Application of tomato SlCSN5A protein or its encoding gene

Abstract

The present invention provides an application of a tomato SlCSN5A protein or a gene encoding it, and relates to the field of biological technology. The application includes (a) regulating the ubiquitination level or stability of proteins in chloroplasts; (b) regulating the ubiquitination level or stability of the chloroplast vesicle protein SlCV; (c) regulating the response of tomatoes to low temperature stress. The present invention found that LNT stress treatment caused a decrease in the abundance and NPQ level of SlPsbS protein in tomato leaves, and SlCSN5A can interact with SlPsbS and promote its ubiquitination and degradation, and can be used to regulate the ubiquitination level or stability of SlPsbS protein and other proteins in chloroplasts. In addition, SlCSN5A is also involved in CV-mediated PsbS degradation and regulates the response of tomatoes to night low temperature stress. The present invention discloses a degradation pathway of chloroplast proteins that is dependent on ubiquitination and is mediated by SlCV and SlCSN5A.

Description

Technical Field

[0001] The present invention relates to the field of biological technology, and in particular to an application of a tomato S1CSN5A protein or a gene encoding the same. Background Art

[0002] Low temperature stress can disrupt almost all major components of photosynthesis, including thylakoid electron transport, carbon fixation, chloroplast morphology, and photoinhibition ( Lu et al., 2020 ; Zhang et al., 2014 ; Liu et al., 2012 ), which in turn affects crop yield and quality.

[0003] Low temperature slows down the metabolic consumption of NADPH and ATP through the Calvin-Benson-Bassham (CBB) cycle and other energy-consuming processes, resulting in excessive accumulation of ATP and NADPH at the end of the light reaction (Gan et al., 2019). Therefore, low temperature disrupts redox and energy dynamic balance and leads to higher photosystem II (PSII) excitation stress. Plants have evolved strategies to respond quickly to adversity. For example, PSII can quickly protect by dissipating excess excitation energy in the form of heat and reducing energy transfer to the reaction center through non-photochemical quenching (NPQ) (Gao et al., 2022; et al., 2017). NPQ is widely considered to be the main factor that rapidly regulates light harvesting to protect the PSII reaction center from photoinhibition (Ruban, 2016; Demmig-Adams et al., 2014). The core of the photoprotection mechanism lies in the response to the transmembrane pH gradient (ΔpH) (Krishnan-Schmieden et al., 2021). The PsbS protein has been identified as the main induced protein activated by NPQ in plants. It is constitutively present on the thylakoid membrane and is a member of the larger LHC superfamily, but unlike all other proteins in this family, it does not specifically bind any chlorophyll or xanthophyll pigment (Li et al., 2000). The PsbS protein contains protonatable amino acids that can sense signals from ΔpH and thylakoid lumen acidification and transmit the signals to the antenna, thereby enabling a switch to a photoprotective state through antenna-photosystem rearrangement (Ruban, 2018; Sacharz et al., 2017). PsbS protein accumulates rapidly and transiently under stress, while long-term or severe stress accelerates the degradation of PsbS (Redekop et al., 2020; Wang et al., 2018; Tibiletti et al., 2016; Redekop et al., 2020; Wang et al., 2018). At the same time, PsbS mutant plants have reduced NPQ capacity and are more susceptible to high light stress (Acebron et al., 2021). In summary, the stability of PsbS protein plays an important role in the response of plants to abiotic stress. However, the degradation mechanism of PsbS protein is still poorly understood.

[0004] Chloroplasts are highly sensitive to cold stress and have been considered as central sensors and responders to low temperatures (Gao et al., 2022). Under stress conditions, efficient photosynthesis and other metabolic reactions require the normal turnover of damaged chloroplasts and their specific components. Therefore, damaged chloroplasts or their specific components need to be effectively degraded to ensure normal cellular function (Yang et al., 2019). Recent studies have emphasized the relationship between ubiquitination and chloroplast-associated protein degradation. Under moderate stress conditions, the inhibitor of PPI1 site 1 (SP1) E3 ligase interacts with the Omp85-type barrel channel protein SP2 and cell division cycle 48 (CDC48A) to mediate the degradation of specific components of the outer membrane translocation protein of the chloroplast (TOC) complex through the 26S proteasome, while under severe stress conditions, the U-Box 4 (PUB4) E3 ligase mediates the ubiquitination of chloroplast membrane proteins and the subsequent degradation of damaged entire chloroplasts (Ling et al., 2019). In a recent study, CDC48A and its cofactor ubiquitin fusion degradation 1 (UFD1)-nucleoprotein localization 4 (NPL4) were involved in the ubiquitination and degradation of chloroplast proteins rbcL and AtpB ( Li et al., 2022 ).

[0005] The COP9 signalosome (CSN) is an evolutionarily conserved multi-subunit protein complex composed of eight subunits (CSN1-CSN8) (Singh et al., 2019). CSN is a metalloprotease that cleaves covalently linked RUB1 (Related Ubiquitin 1) / NEDD8 (neural precursor cell expressed, developmentally down-regulated 8) from the Cullin-Ring E3 ubiquitin ligase (CRLS) complex (Lyapina et al., 2001; Schwechheimer et al., 2001) and functions in the ubiquitin-proteasome pathway of protein degradation (Barth et al., 2016). This catalytic activity is located in the JAMM (JAB1 / MPN / Mov34 metalloenzyme, also known as MPN) motif of the CSN5 subunit (Cope et al., 2002). CSN5 is involved in a wide range of biological processes (Jin et al., 2014). In Arabidopsis, CSN5 is encoded by two genes, CSN5A and CSN5B (Jin et al., 2014). The csn5a mutant exhibits a reduction in growth, lateral root formation, root hair formation, and flower size, whereas the csn5b mutant does not show any detectable growth abnormalities and is essentially indistinguishable from the wild type (Gusmaroli et al., 2007). While the csn5a mutant displays significant accumulation of neddylated CUL1, csn5b does not have any defect in removing neddylation (Tomoda et al., 1999). Therefore, it is CSN5A, not CSN5B, that plays a major role in plant development (Jin et al., 2014). In addition, CSN5 plays a key role in plant responses to biotic and abiotic stresses by regulating the biosynthesis of auxin, jasmonic acid, and anthocyanins (Luo et al., 2021; Singh et al., 2019; Sang et al., 2019; Schwechheimer et al., 2001). Under high temperature stress, Arabidopsis csn5a mutants showed increased chlorophyll content, carbon dioxide assimilation rate, and photosynthesis activity (Singh et al., 2019). These studies suggest that CSN may be involved in plant responses to abiotic stress by regulating photosynthesis activity. However, the role of CSN5 under low temperature stress and its relationship with chloroplast protein stability have not been reported.

[0006] How proteins in chloroplasts are ubiquitinated remains unclear. One possibility is that ubiquitination may occur inside chloroplasts, but no E3 ubiquitin ligase has been found in chloroplasts. Alternatively, proteins in chloroplasts are modified by E3 ubiquitin ligases outside chloroplasts after being transported outside chloroplasts by an unknown mechanism (Li et al., 2022). Nuclear-encoded chloroplast vesicle (CV) proteins are involved in chloroplast degradation outside the organelle. Stress-induced CVs target chloroplasts and interact with proteins localized in thylakoids, stroma, and capsule. This interaction leads to destabilization of the photosystem and the formation of CV-containing vesicles (CCVs), which transfer stroma and thylakoid proteins to the central vacuole for degradation (Wang and Blumwald, 2014). CVs are involved in the efflux of chloroplast proteins, providing the possibility for contact between proteins in chloroplasts and cytoplasmic components, but it is still unclear whether CVs are involved in the ubiquitination of chloroplast proteins and whether CVs are involved in the photoprotection mechanism under stress conditions. Tomato (Solanum Lycopersicum L) is a widely grown horticultural crop in the world, but its tolerance to cold stress is poor (Lu et al., 2021). Currently, there are few reports on the photoprotection mechanism of tomato under cold stress.

