SlRBP1 promotes translational efficiency via SleIF4A2 to maintain chloroplast function in tomato

Abstract

Many glycine-rich RNA-binding proteins (GR-RBPs) have critical functions in RNA processing and metabolism. Here, we describe a role for the tomato (Solanum lycopersicum) GR-RBP SlRBP1 in regulating mRNA translation. We found that SlRBP1 knockdown mutants (slrbp1) displayed reduced accumulation of total chlorophyll and impaired chloroplast ultrastructure. These phenotypes were accompanied by deregulation of the levels of numerous key transcripts associated with chloroplast functions in slrbp1. Furthermore, native RNA immunoprecipitation-sequencing (nRIP-seq) recovered 61 SlRBP1-associated RNAs, most of which are involved in photosynthesis. SlRBP1 binding to selected target RNAs was validated by nRIP-qPCR. Intriguingly, the accumulation of proteins encoded by SlRBP1-bound transcripts, but not the mRNAs themselves, was reduced in slrbp1 mutants. Polysome profiling followed by RT-qPCR assays indicated that the polysome occupancy of target RNAs was lower in slrbp1 plants than in wild-type. Furthermore, SlRBP1 interacted with the eukaryotic translation initiation factor SleIF4A2. Silencing of SlRBP1 significantly reduced SleIF4A2 binding to SlRBP1-target RNAs. Taking these observations together, we propose that SlRBP1 binds to and channels RNAs onto the SleIF4A2 translation initiation complex and promotes the translation of its target RNAs to regulate chloroplast functions.

The glycine-rich RNA-binding protein SlRBP1 maintains chloroplast functions via regulating translational efficiency of key transcripts associated with chloroplast function.

IN A NUTSHELL.

Background: The nuclear genome encodes most of the proteins that are found in chloroplasts; these proteins are translated in the cytoplasm and imported into the chloroplast. The finely tuned regulation of these nuclear transcripts encoding chloroplast-localized proteins is crucial for chloroplast function. Numerous RNA-binding proteins (RBPs) that regulate nuclear-encoded chloroplast transcripts have been identified in Arabidopsis, rice, and maize; however, the tomato RBPs directly involved in the translation of RNA targets remain largely unknown.

Question: How does the nucleus-encoded protein SlRBP1 specifically control its targets to maintain chloroplast function during tomato growth and development?

Findings: Loss-of-function of SlRBP1 caused dwarf tomato plants with yellow leaves and downregulated photosynthesis, and fruits of different shades of yellow and orange, indicating that SlRBP1 is important for chloroplast function. Moreover, silencing of SlRBP1 dramatically decreased the translation efficiency of its target mRNAs, nuclear transcripts encoding key photosynthesis-related, chloroplast-targeted proteins. This resulted in a dramatic reduction in the accumulation of the proteins encoded by its target mRNAs. SlRBP1 interacts with the eukaryotic translation initiation factor SleIF4A2 and promotes its association with RNA targets. Based on these observations, we propose a model for translational regulation of chloroplast functions by SlRBP1.

Next steps: As RNA structure is important for the binding of RBPs to their target RNA, our future work will investigate the specific secondary structures of target RNAs for the binding of SlRBP1 and explore how SlRBP1 and SleIF4A2 bind in concert to mRNAs to regulate translation.

Introduction

Many important cellular processes take place in the chloroplast, for example, photosynthesis and aspects of lipid, chlorophyll (Chl), and carotenoid biosynthesis (Chan et al., 2010). Moreover, most plastid proteins (>90%) are encoded by nuclear genes (Taylor, 1989). The mRNAs of plastid proteins are transcribed in the nucleus, translated in the cytoplasm, and then imported into plastids (Jung and Chory, 2010). Thus, chloroplast development and function rely on chloroplast-localized proteins encoded by the nuclear genes (Nott et al., 2006).

Recently, RNA-binding proteins (RBPs) have been reported to play essential roles in processing and metabolism of nucleus-transcribed transcripts, including pre-mRNA splicing and mRNA export (Nickelsen, 2003; Lorković, 2009). Identification and functional characterization of RBPs by genetic and biochemical approaches from various species have advanced our understanding of RBPs in different cellular processes (Nocker and Vierstra,1993; Chen et al., 2010; Kim et al., 2010; Kwak et al., 2013; Zhang et al., 2014; Lu et al., 2018).

Glycine-rich RBPs (GR-RBPs) harbor RNA recognition motifs (RRMs) at their N-terminus and Gly-rich regions at their C-terminus (Lee and Kang, 2016; Mahalingam and Walling, 2019). GR-RBPs belong to a superfamily with high protein abundance in a variety of plant species (Mangeon et al., 2010; Magdalena and Michal, 2018) and regulate plant growth and development. For instance, loss of function of Arabidopsis thaliana (At)RZ-1b and AtRZ-1c cause delayed seed germination, reduced stature, and serrated leaves (Wu et al., 2016). AtGRP7 regulates circadian rhythm, stress responses, and flowering timing (Xiao et al., 2015; Meyer et al., 2017). Loss of tomato (Solanum lycopersicum) organelle RRM-containing protein (SlORRM4) causes a delay in fruit ripening, presumably due to the significant changes in C-to-U editing of target RNAs (Yang et al., 2017). In barley (Hordeum vulgare), HvGR-RBP1 controls the timing of anthesis and senescence (Alptekin et al., 2021). However, due to difficulty in identification of GR-RBP-bound RNA targets, regulatory mechanisms for most GR-RBPs remain unknown.

Here, we focused on the tomato GR-RBP SlRBP1 and reported an unexpected finding that SlRBP1 modulates the translation of nucleus-transcribed transcripts encoding chloroplast-localized proteins. We found that SlRBP1 is indispensable for photosynthesis and chloroplast function. Silencing of SlRBP1 caused chloroplast dysfunction and reduced translational efficiency of target RNAs. Further studies showed that SlRBP1 interacts with the eukaryotic translation initiation factor (SleIF4A2) and promotes its association with RNA targets. Therefore, our results revealed a pathway by which SlRBP1 maintains chloroplast function by promoting translatability of photosynthesis-associated nuclear-transcribed transcripts.

Results

VIGS of SlRBP1 impaired coloring of tomato fruits

To study potential roles of RBPs in fruit development and ripening, we surveyed tomato genome annotation in the SOL Genomics Network (http://solgenomics.net/) (Supplemental Figure S1), and identified 116 putative RBPs. Through expression analysis of RNA-seq data from Tomato Genome Consortium (2012) and hierarchical clustering analysis of the putative RBPs, we pinpointed a gene encoding glycine-rich RNA-binding protein (SlRBP1) as its transcript level reaches the highest in different tomato organs and fruit ripening stages (Supplemental Figure S1).

We first conducted virus-induced gene silencing (VIGS) of SlRBP1 on developing tomato fruits. In this system, fruits developed and ripened normally when infiltrated with Tobacco rattle virus (TRV) control. The silencing of the phytoene desaturase gene (PDS) was typically used as the positive control for VIGS because the fruits infiltrated with the TRV-PDS turned red-yellow in a mottled pattern. Notably, VIGS of SlRBP1 resulted in different shades of yellow and white on fruits (Figure�1A). Compared to TRV-control fruits, mRNA levels for PDS or SlRBP1 decreased by ∼80 and 85%, respectively, in the yellow sectors of TRV-PDS and TRV-SlRBP1 fruits (Figure�1, B and C). These results suggested that silencing of SlRBP1 caused uneven coloration of the fruits.

Figure 1.

