Histone H3K27 Demethylase SlJMJ3 Modulates Fruit Ripening in Tomato.

The histone lysine (K) demethylase 4 (KDM4/JHDM3) subfamily of jumonji domain-containing demethylases (JMJs) has been implicated in various aspects of plant development. However, their involvement in regulating the ripening of fleshy fruits remains unclear. Here, we identified SlJMJ3, a member of the KDM4/JHDM3 family, as a H3K27me3 demethylase in tomato (Solanum lycopersicum) that plays an important role in fruit ripening regulation. Overexpression of SlJMJ3 led to accelerated fruit ripening, whereas loss-of-function of SlJMJ3 delayed this process. Furthermore, we determined that SlJMJ3 exerts its regulatory function by modulating the expression of multiple ripening-related genes involved in ethylene biosynthesis and response, carotenoid metabolism, cell wall modification, transcriptional control, and DNA methylation modification. SlJMJ3 bound directly to the promoters of ripening-related genes harboring the CTCTGYTY motif and activates their expression. Additionally, SlJMJ3 reduced the levels of H3K27me3 at its target genes, thereby up-regulating their expression. In summary, our findings highlight the role of SlJMJ3 in the regulation of fruit ripening in tomato. By removing the methyl group from trimethylated histone H3 lysine 27 at ripening-related genes, SlJMJ3 acts as an epigenetic regulator that orchestrates the complex molecular processes underlying fruit ripening.

WUSCHEL-related homeobox transcription factor SlWOX13 regulates tomato fruit ripening.

Fruit ripening is a complex, genetically programmed process involving the action of critical transcription factors (TFs). Despite the established importance of WUSCHEL-related homeobox (WOX) TFs in plant development, the involvement of WOX and its underlying mechanism in the regulation of fruit ripening remain unclear. Here, we demonstrate that SlWOX13 regulates fruit ripening in tomato (Solanum lycopersicum). Overexpression of SlWOX13 accelerates fruit ripening, whereas loss-of-function mutation in SlWOX13 delays this process. Moreover, ethylene synthesis and carotenoid accumulation are significantly inhibited in slwox13 mutant fruit but accelerated in SlWOX13 transgenic fruit. Integrated analyses of RNA-seq and chromatin immunoprecipitation (ChIP)-seq identified 422 direct targets of SlWOX13, of which 243 genes are negatively regulated and 179 are positively regulated by SlWOX13. Electrophoretic mobility shift assay, RT-qPCR, dual-luciferase reporter assay, and ChIP-qPCR analyses demonstrated that SlWOX13 directly activates the expression of several genes involved in ethylene synthesis and signaling and carotenoid biosynthesis. Furthermore, SlWOX13 modulates tomato fruit ripening through key ripening-related TFs, such as RIPENING INHIBITOR (RIN), NON-RIPENING (NOR), and NAM, ATAF1, 2, and CUC2 4 (NAC4). Consequently, these effects promote fruit ripening. Taken together, these results demonstrate that SlWOX13 positively regulates tomato fruit ripening via both ethylene synthesis and signaling and by transcriptional regulation of key ripening-related TFs.

Melatonin Treatment Maintains the Quality of Fresh-Cut Gastrodia elata under Low-Temperature Conditions by Regulating Reactive Oxygen Species Metabolism and Phenylpropanoid Pathway.

The application of melatonin (MT) has been shown to improve the quality during the storage of fruits and vegetables. The primary objective of this study is to investigate the effects of MT on the quality of fresh-cut Gastrodia elata during low-temperature (4 °C) storage. The results indicated that MT treatment not only suppressed the respiratory rate and malondialdehyde content but also slowed down the decline in total acidity and total soluble solids, effectively inhibiting microbial growth and enhancing the product safety of fresh-cut G. elata. The treatment with MT reduced the superoxide anions and hydrogen peroxide production, as well as inhibiting the activity and expression of peroxidase and polyphenol oxidase. Additionally, it led to increased activity and the expression of antioxidant-related enzymes, including superoxide dismutase, catalase, ascorbate peroxidase, glutathione reductase, monodehydroascorbate reductase, and dehydroascorbate reductase, while also resulting in elevated levels of ascorbic acid and glutathione. Furthermore, the treatment with MT induced an increase in the total phenolic and flavonoid content of fresh-cut G. elata and enhanced the activity and expression of key enzymes involved in the phenylpropanoid pathway (phenylalanine ammonia-lyase, cinnamate-4-hydroxylase, 4-coumarate: CoA ligase). In summary, MT enhances the antioxidant capacity by activating both the ROS metabolism and phenylpropanoid pathway, thus maintaining the quality of fresh-cut G. elata.

