Dark-induced Senescence Research Articles

The H2S-responsive transcription factor ERF.D3 regulates tomato abscisic acid metabolism, leaf senescence, and fruit ripening.

Hydrogen sulfide (H2S) is a signaling molecule that regulates plant senescence. In this study, we found that H2S delays dark-induced senescence in tomato (Solanum lycopersicum) leaves. Transcriptome and RT-qPCR analyses revealed an Ethylene Response Factor ERF.D3 is quickly induced by H2S. H2S also persulfidated ERF.D3 at amino acid residues C115 and C118. CRISPR/Cas9-mediated gene editing and gene overexpression analyses showed that ERF.D3 negatively regulates leaf senescence and fruit ripening. Abscisic acid (ABA) levels were reduced by ERF.D3 overexpression, suggesting ERF.D3 might regulate ABA metabolism. Additionally, the abscisic acid 8'-hydroxylase-encoding gene CYP707A2, which is required for ABA degradation, was identified as an ERF.D3 target gene through transcriptome data, RT-qPCR, dual-luciferase reporter assays and electrophoretic mobility shift assays. ERF.D3 persulfidation enhanced its transcriptional activity towards CYP707A2. Moreover, the E3 ligase RNF217 ubiquitinated ERF.D3, which may accelerate fruit ripening during the late stage of fruit development. Overall, our study provides valuable insights into the roles of a H2S-responsive ERF.D3 and its persulfidation state in delaying leaf senescence and fruit ripening and provides a link between H2S and ABA degradation.

OsCNGC7 modulates calcium dynamics and accelerates leaf senescence in rice

Calcium plays a crucial role in regulating plant senescence. However, the specific effects of increased intranuclear calcium versus cytoplasmic calcium on aging remain unclear. Cyclic nucleotide-gated channels (CNGCs), which manage Ca2⁺ levels in plant cells, are particularly significant in this context. These channels are known to relocate between the nuclear envelope and the plasma membrane in response to stress and developmental signals. Through this movement, CNGCs help regulate the balance of cytosolic and intranuclear Ca2⁺. In this study, we categorized the 16 CNGC genes in rice into five subgroups. OsCNGCs are notably expressed in leaves, especially during the reproductive stage. Both OsCNGC6 and OsCNGC7 exhibit dual localization to the plasma membrane and the nuclear envelope. Knockdown of OsCNGC7 led to reduced levels of Ca2⁺ and K⁺ in plants. Conversely, yeast expressing the OsCNGC7 gene showed increased sensitivity to Ca2⁺. Additionally, while the [Ca2⁺]cyt was maintained at relatively low levels in both wild-type and OsCNGC7-RNAi lines, the fluorescence intensity was significantly higher in OsCNGC7-overexpressing lines, particularly in the nucleus of root tips. Overexpression of OsCNGC7 resulted in enhanced stomatal opening and accelerated leaf senescence from the tillering stage to grain filling in rice. Treatment with MeJA rapidly induced OsCNGC7 expression, while knockdown of OsCNGC7 delayed both MeJA-induced and dark-induced leaf senescence. Further analysis revealed that OsCNGC7 interacts with OsKAT2 and OsALMT2. In conclusion, our findings highlight the distinct roles of OsCNGCs in regulating senescence. This knowledge could provide new strategies for manipulating plant senescence and enhancing crop productivity.

RICE LONG GRAIN 3 delays dark-induced senescence by downregulating abscisic acid signaling and upregulating reactive oxygen species scavenging activity.

