A novel NAC transcription factor mediates negative regulation of early ethylene production and ripening in tomato fruits

Effect of ethylene on mRNA abundance of three β-galactosidase genes in wild type and mutant tomato fruit

NAC transcription factors (TFs) are important regulators of expressional reprogramming during plant development, stress responses, and leaf senescence. NAC TFs also play important roles in fruit ripening. In tomato (Solanum lycopersicum), one of the best characterized NACs involved in fruit ripening is NON-RIPENING (NOR), and the non-ripening (nor) mutation has been widely used to extend fruit shelf life in elite varieties. Here, we show that NOR additionally controls leaf senescence. Expression of NOR increases with leaf age, and developmental as well as dark-induced senescence are delayed in the nor mutant, while overexpression of NOR promotes leaf senescence. Genes associated with chlorophyll degradation as well as senescence-associated genes (SAGs) show reduced and elevated expression, respectively, in nor mutants and NOR overexpressors. Overexpression of NOR also stimulates leaf senescence in Arabidopsis thaliana. In tomato, NOR supports senescence by directly and positively regulating the expression of several senescence-associated genes including, besides others, SlSAG15 and SlSAG113, SlSGR1, and SlYLS4. Finally, we find that another senescence control NAC TF, namely SlNAP2, acts upstream of NOR to regulate its expression. Our data support a model whereby NAC TFs have often been recruited by higher plants for both the control of leaf senescence and fruit ripening.

Ethylene biosynthesis controlled by NON-RIPENING: A regulatory conflict between wounding and ripening

The effects of n-propyl gallate (nPG) on postharvest ripening of tomato have been studied. Mature green (MG) stage tomatoes were harvest and treated with 0.5 mM nPG at 25 ± 1C for 12 h. Following the nPG treatment, fruit ripening was delayed by at least 7 days. Cyanide-resistant respiration and ethylene production were suppressed significantly at the first 7 days. nPG treatment decreased levels of H2O2 and increased activities of antioxidant enzymes and chemicals, particularly for superoxide dismutase (SOD), catalase (CAT) and guaiacol peroxidase (GPX) and ascorbic acid (AsA). The decreases of flesh firmness and total chlorophylls were inhibited, and the production of soluble sugars and lycopene were suppressed by nPG either. Alternative oxidase (AOX) and cyanide-resistant respiration play important roles in fruit climacteric and may be correlated with system 2 ethylene production. nPG could be used as an efficient inhibitor to tomato fruit ripening. PRACTICAL APPLICATIONS Control of fruit ripening is of enormous commercial importance. Generally, tomato fruit ripening is controlled through the use of gas, temperature and humidity control. This research has focused on the application of n-propyl gallate (nPG) on postharvest ripening of tomato fruit. The results of the study have implied that nPG treatment could obviously prolong the shelf life and keep the postharvest quality of tomato fruits, and the firmness loss, respiration rate and ethylene production could be better suppressed compared with the control fruits, which shows that nPG is a potentially effective and safe inhibitor for climacteric fruits ripening, such as tomato. Utilization of the results of this research is likely to benefit for the storage technological improvement.

