Chinese flowering cabbage (Brassica campestris L. spp. chinensis var. utilis Tsen et Lee), a leafy vegetable with a flowering stem, is a specialty vegetable mainly produced in southern China and consumed worldwide (Zhang et al., 2013). However, the storage and transportation of Chinese flowering cabbage are restricted because the leaves are prone to yellowing after harvest. Leaf yellowing is a major indicator of leaf senescence. It is the main cause of a decline in its edibility and commodity value, leading to severe postharvest losses of the leafy vegetables (Porat et al., 2018). After harvest, vegetables are usually stored and transported under a weak light or even dark environment. Water loss and light deprivation are the key elements accelerating leaf senescence and strongly decrease shelf-life of vegetables (Liebsch and Keech, 2016). Chloroplast is the first organelle to be dismantled during leaf senescence. Degradation of chlorophyll is a signal involved in the senescence-associated degradation processes (Krieger-Liszkay et al., 2019). As an essential cofactor of the photosystem in chloroplasts, chlorophyll is important for photosynthesis. Research showed that the decline in photosynthetic activity and the downregulation of photosynthesis-associated genes expression are closely related to the increased expression of major senescence-associated genes (SAGs; Hensel et al., 1993). Therefore, the chlorophyll degradation and photosynthesis decline are important events during shade-induced leaf senescence.

Chlorophyll degradation in plant is basically promoted by a biochemical chlorophyll breakdown pathway, termed the “pheophorbide a oxygenase (PAO)/phyllobilin” pathway (Kuai et al., 2018). The pathway involves a series of chlorophyll catabolic enzymes (CCEs), including non-yellow coloring1 (NYC1), NYC1-like (NOL), stay-green (SGR), SGR-like (SGRL), pheophytin pheophorbide hydrolase (PPH), and pheide a oxidase (PAO; Kuai et al., 2018; Aubry et al., 2020). Chlorophyll breakdown process is regulated by internal and external stimuli during leaf senescence. Studies have confirmed that reactive oxygen species (ROS) production (oxidative burst) is closely related to aging metabolism in plants, and H₂O₂ over-accumulation is an important leaf senescence promoter (Guo et al., 2017). H₂O₂ plays a dual role in regulating plant physiological metabolism: It acts as a regulatory signal molecule in plant development and in abiotic or biotic stress responses at low concentrations (Rogers and Munné-Bosch, 2016). Exogenous H₂O₂ treatment can activate antioxidant defense systems and improve plant resistance to abiotic stress (Zhang et al., 2011). Strong antioxidant properties play an important role in delaying cell senescence. Postharvest treatments with H₂O₂ maintains fruit quality by activating the antioxidant defense system (Chumyam et al., 2019). However, to the best of the authors’ knowledge, there has been no research so far using exogenous H₂O₂ treatment preharvest for delaying leaf senescence in vegetables.

The aim of this study was to investigate the possible role of preharvest treatment with H₂O₂ on delaying leaf senescence in Chinese flowering cabbage. We studied the modulatory effects on leaf yellowing, lipid oxidation, cell integrity, chlorophyll content, photochemical efficiency and their relationships to oxidative stress and water loss. We also studied its regulation on proteins and expression levels of related key genes involved in ROS accumulation, chlorophyll synthesis/degradation, and photosynthesis capacity during cabbage storage. The findings of this study provide new insights into the mechanisms underlying exogenous H₂O₂-delayed leaf yellowing in Chinese flowering cabbage.

Leaf senescence is the primary factor affecting commodity value of leafy vegetables during postharvest storage and distribution. Over the past decades, researchers have been attempting to develop techniques to delay leaf senescence. H₂O₂ is an important signaling molecule playing a dual role in regulating plant senescence. Excessive accumulation of H₂O₂ can accelerate leaf senescence (Guo et al., 2017). However, application with low concentration of exogenous H₂O₂ can activate the antioxidant enzymes under environmental stresses (Zhang et al., 2011). The results here showed that preharvest application of 10 mM H₂O₂ to root effectively alleviated leaf yellowing and prolonged the shelf life of Chinese flowering cabbage after harvest. The retardation of leaf senescence during storage was manifested in lower oxidative stress, water loss, and leaf yellowing indexes, as well as higher chlorophyll levels and photosynthetic capacity. To the best of our knowledge, this is the first time showing that treating vegetable roots with low H₂O₂ before harvest can delay leaf senescence during storage.

