Present address: Interfaculty Institute for Biochemistry, Eberhard-Karls-University Tübingen, Hoppe-Seyler-Strasse 4, D-72076 Tübingen, Germany.
TOMATO AGAMOUS-LIKE 1 is a component of the fruit ripening regulatory network
Article first published online: 3 NOV 2009
© 2009 Weizmann Institute of Science. Journal compilation © 2009 Blackwell Publishing Ltd
The Plant Journal
Volume 60, Issue 6, pages 1081–1095, December 2009
How to Cite
Itkin, M., Seybold, H., Breitel, D., Rogachev, I., Meir, S. and Aharoni, A. (2009), TOMATO AGAMOUS-LIKE 1 is a component of the fruit ripening regulatory network. The Plant Journal, 60: 1081–1095. doi: 10.1111/j.1365-313X.2009.04064.x
- Issue published online: 8 DEC 2009
- Article first published online: 3 NOV 2009
- Received 15 September 2009; revised 14 October 2009; accepted 19 October 2009.
- Top of page
- Experimental procedures
- Supporting Information
After fertilization, the expanding carpel of fleshy fruit goes through a phase change to ripening. Although the role of ethylene signalling in mediating climacteric ripening has been established, knowledge regarding the regulation of ethylene biosynthesis and its association with fruit developmental programs is still lacking. A functional screen of tomato transcription factors showed that silencing of the TOMATO AGAMOUS-LIKE 1 (TAGL1) MADS box gene results in altered fruit pigmentation. Over-expressing TAGL1 as a chimeric repressor suggested a role in controlling ripening, as transgenic tomato fruit showed reduced carotenoid and ethylene levels, suppressed chlorophyll breakdown, and down-regulation of ripening-associated genes. Moreover, fruits over-expressing TAGL1 accumulated more lycopene, and their sepals were swollen, accumulated high levels of the yellow flavonoid naringenin chalcone and contained lycopene. Transient promoter-binding assays indicated that part of the TAGL1 activity in ripening is executed through direct activation of ACS2, an ethylene biosynthesis gene that has recently been reported to be a target of the RIN MADS box factor. Examination of the TAGL1 transcript and its over-expression in the rin mutant background suggested that RIN does not regulate TAGL1 or vice versa. The results also indicated RIN-dependent and -independent processes that are regulated by TAGL1. We also noted that fruit of TAGL1 loss-of-function lines had a thin pericarp layer, indicating an additional role for TAGL1 in carpel expansion prior to ripening. The results add a new component to the current model of the regulatory network that controls fleshy fruit ripening and its association with the ethylene biosynthesis pathway.
- Top of page
- Experimental procedures
- Supporting Information
The switch to ripening involves the combined action of hormonal signal transduction cascades, regulatory circuits and environmental cues (Fei et al., 2004; Srivastava and Handa, 2005; Carrari and Fernie, 2006; Seymour et al., 2008). This integrated process triggers a phase change in fruit development, typically characterized by dramatic shifts in primary and secondary metabolism. Although many efforts have been made in order to understand the mechanism behind ripening, the core set of genetic components required for activation of this process have not been yet identified.
To date, most studies regarding the regulatory and signalling pathways of ripening have been performed in tomato, largely through investigation of ripening-deficient mutants. Several of these mutants, such as Never-ripe (Nr; Lanahan et al., 1994; Wilkinson et al., 1995; Chen et al., 2004) and Green ripe (Gr)/Never-ripe2 (Nr2; Kerr, 1981, 1982; Barry and Giovannoni, 2006) were deficient in their ethylene pathway or downstream signal transduction. Other mutants, including ripening inhibitor (rin), non-ripening (nor) and colourless non-ripening (Cnr), exhibit altered transcription factor activity (Thompson et al., 1999; Vrebalov et al., 2002; Manning et al., 2006).
Ethylene is a fundamental signal in climacteric fruit maturation (Yang and Hoffman, 1984; Alba et al., 2005). S-adenosyl-l-methionine (SAM) is converted into 1-aminocyclopropane-1-carboxylic acid (ACC) by ACC synthase (ACS) (Sato and Theologis, 1989), and ACC is further converted into ethylene by ACC oxidase (ACO) (Slater et al., 1985; Hamilton et al., 1990; Bleecker and Kende, 2000). In tomato, both ACS and ACO are part of multi-gene families, with nine copies of ACS (Zarembinski and Theologis, 1994) and three copies of ACO (Bouzayen et al., 1993) so far identified in the tomato genome. Two of the ACS genes (ACS2 and ACS4) have been shown to be up-regulated by ethylene and play an important role in tomato fruit ripening (Olson et al., 1991; Lincoln et al., 1993).
An early model for ethylene biosynthesis by McMurchie et al. (1972) was later extended (Nakatsuka et al., 1998; Barry et al., 2000) to propose that ACS2 and ACS4 initiate ethylene production during advanced ripening stages (termed system 2), while ACS1A and ACS6 participate in ethylene production in green tissues (termed system 1). A recent study suggested that tomato fruit can initiate ethylene system 2 independently of the cumulative effects of system 1, providing evidence that ripening-associated ethylene biosynthesis is regulated by both an auto-catalytic system and by ethylene-independent developmental factors (Yokotani et al., 2009).
Several transcription factors have been reported to act as regulators of tomato ethylene biosynthesis. For example, the homeobox protein HB-1 was recently reported to regulate ACO1 expression (Lin et al., 2008). Mutation in the MADS box factor RIPENING INHIBITOR (RIN) (Vrebalov et al., 2002) stops the characteristic processes associated with ripening of tomato fruit, including auto-catalytic ethylene production (Herner and Sink, 1973). Recently, Ito et al. (2008) showed that RIN might regulate ACS2, as it binds to its promoter.
MADS box transcription factors have been shown to play a significant role in the development of reproductive organs, including dry and fleshy fruit (Becker et al., 2002; Becker and Theissen, 2003; Balanza et al., 2006; Seymour et al., 2008). In addition to the tomato MADS box factor RIN, which belongs to the SEPALLATA (SEP) clade (Vrebalov et al., 2002; Hileman et al., 2006), members of the C-type MADS box group have also been associated with fleshy fruit development and ripening (Diaz-Riquelme et al., 2009; Tadiello et al., 2009).
