SEARCH

SEARCH BY CITATION

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Small Scale Cloning of sRNAs from Tomato Tissues
  5. Large Scale Sequencing of Tomato sRNAs
  6. Function of Specific Tomato miRNAs
  7. Concluding Remarks and Future Directions
  8. References

Short RNAs are 20–24 nucleotide long non-coding RNA molecules generated by one of the Dicer-like enzymes. They recognize specific RNA or DNA sequences and guide the RNA silencing complex to their targets leading to post-transcriptional or transcriptional gene silencing of the target. Most of our knowledge about short RNAs comes from studying the model species Arabidopsis. Recently, however, several reports emerged about short RNAs in tomato, which is a model plant for fleshy fruit development and ripening. Tomato short RNAs have been sequenced and a database was established. Novel non-conserved microRNAs were found that showed differential expression between fruit and other tissues, even during fruit development, suggesting that they may play a role in fruit formation. Several target genes were predicted and validated for both conserved and non-conserved miRNAs and some of these targets are key players in fruit ripening, such as Colourless non-ripening. The present study reviews the current state of tomato short RNAs research and suggests future directions.

inline imageTamas Dalmay (Corresponding author)

Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Small Scale Cloning of sRNAs from Tomato Tissues
  5. Large Scale Sequencing of Tomato sRNAs
  6. Function of Specific Tomato miRNAs
  7. Concluding Remarks and Future Directions
  8. References

Post-transcriptional gene silencing was discovered in the early 1990s (Napoli et al. 1990) in plants and it fundamentally changed how we thought about RNA. In addition to coding for proteins (mRNA) and facilitating protein synthesis (rRNA and tRNA), RNA turned out to regulate gene expression. This function is carried out by a new group of RNAs called short RNAs (sRNAs). sRNAs are 20–24 nucleotides long and generated by one of the Dicer-like (DCL) proteins (Bartel 2004). There are two main types of sRNAs based on biogenesis: microRNAs (miRNAs) and small interfering RNAs (siRNAs).

MicroRNAs are generated from a single strand of RNA precursor that forms partially double stranded stem-loop structures (hairpins) through intramolecular base pairing. The stem part of these hairpins is recognized and cleaved by DCL1 resulting in a short RNA duplex, from which one of the strands (mature miRNA) is incorporated into the RNA-induced silencing complex (RISC). miRNAs anneal to complementary regions in mRNAs through Watson-Crick base-pairs and therefore guide RISC to specific mRNAs. RISC cleaves the targeted mRNA and suppresses translation of uncleaved messages (Reinhart et al. 2002; Brodersen et al. 2008).

The other class of sRNAs involve the synthesis of a complementary strand of RNA by an RNA-dependent RNA-polymerase (RDR). There are several RDRs in plants and the products of the different RDRs are processed by different DCLs. For example the products of RDR6 (Dalmay et al. 2000; Mourrain et al. 2000), which makes double stranded RNA (dsRNA) from miRNA cleaved non-coding single stranded RNA molecules, are processed by DCL4 into 21-mer trans-acting siRNAs (ta-siRNAs) (Peragine et al. 2004; Vazquez et al. 2004). However, the RDR2 generated dsRNAs, usually derived from transposable elements and repeat loci are processed by DCL3 into 24-mer heterochromatin siRNAs (Lu et al. 2006). While ta-siRNAs target mRNAs similarly to miRNAs, heterochromatin siRNAs cause DNA methylation and/or heterochromatin formation that leads to transcriptional gene silencing (Volpe et al. 2002).

Most if not all basic discovery in the sRNA field is made through studying Arabidopsis and initially it was thought that all miRNAs are conserved in flowering plants. However, it became apparent that the evolution of miRNAs did not stop after speciation therefore in addition to conserved miRNAs there are so called “young miRNAs” (Allen et al. 2004). While conserved miRNAs regulate expression of genes involved in basic developmental processes, the non-conserved miRNAs may be involved in the development of traits that are specific for certain taxonomic groups. This hypothesis led us and others to study sRNAs in tomato. Arabidopsis is widely used to investigate many basic developmental processes but its fruit is different from the fruits of many other plant species. Many edible fruits are fleshy where the wall of the ovule becomes a thick, high water content pericarp. The model species for fleshy fruit biology is tomato, which is also an important crop. This review summarizes our current knowledge of sRNAs in tomato fruit.

