In the metabolic pathway of chlorophylls (Chls), an enzyme called STAY-GREEN or SGR catalyzes the removal of the central magnesium ion of Chls and their derivatives to their corresponding free bases, including pheophytins. The substrate specificity of SGR has been investigated through in vitro reactions using Chl-related molecules. However, information about the biochemical properties and reaction mechanisms of SGR and its substrate specificity remains elusive. In this study, we synthesized various Chl derivatives and investigated their in vitro dechelations using an SGR enzyme. Chl-a derivatives with the C3-vinyl group on the A-ring, which is commonly found as a substituent in natural substrates, and their analogs with ethyl, hydroxymethyl, formyl, and styryl groups at the C3-position were prepared as substrates. In vitro dechelatase reactions of these substrates were performed using an SGR enzyme derived from an Anaerolineae bacterium, allowing us to investigate their specificity. Reactivity was reduced for substrates with an electron-withdrawing formyl or sterically demanding styryl group at the C3-position. Furthermore, the Chl derivative with the C8-styryl group on the B-ring was less reactive for SGR dechelation than the C3-styryl substrate. These results indicate that the SGR enzyme recognizes substituents on the B-ring of substrates more than those on the A-ring.

The central ion Mg2+ is responsible for the differences between chlorophyll a and its free base in their reactivity toward metal ions and thus their resistance to oxidation. We present here the results of spectroscopic (electronic absorption and emission, circular dichroism, and electron paramagnetic resonance), spectroelectrochemical, and computational (based on density functional theory) investigations into the mechanism of pheophytin, a degradation that occurs in the presence of Cu ions and O2. The processes leading to the formation of the linear form of tetrapyrrole are very complex and involve the weakening of the methine bridge due to an electron withdrawal by Cu(II) and the activation of O2, which provides protection to the free ends of the opening macrocycle. These mechanistic insights are related to the naturally occurring damage to the photosynthetic apparatus of plants growing on metal-contaminated soils.

Marine algae extracts are an important area of potential drug discovery; however, nearly all studies to date have used non-fluorescent-based methods to determine changes in target cell activity. Many of the most robust immunological and cellular analyses rely on fluorescent probes and readouts, which can be problematic when the algae extract is fluorescent itself. In this study, we identified the fluorescent spectrum of an isolated extract from the marine dinoflagellate Karenia brevis, which included two fluorescing components: chlorophyll α and pheophytin α. When excited at 405 nm and 664 nm, the extract emitted fluorescence at 676 nm and 696 nm, respectively. The extract and its fluorescing components, chlorophyll α and pheophytin α, entered phagocytic RAW 264.7 macrophages and non-phagocytic Vero kidney cells through distinct mechanisms. When incubated with the extract and its main components, both the RAW 264.7 macrophages and the Vero cells accumulated fluorescence as early as 30 min and continued through 48 h. Vero kidney cells accumulated the K. brevis fluorescent extract through a dynamin-independent and acidified endosomal-dependent mechanism. RAW 264.7 macrophages accumulated fluorescent extract through a dynamin-independent, acidified endosomal-independent mechanism, which supports accumulation through phagocytosis. Furthermore, RAW 264.7 macrophages downregulated cell-surface expression of CD206 in response to extract stimulation indicating activation of phagocytic responses and potential immunosuppression of these immune cells. This study represents the first characterization of the cellular update of K. brevis extracts in phagocytic versus non-phagocytic cells. The data suggest the importance of understanding cellular uptake of fluorescing algae extracts and their mechanism of action for future drug discovery efforts.