The tomato AGAMOUS (AG) orthologue, TAG1, has been implicated in tomato fruit ripening in the AGAMOUS C-type lineage. Ishida et al. (1998) showed that sepals of tomato plants ectopically over-expressing TAG1 swell and ripen in vivo, when induced by cold temperature (Bartley and Ishida, 2003, 2007). Without cold induction, sepals of tomato plants over-expressing TAG1 were swollen but did not ripen (Pnueli et al., 1994).
Two MADS box genes of the C-type PLENA lineage, SHATTERPROOF 1 and 2 (AtSHP1 and AtSHP2), have been shown to control valve separation during Arabidopsis fruit dehiscence (Ferrandiz et al., 1999, 2000; Liljegren et al., 2000). A ripening-regulated peach SHP homologue (PpPLENA) was ectopically expressed in tomato (Tani et al., 2007; Tadiello et al., 2009). The sepals of the transgenic fruit developed into carpel-like structures and ripened. Furthermore, PpPLENA-expressing fruit showed accelerated ripening as evidenced by induction of expression of characteristic ripening genes. The SHP-like gene from tomato, TOMATO AGAMOUS-LIKE 1 (TAGL1), was initially reported to be expressed during early stages of fruit development (Busi et al., 2003), and Hileman et al. (2006) later showed that TAGL1 is also highly expressed in later stages of development. Yeast two-hybrid (Y2H) assays implied that TAGL1 might interact with various MADS box proteins, including RIN and JOINTLESS (Leseberg et al., 2008).
A screen using virus-induced gene silencing indicated that silencing of the tomato TAGL1 MADS box transcription factor results in altered fruit pigmentation. Loss of function resulted in reduced carotenoid (e.g. lycopene) and ethylene levels, suppressed chlorophyll breakdown, and down-regulation of a set of ripening-associated genes. The fruit of tomato plants over-expressing TAGL1 exhibited higher lycopene levels, and their sepals were swollen and showed ectopic lycopene production and accumulation of the yellow flavonoid naringenin chalcone. It appears that part of the TAGL1 activity in ripening is executed through regulation of the ACS2 ethylene biosynthesis gene. Examination of TAGL1 over-expression in the rin mutant background highlighted RIN-dependent and -independent ripening processes. Finally, we provide evidence that TAGL1 is also important for expansion of the carpel prior to ripening.
- Top of page
- Experimental procedures
- Supporting Information
Silencing of the TOMATO AGAMOUS-LIKE 1 gene alters fruit pigmentation
In order to identify regulatory genes that are associated with tomato fruit ripening, we screened a set of tomato transcription factors using the virus-induced gene silencing (VIGS) method (Liu et al., 2002). Overall, cDNAs corresponding to 114 putative transcription factors selected based on their differential expression during fruit development (Alba et al., 2005; Mintz-Oron et al., 2008) were used for the infection, and fruit were screened for phenotypes. The most obvious phenotype was detected in a plant silenced with the tomato MADS box transcription factor TOMATO AGAMOUS 1 (TAG1) cDNA, which had fruit that exhibited dark green regions at the mature green stage (Figure 1c). The same fruit showed yellow–orange sectors upon fruit maturation (Figure S1a). As the fragment used corresponded to the full-length transcript, we performed another VIGS assay using a TAG1-specific fragment (Figures 1d and S1b). However, this assay did not result in any effect on fruit pigmentation. Thus, we hypothesized that a gene closely related to TAG1 may have been silenced in the first VIGS assay.
Phylogenetic analysis of tomato MADS box transcription factors that are closely related to TAG1 suggested TOMATO AGAMOUS-LIKE 1 (TAGL1; SGN-U581068) as the best candidate for co-silencing with TAG1 in our original screen (Figure 1j). Indeed, TAGL1 silencing, using either the full-length cDNA or a specific fragment, resulted in altered fruit pigmentation (Figure 1e,f,h,i). These results suggest that the phenotype was due to silencing of the TAGL1 gene.
TAGL1 expression is ripening-regulated and altered in 1-MCP-treated fruit, but is not changed in the rin mutant or by exposure to ethylene
The phenotype obtained through TAGL1 silencing suggested that TAGL1 may be involved in the regulation of ripening in tomato fruit. To examine whether TAGL1 expression correlates with a possible function in fruit maturation, we measured its transcript levels in various tomato organs (Figure 2a). TAGL1 is expressed in fruit tissues (peel, flesh and seeds) from various developmental stages (immature green, IG; mature green, MG; breaker, Br; orange, Or; ripe) and in buds and flowers, but expression was not detected in leaves, roots and pollen. No significant difference in TAGL1 transcript levels was observed between peel and flesh tissues, in which its expression increases during ripening, peaking at the Or stage of fruit development.
The chemical 1-methylcyclopropene (1-MCP) is an inhibitor of ethylene perception and therefore interferes with fruit ripening (Yokotani et al., 2009). To examine whether blocking the ethylene receptors alters TAGL1 expression, fruits of three developmental stages were incubated with 1-MCP, and TAGL1 expression was measured 24 h post-treatment. The results revealed that TAGL1 is significantly induced when 1-MCP is applied to MG fruit compared to non-treated fruit (Figure 2b). TAGL1 expression was also examined in whole fruit tissues of the rin mutant (Figure 2c). No significant difference was observed between rin and wild-type (WT), indicating that TAGL1 expression is not regulated by RIN. We next examined whether TAGL1 is induced by the application of exogenous ethylene. Although expression of the ACS4, which served as a positive control, was significantly up-regulated, no significant alteration in TAGL1 transcript levels was observed upon exposure to ethylene (Figure 2d).
Thus, TAGL1 expression is ripening-regulated but is not changed in the rin mutant, and is altered in fruit upon inhibition of ethylene perception but not upon exposure to ethylene.
