Abstract
Tomato breeding has traditionally prioritized agronomic traits, such as yield and pest resistance, thereby inadvertently reducing volatile organic compounds (VOCs) and negatively affecting flavor perception (Colantonio et al. 2022). Advances in VOC chemistry and biosynthetic gene identification have spurred efforts to restore tomato flavor (Garbowicz et al. 2018, Martina et al. 2021, Villena et al. 2023). However, the mechanisms influencing VOC synthesis remain largely unexplored. STAY-GREEN 1 (SGR1) regulates chlorophyll degradation in the tomato (Hu et al. 2011). Although SGR1 loss improves fruit traits, including carotenoid content, antioxidants, and shelf life (Luo et al. 2013, Kim et al. 2022, Kim et al. 2024), its influence on VOCs remains unknown. To address this, we used CRISPR-Cas9 to generate SlSGR1 (Solyc08g080090) knockout (sgr1) mutants in the “Ailsa Craig” cultivar, a widely used model for tomato fruit research. Eight T₀ lines with biallelic or heterozygous mutations were obtained (Supplementary Fig. S1), and two T₂ homozygous lines (ko-1 and ko-14) with a 1-bp insertion in exon 1 (A and T, respectively), but no T-DNA integration, were selected. These mutations caused a frameshift and introduced a premature stop codon, which is predicted to produce a truncated 12-amino-acid fragment instead of the full-length 272-amino acid protein (Fig. 1a, Supplementary Fig. S2). These lines showed a 30%–55% reduced SlSGR1 expression during fruit ripening (T–R stages) compared with that in the wild type (WT) (Supplementary Fig. S3). The mutants exhibited normal growth but delayed leaf senescence (Supplementary Fig. S4). To assess protein function, WT and mutant SGR1 were fused to a Flag tag and expressed in Nicotiana benthamiana. WT:Flag was detected and triggered senescence, whereas mutant:Flag was undetectable and caused no response (Supplementary Fig. S5), confirming sgr1 as a null mutation. Enhanced VOC production in tomato fruits with SlSGR1 mutation. (a) Illustration showing SlSGR1 gene structure, sgRNA target site with PAM sequences, and the resulting edited sequences in sgr1 knockout plants. (b) Images of fruits under various stages of ripening and the chlorophyll content in WT and sgr1 knockout fruits. (c) Principal component analysis of VOCs detected at six developmental stages in WT and sgr1 knockout fruits. (d) Representative VOCs significantly increased in sgr1 knockout fruits among those depicted in Supplementary Fig. S7. (e) Relative expression levels of genes associated with the production of carotenoid- and fatty acid-derived VOCs in WT and sgr1 knockout fruits, as quantified by qPCR. (f) Quantification of carotenoids and free fatty acids in WT and sgr1 knockout fruits. (g) Plastid number per cell in the outer pericarp of WT and sgr1 knock-out lines across ripening stages. Stacked bars denote the number of chloroplasts (green) and chromoplasts (orange). Representative bright-field images of outer pericarp cells at MG, B, and R stages are shown on the right. Scale bar represents 100 μm. Data in (b), (e), and (f) represent the means (±SE) of three biological replicates. Each replicate consists of a pool of three fruits. Data in (g) represent the means (±SE) of four to six biological replicates. Statistical differences between WT and sgr1 knockout lines were determined using an unpaired t-test: *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. MG, mature green; B, breaker; T, turning; O, orange; R, red; OR, over-ripe; VOCs, volatile organic compounds; WT, wild-type. Leaf phenotype assessments and spectrophotometry revealed that sgr1 lines retained chlorophyll across young, mature, and old leaves until 11 weeks, whereas chlorophyll was fully degraded in the old leaves of the WT by this stage (Supplementary Fig. S6). Similarly, sgr1 knockout fruits maintained higher chlorophyll levels than those in the WT throughout development, with a 5-fold increase after the O stage (Fig. 1b), indicating delayed chlorophyll degradation in both leaves and fruits. VOCs were analyzed across six ripening stages in WT and sgr1 knockout fruits, leading to the identification of 31 distinct VOCs. These compounds were categorized into four groups based on their biosynthetic origins: carotenoid-, fatty acid (FA)-, phenylalanine-, and branched-chain amino acid-derived VOCs (Supplementary Table S2). Principal component analysis of the VOCs indicated that WT fruits clustered by developmental stages, while sgr1 knockout fruits were distinct from the WT at each corresponding stage. Notably, sgr1 knockout fruits at the OR stage differed significantly