A Non-Climacteric Fruit Gene CaMADS-RIN Regulates Fruit Ripening and Ethylene Biosynthesis in Climacteric Fruit

MADS-box genes have been reported to play a major role in the molecular circuit of developmental regulation. Especially, SEPALLATA (SEP) group genes play a central role in the developmental regulation of ripening in both climacteric and non-climacteric fruits. However, the mechanisms underlying the regulation of SEP genes to non-climacteric fruits ripening are still unclear. Here a SEP gene of pepper, CaMADS-RIN, has been cloned and exhibited elevated expression at the onset of ripening of pepper. To further explore the function of CaMADS-RIN, an overexpressed construct was created and transformed into ripening inhibitor (rin) mutant tomato plants. Broad ripening phenotypes were observed in CaMADS-RIN overexpressed rin fruits. The accumulation of carotenoid and expression of PDS and ZDS were enhanced in overexpressed fruits compared with rin mutant. The transcripts of cell wall metabolism genes (PG, EXP1 and TBG4) and lipoxygenase genes (TomloxB and TomloxC) accumulated more abundant compared to rin mutant. Besides, both ethylene-dependent genes including ACS2, ACO1, E4 and E8 and ethylene-independent genes such as HDC and Nor were also up-regulated in transgenic fruits at different levels. Moreover, transgenic fruits showed approximately 1–3 times increase in ethylene production compared with rin mutant fruits. Yeast two-hybrid screen results indicated that CaMADS-RIN could interact with TAGL1, FUL1 and itself respectively as SlMADS-RIN did in vitro. These results suggest that CaMADS-RIN affects fruit ripening of tomato both in ethylene-dependent and ethylene-independent aspects, which will provide a set of significant data to explore the role of SEP genes in ripening of non-climacteric fruits.


Introduction
The ripening of fleshy fruit is a developmental biochemical process including numerous metabolic changes.These changes not only make fruit assisting in seed dispersal, but also provide essential nutrition for human and animal diets [1][2][3].Classically, two classes of fruits have been recognized.In climacteric fruits, such as tomato (Solanum lycopersicum) and banana (Musa acuminata), ethylene synthesis and respiration increased dramatically at the onset of ripening.While in non-climacteric fruits, such as strawberry (Fragaria 6 ananassa) and pepper (Capsicum annuum), these changes are not found [2].However, these two distinct programs usually result in similar ripening-related changes, including changes in color, flavor, aroma and nutrition [1,2].
In climacteric fruits, ethylene plays an important role in triggering the onset of ripening and is an essential factor for the ripening process [4,5].Both the functional ethylene synthesis and the ability of ethylene perception are necessary for fruit ripening.For ethylene synthesis, the expression of ACO1 (1-aminocyclopropane-1-carboxylic acid oxidase 1) and ACO3 (1-aminocyclopropane-1-carboxylic acid oxidase 3) are both significantly increased at the onset of fruit ripening [6].It has been revealed that ethylene production and fruit ripening are strongly inhibited in ACS2 (1-aminocyclopropane-1carboxylic acid synthase 2) RNAi transgenic tomato fruits [6].For ethylene perception and response, it is generally considered that E4 and E8 are two classical genes which are involved in fruit ripening.The promoter of E8 has been characterized and is widely used to drive the expression of exogenous genes in transgenic tomato fruits [7][8][9].
Tomato is generally considered to be a model plant for studying climacteric fruit ripening.To date, a wide range of studies have been performed to uncover the mechanism of fruit ripening of tomato, and a cascade of transcription regulators acting upstream of the ethylene pathway has been revealed [2,10].In recent years, MADS-box genes have been reported to play a major role in the molecular circuit of developmental regulation [2,[11][12][13][14].Several MADS-box genes have been identified and demonstrated to be involved in fruit development in tomato fruit [10,15].The antisense suppression of TAGL1 (TOMATO AGAMOUS-LIKE 1) results in ripening inhibition and pericarp thickness reduction [13].FUL1 and FUL2 are orthologs of Arabidopsis FUL (FRUIT-FULL) gene,both of which are related to fruit ripening [15,16].
And recently we reported a SEP group MADS-box gene, SlMADS1 which regulated fruit ripening as an inhibitor [17].
In non-climacteric fruits, ripening is thought to be ethylene independent [2].However, ethylene may also play roles in ripening of non-climacteric fruits.For instance, grapes have been reported to contain a functional network of ethylene signaling at the onset of ripening [18].A transient increase of endogenous ethylene production occurs before veraison of grape, and during this stage diameter, acidity and anthocyanin change expeditiously [18].It has also been reported that the expression of ACO1 in pineapple is induced in ripening fruit tissue [19].Furthermore, as well as what occurs during climacteric fruit ripening, there is an increasing synthesis of receptors (i.e.FaETR1 and FaERS1) concomitant with the increased synthesis of ethylene in strawberries [20].Nevertheless, to date, the molecular regulation of ripening in non-climacteric fruits and the relationship between ethylene and non-climacteric fruits is still unclear.
Prior studies indicated that SEP genes played a central role in the developmental regulation of ripening in both climacteric and non-climacteric fruits [21].The regulation of SEP genes to the ripening of climacteric fruits is now well established.SlMADS-RIN is a typical SEP group gene which regulates tomato ripening, including both ethylene-dependent and ethylene-independent ripening pathways [12].To date, the transcriptional cascade downstream from SlMADS-RIN has been well researched.SlMADS-RIN has been reported to control fruit softening, carotenoid accumulation, ethylene production and ethylene perception during ripening [22][23][24].Besides, SlMADS-RIN interacts with other MADS-box proteins such as TAGL1, FUL1 and FUL2 which involved in fruit ripening, in vitro [16,25].In addition, four SEP group genes which are highly expressed in fruit have been cloned in banana and MaMADS2 was reported to act in the pulp upstream of the increase in ethylene production similarly to SlMADS-RIN [26].Suppression of the homeologous SEPAL-LATA1/2-like genes in the fleshy fruit apple (Malus 6 domestica) led to greatly reduced fruit flesh.Furthermore, like SlMADS-RIN gene in tomato, MADS9 gene acts as a transcriptional activator of the ethylene biosynthesis enzyme, 1-aminocyclopropane-1-carboxylate (ACC) synthase 1 [27].Nevertheless, the mechanisms underlying the regulation of SEP genes to non-climacteric fruits ripening are still unclear.Therefore, in this study, a SEP gene which is a potential ortholog to SlMADS-RIN has been cloned from a non-climacteric fruit pepper (Capsicum annuum L. cv.Bukang).And its function in complementing the rin tomato mutant has been examined.

