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Trong các tác nhân gây ra sư chín hóa sinh của thực vật, Ethylene được biết như một trong những nhân tố để bắt đầu, điều chỉnh và điều phối sự biểu hiện của hệ thống các Genes tham gia vào quá trình chín. Sự bùng nổ trong sản xuất ethylene là mốc quan trọng cho sự khởi đầu của chín của trái cây thời kỳ. Trong nghiên cứu này nhóm nghiên cứu đã cho thấy một kết quả kahr quan khi có thể kéo dài quá trình bản quản cà chua lên tớ 45 ngày mà không làm ảnh hưởng đến mùi vị và giá trị dinh dưỡng của cà chua.
Journal of Plant Physiology 170 (2013) 987–995 Contents lists available at SciVerse ScienceDirect Journal of Plant Physiology journal homepage: www.elsevier.com/locate/jplph Physiology Delayed ripening and improved fruit processing quality in tomato by RNAi-mediated silencing of three homologs of 1-aminopropane-1-carboxylate synthase gene Aarti Gupta a , Ram Krishna Pal b , Manchikatla Venkat Rajam a,∗ a b Plant Polyamine, Transgenic and RNAi Research Laboratory, Department of Genetics, University of Delhi South Campus, New Delhi 110021, India Division of Post-Harvest Technology, Indian Agricultural Research Institute, New Delhi 110012, India a r t i c l e i n f o Article history: Received October 2012 Received in revised form 18 February 2013 Accepted 18 February 2013 Available online 16 March 2013 Keywords: ACC synthase Delayed ripening Ethylene Fruit quality Polyamines a b s t r a c t The ripening hormone, ethylene is known to initiate, modulate and co-ordinate the expression of various genes involved in the ripening process The burst in ethylene production is the key event for the onset of ripening in climacteric fruits, including tomatoes Therefore ethylene is held accountable for the tons of post-harvest losses due to over-ripening and subsequently resulting in fruit rotting In the present investigation, delayed ripening tomatoes were generated by silencing three homologs of 1aminocyclopropane-1-carboxylate (ACC) synthase (ACS) gene during the course of ripening using RNAi technology The chimeric RNAi-ACS construct designed to target ACS homologs, effectively repressed the ethylene production in tomato fruits Fruits from such lines exhibited delayed ripening and extended shelf life for ∼45 days, with improved juice quality The ethylene suppression brought about compositional changes in these fruits by enhancing polyamine (PA) levels Further, decreased levels of ethylene in RNAi-ACS fruits has led to the altered levels of various ripening-specific transcripts, especially the up-regulation of PA biosynthesis and ascorbic acid (AsA) metabolism genes and down-regulation of cell wall hydrolyzing enzyme genes These results suggest that the down-regulation of ACS homologs using RNAi can be an effective approach for obtaining delayed ripening with longer shelf life and an enhanced processing quality of tomato fruits Also, the chimeric gene fusion can be used as an effective design for simultaneous silencing of more than one gene These observations would be useful in better understanding of the ethylene and PA signaling during fruit ripening and molecular mechanisms underlying the interaction of these two molecules in affecting fruit quality traits © 2013 Elsevier GmbH All rights reserved Introduction Tomato is one of the most valuable fruit crop across the world and is rich in minerals, fibers, vitamins and antioxidants (Rajam et al., 2007) It also serves as a model system for ripening related studies Tomato fruit ripening is initiated with climacteric burst of ethylene, which co-ordinates and regulates the expression of ripening-specific genes in fruit (Osorio et al., 2011) The biosynthesis of ethylene starts with S-adenosylmethionine (SAM), which Abbreviations: ACC, 1-aminocyclopropane-1-carboxylic acid; ACS, ACC synthase; ADC, arginine decarboxylase; NPT-II, neomycine phosphotransferase; ODC, ornithine decarboxylase; PA, polyamine; PLW, physiological loss of water; Put, putrescine; SAMDC, S-adenosylmethionine decarboxylase; Spd, spermidine; Spm, spermine; TAs, titratable acids; TSS, total soluble solids; WT, wild-type ∗ Corresponding author at: Department of Genetics, University of Delhi South Campus, Benito Juarez Road, New Delhi 110021, India Tel.: +91 11 24110866; fax: +91 11 24112437 E-mail address: rajam.mv@gmail.com (M.V Rajam) 0176-1617/$ – see front matter © 2013 Elsevier GmbH All rights reserved http://dx.doi.org/10.1016/j.jplph.2013.02.003 is converted into 1-aminocyclopropane-1-carboxylic acid (ACC) by ACC synthase (ACS), and ACC is finally converted into ethylene by ACC oxidase (ACO) (Adams and Yang, 1979) ACS carries out the rate limiting step in ethylene biosynthesis (Yang and Hoffman, 1984) and it in tomato, is a part of multi-gene family, comprising nine homologs, which are differentially expressed (Cara and Giovannoni, 2008) ACS2 and ACS4 are responsible for climacteric burst of ethylene production at the onset of ripening (termed System-2), while ACS1A and ACS6 maintain basal levels of ethylene in green tissues (termed System-1) (Rottmann et al., 1991; Lincoln et al., 1993) It has been reported that basal ethylene is essential