Fruits of wild tomato species show different ethylene-dependent ripening characteristics, such as variations in fruit color and whether they exhibit a climacteric or nonclimacteric ripening transition. 1-Aminocyclopropane-1-carboxylic acid (ACC) synthase (ACS) and ACC oxidase (ACO) are key enzymes in the ethylene biosynthetic pathway encoded by multigene families. Gene duplication is a primary driver of plant diversification and angiosperm evolution. Here, interspecific variations in the molecular regulation of ethylene biosynthesis and perception during fruit ripening in domesticated and wild tomatoes were investigated. Results showed that the activated ACS genes were increased in number in red-ripe tomato fruits than in green-ripe tomato fruits; therefore, elevated dosage of ACS enzyme promoted ripening ethylene production. Results showed that the expression of three ACS isogenes ACS1A, ACS2, and ACS4, which are involved in autocatalytic ethylene production, was higher in red-ripe tomato fruits than in green-ripe tomato fruits. Elevated ACS enzyme dosage promoted ethylene production, which corresponded to the climacteric response of red-ripe tomato fruits. The data suggest that autoinhibitory ethylene production is common to all tomato species, while autocatalytic ethylene production is specific to red-ripe species. The essential regulators Non-ripening (NOR) and Ripening-Inhibitor (RIN) have experienced gene activation and overlapped with increasing ACS enzyme dosage. These complex levels of transcript regulation link higher ethylene production with spatiotemporal modulation of gene expression in red-ripe tomato species. Taken together, this study shows that bursts in ethylene production that accompany fruit color changes in red-ripe tomatoes are likely to be an evolutionary adaptation for seed dispersal.

Ripening is the last stage of the developmental program in fleshy fruits. During this phase, fruits become edible and acquire their unique sensory qualities and post-harvest potential. Although our knowledge of the mechanisms that regulate fruit ripening has improved considerably over the past decades, the processes that trigger the transition to ripening remain poorly deciphered. While transcriptomic profiling of tomato (Solanum lycopersicum L.) fruit ripening to date has mainly focused on the changes occurring in pericarp tissues between the Mature Green and Breaker stages, our study addresses the changes between the Early Mature Green and Late Mature Green stages in the gel and pericarp separately. The data showed that the shift from an inability to initiate ripening to the capacity to undergo full ripening requires extensive transcriptomic reprogramming that takes place first in the locular tissues before extending to the pericarp. Genome-wide transcriptomic profiling revealed the wide diversity of transcription factor (TF) families engaged in the global reprogramming of gene expression and identified those specifically regulated at the Mature Green stage in the gel but not in the pericarp, thereby providing potential targets toward deciphering the initial factors and events that trigger the transition to ripening. The study also uncovered an extensive reformed homeostasis for most plant hormones, highlighting the multihormonal control of ripening initiation. Our data unveil the antagonistic roles of ethylene and auxin during the onset of ripening and show that auxin treatment delays fruit ripening via impairing the expression of genes required for System-2 autocatalytic ethylene production that is essential for climacteric ripening. This study unveils the detailed features of the transcriptomic reprogramming associated with the transition to ripening of tomato fruit and shows that the first changes occur in the locular gel before extending to pericarp and that a reformed auxin homeostasis is essential for the ripening to proceed.

Ethylene is a plant hormone that has an essential role in fruit ripening (Yang and Hoffman, 1984; Kende, 1993). ACC synthase (S-adenosyl-L-methionine methylethioadenosine-lyase, EC 4.4.1.14), which is encoded by a multigene family, plays a regulatory role in ethylene production. Severa1 genes for ACC synthase have been isolated from tomato (Rottmann et al., 1991), mung bean (Botella et al., 1992, 19931, winter squash (Nakajima et al., 1990; Nakagawa et al., 1991), and Arabidopsis (Liang et al., 1992; Van Der Straeten et al., 1992). Two ACC synthase genes (LE-ACS2 and LE-ACS4, which are identified as a wounding and a ripening inducing gene, respectively) are expressed during ripening of tomato fruits (Olson et al., 1991; Rottmann et al., 1991). An antisense RNA experiment with LEACS2 reduced the levels of mRNAs for LEACS2 and LEACS4 in tomato fruits and caused retardation of initiation of ripening of tomato fruits (Oeller et al., 1991). These results showed that wound-induced ACC synthase also played an important role in the production of ethylene in tomato fruit during ripening. We isolated a cDNA (pMEACS1,2097 bp) for ACC synthase from wounded mesocarp tissue of melon fruits (Cucumis melo L. cv AMS) (Table I). The polypeptide derived from the cDNA in Escherichiu coli had ACC synthase activity. Sequence analysis of this cDNA revealed the presente of an open reading frame of 493 amino acids. This polypeptide contained seven sequences that were conserved among other ACC synthases. pMEACSl showed high homology at the amino acid and nucleotide levels to wound-induced ACC synthase from squash (Nakajima et al., 1990; Sato et al., 1991). RNA blot analysis showed that the level of mRNA for the gene increased in the mesocarp tissue of melon fruits after wounding and also during ripening. Since we could detect cDNA only for MEACSl ACC synthase in a PCR experiment with the mRNA from mesocarp tissue of ripe melon fruits, MEACSl should be the gene that is preferentially expressed during ripening of