Small Scale Cloning of sRNAs from Tomato Tissues

  1. Top of page
  2. Abstract
  3. Introduction
  4. Small Scale Cloning of sRNAs from Tomato Tissues
  5. Large Scale Sequencing of Tomato sRNAs
  6. Function of Specific Tomato miRNAs
  7. Concluding Remarks and Future Directions
  8. References

The most straightforward approach to identify sRNAs in plants is by sequencing cDNA libraries generated from sRNAs. This can be achieved by ligating two different adaptor oligonucleotides to the 5′ and 3′ end of the size purified small RNA fraction, followed by a reverse transcription-polymerase chain reaction (RT-PCR) step using primers homologous to the adaptors. Initially these PCR products were cloned into plasmids (either individually or after generating concatemers). Two such studies were reported for tomato sRNA sequencing (Pilcher et al. 2007; Itaya et al. 2008). Pilcher et al. (2007) sequenced more than 4 000 sRNAs isolated from pericarp of mature green fruits from the cultivar Ailsa Craig. Itaya et al. (2008) generated a slightly smaller library by sequencing 1 210 sRNAs from four different tissues: mature leaf, flower buds, young fruits and mature ripe fruits. Both studies identified several conserved miRNAs and new miRNA candidates, which were validated by northern blots. Some of the novel sRNAs could be folded into stem-loop structures characteristic for miRNAs and some of them even showed differential accumulation between leaves and fruits. Itaya et al. (2008) also established a database for tomato sRNAs that contains all sequences from both studies and also from subsequent reports. The database is publicly available at http://ted.bti.cornell.edu/cgi-bin/TFGD/sRNA/home.cgi.

Large Scale Sequencing of Tomato sRNAs

  1. Top of page
  2. Abstract
  3. Introduction
  4. Small Scale Cloning of sRNAs from Tomato Tissues
  5. Large Scale Sequencing of Tomato sRNAs
  6. Function of Specific Tomato miRNAs
  7. Concluding Remarks and Future Directions
  8. References

Cloning of the PCR products to plasmids became unnecessary with the development of next generation high throughput sequencing technologies. Moxon et al. (2008) reported the first and until now the only high throughput sequencing study on tomato sRNAs. The 454 sequencing platform was used to sequence tomato sRNAs from young leaves and a mixture of young green fruits (1–15 mm) of the cultivar Microtom. More than 700 000 reads were generated for the two samples and after adaptor removal; around 400 000 good-quality short sequences were obtained from fruits and around 170 000 from leaves. After removing redundant sequences, 225 000 and 102 000 unique sequences were found for fruits and leaves, respectively. The 21- and 22-mer sequences showed the highest degree of redundancy, suggesting that these are mainly generated from miRNA genes, which are highly expressed. On the other hand, the 24-mers showed very low level of redundancy indicating that the heterochromatic siRNAs are produced from many different loci but at a relatively low level.

The two libraries contained most of the known conserved miRNA families. Twenty families identified in other species had a perfect homolog in tomato and an additional 10 families could be identified allowing up to two nucleotide mismatches. Interestingly, even miRNAs that were thought to be non-conserved were found in tomato. miR858 (only found in Arabidopsis), miR894 (only found in moss) and miR482 (only found in poplar) were all present in the libraries and were also confirmed by northern blotting. This raises the possibility that the presence/absence of young miRNAs in different taxonomic groups could be a potential tool for phylogenetic studies.

Expression of 13 conserved miRNAs were analyzed by northern blotting in leaves, flower buds and four stages of fruit development across very young fruits (1–3 mm) and mature green. Only three of them (miR165/166, miR403 and miR472) showed constitutive expression; the other 10 accumulated differentially in the different tissues. miRNAs 156/157, 164, 408, 858 and 894 were present at a higher level in leaves and flower buds than in fruits and miR169 showed an opposite pattern. Interestingly, miR390 and miR171 (and also mir171*) showed differential expression across the different stages of fruits, suggesting that they are involved in development of young fruits. Target mRNAs of 12 conserved miRNAs were predicted and validated by 5′ rapid amplification of cDNA ends (RACE) technique. 5′ RACE can be used to validate targets because miRNAs were shown to cleave the targets at a specific position (opposite to the 10–11th nucleotide of the miRNA) and the 3′ cleavage product is relatively stable, and therefore its 5′ end can be mapped. The most important validated target was Colourless never ripe (CNR) that was targeted by miR156/157. CNR is one of the key genes in fruit ripening and this was the first experimental data indicating that fleshy fruit ripening is regulated by miRNAs.