[0007] In view of this, the present invention is proposed. Summary of the invention

[0008] The object of the present invention is to provide a new application of a tomato SlCSN5A protein or a gene encoding the same. The present invention discloses a degradation pathway of chloroplast proteins dependent on ubiquitination mediated by SlCV and SlCSN5A, indicating that SlCV and SlCSN5A play a key role in regulating the stability of chloroplast proteins under abiotic stress (especially low temperature stress), and provides a potential new pathway for chloroplast quality control.

[0009] The technical solution provided by the present invention is as follows:

[0010] In one aspect, the present invention provides a use of a tomato S1CSN5A protein or a gene encoding the same, the use comprising one or more of the following aspects:

[0011] (a) Regulate the ubiquitination level or stability of proteins in chloroplasts;

[0012] (b) regulating the ubiquitination level or stability of the chloroplast vesicle protein SlCV;

[0013] (c) Regulate the response of tomato to low temperature stress.

[0014] The present invention experimentally found that LNT stress treatment caused the abundance and NPQ level of SlPsbS protein in tomato leaves to decrease, and COP9 signaling subunit 5A (SlCSN5A) interacted with SlPsbS to promote its ubiquitination and degradation in the cytoplasm. SlCSN5-RNAi plants showed higher tolerance and stability of SlPsbS protein under LNT stress. Therefore, SlCSN5A can be used to regulate the ubiquitination level or stability of proteins (especially SlPsbS) in chloroplasts.

[0015] In one embodiment, the application is to regulate the ubiquitination level or stability of proteins in chloroplasts and the ubiquitination level or stability of chloroplast vesicle protein SlCV under LNT stress.

[0016] The nucleotide sequence and amino acid sequence of the S1CSN5A gene are known in the prior art and can be downloaded from common websites (such as GENE BANK).

[0017] In one embodiment, the application is to regulate by inactivating the S1CSN5A protein or knocking out the coding gene or reducing the expression level of the coding gene. The regulation is to reduce the expression or activity of the protein encoded by the tomato regulatory gene S1CSN5A by RNAi, gene knockout or gene inactivation. Specifically, the expression of S1CSN5A and / or S1CV or protein can be inhibited by transformation with a recombinant vector, a transgenic cell line containing a recombinant vector or a recombinant bacterium.

[0018] In one embodiment, the application is the application of tomato SlCSN5A gene or protein in improving the low temperature resistance of tomatoes, specifically including reducing or not expressing the expression of the encoding gene, and the methods used include: mutating or knocking out all or part of the sequence of the encoding gene; or constructing an interference vector to interfere with the expression of the encoding gene; or using a gene silencing system to silence the expression of the encoding gene.

[0019] In one embodiment, the intrachloroplast protein comprises the chloroplast SIPsbS protein.

[0020] Whether CSN is involved in the ubiquitination of chloroplast proteins has not been clearly studied. The present invention studied whether SlCSN5A can interact with SlPsbS through yeast two-hybrid (Y2H) experiments. It was found for the first time that SlCSN5A can interact with SlPsbS in yeast cells, and the SlPsbS binding site on SlCSN5A is located in the MPN rather than the ICA domain. In addition, firefly luciferase (LUC) complementary imaging (LCI) detection and immunoprecipitation experiments also showed the interaction between the two. Further analysis of the effect of SlCSN5A on the stability of SlPsbS protein confirmed that SlCSN5A caused the ubiquitination and degradation of SlPsbS.

[0021] Therefore, the tomato SlCSN5A protein or its encoding gene can be used to regulate the ubiquitination level or stability of proteins in chloroplasts. Knocking out the encoding gene (SlCSN5A) or reducing its expression level will reduce the ubiquitination level of proteins in chloroplasts and maintain their stability. The experiments of the present invention show that compared with WT plants, SlCSN5-RNAi#1 and SlCSN5-RNAi#3 plants have higher Fv / Fm, Pn and NPQ levels under LNT stress, while the ROS content is significantly reduced. Under LNT stress, the abundance of SlPsbS protein in SlCSN5-RNAi#1 and SlCSN5-RNAi#3 plants is higher than that in WT plants.

[0022] Prior to the present invention, it was unclear whether chloroplast vesicle (CV) proteins are ubiquitinated with chloroplast proteins and whether CV is involved in the light protection mechanism under stress conditions. The present invention proves through experimental studies that SlCV participates in the transport of chloroplast SlPsbS and promotes the contact between SlCSN5A and the substrate (SlPsbS). SlCV also interacts with SlCSN5A (SlCV interacts with SlCSN5A in yeast cells and plants) and is ubiquitinated and degraded. When SlCV-GFP and SlCSN5A-HA are co-expressed, the ubiquitination level of SlPsbS protein is enhanced. While SlCV silencing reduces light damage to tomato leaves under LNT stress, SlCV overexpression inhibits the production of NPQ in tomato leaves under LNT stress, and leads to degradation of chloroplast and thylakoid membrane proteins in tomato leaves under LNT stress. Therefore, tomato SlCSN5A protein or its encoding gene can be used to regulate the ubiquitination level or stability of chloroplast vesicle protein SlCV.

[0023] In a preferred embodiment, the chloroplast intracellular proteins further include one or more of the thylakoid proteins SlPsbO, SlPsaD, SlFd and SlCP12.

[0024] The present invention also found that SlCSN5A regulates the ubiquitination level of multiple chloroplast proteins. The present invention studied the effect of SlCSN5A on the stability and ubiquitination level of SlPsbO, SlPsaD, SlFd and SlCP12 proteins, indicating that SlCSN5A can promote the degradation of SlPsbO, SlPsaD, SlFd and SlCP12. And SlCV can also interact with multiple chloroplast proteins (SlPsbO, SlPsaD, SlFd, SlCP12). SlCSN5A seems to regulate the ubiquitination level of each chloroplast protein that SlCV interacts with.

[0025] In one embodiment, the tomato S1CSN5A protein mediates the ubiquitination and degradation of proteins in chloroplasts by directly interacting with proteins in chloroplasts.

[0026] In one embodiment, the tomato SlCSN5A protein interacts with the chloroplast vesicle protein SlCV in the cytoplasm to promote the degradation of the chloroplast vesicle protein SlCV and proteins within the chloroplast.

[0027] The present study showed that LNT stress activated the expression of SlCV, and the SlCV protein interacted with the chloroplast protein (SlPsbS) with the chloroplast as the target, inducing the formation of CCV containing the chloroplast protein (SlPsbS), and then the CCV was released from the chloroplast. In the cytoplasm, SlCV and SlPsbS contacted SlCSN5A and an unknown E3 ubiquitin ligase through the extramembrane region, and then were ubiquitinated and degraded by the UPS.

[0028] In one embodiment, the tomato S1CSN5A protein regulates the dissipation of the tomato photosystem capacity under low night temperature stress.

[0029] In one embodiment, the application includes increasing the stability and NPQ value of SlPsbS protein in tomato leaves under low night temperature stress by silencing the SlCSN5 gene, reducing ROS accumulation, and improving the plant's resistance to low temperature. SlCSN5 is involved in the light protection mechanism under stress conditions.

[0030] Under LNT stress, as the stress time prolonged, the tomato plants gradually wilted, the abundance of SlPsbS protein decreased significantly, the Fv / Fm decreased, and O2 -and H2O2 levels increased, and NPQ decreased significantly after 24 hours of LNT stress. In addition, the Slpsbs mutant had extremely low NPQ levels under both normal and LNT conditions. Therefore, SlPsbS plays an important role in the response of tomatoes to LNT stress. A high abundance of SlPsbS protein can have a higher PsbS-mediated non-photochemical quenching (NPQ), which can reduce the dissipation of the photosystem capacity under low night temperature stress. The SlCSN5A protein can promote the ubiquitination and degradation of the SlPsbS protein. The experiment of the present invention also shows that SlCSN5 silencing can increase the NPQ of tomato leaves under LNT stress, reduce the dissipation of the photosystem capacity under low night temperature stress, and improve low temperature resistance. SlCSN5A can affect the photoprotection mechanism by regulating the stability of chloroplast proteins under LNT stress. Silencing the SlCSN5 gene can reduce the dissipation of the photosystem capacity and enhance photoprotection.

[0031] In one embodiment, the tomato SlCSN5A and chloroplast vesicle protein SlCV jointly regulate the stability of tomato chloroplast proteins under low temperature stress.

