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. 2021 Aug 2;187(3):1636–1652. doi: 10.1093/plphys/kiab376

CRISPR/Cas9 mutants of tomato MICRORNA164 genes uncover their functional specialization in development

Suresh Kumar Gupta 1, Abhaypratap Vishwakarma 1, Hawi Deressa Kenea 1,2, Ortal Galsurker 1, Hagai Cohen 1, Asaph Aharoni 3, Tzahi Arazi 1,✉,2
PMCID: PMC8566253  PMID: 34618074

Abstract

Plant MICRORNA164 (miR164) plays diverse regulatory functions by post-transcriptional repression of certain NAM/ATAF/CUC-domain transcription factors. However, the involvement of miR164 in fleshy fruit development and ripening remains poorly understood. Here, de novo prediction of tomato (Solanum lycopersicum) MIR164 genes identified four genes (SlMIR164a-d), of which SlMIR164d has an atypically long pre-miRNA. The roles of the fruit expressed SlMIR164a, b, and d were studied by analysis of their Clustered Regularly Interspaced Short Palindromic Repeats mutants. The slmir164bCR mutant plants exhibited shoot and flower abnormalities characteristic of ectopic boundary specification, whereas the shoot and flower development of slmir164aCR and slmir164dCR mutants were indistinguishable from wild-type. Strikingly, the knockout of SlMIR164a practically eliminated sly-miR164 from the developing and ripening fruit pericarp. The sly-miR164-deficient slmir164aCR fruits were smaller than the wild-type, due to reduced pericarp cell division and expansion, and displayed intense red color and matte, instead of glossy appearance, upon ripening. We found that the fruit skin phenotypes were associated with morphologically abnormal outer epidermis and thicker cuticle. Quantitation of sly-miR164 target transcripts in slmir164aCR ripening fruits demonstrated the upregulation of SlNAM3 and SlNAM2. Specific expression of their miR164-resistant versions in the pericarp resulted in the formation of extremely small fruits with abnormal epidermis, highlighting the importance of their negative regulation by sly-miR164a. Taken together, our results demonstrate that SlMIR164a and SlMIR164b play specialized roles in development: SlMIR164b is required for shoot and flower boundary specification, and SlMIR164a is required for fruit growth including the expansion of its outer epidermis, which determines the properties of the fruit skin.

Tomato MICRORNA164 genes play specialized roles in shoot and flower development and fruit growth, including the differentiation of fruit outer epidermis, which determines its skin properties.

Introduction

MicroRNAs (miRNAs) represent an important class of regulatory 20– to 22-nt long small RNAs. Most mature miRNAs originate from a MICRORNA (MIR) gene that its primary RNA transcript (pri-miRNA) can fold into a characteristic stem–loop structure with partial sequence complementarity between its arms. The RNase III enzyme DICER-LIKE1 (DCL1) cleaves the stem–loop structure out of the pri-miRNA, producing a typical 80- to 300-nt-long miRNA precursor (pre-miRNA). The release of the mature miRNA and its complementary strand (miRNA*) from the pre-miRNA involves the recognition and two-step processing of the pre-miRNA by DCL1 (Park et al., 2002). The liberated miRNA/miRNA* duplex is transported to the cytoplasm probably in a complex with ARGONAUTE1 (AGO1), the central effector of miRNA action (Bologna et al., 2018). In the cytoplasm, the miRNA loaded AGO1, as part of the miRNA-Induced Silencing Complex, negatively regulates the expression of highly complementary target mRNAs via mRNA cleavage, translational repression, or both (Voinnet, 2009).

Plant miRNAs play central regulatory roles in a broad range of biological processes including plant development, responses to phytohormones, nutrient homeostasis, abiotic, and biotic stresses (Chen, 2009; Li et al., 2012; Sunkar et al., 2012). While the majority of miRNAs are clade and even species-specific, other miRNAs show different degrees of conservation and are even deeply conserved among all land plants (Axtell and Bowman, 2008). Conserved miRNAs are typically encoded by multigene families that code for the same or closely related mature miRNA members. The miR164 family of miRNAs was classified as moderately conserved because it is present among angiosperms but was not identified in the more ancient plant clades gymnosperms, lycopods and bryophytes. To date, miR164 family has been identified in at least 43 angiosperm species and contains between 1 and 13 (soybean [Glycine Max]) members (Lunardon et al., 2020). The conservation of miR164 in flowering plants suggests that it has played a major role in shaping their evolution. miR164 exerts its activity by negatively regulating the expression of certain members of the NAM/ATAF/CUC (NAC) domain family of transcription factors.

In Arabidopsis, miR164 is coded by three genomic loci (MIR164A-C) and their functions were investigated with loss-of-function alleles, revealing specialized as well as redundant functions for individual MIR164 genes (Sieber et al., 2007). The Arabidopsis miR164 guides the cleavage of six NAC genes, CUP‐SHAPED COTYLEDONS1 (CUC1), CUC2, NAC1, ORESARA1 (ORE1), NAC4, and NAC5 (Mallory et al., 2004; Guo et al., 2005; Kim et al., 2009; Lee et al., 2017). In Arabidopsis, miR164a controls the extent of leaf serration in a nonredundant manner by regulating the transcript accumulation of CUC2 (Nikovics et al., 2006). Similarly, miR164c restricts the development of extra petals in early developing flowers by downregulating the expression of CUC1 and CUC2 in the second whorl (Baker et al., 2005). In addition, miR164a and miR164b regulate the auxin-mediated lateral root emergence by directing the cleavage of NAC1 (Guo et al., 2005). Besides, miR164 family members display functional redundancy as mir164abc triple mutant exhibits severe defects in phyllotaxis and flower development that were not observed in the single mutants (Sieber et al., 2007). In addition, miR164 was shown to regulate the ethylene-dependent leaf senescence and pathogen-induced cell death by negatively regulating the expression of their positive regulators ORE1 and NAC4, respectively (Kim et al., 2009; Lee et al., 2017). Along with Arabidopsis, a mutant in the MIR164 gene, FveMIR164a, was recently reported in strawberry which is involved in the specification of leaf and floral organ morphology via the posttranscriptional regulation of FveCUC2 (Zheng et al., 2019). In both Arabidopsis and strawberry, the MIR164 gene mutants shade light on the specific and redundant functions of corresponding genes and on the full repertoire of coded miR164 functions. Still, at present, loss-of-function mutants of MIR164 genes have not been reported in any other plant species.