In addition to the conserved miRNAs, Moxon et al. (2008) also identified new tomato miRNAs that are not present in the Arabidopsis genome. More than 80 candidate miRNA genes were identified based on miRNA-like stem-loop structure of the cloned sequences and their flanking regions. However, the ability to form a hairpin is not proof that a sequenced short RNA is indeed a miRNA because of the large amount of siRNAs in plant cells and the high number of possible hairpins in the genome (Meyers et al. 2008). Additional proof is required to validate miRNA genes and the best evidence is to show that the putative miRNA is generated from the hairpin by Dicer. This involves the sequencing of the miRNA* strand (the other strand of the miRNA duplex, which comes from the complementary strand of the stem) that normally shows a two nucleotide overhang at the 3′ end of the miRNA/miRNA* duplex. Only one of the 65 candidates had a miRNA* sequenced in the libraries and this was the first non-conserved bona fide miRNA identified in tomato (miR1916). In addition to biogenesis, the activity of the putative miRNA can be used to validate new miRNAs (and also the fact that they can be found in several independent libraries). Targets were predicted for the candidate miRNAs and 65 mRNAs were identified that showed very strong complementarity to candidate miRNAs. The 5′ RACE technique was applied for all 65 putative targets but only three were validated. One of these targets is very interesting because it is a member of the constitutive triple response (CTR) family and CTR1 is a key negative regulator of ethylene response. It is worth noting that the accumulation of miR1917 (that targets le-CTR4sv2; a splice variant of CTR4) increases during fruit development, which is expected if it suppresses a negative regulator of ethylene response since negative regulation of ethylene response should be suppressed during ripening when the fruit responds to ethylene. The other two validated targets cleaved by miR1918 and mir1919 do not have known homologous genes in other species, therefore their function is not known. The fact that 62 predicted targets could not be validated raises several possibilities. It is possible that these sRNAs are not miRNAs (since no miRNA* sequences were found) therefore they do not act as miRNAs and the predicted targets have complementary sequences just by chance. Another possibility is that these targets are only regulated at the translational level. Brodersen et al. (2008) showed that plant miRNAs can suppress translation in addition to cleavage of mRNAs, although it was not demonstrated that an mRNA can be only suppressed at the translational level without any cleavage.

In addition to conserved and non-conserved miRNAs, Moxon et al. (2008) identified a very abundant group of sRNAs, which derived from TAPIR loci. TAPIR is a transposable element with a relatively high copy number (the exact copy number is not known since the full genome sequence is not available) and it can be folded into a long (approximately 150 base pair), almost perfect stem-loop structure. The interesting observation was that the TAPIR derived sRNAs were not evenly distributed along the stem but showed a miRNA/miRNA*-like pattern, although not with a perfect 2 nucleotide 3′ overhang. However, it is impossible to decide which short read comes from which TAPIR locus because the TAPIR loci are very similar to each other. Nevertheless, the accumulation pattern of sRNAs on the TAPIR elements suggests that some miRNAs are evolved from transposable elements, which was also suggested by other groups (Piriyapongsa and Jordan 2008).

Function of Specific Tomato miRNAs

  1. Top of page
  2. Abstract
  3. Introduction
  4. Small Scale Cloning of sRNAs from Tomato Tissues
  5. Large Scale Sequencing of Tomato sRNAs
  6. Function of Specific Tomato miRNAs
  7. Concluding Remarks and Future Directions
  8. References