Over-expression of TAGL1 as a dominant repressor results in a loss-of-function phenotype that includes altered fruit carotenoids and ethylene levels
A chimeric transcription factor fused to the EAR (ERF-associated amphiphilic repression) motif functions as a dominant repressor in the presence of both endogenous and functionally redundant transcription factors for a target gene (Takase et al., 2007). This strategy has been shown to be valuable for obtaining loss-of-function phenotypes (Hiratsu et al., 2003; Matsui et al., 2004; Takase et al., 2007), and was therefore used in this study. More than 10 independent transgenic lines that express TAGL1 as a dominant repressor (termed TAGL1–SRDX) under the control of the tomato ethylene- and ripening-induced E8 gene promoter were generated. Mature fruit of transgenic plants expressing TAGL1–SRDX did not turn red upon ripening and exhibited an orange colour (Figure 3a). Expression analysis of the TAGL1–SRDX transcript showed high expression in the transgenic fruit (data not shown), which also exhibited significant down-regulation of the endogenous TAGL1 transcript (Figure 3b).
To obtain a better insight into the ripening-associated phenotype of the transgenic TAGL1–SRDX fruit, we analysed the levels of various isoprenoids (i.e. carotenoids, chlorophylls and tocopherols) at the Or stage (Figure 3c,d). Among the carotenoids, lycopene and two of its derivatives, phytofluene and phytoene, were reduced to trace levels in the transgenic fruit. In contrast, the levels of chlorophyll a and b, and their degradation product pheophytin a, were significantly increased in the TAGL1–SRDX-expressing fruit.
To determine whether the phenotype observed in the TAGL1–SRDX-expressing fruit was the result of altered climacteric ripening, we measured ethylene and CO2 (an indicator for respiration) emission from these fruit after the Br stage (Figures 3e and S2). The results showed that TAGL1–SRDX-expressing fruit do not show the increases in both ethylene and respiration (CO2) levels that are typical of WT fruit shortly after the Br stage.
Transcriptome analysis detects down-regulated expression of a large set of ripening-associated transcripts in TAGL1–SRDX-expressing fruit
Microarray analysis was performed in order to examine the effect on gene expression in TAGL1–SRDX-expressing fruit (Br stage). Compared to WT, 37 and 21 genes showed significant down- or up-regulated expression, respectively, in TAGL1–SRDX-expressing fruit (Table S1). The down-regulated transcripts in TAGL1–SRDX-expressing fruit were enriched (26 in total, approximately 70%) in genes previously found to be related to fruit ripening in tomato (or detected as exhibiting ripening-regulated expression in publicly available array data; Table 1).
|Genbank ID||Putative protein||Possible function||Fold changeb||Reference|
|BM413158||Anthranilate synthase (ASA)||Tryptophan biosynthesis||−4.3||Mintz-Oron et al., 2008|
|X71900.1||Histidine decarboxylase (HDC)||Histidine metabolism||−2.7||Picton et al., 1993|
|AW223528a||Phytoene synthase 1 (PSY1)||Carotenoid biosynthesis||−2.4||Bartley et al., 1992|
|AF020390.2||β-galactosidase II (TBG4)||Cell-wall metabolism||−4.4||Smith et al., 1998|
|AY034075.1||Mannan endo-1,4-β-mannosidase (MAN4)||Cell-wall metabolism||−3.1||Carrington et al., 2002|
|AF154421.1||β-galactosidase (TBG3)||Cell-wall metabolism||−2.1||Smith and Gross, 2000|
|BT014190.1a||Pectate lyase (PL)||Cell-wall metabolism||−9.6||Mintz-Oron et al., 2008|
|CN550618||Replication licensing factor||DNA replication||−2.4||Mintz-Oron et al., 2008|
|M63490.1a||1-aminocyclopropane-1-carboxylate synthase (ACS4)||Ethylene biosynthesis||−7.2||Lincoln et al., 1993|
|AY077626.1a||Ethylene response factor 1 (ERF1)||Ethylene signal transduction||−2.7||Li et al., 2007|
|BG123587||β-ketoacyl CoA synthase (CER6)||Fatty acid biosynthesis||−2.5||Leide et al., 2007|
|BT014299.1||ULTRAPETALA1 (ULT1)||Floral determination||−2.0||Mintz-Oron et al., 2008|
|BE437087||l-allo-threonine aldolase||Glycine biosynthesis||−2.1||Mintz-Oron et al., 2008|
|CD002771||Peroxiredoxin||Redox signaling||−2.6||Mintz-Oron et al., 2008|
|BG643920||Cinnamoyl CoA reductase (CCR)||Phenylpropanoid biosynthesis||−3.9||Mintz-Oron et al., 2008|
|BT013266.1||Dehydroquinate dehydratase/shikimate dehydrogenase (DHQD3)||AAA biosynthesis||−3.7||Mintz-Oron et al., 2008|
|AW223174||Phosphoenolpyruvate carboxylase 1 (PEPC1)||Replenishment of TCA cycle||−2.1||Mintz-Oron et al., 2008|
|AY187634.1||Phosphoenolpyruvate carboxylase kinase 2 (PPCK2)||Replenishment of TCA cycle||−5.1||Marsh et al., 2003|
|BI207393||Isocitrate dehydrogenase (IDH)||TCA cycle||−2.8||Mintz-Oron et al., 2008|
|BG627658||High leaf temperature 1||Regulation of stomata movement||−3.7||Mintz-Oron et al., 2008|
|BM535639||Triacylglycerol lipase||Lipid metabolism||−2.3||Alba et al., 2005|
|BT012806.1||Rab GTPase||Signal transduction||−2.4||Mintz-Oron et al., 2008|
|BM410663||Soluble starch synthase (SStS)||Starch biosynthesis||−2.2||Mintz-Oron et al., 2008|
|BG626714||NSHF||Unknown||−2.1||Mintz-Oron et al., 2008|
|AW738056||NSHF||Unknown||−2.1||Mintz-Oron et al., 2008|
|BI208323||WRKY transcription factor||Unknown||−2.0||Mintz-Oron et al., 2008|
Among the ripening-associated down-regulated transcripts in TAGL1–SRDX-expressing fruit were those encoding key enzymes in ethylene (ACS4) and carotenoid (PHYTOENE SYNTHASE 1, PSY1) biosynthesis, as well as cell-wall enzymes (β-GALACTOSIDASE II isoforms 1 and 2 and MANNAN ENDO-1,4-β-MANNOSIDASE). Expression of three genes putatively encoding enzymes associated with the activity of the TCA cycle (ISOCITRATE DEHYDROGENASE, PHOSPHOENOLPYRUVATE CARBOXYLASE 1 and its phosphorylating enzyme PHOSPHOENOLPYRUVATE CARBOXYLASE KINASE 2) was reduced in the TAGL1–SRDX-expressing fruit. ETHYLENE RESPONSE FACTOR 1 (ERF1), which putatively takes part in the ethylene signal transduction during tomato fruit ripening, also exhibited reduced transcript levels.
