Exploring the interplay between angiosperm chlorophyll metabolism and environmental factors.

MAIN CONCLUSION: In this review, we summarize how chlorophyll metabolism in angiosperm is affected by the environmental factors: light, temperature, metal ions, water, oxygen, and altitude. The significance of chlorophyll (Chl) in plant leaf morphogenesis and photosynthesis cannot be overstated. Over time, researchers have made significant advancements in comprehending the biosynthetic pathway of Chl in angiosperms, along with the pivotal enzymes and genes involved in this process, particularly those related to heme synthesis and light-responsive mechanisms. Various environmental factors influence the stability of Chl content in angiosperms by modulating Chl metabolic pathways. Understanding the interplay between plants Chl metabolism and environmental factors has been a prominent research topic. This review mainly focuses on angiosperms, provides an overview of the regulatory mechanisms governing Chl metabolism, and the impact of environmental factors such as light, temperature, metal ions (iron and magnesium), water, oxygen, and altitude on Chl metabolism. Understanding these effects is crucial for comprehending and preserving the homeostasis of Chl metabolism.

A tetraploid-dominated cytochimera developed from a natural bud mutant of the nonapomictic mandarin variety 'Orah'.

Nonapomictic citrus tetraploids are desirable in citrus breeding for the production of triploid, seedless varieties, and polyploid rootstocks. However, only a few lines have been reported, and they were all generated using chemical methods. A 2x + 4 × cytochimera of the nonapomictic citrus variety 'Orah' mandarin, which developed from a bud mutant, was found due to its morphology differing from that of diploid plants and characterised via ploidy analysis combining flow cytometry and chromosome observation. The chimaera was stable, and there were 1.86-1.90 times as tetraploid cells as diploid cells. Anatomical structure observation revealed that the 'Orah' chimaera may be a periclinal chimaera with diploid cells in the L1 layer and tetraploid cells in the L2 and L3 layers. The chimaera showed some typical traits of polyploid plants, including thicker shoots, wider and thicker leaves, larger flowers and fruits, and fewer but larger seeds in fruits than in diploid plants. Almost all the seeds of the chimaera were monoembryonic. Most of the self-pollinated progenies of the chimaera were identified as tetraploids, and some triploid, pentaploid, and hexaploid plants were found. As a female, the chimaera produced allotriploids when crossed with Australian finger lime. In addition, 6 plants developed from polyembryonic seeds of the chimaera were identified as sexual tetraploid progenies with low-level recombinant genomes. Therefore, the 'Orah' 2x + 4 × chimaera can be used as a female parent to produce hybrid triploid and tetraploid citrus plants with high efficiency. Identification of the chimaera demonstrated that tetraploid citrus plants, especially nonapomictic varieties, can be generated from shoot bud mutants. Supplementary Information: The online version contains supplementary material available at 10.1007/s11032-024-01456-x.

Three Alternaria Species, Including a New Species, Causing Leaf Spot Disease of Loquat (Eriobotrya japonica) in China.

Loquat (Eriobotrya japonica) is an economically important subtropical fruit crop in China. Field surveys conducted in different loquat orchards located in Chongqing, Sichuan, and Fujian provinces between 2017 and 2020 resulted in a collection of 56 Alternaria-like isolates from trees exhibiting symptoms of loquat leaf spot. Multigene phylogenetic analyses using seven gene regions, namely, ITS, gapdh, RPB2, tef1, Alt a 1, endoPG, and OPA10-2, showed that all the isolates belonged to the genus Alternaria, and supporting morphological analysis identified them as members of species A. alternata, A. gaisen, and A. chongqingensis sp. nov. In vitro and in vivo pathogenicity tests showed all the identified species to be pathogenic and able to cause leaf spot disease on loquat. Moreover, comprehensive phylogenetic analyses employing all combinations of the above seven gene sequences revealed the capability of Alt a 1-tef1-endoPG to provide a well-resolved gene tree for Alternaria spp. at the species level. This study adds to the current knowledge on an unknown species (A. chongqingensis sp. nov.) and is the first report of A. gaisen in loquat worldwide.

NpPP2-B10, an F-Box-Nictaba Gene, Promotes Plant Growth and Resistance to Black Shank Disease Incited by Phytophthora nicotianae in Nicotiana tabacum.