Defense response of tomato fruit at different maturity stages to salicylic acid and ethephon

The nitric oxide (NO) treatment on tomato (Solanum lycopersicum L. cv. ‘Myrock’) fruits delayed the burst of ethylene production and color development in both mature green (MG) and breaker (BR) stage fruits. However, no clear differences were observed in pink and full red stage fruits during storage. The gene expressions of 1-aminocyclopropane-1-carboxylic acid synthase (ACS) and 1-aminocyclopropane-1-carboxylic acid oxidase (ACO) were determined in both MG and BR stage fruits using the reverse transcription-polymerase chain reaction method. The analyzed ACS genes include LeACS2, LeACS4, LeACS6, LeACSH6A, and LeACSH6B. The gene expression of ACO was investigated on LeACO1, LeACO2, LeACOH2, LeACO4, and LeACOH4. The ACS genes in NO-treated and untreated fruits were expressed extensively over the storage period in both MG and BR stage fruits, and the expression patterns were similar. However, the expression of ACO genes was different at different ripening stages. In NO-treated fruit, expression of LeACO1, LeACOH2, and LeACO4 was decreased and delayed. These results implicate that NO might control the postharvest metabolism of crops, dependent on either dose level or commodities.

Climacteric fruit ripening, as it occurs in many fruit crops, depends on a rapid, autocatalytic increase in ethylene production. This agriculturally important process has been studied extensively, with tomato simultaneously acting both as a model species and target crop for modification. In tomato, the ethylene biosynthetic genes ACC SYNTHASE2 (ACS2) and ACS4 are highly expressed during fruit ripening, with a combined loss of both ACS2 and ACS4 activity preventing generation of the ethylene burst necessary for fruit ripening. However, the individual roles and importance of ACS2 and ACS4 have not been determined. In this study, we examined specifically the role of ACS4 by comparing the phenotype of an acs4 mutant firstly with that of the wild-type, and secondly with two novel ripening-inhibitor (rin) mutants. Ethylene production during ripening was significantly reduced in both acs4-1, and rin lines, with rin genotypes showing the weaker ethylene burst. Also i) the time between anthesis and the start of fruit ripening and ii) the time required to progress through ripening were significantly longer in acs4-1 than in the wild type, but shorter than in the strongest rin mutant. The delay in ripening was reflected in the lower expression of ripening-related transcripts during the mature green and light red ripening stages. Furthermore, expression of ACS2 and ACS4 was strongly dependent on a functional RIN gene, while ACS2 expression was largely independent of ACS4. Altogether, we show that ACS4 is necessary for normal progression of tomato fruit ripening and that mutation of this gene may provide a useful means for altering ripening traits.