We performed quantitative real-time PCR in order to corroborate the array data and evaluate the expression of additional genes associated with carotenoids (Figure 4a), ethylene (Figure 4b) and other ripening-related processes (Figure 4c). The expression of two out of 10 examined carotenoid pathway genes was significantly altered. These included PSY1, which was down-regulated in TAGL1–SRDX-expressing fruit, and LCY-e, which was up-regulated. Among the ethylene-related genes, expression of ACS4 and ERF1 was significantly reduced in TAGL1–SRDX-expressing fruit, as revealed by the array results. The expression of ACS2 was also significantly down-regulated in TAGL1–SRDX-expressing fruit. However, no significant alteration in gene expression was observed in the case of ACO1 and S-adenosyl methionine synthase 1 (SAMS1), both of which are involved in ethylene biosynthesis.
Among the ripening-related genes, no significant alteration in transcript levels was detected for the RIN, TDR8, ACID INVERTASE (WIV-1) and LYPOXYGENASE (LOXC) genes. However, PECTATE LYASE (PL), which is associated with fruit softening (Marin-Rodriguez et al., 2002), was strongly down-regulated.
TAGL1 over-expression induces swelling and ripening of sepals
TAGL1 was subsequently over-expressed under the control of the 35S CaMV promoter in tomato (TAGL1oe lines). The increased expression of TAGL1 resulted in sepal swelling, yellowing and the appearance of red regions, indicating the presence of lycopene (Figures 5e, S3c,e,f and S7). HPLC analysis of TAGL1oe ripe fruit sepals confirmed the presence of the red carotenoid, lycopene, which does not accumulate in WT sepals (Figure 6a). However, apart from lycopene, no alteration in the accumulation of other isoprenoids was detected (Figure S4). These results suggest that the yellowing of the sepals is not caused by accumulation of carotenoids. Tomato fruit, particularly its peel, typically accumulates high levels of the yellow flavonoid naringenin chalcone (NarCh). We therefore examined NarCh levels in TAGL1oe sepals, and detected a dramatic increase in its levels compared to WT sepals (Figure 6b).
Expression analysis of ripening-related genes was subsequently performed in young sepals of TAGL1oe fruit (IG stage; Figure 6c). Over-expression of TAGL1 had opposite effects on two key genes from the ethylene biosynthetic pathway. While ACS4 was significantly up-regulated, the ACO1 gene was significantly down-regulated in sepals of the transgenic plants. Expression of PSY1 was also significantly down-regulated in young sepals of the TAGL1oe fruit. In accordance with the detected increase in NarCh accumulation, TAGL1oe sepals showed a significant induction of CHALCONE SYNTHASE (CHS), which encodes the enzyme that catalyses biosynthesis of this flavonoid.
Effect of TAGL1 over-expression on sepals in the rin mutant background
To examine the effect of over-expressing TAGL1 on phenotypes observed in the rin mutant, we transformed rin plants with the same TAGL1 over-expression construct. This experiment was designed to provide evidence for processes that are RIN-dependent and those that can be executed by TAGL1 in the absence of RIN. TAGL1 over-expression was found to induce sepal swelling and ripening in the rin background (Figures 5k and S3d). NarCh levels in rin/TAGL1oe sepals showed a sharp increase compared to rin sepals (Figure 6d). The gene expression changes in the rin/TAGL1oe sepals resembled those detected in TAGL1oe, with ACS4 and CHS expression being induced and PSY1 expression being reduced (Figure 6e). However, no significant change in ACO1 expression was detected in the rin/TAGL1oe sepals.
HPLC analysis of rin/TAGL1oe sepals revealed a significant reduction in the levels of all the isoprenoids examined (i.e. neoxanthin, α-tocopherol, violaxanthin, chlorophyll a and b, lutein, β-carotene and zeaxanthin) (Figure 6f). However, we did not detect the lycopene-accumulating regions in rin/TAGL1oe sepals that were observed in TAGL1oe sepals (Figures 5e and S3c,e,f), suggesting that carotenoid accumulation could not be induced by TAGL1 independently of RIN activity.
Effect of TAGL1 over-expression on fruit in the wild-type and rin mutant backgrounds
Fruit pigmentation in the TAGL1oe plants appeared to be more intense in comparison with WT fruit at the same developmental stage (Figure 5d,e). HPLC analysis indicated that ripe TAGL1oe fruit showed a mild increase in lycopene levels (n = 3, P = 0.055) compared to those of the WT (Figure 7a). In addition, TAGL1oe fruit showed a significant increase in phytoene and phytofluene levels (Figure 7b). This indicated that TAGL1 over-expression induces part of the fruit carotenoid biosynthesis pathway.
We also examined the fruit of plants over-expressing TAGL1 in the rin mutant background, in which chlorophylls are normally accumulated due to inhibition of ripening. Isoprenoid analysis in ripe rin/TAGL1oe fruit revealed reduced accumulation of chlorophylls a and b and lutein, but no accumulation of lycopene (Figure 7c). Thus, TAGL1 over-expression can induce chlorophyll breakdown in the rin mutant background. We also noted that the yellow colour of mature rin/TAGL1oe fruit was more intense than that of a typical rin fruit (Figure 5j,k). As we did not detect increased accumulation of NarCh in the rin/TAGL1oe fruit (in contrast to sepals; data not shown), this suggests that the degradation of chlorophylls (Figure 7c) exposed the yellow colour of the rin peel.