The biological function of miRNAs in tomato is largely unknown. The only area where we have some understanding of their role is leaf formation. It was shown in Arabidopsis that miR319 targets several members of the TEOSINTE BRANCHED/CYCLOIDEA/PCF (TCP) transcription factor family (Palatnik et al. 2003). One of the tomato TCP genes is LANCEOLATE (LA) and a partially dominant mutation in this gene causes the dose-dependent gradual conversion of compound leaves into small, simple leaves. Ori et al. (2007) found that five different La mutants all contained point mutations in the conserved mir319 target site on the LA mRNA, suggesting that the phenotype in these mutants was due to the lack of mir319 regulation of La. Interestingly, it was shown that mutant La mRNAs were still cleaved by miR319 but with less efficiency than the wild type LA mRNA. The reduced sensitivity to miR319 resulted in an elevated LA expression in very young leaf primordia and accelerated differentiation of leaf margins. On the other hand, increased expression of miR319 led to larger leaflets and continuous growth of leaf margins. These observations suggest that a certain level of LA and LA-like proteins is required for differentiation of leaf margins. Where and when LA expression is downregulated by miR319 leaflet-initiation programs can occur, which is supported by the complementary spatial and temporal expression of LA and mir319. This model explains why a higher level of LA causes simple leaf formation and overexpression of mir319 leads to enlarged leaflets with highly lobed margins.

Another conserved miRNA with a known biological function in tomato is miR164. This miRNA targets members of the no apical meristem/ATAF1/cup-shaped cotyledon (NAC) transcription factor family in Arabidopsis (Mallory et al. 2004). One of its target mRNAs in tomato is encoded by the GOBLET (GOB) gene. Mutations in GOB and overexpression of mir164 led to similar phenotype: loss of secondary-leaflet initiation and smooth leaflet margin (Berger et al. 2009). Expression of GOB and mir164 is complementary similarly to LA and mir319. Based on these data Berger et al. (2009) concluded that GOB marks leaflet boundaries and its spatial and temporal accumulation defines leaf elaboration and this process is regulated by mir164.

Concluding Remarks and Future Directions

  1. Top of page
  2. Abstract
  3. Introduction
  4. Small Scale Cloning of sRNAs from Tomato Tissues
  5. Large Scale Sequencing of Tomato sRNAs
  6. Function of Specific Tomato miRNAs
  7. Concluding Remarks and Future Directions
  8. References

The complexity of sRNAs in tomato has just been realized and the only deep sequencing study so far proved to be unsaturated despite producing more than 700 000 reads. In addition, the analysis of the data was limited by the lack of a finished genome sequence. The full sRNA content of tomato tissues will be achieved when more effective platforms (such as Solexa and SOLID) will be used and the full genome sequence will be available. Northern blot analysis suggests that several miRNAs are differentially expressed in different tomato tissues. It will be interesting to see whether miRNA accumulation changes during fruit development. Profiling short RNAs by deep sequencing at different developmental stages of fruit will answer these questions and will establish groups of miRNAs with similar temporal expression patterns. Expression profiles of miRNAs during fruit development can be compared with expression profiles of mRNAs at the same developmental stages and look for negative correlation between miRNAs and predicted mRNA targets. Although, Kawashima et al. (2009) showed that accumulation of miRNAs and their targets do not always show negative correlation, many targets could be validated this way. Another way to identify/validate targets is to carry out a 5′ RACE at a genomic scale. German et al. (2008) and Addo-Quaye et al. (2008) reported high throughput sequences of libraries generated from miRNA cleaved mRNAs. This approach should also be applied for tomato tissues.

Understanding the biological functions of miRNAs involved in fruit development requires the generation of transgenic tomato plants similar to the ones that helped decipher the function of miR319 and miR164 (Ori et al. 2007; Berger et al. 2009). miRNA overexpressing lines either from the constitutive 35S promoter or from spatial and/or temporal-specific promoters represent one approach. The other approach is to suppress miRNA activity but it is difficult to manipulate the activity of the miRNA directly. To overcome this problem, miRNA-resistant target genes can be expressed ectopically, if the target gene is known and validated. If the target gene is not known or there are several of them, a slightly different technique can be used. The so-called decoy approach (or miRNA mimicry) takes advantage of a natural non-coding RNA that suppresses miRNA activity (Franco-Zorrilla et al. 2007). The IPS1 (INDUCED BY PHOSPHATE STARVATION 1) non-coding RNA was shown to be targeted by miR399 but due to mismatches close to the expected cleavage site the target RNA is not cleaved. It is still recognized by the miRNA but because RISC does not cleave the RNA, the miRNA-RISC complex stays attached to the target RNA. This leads to reduced activity of the miRNA on the “real” mRNA targets. Artificial miRNA decoys can be designed and ectopically expressed to achieve a reduced miRNA activity on unknown target mRNAs or on several target genes. Applying these approaches to conserved and non-conserved tomato miRNAs will probably uncover interesting regulatory pathways during fleshy fruit development and other processes.

