Transcription-related metabolic regulation in grafted lemon seedlings under magnesium deficiency stress.

Magnesium is one of the essential nutrients for plant growth, and plays a pivotal role in plant development and metabolism. Soil magnesium deficiency is evident in citrus production, which ultimately leads to failure of normal plant growth and development, as well as decreased productivity. Citrus is mainly propagated by grafting, so it is necessary to fully understand the different regulatory mechanisms of rootstock and scion response to magnesium deficiency. Here, we characterized the differences in morphological alterations, physiological metabolism and differential gene expression between trifoliate orange rootstocks and lemon scions under normal and magnesium-deficient conditions, revealing the different responses of rootstocks and scions to magnesium deficiency. The transcriptomic data showed that differentially expressed genes were enriched in 14 and 4 metabolic pathways in leaves and roots, respectively, after magnesium deficiency treatment. And the magnesium transport-related genes MHX and MRS2 may respond to magnesium deficiency stress. In addition, magnesium deficiency may affect plant growth by affecting POD, SOD, and CAT enzyme activity, as well as altering the levels of hormones such as IAA, ABA, GA3, JA, and SA, and the expression of related responsive genes. In conclusion, our research suggests that the leaves of lemon grafted onto trifoliate orange were more significantly affected than the roots under magnesium-deficient conditions, further indicating that the metabolic imbalance of scion lemon leaves was more severe.

Pangenome analysis provides insight into the evolution of the orange subfamily and a key gene for citric acid accumulation in citrus fruits.

The orange subfamily (Aurantioideae) contains several Citrus species cultivated worldwide, such as sweet orange and lemon. The origin of Citrus species has long been debated and less is known about the Aurantioideae. Here, we compiled the genome sequences of 314 accessions, de novo assembled the genomes of 12 species and constructed a graph-based pangenome for Aurantioideae. Our analysis indicates that the ancient Indian Plate is the ancestral area for Citrus-related genera and that South Central China is the primary center of origin of the Citrus genus. We found substantial variations in the sequence and expression of the PH4 gene in Citrus relative to Citrus-related genera. Gene editing and biochemical experiments demonstrate a central role for PH4 in the accumulation of citric acid in citrus fruits. This study provides insights into the origin and evolution of the orange subfamily and a regulatory mechanism underpinning the evolution of fruit taste.

[Fruit variation and geographical distribution of citron].

The ripe dried fruit of citron(Citrus medica) is one of the important sources of Chinese herb Citri Fructus. At the same time, it is also grown for edible and ornamental uses. There are many species and abundant genetic variation. To clarify the intraspecific variation and resource distribution of citron, this study investigated the variation in 11 citron fruits, basically covering the main species in China, including Xiaoguo citron(C. medica var. ethrog), Goucheng(C. medica var. yunnanensis), Muli citron(C.medica var. muliensis), Dehong citron(C.medica×Citrus spp.), Fuzhou citron(C.medica×C.grandis?), Mawu(C.medica×C.grandis?), Cangyuan citron, Binchuan citron, Sweet citron, Big citron, and Small citron. The natural communities of citron were proved to be mainly distributed in the southwestern and western Yunnan and southeastern Tibet of China, with Yunnan, Sichuan, Guangxi, Chongqing, Hubei, and Zhejiang identified as the main production areas. Citron has also been widely grown in India, the Mediterranean region, and the Caribbean coast countries. The field investigation revealed the large-scale intraspecific variation of citron fruits. Most of the fruits are oval-like or sphere-like in shape. The fruits are green when raw and yellow when ripe, with oil cell dots on the skin, stripe-likes running from top to bottom, and bulge at the top. Usually, in the smaller citron fruits, the pulp and juice vesicles are better developed and the central columella is tighter. By contrast, the juice vesicles and central columella in larger fruits became more vacant, with carpels visible, and the apex segregation and development of the carpels is one of the reasons for variation. These variations should be given top priority in the future variety selection and breeding, and the quality differences of different citron species and their mechanisms should be further studied. In particular, variety selection and classification management according to their medicinal or edible purposes will provide scientific and technological supports for the orderly, safe, and effective production of citron products consumed as food and medicine.