Leaf senescence, regarded as the final stage of leaf development, is a highly controlled process requiring massive transcriptional regulations (Liebsch and Keech, 2016). The upregulated genes involves in the senescence process have been designated as SAGs, among which SAG12 is a senescence-specific marker gene and be widely used as the reference for evaluating the leaf senescence (Carrión et al., 2013). The expression of BrSAG12 in Chinese flowering cabbage is markedly upregulated as leaf got yellowed during storage. However, the upregulation of BrSAG12 was notably suppressed in H₂O₂ treatment group in comparison to control group (Figures 1A–D). Leaf yellowing, in considered a symptom of senescence, involves disassembly of chloroplast and breakdown of related protein complexes (Brouwer et al., 2012). Chloroplast is the first organelle to be dismantled during leaf senescence, and chlorophyll degradation is one of the most important events (Schippers et al., 2015). In the present study, the contents of chlorophyll a and b in leaves during storage declined with the upregulating of BrSAG12. However, both the chlorophyll a and b were preserved in the H₂O₂-treated plants (Figures 4A,B). These results suggest that preharvest treatment with H₂O₂ delay leaf senescence and alleviate leaf yellowing during storage.

During leaf senescence, the chlorophyll generally decline as the chlorophyll catabolic genes are preferentially enhanced and chlorophyll biosynthesis genes are down-regulated (Wang et al., 2020). The “PAO/phyllobilin pathway” is a dedicated pathway responsible for the degradation of chlorophyll (Figure 7A), which is associated with SGRs and some CCEs, including NOL, NYC, and PAO (Kuai et al., 2018). Studies have demonstrated that SGRs or NYC mutants showed a stay-green phenotype in Arabidopsis, while the over-expression of SGRs induces premature yellowing in leaves, suggesting that SGRs and NYC are the critical genes for accelerating chlorophyll degradation (Yamatani et al., 2013; Sakuraba et al., 2014). Here, the expression levels of BrNYC1 and BrSGRs (BrSGR1, BrSGR2, and BrSGRL) were significantly suppressed in the H₂O₂ treatment group in comparison to that in the control group (Supplementary Figure S.3). Furthermore, accumulation of NYC1 and SGRs proteins in the H₂O₂ treatment group showed the same trend (Figure 3E). These results indicate that preharvest treatment with H₂O₂ maintained higher level of chlorophyll is correlated with the regulation on PAO/phyllobilin pathway.

Nevertheless, changes of chlorophyll content in senescent leaves is determined by the relative rates of chlorophyll degradation and synthesis processes that occur in chloroplasts (Hörtensteiner, 2006). Chlorophyll synthesis is still one of the critical processes for maintaining chlorophyll content, because chlorophyll synthesis process is detectable until the final stage of the lifespan in leaves (Hörtensteiner, 2006). The chlorophyll synthesis process in higher plants occurs through a specific pathway, in which Hems (HemA, L, B, and C), Chls (ChlE, M, I, and G), and CAO proteins are the key enzymes involved in the process (Figure 7B; Wang and Grimm, 2015). Our results showed that the main DEPs including HemA, ChlE, ChlM, ChlI, and CAO proteins were markedly upregulated in H₂O₂ treatment group in comparison with control group (Figure 3E), suggesting preharvest H₂O₂ treatment maintained leaf chlorophyll biosynthesis during storage. The results here indicate that preharvest treatment with exogenous H₂O₂ maintained chlorophyll content by simultaneously suppressing chlorophyll degradation and preserving chlorophyll synthesis in harvested cabbage. This can account for the higher chlorophyll a and b content in H₂O₂-treated Chinese flowering cabbage (Figures 4A,B).