for progression of system ethylene Tomato fruit can also initiate system ethylene independently of system 1, which proves that ripening-associated ethylene biosynthesis is regulated by both auto-catalytic system and ethylene-independent factors (Yokotani et al., 2009) A number of physiological effects of ethylene in plants seem to be antagonized by polyamines [PAs: putrescine (Put), spermidine (Spd) and spermine (Spm)] by modulating genes involved 988 A Gupta et al / Journal of Plant Physiology 170 (2013) 987–995 Fig T-DNA map of RNAi-ACS binary vector Antisense chimera was designed to be 50 bp shorter than the sense chimera, such that after transcription the antisense RNA folds back and complements with sense RNA to form dsRNA molecules with loop in between in the ethylene signaling and biosynthetic pathways (Apelbaum et al., 1981; Handa et al., 2011) Increased levels of PAs have shown to result in delayed fruit ripening, increased fruit firmness, prolonged shelf life, reduced ethylene and respiration rate emissions (Bregoli et al., 2002; Nambeesan et al., 2010) In plants, Put is either synthesized directly from ornithine via the action of ornithine decarboxylase (ODC) or indirectly from arginine via arginine decarboxylase (ADC) Higher PAs (Spd and Spm) are synthesized by the sequential addition of aminopropyl groups to Put by Spd synthase (SPDSYN) and Spm synthase (SPMSYN) The aminopropyl groups are provided from decarboxylated S-adenosylmethionine (dcSAM) which is formed by decarboxylation of SAM by SAM decarboxylase (SAMDC) SAM acts as the common precursor for both PA and ethylene biosynthesis pathways, suggesting it to be a constraint for either of the biosynthetic pathway (Tiburcio et al., 1990) The green tomato transforms into ripe fruit while it accumulates carotenoid pigments and turns red, develops flavor and aroma with alteration in sugars, acids and volatile profile with soft texture (Giovannoni, 2001) Ripening-associated softening is a major factor limiting fruit shelf life and storage and thus, contributes to the tons of losses of this crop In fact, several attempts have been made to control ripening process For delayed ripening, major focus has been on the manipulation of ethylene production, perception and action employing either sense or antisense technology (Hamilton et al., 1990; Oeller et al., 1991; Theologis et al., 1993; Ye et al., 1996; Wilkinson et al., 1997; Xiong et al., 2003, 2005) All these researchers have been successful in obtaining low ethylene producing tomatoes, displaying an extended shelf life but with compromised fruit quality traits However, selective nature of RNAi can be more specific for suppression and is more effective than either sense or an antisense RNA (Fire et al., 1998) Additionally, suppression of more than one homolog would be more effective over shut-down of single homolog From the perusal of literature, it is apparent that PA-ethylene nexus plays a crucial role in fruit ripening Thus, looking at the multifunctional and regulatory aspects of PA and ethylene, it is possible that controlled manipulation of these key regulators rather than structural or regulatory gene operating in a single branch of biosynthesis pathway may result in better improvement of fruit shelf life and quality traits In the present study, in order to modulate PA-ethylene levels, we have suppressed the expression of three homologs of ACS gene (ACS6, ACS1A and ACS2), thereby targeting system and production of ethylene simultaneously at the onset of fruit ripening, using RNAi approach Fruit-specific down-regulation of these genes was accomplished by 2A11 promoter from tomato Materials and methods In silico analysis The nucleotide sequences of fruit-specific homologs of ACS, viz., ACS6 (GenBank ID, AF179249), ACS1A (GenBank ID, U18056.2) and ACS2 (GenBank ID, NM 001247249.1) from Solanum lycopersicum were compared by pair-wise alignment using NCBI BLAST2 Seq To avoid unintended targeting by RNAi in transformed tomato, the selected ACS homologs were evaluated for their probable off-target in silico The complete mRNA sequences for all three ACS genes were used for homology searching with all the available sequences in nr database using NCBI BLAST The individual mRNA sequences were also checked for probable off-targets by splitting it into 21 bp fragment imitating the siRNA The off-target free partial mRNA sequence of ACS6, ACS1A and ACS2 were chosen for preparing RNAi construct The primer sequences designed for the amplification of ACS homologs, 2A11 promoter and antisense ACS (Chimeric) are given in Table S1 Generation of tomato transformants with RNAi-ACS (chimera) construct The partial cDNA of three ACS homologs ACS6 (201 bp), ACS1A (286 bp) and ACS2 (506 bp) were amplified from tomato (fruit pericarp) total RNA by semi-quantitative RT-PCR using specific set of primers designed with restriction sites ACS1A and ACS2 were cloned into pBSK vector sequentially using KpnI–EcoRI and EcoRI–BamHI restriction sites respectively The 2A11 promoter (1.35 kb – GenBank ID, DQ453963) was isolated from tomato and cloned into pGEM-T easy Following sequencing, 2A11 promoter was excised by