Dataset Information

Characterization under quasi-native conditions of the capsanthin/capsorubin synthase from Capsicum annuum L.

ABSTRACT: Chromoplasts are typical plastids of fruits and flowers, deriving from chloroplasts through complex processes of re-organisation and recycling. Since this transition leads to the production of reactive species, chromoplasts are characteristic sites for biosynthesis and accumulation of carotenoids and other antioxidants. Here, we have analysed the chromoplast membranes from Capsicum annuum L. fruits, finding a significant expression of the capsanthin/capsorubin synthase. This enzyme was isolated by a very mild procedure allowing its analyses under quasi-native conditions. The isolated complex appeared as a red coloured homo-trimer, suggesting the retention of at least one of the typical carotenoids from C. annuum. Moreover, the protein complex was co-purified with a non-proteinaceous fraction of carotenoid aggregates carrying a high molecular weight and separable only by Size Exclusion Chromatography. This last finding suggested a relationship between the carotenoids synthesis on chromoplast membranes and the presence of organised carotenoids aggregates typical for chromoplasts. Further MS analyses also provided important hints on the interactome network associated to the capsanthin/capsorubin synthase, confirming its functional relevance during ripening. Results are discussed in the frame of the primary role played by carotenoids in quenching the growing oxidative stress during fruits ripening.

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Capsicum Annuum

TISSUE(S): Plant Cell, Fruit Body

SUBMITTER: Joanna Kirkpatrick

LAB HEAD: Domenica Farci

PROVIDER: PXD013617 | Pride | 2019-09-16

REPOSITORIES: Pride

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Dataset's files

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			Action	DRS
	180731_JMK_DP_BN_gel_band1_5uL.raw	Raw
	180731_JMK_DP_BN_gel_band2_5uL.raw	Raw
	180731_JMK_DP_SDS_gel_band1_5uL.raw	Raw
	180731_JMK_DP_SDS_gel_band2_5uL.raw	Raw
	combined.zip	Other

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Characterization under quasi-native conditions of the capsanthin/capsorubin synthase from Capsicum annuum L.

Piano Dario D Cocco Emma E Guadalupi Giulia G Kalaji Hazem M HM Kirkpatrick Joanna J Farci Domenica D

Plant physiology and biochemistry : PPB 20190905

Chromoplasts are typical plastids of fruits and flowers, deriving from chloroplasts through complex processes of re-organization and recycling. Since this transition leads to the production of reactive species, chromoplasts are characteristic sites for biosynthesis and accumulation of carotenoids and other antioxidants. Here, we have analysed the chromoplast membranes from Capsicum annuum L. fruits, finding a significant expression of the capsanthin/capsorubin synthase. This enzyme was isolated ...[more]

PMID: 31505449

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Project description:Disruptions of protein homeostasis in the endoplasmic reticulum (ER) elicit activation of the unfolded protein response (UPR), a translation- and transcription-coupled proteostatic stress response pathway. The primary translational control arm of the UPR is initiated by the PERK-dependent phosphorylation of eIF2Î±, leading to a large-scale reprogramming of translation and subsequent activation of an ATF4-mediated transcriptional program. It has remained challenging, however, to accurately evaluate the contribution of each component of the eIF2Î±/ATF4 pathway to the remodelling of transcription and translation. Here, we have used mouse embryonic fibroblasts containing either a knock-in of the non-phosphorylatable eIF2Î± S51A mutant or knock-out for ATF4 by ribosome profiling and mRNA-seq to define the specific contributions of eIF2Î± phosphoryation and ATF4 in controlling the translational and transcriptional components of the UPR. These studies show that the transcriptional and translational targets of each P-eIF2Î±, ATF4, and the other UPR gene expression programs overlapped extensively, leading to a core set of UPR genes whose robust expression in response to ER stress is driven by multiple mechanisms. The identification of other, more factor-specific targets illustrated the potential for functional specialization of the UPR. As the UPR progressed temporally, cells employed distinct combinations of transcriptional and translational mechanisms, initiated by different factors, to adapt to ongoing stress. These effects were accompanied by a buffering effect where changes in mRNA levels were coupled to opposing changes in ribosome loading, a property which makes cooperation between transcription and translation essential to confer robust protein expression. Translational analysis by ribosome profiling and mRNA-seq of PERK pathways mutants during the UPR. Mouse embryonic fibroblasts (MEFs) lacking components of the PERK pathway (eIF2a phosphorylation and ATF4) were subjected to ER stress and analyzed by ribosome profiling.