[0032] The present invention shows that LNT activates tomato chloroplast vesicle protein (SlCV), and the SlCV protein targets chloroplasts to interact with SlPsbS, induces the formation of vesicles (CCV) containing SlPsbS, and then exports CCV from chloroplasts under LNT stress. Subsequently, SlCV and SlPsbS interact with SlCSN5A in the cytoplasm, and their interaction promotes the degradation of SlCV and SlPsbS. It can be seen that SlCSN5A and SlCV jointly mediate the degradation pathway of chloroplast proteins dependent on ubiquitination, and jointly regulate the stability of chloroplast proteins in tomatoes under low temperature stress.

[0033] In another aspect, the present invention provides a method for improving the low temperature resistance of tomatoes, the method comprising the step of reducing the expression level of the SlCSN5A gene in tomatoes; preferably, the expression level of the SlCSN5A gene in tomatoes is reduced by RNAi, CRISPR / Cas9 gene knockout or knockdown methods.

[0034] A method for improving the low temperature resistance of tomatoes, comprising the following steps: (1) designing a target sequence sgRNA of the gene SlCSN5A and constructing a CRISPR / Cas9 vector; (2) constructing an Agrobacterium genetically engineered bacterium containing the CRISPR / Cas9 vector; and (3) transforming tomato cotyledons with the genetically engineered bacterium to obtain a homozygous mutant strain with stable inheritance.

[0035] In one embodiment, the method further comprises reducing the expression level of the SlCV gene in tomatoes by gene knockout or knockdown methods, so that the low temperature resistance of the tomato mutant is enhanced.

[0036] In one embodiment, CRISPR-Cas9 gene editing technology is used to knock out the SlCSN5A gene and / or the SlCV gene in tomatoes, and ultimately screen out cold-resistant tomato plants.

[0037] In one embodiment, the cold resistance of tomato mutants is improved by knocking out the SlCSN5 gene (including SlCSN5A and SlCSN5B. Since SlCSN5A and SlCSN5B have high sequence similarity, it is impossible to silence them independently in tomatoes) and / or the SlCV gene to obtain mutants, or by hybridizing the mutants to obtain homozygous double mutants.

[0038] Beneficial effects:

[0039] The present invention is the first to study and discover the role of tomato SlCSN5A in regulating the stability of chloroplast proteins through UPS, especially by participating in the ubiquitination degradation of SlCV and SlPsbS, regulating the response of tomatoes to low temperature stress; by silencing tomato SlCSN5A, the stability of SlPsbS protein in tomato leaves under night low temperature stress can be improved, further improving the plant's stress resistance, and can also be used to promote the yield and quality of tomato crops under low temperature stress.

[0040] The present invention provides the mechanism of tomato SlCSN5A protein and tomato chloroplast vesicle protein (SlCV) in regulating the low temperature resistance of tomatoes, which provides an important direction for cultivating tomato low temperature resistant germplasm resources and improving the low temperature resistance of tomatoes.

[0041] The present invention confirms that SlCV and SlCSN5A play a key role in the ubiquitination and degradation of proteins in chloroplasts. The SlCV and SlCSN5A pathways appear to have a broad impact on the stability of proteins in chloroplasts, which also provides a basis for understanding the regulatory mechanism of chloroplast protein quality control.

[0042] The present invention studies and confirms the gene that regulates the stability of tomato chloroplast protein under low temperature adversity, and obtains transgenic tomatoes by reducing or eliminating the SlCSN5A and / or SlCV or protein content in the target tomatoes, which can provide a new way to improve the yield of tomato cultivation under low temperature. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0044] Figure 1 The LNT provided by the present invention inhibited the NPQ of tomato leaves and promoted the degradation of PsbS;

[0045] Figure 2 The results provided by the present invention show that silencing of SlPsbS increases the sensitivity of tomatoes to LNT stress;

[0046] Figure 3 The SlCSN5A provided by the present invention leads to the ubiquitination and degradation of SlPsbS;

[0047] Figure 4 The present invention provides that silencing of SlCSN5 increases the NPQ results of tomato leaves under LNT stress;

[0048] Figure 5 The subcellular localization results of SlPsbS and SlCSN5A provided by the present invention;

[0049] Figure 6 The expression amount of SlCV and the subcellular localization results of SlCV under LNT stress provided by the present invention;

[0050] Figure 7 The SlCV provided by the present invention interacts with SlPsbS and promotes the degradation of SlPsbS;

[0051] Figure 8 The co-localization analysis results of SlCV, SlPsbS and vacuolar precursor AtRab2a provided by the present invention;

[0052] Fig. 9 The SlCSN5A provided by the present invention leads to the ubiquitination and degradation of SlCV;

[0053] Fig.10 The co-localization analysis results of SlCV and SlCSN5A under LNT stress provided by the present invention;

[0054] Fig.11 The SlCV silencing provided by the present invention reduces the light damage results of tomato leaves under LNT stress;

[0055] Fig.12 The results of the present invention showing that overexpression of SlCV inhibits the production of NPQ in tomato leaves under LNT stress;

[0056] Fig.13 The results of the effects of SlCV overexpression on the chloroplast ultrastructure and thylakoid protein complex of tomato leaves under LNT stress provided by the present invention;

[0057] Fig.14The results of the interaction between SlCV and SlCSN5A and multiple thylakoid proteins provided by the present invention;

[0058] Fig.15 The results of the ubiquitination and degradation analysis of thylakoid proteins by SlCSN5A provided by the present invention;

[0059] Fig.16 The results of the present invention provide that SlCSN5A regulates the stability of SlPsbS in SlCV overexpressing plants;

[0060] Fig.17 A schematic diagram of SlCSN5A provided by the present invention regulating the stability of SlPsbS protein under LNT stress through the ubiquitin-proteasome system. DETAILED DESCRIPTION

[0061] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0062] 1. Experimental Materials and Methods

[0063] 1.1 Plant materials and growth conditions

[0064] According to the method of Shimatani et al. (2017), the Slpsbs and Slcv mutants were obtained by CRISPR / Cas9 technology. To obtain transgenic tomato SlCV overexpression plants (SlCV-OE), the full-length coding region of SlCV (without the stop codon) was inserted into the pHSN6A01 vector (containing 4× minimal VP16 activation domain). SlCSN5-RNAi plants were generously provided by Professor Taotao Wang (Huazhong Agricultural University, China) (Luo et al., 2021). Virus-induced gene silencing (VIGS) of the tomato SlCSN5A gene was performed using the pTRV1 / 2 vector according to the method of Singh et al. (2022). The wild-type (WT) tomato plants used in the present invention were the 'Ailsa Craig' ecotype, and transgenic tomato plants were obtained by Agrobacterium-mediated method. Tomato Slpsbs and Slcv mutants were identified by PCR and DNA sequencing was performed. qRT-PCR was used to quantify the growth of CV-OE, SlCSN5-RNAi, and pTRV-SlCSN5A plants. Tomato seeds were grown at 28°C:18°C (day and night), relative humidity 60% (Beijing Kulan), and cool white fluorescent light (600 μmol m -2 s -1, photoperiod 12h) to germinate and grow.

[0065] Tomato seedlings with five leaves and one heart were transferred to a dark growth chamber with an air temperature of 4°C and treated with LNT for 24 h (18:00-18:00 [next day]), and 18°C ​​was used as a control. During the experimental period, all measurements were performed on the fourth fully expanded functional leaf. Tomato leaves were collected immediately after treatment for physiological experiments, and the remaining leaves were collected in liquid nitrogen and stored at -80°C for physiological and molecular biology experiments. The experiment was repeated five times independently.

[0066] 1.2 Measurement of Pn, Fv / Fm and NPQ

[0067] Actinic light (228 μmol m -2 s -1 , 635nm). The CO2 concentration was 600ppm and the temperature was about 25℃ (Lu et al., 2020). After 30min in darkness, Fv / Fm and NPQ were measured using MAXI-Imaging-PAM (Heinz Walz, Effeltrich, Germany). After the measurement started, a saturation pulse (10,000μmol m -2 s -1 , 300 ms to obtain the maximum fluorescence (Fm). -2 s -1 , 635nm), and the maximum fluorescence (FM and FM') of dark adaptation and light adaptation was obtained by saturation pulse. -2 s -1 The initial fluorescence (Fo and Fo') of dark adaptation and light adaptation was measured by 30 μm / s (0.6 kHz, frequency 0.6 kHz). The calculation formula is Fv / Fm = (Fm-Fo) / Fm, NPQ = (Fm-Fm') / Fm'.