Black shank, a devastating disease affecting tobacco production worldwide, is caused by Phytophthora nicotianae. However, few genes related to Phytophthora resistance have been reported in tobacco. Here, we identified NpPP2-B10, a gene strongly induced by P. nicotianae race 0, with a conserved F-box motif and Nictaba (tobacco lectin) domain, in the highly resistant tobacco species Nicotiana plumbaginifolia. NpPP2-B10 is a typical F-box-Nictaba gene. When it was transferred into the black shank-susceptible tobacco cultivar 'Honghua Dajinyuan', it was found to promote resistance to black shank disease. NpPP2-B10 was induced by salicylic acid, and some resistance-related genes (NtPR1, NtPR2, NtCHN50, and NtPAL) and resistance-related enzymes (catalase and peroxidase) were significantly upregulated in the overexpression lines after infection with P. nicotianae. Furthermore, we showed that NpPP2-B10 actively regulated the tobacco seed germination rate, growth rate, and plant height. The erythrocyte coagulation test of purified NpPP2-B10 protein showed that NpPP2-B10 had plant lectin activity, and the lectin content in the overexpression lines was significantly higher than that in the WT, which could lead to accelerated growth and improved resistance of tobacco. SKP1 is an adaptor protein of the E3 ubiquitin ligase SKP1, Cullin, F-box (SCF) complex. We demonstrated that NpPP2-B10 could interact with the NpSKP1-1A gene in vivo and in vitro through yeast two-hybrid (Y2H) and bimolecular fluorescence complementation (BiFC), indicating that NpPP2-B10 likely participates in the plant immune response by mediating the ubiquitin protease pathway. In conclusion, our study provides some important insights concerning NpPP2-B10-mediated regulation of tobacco growth and resistance.

EjFAD8 Enhances the Low-Temperature Tolerance of Loquat by Desaturation of Sulfoquinovosyl Diacylglycerol (SQDG).

Loquat (Eriobotrya japonica Lindl.) is an evergreen fruit tree of Chinese origin, and its autumn-winter flowering and fruiting growth habit means that its fruit development is susceptible to low-temperature stress. In a previous study, the triploid loquat (B431 × GZ23) has been identified with high photosynthetic efficiency and strong resistance under low-temperature stress. Analysis of transcriptomic and lipidomic data revealed that the fatty acid desaturase gene EjFAD8 was closely associated with low temperatures. Phenotypic observations and measurements of physiological indicators in Arabidopsis showed that overexpressing-EjFAD8 transgenic plants were significantly more tolerant to low temperatures compared to the wild-type. Heterologous overexpression of EjFAD8 enhanced some lipid metabolism genes in Arabidopsis, and the unsaturation of lipids was increased, especially for SQDG (16:0/18:1; 16:0/18:3), thereby improving the cold tolerance of transgenic lines. The expression of ICE-CBF-COR genes were further analyzed so that the relationship between fatty acid desaturase and the ICE-CBF-COR pathway can be clarified. These results revealed the important role of EjFAD8 under low-temperature stress in triploid loquat, the increase expression of FAD8 in loquat under low temperatures lead to desaturation of fatty acids. On the one hand, overexpression of EjFAD8 in Arabidopsis increased the expression of ICE-CBF-COR genes in response to low temperatures. On the other hand, upregulation of EjFAD8 at low temperatures increased fatty acid desaturation of SQDG to maintain the stability of photosynthesis under low temperatures. This study not only indicates that the EjFAD8 gene plays an important role in loquat under low temperatures, but also provides a theoretical basis for future molecular breeding of loquat for cold resistance.

Photosynthetic Efficiency and Glyco-Metabolism Changes in Artificial Triploid Loquats Contribute to Heterosis Manifestation.

