Association of T2/S-RNase With Self-Incompatibility of Japanese Citrus Accessions Examined by Transcriptomic, Phylogenetic, and Genetic Approaches

Several citrus varieties show gametophytic self-incompatibility (GSI), which can contribute to seedless fruit production in several cultivars. This study investigated the genes regulating this trait through RNA-seq performed using styles collected from the flowers of Japanese citrus cultivars ‘Hyuganatsu,' ‘Tosabuntan,' ‘Hassaku,' ‘Banpeiyu,' and ‘Sweet Spring'. We screened the transcripts of putative T2 RNases, i.e., the protein family including all S-RNases from S-RNase-based GSI plants, and constructed a phylogenetic tree using the screened T2 RNases and S-RNases retrieved from citrus genome databases and a public database. Three major clusters (class I–III) were formed, among which, the class III cluster contained family specific subclusters formed by S-RNase and a citrus-specific cluster monophyletic to the S-RNase clusters. From the citrus class III cluster, six transcripts were consistent with the S haplotypes previously determined in Japanese citrus accessions, sharing characteristics such as isoelectric point, extracellular localization, molecular weight, intron number and position, and tissue-specific expression with S-RNases. One T2 RNase gene in self-incompatible Hyuganatsu was significantly down-regulated in the styles of a self-compatible mutant of Hyuganatsu in RNA-seq and qPCR analyses. In addition, the inheritance pattern of some T2 RNase genes was consistent with the pattern of the S haplotype in the progeny population of Hyuganatsu and Tosabuntan. As all results supported citrus self-incompatibility being based on S-RNase, we believe that six T2 RNase genes were S-RNases. The homology comparison between the six T2 RNases and S-RNases recently reported in Chinese citrus revealed that three out of six T2 RNases were identical to S-RNases from Chinese citrus. Thus, the other three T2 RNases were finally concluded to be novel citrus S-RNases involved in self-incompatibility.

Type I H+-pyrophosphatase regulates the vacuolar storage of sucrose in citrus fruit

The aim of this work was to evaluate the general role of the vacuolar pyrophosphatase proton pump (V-PPase) in sucrose accumulation in citrus species. First, three citrus V-PPase genes, designated CsVPP-1, CsVPP-2, and CsVPP-4, were identified in the citrus genome. CsVPP-1 and CsVPP-2 belonging to citrus type I V-PPase genes are targeted to the tonoplast, and CsVPP-4 belonging to citrus type II V-PPase genes is located in the Golgi bodies. Moreover, there was a significantly positive correlation between transcript levels of type I V-PPase genes and sucrose, rather than hexose, content in fruits of seven citrus cultivars. Drought and abscisic acid treatments significantly induced the CsVPP-1 and CsVPP-2 transcript levels, as well as the sucrose content. The overexpression of type I V-PPase genes significantly increased PPase activity, decreased pyrophosphate contents, and increased sucrose contents, whereas V-PPase inhibition produced the opposite effect in both citrus fruits and leaves. Furthermore, altering the expression levels of type I V-PPase genes significantly influenced the transcript levels of sucrose transporter genes. Taken together, this study demonstrated that CsVPP-1 and CsVPP-2 play key roles in sucrose storage in the vacuole by regulating pyrophosphate homeostasis, ultimately the sucrose biosynthesis and transcript levels of sucrose transport genes, providing a novel lead for engineering or breeding modified taste in citrus and other fruits.

Graft-induced Changes in MicroRNA Expression Patterns in Citrus Leaf Petioles

Grafting is an important, widely used plant propagation technique but its physiological effects are as yet insufficiently understood. Recent studies indicate that movement of proteins and small RNAs through the graft union might be involved. MicroRNAs are known to play a significant role in regulation of higher plants’ developmental and metabolic traits. Extending this logic, we hypothesize that changes in activity of specific microRNAs are one of the mechanisms involved in physiological effects of grafting. The objective of the present study was to test this hypothesis. We determined the expression of a broad range of microRNAs in Citrus leaf petioles, as affected by grafting. Four stock/scion combinations (‘Merav’ mandarin and ‘Star Ruby‘ grapefruit scions X ‘Troyer’ citrange and ‘Volkamer’ lemon rootstocks), rootstock auto-grafts and plants of the variety used as rootstock (= non-grafted) were examined. Grafting caused a dramatic reduction in the expression of the major microRNAs, miR156 (and miR157), which appear to be associated with reduction of juvenility in perennial woody plants. This effect was strongest in hetero-grafts but evident also in auto-grafts. Expression of miR894 also declined upon grafting. Differences in the expression of miR397 were found among grafted scion cultivars, while in non-grafted rootstocks expression of miR397 was barely detectable. Bioinformatic analysis confirmed the presence of miR397 in the citrus genome, validated its sequence and demonstrated its ability to form a stem loop. The differences in miR397 expression might be related to specific copper and other micronutrient requirements of citrus stock-scion combinations.Thus, our results support the hypothesis, indicating the involvement of specific microRNAs in engendering physiological effects of grafting in Citrus. The precise, underlying mechanism needs to be elucidated.