Leaf senescence is a complex developmental process influenced by abscisic acid (ABA) and reactive oxygen species (ROS), both of which increase during senescence. Understanding the regulatory mechanisms of leaf senescence can provide insights into enhancing crop yield and stress tolerance. In this study, we aimed to elucidate the role and mechanisms of rice (Oryza sativa) LONG GRAIN 3 (OsLG3), an APETALA2/ETHYLENE RESPONSIVE FACTOR (AP2/ERF) transcription factor, in orchestrating dark-induced leaf senescence. The transcript levels of OsLG3 gradually increased during dark-induced and natural senescence. Transgenic plants overexpressing OsLG3 exhibited delayed senescence, whereas CRISPR/Cas9-mediated oslg3 mutants exhibited accelerated leaf senescence. OsLG3 overexpression suppressed senescence-induced ABA signaling by downregulating OsABF4 (an ABA-signaling-related gene) and reduced ROS accumulation by enhancing catalase activity through upregulation of OsCATC. In vivo and in vitro binding assays demonstrated that OsLG3 downregulated OsABF4 and upregulated OsCATC by binding directly to their promoter regions. These results demonstrate the critical role of OsLG3 in fine-tuning leaf senescence progression by suppressing ABA-mediated signaling while simultaneously activating ROS-scavenging mechanisms. These findings suggest that OsLG3 could be targeted to enhance crop resilience and longevity.

Arabidopsis GDH1 and GDH2 genes double knock-out results in a stay-green phenotype during dark-induced senescence.

Yellowing is the first visually observable sign of plant leaf senescence. We found that Arabidopsis double knockout mutant gdh1gdh2 for genes of NAD(H)-dependent glutamate dehydrogenase retains green color of the leaves (stay-green phenotype) during a dark-induced senescence, in contrast to wild-type plants, whose leaves turn yellow. When the gdh1gdh2 plants are exposed to the dark more than four days, they demonstrate slower chlorophyll degradation than in the wild-type plants under the same conditions, as well as dysregulation of chlorophyll breakdown genes encoding chlorophyll b reductase, Mg-dechelatase, pheophytinase and pheophorbide a oxygenase. The slowed degradation of chlorophyll b in gdh1gdh2 plants significantly alters the chlorophyll a/b ratio. Ion leakage in the mutant plants increases significantly from four to eight days in the darkness, correlating with their premature death during this period. The discovered facts suggest a functional connection between activity of NAD(H)-dependent glutamate dehydrogenase and dark-induced senescence progress in Arabidopsis.

Expansion and diversification of the Glycine max (Gm) ERD15-like subfamily of the PAM2-like superfamily.

Despite modulating senescence and drought responses, the GmERD15-like subfamily members are differentially induced by multiple stresses and diverge partially in stress signaling functions. The PAM2 motif represents a binding site for poly (A)-binding proteins (PABPs), often associated with RNA metabolism regulation. The PAM2-containing protein ERD15 stands out as a critical regulator of diverse stress responses in plants. Despite the relevance of the PAM2 motif, a comprehensive analysis of the PAM2 superfamily and ERD15-like subfamily in the plant kingdom is lacking. Here, we provide an extensive in silico analysis of the PAM2 superfamily and the ERD15-like subfamily in soybean, using Arabidopsis and rice sequences as prototypes. The Glycine max ERD15-like subfamily members were clustered in pairs, likely originating from DNA-based gene duplication, as the paralogs display high sequence conservation, similar exon/intron genome organization, and are undergoing purifying selection. Complementation analyses of an aterd15 mutant demonstrated that the plant ERD15-like subfamily members are functionally redundant in response to drought, osmotic stress, and dark-induced senescence. Nevertheless, the soybean members displayed differential expression profiles, biochemical activity, and subcellular localization, consistent with functional diversification. The expression profiles of Glyma04G138600 under salicylic acid (SA) and abscisic acid (ABA) treatments differed oppositely from those of the other GmERD15-like genes. Abiotic stress-induced coexpression analysis with soybean PABPs showed that Glyma04G138600 was clustered separately from other GmERD15s. In contrast to the AtERD15 stress-induced nuclear redistribution, Glyma04G138600 and Glyma02G260800 localized to the cytoplasm, while Glyma03G131900 fractionated between the cytoplasm and nucleus under normal and stress conditions. These data collectively indicate that despite modulating senescence and drought responses, the GmERD15-like subfamily members are differentially induced by multiple stresses and may diverge partially in stress signaling functions.