A NAC transcriptional factor BrNAC029 is involved in cytokinin-delayed leaf senescence in postharvest Chinese flowering cabbage.

Both cytokinin and NAC transcription factors were reported to involve in leaf senescence. However, the mechanism of NAC transcription factors how to regulate cytokinin-delayed leaf senescence is still unknown. In this study, application of N-(2-chloro-4-pyridyl)-N'-phenylurea (CPPU), a cytokinin analogue, significantly delayed leaf senescence and maintained cytokinin content of Chinese flowering cabbage during storage. Meanwhile, the expression of an NAC transcriptional activator (BrNAC029) was increased but suppressed by CPPU treatment. Furthermore, BrNAC029 activated the expressions of chlorophyll catabolic genes BrPAO and BrSGR2, cytokinin oxidase gene BrCKX1 and senescence maker gene BrSAG113 by binding to their promoters. Additionally, overexpressions of BrNAC029 in tobacco and Arabidopsis accelerated leaf senescence and up-expressed the related genes. Taken together, it was suggested that BrNAC029 may serve as a transcriptional activator to activate the transcriptions of these related genes to eventually accelerate leaf senescence of Chinese flowering cabbage by promoting chlorophyll degradation and reducing endogenous cytokinin level.

Proteome-wide identification of non-histone lysine methylation in tomato during fruit ripening.

INTRODUCTION: Histone and non-histone methylations are important post-translational modifications in plants. Histone methylation plays a crucial role in regulating chromatin structure and gene expression. However, the involvement of non-histone methylation in plant biological processes remains largely unknown. METHODS: The methylated substrates and methylation sites during tomato fruit ripening were identified by LC-MS/MS. Bioinformatics of lysine methylated proteins was conducted to analyze the possible role of methylated proteins. The effects of methylation modification on protein functions were preliminarily investigated by site-directed mutation simulation. RESULTS: A total of 241 lysine methylation (mono-, di- and trimethylation) sites in 176 proteins were identified with two conserved methylation motifs: xxxxxxExxx_K_xxxExxxxxx and xxxxxxExxx_K_xxxxxxxxxx. These methylated proteins were mainly related to fruit ripening and senescence, oxidation reduction process, signal transduction, stimulus and stress responses, and energy metabolism. Three representative proteins, thioredoxin (Trx), glutathione S-transferase T1 (GST T1), and NADH dehydrogenase (NOX), were selected to investigate the effect of methylation modifications on protein activity. Mimicking demethylation led to decreased Trx activity but increased GST T1 and NOX activities. In addition, RT-qPCR exhibited that the expression of many genes that encode proteins subjected to methylation was upregulated during fruit ripening. CONCLUSION: Our study suggests that tomato fruit ripening undergo non-histone lysine methylation, which may participate in the regulation of fruit ripening. It is the first report of methyl proteome profiling of non-histone lysine in horticultural crops.

Histone H3K27 demethylase SlJMJ4 promotes dark- and ABA- induced leaf senescence in tomato.

Leaf senescence is a highly-programmed developmental process during the plant life cycle. ABA plays an important role in leaf senescence. However, the mechanism underlying ABA-mediated leaf senescence, particularly the upstream epigenetic regulatory network, remains largely unclear. Here, we identified that SlJMJ4, a Jumonji C (jmjC) domain-containing protein in tomato, specifically demethylates di- and tri-methylations of lysine 27 of histone H3 (H3K27) in vitro and in vivo. Overexpression of SlJMJ4 results in premature senescence phenotype and promotes dark- and ABA-induced leaf senescence in tomato. Under dark condition, SlJMJ4-promoted leaf senescence is associated with upregulated expression of transcription factors (SlORE1 and SlNAP2) and senescence-associated genes (SlSAG113, SlSAG12) via removal of H3K27me3. In responses to ABA, overexpression of SlJMJ4 increases its binding at the loci of SlORE1, SlNAP2, SlSAG113, SlSAG12, SlABI5 and SlNCED3 and decreases their H3K27me3 levels, and therefore activates their expression and mediates ABA-induced leaf senescence in tomato. Taken together, these results demonstrate that SlJMJ4 plays a positive role in leaf senescence in tomato and is implicated in ABA-induced leaf senescence by binding to many key genes related to ABA synthesis and signaling, transcription regulation and senescence and hence promoting their H3K27me3 demethylation.