TAGL1 co-suppression inhibits ripening and alters fruit isoprenoid levels
Several TAGL1oe and rin/TAGL1oe lines showed a different fruit phenotype compared to the typical over-expressing ones, suggesting the possibility of TAGL1 co-suppression (TAGL1co-sup and rin/TAGL1co-sup lines). This was confirmed by TAGL1 expression analysis in fruit (Figure S5). Fruit of TAGL1co-sup plants were dark green and firm at an early stage of development (Figure 5c). Later in development (ripe stage), TAGL1co-sup fruit showed patchy yellow regions on a red background, suggesting that only these yellow parts exhibited co-suppression (Figure 5f). Sepals of TAGL1co-sup fruit appeared normal, but stayed green and firm after fruit ripening (Figure 5f). HPLC analysis of TAGL1co-sup ripe fruit revealed a significant increase in α-tocopherol, lutein and β-carotene, as well as phytoene and phytofluene levels (Figure 7b). Lycopene levels did not change in this fruit.
The rin/TAGL1co-sup lines were dark green compared to the rin fruit (at the MG stage) (Figure 5i,g), and resembled the fruit of co-suppression lines in the WT background (Figure 5c). Upon maturation, rin/TAGL1co-sup fruit showed a green–yellow colour, had a thin pericarp and remained small in size (Figure 5l). Isoprenoid analysis showed a significant increase in the levels of chlorophyll b, α-tocopherol and lutein in the rin/TAGL1co-sup ripe fruit compared to control rin fruit (Figure 7c).
TAGL1 activates the ACS2 promoter in a transient expression system
The down-regulation of both ACS2 and ACS4 in the TAGL1–SRDX-expressing fruit suggested that TAGL1 might induce ripening through direct activation of this pair of ethylene biosynthesis genes. We therefore used a luciferase-based transient expression system (Hellens et al., 2005) to evaluate activation of the ACS gene promoter regions by the TAGL1 transcription factor (Figure 8), and found that TAGL1 is able to activate the promoter of ACS2 but not that of ACS4.
- Top of page
- Experimental procedures
- Supporting Information
TAGL1 participates in the developmental regulation of tomato fruit ripening
The gaseous hormone ethylene is a major factor in climacteric fruit ripening. Switching on the ethylene biosynthetic pathway in a temporal and spatial manner is crucial for initiating the ripening process during climacteric fruit development. Several transcription factors belonging to various gene families have been proposed to link developmental programs and ethylene biosynthesis in fruit. Characterization of plants with various mutations of these regulators indicated that additional factors are required for the full execution of climacteric ripening (Vrebalov et al., 2002; Giovannoni et al., 2004; Manning et al., 2006). Moreover, some of these proteins, for example those belonging to the MADS domain family, function as components of larger multimeric complexes. In this study, we have identified the TOMATO AGAMOUS-LIKE 1 MADS box protein as an additional element of the regulatory network that mediates between fruit development and activation of ethylene biosynthesis for triggering ripening.
Several lines of evidence confirm the role of TAGL1 in the ripening process. The most significant of these are the effects of its modification on the typical metabolism and gene expression associated with ripening. For example, over-expressing TAGL1 as a chimeric repressor resulted in a dramatic decrease in the levels of several carotenoids, including phytoene, phytofluene, lycopene and two of its derivatives. The same fruit also contained high levels of chlorophylls (suggesting decelerated chlorophyll degradation) and did not show climacteric elevated ethylene synthesis. Approximately 70% of the genes that showed reduced expression in these fruit were associated with ripening. The putative functions of the down-regulated genes indicated a major effect on cell-wall metabolism associated with fruit softening, on ethylene biosynthesis and signalling (ACS2, ACS4 and ERF1), and on biosynthesis of secondary metabolites and their central/primary metabolite precursors.
The association between ethylene biosynthesis during ripening and TAGL1 activity
Direct activation of key genes in the ethylene biosynthetic pathway is a straightforward route for inducing ethylene production and triggering expression of the downstream ethylene-dependent ripening genes. Assays using the ACS2 and ACO1 gene promoters showed that they were bound by the RIN and HB-1 proteins, respectively (Ito et al., 2008; Lin et al., 2008). Down-regulated expression of ACS2 and ACS4 in TAGL1–SRDX-expressing fruit and the absence of the ripening ethylene peak suggested that TAGL1 exerts its effect on ripening (or at least part of it) by regulating ACS expression. ACO1 transcript levels were not altered in TAGL1–SRDX-expressing fruit, and are therefore not likely to be controlled by TAGL1.
Using transient expression assays in Nicotiana benthamiana leaves, we examined the capacity of TAGL1 to activate the promoters of ACS2 and ACS4. This assay showed that TAGL1 could activate the promoter of ACS2 but not that of ACS4. It could be that the significant changes in ACS4 expression in both the TAGL1–SRDX-expressing fruit and the swollen/ripening sepals (in both the WT and rin backgrounds) resulted from feedback prompted by the changes in ethylene levels in these two organs. Nevertheless, due to the limitation of this transient assay (a heterologous system), the possibility cannot be ruled out that ACS4 expression is directly controlled by TAGL1.
The association between TAGL1 and ethylene is similar to that observed for other known developmental ripening regulators in aspects additional to those described above. As detected previously in the case of RIN (Ito et al., 2008), although TAGL1 is likely to activate ethylene biosynthesis, its expression is not significantly induced by exposure of fruit to ethylene. Moreover, application of exogenous ethylene to the rin, nor and Cnr mutants does not restore fruit ripening (Herner and Sink, 1973; Tigchelaar et al., 1978; Thompson et al., 1999), and the same result was observed here when TAGL1–SRDX-expressing fruit were exposed to ethylene (data not shown). This indicated that TAGL1 is likely to control both ethylene-dependent and -independent ripening pathways. Adams-Phillips et al. (2004) suggested that the latter mode of control might represent conserved mechanisms of ripening between climacteric and non-climacteric fruit. However, TAGL1 was responsive to the ethylene perception inhibitor 1-MCP, as its expression was induced in MG stage fruit treated with 1-MCP.