Tomato (Solanum lycopersicum) is the nonstarchy vegetable most consumed worldwide and a major model crop for the study of fleshy fruit development. Previous annotations of tomato small RNAs suggested that miR164 is coded by two genomic loci (SlMIR164a and SlMIR164b) that express an identical mature miRNA species (sly-miR164a/b; Hendelman et al., 2013). It was shown that sly-miR164a/b negatively regulates the CUC2-like GOBLET (GOB) and NAC-domain containing SlNAM2 that are involved in organ-boundary formation (Berger et al., 2009; Hendelman et al., 2013). In addition, sly-miR164a/b was found to guide the cleavage of two additional NAC-domain transcription factors with unknown functions, SlNAC1, a homolog of NAC1 and SlNAM3, which is most similar to ORE1 (Hendelman et al., 2013). Sly-miR164a/b is abundant in vegetative and floral tissues and interestingly, also in the fruit pericarp, especially during ripening (Mohorianu et al., 2011; Hendelman et al., 2013; Gao et al., 2015). Similarly, miR164 ripening-associated expression patterns were also found in the nonclimacteric orange (Liu et al., 2014) and pepper (Hwang et al., 2013) fruits, suggesting that the roles of miR164 in the pericarp of ripening fruits are conserved in additional fleshy fruit species. Constitutive expression of miR164 in Micro-Tom tomato modified the schedule of tomato fruit development and maturation (Rosas Cárdenas et al., 2017). Nevertheless, the specific functions of tomato MIR164 genes and their mature miR164 in the fruit remain obscure.

In this study, we exploited public high-volume small RNA data to identify all transcriptionally active sly-miR164 coding loci in the tomato genome. This analysis led to the identification of two previously undisclosed SlMIR164 genes (SlMIR164c and SlMIR164d), which code for two unique sly-miR164 members. We then used Short Tandem Target Mimic (STTM) to silence mature miR164 activities as well as Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) technology to knockout specific SlMIR164 coding genes. By dissecting generated mutant plants, we demonstrated their specialization in development and uncovered previously unidentified roles for miR164 in fruit development.

Results

The tomato miR164 family is larger than earlier reported

Previously, we have identified two sly-miR164 coding loci located on chromosome 9 (SlMIR164a) and chromosome 1 (SlMIR164b; Hendelman et al., 2013; Figure 1A). Both loci code for the same mature sly-miR164 species (henceforth will be called sly-miR164a/b). De-novo MIR loci prediction using high-volume 20–22 nt small RNA data, assembled from 178 public small RNA data sets representing various healthy and pathogen-infected vegetative, as well as reproductive tissues, was used to inform on all the transcriptionally active MIR164 loci in the tomato genome (Supplemental Table S1). This analysis identified four SlMIR164 genes including two previously undisclosed ones located on chromosome 6 (SlMIR164c) and chromosome 10 (SlMIR164d; Figure 1A). The frequency of identified sly-miR164 members in the small RNA data sets indicated that sly-miR164a/b is the most abundant and present in most of the datasets (175/178), sly-miR164d is the second most abundant and was present in 134/178 datasets and sly-miR164c is the least abundant and was present only in 40/178 datasets, of which 30 represent root small RNAs. Despite sly-miR164c and sly-miR164d limited abundance, their star strands were present in 53/178 and 61/178 datasets, respectively, (Supplemental Table S1) strongly supporting their authenticity. The mature sly-miR164c and sly-miR164d differ from each other, and from sly-miR164a/b, by 2 nt (Figure 1B). Nevertheless, psRNATarget (Dai and Zhao, 2011) analysis of sly-miR164c and sly-miR164d predicted the same NAC-domain transcription factor targets with high probability (expect ≤2.5) as targeted by sly-miR164a/b (Supplemental Table S2), supporting their functionality in tomato. The pre-miR164a (202 nt), pre-miR164b (134 nt), and pre-miR164c (117 nt) foldbacks are predicted to be shorter than 300 nt, a length that conforms with the recommended criteria for plant miRNA annotation (Axtell and Meyers, 2018). Remarkably, the pre-miR164d hairpin spans 645 nt of which 77 nt comprise the stem containing the miR164/miR164* duplex, and 568 nt comprise an elaborate loop structure (Figure 1A). Except for its unusually large size, pre-miR164d follows the other criteria for plant miRNA annotation. It codes for RNA-seq verified single 21 nt miRNA:miRNA* duplex, which is not interrupted by secondary stems or large loops and contains up to five mismatched positions. Moreover, alignment between pre-miR164d and pre-miR164a-c stem sequences revealed strong identities between the 5′ (84% identity) and 3′ (91% identity) arms of pre-miR164d and pre-miR164a (Figure 1, C and D). Phylogenetic analysis of miR164s from tomato and selected angiosperm species revealed sly-miR164a/b-identical miRNAs in both dicots and monocots and sly-miR164c-identical miRNAs in certain dicots but did not find sly-miR164d-identical sequence in any of the tested species (Figure 1E). To inquire whether sly-miR164d is conserved in Solanaceae, we queried the genomes of potato (Soanum tuberosum), pepper (Capsicum annum), eggplant (Capsicum annum), tobacco (Nicotiana tabacum), Nicotiana benthamiana, and petunia (Petunia axillaris), with sly-miR164d and sly-miR164d* sequences. This search identified a single locus that code for identical sly-miR164d and sly-miR164d* in potato, pepper, eggplant, and petunia, and two loci that code for sly-miR164d and sly-miR164d* in Nicotiana tabacum. Furthermore, querying the genome of coffee, which belongs to the Rubiaceae family and is also found in the Asterid clade like Solanaceae, did not identify sly-miR164d and sly-miR164d* similar sequences. RNAfold of the miR164d flanking genomic sequences predicted that all can fold into a short stem, which contains perfectly aligned miR164d/miR164d* sequences, and an elaborate loop, reminiscent of the noncanonical structure of tomato pre-miR164d (Supplemental Figure S1). This corroborates the authenticity of sly-miR164d and suggests that it is a Solanaceae-specific miRNA. Taken together, our data suggest that the tomato miR164 family is larger than earlier reported and contains at least four members.

Figure 1.

Figure 1

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The miR164 coding loci in tomato. A, Hairpin secondary structures of sly-miR164 precursors as predicted by RNAfold (http://rna.tbi.univie.ac.at/cgi-bin/RNAWebSuite/RNAfold.cgi). The position of each precursor in the tomato genome is indicated. Mature sly-miR164 and respective sly-miR164* sequences, which were identified within the small RNA libraries, are marked by red and blue, respectively. Arrowheads mark the predicted cleavage sites of Cas9. B, Multiple nucleotide sequence alignment of tomato mature miR164 members. C and D, Multiple nucleotide sequence alignment of tomato pre-miR164 5′ (C) and 3′ (D) stem sequences. Numbers indicate the positions in the predicted respective pre-miRNA. Asterisks mark the sequence of miR164 (C) and miR164* (D). E, A rooted phylogenetic tree of miR164 from tomato (sly) and selected angiosperms as follows: Vitis vinifera (vvi), Populus trichocarpa (ptc), Zea mays (zma), Sorghum bicolor (sbi), Oryza sativa (osa), and Glycine max (gma). The miR164 sequences were retrieved from miRBase (release 22). For convenience miR164 members with identical sequences were grouped together (/). Sly-miR160a served as an outgroup. The tree was constructed using MEGA × maximum likelihood tree with default parameters (Kumar et al., 2018).