Chlorophyll was extracted and microencapsulated using different carrier agents. Subsequently, in vitro digestion was performed, and the bioaccessibility of chlorophyll in the different encapsulation systems was carried out. The zeta potential, particle size, and PDI were significantly modified after the micellarization of digested microcapsules. I-W-Chl presented with the highest total chlorophyll recovery and micellarization rate of 54% and 43%, respectively. In the aqueous micellar fraction, the different encapsulation systems had total chlorophylls, pheophytins, and pheophorbides ranging from 13 to 49%, 42 - 77%, and 3 - 22% respectively. The bioaccessibility of total chlorophyll pigment ranging from 7% to 20% is given in the following order: I-W-Chl > WPI-Chl > Z-Chl > Ca-Chl > SCChlV > SCChlC. The result established in this study shows that the carrier agent type could inhibit or mediate the bioaccessibility of chlorophyll with the potential to be an efficient delivery system for health promoting compounds.

The type of carrier agent could impact pheophytin stability and bioaccessibility. Hence, it is important to have an elaborate understanding on the extent and type of pheophytin transformation during in vitro digestion of microcapsules. Four kinds of protein/polysaccharides complex were used to fabricate pheophytin microcapsules and investigated for pigments bioaccessibility. With different carriers, pheophytin pigments showed new characteristics influencing particle size and zeta potential during in vitro digestion. Pheophytin b was widely transformed to pheophorbide b, confirming pheophorbidation of the b series in proper condition. No 151-hydroxy lactone chlorophyll or pheophytin derivatives were detected, indicating some protective effect of microencapsulation. Pheophytins loaded in gelatin-pectin complex exhibited a relatively higher recovery rate, micellarization rate, and bioaccessibility index. The result presented in this study shows that the type of carrier agent could initiate the removal of phytyl groups in pheophytins and also inhibit or mediate their bioaccessibility.

The preparation of dephytylated chlorophyll standards is inefficient and the process is complicated, which hinders chlorophyll determination and related bioactive property investigation. In this paper, chlorophyll derivatives from four phytylated chlorophylls (chlorophyll a, chlorophyll b, pheophytin a, and pheophytin b) before and after the enzymatic reaction were qualitatively and quantitatively characterized by UPLC-DAD-MS. A simple index was proposed to characterize chlorophyll pigments from their oxidized counterparts by the λmax of the typical peak of chlorophyll derivatives in UV-visible spectrum and their signal intensity ratios. The optimal reaction conditions for the enzymatic reaction of four chlorophyll pigments were optimized, and kinetic models were fitted. The results showed that the optimal temperatures for the enzymatic reactions of chlorophyll a, chlorophyll b, pheophytin a, and pheophytin b were 30, 30, 60, and 60°C, respectively, and their optimal reaction time was 2, 3, 1, and 3 h, respectively. Kinetic models were fitted under optimal reaction conditions to study the Km and Vm values of the enzymatic reactions. PRACTICAL APPLICATION: Dephytylated chlorophylls, such as chlorophyllide and pheophorbide, are frequently determined in food industry and are always required to be prepared in lab with acetone powder from plant tissue. Moreover, chlorophyll pigments are easy to undergo oxidations, which make the characterization of dephytylated chlorophyll pigments more complicated and difficult. In this paper, four types of phytylated chlorophylls were investigated respectively about the dephytinization process with the citrus acetone powder, and the reaction mixture was analyzed with UPLC-DAD-MS, which can provide an important reference for relevant chlorophyll determination studies and the development of chlorophyll identification protocols.

CRISPR/dCas9 is an important DNA modification tool in which a disarmed Cas9 protein with no nuclease activity is fused with a specific DNA modifying enzyme. A previous study reported that overexpression of the TET1 catalytic domain (TET1cd) reduces genome-wide methylation in Arabidopsis. A spontaneous naturally occurring methylation region (NMR19-4) was identified in the promoter region of the PPH (Pheophytin Pheophorbide Hydrolase) gene, which encodes an enzyme that can degrade chlorophyll and accelerate leaf senescence. The methylation status of NMR19-4 is associated with PPH expression and leaf senescence in Arabidopsis natural accessions. In this study, we show that the CRISPR/dCas9-TET1cd system can be used to target the methylation of hypermethylated NMR19-4 region to reduce the level of methylation, thereby increasing the expression of PPH and accelerating leaf senescence. Furthermore, hybridization between transgenic demethylated plants and hypermethylated ecotypes showed that the demethylation status of edited NMR19-4, along with the enhanced PPH expression and accelerated leaf senescence, showed Mendelian inheritance in F1 and F2 progeny, indicating that spontaneous epialleles are stably transmitted trans-generationally after demethylation editing. Our results provide a rational approach for future editing of spontaneously mutated epialleles and provide insights into the epigenetic mechanisms that control plant leaf senescence.

