3.17 西红柿

Enhancement of fruit shelf life by suppressing N-glycan processing enzymes Vijaykumar S. Meli1, Sumit Ghosh1, T. N. Prabha, Niranjan Chakraborty, Subhra Chakraborty2, and Asis Datta2 National Institute of Plant Genome Research, New Delhi 110067, India Edited by Roger N. Beachy, Donald Danforth Plant Sciences Center, St. Louis, MO, and approved January 5, 2010 (received for review August 19, 2009) In a globalized economy, the control of fruit ripening is of strategic importance because excessive softening limits shelf life. Efforts have beenmade to reduce fruit softening in transgenic tomato through the suppression of genes encoding cell wall–degrading proteins. How- ever, these havemetwith very limited success.N-glycans are reported to play an important role during fruit ripening, although the role of any particular enzyme is yet unknown. We have identified and targeted two ripening-specific N-glycoprotein modifying enzymes, α-mannosidase (α-Man) and β-D-N-acetylhexosaminidase (β-Hex). We show that their suppression enhances fruit shelf life, owing to the reduced rate of softening. Analysis of transgenic tomatoes revealed≈2.5- and≈2-fold firmer fruits in the α-Man and β-Hex RNAi lines, respectively, and ≈30 days of enhanced shelf life. Overexpres- sionofα-Manorβ-Hexresulted inexcessivefruit softening.Expression ofα-Man and β-Hex is induced by the ripening hormone ethylene and is modulated by a regulator of ripening, rin (ripening inhibitor). Fur- thermore, transcriptomic comparative studies demonstrate thedown- regulationof cellwalldegradation-andripening-relatedgenes inRNAi fruits. It is evident from these results that N-glycan processing is involved in ripening-associated fruit softening. Genetic manipulation ofN-glycan processing can be of strategic importance to enhance fruit shelf life, without any negative effect on phenotype, including yield. α-mannosidase | β-D-N-acetylhexosaminidase | fruit softening | RNAi The postharvest losses of fruits and vegetables in the developingcountries account for almost 50% of the produce. India, the world’s second largest producer of fruits and vegetables, loses 35– 40% of produce because of excessive softening. The softening that accompanies ripening of fruits exacerbates damage during shipping and handling processes. It plays a major role in determining the cost factor, because it has a direct impact on palatability, consumer acceptability, shelf life, and postharvest disease/pathogen resistance (1–3). Generally, reduction in fruit firmness due to softening is accompaniedby increased expressionof cellwall–degrading enzymes actinguponproteinsandcarbohydrates (4).However,manyefforts to suppress expression of cell wall–degrading enzymes have not pro- vided the insight needed to genetically engineer fruits whose soft- ening can be adequately controlled (5–11). Previous studies have shown that polygalacturonase, pectin methylesterase, β-glucanase, and β-galactosidase are not sufficient to significantly impact texture (5–10, 12). Thismaybedue to thepresenceof functionally redundant components of a complicated metabolic process (6, 7, 10, 13). It also suggests that the suppression of enzymes acting on cellulose, hemi- cellulose, and pectin is not sufficient to prevent softening. The improvement in fruit shelf life achieved to date is not adequate, and therefore the identification of new targets is required. N-glycoproteins are commonly found in plant cell walls, and free N-glycans occur as the precursors of glycosylation or glycoprotein proteolysis. The biologic activity of freeN-glycans has been noted: injection of Man3(Xyl)GlcNAc(Fuc)GlcNAc and Man3GlcNAc into mature green tomatoes stimulated ripening, as measured by the red coloration and ethylene production (14). Free N-glycans constitute a significant fraction of the soluble oligosaccharide pool in the tomato pericarp. They are present in the pericarp tissue at all stages of tomato development, and the amount increases partic- ularly