Identification of nlg1 mutant
The nlg1 was isolated from a mutagenesis population of Japonica rice cultivar YunDao32 (YD32) induced by EMS. Under natural field environment, nlg1 exhibited pleiotropic phenotypes, including narrow leaves, slender grains, tiller number increased and plant dwarfism (Fig. 1A-C, E-H). Compared with the wild type, the flag leaf, second leaf and third leaf width in nlg1 were reduced by approximately 22.6%, 22.4% and 20.3%, and the leaf length were reduced by about 57.9%, 47.5% and 38.6%, respectively (Fig. 1G, H). The panicle size, grain number per panicle and grain width were significantly decreased (Fig. 1C, I). Reduction of mutant plant height was mainly caused by the decrease in first to seventh internode length in 46.1%, 44.8%, 40.7%, 29.8%, 25.1%, 22% and 21.4%, respectively (Fig. 1D, J). We also investigated the dynamic characteristics of nlg1 seedling from 10 days to 35 days, and found that the leaf width difference began to appear around 10 days, and the difference of tiller number emerged around 30 days after sowing (Figure S1A-F). In general, the mutation of NLG1 gene affects multiple growth and development process, especially in leaf width, grain size and plant height.
NLG1 affects the development of vascular bundles and spikelet cell size
To reveal the potential cytological characteristics, the vascular bundle characteristics and cell morphology in leaf, culm and spikelet were analyzed by paraffin section and SEM. The cross section of leaves showed the number of large veins and small veins were significantly decreased, and the abaxial sclerenchyma of small veins were disappeared in nlg1 (Fig. 2A-E, J-K). The cross section of culms revealed that the number of vascular bundles decreased, and the thickness of culm and number of cell layers were increased in nlg1 than that of YD32 (Fig. 2F-I, K-N). We noticed that spikelet hull width of nlg1 was narrower than YD32 (Fig. 3A-B, K). The paraffin sectioning and SEM observation further showed that spikelet perimeter was shorter, the cell length and width of inner and outer glume and number of outer parenchymal cell were decreased in nlg1 than YD32 (Fig. 3C-J, L-O). Taken together, these results demonstrated that the narrow leaves of nlg1 was caused by the reduction of large veins and small veins, while the slender grains was due to the decrease of cell proliferation and cell expansion.
Map-Based cloning NLG1
To isolate the target gene NLG1, we employed a map-based cloning approach using 1856 F2 individuals derived from the cross between nlg1 and TN1. NLG1 was primitively located on chromosome 3 and finally narrowed in an interval of 42.1kb, which contained 6 open reading frames (ORFs) based on the Rice Genome Annotation Project database (http://rice.plantbiology.msu.edu/) (Fig. 4A-C, Table S1). The 10 ORFs were sequenced and a G to A single base-pair substitution were found in the seventh exon of LOC_Os03g14890, causing the 227th residue alteration from valine (Val) to methionine (Met) (Fig. 4D-E). To verify the candidate gene, the entire coding sequence of LOC_Os03g14890 harboring 2302bp upstream of ATG and 412bp downstream were constructed into pCAMBIA1300 and transformed into the nlg1 mutants. A total of 14 independent transgenic lines were obtained, and their mutant traits were all restored as YD32 (Fig. 4F, G, J). In addition, we performed an RNAi suppression of NLG1 in YD32 and found that the positive lines also presented a narrow leaves and slender grains phenotype similar to nlg1 (Fig. 4F, H, J). However, the phenotypes of NLG1 overexpression lines were not significantly different from that of wild type (Fig. 4F, I, J). We further detected the transcription level of NLG1 in these lines and found that NLG1 were up-regulated in overexpression and complementary lines and were down-regulated in RNAi lines (Fig. 4K, Figure S2F, G). These results indicated that LOC_Os03g14890 was identical to NLG1 (Figure S2A-E, Figure S3A-C).
NLG1 encodes a mitochondrial import inner membrane translocase Tim21
Sequence analysis revealed that NLG1 encodes a mitochondrial import inner membrane translocase Tim21. To further analyze the structure and function, NLG1 protein and 21 corresponding orthologs were used for phylogenetic analysis. The result indicated that NLG1 has the closest evolutionary relation with Zizania palustris, an aquatic wild rice with large grains growing in North America. Although SD3 (NP_001031562.1) is known as a Tim21 in Arabidopsis, it has a distinct evolutionary relationship with NLG1 (Fig. 5A) [32], revealing that the Tim21 functions may be differentiated in different species. Further protein alignment showed that these orthologs contained a highly conserved mitochondrial transmembrane region and Tim21 domain (Figure S4A). Tertiary structure model analysis between NLG1 and nlg1 revealed that substitution of Met227 for Val227 in Tim21 domain caused a peptide bonds alteration, which may explain the disruptive function of nlg1 (Fig. 5B).