F-box protein EBF1 and transcription factor ABI5-like regulate banana fruit chilling-induced ripening disorder.

Cold stress adversely affects plant production, both qualitatively and quantitatively. Banana (Musa acuminata) is sensitive to cold stress and suffers chilling injury (CI) when stored under 11°C, causing abnormal fruit softening. However, the mechanism underlying the abnormal fruit softening due to CI remains obscure. This study uncovered the coordinated transcriptional mechanism of ethylene F-box (EBF1) protein and abscisic acid-insensitive 5 (ABI5)-like protein in regulating chilling-induced softening disorders of Fenjiao banana. Cold stress severely inhibited the transcript and protein levels of EBF1, ABI5-like, and fruit softening-related genes. The ABI5-like protein bound to the promoters of key starch and cell wall degradation-related genes such as ß-amylase 8 (BAM8), pectate lyase 8 (PL8), and ß-D-xylosidase23-like (XYL23-like) and activated their activities. EBF1 physically interacted with ABI5-like and enhanced the transcriptional activity of the key starch and cell wall degradation-related genes but did not ubiquitinate or degrade ABI5-like protein. This promoted fruit ripening and ameliorated fruit CI in a manner similar to the effect of exogenous abscisic acid treatment. The ectopic and transient overexpression of EBF1 and ABI5-like genes in tomato (Solanum lycopersicum) and Fenjiao banana accelerated fruit ripening and softening by promoting ethylene production, starch and cell wall degradation, and decreasing fruit firmness. EBF1 interacted with EIL4 but did not ubiquitinate or degrade EIL4, which is inconsistent with the typical role of EBF1/2 in Arabidopsis (Arabidopsis thaliana). These results collectively highlight that the interaction of EBF1 and ABI5-like controls starch and cell wall metabolism in banana, which is strongly inhibited by chilling stress, leading to fruit softening and ripening disorder.

SlJMJ7 orchestrates tomato fruit ripening via crosstalk between H3K4me3 and DML2-mediated DNA demethylation.

The ripening of fleshy fruits is a unique developmental process that Arabidopsis and rice lack. This process is driven by hormones and transcription factors. However, the critical and early regulators of fruit ripening are still poorly understood. Here, we revealed that SlJMJ7, an H3K4 demethylase, is a critical negative regulator of fruit ripening in tomato. Combined genome-wide transcription, binding sites, histone H3K4me3 and DNA methylation analyses demonstrated that SlJMJ7 regulates a key group of ripening-related genes, including ethylene biosynthesis (ACS2, ACS4 and ACO6), transcriptional regulation (RIN and NOR) and DNA demethylation (DML2) genes, by H3K4me3 demethylation. Moreover, loss of SlJMJ7 function leads to increased H3K4me3 levels, which directly activates ripening-related genes, and to global DML2-mediated DNA hypomethylation in fruit, which indirectly prompts expression of ripening-related genes. Together, these effects lead to accelerated fruit ripening in sljmj7 mutant. Our findings demonstrate that SlJMJ7 acts as a master negative regulator of fruit ripening not only through direct removal of H3K4me3 from multiple key ripening-related factors, but also through crosstalk between histone and DNA demethylation. These findings reveal a novel crosstalk between histone methylation and DNA methylation to regulate gene expression in plant developmental processes.

Benzothiazole Treatment Regulates the Reactive Oxygen Species Metabolism and Phenylpropanoid Pathway of Rosa roxburghii Fruit to Delay Senescence During Low Temperature Storage.

Rosa roxburghii fruit were used as research objects to study the effects of different concentrations of benzothiazole (BTH) treatment on quality parameters, reactive oxygen species (ROS) metabolism, and the phenylpropanoid pathway during storage at 4°C for 14days. Results showed that BTH effectively delayed senescence with lower decay incidence, weight loss, and lipid peroxidation level and maintained the quality with higher contents of total soluble solid (TSS) content, titratable acidity (TA) in R. roxburghii fruit. Moreover, BTH increased hydrogen peroxide (H2O2) content, superoxide anion (O2 •-) production rate, and the activities and expression of superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX), glutathione (GSH) reductase (GR), monodehydroascorbate reductase (MDHAR), dehydroascorbate reductase (DHAR), and peroxidase (POD), and the contents of GSH and ascorbic acid (AsA), but reduced the oxidized GSH (GSSG) content. In addition, the activities and gene expression of phenylalanine ammonia lyase (PAL), cinnamate 4-hydroxylase (C4H), and 4-coumarate-CoA ligase (4CL) and the concentrations of flavonoids, total phenols, and lignin were significantly elevated by BTH. These findings imply that BTH can delay senescence and maintain the quality of R. roxburghii fruit by modulating ROS metabolism and the phenylpropanoid pathway under low-temperature conditions.