EcoRI and SacI and ligated into pCAMBIA2300 binary vector ACS6 was cloned in pCAMBIA2300 downstream to 2A11 promoter using SacI and KpnI restriction sites followed by ligation of ACS1A + (from pBSK) with KpnI and BamHI restriction sites Sense ACS6 + ACS1A + ACS2 was used as a template in PCR reaction to amplify an antisense chimera, which is 50 bp shorter than the sense one It was cloned into pCAMBIA2300 binary vector harboring 2A11 promoter, polyA signal and sense ACS6 + 1A + by using BamHI and XbaI restriction sites The polyA signal from pRT101 vector was also cloned into pCAMBIA2300 vector using XbaI and PstI restriction sites (Fig 1) RNAi-ACS construct was mobilized into Agrobacterium tumefaciens LBA4404 strain by chemical transformation, and was then utilized to transform cotyledons of tomato (S lycopersicum Mill cv Pusa Early Dwarf) by a procedure described by Madhulatha et al (2007) Genomic DNA isolation and transgene integration in tomato transformants Genomic DNA was isolated from the leaves of wild-type (WT) and transformants using CTAB method (Doyle and Doyle, 1990) The transformants were screened by PCR for the presence of transgene Primer pairs for the amplification of 750 bp fragment of NPT-II gene are F, -TCAGAAGAACTCGTCAAGAA-3 and R, ATGGGGATTGAACAAGATGG-3 Genomic DNA (10 g) was digested with EcoRI enzyme and subjected to DNA hybridization to confirm the integration and copy number of transgene using radiolabeled NPT-II gene probe Blots were prepared by standard protocol (Sambrook et al., 1989) using A Gupta et al / Journal of Plant Physiology 170 (2013) 987–995 nylon membrane (MDI, India) Probe was prepared by using random primer kit following the manufacturer’s guidelines (BRIT, India) Pre-hybridization and hybridization were carried out as described by Sambrook et al (1989) A total of thirty RNAi-ACS (chimeric) transformants, confirmed for transgene integration by PCR and DNA hybridization (Fig S2) were maintained both in transgenic green-house/net-house conditions Expression analysis Pericarp tissue (50 mg) of mature green (MG), breaker red (BR), pink red (PR) and red ripe (RR) fruit was pulverized to homogenate powder The homogenate was used for isolation of total RNA according to the manufacturer’s guidelines with TriZol reagent (Invitrogen, USA) with subsequent RNase free DNase (Fermentas, Canada) treatment to remove any DNA contamination Semi-quantitative RT-PCR was performed to check transcript levels of the target gene and various genes involved in fruit ripening, in RNAi-ACS and WT tomato plants DNA-free RNA (200 ng) was then used for one-step RT-PCR reaction by following the manufacturer’s guidelines (Taurus-Scientific, India) The products were analyzed on ethidium bromide stained 1.5% agarose gel Primers used in the study were designed with reference to nucleotide sequences registered in the Genbank database The reaction conditions were as follows: 45 ◦ C for 50 min, followed by 94 ◦ C for min, n cycles of 94 ◦ C for 30 s, annealing temperature for 30 s and 72 ◦ C for 30 s, final extension at 72 ◦ C for 15 All semi-quantitative-RT-PCR experiments were carried out thrice in three independent experiments by using the pulverized tissue of three fruits (at same stage) from same plant (pooled) The results presented are relative The accession numbers for ripening-associated genes selected for transcript analysis are: ornithine decarboxylase (SlODC, NM 001247687.1), arginine decarboxylase (SlADC1, NM 001247135.1), S-adenosylmethionine decarboxylase (SlSAMDC1, EU196515.1; SlSAMDC2, EU196516.1; SlSAMDC3, EU196517.1), spermidine synthase (SlSPDSYN, NM 001247564.1), ACS6 (AF179249), SlACS1A (U18056.2), SlACS2 (NM 001247249.1), SlACS4 (NM 001247351), SlACO1 (NM 001247095), SlE8 (X13437.1), 1-deoxy-d-xylulose-5phosphate synthase (SlDXS1, AF143812), (SlPSY1, EF157835.1), lycopene-epsilon cyclase (SlLES, Y14387), SlTAGL1 (AY098735.2), polygalacturonase (SlPG, X05656), expansin (SlEXP1, U82123), -galactosidase (SlTBG4, AF020390), ␣-xyloglucan endotransglucosylase/hydrolase (SlXTH5, AY497475), l-galactono-1,4-lactone dehydrogenase (SlGLDH, NM 001247674.1), ascorbate oxidase (SlAO, AY971876.1), dehydroascorbate reductases (SLDHAR1, AY971873.1; SLDHAR2, AY971874; SlMDHAR, NM 001247084.1) and actin (SlActin, BT012695) The details of primers are provided in Table S2 Isolation of small RNAs and RNA hybridization for detection of siRNAs in RNAi-ACS tomato lines Total RNA (200–400 g) from BR fruit was enriched for low molecular weight RNA as per the method described by Lu et al (2007) About 90 g of the low molecular weight RNA was fractionated to separate small RNAs (20–25 nt) using 15% denaturing polyacrylamide gel according to Lu et al (2007) RNA fractions were electroblotted onto the positively charged nylon membrane (MDI, India) using semi-dry blot (Benchtop Labsystems, India) and the membrane was incubated for overnight at 42 ◦ C in a prehybridization buffer (Sambrook et al., 1989) Hybridization was carried out at 36 ◦ C for 20 h using radiolabeled ACS1A, ACS2 and ACS6 DNA as probe 989 Estimation of rate of ethylene production in tomato fruits Three red fruits were enclosed in 100 mL