Narrow Leaf21, encoding ribosomal protein RPS3A, controls leaf development in rice

A ribosomal protein controls rice leaf width through translational regulation of auxin response factors and a key transcription factor regulating blade lateral outgrowth.

Abstract

Leaf morphology influences photosynthesis, transpiration, and ultimately crop yield. However, the molecular mechanism of leaf development is still not fully understood. Here, we identified and characterized the narrow leaf21 (nal21) mutant in rice (Oryza sativa), showing a significant reduction in leaf width, leaf length and plant height, and increased tiller number. Microscopic observation revealed defects in the vascular system and reduced epidermal cell size and number in the nal21 leaf blade. Map-based cloning revealed that NAL21 encodes a ribosomal small subunit protein RPS3A. Ribosome-targeting antibiotics resistance assay and ribosome profiling showed a significant reduction in the free 40S ribosome subunit in the nal21 mutant. The nal21 mutant showed aberrant auxin responses in which multiple auxin response factors (ARFs) harboring upstream open-reading frames (uORFs) in their 5′-untranslated region were repressed at the translational level. The WUSCHEL-related homeobox 3A (OsWOX3A) gene, a key transcription factor involved in leaf blade lateral outgrowth, is also under the translational regulation by RPS3A. Transformation with modified OsARF11, OsARF16, and OsWOX3A genomic DNA (gDNA) lacking uORFs rescued the narrow leaf phenotype of nal21 to a better extent than transformation with their native gDNA, implying that RPS3A could regulate translation of ARFs and WOX3A through uORFs. Our results demonstrate that proper translational regulation of key factors involved in leaf development is essential to maintain normal leaf morphology.

Introduction

Leaf is a vital organ of a plant required for photosynthesis, respiration, and transpiration, which also affects overall plant architecture (Govaerts et al., 1996). Leaf size and morphology vary from species to species (Efroni et al., 2010). Leaf development starts with the initiation of leaf primordia, which are a group of histologically distinct cells located at the peripheral zone of the shoot apical meristem. Leaf primordia further develop into leaf along the adaxial–abaxial, proximodistal, and mediolateral axes (Satterlee and Scanlon, 2019).

Leaf width is an important indicator of leaf morphology and a complex quantitative trait controlled by multiple genes (such as Narrow Leaf1 (NAL1), NAL2, NAL7, NAL9, and NAL11), as well as abiotic factors (Fujino et al., 2008; Qi et al., 2008; Li et al., 2013; Jiang et al., 2015; Wu et al., 2016). Allelic genes, that is, Abnormal Vascular Bundles (AVBs), Semi-Rolled Leaf2 (SRL2), and Narrow and Rolled Leaf2 (NRL2) promote leaf blade outgrowth by regulating procambium cell establishment, cell proliferation, and vein patterning in rice (Oryza sativa; Hu et al., 2010; Liu et al., 2016; Zhao et al., 2016; Ma et al., 2017). Transcription factors AUXIN RESPONSE FACTORS (ARFs) family and WUSCHEL-RELATED HOMEOBOX (WOX) genes also play important roles in leaf development (Sarkar et al., 2007). In Arabidopsis, WOX3 and WOX1 control leaf blade development. There are three WOX3 homologous genes in the rice genome, i.e. the duplicated OsWOX3A genes on the short arms of chromosomes 11 and 12, which are designated as NARROW LEAF2 (NAL2) and NAL3, respectively, and OsWOX3B on chromosome 5 (Cho et al., 2013; Ishiwata et al., 2013; Honda et al., 2018). OsWOX3A is a key regulator of leaf lateral development. The OsWOX3A loss-of-function mutant showed extremely narrow leaf, while OsWOX3A-overexpressing plants exhibit twisted and knotted leaves (Cho et al., 2016). Interestingly, ribosomes, the machinery of protein synthesis, also regulate leaf development specifically. In Arabidopsis, mutants in the large subunit of ribosomal proteins such as pgy1, pgy2, pgy3, ae5, ae6, oli5, oli7, rpl4a/d, and rpl5, and small subunit such as pfl1, pfl2, and aml1 display defects in leaf development (Van Lijsebettens et al., 1994; Weijers et al., 2001; Nishimura et al., 2005; Pinon et al., 2008; Yao et al., 2008; Fujikura et al., 2009; Ito et al., 2010; Rosado et al., 2012). Similarly, the rice rml1 mutant in the large subunit RPL3B, the only ribosomal mutant ever reported in rice, also showed retarded growth and narrow leaf (Zheng et al., 2016). However, the regulation mechanism in leaf development is unknown for most of these ribosomal proteins.

The phytohormone auxin plays a vital role in various plant morphogenetic processes and physiological responses throughout the life of a plant (Friml et al., 2003). In rice, auxin stimulus is controlled by a complex network including 25 ARFs and 31 Aux/IAA interacting partners, in which they induce or repress the transcription of auxin responsive genes containing auxin response elements (Jain et al., 2006; Wang et al., 2007). These elements control the translation of auxin signal into gene expression and developmental processes (Hagen and Guilfoyle, 2002). In Arabidopsis, ARF5/MONOPTEROS (MP) and ARF7/NONPHOTOTROPIC HYPOCOTYL4 (NPH4) genes control axis formation, cell expansion, vascular and body pattern in embryonic and post-embryonic development (Hardtke et al., 2004). The arf5 and arf7 mutants exhibited abnormalities in leaf shape due to defects in vascular tissue formation and auxin-dependent cell expansion (Przemeck et al., 1996). Similarly, in rice, the loss-of-function mutant of OsARF11, an ortholog of AtARF5, also showed the narrow leaf phenotype (Sakamoto et al., 2013). Similar phenotypes have also been reported in auxin biosynthesis and polar transport mutants such as nal1 and nal7 in rice (Fujino et al., 2008; Qi et al., 2008; Jiang et al., 2015).

Translational regulation contributes to the specific gene expression associated with plant physiological and environmental responses, but its role in development is still not very clear. Translational regulation depends on multiple control elements such as upstream open-reading frame (uORF) found in the 5′-untranslated region (5′-UTR) of both eukaryotic and prokaryotic mRNA. A uORF may contribute independently or in a coordinated manner in the regulation of translation of the downstream major ORF (mORF; Yaman et al., 2003; Nishimura et al., 2005). During the scanning of mRNA, once ribosome encounters with a uORF, translation will terminate at the stop codon, and translation of downstream mORF will need translation reinitiation. For this purpose, the ribosome must shape another initiation complex toward the start codon AUG of the downstream mORF (Nishimura et al., 2005; Malygin et al., 2013). Translation efficiency of the downstream mORF can be repressed through inefficient reinitiation (Roy et al., 2010). Bioinformatics studies predicted that 20%–30% of genes in Arabidopsis contain uORFs in the 5′-UTR, and the uORFs-containing transcripts are enriched for transcription factors (Rosado et al., 2012). However, knowledge about the number of functional uORFs and their role in the regulatory mechanism is minimal.

Studies related to ribosomal mutants and defects in translation reinitiation have been reported in Arabidopsis and the auxin-signaling pathway has been proposed as a candidate for translation through uORFs (Zhou et al., 2010; Horiguchi et al., 2012). In vivo studies have shown that the presence of multiple uORFs at 5′-leader can regulate the translation of multiple ARFs (Zhou et al., 2010). Still, some issues such as involvement of single or numerous ribosomal proteins in the translational control of the auxin responses, the relative contribution of large and small subunits of ribosomal protein in translation reinitiation, specific ARFs controlled by uORFs in different developmental processes, and conservation of this mechanism in other plant species remain unknown.