Molecular Characterization of CaMADS-RIN
The full-length cDNA of CaMADS-RIN was cloned previously by our laboratory from pepper and deposited into genbank (accession number: DQ999998).Gene sequence analysis showed that CaMADS-RIN contained an ORF of 732 bp, a 59-UTR (untranslated region) of 74 bp and a 39-UTR (untranslated region) of 236 bp.The predicted CaMADS-RIN protein had 243 amino acids with an estimated molecular mass of 28 kD.Alignment analysis of amino acid sequences of MADS-box genes exhibited that CaMADS-RIN had conserved MADS-box domains (MADS domain, I domain and K domain) and its C-terminal region was highly divergent from other MADS-box sequences.Additionally, phylogenetic analysis revealed that CaMADS-RIN belonged to the SEP clade and showed the highest similarity to SlMADS-RIN (Figure S1).

CaMADS-RIN Exhibited Elevated Expression at the Onset of Ripening of Pepper
Real-time PCR was performed for analysis the accumulation of CaMADS-RIN transcripts in roots, stems, leaves, flowers, and a series of stages of fruits to explore the expression profile of CaMADS-RIN in pepper.Low level expression of CaMADS-RIN was observed in roots, stems, leaves and flowers (Figure 1).For pepper fruits, the CaMADS-RIN expression was low or barely detectable in green fruits, while the transcript showed high expression at the onset of ripening (Figure 1).This expression pattern was similar to the previous reported SlMADS-RIN [12], and indicated that CaMADS-RIN might be involved in fruit ripening of pepper.