Previous studies indicated that extensive genetic variations could be generated due to polyploidy, which is considered to be closely associated with the manifestation of polyploid heterosis. Our previous studies confirmed that triploid loquats demonstrated significant heterosis, other than the ploidy effect, but the underlying mechanisms are largely unknown. This study aimed to overcome the narrow genetic distance of loquats, increase the genetic variation level of triploid loquats, and systematically illuminate the heterosis mechanisms of triploid loquats derived from two cross combinations. Here, inter-simple sequence repeats (ISSRs) and simple sequence repeats (SSRs) were adopted for evaluating the genetic diversity, and transcriptome sequencing (RNA-Seq) was performed to investigate gene expression as well as pathway changes in the triploids. We found that extensive genetic variations were produced during the formation of triploid loquats. The polymorphism ratios of ISSRs and SSRs were 43.75% and 19.32%, respectively, and almost all their markers had a PIC value higher than 0.5, suggesting that both ISSRs and SSRs could work well in loquat assisted breeding. Furthermore, our results revealed that by broadening the genetic distance between the parents, genetic variations in triploids could be promoted. Additionally, RNA-Seq results suggested that numerous genes differentially expressed between the triploids and parents were screened out. Moreover, KEGG analyses revealed that "photosynthetic efficiency" and "glyco-metabolism" were significantly changed in triploid loquats compared with the parents, which was consistent with the results of physiological indicator analyses, leaf micro-structure observations, and qRT-PCR validation. Collectively, our results suggested that extensive genetic variations occurred in the triploids and that the changes in the "photosynthetic efficiency" as well as "glyco-metabolism" of triploids might have further resulted in heterosis manifestation in the triploid loquats.

Volatile composition changes in lemon during fruit maturation by HS-SPME-GC-MS.

BACKGROUND: Volatiles are determinants of fruit aroma and flavor characteristics and also provide valuable information for lemon as ingredient for the food and drinks industry. Volatiles in 'Eureka' lemon and 'Xiangshui' lemon pulps from 130 to 186 days after flowering were enriched by headspace-solid-phase microextraction (HS-SPME), and analyzed by gas chromatography-mass spectrometry (GC-MS). RESULTS: Seventy-seven volatiles of two lemon cultivars at the different ripening stages were identified and divided into six categories. Varieties and ripening stages had significant effects on individual volatiles in each category. The proportion of monoterpenes was found to be higher in 'Eureka' lemon, while 'Xiangshui' lemon had a higher proportion of sesquiterpenes, aldehydes and alcohols. The proportion of monoterpene fluctuation decreased during fruit ripening, while fluctuation of sesquiterpenes, alcohols, aldehydes and esters increased. Among the hydrocarbons, monoterpenes decreased their relative abundance from 91.67% to 81.04% in 'Eureka' lemon, and from 83.01% to 60.04% in 'Xiangshui' lemon; conversely, sesquiterpenes increased from 0.73% to 2.89% in 'Eureka' lemon, and from 3.21% to 8.48% in 'Xiangshui' lemon. Among the oxygenated volatiles, the proportions of alcohols, aldehydes and esters were higher at 186 days after flowering in both two cultivars. CONCLUSION: The volatile organic compounds during fruit ripening of lemon varieties with different resistance were elucidated. The proportion of oxygenated volatiles increased during fruit ripening, and disease-resistant varieties had a higher proportion. These results provided important theoretical support for the utilization of lemon fruits and the innovation of disease-resistant germplasm resources. © 2021 Society of Chemical Industry.

Integrated metabolic profiling and transcriptome analysis of pigment accumulation in Lonicera japonica flower petals during colour-transition.

BACKGROUND: Plants have remarkable diversity in petal colour through the biosynthesis and accumulation of various pigments. To better understand the mechanisms regulating petal pigmentation in Lonicera japonica, we used multiple approaches to investigate the changes in carotenoids, anthocyanins, endogenous hormones and gene expression dynamics during petal colour transitions, i.e., green bud petals (GB_Pe), white flower petals (WF_Pe) and yellow flower petals (YF_Pe). RESULTS: Metabolome analysis showed that YF_Pe contained a much higher content of carotenoids than GB_Pe and WF_Pe, with α-carotene, zeaxanthin, violaxanthin and γ-carotene identified as the major carotenoid compounds in YF_Pe. Comparative transcriptome analysis revealed that the key differentially expressed genes (DEGs) involved in carotenoid biosynthesis, such as phytoene synthase, phytoene desaturase and ζ-carotene desaturase, were significantly upregulated in YF_Pe. The results indicated that upregulated carotenoid concentrations and carotenoid biosynthesis-related genes predominantly promote colour transition. Meanwhile, two anthocyanins (pelargonidin and cyanidin) were significantly increased in YF_Pe, and the expression level of an anthocyanidin synthase gene was significantly upregulated, suggesting that anthocyanins may contribute to vivid yellow colour in YF_Pe. Furthermore, analyses of changes in indoleacetic acid, zeatin riboside, gibberellic acid, brassinosteroid (BR), methyl jasmonate and abscisic acid (ABA) levels indicated that colour transitions are regulated by endogenous hormones. The DEGs involved in the auxin, cytokinin, gibberellin, BR, jasmonic acid and ABA signalling pathways were enriched and associated with petal colour transitions. CONCLUSION: Our results provide global insight into the pigment accumulation and the regulatory mechanisms underlying petal colour transitions during the flower development process in L. japonica.