of an air-tight container for h at room temperature The headspace atmosphere (3 mL) of the container was withdrawn and injected into gas chromatograph (HP 5890, Hewlett Packard, USA) for ethylene estimation (Singh and Pal, 2008) The experiment was carried out thrice with twenty replicates in each Measurement of respiratory activity in tomato fruits The individual fruits were sealed in an air-tight container for h Respiratory activity of the harvested fruits was determined by head space gas analysis of jar using CO2 /O2 analyzer (Checkmate 9900 O2 /CO2 , PBI Dansensor, Denmark) (Singh and Pal, 2008) The experiment was carried out thrice with twenty replicates each Determination of physiological loss of water (PLW) PLW in RR fruits during first ten days of their storage (at room temperature) was estimated by subtracting the sample weights from their previous recorded weights and was represented as % PLW/day compared to the initial weight Data was recorded for twenty biological replicates each in three independent sets Polyamine analysis About 100 mg of pericarp tissue (from different stages of fruit ripening, viz., BR and RR stages) was homogenized in mL of 10% perchloric acid The homogenate of three fruits (with same age, from same plant) was pooled The extract was fractionated, dansylated, chromatographed and quantified by the method described by Bajaj and Rajam (1996) using dual wavelength fluorometer (BioRad, VersaFluor, USA) with an excitation wavelength of 350 nm and an emission wavelength of 495 nm The Polyamine content was estimated in three fruits each in three independent experiments Determination of on-vine ripening period Flowers were tagged at anthesis and days were noted for fruit formation in three independent sets with twenty replicates in each MG fruits displaying first sign of color change were identified as BR stage Days for BR to reach RR were noted for RNAi-ACS, WT and unrelated (UR) control fruits to determine the on-vine ripening period Determination of fruit shelf life RR fruits (twenty replicates) of RNAi-ACS, WT and UR plants were kept at room temperature and time was noted for visual signs of shriveling The experiment was carried out thrice Measurement of total soluble solids (TSS) content Tomato fruit (RR stage) homogenate was utilized for measuring TSS content by a hand refractrometer (model: Fisher, Japan) (Singh and Pal, 2008) The homogenate of three fruits (from same plant) was pooled TSS content was measured in nine fruits each in three independent experiments Estimation of titratable acidity The homogenate of three fruits (RR stage) from same plant were pooled Total titratable acidity (TA) was then determined by titrating the homogenate against 0.1 N NaOH solution using phenolphthalein as an indicator to the end point at pH 8.1 (Singh and 990 A Gupta et al / Journal of Plant Physiology 170 (2013) 987–995 Pal, 2008) TA was measured in nine fruits in three independent experiments Determination of ascorbic acid (AsA) content AsA content in tomato fruits at RR stage was estimated titrimetrically using 2,6-dichlorophenol indophenol as an indicator dye AsA standard was prepared by dissolving 100 mg of l-AsA in 100 mL of 1% HPO3 (Singh and Pal, 2008) The pooled homogenate of three fruits (from same plant) was utilized for the estimation AsA content was measured in nine fruits in three independent experiments Quantification of lycopene content Lycopene fractions of the homogenized pericarp tissue (RR stage) were determined using spectrophotometric method as described by AOAC (2000) The pooled homogenate of three fruits (from same plant) were utilized in each experiment Lycopene fractions were estimated in nine fruits in three independent experiments Data analysis All results were obtained from at least three independent experiments Data presented are average (mean) with the standard error from all the experiments and significant differences were determined by Student’s t-test (P < 0.05) Results and discussion Three homologs of ACS, viz., ACS1A, ACS2, and ACS6 were considered in tandem for RNAi-mediated down-regulation to eliminate any redundancy associated with gene function and to achieve maximum suppression of the autocatalytic burst in ethylene production However, ACS4 was not considered for present study due to the unavailability of any off-target free region and its homology with other tomato genes like expansin (data not shown) To alleviate the pleiotropic effects which might arise due to constitutive downregulation of ethylene, fruit-specific expression of RNAi-ACS was achieved by 2A11 promoter (Fig 1) This promoter shows low levels of activity throughout fruit development but gets induced to high activity at the onset of ripening (Pear et al., 1989) Previous experiments in our lab have shown that tomato plants transformed with 2A11-GUS construct exhibited GUS expression exclusively in ripening fruit with pronounced activity in pericarp, vascular bundles, placental tissue and seed tegument (Fig S1) These results were also supported by data obtained by Lin et al (2006) to PR stage and declined dramatically at RR stage (Fig 3A) The observed decline in ACS transcripts