Proteomics analysis of broccoli after harvest
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We observed distinct differences between broccoli stored at 4 and 25 °C. At 4 °C, the broccoli maintained its green color and firmness for a longer period compared to those stored at 25 °C. In contrast, broccoli stored at 25 °C exhibited faster yellowing and wilting, indicative of accelerated senescence. We have performed physiological parameters such as water loss, chlorophyll measurement in our previous research on transcriptomics of stored broccoli[4]. To understand the proteomic responses of storage temperatures, we conducted proteomics analysis. Farm-grown broccoli in three biological replicates was stored at two different temperatures, 25 and 4 °C, for 1, 3, and 5 d before proteomic profiling was conducted to understand the onset and progression of senescence using a TMT-based method. The total protein was isolated from broccoli florets and digested into peptides that were subsequently labelled with isobaric reagents to be run and analyzed using LC-MS/MS.
Two sets of internal controls were used to normalize the data in order to determine the differentially expressed proteins (DEPs). Sample name descriptions are detailed in Supplemental Tables S1 & S2. For instance, to identify the DEPs between refrigerated and room temperature storage, all samples from day 1 stored at 4 °C were pooled for use as the control (Supplemental Table S1). We then used a fold-change cut-off (cut-off score = 2) to select 1714 DEPs from the broccoli stored as 25 °C for 1 d and identified them using sequences available from Uniprot (www.uniprot.org) (Supplemental Table S2). Similarly, we pooled all samples from day 1 stored at 25 °C and selected 2,276 DEPs from the broccoli stored at 4 °C. There were a total of 2,395 proteins expressed only at either 25 or 4 °C (Supplemental Tables S1 & S2). To determine the upregulated and downregulated DEPs for a specific storage condition, we conducted Venn Diagram analysis (Fig. 1a, b). Among the upregulated DEPs, there were 156 proteins detected at 4 °C and 221 proteins at 25 °C. A higher number of proteins were upregulated on day 3 and day 5 of storage at 25 °C in comparison to storage at 4 °C, suggesting that a larger number of proteins related to stress, growth, and development were induced during the more rapid senescence that occurred at the higher temperature. In contrast, the samples stored for 5 d at 4 °C had a larger number of down-regulated DEPs than those stored at 25 °C. There were 231 DEPs identified during storage at 25 °C, out of which 71 were up-regulated only on day 3 and 67 were up-regulated only on day 5; however, 93 proteins were shared between days 3 and 5 (Fig. 1c, Supplemental Table S1). On the other hand, 169 DEPs were identified during storage at the lower temperature, of which only 57 were downregulated on day 3 and 29 on day 5. Additionally, the higher number of DEPs following storage at 25 °C suggests that protein expression significantly increased during the 5 d of storage, resulting in an accumulation of protein, leading to faster progression of senescence at the higher temperature.
There were some proteins that were differentially regulated on both day 3 and day 5 under both 25 and 4 °C, suggesting that certain proteins underwent biological programs during senescence regardless of the temperature (Fig. 1a, b). There were 54 upregulated DEPs that were present in both warm and cold-stored samples. These included proteins encoded by the ARGONAUTE4 (AGO4), DICER-LIKE3 (DCL3), and SUPPRESSOR OF GENE SILENCING3 (SGS3) genes. Several DEPs in this group were also involved in silencing transcriptional genes as components of the RISC complex associated with the small interfering RNA (siRNA) pathway. These results suggested that siRNA-involved molecular processes modulate tissue senescence. Proteins encoded by 36 genes were downregulated under both warm and cold storage. Among them, 30% were ribosomal proteins, suggesting that ribosome biosynthesis was reduced during senescence.
