Pathogen-Induced Leaf Chlorosis: Products of Chlorophyll Breakdown

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Pathogen-Induced Leaf Chlorosis: Products of Chlorophyll Breakdown Found in Degreened Leaves of Phytoplasma-Infected Apple (Malus x domestica Borkh.) and Apricot (Prunus armeniaca L.) Trees Relate to the Pheophorbide a Oxygenase / Phyllobilin Pathway Cecilia Mittelberger, Hacer Yalcinkaya, Christa Pichler, Johanna Gasser, Gerhard Scherzer, Theresia Erhart, Sandra Schumacher, Barbara Holzner, Katrin Janik, Peter Robatscher, Thomas Müller, Bernhard Kräutler, and Michael Oberhuber J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b05501 • Publication Date (Web): 07 Mar 2017 Downloaded from http://pubs.acs.org on March 18, 2017

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Journal of Agricultural and Food Chemistry

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Pathogen-Induced Leaf Chlorosis: Products of Chlorophyll Breakdown Found in

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Degreened Leaves of Phytoplasma-Infected Apple (Malus x domestica Borkh.) and

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Apricot (Prunus armeniaca L.) Trees Relate to the Pheophorbide a Oxygenase /

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Phyllobilin Pathway

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Cecilia Mittelbergera, Hacer Yalcinkayab, Christa Pichlera, Johanna Gasserb, Gerhard

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Scherzerb, Theresia Erhartb, Sandra Schumachera, Barbara Holznera, Katrin Janika, Peter

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Robatschera, Thomas Müllerb, Bernhard Kräutlerb*, and Michael Oberhubera*

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a

Laimburg Research Center for Agriculture and Forestry, Laimburg 6 - Pfatten (Vadena),

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39040 Auer (Ora), BZ, Italy b

Institute of Organic Chemistry and Center of Molecular Biosciences, University of Innsbruck, Innrain 80/82, 6020 Innsbruck, Austria

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*Corresponding authors:

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E-mail: [email protected]

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E-mail: [email protected]

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ACS Paragon Plus Environment

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Abstract

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Phytoplasmoses such as Apple Proliferation (AP) and European Stone Fruit Yellows (ESFY)

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cause severe economic losses in fruit production. A common symptom of both phytoplasma

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diseases is the early yellowing or leaf chlorosis. Even though chlorosis is a well-studied

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symptom of biotic and abiotic stress, its biochemical pathways are hardly known. In

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particular, in this context, a potential role of the senescence-related pheophorbide a

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oxygenase/phyllobilin (PaO/PB) pathway is elusive, which degrades chlorophyll (Chl) to

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phyllobilins (PBs), most notably to colorless non-fluorescent Chl catabolites (NCCs). In this

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work, we identified the Chl catabolites in extracts of healthy senescent apple and apricot

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leaves. In extracts of apple tree leaves, a total of 12 Chl catabolites were detected, in extracts

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of leaves of the apricot tree 16 Chl catabolites were found. The major seven NCC fractions in

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the leaves of both fruit tree species were identical, and displayed known structures. All of the

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major Chl catabolites were also found in leaf extracts from AP- or ESFY-infected trees,

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providing the first evidence that the PaO/PB pathway is relevant also for pathogen-induced

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chlorosis. This work supports the hypothesis that Chl breakdown in senescence and

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phytoplasma infection proceeds via a common pathway in some members of the Rosaceae

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family.

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Keywords: Chlorophyll, Chlorophyll Catabolites, Phytoplasma, Apple Proliferation,

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European Stone Fruit Yellows

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Introduction

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Phytoplasma infections cause severe losses in orchards worldwide1. One of the most common

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symptoms of infection is early leaf yellowing or chlorosis1,2 caused by the disappearance of

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chlorophyll (Chl). Superficially, chlorosis resembles yellowing of senescent leaves in fall, but

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senescence-induced and pathogen-induced degreening may not proceed via the same

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biochemical pathways3. Breakdown of Chl associated with autumnal leaf coloration has only

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recently been elucidated on a molecular basis4: During senescence Chl is metabolized via the

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pheophorbide a oxygenase/phyllobilin (PaO/PB) pathway. It furnishes 1-formyl-19-oxobilin-

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type Chl catabolites, or ‘type I’ PBs, represented by the non-fluorescent Chl catabolites

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(NCCs, see Figure 1)4, or ‘type II’ PBs, such as the 1,19-dioxobilin-type NCCs (DNCCs)5.

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PaO, a Rieske-type iron-sulfur monooxygenase, is the key enzyme in breakdown of Chl,

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catalyzing the opening of the chlorin macrocyle6,7. The colorless NCCs and DNCCs are

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considered the major products of Chl breakdown that accumulate in senescent leaves. NCCs

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and DNCCs have been isolated with varying peripheral functionalizations from senescent

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leaves of a series of higher plants4. In extracts of leaves NCCs are readily oxidised in the

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presence of air to yellow Chl catabolites (YCCs)8 and pink Chl catabolites (PiCCs)9, some of

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which have been directly detected in small amounts in fresh leaf extracts, as well (Figure

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1)10,11.

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Even though chlorosis is a well-studied symptom of biotic and abiotic stress, its biochemical

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pathways are less well understood6. In particular, very little is known about the role of the

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senescent PaO/PB pathway of Chl breakdown during biotic stress situations and pathogen

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defense. While first data showed PaO activity only during senescence6, more recent work

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found some PaO activity also in pre-senescent tissues, during wounding and infection with

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Pseudomonas syringae6,7,12. Plant defense mechanisms include the production of reactive

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oxygen species (ROS) that are capable of damaging the cells of invading organisms13. Beside


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the production of apoplastic ROS, also chloroplastic ROS production is linked to plant

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defense strategies14,15 and, thus, a target for manipulation by pathogens14. Indeed, Chl

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catabolites such as NCCs and DNCCs are known to be effective antioxidants16. Ougham et

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al.3 distinguish senescence from non-physiological bleaching, which might occur non-

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enzymatically by the action of ROS in infected plants; therefore, leaf yellowing is considered

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a common symptom caused by very different pathogens. However, it remains to be

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demonstrated whether these visual signs of Chl breakdown emerge via non-physiological

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bleaching or well-orchestrated biochemical processes like the PaO/PB pathway. Pathogen

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infection, but also abiotic factors, can alter leaf senescence17 and lead to premature yellowing.