during ripening. Moreover, the blocking of N-glycosylation delayed fruit ripening, which suggests that N-glycan processing may be important in the ripening process (15). Therefore, among the suite of enzymes involved in carbohydrate metabolism, we tar- geted theN-glycan processing enzymes α-mannosidase (α-Man; EC 3.2.1.24) and β-D-N-acetylhexosaminidase (β-Hex; EC 3.2.1.52). α-Manandβ-Hex,membersof glycosyl hydrolase families 38 and20, respectively, are known to break the glycosidic bonds between car- bohydrates, as well as between carbohydrate and noncarbohydrate (16, 17). α-Man cleaves the terminal α-mannosidic linkages from both the high mannose type and plant complex type N-glycans present in glycoproteins (18), whereas, β-Hex cleaves the terminal N-acetyl-D-hexosamine residues and generates the paucimannosi- dic N-glycans present in most plant glycoproteins (19, 20). More- over,α-Manand β-Hexare present at high levels during the ripening of many fruits, including the climacteric fruit tomato (16, 17). However, their molecular function remains to be elucidated. Here we report the isolation and functional characterization of α-Man and β-Hex and their use to produce transgenic fruits with enhanced shelf life. Results and Discussion Identification and Cloning of Tomato Ripening–Specific α-Man and β-Hex. One of the strategies to elucidate fruit softening is to iden- tify and characterize proteins expressed during ripening and whose biochemical activities can be mechanistically related to the observed cell wall changes. Using p-nitrophenyl-α-D-mannopyranoside (pNP- Man, Km 4.6 mM) and p-nitrophenyl-β-D-N-acetylglucosaminide (pNP-GlcNAc, Km 0.225 mM) as substrates, we found the maximum activity of α-Man and β-Hex at the breaker and pink stages of tomato ripening, respectively (Figs. 1A and 2A). However, α-Man and β-Hex activities were not detected in other parts of the plant (e.g., stem, leaves, and roots). To correlate specific activity with protein accumulation patterns during ripening, immunoblot analysis was performed using polyclonal antibodies raised against β-Hex and α-Man. The analysis revealed maximum accumulation of α-Man and β-Hex proteins at the breaker and pink stages, respectively (Figs. 1B and 2B). Ripening-related changes like climacteric ethyl- ene production, chlorophyll degradation, lycopene synthesis, and cell wall disassembly start at the breaker stage, and subsequently an increase in expression of ripening-related cell wall hydrolases is evident (13, 21). The accumulation of α-Man and β-Hex at the critical stage of tomato ripening strengthened our hypothesis that they are involved in ripening and/or softening. To address this issue, Author contributions: V.S.M., S.G., S.C., and A.D. designed research; V.S.M., S.G., and T.N.P. performed research; V.S.M., S.G., N.C., S.C., and A.D. analyzed data; and V.S.M., S.G., S.C., and A.D. wrote the paper. The authors declare no conflict of interest. This article is a PNAS Direct Submission. Data deposition: The sequences reported in this paper have been deposited in the Na- tional Center for Biotechnology Information GeneBank database, www.ncbi.nlm.nih.gov (accession nos. EU244853 and EU244854). 1V.S.M. and S.G. contributed equally to this work. 2To whom correspondence may be addressed. E-mail: asisdatta@hotmail.com or subhrac@ hotmail.com. This article contains supporting information online at www.pnas.org/cgi/content/full/ 0909329107/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.0909329107 PNAS Early Edition | 1 of 6 A G RI CU LT U RA L SC IE N CE S we purified (Table S1) and characterized both α-Man and β-Hex from tomato pericarp. Purified α-Man and β-Hex constituted ≈360- and ≈300-kDa proteins, respectively, on nondenaturing PAGE (Figs. 1C and 2C). However, the molecular masses of α-Man and β-Hex on superdex 200 analytical gel filtration column were found to be ≈290 and ≈206 kDa, respectively. This discrepancy in molecular mass determined by gel