There were 36 DEPs that were upregulated at 25 °C, but downregulated at 4 °C (Fig. 1d). Many known proteasome components were present in this group, for example, Embryo Defective 2719 (EMB2719), 20S Proteasome Beta Subunit (PBG1), Regulatory Particle Non-ATPASE 12A (RPN12a), ATS9, and Regulatory Particle Non-ATPASE (RPN10). The 26S proteasome mediates ubiquitin-dependent protein degradation, and the 20S proteasome mediates ubiquitin-independent protein degradation[17]. These studies confirmed that both 26S and 20S proteasome activities were inhibited at low temperature, which would likely slow down the progression of senescence. A further 15 DEPs were downregulated at 25 °C but upregulated at 4 °C (Fig. 1d). These included several Nascent polypeptide-Associated Complex subunit (NAC) proteins. NAC proteins are involved in the autophagy pathway and in regulating protein homeostasis during tissue senescence[9]. This indicated that high temperature influenced the autophagy pathway and inhibited protein homeostasis under stress conditions.
Functional categorization of the DEPs
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To determine the properties and functional classifications of the DEPs in postharvest broccoli, the proteins were linked to gene ontology (GO) terms within the database, and the GO was analyzed to categorize the DEPs into cell components, molecular functions, and related biological processes (Fig. 2, Supplemental Table S4). At room temperature, several GO terms related to biological activities, such as stress responses, abiotic stimuli, chemical responses, and transport mechanisms, were enriched. The DEPs were mainly involved in organellar and cytoplasmic development processes responsible for RNA and protein binding, which could lead to structural modifications during postharvest senescence.
The GO enrichment studies from cold storage identified similar processes, yet with lower rates of enrichment for GO terms related to signal processing compared to those related to metabolic processes, cellular components, and anatomical development, with an additional increase in antioxidant activity and oxidoreductase activity. Overall, the percentage response to the functions related to room temperature storage of broccoli was higher relative to cold storage. Therefore, the rate of progression of postharvest senescence was higher at room temperature due to higher biological activity, with a steep decline in energy related to these biological processes.
Identification of oppositely regulated proteins during cold and room temperature storage
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To discover regulators of temporal postharvest stress responses, we examined postharvest proteins that could lead to a better understanding of distinct gene expression over storage time. Such genes could possibly serve as indicators of the different stages of senescence and be used to monitor the physiological status of broccoli. We sought candidate transcripts associated with fundamental physiological processes or biochemical pathways likely to be highly conserved over a wide range of conditions. We identified proteins that were oppositely regulated under room temperature and cold storage and that also showed stage-specific expression patterns, including proteins upregulated after 5 d of storage at room temperature but downregulated under the other conditions and at the other time points (Fig. 3a, b). These criteria identified transcripts that showed distinctive, repeatable patterns of change during the period of senescence before senescence symptoms such as yellowing were visible and also groups of transcripts that changed in contrasting ways. A heat map of protein expression patterns illustrated the proteins that were oppositely regulated at room temperature or cold storage (Fig. 3c). Our analysis revealed four indicators of senescence: calmodulin-domain protein kinase 5 (CDPK5, LOC106307255), mitogen-activated protein kinase 8 (MPK8, LOC106325233), a growth regulating factor (GRF5, LOC106266836), and a RING/PHD zinc finger transcription factor (LOC106295767) as proteins induced by storage under higher temperature for 5 d (Fig. 3c). These protein kinases and transcription factors are known to play crucial roles in switching genes on or off during developmental transitions due to their ability to regulate various cellular processes, including gene expression, signal transduction, and protein activity.
It is worth noting that protein kinases can also trigger a cascade of post-translational modifications, such as ubiquitination, SUMOylation, and acetylation. These modifications can further influence protein interactions and localization, impacting gene regulation and developmental transitions. Several proteins related to these steps of signal transduction were identified among those upregulated after 3 d of storage at 25 °C (Fig. 3d, e; Supplemental Table S5), including an adenylate kinase (ADK1, LOC), a dehydrin (HIRD11), an early nodulin-like protein (ENODL 15), and a tetraketide alpha-pryone reductase (TKPR2). Those protein kinases and dehydrin proteins act as intermediaries in signal transduction pathways, receiving and transmitting these signals to the nucleus, where they can influence gene expression. We also found proteins up-regulated at day 3 during room temperature storage while down-regulated at day 3 and day 5 during cold storage. Those proteins can be used as marker genes for day 3 during room temperature storage. A list of those protein-associated genes are included in Supplemental Table S5.