Silencing of SlRBP1 caused uneven coloring of tomato fruits. A, Phenotype of tomato fruits infiltrated with TRV-control, TRV-PDS, and TRV-SlRBP1. B, RT-qPCR assay shows PDS transcript levels in TRV-control fruits and TRV-PDS fruits. C, RT-qPCR assay shows SlRBP1 transcript levels in TRV-control fruits and TRV-SlRBP1 fruits. Actin was used as a reference in (B) and (C). The relative transcript levels in control fruits were set to 1. Error bars indicate �sd of three biological replicates. Asterisks indicate a significant difference, as determined by Student’s t test (***P < 0.001). D, Confocal imaging shows subcellular localization of SlRBP1 in N. benthamiana leaves. GFP localization was observed in N. benthamiana leaves infiltrated with either 35S:SlRBP1-GFP or a 35S:GFP control. As RIN is a transcription factor, RIN-tdTomato is selected as a control for nuclear localization. Bars, 50 μm. E, Immunoblot analysis of nuclear and cytoplasmic fractions from SlRBP1_pro:FM-SlRBP1. Nuclear Histone H3 and cytoplasmic Actin were used as controls.

Phylogenetic analysis of GR-RBPs in 12 plant species revealed that SlRBP1 was most closely related to potato (Solanum tuberosum) StGRP2 and pepper (Capsicum annuum) CaGRP1 (Supplemental Figure S2; Supplemental Table S1). Confocal microscopy imaging showed that SlRBP1 accumulated in the nucleus and cytoplasm (Figure�1D). We also conducted nucleocytoplasmic fractionations using the transgenic lines expressing SlRBP1 tagged with Flag and Myc tags under the control its native promoter (SlRBP1_pro:FM-SlRBP1; Figure�1E). Immunoblot assays indicated again that SlRBP1 resides in both the nucleus and cytoplasm, referring that the protein might function in two organelles.

Knockdown of SlRBP1 results in dwarf tomato plants with yellow leaves

We next generated knockdown mutants of SlRBP1 by artificial miRNAs (amiRNA) in the S. lycopersicum cultivar Micro-Tom (Figure�2A). Small RNA (sRNA) blot analysis validated the successful processing of amiR-SlRBP1 in the transgenic plants (Figure�2B), as the SlRBP1 transcripts were reduced significantly in amiR-SlRBP1 relative to wild-type (WT; Figure�2C). Notably, the amiR-SlRBP1 line exhibited a dwarf phenotype with small yellow leaves and much shorter internodes, which was reflected in the average plant height of only 6.1 cm for amiR-SlRBP1 plants compared to 15.8 cm of WT plants (Figure�2, D and E). Furthermore, the amiR-SlRBP1 line showed severe flower abscission, a lower fruit-setting ratio relative to WT, and fruits in different shades of yellow and orange (Figure�2F), all indicative of plant growth retardation.

Figure 2.

Knockdown of SlRBP1 affects vegetative and reproductive growth in tomato. Representative images of 5-week-old plants (A), leaves, flowers (D), and fruits (F) of WT (S. lycopersicum cv. Micro-Tom) and amiR-SlRBP1 plants. Bars, 1 cm. B, sRNA blot analysis of amiRNA in amiR-SlRBP1 plants. The U6 RNA was used as a loading control. C, RT-qPCR assay shows highly efficient silencing of SlRBP1 in amiR-SlRBP1 plants. Actin was used as a reference. Relative expression of SlRBP1 in WT was set to 1. Error bars indicate � sd of three biological replicates with each measured in triplicate. E, Plant height of WT and amiR-SlRBP1. Values are means �sd (n = 10). G, Total Chl content in young leaves of WT and amiR-SlRBP1 plants. H, TEM imaging shows difference in chloroplast ultrastructure in the leaves of WT and amiR-SlRBP1 plants. Black arrows point to thylakoid. Bars, 500 nm or 1 μm. All asterisks indicate a significant difference as determined by Student’s t test (**P < 0.01; ***P < 0.001).

Intriguingly, total Chl in amiR-SlRBP1 leaves was much lower than that in WT (Figure�2G). In addition, chloroplasts in amiR-SlRBP1 leaves had irregular shapes and empty internal structures, with missing thylakoids, whereas a normal chloroplast in WT took on the shape of an ellipsoid, with intact thylakoid structure (Figure�2H). Thus, chloroplast ultrastructure was significantly impaired in amiR-SlRBP1 leaves.

For amiR-mediating knockdown of SlRBP1, we also obtained three independent T₀ transgenic lines in S. lycopersicum cv. Ailsa Craig (AC; Figure�3, A and B). The net photosynthesis rate (A) and the maximal PSII quantum yield (Fv/Fm) were largely reduced in amiR-SlRBP1 compared with WT, suggesting the downregulation of photosynthesis in amiR-SlRBP1 (Figure�3, C and D).

Figure 3.

Silencing of SlRBP1 caused photosystem defect. A, Phenotype of WT (S. lycopersicum cv. AC) and amiR-SlRBP1 seedlings. Bars, 2 cm. B, RT-qPCR assay shows SlRBP1 expression level in different amiR-SlRBP1 transgenic lines compared to WT. Actin was used as a reference. The relative transcript levels in WT leaves were set to 1. Error bars indicate �sd of three biological replicates. C, The Fv/Fm value of WT and amiR-SlRBP1 leaves. Error bars indicate �sd of three biological replicates. D, Photosynthesis parameter assay shows the net A of WT and amiR-SlRBP1 leaves. Error bars indicate �sd of three biological replicates. All asterisks indicate a significant difference, as determined by Student’s t test (*P < 0.05; **P < 0.01; ***P < 0.001).

We also designed an RNA interference (RNAi) construct that targets the coding sequence (CDS) and 3′-untranslated region (UTR) of SlRBP1 (515–810 bp). shRNA molecules were processed within the cell to generate siRNAs which in turn knocked down SlRBP1 expression. Importantly, the silencing efficiency of SlRBP1 correlated well with the yellow leaves in SlRBP1-RNAi lines (Supplemental Figure S3). These results again indicated that loss-of-function of SlRBP1 led to chloroplast dysfunction.

Downregulation of SlRBP1 alters expression of key genes involved in photosynthesis and chloroplast function in leaves

To elucidate how SlRBP1 affected Chl accumulation and chloroplast function, we analyzed global gene expression profiling by RNA-seq with amiR-SlRBP1 seedlings (Figure�4A). We observed that 442 and 380 genes were downregulated and upregulated, respectively, between amiR-SlRBP1 versus WT leaves (Fold-change [FC] > 2 and adjusted P < 0.05; Figure�4B; Supplemental Dataset 1). Gene ontology (GO) enrichment analysis showed that the downregulated genes were significantly enriched in the genetic pathways for light-harvesting (biological process), membrane part (cellular components), pigment binding, and tetrapyrrole binding (molecular functions; Figure�4C). On the other hand, upregulated genes were significantly involved in plastid transcription (Supplemental Figure S4A). According to Kyoto Encyclopedia of Genes and Genomes (KEGG) annotations, two biological pathways were significantly affected, including photosynthesis-antenna proteins and circadian rhythm (Figure�4D; Supplemental Figure S4B). Moreover, some downregulated genes were also the key genes associated with the Chl accumulation and chloroplast function exemplified by Chl a-b binding protein (CAB), ethylene-dependent gravitropism-deficient and yellow-green-like 3 (EGY3; Figure�4E). In summary, SlRBP1 plays a vital role in plant development by affecting key genes related to photosynthesis and chloroplast function.

Figure 4.