Ribosomal proteins are a conserved family in biological evolution. Ribosomal protein small subunit 3A (RPS3A) is only present in eukaryotic and archaebacterial ribosomes (Malygin et al., 2013). Monospecific polyclonal antibodies analyses showed that ribosomal proteins S3a and S24 are present on the outer layer of the 40S ribosomal subunit and are part of the domain which cooperates with the 3′ region of 18S rRNA, mRNA, initiation factors (eIF-2 and eIF-3), elongation factors (EF-1 and EF-2), and enhances the attachment of eIF-2 to ribosomes (Bommer et al., 1988; Kashuba et al., 2005). These previous studies demonstrated that RPS3A plays a pivotal role in translation initiation and reinitiation.

In this study, we report the function of RPS3A in leaf development through translational regulation of ARFs and WOX3A. The loss-of-function mutant of RPS3A (named as nal21) showed narrow leaves and dwarf phenotype. We further analyzed the change in auxin responses due to the reduced translational efficiency of multiple uORF-containing ARFs in nal21. To the best of our knowledge, no previous study reports the genetic basis of translational regulation of ARFs and WOX3 via uORFs, and the specific ribosome subunit-mediated translational control of leaf development in rice.

Results

Phenotypic characterization of the nal21 mutant

We isolated a nal21 mutant from the ethyl methanesulfonate mutagenesis-derived mutant library of the japonica rice variety Shengdao 16. The plants of nal21 showed approximately 50% reduction in the leaf width, also reduced plant height and leaf length, and increased number of tillers (Figure 1, A–H). Reduction in mutant plant height was mainly due to the reduced lengths of each internode and lacking of a basal internode compared with the wild type (WT; Supplemental Figure S1, A, B, and E). The mutation also had mild effects on the reproductive organs as the panicle became shorter and seed length was slightly reduced (Supplemental Figure S1, B–G).

Figure 1.

Gross morphology of WT and the nal21 mutant. A, WT and nal21 plants at the grain-filling stage, scale bar = 10 cm. B–D, Morphology of the top, second, and third leaf between WT and the nal21 mutant. Scale bar = 5 cm. E–H, Quantification data of plant height (E), number of tillers (F), leaf length (G), and leaf width (H) of WT and the nal21 mutant. Data are mean ± sd (Student’s t test, **P < 0.01, n = 20).

The shape of a leaf is more critical because it affects the crop biomass and yield. Vascular bundles patterning and cell number and size are the major cytological factors controlling leaf shape (Qi et al., 2008; Li et al., 2013). To reveal the potential changes in leaf vascular bundles patterning, leaf cross-sectioning was carried out (Figure 2, A–F), which showed that the number of both large veins and small veins was reduced significantly in the nal21 mutant leaves compared with the WT (Figure 2, M and N). We further studied the relationship between leaf narrowing and leaf epidermal cell number and size using microscopic approach. A significant reduction in cell width and number of cells along the leaf-width direction was recorded in nal21 (Figure 2, G–L, O, and P), indicating that both cell division and expansion were affected in the mutant.

Figure 2.

Microscopic comparison of WT and the nal21 mutant leaf. A, C, and E, Cross section of WT leaf. Scale bars = 1 mm. B, D, and F, Cross section of mutant leaf. Scale bars = 1 mm. Asterisks represent large veins and solid circles represent small veins. G–I, Epidermal cells of the WT leaves, including the top first leaf, second leaf, and third leaf. Scale bars = 50 μm. J–L, Epidermal cells of the nal21 mutant leaves. Scale bars = 50 μm. M, Number of large veins in the top, second, and third leaf. N, Number of small veins in the top, second, and third leaf. O, Epidermal cell width. P, Epidermal cell number (along leaf-width axis). Data are mean ± sd (Student’s t test, **P < 0.01, n = 15).

Map-based cloning and functional confirmation of NAL21

To work out the genetics underlying the altered phenotype, a cross was made between the nal21 mutant and WT. All the plants in F₁ generation had normal leaf phenotype like WT (Supplemental Figure S2, A–D). In the F₂ generation consisting of 200 plants, we observed 154 WT and 46 narrow leaf plants, respectively, fitting a chi-square ratio of 3:1 (χ² = 0.28 < χ² = 3.84, P > 0.05), suggesting that phenotype of the nal21 mutant is controlled by a single recessive gene. Mutant plants in F₂ population derived from the cross between nal21 and an indica rice variety Dular was used for map-based cloning of the NAL21 gene. The NAL21 gene was primarily mapped to the region between the insertion–deletion (InDel) markers R3-2 and R3-3 on the short arm of chromosome 3 using 22 mutant plants. After fine-mapping with 232 mutant plants and newly developed InDel markers (Supplemental Table S1), the target region was narrowed down to 59 kb between the markers C3-5 and C3-6 (Figure 3, A).

Figure 3.

Map-based cloning of the NAL21 gene and transgenic complementation test. A, Primary and fine mapping of the NAL21 gene on chromosome 3. The NAL21 locus was narrowed to a 59-kb region. B, The structure of candidate gene and mutation site (black boxes and lines represent exon and intron, respectively, and red arrow represents mutation site). C, PCR amplification of NAL21 from WT gDNA (1), WT cDNA (2), and nal21 cDNA (3). Red arrows represent two transcripts amplified from the nal21 mutant. D, Sequencing of WT and nal21 cDNA revealed two cDNA isoforms I and II. Green color represents intron retention and a premature stop codon, while pink color represents alternative splicing and change in cDNA size. E–H, Phenotypes of the NAL21 gDNA complementation lines (B285). Whole plant (E), top leaf (F), second leaf (G), and third leaf (H) were shown. Scale bar = 5 cm.

According to the rice genome annotation project (http://rice.plantbiology.msu.edu/cgi-bin/gbrowse/rice/), a total of 15 putative genes were predicted in the target region, 7 of which encode protein with known biochemical functions (Supplemental Table S2). Sequencing analysis of all the predicted genes identified a point mutation from G to A at the splicing site between exon 2 and intron 2 in the LOC_Os03g10340 gene (Figure 3, B). Reverse transcription polymerase chain reaction (RT-PCR) analysis was used to check the effects of mutation on splicing and two isoforms were detected in the nal21 mutant. In the large isoform (Isoform I in Figure 3, C), the intron 2 was retained in the mRNA and a premature stop codon was introduced by the retained intron, producing a truncated protein of 56 amino acids with the last amino acid encoded by the retained intron. In the small isoform (Isoform II in Figure 3, C), an alternative splicing site was detected at 8 bp upstream of the original splicing site. This caused the deletion of 8 bp in the cDNA which is, however, difficult to differentiate from that of WT on the agarose gel. The frame-shift mutation results in a truncated protein of 55 amino acids with the last three amino acids encoded by the mis-jointed exons 2 and 3 (Figure 3, D).

To confirm whether the loss of function of LOC_Os03g10340 causes the nal21 phenotype, genomic DNA (gDNA) fragment of 5,294 bp (including the 2,386-bp promoter region before the start codon) was introduced into the nal21 mutant. Seven independent transgenic lines were obtained, all of which showed complete complementation of the leaf phenotype as well as plant height (Figure 3, E–H and Supplemental Table S3). This result clearly demonstrates that the single base substitution in LOC_Os03g10340 (hereafter known as NAL21) is responsible for the altered phenotype of the nal21 mutant.

Blast analysis showed that NAL21 encodes a RPS3aA. There are two homologs of RPS3a in rice, i.e. RPS3aB and RPS3aC encoded by LOC_Os12g21798 and LOC_Os02g18550, respectively, and two putative orthologs in Arabidopsis. Sequence alignment indicated that RPS3a homologs are highly conserved in most parts of the protein except the highly divergent C-terminal end (Supplemental Figure S3, A). In the truncated proteins encoded by the two aberrant isoforms in the nal21 mutant, the conserved region after the 52nd and 55th amino acid was completely lost (Supplemental Figure S3, A). In addition, a phylogenetic tree was constructed using ortholog genes from different species of monocots, dicots, fungi, animals, and amoeba which indicated that they are closely related except amoeba (Supplemental Figure S3, B).