The first target genes identified for plant MADS box transcription factors, e.g. DEFIECIENS, GLOBOSA, PISTILLATA, AGAMOUS and APETALA3, were the MADS genes themselves (Schwarz-Sommer et al., 1992; Trobner et al., 1992; Jack et al., 1994; Chen et al., 2000; Gomez-Mena et al., 2005). Such positive auto-regulatory loops are widespread mechanisms that maintain expression patterns of genes (de Folter and Angenent, 2006). Analysis of the TAGL1–SRDX-expressing fruit showed that the level of endogenous TAGL1 transcript was significantly reduced. This suggested that TAGL1 might bind and activate its own promoter, and therefore down-regulates its own transcript when over-expressed as a chimeric repressor.
TAGL1 and RIN may interact in fruit to activate ethylene biosynthesis but have separate roles in the regulation of other ripening processes
In order to position the TAGL1 protein in the tomato ripening regulatory network, we examined its relationship to the RIN protein. Examination of RIN transcripts in the TAGL1–SRDX-expressing fruit and TAGL1 expression in the rin mutant background suggested that they do not regulate one another. The similar expression pattern of both genes during fruit development and their association with the ACS2 promoter supports the possibility of interaction between these two MADS box proteins through cooperative binding of the ACS2 gene upstream region. Further support for such putative interaction was provided recently by a two-hybrid assay in yeast (Leseberg et al., 2008).
The rin mutant displays strong inhibition of ripening, including altered carotenoid profile (primarily decreased lycopene levels), reduced fruit softening and flavour production, suppressed climacteric respiration and lack of the characteristic ethylene production profile (Herner and Sink, 1973). Over-expression of TAGL1 in the rin mutant background provided us with additional information regarding the relationship between these two proteins, and the level and points of overlap in controlling ripening. The results of this experiment indicated that TAGL1 requires RIN activity for the induction of lycopene accumulation in fruit and in the swollen/ripening sepals.
In contrast to lycopene accumulation, chlorophyll breakdown during the transition from the MG to the Br stage of fruit development appears to be independent of RIN activity and could be activated by TAGL1 only. Over-expression of TAGL1 in the rin background resulted in reduced chlorophyll levels in both fruit and swollen/ripening sepals compared to their levels in rin plants. Moreover, fruit over-expressing TAGL1–SRDX accumulated higher levels of chlorophylls. In addition, the fruit of plants in which co-suppression occurred showed enhancement of the rin phenotype, with a darker green appearance (compared to the yellowish colour of rin fruit), higher chlorophyll levels, and smaller size. Interestingly, the fruit of rin/TAGL1oe plants were softer and their peel could be removed more easily than that of rin fruit, suggesting that the activity of genes encoding enzymes responsible for cell-wall degradation and fruit softening might also be regulated by TAGL1 in a RIN-independent manner.
The phenylpropanoid/flavonoid pathway and its regulation by TAGL1
The flavonoid NarCh provides a yellow appearance to tomato fruit peel, accumulating to approximately 1% of the cuticular layer in the Or stage before decreasing in the ripe fruit. Recently, Mintz-Oron et al. (2008) demonstrated co-expression of genes associated with the biosynthesis of NarCh and its precursors during the Br and Or stages of fruit development. While tomato fruit flavonoids have been investigated to a reasonable extent, knowledge regarding their association with the ripening program is limited. The accumulation of NarCh in the swollen/ripening sepals of fruit over-expressing TAGL1 was therefore intriguing. The accumulation of NarCh in the swollen/ripening sepals of rinTAGL1oe plants suggests that, at least in tomato sepals, the flavonoid pathway could be activated by TAGL1, with no involvement of RIN activity. This was further supported by the significant induction of CHS expression (encoding the enzyme catalysing NarCh biosynthesis) in the swollen/ripening sepals.
In contrast to Saladie et al. (2007), we detected a severe reduction in NarCh levels in rin fruit (Figure S6). Both Bargel and Neinhuis (2004) and Saladie et al. (2007) reported that isolated cuticles of the nor mutant display reduced pigmentation. Gene expression analysis of the rin mutant and treatment of tomato fruit with 1-MCP showed down-regulation of multiple genes associated with flavonoid biosynthesis (A. Aharoni and A. Adato, Department of Plant Sciences, Weizmann Institute of Science, Israel, unpublished results). In contrast to the results described above, we did not observe accumulation of NarCh in fruit of plants over-expressing TAGL1 or down-regulation of flavonoid biosynthesis genes in the array assay of TAGL1–SRDX-expressing fruit. It therefore remains to be examined in detail whether flavonoids are part of the biochemical processes controlled by TAGL1 and other developmental regulators, or alternatively are independent of climacteric ethylene.
Functional conservation of TAGL1 and its homologues in dry and fleshy fruit development
In Arabidopsis, a number of MADS box genes are required for proper development of the dehiscence zone and normal cell division, expansion and differentiation during silique morphogenesis (Becker and Theissen, 2003). Characterization of RIN and TAGL1 in this study suggests that the function of genes associated with dry fruit development evolved to regulate fleshy fruit formation and ripening. Thus, even though a basic similarity exists in the function of these homologous genes with regard to dry and fleshy fruit formation, it remains to be examined whether mutants such as SHP1/2 (the Arabidopsis homologues of TAGL1) could be fully complemented by TAGL1. Combined changes in the number, expression pattern and interaction of such key regulatory factors most likely facilitated the alteration of their function in fruit development.
Among fleshy fruit species, functional conservation between these regulatory genes is expected to be higher, as described recently by Tadiello et al. (2009) for the TAGL1 homologue of peach (i.e. PpPLENA). Ectopic expression of the ripening-regulated PpPLENA gene in tomato resulted in the transformation of sepals into fleshy and ripening carpel-like structures, and fruit exhibiting accelerated ripening. The authors suggested that PpPLENA interfered with the endogenous activity of TAGL1. This possibility is further corroborated by this work in which similar phenotypes were obtained upon modulation of TAGL1 expression.