Project description:Experiments were carried out in 2008 on 8-year-old apple trees (cv. Golden Delicious/M9) trained with standard horticultural practices at the experimental farm of the IASMA (Trento, Italy). Populations of fruits with different abscission potentials (abscising fruitlets versus persisting fruitlets), were established as described by Dal Cin et al. (2005, 2007, 2009), Angeli et al. (2002), and other preliminary experiments (unpublished data). Briefly, the abscising population was made up of lateral fruitlets treated with benzylaminopurine (BA) at 200 ppm (commercial name Brancher-Dirado), when fruits had an average size of 13 mm (about 15 days after petal fall, DAPF). The population of central persisting fruitlets was generated by removing all the laterals from each cluster at petal fall, and leaving exclusively the central flower that had been hand-pollinated at full bloom with compatible pollen (cv. Stark Red). Samples of the two populations were collected at defined time points from groups of twenty homogeneous trees randomly distributed in the orchard in four blocks. Fruits were collected and categorised into three classes of size (class 1, smaller fruits; class 2, medium fruits; class 3, bigger fruits), two classes related to the position within the clusters (lateral versus central fruits), and two classes on the base of the treatment (BA-treated versus untreated fruits). Fruits of the intermediate size (class 2 fruits in Figure 1) were not considered for sampling and subsequent molecular analyses, and only fruits of the two more divergent size classes 1 and 3 were kept. This resulted in a combined categorisation of fruits into 4 classes: 1) untreated lateral fruitlets, 2) lateral fruitlets treated with BA, 3) untreated central fruitlets, and 4) central fruitlets treated with BA (Figure 1). Each class was further distinguished into the two size categories 1 (small fruits) and 3 (bigger fruits), for a total of eight experimental theses. Fruitlet shedding and ethylene evolution were monitored throughout the physiological drop from the beginning of the experiments up to 46 DAPF in all fruitlet classes, separately. Seed and cortex (including epidermis) samples were collected from all classes of fruitlets at 0 (T0), 1 (T1), 2 (T2), 4 (T3), 6 (T4), and 8 (T5) days after the BA treatment from control and treated trees, and according to their position within the clusters (central vs lateral) and size (small vs big), as described. The latter parameter was decided at each sampling date based upon the mean cross diameter of the whole population of lateral fruits, calculated over a sample of 100 fruits measured randomly on twenty trees. The small ones had a cross diameter below the mean - s.d. (standard deviation), whereas the big ones had a cross diameter above the mean + s.d. Lateral fruitlets were collected from intact clusters showing a clear hierarchy in terms of fruit size (i.e. with a clearly distinguishable central fruit, bigger than any lateral). Acronyms were ascribed to samples according to the following code: the first letter(s) describes fruitlet position within the cluster and the presence of BA treatment (L=untreated lateral, C=untreated central, LB=treated lateral, CB=treated central), then a digit to describe the size (1=small, 3=big), and finally a digit to described the time point (0=time of the treatment, 1=1 day after treatment, 2=2 days after treatment, 3=4 days after treatment, etc.). For example, LB32 is a lateral fruit, treated with BA, big sized, 2 days after treatment.<br>For microarray hybridizations, only samples of T0, T2, and T3 were used.

Dataset Information

Characterization under quasi-native conditions of the capsanthin/capsorubin synthase from Capsicum annuum L.

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Publications

Characterization under quasi-native conditions of the capsanthin/capsorubin synthase from Capsicum annuum L.

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