Expression pattern and subcellular localization of NAL21

Reverse transcription quantitative polymerase chain reaction (RT-qPCR) was used to study the spatio-temporal expression pattern of NAL21 in different tissues of WT. NAL21 was expressed in all tissues. However, the expression level in the root, young stem, leaf blade, leaf sheath, and young panicle was relatively higher than that from the seed, mature stem, and glume (Figure 4, A). To observe the spatiotemporal expression pattern of NAL21 visually, a β-glucuronidase (GUS) gene driven by the NAL21 promoter (∼2.5 kb upstream of the translation start site) was introduced into calli of cv Shiokari. Strong GUS activity was detected in young leaf blade (Figure 4, B), root tip and lateral root primordium (Figure 4, D–E), young stem (Figure 4, G), tiller bud (Figure 4, I), and young panicle (Figure 4, J–L), but GUS signal was very weak in the old tissues such as leaf sheath, stem, seed, and panicle (Figure 4, C, F, H, and M). These results were almost consistent with that of RT-qPCR. Expression of NAL21 in tissues with high cell division activity was consistent with the typical expression pattern of several ribosome protein genes in Arabidopsis (Nishimura et al., 2005; Pinon et al., 2008; Rosado et al., 2010).

Figure 4.

Expression pattern and subcellular localization of NAL21. A, RT-qPCR analysis of NAL21 expression in different tissues of WT normalized to actin. Bars represent mean of three biological repeats ± sd. B–M, Histochemical staining of different tissues of the NAL21 promoter-GUS transgenic lines. B, Leaf blade; C, leaf sheath; D and E, root tip and lateral roots; F, seed; G and H, young and mature stem, respectively; I, young and mature tiller bud; J–M, comparison of young and mature panicle. N, Subcellular localization of NAL21-GFP in the transgenic rice leaf protoplasts. ER-Tracker Red is used as a marker of ER. Bars: (B–G) 1 mm; (H–I) 2 mm; (J–M) 500 µm; and (N) 5 µm.

In eukaryotic cells, ribosomes are divided into free ribosomes that are freely distributed in the cytoplasm, and others that are attached to the surface of the endoplasmic reticulum (ER; Miller and Zachary, 2017). Using the online software SLP-Local (http://sunflower.kuicr.kyoto-u.ac.jp/∼smatsuda/slplocal.html), the NAL21 protein is predicted to locate on the secretory pathway (ER). To validate the subcellular localization of NAL21, the GFP gene was fused to the C-terminus of NAL21 coding sequence and transformed into the nal21 mutant. The NAL21-GFP construct could rescue the mutant phenotype to the WT level (Supplemental Figure S4). Protoplasts isolated from the transgenic plants were observed under the confocal microscope. Some of the NAL21-GFP fluorescence was detected in a necklace-like structure which could overlay with the ER marker ER-Tracker Red, whereas other signal was freely distributed in the cytosol. This result suggested that NAL21-GFP protein was localized to both ER-bound and free ribosomes (Figure 4, N).

Ribosomal structure alteration in the nal21 mutant

Ribosomal genes are involved in the development of different vegetative organs through controlling protein synthesis (Garreau de Loubresse et al., 2014; Saha et al., 2017). Some reported ribosome mutants like rpl3p, rml1, apum23, and rpl4 showed different sensitivity to translational inhibitors due to alterations on ribosome structure and function (Abbasi et al., 2010; Hang et al., 2014; Zheng et al., 2016). The mode of action of the antibiotics is variable from prokaryotes to eukaryotes, and this depends on their shapes and binding sites on ribosome (Garreau de Loubresse et al., 2014). We compared the response of WT and nal21 to different known ribosome-targeting antibiotics by measuring root and shoot lengths. Among these antibiotics, cycloheximide binds the E-site on the large subunit in eukaryotic cell, which is the tRNA binding site (Garreau de Loubresse et al., 2014). Hygromycin B and geneticin (G418) bind near the decoding center on the small subunit and cause misreading of mRNA in both prokaryotic and eukaryotic cells (Brodersen et al., 2000; Garreau de Loubresse et al., 2014; Yoruk and Albayrak, 2015). The nal21 mutant displayed resistance to hygromycin B and geneticin (G418) when compared with the WT (Figure 5, A–H). It also showed less sensitivity to cycloheximide (Supplemental Figure S5, A–D). As a control, the prokaryotic-specific antibiotics chloramphenicol, erythromycin, and streptomycin had no effects on the shoot and root growth in both WT and nal21 (Supplemental Figure S5, E–J). This mild resistance to antibiotics is probably due to aberrantly assembled ribosome structure which blocks the proper binding of specific antibiotics.

Figure 5.

Antibiotics resistance assays and ribosome profiling analysis. Seedlings of WT and nal21 were grown in hydroponic culture with or without antibiotics. WT (A, E) and the nal21 mutant (B, F) plants were treated with different concentrations of hygromycin B and geneticin (G418) exogenously (Bars = 1 cm). Root length (C, G) and shoot length (D, H) were recorded after 10 d. Data indicate mean ± sd (n = 16). I, The ribosome profile monitored by absorbance at 260 nm over 5%–50% linear sucrose gradient. Absorbance peaks at 260 nm represent free 40S and 60S subunits, 80S monosomes and polysomes. The ribosome profiling assay was repeated three times with similar results. The green boxed region corresponding to polysomes was shown at a fine scale in the inset.

To further explore the potential role of NAL21 in ribosomal biogenesis, we examined ribosome profile in WT and the nal21 mutant by detecting absorbance at 260 nm after sucrose density gradient ultracentrifugation. Compared with WT, the nal21 mutant showed decreased levels of the free 40S subunits, 80S monosomes, and polysomes while increased levels of free 60S subunits (Figure 5, I). This could be due to the fact that 40S subunits are not well assembled, or abnormal ribosomes are degraded rapidly, leading to a decrease in the level of 40S. Due to the insufficient amount of 40S subunits to assemble with 60S subunits to form the monosomes and polysomes, more free 60S accumulated in the nal21 mutant. The decreased levels of polysomes indicated that translation initiation might be defective leading to reduced translation efficiency in the nal21 mutant (Garcia-Gomez et al., 2014). According to the model of ribosome function (Horiguchi et al., 2012), we assume that ribosome aberrancy and insufficiency, or defective translation initiation resulted in the abnormal development in the nal21 mutant.

The reduced response of the ribosomal mutant nal21 to auxin

In Arabidopsis, phenotypes of ribosomal protein mutants are often related with auxin (Degenhardt and Bonham-Smith, 2008; Rosado et al., 2010). In rice, the auxin biosynthesis mutant nal7 and auxin polar transport mutant nal1 showed a narrow leaf phenotype with reduced vascular bundles (Fujino et al., 2008; Qi et al., 2008; Jiang et al., 2015). In the RPS3A mutant nal21, we also observed similar leaf phenotype as nal1 and nal7. To examine whether nal21 is also related to auxin, we explored changes in auxin sensitivity in the nal21 mutant. We compared the effect of exogenously applied auxin on WT and the nal21 mutant, by measuring shoot length, primary root elongation, lateral root initiation, and number of crown roots, which are typically regulated by auxin. For this purpose, nal21 and WT plants were germinated and grown on half-strength Murashige and Skoog (MS) medium containing different concentrations of indole-3-acetic acid (IAA) for 10 d.

IAA treatment inhibited nal21 root and shoot elongation to a less extent than it did in WT, indicating that the nal21 mutant responded inappropriately to exogenously applied IAA (Figure 6, A–D). Moreover, IAA promoted lateral root formation and increased the number of crown roots in WT plants; however, such effects could not be seen in the nal21 mutant (Figure 6, E and F). To visualize the postulated auxin response defects at the in situ level, the auxin-responsive DR5: GUS reporter gene (Ulmasov et al., 1997) was transformed into both the WT and nal21 mutant. Histochemical analysis of DR5: GUS activity revealed that the nal21 mutant showed weaker signal in the root and shoot tips as well as at the entire root and young leaf blade when compared with WT (Figure 6, G–L). The induction rate of some early auxin-responsive genes, such as OsIAA20, OsSAUR13, and OsGH3.1 (Sakamoto et al., 2013), decreased significantly in the nal21 mutant (Figure 6, M). These results suggested that the nal21 mutant is less responsive to auxin.