filtration and nondenaturing PAGE analyses could be due to the glycoprotein nature, which was confirmed by periodic acid-Schiff (PAS) staining and deglycosylation with endo- glycosidase H (Fig. S1 A–D). Further, when separated on SDS/ PAGE, α-Man and β-Hex resolved into two (70 and 45 kDa) and a single (80 kDa) polypeptide(s), respectively, suggesting that they function as oligomeric proteins in tomato cells (Figs. 1D and 2D). On SDS/PAGE, purified α-Man was resolved into two subunits of 70 and 45 kDa, under reducing (with β-mercaptoethanol; Fig. 1D) as well as nonreducing conditions (without β-mercaptoethanol; Fig. S1E). This indicated that the subunits are associated with the hy- drophobic interaction rather than the interdisulfide bonds. These two subunits are probably derived by cleavage of the 114-kDa precursor polypeptide (Fig. S2A), which is encodedbyα-ManmRNA(3,090nt). This assumptionwasmade on the basis of the fact that the amino acid sequencesof70- and45-kDapolypeptideswereactuallymatchingwith the N-terminal and C-terminal, respectively, of the encoded polype- ptide (mass spectrometry analysis). Northern blot analysis using 32P- labeled 3′ or 5′ end sequence (≈500 bp) of α-Man identified a single mRNA species of ≈3 kb (Fig. 1F). This suggests that the subunits are the consequence of posttranslational protease cleavage rather than posttranscriptional modification. The roles of α-Man and β-Hex in ripening and/or softening were examined by cloning the genes from tomato using degenerate primers and then systematically testing their functions. In silico analysis of the α-Man sequence revealed the coding region to be 3,090 bp long, encoding a polypeptide of 1,029 aa, with a calculated molecular mass of 114 kDa (Fig. S2A). This is comparable to the combinedmolecular mass of the α-Man subunits (115 kDa), as determined by SDS/PAGE (Fig. 1D). Theproteinhas three glycosyl hydrolase domains (Fig. S2B) and showed69% identitywithVitis viniferaα-Man (XP_002276092.1). The coding regionofβ-Hex is 1,728bp long, encoding apolypeptideof 575 aawith a calculatedmolecularmass of 64 kDa (Fig. S2C), which is less than the molecular mass determined by SDS/PAGE (80 kDa). This was attributed to posttranslational modifications because it has eight probable N-glycosylation sites (Fig. S2C), and the glycoproteic nature of the protein was confirmed by PAS staining (Fig. S1A). However,deglycosylationof thepurifiedproteinwithEndoHrevealed a≈5-kDa glycans moiety (Fig. S1C). This suggests that there could be an involvement of other kinds of posttranslational modification(s) in addition to the glycosylation. β-Hex has two domains related to gly- cosyl hydrolase 20 (Fig. S2D) and showed 68% identity with the V. vinifera ortholog (XP_002266897.1). To verify gene expression patterns and to corroborate earlier results, Northern blot analysis was performed, which revealed that α-Man and β-Hex transcripts were most abundant at the breaker and pink stages of ripening, respectively (Figs. 1F and 2F). α-Man and β-Hex Are the Cell Wall Proteins Involved in N-glycan Processing. During ripening, many fruits, including tomato, dis- assemble the components of the cell wall, resulting in changes in the cell wall rheologic properties and softening of the ripe fruit (3, 4). The subcellular localization revealed both α-Man and β-Hex to be cell wall proteins (Fig. S3 A and B). Free N-glycans in the pericarp account for >1 μg/g of the fresh weight of tomato, which further increases during the ripening process (15). Moreover, blocking ofN-glycosylation with tunicamycin delays fruit ripening. Further, when injected in fruits,N-glycans are known to stimulate red coloration and ethylene production (14). To determine the N-glycan processing ability of α-Man and β-Hex, the purified enzymeswere incubatedwith differentN-glycans commonly found in fruit pericarp. Further, release of mannose or GlcNAc was determined by high-performance anion exchange chromatog- raphy (Figs. 1E and 2E). Cell