Another potentially interesting and so far unexplored area is the extent of gene expression regulation by ta-siRNAs in tomato. Until now ta-siRNAs were identified only in Arabidopsis and the complexity of ta-siRNAs in other species is not known. The fact that only one of the trans acting siRNA (TAS) RNAs (TAS3) is found in other species (Allen et al. 2005) suggests that other species–including tomato–also encode for non-conserved TAS RNAs. Since ta-siRNAs are produced by subsequent cleavage of TAS RNAs by DCL4, they can be predicted by searching for sRNAs with phased patterns (Chen et al. 2007). The candidates can be validated if a mutant is available in the ta-siRNA pathway (such as RDR6, SGS3 or DCL4) by demonstrating the accumulation of the potential ta-siRNA in wild type plants and the absence of accumulation in the mutant.

(Co-Editor: Xiaofeng Cao)

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Small Scale Cloning of sRNAs from Tomato Tissues
  5. Large Scale Sequencing of Tomato sRNAs
  6. Function of Specific Tomato miRNAs
  7. Concluding Remarks and Future Directions
  8. References
  • Addo-Quaye C, Eshoo TW, Bartel DP, Axtell MJ (2008) Endogenous siRNA and miRNA targets identified by sequencing of the Arabidopsis degradome. Curr. Biol. 18, 758762.
  • Allen E, Xie Z, Gustafson AM, Sung GH, Spatafora JW, Carrington JC (2004) Evolution of microRNA genes by inverted duplication of target gene sequences in Arabidopsis thaliana. Nat. Genet 36, 12821290.
  • Allen E, Xie Z, Gustafson AM, Carrington JC (2005) microRNA-directed phasing during trans-acting siRNA biogenesis in plants. Cell 121, 207221.
  • Bartel DP (2004) MicroRNAs: genomics, biogenesis, mechanism, and function. Cell 116, 281297.
  • Berger Y, Harpaz-Saad S, Brand A, Melnik H, Sirding N, Alvarez JP, Zinder M, Samach A, Eshed Y, Ori N (2009) The NAC-domain transcription factor GOBLET specifies leaflet boundaries in compound tomato leaves. Development 136, 823832.
  • Brodersen P, Sakvarelidze-Achard L, Bruun-Rasmussen M, Dunoyer P, Yamamoto YY, Sieburth L, Voinnet O (2008) Widespread translational inhibition by plant miRNAs and siRNAs. Science 320, 11851190.
  • Chen HM, Li YH, Wu SH (2007) Bioinformatic prediction and experimental validation of a microRNA-directed tandem trans-acting siRNA cascade in Arabidopsis. Proc. Natl. Acad. Sci. USA 104, 33183323.
  • Dalmay T, Hamilton A, Rudd S, Angell S, Baulcombe D (2000) An RNA-dependent RNA polymerase is required for posttranscriptional gene silencing mediated by a transgene but not by a virus. Cell 101, 543553.
  • Franco-Zorrilla JM, Valli A, Todesco M, Mateos I, Puga MI, Rubio-Somoza I, Leyva A, Weigel D, García JA, Paz-Ares J (2007) Target mimicry provides a new mechanism for regulation of microRNA activity. Nat. Genet. 39, 10331037.
  • German MA, Pillay M, Jeong DH, Hetawal A, Luo S, Janardhanan P, Kannan V, Rymarquis LA, Nobuta K, German R, De Paoli E, Lu C, Schroth G, Meyers BC, Green PJ (2008) Global identification of microRNA-target RNA pairs by parallel analysis of RNA ends. Nat. Biotechnol. 26, 941946.
  • Itaya A, Bundschuh R, Archual AJ, Joung JG, Fei Z, Dai X et al. (2008) Small RNAs in tomato fruit and leaf development. Biochim. Biophys. Acta 1779, 99107.
