Transcriptome differential expression analysis of defoliation of two different lemon varieties.

'Allen Eureka' is a bud variety of Eureka lemon with excellent fruiting traits. However, it suffers from severe winter defoliation that leads to a large loss of organic nutrients and seriously affects the tree's growth and development as well as the yield of the following year, and the mechanism of its response to defoliation is still unclear. In order to investigate the molecular regulatory mechanisms of different leaf abscission periods in lemon, two lemon cultivars ('Allen Eureka' and 'Yunning No. 1') with different defoliation traits were used as materials. The petiole abscission zone (AZ) was collected at three different defoliation stages, namely, the pre-defoliation stage (CQ), the mid-defoliation stage (CZ), and the post-defoliation stage (CH). Transcriptome sequencing was performed to analyze the gene expression differences between these two cultivars. A total of 898, 4,856, and 3,126 differentially expressed genes (DEGs) were obtained in CQ, CZ, and CH, respectively, and the number of DEGs in CZ was the largest. GO analysis revealed that the DEGs between the two cultivars were mainly enriched in processes related to oxidoreductase, hydrolase, DNA binding transcription factor, and transcription regulator activity in the defoliation stages. KEGG analysis showed that the DEGs were concentrated in CZ and involved plant hormone signal transduction, phenylpropanoid biosynthesis, glutathione metabolism, and alpha-linolenic acid metabolism. The expression trends of some DEGs suggested their roles in regulating defoliation in lemon. Eight gene families were obtained by combining DEG clustering analysis and weighted gene co-expression network analysis (WGCNA), including ß-glucosidase, AUX/IAA, SAUR, GH3, POD, and WRKY, suggesting that these genes may be involved in the regulation of lemon leaf abscission. The above conclusions enrich the research related to lemon leaf abscission and provide reliable data for the screening of lemon defoliation candidate genes and analysis of defoliation pathways.

Response of carbon fixation, allocation, and growth to source-sink manipulation by defoliation in vegetative citrus trees.

Source-sink balance in plants determines carbon distribution, and altering it can impact carbon fixation, transport, and allocation. We aimed to investigate the effect of altered source-sink ratios on carbon fixation, transport, and distribution in 'Valencia' sweet orange (Citrus x sinensis) by various defoliation treatments (0%, 33%, 66%, and 83% leaf removal). Gas exchange parameters were measured on 0 and 10 days after defoliation using A/Ci response curves, and leaf export was measured two days after defoliation using radioisotope tracer techniques. Greater defoliation increased the maximum rate of carboxylation (Vcmax), electron transport rate (J1200), and triose-phosphate utilization rate (TPU). Leaf export was unaffected by defoliation but increased in leaves closer to the shoot apex. Basipetal translocation velocity in the trunk remained unaltered, indicating that more photosynthates remained in the shoot rather than being transported directly to the root sink. Defoliated plants initiated more new flush shoots but accumulated less shoot biomass per plant after 8 weeks. Carbon allocation to fine roots was smaller in defoliated plants, suggesting defoliation led to retention of carbohydrates in aboveground organs such as the trunk and other shoots from previous growing cycles. In conclusion, the low source-sink ratio increased carbon fixation without impacting individual leaf export in citrus. The results suggest that intermediate sinks such as the aboveground perennial organs play a role in mediating the translocation velocity. Further research is necessary to better understand the dynamics of source-sink regulation in citrus trees.

Intercropping fruit trees in tea plantation improves soil properties and the formation of tea quality components.

Intercropping has been recommended as a beneficial cropping practice for improving soil characteristic and tea quality. However, there is limited research on the effects of intercropping fruit trees on soil chemical properties, soil aggregate structure, and tea quality components. In this study, intercropping fruit trees, specifically loquats and citrus, had a significant impact on the total available nutrients, AMN, and AP in soil. During spring and autumn seasons, the soil large-macroaggregates (>2 mm) proportion increased by 5.93% and 19.03%, as well as 29.23% and 19.14%, respectively, when intercropping loquats and citrus. Similarly, intercropping waxberry resulted in a highest small-macroaggregates (0.25 mm-2 mm) proportion at 54.89% and 77.32%. Soil aggregate stability parameters of the R0.25, MWD, and GMD were generally considered better soil aggregate stability indicators, and significantly improved in intercropping systems. Intercropping waxberry with higher values for those aggregate stability parameters and lower D values, showed a better soil aggregate distribution, while intercropping loquats and citrus at higher levels of AMN and AP in different soil aggregate sizes. As the soil aggregate sizes increased, the AMN and AP contents gradually decreased. Furthermore, the enhanced levels of amino acids were observed under loquat, waxberry, and citrus intercropping in spring, which increased by 27.98%, 27.35%, and 26.21%, respectively. The contents of tea polyphenol and caffeine were lower under loquat and citrus intercropping in spring. These findings indicated that intercropping fruit trees, specifically loquat and citrus, have immense potential in promoting the green and sustainable development of tea plantations.

ACC SYNTHASE4 inhibits gibberellin biosynthesis and FLOWERING LOCUS T expression during citrus flowering.

