Acetylome reprograming participates in the establishment of fruit metabolism during polyploidization in citrus.

Polyploidization leads to novel phenotypes and is a major force in evolution. However, the relationship between the evolution of new traits and variations in the post-translational modifications (PTM) of proteins during polyploidization has not been studied. Acetylation of lysine residues is a common protein PTM that plays a critical regulatory role in central metabolism. To test whether changes in metabolism in citrus fruit is associated with the reprogramming of lysine acetylation (Kac) in non-histone proteins during allotetraploidization, we performed a global acetylome analysis of fruits from a synthetic allotetraploid citrus and its diploid parents. A total of 4,175 Kac sites were identified on 1,640 proteins involved in a wide range of fruit traits. In the allotetraploid, parental dominance (i.e. resemblance to one of the two parents) in specific fruit traits, such as fruit acidity and flavonol metabolism, was highly associated with parental Kac level dominance in pertinent enzymes. This association is due to Kac-mediated regulation of enzyme activity. Moreover, protein Kac probably contributes to the discordance between the transcriptomic and proteomic variations during allotetraploidization. The acetylome reprogramming can be partially explained by the expression pattern of several lysine deacetylases (KDACs). Overexpression of silent information regulator 2 (CgSRT2) and histone deacetylase 8 (CgHDA8) diverted metabolic flux from primary metabolism to secondary metabolism and partially restored a metabolic status to the allotetraploid, which expressed attenuated levels of CgSRT2 and CgHDA8. Additionally, KDAC inhibitor treatment greatly altered metabolism in citrus fruit. Collectively, these findings reveal the important role of acetylome reprogramming in trait evolution during polyploidization.

Identification of a delayed leaf greening gene from a mutation of pummelo.

Delayed greening of young leaves is an unusual phenomenon of plants in nature. Citrus are mostly evergreen tree species. Here, a natural mutant of "Guanxi" pummelo (Citrus maxima), which shows yellow leaves at the young stage, was characterized to identify the genes underlying the trait of delayed leaf greening in plants. A segregating population with this mutant as the seed parent and a normal genotype as the pollen parent was generated. Two DNA pools respectively from the leaves of segregating seedlings with extreme phenotypes of normal leaf greening and delayed leaf greening were collected for sequencing. Bulked segregant analysis (BSA) and InDel marker analysis demonstrated that the delayed leaf greening trait is governed by a 0.3 Mb candidate region on chromosome 6. Gene expression analysis further identified a key candidate gene (Citrus Delayed Greening gene 1, CDG1) in the 0.3 Mb region, which showed significantly differential expression between the genotypes with delayed and normal leaf greening phenotypes. There was a 67 bp InDel region difference in the CDG1 promoter and the InDel region contains a TATA-box element. Confocal laser-scanning microscopy revealed that the CDG1-GFP fusion protein signals were co-localized with the chloroplast signals in the protoplasts. Overexpression of CDG1 in tobacco and Arabidopsis led to the phenotype of delayed leaf greening. These results suggest that the CDG1 gene is involved in controlling the delayed leaf greening phenotype with important functions in chloroplast development.

PtSVP, an SVP homolog from trifoliate orange (Poncirus trifoliata L. Raf.), shows seasonal periodicity of meristem determination and affects flower development in transgenic Arabidopsis and tobacco plants.