Knockdown of sly-miR164 by STTM

To analyze the functions of sly-miR164 in tomato fruit development, we initially employed the STTM approach (Yan et al., 2012) to silence all the mature sly-miR164 members. To that end, we have generated transgenic responder lines (OP:STTM164) that contained an STTM164 fragment, which is predicted to bind and silence all identified sly-miR164 members, under the pOP promoter (Figure 2A). The pOP promoter is inactive and can be trans-activated only by its artificial pOP activator LhG4. Crossing the generated OP:STTM164 responder lines with the 35S:LhG4 driver line, resulted in constitutive trans-activation (≫) of the OP:STTM164 transgene in their F1 progeny that exhibited excess cotyledons and leaves compared with control. The strongest phenotype was observed in 35S≫STTM164-4 seedlings that exhibited supernumerary abnormal leaves (Figure 2B). Quantitation of sly-miR164a/b in the shoot apices of 35S≫STTM164 seedlings showed 65% reduction in its accumulation compared with control apices (Figure 2C). Through RT semi-quantitative PCR the levels of sly-miR164 target transcripts were estimated in 35S≫STTM164 shoot apices. This analysis revealed the upregulation of all measured targets. The highest upregulation of GOB was observed in 35S≫STTM164-4, which displayed the strongest phenotype (Figure 2D). Nevertheless, the shoot development of 35S≫STTM164 seedlings was arrested after the formation of only a few leaves, making them noninformative for studying later stages of development.

Figure 2.

Figure 2

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Characterization of 35S≫STTM164 seedlings. A, Schematic representation of the binary STTM164 responder construct. Target mimic sequences, their Watson–Crick pairing to sly-miR164a/b, and the three base pair bulge (boldface) are shown in the expanded region. OCS, octopine synthase; NOS, nopaline synthase. B, Phenotypes of 15-d-old trans-activated 35S≫STTM164 seedlings of indicated responder lines compared with the driver line control (35S::LhG4). Scale bar = 2 mm. L, leaf; C, cotyledon. C, Quantitation of mature sly-miR164a/b in the shoot apices of 35S::LhG4 and 35S≫STTM164-12 seedlings by RT-qPCR. U6 was used as a reference gene. Error bars indicate ±SD over two biological replicates. The P-value was determined by Student’s t test. D, Quantitation of sly-miR164 target transcripts in the shoot apices of indicated genotypes by RT semi-quantitative PCR. The TIP41 transcript was used as a reference gene. The numbers above the bands indicate expression level relative to TIP41. The numbers in parenthesis indicate used PCR cycles in respective gels.

Generation of SlMIR164 mutants by CRISPR/Cas9

To further investigate sly-miR164 functions in the fruit, we sought to eliminate the activity of specific sly-miR164 members instead of all, hoping that the resulted mutant plants will inform on later development. To this end, CRISPR/Cas9-mediated mutagenesis was utilized, targeting the sly-miR164 sequence and pre-miRNA stem of each identified SlMIR164 gene with 2–3 gene-specific guide RNAs (gRNAs) (Figures 1, A and 3, A–D). This approach resulted in the isolation of SlMIR164a, SlMIR164b, and SlMIR164d CRISPR mutants (slmir164CR). However, analysis of generated transgenic plants and their progeny did not identify any slmir164cCR mutant among them, possibly due to corresponding locus inaccessibility by Cas9. The generated slmir164aCR-21, slmir164dCR-13 and slmir164dCR-15 mutant alleles lacked the mature miR164 sequence and different parts of its precursor thus rendering them as null alleles (Figure 3, E and G). In contrast, the slmir164bCR-1 and slmir164bCR-17 mutant alleles contained intact sly-miR164 sequence, but harbored one and four bp deletions, respectively, in the sequence that forms the pre-miRNA lower stem, which is located upstream to the mature miRNA (Figure 3, F). Previously, mutations in pre-miR160a lower stem caused a reduction in miR160a levels probably by disturbing its biogenesis (Damodharan et al., 2018), raising the possibility for a similar effect on the biogenesis of sly-miR164b.

Figure 3.

Figure 3

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The SlMIR164 CRISPR mutants. A–D, Schematic illustrations of the SlMIR164 gene loci (location of each in the tomato genome is indicated). Black, white, and gray boxes indicate the pre-miR164, mature sly-miR164 and miR164*, respectively. Half arrows indicate primers used for genotyping. In the expanded regions, sly-miR164 is italicized, gRNA-targeted sequences are indicated, Cas9 cleavage sites are indicated by arrowheads, and PAM sequences are colored in orange. In (B) the NlaIII restriction enzyme site is indicated, which was used for genotyping. E–G, Nucleotide sequence alignments between SlMIR164 genes and their corresponding CRISPR mutants. The sly-miR164 sequence is indicated. Tilde symbol indicates omitted nucleotides. The number after open triangle indicates the size of deletion in bp.

Sly-miR164b is required for normal shoot and flower development

During vegetative development, both slmir164aCR-21 and slmir164dCR-13 plants did not differ from wild-type indicating their redundancy or no function in shoot development (Figure 4, A–E). In contrast, slmir164bCR-1 and slmir164bCR-17 plants displayed very similar abnormal vegetative development. Mutant seedlings frequently produced supernumerary cotyledons (Figure 4A; Supplemental Figure S2, A and B), and leaf primordia (Figure 4B). Mutant leaves were smaller and less compound (Figure 4C). In addition, slmir164bCR plants had very short primary stems, owing to reduction in internode length, and excess branches, both of which contributed to their bushy architecture (Figure 4, D and E; Supplemental Figure S2C). Quantitation of selected sly-miR164 targets in slmir164bCR-17 shoot apices revealed their strong upregulation (Figure 4F), which was consistent with a significant 34% reduction in sly-miR164a/b levels in this tissue (Figure 4G). Conversely, significant changes in the levels of sly-miR164 target transcripts were not detected in the shoot apices of slmir164aCR-21 loss-of-function mutant (Figure 4F). Accordingly, the levels of sly-miR164a/b were only slightly reduced in slmir164aCR-21 (Figure 4G). In wild-type M82 shoot apices, sly-miR164d is expressed around eight-fold less than sly-miR164a/b (Supplemental Table S1, small RNA dataset #38). Because of sly-miR164d low levels in the shoot apex, its elimination in slmir164dCR-21 was not expected to significantly affect the pool of sly-miR164. In line with that, quantification of selected sly-miR164 target mRNAs in slmir164dCR-13 shoot apex revealed no significant upregulation, in agreement with its wild-type phenotype (Figure 4F). Like their mutant shoots, the slmir164aCR-21 and slmir164dCR-13 mutant flowers did not exhibit any wild-type deviating phenotypes (Figure 4, H–O). In contrast, slmir164bCR-17 plants rarely flowered and produced solitary, misshapen infertile flowers that exhibited fewer sepals and petals, supernumerary deformed stamens and multiple pistils (Figure 4, H–M). The mutant pistils contained abnormal sterile ovaries and uncovered ovules (Figure 4, L and N). In addition, slmir164bCR-17 flowers also developed sepaloids (Figure 4M inset) and their pedicle abscission zone (AZ) was considerably larger than that of the wild-type and other mutants (Figure 4O). Due to their abnormal flower ovaries, the slmir164bCR-17 plants never set fruits.

Figure 4.