CaMADS-RIN Overexpressed rin Fruits Showed Ripening Phenotype
To gain further insight into the function of CaMADS-RIN, an overexpressed construct was created and transformed into rin mutant tomato plants via Agrobacterium tumefaciens-mediated T-DNA transfer.Five independent transgenic lines were produced and integration transgene was confirmed by PCR.Quantitative real-time PCR resulted that abundant CaMADS-RIN transcripts were observed in the transgenic lines, while no expression of CaMADS-RIN was detected in rin mutant (Figure 2a).The expression level of pepper and tomato MADS-RIN in wild type, rin mutant tomato and transgenic lines was also detected by a pair of conserved primers, CaSlRIN (RT)-F and CaSlRIN (RT)-R, which specially targeted to CaMADS-RIN, SlMADS-RIN and SlMADS-RIN mutant (Figure 2b).The results suggested that transgenic fruits showed approximately 8-fold increases in MADS-RIN expression compared to rin mutant and wild type.
As shown in Figure S2, no obvious changes of the roots, stems, leaves and flowers were detected in the transgenic plants compared with rin.The most striking phenotype of CaMADS-RIN overexpressed rin lines was that the fruit ripening could be partially restored in transgenic fruits (Figure 2c).Fruits had no color change at the onset of ripening in the rin mutant, while the CaMADS-RIN overexpressed rin fruits had an obvious color change and became yellow-orange at that time (Figure 2c).Flowers were The expression of CaMADS-RIN in Rt, roots; St, stems; Le, leaves; Fl, flowers; Sf, fruits of 1 cm; Bf, fruits of 6 cm; Of, orange fruits; Rf, red fruits.Expression was determined by Q-RT-PCR as relative quantification.Results are of a representative experiment, and are an average of three repetitions 6SD.doi:10.1371/journal.pone.0095559.g001tagged at anthesis, and the time to ripening from anthesis stage was measured for wild type, rin and the transgenic rin lines.It was observed that ripening time of transgenic rin tomato fruits was consistent with wild type, while accelerated about 10 d compared with rin mutant (Table 1).

Carotenoid Accumulation in Transgenic Fruits were Partially Restored
As shown in the Figure 2c, the CaMADS-RIN overexpressed rin lines developed yellow-orange ripening fruits.Carotenoids are the mainly accumulated color during tomato fruit ripening, thus the total carotenoids in transgenic and control fruits at stages of B (breaker), B+4 (four days after breaker) and B+7 (seven days after breaker) were extracted and determined.The results showed that the accumulation of carotenoid in transgenic lines was higher than rin mutant, while was just 30% of wild type tomato AC ++ (Figure 3a).In order to gain further insight into this phenotype, expression of several carotenoid biosynthesis genes were detected by real-time PCR.The results suggested that PDS (phytoene desaturase) and ZDS (zeta-carotene desaturase) were both up-regulated  in the overexpressed lines compared with rin mutant; the transcripts of PDS in B+4 and B+7 stage fruits and ZDS in B stage fruits accumulated even higher than wild type (Figure 3c and  d).However, for PSY1 (Phytone synthease 1), the rate-limiting enzyme for carotenoid biosynthesis in tomato, the expression was depressed in overexpressed lines compared with rin mutant and wild type (Figure 3b).

Expression of Cell Wall Metabolism-related Genes in
CaMADS-RIN Overexpressed rin Fruits During the ripening of fruits, the transgenic fruits became as soft as wild type, while the rin mutant fruits did not.Thus, the expression of a set of cell wall metabolism-related genes was examined by real-time PCR.The results showed that the critical determinant of cell wall metabolism, PG (polygalacturonase) [28] was dramatically up-regulated in transgenic fruits compared with rin, although it still did not restored to the level of wild type (Figure 4a).Another two genes, TBG4 (b-Galactosidase 4) [29] and EXP1 (a-Expansin 1) [30] showed significant elevation in ripening fruit of transgenic fruits compared to rin and nearly recovered to wild type level (Figure 4c, d).It was confirmed that CaMADS-RIN overexpression had induced cell wall metabolism in rin mutant.

Expression of Lipoxygenase Genes in CaMADS-RIN Overexpressed rin Fruits
In order to detect the effect of overexpressing CaMADS-RIN on flavor volatiles, two ripening-related genes, TomloxB and TomloxC, which encode lipoxygenase that transforms polyunsaturated fatty acids into hydroperoxides were detected.The results showed that both of their transcripts were markedly higher in CaMADS-RIN overexpressed rin fruits than that in rin mutant fruits (Figure 5a and  b).Furthermore, TomloxC even had higher expression level in transgenic fruits at B+4 and B+7 stages compared to wild type (Figure 5b).