Exogenous 2,4-Epibrassinolide Treatment Maintains the Quality of Carambola Fruit Associated With Enhanced Antioxidant Capacity and Alternative Respiratory Metabolism.

Brassinosteroids act by delaying fruit ripening. The effects of different concentrations of 2,4-epibrassinolide (eBL) treatments on carambola fruit ripening were investigated. The results show that treatment of 2.8 mg L-1, eBL with 10 min effectively delays ripening and maintains the quality of carambola fruit. This is achieved by retarding color changes and firmness losses while maintaining high level of soluble protein content and vitamin C, and low organic acid content. eBL-delayed senescence may be due to the inhibition of respiration rate and enhanced antioxidant system. It is noteworthy that eBL treatment markedly reduces the content of fructose-6-phosphate (6-P-F) and enhances the activity of cytochrome oxidase (CCO), and the total activity of glucose-6-phosphate dehydrogenase (G-6-PDH) and 6-phosphate gluconate dehydrogenase (6-PGDH). eBL treatment induces the IAA and GA contents but reduces that of ABA. In general, senescence retardation and quality improvement by eBL treatment may be due to the enhanced antioxidant capacity and altered respiratory pathways.

BTH Treatment Delays the Senescence of Postharvest Pitaya Fruit in Relation to Enhancing Antioxidant System and Phenylpropanoid Pathway.

The plant resistance elicitor Benzo (1,2,3)-thiadiazole-7-carbothioic acid S-methyl ester (BTH) can enhance disease resistance of harvested fruit. Nonetheless, it is still unknown whether BTH plays a role in regulating fruit senescence. In this study, exogenous BTH treatment efficiently delayed the senescence of postharvest pitaya fruit with lower lipid peroxidation level. Furthermore, BTH-treated fruit exhibited lower hydrogen peroxide (H2O2) content, higher contents of reduced ascorbic acid (AsA) and reduced glutathione (GSH) levels and higher ratios of reduced to oxidized glutathione (GSH/GSSG) and ascorbic acid (AsA/DHA), as well as higher activities of ROS scavenging enzymes, including superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX), peroxidase (POD) and glutathione reductase (GR) in comparison with control fruit. Moreover, BTH treatment enhanced the activities of phenylpropanoid pathway-related enzymes, including cinnamate-4-hydroxylase (C4H), phenylalanine ammonia-lyase (PAL) and 4-coumarate/coenzyme A ligase (4CL) and the levels of phenolics, flavonoids and lignin. In addition, BTH treatment upregulated the expression of HuSOD1/3/4, HuCAT2, HuAPX1/2 and HuPOD1/2/4 genes. These results suggested that application of BTH delayed the senescence of harvested pitaya fruit in relation to enhanced antioxidant system and phenylpropanoid pathway.

Histone demethylase SlJMJ6 promotes fruit ripening by removing H3K27 methylation of ripening-related genes in tomato.

Fruit ripening is governed by a complex regulatory network. Reversible histone methylation and demethylation regulate chromatin structure and gene expression. However, little is known about the involvement of histone demethylases in regulating fruit ripening. Here, we found that the tomato (Solanum lycopersicum) SlJMJ6 encodes a histone lysine demethylase that specifically demethylates H3K27 methylation. Overexpression of SlJMJ6 accelerates tomato fruit ripening, which is associated with the upregulated expression of a large number of ripening-related genes. Integrated analysis of RNA-seq and chromatin immunoprecipitation followed by sequencing identified 32 genes directly targeted by SlJMJ6 and transcriptionally upregulated with decreased H3K27m3 in SlJMJ6-overexpressed fruit. Numerous SlJMJ6-regulated genes are involved in transcription regulation, ethylene biosynthesis, cell wall degradation and hormone signaling. Eleven ripening-related genes including RIPENING INHIBITOR (RIN), 1-aminocyclopropane 1-carboxylate synthase-4 (ACS4), 1-aminocyclopropane-1-carboxylate oxidase 1 (ACO1), pectate lyase (PL) and beta-galactosidase 4 (TBG4), and a DNA demethylase DML2, were confirmed to be regulated directly by SlJMJ6 through removing H3K27me3. Our results demonstrate that SlJMJ6 is a ripening-prompting H3K27me3 demethylase that activates the expression of the ripening-related genes by modulating H3K27me3, thereby facilitating tomato fruit ripening. Our work also reveals a novel link between histone demethylation and DNA demethylation in regulating fruit ripening. To our knowledge, this is the first report of the involvement of a histone lysine demethylase in the regulation of fruit ripening.