Figure 4

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Characterization of vegetative and flower development of slmir164CR mutants. A–E, Pictures of representative wild-type and slmir164CR mutants as indicated; 8-d-old seedlings (A), their shoot apices (B), fifth leaf (C), 45-d-old plants (D), their lower stem (E). Scale bars = 1 cm (A), 200 µm (B), 5 cm (C–E). F and G, Quantitation of indicated sly-miR164 target transcripts (F) and mature sly-miR164 (G) in the shoot apex of 8-d-old wild-type and mutant seedlings by RT-qPCR. SlTIP41 (F) and U6 (G) were used as reference genes. Error bars indicate ±sd over three biological replicates. Different letters indicate statistically significant differences as determined by Tukey–Kramer multiple comparison test (P ≤ 0.05). H, Pictures of representative anthesis flowers of indicated genotypes. I to N, isolated anthesis flower organs; sepals (I), petals (J), stamens (K), pistils (L), styles and in inset sepaloids (M), manual cross-sections of ovaries (N). In (L) the arrow marks exposed ovules. O, Pictures of flower’s pedicel abscission zone (marked by arrow heads) of indicated genotypes. Scale bars = 1 mm (H–O).

Sly-miR164a is predominant in fruit and is required for its proper development

In silico expression analysis of sly-miR164 members in the pericarp of MicroTom developing fruits indicated that the mature sly-miR164a/b species, which is coded by SlMIR164a and SlMIR164b genes, was several orders of magnitude more prevalent than sly-miR164d and sly-miR164c (Figure 5A). Sly-miR164a/b was relatively abundant in anthesis flowers, its levels dropped in young immature fruits and steadily increased until the breaker stage after which it was substantially upregulated in the ripening pericarp (Figure 5A). Previously, analysis of pri-miR164a and pri-miR164b transcript levels in developing fruit pericarp indicated that SlMIR164a expression level is approximately 18-fold higher than SlMIR164b (Gao et al., 2015). Consistent with that, quantitation of sly-miR164a/b in the pericarp of slmir164aCR-21 developing and ripening fruits demonstrated at least 90% reduction in its levels (Figure 5B), indicating that SlMIR164a is the major contributor of sly-miR164a/b in the fruit pericarp. Despite the dramatic reduction in sly-miR164a levels in mutant fruits, their set and growth schedule appeared invariant and they attained maturity and ripening at the same time as wild-type fruits (Figure 5C). However, slmir164aCR-21 fruits appeared smaller compared with wild-type fruits starting from ca. 15 d post-anthesis (DPA) (Figure 5C). Accordingly, analysis of mutant red ripe fruits size showed a significant reduction in their perimeter and area (Figure 5, D and E), as well as in their weight (Figure 5F), in comparison with wild-type. In addition, we frequently noticed that mutant red ripe fruits displayed deeper red color and matte instead of glossy appearance (Figure 5C). These observations were confirmed by analysis of mutant red ripe fruits skin through Chroma meter that revealed reductions in lightness, intensity, and hue values (Figure 5G). The slmir164aCR-21 red ripe fruits were also analyzed for firmness, brix, titratable acidity, ethylene, and CO2 emission (Supplemental Figure S3, A–E). These analyses indicated no change than wild-type fruits, except that the mutant fruits were firmer.

Figure 5.

Figure 5

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SlMIR164a knockout affects fruit size and appearance. A, Abundance of mature sly-miR164 family members in tomato cv MicroTom anthesis flowers and in total pericarp of fruits at indicated stage. Mature sly-miR164a and sly-miR164b have identical sequences and hence shown as sly-miR164a/b. The expression data were retrieved from the Tomato Functional Genomics Database (http://ted.bti.cornell.edu/). B, Quantitation by RT-qPCR of sly-miR164a/b in wild-type and mutant anthesis ovaries and total pericarp of fruits at indicated stages. MG, mature green; OR, orange; RR, red ripe. U6 was used as a reference gene. Error bars indicate ±sd over two (10 and 15 DPA) or three (other stages) biological replicates. Each replicate contained pericarp samples from five different fruits harvested from five independent plants. The P-value was determined by Student’s t test. C, Representative pictures of wild-type and slmir164aCR-21 fruits at indicated DPA. IG, immature green; MG, mature green; OR, orange; RR, red ripe. Scale bars = 5 cm. D–G, Measurements of perimeter (D), area (E), fresh weight (F), skin color lightness, intensity, and hue (G) of red ripe wild-type and mutant fruits. In D–G, box center line, median; average, + sign; box limits, upper and lower quartiles; whiskers, min and max values; points, individual values; n = number of fruits; the P-value was determined by Student’s t test.

A more in-depth examination of developing and ripening pericarp using histology indicated that slmir164aCR-21 fruit pericarp was slightly thinner than that of wild-type already at 10 and 15 DPA (Figure 6A). Accordingly, at these stages, the mutant pericarp contained fewer cell layers than respective wild-type pericarp (Figure 6B). Moreover, analysis of mesocarp cell volume and numbers indicated that compared with wild-type, mutant cells volume was reduced and accordingly their numbers were significantly increased at 15 DPA (Figure 6, C and D). Thus, the reduced size of mutant fruits was caused by reduced cell division and expansion of their mesocarp cells. Moreover, mutant epicarp cells had a similar shape as wild-type in the immature green pericarp (10–15 DPA) and later adopted a conical instead of flat shape in the mature and ripening pericarp, suggesting reduced cell expansion. Furthermore, the mutant epicarp cells stained more extensively with the lipid stain Sudan IV indicating excess deposition of cuticle on epidermal cells compared with wild-type (Figure 6E). Indeed, gas chromatography–mass spectrometry (GC–MS) analysis of the cuticles of slmir164aCR-21 fruits at orange and red ripe stages found higher levels of C16-hydroxy and C16-10,16 dihydroxy acids, the two predominant monomers of the cutin polymer (Supplemental Figure S4).

Figure 6.

Figure 6

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The pericarp of developing and ripening slmir164aCR-21 fruits. A, Representative images of wax embedded wild-type and mutant pericarp cross-sections from ovaries (0) and fruits at indicated DPA stained with fast green and safranin. The representative outer pericarp is boxed. The insets in 10 and 15 DPA images show a higher magnification of the boxed pericarp. Pericarp cell layers are indicated on the right side of each image. E1, outer epidermis (epicarp); E2 and E3, sub-epidermal layers; M, mesocarp cell layers; yellow rectangles mark the mesocarp boundaries; I1 and I2, inner epidermis and adjacent layer, respectively. The nomenclature of pericarp layers is adopted from (Renaudin et al., 2017). Red circles mark the regions of vascular bundles excluded from the cellular analyses. Scale bars = 50 µm (images), 20 µm (insets). B–D, Measurement of pericarp cell layer number (B), mesocarp cell volume (C), and mesocarp cell number (D) of wild-type and mutant fruit. In B–D, center line, median; average, + sign; box limits, upper and lower quartiles; whiskers, min and max values; points, individual values; n = number of pericarp samples (B and C) and cells (D); the P-value was determined by Student’s t test. E, Representative images of wax embedded wild-type and mutant pericarp cross-sections from fruits at indicated DPA stained with Sudan IV. C, cuticle; E, epidermal cell; SD, sub-epidermal deposit. Scale bars = 50 µm.