Transcriptome and Metabolome Comparison of Smooth and Rough Citrus limon L. Peels Grown on Same Trees and Harvested in Different Seasons.

Background: Farmers harvest two batches fruits of Lemons (Citrus limon L. Burm. f.) i.e., spring flowering fruit and autumn flowering fruit in dry-hot valley in Yunnan, China. Regular lemons harvested in autumn have smooth skin. However, lemons harvested in spring have rough skin, which makes them less attractive to customers. Furthermore, the rough skin causes a reduction in commodity value and economical losses to farmers. This is a preliminary study that investigates the key transcriptomic and metabolomic differences in peels of lemon fruits (variety Yuning no. 1) harvested 30, 60, 90, 120, and 150 days after flowering from the same trees in different seasons. Results: We identified 5,792, 4,001, 3,148, and 5,287 differentially expressed genes (DEGs) between smooth peel (C) and rough peel (D) 60, 90, 120, and 150 days after flowering, respectively. A total of 1,193 metabolites differentially accumulated (DAM) between D and C. The DEGs and DAMs were enriched in the mitogen-activated protein kinase (MAPK) and plant hormone signaling, terpenoid biosynthesis, flavonoid, and phenylalanine biosynthesis, and ribosome pathways. Predominantly, in the early stages, phytohormonal regulation and signaling were the main driving force for changes in peel surface. Changes in the expression of genes associated with asymmetric cell division were also an important observation. The biosynthesis of terpenoids was possibly reduced in rough peels, while the exclusive expression of cell wall synthesis-related genes could be a possible reason for the thick peel of the rough-skinned lemons. Additionally, cell division, cell number, hypocotyl growth, accumulation of fatty acids, lignans and coumarins- related gene expression, and metabolite accumulation changes were major observations. Conclusion: The rough peels fruit (autumn flowering fruit) and smooth peels fruit (spring flowering fruit) matured on the same trees are possibly due to the differential regulation of asymmetric cell division, cell number regulation, and randomization of hypocotyl growth related genes and the accumulation of terpenoids, flavonoids, fatty acids, lignans, and coumarins. The preliminary results of this study are important for increasing the understanding of peel roughness in lemon and other citrus species.

Characterization of the complete chloroplast genome of 'Yunning No.1' lemon (Citrus limon).

'Yunning No.1' lemon, a mutant of Eureka lemon, is originally found in Yunnan province of China and is the main cultivated lemon variety there. In this study, we assembled and annotated its chloroplast genome using Illumina Hiseq-2500 whole genome re-sequencing data. Its chloroplast genome is 160,141 bp in size, containing a 87,754 bp large single copy region, a 18,385 bp small single copy region and a pair of 27,001 bp inverted repeat region. Like many citrus species, 114 unique genes (including 80 protein-coding genes, 30 tRNAs and 4 rRNAs) could be identified from the chloroplast genome of 'Yunning No.1'. Phylogenetic analysis revealed that the 'Yunning No.1' chloroplast genome was closest to Citrus maxima.

Variations of membrane fatty acids and epicuticular wax metabolism in response to oleocellosis in lemon fruit.

Oleocellosis is a physiological disorder causing blemishes on fruit surface. This study investigated the influence of oleocellosis on the membrane fatty acids and wax in lemon fruit rinds at the morphological, physiological, metabolic and molecular levels by using a variety with a high incidence rate of oleocellosis (green lemon). Oleocellosis-damaged rinds showed loose and flaky wax layers with more fissures on the surface, as well as higher contents of C16 and C18 fatty acids and very long chain (VLC) fatty alkanes while lower contents of VLC fatty aldehydes. The main differentially expressed genes, including FabZ, FAD2 and SAD6 involved in the accumulation of C16 and C18 fatty acids and CER1 involved in the transformation of VLC fatty aldehydes to VLC fatty alkanes, were up-regulated by oleocellosis. These results indicate that oleocellosis accelerates the accumulation of membrane free fatty acids and transformation of VLC fatty aldehydes to VLC fatty alkanes.

