Citric acid is an important contributor to fruit flavor. The extremely significant differences in citric acid levels are observed among citrus species and varieties. The molecular basis of citric acid accumulation remains largely unknown on the inter-species level. Here, we performed transcriptome analysis of five pairs of acidic and acidless variety fruits from kumquat (Citrus crassifolia), lemon (C. limon), pummelo (C. maxima), mandarin (C. reticulata), and sweet orange (C. sinensis) to investigate the common and specific genes related to citric acid accumulation in these citrus species. Transcript profiles of the citrate metabolism genes revealed that the mechanism of citric acid accumulation in kumquat differed from that in the other four species. The comparative analysis and weighted gene co-expression network analysis identified 61 candidate genes from lemon, pummelo, mandarin, and sweet orange. Vacuolar acidification regulated by CitPH4-CitAN1-CitPH5 was a common mechanism underlying citric acid accumulation in these four species. In addition, gene network analysis indicated that CitPH4 was a central regulator, exhibiting a strong correlation with the genes involved in vacuolar trafficking and potassium (K⁺) transport. The co-expression and functional enrichment analyses showed that active sugar metabolism and increased citrate synthesis were mainly responsible for the accumulation of citric acid in kumquat. Twenty-four transcription factors were identified as candidate regulators, of which CitbHLH28 was found to regulate acidity level via transactivating CWINV2 and PK3 expressions. Overall, this study provides an insight into the mechanism of citric acid accumulation in citrus fruit and provides useful references for fruit quality improvement through acidity control in different citrus species.

Phase transition and floral induction in citrus requires several years of juvenility after germination. Such a long period of juvenility has been a major hindrance to its genetic improvement program. Studies have shown that miR156 along with its downstream genes SQUAMOSA PROMOTER BINDING PROTEINS (SBP) and SBP-LIKE (SPL) mediate the phase transition and floral induction process in plants. Our current study has systematically analyzed SPLs in 15 different citrus-related species, systematically annotated them based on their close homology to their respective Arabidopsis orthologs, and confirmed the functional attributes of the selected members in floral precocity. The majority of the species harbored 15 SPLs. Their cis-element assessment suggested the involvement of the SPLs in diverse developmental and physiological processes in response to different biotic and abiotic cues. Among all, SPL5, SPL9, and SPL11 stood out as consistently differentially expressed SPLs in the adult and young tissues of different citrus-related species. Independent overexpression of their F. hindsii orthologs (FhSPL5, FhSPL9, and FhSPL11) brought an enhanced expression of endogenous FLOWERING LOCUS T leading to the significantly precocious flowering in transgenic Arabidopsis lines. Future study of the genes in the citrus plant itself is expected to conclude the assessments made in the current study.

As an important spice species in Rutaceae, the Sichuan pepper (Zanthoxylum armatum) can provide pungent and numbing taste, as well as aroma in its mature fruit. Here we assembled a chromosome-level genome of green prickly ash which was widely cultivated in a major production area including Chongqing and Sichuan province, China. We generated 712 Gb (~112 ×) PacBio long reads and 511 Gb (~82 ×) Hi-C data, and yielded an assembly of 99 pseudochromosomes with total size of 5.32 Gb and contig N50 of 796 kb. The genomic analyses and cytogenetic experiments both indicated that the cultivar ‘Zhuye Huajiao’ was a triploid. We identified a Zanthoxylum-specific whole genome duplication event emerging about 24.8 million years ago (Mya). We also detected a transposition burst event (0.3–0.4 Mya) responsible for the large genome size of Z. armatum. Metabolomic analysis of the Zanthoxylum fruits during development stages revealed profiles of 39 volatile aroma compounds and 528 secondary metabolites, from which six types of sanshools were identified. Based on metabolomic and transcriptomic network analysis, we screened candidate genes encoding long chain acyl-CoA synthetase, fatty acid desaturase, branched-chain amino acid aminotransferase involved in sanshool biosynthesis and three genes encoding terpene synthase during fruit development. The multi-omics data provide insights into the evolution of Zanthoxylum and molecular basis of numbing and aroma flavor of Sichuan pepper.