[0068] 1.3 Hydrogen peroxide (H2O2), superoxide anion (O2 - ) staining and reactive oxygen species (ROS) fluorescence analysis

[0069] Detection of intracellular O2 by Nitroblue tetrazolium (NBT) and Diaminobenzidine (DAB) staining - and H2O2 accumulation. Fresh tomato leaves were immersed in 10 μg·mL -1The samples were incubated in 0.2% DAB and 0.2% NBT solution at room temperature for 12 h, boiled in 95% ethanol for dehydration, and scanned for observation.

[0070] ROS fluorescence was observed under a confocal microscope (LSM880NLO, Zeiss, Germany) using 2',7'-dichlorodihydrofluorescein diacetate (H2DCFDA, 10InvM, Invitgen) (Yu et al., 2022). The excitation wavelength of ROS fluorescence was 488 nm and the absorption wavelength was 525 nm.

[0071] 1.4 Analysis of chloroplast ultrastructure

[0072] At the end of the treatment, the fourth fully expanded leaf from the top of the plant was randomly selected and examined by electron microscopy. Leaf tissue excluding the main vein and leaf margin was excised and fixed in McDowell's solution (McDowell and Trump, 1976) and prepared as previously described (Hricová et al., 2006). Samples were observed using a transmission electron microscope (H7650 model, Hitachi, 75 kV, Japan). Relevant measurements were obtained from the images using ImageJ (version 2.0.0) software.

[0073] 1.5 Total RNA extraction and qRT-PCR

[0074] Total RNA from tomato leaves was extracted using a plant total RNA extraction kit (Tiangen, Beijing, China) according to the manufacturer's instructions. The first-strand complementary DNA was synthesized using a PrimeScriptTMRT kit (Takara, Dalian, China). Quantitative RT-PCR and gene expression analysis were performed using SYBR@PreMix ExTaqTM (Takara, Dalian, China), ABI7500 real-time polymerase chain reaction system (Applied Biosystems, Foster City, CA) and software 7500V2.0.6.

[0075] 1.6 Subcellular localization

[0076] The coding regions of SlPsbS, SlCSN5A and SlCV (without stop codon) were cloned and inserted into the BamHI and SalI sites upstream of GFP in the pCAMBIA1300 vector. SlPsbS, SlCSN5A, AtHPR2 (cytoplasmic localized protein) (Ye et al., 2014) and AtRab2a (prevacuolar compartment rab5 GTPase Rha1) were ligated into the pCAMBIA1300-mCherry expression vector. The constructed recombinant vectors were then transformed into the Agrobacterium EHA105 strain and infiltrated into the leaves of 3-week-old tobacco plants. After infiltration, tobacco plants were kept in the dark for 48 h. Some plants were treated with low night temperature (4 °C) for 24 h, with room temperature as the control. Fluorescence signals were detected using a confocal laser scanning microscope (TCS SP8, Leica Wetzlar, Germany). The excitation wavelength for GFP observation is 488nm, and the emission wavelength is 520-540nm; the excitation wavelength for mCherry observation is 561nm, and the emission wavelength is 610-630nm.

[0077] 1.7 Yeast two-hybrid

[0078] The SlCSN5A and SlCV coding sequences were inserted into the pGBKT7 vector, and the SlPsbS and SlCSN5A gene fragments were cloned into the pGADT7 vector. The fusion expression vector was co-transformed into the Y2H Gold strain. The transformed yeast cells were cultured on SD-Trp-Leu (DDO) medium for 3 days. Then they were transferred to SD-Trp-LeuHis-Ade (QDO) medium containing X-galactose (5-bromo-4-chloro-3-indolyl-bD-galactopyranoside) for color development. The QDO culture medium contained 40μg-α-Gal and 100ng-1 aurobacitin A (ABA).

[0079] 1.8 Bimolecular fluorescence complementation

[0080] The CDS of SlCSN5A and SlCV were connected to the pSPYNE-35S vector. The CDS of SlPsbS and SlCSN5A were amplified and cloned into the pSPYCE-35S vector. The constructed recombinant expression vector was introduced into the Agrobacterium EHA105 strain and co-transformed into tobacco leaves. YFP fluorescence was observed under a laser confocal microscope (TCS SP8, Leica Wetzlar, Germany) after 72 hours. The excitation wavelength was 488 nm and the emission wavelength was 520-540 nm.

[0081] 1.9 Luciferase complementation assay

[0082] The coding sequence of SlCSN5A and SlCV without stop codon is cloned in the pCAMBIA-NLuc between BamHI and SalI.Utilize BamHI and PstI restriction site that SlPsbS and SlCSN5A CDS are inserted in the pCAMBIA1300-CLuc carrier.Agrobacterium infiltration test is carried out as above.Use Night SHADELB 985 imaging system (Berthold Technologies) to detect luciferase activity in 72 hours after infiltration.Detection first 30 minutes, the potassium luciferin (Gold Biotech Inc., StLouis, MO, USA) of 0.2mM is sprayed to the position identical with agrobacterium infiltration.

[0083] 1.10 Immunoblotting and blue-green mild electrophoresis (BN-PAGE)

[0084] Plant leaf tissues were weighed, frozen in liquid nitrogen, and ground in extraction buffer (Sang et al., 2019). Protein concentration was measured using a BCA protein assay kit (Thermo Science, Waltham, MA, USA). 50 μg of total protein was separated on a 10% sodium dodecyl sulfate-polyacrylamide gel, transferred to a nitrocellulose membrane (BioRad), and incubated with anti-PsbS antibody (PhytoAB, USA) or GFP antibody (Solarbio, China). After incubation with goat anti-rabbit or anti-mouse horseradish peroxidase antibodies (Solarbio, China), the labeled proteins were detected by enhanced chemiluminescence. Actin antibody (CWBIO, China) was used as a control.

[0085] For BN-PAGE, the method described by Sárvári et al. (2022) was followed. Thylakoid membranes were isolated from freshly collected tomato leaves, and the extracted thylakoid membrane proteins were diluted with 1% DDM at 1 mg ml -1 After standing at 4°C for 30 minutes, the samples were separated using a gradient gel from 4% to 12%. Images of the BN-PAGE gel were acquired using an image scanner.

[0086] 1.11 In vivo protein degradation and ubiquitination detection

[0087] The amplified SlCSN5A and SlCVCDS fragments were inserted into the plant expression vector pRI101 with an HA tag. The SlPsbS and SlCV coding sequences were ligated into the pCAMBIA1300-GFP vector. Agrobacterium strains carrying the E3 ligase, substrate, and p19 gene constructs and the internal control plasmid were co-infiltrated into Nicotiana benthamiana leaves. Three days after infiltration, samples were collected for immunoblot analysis. 50 μΜ MG132 (26S proteasome inhibitor) was injected 12 hours before sample collection (Liu et al., 2019).

[0088] Tobacco leaves co-expressing SlCSN5A-HA / SlCV-HA and SlPsbS-GFP were collected for immunoprecipitation analysis. Total proteins were extracted in IP buffer (Solarbio, China). Cell lysates were then incubated with GFP-Trap magnetic beads (Sigma, USA) at 4°C for 4 h. Immunoblotting analysis was performed using HA (Solarbio, China) and GFP (Solarbio, China) antibodies, respectively. Ubiquitinated proteins were immunodetected using anti-Ub antibodies (Millipore, USA) (Belda-Palazon et al., 2016; Luo et al., 2021).

[0089] 1.12 In vitro ubiquitination assay

[0090] After connecting the HA / GFP tag, the full-length sequences of SlCSN5A, SlPsbS and SlCV were inserted into the pET-51b vector. The constructed recombinant expression vector was transformed into Escherichia coli BL21 (DE3) competent cells. The recombinant protein was purified by BeyotimeHis-Tag purification resin. The purified product was incubated with ubiquitin activating enzyme (E1), ubiquitin conjugating enzyme (E2) and ubiquitin (Ub) (30℃, 1.5h). It was separated on SDS-PAGE gel and immunoblotted using GFP (Solarbio, China) and HA (Solarbio, China) antibodies. Ubiquitin was detected with anti-Ub antibody (Solarbio, China).