is collinear with the onset of 2A11 promoter activity RNAi-ACS lines releasing low levels of ethylene, displayed drastic reduction in ACS2 transcript levels This decline in transcript levels was more pronounced in RNAi-ACS81 line at all ripening stages and subtled in RNAi-ACS123 and RNAiACS125 In WT tomato, ACS1A and ACS6 transcript levels were substantially lower than ACS2 mRNA levels (as were detected in WT tomatoes with 30 cycles of amplification in contrast to 25 cycles for ACS2) Previously, it was reported that the expression of ACS6 and ACS1A genes is restricted to the early fruit developmental stages (Nakatsuka et al., 1998; Barry et al., 2000), but our results revealed that the ACS1A and ACS6 transcripts persisted through MG to RR stage in WT tomatoes In RNAi-ACS tomato lines, the expression of ACS6 and ACS1A genes followed the same trend as that of ACS2 These results showed a correlation between transcript abundance and rate of ethylene evolution in RNAi-ACS fruits siRNAs specific to ACS1A, ACS6 and ACS2 were detected at BR stage in fruits of RNAi-ACS lines RNAi-ACS81 line has exhibited high levels of siRNAs with undetectable levels of transcripts of all the targeted genes (Fig 3B) Hence, decline in ACS transcripts in RNAi-ACS fruits expressing dsRNA is an RNAi manifestation On the basis of RT-PCR and siRNA detection results, RNAi-ACS lines were categorized into highly silenced lines (RNAi-ACS60 and RNAi-ACS81) and moderately silenced lines (RNAi-ACS123 and RNAi-ACS125) RNAi-ACS81 and RNAi-ACS123 displaying high and moderate silencing, along with WT were utilized for further work on the expression analysis of ripening-related genes Decline in transcripts levels of ACS4, ACO1 and E8 was noted in case of both the RNAi-ACS lines studied, over WT fruits (Fig 3C) A plausible explanation to this observation is the positive feedback regulation of these genes by ethylene (Barry et al., 1996; Nakatsuka et al., 1998) Decline in ACS4 gene expression may not be solely due to the ethylene regulation, it might also be due to the shared homology between ACS2 and ACS4 gene sequences which could have caused the off-targeting of ACS4 gene Moreover, conversion of SAM to ACC by ACC synthase marks the rate limiting step for ethylene biosynthesis which possibly explains that ACS gene repression has lead to low levels of ACO1 transcripts in RNAi-ACS81 line Transcription of E8 in fruit has been shown to be regulated and stimulated by ethylene, which is well evident by the presence of ethylene regulated sequences in E8 gene promoter (Deikman et al., 1998) TAGL1 codes for transcription factor, which activates ethylene biosynthesis by binding to ACS2 promoter (Vrebalov et al., 2009) The expression of this gene was analyzed and we could not see any significant difference in TAGL1 transcript accumulation in fruits of RNAi-ACS lines over WT, suggesting that alterations in ethylene levels in RNAi fruits did not affect the expression of this gene (Fig 3C) Ethylene suppression in RNAi-ACS fruits Fruits from all thirty RNAi-ACS tomato lines along with unrelated control (UR, other RNAi lines from lab) and WT plants were analyzed for rate of ethylene evolution Results presented in Fig show that fruits from RNAi-ACS lines liberated reduced levels of ethylene Ethylene liberation was found to be least in RNAi-ACS60 and RNAi-ACS81, releasing only 4–5% when compared with controls (WT and UR) Fruits of other RNAi-ACS lines showed 10–70% of ethylene evolution to that of control fruits This difference in ethylene evolution among RNAi-ACS lines could be attributed to variable suppression of the target gene This in turn is ascribable to differential abundance of the introduced dsRNA, as influenced by site of integration and dosage effect of the introduced genes (Fig S2B) (Kohli et al., 2003; Kerschen et al., 2004) Transcript levels of the targeted homologs, viz., ACS1A, ACS2 and ACS6, were checked by semi-quantitative RT-PCR Results showed that levels of ACS2 transcripts in WT fruits were high through MG Polyamine accumulation in RNAi-ACS lines of tomato with reduced ethylene levels In WT tomato fruits at BR and RR stages of ripening, Put levels were found to be highest among three PAs followed by Spd and Spm titers Put and Spm were mainly present in free fractions followed by conjugated and bound forms But Spd pools were maintained by almost equal fractions of free and conjugated forms followed by bound form As the fruit attains RR stage, PA levels were reduced to almost half of the initial levels at BR stage (Fig 4) RNAi fruits at BR stage exhibited 13–25% increase in Put, 15–40% increase in Spd and 15–45% increase in Spm over WT In contrast to WT RR tomatoes, RNAi-ACS81 tomato showed higher levels of PAs at RR than at BR, with 100% increase in Put and 150% increase in Spd and Spm levels Expression pattern of ODC and ADC genes did not alter, while SAMDC1 and SPDSYN mRNA levels were increased in both RNAiACS81 and RNAi-ACS123 fruits over WT (Fig 5) There was also A Gupta et al / Journal of Plant Physiology 170 (2013) 987–995 991 Fig Ethylene levels in fruits of RNAi-ACS lines and controls Bars represent the means of twenty