Chlorophyll (Chl) breakdown is a diagnostic visual process of leaf senescence, which furnishes phyllobilins (PBs) by the PAO/phyllobilin pathway. As Chl breakdown disables photosynthesis, it appears ...to have no role in photoactive green leaves. Here, colorless PBs were detected in green, non-senescent leaves of
Arabidopsis thaliana
. The PBs from the green leaves had structures entirely consistent with the PAO/phyllobilin pathway and the mutation of a single Chl catabolic enzyme completely abolished PBs with the particular modification. Hence, the PAO/phyllobilin pathway was active in the absence of visible senescence and expression of genes encoding Chl catabolic enzymes was observed in green
Arabidopsis
leaves. PBs accumulated to only sub-% amounts compared to the Chls present in the green leaves, excluding a substantial contribution of Chl breakdown from rapid Chl turnover associated with photosystem II repair. Indeed, Chl turnover was shown to involve a Chl
a
dephytylation and Chl
a
reconstitution cycle. However, non-recyclable pheophytin
a
is also liberated in the course of photosystem II repair, and is proposed here to be scavenged and degraded to the observed PBs. Hence, a cryptic form of the established pathway of Chl breakdown is indicated to play a constitutive role in photoactive leaves.
The typical main products of chlorophyll (Chl) breakdown in higher plants are non‐fluorescent, colorless phyllobilins, named phylloleucobilins. These long elusive Chl‐catabolites are linear ...tetrapyrroles, whose structure elucidation has required thorough spectroscopic analyses. Interestingly, in recent LC/MS studies of leaf extracts, isomeric forms of phylloleucobilins were detected. The existence of isomeric phyllobilins may suggest incomplete stereo‐selectivity of catabolic processes, or isomerization processes in plant cells or in the analytes. Here we report a study with the phylloleucobilin NCC‐1, a basic Chl‐catabolite in extracts of leaves and fruit. NCC‐1 and its main isomerization product in aqueous solution were identified as 82‐epimers. Formation of 82‐epi‐NCC‐1 from NCC‐1 implies an unstable enol(ate)‐intermediate, which reverts to NCC‐1 or converts to 82‐epi‐NCC‐1. Such reversible epimerization reactions are a non‐biological in vitro feature of typical phylloleucobilins, and probably also take place in vivo.
Phytoplasmoses such as apple proliferation (AP) and European stone fruit yellows (ESFY) cause severe economic losses in fruit production. A common symptom of both phytoplasma diseases is early ...yellowing or leaf chlorosis. Even though chlorosis is a well-studied symptom of biotic and abiotic stresses, its biochemical pathways are hardly known. In particular, in this context, a potential role of the senescence-related pheophorbide a oxygenase/phyllobilin (PaO/PB) pathway is elusive, which degrades chlorophyll (Chl) to phyllobilins (PBs), most notably to colorless nonfluorescent Chl catabolites (NCCs). In this work, we identified the Chl catabolites in extracts of healthy senescent apple and apricot leaves. In extracts of apple tree leaves, a total of 12 Chl catabolites were detected, and in extracts of leaves of the apricot tree 16 Chl catabolites were found. The seven major NCC fractions in the leaves of both fruit tree species were identical and displayed known structures. All of the major Chl catabolites were also found in leaf extracts from AP- or ESFY-infected trees, providing the first evidence that the PaO/PB pathway is relevant also for pathogen-induced chlorosis. This work supports the hypothesis that Chl breakdown in senescence and phytoplasma infection proceeds via a common pathway in some members of the Rosaceae family.
The tetrapyrrolic chlorophyll catabolites (or phyllobilins, PBs) were analyzed in yellow fall leaves of the grape Chardonnay, a common Vitis vinifera white wine cultivar. The major fractions in leaf ...extracts of V. vinifera, tentatively assigned to PBs, were isolated and their structures elucidated. The dominant fraction is a dioxobilin‐type non‐fluorescent Chl‐catabolite of a previously observed type. Two less polar fluorescent PBs were characterized as a novel dioxobilin‐type fluorescent Chl‐catabolite with a bicyclo‐1′,6′‐glycosyl architecture, and its new fluorescent formyloxobilin‐type analogue. The discovery of persistent hypermodified fluorescent PBs with the architecture of bicyclo‐17.3.1‐PBs (bcPBs), suggests the activity of an unknown enzyme that forges the 20‐membered macroring at the tetrapyrrolic core of a fluorescent PB. bcPBs may play specific physiological roles in grapevine plants and represent endogenous anti‐infective agents, as found similarly for other organic bicyclo‐n.3.1‐1′,6′‐glycosyl derivatives.
The colors of the fall: Blue fluorescent chlorophyll catabolites with a unique bicyclo‐1′,6′‐glycosyl architecture occur in golden yellow fall leaves of Chardonnay, a common white wine cultivar. This discovery implies unknown enzymes that assemble a 20‐membered macroring at a tetrapyrrole to generate exceptionally structured chlorophyll catabolites. Possibly, these may play specific physiological roles and/or represent anti‐infective agents in the grapevine leaves.