  • Kawashima CG, Yoshimoto N, Maruyama-Nakashita A, Tsuchiya YN, Saito K, Takahashi H, Dalmay T (2009) Sulphur starvation induces the expression of microRNA-395 and one of its target genes but in different cell types. Plant J. 57, 313321.
  • German MA, Pillay M, Jeong DH, Hetawal A, Luo S, Janardhanan P, Kannan V, Rymarquis LA, Nobuta K, German R, De Paoli E, Lu C, Schroth G, Meyers BC, Green PJ (2006) MicroRNAs and other small RNAs enriched in the Arabidopsis RNA-dependent RNA polymerase-2 mutant. Genome Res. 16, 12761288.
  • Mallory AC, Dugas DV, Bartel DP, Bartel B (2004) MicroRNA regulation of NAC-domain targets is required for proper formation and separation of adjacent embryonic, vegetative, and floral organs. Curr. Biol. 14, 10351046.
  • Meyers BC, Axtell MJ, Bartel B, Bartel DP, Baulcombe D, Bowman JL, Cao X, Carrington JC, Chen X, Green PJ, Griffiths-Jones S, Jacobsen SE, Mallory AC, Martienssen RA, Poethig RS, Qi Y, Vaucheret H, Voinnet O, Watanabe Y, Weigel D, Zhu JK (2008) Criteria for annotation of plant MicroRNAs. Plant Cell 20, 31863190.
  • Mourrain P, Béclin C, Elmayan T, Feuerbach F, Godon C, Morel JB, Jouette D, Lacombe AM, Nikic S, Picault N, Rémoué K, Sanial M, Vo TA, Vaucheret H (2000) Arabidopsis SGS2 and SGS3 genes are required for posttranscriptional gene silencing and natural virus resistance. Cell 101, 533542.
  • Moxon S, Jing R, Szittya G, Schwach F, Rusholme Pilcher RL, Moulton V, Dalmay T (2008) Deep sequencing of tomato short RNAs identifies microRNAs targeting genes involved in fruit ripening. Genome Res. 18, 16021609.
  • Napoli C, Lemieux C, Jorgensen R (1990) Introduction of a chimeric chalcone synthase gene into petunia results in reversible co-suppression of homologous genes in trans. Plant Cell 2, 279289.
  • Ori N, Cohen AR, Etzioni A, Brand A, Yanai O, Shleizer S, Menda N, Amsellem Z, Efroni I, Pekker I, Alvarez JP, Blum E, Zamir D, Eshed Y (2007) Regulation of LANCEOLATE by miR319 is required for compound-leaf development in tomato. Nat Genet. 39, 787791.
  • Palatnik JF, Allen E, Wu X, Schommer C, Schwab R, Carrington JC, Weigel D (2003) Control of leaf morphogenesis by microRNAs. Nature 425, 257263.
  • Peragine A, Yoshikawa M, Wu G, Albrecht HL, Poethig RS (2004) SGS3 and SGS2/SDE1/RDR6 are required for juvenile development and the production of trans-acting siRNA in Arabidopsis. Genes Dev. 18, 23682379.
  • Pilcher RL, Moxon S, Pakseresht N, Moulton V, Manning K, Seymour G, Dalmay T (2007) Identification of novel small RNAs in tomato (Solanum lycopersicum). Planta 226, 709717.
  • Piriyapongsa J, Jordan IK (2008) Dual coding of siRNAs and miRNAs by plant transposable elements. RNA 14, 814821.
  • Reinhart BJ, Weinstein EG, Rhoades MW, Bartel B, Bartel DP (2002) MicroRNAs in plants. Genes Dev. 16, 16161626.
  • Vazquez F, Vaucheret H, Rajagopalan R, Lepers C, Gasciolli V, Mallory AC, Hilbert JL, Bartel DP, Crété P (2004) Endogenous trans-acting siRNAs regulate the accumulation of Arabidopsis mRNAs. Mol Cell 16, 6979.
  • Volpe TA, Kidner C, Hall IM, Teng G, Grewal SI, Martienssen RA (2002) Regulation of heterochromatic silencing and histone H3 lysine-9 methylation by RNAi. Science 297, 18331837.