First report of Fusarium solani Species Complex Causing Root Rot of Loquat (Eriobotrya japonica) in China.

Loquat (Eriobotrya japonica), a native fruit tree to China, is a popular edible fruit with medicinal properties (Badenes et al. 2013). A 2016-2019 field survey of ~13,000 loquat trees in two orchards in Chongqing and Fujian provinces showed about 5 to 10% root rot disease incidence. The disease symptoms included leaf yellowing, wilting, rotting of main root, and cracking of lateral roots, eventually leading to defoliation and death. To determine the causative agent, diseased roots from six trees were collected, washed in tap water, cut into 2-3 mm pieces, and disinfected for 3 min in 75% (v/v) EtOH. After rinsing in sterilized water, the root pieces were soaked in 10% NaClO (w/v) for 5-10 min, rinsed thrice in sterile water, and plated on potato dextrose agar (PDA). After 7 days of incubation at 25°C, individual spores were collected from the fungal colonies and replated. Single spore cultures growing on PDA gave rise to woolly-cottony, cream-white colored aerial mycelium and a yellowish pigmented mycelium. The average colony growth rate was 8.6 mm day-1 (n=3). Microscopic observation of the mycelium revealed septate and hyaline hyphae and long cylindrical monophialides. Macroconidia were moderately curved, stout, 3-4 septate, measuring 20.79-48.70 µm × 4.16-10.14 µm (n=50). Microconidia produced from long phialides were kidney-shaped, 0-2 septate, and 5.72-17.28 µm × 2.29-6.51 µm (n=50) in size. The mycelial characteristics and reproductive structures of the isolates fit the morphological description of Fusarium sp. (Summerell et al. 2003). To confirm this identification, translation elongation factor (EF-1α) and RNA polymerase I beta subunit (RPB1) and RNA polymerase II beta subunit (RPB2) regions of the genome were PCR amplified from 3 separate isolates (R2, R4 and R5) using EF1/ EF2, RPB1-Fa/G2R, RPB2-5f2/7cR & RPB2-7cF/11aR primer pairs (O'Donnell et al. 2010) and sequenced. BLASTn comparison of the EF-1α (MT976167), RPB1 (MT967271) and RPB2 (MW233052) regions from isolate R4 showed 99% identity with the EF-1α (GU170620, 675/676 bp), RPB1 (KC808270, 1543/1545 bp) and RPB2 (MK4419902, 1637/1638 bp) sequences of Fusarium solani species complex (FSSC) in GenBank database. The same species level identification was also found using FUSARIUM-ID and FUSARIUM-MLDT databases. Two-year-old seedlings (n=3) of two different cultivars, 'Hunanzaoshu' and 'Huabai No. 1', growing in pots indoors at 25-27 °C were inoculated by drenching the soil with a conidial suspension of isolate R4 (40 mL, 106 conidia mL-1 obtained from 6-10 day old cultures). Control plants (n=3) were inoculated with sterilized water. At 20 days after inoculation (DAI) the leaves of inoculated plants became chlorotic and wilted, defoliated over time, and by 53 DAI 91.67% of plants died. The taproot and lateral roots of inoculated plants appeared brown to black in color and most lateral roots died and decomposed at 53 DAI, whereas the control plant roots remained healthy. All control plants remained symptomless. Based on morphological and molecular characters (TEF-1, RPB1 and RPB2), the re-isolated pathogen from diseased plants was identical to the R4 isolate used for inoculation and the disease assays were repeated thrice. FSSC was recently reported to cause fruit rot disease on loquat in Pakistan (Abbas et al. 2017). Identifying Fusarium solani species complex as a disease agent in Chinese loquat will assist in future development of improved germplasm for this important worldwide tree crop.