Accumulation of SlNAM2 and SlNAM3-encoding transcripts is associated with abnormal pericarp development

Further study was carried out to find which sly-miR164 target mRNAs (Hendelman et al., 2013) are negatively regulated by sly-miR164a in the fruit pericarp. In developing and ripening wild-type fruit pericarp, sly-miR164 target mRNAs were expressed at very low levels (Figure 7A). In line with their low expression, we could not reliably quantitate them by RT-qPCR in the total pericarp of wild-type and slmir164aCR-21 immature green fruits. Moreover, significant upregulation of any sly-miR164 target was not detected in mature green fruit pericarp. However, their quantitation in the total pericarp of ripening fruits detected upregulation of 7.5- and 5.5-fold of SlNAM3, and 2.5- and 1.69-fold of SlNAM2, in the orange and red ripe stages, respectively, but no change in the levels of SlNAC1 and GOB (Figure 7, B–E). This indicated that SlNAM3 and to a lesser extent SlNAM2 are the primary target transcripts of sly-miR164a in the ripening pericarp.

Figure 7.

Figure 7

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Quantitation of miR164 target transcripts in tomato fruit pericarp. A, Tomato Expression Atlas cube (Shinozaki et al., 2018) of indicated miR164 targets in total pericarp and pericarp tissues of M82 fruit. White and red indicate no and high expression, respectively, in Reads Per Million. The cube was drawn via the Tomato Expression Atlas website (https://tea.solgenomics.net/expression_viewer/input). B–E, Expression levels of miR164 targets in the total pericarp of M82 and slmir164aCR-21 mutant fruits at indicated stages, as determined by RT-qPCR; SlNAM3 (A), SlNAM2 (B), SlGOB (C), and SlNAC1 (D). SlTIP41 was used as a reference gene. Error bars indicate ±sd over three biological replicates. Different letters indicate statistically significant differences as determined by Tukey–Kramer multiple comparison test (P ≤ 0.01). MG, mature green; OR, orange; RR, red ripe.

To inquire whether SlNAM2 and SlNAM3 upregulation in slmir164aCR-21 may be responsible for its fruit phenotypes (Figures 5 and 6), we specifically overexpressed their sly-miR164a-resistant versions (hereafter mSlNAM2 and mSlNAM3) in the fruit pericarp and characterized the resulting fruit phenotypes. To that end, we utilized a previously generated (Hendelman et al., 2013) responder line of mSlNAM2 (OP::mSlNAM2) and a responder line generated here for mSlNAM3 (Figure 8A). The expression of mSlNAM2 and mSlNAM3 was then transactivated by crossing OP::mSlNAM2 and OP::mSlNAM3 responder lines with a p2A11::LhG4 driver line (Figure 8B). The p2A11::LhG4 driver line drives LhG4 expression under the 2A11 fruit-specific promoter (Van Haaren and Houck, 1993), resulting in its weak and strong expression in the developing and ripening fruit pericarp, respectively (Supplemental Figure S5). The fruits of the transactivated p2A11≫mSlNAM2 and p2A11≫mSlNAM3 F1 progeny were much smaller than wild-type and slmir164aCR-21 fruits and occasionally their pericarp cracked. In addition, the pericarp of both did not attain red color at the same time as the fruit pericarp of wild-type and slmir164aCR-21 (Figure 8, C–G). In line with their small size, histology of the p2A11≫mSlNAM3 fruit pericarp indicated an exaggerated reduction in its thickness compared with wild-type and slmir164aCR-21, due to reduction in the size of its mesocarp cells (Figure 8H). In addition, the p2A11≫mSlNAM3 fruit epicarp cells adopted an extremely conical shape compared with the slightly conical and elongated epicarp cells of slmir164aCR-21 and wild-type, respectively (Figure 8I). Staining the epicarp layer with the lipid stain Sudan IV showed increased staining compared with wild-type, but comparable staining with that of the slmir164aCR-21 pericarp (Figure 8J). The abnormal pericarp phenotypes of p2A11≫mSlNAM2 and p2A11≫mSlNAM3 fruits were associated with a strong increase in SlNAM2 and SlNAM3 transcript accumulation, respectively, compared with wild-type and slmiR164aCR-21 fruits (Figure 8, K and L).

Figure 8.

Figure 8

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Phenotypic characterization of p2A11≫mSlNAM2 and p2A11≫mSlNAM3 fruits. A, Schematic representation of the OP::mSlNAM3 responder binary construct. The sly-miR164a/b sequence, its target SlNAM3 nucleotide sequence and a corresponding amino acid sequence, and the silent mutations (lowercase bold nucleotides) incorporated in mSlNAM3, are shown in the expanded region. B, Schematic representation of the p2A11::LhG4 driver binary construct. OCS, octopine synthase; NOS, nopaline synthase. C–E, Representative pictures of indicated genotypes fruits at the mature green stage (C), 9 d post breaker stage (D) and their half cross-sections (E). Scale bars = 2 cm. F and G, Measurements of fruit area (F) and perimeter (G), of indicated genotypes. Box center line, median; average, + sign; box limits, upper and lower quartiles; whiskers, min and max values; points, individual values; n = number of fruits. Different letters indicate statistically significant differences as determined by Tukey–Kramer multiple comparison test (P ≤ 0.001). H–J, Representative images of wax embedded pericarp cross-sections from indicated genotype orange fruits stained with fast green—safranin (H), their respective close-ups (I), and stained with Sudan IV (J). E, epidermis; E1, the outer epidermis (epicarp); E2 and E3, sub-epidermal cell layers; M, mesocarp cell layers, yellow rectangles mark the mesocarp boundaries; I, inner epidermis; C, cuticle; SD, sub-epidermal deposit. Scale bars = 500 µm (E), 50 µm (G and H). K, L, Quantitation by RT-qPCR of SlNAM2 (K) and SlNAM3 (L) transcript levels, in orange fruit pericarp of indicated genotypes. SlTIP41 was used as a reference gene. Error bars indicate ±sd over three biological replicates. The P-value was determined by Student’s t test.

Functional redundancy among sly-miR164a/b coding genes

In view of the abundance and major role of sly-miR164a/b in shoot, flower, and fruit development, we further analyzed the potential for functional redundancy among its coding genes by investigating the phenotypic consequences of co-inactivation them. Compared with the phenotype of slmir164aCR-21 and slmir164bCR-17 single mutant seedlings (Figure 4A), slmir164aCR-21slmir164bCR-17 double mutant seedlings exhibited a more severe phenotype. They failed to develop cotyledons and their leaf initiation ceased after the formation of only a few abnormal leaves, likely due to the consumption of their shoot apical meristem (SAM) (Figure 9A; Supplemental Figure S2D). The increase in phenotypic severity suggested functional redundancy between SlMIR164a and SlMIR164b. As slmir164aCR-21slmir164bCR-17 seedlings were developmentally impaired, the redundancy during the reproductive stage was tested using the hypomorphic slmir164aCR-21slmir164bCR-17−/+ mutant. The vegetative and flower development of this mutant were similar to those in the single mutant parent genotypes (Figure 9B), but its red ripe fruits were smaller than both (Figure 9, C–E), suggesting that SlMIR164b plays a minor redundant role in the regulation of fruit growth.

Figure 9.