BpTCP19 targets BpWRKY53 to negatively regulate jasmonic acid- and dark-induced leaf senescence in Betula platyphylla

TCP (TEOSINTE-LIKE1, CYCLOIDEA, and PROLIFERATING CELL FACTOR1) is a plant-specific transcription factor that has garnered significant attention due to its wide-ranging involvement in the regulation of plant growth or developmental processes. However, the molecular mechanisms through which TCP genes orchestrate leaf senescence have not been extensively elucidated. BpTCP19, a member of the PCF subfamily in Betula platyphylla, and has high homology to AtTCP19. BpTCP19 displayed pronounced downregulation in response to methyl jasmonate (MeJA) and dark treatment. Overexpressing BpTCP19 in Betula platyphylla led to a delay in leaf senescence, resulting in prolonged leaf greenness under both MeJA and dark conditions. Transcriptome analysis revealed that overexpression of BpTCP19 induced alterations in the expression levels of genes linked to cell proliferation, hormone signaling transduction, and leaf senescence, including the early responsive factor BpWRKY53. Furthermore, through Yeast one-hybrid assays and GUS analysis, BpTCP19 was shown to bind to the promoter region of BpWRKY53, suppressing its expression and thereby retarding leaf senescence. This study elucidates the physiological and molecular functions of BpTCP19 as a central transcriptional regulatory module in leaf senescence and provides a potential target gene for delaying leaf senescence by mitigating sensitivity to external aging signals such as Jasmonic acid (JA) and darkness.

Genome-wide association study of image-based trait reveals the genetic architecture of dark-induced leaf senescence in rice

Genome-wide association study of image-based trait reveals the genetic architecture of dark-induced leaf senescence in rice

Unveiling the role of epigenetics in leaf senescence: a comparative study to identify different epigenetic regulations of senescence types in barley leaves

BackgroundDevelopmental leaf senescence (DLS) is an irreversible process followed by cell death. Dark-induced leaf senescence (DILS) is a reversible process that allows adaptations to changing environmental conditions. As a result of exposure to adverse environmental changes, plants have developed mechanisms that enable them to survive. One of these is the redirection of metabolism into the senescence pathway. The plant seeks to optimise resource allocation. Our research aims to demonstrate how epigenetic machinery regulates leaf senescence, including its irreversibility.ResultsIn silico analyses allowed the complex identification and characterisation of 117 genes involved in epigenetic processes in barley. These genes include those responsible for DNA methylation, post-translational histone modifications, and ATP-dependent chromatin remodelling complexes. We then performed RNAseq analysis after DILS and DLS to evaluate their expression in senescence-dependent leaf metabolism. Principal component analysis revealed that evaluated gene expression in developmental senescence was similar to controls, while induced senescence displayed a distinct profile. Western blot experiments revealed that senescence engages senescence-specific histone modification. During DILS and DLS, the methylation of histone proteins H3K4me3 and H3K9me2 increased. H3K9ac acetylation levels significantly decreased during DILS and remained unchanged during DLS.ConclusionsThe study identified different epigenetic regulations of senescence types in barley leaves. These findings are valuable for exploring epigenetic regulation of senescence-related molecular mechanisms, particularly in response to premature, induced leaf senescence. Based on the results, we suggest the presence of an epigenetically regulated molecular switch between cell survival and cell death in DILS, highlighting an epigenetically driven cell survival metabolic response.