from WT fruits at all ripening stages (Fig. 1c). Moreover, a heatmap analysis demonstrated that the majority of VOCs accumulated more in sgr1 knockout fruits than in WT fruits during ripening (Supplementary Fig. S7). Carotenoid- and FA-derived VOCs increased markedly in the knockout fruits, peaking at the OR stage (Fig. 1d). These findings provide the first evidence of SlSGR1’s essential role in affecting VOC production in tomato fruits. To determine the cause of increased VOC accumulation in sgr1 knockout fruits, we analyzed gene expression related to carotenoid- and FA-derived VOCs. For genes involved in carotenoid synthesis, sgr1 knockout fruits exhibited elevated expression of phytoene synthase 1 (PSY1) and lycopene-β-cyclase (CYC-B), while cleavage dioxygenase 1B (CCD1B) remained unchanged, and CCD1A was reduced at most ripening stages, likely due to feedback regulation (Fig. 1e, Supplementary Fig. S8a). For FA-derived VOCs, among lipase 1 (LIP1) and LIP8, which contribute to FA production—precursors of FA-derived VOCs—the expression of LIP1 (Garbowicz et al. 2018) was substantially higher in sgr1 knockout fruits at the O and R stages than in WT fruits. However, the expression of LIP8 and other downstream genes did not differ between sgr1 knockout and WT fruits (Fig. 1e, Supplementary Fig. S8b). We further investigated whether increased PSY and CYC-B expression affected carotenoid levels and found that sgr1 knockout fruits accumulated higher levels of lycopene, β-carotene, and lutein than WT fruits at the R and OR stages (Fig. 1f). Additionally, we measured the FA content to assess whether increased LIP1 expression boosted FA production. The levels of 18:0, 18:1, and 18:2 FA forms were higher in sgr1 knockout fruits than in the WT, particularly at the OR stage (Fig. 1f), suggesting that SlSGR1 deficiency leads to altered expression of multiple genes involved in the synthesis of substrates for carotenoid- and FA-derived VOC production. Previous studies have shown that tomato mutants with more plastids, like hp1 and hp2, produce more chlorophyll and carotenoids, which enhances the levels of fatty acid-derived VOCs (Kovács et al. 2009). Similarly, our study found that sgr1 knockout fruits have more chloroplasts than WT fruits throughout all ripening stages (Fig. 1g). From the orange stage onward, sgr1 fruits also exhibited faster chloroplast-to-chromoplast conversion (Fig. 1g). This suggests that the increased plastid number and accelerated differentiation in sgr1 fruits contribute to their higher levels of carotenoids, fatty acids, and related VOCs. Future research should elucidate the precise mechanism by which SGR1 deficiency affects chloroplast proliferation. Improving flavor, a key factor in increasing tomato appeal to consumers, represents a significant challenge in plant breeding. This work provides direct evidence of the pivotal role of SlSGR1 in the production of VOCs, particularly carotenoid- and FA-derived VOCs. Furthermore, we demonstrate that the increased VOCs result from changes in the expression of genes involved in VOC synthesis, which enhances precursor production. These findings provide valuable insights into the influence of SlSGR1 on VOC synthesis and establish a crucial foundation for leveraging SlSGR1 to enhance tomato flavor and overall fruit quality, contributing to the development of high-value tomato varieties. J.-I.C., G.-H.H., and J.-H.K. designed the experiments. J.-I.C., G.-H.H., S.-Y.K., S.-M.K., G.-J.K., B.K., and J.Y.C. performed the experiments. J.-I.C., G.-H.H., H.-Y.K., J.K.K., and J.-H.K. analyzed the results. J.-H.K. wrote the manuscript with input from all co-authors. All authors read and approved of the manuscript. None declared. This work was supported by grants from the New Breeding Technologies Development Program (RS-2024-00322125) and from the Basic Science Research Program (NRF-2022R1A2C1008643) of the Republic of Korea. All data are incorporated into the article and its online supplementary material.
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Claims 20
Claim · model-written summary
Two homozygous sgr1 lines had one-base insertions without T-DNA integration.
- Taxon
- tomato
Solanum lycopersicumt0 - Material
- ko-1 and ko-14e0
- Outcome
- a 1-bp insertion in exon 1e0
- Value
- 1-bpe0
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
t0“tomato”
e0“two T₂ homozygous lines (ko-1 and ko-14) with a 1-bp insertion in exon 1 (A and T, respectively), but no T-DNA integration”
Claim · model-written summary
The insertions caused a frameshift and premature stop codon, predicting a truncated protein.