Ethylene Production Ability and Ethylene Biosynthetic Genes were Partially Recovered in Transgenic Lines
It has been proven that ethylene is an essential factor in triggering the onset of ripening and in regulating the ripening process in climacteric fruits [4,5].However, in non-climacteric fruits the role of ethylene is still unclear.Thus, in order to investigate the relation between ethylene and CaMADS-RIN of a non-climacteric fruit pepper, we measured ethylene production in rin mutant, transgenic and AC ++ fruits.Although the ethylene production of transgenic lines were still less than AC ++ , transgenic fruits produced approximately 1-3 fold more ethylene than rin mutant during fruit ripening (Figure 6a).
To further characterize the molecular mechanism of the ethylene biosynthesis in CaMADS-RIN overexpressed rin tomatoes, a set of ethylene biosynthetic genes were detected in wild type AC ++ , rin mutant and transgenic tomato fruits.Compared with rin mutant, CaMADS-RIN overexpressed lines exhibited that two ethylene biosynthetic genes, ACS2 and ACO1 were dramatically up-regulated in B stage of fruits and slightly up-regulated in B+4 and B+7 stages of fruits (Figure 6b and c), suggesting that, like SlMADS-RIN, CaMADS-RIN could regulate ethylene production by impacting ethylene biosynthetic genes in tomato.

CaMADS-RIN Overexpressed rin Fruits
As shown above, ethylene producing ability was partially recovered in transgenic lines.And it's reported that a number of genes expression is influenced by ethylene levels.Among them we focused on two genes E4 and E8 and the expression of these genes were detected in wild type, rin and the transgenic lines.E4 was markedly increased in transgenic fruits at B and B+4 stages, and slightly induced at B+7 stage (Figure 6d).While the expression of E8 was significantly higher in all the stages of CaMADS-RIN overexpressed rin fruits than that in rin mutant fruits (Figure 6e).Compared with wild type, CaMADS-RIN overexpressed fruits displayed lower expression of both E4 and E8 at B stage, while E4 had higher expression level at B+4 and B+7 stages of ov-01 transgenic fruits (Figure 6).

Expression of Ethylene-independent Ripening Related Genes in CaMADS-RIN Overexpressed rin Fruits
Transcriptome analysis of promoters of differentially regulated genes have provided that SlMADS-RIN not only regulates ethylenedependent aspects but also impacts ethylene-independent aspects of ripening in tomato [31].In order to detect whether pepper MADS-RIN had the same role, we focused on a histidine metabolism gene, HDC which is insensitive to ethylene and regulated by SlMADS-RIN [32].The real-time PCR resulted that the transcripts of HDC accumulated much higher than rin, but was still not rescued to wild type level (Figure 7a).An ethyleneindependent ripening related transcript factor Nor, whose promoter is associated with SlMADS-RIN [33,34] was also detected in transgenic fruits.The results showed that Nor was markedly upregulated compared to both rin and wild type (Figure 7b).

CaMADS-RIN Exhibited the Same Interaction Mode as SLMADS-RIN In vitro
It is reported that hetero-or homo-dimers are often detected in MADS domain proteins [25].The tomato MADS-box protein SlMADS-RIN whose mutant is rin interacts with other MADS-box proteins such as TAGL1, FUL1 and FUL2 which are involved in fruit ripening, in vitro [16,25].To further test whether CaMADS-RIN could take the place of SlMADS-RIN in tomato, yeast twohybrid assay was performed.The open reading frame of CaMADS-RIN and SlMADS-RIN were amplified and cloned into pGBKT7 as the baits.Self-activation of pGBKT7-CaRIN and pGBKT7-SlRIN were tested and the results are minus (Figure 8a).While the open reading frames of TAGL1, FUL1, CaMADS-RIN and SlMADS-RIN were amplified and cloned into pGADT7 as the prey respectively.Figure 8b showed that SlMADS-RIN could interact with TAGL1, FUL1 and itself in vitro.And as expected, CaMADS-RIN exhibited the same interaction mode as SlMADS-RIN had (Figure 8).