Figure 6.

Response of WT and the nal21 mutant to exogenous auxin treatment. A and B, WT and mutant plants treated with different concentrations of IAA (Bar = 1 cm). Length of primary roots (C), shoot length (D), number of crown roots (E), and number of lateral roots (F) measured after 10 d. The nal21 mutant showed resistance to exogenously applied IAA. G–L, DR5: GUS expression in root tip, leaf blade, and leaf tip of WT (G, I, and K) and the nal21 mutant (H, J, and L), respectively (Bar = 1 mm). M, RT-qPCR analysis of early auxin-responsive genes OsIAA20, OsSAUR13, and OsGH3.1 expression in WT and the nal21 mutant plants. Actin was used as an internal control. Values are the mean ± sd of three biological repeats. Bars labeled with asterisks differ significantly (**P < 0.01, Student’s t test) from the WT plants.

RPS3A facilitates translation of multiple ARFs containing 5′-uORF

Several Arabidopsis ribosomal large subunit proteins such as RPL24B, RPL4A, RPL4D, and RPL5A have been reported to modulate auxin response by translational regulation of multiple uORF-containing ARFs. The number and length of uORFs are negatively related to protein translation efficiency of the downstream main ORF (Nishimura et al., 2005; Zhou et al., 2010; Rosado et al., 2012). We postulated that this translational regulatory mechanism might also be responsible for the reduced auxin response in the rice nal21 mutant harboring mutation in the ribosomal small subunit protein RPS3a. There are 25 ARF-related genes in the rice genome, and full-length cDNA sequences are available for all except two ARFs in the GenBank database (Wang et al., 2007). Among them, 15 ARF genes contain one or more uORFs in their 5′-UTR (Supplemental Table S4). We examined whether the translation efficiency of these uORF-containing ARFs decreased in the nal21 mutant by a transient expression assay in rice leaf protoplasts. The respective 5′-UTR of each ARF was fused with a firefly luciferase (LUC) coding sequence and transcribed from a CaMV35S promoter (Supplemental Figure S6, A and B). OsARF2 and OsARF24, which do not have an uORF, were used as controls. The transient expression vectors were transformed into the protoplasts of WT and nal21. Among the 15 OsARF genes, 9 showed reduced LUC activity in the nal21 mutant compared with WT (Figure 7, B and C and Supplemental Figure S6, C). This suggests that multiple ARF family transcription factors tend to be translationally repressed in the nal21 mutant.

Figure 7.

The effect of 5′-uORFs of ARF11 and ARF16 on the expression of the downstream ORF. A, Schematic view of the transient expression vectors. The 5′-UTR containing intact uORFs or mutated form was inserted between the CaMV 35S promoter and the firefly LUC gene. The mutant version was generated by substitution of the uORF start codon ATG to TTG. B and C, Transient LUC expression vectors driven by the CaMV 35S promoter (1), intact or mutated uORFs of ARF11 (2 and 3), and ARF16 (4 and 5) were transformed into leaf protoplasts of WT and the nal21 mutant, respectively. The RLUC driven by the 35S promoter was co-transformed and used as an internal control. Transient expression was measured and calculated by the ratio of LUC/RLUC. Each assay was performed at least four times and data are mean ± sd. Student’s t test was used to calculate significance (**P < 0.01, ns = non-significance). D–M, ARF16-Prom: GUS expression in different tissues of WT and the nal21 mutant: D and I, root tips; E and J, mature zone of root; F and K, shoot base; G and L, leaf sheath; and H and M, leaf blade. Bars: (D, H–J, and L) 2 µm; (F and K) 1 mm; and (E and G) 500 µm. N, Western blot analysis showed that ARF11 and ARF16 protein levels were reduced in the nal21 mutant. Three biological replicates of WT and the nal21 mutant were used and anti-actin was run as a loading control. The band intensity was measured using Image J and the ratio of target protein to loading control was shown below the blot.

We further examined whether these uORFs regulate the translation efficiency of their downstream mORFs by eliminating uORF from the transient expression vectors. OsARF11 and OsARF16 are orthologs of Arabidopsis AtARF5/ MP and AtARF7/NPH4, respectively (Wang et al., 2007). Both AtARF5 and AtARF7 play a crucial role in auxin-induced leaf expansion in Arabidopsis (Hardtke et al., 2004; Wilmoth et al., 2005). The loss-of-function mutant of OsARF11 caused by insertion of a Tos17 retrotransposon showed a mild narrow-leaf phenotype (Sakamoto et al., 2013). The OsARF11 and OsARF16 knockout mutants created by the CRISPR-Cas9-based genome editing technology also showed the narrow leaf phenotype along with reduced plant height and increased tiller number (Supplemental Figures S7, S8). So, we selected OsARF11 and OsARF16 for this study. There are one and two uORFs in the 5′-UTR of OsARF11 and OsARF16, respectively. Through site-directed mutagenesis, the start codon ATG of uORFs was substituted to TTG (Figure 7, A). Both vectors harboring the intact uORF and eliminated version were transformed into the WT and the nal21 leaf protoplasts, respectively. Compared with the control vector (35S-LUC), the transcriptional fusion of 5′-UTR of both OsARF11 and OsARF16 significantly reduced the LUC activity. However, the LUC activity was partially recovered after removal of the uORF from the 5′-UTR (Figure 7, B and C). The similar trend was observed in both the WT and nal21 mutant protoplasts. Moreover, the LUC activity in nal21 increased to a comparable level with that in WT after removal of uORFs. Therefore, uORFs present in the 5′-UTR of OsARF11 and OsARF16 impede translation of the mORF in transfected protoplasts.

To confirm that translational regulation of OsARF11 and OsARF16 was affected by the ribosome mutation in planta, we transformed the GUS reporter gene driven by the OsARF11 and OsARF16 promoter containing uORFs into nal21 and WT, respectively. Histochemical staining revealed decreased GUS signals in the root tip, root stele, shoot base, leaf sheath, and blade in the nal21 mutant compared with WT (Figure 7, D–M and Supplemental Figure S9), which indicated that translation repression was induced in the nal21 mutant. Further, western blot analysis directly showed a decreased level of the OsARF11 and OsARF16 protein in nal21 compared with WT (Figure 7, N). Therefore, the OsARF11 and OsARF16 transcripts are subjected to translation regulation mediated by the ribosomal proteins.

Translational defects of ARFs mediated by uORFs are at least partially responsible for the nal21 phenotype

To confirm that the decreased protein levels of OsARF11 and OsARF16 contribute for the narrow leaf phenotype in the nal21 mutant, we transformed the nal21 mutant with the OsARF11 or OsARF16 gDNA fragments containing functional and nonfunctional uORFs (OsARF11 gDNA, OsARF11 gDNA ΔuORFs, OsARF16 gDNA, and OsARF16 gDNA ΔuORFs, Figure 8, A). In the nonfunctional uORFs, the start codon ATG of uORFs was mutated to TTG. The OsARF11 and OsARF16 gDNA with both functional and nonfunctional uORFs could rescue the narrow-leaf phenotype of the nal21 mutant (Figure 8, B–F and H). However, the transgenic lines harboring OsARF11 and OsARF16 gDNA with nonfunctional uORFs showed broader leaf when compared with those harboring functional uORFs. Besides the leaf phenotype, we also observed the root phenotype (Supplemental Figure S10, A–D). Similarly, the transgenic lines harboring OsARF11 and OsARF16 genomic fragment with nonfunctional uORFs increased the length of primary root, number of crown roots, and number and length of lateral root to a larger extent when compared with plants harboring functional uORFs (Supplemental Figure S10, E–L). RT-qPCR and western blot analysis revealed that transgenic lines harboring nonfunctional uORFs accumulated more ARF11 and ARF16 protein than those harboring functional uORFs although the mRNA transcript levels were similar (Figure 8, G and I–K). The partial complementation of the nal21 mutant phenotypes by the OsARF11 and OsARF16 gDNA with functional uORFs might be due to the increased mRNA transcript levels by introducing additional copies of OsARF11 and OsARF16 gDNA into the genome. Similar phenomena have been reported when introducing the AtARF3 gDNA with functional uORFs into the Arabidopsis ribosomal large subunit mutants rpl4d, rpl5a, and rpl24 (Nishimura et al., 2005; Rosado et al., 2012). These results suggested that translational defects of the ARF11 and ARF16 mRNAs mediated by uORFs in their 5′-UTR are at least partially responsible for narrow leaf phenotype in the nal21 mutant.

