RNA sequencing was conducted on an Illumina HiSeq platform. The RNA-Seq clean reads were aligned to the TIGA rice genome using TopHat (v2.1.0). The RNA-Seq data were analysed using a previously described method. Differentially expressed genes were detected using the DESeq R package (v1.10.1) with a relative change threshold of two (P < 0.05, false discovery rate < 0.01). Gene ontology (GO) categories were identified using the GOseq R package. KEGG pathways were assigned using the KEGG database (http://www.kegg.jp/) and were considered significant at P < 0.05.
Free IAA Measurement
Leaf tissue (100 fresh weight [FW]) sampled at the tillering stage was homogenized in liquid nitrogen, then 1 mL precooled sodium phosphate buffer was added and the homogenate was incubated at 4 °C overnight in the dark. After centrifugation at 8000 g for 10 min, the residue was diluted with 0.5 mL precooled sodium phosphate buffer for 2 h and then evaporated under N2 gas at 40 °C until the organic phase had disappeared. Petroleum ether (0.5 mL) was added to extract the residue three times, and the lower layer was evaporated to dryness under N2 gas at 40 °C. The residue was dissolved in 0.5 mL of the mobile phase (600 mL ultrapure water, 6 mL acetic acid, and 400 mL methanol) and then filtered. Free IAA in the extract was analysed using an Agilent 1100 HPLC (Agilent, USA) and Kromasil C18 reversed-phase column (250 mm * 4.6 mm, 5 μm; Waters, USA) (Krisantini et al. 2009).
Extraction and Measurement of Amino Acids
Leaf tissue (10 mg; FW) sampled at the tillering stage was homogenized in liquid nitrogen and extracted with 1 mL precooled extraction mixture (1% formic acid–methanol) for 10 min at 25 °C. After centrifugation at 13,000 g at 4 °C for 5 min, the supernatant was diluted fivefold. An aliquot (100 μL) of diluted supernatant was added to 100 μl of 100 ppb double isotope internal standard (Phe-1-13C and Val-1-13C) and vortexed for 30 s. After filtration, the supernatant was analysed by liquid chromatography–mass spectrometry (LC/MS) with regard to amino acid standards. An ACQUITY™ UPLC™ BEH C18 column (2.1 × 100 mm, 1.7 μm; Waters, USA) and electrospray positive ionization (ESI, USA) source was used for LC/MS (Liyanaarachchi et al. 2017).
Treatment with Mitochondrial Electron Transport Inhibitors
The antimycin A (ShangHai Maokang Biotechnology, China) and oligomycin (ShangHai Maokang Biotechnology, China) used were from acqueous dilutions of DMSO stock solutions (150 mM antimycin A; 20 mM oligomycin) to 1.25 μM antimycin A and 0.5 μM oligomycin. Wild-type and dvb1 mutant plants were treated with the inhibitor for about 4 days. The corresponding solution of water mixed with DMSO was used as the control. After treatment, the water, ATP and IAA contents were measured and analysed. The treated plants were photographed using a Canon 5DIII digital camera (Tokyo, Japan). All experiments were performed using at least six independent biological replicates.
Sequence data from this article can be found in the GenBank/EMBL libraries under the following accession numbers: OsDVB1 (LOC_03g62420), OsFIB (LOC_01g07500), OsTAR1 (LO05g07720), OsTDD1 (LOC_04g38950), OsCOW1 (LOC_03g06654).