[0091] 1.13 Statistical analysis

[0092] The experimental design was a completely randomized design. SPSS22 software (IBM SPSS STATISTICS, USA) was used to analyze the data. P < 0.05 was considered statistically significant. Origin 2022 software (Origin Lab, Northampton, MA, USA) was used to draw the graph.

[0093] 2 Results Analysis

[0094] 2.1 LNT inhibits NPQ in tomato leaves and promotes SlPsbS degradation

[0095] Under LNT stress, as the stress time increases, tomato plants gradually wilt ( Figure 1 Middle A), Fv / Fm decreases ( Figure 1 Middle B) NBT and DAB histochemical staining showed that compared with the control, the O2 - and H2O2 levels increased ( Figure 1 Within 12 h of LNT stress, NPQ was significantly induced, while after 24 h of LNT stress, NPQ was significantly decreased ( Figure 1 D). Similar to NPQ, LNT stress also caused a significant decrease in the abundance of SlPsbS protein ( Figure 1 (E). Chloroplast ultrastructural analysis showed that chloroplasts in tomato leaves grown under normal conditions were slender, with neatly arranged and tightly packed grana and stroma lamellae, and a small number of osmophilic granules. However, LNT stress caused chloroplast swelling, a decrease in the number of grana lamellae, a significant increase in grana diameter / height, broken stroma lamellae, and an increase in the accumulation of osmophilic granules ( Figure 1 Middle F).

[0096] 2.2SlPsbS silencing increases the sensitivity of tomato plants to LNT stress

[0097] To investigate the role of SlPsbS in the response of tomato to LNT stress, we used CRISPR / Cas9 gene editing technology to generate the Slpsbs mutant. Western blot analysis showed that SlPsbS protein did not accumulate in the Slpsbs mutant ( Figure 2 (A). Subsequently, we subjected the Slpsbs mutant to LNT stress. As expected, the Slpsbs mutant had a lower tolerance to LNT. Compared with WT plants, the Slpsbs mutant plants wilted more severely under LNT stress, with significantly reduced Fv / Fm and Pn, and significant accumulation of ROS ( Figure 2 In addition, unlike WT plants that had higher NPQ values ​​under LNT stress, the Slpsbs mutant had extremely low NPQ levels under both normal and LNT conditions ( Figure 2 Middle F).

[0098] 2.3SlCSN5A causes SlPsbS ubiquitination and degradation

[0099] A previous study showed that chloroplast proteins can be ubiquitinated and degraded (Li et al., 2022). CSN is thought to regulate the ubiquitin / proteasome system (UPS), and whether CSN is involved in the ubiquitination of chloroplast proteins is an interesting question. First, we used yeast two-hybrid (Y2H) experiments to investigate whether SlCSN5A can interact with SlPsbS and found that SlCSN5A can interact with SlPsbS in yeast cells ( Figure 3 In addition, we found that the SlPsbS binding site on SlCSN5A is located in the MPN rather than the ICA domain ( Figure 3 To confirm this result in vivo, we performed firefly luciferase (LUC) complementation imaging (LCI) assays. When recombinant vectors containing SlCSN5A fused to the N-terminus of LUC (SlCSN5A-nLUC), and SlPsbS tagged with the C-terminus of LUC (cLUC-SlPsbS) were co-infiltrated into tobacco leaves, a strong luminescence signal was detected, but no signal was detected in the negative control ( Figure 3 Co-immunoprecipitation experiments showed that when protein extracts were immunoprecipitated with GFP antibodies, SlCSN5A-HA was detected in the SlPsbS-GFP immunoprecipitates, while the negative control was not ( Figure 3 Middle E).

[0100] Next, we further analyzed the effect of SlCSN5A on the stability of SlPsbS protein. SlPsbS and SlCSN5A were connected to pCAMBIA1300-GFP and PRI101-HA vectors, respectively, and driven by the CaMV35S promoter. After SlPsbS-GFP and SlPsbS-GFP+SlCSN5A-HA vectors were transiently transferred into tobacco leaves, the anti-GFP antibody could hardly detect the protein band of SlPsbS-GFP. However, in the presence of MG132, SlPsbS-GFP protein resumed accumulation ( Figure 3 To investigate whether SlCSN5A affects the ubiquitination of SlPsbS protein, we immunoprecipitated SlPsbS-GFP and detected the ubiquitination of SlPsbS after immunoblotting analysis using anti-Ub ( Figure 3 Middle E).

[0101] 2.4SlCSN5 silencing increases NPQ in tomato leaves under LNT stress

[0102] LNT stress induced a significant increase in the expression level of SlCSN5A ( Figure 4To further investigate the role of SlCSN5A in the response of tomato to LNT stress, we used RNA interference to downregulate the expression of SlCSN5 (including SlCSN5A and SlCSN5B) in stably transformed plants ( Figure 4 (A). Since SlCSN5A and SlCSN5B have high sequence similarity, it is impossible to silence them independently in tomato (Luo et al., 2021). SlCSN5-RNAi (SlCSN5-RNAi#1, SlCSN5-RNAi#3) plants showed reduced sensitivity to LNT stress. Compared with WT plants, SlCSN5-RNAi#1 and SlCSN5-RNAi#3 plants had higher Fv / Fm, Pn and NPQ levels under LNT stress, while ROS content was significantly reduced ( Figure 4 In addition, under LNT stress, the abundance of SlPsbS protein in SlCSN5-RNAi#1 and SlCSN5-RNAi#3 plants was higher than that in WT plants ( Figure 4 Middle G).

[0103] 2.5SlPsbS and SlCSN5A interact in the cytoplasm

[0104] Subcellular localization analysis showed that SlPsbS was localized in chloroplasts ( Figure 5 However, the green fluorescence signal of SlCSN5A-GFP transiently expressed in wild-type tobacco leaves overlaps with the cytoplasmic HPR2-mCherry signal ( Figure 5 B), indicating that SlCSN5A is localized in the cytoplasm. Bimolecular fluorescence complementation (BIFC) analysis showed that when SlCSN5A-nYFP and cYFP-SlPsbS were co-expressed in tobacco leaves, a YFP fluorescence signal was generated in the cytoplasm ( Figure 5 Middle C).

[0105] 2.6 SlCV interacts with SlPsbS and mediates SlPsbS degradation

[0106] PsbS is located in the thylakoid membrane (Daskalakis and Papadatos, 2017), while CSN5A is localized in the cytoplasm (Mo et al., 2021). Since chloroplasts are organelles surrounded by double membranes, whether and how SlPsbS translocates from chloroplasts and contacts SlCSN5A and the UPS complex in the cytoplasm is a mysterious question. The study by Wang and Blumwald (2014) showed that CV proteins can induce the formation of CCV, which is released from chloroplasts to the vacuole for protein degradation. Therefore, we speculate that CCV containing SlPsbS is secreted from chloroplasts, and in the cytoplasm, CCV facilitates the contact between SlPsbS and SlCSN5A. To prove this conjecture, we first detected the expression level of SlCV under LNT stress. As expected, the expression level of SlCV increased significantly with the aggravation of the stress level and was upregulated 356-fold ( 24 h after stress). Figure 6 To further analyze the function of SlCV, we studied its subcellular localization. Laser confocal scanning microscopy showed that the green fluorescence signal of SlCV-GFP driven by the CaMV35S promoter was consistent with the autofluorescence of chloroplasts, indicating that SlCV is localized in chloroplasts ( Figure 6 Interestingly, after LNT treatment of tobacco plants expressing SlCV-GFP, we found green fluorescence signals in some unknown compartments outside the chloroplasts ( Figure 6 Middle C).