Fruit ripening is a developmental process that is associated with increased susceptibility to the necrotrophic pathogen Botrytis cinerea. Histochemical observations demonstrate that unripe tomato (Solanum lycopersicum) fruit activate pathogen defense responses, but these responses are attenuated in ripe fruit infected by B. cinerea. Tomato fruit ripening is regulated independently and cooperatively by ethylene and transcription factors, including NON-RIPENING (NOR) and RIPENING-INHIBITOR (RIN). Mutations in NOR or RIN or interference with ethylene perception prevent fruit from ripening and, thereby, would be expected to influence susceptibility. We show, however, that the susceptibility of ripe fruit is dependent on NOR but not on RIN and only partially on ethylene perception, leading to the conclusion that not all of the pathways and events that constitute ripening render fruit susceptible. Additionally, on unripe fruit, B. cinerea induces the expression of genes also expressed as uninfected fruit ripen. Among the ripening-associated genes induced by B. cinerea are LePG (for polygalacturonase) and LeExp1 (for expansin), which encode cell wall-modifying proteins and have been shown to facilitate susceptibility. LePG and LeExp1 are induced only in susceptible rin fruit and not in resistant nor fruit. Thus, to infect fruit, B. cinerea relies on some of the processes and events that occur during ripening, and the fungus induces these pathways in unripe fruit, suggesting that the pathogen itself can initiate the induction of susceptibility by exploiting endogenous developmental programs. These results demonstrate the developmental plasticity of plant responses to the fungus and indicate how known regulators of fruit ripening participate in regulating ripening-associated pathogen susceptibility.

Phytosterol content and composition and sterol C-22 desaturase (LeSD1; CYP710A11) transcript levels in pericarp tissue of 'Rutgers' tomato fruit were compared in the wild-type (wt) and isogenic lines of the nonripening mutants nor and rin at four stages of ripening/aging. Wild-type fruit were harvested at the mature-green (MG), breaker (BK), breaker plus 3 days (B + 3), and breaker plus 6 days (B + 6) stages, whereas nor and rin fruits were harvested at comparable chronological ages (days after pollination). At the MG stage, wt and mutant fruits had closely similar sterol contents, compositions, and conjugations, with >91% of the total sterols in the acylated steryl glycoside plus steryl glycoside (ASG + SG) fraction. During ripening/aging, there were substantial increases in total sterols and the percentage of sterols in the free plus esterified (FS + SE) fraction. Both changes were greater in wt than in nor or rin. In fruit of wt, rin, and nor, respectively, the increases in total sterols between MG and B + 6 were 2.1-, 1.9-, and 1.5-fold, and at B + 6 the percentages of total sterols in FS + SE were 42, 21, and 24. Among all sterol lipids (ASG, SG, FS, and SE), the ratio of stigmasterol (stigmasta-5,22-dien-3beta-ol) to beta-sitosterol (stigmast-5-en-3beta-ol), the two major sterols in tomato, increased 2.3-fold during ripening of wt fruit but declined slightly during comparable aging of nor and rin fruits. In accord with these changes, the abundance of LeSD1 transcript increased 4-fold in pericarp of ripening wt fruit, peaking at B + 3, whereas transcript levels in nor and rin fruits fluctuated but never exceeded the abundance in wt fruit at the MG stage. These findings indicate that the ripening-specific increase in stigmasterol in wt fruit results from a marked increase in LeSD1 transcription and translation, which accelerates C-22 desaturation of the precursor sterol, beta-sitosterol.

This review contains functional roles of NAC transcription factors in the transcriptional regulation of ripening in tomato fruit, describes the interplay between ABA/ethylene and NAC TFs in tomato fruit ripening. Fruit ripening is regulated by a complex network of transcription factors (TFs) and genetic regulators in response to endogenous hormones and external signals. Studying the regulation of fruit ripening has important significance for controlling fruit quality, enhancing nutritional value, improving storage conditions and extending shelf-life. Plant-specific NAC (named after no apical meristem (NAM), Arabidopsis transcription activator factor 1/2 (ATAF1/2) and Cup-shaped cotyledon (CUC2)) TFs play essential roles in plant development, ripening and stress responses. In this review, we summarize the recent progress on the regulation of NAC TFs in fruit ripening, discuss the interactions between NAC and other factors in controlling fruit development and ripening, and emphasize how NAC TFs are involved in tomato fruit ripening through the ethylene and abscisic acid (ABA) pathways. The signaling network regulating ripening is complex, and both hormones and individual TFs can affect the status or activity of other network participants, which can alter the overall ripening network regulation, including response signals and fruit ripening. Our review helps in the systematic understanding of the regulation of NAC TFs involved in fruit ripening and provides a basis for the development or establishment of complex ripening regulatory network models.