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Diagnostic tools for detecting pathogen infections are highly desirable, and premature

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emergence of Chl catabolites before onset of actual senescence could be used as biomarkers

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for pathogen infection long before a visible sign of degreening.

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Apple proliferation (AP), caused by Candidatus Phytoplasma mali (Ca. P. mali) and

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European Stone Fruit Yellows (ESFY), caused by Candidatus Phytoplasma prunorum (Ca. P.

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prunorum), are two main threats in apple and apricot production in South Tyrol18. The

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economically most important symptoms in both diseases are the production of small, tasteless

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and colorless fruit2. In addition both diseases lead to premature leaf degreening2,19.

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Despite the significant body of knowledge on both pathogen-induced leaf chlorosis and leaf

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senescence a role for the PaO/PB pathway in pathogen-induced degreening is yet to be

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established. In this work, we report on the detection of phyllobilins in prematurely degreened

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leaves of Ca. P. mali-infected apple and Ca. P. prunorum-infected apricot trees, providing

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unprecedented evidence for the PaO/PB pathway of Chl breakdown in phytoplasma-induced

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leaf chlorosis.

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Material and Methods

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Reagents


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CaCO3 and ethanol were obtained from Sigma-Aldrich (Milan, Italy), HPLC- and LC-MS-

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grade methanol (MeOH) from VWR (Milan, Italy), and NH4OAc for LC-MS analysis,

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K2HPO4, and KH2PO4 from Fisher Scientific (Illkirch, France). A Cj-NCC-1 reference sample

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was essentially obtained as described before20,21.

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Instrumentation

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Analytical HPLC: Agilent 1260 Infinity with on-line degasser, quaternary pump, column

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oven, and PDA detector. Precolumn: Phenomenex SecurityGuard Cartridge C18, 4 x 3 mm;

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column: Phenomenex HyperClone 5µm, ODS C18 120A; 250 x 4.6 mm; column temperature:

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25 °C.

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LC-MS: Triple quadrupole mass spectrometer. Thermo Fisher, Accela 1250 Pump, with on-

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line degasser, quaternary pump, column oven, and Accela PDA Detector, TSQ Quantum

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Access Max mass analyzer. Precolumn: Phenomenex SecurityGuard Cartridge C18, 4 x 3

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mm; column: Phenomenex HyperClone column, 5µm, ODS C18 120A; 250 x 4.6 mm;

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column temperature: 25 °C.

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LC-Q-TOF-MS: Quadrupole-time of flight mass spectrometer. Thermo Fisher, Ultimate 3000

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RSLC Pump, with on-line degasser, binary pump, column oven, and 3000RS DAD Detector,

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Bruker Impact HD high resolution mass analyzer. Precolumn: Phenomenex SecurityGuard

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Cartridge C18, 4 x 3 mm; column: Phenomenex HyperClone column, 5µm, ODS C18 120A;

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250 x 4.6 mm; column temperature: 25 °C.

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Sample Set and Sample Preparation

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Apple (Malus x domestica Borkh.) leaf samples were taken from a commercial orchard with

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the cultivar ‘Golden Delicious’ (planting year: 2000) located at 380 m a.s.l. near Meran,

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South Tyrol (Italy), managed according to the guidelines for the integrated fruit production in

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South Tyrol22. From June 2013 to December 2013 five Ca. P. mali-infected and seven healthy

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trees were sampled every two weeks (Table S1, Supporting Information).


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For apricot (Prunus armeniaca L.) samples a commercial orchard with mixed cultivars

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located at 260 m a.s.l. in Bozen, South Tyrol, (Italy) was chosen. As ESFY (Ca. P.

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prunorum)-infected trees had been replaced continuously in the orchard's history, the planting

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year was not uniform. Samples were taken from the following cultivars: Orange Ruby,

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Augusta, Aurora and an unknown cultivar. Three healthy trees (cv. Orange Ruby, Augusta),

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three infected non-symptomatic trees (cv. Orange Ruby, Aurora) and six infected

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symptomatic trees (cv. Orange Ruby, Augusta, unknown cultivar) were sampled every three

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weeks, beginning in June until December 2013. All sampled trees were analyzed by PCR23–25

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to verify infection with Ca. P. mali or Ca. P. prunorum, respectively.

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Six leaves per apple and apricot tree were collected from newly grown shoots around the tree

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and transported to the laboratory at -20° C. Two discs were punched from each side of the

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frozen leaves using the snap-cap of a microcentrifuge tube (2,5 mL, d = 10 mm), weighed and

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stored at -80° C until analysis (max. 2 weeks after weighing) in centrifuge tubes (Falcon, 15

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mL). During sample preparation care was taken to protect the leaves from light and

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temperature by keeping them frozen. The discs from the right side of the leaves were pooled

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for each group (healthy, infected symptomatic and infected non-symptomatic) and used for

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HPLC-DAD analysis, while those from the left side were used for Chl content determination

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for each single tree.

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Chlorophyll Content

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The sample discs were homogenized in MeOH (2 mL) under liquid nitrogen for approx. 30

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sec after the addition of a spatula tip of CaCO3 using a mortar and pestle. Mortar and pestle

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were washed with MeOH (3 x 2 mL), the suspension was centrifuged (3 min, 4,500 g), and

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the pellet was washed with MeOH (3 x 5 mL). The supernatants were transferred into a

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volumetric flask (25 mL), protected from light, kept at 0° C and brought up to volume with

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MeOH. The volume of MeOH was reduced with decreasing green color in the leaves: Fully

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senescent leaves were extracted in 10 mL MeOH (4 x 2 mL and diluted to 10 mL). An aliquot ACS Paragon Plus Environment

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(2 mL) was transferred into a microcentrifuge tube (2 mL) and, if necessary, stored at -80° C

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(protected from light). After centrifugation (1 min, 7,200 g), 500 µL of the supernatant were

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analyzed by UV/Vis spectrometry using Lambert-Beer’s law. The concentration of Chl a and

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Chl b was determined according to Porra et al. (1989)26 and the carotenoid content according

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to Lichtenthaler (1987)27.