Expression Pattern And Subcellular Localization Of Nlg1
To identify the spatio-temporal expression patterns of NLG1, we quantified the NLG1 transcription level in various tissues and organs by RT-qPCR. The results suggested that NLG1 was expressed abundantly in root, culm, leaf, leaf sheath, panicle and seedling, especially higher in young leaf and panicle (Fig. 5C). GUS staining assay were performed to determine the specific promoter activity of NLG1, and the staining tissues was consistent with RT-qPCR results. Moreover, the cross section of root showed a specific staining in vascular bundles (Fig. 5D-K). Therefore, NLG1 functions in a constitutive expression pattern. To examine the subcellular localization of NLG1, the pubi::NLG1-GFP and pubi::nlg1-GFP fusion protein was expressed in rice protoplasts respectively and the Mitochondria-RFP were used as mitochondria marker. The GFP signals were overlapped with Mitochondria-RFP signals, which manifested that NLG1 and nlg1 are located in the mitochondria (Fig. 5L), and the mutation in nlg1 did not affect the localization.
Mitochondrial Structure Defection and Compromised ATP Content in nlg1
To determine the effect of NLG1 on mitochondrial development, the TEM were conducted to observe the ultrastructure in mitochondria of YD32 and nlg1. Compared with the wild type, the mitochondria of nlg1 displayed abnormal and degraded cristae, which blurred the boundary of inner membranes (Fig. 6A). It is well known that mitochondrial is the site of plant oxidative respiration, organics decomposition and ATP production. So, we measured the ATP content of flag leaf in YD32 and nlg1 at heading stage and found that the ATP content of nlg1 was only half of YD32 (Fig. 6B). Moreover, we also observed the leaf epidermal cells via SEM. The results showed that the stomata density of nlg1 was lower than YD32, which implied that aberrant mitochondria development may lead to a weaker respiration in nlg1 (Fig. 6C-D).
Antimycin A and oligomycin are inhibitors of cytochrome c reductase and play a role in inhibiting electron transport from ubiquinone to cytochrome c, which may decrease ATP contents in mitochondria [38]. The 14-day-old seedlings of YD32 and nlg1 were treated with antimycin A (1 µM) and oligomycin (0.2 µM), respectively, and survival rates were calculated to assess the sensitivity to antimycin A and oligomycin. The results showed that both YD32 and nlg1 showed various degrees of withered leaves after treatment. However, nlg1 mutants showed more severe growth defects than that of YD32, indicating that nlg1 were more sensitive to mitochondrial electron transport chain inhibitors (Figure S5).
NLG1 Influences Auxin Response and Mitochondrial membrane development
To reveal the function of NLG1 in regulating leaf width and grain size development, we conducted a RNA sequencing (RNA-seq) analysis. Compared with YD32, a total of 596 up and 970 down regulated differentially expressed genes (DEGs, threshold of twofold change ≥ 1 and p value ≤ 0.05) were detected in nlg1 (Fig. 7A). These DEGs included many genes related to the mitochondrial membrane translocase (Os03g0305600 (Tim17), Os02g0672500 (Tim23) and Os02g0131600 (TOM22), cellulose synthase (Os07g0424400, Os06g0230100, Os08g0160500, Os03g0770800 and Os03g0837100), auxin response and transport (IAA2, IAA8 and Os04g0671900), and ATP synthase (Os04g0117100 and Os08g0250200) (Fig. 7B, Table S3). These results suggested that NLG1 may participate in mitochondrial membrane development and ATP metabolism, and regulate leaf width and grain size by affecting cellulose synthesis and auxin transport. Moreover, we further investigated the expression of a number of genes related to channel proteins located in mitochondrial membrane and respiratory chain complex. The results revealed that the expression level of translocase of the inner membrane gene Tim17, outer membrane genes Tom40 and Tom22, and membrane channel protein gene VDAC1 were significantly up-regulated, while respiratory function related genes AOX1a and COX11 were decreased markedly in nlg1 (Fig. 7E).
Narrow leaf and slender grain are generally associated with the synthesis and transduction of auxin signal. The auxin biosynthesis mutant fib, polar auxin transporter (PAT) mutant nal1 and auxin responser mutant nal21 all presented narrow leaves and small panicles [5, 11, 39]. Considering phenotypic similarity of nlg1 with fib, nal1 and nal21, the internal IAA levels of YD32 and nlg1 were measured. As expected, the internal IAA content were 59.7% reduction in nlg1 than YD32 (Fig. 7C). We further detected the expression of auxin biosynthesis, response and transport genes, and found that auxin response genes ARF1, ARF7, ARF8, ARF9 and ARF11, auxin efflux carrier genes PIN3, PIN5a and PIN5c were significantly down-regulated in nlg1, which is consistent with the RNA-seq consequence (Fig. 7D). Meanwhile, the expression levels of narrow leaf, grain with and cell cycle genes were also detected. The results showed that the NAL1, NAL2, NAL3, GW8, TAD1 and OsAPC6 were markedly down-regulated, while GW7 expression was highly up-regulated in nlg1 than that of YD32 (Fig. 7E), indicating that these genes may be responsible for nlg1 mutant phenotype.