Figure 9

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Vegetative and fruit phenotypes of slmir164CR double mutants. A, Pictures of 2-week-old mutant seedlings of indicated genotypes. Inset shows slmir164aCR-21slmir164bCR-17 seedling apex at a higher magnification. Scale bars = 2 cm (picture), 1 mm (inset). B, Pictures of 60-d-old mutant plants of indicated genotypes. Scale bars = 5 cm. C, Pictures of wild-type and mutant red ripe fruits of indicated genotypes. Scale bar = 5 cm. D and E, Measurement of red ripe fruit perimeter (D) and area (E) of indicated genotypes. In D and E, center line, median; average, + sign; box limits, upper and lower quartiles; whiskers, min and max values; points, individual values; n = number of fruits. Different letters indicate statistically significant difference as determined by Tukey–Kramer multiple comparison test, P ≤ 0.01 for (D) and P ≤ 0.001 (E).

Discussion

The tomato miR164 family

De novo prediction of tomato MIR164 genes, which was based on a large-scale small RNA data and an updated tomato genome (SL3.0), led to the identification of two previously undiscovered MIR164 coding genes, SlMIR164c and SlMIR164d, bringing the number of tomato MIR164 genes to four, which is one more than in Arabidopsis but much less than the 13 members of the soybean miR164 family (Lunardon et al., 2020).

The current criteria for miRNA annotation limit the pre-miRNA length to 300 nt or less (Axtell and Meyers, 2018). Nevertheless, by allowing for a longer pre-miRNA prediction, we were able to identify SlMIR164d that its transcribed pre-miR164d hairpin is predicted to span 645 nt. Except for its atypical length, pre-miR164d conforms to other MIR gene criteria. This and the conservation of sly-miR164d in the genomes of additional Solanaceae species support SlMIR164d as an authentic MIR164 gene. Our results indicate that sly-miR164a/b is conserved in both dicots and monocots, sly-miR164c is conserved in dicots and sly-miR164d is only conserved in Solanaceae species. The limited conservation and relatively weak expression of sly-miR164d together suggest that it represents a “young” MIR gene that was recently evolved (Cuperus et al., 2011). Few routes have been proposed for the evolution of new MIR genes. Experimental evidence suggests that a MIR gene can form from inverted duplication of its target gene (Allen et al., 2004), modified transposable element (Piriyapongsa and Jordan, 2008), randomly formed complementary foldback sequence (Felippes et al., 2008), or duplication of an existing MIR gene (Xia et al., 2013). The high sequence identity between the stems of pre-miR164a and pre-miR164d supports the latter route, namely that SlMIR164d probably originated from a duplication of SlMIR164a after which it acquired its elaborate loop.

Roles of miR164 in shoot and flower development

Despite multiple transformation cycles, a SlMIR164C CRISPR mutant was not recovered by us, thus hindering the investigation of sly-miR164c biological roles. Nevertheless, its presence mainly in roots raises the possibility that it has a specialized role in this organ. In contrast, the isolation of SlMIR164a, SlMIR164b, and SlMIR164d CRISPR mutants enabled us to investigate the functions of corresponding miR164 family members. Our results indicate that inactivation of SlMIR164a and SlMIR164d did not affect any obvious aspect of shoot and flower development, suggesting that sly-miR164a and sly-miR164d are not required for their normal development. Still, the more severe defects in the shoot apices of slmir164aCR-21slmir164bCR-17 seedlings compared with slmir164bCR-17 single mutant suggest that sly-miR164a plays a redundant but minor role with sly-miR164b in the regulation of meristem-organ boundary formation.

In contrast to slmir164aCR-21 and slmir164dCR mutants, the slmir164bCR mutants exhibited various shoot and flower developmental defects. Several mutant phenotypes were reminiscent but more severe than the respective phenotypes of the tomato mutant Gob-4d (Berger et al., 2009). Like Gob-4d, slmir164bCR mutants produced supernumerary cotyledons, leaves, stamens, and pistils, as well as, short stem internodes. The Gob-4d phenotypes were caused by over-accumulation of the mutant miR164-resistant CUC2 homolog SlGOB, which regulates meristem–organ and organ–organ boundary specification (Berger et al., 2009; Maugarny-Calès et al., 2019). Consistent with that, SlGOB was upregulated in the slmir164bCR mutants resulting in the specification of ectopic boundaries. In Arabidopsis, the strong expression of a miR164-resistant version of CUC2 (CUC2g-m4) resulted in the production of round instead of highly serrated leaves due to reduced leaf boundaries, and unfused ovaries instead of intact (Nikovics et al., 2006). Contrary to Gob-4d and similar to CUC2g-m4 plants, the slmir164bCR plants produced leaflets with fewer lobes and flowers with a reduced number of sepals and petals, likely due to reduced boundary formation, and ovaries with carpel fusion defects. This suggests that the differences between slmir164bCR and Gob-4d phenotypes are possibly due to the parallel upregulation of the other sly-miR164 target mRNAsin slmir164bCR, including that of the boundary gene SlNAM2 (Hendelman et al., 2013). Target mRNAs upregulation was due to reduced sly-miR164b levels, likely as a result of inefficient processing of the mutant pre-miR164b because of its mutated lower stem, which was shown to be required for correct pre-miRNA processing (Mateos et al., 2010). It was suggested that SlGOB functions as a positive regulator of pedicel AZ development (Nakano et al., 2011). In line with that, the slmir164bCR flowers displayed an overgrown pedicel AZ, likely as a result of upregulation of SlGOB. Taken together our results demonstrate that sly-miR164b is the primary miR164 species that regulates meristem-organ and organ-organ boundary specification, stem internode elongation, and flower AZ development.

Roles of miR164 in fleshy fruit development and ripening

Tomato fruit growth can be divided into two distinct phases. Following fruit set until 8–10 DPA fruit growth is characterized by cell division and cell expansion (Phase I), after which fruit growth is primarily done by cell expansion (Phase II), which contributes most to its final size (Renaudin et al., 2017). Our data showed that knockout of slmir164aCR reduced sly-miR164a/b levels by ca. 90% indicating that it is the primary contributor of sly-miR164 in the developing and ripening fruit pericarp. As opposed to slmir164aCR-21 normal shoot and flower phenotypes, its mutant fruits, were smaller, due to reduced pericarp cell division and cell expansion. It was shown that miR164-regulated CUC genes function as suppressors of cell division (Sieber et al., 2007) and expansion (Pei et al., 2013). Consistent with that, overexpression of miR164-resistant SlNAM2 and SlNAM3 in the fruit pericarp strongly inhibited fruit pericarp growth. This suggests that the regulatory activity of sly-miR164 is crucial for Phases I and II of fruit growth. Since we could not detect the upregulation of any sly-miR164a target mRNA in the growing and maturing pericarp, at present, we cannot pinpoint which mRNA is negatively regulated by sly-miR164a in corresponding fruits. One possibility for our inability to detect the upregulation of target mRNAs may be a weaker effect of sly-miR164a depletion in the growing and maturing pericarp compared with ripening pericarp, due to its relatively low levels. Another possibility is that sly-miR164a negative regulation of its targets may take place in specific cells of the growing and maturing pericarp and hence the effect of its depletion is diluted when measured in the total pericarp. Another less likely possibility is that sly-miR164a inhibits the translation of its targets in the growing and maturing fruit pericarp.