Key DEGs are related to plant photosynthesis in amiR-SlRBP1 seedlings. A, Representative images of 2-weeks-old WT and amiR-SlRBP1 seedlings used for comparative transcriptome analysis. B, Volcano plot visualization of transcriptome deep sequencing (RNA-seq) data. Each dot corresponds to a DEG. NS, not significant; UP, up-regulated; DN, down-regulated. SlRBP1 is labeled in black. The filters of FC > 2 and adjust P < 0.05 are marked with purple lines. GO enrichment (C) and KEGG enrichment analysis (D) of downregulated DEGs in amiR-SlRBP1 seedlings compared to WT. E, Heat map representing the transcript abundance of SlRBP1, CAB1c, CAB1a, CAB1b, CAB7, CAB1d, and EGY3 in WT and amiR-SlRBP1 seedlings.

RNA targets of SlRBP1 were closely related to chloroplast function

RNA-binding proteins interact with RNAs to fulfill their functions. To identify RNAs associated with SlRBP1 in vivo, we conducted native RNA immunoprecipitation (IP) followed by high-throughput sequencing (nRIP-seq) of SlRBP1 using the SlRBP1_pro:FM-SlRBP1 line (Supplemental Figure S5A). In parallel, we performed IP from WT plants as negative control (Supplemental Figure S5B). The nRIP-seq analysis recovered 218 putative transcripts that were exclusively enriched in the immunoprecipitants of SlRBP1_pro:FM-SlRBP1 and not that of WT (Supplemental Figure S5C).

A comparison between the lists of 218 enriched genes from nRIP-seq and 822 differentially expressed genes (DEGs) from our RNA-seq data yielded a very limited overlap of five genes (Supplemental Figure S5C; Supplemental Table S2), suggesting that SlRBP1 might not regulate the accumulation of target RNAs at the transcriptional or posttranscription level. We next increased the stringency and filtered out the SlRBP1-bound transcripts with low expression levels (<50 RPKM). Thus, a total set of 61 transcripts were obtained and defined as the putative target RNAs of SlRBP1. Again, GO and KEGG analysis revealed that these 61 target genes were largely enriched in photosynthesis metabolic pathways and chloroplast structure, including photosystem I, thylakoid part, and membrane protein complex (Supplemental Figure S6, A and B; Supplemental Dataset 2).

These transcripts were bona fide targets of SlRBP1, rather than being identified due to nonspecific binding of Myc portion of Myc-SlRBP1 fusion because quantitative reverse transcription PCR (RT-qPCR) assays showed the enrichment of target RNAs in RIP with from SlRBP1_pro:FM-SlRBP1 but not in the control RIP with WT lines (Figure�5A). In contrast, nontarget genes SlRIP1b (Yang et al., 2020), Actin, Ubiquitin-3 and β-tubulin were comparable in RIP-qPCR assays between two samples.

Figure 5.

nRIP-seq identified numerous SlRBP1-bound RNA targets. A, The percentage of nRIP-enriched RNA relative to input sample, as determined by RT-qPCR. Yellow and gray columns represent IP from SlRBP1_pro:FM-SlRBP1 and WT with an anti-Myc antibody, respectively. Error bars represent � sd from biological triplicates. Actin, SlRIP1b, Ubiquitin 3, and β-Tubulin served as the negative controls. Asterisks indicate a significant difference as determined by Student’s t test (*P < 0.05; **P < 0.01; ***P < 0.001; NS, not significant). B, Integrated Genome Browser (IGB) view tracks displaying nRIP-seq read distributions in Actin, βCA1, RCA, and PsaD transcripts in WT and SlRBP1_pro:FM-SlRBP1 plants. C, Testing the binding of SlRBP1 against its target RNAs. His tag (His) or His-SlRBP1 were tested for binding to the βCA1, RCA, and PsaD transcripts, followed by RT-PCR. Actin and SlRIP1b served as negative control transcripts that are not bound by SlRBP1. D, Biotin-labeled βCA1 mRNA fragments were tested for SlRBP1 binding in vivo. Biotin labeled SlRIP1b mRNA and GST protein were used as negative controls. E, RT-qPCR analysis of PDS, βCA1, and RCA transcript levels in TRV-control, TRV-PDS, TRV-βCA1 leaves and TRV-RCA leaves. Asterisks indicate a significant difference, as determined by Student’s t test (***P < 0.001). F, Tomato leaves infiltrated with TRV-RCA and TRV-βCA1 exhibit yellow color compared to TRV-control tomato leaves. Bars, 1 cm.

Interestingly, some key genes related to photosynthetic metabolic pathways that were dramatically enriched in SlRBP1 RIP-seq even exhibited an enrichment rate (IP/Input) greater than 10. These transcripts are Photosystem I reaction center subunit II (PsaD), Rubisco activase (RCA), and β-Carbonic anhydrase 1 (βCA1), and their encoded proteins are chloroplast-targeted (Figure�5B; Supplemental Table S2; Ferreira et al., 2008; Fukayama et al., 2012). nRIP-qPCR and protein-binding RNA assays further validated that SlRBP1 is specifically bound to the three targets in vivo and in vitro (Figure�5, B and C). In addition, RNA pull down assay showed that SlRBP1 was associated with CDS and 3′-UTR of βCA1 in vitro (Figure�5D).

In light of our finding that SlRBP1 predominantly bound to βCA1 and RCA, we hypothesized that SlRBP1 modulates chloroplast function through its target RNAs. To test this hypothesis, we silenced βCA1 and RCA in tomato leaves by VIGS with PDS as a control. Consistent with the observations in the mutants, the PDS, βCA1, and RCA mRNA levels in the (white) yellow areas of the TRV-PDS, TRV-βCA1, and TRV-RCA leaves decreased by ∼88%, 82%, and 77%, respectively, compared with TRV leaves (Figure�5E). Indeed, both TRV-βCA1- and TRV-RCA-infected leaves showed mottled yellow areas, which were reminiscent of amiR-SlRBP1 phenotype (Figure�5F). Overall, our results indicated that SlRBP1 maintains chloroplast function via directly binding to the nuclear transcripts encoding chloroplast-targeted proteins in vivo.

SlRBP1 affects protein accumulation of its target genes but not their transcript levels

Since many RBPs are key regulators in posttranscriptional control of gene expression, we investigated whether SlRBP1 would affect the expression of its bound transcripts. To this end, we re-visited RNA-seq data from WT, amiR-SIRBP1, and SIRBP1_pro:FM-SIRBP1 that harbors an extra copy of the SIRBP1 gene. Of note, the steady-state transcript levels of βCA1, RCA, and PsaD were comparable in SlRBP1_pro:FM-SlRBP1 or amiR-SlRBP1 seedlings relative to WT (Figure�6A; Supplemental Figure S7). This result was further validated by PCR, which indicated that SIRBP1 did not alter the accumulation of its bound mRNAs (Figure�6B).

Figure 6.

Silencing of SlRBP1 dramatically decreased the levels of the proteins encoded by its bound targets. A, IGB view of mapped reads for SlRBP1 and selected SlRBP1-bound target transcripts in WT, amiR-SlRBP1 and SlRBP1_pro:FM-SlRBP1 plants. Note: amiR-SlRBP1 plants accumulate few SlRBP1 mRNAs, while SlRBP1 is highly expressed in SlRBP1_pro:FM-SlRBP1 compared to WT. B, PCR analysis of the expression of SlRBP1 and target genes in WT, amiR-SlRBP1 and SlRBP1_pro:FM-SlRBP1 plants. Actin was used as a reference. Accumulation of target RNA-encoded proteins in WT, amiR-SlRBP1 (C) and SlRBP1_pro:FM-SlRBP1 (D) plants. βCA1, PsaD, and RCA are protein encoded by SlRBP1 target RNAs. Actin was used as an internal control. E, Treatment of seedlings with the proteasome inhibitor MG132 does not affect the accumulation of RCA, PsaD, or βCA1 proteins. Actin serves as a loading control. SlREM1 is a positive control for MG132 treatment.