Variations of Flavonoid Composition and Antioxidant Properties among Different Cultivars, Fruit Tissues and Developmental Stages of Citrus Fruits.

A large number of biologically active compounds are present in ripe citrus fruits. However, few studies have been focused on the changes in flavonoids and the evolution of antioxidant activity during citrus fruit growth. In this study, fruits of five citrus cultivars cultivated in China were sampled at 60-210 days post-anthesis (DPA) at intervals of 30 days. The amounts of main flavonoids in the peel and pulp were analyzed by HPLC and their activities were studied by DPPH, ABTS and FRAP. The results showed that the contents of hesperidin, diosmin, eriodictyol, rutin and nobiletin increased before 90 DPA and then decreased with the growth and development of fruits, but an opposite tendency was observed for naringin and narirutin. The antioxidant activities in citrus peel and pulp were found to be significantly correlated with some flavonoids. The results may be of guiding values in citrus production and utilization of citrus fruit by-products.

Identification of Genes Associated with Lemon Floral Transition and Flower Development during Floral Inductive Water Deficits: A Hypothetical Model.

Water deficit is a key factor to induce flowering in many woody plants, but reports on the molecular mechanisms of floral induction and flowering by water deficit are scarce. Here, we analyzed the morphology, cytology, and different hormone levels of lemon buds during floral inductive water deficits. Higher levels of ABA were observed, and the initiation of floral bud differentiation was examined by paraffin sections analysis. A total of 1638 differentially expressed genes (DEGs) were identified by RNA sequencing. DEGs were related to flowering, hormone biosynthesis, or metabolism. The expression of some DEGs was associated with floral induction by real-time PCR analysis. However, some DEGs may not have anything to do with flowering induction/flower development; they may be involved in general stress/drought response. Four genes from the phosphatidylethanolamine-binding protein family were further investigated. Ectopic expression of these genes in Arabidopsis changed the flowering time of transgenic plants. Furthermore, the 5' flanking region of these genes was also isolated and sequence analysis revealed the presence of several putative cis-regulatory elements, including basic elements and hormone regulation elements. The spatial and temporal expression patterns of these promoters were investigated under water deficit treatment. Based on these findings, we propose a model for citrus flowering under water deficit conditions, which will enable us to further understand the molecular mechanism of water deficit-regulated flowering in citrus. HIGHLIGHT: Based on gene activity during floral inductive water deficits identified by RNA sequencing and genes associated with lemon floral transition, a model for citrus flowering under water deficit conditions is proposed.

Identification of pummelo cultivars by using a panel of 25 selected SNPs and 12 DNA segments.

Pummelo cultivars are usually difficult to identify morphologically, especially when fruits are unavailable. The problem was addressed in this study with the use of two methods: high resolution melting analysis of SNPs and sequencing of DNA segments. In the first method, a set of 25 SNPs with high polymorphic information content were selected from SNPs predicted by analyzing ESTs and sequenced DNA segments. High resolution melting analysis was then used to genotype 260 accessions including 55 from Myanmar, and 178 different genotypes were thus identified. A total of 99 cultivars were assigned to 86 different genotypes since the known somatic mutants were identical to their original genotypes at the analyzed SNP loci. The Myanmar samples were genotypically different from each other and from all other samples, indicating they were derived from sexual propagation. Statistical analysis showed that the set of SNPs was powerful enough for identifying at least 1000 pummelo genotypes, though the discrimination power varied in different pummelo groups and populations. In the second method, 12 genomic DNA segments of 24 representative pummelo accessions were sequenced. Analysis of the sequences revealed the existence of a high haplotype polymorphism in pummelo, and statistical analysis showed that the segments could be used as genetic barcodes that should be informative enough to allow reliable identification of 1200 pummelo cultivars. The high level of haplotype diversity and an apparent population structure shown by DNA segments and by SNP genotypes, respectively, were discussed in relation to the origin and domestication of the pummelo species.