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Qualitative and Quantitative Chlorophyll Catabolite Determination

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Leaf discs were extracted as described for Chl content determination, but the combined

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supernatants were evaporated to dryness (40 mbar, 20° C water bath, protected from light).

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The residue was reconstituted in MeOH (2 mL, solvent B) and stored in a microcentrifuge

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tube at -80° C until analysis. An aliquot was diluted with aq. potassium phosphate buffer

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(solvent A, 50 mM, pH 7.0) 1:2 v/v and centrifuged (1 min, 7,200 g, 4° C). The supernatant

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was analyzed by HPLC-DAD (flow rate: 0.5 mL min-1 at 20° C) using the following solvent

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gradient: 0-5 min: 20% B; 55 min: 70% B; 60 min: 100% B; 70 min: 100% B; 75 min: 20%

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B; 85 min: 20% B. Depending on signal intensity of the chromatogram, 20 µL or 100 µL were

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injected, and UV/Vis traces at 320 nm and 420 nm were recorded. Chl catabolites were

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tentatively identified by their retention times and UV/Vis spectra28,29.

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For a semi-quantitative analysis of Chl catabolites, HPLC-DAD traces were calibrated based

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on peak height with purified Cj-NCC-1 (polar NCC from Cercidiphyllum japonicum

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Aliquots of Cj-NCC-1 were dissolved in MeOH to a final UV/Vis absorption of 0.4-0.8 at 312

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nm, quantified using Lambert-Beer’s law (ε312

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calibration (R2 = 0.9938).

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Purification of Chlorophyll Catabolites and Mass Spectrometric Analysis

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A sample of 3-4 g senescent leaf material was ground in mortar and pestle under liquid

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nitrogen as described before. The mixture was centrifuged (6 min, 7,200 g, 4° C), and the

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combined supernatants were stored at -80° C. An aliquot was centrifuged (1 min, 7,200 g),

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diluted (1:1 v/v) with aq. potassium phosphate (solvent A, 50mM, pH 7.0) and centrifuged

nm(Cj-NCC-1

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).

= 4.2521) and used for a linear


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again (1 min, 7,200 g, 4° C). The supernatant (100 µL) was purified on the analytical HPLC

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with a longer solvent gradient (0-5 min, 20% B; 80 min, 70% B; 85 min, 100% B; 95 min,

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100% B; 100 min, 20% B; 110 min, 20% B).

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The HPLC fractions were injected (20 µL) without further sample preparation on a LC-MS

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system, set at 4.0 kV spray voltage in positive mode, using ammonium acetate (solvent A, 4

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mM, pH 7.0) and MeOH (solvent B) as eluents. Flow and gradient: 0.5 mL min-1, 0-5 min,

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20% B; 30 min, 70% B; 35 min, 100% B; 50 min, 100% B; 51 min, 20% B; 57 min, 20% B.

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Samples for LC-Q-TOF-MS were prepared as described above for HPLC-DAD analysis. The

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leaf extracts were diluted with solvent A (1:1 v/v) and analyzed by LC-Q-TOF-MS (injection

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volume: 20 µl; flow rate: 0.5 mL min-1 at 20° C) using the following solvent gradient: 0-5

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min: 20% B; 55 min: 70% B; 60 min: 95% B; 70 min: 95% B; 75 min: 20% B; 85 min: 20%

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B.

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DNA Extraction and PCR Analysis

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DNA was extracted from root phloem tissue, as described by Baric et al.23, using the DNeasy

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Plant Mini Kit (Qiagen) according to manufacturer’s instructions with the following

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modifications: The tissue (200 mg) was covered with 400 µL of CTAB buffer (2.5%

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cetyltrimethylammonium bromide, 100 mM Tris (tris(hydroxymethyl)aminomethane) pH 8.0,

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1.4 M NaCl, 50 mM EDTA pH 8.0, 1% PVP 40 (polyvinylpyrrolidone), 0.5% ascorbic acid)

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and disrupted in a ball mill (30.0 Hz, Retsch GmbH). The DNeasy Mini spin columns were

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washed according to protocol with buffer AW2 and, in addition, with 70% (v/v) EtOH (500

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µL) and centrifuged again for 2 min at 20,000 g. DNA was eluted with 100 µL TE-elution

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buffer (10 mM Tris-Cl, 0.5 mM EDTA, pH 9.0) and stored at -80° C until PCR analysis.

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Duplex real-time PCR analysis of apple DNA was performed as described by Baric et al.23,

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using the AP specific primers qAP-16S-F/qAP-16S-R with the probe qAP-16S. For internal

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control following primers and probe were used: qMd-cpLeu-F/qMd-cpLeu-R and qMd-

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cpLeu23. Real-time PCR was performed in 20 µL reaction volume containing 2 µL of ACS Paragon Plus Environment

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template DNA, 1X TaqMan® Universal PCR Master Mix (Invitrogen®), 900 nM of both qAP-

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16S primers, 100 nM of both qMd-cpLeu primers and 200 nM of each probe. Amplification

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and detection were performed using a 7500 Fast Real-Time PCR System (Applied

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Biosystems®) applying following cycle conditions: 95° C, 5 min (1x); 95° C, 15 sec and 57

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°C, 1 min (45x); 95 °C, 1 min (1x); 65° C, 1 min (1x).

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Similarly, for apricot DNA a TaqMan® realtime PCR was carried out using the specific

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primers qAP-16S-F/qAP-16S-R24 and the hybridization probe qESFY16S30. For internal

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control primers 18S-F/18S-R25 and probe 18S31 were applied with following cycle conditions:

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95° C, 10 min (1x); 95° C, 15 sec and 60° C, 1 min (40x).

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Data Analysis

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All data were analyzed with SPSS 20.0 (SPSS Inc., Chicago, IL, USA) using analysis of

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variance (ANOVA) followed by Tukey (α = 0.05) post-hoc test.

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Results

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Chlorophyll Catabolites in Leaves of Infected and Healthy Apple Trees (Cultivar ‘Golden

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Delicious’)

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All trees included in this study were tested for infection with Ca. P. mali and Ca. P. prunorum

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by PCR23–25. Five non-symptomatic apple trees were confirmed negative for Ca. P. mali,

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while seven apple trees were tested positive and showed symptoms in June 2013. In three out

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of six asymptomatic apricot trees Ca. P. prunorum could be detected, allowing to include

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three infected, but non-symptomatic apricot trees in this study (for a detailed description of

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the sample set, see Table S1, Supporting Information).