wall localization and N-glycan pro- cessing abilities of α-Man and β-Hex suggest their participation in the degradation of cell wallN-glycoproteins and the generation of free N-glycans, which further stimulate ripening, possibly by interacting with the protein(s) to transduce the potential ripening signal. Expression of α-Man and β-Hex Is Inhibited in Tomato Ripening– Impaired Mutants and Regulated by Ethylene. The expression of α-Man and β-Hex particularly during ripening led us to examine the ripening-impaired mutants rin, nor, and Nr. Expression analyses revealed that α-Man transcript levels were ≈10% and 70% of wild type in rin andNrmutants, respectively, whereas, α-Man transcript level was similar to wild type in the nor mutant (Fig. 1G). The transcript levels ofβ-Hexwere≈20%and10%ofwild type in rinand nor fruits, respectively. However, in the case of Nr fruits, β-Hex transcript levels were ≈40% and ≈10% at pink and red ripe stages, respectively (Fig. 2G). These mutants are deficient in ripening- associated ethylene biosynthesis or ethylene perception, and they exhibit delayed fruit softening (22–24). The reduced expression of α-Man and β-Hex in these mutants strongly suggests their involve- ment in fruit softening and regulation by ethylene (25), through the Fig. 1. Identification and isolation of ripening–specific tomato α-Man. (A) α-Man activity at ripening stage of tomato. MG, mature green; B, breaker; P, pink; RR, red ripe. Data are mean ± SEM, n = 4. (B) Immunoblot analysis of α-Man at different stages of ripening with purified enzyme as positive control. (C) Purified α-Man resolved on 6% nondenaturing PAGE (lane 1). M, marker (kDa). (D) Purified protein denatured and separated on 12.5% SDS/ PAGE (lane 1). (E) High-performance anion exchange chromatograms show the N-glycan processing ability of α-Man. Arrow indicates the release of mannose residues. (F) Northern blot shows expression of α-Man gene in wild-type tomato at different stages of ripening. (G) qRT-PCR analysis showing the relative expression of α-Man in rin (ripening inhibitor), nor (nonripening), and Nr (never ripe) at the same chronological age of wild type (AC, Ailsa Craig). Data are mean ± SEM (n = 3). (H) Inducibility of α-Man by ACC, as revealed by qRT-PCR analysis. Data are mean ± SEM (n = 4). 2 of 6 | www.pnas.org/cgi/doi/10.1073/pnas.0909329107 Meli et al. NEVERRIPE (NR) receptor.We show that 1-aminocyclopropane 1-carboxylic acid (ACC), the precursor of ethylene, induces α-Man and β-Hex in tomato seedlings≈2.5- and≈4-fold, respectively (Figs. 1H and 2H). These observations indicate that α-Man and β-Hex are regulated by ethylene and act downstream of ripening regulators. Silencing of α-Man or β-Hex Resulted in Firmer Fruits with Reduced Softening and Enhanced Shelf Life. For functional characterizations of α-Man and β-Hex, we resorted to the “knockdown” approach to demonstrate their roles in ripening and/or softening. Endog- enous expression of α-Man and β-Hex was silenced in tomato by expression of gene-specific hairpin RNAs under the control of CaMV 35S promoter (26) (Fig. S2E). Stable RNAi along with the antisense and overexpression lines were raised by Agrobacterium-mediated transformation of tomato cotyledons. To confirm and quantitate suppression of genes in RNAi fruits, quantitative real-time RT-PCR (qRT-PCR) was performed, which revealed up to 99% suppression of α-Man and β-Hex expression at the breaker and pink stages, respectively. However, antisense lines showed 55–80% suppression, and overexpression lines had up to 30-fold more transcript level than control (Fig. S4 A–C). Furthermore, we confirmed the generation of α-Man and β-Hex specific 21–23 mer siRNAs, which is the hallmark of RNAi-mediated silencing (Fig. 3C). Prolonging the desirable texture during ripening is the key to increasing fruit shelf life (27). For texture analysis and shelf life determination, transgenic and control (only vector transformed/ nontransformed) fruits