Self-incompatibility (SI) is an important mechanism that prevents self-fertilization and avoids inbreeding in flowering plants. The most widespread SI system utilizes S -ribonucleases ( S-RNase s) and S -locus F-boxes ( SLF s) as S -determinants. In citrus, SI is ancestral; Citrus maxima (pummelo) is self-incompatible, while Citrus reticulata (mandarin) and its hybrids are self-compatible (SC). Here we identified nine highly polymorphic pistil-specific, developmentally expressed S-RNase s from pummelo that segregate with S -haplotypes in a gametophytic manner and cluster with authentic S-RNase s. We provide evidence that these S-RNase s function as the female S -determinants in citrus. Moreover, we found that each S-RNase is linked to ~nine SLF s. Analysis of 117 citrus SLF/SLFL genes revealed clustering into 12 types and evidence that the S-RNase s and intra-haplotypic SLFs/SLFL s co-evolved. Our data are consistent with citrus having an S -locus comprising a S-RNase and several SLF s that fit the non-self-recognition model. We identified a predominant single nucleotide mutation, S m - RNase , in SC citrus, which provides a ‘natural’ loss of function. We present evidence that SI-SC transitions due to the S m -RNase, initially arose in mandarin, spreading to its hybrids and became fixed. Identification of an evolutionarily distant new genus utilizing the S -RNase-based SI system, >100 million years separated from the nearest S -RNase family, is a milestone for evolutionary comparative studies.

Mandarin (Citrus reticulata) is one of the most important citrus crops worldwide. Its domestication is believed to have occurred in South China, which has been one of the centers of mandarin cultivation for four millennia. We collected natural wild populations of mandarin around the Nanling region and cultivated landraces in the vicinity. We found that the citric acid level was dramatically reduced in cultivated mandarins. To understand genetic basis of mandarin domestication, we de novo assembled a draft genome of wild mandarin and analyzed a set of 104 citrus genomes. We found that the Mangshan mandarin is a primitive type and that two independent domestication events have occurred, resulting in two groups of cultivated mandarins (MD1 and MD2) in the North and South Nanling Mountains, respectively. Two bottlenecks and two expansions of effective population size were identified for the MD1 group of cultivated mandarins. However, in the MD2 group there was a long and continuous decrease in the population size. MD1 and MD2 mandarins showed different patterns of interspecific introgression from cultivated pummelo species. We identified a region of high divergence in an aconitate hydratase (ACO) gene involved in the regulation of citrate content, which was possibly under selection during the domestication of mandarin. This study provides concrete genetic evidence for the geographical origin of extant wild mandarin populations and sheds light on the domestication and evolutionary history of mandarin.

Citrus sinensis seedlings were irrigated with nutrient solution at a concentration of 0 (Mg-deficiency) or 2 (Mg-sufficiency) mM Mg (NO3)2 for 16 weeks. Mg-deficiency-induced interveinal chlorosis, vein enlargement and corkiness, and alterations of gas exchange, pigments, chlorophyll a fluorescence (OJIP) transients and related parameters were observed in middle and lower leaves, especially in the latter, but not in upper leaves. Mg-deficiency might impair the whole photosynthetic electron transport, including structural damage to thylakoids, ungrouping of photosystem II (PSII), inactivation of oxygen-evolving complex (OEC) and reaction centers (RCs), increased reduction of primary quinone electron acceptor (QA) and plastoquinone pool at PSII acceptor side and oxidation of PSI end-electron acceptors, thus lowering energy transfer and absorption efficiency and the transfer of electrons to the dark reactions, hence, the rate of CO2 assimilation in Mg-deficiency middle and lower leaves. Although potassium, Mg, manganese and zinc concentration in blades displayed a significant and positive relationship with the corresponding element concentration in veins, respectively, great differences existed in Mg-deficiency-induced alterations of nutrient concentrations between leaf blades and veins. For example, Mg-deficiency increased boron level in the blades of upper leaves, decreased boron level in the blades of lower leaves, but did not affect boron level in the blades of middle leaves and veins of upper, middle and lower leaves. To conclude, Mg-deficiency-induced interveinal chlorosis, vein enlargement, and corkiness, and alterations to photosynthesis and related parameters increased with increasing leaf age. Mg-deficiency-induced enlargement and corkiness of veins were not caused by Mg-deficiency-induced boron-starvation.

Self-incompatibility (SI) substantially restricts the yield and quality of citrus. Therefore, breeding and analyzing self-compatible germplasm is of great theoretical and practical significance for citrus. Here, we focus on the mechanism of a self-compatibility mutation in ‘Guiyou No. 1’ pummelo (Citrus maxima), which is a spontaneous mutant of ‘Shatian’ pummelo (Citrus maxima, self-incompatibility). The rate of fruit set and the growth of pollen tubes in the pistil confirmed that a spontaneous mutation in the pistil is responsible for the self-compatibility of ‘Guiyou No. 1’. Segregation ratios of the S genotype in F1 progeny, expression analysis, and western blotting validated that the reduced levels of S2-RNase mRNA contribute to the loss of SI in ‘Guiyou No. 1’. Furthermore, we report a phased assembly of the ‘Guiyou No. 1’ pummelo genome and obtained two complete and well-annotated S haplotypes. Coupled with an analysis of SV variations, methylation levels, and gene expression, we identified a candidate gene (CgHB40), that may influence the regulation of the S2-RNase promoter. Our data provide evidence that a mutation that affects the pistil led to the loss of SI in ‘Guiyou No. 1’ by influencing a poorly understood mechanism that affects transcriptional regulation. This work significantly advances our understanding of the genetic basis of the SI system in citrus and provides information on the regulation of S-RNase genes.

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.