The Involvement of the Banana F-Box Protein MaEBF1 in Regulating Chilling-Inhibited Starch Degradation through Interaction with a MaNAC67-Like Protein.

Low-temperature storage is a common strategy for preserving and transporting vegetables and fruits. However, many fruits are hypersensitive to chilling injury, including bananas. In the present study, storage conditions of 11 °C delayed the ripening of Fenjiao (Musa ABB Pisang Awak) banana, and the pulp could be softened after ethephon treatment. Storage conditions of 7 °C prevented fruit from fully softening, and fruit contained a significantly higher starch content and lower soluble sugar content. MaEBF1, a critical gene component in the ethylene signaling pathway, was repressed during ripening after fruit had been stored for 12 days at 7 °C. The expression of a series of starch degradation-related genes and a MaNAC67-like gene were also severely repressed. Both MaEBF1 and MaNAC67-like genes were ethylene-inducible and localized in the nucleus. MaNAC67-like protein was able to physically bind to the promoter of genes associated with starch degradation, including MaBAM6, MaSEX4, and MaMEX1. Yeast two-hybrid, GST-pull down, and BiFC assays showed that MaEBF1 interacted with the MaNAC67-like protein, and their interaction further activated the promoters of MaBAM6 and MaSEX4. The current study indicates that MaNAC67-like is a direct regulator of starch degradation and potential for involvement in regulating chilling-inhibited starch degradation by interacting with the ethylene signaling components in banana fruit. The present work paves the way for further functional analysis of MaEBF1 and MaNAC67-like in banana, which will be useful for understanding the regulation of banana starch metabolism and fruit ripening.

Transcriptomic analysis reveals key factors in fruit ripening and rubbery texture caused by 1-MCP in papaya.

BACKGROUND: Ethylene promotes fruit ripening whereas 1-methylcyclopropene (1-MCP), a non-toxic antagonist of ethylene, delays fruit ripening via the inhibition of ethylene receptor. However, unsuitable 1-MCP treatment can cause fruit ripening disorders. RESULTS: In this study, we show that short-term 1-MCP treatment (400 nL•L- 1, 2 h) significantly delays papaya fruit ripening with normal ripening characteristics. However, long-term 1-MCP treatment (400 nL•L- 1, 16 h) causes a "rubbery" texture of fruit. The comparative transcriptome analysis showed that a total of 5529 genes were differently expressed during fruit ripening compared to freshly harvested fruits. Comprehensive functional enrichment analysis showed that the metabolic pathways of carbon metabolism, plant hormone signal transduction, biosynthesis of amino acids, and starch and sucrose metabolism are involved in fruit ripening. 1-MCP treatment significantly affected fruit transcript levels. A total of 3595 and 5998 differently expressed genes (DEGs) were identified between short-term 1-MCP, long-term 1-MCP treatment and the control, respectively. DEGs are mostly enriched in the similar pathway involved in fruit ripening. A large number of DEGs were also identified between long-term and short-term 1-MCP treatment, with most of the DEGs being enriched in carbon metabolism, starch and sucrose metabolism, plant hormone signal transduction, and biosynthesis of amino acids. The 1-MCP treatments accelerated the lignin accumulation and delayed cellulose degradation during fruit ripening. Considering the rubbery phenotype, we inferred that the cell wall metabolism and hormone signal pathways are closely related to papaya fruit ripening disorder. The RNA-Seq output was confirmed using RT-qPCR by 28 selected genes that were involved in cell wall metabolism and hormone signal pathways. CONCLUSIONS: These results showed that long-term 1-MCP treatment severely inhibited ethylene signaling and the cell wall metabolism pathways, which may result in the failure of cell wall degradation and fruit softening. Our results reveal multiple ripening-associated events during papaya fruit ripening and provide a foundation for understanding the molecular mechanisms underlying 1-MCP treatment on fruit ripening and the regulatory networks.