Flowering is an essential process in fruit trees. Flower number and timing have a substantial impact on the yield and maturity of fruit. Ethylene and gibberellin (GA) play vital roles in flowering, but the mechanism of coordinated regulation of flowering in woody plants by GA and ethylene is still unclear. In this study, a lemon (Citrus limon L. Burm) 1-aminocyclopropane-1-carboxylic acid synthase gene (CiACS4) was overexpressed in Nicotiana tabacum and resulted in late flowering and increased flower number. Further transformation of citrus revealed that ethylene and starch content increased, and soluble sugar content decreased in 35S:CiACS4 lemon. Inhibition of CiACS4 in lemon resulted in effects opposite to that of 35S:CiACS4 in transgenic plants. Overexpression of the CiACS4-interacting protein ETHYLENE RESPONSE FACTOR3 (CiERF3) in N. tabacum resulted in delayed flowering and more flowers. Further experiments revealed that the CiACS4-CiERF3 complex can bind the promoters of FLOWERING LOCUS T (CiFT) and GOLDEN2-LIKE (CiFE) and suppress their expression. Moreover, overexpression of CiFE in N. tabacum led to early flowering and decreased flowers, and ethylene, starch, and soluble sugar contents were opposite to those in 35S:CiACS4 transgenic plants. Interestingly, CiFE also bound the promoter of CiFT. Additionally, GA3 and 1-aminocyclopropanecarboxylic acid (ACC) treatments delayed flowering in adult citrus, and treatment with GA and ethylene inhibitors increased flower number. ACC treatment also inhibited the expression of CiFT and CiFE. This study provides a theoretical basis for the application of ethylene to regulate flower number and mitigate the impacts of extreme weather on citrus yield due to delayed flowering.

Efficiency of exogenous zinc sulfate application reduced fruit drop and improved antioxidant activity of 'Kinnow' mandarin fruit / A eficiência da aplicação de sulfato de zinco exógeno reduziu a queda da fruta e melhorou a atividade antioxidante da tangerina 'Kinnow'

'Kinnow' mandarin (Citrus nobilis L.× Citrus deliciosa T.) is an important marketable fruit of the world. It is mainstay of citrus industry in Pakistan, having great export potential. But out of total production of the country only 10% of the produce meets the international quality standard for export. Pre-harvest fruit drop and poor fruit quality could be associated with various issues including the plant nutrition. Most of the farmers do not pay attention to the supply of micro nutrients which are already deficient in the soil. Furthermore, their mobility within plants is also a question. Zinc (Zn) is amongst those micronutrients which affect the quality and postharvest life of the fruit and its deficiency in Pakistani soils is already reported by many researchers. Therefore, this study was carried out to evaluate the influence of pre-harvest applications of zinc sulfate (ZnSO4; 0, 0.4%, 0.6% or 0.8%) on pre-harvest fruit drop, yield and fruit quality of ‘Kinnow’ mandarin at harvest. The treatments were applied during the month of October i.e. 4 months prior to harvest. The applied Zn sprays had significant effect on yield and quality of the "Kinnow" fruit. Amongst different foliar applications of ZnSO4applied four months before harvest, 0.6% ZnSO4 significantly reduced pre-harvest fruit drop (10.08%) as compared to untreated control trees (46.45%). Similarly, the maximum number of fruits harvested per tree (627), fruit weight (192.9 g), juice percentage (42.2%), total soluble solids (9.5 °Brix), ascorbic acid content (35.5 mg 100 g-¹) and sugar contents (17.4) were also found significantly higher with 0.6% ZnSO4 treatment as compared to rest of treatments and control. Foliar application of 0.6% ZnSO4 also significantly improved total antioxidants (TAO) and total phenolic contents (TPC) in fruit. In conclusion, foliar [...].

A tangerina 'Kinnow' (Citrus nobilis L. × Citrus deliciosa T.) é uma importante fruta comercializável do mundo. É o esteio da indústria cítrica no Paquistão, com grande potencial de exportação. Mas, da produção total do país, apenas 10% da produção atendem o padrão internacional de qualidade para exportação. A queda da fruta antes da colheita e a baixa qualidade da fruta podem estar associadas a vários problemas, incluindo a nutrição da planta. A maioria dos agricultores não se preocupa com o fornecimento de micronutrientes que já são deficientes no solo. Além disso, sua mobilidade dentro das plantas também é uma questão. O zinco (Zn) está entre os micronutrientes que afetam a qualidade e a vida pós-colheita da fruta, e sua deficiência em solos paquistaneses já é relatada por diversos pesquisadores. Portanto, este estudo foi realizado para avaliar a influência da aplicação pré-colheita de sulfato de zinco (ZnSO4; 0, 0,4%, 0,6% ou 0,8%) na queda dos frutos na pré-colheita, produtividade e qualidade dos frutos da tangerina 'Kinnow' em colheita. Os tratamentos foram aplicados durante o mês de outubro, ou seja, 4 meses antes da colheita. As pulverizações de Zn aplicadas tiveram efeito significativo no rendimento e na qualidade da fruta 'Kinnow'. Entre as diferentes aplicações foliares de ZnSO4 efetuadas quatro meses antes da colheita, 0,6% de ZnSO4 reduziu significativamente a queda de frutos antes da colheita (10,08%) em comparação com as árvores de controle não tratadas (46,45%). Da mesma forma, número máximo de frutos colhidos por árvore (627), peso do fruto (192,9 g), porcentagem de suco (42,2%), sólidos solúveis totais (9,5 ° Brix), teor de ácido ascórbico (35,5 mg / 100 g-¹) e os teores de açúcar (17,4) também foram significativamente maiores com o tratamento com 0,6% de ZnSO4 em comparação com o restante dos tratamentos e o controle. A aplicação foliar de 0,6% de ZnSO4 também melhorou significativamente os [...].