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Chl catabolites were tentatively identified in HPLC-DAD traces based on their retention time

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and the characteristic UV/Vis spectra of NCCs20,21,28,32,33, YCCs8 and PiCC9 (see Figure S1).

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Where mass-spectral data was available, matching molecular ions, UV/Vis spectra and

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retention times were found sufficient to identify fractions with previously known Chl


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catabolites. Fractions were named as described by Moser et al. (2012)29 using a combination

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of the plant source (e.g. Md for Malus x domestica), the chromophore type (NCC for non-

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fluorescent Chl catabolite, YCC for yellow Chl catabolite, and PiCC for pink Chl catabolite)

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and a standardized HPLC-retention time.

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In 2013, on healthy apple trees the first degreened leaves containing Chl catabolites appeared

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in November. In extracts of senescent leaves from healthy apple trees ten NCCs, one YCC

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and one PiCC were detected (see Figure 2, Table 1, and Figure S1, S2 and S3, Supporting

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Information). Nine of them were described previously, allowing for their pair wise tentative

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identification on the basis of UV/Vis and mass spectra4. Based on this and on the identical

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HPLC retention time, the catabolite Md-NCC-29 was identified with a recently described

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catabolite from plum trees (Prunus domestica)34. Md-NCC-29 showed a pseudo-molecular

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ion at m/z 1003.4 [M+H]+, consistent with the molecular formula of C47H62N4O20, and

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fragment ions at m/z 841.4, and 679.3 that indicated the sequential loss of two sugar moieties.

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Likewise, Md-NCC-31 (m/z 841.2) was identified tentatively with Zm-NCC-133. Md-NCC-35

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(m/z 679.0) with So-NCC-235, Md-NCC-47 with Nr-NCC-228, Md-NCC-50 and Md-NCC-49

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(both m/z 645.2) with Cj-NCC-1 from senescent Cercidiphyllum japonicum leaves

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C-82 epimer10,35, respectively. The less polar fraction Md-NCC-58 (m/z 629.2) had matching

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chromatographic and spectroscopic data with Cj-NCC-221. The yellow catabolite Md-YCC-54

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(m/z 643.1) was previously also found (as Cj-YCC-28, Figure S1, Supporting Information) in

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C. japonicum leaves. Finally, the extracts of the apple leaves contained traces of the pink

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catabolite Md-PiCC-63 (m/z 641.0) corresponding to Cj-PiCC9 (Figure S1, Supporting

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Information).

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Several additional minor Chl catabolites were identified by LC-MS and LC-Q-TOF-MS: For

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Md-NCC-42 a base peak was detected at m/z 661.3, consistent with an oxidative addition of

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an oxygen atom to the C15 position of Md-NCC-49, as described previously36. The mass

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spectrum of Md-NCC-44 indicated a pseudo-molecular ion at m/z 675.2 and fragments at ACS Paragon Plus Environment

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and its

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643.2 and 552.2, corresponding to the loss of MeOH and ring D, respectively (all consistent

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with an addition of a methoxy group to the C15 position of Md-NCC-49, as also described

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previously36).

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In 2013, the first degreened leaves on infected apple trees already appeared in October,

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instead of November on healthy trees. Most notably, we found the same Chl catabolites in

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degreened leaves from infected apple trees as in healthy leaves (Table 1), albeit in somewhat

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different amounts. Chl catabolites in healthy and infected apple leaves accounted for approx.

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15-17% of the degraded Chl.

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Chlorophyll Catabolites in Leaves of Infected and Healthy Apricot Trees

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On healthy apricot trees the first senescent leaves were observed in October 2013, while, on

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infected trees, first degreened leaves were detected already in July 2013. Leaves from infected

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non-symptomatic trees showed slight degreening in August 2013. In total 16 different Chl

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catabolites were detected by their characteristic UV/Vis spectra in apricot leaf extracts, among

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them 12 NCCs and three YCCs. In addition, one DNCC was identified tentatively (see Figure

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2, Tables 1 and 2, Figure S4 and S5, Supporting Information). The chromatographic, UV/Vis

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and mass-spectral data of eight Chl catabolites were consistent with those reported in other

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plants: Along these lines, the catabolite Pa-NCC-29 showed a pseudo-molecular ion at m/z

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1003.4 [M+H]+, consistent with the molecular formula of C47H62N4O20. It exhibited the same

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retention time, UV/Vis and mass spectral data as Md-NCC-29. Pa-NCC-31 (m/z 841.0) was

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again identified with Zm-NCC-133, Pa-NCC-35 (m/z 679.0) corresponded to So-NCC-235. Pa-

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NCC-47 (m/z 807.1) was identified with Nr-NCC-228, Pa-NCC-50 and Pa-NCC-49 (both

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645.2) were identified as Cj-NCC-1 and its C82 epimer, and the yellow catabolite Pa-YCC-54

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(m/z 643.1) was identified with Cj-YCC-28. Based on the retention time and the characteristic

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UV/Vis spectrum Pa-NCC-58 was tentatively identified with the less polar Cj-NCC-221.

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Thus, the major (seven) NCC fractions in leaves of apricot and apple trees were tentatively


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identified pairwise. They also showed the same UV/Vis- and mass spectrometric

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characteristics as corresponding NCCs found in leaves of the plum tree34.

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Interestingly, in extracts of the leaves of the apricot tree, a minor additional fraction was

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detected that showed the UV/Vis-characteristics of 1,19-dioxobilin-type nonfluorescent Chl

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catabolite (or DNCC), i.e. that represents a type-II PB5, and which was provisionally named

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Pa-DNCC-45 The mass spectroscopic data for Pa-DNCC-45 indicated it to be a known 1,19-

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dioxobilin, and to represent the formal deformylation product of Pa-NCC-50. It could also be

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detected in the leaves of the infected apricot trees.