In cold extracts of senescent leaves of the plum tree (Prunus domestica ssp. domestica), six colorless non‐fluorescent chlorophyll catabolites (NCCs) were characterized, named Pd‐NCCs. In addition, ...several minor NCC fractions were tentatively classified. The structure of the most polar one of the NCCs, named Pd‐NCC‐32, featured an unprecedented twofold glycosidation pattern. Three of the NCCs are also functionalized at their 32‐position by a glucopyranosyl group. In addition, two of these glycosidated NCCs carry a dihydroxyethyl group at their 18‐position. In the polar Pd‐NCC‐32, the latter group is further glycosidated at the terminal 182‐position. Four other major Pd‐NCCs and one minor Pd‐NCC were identified with five NCCs from higher plants known to belong to the ‘epi’‐series. In addition, tentative structures were derived for two minor fractions, classified as yellow chlorophyll catabolites, which represented (formal) oxidation products of two of the observed Pd‐NCCs. The chlorophyll catabolites in leaves of plum feature the same basic structural pattern as those found in leaves of apple and pear trees.
NESP55 (neuroendocrine secretory protein with
M
r 55,000) comprises a novel chromogranin-like protein, which is paternally imprinted at the genomic level. We used antisera raised against GAIPIRRH, a ...peptide present at the C-terminus of this protein, and against TC-14, a peptide located in the N-terminal half of NESP55. Radioimmunoassay, gel-filtration chromatography and immunoblotting were used to determine the levels and the molecular forms of NESP55 in different bovine organs. The tissues with the highest levels of GAIPIRRH immunoreactivity were, in decreasing order: the adrenal medulla, the anterior pituitary, the posterior pituitary, various brain regions, and the intestine. The degree of proteolytic processing revealed differences among the tissues analyzed. The lowest processing was detected in the anterior pituitary and in the brain where only a peak corresponding to the intact precursor was present. This was also true for cerebrospinal fluid (CSF). In the posterior pituitary and in the intestine, the free peptide GAIPIRRH was the predominant molecular form. GAIPIRRH-IR, as in the CSF, is present in serum mainly as an intact precursor. A relatively high concentration of GAIPIRRH-IR was found in the kidney medulla, probably due to an endocytotic re-uptake of this molecule from the tubuli after filtration in the glomeruli. The present study is consistent with the concept that NESP55, like the other chromogranins, becomes proteolytically processed. The function of this new chromogranin-like protein, therefore, might be to represent a precursor of biologically active peptides.
Secretoneurin is a 33-amino-acid peptide produced in vivo from secretogranin II. An antiserum raised against this peptide recognizes both the free peptide and its precursors. By HPLC and ...radioimmunoassay we characterized the immunoreactive molecules and determined the levels of immunoreactivity in various rat organs. In adrenal medulla and to a lesser degree in the anterior pituitary processing of secretogranin II to secretoneurin was very limited, whereas in all other organs studied (brain, intestine, endocrine pancreas, thyroid gland, and posterior pituitary) a high degree of processing was apparent. Thus, practically all of the immunoreactivity was present as free secretoneurin. This was also true for serum. When the total amount of secretoneurin immunoreactivity was calculated for the various organs, the largest pools in descending order were in the intestine, CNS, anterior pituitary, pancreas, and adrenal gland. This makes it likely that secretoneurin in serum is mainly derived from the intestine. The high degree of processing of secretogranin II in most organs is consistent with the concept that this protein acts as a precursor of a functional peptide, i.e., secretoneurin.
Single molecule localization microscopy (SMLM) has enormous potential for resolving subcellular structures below the diffraction limit of light microscopy: Localization precision in the low digit ...nanometer regime has been shown to be achievable. In order to record localization microscopy data, however, sample fixation is inevitable to prevent molecular motion during the rather long recording times of minutes up to hours. Eventually, it turns out that preservation of the sample's ultrastructure during fixation becomes the limiting factor. We propose here a workflow for data analysis, which is based on SMLM performed at cryogenic temperatures. Since molecular dipoles of the fluorophores are fixed at low temperatures, such an approach offers the possibility to use the orientation of the dipole as an additional information for image analysis. In particular, assignment of localizations to individual dye molecules becomes possible with high reliability. We quantitatively characterized the new approach based on the analysis of simulated oligomeric structures. Side lengths can be determined with a relative error of less than 1% for tetramers with a nominal side length of 5 nm, even if the assumed localization precision for single molecules is more than 2 nm.
Fourier reconstruction algorithms significantly outperform conventional backprojection algorithms in terms of computation time. In photoacoustic imaging, these methods require interpolation in the ...Fourier space domain, which creates artifacts in reconstructed images. We propose a novel reconstruction algorithm that applies the one-dimensional nonuniform fast Fourier transform to photoacoustic imaging. It is shown theoretically and numerically that our algorithm avoids artifacts while preserving the computational effectiveness of Fourier reconstruction.
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