We also assessed if SlRBP1 binding affected the stability of target RNAs. We treated WT and amiR-SlRBP1 plants with the transcriptional inhibitor cordycepin. RT-qPCR assays showed that there was a marginal difference in the degradation rate of βCA1 and RCA transcripts, indicating that SIRBP did not alter the half-life of selected targets (Supplemental Figure S8).

We next hypothesized that SlRBP1 might influence protein accumulation of its targets. Indeed, βCA1, PsaD, and RCA proteins, in contrast to the control Actin protein, were barely detectable in amiR-SlRBP1 leaves (Figure�6C). Moreover, the protein accumulation of selected targets was slightly increased in the SlRBP1_pro:FM-SlRBP1 line relative to that of WT plants (Figure�6D). Thus, SlRBP1 largely impacted the protein levels of its targets but not the steady-state levels of target RNAs.

In addition, we assessed the impact of SIRBP1 on protein stability of βCA1, PsaD, and RCA. We found that the accumulation of these proteins remained fairly constant with or without the proteasome inhibitor MG132 (Figure�6E). This was different from the ubiquitin-targeted protein SlREM1, which increased in the presence of MG132 (Cai et al., 2018). This result clearly indicated that SlRBP1 enhancement of the accumulation of the proteins encoded by its bound transcripts is not related to protein stability.

Knockdown of SlRBP1 confers alterations in translational efficiency

To further investigate the role of SlRBP1 in translation regulation, we performed polysome profiling assays of WT and amiR-SlRBP1 leaves. We observed that knockdown of SlRBP1 caused considerable and consistent decreases in the levels of the 40S subunits and polysomes relative to WT plants (Supplemental Figure S9). This result indicated the presence of a defect in translation, potentially at the initiation step in amiR-SlRBP1 versus WT plants (Figure�7A).

Figure 7.

Silencing of SlRBP1 reduced the translation efficiency and conferred an imbalanced polysome profile. A, Quantitative sucrose density gradient analysis showing the imbalanced polysome profile in amiR-SlRBP1 compared to WT plants. B, RT-qPCR assays show the abundance of SlRBP1 target RNAs in polysome fractions from amiR-SlRBP1 and WT plants. Actin, Ubiquitin 3, and β-Tubulin served as the negative controls. Error bars indicate �sd of three technical replicates. Asterisks indicate a significant difference as determined by Student’s t test (*P < 0.05; **P < 0.01; ***P < 0.001).

Moreover, we recovered total and polysome-bound mRNA for RT-qPCR assays to assess translation efficiency of SIRBP1-bound targets in the WT and amiR-SlRBP1 leaves. To ensure sample representativity, we randomly selected 18 out of the 61 targets and a similar number of nontargets as controls (Figure�7B; Supplemental Figures S10 and S11). We observed that only 1 of 15 nontargets showed decrease in polysome occupancy (Figure�7B; Supplemental Figure S11). However, among 18 selected targets, 16 displayed substantial decrease of the polysome occupancy in amiR-SlRBP1 compared to WT (Figure�7B; Supplemental Figure S10). Notably, eight of the target RNAs are associated with photosynthesis metabolic pathways such as βCA1, RCA, PsaD, Photosystem II core complex proteins (PsbY), Photosystem I reaction center subunit VI-1 (PsaH), Photosystem I reaction center subunit IV (PsaE), Ferredoxin 1, and CAB4 (Figure�7B). Overall, these results strongly indicated the silencing of SlRBP1 reduced the translation efficiency of target RNAs, especially for the transcripts involved in photosynthesis.

SlRBP1 interacts with SleIF4A2 to regulate translation initiation of targets

To further understand how SlRBP1 regulates the translation of its bound RNAs, we performed sequential Co-IP combined with mass spectrometry (IP-MS) of SlRBP1_pro:FM-SlRBP1 and WT (negative control), aiming at identifying SIRBP1-interacting proteins (Figure�8A;Supplemental Figure S12, A and B). As illustrated by a volcano plot, candidates 111 protein partners were significantly enriched in SlRBP1_pro:FM-SlRBP1 samples compared with WT control (FC > 3, peptide-spectrum match > 4 and P < 0.05; Figure�8A;Supplemental Dataset 3). SlRBP1 was among the most abundant proteins recovered in IPs from SlRBP1_pro:FMF-SlRBP1 samples, validating the physiological relevance of the IP-MS assay (Figure�8A).

Figure 8.

SlRBP1 interacted with SleIF4A2 to regulate translation of targets. A, Volcano plot illustrating log₂ FC (x-axis) and statistical significance distribution (y-axis) of the proteomic data set. The dashed line indicates the threshold above which proteins are significantly enriched (FC > 3 and P < 0.05). Detailed diagram of informatics pipeline for the identification of putative SlRBP1-interacting proteins. B, BiFC assay confirmed the interaction of SlRBP1 with SleIF4A2. SlRBP1 was fused with the C-terminal end of mCherry, the SleIF4A2 were fused with the N-terminal end of mCherry. As NOR is a transcription factor, NOR-GFP is selected as a marker protein for the nucleus. The constructs used for transformation are indicated (left). Bars, 50 μm. C, Co-IP validation of the interaction between SlRBP1 and SleIF4A2. Various combinations of SleIF4A2-6Myc and SlRBP1-GFP fusion proteins as indicated were transiently expressed in N. benthamiana leaves followed by IP using anti- C-Myc Magnetic Beads. D, LUC complementation assay validation of SlRBP1 interaction with SleIF4A2 in vivo. SlRBP1 fused with the N-terminus of Luc (SlRBP1-Nluc) were coexpressed with SleIF4A2 fused with the C-terminus of Luc (SleIF4A2-Cluc) in N. benthamiana leaves. E, RIP-qPCR showed that the RNA-binding capacity (RCA and βCA1) of SleIF4A2 was decreased in amiR-SlRBP1 leaves compared to WT. For the RIP assay, the protein–RNA complexes were extracted from WT and amiR-SlRBP1 leaves and subjected to IP with anti-SleIF4A2 polyclonal antibody or rabbit IgG antibody (negative control). Asterisks indicate a significant difference, as determined by Student’s t test; **P < 0.01; ***P < 0.001. The GAPDH was used as a negative control.

Among the SlRBP1-interacting proteins, we recovered heat shock protein 90 (HSP90) and SleIF4A2 (Solyc12g095990) with the high-abundance proteins in the IP-MS datasets (Supplemental Figure S13). HSP90 is a highly conserved molecular chaperone involved in many client proteins folding and stabilization in plants (Shigeta et al., 2014). How SIRBP1 impacts HSP90 function, or vice versa, will be studied in the future and not discussed here.

Rather, we focused on SleIF4A2, an eukaryotic translation initiation factor (Supplemental Figure S14) (Parsyan et al., 2011). First, we further validated if SleIF4A2 is a partner of SlRBP1 in vivo. To this end, we conducted bimolecular fluorescence complementation (BiFC) assays. We observed that co-transfection of SlRBP1-mCherry^C and mCherry^N-SleIF4A2 in Nicotiana benthamiana leaves produced strong mCherry fluorescence signals indicating that SlRBP1 indeed interacted with SleIF4A2 in vivo (Figure�8B). This interaction is specific because SlORRM4, another protein in GR-RBP family did not show a BiFC signal (Yang et al., 2020).