biological replicates and three independent experiments, with standard error values as error bars * Significant at P < 0.05 between controls and RNAi lines no change in transcript levels of SAMDC2 and SAMDC3 genes These two homologs exhibited differential transcript accumulation, with SAMDC2 mRNA levels being higher in BR stage than RR, while SAMDC3 transcripts, although detected in BR stage vanished at RR stage Interestingly, transcript levels of SAMDC2 and SAMDC3 were found to be lower than SAMDC1 mRNA levels Thus, SAMDC1 seems to be predominantly responsible for SAMDC activity in ripening fruits The increased accumulation of SAMDC1 and SPDSYN transcripts was therefore accountable for enhanced Spd and Spm content in RNAi-ACS fruits Elevated SAMDC and SPDSYN transcripts accumulation in turn may be because of diversion of SAM pools toward PA biosynthetic pathway (due to reduction in ethylene biosynthesis) Henceforth, we hypothesize a positive feedback regulation of SAMDC and SPDSYN gene transcription by their upstream precursor, i.e., SAM, which is also supported by the experiments carried out in our lab, i.e., a significant decrease in ethylene production was observed with over-expression of PA biosynthesis genes during fruit ripening (unpublished results) Thus, in response to higher availability of precursor (SAM), PA biosynthesis pathway is activated causing higher accumulation of Spd and Spm The Fig Expression pattern of ethylene biosynthesis and related genes in tomato fruit during ripening (A) Semi-quantitative RT-PCR analysis of ACS transcript levels in WT and RNAi-ACS tomato lines at different stages of fruit ripening; (B) detection of ACS specific siRNAs in RNAi-ACS transformants by modified RNA hybridization; (C) semiquantitative RT-PCR analysis for expression of ethylene biosynthesis and related genes, at BR and RR stages of fruit ripening ‘n’ denote no of cycles in semi-quantitative RT-PCR analysis 992 A Gupta et al / Journal of Plant Physiology 170 (2013) 987–995 Free Conj Bound 600 *†‡ Total PA levels (n mol/g fresh wt) 500 400 300 *† *† *† *†‡ *†‡ *†‡ *†‡*†‡ *† *† *† 200 100 RNAi-ACS lines Fig The levels of free, conjugated and bound PAs in WT and RNAi-ACS tomato fruits Bars represent the means of three biological replicates and three independent experiments, with standard error values as error bars *, †, ‡ as Significant differences in free, conjugated and bound PAs, respectively at P < 0.05 as compared to their respective controls increase in Put amount in fruits of RNAi-ACS lines can be due to the enhanced inter-conversion of excess pools of higher PAs, viz., Spd/Spm to Put through acetylation mechanism by Spd/Spm N1acetyl transferase ‘SSAT’ (Seiler, 2004; Hazarika and Rajam, 2011) Our data indicates that interference with ethylene biosynthesis in tomato fruits of RNAi-ACS lines had resulted in the accumulation of PAs in such fruits, which supports the competitive interaction between ethylene and PA biosynthetic pathways Delayed ripening and enhanced shelf life of fruits in RNAi-ACS lines of tomato Reduction in ethylene levels has led to significant reduction in CO2 evolution (marker for respiration) in RNAi-ACS tomato lines over controls (WT and UR) Among the various lines analyzed for rate of respiration, RNAi-ACS60 and RNAi-ACS81 were found to exhibit ∼50% reduction in respiratory activity in harvested fruits over control fruits, while rest of the RNAi-ACS lines showed up to 30% reduction in rate of respiration (Table 1) The variation in respiratory activity of different fruits corresponds to the different levels of ethylene liberated by such fruits The results are in accordance with the observations made by Defilippi et al (2004) in apple and Wang et al (2010) in tomato Results on physiological loss of water (PLW) showed almost similar trends of reduced PLW percentage among the RNAi-ACS tomatoes over WT fruits (Table 1) The reduction was prominent with up to 40% reduction in the rate of PLW in RNAi-ACS tomatoes over controls Such a reduction in respiratory activity, PLW and thus slower metabolic rate has delayed ripening and extended the shelf life of RNAi-ACS tomato fruits A significant delay of on vine ripening for RNAi-ACS tomatoes was recorded as compared to control fruits On vine ripening period (BR to RR) was delayed for ∼45 days in RNAi-ACS60 and RNAiACS81 over controls (Table 1) RR fruits harvested from controls and RNAi-ACS lines showed significant difference in their shelf life under storage condition (room temperature) Control fruits kept at room temperature started rotting after 8–10 days of harvest, while most promising RNAi-ACS lines showed extended shelf life of about 45 days beyond normal shelf life before decaying (Table 1; Fig 6A) Guillén et al (2007) have demonstrated a similar correlation between degree of ethylene inhibition and rate of ripening with dose- and time-dependent application of 1-MCP in tomato Fig Semi-quantitative RT-PCR analysis for expression of PA biosynthesis genes in WT and RNAi-ACS81 and RNAi-ACS123 fruits ‘n’ represents no of cycles in semiquantitative RT-PCR analysis Cell wall components including cellulose, hemicellulose and pectin