CaMADS-RIN Overexpressed rin Fruits Exhibited Uncompleted Ripening Phenotype
In this study, we observed that CaMADS-RIN overexpressed rin fruits appeared yellow-orange (Figure 2c).The accumulation of carotenoid in transgenic lines was higher than that in rin mutant, but lower than that in wild type (Figure 3a).It's reported that the formation of carotenoids is a characteristic of both tomato and pepper fruits, although the other end products that form differ between fruits of the two species [35].Prior study showed that the expression of PSY1, PDS and ZDS was all reduced dramatically in rin mutant and the mutant fruits were green [12,34].In our transgenic fruits, PDS and ZDS expression was both restored or even higher than wild type, which may cause the slightly higher carotenoid accumulation.However, phytone synthease 1 catalyzes a highly influential step for total carotenoid synthesis [36,37].A mutation in PSY1 causes a yellow-fresh phenotype and an absence of carotenoids in ripe fruit [38,39].Our result of Figure S3 suggested that PSY1 had an analogous expression profile in pepper as in tomato, indicating that PSY1 has the same role in pepper as in tomato.For PSY1, a rate-limiting enzyme for carotenoid biosynthesis, its expression was depressed in overexpressed lines (Figure 3b), which might be responsible for the orange color of transgenic fruit.
In addition, compared with wild type, most of the cell wall metabolism and lipoxygenase genes displayed lower expression in transgenic fruits, although they all had higher expression level compared to rin (Figure 4 and 5).These results suggested that broad ripening phenotypes of rin mutant were not completely rescued by heterologously expressing CaMADS-RIN, which might be a consequence of reduced expression of CaMADS-RIN gene compared with wild type.Considered this, a pair of conserved primers which specially targeted to CaMADS-RIN, SlMADS-RIN and SlMADS-RIN mutant was designed to detect the expression level of MADS-RIN in wild type, rin mutant tomato and transgenic lines.The results showed that approximately 8-fold increases of MADS-RIN expression in transgenic fruits compared to rin mutant and wild type (Figure 2b), indicating that the partial ripening phenotype of transgenic lines were not a consequence of reduced Recently, two grapevine SEP genes, VviSEP3 and VviSEP4, are heterologously expressed in tomato rin mutant, which are also just capable of partially complementing the non-ripening phenotype of rin [40].Furthermore, heterologously expression of two banana SEP genes, MaMADS1 and MaMADS2, in rin tomato fruit could not complement ripening [26].These results indicate that though MADS-RINs in pepper, tomato or other species are highly homologous, there are some different special roles respectively in the regulation of fruit ripening.

CaMADS-RIN is Involved in Both Ethylene-dependent and Ethylene-independent Fruit Ripening
Ethylene plays an essential role during ripening, especially climacteric fruits ripening [5,6].The mechanism of ethylenerelated ripening in climacteric fruits to date has been well established [2,6,12].Both the normal function of ethylene biosynthesis and the ability of ethylene response are required for ripening process [2,6,12].The fruit of RNAi repression of ACS2 could not ripen normally [41].Previous studies also indicated that RNAi inhibition of ACO1 delays ripening of climacteric fruits [11,42,43].In our study, both of the two ethylene biosynthetic genes were induced significantly in transgenic fruits (Figure 6b and  c), indicating that CaMADS-RIN regulates ethylene biosynthesis.This result was further confirmed by the higher ethylene production of transgenic fruits.For ethylene response, E4 and E8 are two classical genes involved in fruit ripening [44].Our results showed that the transcripts of the two genes accumulated higher than rin mutant (Figure 6d and e), suggesting that besides the functional ethylene synthesis, the ability of ethylene perception and response are also regulated by CaMADS-RIN.
Limited information has been published on the mechanism of ethylene-independent fruit ripening.Primary metabolism genes are important members that belong to ethylene-independent group.Among them we focused on HDC, whose transcripts are not restored by treatment of fruit with ethylene but involved in fruit ripening [32].Its transcripts are not detected in tomato leaves and unripe fruit, accumulate during early fruit ripening and then decline [32].Our real-time PCR showed that HDC was significantly up-regulated in transgenic fruits (Figure 7a).Moreover, an ethylene-independent ripening-related transcriptional factor gene, Nor belonged to NAC family whose mutant exhibited non-ripening phenotypes [33] was also detected.The results showed that significant induction was detected in transgenic fruits compared with both rin and wild type (Figure 7b).In addition, broad ripening phenotypes including carotenoids biosynthesis (Figure 3), cell wall metabolism (Figure 4) and fatty acid-derived flavor compounds metabolism (Figure 5) were all regulated by CaMADS-RIN.These results suggest that CaMADS-RIN is involved in both ethylene-dependent and ethylene-independent fruit ripening.