OsRopGEF10 Attenuates Cytokinin Signaling to Regulate Panicle Development and Grain Yield in Rice

Cytokinins, which play crucial roles in shoot development, substantially affect grain yield. In rice, the OsRopGEF10-OsRAC3 module is associated with cytokinin signaling and crown root development. However, the effects of RopGEF-mediated cytokinin signaling on rice shoot development and grain yield remain unclear. In this study, we investigated the role of OsRopGEF10 in SAM development and the underlying mechanism. We showed that overexpression of OsRopGEF10 inhibited SAM and panicle development, leading to decreased grain yield. Intriguingly, the overexpression of a specific amino acid mutant of OsRopGEF10, designated gef10-W260S, was found to promote panicle development and grain yield. Further analysis using the BiFC assay revealed that the gef10-W260S mutation disrupted the recruitment of rice histidine phosphotransfer proteins (OsAHP1/2) to the plasma membrane (PM), thereby promoting cytokinin signaling. This effect was corroborated by a dark-induced leaf senescence assay, which revealed an increased cytokinin response in the gef10-W260S ectopic expression lines, whereas the overexpression lines presented a suppressed cytokinin response. Moreover, we revealed that the enhanced panicle development in the gef10-W260S lines was attributable to the upregulated expression of several type-B response regulators (RRs) that are crucial for panicle development. Collectively, these findings revealed the negative regulatory function of OsRopGEF10 in the development of the shoot apical meristem (SAM) via interference with cytokinin signaling. Our study highlights the promising role of OsRopGEF10 as a potential target for regulating SAM and panicle development in rice, revealing a valuable breeding strategy for increasing crop yield.

Natural variation in photosynthetic electron transport of wheat flag leaves in response to dark-induced senescence

Early leaf senescence affects photosynthetic efficiency and limits growth during the late production stage of winter wheat (Triticum aestivum). Natural variation in photosystem response to senescence represents a valuable resource for improving the aging traits of flag leaves. To explore the natural variation of different phases of photosynthetic electron transport in modern wheat cultivars during senescence, we exposed the flag leaves of 32 wheat cultivars to dark conditions to induce senescence process, and simultaneously measured prompt fluorescence and modulated 820 nm reflection. The results showed that the chlorophyll content, activity of PSII donor side, PSI and electron transfer between PSII and PSI were all decreased during dark-induced senescence, but they showed different sensitivity to dark-induced senescence. Furthermore, natural variation in photosynthetic parameters among the 32 wheat cultivars were also observed and showed by variation coefficient of the different parameters. We observed that PSII and PSI activity showed less sensitivity to dark-induced senescence than electron transfer between them, while PSII and PSI activity exhibit greater natural variation than electron transport between PSII and PSI. It suggests that Cytb6f might degrade faster and have less variation than PSII and PSI during dark-induced senescence.

Discovery of anisiflupurin, an inhibitor of cytokinin dehydrogenase that mitigates heat-induced yield reduction in rice.

In a screening of anilinopurine, anisiflupurin was identified as potent inhibitor of cytokinin dehydrogenase/oxidase (CKX). Inhibitors of CKX have been supposed to be potent plant growth regulators to alleviate the detrimental effects of abiotic stress on crop production. The aim of the study was to profile anisiflupurin in a set of physiological assays and to evaluate its potential for heat stress mitigation in rice field trials. Anisiflupurin delayed dark-induced senescence and increased transpiration in detached maize leaves in a dose-dependent manner. Similarly, the transpiration of young rice plants under heat stress was increased for several days after application with anisiflupurin. Application of anisiflupurin during early phases of generative growth not only restored heat-induced pollen alterations it increased grain yield in field grown rice under heat conditions as demonstrated in a large field program conducted in southeast Asia. Thereby, efficacy of anisiflupurin was rate-dependent and most effective when applied during early generative growth phases prior heat stress. Application of anisiflupurin secures seed setting by protecting pollen development and enhances grain weight under heat stress conditions in rice. The results of this research opens up a promising avenue for mitigating the adverse effects of heat stress in rice cultivation. © 2024 Society of Chemical Industry.

Overexpression of cassava melatonin receptor PMTR1 plays dual roles in development under light and dark conditions in Arabidopsis.

