The Arabidopsis EH proteins (AtEH1/Pan1 and AtEH2/Pan1) are components of the endocytic TPLATE complex (TPC) which is essential for endocytosis. Both proteins are homologues of the yeast ARP2/3 complex activator, Pan1p. Here, we show that these proteins are also involved in actin cytoskeleton regulated autophagy. Both AtEH/Pan1 proteins localise to the plasma membrane and autophagosomes. Upon induction of autophagy, AtEH/Pan1 proteins recruit TPC and AP-2 subunits, clathrin, actin and ARP2/3 proteins to autophagosomes. Increased expression of AtEH/Pan1 proteins boosts autophagosome formation, suggesting independent and redundant pathways for actin-mediated autophagy in plants. Moreover, AtEHs/Pan1-regulated autophagosomes associate with ER-PM contact sites (EPCS) where AtEH1/Pan1 interacts with VAP27-1. Knock-down expression of either AtEH1/Pan1 or VAP27-1 makes plants more susceptible to nutrient depleted conditions, indicating that the autophagy pathway is perturbed. In conclusion, we identify the existence of an autophagy-dependent pathway in plants to degrade endocytic components, starting at the EPCS through the interaction among AtEH/Pan1, actin cytoskeleton and the EPCS resident protein VAP27-1.

Carotenoids play vital roles in the colouration of plant tissues and organs, particularly fruits; however, the regulation of carotenoid metabolism in fruits during ripening is largely unknown. Here, we show that red light (RL) promotes fruit colouration by inducing accelerated degreening and carotenoid accumulation in kumquat fruits. Transcriptome profiling reveals that a NAC family transcription factor, FcrNAC22 is specifically induced in RL-irradiated fruits. FcrNAC22 localizes to the nucleus, and its gene expression is up-regulated as fruits change colour. Results from dual luciferase, yeast one-hybrid (Y1H) assays and electrophoretic mobility shift assays (EMSAs) indicate that FcrNAC22 directly binds to and activates the promoters of three genes (i.e. FcrLCYB1, FcrBCH2 and FcrNCED5) encoding key enzymes in carotenoid metabolic pathway. Moreover, FcrNAC22 overexpression in citrus and tomato fruits as well as in citrus callus enhances expression of a dozen of carotenoid biosynthetic genes including the aforementioned three, accelerates plastid conversion into chromoplasts, and promotes colour change. Knock down of FcrNAC22 expression in transient transformed citrus fruits attenuates fruit colouration induced by red light. Taken together, our results demonstrate that FcrNAC22 is an important transcription factor that mediates RL-induced fruit colouration via upregulation of carotenoid metabolism in citrus and perhaps in other plants.

Elaioplasts of citrus peel are colorless plastids which accumulate significant amounts of terpenes. However, other functions of elaioplasts have not been fully characterized to date. Here, a LC–MS/MS shotgun technology was applied to identify the proteins from elaioplasts that were highly purified from young fruit peel of kumquat. A total of 655 putative plastid proteins were identified from elaioplasts according to sequence homology in silico and manual curation. Based on functional classification via Mapman, ~50% of the identified proteins fall into six categories, including protein metabolism, transport, and lipid metabolism. Of note, elaioplasts contained ATP synthase and ADP, ATP carrier proteins at high abundance, indicating important roles for ATP generation and transport in elaioplast biogenesis. Additionally, a comparison of proteins between citrus chromoplast and elaioplast proteomes suggest a high level of functional conservation. However, some distinctive protein profiles were also observed in both types of plastids notably for isoprene biosynthesis in elaioplasts, and carotenoid metabolism in chromoplasts. In conclusion, this comprehensive proteomic study provides new insights into the major metabolic pathways and unique characteristics of elaioplasts and chromoplasts in citrus fruit.

Although multiple microscopic techniques have been applied to horticultural research, few studies of individual organelles in living fruit cells have been reported to date. In this paper, we established an efficient system for the transient transformation of citrus fruits using an Agrobacterium-mediated method. Kumquat (Fortunella crassifolia Swingle) was used; it exhibits higher transformation efficiency than all citrus fruits that have been tested and a prolonged-expression window. Fruits were transformed with fluorescent reporters, and confocal microscopy and live-cell imaging were used to study their localization and dynamics. Moreover, various pH sensors targeting different subcellular compartments were expressed, and the local pH environments in cells from different plant tissues were compared. The results indicated that vacuoles are most likely the main organelles that contribute to the low pH of citrus fruits. In summary, our method is effective for studying various membrane trafficking events, protein localization, and cell physiology in fruit and can provide new insight into fruit biology research.

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