A WRKY Transcription Factor, EjWRKY17, from Eriobotrya japonica Enhances Drought Tolerance in Transgenic Arabidopsis.

The WRKY gene family, which is one of the largest transcription factor (TF) families, plays an important role in numerous aspects of plant growth and development, especially in various stress responses. However, the functional roles of the WRKY gene family in loquat are relatively unknown. In this study, a novel WRKY gene, EjWRKY17, was characterized from Eriobotrya japonica, which was significantly upregulated in leaves by melatonin treatment during drought stress. The EjWRKY17 protein, belonging to group II of the WRKY family, was localized in the nucleus. The results indicated that overexpression of EjWRKY17 increased cotyledon greening and root elongation in transgenic Arabidopsis lines under abscisic acid (ABA) treatment. Meanwhile, overexpression of EjWRKY17 led to enhanced drought tolerance in transgenic lines, which was supported by the lower water loss, limited electrolyte leakage, and lower levels of reactive oxygen species (ROS) and malondialdehyde (MDA). Further investigations showed that overexpression of EjWRKY17 promoted ABA-mediated stomatal closure and remarkably up-regulated ABA biosynthesis and stress-related gene expression in transgenic lines under drought stress. Overall, our findings reveal that EjWRKY17 possibly acts as a positive regulator in ABA-regulated drought tolerance.

Ectopic expression of an Eriobotrya japonica APETALA3 ortholog rescues the petal and stamen identities in Arabidopsis ap3-3 mutant.

APETALA3: (AP3) encodes a floral homeotic class B-function MADS-box protein and plays crucial roles in petal and stamen development. To better understand the functional roles of AP3 orthologs in Eriobotrya, we isolated and identified an AP3 ortholog, referred to as EjAP3, from Eriobotrya japonica. Analyses of protein sequence and phylogenetic tree showed that the EjAP3 was assigned to the rosids euAP3 lineage and included a distinctive PI-derived and euAP3 motifs at the C-terminal domain. Subcellular localization of EjAP3 was determined to be in the nucleus. Expression analysis suggested that EjAP3 expression was restricted only in petals and stamens, but not in sepals and carpels. Importantly, during the floral development, EjAP3 expression level was the highest at the stage of visible floral bud. Furthermore, ectopic expression of EjAP3 in Arabidopsis ap3-3 mutant rescued the second whorl petals and the third whorl stamens. The expression pattern and function characterization of EjAP3 contribute to better understand the roles of AP3 orthologs in Eriobotrya.

Homeotic transformation from stamen to petal in Eriobotrya japonica is associated with hormone signal transduction and reduction of the transcriptional activity of EjAG.

Double-flower loquat (Eriobotrya japonica) is a new germplasm with homeotic transformation of stamen into petal in whorl 3. However, little information is available on the molecular mechanism of this transformation. Herein, we analyzed the transcriptome, candidate genes and endogenous hormones to investigate the mechanisms underlying this homeotic transformation. Some transcription factors, such as MADS-box, TCP and MYB, were significantly differentially expressed. Importantly, we confirmed that one of these (DN39625_c0_g1), which encoded a C-class floral homeotic protein referred to as AGAMOUS ortholog (EjAG), was significantly downregulated. Subcellular localization of EjAG was found to be in the nucleus. Ectopic expression of EjAG rescued the development of stamens and carpels from the double-flower phenotype in an Arabidopsis ag mutant, suggesting that EjAG expression is associated with double-flower formation. Meanwhile, enrichment analyses showed that the differentially expressed genes (DEGs) were mainly involved in the metabolic pathways of hormone signal transduction. The DEGs of auxin, gibberellin A (GA) and cytokinin signaling pathways were mainly upregulated. However, the DEGs of abscisic acid (ABA) and the ethylene signaling pathway were mainly downregulated. Accordingly, the concentrations of indoleacetic acid, kinetin and GA3 were high at the petaloid stamen stage, but the ABA concentration remained low. The identified genes and pathways provide abundant sequence resources for studying the mechanisms underlying the homeotic transformation in loquat and other Rosaceae species.