CaMADS-RIN May Play an Important Role in Pepper Fruit Ripening
Prior studies have indicated that rin mutation displays enlarged sepals and inhibited fruit ripening.This mutant phenotype has been attributed to a function of two MADS-box transcriptional factors, SlMADS-RIN and SlMADS-MC.SlMADS-RIN regulates fruit ripening and SlMADS-MC involves in sepal development [12].The transcriptional cascade downstream from SlMADS-RIN has already been well researched at present.It has been revealed that SlMADS-RIN bound to the cis-element of ACS2 [24], then controlled fruit softening, carotenoid accumulation, ethylene production and ethylene perception [22,24].Additional ripening-related genes such as E4, E8, PG, EXP1 and TBG4 were also regulated by SlMADS-RIN directly [22].In our study, CaMADS-RIN overexpressed rin tomato exhibited ripening phenotype (Figure 2).The expression of most target genes of SlMADS-RIN was up-regulated in CaMADS-RIN overexpressed fruits compared with rin mutant (Figures 4, 5, 6, and 7).Additionally, the transgenic lines produced more ethylene than rin mutant (Figure 6a).Yeast two-hybrid assay displayed that CaMADS-RIN exhibited the same interaction mode as SlMADS-RIN in vitro (Figure 8).These results suggest that CaMADS-RIN plays a positive role in ethylene biosynthesis and fruit ripening of tomato.
Phylogenetic analysis suggested that CaMADS-RIN belonged to SEP clad (Figure S1).SEP genes not only have been reported to have a function on fruit ripening in climacteric fruits, but also play a central role in the developmental regulation of ripening in nonclimacteric fruits, such as FaMADS9 in strawberry [21].It is reported that FaMADS9 could lead to the inhibition of normal development and ripening in the petal, achene, and receptacle tissues [21].Besides, heterologously expressing a grapevine SEP gene, VviSEP4, was capable to partially complement the nonripening phenotype of the tomato rin mutant [40].In pepper, MADS-box genes also have been cloned and characterized to be involved in fruit ripening.Two pepper MADS-box genes, CaMADS1 and CaMADS6 were reported to play a regulatory role for flower and fruit development through interaction of the two genes products [45].Our results suggested that the expression pattern of CaMADS-RIN in pepper was similar to that of SlMADS-RIN, which expressed at the onset of ripening (Figure 1).Combining the prior researches and our results, it could be concluded that CaMADS-RIN commits its function in pepper fruit ripening, the same way as in tomato.Although transgenic pepper with reduced expression levels of CaMADS-RIN should be required to test this hypothesis, our study about CaMADS-RIN will supply a set of significant data for pepper fruit ripening study.Also with the assistant of this study, we can further have more robust conclusions and perform the studies with insight into the role of SEP genes in ripening of non-climacteric fruits and the relation between ethylene and non-climacteric fruits ripening.

Plant Materials and Treatments
In this experiment, rin mutant tomato (Solanum lycopersicon Mill.cv.ripening inhibitor) and wild type tomato AC ++ (Solanum lycopersicon Mill.cv.Ailsa Craig) were used.The plants were planted in greenhouse and watered daily.Transgenic cultures grew under standard greenhouse conditions (16 h-day/8 h-night cycle, 25/ 18uC day/night temperature, 80% humidity, and 250 mmol m 22 s 21 light intensity).Two generations of tomato plants were used in experiments.The plants of first generation (T0) came from tissue culture and plants of the second generation (T1) were from seedlings.Flowers were tagged at anthesis.The ripening stages of tomato fruits were divided according to days after anthesis (dpa) and fruit color.In wild type, IMG (Immature green) fruits were defined as 28 dpa.MG (Mature green) fruits were defined as 35 dpa and were characterized as being green and shiny with no obvious color change.At B (Breaker) stage, fruits color change from green to yellow.After breaker the fruit stages were divided into B+4 (4 days after Breaker) and B+7 (7 days after Breaker).All plant samples were immediately frozen with liquid nitrogen, mixed, and stored at -80uC until further use.

Phylogenetic Analysis
Full-length cDNA of CaMADS-RIN was cloned by screening a cDNA library using the tomato MADS-box gene SlMADS-RIN as a probe, and deposited into genbank (accession number: DQ999998).A phylogenetic tree was constructed with the sequence of CaMADS-RIN and the other 19 MADS-box genes by MEGA 3.1.The neighbor-joining method contains the following parameters: poisson model,pairwise deletion and bootstrap analysis of 1000 replicates.The numbers at the nodes indicate the bootstrap values.The bar at the bottom indicates the relative divergence of the sequences examined.

Complementation of rin Mutants
Full-length cDNA of CaMADS-RIN was amplified with primers CaRINov-F (59 CGG GAT CCA TGG GTA GAG GGA AAG TAG A 39) and Oligo d(T)18 (59 CCC GAG CTC TTT TTT TTT TTT TTT TTT 39) through high fidelity PCR (Prime STAR TM HS DNA polymerase, Takara, China).Then the amplified products were digested with BamH I and Sac I respectively, and linked into pBI121 plasmid at BamH I and Sac I restriction sites.
The generated binary plasmids were translated into Agrobacterium LBA4404 strain and introduced into rin tomato mutant by Agrobacterium-mediated transformation described previously [46].The transgenic plants were detected with primers NPTII-F (59 GAC AAT CGG CTG CTC TGA 39) and NPTII-R (59 AAC TCC AGC ATG AGA TCC 39).The positive transgenic plants were selected and used for subsequent experiments.