Validation of DEPs through Real-Time PCR
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Changes in mRNA expression levels may correlate with changes in protein expression. Therefore, comparing qPCR results with proteomics data can provide a more comprehensive understanding of gene expression regulation. To validate the proteomics results, we investigated the transcript levels for the top 12 DEPs in broccoli over time during storage. The genes that encoded the 12 DEPs were identified, and their transcript levels were examined during storage by qRT-PCR (Fig. 4). It's crucial to acknowledge that mRNA levels do not always correlate perfectly with protein levels due to post-transcriptional and translational modifications, and protein stability factors.
The 12 DEPs selected for qRT-PCR evaluation included a thioredoxin, cytochrome P450, a subunit from the light harvesting complex PS II, a plastid protein, and a serine methyltransferase. Consistent with the proteomics data, the transcript levels of those genes were higher after 3 and 5 d of storage at room temperature (25 °C) in comparison to the control (day 1), but were unchanged during 5 d of cold storage (4 °C). Several of the DEPs are predicted to be part of the nucleosome complex, namely the DEK domain chromatin-associated protein, homeobox-3, and a zinc finger protein, but their transcript levels varied under both storage temperatures. For instance, transcript levels of the DEK domain protein decreased from day 1 to day 3 but increased on day 5 at 25 °C, while at 4 °C, the levels increased by day 3 but declined by day 5. Additionally, the homeobox gene exhibited relatively higher expression levels on day 5 in comparison to day 1 at both temperatures. The transcript levels of a few genes involved in developmental regulation, including embryogenesis-related genes, a TCP chaperonin gene, a temperature-induced lipocalin, and SNF7, were also validated (Fig. 4). The transcript levels of lipocalin were lower on day 5 at room temperature compared to cold storage. The chaperonin and LEA transcripts were high at room temperature and showed a significant transition from day 1 to day 5, but remained unchanged in cold storage.
Identification of the key pathways involved in postharvest senescence
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To further identify the key pathways and crucial proteins during tissue senescence, we utilized the Kyoto Encyclopedia of Genes and Genomes (KEGG) database and conducted pathway enrichment analysis (Supplemental Table S5). We found several networks that are directly related to senescence, for example, ribosome synthesis, the spliceosome, and carbon metabolism.
Ribosomal protein changes
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Our pathway enrichment analysis suggested that ribosome proteins were downregulated during storage at the lower temperature, resulting in slower protein synthesis during postharvest senescence (Fig. 5). Ribosomal proteins are involved in translation efficiency, ribosome stability, and other cellular processes, including DNA repair, apoptosis, and regulation of gene expression[16]. Our study identified numerous ribosomal proteins associated with both the smaller and larger subunits that were differentially regulated (Fig. 5). This finding suggested that ribosomal protein synthesis is inhibited at the lower temperature. However, determination of the precise functions of these small and large ribosomal protein subunits in postharvest senescence requires further research. Additionally, photosynthesis and plant-pathogen interaction pathways were also down-regulated in the lower temperature storage conditions (Supplemental Table S5).