An Integrative Analysis of Transcriptome, Proteome and Hormones Reveals Key Differentially Expressed Genes and Metabolic Pathways Involved in Flower Development in Loquat.

Flower development is a vital developmental process in the life cycle of woody perennials, especially fruit trees. Herein, we used transcriptomic, proteomic, and hormone analyses to investigate the key candidate genes/proteins in loquat (Eriobotrya japonica) at the stages of flower bud differentiation (FBD), floral bud elongation (FBE), and floral anthesis (FA). Comparative transcriptome analysis showed that differentially expressed genes (DEGs) were mainly enriched in metabolic pathways of hormone signal transduction and starch and sucrose metabolism. Importantly, the DEGs of hormone signal transduction were significantly involved in the signaling pathways of auxin, gibberellins (GAs), cytokinin, ethylene, abscisic acid (ABA), jasmonic acid, and salicylic acid. Meanwhile, key floral integrator genes FLOWERING LOCUS T (FT) and SUPPRESSOR OF OVEREXPRESSION OF CONSTANS1 (SOC1) and floral meristem identity genes SQUAMOSA PROMOTER BINDING LIKE (SPL), LEAFY (LFY), APETALA1 (AP1), and AP2 were significantly upregulated at the FBD stage. However, key floral organ identity genes AGAMOUS (AG), AP3, and PISTILLATA (PI) were significantly upregulated at the stages of FBE and FA. Furthermore, transcription factors (TFs) such as bHLH (basic helix-loop-helix), NAC (no apical meristem (NAM), Arabidopsis transcription activation factor (ATAF1/2) and cup-shaped cotyledon (CUC2)), MYB_related (myeloblastosis_related), ERF (ethylene response factor), and C2H2 (cysteine-2/histidine-2) were also significantly differentially expressed. Accordingly, comparative proteomic analysis of differentially accumulated proteins (DAPs) and combined enrichment of DEGs and DAPs showed that starch and sucrose metabolism was also significantly enriched. Concentrations of GA3 and zeatin were high before the FA stage, but ABA concentration remained high at the FA stage. Our results provide abundant sequence resources for clarifying the underlying mechanisms of the flower development in loquat.

EjFRI, FRIGIDA (FRI) Ortholog from Eriobotrya japonica, Delays Flowering in Arabidopsis.

In the model species Arabidopsis thaliana, FRIGIDA (FRI) is a key regulator of flowering time and can inhibit flowering without vernalization. However, little information is available on the function in the Rosaceae family. Loquat (Eriobotrya japonica) belongs to the family Rosaceae and is a distinctive species, in which flowering can be induced without vernalization, followed by blooming in late-autumn or winter. To investigate the functional roles of FRI orthologs in this non-vernalization species, we isolated an FRI ortholog, dubbed as EjFRI, from loquat. Analyses of the phylogenetic tree and protein sequence alignment showed that EjFRI is assigned to eurosids I FRI lineage. Expression analysis revealed that the highest expression level of EjFRI was after flower initiation. Meanwhile, EjFRI was widely expressed in different tissues. Subcellular localization of EjFRI was only detected to be in the nucleus. Ectopic expression of EjFRI in wild-type Arabidopsis delayed flowering time. The expression levels of EjFRI in transgenic wild-type Arabidopsis were significantly higher than those of nontransgenic wild-type lines. However, the expression levels of AtFRI showed no significant difference between transgenic and nontransgenic wild-type lines. Furthermore, the upregulated AtFLC expression in the transgenic lines indicated that EjFRI functioned similarly to the AtFRI of the model plant Arabidopsis. Our study provides a foundation to further explore the characterization of EjFRI, and also contributes to illuminating the molecular mechanism about flowering in loquat.

Molecular cytogenetic analysis of genome-specific repetitive elements in Citrus clementina Hort. Ex Tan. and its taxonomic implications.