Figure 8.

Transformation of OsARF11 and OsARF16 gDNA containing functional and non-functional uORF complemented the leaf phenotype in the nal21 mutant. A, Schematic map of the OsARF11 and OsARF16 constructs used for transformation. The mutant version was generated by substitution of the uORF start codon ATG to TTG. B and C, Gross morphology of the nal21 mutant transformed with the OsARF11 gDNA (B437), OsARF11 ΔuORF (B438), OsARF16 gDNA (B439), and OsARF16 ΔuORF (B440; Bar = 10 cm). D and E, Leaf phenotype of complemented plants (Bar = 5 mm). F and H, Quantification of leaf width. Values are the mean ± sd of 15 leaves. ^**P < 0.01 (Student’s t test). G and I, Relative expression of OsARF11 and OsARF16 in WT, nal21, and transgenic plants. J–K, Western blot shows that ARF11 and ARF16 protein level are recovered in the complemented plants. Anti-actin was used as a loading control. The band intensity was measured using Image J and the ratio of target protein to loading control was shown below the blot.

OsWOX3A is another target of NAL21-mediated translational regulation

Besides the ARF gene family, are there any other leaf width-related genes subjected to translational regulation through a uORF-dependent mechanism? A bioinformatic study revealed that transcription factor-encoding genes are preferred targets of the uORF-mediated translational regulation (Hayden and Jorgensen, 2007). Among the genes previously cloned from the rice narrow-leaf mutants, OsWOX3A encoded by the NAL2/3 gene belongs to the WOX transcription factor family (Cho et al., 2013; Ishiwata et al., 2013). There are three uORFs in the 5′-UTR of OsWOX3A (Figure 9, A). Therefore, we selected OsWOX3A for further analysis.

Figure 9.

OsWOX3A 5′-uORFs affect the expression of downstream main ORF. A, Schematic maps of the transient expression vectors. The OsWOX3A 5′-UTR containing intact uORFs or mutated form was inserted between the CaMV 35S promoter and the firefly LUC gene. The start codon of uORF was substituted (ATG to TTG) to create nonfunctional uORF. B, Transient LUC expression vector driven by the CaMV 35S promoter (1), intact or mutated uORFs of OsWOX3A (2 and 3, respectively) were transformed into leaf protoplasts of WT and the nal21 mutant, showing that translation efficiency was reduced in the nal21 mutant. Co-transformation of CaMV 35S-driven RLUC was used as an internal control. Transient expression was measured and calculated by the ratio of LUC/RLUC; data are mean ± sd (n = 4, **P < 0.01, ns = non-significant, Student’s t test). C–V, Histochemical staining of the OsWOX3A Prom: GUS transgenic plants in the WT (C–L) and the nal21 mutant (M–V) backgrounds. C and M, roots; D and N, root mature zone; E and O, root tips; F and P, lateral roots; G and Q, shoot base; H and R, tiller bud; I and S, lamina joint; J and T, leaf tip; K and U, leaf blade; and L and V, leaf sheath. Bars: (C–E, M–O, H, R, I–J, S–T, K, and U) 500 µm; (F and P) 200 µm; and (G, Q, L, and V) 1 mm. W, Western blot analysis showed a decreased protein level of OsWOX3A in the nal21 mutant compared with WT. Three replicates of WT and mutant were used, and anti-HSP82 was used as a loading control. The band intensity was measured using Image J and the ratio of target protein to loading control was shown below the blot.

Transient expression assay in rice leaf protoplasts showed that translation efficiency of the LUC gene fused with the 5′-UTR of OsWOX3A was significantly lower in the nal21 mutant compared with the WT. After elimination of uORFs from the 5′-UTR of OsWOX3A, the translation efficiency was partially raised and became comparable between WT and the nal21 mutant (Figure 9, B). These results indicate that uORFs present in the 5′-UTR of OsWOX3A might obstruct translation of the mORF in transfected protoplasts. For confirmation of translational regulation of OsWOX3A affected by the ribosome mutation in planta, we also transformed the GUS reporter gene driven by the OsWOX3A promoter containing uORFs into WT and the nal21 mutant. Histochemical staining revealed decreased GUS signals in the root, coleoptile, leaf tip, leaf blade and sheath, and laminar joint in the nal21 mutant compared with WT (Figure 9, C–V), indicating that translation repression was induced in the nal21 mutant. Furthermore, western blot analysis directly showed a decreased protein level of OsWOX3 in the nal21 mutant compared with WT (Figure 9, W).

To confirm the functional relationship between the NAL21 and OsWOX3A gene in leaf width development, we transformed OsWOX3A gDNA fragment containing functional (OsWOX3A gDNA) and nonfunctional uORFs (OsWOX3A gDNA ΔuORFs) into the nal21 mutant background (Figure 10, A). The transgenic plants harboring functional uORFs rescued leaf phenotype up to a lesser extent when compared with nonfunctional uORFs transgenic plants (Figure 10, B–E). Western blot analysis also showed that more WOX3A protein accumulated in transgenic plants harboring nonfunctional uORFs than those harboring functional uORFs although similar WOX3A mRNA transcript levels were detected between them (Figure 10, E and F). Interestingly, the transgenic plants were dwarf with twisted and knotted leaves (Figure 10, B and C). This phenomenon was probably due to the increased WOX3A protein levels, because similar phenotype has been reported for the OsWOX3A overexpression lines (Cho et al., 2016). Therefore, besides the ARF family transcription factors, OsWOX3A is also subjected to the translational regulation mediated by NAL21/RPS3A.

Figure 10.

Genetic analysis of NAL21 and OsWOX3A. A–E, Transformation of OsWOX3A gDNA with and without uORF complemented the leaf phenotype in the nal21 mutant. A, Schematic map of the OsWOX3A gDNA containing functional and non-functional uORFs (ATG was mutated to TTG). B, Phenotype of complemented plants with the OsWOX3A gDNA containing functional (B497) and nonfunctional uORF (B498). Bar = 10 cm. C, Leaf blade of complemented plants (Bar = 5 mm). D, Measurement of leaf width. The significance of data was tested with Student’s t tests (**P < 0.01, n = 15, data are mean ± sd). E, The expression level of OsWOX3A in the transgenic lines. The significance of data was tested with Student’s t tests (**P < 0.01, n = 3, data are mean ± se). F, Western blot showed that the WOX3A protein level was recovered in the complemented plants. Anti-HSP82 was used as a loading control. The band intensity was measured using Image J and the ratio of target protein to loading control was shown below the blot.