A MADS-box gene was isolated using the suppressive subtractive hybridization library between early-flowering mutant and wild-type trifoliate orange (Poncirus trifoliata L. Raf.). This gene is highly homologous with Arabidopsis SHORT VEGETATIVE PHASE (SVP). Based on real-time PCR and in situ hybridization during bud differentiation, PtSVP was expressed intensively in dormant tissue and vegetative meristems. PtSVP transcripts were detected in apical meristems before floral transition, then down-regulated during the transition. PtSVP expression was higher in differentiated (flower primordium) than in undifferentiated cells (apical meristems). The PtSVP expression pattern during apical meristem determination suggested that its function is not to depress flower initiation but to maintain meristem development. Transcription of PtSVP in Arabidopsis svp-41 showed partially rescued SVP function. Ectopic overexpression of PtSVP in wild-type Arabidopsis induced late flowering similar to the phenotypes induced by other SVP/StMADS-11-like genes, but transformants produced additional trichomes and floral defects, such as flower-like structures instead of carpels. Ectopic expression of PtSVP in tobacco also caused additional florets. Overexpression of PtSVP in tobacco inhibited early transition of the coflorescence and prolonged coflorescence development, thus causing additional florets at the later stage. A yeast two-hybrid assay indicated that PtSVP significantly interacted with PtAP1, a homolog of Arabidopsis APETALA1 (AP1). These findings suggest that citrus SVP homolog genes are involved in flowering time regulation and may influence inflorescence meristem identity in some conditions or genetic backgrounds. SVP homologs might have evolved among plant species, but the protein functions are conserved between Arabidopsis and citrus.

Involvement of polyamine biosynthesis in somatic embryogenesis of Valencia sweet orange (Citrus sinensis) induced by glycerol.

Culture of Citrus sinensis embryogenic callus on the embryo-inducing medium (EIM) containing glycerol gave rise to a large number of embryos, whereas very few embryos were observed on the callus growth medium (CGM). In the current paper, attempts were made to investigate whether polyamine biosynthesis was involved in glycerol-mediated somatic embryogenesis. Quantification of free polyamines by high-performance liquid chromatography showed that the cultures on EIM had less putrescine than those on CGM. However, increase in spermidine and spermine was detected in cultures on EIM during the first 20d of culture, coincident with abundant somatic embryogenesis. The globular embryos contained more polyamines than embryos at other stages. Semi-quantitative reverse transcriptase-polymerase chain reaction assay showed that expression levels of all of the five key genes involved in polyamine biosynthesis, with the exception of S-adenosylmethionine decarboxylase, were induced in cultures on EIM, and that their transcriptional levels were increased with maturation of the embryos. Addition of alpha-difluoromethylornithine, a polyamine biosynthesis inhibitor, to EIM resulted in remarkable inhibition of somatic embryogenesis, concurrent with notable reduction of endogenous putrescine and spermidine, particularly at higher concentrations. Exogenous application of 1mM putrescine to EIM together with 5mM alpha-difluoromethylornithine led to dramatic enhancement of endogenous polyamines, which successfully restored somatic embryogenesis. All of these, collectively, demonstrated that free polyamines, at least spermidine and spermine herein, were involved in glycerol-mediated promotion of somatic embryogenesis, which will open a new avenue for establishing a sophisticated system for somatic embryogenesis based on the modulation of endogenous polyamines.

Relationship between ploidy variation of citrus calli and competence for somatic embryogenesis.

This study focuses on the relationship between the genetic variation of calli and the competence for somatic embryogenesis in citrus. The DNA content of 35 citrus calli of different genotypes was measured three times by flow cytometry during a period of four years. The results showed that 71.4 % of the genotypes had a progressive increase of varied cells, while those of Page tangelo, Shamouti sweet orange, Russ navel orange and Cleopatra decreased; significant difference in the variation degree (percentages) existed among genotypes. Studies carried out on the induction of somatic embryogenesis revealed that 9 out of the 35 genotypes had still kept the competence of somatic embryogenesis, and the rest 26 had lost the competence. Correlation analysis indicated that there was no significant relationship between the variation degree and the embryogenesis competence r = -0.10 (P < 0.01), neither for the relationship between the subculture duration and the regeneration capacity.

[Creation and hybrid identification of the sexual progenies using citrus somatic hybrid as pollen parent].

110 plantlets were obtained from the cross between citrus allotetraploid somatic hybrid [Hamlin sweet orange (Citrus sinensis Osback) + Rough Lemon (C. jambhiri Luss)] (as the pollen parent) and mono-embryonic diploid type Iben No.4 [Huanongbendizao tangerine (C. reticulata Blanco) x Ichang papeda (C. ichangensis Swingle)] by the means of young embryo culture in vitro, and there were 93 triploids and 17 diploids through chromosome counting and the ploidy analyser identification. RAPD analyses showed that all sexual progenies were hybrid.