- Taxon
- tomatoSolanum lycopersicumt0
- Gene
- SlSGR1e1
- Outcome
- a frameshift and introduced a premature stop codone0
- Value
- 12-amino-acid fragmente0
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
t0“tomato”
e0“These mutations caused a frameshift and introduced a premature stop codon, which is predicted to produce a truncated 12-amino-acid fragment instead of the full-length 272-amino acid protein”
e1“SlSGR1 (Solyc08g080090) knockout”
Claim · model-written summary
SlSGR1 expression was 30%–55% lower during fruit ripening in mutants than in WT.
- Taxon
- tomatoSolanum lycopersicumt0
- Organ / stage
- fruit ripening (T–R stages)e0
- Gene
- SlSGR1e0
- Outcome
- SlSGR1 expressione0
- Direction
- reducede0
- Value
- 30%–55%e0
- Comparator
- wild type (WT)e0
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
t0“tomato”
e0“These lines showed a 30%–55% reduced SlSGR1 expression during fruit ripening (T–R stages) compared with that in the wild type (WT)”
Claim · model-written summary
Mutant plants had normal growth but delayed leaf senescence.
- Taxon
- tomatoSolanum lycopersicumt0
- Organ / stage
- leafe0
- Outcome
- delayed leaf senescencee0
- Direction
- delayede0
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
t0“tomato”
e0“The mutants exhibited normal growth but delayed leaf senescence”
Claim · model-written summary
WT SGR1 protein was detected and triggered senescence when expressed in Nicotiana benthamiana.
- Taxon
- tomatoSolanum lycopersicumt0
- Interactor
- Nicotiana benthamiana
Nicotiana benthamianae1other - Gene
- WT:Flage0
- Outcome
- triggered senescencee0
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
t0“tomato”
e0“WT:Flag was detected and triggered senescence, whereas mutant:Flag was undetectable and caused no response”
e1“expressed in Nicotiana benthamiana”
Claim · model-written summary
Mutant SGR1 protein was undetectable and caused no senescence response.
- Taxon
- tomatoSolanum lycopersicumt0
- Gene
- mutant:Flage0
- Outcome
- mutant:Flag was undetectable and caused no responsee0
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
t0“tomato”
e0“WT:Flag was detected and triggered senescence, whereas mutant:Flag was undetectable and caused no response”
Claim · model-written summary
Mutant leaves retained chlorophyll until 11 weeks, unlike old WT leaves.
- Taxon
- tomatoSolanum lycopersicumt0
- Organ / stage
- leavese0
- Outcome
- retained chlorophylle0
- Value
- 11 weekse0
- Comparator
- WTe0
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
t0“tomato”
e0“sgr1 lines retained chlorophyll across young, mature, and old leaves until 11 weeks, whereas chlorophyll was fully degraded in the old leaves of the WT by this stage”
Claim · model-written summary
Knockout fruits maintained higher chlorophyll, with a fivefold increase after the orange stage.
- Taxon
- tomatoSolanum lycopersicumt0
- Organ / stage
- fruitse0
- Outcome
- chlorophyll levelse0
- Direction
- highere0
- Value
- 5-folde0
- Comparator
- WTe0
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
t0“tomato”
e0“sgr1 knockout fruits maintained higher chlorophyll levels than those in the WT throughout development, with a 5-fold increase after the O stage”
Claim · model-written summary
Thirty-one distinct VOCs were identified across fruit ripening stages.
- Taxon
- tomatoSolanum lycopersicumt0
- Organ / stage
- fruitse0
- Outcome
- distinct VOCse0
- Value
- 31e0
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
t0“tomato”
e0“VOCs were analyzed across six ripening stages in WT and sgr1 knockout fruits, leading to the identification of 31 distinct VOCs”
Claim · model-written summary
Knockout fruit VOC profiles differed from WT at corresponding stages, especially at over-ripe stage.
- Taxon
- tomatoSolanum lycopersicumt0
- Organ / stage
- fruitse0
- Outcome
- sgr1 knockout fruits were distinct from the WTe0
- Comparator
- WTe0
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
t0“tomato”
e0“sgr1 knockout fruits were distinct from the WT at each corresponding stage. Notably, sgr1 knockout fruits at the OR stage differed significantly from WT fruits at all ripening stages”
Claim · model-written summary
Most VOCs accumulated more in knockout fruit during ripening.