Discussion

Function of RPS3a in translational reinitiation in rice

It is known that ribosome consists of a 60S large subunit with ∼47 ribosomal proteins (designated RPL) and a 40S small subunit with ∼33 ribosomal proteins (designated RPS). During translation process, 40S performs a key role in the initiation of translation by scanning of start codon AUG of mRNA (Bommer et al., 1988), while 60S plays a key role in the elongation by catalyzing the peptide bond formation between amino acids of the nascent protein (Nishimura et al., 2005; Malygin et al., 2013; Hronova et al., 2017). Translation of polycistronic gene is subjected to regulation by the uORFs in the 5′-UTR, because translation of the main ORF has to be reinitiated after finishing translation at the stop codon of uORF (Zhou et al., 2010; Rosado et al., 2012). It has been reported that reinitiation requires large subunits because slow elongation on uORF affects the efficiency of reinitiation (Doudna and Rath, 2002). Indeed, several Arabidopsis ribosomal large subunit proteins, such as RPL4A, RPL4D, RPL5A, and RPL24B, have been implicated in the translational reinitiation of multiple uORF-containing ARFs (Nishimura et al., 2005; Zhou et al., 2010; Rosado et al., 2012). However, whether this mechanism is conserved in other plant species, such as rice (O. sativa), the model plant of monocots, is still not known (Zheng et al., 2016). Moreover, given the key role in the initiation process, the ribosomal small subunit protein should promote reinitiation process by resumption of scanning of start codon AUG of the mORF after the translation of uORFs (Bommer et al., 1988; Szamecz et al., 2008). However, no ribosomal small subunit proteins have been reported to promote the reinitiation process up to now.

In this study, we reported that ribosomal small subunit promotes translational reinitiation of uORF-containing genes. We characterized the rice narrow leaf mutant nal21, a loss-of-function mutant of the ribosomal small subunit protein RPS3A. We confirmed that translation reinitiation of the multiple uORF-containing genes is compromised in the rps3a mutant by measuring translation efficiency in rice protoplasts and detecting protein level using GUS reporter gene and western blotting (Figures 7, 9 and  Supplemental Figure S6). The translation efficiency and protein level of OsARF11, OsARF16, and OsWOX3A, three transcription factor genes controlling leaf width, was lower in the rps3a mutant, compared with WT. This could be due to the presence of multiple uORFs at their 5′-UTR and lower reinitiation efficiency caused by the absence of the RPS3A protein. Indeed, when these uORFs were eliminated, the translation efficiency of OsARF11, OsARF16, and OsWOX3A with nonfunctional uORFs was significantly increased and could rescue the narrow leaf phenotype of the nal21 mutant (Figures 8, 10). These results suggest that RPS3A is required for translation reinitiation and confirm that the translational control of ARFs by upstream ORFs is a conserved mechanism.

RPS3a is specific to archaebacterial and eukaryotic ribosomes and directly involved in the translation initiation process (Malygin et al., 2013). RPS3a, along with RPS19, S13, S16, and S24 are present on the outer layer of the 40S ribosomal subunit and form the binding domain for the translation initiation factors such as eIF-2 and eIF-3 (Bommer et al., 1988, 1991; Kashuba et al., 2005). eIF-2 plays a central role in translation initiation (Bommer et al., 1988), whereas eIF-3 promotes reinitiation after translation of uORFs (Szamecz et al., 2008; Roy et al., 2010; Schepetilnikov et al., 2013; Hronova et al., 2017). In the ribosome profiling assay, the peak of the 40S ribosomal subunit was clearly reduced in the nal21 mutant (Figure 5, I), suggesting that RPS3A is required for the stability or proper functioning of the 40S ribosomal subunit. The binding of eukaryotic initiation factors eIF-2 and eIF-3 to ribosome is presumably reduced due to the lacking of RPS3A protein, which may explain the reduced translation efficiency observed in the nal21 mutant (Figures 7, 9 and Supplemental Figure S6).

Role of ribosome in auxin signaling and leaf development

Besides protein synthesis, ribosomes also play specific roles in the growth and development of different organs (Wool, 1996; Byrne, 2009). Many studies showed that the response of auxin and functions of ribosomes are closely linked (Li et al., 2015). In Arabidopsis, several ribosomal large subunit proteins such as RPL4A, RPL4D, RPL5A, and RPL24B have been involved in auxin-regulated developmental processes like cell proliferation, cell expansion, polarity development in leaves, and apical–basal patterning of gynoecium (Nishimura et al., 2005; Byrne, 2009; Fujikura et al., 2009; Zhou et al., 2010; Rosado et al., 2012). In this study, mutation in the ribosomal small subunit protein RPS3A also caused specific auxin-related defects such as reduced leaf width and vein number, increased tiller number, reduced lateral root, and crown root formation, and delayed expression of auxin-responsive genes (Figures 1, 2, 6). At the cellular level, both the number and size of leaf epidermal cells was reduced in the nal21 mutant (Figure 2). Similarly, Arabidopsis ribosome proteins RPL5A and RPL5B coordinate both cell proliferation and cell expansion in the determination of leaf size (Fujikura et al., 2009). These results suggest that aberrant auxin responses are a common feature of mutants of both large and small subunits of the ribosome in different plant species.

To explain the aberrant auxin responses in the nal21 mutant at the molecular level, we focused on posttranscriptional regulation of the TIR1/AFB-Aux/IAA-ARF signal transduction pathway by ribosome. One post-transcriptional regulation is mediated by uORF, which interacts with ribosomal components and hinders the translation of mORF (Nishimura et al., 2005; Zhou et al., 2010). These uORFs are common for ARF family members but uncommon among the TIR1/AFB and Aux/IAA family members (Zhou et al., 2010). In our study, we revealed that the translation efficiency of nine uORF-containing ARFs was reduced in the nal21 mutant (Figure 7, B and C and Supplemental Figure S6, C). We further demonstrated that protein level of OsARF11 and OsARF16, potentially regulating leaf width was lower in the nal21 mutant compared with WT (Figure 7, N). Interestingly, mRNA level of OsARF11 and OsARF16 was not changed in the nal21 mutant under both the steady-state condition (Figure 8, G and I) and treatment with exogenously applied auxin (Supplemental Figure S11), excluding the potential effect of uORF on mRNA stability (Vilela et al., 1999). This change in protein level should be due to the presence of uORF. Removal of uORF from OsARF11 and OsARF16 have largely rescued auxin-related phenotypes in the nal21 mutant background (Figure 8 and Supplemental Figure S10), suggesting that uORF-mediated down-regulation of OsARF11 and OsARF16 protein levels is responsible for observed phenotypic defects in the nal21 mutant. Therefore, ribosomal components are essential for a translational regulation mechanism to intensify auxin signals.

On the other hand, because ribosomes are the factory for protein synthesis, it is possible that auxin might make efficient use of ribosomes to induce various responses during plant development. Previous studies have shown that auxin can induce the expression of ribosomal protein mRNAs to increase ribosomal protein level (Gantt and Key, 1985; Beltránpeña et al., 2010), and change the modification pattern of ribosomal proteins (Franziska et al., 2004). In this study, exogenously applied auxin could also induce expression of RPS3A in the WT (Supplemental Figure S12). Based on our results and previously reported studies (Rosado et al., 2010, 2012), we presume a link between ribosome and auxin, where auxin induces the transcription of ribosomal components while ribosome regulates the translation of ARFs in turn.

OsWOX3A is also a target regulated by ribosome protein

Upstream ORFs, which regulate translation of the downstream main ORF, have been found in the 5′-UTR of some specific mRNAs (Hayden and Jorgensen, 2007). Initially, multiple ARF transcription factors were identified as targets of the uORF-mediated translational regulation in Arabidopsis, which is consistent with the auxin-specific phenotypes of a series of ribosomal protein mutants (Nishimura et al., 2005; Rosado et al., 2012). Later on, the sucrose-regulated transcription factor bZIP11 and several genes in the lipid metabolism pathway were also found to be direct downstream targets regulated by ribosomal complex in Arabidopsis (Rahmani et al., 2009; Li et al., 2015). These studies suggest that the targets of the uORF-mediated translation regulation might be more diverse.