It was shown that the growth of tomato fruits continues by cell expansion till the red ripe stage, although at a much slower rate than in the previous growth phase (Domínguez et al., 2012). Consistent with that, the number of epidermal cells per surface area was smaller in ripe compared with mature green fruits, due to an increase in tangential width (España et al., 2014; Segado et al., 2016). The conical rather than elongated epidermis cells in the epicarp of slmir164aCR-21 and p2A11≫mSlNAM3 ripening fruits, raise the possibility that sly-miR164a regulates pericarp cell expansion during ripening, through the negative regulation of SlNAM3 and most likely SlNAM2. In tomatoes, it was reported that abnormal epidermal cell development was associated with the formation of irregular outer surface resulting in fruits with dull skin (Petit et al., 2014). Therefore, the less glossy appearance of slmir164aCR-21 fruits could result from the irregularities in outer surface formation due to improper epidermal cell expansion that probably also affected cuticle deposition.

MiR164 has been implicated in the regulatory network of ethylene and its transcription is negatively regulated by ethylene treatment in Arabidopsis seedlings and rose petals (Kim et al., 2009; Li et al., 2013; Pei et al., 2013). Ethylene is an endogenous modulator of several aspects of plant development including fruit ripening. The climacteric tomato fruit is highly sensitive to ethylene and accordingly is regulated by it. At the onset of ripening, the ethylene levels in the fruit begin to increase and reach maximum levels in the ripened fruit (Poel et al., 2012). A similar trend was also found for sly-miR164 expression in the fruit pericarp (Hendelman et al., 2013), raising the possibility that it is induced by ethylene and maybe regulates climacteric tomato fruit ripening. However, sly-miR164a/b was not induced following ethylene treatment of fruits (Gao et al., 2015), indicating that its upregulation at the onset of fruit ripening is not dependent on ethylene. Our results indicated that almost complete depletion of miR164 from the pericarp of slmiR164aCR-21 ripening fruit, which was associated with relatively mild upregulation of SlNAM2 and SlNAM3 in the ripening pericarp, did not alter ripening duration and ethylene-mediated biochemical changes. This strongly suggests that sly-miR164 is not a major regulator of climacteric tomato ripening. On the other hand, we observed a delay in red color development in p2A11≫mSlNAM2 and p2A11≫mSlNAM3 small fruits, which accumulated very high levels of SlNAM2 and SlNAM3 encoding transcripts in their pericarp, suggesting that their negative regulation by sly-miR164 may promote ripening. However, further investigation is required to determine if tomato fruit ripening was directly inhibited by SlNAM2 and SlNAM3, or indirectly, due to their effect on pericarp development.

Conclusion

This study is a continuation of our initial functional characterization of the miR164 regulatory module in tomato (Hendelman et al., 2013). Our results reveal a larger tomato miR164 family than previously known and uncover redundancy and specialization among the corresponding SlMIR164 genes in the regulation of shoot, flower, and in particular fleshy fruit development and ripening. We found that SlMIR164a is the major contributor of sly-miR164 in the fruit pericarp and is required for pericarp growth including the expansion of the epicarp, which determines the properties of the fruit skin.

Materials and methods

De novo prediction of miR164 coding genes

De novo prediction of tomato (Solanum lycopersicum) MIR164 loci was performed using 178 tomato small RNA sequence datasets that are available at the Sequence Read Archive (listed in Supplemental Table S1). Following adaptor trimming, size selection and mapping to the tomato genome (SL3.0), a total of 499,404,477 20- to 22-nt long small RNAs were used as input for ShortStack 3.8.3 (Axtell, 2013). Default parameters were used except that the “foldsize” parameter was set to the maximum 1,000 nt to enable the prediction of MIR genes that code for pre-miRNAs longer than 300 nt. Each predicted pre-miRNA sequence was further manually inspected for a characteristic hairpin structure and lack of identity to tomato repetitive elements.

Plant material and growth conditions

The tomato cultivar M82 was used in this study for constructing the CRISPR mutants as well as the OP::STTM164, OP::mSlNAM3, and p2A11::LhG4 transgenic lines. The 35S::LhG4 driver line and OP::mSlNAM2 responder line have been described in Hendelman et al. (2013). The CRISPR double mutants slmir164aCR-21slmir164bCR-17/1 and slmir164aCR-21slmir164dCR-13 were obtained by crossing. The 35S≫STTM164 and p2A11≫mSlNAM3 trans-activated plants were obtained by crossing the respective driver lines to indicated responder lines, and PCR genotyping their F1 progeny. Germination and seedling growth were performed as described in Hendelman et al. (2013).

Plasmid constructions

For the OP::STTM164 responder construct, an STTM164 fragment consisting of two 24 bp miR164 target mimic sequences separated by 88-bp spacer and delimited by 5′-XhoI and 3′-HindIII was artificially synthesized (GENEWIZ, USA) and cloned into pUC57, digested with XhoI and HindIII and the released STTM164 fragment was subcloned into the corresponding sites of pART27-OP binary vector under the control of the pOP promoter, to generate pART27-OP::STTM164. For the CRISPR/Cas9 mediated mutagenesis of SlMIR164 genes, gene-specific gRNAs targeting the mature sly-miR164 and 5ʹ adjacent sequences were designed (The gRNA sequences are listed in Supplemental Table S3), and each incorporated in silico into an sgRNA consisting of the respective gRNA followed by a 76-bp generic scaffold and a 7xT Polymerase III terminator sequence. Then constructs containing two (SlMIR164a, SlMIR164b, and SlMIR164c) or three (SlMIR164d) sgRNAs in tandem each under the control of the synthetic Arabidopsis U6 promoter, delimited by 5′-MluI and 3′-SalI (SlMIR164a), 5′-SalI, and 3′-HindIII (SlMIR164b) or 5′-MluI and 3′-HindIII (SlMIR164c and SlMIR164d), were artificially synthesized (GENEWIZ, USA) and cloned into pUC57, digested with respective restriction enzymes, and the released sgRNAs fragments were ligated into the compatible sites of the pRCS binary vector alongside the plant codon-optimized version of Streptococcus pyogenes Cas9 (Damodharan et al., 2018) expressed under the constitutive CaMV 35S promoter. For the p2A11::LhG4 driver construct, the 2A11 4051 bp promoter region (SL4.0ch07:59287072-59291122) was amplified from tomato genomic DNA using the KpnI-2A11-fwd and BamH1-2A11-rev primer pair. The amplified PCR product was restricted with KpnI/BamHI and cloned into corresponding sites of the LhG4-BJ36 plasmid. Furthermore, the NotI restricted fragment of 2A11::LhG4 was mobilized into pART27 vector to generate pART27-2A11::LhG4 construct. For the OP::mSlNAM3 responder construct, four silent mutations were inserted in the SlNAM3 miR164 target site using two-step PCR mutagenesis. Firstly, the coding region of SlNAM3 was PCR amplified with SalI-SlNAM3-fwd and HindIII-SlNAM3-rev primer pair. The amplified fragment was then digested with SalI and HindIII and cloned into respective sites of the OP-TATA-BJ36 plasmid to generate OP::SlNAM3. A fragment containing the four silent mutations was amplified from OP::SlNAM3 using PmlI-mSlNAM3-miR164 and HindIII-SlNAM3-rev primer pair, restricted with PmlI/HindIII and cloned into the identical sites of OP::SlNAM3, replacing the respective wild-type SlNAM3 fragment to generate OP::mSlNAM3. Following sequence validation, the NotI fragment of OP::mSlNAM3 was mobilized into the pART27 binary vector to generate pART27-OP::mSlNAM3.