[0107] To determine the role of SlCV in the degradation of SlPsbS, we used Y2H to analyze the interaction between SlCV and SlPsbS. The results showed that SlCV interacted with SlPsbS in yeast cells ( Figure 7 LCI experiments also further confirmed the interaction between SlCSN5A and SlPsbS in plants ( Figure 7 (B). BIFC analysis revealed that when SlCV-nYFP and cYFP-SlPsbS were co-expressed in tobacco leaves, YFP fluorescence signals were detected in chloroplasts ( Figure 7 In co-immunoprecipitation analysis, we found that SlCV-HA could be co-immunoprecipitated with SlPsbS-GFP ( Figure 7 In addition, we transiently co-expressed SlCV-GFP and SlPsbS-mCherry in wild-type tobacco leaves. SlCV-GFP and SlPsbS-mCherry co-localized in chloroplasts at room temperature, and after LNT treatment, we detected SlCVGFP and SlPsbS-mCherry fluorescence signals outside the chloroplasts ( Figure 8Middle A). Previous studies have shown that CCV is ultimately transported to the vacuole (Wang and Blumwald, 2014). Therefore, we expressed SlCV-GFP / AtRab2a-mCherry and SlPsbS-GFP / AtRab2amCherry (prevacuolar compartment rab5 GTPase Rha1) in wild-type tobacco leaves, respectively (Foresti et al., 2010), to assess whether CCV also moves SlPsbS to the central vacuole. After LNT treatment of tobacco plants, the SlCV-GFP signal was found to be consistent with the AtRab2a-mCherry signal. Similarly, we detected the SlPsbS-GFP signal in the prevacuolar compartment ( Figure 8 These results suggest that SlCV interacts with SlPsbS and changes the subcellular location of SlPsbS by forming CCV.

[0108] In addition, we investigated whether SlCV is directly involved in the ubiquitination and degradation of SlPsbS. When SlCV-HA and SlPsbS-GFP were co-infected in tobacco leaves, SlCV promoted the degradation of SlPsbS. In the presence of MG132, the accumulation of SlPsbS protein was partially restored ( Figure 7 D), indicating that SlCV may degrade SlPsbS through the UPS. However, no ubiquitination band of SlPsbS was detected in the immunoprecipitates of SlCV-HA and SlPsbS-GFP. In contrast, when SlCSN5A-HA was present, a ubiquitination band of SlPsbS was detected ( Figure 7 Middle E), indicating that SlCV can promote UPS-mediated degradation of SlPsbS through SlCSN5A.

[0109] 2.7SlCSN5A causes SlCV ubiquitination and degradation

[0110] To better understand the relationship between SlCV, SlCSN5A, and SlPsbS, we next investigated possible protein-protein interactions between SlCV and SlCSN5A. We demonstrated by Y2H and LCI assays that SlCV interacts with SlCSN5A in yeast cells and plants ( Fig. 9 In addition, the SlCV binding site on SlCSN5A is located in the MPN rather than the ICA domain ( Fig. 9 In the BIFC assay, SlCV-nYFP and cYFP-SlCSN5A produced YFP fluorescence in the cytoplasm ( Fig. 9(C). The interaction between SlCV and SlCSN5A in the cytoplasm may be related to the location change of SlCV under stress conditions. Therefore, we co-infiltrated SlCV-GFP and SlCSN5A-mCherry into tobacco leaves. After LNT treatment, the red and green fluorescence signals overlapped in the cytoplasm ( Fig.10 ). In addition, we used co-immunoprecipitation experiments to confirm the interaction between SlCSN5A and SlCV. SlCSN5A-HA and SlCV-GFP were co-transformed into tobacco leaves, immunoprecipitated with GFP antibody, and analyzed by immunoblotting with HA antibody. SlCSN5A-HA could co-immunoprecipitate with SlPsbS-GFP, while the negative control did not ( Fig. 9 E).

[0111] As expected, no accumulation of SlCV-GFP protein was detected when SlCV-GFP and SlCSN5A-HA were co-expressed in tobacco leaves ( Fig. 9 D), but after adding MG132, the abundance of SlCV protein increased ( Fig. 9 Next, we used Ub antibody to detect the ubiquitination level of SlPsbS. The results showed that when co-expressed with SlCSN5A-HA, the ubiquitination level of SlPsbS protein was enhanced ( Fig. 9 Middle E).

[0112] 2.8SlCV silencing reduces light damage in tomato leaves under LNT stress

[0113] We used CRISPR / Cas9 gene editing technology to obtain Slcv mutant plants to further study the role of SlCV in LNT stress ( Fig.11 Middle A). Under LNT stress, SlCV mutant plants maintained normal plant morphology and lower ROS levels compared with WT plants ( Fig.11 (B, C). The Slcv mutant also showed an increase in Fv / Fm and Pn under LNT ( Fig.11 Meanwhile, LNT induced higher NPQ values ​​and SlPsbS protein abundance in Slcv mutants than in WT plants ( Fig.11 Middle F).

[0114] 2.9SlCV overexpression inhibits NPQ production in tomato leaves under LNT stress

[0115] Under the control of the CaMV35S constitutive promoter, SlCV overexpression (SlCV-OE#91, SlCV-OE#92) transgenic tomato plants were obtained. Under normal circumstances, SlCV-OE#91 and SlCV-OE#92 plants are significantly shorter than WT plants ( Fig.12In addition, SlCV-OE#91 and SlCV-OE#92 showed increased sensitivity to LNT stress, as evidenced by more severe plant wilting, significantly reduced Fv / Fm and Pn, and a large accumulation of ROS levels ( Fig.12 Unlike the higher NPQ value of WT plants, the NPQ levels of SlCV-OE#91 and SlCV-OE#92 plants were lower ( Fig.12 In addition, we also analyzed the changes in SlPsbS protein abundance in SlCV overexpressing plants. Under control conditions, SlCV-OE#91 and SlCV-OE#92 plants showed lower SlPsbS levels, and further aggravated the degradation of SlPsbS protein after LNT treatment ( Fig.12 Middle F).

[0116] 2.10SlCV overexpression leads to degradation of chloroplast and thylakoid membrane proteins in tomato leaves under LNT stress

[0117] To investigate whether SlCV overexpression affects the underlying chloroplast organelles, transmission electron microscopy was used to study the ultrastructure of chloroplasts in WT, SlCV-OE#91, and SlCV-OE#92 before and after LNT treatment. Under normal temperature conditions, the chloroplasts of SlCV overexpressing plants were significantly swollen and deformed, but the matrix and grana were still clearly visible ( Fig.13 After LNT treatment, the chloroplasts of SlCV overexpressing plants expanded further compared with those of WT plants ( Fig.13 A), the number of osmiophilic granules increased significantly ( Fig.13 The number of chloroplast grana and grana lamellae decreased significantly ( Fig.13 A, B). The grana swells into a nearly spherical shape ( Fig.13 A, C), thylakoid membrane degradation, lamellae blur, chloroplasts are on the verge of collapse and degradation ( Fig.13 (A).

[0118] Chloroplast thylakoids contain most of the proteins involved in the light reactions of photosynthesis. To evaluate the effect of SlCV overexpression on the abundance of thylakoid proteins under LNT stress, we separated and detected the levels of thylakoid protein complexes by BN-PAGE. Under normal conditions, there was no difference in the chloroplast protein content between WT and SlCV-OE#91 plants, while the content of PSII dimers and PSI and ATP synthase was significantly reduced in SlCV-OE#92 plants ( Fig.13 (C). Under LNT stress, the abundance of PSII-LHCII supercomplex, PSII dimer and PSI, ATP synthase, cytochrome b6f complex, and LCII trimer and monomer were reduced in SlCV-OE#91 and SlCV-OE#92 plants compared with WT plants ( Fig.13 Middle C), indicating that SlCV overexpression promoted the degradation of chloroplast proteins in tomato leaves under LNT stress.

[0119] 2.11SlCSN5A regulates the ubiquitination levels of multiple chloroplast proteins

[0120] Given the effects of SlCV on chloroplast structure and thylakoid protein abundance, we further evaluated the relationship between SlCV and chloroplast proteins. Y2H showed that SlCV can interact with SlPsbO, SlPsaD, SlFd, and SlCP12 in yeast cells ( Fig.14 BIFC analysis also found that when SlCV-nYFP was co-transformed with cYFP-PsbO, cYFP-PsaD, cYFP-Fd and cYFP-CP12 into tobacco leaves, YFP fluorescence signals were detected in chloroplasts ( Fig.14 Interestingly, Y2H experiments further showed that SlCSN5A interacts with SlPsbO, SlPsaD, SlFd, and SlCP12 in yeast cells, and the binding site is located in the MPN domain of SlCSN5A ( Fig.14 (in CF).