- Taxon
- tomatoSolanum lycopersicumt0
- Organ / stage
- fruitse0
- Outcome
- VOCse0
- Direction
- moree0
- Comparator
- WT fruitse0
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
t0“tomato”
e0“the majority of VOCs accumulated more in sgr1 knockout fruits than in WT fruits during ripening”
Claim · model-written summary
Carotenoid-derived VOCs increased markedly and peaked at over-ripe stage.
- Taxon
- tomatoSolanum lycopersicumt0
- Organ / stage
- fruitse0
- Outcome
- Carotenoid- and FA-derived VOCse0
- Direction
- increasede0
- Qualifier
- OR stagee0
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
t0“tomato”
e0“Carotenoid- and FA-derived VOCs increased markedly in the knockout fruits, peaking at the OR stage”
Claim · model-written summary
PSY1 and CYC-B expression was elevated in knockout fruit.
- Taxon
- tomatoSolanum lycopersicumt0
- Gene
- phytoene synthase 1 (PSY1) and lycopene-β-cyclase (CYC-B)e0
- Outcome
- elevated expressione0
- Direction
- elevatede0
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
t0“tomato”
e0“sgr1 knockout fruits exhibited elevated expression of phytoene synthase 1 (PSY1) and lycopene-β-cyclase (CYC-B)”
Claim · model-written summary
CCD1B expression was unchanged in knockout fruit.
- Taxon
- tomatoSolanum lycopersicumt0
- Gene
- cleavage dioxygenase 1B (CCD1B)e0
- Outcome
- cleavage dioxygenase 1B (CCD1B)e0
- Direction
- unchangede0
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
t0“tomato”
e0“cleavage dioxygenase 1B (CCD1B) remained unchanged”
Claim · model-written summary
CCD1A expression was reduced at most ripening stages.
- Taxon
- tomatoSolanum lycopersicumt0
- Gene
- CCD1Ae0
- Outcome
- CCD1Ae0
- Direction
- reducede0
- Qualifier
- most ripening stagese0
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
t0“tomato”
e0“CCD1A was reduced at most ripening stages”
Claim · model-written summary
LIP1 expression was substantially higher at orange and red stages in knockout fruit.
- Taxon
- tomatoSolanum lycopersicumt0
- Organ / stage
- fruitse0
- Gene
- LIP1e0
- Outcome
- expression of LIP1e0
- Direction
- highere0
- Comparator
- WT fruitse0
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
t0“tomato”
e0“the expression of LIP1 (Garbowicz et al. 2018) was substantially higher in sgr1 knockout fruits at the O and R stages than in WT fruits”
Claim · model-written summary
LIP8 and other downstream gene expression did not differ between knockout and WT fruit.
- Taxon
- tomatoSolanum lycopersicumt0
- Gene
- LIP8e0
- Outcome
- expression of LIP8 and other downstream genese0
- Direction
- did not differe0
- Comparator
- WT fruitse0
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
t0“tomato”
e0“the expression of LIP8 and other downstream genes did not differ between sgr1 knockout and WT fruits”
Claim · model-written summary
Lycopene, β-carotene and lutein levels were higher in knockout fruit at red and over-ripe stages.
- Taxon
- tomatoSolanum lycopersicumt0
- Outcome
- levels of lycopene, β-carotene, and luteine0
- Direction
- highere0
- Comparator
- WT fruitse0
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
t0“tomato”
e0“sgr1 knockout fruits accumulated higher levels of lycopene, β-carotene, and lutein than WT fruits at the R and OR stages”
Claim · model-written summary
The 18:0, 18:1 and 18:2 fatty acid forms were higher in knockout fruit.
- Taxon
- tomatoSolanum lycopersicumt0
- Outcome
- levels of 18:0, 18:1, and 18:2 FA formse0
- Direction
- highere0
- Comparator
- WTe0
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
t0“tomato”
e0“The levels of 18:0, 18:1, and 18:2 FA forms were higher in sgr1 knockout fruits than in the WT, particularly at the OR stage”
Claim · model-written summary
Knockout fruits had more chloroplasts and faster chloroplast-to-chromoplast conversion.
- Taxon
- tomatoSolanum lycopersicumt0
- Outcome
- more chloroplastse0
- Direction
- moree0
- Comparator
- WT fruitse0
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
t0“tomato”
e0“sgr1 knockout fruits have more chloroplasts than WT fruits throughout all ripening stages (Fig. 1g). From the orange stage onward, sgr1 fruits also exhibited faster chloroplast-to-chromoplast conversion”