were harvested at the pink stage and stored at room temperature (23–25 °C and55–60%relative humidity). To quantify texture, we analyzed the firmness of the fruit (Materials and Methods), which revealed enhancement of firmness in α-Man and β-Hex RNAi fruits. Ten days after the pink stage, α-Man and β-HexRNAi fruits were≈2.5-fold and≈2-fold firmer than control, respectively (Fig. 3B), and showed no signs of deterioration up to 45 days (Fig. 3A). The α-Man and β-Hex antisense fruits retained the texture for 25–30 days and were ≈1.5-fold firmer than control. Overexpression lines showed early signs of fruit deterioration and were ≈30% softer than their counterpart at the pink stage (Fig. 3B). The firmness of T1 and T2 fruits of α-Man and β-Hex RNAi plants was compared with that of T0 fruits, which revealed a stable and heritable transfer of the character (Fig. S5 A and B). The RNAi lines had no negative effect on vegetative growth, fruit development, days to maturity, seed production, and yield (Table S2). Moreover, RNAi fruits essentially underwent normal cli- macteric ripening and color development while attached to the plant but held their texture and showed longer vine life. Further, time-lapse photography revealed that RNAi fruits, harvested at the pink stage, retained their texture and firmness up to 45 days, whereas control started shrinking and losing their texture after 15days (Fig. 3A).We then investigated the cell wall changes during ripening of transgenic fruits and found that the cell wall of α-Man RNAi fruits was much more compact with more polysaccharide deposition on the wall than control (Fig. 3D). However, reduced cell separation was observed in β-Hex RNAi fruits as compared with control (Fig. 3E). The enzymes α-Man and β-Hex target gly- coproteins and cleave the terminal α-mannose and GlcNAc resi- dues, respectively, present inN-linked glycans. Therefore, to know the status of the α-mannose- and GlcNAc-containing glyco- proteins in RNAi fruits, we performed lectin blotting using Gal- anthus nivalis agglutinin and wheat germ agglutinin. The analysis revealed enhanced levels of α-mannose- and GlcNAc-containing glycoproteins in the fruit of α-man and β-Hex RNAi lines, respectively (Fig. S6 A and B). To substantiate these results, we isolated theN-glycans linked to glycoproteins and found increased levels in RNAi fruits compared with control (Fig. S6C). These results suggest that the intact cell wall polysaccharides are broken down to a lesser extent in RNAi fruits as compared with control. Suppression of α-Man or β-Hex Leads to Down-regulation of Ripening- Related Genes.Overall, the results demonstrate a substantial im- provement in fruit shelf life by targeting N-glycan-processing enzymes. Furthermore, N-glycan processing significantly affects ripening-associated changes in the cell wall: transgenic fruits showed reduced cell separation and compact cell wall compared with control (Fig. 3 D and E). Therefore, we were interested in knowing whether N-glycans, generated by α-Man and β-Hex activities, could play a physiologic role in the regulation of gene expression patterns, related to fruit ripening processes. For this, we performed comparative transcriptomic studies of pink-stage β-Hex RNAi fruits and breaker-stage α-Man RNAi fruits vs. control (only vector transformed). The analyses revealed down- regulation of many genes that are associated with fruit ripening and cell wall degradation in β-Hex and α-Man RNAi fruits (Table S3). Further, to validate these results, qRT-PCR was performed for a few genes related to fruit ripening and/or soft- ening (Fig. 4 A and B). The RNAi fruits showed down-regulation of genes that encode certain cell wall–degrading proteins, such as pectin methylesterase, glucan endo-1,3-β-D-glucosidase, β-1,3- glucanase, endo-xyloglucan transferase, pectinesterase, expansin, pectinacetylesterase, α-galactosidase, pectate lyase, (1-4)-β-mannan endohydrolase, and β-galactosidase. Therefore, suppress