MePMTR1 is involved in plant development and production as well as photosynthesis in plant. Melatonin is widely involved in plant growth and development as well as stress responses. Compared with the extending studies of melatonin in stress responses, the direct link between melatonin and plant development in the whole stages remains unclear. With the identification of phytomelatonin receptor PMTR1 in plants, melatonin signalling is becoming much clearer. However, the function of MePMTR1 in tropical crop cassava remains elusive. In this study, we found that overexpression of MePMTR1 showed larger biomass than wild type (WT), including higher number and area of leaves, weight, and accompanying with higher photosynthetic efficiency. Consistently, exogenous melatonin accelerated photosynthetic rate in Arabidopsis. In addition, MePMTR1-overexpressed plants exhibited more resistance to dark-induced senescence compared with WT, demonstrated by higher chlorophyll, lower hydrogen peroxide and superoxide content. In summary, this study illustrated that melatonin and its receptor regulate growth, development and senescence in plants, highlighting the potential application of melatonin and its receptor in improving crop yield and photosynthesis.

Rice melatonin deficiency causes premature leaf senescence via DNA methylation regulation

In a study of DNA methylation changes in melatonin-deficient rice mutants, mutant plants showed premature leaf senescence during grain-filling and reduced grain yield. Melatonin deficiency led to transcriptional reprogramming, especially of genes involved in chlorophyll and carbon metabolism, redox regulation, and transcriptional regulation, during dark-induced leaf senescence. Hypomethylation of mCG and mCHG in the melatonin-deficient rice mutants was associated with the expression change of both protein-coding genes and transposable element-related genes. Changes in gene expression and DNA methylation in the melatonin-deficient mutants were compensated by exogenous application of melatonin. A decreased S-adenosyl-L-methionine level may have contributed to the DNA methylation variations in rice mutants of melatonin deficiency under dark conditions.

Leaf Senescence Regulation Mechanism Based on Comparative Transcriptome Analysis in Foxtail Millet.

Leaf senescence, a pivotal process in plants, directly influences both crop yield and nutritional quality. Foxtail millet (Setaria italica) is a C4 model crop renowned for its exceptional nutritional value and stress tolerance characteristics. However, there is a lack of research on the identification of senescence-associated genes (SAGs) and the underlying molecular regulatory mechanisms governing this process. In this study, a dark-induced senescence (DIS) experimental system was applied to investigate the extensive physiological and transcriptomic changes in two foxtail millet varieties with different degrees of leaf senescence. The physiological and biochemical indices revealed that the light senescence (LS) variety exhibited a delayed senescence phenotype, whereas the severe senescence (SS) variety exhibited an accelerated senescence phenotype. The most evident differences in gene expression profiles between these two varieties during DIS included photosynthesis, chlorophyll, and lipid metabolism. Comparative transcriptome analysis further revealed a significant up-regulation of genes related to polysaccharide and calcium ion binding, nitrogen utilization, defense response, and malate metabolism in LS. In contrast, the expression of genes associated with redox homeostasis, carbohydrate metabolism, lipid homeostasis, and hormone signaling was significantly altered in SS. Through WGCNA and RT-qPCR analyses, we identified three SAGs that exhibit potential negative regulation towards dark-induced leaf senescence in foxtail millet. This study establishes the foundation for a further comprehensive examination of the regulatory network governing leaf senescence and provides potential genetic resources for manipulating senescence in foxtail millet.

Rice ONAC016 promotes leaf senescence through abscisic acid signaling pathway involving OsNAP

Senescence-induced NAC (senNAC) TFs play a crucial role in senescence during the final stage of leaf development. In this study, we identified a rice senNAC, ONAC016, which functions as a positive regulator of leaf senescence. The expression of ONAC016 increased rapidly in rice leaves during the progression of dark-induced and natural senescence. The onac016-1 knockout mutant showed a delayed leaf yellowing phenotype, whereas the overexpression of ONAC016 accelerated leaf senescence. Notably, ONAC016 expression was upregulated by abscisic acid (ABA), and thus detached leaves of the onac016-1 mutant remained green much longer under ABA treatment. Quantitative RT-PCR analysis showed that ONAC016 upregulates the genes associated with chlorophyll degradation, senescence, and ABA signaling. Yeast one-hybrid and dual-luciferase assays revealed that ONAC016 binds directly to the promoter regions of OsNAP, a key gene involved in chlorophyll degradation and ABA-induced senescence. Taken together, these results suggest that ONAC016 plays an important role in promoting leaf senescence through the ABA signaling pathway involving OsNAP.