Ripening is the main physiological process affecting banana (Musa spp.) fruit quality traits. The progress of banana ripening process differs for fruit ripened on the plant versus green harvested bunches and depends on the treatment of the fruit after harvest. We investigated the effect of the mode of ripening on ethylene biosynthesis of 'IDN 110' (Musa acuminata, AA genome) banana fruit ripened (a) in planta (On-Plant); (b) ex planta in air (Air-Fruit); or (c) after acetylene treatment (Ace-Fruit). The levels of ethylene production of the whole fruit and, those of 1-aminocyclopropane-1-carboxylic acid (ACC) in pulp, and ACC oxidase (MA-ACO1 and MA-ACO2) and ACC synthase (MA-ACS1) mRNA in both peel and pulp tissues were examined. From harvesting at mature-green stage, the ripening speed of fruit was not correlated with ethylene production. Ace-Fruit took 10 days to reach overripe stage with a maximum of 22.6 μl kg -1 h -1 of ethylene production, whereas Air-Fruit and On-Plant fruit took 27 and 33 days to reach overripe stage, respectively, and produced 11.5 and 29.6 μl kg -1 h -1 of ethylene, respectively. During ripening, ACC accumulated differentially; except for On-Plant fruit, ACC level increased during ripening and concomitantly with ethylene production and MA-ACS1 mRNA level. Whatever the mode of ripening, the level of MA-ACO1 mRNA was 100-fold higher than that of MA-ACO2. The mRNA level of MA-ACO1 and MA-ACO2 were accumulated sequentially during fruit development and ripening. MA-ACO1 gene was transiently induced between mature green and ripe stages while that of MA-ACO2 increased mainly at the overripe stage. The pattern of MA-AC02 gene expression was correlated with that of ethylene production whatever the mode of ripening while this correlation was observed with MA-ACO1 only On-Plant fruit. These results suggest that: (a) the level of ripening-ethylene production of the whole fruit is not the sole factor controlling the speed of fruit ripening in planta; (b) this level is regulated at the downstream step of ACC biosynthesis mediated by the MA-ACS1 gene; and (c) the product of MA-ACO2 might be involved in this regulation in pulp tissue. These findings are also discussed in regard with improvement of banana quality traits project throughout breeding programs.

To study ripening-related chilling injury in pepper ( Capsicum annuum L.), chilling-tolerant ‘Buchon’ and chilling-sensitive ‘Nockgwang’ fruit were harvested at mature green (MG), breaker (BR), and red-ripe (RR) stages and stored at 1, 5, and 10 °C for 21 d. ‘Buchon’ did not show surface pitting (SP) regardless of ripeness stage and storage temperature, whereas ‘Nockgwang’ at MG and BR exhibited SP at 1 and 5 °C. After 14 days of storage at 1 °C, chilling-sensitive ‘Nockgwang’ did not show SP when fruit were at the RR stage. Compared with ‘Buchon’, ‘Nockgwang’ at MG and BR had more electrolyte leakage increase during storage at 1 and 5 °C. ‘Buchon’ at all ripeness stages showed significantly higher ethylene production during storage regardless of storage temperatures. Contents of β-carotene and lycopene increased in both cultivars as ripening progressed. The contents of β-carotene and lycopene were similar between the two cultivars regardless of storage temperatures and ripeness stages. Susceptibility of pepper fruit to chilling appeared to be related to superoxide dismutase (SOD) and catalase (CAT). Activities of SOD and CAT were much higher in ‘Buchon’ than ‘Nockgwang’, more apparently at MG and BR. The results suggest that chilling-tolerant ‘Buchon’ and fruit at RR could have been equipped with a more efficient antioxidizing system, even if it was not clear whether oxidative stress is a cause or an effect of the CI in pepper.