[0121] Next, we investigated the effect of SlCSN5A on the stability and ubiquitination levels of SlPsbO, SlPsaD, SlFd, and SlCP12 proteins. As expected, SlCSN5A promoted the degradation of SlPsbO, SlPsaD, SlFd, and SlCP12, while MG132 significantly increased the abundance of the proteins in chloroplasts. Similarly, SlCSN5A also regulated the ubiquitination levels of SlPsbO, SlPsaD, SlFd, and SlCP12 ( Fig.15 ).

[0122] 2.12SlCSN5A is involved in SlCV-mediated SlPsbS degradation

[0123] To further investigate the roles of SlCSN5A and SlCV thylakoid protein SlPsbS in ubiquitination and degradation, we silenced SlCSN5A in SlCV-OE#91 and SlCV-OE#92 plants. Under LNT stress, the abundance of Fv / Fm, Pn, NPQ, and SlPsbS proteins in pTRV in SlCV-OE#91 and SlCV-OE#92 backgrounds was significantly lower than that in pTRV in WT background ( Fig.16 However, silencing SlCSN5A in SlCV-OE plants significantly improved the tolerance of SlCV-OE plants to LNT stress ( Fig.16Compared with pTRV plants in SlCV-OE#91 and SlCV-OE#92 backgrounds, pTRV-SlCSN5A plants in SlCV-OE#91 and SlCV-OE#92 backgrounds had higher Fv / Fm, Pnh and NPQ under LNT stress ( Fig.16 Analysis of SlPsbS protein levels revealed that silencing the SlCSN5A gene delayed the degradation of SlPsbS protein in SlCV-OE plants under LNT stress ( Fig.16 Middle E).

[0124] 3 Discussions

[0125] Chloroplasts and photosynthesis are highly sensitive to stress conditions. The thylakoid membrane of the chloroplast contains the photosystem complex, also known as the photosynthetic membrane, which is the site of the photosynthetic light reaction. The degradation of photosynthetic membrane coupling proteins and the inhibition of protein synthesis under low temperature destroy the integrity of the photosynthetic membrane, thereby impairing the light reaction of the photosynthetic membrane (Gan et al., 2019). PSII is distributed on the thylakoid membrane. It is extremely sensitive to temperature and is easily inactivated, and the photosynthetic efficiency decreases rapidly, which is called photoinhibition (Philzenberg et al., 2020). LNT stress can destroy the chloroplast structure, resulting in a significant decrease in Fv / Fm and accumulation of ROS, which affects the activity of the photosystem, reduces the efficiency of photosynthesis, and causes severe photoinhibition of the photosystem (Lu et al., 2020; Lu et al., 2021). However, evolution has brought about a mechanism to dissipate excess light energy in the form of heat through the light-harvesting complex of the photosystem to prevent ROS damage, which is one of the most important rapid photoprotection mechanisms. This process is called NPQ (Ruban, 2018). The mechanism of action of NPQ lies in the response of the molecule to ΔpH. PsbS protein has been identified as a key factor in NPQ activation in plants (Krishnan-Schmieden et al., 2021). Stress promotes the accumulation of PsbS protein (Nawrocki et al., 2022), but long-term or severe stress leads to a decrease in the abundance of PsbS protein (Wang et al., 2018). In the present experiment, LNT stress led to a decrease in NPQ values, which may be related to the decrease in the abundance of SlPsbS protein in the later stage of stress ( Figure 1 (D, E). The Slpsbs mutant showed a slower increase in NPQ, severe photoinhibition, and was more susceptible to LNT stress ( Figure 2 ), which is consistent with the findings in Chlamydomonas reinhardtii and Arabidopsis thaliana (Acebron et al., 2021; Redekop et al., 2020). These results indicate that PsbS-mediated NPQ plays an important role in photoprotection. Therefore, it is of great significance to improve plant stress resistance by improving the stability of PsbS protein.

[0126] The UPS is a rapid regulatory mechanism for selective protein degradation in plants and plays a vital role in growth and development (Xu and Xue, 2019). When chloroplast development is affected, abnormal organelles are degraded and recycled in a process involving ubiquitination of chloroplast proteins (Jeran et al., 2021). Ubiquitinome studies have shown that chloroplast proteins account for a large proportion of the total pool of ubiquitinated proteins in various plants (Rosnoblet et al., 2021; Lu et al., 2019). Current studies suggest that ubiquitin modification of proteins located on the outer membrane of chloroplasts and on photodamaged chloroplasts plays an important role (Jeran et al., 2021; Ling et al., 2012). A recent study revealed the biological and biochemical functions of CDC48A and its cofactors UFD1 and NPL4 in controlling the degradation of ubiquitin-modified proteins in Arabidopsis chloroplasts (Li et al., 2022). In this study, we found that SlCSN5A mediated the ubiquitination and degradation of proteins in chloroplasts, which provides new insights into the regulatory mechanisms of chloroplast protein quality control.

[0127] CSN is an evolutionarily conserved protein complex (Singh et al., 2019). CSN is best known as a regulator of the CRL family, which catalyzes key steps in protein ubiquitination and degradation through the UPS (Lyapina et al., 2001; Schwechheimer et al., 2001). CSN is involved in almost all processes of plant growth, development, and environmental response. CSN5 regulates Arabidopsis seed germination by promoting protein degradation of RGL2 and ABI5 (Jin et al., 2018). In tomato, SlCSN5 also regulates the biosynthesis of ASA and anthocyanins by affecting the stability of SlZF3 and SlBBX20 (Luo et al., 2021; Li et al., 2018). Under high temperature stress, the Arabidopsis csn5a mutant showed an increase in CO2 assimilation rate and photosynthesis activity, indicating that CSN5A is involved in chloroplasts and photosynthesis (Singh et al., 2019). In this study, SlCSN5A can regulate the ubiquitination level of SlPsbS and promote its degradation ( Figure 3 Silencing of SlCSN5 increased the stability and NPQ value of SlPsbS protein in tomato leaves under LNT stress, avoided excessive accumulation of ROS, and improved the stress resistance of plants ( Figure 4), indicating that SlCSN5A can affect the photoprotection mechanism by regulating the stability of chloroplast proteins under LNT stress. CSN5A is highly conserved between plants and animals, and the homologous proteins in Arabidopsis and humans have >60% amino acid similarity. In plants, animals, and fungi, CSN regulates the UPS to direct ubiquitinated proteins to the 26S proteasome for degradation (Schwechheimer, 2001). Our results provide direct evidence that CSN5A is involved in chloroplast protein turnover, revealing a chloroplast protein degradation pathway that is different from previous studies and expanding the existing CSN5A functional model.

[0128] UPS-mediated protein degradation requires a physical interaction between the degradation machinery and its substrates. SlCSN5A directly interacts with SlPsbS both in vitro and in vivo ( Figure 3 ). However, existing studies have shown that CSN5A does not have E3 ubiquitin ligase activity. Therefore, the E3 ubiquitin ligases that modify chloroplast proteins associated with CSN5A remain largely unknown. Importantly, PsbS is constitutively present in the thylakoid membrane, and no evidence has been found that it is associated with the chloroplast envelope (Li et al., 2000). A large number of studies have shown that CSN5 is localized in the nucleus and cytoplasm (Mo et al., 2021; Luo et al., 2021; Gusmaroli et al., 2004). Our results indicate that SlPsbS interacts with SlCSN5A in the cytoplasm ( Figure 3 How the substrate is transferred from the chloroplast to the cytoplasm to contact CSN5A is a new question.