are the major contributors for flesh firmness and hence the shelf life During ripening, cell wall undergoes substantial disassembly caused by increased expression of various cell wall degrading enzymes like polysaccharide hydrolases/glycoside hydrolase, transglycosylases, lyases and expansins (Brummell, 2006) EXP1, TBG4, PG and XTH5 genes have been shown to be specifically expressed during fruit ripening and play major role in fruit softening (Pirrello et al., 2009) Here, comparative expression analysis of genes involved in cell wall hydrolysis indicated considerable reduction in transcripts of EXP1, TBG4, PG and XTH5 genes during fruit ripening in RNAi-ACS lines over WT fruits (Fig 6B) This could be due to the reduced ethylene levels in these tomatoes as expression of these genes has been reported to be ethylene responsive (Maclachlan and Brady, 1994; Smith and Gross, 2000; Zhaohui et al., 2009) In RNAi-ACS lines, the inhibition of EXP1, TBG, PG and XTH expression is consistent with the delay of fruit ripening and prolonged shelf life of fruits influenced by reduction in ethylene levels Thus, decline in respiratory activity and lower transcript abundance of cell wall degrading genes has led to the delayed ripening and enhanced shelf life in RNAi-ACS tomato lines In addition, increased PA accumulation in fruits of RNAi-ACS lines may have also influenced the enhanced shelf life possibly by stabilizing the membranes PAs have been reported as modulator of supra-molecular conformation of pectin Evidences are available supporting that PA can bound covalently with cell wall and inhibition of PA biosynthesis interferes with cell wall formation making it amorphous and its exogenous application reverses the changes (Berta et al., 1997; Messiaen et al., 1997) Improved fruit quality in RNAi-ACS tomato lines Total soluble solids (TSS) and titratable acids (TAs) are of special significance for processing industry TSS of a produce comprises of sugar, mineral and acid contents and reflects its specific gravity or density It was highly encouraging to observe that RNAi-ACS tomatoes releasing traces of ethylene recorded very high level of TSS with ∼40–45% increase (Table 2) Although previous reports (Opiyo and Ying, 2005) have suggested TSS to be ethylene independent but our results indicate a correlation between accumulation of TSS and ethylene reduction, and the underlying mechanism for this still to be worked out A Gupta et al / Journal of Plant Physiology 170 (2013) 987–995 993 Table Storage attributes of fruits from control and RNAi-ACS lines of tomato RNAi lines Respiration rate/CO2 (nL/g fresh wt./h) Wild-type Unrelated control RNAi-ACS60 RNAi-ACS81 RNAi-ACS71.2 RNAi-ACS93 RNAi-ACS123 RNAi-ACS128 RNAi-ACS109 RNAi-ACS124 RNAi-ACS125 RNAi-ACS86 RNAi-ACS88 RNAi-ACS87 RNAi-ACS83 RNAi-ACS17.2 RNAi-ACS79 RNAi-ACS74 RNAi-ACS2 RNAi-ACS127 RNAi-ACS102 RNAi-ACS1A 8.93 8.71 4.12 4.88 5.96 4.99 5.19 5.27 5.72 6.10 6.29 6.17 6.66 6.86 6.64 9.21 8.11 7.38 8.13 7.64 8.65 5.91 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 1.13 0.92 0.31* 0.37* 0.55* 0.47* 0.56* 0.81* 1.02* 0.62* 0.64* 0.69* 0.85 0.62 1.05 0.83 0.98 0.95 0.51 0.57 0.80 0.75* PLW (%/day) 1.21 1.27 0.48 0.50 0.62 0.79 0.53 0.68 0.76 0.61 0.71 0.75 0.95 0.83 0.90 1.09 0.95 0.94 1.21 0.62 0.98 0.73 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 0.20 0.15 0.09* 0.09* 0.10* 0.06* 0.03* 0.08* 0.11* 0.07* 0.05* 0.07* 0.11 0.07 0.05 0.09 0.06 0.04 0.05 0.09* 0.08 0.05* On vine ripening (days) 15.36 14.59 56.66 57.00 45.83 36.66 43.16 45.00 37.00 41.83 29.11 15.66 24.50 23.83 20.16 18.33 18.50 19.66 15.33 16.01 14.66 30.50 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 0.70 0.94 0.72* 0.71* 0.74* 0.63* 0.74* 0.68* 0.81* 0.67* 0.53* 0.76 0.95* 0.64* 0.85* 0.57* 1.03* 0.92 0.81 0.96 4.42 0.61* Shelf life (days) 9.63 9.95 53.72 55.98 37.85 21.48 41.65 39.44 27.35 39.15 25.55 15.75 20.53 19.83 15.16 13.00 12.57 10.05 11.40 9.25 11.67 33.70 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 0.60 0.83 0.93* 0.95* 1.00* 0.52* 0.79* 0.87* 0.74* 0.85* 1.23* 0.89* 0.84* 1.14* 1.20* 1.13* 0.91* 0.89 1.20 1.06 0.94 2.29* Data is the mean ± standard error, based on at least three independent experiments with twenty replicates in each experiment * Significant at P < 0.05 between controls and RNAi lines RNAi-ACS fruits were found to bear ∼1.5–2.0 fold increase in TAs over controls (Table 2) Lower rate of respiration in RNAiACS fruits explains higher accumulation of TAs in these fruits, since organic acids (e.g citric acid) have been established as substrates of respiration, and is an ethylene-dependent factor (Defilippi et al., 2004) The higher levels of TSS with simultaneous increase in TAs might provide a characteristic flavor for RNAi-ACS fruits In plant cells, ascorbic acid (AsA) is continuously oxidized and reduced where ascorbic free radicals and dehydroascorbate (DHA) are the oxidation products, which can be reconverted back to AsA Ascorbate has been established as an important co-factor for in vitro activity of ACO (Smith et al., 1992) As a general trend, it has been seen that AsA levels decline during ripening and senescence, and has been