We speculated that the lower temperature inhibited photosynthesis and the activities of protein-proteins interaction to slow down senescence and to reduce pathogen infection. We also found that spliceosome proteins were up-regulated on day 3 of storage at the higher temperature. Recently, alternative splicing has been recognized as a critical contributor to senescence and aging[18]. The RNA-protein complexes that combine with unmodified pre-mRNA and various other proteins to form a spliceosome are known as snRNPs. The spliceosome consists of five snRNPs and over 200 additional proteins (Supplemental Fig. S1a). The spliceosome controls splicing of introns and requires the recognition and subsequent cleavage of genes at the 5' donor and 3' acceptor sites[19]. Splicing is one of the post-transcriptional, pre-processing steps of mRNA maturation, allowing translation into proteins. Splicing of pre-mRNAs is a key step in the regulation of gene expression, transcriptome plasticity, and proteome diversity in eukaryotes[20]. Several spliceosome components, including pre-mRNA splicing proteins, were up-regulated during storage at 25 °C, suggesting up-regulation of splicing during senescence (Supplemental Table S6). These proteins could be used as potential markers for the early stages of senescence. Several enriched genes were also involved in translation elongation factor activity, GTPase activity, GTP binding, and RNA transport (Supplemental Table S6).
Carbon metabolism
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Several proteins associated with carbon metabolism and carbon fixation in photosynthetic organisms and protein processing in the endoplasmic reticulum (ER) were up-regulated during storage at 25 °C (Supplemental Fig. S1b, Supplemental Table S5). Most of the protein synthesis machinery occurs in the ER, a localized subcellular organelle where protein folding occurs via chaperones. Our results indicated that misfolded proteins were retained within the ER lumen during senescence. It also suggested that protein production during stress-induced senescence produces proteins that are terminally misfolded and directed toward degradation through the proteasome. This accumulation of misfolded proteins in the ER activated the unfolded protein response signaling pathway, leading to protein degradation. Additionally, we observed in our data that most of the carbon sequestering and fixation-associated genes were differentially regulated (Supplemental Fig. S1b). These DEPs were involved in several energy-requiring processes, suggesting the active processes speed up the progression of senescence. The affected metabolic pathways included carbon metabolism, glycolysis, and the Kreb's cycle. During the ongoing senescence process, when subjected to prolonged storage at room temperature, fresh broccoli florets underwent a series of complex physiological processes, allowing the activation of stress-associated genes and proteins, leading to degradation of chloroplasts and reduction in other accessory pigments[13].
ROS signaling
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There were several DEPs related to oxidative phosphorylation pathways that were downregulated during storage at 25 °C. Senescence involves accumulation of reactive oxygen species (ROS), increasing energy metabolism and carbohydrate metabolism during storage. Accumulation of ROS in the cells generates free radicals, which cause damage to cells and macromolecules, including DNA, proteins, and nucleic acids. Under stress, pathways associated with protein modification are upregulated to counter any loss or damage that occurs during the changing environmental conditions. Most of the proteins associated with protein metabolism belong to the thioredoxin family, which are responsible for maintaining the redox status of proteins targeted during development[21]. Thioredoxins function in developing plant resistance toward stress conditions. Differential expression of certain proteins, including Rubisco, suggested that most of the carbohydrate pathways were involved in the senescence process. We observed certain DEPs involved in carbohydrate-related metabolism during biosynthesis of glycolysis components and pyruvate metabolism, and in the functioning of photosystems during energy biosynthesis (Supplemental Table S5). In this study we found that the cellular machinery involved in stress metabolism was differentially expressed during room temperature storage.
Differentially expressed proteins and altered chemical interaction networks observed during postharvest storage
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We predicted potential relationships among the DEPs in broccoli stored under the two different storage temperatures using the STRING web tool (https://string-db.org/), which provides an integrated, differential network of proteins from metabolic pathways as well as chemical-protein and protein-protein interactions. Relationships observed during postharvest senescence were mapped (Fig. 6). Cysteine was the most prominent amino acid observed to interact with diverse DEPs (Bra000159, Bra34356, Bra032828, Bra002411, and Bra40980), forming a strong network and suggesting that cysteine regulates a wide variety of metabolic processes, including protein biosynthesis. It is also responsible for regulating Coenzyme A, which is an important intermediate for the electron transport chain, photosynthetic complexes, and the C3 cycle.