Tomato transformations

The binary constructs pRCS-35S::Cas9-SlMIR164a-d, pART27-OP::STTM164, pART27-OP::mSlNAM3, and pART27-p2A11::LhG4 were transformed into tomato cultivar M82 by co-cultivation of cotyledons derived from 12-d-old seedlings with Agrobacterium tumefaciens strain GV3101 as described previously (Damodharan et al., 2018), followed by regeneration on selective 100 mg/L kanamycin-containing media, where only transgenic seedlings developed a branched root system. The transgenic status of the kanamycin-resistant seedlings was further validated by genomic DNA PCR with transgene specific primer pairs to detect the presence of the Cas9, responder (OP::gene), and driver (promoter::LhG4) transgenes.

Isolation of SlMIR164 CRISPR mutants

To isolate CRISPR mutants in SlMIR164 genes (slmir164CR), the primary (T0) transformants were screened by genomic DNA PCR with specific primers flanking the gRNAs targeted sequences followed by agarose gel electrophoresis to detect amplicons containing relatively large indels or Restriction Fragment Length Polymorphism (RFLP) analysis to detect those with small indels. The electrophoresis analysis led to the identification of T0 plants harboring slmir164aCR-21, slmir164dCR-13, and slmir164dCR-15 mutant alleles and the RFLP analysis with NlaIII led to the identification of slmir164bCR-1 and slmir164bCR-17 mutant alleles. Identified T0 plants carrying indels were backcrossed to M82 wild-type, and the resulting F1 progeny were genotyped as described above to detect the CRISPR heterozygous mutants. These were selfed, and the resulting F2 progeny were genotyped as described above, followed by sequencing of the mutant loci amplicons. The verified F2 homozygous CRISPR mutants (slmir164CR) were used in the study.

Total RNA extraction and RT-qPCR

Total RNA was extracted from different tomato tissues (shoot apices, leaf, and fruit) with Bio-Tri RNA reagent (Bio-Lab, Jerusalem, Israel) according to the manufacturer’s protocol. cDNA preparation and RT-qPCR assays for sly-miR164 target genes were performed as previously described in Hendelman et al., 2016. Mature miRNA levels were quantified using hairpin/stem–loop cDNA RT-qPCR as described in Turner et al., 2013. The relative expression level of sly-miR164 was calculated either using the standard-curve method (Figure 4, G) or ΔΔCT methods (others) by normalizing U6 as the reference gene.

Fruit analysis

Fruit chronological age was determined by tagging the flower at the anthesis stage that is considered as 0 DPA. The different stages of fruit ripening and their corresponding age were determined by visually monitoring the fruit color from the day of anthesis. Fruit shape parameters were measured using the Tomato Analyzer software Version 3.0 (Brewer et al., 2006). The skin color properties of wild-type and slimir164aCR-21 fruits were measured at the red ripe stage using the CR-400 Chroma Meter (Konica Minolta, Japan).

Histological and cuticle analyses

A pericarp of approximately 125 cubic mm was excised from the equatorial region of transverse crossed fruit (at least 5 fruit samples per genotype). The excised tissue was immediately fixed in FAA solution (3.7% formaldehyde, 5% acetic acid, 50% EtOH, v/v/v), placed under vacuum for 1 hour and then incubated overnight at room temperature. The next day, FAA was aspirated and fixed tissues were washed in an increasing gradient of ethanol (up to 100%) and embedded in paraffin. The deparaffinized 10-µm thick pericarp sections were stained with Safranin and Fast green as described in Hendelman et al. (2016). Mesocarp cell number and cell volume determination were performed as described in Xiao et al. (2009) and detailed in Supplemental Figure S6. For analysis of cuticle thickness, 14-µm thick sections of paraffin fixed pericarp were stained with Sudan IV (MP biomedicals, USA), as described previously in Buda et al. (2009), and following mounting in distilled water and examined immediately under bright-field using an Olympus DP73 microscope equipped with a digital camera.

PCR and RT-qPCR primers

The sequences of all the primers used in this study are listed in Supplemental Table S3.

Accession numbers

Sequence data from this article can be found in the GenBank/EMBL data libraries under accession numbers SlNAM2: Solyc03g115850; SlNAM3: Solyc06g069710; GOB: Solyc07g062840; SlNAC1: Solyc07g066330.

Supplemental data

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

Supplemental materials and methods .

Supplemental Figure S1 . Hairpin secondary structure of miR164d in various Solanaceae species.

Supplemental Figure S2 . Additional vegetative phenotypes of slmir164CR mutants.

Supplemental Figure S3 . Additional characterization of slmir164aCR-21 red ripe fruits.

Supplemental Figure S4 . GC-MS analysis of individual cutin monomers in skin tissue of wild-type and slmir164aCR-21 mature green (MG), orange (OR) and red ripe (RR) fruits.

Supplemental Figure S5 . Quantitation of the artificial OP activator LhG4 transcript in the pericarp of p2A11::LhG4 developing and ripening fruits by semi-quantitative PCR.

Supplemental Figure S6 . Measurements of mesocarp cell numbers and cell volume.

Supplemental Table S1 . Details of the tomato SRA small RNA data sets used for de novo prediction of SlMIR164 genes and the frequencies of sly-miR164 and respective star strand in them.

Supplemental Table S2 . sly-miR164 target genes predicted by psRNAtarget.

Supplemental Table S3 . List of primers and gRNAs used in this study.

Supplementary Material

kiab376_Supplementary_Data
Click here for additional data file. (2.7MB, zip)

Acknowledgments

We thank Amnon Lers (Institute of Postharvest and Food Sciences, ARO) for his assistance in characterization of ripened fruits; Stephanie Jaubert and Bruno Favery (Institut Sophia Agrobiotech, France), and Jackson Khedia from the Arazi laboratory for critical reading of the manuscript.

Funding

S.K.G. and A.V. are receivers of PBC ARO Israel-India fellowship.

Conflict of interest statement. None declared.

 

T.A.: supervised the research; T.A. and S.K.G.: designed the experiments, analyzed the data and wrote the manuscript; S.K.G.: carried out most experiments; H.D.K.: generated the SlMIR164c CRISPR transgenic plants and quantified sly-miR164 in the 35S≫STTM164-12 mutant; A.V.: generated the OP::mSlNAM3 transgenic line; O.G.: quantitated miR164 targets in the SlMIR164d mutant; H.C. and A.A.: performed and analyzed metabolite profiling assays.

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: Tzahi Arazi (tarazi@agri.gov.il).

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