We also conducted Co-IP assays. When SlRBP1-GFP and SleIF4A2-6Myc fusion proteins were transiently coexpressed in N. benthamiana leaf cells, the SleIF4A2-6Myc fusion protein could be easily coimmunoprecipitated with SlRBP1-GFP (Figure�8C). Finally, we performed split Luciferase Complementation Imaging (LCI) assays. Again, SleIF4A2 and SIRBP1 demonstrated clear LCI signal. Altogether, all these three independent assays clearly indicated that SleIF4A2 is a bona fide partner of SlRBP1 (Figure�8D). In addition, SlRBP1 could form homodimers in vivo (Supplemental Figure S15).

Given that SlRBP1 specifically interacts with SleIF4A2, and that SleIFA2 is a canonical factor essential for protein synthesis, we hypothesized that SlRBP1 might increase the affinity between its target RNAs and SleIF4A2. To test this, we performed the IP of SleIF4A2-RNAs complex by a polyclonal antibody against SleIF4A2 in different backgrounds (Supplemental Figure S16). Indeed, silencing of SlRBP1 dramatically decreased the association of SleIF4A2 with βCA1 and RCA, but not a control (Glyceraldehyde 3-phosphate dehydrogenase [GAPDH]; Figure�8E). Taken together, our data showed that SlRBP1 promotes the association of SleIF4A2 with its specific RNA targets, which is a prerequisite for translational initiation.

Discussion

GR-RBPs have been known for their important roles in RNA processing and metabolism (Sachetto-Martins et al., 2000; Yang et al., 2014; Lee et al., 2012). Here, we revealed a regulatory role of SlRBP1 (GR-RBP) in the translation of its bound targets, rather than in RNA processing. Through its role in translation, SlRBP1 maintains chloroplast functions for optimal plant growth and development. Several pieces of evidence supported our notion: first, knockdown mutations of SlRBP1 caused severe chloroplast dysfunction, resulting in dwarf tomato plants with yellow leaves (Figures�2 and 3). Second, SlRBP1 associated with 61 target RNAs, most of which are key photosynthesis-related nucleus transcripts encoding chloroplast-targeted proteins (Figure�5;Supplemental Figure S6). Third, reduced expression of SlRBP1 dramatically decreased the translation efficiency of target RNAs resulting in a dramatic reduction in the protein accumulation of its targets (Figures�6C and 7B). Fourth, the translational initiation factor, SlelF4A2, is a bona fide partner of SlRBP1 (Figure�8, B–D). Last but more importantly, silencing of SlRBP1 significantly reduced the association of SleIF4A2 with its RNA targets, and led to less occupancy of polysomes on RNA targets (Figure�8E).

Based on these observations, we propose a model of translational regulation on chloroplast functions by SlRBP1 (Figure�9). SlRBP1 recruits its RNA targets to SleIF4A2, increasing the efficiency of translation initiation. Consequently, more ribosomes are launched onto SlRBP1-bound mRNAs, allowing more active synthesis of the proteins, which are in turn targeted to chloroplasts for photosynthesis and other chloroplast functions. This is a unique function and mechanism of plant RBP protein as other similar case has been only reported in mammalian cells (Zhang et al., 2019b). Thus, our results here and a previous report in animal indicated that RNA-binding proteins act cooperatively with translation factors to influence the translation of their RNA targets in eukaryotes.

Figure 9.

Working model of SlRBP1 function in modulating translation of target RNAs to maintain the chloroplast function. SlRBP1 binds to and channels RNAs onto the translation initiation SleIF4A2 complex to regulate translation initiation of targets.

During evolution, numerous genes have been transferred from the organelles to the nucleus in eukaryotes. Thus, the organelles, exemplified by the chloroplast here, harbor small genomes that could not encode enough proteins to support basic chloroplast function (Dyall et al., 2004; Stern et al., 2010; Allen et al., 2011; Pogson et al., 2015). A majority of proteins that are required for the maintenance of chloroplast functions, are nuclear genome-encoded and translated into the cytoplasm, and subsequently imported into the chloroplast (Millar et al., 2006; Scharff and Bock, 2014; Sjuts et al., 2017). If the chloroplast super-complex is regarded as a machine, the “domestic parts” (the polypeptide subunits encoded by the chloroplast genome) and the “imported parts” (nuclear genome-encoded subunits) of this machine must match perfectly regarding quantity and quality to ensure the effective function of chloroplast. Therefore, the fine-tune regulation of these “imported parts” is crucial for the chloroplast maintenance. βCA is among the vital proteins that provide the necessary rate of CO₂ supply for carboxylation of ribulose bisphosphate (RuBP) in Calvin cycle (Ignatova et al., 2019). RCA is a chaperone that functions in Rubisco repair and carboxysome organization and is thus critical for plant development (Flecken et al., 2020). Some evidence showed that βCA is regulated at transcriptional level (Huang et al., 2017; Hu et al., 2021). In this study, SlRBP1 predominantly bound to the βCA and RCA transcripts, and regulated them at the translation level. In sum, our results reveal a regulatory mechanism of how a nucleus-encoded protein SlRBP1 specifically controls the accumulation of the “imported parts” to maintain chloroplast function.

How exactly SlRBP1 recognizes and specifically binds to its target transcripts awaits future investigation. One possibility is that the target mRNAs might harbor certain consensus sequence motifs, which can be recognized by SlRBP1. Another possibility is that SlRBP1 may bind specific secondary structures of its target RNAs. Recently, an elegant study showed RNA structure is important for the binding of RBP to its target RNA (Liu et al., 2020; Zhu et al., 2021). For instance, the human 15.5K protein specifically binds to Kink-turn RNAs and stabilizes their structures (Cojocaru et al., 2015). Further investigation of how SlRBP1 selectively binds to its targets and how SlRBP1 and SleIF4A2 concertedly bind to the mRNAs to regulate translation will not only advance our understanding of translation regulatory mechanism, but also provide new tools to enhance the function of chloroplast for plant growth and yield.

Materials and methods

Plant materials and growth conditions

WT S. lycopersicum cv. Micro-Tom and cv. AC and transgenic tomato plants were grown in a greenhouse under standard conditions (16 h of light at 26�C followed by 8 h of darkness at 20�C) with T5 fluorescent light tubes. The same growth condition was used for N. benthamiana. Samples from transgenic lines and WT plants were harvested and immediately frozen in liquid nitrogen following storage at −80�C.

Cloning of the full-length SlRBP1

Total RNA was prepared and used for cDNA synthesis with HiScript III 1st Strand cDNA Synthesis Kit (Vazyme Biotech, Beijing, China; cat. no. R312). A FirstChoice RLM-RACE kit (Invitrogen, Waltham, MA, USA; cat. no. AM1700) was used for cloning the 3′-poly A tail sequences. Primers were designed to amplify 5′- and 3′-UTR based on the RNA-seq data. We also designed primers to amplify the full-length SlRBP1 cDNA. The amplified fragment was finally determined by Sanger sequencing. The full-length sequence of SlRBP1 was submitted to the NCBI database (MG518522). The primers are listed in Supplemental Dataset 4.

RNA isolation and RT-qPCR analysis

Total RNA was isolated from leaves as previously described (Zhu et al., 2015). RNA concentration was measured using a Nano-300 spectrophotometer (Allsheng, Hangzhou, China). After removing genomic DNA contaminants by DNase I treatment, total RNA was treated with gDNA Eraser and then used as template for first-strand cDNA synthesis using HiScript II 1st Strand cDNA Synthesis Kit (Vazyme Biotech, Beijing, China; cat. no. R212) according to the manufacturer’s protocol. RT-qPCR was performed with SYBR Green PCR Master Mix (TransGen Biotech, Beijing, China; cat. no. AQ131) on a CFX96 Real-Time PCR System (Bio-Rad, Hercules, CA, USA). Actin was used as endogenous control. Every experiment included three biological repeats. RT-qPCR primers are listed in Supplemental Dataset 4.