[0129] Studies have shown that aging and abiotic stress activate CV transcription (Umnajkitikorn et al., 2020; Sade et al., 2018; Wang and Blumwald, 2014). Similarly, our results also showed that LNT stress induced high expression of SlCV and promoted the translocation of SlCV from chloroplasts ( Figure 6 ). In Arabidopsis, CV destabilizes chloroplasts and induces the formation of CCV. CCV carries matrix proteins, coat proteins, and thylakoid membrane proteins and is ultimately released from the chloroplast into the cytoplasm (Wang and Blumwald, 2014). Based on these results, we speculated that SlCV is a bridge connecting SlPsbS and SlCSN5A. Therefore, we analyzed the relationship between SlCV, SlPsbS, and SlCSN5A. We found that SlCV can directly interact with SlPsbS and promote the degradation of SlPsbS ( Figure 7 In addition, after LNT stress, we observed colocalization of SlCV and SlPsbS outside the chloroplast ( Figure 8). In addition, we detected the interaction between SlCV and SlCSN5A in vivo and in vitro, and after LNT stress, the SlCV-GFP signal overlapped with the SlCSN5A-mCherry signal in the cytoplasm ( Fig. 9 , 10). These data strongly suggest that SlCV can be translocated from chloroplasts to the cytoplasm together with SlPsbS and that SlCV helps facilitate the contact of substrates (chloroplast proteins) with SlCSN5A and an unknown E3 ubiquitin ligase. In addition to SlPsbS, SlCV can also interact with a variety of chloroplast proteins (SlPsbO, SlPsaD, SlFd, SlCP12) ( Fig.14 Interestingly, SlCSN5A appears to regulate the ubiquitination level of each SlCV-interacting chloroplast protein ( Fig.14 and 15 These results further confirm that SlCV and SlCSN5A play a key role in the ubiquitination and degradation of proteins within chloroplasts and that the SlCV and SlCSN5A pathways appear to have a broad impact on the stability of proteins within chloroplasts.

[0130] CCV can carry CV and chloroplast proteins out of chloroplasts and accumulate in the cytoplasm of mesophyll cells (Wang and Blumwald, 2014). In this study, SlCV could not ubiquitinate SlPsbS ( Figure 7 ), but SlCSN5A can regulate the ubiquitination levels of SlCV and SlPsbS ( Figure 3 and 9 ). Therefore, SlCSN5A may be involved in the ubiquitination and degradation of CCV containing SlCV and chloroplast proteins. Before CCV formation, CV proteins are mainly associated with the thylakoid membrane and the capsule, and CCV is considered to be a vesicle structure formed directly by the chloroplast membrane destroyed by CV (Wang and Blumwald, 2014). CV homologs are present in all plant species sequenced so far and contain a conserved transmembrane domain (Wang and Blumwald, 2014). The binding of SlCV and SlPsbS to SlCSN5A in CCV may be related to its predicted transmembrane domain. As observed in Arabidopsis, stress induces the translocation of SlCV and SlPsbS to the vacuolar precursor, suggesting that SlCV and SlPsbS may also be mobilized to the vacuole for protein degradation ( Figure 8)(Wang and Blumwald, 2014). Recent studies have found that in addition to the 26S proteasome pathway, ubiquitination-mediated vacuolar degradation is also involved in the regulation of ABA signaling. Two major components of ESCRT-I, vacuolar protein sorting 23A (VPS23A) and FYVE1 / FYVE domain-containing protein (FREE1), are used to recognize PYL4 ubiquitinated by the E3 ligase RSL1 and promote the transport of PYL4 to the vacuole for degradation (Belda-Palazon et al., 2016; Yu et al., 2016; Bueso et al., 2014). Therefore, SlCSN5A-mediated ubiquitination and degradation of SlCV and SlPsbS is not inconsistent with the vacuolar protein degradation of CCV. On the contrary, this may indicate that the vacuolar protein degradation of CCV may be related to ubiquitination.

[0131] During aging and abiotic stress, CV plays a key role in the destabilization of the photosynthetic apparatus. Overexpression of CV disrupts the dynamic balance of ROS, leading to excessive accumulation of ROS and reducing the stability of chloroplasts and chloroplast proteins (Yu et al., 2022; Wang and Blumwald, 2014), while silencing of CV delays abiotic stress-induced chloroplast turnover and aging, and enhances source fitness (i.e., carbon and nitrogen assimilation) and photorespiration (Umnajkitikorn et al., 2020; Sade et al., 2018; Wang and Blumwald, 2014). Under LNT stress, overexpression of SlCV leads to a significant decrease in NPQ, accelerated degradation of SlPsbS, and reduced abundance of thylakoid protein complexes ( Fig.12 ), while the Slcv mutant had a higher NPQ value ( Fig.11 ), indicating that CV regulates NPQ levels and plays a role in photoprotection in response to stress. Under heat stress, CSN5-silenced plants improve heat tolerance by regulating auxin signaling (Singh et al., 2019). Similarly, SlCSN5-RNAi plants also showed enhanced resistance and stability of SlPsbS protein under LNT stress ( Figure 4 ). This result suggests that SlCSN5A is more destructive to the degradation of chloroplast proteins than SlCV. SlCV may play a more important role as a connector between SlCSN5A and chloroplast proteins. It is worth noting that silencing SlCSN5A in tomato SlCV-OE plants partially rescued the LNT-sensitive phenotype of SlCV-OE plants and promoted the accumulation of SlPsbS protein ( Fig.16 These genetic observations suggest that SlCV-induced stress sensitivity and chloroplast protein instability in tomato plants may be partially dependent on SlCSN5A.

[0132] Based on the present and previous findings, we constructed a model to describe the role of SlCSN5A in regulating chloroplast protein stability through the UPS. LNT stress activates the expression of SlCV, which targets chloroplasts to interact with chloroplast proteins (SlPsbS), inducing the formation of CCV containing chloroplast proteins (SlPsbS), which are then released from chloroplasts. In the cytoplasm, SlCV and SlPsbS contact SlCSN5A and an unknown E3 ubiquitin ligase through the extramembrane region and are then ubiquitinated and degraded by the UPS. A decrease in the abundance of SlPsbS protein leads to a decrease in NPQ values, which in turn aggravates light damage to the photosystem and reduces the stress resistance of plants ( Fig.17 ). Altogether, our work reveals a potential new pathway for regulating chloroplast protein stability under stress conditions.

[0133] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. Application of tomato S1CSN5A protein or its encoding gene, characterized in that: The application includes one or more of the following aspects: (a) Reduce the ubiquitination level of proteins in chloroplasts or regulate the stability of proteins in chloroplasts; (b) reduce the ubiquitination level of chloroplast vesicle protein SlCV or regulate the stability of chloroplast vesicle protein SlCV; (c) regulating the response of tomato to low temperature stress; Wherein, the application is to regulate by inactivating the SlCSN5A protein or knocking out the encoding gene or reducing the expression level of the encoding gene; In step (c), the regulation of the response of tomatoes to low temperature stress is to reduce the dissipation of the photosystem capacity of tomatoes under low night temperature stress; The tomato SlCSN5A and chloroplast vesicle protein SlCV jointly enhance the stability of tomato chloroplast proteins under low temperature stress.

2. The use according to claim 1, characterized in that: The chloroplast internal protein is one or more of the chloroplast SlPsbS protein or the thylakoid proteins SlPsbO, SlPsaD, SlFd and SlCP12.

3. The use according to claim 1, characterized in that: The tomato SlCSN5A protein mediates the ubiquitination and degradation of proteins in chloroplasts by directly interacting with proteins in chloroplasts.

4. The use according to claim 1, characterized in that: The tomato SlCSN5A protein interacts with the chloroplast vesicle protein SlCV in the cytoplasm, promoting the degradation of the chloroplast vesicle protein SlCV and proteins in the chloroplast.

5. The use according to claim 1, characterized in that: The application is to improve the stability and NPQ value of SlPsbS protein in tomato leaves under low night temperature stress by silencing the SlCSN5 gene, reduce ROS accumulation, and improve the low temperature resistance of plants.

6. A method for improving the low temperature resistance of tomatoes, characterized in that: The method comprises the step of reducing the expression level of the SlCSN5A gene in tomatoes; wherein the step of reducing the expression level of the SlCSN5A gene in tomatoes comprises: reducing the expression level of the SlCSN5A gene in tomatoes by using RNAi, CRISPR / Cas9 gene knockout or knockdown methods, or By reducing the expression level of the SlCV gene in tomatoes through gene knockout or knockdown methods, the low temperature resistance of tomato mutants is enhanced.

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