Several key protein networks were identified from this analysis (Fig. 6a). For instance, Bra040980, which encodes a chloroplast ribosomal protein that is a constituent of the large subunit of the ribosomal complex, was a hub for protein interactions. Furthermore, some stress-responsive proteins, such as the antioxidant proteins superoxide dismutase, peroxidase, and thioredoxin, were also included in this interaction network. Another protein, Bra021125, which encodes Glutaredoxin 4 (GRX4), a metal ion binding protein that is involved in iron homeostasis, had several contacts with several antioxidant proteins including Bra029879, which encodes a manganese superoxide dismutase 1; Bra018436, a hydrogen peroxide catabolic gene; and Bra006413, a redox stress related gene (Fig. 6a). In addition, some proteins involved in RNA binding and translation initiation were also part of this network.
Some dehydrin proteins were also activated during room temperature storage (Fig. 6b). Dehydrins are late embryogenesis abundant (LEA) proteins involved in environmental stress responses in higher plants[22]. The expression of dehydrins occurs in response to diverse stresses such as cold, drought, high salinity, and pathogen infection[23,24]. These are mostly plant-specific proteins, although some LEA proteins have been found in other kingdoms accumulating in the cytoplasm, nucleus, plastid, mitochondrion, ER, and plasma membrane[25]. Functional genomics studies have shown the participation of dehydrins in promoting stress tolerance. Dehydration-responsive proteins such as Brassica rapa cysteine protease 3 (BrCP3), a cysteine-type peptidase involved in various stress response mechanisms, was also present in this network (Fig. 6b). BrCP3 was a hub for this protein interaction network. Within this network, we found BrCP3 interacts with a programmed cell death-related protein, Bra015607 (a homolog of AtSERPIN1, At1g47710), and a serpin family Bra031527. These proteins are parts of intricate regulatory networks that showcase the remarkable adaptability of plants to their environment[26]. Understanding their functions and interactions can provide insights into the molecular pathways that plants use to counteract the negative effects of dehydration and other abiotic stresses (Fig. 6b).
Overexpression of dehydrins promotes tissue senescence
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To test the function of those dehydrin proteins that are activated during broccoli storage, we identified the Arabidopsis homologs of the broccoli gene HIRD1 (AT1G54410) and harpin (HrpN)-interacting protein (a homolog of Arabidopsis gene AT1G15395). Both of those proteins were found in this study to be significantly differentially expressed (Supplemental Table S5). The genes were overexpressed through transient transformation of tobacco leaves, and the resulting phenotypes were analyzed to determine if any of the genes were involved in tissue senescence. We hypothesized that overexpression of the identified genes would accelerate leaf senescence in tobacco epidermal tissue (Fig. 7). In this assay, the negative control did not show obvious changes until 9 d after injection. We selected as a positive control little zipper 3 (ZPR3), which showed a strong senescence pattern in our previous study[4]. Similarly, we found that the dehydrin protein HIRD11 and the HrpN-interacting protein caused bleaching of leaf color after infiltration, suggesting that these two genes are involved in tissue senescence (Fig. 7). Dehydrins, specifically the Arabidopsis dehydrin HIRD11, exhibit cryoprotective activities, protecting freeze-sensitive enzymes such as lactate dehydrogenase (LDH) from damage during freezing conditions, and this cryoprotective ability is attributed to the presence of hydrophobic amino acids and certain conserved segments within the dehydrin protein[27]. One of the typical phenomena observed in transgenic plants expressing dehydrins is the reduction of lipid peroxidation under stress conditions[28]. While dehydrins protect the plants against cold stress[29], their in vivo mechanisms remain mostly unknown. Mutation of hrpN caused a significant decrease in virulence of the plant pathogen Erwinia amylovora[30]. When externally applied to Arabidopsis, a non-host plant, partially purified HrpN promoted plant growth[31].