VIGS

pTRV vectors were used for VIGS. About 300- to 500-bp fragments of SlRBP1, βCA1, RCA, and PDS were designed using the VIGS tool (http://solgenomics.net/tools/vigs) to avoid off-target silencing. Specific cDNA fragments were amplified and inserted into pTRV2 vectors that were transferred to Agrobacterium strain GV3101. VIGS of SlRBP1 and PDS on MT fruits was performed according to previous study (Yang et al., 2017), and VIGS of βCA1, RCA, and PDS on MT leaves was according to previous study (Liu et al., 2002; Fu et al., 2005). Oligonucleotide primers are listed in Supplemental Dataset 4.

Generation of transgenic tomato plants

The CaMV 35S promoter in the pCAMBIA-Flag-Myc (Zhu et al., 2011) was replaced with the SlRBP1 promoter (from −2,500 bp to the base before the translation start codon). Then the CDS of SlRBP1 was inserted to generate the pCAMBIA-SlRBP1pro: Flag-Myc-SlRBP1 construct. The amiRNA targeting SlRBP1 was designed with Web MicroRNA Designer (http://wmd3.weigelworld.org/cgi-bin/webapp.cgi). The SlRBP1 amiRNA precursor was synthesized by Genescript (Nanjing, China), using the A. thaliana MIR390 precursor as a backbone (Ju et al., 2017) and then cloned into pCAMBIA1300 to generate pCAMBIA-amiR-SlRBP1. To construct the SlRBP1 RNAi vector, we amplified a 250 bp fragment targeting the CDS and 3′-UTR of SlRBP1 and cloned the resulting PCR product into the pHELLSGATE vector. The final binary vectors were transformed into tomato plants using Agrobacterium strain GV3101. All primers for plasmid construction are listed in Supplemental Dataset 4.

Phylogenetic analysis

The alignment of protein sequence was performed using ClustalX 2.1 with default parameters. A rooted phylogenetic tree was constructed through MEGA version 5.2 by neighbor-joining method with 1,000 bootstrap replicates and visualized with the tool iTOL. Alignments used for phylogenetic analysis are provided as Supplemental Files S1–S4.

Subcellular localization of SlRBP1

The SlRBP1 CDS without stop codon was cloned into the green fluorescent protein (GFP) expression vector using a one-step cloning kit (Vazyme Biotech, Beijing, China; cat. no. C112). tdTomato was fused to the C terminus of the transcription factor RIPENING INHIBITOR (RIN), which is as the positive control. These fusion constructs and the control 35S:GFP construct were introduced into Agrobacterium strain GV3101, infiltrated into N. benthamiana leaves, and allowed to produce the fluorescent proteins for 48 h. Two days after infiltration, N. benthamiana leaves were observed under a Nikon A1RMPsi laser-scanning confocal microscope (Tokyo, Japan). The primers used above are listed in Supplemental Dataset 4.

sRNA blot analysis

Total RNA was extracted from 14-day-old seedlings of amiR-SlRBP1 plants. Northern blots were performed as described with some modifications (Zhu et al., 2013). Each lane contained 20 μg of total RNA. Two DNA oligonucleotides complementary to S. lycopersicum U6 RNA and amiR-SlRBP1 and separately labeled by DIG Oligonucleotide Tailing Kit (Roche, Mannheim, Germany; cat. no. 03353583910) were used as probes for hybridization. Blots were hybridized with DIG-labeled oligonucleotide probes complementary to amiR-SlRBP1. U6 served as the loading control in blots. Hybridization was carried out in PerfectHyb Plus Hybridization buffer (Sigma-Aldrich, St Louis, MO, USA; cat. no. H7033) at 37�C in an LF-III molecular hybridization oven (Scientz, Ningbo, China). RNA blots were detected with the DIG Wash and Block Buffer Kit (Roche, Mannheim, Germany; cat. no. 11585762001) followed by imaging on a Tanon-5200 imager (Tanon Science & Technology, Shanghai, China). Primers and probes used are listed in Supplemental Dataset 4.

Photosynthetic parameter measurement

To evaluate the maximum quantum yield of photosystem II photochemistry (Fv/Fm), all the seedlings were adapted under darkness for 30 min and then imaged using Imaging-PAM Chl fluorescence system (MAXI, Heinz Walz, Effeltrich, Germany). The net A was performed with the LI-6800 photosynthesis system (Li-Cor Inc., Lincoln, NE, USA) with the leaf chamber fluorometer (6800-01A, area 2 cm²), in which a mixture of red (90%) and blue (10%) LEDs with peak intensities of 625 and 475 nm, respectively, was provided. During measurement, photosynthetic photon flux density was 200-μmol m⁻² s⁻¹, CO₂ concentration was 400-μmol mol⁻¹, leaf temperature was 25�C, leaf-to-air vapor pressure deficit (leaf-air) was around 1.0 kPa, and the flow rate of air through the system was 500-μmol s⁻¹. Upon net photosynthetic rate reaching steady-state condition (10 min), gas exchange parameters were logged (Zhang et al., 2019a).

Protein extraction and immunoblot analysis

Total proteins were extracted according to the method described previously (Yang et al., 2017). The primary antibodies against c-Myc (Sigma-Aldrich, St Louis, MO, USA; cat. no. C3956), His tag (Abmart, Shanghai, China; cat. no. M30111M), GST (Easybio, Beijing, China; cat. no. BE2013), Actin (Cowin Biosciences, Beijing, China; cat. no. CW0264M), Histone H3 (Easybio, Beijing, China; cat. no. BE3015), GFP (Abmart, Shanghai, China; cat. no. M20004M), RCA (Agrisera, V�nn�s, Sweden; cat. no. AS10 700), eIF4A (Agrisera, V�nn�s, Sweden; cat. no. AS19 4251), βCA1 (Agrisera, V�nn�s, Sweden; cat. no. AS19 4321), and IgG (Abmart, Shanghai, China; cat. no. B30011M) were all used at 1:5,000 dilution. The antibodies against PsaD (PhytoAB, San Jose, CA, USA; cat. no. PHY0056) were at a dilution of 1: 2,000. Secondary goat anti-rabbit IgG (MBL, Nagoya, Japan; cat. no. 458) and anti-mouse IgG (MBL, Nagoya, Japan; cat. no. 330) were at a dilution of 1:10,000. The blots were visualized using an enhanced chemiluminescence kit (Absin, Shanghai, China; cat. no. abs920) followed by imaging on a Tanon-5200 imager (Tanon Science & Technology, Shanghai, China).

Nuclear–cytoplasmic fractionation

Nuclear and cytoplasmic fractions were prepared as described previously (Wang et al., 2011), using leaves from transgenic lines SlRBP1pro: SlRBP1. Histone H3 and Actin were used as the internal controls for nuclear and cytoplasmic fractions, respectively.

Transcriptome deep sequencing (RNA-seq) and analysis

Total RNA samples were extracted from WT, amiR-SlRBP1, and SlRBP1_pro:FM-SlRBP1 tomato leaves (three replicates per sample). RNA-seq libraries were constructed and sequenced (Novogene, Tianjin, China). Reads with low quality and adapter sequences were removed. Clean reads were aligned to the Tomato reference genome (version SL2.50) using Tophat (version 1.4.6; Tomato Genome Consortium, 2012; Liao et al., 2014). Differential gene expression was determined with the DEseq2 using the criteria of FC > 2 and adjusted P < 0.05.