BACKGROUND: Clementine mandarin (Citrus clementina Hort. ex Tan.) is one of the most famous and widely grown citrus cultivars worldwide. Variations in relation to the composition and distribution of repetitive DNA sequences that dominate greatly in eukaryote genomes are considered to be species-, genome-, or even chromosome-specific. Repetitive DNA-based fluorescence in situ hybridization (FISH) is a powerful tool for molecular cytogenetic study. However, to date few studies have involved in the repetitive elements and cytogenetic karyotype of Clementine. RESULTS: A graph-based similarity sequence read clustering methodology was performed to analyze the repetitive DNA families in the Clementine genome. The bioinformatics analysis showed that repetitive DNAs constitute 41.95% of the Clementine genome, and the majority of repetitive elements are retrotransposons and satellite DNAs. Sequential multicolor FISH using a probe mix that contained CL17, four satellite DNAs, two rDNAs and an oligonucleotide of (TTTAGGG)3 was performed with Clementine somatic metaphase chromosomes. An integrated karyotype of Clementine was established based on unequivocal and reproducible chromosome discriminations. The distribution patterns of these probes in several Citrus, Poncirus and Fortunella species were summarized through extensive FISH analyses. Polymorphism and heterozygosity were commonly observed in the three genera. Some asymmetrical FISH loci in Clementine were in agreement with its hybrid origin. CONCLUSIONS: The composition and abundance of repetitive elements in the Clementine genome were reanalyzed. Multicolor FISH-based karyotyping provided direct visual proof of the heterozygous nature of Clementine chromosomes with conspicuous asymmetrical FISH hybridization signals. We detected some similar and variable distribution patterns of repetitive DNAs in Citrus, Poncirus, and Fortunella, which revealed notable conservation among these genera, as well as obvious polymorphism and heterozygosity, indicating the potential utility of these repetitive element markers for the study of taxonomic, phylogenetic and evolutionary relationships in the future.

Cis-regulated additively expressed genes play a fundamental role in the formation of triploid loquat (Eriobotrya japonica (Thunb.) Lindl.) Heterosis.

Triploid loquat (Eriobotrya japonica (Thunb.) Lindl.) has greater vigor than their respective diploid and tetraploid parents, but the molecular basis of this triploid heterosis remains unclear. Recent studies have suggested that DNA methylation is involved in heterosis, which is a recognized method of suppressing gene expression. However, our previous studies revealed a trend of increased DNA methylation in triploid loquat hybrids compared to their parents. To elucidate the mechanism of triploid loquat heterosis, we investigated the levels and regulation of relative gene expression between hybrid and parental lines using RNA-Seq technology. We found that gene expression in the hybrid lines was down-regulated and gene expression analysis revealed that approximately 94.56 and 86.97% were expressed additively in triploid-A and triploid-B, respectively. Analyses of the allele-specific gene expression in the hybrids revealed significantly more Longquan-1 alleles were preferentially expressed in the two hybrid lines. Further analysis of cis- and trans-regulatory effects showed that gene expression variation between parental alleles is largely attributable to cis-regulatory variation in triploid loquat and analyses of genes belonging to cis-regulatory variation showed that 88-90% of cis genes contributed to an additive expression pattern. Taken together, our results suggest that gene expression variation in triploid loquat fundamentally cis-regulated may play a dominant role in triploid loquat heterosis.

Extensive genetic and DNA methylation variation contribute to heterosis in triploid loquat hybrids.

We aim to overcome the unclear origin of the loquat and elucidate the heterosis mechanism of the triploid loquat. Here we investigated the genetic and epigenetic variations between the triploid plant and its parental lines using amplified fragment length polymorphism (AFLP) and methylation-sensitive amplified fragment length polymorphism (MSAP) analyses. We show that in addition to genetic variations, extensive DNA methylation variation occurred during the formation process of triploid loquat, with the triploid hybrid having increased DNA methylation compared to the parents. Furthermore, a correlation existed between genetic variation and DNA methylation remodeling, suggesting that genome instability may lead to DNA methylation variation or vice versa. Sequence analysis of the MSAP bands revealed that over 53% of them overlap with protein-coding genes, which may indicate a functional role of the differential DNA methylation in gene regulation and hence heterosis phenotypes. Consistent with this, the genetic and epigenetic alterations were associated closely to the heterosis phenotypes of triploid loquat, and this association varied for different traits. Our results suggested that the formation of triploid is accompanied by extensive genetic and DNA methylation variation, and these changes contribute to the heterosis phenotypes of the triploid loquats from the two cross lines.

Genetic enhancement of oil content in potato tuber (Solanum tuberosum L.) through an integrated metabolic engineering strategy.