The results from both studies implicate functioning of these two C-type MADS box proteins (i.e. TAGL1 and PpPLENA) not only in ripening, the ultimate step of carpel development, but also in the preceding phases in which the carpel is formed and expands to almost its final size. Swelling of the sepals (or calyx) by TAGL1 over-expression may involve interaction with JOINTLESS, the closest homologue of the MPF2 protein that is essential for the inflated calyx syndrome in Physalis pubescens (Mao et al., 2000; He and Saedler, 2005). Interestingly, Y2H assays recently showed that JOINTLESS may interact with TAGL1 and TAG1 (Leseberg et al., 2008), while over-expressing TAG1 results in sepal swelling and ripening in vivo (Bartley and Ishida, 2003). In both cases (i.e. TAG1 and TAGL1), sepal swelling and ‘ripening’ could be a result of either direct activation of the ripening process or homeotic conversion of the sepal to a carpeloid organ that expands and ripens. Future work may find that the interaction between these two MADS box factors is significant for tomato carpel expansion.
This study provides evidence for the involvement of TAGL1 in controlling tomato carpel ripening and perhaps its expansion earlier during its development. A scheme representing the proposed model for TAGL1 function in the network of regulatory factors controlling fruit ripening is presented in Figure 9. It is apparent that many additional genetic and environmental components of the comprehensive network that mediates fleshy fruit development and ripening await discovery.
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- Experimental procedures
- Supporting Information
Tomato plants (Solanum lycopersicum) cv. Ailsa Craig (AC) (obtained from the Tomato Genetics Resource Center; http://tgrc.ucdavis.edu) and cv. MicroTom (obtained from Avi Levy, Plant Sciences Department, Weizmann Institute of Science, Isreal) were grown in a climate-controlled greenhouse at 24°C during the day and 18°C during the night, with natural light. The fruit stages used were immature green (IG), mature green (MG), breaker (Br), orange (Or) and ripe, which were picked on average 10, 35, 38, 41 and 44 days post-anthesis, respectively. Sepals were collected based on the fruit ripening stage.
Generation of transgenic tomato plants
The 35S::TAGL1 construct was generated by cloning of the TAGL1 cDNA (using NcoI and BamHI sites) into pAA100-35S between the 35S CaMV promoter and a nopaline synthase (NOS) terminator, extracting the 35S::TAGL1::tNOS cassette (using PacI and AscI sites), and cloning into pBIN-PLUS (van Engelen et al., 1995). A dominant repressor construct was created by generating a translational fusion between the EAR repression domain (SRDX; Hiratsu et al., 2003) and the 3′ end of the TAGL1 cDNA, introducing TAGL1–SRDX into pAA100 (using NcoI and SacI sites) containing the E8 promoter (the 35S CaMV in pAA100 was replaced previously by the E8 promoter through BamHI and NcoI cloning), and transfer of the E8::TAGL1-SRDX cassette to pBIN-PLUS (using PacI and AscI sites). Constructs were transformed into cv. MicroTom as described by Meissner et al. (1997, 2000). Oligonucleotides used in this study as listed in Table S2.
Virus-induced gene silencing (VIGS)
ESTs putatively corresponding to 114 transcription factors that were found to be differentially expressed during tomato fruit maturation were generously provided by the Tomato Molecular Resource Distribution Center (Boyce Thompson Institute, Cornell University, Ithaca, NY). ESTs were individually amplified, cloned (AscI and NotI) into pENTR/D-TOPO (Invitrogen, http://www.invitrogen.com/) and introduced into pTRV2-AttR1-AttR2 (Liu et al., 2002) using the Gateway LR clonase enzyme kit (Invitrogen). The pTRV2-AttR1-AttR2 plasmid was transformed into Escherichia coli, and the insertion sequence was verified, and subsequently transferred to Agrobacterium tumefaciens strain AGLO. A PHYTOENE DESATURASE (PDS) gene fragment was introduced into pTRV2-AttR1-AttR2 to serve as a positive control. For plant inoculation, Agrobacterium containing pTRV1 and pTRV2 (empty or containing the insert) was grown as described by Liu et al. (2002), and when the bacteria reached an absorbance of 1, they were mixed in a 1:1 ratio, shaken for 4 h, and concentrated to an absorbance of 10, before inoculation of 3-week-old seedlings by stabbing using a wooden toothpick in three or four places along the stem. Fruit were examined visually several times during ripening, and positive clones were re-tested three times.
Ethylene and CO2 measurements
Br stage fruit were kept for 1 day in 250 ml flasks at room temperature (pool of 3–7 fruit in each of the three biological replicates), flasks were sealed for 4 h, ethylene and CO2 were measured in the headspace by sampling using a syringe through a septum in the flask lid), and the flasks were left open for 20 h each day (11 days). Measurements were performed as described by Fallik et al. (2003) with slight modifications (for details, see Appendix S1).
Ethylene and 1-MCP treatments
Fruit (cv. Ailsa Craig) at the MG, Br and Or stages were incubated with 1 ppm of 1-MCP for 19 h, moved to open air for 24 h, and subsequently frozen. MG fruit (cv. MicroTom) were incubated in 40 ppm ethylene for 16 h, followed by 8 h of aeration at room temperature before snap freezing. Control fruit were incubated in air instead of ethylene or 1-MCP.
Isoprenoid and flavonoid extraction and analyses
Isoprenoid extraction was performed as described by Bino et al. (2005). Analysis was performed using an HPLC-PDA detector (Waters, http://www.waters.com) and an YMC C30 column (YMC Co. Ltd., http://www.ymc.co.jp/en/) as described by Fraser et al. (2000). Flavonoids were extracted and profiled by UPLC-QTOF-MS as described previously by Mintz-Oron et al. (2008). Peak areas of the compounds were determined according to the spectral characteristic, wavelength and the retention time given in Table S4.