TEM analysis

Transmission electron microscopy (TEM) was performed with 4-week-old leaves of WT and amiR-SlRBP1 according to the method described before (Yang et al., 2017).

nRIP-seq and nRIP-qPCR

SlRBP1-nRIP was performed as previously described with some modifications (Yang et al., 2020). Lysates were extracted from the leaves of SlRBP1_pro:FM-SlRBP1 and WT plants using lysis buffer (20-mM Tris–HCl pH 7.5, 300-mM KCl, 5-mM MgCl₂, 5-mM DTT, 0.2% Triton X-100, 2% glycerol, 1-mM PMSF, 1 tablet mL⁻¹ protease inhibitor cocktail tablet (Roche, Mannheim, Germany; cat. no. 04693132001) and 1 unit �L⁻¹ RNase inhibitor (Vazyme Biotech, Beijing, China; cat. no. R301). The supernatant immunoprecipitated with anti-C-Myc Magnetic Beads (Thermo Fisher Scientific, Waltham, MA, USA; cat. no. 88843).

SleIF4A-nRIP was performed as previously described (Yang et al., 2020). Lysates were extracted from the leaves of WT and amiR-SlRBP1 plants and then precleared with Dynabeads Protein A/G (Thermo Fisher Scientific, Waltham, MA, USA; cat. no. 88803). Endogenous SleIF4A protein complex was immunoprecipitated with a monoclonal anti-SleIF4A antibody (Agrisera, V�nn�s, Sweden; cat. no. AS19 4251) together with Dynabeads Protein A/G.

Protein–RNA complexes were washed 5 times with lysis buffer and then eluted from the beads with elution buffer (100-mM Tris pH 6.8, 4% Sodium dodecyl sulfate). After treatment with proteinase K (Thermo Fisher Scientific, Waltham, MA, USA; cat. no. 100005393) and DNase I (TransGen Biotech, Beijing, China; cat. no. GD201-01), the enriched RNAs were extracted using acid-phenol: chloroform (pH 4.5) followed by ethanol precipitation.

For SlRBP1 RIP-seq, the RNA (input and immunoprecipitate) libraries were constructed and sequenced on an Illumina HiSeq2500 platform (Novogene, Tianjin, China). Clean reads were mapped to the tomato reference genome (version SL2.50) using Tophat (version 1.4.6). The target binding regions of SlRBP1 were identified using MACS2 software (version 2.1.1) with default options except for “–nomodel.” A stringent cutoff threshold for false discovery rate P < 0.005 was used to obtain high-confidence binding regions of SlRBP1.

For nRIP-qPCR, reverse transcription was performed with TransScript One-Step gDNA Removal and cDNA Synthesis SuperMix Kit (TransGen Biotech, Beijing, China; cat. no. AT311-03) with random hexamers, followed by RT-qPCR analysis. The primers used for RIP-qPCR are listed in Supplemental Dataset 4.

mRNA stability assay

For target RNA stability assay as previously described with minor modifications (Zhao et al., 2019), seeds from WT and amiR-SlRBP1 transgenic plants were germinated and grown on solid MS medium for 14 days before transfer to 200-mL incubation buffer (1-mM PIPES pH 6.25, 1-mM sodium citrate, 1-mM KCl, 15-mM sucrose) with 0.5-mM cordycepin (Targetmol, Shanghai, China; cat. no. T2993). The samples were placed under vacuum for 15 min and then incubated at room temperature for the indicated times (0, 0.5, 1, 3, and 6 h) and quickly frozen in liquid nitrogen. Total RNA extraction was performed as described above, followed by RT-qPCR analysis with primers specific for eIF4A1 as control for mRNAs with high stability (Fedak et al., 2016). Primers used are listed in Supplemental Dataset 4.

Protein binding RNA assay

Protein binding RNA assay was prepared as described previously (Tian et al., 2019). The SlRBP1 CDS was cloned into the pET-46 vector using a one-step cloning kit (Vazyme Biotech, Beijing, China; cat. no. C112-02). The recombinant vector was introduced into Escherichia coli BL21 (DE3) cells for His-SlRBP1 protein expression and purification. Soluble His-SlRBP1 protein was purified using a His-Tagged Protein Purification Kit (Cowin Biosciences, Beijing, China; cat. no. CW0894S) according to the manufacturer’s instructions. Total RNA, which was extracted from 4-week-old leaves of WT, was heated to 95�C for 2 min, then slowly cooled down to room temperature for annealing. The total RNA was precleared with empty Dynabeads His-Tag Isolation (Thermo Fisher Scientific, Waltham, MA, USA; cat. no. 10103D) in pull-down buffer (6.5-mM sodium phosphate pH 7.4, 140-mM NaCl, 0.02% Tween-20, and 40-U/mL RNase inhibitor). For binding of RNAs by SlRBP1 protein, 2 μg of His-SlRBP1 was bound to Dynabeads subsequently washed with washing buffer (100-mM sodium-phosphate pH 7.5, 600-mM NaCl, 1-mM PMSF, and 0.02% Tween-20). The SlRBP1–RNA complexes were eluted with buffer (50-mM sodium-phosphate pH 8.0, 300-mM NaCl, 0.01% Tween-20, and 300-mM imidazole) at 37�C for 10 min. The enriched RNAs were recovered by phenol/chloroform extraction and ethanol precipitation following RT-qPCR. Primers used are listed in Supplemental Dataset 4.

RNA pull-down assay

RNA pull-down assay was prepared as described previously (Tian et al., 2019). The different parts of βCA RNA were transcribed in vitro with RNA Production Systems-T7 (Promega, Beijing, China; cat. no. P1300). The 3′-terminus of RNA were labeled with biotin using the Pierce RNA 3′-end Desthiobiotinylation Kit (Thermo Fisher Scientific, Waltham, MA, USA; cat. no. 20163). The biotinylated RNAs (50 pmol) were heated to 95�C for 2 min and slowly cooled down to room temperature for annealing. After the annealed biotinylated RNAs were associated with the beads (Thermo Fisher Scientific, Waltham, MA, USA; cat. no. 88816), the labeled RNAs were gently mixed with soluble protein (2–3 μg) for 1 h at 4�C. The beads were washed 8 times with 50-mM Tris (pH 7.5), 150-mM NaCl, 1-mM DTT, 0.01% Tween-20, and RNase inhibitor (40 U/mL). Proteins were immunoblotted with appropriate antibodies. The biotinylated RNAs were detected using Chemiluminescent Nucleic Acid Detection Module Kit (Thermo Fisher Scientific, Waltham, MA, USA; cat. no. 89880). The primers used above are described in Supplemental Dataset 4.

MG132 treatment

To assess ubiquitin-proteasome system-mediated degradation of target proteins, six-leaf-stage WT plants were divided into two parts: one set was infiltrated with liquid MS medium supplemented with MG132 (Sigma-Aldrich, St Louis, MO, USA; cat. no. 474787), while the other set was infiltrated with liquid MS medium only (Li et al., 2020). After 12 h, protein abundance was determined by immunoblot analysis.

Polysome profiling

Polysome from both WT and amiR-SlRBP1 transgenic leaves were fractionated over sucrose gradients as previously described with some modifications (Zhu et al., 2016). Briefly, 0.5 g tissue were lysed by incubation for 15 min on ice. After centrifuging at 15,000g for 30 min at 4�C, the supernatant was filtered through Mira-cloth (Cal BioChem, San Diego, CA, USA). A total of 5,000 A260 units of the supernatant were layered onto a linear 5%–50% (w/v) sucrose gradient poured with the Gradient Master 108 (BioComp Instruments, Fredericton, Canada). After ultracentrifugation in a Hitachi P40ST rotor at 35,000g for 3 h at 4�C, each fraction was analyzed using the Piston Gradient Fractionator (BioComp Instruments) attached to a Model EM-1 Econo UV Monitor (Bio-Rad) for continuous measurement of the absorbance at 260 nm. All 61 fractions were collected using a Gilson 203B Fraction Collector. Three biological replications were performed with similar outcome; thus Figure�7A only shows the result of one of the three replications.