Quantitative Real-time PCR Analysis
Total RNA from pepper (Capsicum annuum L. cv.Bukang), rin and transgenic lines were extracted using Trizol (Invitrogen, USA) according to the manufacturer's instructions.Quantitative realtime PCR analysis was carried out using the CFX96 TM Real-Time System (C1000 TM Thermal Cycler).All reactions were performed using the SYBRH Premix Ex Taq II kit (TaKARA, China) in a 10 mL total sample volume (5.0mL 26SYBR Premix Ex Taq, 1.0 mL primers, 1.0 mL cDNA, 3.0mL ddH2O).To remove the effect of genomic DNA and the template from environment, NTC (no template control) and NRT (no reverse transcription control) were performed.Additionally, three replications for each sample were used and standard curves were run simultaneously.Tomato SlCAC gene (Table S1) and pepper b-actin gene (Table S1) were used as internal standard.The primers CaRIN (RT)-F and CaRIN (RT)-R (Table S1) were used to determine the expression level of CaMADS-RIN in pepper, rin mutant tomato and transgenic lines.The expression level of CaMADS-RIN in wild type, rin mutant tomato and transgenic lines were also detected by CaSlRIN(RT)-F and CaSlRIN(RT)-R which is a pair of special primer targeted to CaMADS-RIN, SlMADS-RIN and SlMADS-RIN mutant.Furthermore, the expression levels of fruit ripening and ethylene biosynthesis pathway genes, including E4 [44,47], E8 [48], PSY1, PDS, ZDS [36,[49][50][51], ACO1 and ACS2 [6,52], PG, EXP1 and TBG4 [28][29][30], TomloxB and TomloxC [46,52,53], HDC [32] were determined simultaneously.Primers were shown in table S1.

Carotenoid Extraction
A 1.0 g sample of each line was cut from pericarp in a 5 mm wide strip around the equator of B, B+4 and B+7 fruits, respectively.Then 10 mL of 60:40 (v/v) hexane-acetone was added respectively and total carotenoids of wild type (AC ++ ), rin mutant and transgenic lines fruits were extracted.The extract was centrifuged at 4000 g for 5 min and the absorbance of supernatant was measured at 450 nm.Carotenoid content was calculated with the following equations: total carotenoid mg mL 21 = 4*(OD 450)*10 mL/1 g [49,54].Three independent experiments were performed for each sample.

Ethylene Measurements
Fruits of rin mutant, AC ++ and transgenic fruits at B, B+4, B+7 stages were harvested and placed in open 100 mL jars for 3 h to minimize the effect of wound ethylene caused by picking.Jars were then sealed and incubated at room temperate for 24 h, 1 mL of headspace gas was injected into a Hewlett-Packard 5890 series gas chromatograph equipped with a flame ionization detector (FID).Samples were compared with reagent grade ethylene standards of known concentration and normalized for fruit weight [35].