The WOX genes play a key role in leaf blade outgrowth (Nakata et al., 2012; Cho et al., 2013; Ishiwata et al., 2013). The presence of three uORFs in the upstream of OsWOX3A gene suggests that OsWOX3A may be a target of the uORF-mediated translational regulation. This hypothesis is supported by several lines of evidence. First, the translation efficiency of OsWOX3A was reduced due to presence of uORFs in the 5′-UTR (Figure 9, A and B). Second, reduction of the OsWOX3A promoter: GUS signals and OsWOX3A protein level in the nal21 also confirm that translational regulation of OsWOX3A was affected by the ribosome mutation in planta (Figure 9, C–W). Third, the narrow leaf phenotype of nal21 was complemented by transformation with the OsWOX3A gDNA with nonfunctional uORFs. Consistently, increased protein level of OsWOX3A was detected in the transgenic plants than WT (Figure 10, F). These findings indicate that OsWOX3A, a key factor in leaf blade lateral expansion, is another target of translational regulation by ribosome proteins.

Several studies in Arabidopsis have reported the relationship between ARF and WOX genes, two kinds of important regulator of leaf blade lateral expansion. On the one hand, AtARF5/MP directly binds to the WOX1 and PRS/WOX3 promoters and activates their expression (Guan et al., 2017; Xiong and Jiao, 2019). On the other hand, WUSCHEL directly binds to the promoter of AtARF5/MP and suppresses its expression (Ma et al., 2019; Lanctot and Nemhauser, 2020). To examine whether similar relationship exists between ARF11/16 and WOX3A in rice, we checked the mRNA level of OsWOX3A in WT and CRISPR-Cas9-mediated knockout lines of OsARF11 and OsARF16 (Supplemental Figures S7, S8). Similarly, mRNA levels of OsARF11 and OsARF16 were also checked in WT and the oswox3a mutant. As expected, the transcripts level of OsWOX3A was reduced in the osarf11 and osarf16 mutants, while OsARF11 and OsARF16 were upregulated in the oswox3a mutant when compared with WT (Supplemental Figure S13).

Based on the current findings, we proposed a working model for RPS3A (Figure 11). RPS3A and other ribosomal proteins regulate leaf development through uORF-mediated translational control of ARFs and the key regulator of leaf blade lateral expansion OsWOX3A. The intact ribosome can overcome the translational repression imposed by uORFs in the 5′-UTR. Enough amount of ARF and OsWOX3A protein required for normal leaf development is obtained. However, in the rps3a mutant, translation of ARFs and OsWOX3A is repressed due to the inefficient reinitiation after translation of the 5′-uORF. As a result, the amount of ARFs and OsWOX3A protein is reduced and the narrow leaf phenotype is observed. In summary, our study reveals the important role of the uORF-mediated translational regulation of ARFs and WOX3A gene in proper auxin signaling and normal leaf development. This additional regulatory step in leaf blade lateral expansion will provide an area for future research to elaborate ribosome function.

Figure 11.

Working model for RPS3A and other ribosome proteins in regulation of leaf development through uORF-mediated translational control. A, 5′-uORFs of ARFs and the key regulator of leaf blade lateral expansion OsWOX3A repress the translation of main ORFs. In WT plant, the intact ribosome can initiate the translation at the start codon of uORF. After termination of translation at the stop codon of uORF, the 60S large subunit disassociates from the mRNA whereas the 40S small subunit continues scanning along the mRNA to find the start codon of the main ORF and start the translation again (reinitiation). The translational repression imposed by uORFs is overcomed, and sufficient ARF and OsWOX3A protein required for normal leaf development is obtained. B, In the nal21 mutant, the RPS3A protein is lost, translation of ARFs and OsWOX3A is repressed due to the inefficient reinitiation after translation of the 5′-uORF. As a result, the amount of ARFs and OsWOX3A (pink pentagons and brown ovals) protein is reduced and the narrow leaf phenotype is observed.

Materials and methods

Plant materials and growth conditions

The nal21 mutant was isolated from an EMS-mutagenized M₂ population of japonica rice (O. sativa) cv Shengdao 16. All the plant materials were grown in paddy fields in Beijing, Shandong, and Hainan provinces in China under natural growing conditions. For auxin and antibiotics treatment, healthy seeds of WT and the nal21 mutant were germinated in water. Uniform seedlings were selected and grown hydroponically with different concentrations of various antibiotics or IAA for 10 d. For examining expression of early auxin-responsive genes, sampling was done at different time points after treatment of 10-d-old seedlings with 1 µM IAA.

Microscopic analysis

For measurement of leaf veins and number of cells, samples were prepared using a previously described protocol (Vasco et al., 2014; Jiang et al., 2015; Guo et al., 2019). After bleaching with ethanol, pictures were taken under an optical microscope (BX43, Olympus, Tokyo, Japan) using cellSens software.

Map-based cloning

To map the genomic location of NAL21, 232 mutants were selected from the F₂ population of the cross between nal21 and an Indica cv Dular. Based on the polymorphism between the Indica and Japonica genome sequence, InDel markers were developed to map the gene (Supplemental Table S1). The candidate genes were PCR-amplified and sequenced to detect the mutation site.

Complementation and CRISPR-Cas9 vectors construction

For the NAL21-gDNA-C, OsARF11-gDNA-C, OsARF16-gDNA-C, OsWOX3-gDNA-C complementation vectors, gDNA fragments were amplified through PCR and cloned into the binary vector pCAMBIA1305.1 using In-fusion HD cloning kit (Takara Bio USA, Inc.). The ATG to TTG mutations in OsARF11-gDNA ΔuORF-C, OsARF16 gDNA ΔuORF-C, and OsWOX3 gDNA ΔuORF-C were induced using Q5 site-directed mutagenesis kit (NEB Cat. No. E0554S). These complementation vectors were introduced into the nal21 mutant callus by Agrobacterium-mediated transformation. To construct the CRISPR-Cas9 vectors for the OsARF11 and OsARF16 gene, the target sequences of sgRNAs were selected based on the prediction of the web-based tool CRISPR-P (Lei et al., 2014). The sgRNA expression cassettes OsU3a-ARF11T1, OsU6a-ARF11T2, OsU6a-ARF16T1, and OsU6b-ARF16T2 were assembled in the plant binary vector pYLCRISPR/Cas9 Pubi-H as described previously (Ma et al., 2015). The CRISPR-Cas9 vectors were introduced into the WT rice variety Shengdao 16 by Agrobacterium-mediated transformation. All the primers for the above-mentioned vectors are listed in Supplemental Table S1.

Subcellular location of the NAL21 protein

To validate the subcellular location of NAL21, protoplasts from NAL21-GFP transgenic plants (driven by the rice Actin1 promoter) rescuing the nal21 phenotype were isolated. The GFP fluorescence was examined by a laser scanning confocal microscope (LSM 700; Carl Zeiss, Germany). ER-Tracker Red (E34250, Thermo Fisher, Waltham, MA, USA) was used as a marker to confirm the ER localization. Argon and HeNe lasers were used, and the excitation/emission wavelengths for GFP and ER-Tracker Red were 488/550 and 587/615 nm, respectively.

Promoter-GUS assay

Tissue samples from transgenic plants expressing the GUS gene driven by the promotor of NAL21, OsARF11, OsARF16, OsWOX3, or auxin reporter DR5 were collected at different developmental stages and stained according to the protocol described by Jefferson (1989). Photographs were taken using a stereomicroscope as described previously (Zafar et al., 2020).

Sequence alignment of the NAL21 protein and phylogenetic analysis

Protein sequences were retrieved from NCBI database using blastp program (https://blast.ncbi.nlm.nih.gov/Blast.cgi) and aligned using ClustalW. Phylogenetic tree was constructed using the MEGA6 software by employing the neighbor-joining method with 1,000 bootstrap replicates.

RNA extraction and RT-qPCR

Total RNA was extracted from different tissues using the RNAprep Pure Plant Kit (Tiangen, Beijing, China) following the manual instructions. One microgram of RNA was reverse-transcribed into cDNA using Superscript III Reverse Transcription Kit (Invitrogen, Carlsbad, CA, USA). The RT-qPCR was run in Applied Biosystems Prism 7500HT Real-time Sequence Detection System using 2 × SYBR Green PCR Master Mix (TAKARA, Kyoto, Japan). The rice ACTIN1 gene was used as internal control. All primers used for RT-qPCR are listed in Supplemental Table S5.