Chlorophyll degradation during leaf senescence is a highly complex and ordered process can be triggered by internal age-dependent factors and external biotic and/or abiotic stresses (Zhang et al., 2021). After harvest, leafy vegetables are particularly prone to rapid dehydration due to their large surface area, and retention of water is crucial for their storage. It has been shown that excessive water loss can initiate chlorophyll degradation and induce rapid senescence in leaves, whereas an increase in proline can reduce water loss and delay leaf senescence (Jafari et al., 2019; Hanif et al., 2021). H₂O₂ acts as a signal molecule is involved in plant physiological metabolism, such as guard cell physiology, and helps plants against drought stress (Singh et al., 2017). It was showed that H₂O₂ plays an important role in ABA-induced stomatal closure and water retention (Watkins et al., 2017). The results here showed that water loss of H₂O₂ treatment group was lower than that of control group during storage (Figure 5A). The results were consistent with the regulations of proline content and leaf stomatal aperture in the H₂O₂ treatment group (Figures 5B–D). The biological process annotation also revealed that the main DEPs induced by H₂O₂ treatment were involved in regulation of stomatal opening and response to water deprivation (Figure 2D). These findings indicate that preharvest H₂O₂ treatment mediated leaf senescence during storage at least partly by reducing water loss.

In addition to water loss, shading should be another major cause accelerating chlorophyll degradation and leaf yellowing when cabbages were stored under a weak light condition after harvest. It has been shown that weak light or darkness can initiate chlorophyll degradation and induce leaf senescence (Liebsch and Keech, 2016). Under dark environment, leaf senescence was manifested in an increase in ROS level followed by a subsequent transcriptional regulation of senescence marker genes SAG12 (Rosenvasser et al., 2006). ROS is considered as the main factor to initiate the senescence program and chlorophyll degradation, and the expression of Rbohs were upregulated for production of endogenous ROS during the leaf aging process (Yu et al., 2019). ROS accumulation in cells can cause oxidative damage to membranes, lipids, proteins, and other macromolecules including DNA and RNA (Niu et al., 2020). This was confirmed in the present study that endogenous H₂O₂ in leaves gradually increased during storage and causes lipid peroxidation and membrane damage (Figures 6A–E). However, alleviation of water loss and oxidative stress are beneficial for maintaining membrane function and reduce chloroplast destruction (Figures 4–6). Undoubtedly, a high content of H₂O₂ may cause oxidative damage, leading to chlorophyll degradation, organelle dysfunction and plant senescence (Zhang et al., 2015). However, studies have also demonstrated that H₂O₂ at a low concentration serves as a secondary messenger in signaling pathways (Rogers and Munné-Bosch, 2016). Exogenous H₂O₂ can act as an activator to stimulate the antioxidant defense system, helping to reduce oxidative damage and cell senescence (Chumyam et al., 2019). Our experiments demonstrated that leaf yellowing of Chinese flowering cabbage was accelerated by high concentration of H₂O₂ application, whereas it was delayed by low concentrations (<50 mM) of exogenous H₂O₂ application to roots (Supplementary Figure S.1). It was showed that preharvest treatment with 10 mM H₂O₂ markedly delays the leaf senescence during storage. The treatment suppressed the accumulation of endogenous H₂O₂, reduced lipid peroxidation and protected the cell integrity (Figures 6A–F). These results may be attributed to the attenuation of oxidative stress that result from the activating of antioxidant defense system by preharvest treatment with low concentration of H₂O₂. It can be accounted for by the upregulated expression of ROS scavenging-related genes (BrDHAR, BrMDHAR, BrSOD, BrCAT, and BrAPX) and the suppression of ROS biosynthesis-related genes (BrRbohB, BrRbohC, and BrRbohD; Figures 6G,H). The above results are consistent with the GO functional annotation in proteomic analysis, which showed that the main DEPs induced by H₂O₂ treatment were involved in catalytic activity, antioxidant activity, and transmembrane transporter activity (Figure 2; Supplementary Figure S.2). The activation of antioxidant defense system in leaf tissues can be beneficial for reducing membrane lipid oxidation and suppressing the up-regulation of SAG12 expression and finally delaying leaf senescence during storage (Figure 8).

