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.

Eureka lemon (Citrus limon) is an economically important crop in China. In 2009, a new disease was observed on Eureka lemon in an orchard of Ruili city in Yunnan Province. Nearly 10% of the plants were symptomatic with yellow vein clearing on lateral veins, combined with leaf crinkling and warping of young leaves. Water-soaked appearances were also seen on the adaxial leaf veins. The symptoms were present on mature leaves, resulting in almost 20% yield reduction. The symptoms on leaves were more severe in spring and autumn than in summer. This disease was transmitted by grafting from infected Eureka lemon to six lemon (C. limon) varieties (Femminello, Fino, Villafranca, Verna, Kutdiken, and Monachello) and two sour orange (C. aurantium) varieties (Daidai, Morocco), with inoculated plants showing similar symptoms. Variable symptoms were observed on other hosts, including severe yellowing and vein clearing on Dweet tangor (C. reticulata × C. sinensis), slight vein clearing on Tongshui 72-1 sweet orange (C. sinensis) and Marsh grapefruit (C. paradisi), but no symptoms were observed on Etrog citron (C. medica), Mexican lime (C. aurantifolia), or Ponkan (C. reticulata). When infected branches from Etrog citron, Mexican lime, and Ponkan were grafted onto healthy Morocco sour orange, typical yellowing and vein clearing symptoms were observed. In the mechanical transmission trials using eight herbaceous plant species, leaf rolling, shrinking, and pointed necrosis were observed on Vigna unguiculata, slight leaf rolling symptoms were observed on Capsicum frutescens, whereas Lycopersicum esculenum, Nicotiana benthamiana, Momordica charantia, Solanum melongena, Cucumis sativus, and Sesamum indicum remained symptomless. Symptom expression was influenced by temperature, being visible between 18 and 24°C but disappearing over 32°C. Crude extracts from young symptomatic lemon leaves were negatively stained for transmission electron microscopy observation (1) and showed filamentous virus particles of 13 to 15 × 400 to 1,000 nm. The described combination of symptoms in citrus species and herbaceous indicators, and virus morphology revealed by electron microscopy, suggested presence of Citrus yellow vein clearing virus (CYVCV). RNA extractions were made from 52 samples of Eureka lemon with yellow vein clearing symptoms. A one-step RT-PCR using primer pair (sense: 5′-TACCGCAGCTATCCATTTCC-3′ and antisense: 5′-GCAGAAATCCCGAACCACTA-3′), designed from coat protein gene of CYVCV genome (GenBank Accession No. JX040635) (1), was performed. All of the symptomatic samples yielded the expected cDNA fragment and no amplicon was obtained from healthy plants. Three PCR amplicons were selected for cloning and sequencing. BLAST analysis showed that these nucleotide sequences (KJ859679 to 81) had greater than 97% nucleotide identity with the corresponding region of CYVCV reference genome (2), and therefore, this disease was associated with CYVCV. Symptoms resembling CYVCV infection were observed in China before (3); however, to our knowledge, this is the first proven report of CYVCV present in China. The identification of the pathogen now allows for appropriate field management and control measures. References: (1) V. Grimaldi et al. 13th IOCV Conference, 343-345, 1996. (2) G. Loconsole et al. Phytopathology 102:1168, 2012. (3) C. Zhou et al. 18th IOCV Conference, 44, 2010.

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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.

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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.

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