Yeast Two-hybrid Assay
Yeast two-hybrid was performed using the MATCHMAKER TM GAL4 Two-Hybrid System III according to the manufacturer's protocol (Clontech).The open reading frame of SlMADS-RIN and CaMADS-RIN were amplified by PCR respectively with the primer pairs SlMADS-RIN(Y)-F (59 CCG GAA TTC ATG GGT AGA GGG AAA GTA GA 39) and SlMADS-RIN(Y)-R (59 CGC GGA TCC GTC AAA GCA TCC ATC CAG GT 39); SlMADS-RIN(Y)-F (59 CCG GAA TTC ATG GGT AGA GGG AAA GTAG 39) and SlMADS-RIN(Y)-R (59 CGC GGA TCC GTC AAA GCA TCC ATC CAG G 39).The PCR products were digested using EcoR I and Sal I and cloned into the EcoR I/Sal I site of the pGBKT7 bait vector to obtain the vector pGBKT7-SlRIN and pGBKT7-CaRIN.Then the vectors were translated into Y2HGold.The Y2HGold with baits were plated on SD medium lacking Trp (SDO) and SD medium lacking Trp, His, Ade (TDO) to test self-activation of pGBKT7-SlRIN and pGBKT7-CaRIN.In parallel, the open reading frame of SlTAGL1 and TFUL1 were also amplified by primers SlTAGL1 (Y)-F (59 CCG GAA TTC ATG GTT TTT CCT ATT AAT C 39) and SlTAGL1 (Y)-R (59 CGC GGA TCC GTC AGA CAA GCT GGA GAG G 39); SlFUL1 (Y)-F (59 CCG GAA TTC ATG GGA AGA GGA AGA GTC C 39) and SlFUL1 (Y)-R (59 CGC GGA TCC GTC ACA GTA TTA TTA GCT G 39).These products were cloned into the pGADT7 vector, and translated into Y187.In order to test whether SlRIN and CaRIN could interact with each other, these two genes were also cloned into the pGADT7 vector, and translated into Y187.Subsequently, Y2HGold with baits and Y187 with preys were cultured together respectively in 26YPDA medium for 24 h at 30uC.After that these cultures were cultured on SD medium lacking Trp, Leu (DDO) to select for diploids containing prey and bait vectors.After 2 to 5 days, fresh diploid cells were plated on SD medium lacking Trp, Leu, and His, Ade, with X-a-Gal (QDO/X) to judge the proteins interaction.Plates were incubated for 3 to 7 days at 30uC.An empty prey and bait vector were used as negative controls with each bait and prey construct, respectively.Meanwhile, positive control was transformed and cultured.The assays were repeated at least three times with fresh transformants.

Figure 1 .
Figure 1.Expression profile of CaMADS-RIN in tissues of pepper.The expression of CaMADS-RIN in Rt, roots; St, stems; Le, leaves; Fl, flowers; Sf, fruits of 1 cm; Bf, fruits of 6 cm; Of, orange fruits; Rf, red fruits.Expression was determined by Q-RT-PCR as relative quantification.Results are of a representative experiment, and are an average of three repetitions 6SD.doi:10.1371/journal.pone.0095559.g001

Figure 2 .
Figure 2. Heterologous expression of CaMADS-RIN in rin tomato fruit complemented ripening.(a).Expression of CaMADS-RIN in overexpressed lines, rin mutant and wild type.RNAs were extracted for qPCR assay from B fruits of overexpressed lines and rin mutant.Three replications for each sample were performed.(b).Expression of pepper and tomato MADS-RIN in overexpressed lines, rin mutant and wild type.RNAs were extracted for qPCR assay from B fruits of overexpressed lines, rin mutant and wild type.Three replications for each sample were performed.(c).Phenotype of transgenic fruits.Wild type AC ++ , rin mutant and transgenic fruits were tagged at anthesis and designated as breaker stage at the same age as wild type fruit which showed the first signs of color change.IMG, MG, B, B+4 and B+7 of rin tomato fruits, wild type fruits and CaMADS-RIN overexpressed rin fruits were shown.doi:10.1371/journal.pone.0095559.g002

Figure 6 .
Figure 6.Ethylene production and ethylene biosynthetic and responsive genes expression in control and CaMADS-RIN overexpressed lines.(a).Production of ethylene in control and transgenic lines.Fresh fruits of B, B+4 and B+7 were sealed in airtight vials and 1 mL of gas was sampled from the headspace after 24 h.Values represent means of at least three individual fruits.Vertical bars represent standard error.(b) and (c).Expression of ethylene biosynthetic genes, ACS2 (b) and ACO1 (c) in control and transgenic lines.RNAs were extracted for qPCR assay from B, B+4 and B+7 fruits of overexpressed lines, rin mutant and wild type.Three replications for each sample were performed.(d) and (e).Expression of ethylene responsive genes, E4 (d) and E8 (e) in overexpressed lines, rin mutant and wild type.RNAs were extracted for qPCR assay from B, B+4 and B+7 fruits of overexpressed lines, rin mutant and wild type.Three replications for each sample were performed.doi:10.1371/journal.pone.0095559.g006

Figure 7 .
Figure 7. Expression of ethylene-independent genes in overexpressed lines, rin mutants and wild type fruits.(a).Expression of a histidine metabolism gene HDC in overexpressed lines, rin mutants and wild type fruits.(b).Expression of Nor in overexpressed lines, rin mutants and wild type fruits.RNAs were extracted for qPCR assay from B, B+4 and B+7 fruits of overexpressed lines, rin mutant and wild type.Three replications for each sample were performed.doi:10.1371/journal.pone.0095559.g007