Ribosome profile analysis

Ribosome profiling was done using 7-d-old rice seedlings as described previously (Mustroph et al., 2009; Rivera et al., 2015) with minor modifications. Leaf tissues were washed with DEPC-treated RNase-free water and grinded in liquid nitrogen. One gram of grinded powder was taken and double volume (1.8 mL) of freshly prepared polysome extraction buffer was added to make a homogeneous mixture using a 15-mL homogenizer (Bjbalb, Cat. No. QN3054). The recipe for the polysome extraction buffer was as follows: (0.2 M Tris–HCl [pH 9.0], 0.2 M KCl, 0.025 M EGTA, 0.035 M MgCl₂, 1% [v/v] Detergent mix, 1% [w/v] sodium deoxycholate, 1% [v/v] polyoxyethylene 10 tridecyl ether, 5 mM dithiothreitol [DTT], 1 mM phenylmethylsulfonyl fluoride, 50 µg/mL cycloheximide, 50 µg/mL chloramphenicol, and 100 U/mL RNase inhibitor). To make the Detergent mix, the following reagents were added: 10 mL of Triton X-100, 10 g of Brij-35, 10 mL of Tween-20, 10 mL of octylphenyl-polyethylene glycol (Igepal CA or NP-40) and ddH₂O with final volume up to 50 mL. The homogenized mixture was shifted to 2 mL tube and centrifuged (Eppendorf 5424R) at 16,000 g and 4°C for 45 min. Supernatant was filtered through four layers of Mira-cloth (Cal BioChem, La Jolla, CA, USA) into a new 1.5 mL tube. Concentration was checked using a Nanodrop spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA). A total of 3,500 A260 units of the supernatant was layered onto 12 mL of linear 5%–50% (w/v) sucrose gradient (50 mM Tris–HCl [pH 8.4], 20 mM KCL, 10 mM MgCl₂, 1 mM DTT, 10 µg/mL cycloheximide, 5 µg/mL chloramphenicol, 1× proteinase inhibitor, 100 U/mL RNase inhibitor and sucrose [5%–50%] [w/v]) poured on a Gradient Master 108 (Biocomp Instruments Inc., Fredericton, Canada; version 5.3) then centrifuged in a HITACHI ultracentrifuge (Himac CP80NX) using 13 mL tube (Cat. No. 32901A) in P40ST-2514 rotor at 40,000 rpm for 2.5 h at 4°C. Absorbance of each fraction was measured at 260 nm using the Piston Gradient Fractionator (Biocomp Instruments Inc.) attached to the Model PROBEver4.202 UV Monitor (Biocomp Instruments Inc.).

Transient expression assay

Rice protoplasts were isolated as described earlier (Jabnoune et al., 2015). 35S Prom: ARF/WOX3 5′-UTR: firefly LUC transient expression vectors were constructed (Supplemental Table S6). The renilla LUC (RLUC) driven by the 35S promoter was used as an internal control. Plasmids carrying reporter and internal control were co-transformed into protoplasts of WT and the nal21 mutant with the help of PEG-4000. Activities of the reporter and internal control were measured using the Dual-LUC reporter assay system (E1910; Promega, Madison, WI, USA) with a Lumet LB9507 luminometer (Berthold Technology, Oak Ridge, TN, USA). Mean ratios of LUC/RLUC from at least three biological replicates were used to compare the translation efficiency between WT and mutant.

Development of antibodies

A 504-bp cDNA fragment of OsARF11 (encoding the 409–576^th amino acids in the central region), a 960-bp cDNA fragment of OsARF16 (encoding the 541–860^th amino acids in the central region), and a 387-bp cDNA fragment of OsWOX3 (encoding the 76–203^th amino acids in the C-terminal region) were amplified (Supplemental Table S5). PCR products were cloned into the NdeI and BamHI sites of the Escherichia coli expression vector pET-28a (Novagen, http://www.novagen.com). Then His-tagged recombinant proteins were expressed in BL21 (DE3) cells and purified using Ni-NTA agarose beads (BioDee; Cat. No. DE-NKY-30210) and separated by SDS–PAGE. The appropriate band for each protein was excised from gel and used directly for production of mice polyclonal antibodies. The serum was used for immunoblot detection.

Western blot analysis

Total proteins were extracted from WT and mutant seedlings as described previously (Morris et al., 2014). Total proteins were separated by SDS–PAGE gels (8%–15%) and transferred to the polyvinylidene difluoride membranes (0.45 μm; Merck Millipore, Burlington, MA, USA) by electroblotting. The membrane was first incubated with specific primary antibodies anti-OsARF11, OsARF16, and OsWOX3, and then with goat anti-mouse IgG (horseradish peroxidase) as a secondary antibody (CWBIO; Cat. No. CW0102S). Anti-actin (CWBIO; Cat. No. CW0096M) and anti-HSP82 (Beijing Protein Innovation; Cat. No. abM51099-31-PU) were used as the loading control. A high-sig ECL reagent (Tanon; Cat. No. 180-5001) was used for imaging.

Accession numbers

Sequence data from this article can be found in the Rice Genome Annotation Project website ((http://rice.plantbiology.msu.edu/) under the following accession numbers: NAL21, LOC_Os03g10340; OsARF11, LOC_Os04g56850; OsARF16, LOC_Os06g09660; OsWOX3A, LOC_Os11g01130, and LOC_Os12g01120.

Supplemental data

The following supplemental materials are available in the online version of this article.

Supplemental Figure S1. Panicle and seed morphology and internode length.

Supplemental Figure S2. Phenotypic comparison of WT, heterozygotes, and the nal21 mutant.

Supplemental Figure S3. Amino acid sequence alignment and phylogenetic tree of NAL21 and homologous proteins.

Supplemental Figure S4. The Actin1_pro-NAL21-GFP construct complemented the nal21 mutant phenotype.

Supplemental Figure S5. Resistance assay using ribosome-targeting inhibitors in WT and nal21.

Supplemental Figure S6. Translation efficiency of many uORF-containing OsARF genes was reduced in the nal21 mutant.

Supplemental Figure S7. CRISPR-Cas9-based knockout of OsARF11 in WT.

Supplemental Figure S8. CRISPR-Cas9-based knockout of OsARF16 in WT.

Supplemental Figure S9. ARF11-Prom: GUS expression in different tissues of WT and the nal21 mutant.

Supplemental Figure S10. Transformation of ARF11 and ARF16 gDNA with and without functional uORFs rescues the root phenotype in the nal21 mutant.

Supplemental Figure S11. Expression analysis of OsIAA20, OsARF11, and OsARF16 under 1 µM IAA.

Supplemental Figure S12. Effect of IAA treatment on NAL21 expression.

Supplemental Figure S13. Relationship between OsARF11, OsARF16, and OsWOX3.

Supplemental Table S1. List of primers used for map-based gene cloning and complementation tests.

Supplemental Table S2. Gene function annotation in the fine-mapped region.

Supplemental Table S3. Agronomic parameters of the complemented transgenic plants.

Supplemental Table S4. List of uORFs of OsARF genes in rice.

Supplemental Table S5. Primers for promoter: GUS, RT-qPCR, and E. coli expression vectors for antibody development.

Supplemental Table S6. Primers for 5′-UTR: LUC transient expression assay.

 

X.L. and M.U. designed the experiments. M.U., H.L., S.A.Z., S.B.P., Y.C., L.L., J.F., J.Z., L.P., and S.Y. performed the experiments. M.U. analyzed the data and wrote the original manuscript. X.L., S.A.Z., and M.U. revised the manuscript. X.L. supervised the whole study.

The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (https://academic.oup.com/plphys/pages/general-instructions) is: Xueyong Li (lixueyong@caas.cn).