The interaction of CpEBF1 with CpMADSs is involved in cell wall degradation during papaya fruit ripening.

Ethylene plays a pivotal role in climacteric fruit ripening; whereas 1-MCP, a non-toxic antagonist of ethylene, prevents ethylene-dependent responses and fruit ripening. In this study, a short-term treatment (1 h) with 400 nL L-1 1-MCP delayed the ripening of harvested papaya. However, long-term application of 1-MCP (400 nL L-1, 16 h) resulted in abnormal fruit ripening, with the fruits exhibiting normal yellowing without softening, significantly higher cellulose and lignin contents, and intact cell walls (CW). Furthermore, we found that long-term treatment with 1-MCP significantly inhibited the expression of CpEBF1, an EIN3-binding F-box-1 gene. A protein interaction analysis using yeast two-hybrid, BiFC and GST pull-down assays showed that CpEBF1 interacts with the CpMADS1/3 and CpEIL1 proteins. The interaction of CpEBF1 with CpMADS1/3 further activated the activities of CW-degradation gene promoters. Subcellular localization showed that these proteins were localized in the nucleus. Additionally, the expression levels of CpMADS1/3, CpEIL1, and several CW-degradation-related genes were significantly downregulated by long-term 1-MCP treatment. Therefore, we propose that the inhibited expression of CpEBF1 and CpMADS1/3 resulted in the repressed activation of CW-degradation-related genes via their interaction, thereby resulting in fruit softening disorders.

Bowel Perforation in Premature Infants with Necrotizing Enterocolitis: Risk Factors and Outcomes.

We aim to determine risk factors and clinical outcomes for bowel perforation in premature infants with NEC. We analyzed clinical data of 57 cases of premature infants with NEC at our NICU between January 2010 and December 2012. Based on the presence of bowel perforation, we divided these infants into two groups: perforated NEC group (n = 10) and nonperforated NEC group (n = 47). We compared general information, clinical characteristics, and laboratory findings between groups. The perforated NEC group, compared to the nonperforated NEC group, had significantly lesser gestational age, lower birth weight, higher prevalence of apnea, mechanical ventilation, sepsis and shock, lower blood pH, higher levels of blood glucose, abnormal WBC count and thrombocytopenia, and elevated CRP (all P < 0.05). Moreover, the perforated NEC group had significantly longer durations of fasting and TPN usage, higher incidences of EUGR and cholestasis, longer duration of antibiotics, higher frequency of advanced antibiotics use, and poorer prognosis than the nonperforated NEC group (all P < 0.05). Bowel perforation in premature infants with NEC was associated with multiple risk factors. Early identification of some of these risk factors in premature infants with NEC may help implement early intervention to reduce the incidence of bowel perforation and thereby improve the prognosis.

Direct crystallographic observation of an acyl-enzyme intermediate in the elastase-catalyzed hydrolysis of a peptidyl ester substrate: Exploiting the "glass transition" in protein dynamics.

The crystal structure of the acyl complex of porcine pancreatic elastase with its peptidyl ester substrate N-acetyl-ala-ala-ala-methyl ester (Ac(Ala)3OMe) has been determined at 2.5 A resolution. The complex was stabilized by exploiting the "glass transition" in protein dynamics that occurs at around -53 degrees C (220 K). Substrate was flowed into the crystal in a cryoprotective solvent above this temperature, and then the crystal was rapidly cooled to a temperature below the transition to trap the species that formed. The use of a flow cell makes the experiment a kinetic one and means that the species prior to the rate determining transition state has a chance to accumulate. The resulting crystal structure shows an acyl-enzyme intermediate in which the leaving group is absent and the carbonyl carbon of the C-terminal alanine residue is covalently bound to the gamma oxygen of the active site serine. The ester carbonyl shows no significant distortion from planarity, with the carbonyl oxygen forming one hydrogen bond with the oxyanion hole. The tripeptide is bound in an extended antiparallel beta-sheet with main chain residues of the enzyme. The geometry and interactions of this acyl-enzyme suggest that it represents a productive intermediate. To test this hypothesis, the same crystal was then warmed above the glass transition temperature and a second data set was collected. The resulting electron density map shows no sign of the substrate, indicating hydrolysis of the intermediate followed by product release. This experiment provides direct evidence for the importance of dynamic properties in catalysis and also provides a blueprint for the stabilization of other short-lived species for direct crystallographic observation.