Cryopreservation of shoot-tips of citrus using vitrification: effect of reduced form of glutathione.

A commercial citrus scion cultivar, '439' tangor [C. Suavissima Hort. et Tanaka x C. sinensis (L.) Osbesk cv.Gailiangcheng] was used to investigate whether GSH (reduced form of glutathione) could improve survival of a vitrification procedure. The optimal pre-growth treatment was a 3-day pre-culture on basal pre-culture medium (BPM: MT basal medium containing 0.5 mol/L glycerol and 5 % sucrose at pH 5.8). GSH of 40 mg/L in the pre-culture medium improved shoot tip survival after cryopreservation. GSH in the recovery medium also improved survival, with 10 mg/L giving the best result. GSH of 40 mg/L in the loading and vitrification solutions also improved survival. The optimal cryopreservation protocol was successfully applied to 12 other citrus cultivars. This is the first report on the successful cryopreservation of shoot-tips from commercial citrus scion cultivars.

[Genetic variation of Citrus calli revealed by the ploidy analyser].

The cell DNA content of forty-eight Citrus calli of different genotype were measured by using the Ploidy Analyser. The results showed that 93.8% out of forty-eight Citrus calli had double DNA content except that Ruby. Weizhang and Kinnow had little varied cells and did not show second peak. Moreover, Pineapple, Meiwa kumquat, Changsha, Russ navel, Guoqing No. 4 and Carter had DNA varied cells including triploids and aneuploids based on the DPAC software analyse. Among the forty-eight Citrus calli, Pineapple had the highest of the DNA varied cells, which was up to 18.57%, Anliucheng had the lowest, 4.07%. There existed significant difference in the variation among genotypes by Duncan Analyse. At the same subcultured medium and at the same subcultured period, the effect of cultural duration on the variation of calli was not significant.

[Inheritance of organelle genomes of the somatic hybrid between Cleopatra mandarin (Citrus reticulata) and flying dragon (Poncirus trifoliata)].

Cleaved Amplified Polymorphic Sequence (CAPS) was successfully applied to analyze the organelle composition of three eight-year-old trees of the somatic hybrid between Cleopatra mandarin (Citrus reticulata) and Flying Dragon (Poncirus trifoliata). Five chloroplast and five mitochondrial universal primer pairs were used. All chloroplast primer pairs (rbcL-rbcL, rbcL-PSA I, TrnH-Trnk, TrnD-TrnT, TrnK-TrnK) and three (nad 1 exon B-nad 1 exon C, 18S rRNA-5S rRNA, nad 4 exon 1-nad 4 exon 2) of the five mitochondrial primer pairs, were efficiently amplified, but no polymorphism was detected, when the PCR products were digested by eleven restriction endonucleases, including, Hin6 I, Bus RI, Taq I, Msp I, HinfI, AluI, Dra I, EcoR I, Hind III, BamH I and Pst I respectively, three polymorphic cpDNA-CAPS markers (rbcL-rbcL/Hin 6 I, TrnD-TrnT/BusR I, TrnD-TrnT/Taq I) and one mtDNA-CAPS marker (nad 1-nad1/Msp I) were found. The results showed that cpDNA in the somatic hybrid plants came from Flying Dragon, the mesophyll parent, and mtDNA from Cleopatra mandarin, the embryogenic suspension parent uniformly. In order to prove the reliability of CAPS results, and to get more detailed information about the mtDNA inheritance, RFLP analyses was conducted. Genomic DNA of the somatic hybrids and their corresponding parents were digested by five restriction endonucleases (Dra I, EcoR I, Hind III, BamH I and Pst I), and hybridized with five mitochondrial probes (Cob, Pro 2, Pro I, atp 6, 26S rRNA) as well as one chloroplast probe, i.e. the PCR product of Flying Dragon with the primer pair of trnd 1-trnt 1. The results were in line with those of CAPS, and no novel bands were detected, which indicated that no organelle DNA recombination or rearrangement have been detected in the hybrid plants. The research showed that novel pattern of nuclear-mitochondria-chloroplast interaction could be reached via protoplast fusion.