For polysome-RNA isolation, fractions 51–55 were performed using TRIzol LS reagent (Thermo Fisher Scientific, Waltham, MA, USA; cat. no. 91254901) according to the manufacturer’s protocol. Total RNA extracted from three peak fractions (ribosome fractions). The levels of 18S and 25S rRNA in the three peak fractions (ribosome fractions) were analyzed by RT-PCR (Bai et al., 2019). Each targets and nontarget expressions in polysomal fraction were calculated as the percentage of its expression in total RNA (Wu et al., 2020). The primers used for RT-qPCR are described in Supplemental Dataset 4.

IP-MS

FM–SlRBP1 protein complexes were sequential immunoprecipitated (IP-re-IP) from 5-week-old leaves of WT and SlRBP1_pro:FM-SlRBP1 plants, according to methods of our previous study (Wang et al., 2015). The enriched proteins were then sent to the Institute of Virology and Biotechnology (Hangzhou, Zhejiang, China) for MS analysis with three independent biological replicates. The missing values were imputated using random drawing from a left-censored normal distribution with impute. MinDet function (q = 0.01) function in R package imputeLCMD, and statistical testing was performed using limma package in the R environment (version 4.1.1) at protein levels.

BiFC

The full-length CDSs of SleIF4A2 and SlRBP1 were fused with CDSs of C-mCherry^N and the N-terminal portion of mCherry^C, respectively. Primers used to generate these constructs are shown in Supplemental Table S3. The successful constructs were introduced into Agrobacterium tumefaciens strain GV3101 (Yang et al., 2020). Transfection was performed according to the above method. We also co-infiltrated N. benthamiana leaves with a GFP fusion with the nucleus protein Non-Ripening (NOR) to specifically mark nucleus (Gao et al., 2020). Two days after infiltration, tissue was visualized with a confocal microscope (A1RMPSi).

Split LCI assays

To generate constructs for LCI assays, the CDSs of SlRBP1 and SleIF4A2 were ligated into split luc vector pCAMBIA1300-Cluc/Nluc to produce SlRBP1-Nluc and SleIF4A2-Cluc, respectively. The successful constructs were introduced into A. tumefaciens strains GV3101 and transiently expressed in N. benthamiana leaves as described above. Two days after infiltration, leaves were added with 1-mM luciferin (Promega, Beijing, China; Cai et al., 2018). The resulting luciferase signals were collected using the Tanon-5200 image system. The primers used for LCI are listed in Supplemental Dataset 4.

Co-IP

Co-IP assays were performed as previously described (Zhu et al., 2013) with minor modifications. SlRBP1-GFP, SleIF4A2-Myc, and HSP90-Myc proteins were transiently expressed in N. benthamiana leaves. The infiltrated leaves were collected 48 h after infiltration. After protein extraction, the extracts were coimmunoprecipitated with anti-Myc bead (Thermo Fisher Scientific, Waltham, MA, USA; cat. no. 88843) for at least 2 h at 4�C. Beads were washed 5 times with the lysis buffer. Proteins associated with the Myc fusion protein were eluted by adding 100-�L elution buffer (0.1-M Glycine, pH 2.0) and heating at 95�C for 10 min. The primers used for the vectors used for Co-IP are listed in Supplemental Dataset 4.

Statistical analysis

Significance analysis of the data was conducted using SPSS (version 20.0) software. For two data sets, statistical significance was computed using Student’s t test (*P < 0.05; **P < 0.01; ***P < 0.001). Summary statistics are given in Supplemental Dataset 5.

Accession numbers

The full-length sequence of SlRBP1 can be found in the NCBI database under accession number MG518522. Raw sequencing data from the RNA-seq, and RIP-seq have been deposited at the NCBI SRA (http://www.ncbi.nlm.nih.gov/sra/) under accession number PRJNA606015. Supplemental Table S1 contains the accession numbers of protein used in phylogenetic analyses.

Supplemental data

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

Supplemental Figure S1. Heat map representing the normalized abundance of 116 putative RNA binding proteins related to fruit development and ripening.

Supplemental Figure S2. Phylogenetic tree based on the alignment of protein sequences of GRPs from 12 plant species.

Supplemental Figure S3. Characterization of SlRBP1-silenced plants.

Supplemental Figure S4. GO and KEGG enrichment analysis of upregulated DEGs in amiR-SlRBP1 plants compared to WT.

Supplemental Figure S5. Determination of SlRBP1-bound RNA targets by nRIP-seq.

Supplemental Figure S6. GO and KEGG enrichment analysis of 61 putative SlRBP1 target genes.

Supplemental Figure S7 . The relative expression level of SlRBP1 target RNAs in amiR-SlRBP1 and WT plants.

Supplemental Figure S8 . Comparable degradation rates of βCA1 and RCA between WT and amiR-SlRBP1 plants.

Supplemental Figure S9 . Polysome profile of global translation efficiency in WT and amiR-SlRBP1 leaves.

Supplemental Figure S10 . RT-qPCR assays show the abundance of SlRBP1 nontarget RNAs in polysome fractions from amiR-SlRBP1 and WT plants.

Supplemental Figure S11 . RT-qPCR assays show the abundance of SlRBP1 nontarget RNAs in polysome fractions from amiR-SlRBP1 and WT plants.

Supplemental Figure S12 . SlRBP1 protein accumulation in Input and IP samples from WT and SlRBP1_pro:FM-SlRBP1 plants for sequential IP-MS.

Supplemental Figure S13 . Co-IP validation of the interaction between SlRBP1 and HSP90.

Supplemental Figure S14 . Phylogenetic tree based on the alignment of protein sequences of eIF4A from four species.

Supplemental Figure S15 . BiFC assay to detect dimerization of SlRBP1.

Supplemental Figure S16 . Immunoblot analysis of SleIF4A protein accumulation in Input and IP samples from WT and amiR-SlRBP1 plants for nRIP.

Supplemental Table S1. Accession numbers of protein used in phylogenetic analyses.

Supplemental Table S2 . nRIP-seq analysis of SlRBP1 RNA target.

Supplemental Dataset 1. List of DEGs in amiR-SlRBP1 compared with WT.

Supplemental Dataset 2. GO analysis of SlRBP1 61 target RNAs.

Supplemental Dataset 3. List of 111 candidate SlRBP1-interacting proteins.

Supplemental Dataset 4. A List of primers used in this study.

Supplemental Dataset 5. Statistical analysis using Student’s t test.

Supplemental Files S1–S4. Alignments used to generate phylogenetic trees.

 

L.M. and H.Z. designed experiments. L.M. performed most of the experiments and analyzed most of data. Y.Y. performed VIGS of SlRBP1 in tomato fruits. K.C. carried out BiFC and LCI assays of SlRBP1. X.Z. performed the purification of His-SlRBP1 in vitro. K.C. and J.Z. performed VIGS of βCA1, RCA, and PsaD in tomato leaves. L.M., K.C., and Y.G. performed polysome profiling. R.L., L.Z., K.W., J.L., N.Z., Z.D., G.Q., B.Z., D.F., Y.L., Y.W., and D.Z. provided materials and intellectual input for the work. L.M. and H.Z. wrote the manuscript.

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/plcell) is: Hongliang Zhu (hlzhu@cau.edu.cn).