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As in apple, Chl catabolites were found in extracts from both symptomatic and infected non-

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symptomatic apricot leaves; however, in extracts from symptomatic leaves two Chl

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catabolites were not found: Pa-NCC-33 (isomer of Pa-NCC-31) and Pa-NCC-35. When

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using the peak height to estimate the total catabolite content, we noticed that non-

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symptomatic leaves (both of healthy and infected trees) contained 1.08 nmol/mg fresh mass

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(FM) and 1.03 nmol/mg FM Chl catabolites, respectively, whereas symptomatic leaves

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contained only 0.46 nmol/mg FM at the end of the vegetation period. The content of these

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PBs in leaves harvested on Nov. 18th, 2013 accounted for approx. 50% of the degraded Chl

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content on healthy trees, whereas PBs were only found in a relative amount of approx. 46% in

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infected non-symptomatic trees and approx. 39% in symptomatic trees, respectively.

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Chlorophyll Degradation During the Vegetation Period

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When analyzing the chlorophyll content during the vegetation period and senescence, we

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noticed a remarkable difference between healthy and infected leaves, in particular of apricot

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trees. In healthy leaves, the Chl content remained constant during summer, with a sharp

287

decline during senescence; in contrast infected leaves contained less Chl and degradation had

288

an earlier onset (Figure 3). For instance, Chl content in infected apple leaves was significantly

289

lower at the beginning of October (Oct. 7th, 2013; P = 0.001, one way ANOVA and Tukey

290

post-hoc test) compared to healthy leaves, where degradation started at the end of October ACS Paragon Plus Environment

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(Oct. 21st, 2013). In healthy apple leaves only 39% of the total Chl was degraded, while in

292

infected leaves 51% of the total Chl disappeared. In both apple and apricot Chl was not fully

293

degraded because a frost event stopped Chl degradation, but in apricot leaves Chl degradation

294

started earlier and, thus, was more advanced than in apple leaves.

295

Already at the beginning of August infected symptomatic apricot leaves had significantly

296

(P = 0.05, one way ANOVA and Tukey post-hoc test) lower Chl content than healthy leaves,

297

and showed a slight decrease of 61% of the total Chl from mid-September (Sep. 16th, 2013)

298

until mid-November (Figure 4). The Chl content of infected non-symptomatic leaves was at

299

an intermediate level between the content of healthy and symptomatic leaves but did not

300

differ significantly from the two after Aug. 26th, showing a steady further decrease of 83% of

301

the whole Chl until the end of the vegetation period. In healthy apricot leaves 76% of the total

302

Chl was degraded.

303

Discussion

304

Chlorosis is a common and well-studied sign of biotic or abiotic stress. In this study, we have

305

investigated the disappearance of Chl and the appearance of NCCs, YCCs, and of a PiCC in

306

extracts of phytoplasma-infected vs. healthy senescent leaves of apple and apricot. In an

307

earlier study, dealing with Chl breakdown in ripening fruit and in senescent leaves of apple

308

and pear trees, only two (apple) NCCs were described37. Furthermore, Chl breakdown in these

309

ripening fruit was suggested occurring by the same PaO/PB path,37 as established for

310

senescent leaves4,6. Indeed, NCCs and DNCCs have been found in ripening loquat38 and

311

soybeans39. In contrast, in bananas different Chl catabolites are formed in the ripening

312

fruit29,40 and in degreening leaves41. In the present study, we found the same Chl catabolites in

313

both healthy and infected leaves of apple and apricot trees, two Rosaceae family members

314

(see Table 1). This work provides the first evidence that the PaO/PB pathway is also

315

occurring in infected leaves and is relevant for chlorotic symptoms. Based on HPLC, UV/Vis


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316

and MS data, six major NCCs, one YCC and one PiCC were identified with known Chl

317

catabolites from the Katsura tree (C. japonicum)8,9,20,21, spinach (S. oleracea)35, maize (Z.

318

mays)33, tobacco (N. rustica)28 and plum trees (Prunus domestica)34 (Figure 2 and Table 1).

319

The PaO/PB pathway has been established as a conserved general pathway of Chl breakdown

320

in higher plants6. However, the NCCs formed may differ by their stereochemistry at C164 and

321

by their peripheral functionalization4,6. The more recently discovered branch of the PaO/PB

322

pathway42, giving rise to 1,19-dioxobilin-type Chl catabolites (DNCCs or ‘type II’ PBs)4,5,38,43

323

seems not to be relevant for leaves of the apple tree, where only 1-formyl-19-oxobilin-type

324

Chl catabolites (NCCs or ‘type I’ PBs) were found. Likewise, in leaves of apricot trees,

325

predominantly type-I PBs were found, and one DNCC was tentatively identified as a minor

326

component only. Even though we could not directly deduce stereochemical details from our

327

spectroscopic data, Md-NCC-50 was assigned earlier as belonging to the ‘epi’-series (‘epi’-

328

configuration at C16)37. The identical retention times of Md-NCC-50, Pa-NCC-50 and Cj-

329

NCC-1 suggest that Pa-NCCs belong to the C16 ‘epi’-series also, as do the NCCs from plum

330

tree (Prunus domestica, which is closely related to apricot)34.

331

In apricot leaves two additional YCCs were found, which were not detected in apple leaves:

332

Pa-YCC-31 (exhibiting its pseudomolecular ion at m/z = 1001.4) and Pa-YCC-51 (with m/z =

333

805.1). More work is necessary to characterize these fractions, which are suggested here to

334

represent oxidation products of Pa-NCC-29 and Pa-NCC-47, respectively. In only one

335

healthy senescent apricot leaf sample an additional YCC was detected (r.t. = 33 min.; m/z =

336

839.1), a presumable oxidation product of Pa-NCC-31.

337

This investigation has revealed the remarkably close structural relationship and frequent

338

identity of Chl catabolites from senescent leaves of several representatives of the

339

Spiraeoideae subfamily of the Rosaceae. Furthermore, this is the first study identifying PBs

340

in non-senescent chlorotic leaves. Previously, different pathways were taken into

341

consideration for senescence-induced Chl breakdown and pathogen-induced degreening: ACS Paragon Plus Environment

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Ougham et al.3 suggested that light-dependent degreening during hypersensitive response, a

343

form of cell death that forms at the point of attempted pathogen ingress44, occurs via different

344

processes which lead to cell death, referred to as ‘pseudosenescence’. Low-temperature-

345

induced chlorosis of Lolium genotypes that were deficient in the PaO/PB pathway was

346

explained by ‘pseudosenescence’3. While our work provides first evidence that phytoplasma

347

infections can trigger the PaO/PB pathway, the relevance of other pathways for Chl

348

degradation during leaf chlorosis would remain to be demonstrated.