correlated with its consumption in ethylene biosynthesis pathway Although in our case comparable level of AsA was noted in all RNAi-ACS lines screened (Table 2), in spite of the blockage of ethylene biosynthesis pathway GLDH oxidizes Lgalactono-1,4-lactone to AsA DHAR1, DHAR2, MDHAR and AO are involved in AsA oxidation and recycling (Stevens et al., 2007) When transcript profile of genes involved in AsA biosynthesis and recycling pathway was analyzed, GLDH1 and AO genes showed an enhanced expression at BR stage while the other genes, viz., DHAR1, DHAR2, MDHAR were unaltered in their expression pattern (Fig 7) The up-regulated expression of both biosynthetic as well as oxidation genes, maintains AsA pools in RNAi-ACS tomato fruits and also points toward their regulation by ethylene Decrease in lycopene content in RNAi-ACS lines of tomato Lycopene content showed significant reduction in its levels in RR fruits of RNAi-ACS lines over controls, ranging from 10 to 40% reduction (Table 3) Fruits from RNAi-ACS lines – RNAi-ACS60, RNAi-ACS81 and RNAi-ACS71.2, releasing traces of ethylene exhibited light red color even after ∼70 days of their harvest (at BR stage) Tomatoes from these lines displayed ∼40% reduced lycopene content over the controls Expression pattern of lycopene biosynthesis gene, PSY1 showed significant reduction in transcript level in BR and RR stages of fruit ripening while DXS1 and LES which is involved in catabolism of lycopene, showed no significant difference from that Fig Storage attributes of tomato fruits from controls and RNAi-ACS lines (A) Demonstration of extended shelf-life of RNAi-ACS tomatoes at room temperature after 70 days of their harvest at BR stage; (B) transcript expression analysis of cell wall hydrolyzing genes ‘n’ represents no of cycles in semi-quantitative RT-PCR analysis 994 A Gupta et al / Journal of Plant Physiology 170 (2013) 987–995 Table Lycopene content in control and RNAi-ACS fruits Table Fruit quality traits in control and RNAi-ACS lines of tomato RNAi lines TA (g/100 g) Wild-type Unrelated control RNAi-ACS60 RNAi-ACS81 RNAi-ACS71.2 RNAi-ACS93 RNAi-ACS123 RNAi-ACS128 RNAi-ACS109 RNAi-ACS124 RNAi-ACS1A RNAi-ACS125 RNAi-ACS86 RNAi-ACS88 RNAi-ACS87 RNAi-ACS83 RNAi-ACS17.2 RNAi-ACS79 RNAi-ACS74 RNAi-ACS2 RNAi-ACS127 RNAi-ACS102 0.42 0.39 0.97 0.87 0.94 0.89 0.81 0.73 0.72 0.73 0.70 0.55 0.60 0.57 0.61 0.51 0.40 0.44 0.46 0.32 0.41 0.43 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 0.08 0.19 0.13* 0.10* 0.10* 0.16* 0.13* 0.11* 0.10* 0.09* 0.07* 0.14 0.09 0.09 0.05 0.20 0.15 0.16 0.10 0.10 0.09 0.12 AsA (mg/100 g) 27.97 27.13 26.35 26.54 27.21 27.63 30.26 26.86 25.88 29.13 27.14 25.91 28.39 28.67 28.02 25.27 26.34 25.68 27.56 26.59 28.00 27.19 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 0.84 0.76 1.32 0.33 1.32 1.16 1.60 1.04 0.75 0.86 1.24 2.35 1.50 2.93 1.46 2.01 1.44 1.57 0.65 2.99 1.89 2.23 TSS (◦ BRIX) 5.62 5.44 8.03 7.17 7.06 7.19 7.77 6.98 7.31 7.08 7.67 6.70 6.75 6.33 6.78 6.88 5.34 7.00 6.82 5.11 5.39 5.03 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 0.13 0.50 0.19* 0.32* 0.18* 0.27* 0.19* 0.59* 0.63* 0.51* 0.80* 0.18* 0.14* 0.13 0.29 0.06* 0.28 0.04* 0.09* 0.15 0.14 0.43 Data is the mean ± standard error, based on at least three independent experiments with nine replicates in each experiment * Significant at P < 0.05 between controls and RNAi lines RNAi lines Lycopene (mg/100 g) Wild-type Unrelated control RNAi-ACS1A RNAi-ACS2 RNAi-ACS17.2 RNAi-ACS60 RNAi-ACS71.2 RNAi-ACS74 RNAi-ACS79 RNAi-ACS81 RNAi-ACS83 RNAi-ACS86 RNAi-ACS87 RNAi-ACS88 RNAi-ACS93 RNAi-ACS102 RNAi-ACS109 RNAi-ACS123 RNAi-ACS124 RNAi-ACS125 RNAi-ACS127 RNAi-ACS128 9.95 10.04 4.80 9.52 9.26 4.77 4.83 8.70 9.77 4.92 7.87 7.18 6.37 8.18 6.56 8.63 7.22 5.38 5.64 6.30 8.94 4.26 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 0.67 1.34 0.13* 0.27 0.26 0.13* 0.17* 0.29 0.45 0.53* 0.89 0.42* 0.40* 0.59 0.35* 0.56 0.68* 0.32* 0.25* 0.71* 0.28 0.15* Data is the mean ± standard error, based on at least three independent experiments with nine replicates in each experiment * Significant at P < 0.05 between controls and RNAi lines Acknowledgements This work was generously supported by a grant from Department of Biotechnology (BT/PR8657/PBD/16/738/2007), New Delhi Senior Research Fellowship to Aarti Gupta by the Council of Scientific and Industrial Research, New Delhi is acknowledged We also thank University Grants Commission for Special Assistant Program and Department of Science and Technology, New Delhi for FIST program Appendix A Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.jplph 2013.02.003 Fig Expression profile analysis of ascorbic acid biosynthesis and recycling genes ‘n’ represents no of cycles in semi-quantitative RT-PCR analysis of WT in terms of their mRNA levels (Fig 8) Reduction in lycopene content in fruits of RNAi-ACS lines over controls can be attributed to reduced PSY1 mRNA levels, which is possibly a manifestation of a drop in ethylene release shown by these RNAi-ACS lines Fig Expression profile of lycopene metabolic genes ‘n’ represents no of cycles in semi-quantitative RT-PCR analysis 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