M6A RNA methylation counteracts dark-induced leaf senescence in Arabidopsis.

Senescence is an important physiological process which directly affects many agronomic traits in plants. Senescence induces chlorophyll degradation, phytohormone changes, cellular structure damage, and altered gene regulation. Although these physiological outputs are well defined, the molecular mechanisms employed are not known. Using dark-induced leaf senescence (DILS) as the experimental system, we investigated the role of N6-methyladenosine (m6A) mRNA methylation during senescence in Arabidopsis (Arabidopsis thaliana). Plants compromised in m6A machinery components like METHYLTRANSFERASE A (mta mutant) and VIRILIZER1 (vir-1 mutant) showed an enhanced DILS phenotype. This was accompanied by compromised chloroplast and photosynthesis performance in mta as well as accumulation of senescence-promoting camalexin and phytohormone jasmonic acid after dark treatment. m6A levels increased during DILS and destabilized senescence-related transcripts thereby preventing premature aging. Due to inefficient decay, senescence-related transcripts like ORESARA1 (ORE1), SENESCENCE-ASSOCIATED GENE 21 (SAG21), NAC-like, activated by AP3/PI (NAP), and NONYELLOWING 1 (NYE1) over-accumulated in mta thereby causing accelerated senescence during DILS. Overall, our data propose that m6A modification is involved in regulating the biological response to senescence in plants, providing targets for engineering stress tolerance of crops.

Rice Basic Helix-Loop-Helix 079 (OsbHLH079) Delays Leaf Senescence by Attenuating ABA Signaling

Leaf senescence represents the final phase of leaf development and is characterized by a highly organized degenerative process involving the active translocation of nutrients from senescing leaves to growing tissues or storage organs. To date, a large number of senescence-associated transcription factors (sen-TFs) have been identified that regulate the initiation and progression of leaf senescence. Many of these TFs, including NAC (NAM/ATAF1/2/CUC2), WRKY, and MYB TFs, have been implicated in modulating the expression of downstream senescence-associated genes (SAGs) and chlorophyll degradation genes (CDGs) under the control of phytohormones. However, the involvement of basic helix-loop-helix (bHLH) TFs in leaf senescence has been less investigated. Here, we show that OsbHLH079 delays both natural senescence and dark-induced senescence: Overexpression of OsbHLH079 led to a stay-green phenotype, whereas osbhlh079 knockout mutation displayed accelerated leaf senescence. Similar to other sen-TFs, OsbHLH079 showed a gradual escalation in expression as leaves underwent senescence. During this process, the mRNA levels of SAGs and CDGs remained relatively low in OsbHLH079 overexpressors, but increased sharply in osbhlh079 mutants, suggesting that OsbHLH079 negatively regulates the transcription of SAGs and CDGs under senescence conditions. Additionally, we found that OsbHLH079 delays ABA-induced senescence. Subsequent RT-qPCR and dual-luciferase reporter assays revealed that OsbHLH079 downregulates the expression of ABA signaling genes, such as OsABF2, OsABF4, OsABI5, and OsNAP. Taken together, these results demonstrate that OsbHLH079 functions in delaying leaf yellowing by attenuating the ABA responses.

Target of rapamycin (TOR) plays a role in regulating ROS-induced chloroplast damage during cucumber (Cucumis sativus) leaf senescence.