Microarray and bioinformatics analysis
Total RNA was extracted from three pools of five or six Br fruits using the hot phenol method (Verwoerd et al., 1989), and treated with DNase I (Sigma, http://www.sigmaaldrich.com/). Biotinylated cRNA was fragmented and hybridized to the Affymetrix GeneChip® Tomato Genome Array as described in the Affymetrix technical manual (available at http://www.affymetrix.com). Statistical analysis of microarray data was performed using the Partek® Genomics Suite (http://www.partek.com) and the robust microarray averaging (RMA) algorithm (Irizarry et al., 2003). Changes in expression level were determined by anova analysis. The false discovery rate (FDR) was applied to correct for multiple comparisons (Benjamini and Hochberg, 1995). Differentially expressed genes were chosen based on an FDR < 0.15 and a twofold change between genotypes and signal above background in at least one microarray. Functional annotation analysis was performed manually using publicly available databases.
Quantitative real-time PCR
RNA isolation from fruit (without placenta and seeds) was performed by the hot phenol method (Verwoerd et al., 1989), from seeds (cleaned of gel) as described by Ruuska and Ohlrogge (2001), and from all other tissues by the Trizol method (Sigma). DNase I-treated RNA was reverse-transcribed using a high-capacity cDNA reverse transcription kit (Applied Biosystems, http://www.appliedbiosystems.com/) and cDNA was used for real-time PCR analysis performed as described by Mintz-Oron et al. (2008). Gene-specific oligonucleotides were designed using Primer Express 2 software (Applied Biosystems). The CAC gene (Exposito-Rodriguez et al., 2008) was used as an endogenous control.
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- Experimental procedures
- Supporting Information
We thank Danny Gamrasni (Fruit Storage Research Laboratory, Kiryat Shmona, Israel) for the 1-MCP-treated fruit; Elazar Fallik and Sharon Alkalai-Tuvia (Department of Postharvest Science, Agricultural Research Organization, Volcani Center, Israel) for ethylene measurements; Ester Feldmesser and Dena Leshkowitz (Bioinformatics and Biological Computing Unit, Weizmann Institute of Science, Israel) for array data analysis; James Giovannoni and Ruth White – Tomato Molecular Resource Distribution Center (Boyce Thompson Institute, Cornell University, NY), for the EST clones; Savithramma Dinesh-Kumar (Molecular, Cellular & Developmental Biology, Yale University, CT) for the pTRV vectors; Alexander Vainstein and Marianna Ovadis (The Robert H. Smith Institute of Plant Sciences and Genetics in Agriculture, The Hebrew University of Jerusalem, Israel) for the help with VIGS; Rivka Barg (Department of Vegetable Research, Agricultural Research Organization, Volcani Center, Israel) for the E8 promoter; Roger Hellens (HortResearch, Mt Albert Research Centre, New Zealand) for the transient assay vector, and Arie Tishbee and Riri Kramer (Department of Organic Chemistry, Weizmann Institute of Science, Israel) for help with UPLC–QTOF–MS analysis. We also thank Avital Adato for critical reading of the manuscript and fruitful discussions. A.A. is the incumbent of the Adolpho and Evelyn Blum Career Development Chair of Cancer Research. The work in the A.A. laboratory was supported by the Minerva foundation and the Benoziyo Institute.
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- Experimental procedures
- Supporting Information
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- Experimental procedures
- Supporting Information
Figure S1. Virus Induced Gene Silencing (VIGS) of TAG1 resulted in yellow-orange sectors upon fruit maturation. (a) Ripe fruit of a plant infected with a vector containing the full TAG1 (b) Ripe fruit of plant infected with vector carrying a full TAG1 sequence fragment. (c) Ripe fruit of plant infected with the positive control vector, carrying a specific fragment of the PHYTOENE DESATURASE (PDS).
Figure S2. Overexpression of TAGL1 as a chimeric repressor (TAGL1-SRDX) resulted in a reduction in CO2 emission from fruit of transgenic plants. CO2 emission from TAGL1-SRDX and wild type (wt) fruit was measured as described in Experimental Procedures. Asterisk indicates p-value <0.05 (Stuudent’s t-test; n = 3).
Figure S3.TAGL1 overexpression in fruit induces inflation and ripening of sepals. (a) Ripe wild type fruit. (b) Ripe rin fruit. (c) TAGL1 overexpression (TAGL1oe) on wt background results in inflated sepals with reddish regions of lycopene accumulation (indicated by an arrow; see Figure 6a) (d) rinTAGL1oe (rin mutant background) results in inflated sepals with yellow regions of naringenin chalcone accumulation (indicated by an arrow; see Figure 6d). (e) and (f) Ripe fruits of two F1 segregating lines derived from the cross between rinTAGL1oe and wild type.
Figure S4. Overexpression of TAGL1 in the wt background has no effect on the levels of most isoprenoids in sepals. The levels of isoprenoids in sepals of TAGL1oe compared to wt stay unaltered, except for lycopene (see Figure 6a). This might be related to the mild phenotype of the analysed sepals. Isoprenoids were extracted and measured as described in Experimental Procedures from pools of MicroTom Ripe fruit (n = 2–7). Compounds were identified using UV/visible spectra of standards and known retention time.
Figure S5.TAGL1 relative transcript levels in wt, rin mutant and transgenic lines in the rin background. TAGL1 transcript levels are significantly altered in fruit (rin background; Br) of transgenic lines demonstrating overexpression and co-suppression phenotypes (see Figure 6.). Similar TAGL1 expression levels were observed in Br fruits of wt and rin in the cv. MicroTom background.
Figure S6. Levels of naringenin chalcone (NarCh) in the peel of wt and rin ripe fruit. The levels of naringenin chalcone are significantly higher in the peel of wt fruit compared to the peel of rin fruit.
Figure S7. Sepals of TAGL1 overexpressing lines remain fused all along flowering. (a) Wild type flowers. (b) The rin mutant flowers. (c) The TAGL1oe flowers. (d) The rinTAGL1oe flowers. Fused sepals in transgenic plants are indicated with an arrow.
Table S1. List of genes showing altered expression in TAGL1-SRDX breaker fruit.
Table S2. Oligonucleotides that were used in this study.
Table S3. Full names and identifiers of protein sequences used in the phylogenetic analysis.
Table S4. Isoprenoids detected by HPLC analysis.
Appendix S1. Supplementary experimental procedures.
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