349

The question remains whether the effect on Chl degradation is part of the plant’s immune

350

response45, a consequence of indirect stress caused by the infection46 or if it is a virulence

351

strategy from the pathogen attacking the chloroplast, e.g. to obtain nutrients accumulated in

352

the chloroplast or to evade the host immune response14. Chloroplasts have lately drawn

353

attention as a signaling hub during plant immune defense14,45. The photosystem II (PSII) plays

354

a pivotal role during the plant immunity by producing ROS, such as H2O2, against invading

355

pathogens. Interestingly, the function of PSII is inhibited during AP infection2. Just recently,

356

an effector protein of Ca. P. mali was characterized, which binds the Malus x domestica

357

transcription factor MdTCP2447, a homologue of the Arabidopsis thaliana L. nuclear-

358

encoded, chloroplast-located Plastide Transcription Factor 1 (PTF1)48. PTF1 regulates

359

chloroplast expression via the plastid-encoded polymerase (PEP), which is the major

360

chloroplast transcriptase in green tissues. PEP controls the expression of psbD, encoding the

361

reaction center protein D2 of the PSII in A. thaliana49. It is thus likely that Ca. P. mali by

362

targeting PTF1 actively manipulates the chloroplast-regulated defense mechanisms by

363

inhibiting the composition of the PSII complex to prevent excessive H2O2 production. The

364

presence of AP phytoplasma in the canopy of infected apple trees underlies seasonal

365

fluctuation, and significant colonization of the canopy starts in late summer/fall23. As soon as

366

Ca. P. mali is present in higher numbers in the leaves, the pathogen might trigger the above

367

mentioned process and lead to perturbations of the PSII complex. As a consequence, ACS Paragon Plus Environment

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accumulated Chl must be degraded to be detoxified6 and therefore, Chl degradation starts

369

earlier than in non-infected trees. The subsequent progress of Chl degradation could be a

370

consequence of the effect of Ca. P. mali on jasmonate expression. Jasmonate seems to play a

371

role in inducing leaf senescence (which is characterized by Chl degradation) in different plant

372

species50, and during AP infection the seasonal increase of jasmonate in leaves is hampered47.

373

Altered jasmonate levels might thus be -at least partially- involved in the observed course of

374

Chl degradation during infection.

375

We noticed that the amounts of PBs found in leaves did not account for the total content of

376

Chl in green leaves. Even though the low abundance of Chl catabolites and occasional overlap

377

of the HPL-chromatographic fractions limit our HPLC quantification method, we found

378

generally lower amounts of Chl catabolites in apple leaves (Figure 5), when compared to

379

those of the apricot tree (Figure 6). We hypothesize that the physiological state of the apple

380

trees delayed senescence. Apricot leaves degreened earlier and more completely, which could

381

be caused by agricultural practices, including fertilization. Indeed, commercial apple orchards

382

frequently require spraying of Mg/Mn to induce leaf abscission in late fall. In leaves of

383

healthy and infected apple trees the content of PBs accounted for approx. 15-17% of the

384

degraded Chl content. In healthy apricot leaves, Chl catabolites accounted for approx. 50% of

385

the degraded Chl content, in infected non-symptomatic leaves for 46% and in infected

386

symptomatic leaves for 39%. Previous studies also reported an incomplete recovery of Chl

387

catabolites in senescent leaves, when compared to the amount of Chl in green leaves9,20,43.

388

Müller et al.43 suggested diverging pathways, yet undiscovered catabolites or further

389

metabolization of known PBs to still unknown products as possible causes. The apparent

390

influence of pathological events on the yield of Chl breakdown, however, is unprecedented

391

and its quantification requires further investigation.

392

The Chl content remained constant during summer, but was generally lower in infected

393

leaves. While Chl was only slightly reduced in infected apple leaves (Figure 3), infected ACS Paragon Plus Environment

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apricot leaves contained approx. 37% less Chl than healthy ones (Figure 4). Phytoplasma

395

infections have been reported to affect the carbohydrate household causing an inhibition of

396

photosynthesis and chlorosis2. Again, we hypothesize that the highly optimized fertilization in

397

commercial apple orchards mitigates the pathogenic effects, delaying senescence as well as

398

pathogen-induced Chl breakdown.

399

Once Chl has started to break down, Chl degradation proceeded rapidly, within approximately

400

two weeks, in healthy leaves of both apple and apricot trees, confirming earlier observations6.

401

Despite the rapid loss of Chl in healthy apple leaves after Nov 4th, only 39% of the total Chl

402

was degraded, while in healthy apricot leaves 76% of the total Chl degraded. In infected

403

leaves, however, Chl started to disappear earlier, without an immediate rapid loss of Chl.

404

Infected non-symptomatic apricot leaves started to degreen already at the end of July,

405

proceeded to degreen slowly until the end of October with visual symptoms in August, and

406

ended with a fast decline of the remaining Chl within two weeks. Total Chl content in

407

infected apricot leaves was already reduced (to approx. 2 nmol/mg FM with visual chlorosis)

408

in June 2013, remained stable until mid September and decreased slowly until mid November

409

(Figures 4). In line with the Chl content, healthy and infected non-symptomatic apricot leaves

410

contained more than 1.0 nmol/mg FM of Chl catabolites, while infected symptomatic leaves

411

contained only 0.5 nmol/mg FM. From the onset of Chl breakdown Pa-NCC-49 was the most

412

relevant single Chl catabolite in symptomatic leaves. Infected non-symptomatic leaves,

413

instead, contained more Pa-NCC-29 and Pa-NCC-47, reaching a similar relative distribution

414

of NCCs as healthy senescent leaves (Nov. 18th, 2013; Figure 6). Obviously, the infection

415

status had a yet unknown impact on the glycosylation pattern at the FCC level. Chlorosis is a

416

well-known symptom of phytoplasma infection, and similar observations have been made

417

previously1, but the desired deeper understanding of the molecular events that regulate the

418

PaO/PB pathway during infection need further investigation.