In cucumber production, delaying leaf senescence is crucial for improving cucumber yield and quality. Target of rapamycin (TOR) is a highly conserved serine/threonine protein kinase in eukaryotes, which can integrate exogenous and endogenous signals (such as cell energy state levels) to stimulate cell growth, proliferation, and differentiation. However, no studies have yet examined the regulatory role of TOR signalling in cucumber leaf senescence. In this study, the effects of TOR signalling on dark-induced cucumber leaf senescence were investigated using the TOR activator MHY1485 and inhibitor AZD8055 combined with transient transformation techniques. The results indicate that TOR responds to dark-induced leaf senescence, and alterations in TOR activity/expression influence cucumber leaf resistance to dark-induced senescence. Specifically, in plants with elevated TOR activity/expression, we observed reduced expression of senescence-related genes, less membrane lipid damage, decreased cell apoptosis, lower levels of reactive oxygen species production, and less damage to the photosynthetic system compared to the control. In contrast, in plants with reduced TOR activity/expression, we observed higher expression of senescence-related genes, increased membrane lipid damage, enhanced cell apoptosis, elevated levels of reactive oxygen species production, and more damage to the photosynthetic system. These comprehensive results underscore the critical role of TOR in regulating dark-induced cucumber leaf senescence. These findings provide a foundation for controlling premature leaf senescence in cucumber production and offer insights for further exploration of leaf senescence mechanisms and the development of more effective control methods.

COG-imposed Golgi functional integrity determines the onset of dark-induced senescence.

Plant survival depends on dynamic stress-response pathways in changing environments. To uncover pathway components, we screened an ethyl methanesulfonate-mutagenized transgenic line containing a stress-inducible luciferase construct and isolated a constitutive expression mutant. The mutant is the result of an amino acid substitution in the seventh subunit of the hetero-octameric conserved oligomeric Golgi (COG) complex of Arabidopsis thaliana. Complementation studies verified the Golgi localization of cog7, and stress tests established accelerated dark-induced carbon deprivation/senescence of the mutant compared with wild-type plants. Multiomics and biochemical analyses revealed accelerated induction of protein ubiquitination and autophagy, and a counterintuitive increased protein N-glycosylation in senescencing cog7 relative to wild-type. A revertant screen using the overexpressor (FOX)-hunting system established partial, but notable rescue of cog7 phenotypes by COG5 overexpression, and conversely premature senescence in reduced COG5 expressing lines. These findings identify COG-imposed Golgi functional integrity as a main player in ensuring cellular survival under energy-limiting conditions.

Pyrroline-5-carboxylate metabolism protein complex detected in Arabidopsis thaliana leaf mitochondria.

Proline dehydrogenase (ProDH) and pyrroline-5-carboxylate (P5C) dehydrogenase (P5CDH) catalyse the oxidation of proline into glutamate via the intermediates P5C and glutamate-semialdehyde (GSA), which spontaneously interconvert. P5C and GSA are also intermediates in the production of glutamate from ornithine and α-ketoglutarate catalysed by ornithine δ-aminotransferase (OAT). ProDH and P5CDH form a fused bifunctional PutA enzyme in Gram-negative bacteria and are associated in a bifunctional substrate-channelling complex in Thermus thermophilus; however, the physical proximity of ProDH and P5CDH in eukaryotes has not been described. Here, we report evidence of physical proximity and interactions between Arabidopsis ProDH, P5CDH, and OAT in the mitochondria of plants during dark-induced leaf senescence when all three enzymes are expressed. Pairwise interactions and localization of the three enzymes were investigated using bimolecular fluorescence complementation with confocal microscopy in tobacco and sub-mitochondrial fractionation in Arabidopsis. Evidence for a complex composed of ProDH, P5CDH, and OAT was revealed by co-migration of the proteins in native conditions upon gel electrophoresis. Co-immunoprecipitation coupled with mass spectrometry analysis confirmed the presence of the P5C metabolism complex in Arabidopsis. Pull-down assays further demonstrated a direct interaction between ProDH1 and P5CDH. P5C metabolism complexes might channel P5C among the constituent enzymes and directly provide electrons to the respiratory electron chain via ProDH.