STAY-GREEN and Chlorophyll Catabolic Enzymes Interact at Light-Harvesting Complex II for Chlorophyll Detoxification during Leaf Senescence in Arabidopsis

Abstract

During leaf senescence, plants degrade chlorophyll to colorless linear tetrapyrroles that are stored in the vacuole of senescing cells. The early steps of chlorophyll breakdown occur in plastids. To date, five chlorophyll catabolic enzymes (CCEs), NONYELLOW COLORING1 (NYC1), NYC1-LIKE, pheophytinase, pheophorbide a oxygenase (PAO), and red chlorophyll catabolite reductase, have been identified; these enzymes catalyze the stepwise degradation of chlorophyll to a fluorescent intermediate, pFCC, which is then exported from the plastid. In addition, STAY-GREEN (SGR), Mendel's green cotyledon gene encoding a chloroplast protein, is required for the initiation of chlorophyll breakdown in plastids. Senescence-induced SGR binds to light-harvesting complex II (LHCII), but its exact role remains elusive. Here, we show that all five CCEs also specifically interact with LHCII. In addition, SGR and CCEs interact directly or indirectly with each other at LHCII, and SGR is essential for recruiting CCEs in senescing chloroplasts. PAO, which had been attributed to the inner envelope, is found to localize in the thylakoid membrane. These data indicate a predominant role for the SGR-CCE-LHCII protein interaction in the breakdown of LHCII-located chlorophyll, likely to allow metabolic channeling of phototoxic chlorophyll breakdown intermediates upstream of nontoxic pFCC.

A Role for TIC55 as a Hydroxylase of Phyllobilins, the Products of Chlorophyll Breakdown during Plant Senescence

“…The steps of chlorophyll to pFCC conversion have been shown to take place in gerontoplasts (Sakuraba et al, 2012). In agreement with this, fluorescent HPLC fractions corresponding to pFCC and to epi-pFCC have been identified in extracts of gerontoplasts of oilseed rape (Brassica napus) and barley (Schellenberg et al, 1993;Ginsburg et al, 1994;Moser and Matile, 1997 Figure 1A).…”

“…First, we reanalyzed their predicted localization in the envelope (Bartsch et al, 2008) by transiently expressing GFP fusion proteins in mesophyll protoplasts isolated from senescent Arabidopsis wild-type leaves ( Figure 5B). Indeed, GFP signals for both TIC55 and PTC52 surrounded the chloroplasts, i.e., indicating envelope localization like the envelope positive control, TIC110, while PAO colocalized with chlorophyll autofluorescence, i.e., confirming thylakoid localization (Sakuraba et al, 2012).…”

“…However, the subchloroplast location of respective reactions remained largely unclear until recently, in particular because of the inconsistency with regard to the presence or absence of transmembrane domains in these proteins. Thus, for example, PAO is an integral membrane protein with two predicted transmembrane domains ( Figure 5A) (Sakuraba et al, 2012), while RCCR, with which it physically interacts during catalysis (Rodoni et al, 1997;Pruzinská et al, 2007), is a soluble protein (Wüthrich et al, 2000). Recently, based on protein interaction studies, we proposed the model that all chloroplastlocated chlorophyll catabolic enzymes form a highly dynamic multiprotein complex at the thylakoid membrane with interaction with light harvesting complex II proteins (Sakuraba et al, 2012).…”

“…Acor, Aquilegia coerulea; Alyr, Arabidopsis lyrata; Atha, Arabidopsis thaliana; Atri, Amborella trichopoda; Bdis, Brachypodium distachon; Brap, Brassica rapa; Bsta, Brachypodium stacei; Bstr, Boechera stricta; Ccle, Citrus clementina; Cgra, Capsella grandiflora; Cpap, Carica papaya; Crub, Capsella rubella; Csat, Cucumis sativus; Csin, Citrus sinensis; Egra, Eucalyptus grandis; Esal, Eutrema salsugineum; Fves, Fragaria vesca; Gmax, Glycine max; Grai, Gossypium raimondii; Kfla, Klebsormidium flaccidum; Kmar, Kalanchoe marnieriana; Lusi, Linum usitatissimum; Macu, Musa acuminata; Mdom, Malus domstica; Mesc, Manihot esculenta; Mgut, Mimulus guttatus; Mtru, Medicago truncatula; Osat, Oryza sativa; Phal, Panicum hallii; Ppat, Physcomitrella patens; Pper, Prunus persica; Ptri, Populus trichocarpa; Pvir, Panicum virgatum; Pvul, Phaseolus vulgaris; Rcom, Ricinus communis; Sbic, Sorghum bicolor; Sfal, Sphagnum fallax; Sita, Setaria italica; Slyc, Solanum lycopersicum; Some, Selaginella moellendorffii; Spol, Spirodela polyrhiza; Spur, Salix purpurea; Stub, Solanum tuberosum; Svir, Setaria viridis; Tcac, Theobroma cacao; Vvin, Vitis vinifera; Zmay, Zea mays. phototoxicity (Sakuraba et al, 2012), is reached with the formation of pFCC. A pFCC derivative was recently shown to be able to efficiently produce singlet oxygen in vitro when excited with UV light (Jockusch et al, 2014).…”

“…Again, other species, such as C. japonicum, exclusively produce phyllobilins that carry the intact carboxymethyl group, indicating that they lack a MES16 ortholog. Interestingly, these modifying reactions are localized in the cytosol (MES16) (Christ et al, 2012) and at the endoplasmic reticulum (CYP89A9) (Christ et al, 2013), while all enzymes identified to date that are required for the conversion of chlorophyll to pFCC are located within the plastid (Sakuraba et al, 2012). This led to the concept that reactions that commonly occur during chlorophyll breakdown take place in senescing chloroplasts, while species-specific modifications occur outside.…”