Decreased sea ice cover in the northern Bering Sea has altered annual phytoplankton phenology owing to an expansion of open water duration and its impact on ocean stratification. Limitations of satellite remote sensing such as the inability to detect bloom activity throughout the water column, under ice, and in cloudy conditions dictate the need for shipboard based measurements to provide more information on bloom dynamics. In this study, we adapted remote sensing land cover classification techniques to provide a new means to determine bloom stage from shipboard samples. Specifically, we used multiyear satellite time series of chlorophyll a to determine whether in-situ blooms were actively growing or mature (i.e., past-peak) at the time of field sampling. Field observations of chlorophyll a and pheophytin (degraded and oxidized chlorophyll products) were used to calculate pheophytin proportions, i.e., (Pheophytin/(Chlorophyll a + Pheophytin)) and empirically determine whether the bloom was growing or mature based on remotely sensed bloom stages. Data collected at 13 north Bering Sea stations each July from 2013-2019 supported a pheophytin proportion of 28% as the best empirical threshold to distinguish a growing vs. mature bloom stage. One outcome was that low vs. high sea ice years resulted in significantly different pheophytin proportions in July; in years with low winter-to-spring ice, more blooms with growing status were observed, compared to later stage, more mature blooms following springs with abundant seasonal sea ice. The detection of growing blooms in July following low ice years suggests that changes in the timing of the spring bloom triggers cascading effects on mid-summer production.

Microalgae and microalgae-derived compounds have great potential as supplements in the human diet and as a source of bioactive products with health benefits. Spirulina (Arthrospira platensis (Nordstedt) Gomont, or Spirulina platensis) belongs to the class of cyanobacteria and has been studied for its numerous health benefits, which include anti-inflammatory properties, among others. This work was aimed at comparing some spirulina products available on the Italian market. The commercial products here analyzed consisted of spirulina cultivated and processed with different approaches. Single-component spirulina products in powder and flake form, free of any type of excipient produced from four different companies operating in the sector, have been analyzed. The macro- and micromorphological examination, and the content of pigments, phycobiliproteins, phenols, and proteins have shown differences regarding the morphology and chemical composition, especially for those classes of particularly unstable compounds such as chlorophylls and carotenoids, suggesting a great influence of both culture conditions and processing methods.

Nowadays, much available processed and highly palatable food such as cream products and fried and convenient food, which usually showed a high energy density, had caused an increase in the intake of dietary lipids, further leading to significant growth in the prevalence of obesity. Chlorophyll, widespread in fruits and vegetables, was proven to have beneficial effects on alleviating obesity. This study investigated the effects of chlorophyll on the digestive characteristics of lipids under in vitro simulated adult and infant gastrointestinal systems. Chlorophyll decreased the release rate of free fatty acid (FFA) during in vitro adult and infant intestinal digestion by 69.2% and 60.0%, respectively. Meanwhile, after gastrointestinal digestion, chlorophyll changed the FFA composition of soybean oil emulsion and increased the particle size of oil droplets. Interestingly, with the addition of chlorophyll, the activity of pancreatic lipase was inhibited during digestion, which may be related to pheophytin (a derivative of chlorophyll after gastric digestion). Therefore, the results obtained from isothermal titration calorimetry and molecular docking further elucidated that pheophytin could bind to pancreatic lipase with a strong affinity of (4.38 ± 0.76) × 107 M-1 (Ka), while the binding site was amino acid residue Trp253. The investigation not only explained why chlorophyll inhibited digestive enzyme activity to reduce lipids digestion but also provided exciting opportunities for developing novel chlorophyll-based healthy products for dietary application in preventing obesity.