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419

We have detected a total of 17 Chl catabolites in extracts of leaves from apple and apricot

420

trees: the seven major NCC fractions were common in both types of leaves and were known

421

from senescent leaves of other plants, of the plum tree, in particular34. In addition, minor

422

fractions of NCC epimers and NCC oxidation products (YCCs and a PiCC, in particular) were

423

also found along with traces of one tentatively identified DNCC. Most notably, we have

424

identified the same Chl catabolites in extracts of leaves from AP- or ESFY-infected trees,

425

providing the first evidence that PaO/PB pathway is relevant also for pathogen-induced

426

chlorosis. Even though the relative amount of Chl catabolites varied among the leaves, this

427

work supports the hypothesis that Chl breakdown in senescence and phytoplasma infection

428

proceeds via a common pathway in some members of the Rosaceae family. More work is

429

required to understand the generality of this concept and the details of pathogen-induced Chl

430

breakdown, including the molecular events that trigger the PaO/PB pathway. In addition, it

431

remains to be demonstrated whether chlorosis is an integral part of host defense or a side

432

effect to mitigate perturbations of the PS II complex, preventing damage from unbound Chl

433

during infection.

434

Abbreviations Used

435

AP, Apple Proliferation; ESFY, European Stone Fruit Yellows; PaO, phaeophorbide a

436

oxigenase; PB, phyllobilin; Chl, chlorophyll; NCC, non-fluorescent chlorophyll catabolite;

437

DNCC, 1,19-dioxobilin-type chlorophyll catabolite; YCC, yellow chlorophyll catabolite;

438

PiCC, pink chlorophyll catabolite; ROS, reactive oxygen species; Ca. P. mali, Candidatus

439

Phytoplasma mali; Ca. P. prunorum, Candidatus Phytoplasma prunorum; RP-HPLC-DAD,

440

reverse phase – high performance liquide chromatography – diode array detector; LC-MS,

441

liquid chromatography – mass spectrometry; LC-Q-TOF-MS, liquid chromatography –

442

quadrupole – time of flight – mass spectrometry; UV/Vis, ultraviolet/visible adsorption;

443

CTAB, cetyltrimethylammonium bromide; Tris, tris(hydroxymethyl)aminomethane; PVP,


18

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444

polyvinylpyrrolidone; FM, fresh mass; PSII, photosystem II; PTF1, Plastide Transcription

445

Factor 1; PEP, plastid-encoded polymerase;

446

Acknowledgment

447

We thank Stephan Hillebrand, Stefan Dipauli, Peter Innerebner, Michael Unterthurner, Evi

448

Mitterrutzner and Massimo Zago for their support in providing the leaf samples. We thank

449

Josep Valls and Mattia Bosello for their support in mass spectrometric analysis and Andreas

450

Gallmetzer and Yazmid Reyes Domínguez for their support in PCR analysis. Financial

451

support from Interreg IV Italy-Austria program (ERDF; project “Biophytirol” Nr. 5345 CUP:

452

B25E11000300007), by the Austrian Science Foundation (FWF, projects No. L-472 B11 and

453

P-28522-N28) and the Autonomous Province of Bozen (Bolzano) is gratefully acknowledged.

454

Supporting Information Available:

455

Supporting Information includes HPLC-DAD traces of healthy and infected senescent apple

456

and apricot leaf extracts, online (DAD)-UV/Vis spectra of Chl catabolite fractions, changes in

457

chlorophyll and catabolite content during the vegetation period in healthy and infected apple

458

and apricot trees, as well as UV/Vis and mass spectroscopic data of Chl catabolites. Detailed

459

information on sample set and infection status of plants included in this study are provided.

460

Materials and methods are described here.

461

This material is available free of charge via the Internet at http://pubs.acs.org.

462


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463

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(43) Müller, T.; Rafelsberger, M.; Vergeiner, C.; Kräutler, B. A Dioxobilane as Product of a

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Divergent Path of Chlorophyll Breakdown in Norway Maple, Angew. Chem. Int. Ed. Engl.

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2011, 50, pp. 10724–10727.

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(44) Mur, L. A. J.; Kenton, P.; Lloyd, A. J.; Ougham, H.; Prats, E. The Hypersensitive

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Response; the Centenary is Upon us But How Much do we Know?, J. Exp. Bot. 2008, 59,

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pp. 501–520.


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(45) Göhre, V. Immune Responses: Photosynthetic Defence, Nat. Plants. 2015, 1, p. 15079.

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(46) Kariola, T.; Brader, G.; Li, J.; Palva, E. T. Chlorophyllase 1, a Damage Control Enzyme,

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Affects the Balance between Defense Pathways in Plants, Plant Cell. 2005, 17, pp. 282–294.

588

(47) Janik, K.; Mithöfer, A.; Raffeiner, M.; Stellmach, H., et al. An Effector of Apple

589

Proliferation Phytoplasma Targets TCP Transcription Factors - a generalized Virulence

590

Strategy of Phytoplasma?, Mol. Plant Pathol. 2016, doi: 10.1111/mpp.12409.

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(48) Baba, K.; Nakano, T.; Yamagishi, K.; Yoshida, S. Involvement of a Nuclear-Encoded

592

Basic Helix-Loop-Helix Protein in Transcription of the Light-Responsive Promoter of psbD1,

593

Plant Physiol. 2001, 125, pp. 595–603.

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(49) Yamburenko, M. V.; Zubo, Y. O.; Borner, T. Abscisic Acid Affects Transcription of

595

Chloroplast Genes via Protein Phosphatase 2C-Dependent Activation of Nuclear Genes:

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Repression by Guanosine-3’-5’-Bisdiphosphate and Activation by Sigma Factor 5, Plant J.

597

2015, 82, pp. 1030–1041.

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(50) Shan, X.; Wang, J.; Chua, L.; Jiang, D., et al. The Role of Arabidopsis Rubisco Activase

599

in Jasmonate-Induced Leaf Senescence, Plant Physiol. 2011, 155, pp. 751–764.

600


25


Page 26 of 33

601

Figure captions

602

Figure 1. Abridged outline of the PaO/PB pathway of Chl breakdown in senescent leaves4,6.