Potato tuber is a high yielding food crop known for its high levels of starch accumulation but only negligible levels of triacylglycerol (TAG). In this study, we evaluated the potential for lipid production in potato tubers by simultaneously introducing three transgenes, including WRINKLED 1 (WRI1), DIACYLGLYCEROL ACYLTRANSFERASE 1 (DGAT1) and OLEOSIN under the transcriptional control of tuber-specific (patatin) and constitutive (CaMV-35S) promoters. This coordinated metabolic engineering approach resulted in over a 100-fold increase in TAG accumulation to levels up to 3.3% of tuber dry weight (DW). Phospholipids and galactolipids were also found to be significantly increased in the potato tuber. The increase of lipids in these transgenic tubers was accompanied by a significant reduction in starch content and an increase in soluble sugars. Microscopic examination revealed that starch granules in the transgenic tubers had more irregular shapes and surface indentations when compared with the relatively smooth surfaces of wild-type starch granules. Ultrastructural examination of lipid droplets showed their close proximity to endoplasmic reticulum and mitochondria, which may indicate a dynamic interaction with these organelles during the processes of lipid biosynthesis and turnover. Increases in lipid levels were also observed in the transgenic potato leaves, likely due to the constitutive expression of DGAT1 and incomplete tuber specificity of the patatin promoter. This study represents an important proof-of-concept demonstration of oil increase in tubers and provides a model system to further study carbon reallocation during development of nonphotosynthetic underground storage organs.

RNA Silencing in Plants: Mechanisms, Technologies and Applications in Horticultural Crops.

Understanding the fundamental nature of a molecular process or a biological pathway is often a catalyst for the development of new technologies in biology. Indeed, studies from late 1990s to early 2000s have uncovered multiple overlapping but functionally distinct RNA silencing pathways in plants, including the posttranscriptional microRNA and small interfering RNA pathways and the transcriptional RNA-directed DNA methylation pathway. These findings have in turn been exploited for developing artificial RNA silencing technologies such as hairpin RNA, artificial microRNA, intrinsic direct repeat, 3' UTR inverted repeat, artificial trans-acting siRNA, and virus-induced gene silencing technologies. Some of these RNA silencing technologies, such as the hairpin RNA technology, have already been widely used for genetic improvement of crop plants in agriculture. For horticultural plants, RNA silencing technologies have been used to increase disease and pest resistance, alter plant architecture and flowering time, improve commercial traits of fruits and flowers, enhance nutritional values, remove toxic compounds and allergens, and develop high-value industrial products. In this article we aim to provide an overview of the RNA silencing pathways in plants, summarize the existing RNA silencing technologies, and review the current progress in applying these technologies for the improvement of agricultural crops particularly horticultural crops.

Genome-wide identification of R2R3-MYB family in Eriobotrya japonica and functional analysis of EjMYB5 involved in proanthocyanidin biosynthesis.

Loquat (Eriobotrya japonica Lindl.) is a popular fruit and medicinal plant. Proanthocyanidins (PAs), as one of the main types of flavonoids, are the key components of loquat fruit quality and medicinal properties. However, the identification of transcription factors (TFs) involved in PA accumulation in loquat remains limited. R2R3-MYB TFs play key regulatory role in PA accumulation in plants. In this study, 190 R2R3-MYB TFs were identified in loquat genome. Combined with transcriptome data, R2R3-MYB TF EjMYB5 involved in PA accumulation in loquat was isolated. EjMYB5 was transcriptional activator localized to nucleus. Expression of EjMYB5 was closely related to PA accumulation in loquat fruits. Heterogenous overexpression of EjMYB5 in tomato (Solanum lycopersicum) inhibited anthocyanin accumulation and promoted PA accumulation. Additionally, transient overexpression of EjMYB5 in tobacco (Nicotiana benthamiana) leaves promoted PA accumulation by upregulating flavonoid biosynthesis genes (NtDFR, NtANS, and NtLAR). Transcriptome analysis of EjMYB5-overexpressing tomato fruits suggested that EjMYB5 was involved in several biological pathways, including lipid metabolism, MAPK signaling, phenylpropanoid biosynthesis, and flavonoid biosynthesis. Collectively, our findings provided basic data for further analysis the function of R2R3-MYB TFs in loquat, and revealed that EjMYB5 functioned as PA accumulation in loquat.