Chloroplast is one of the most extensive membrane systems found in nature, and chloroplast membranes are rich in lipids and are susceptible to oxidative stress (Cook et al., 2021). Excessive accumulation of ROS is a major cause of chloroplast membranes destruction. The oxidative damage in thylakoid membranes will affect the photosynthetic capacity and accelerate senescence-associated degradation processes in leaves (Krieger-Liszkay et al., 2019). Maintaining strong antioxidant capacity should be a guarantee of the normal membranes function and photosynthetic activities (Exposito-Rodriguez et al., 2017). The decline of photosynthesis in leaves is correlated with the destruction of membranes and degradation of chlorophyll, as chlorophyll is the main component of thylakoid membrane–protein complexes such as PS II, PS I and the cytochrome b6f complex (Figures 7C,D; Sato et al., 2009). The proteins Lhcbs and Lhcas in PS II and PS I, combined with chlorophyll, function as antenna pigment coordinators that play an important role in light harvesting (Wei et al., 2016). During leaf senescence, the breakdown of light-harvesting chlorophyll protein complexes of LHCII is associated with the degradation of chlorophyll (Sakuraba et al., 2015). It was shown that chlorophyll b degradation catalyzed by CCEs in senescent leaves is required for the degradation of LHCII and thylakoid grana. In contrast, in the stay-green phenotype NYC1 mutant, LHCII was selectively retained due to the chlorophyll b degradation significantly inhibited (Sato et al., 2009). The results here show that degradation of the light-harvesting complexes (Lhcb2, Lhcb4, Lhcb6, Lhca1, Lhca3, and Lhca4) in the control group is closely related to the decline of chlorophyll, which indicate abnormal chloroplast function and decline of photosynthetic capacity during leaf senescence. However, the Lhc proteins in leaves during storage were preserved by preharvest treatment with H₂O₂ (Figures 3C,D). These results suggest that protecting membranes from oxidative damage have crucial implications for the light-harvesting and photosynthetic capacity of leaves.

Moreover, the subunits in PS II and PS I, including PsbC, PsbE, PsbR, Psb27, and PsbG were also protected by preharvest H₂O₂ treatment (Figures 3C,D). These subunits are the core components of photosynthetic system. They embedded in membranes and assemble into photosynthetic complexes in plants (Figures 7C,D). Photosynthetic capacity in plants is closely related to some subunits in PS II, PS I, and ATP synthase (Suga and Shen, 2020). F-type ATPases are membrane integrated enzymes localized in the thylakoid membranes and act primarily as ATP synthases that generate the largest amount of ATP for photosynthesis activity (Kühlbrandt, 2019). In this study, preharvest 10 mM H₂O₂ treatment protected F-type ATPases (ATPF0B, ATPF1D; Figure 3C), implying more energy supply for leaf photosynthesis activity. The results of cellular components annotation in proteomic analysis revealed that the upregulated DEPs observed in the H₂O₂ treatment group are involved in components of chloroplast membranes (Figures 2F,G), and the KEGG pathways of light harvesting and photosynthesis (Figures 3A,B). This can account for the higher Fv/Fm value in H₂O₂ treatment group relative to the control group (Figures 4C,D), suggesting preharvest H₂O₂ treatment protect chloroplast function of Chinese flowering cabbage during storage. It was showed that the decline in photosynthesis-associated proteins and photosynthetic activity are the first events occurring at the onset of shade-induced leaf senescence (Brouwer et al., 2012; Krieger-Liszkay et al., 2019). Therefore, the protection in chloroplast function and photosynthesis activity in leaves can be beneficial for attenuating leaf senescence during storage under a weak light environment. These results were consistent with the expression level of senescence marker gene BrSAG12 in leaves during storage (Figure 1D).

In conclusion, preharvest application of low H₂O₂ to roots reduced water loss by modulating proline and the stomatal aperture in Chinese flowering cabbage during storage. The treatment also activated the antioxidant defense system, reduced lipid peroxidation, protected cell membranes system, maintained cell integrity, and then suppressed the upregulation of senescence marker gene BrSAG12. Therefore, the treatment suppressed chlorophyll degradation, preserved chlorophyll content, protected the chloroplast function, and finally delayed the leaf yellowing (Figure 8). These results suggest that preharvest H₂O₂ treatment delay leaf yellowing during storage by alleviating water loss and oxidative stress. The findings provide possibility for developing new techniques to extend the shelf life of Chinese flowering cabbage or other leafy vegetables.

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

GW and SZ contributed to conception and design of the study and wrote sections of the manuscript. MP and YW organized the database. MP and ZC performed the statistical analysis. GW wrote the first draft of the manuscript. All authors contributed to the article and approved the submitted version.

This work was financially supported by the National Natural Science Foundation of China (31901751), Natural Science Foundation of Guangdong Province (2020A1515010024), and Colleges Innovation Project of Guangdong (2018KQNCX017).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.