603

Chlorophylls are degraded via characteristic ring-opening by the monooxygenase PaO to

604

type-I PBs, from which primary fluorescent Chl catabolites, pFCC or epi-pFCC, are

605

subsequently furnished in the chloroplast. pFCC and epi-pFCC are epimers differing by their

606

configuration at C16. From these first colorless PBs other type-I PBs are generated, such as

607

NCCs, YCCs or PiCCs, or, alternatively, type-II PBs arise (such as DNCCs) via oxidative

608

loss of the characteristic formyl group and further peripheral transformations.

609

Figure 2. Top. General chemical formula of NCCs and derived formulas of the six major

610

NCCs in leaves of apple and apricot trees, classified as Md-NCCs and Pa-NCCs, respectively,

611

and specified according to their retention time under standard RP-HPL-chromatographic

612

conditions. Bottom: General chemical formula (left) of YCCs and tentative specification of

613

the two YCCs found in leaves of apple and apricot trees and classified, similarly, as Md-

614

YCCs and Pa-YCCs, respectively, and (right) tentative chemical formula of Pa-DNCC-45.

615

Figure 3. Chlorophyll content in healthy (solid line) and infected apple leaves (dashed line).

616

Bars show the standard error of the mean. FM = Fresh mass

617

Figure 4. Chlorophyll content in healthy (solid line), infected non symptomatic (dotted line)

618

and infected apricot leaves (dashed line). Bars show the standard error of the mean. FM =

619

fresh mass

620

Figure 5. Chl catabolite content in apple leaves in 2013. Md-NCC-50 is the most relevant

621

catabolite in healthy (h) and infected (i) apple leaves.

622

Figure 6. Chlorophyll catabolite content in healthy (h), infected non-symptomatic (i-ns) and

623

infected symptomatic (i-s) apricot leaves during 2013. Pa-NCC-29, Pa-NCC-47 and Pa-

624

NCC-50 are the most relevant Chl catabolites in apricot leaves. ACS Paragon Plus Environment

26

Page 27 of 33


Tables Table 1. In extracts of leaves of apple and apricot trees nine chlorophyll catabolites were identified with known Chl catabolites by HPLC-DAD and mass spectrometry (m/z of pseudomolecular ion listed). § = Identification by HPLC co-injection experiments, h = healthy, i = infected, i-ns = infected non-symptomatic. NCC, YCC and PiCC classified on the basis of their UV/Vis spectra. For constitutional formulas and spectra, see Figures 2 and S1. Fraction

m/z [M+H]+

Identified with

Previously reported

Md-NCC-29 1003.4

Pd-NCC-32

Zm-NCC -1

So-NCC-2

+

+

+ +

+ +

+

+ +

+ +

+

+

-

+ +

Oberhuber et al. 35

Pa-NCC-35 Md-NCC-47 § 807.1

Nr-NCC-2

Berghold et al. 28

+

+

C-82 epimer of Cj-NCC-1

assigned tentatively, see Oberhuber et al.35 and Scherl et al.10

+

+

645.2

+ +

+ +

+

Cj-NCC-1

Curty and Engel20 + +

+ +

+

+

+

+ +

Pa-NCC-47 Md-NCC-49 Pa-NCC-49 Md-NCC-50 § 645.2 Pa-NCC-50 Md-YCC-54 643.1

Cj-YCC-2

Moser et al. 8

Pa-YCC-54 Md-NCC-58 § 629.3

Cj-NCC-2

Oberhuber et al. 21

+ +

+ +

641.0

Cj-PiCC

Ulrich et al. 9

+

+

Pa-NCC-58 Md-PiCC-63

i-ns

Berghold et al. 33

Pa-NCC-31 Md-NCC-35 679.0

i

Erhart et al. 34

Pa-NCC-29 Md-NCC-31 841.2

h

Table 2. Additional type-I PBs as well as one type-II PB were detected in leaf extracts of apple trees (three fractions) as well as apricot trees (seven fractions) and were tentatively identified by HPLC-DAD UV/Vis spectra and, in part, by mass spectrometry. MS = mass spectral data available, h = healthy, i = infected, i-ns = infected non-symptomatic. NCC, DNCC and YCC classified on the basis of their UV/Vis spectra (see Figure S1 and references Losey et al. (2001)5, Christ et al. (2013)42 and Müller et al. (2011)43. For constitutional formulae, see Figures 2 and S1. ACS Paragon Plus Environment

27


MS

h

i

i-ns

Pa-YCC-31

+

+

+

+

Pa-NCC-33

+

+

-

+

Md-NCC-42

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

Md-NCC-53

+

+

Pa-NCC-53

+

+

Fraction

Pa-NCC-42 Md-NCC-44

+

Pa-NCC-44 Pa-DNCC-45

+

Pa-NCC-48 Pa-YCC-51

+

Page 28 of 33

+

+


28

Page 29 of 33


Figure graphics Figure 1.

PaO/phyllobilin pathway

N

N

H

16

5

N CH3

O

CO2CH3

H3C

CH3

O

HN

NH

N

O

C

1

19

Mg N

O

H O

R

H O CO2CH3

HO2C

CH3

HN

pFCC / epi-pFCC CH3

chlorophyll a (R = CH3) chlorophyll b (R = HC=O) R1 OO H

16

O

1

19

HN

NH

4

H

2

R

5

16

NH HN

CO2R3

A

HN

NH

C

10

HO2C

1

D 19

H

O

H

OH

4 5

NH HN 10

E

H

O

B

O CO2CH3

HO2C

NCC

DNCCs: type-II phyllobilins

O NH 15

N 10

H O

CO2CH3 O

O

HO2C H

HN HN

5

NH HN 10

5

OH HO2C

O

OH

HN

NH 15

O CO2CH3 YCC

PiCC


29


Page 30 of 33

Figure 2.


30

Page 31 of 33


Figure 3.

Figure 4.


31


Page 32 of 33

Figure 5.

Figure 6.


32

Page 33 of 33


For Table of Contents Only

The entire Table of Contents graphic is original and was created by the coauthors, using ChemDraw for chemical structures and photographs made by Cecilia Mittelberger for chlorotic leaves. The Table of Contents graphic was assembled using Adobe Illustrator CS4.


33

Pathogen-Induced Leaf Chlorosis: Products of Chlorophyll Breakdown

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