Liquid Polymer/Metallic Salt-Based Stretchable Strain Sensor to Evaluate Fruit Growth.

Conventional methods for measuring the various dimensions of a fruit vary from vernier calipers to machine vision systems. This accounts for system bulkiness, high installation charges, and miscellaneous difficulties in continuous and precise monitoring. Considering the limitations, this paper reveals an inventive liquid-state stretchable strain sensor by incorporating poly(ethylene glycol) (PEG) and silver nitrate into an indigenous transparent polymer band. The combination of poly(dimethylsiloxane) (PDMS) and Ecoflex having an optimal mixing ratio (20:80) realized the equilibrium between a large strain, low stress, and less stickiness. The inclusion of a liquid polymer promoted high viscosity and chemical stability, while the addition of a metallic salt enhanced the electrical conductivity of the sensor. The correlation between strain and resistance showed high sensitivity and good repeatability of the PEG-silver nitrate composite. Linear resistance changes were noted with high coefficients of determination (R2 > 0.99) at least up to the strain of 30%. The performance test as a dendrometer on fruits of two different species demonstrated excellent stability of the sensor with increasing ratios from 1.7 to 3.9 kΩ/mm. This tunable elastic band sensor opened up a route toward long-term evaluation-targeted versatile applications such as fruit growth monitoring.

Evaluation on the phenotypic diversity of Calamansi (Citrus microcarpa) germplasm in Hainan island.

Calamansi or Philippine lime (Citrofortunella macrocarpa) is an important crop for local economic in Hainan Island. There is no study about Calamansi germplasm evaluation and cultivar development. In this study, Calamansi data were collected from 151 of Calamansi seedling trees, and 37 phenotypic traits were analyzed to investigate their genetic diversities. The cluster analysis and principal component analysis were conducted aiming to provide a theoretical basis for the Calamansi genetic improvement. The results of the diversity analysis revealed: (1) the diversity indexes for qualitative traits were ranged from 0.46-1.39, and the traits with the highest genetic diversity level were fruit shaped and pulp colored (H' > 1.20); and the diversity indexes for quantitative traits ranged from 0.67-2.10, with the exception of a lower in fruit juice rate (1.08) and lower in number of petals (0.67). (2) The clustering analysis of phenotypic traits have arranged the samples into 4 categories: the first group characterized by fewer flesh Segment number per fruit (SNF) and more Oil cell number (OCN); the second group had 7 samples, all characterized with larger Crown breadth (CB), higher Yield per tree (YPT), the lager leaf, the higher Ascorbic acid (AA), and less Seed number per fruit (SNPF); the third group had 25 samples characterized by smaller Tree foot diameter (TFD),smaller Fruit shape index (FSI) and higher Total soluble solids (TSS) contain; the fourth group had 87 samples, they were characterized by shorter Petiole length (PEL), larger fruit, higher Juice ratio (JR), multiple Stamen number (SN) and longer Pistil length (PIL). (3) The principal component analysis showed the values of the first 9 major components characteristic vectors were all greater than 3, the cumulative contribution rate reach 72.20%, including the traits of single fruit weight, fruit diameter, tree height, tree canopy width etc. Finally, based on the comprehensive main component value of all samples, the Calamansi individuals with higher testing scores were selected for further observation. This study concludes that Calamansi seedling populations in the Hainan Island holds great genetic diversity in varies traits, and can be useful for the Calamansi variety improvements.

Metabolomic analysis elucidates how shade conditions ameliorate the deleterious effects of greening (Huanglongbing) disease in citrus.

Under tropical and subtropical environments, citrus leaves are exposed to excess sunlight, inducing photoinhibition. Huanglongbing (HLB, citrus greening), a devastating phloem-limited disease putatively caused by Candidatus Liberibacter asiaticus, exacerbates this challenge with additional photosynthetic loss and excessive starch accumulation. A combined metabolomics and physiological approach was used to elucidate whether shade alleviates the deleterious effects of HLB in field-grown citrus trees, and to understand the underlying metabolic mechanisms related to shade-induced morpho-physiological changes in citrus. Using metabolite profiling and multinomial logistic regression, we identified pivotal metabolites altered in response to shade. A core metabolic network associated with shade conditions was identified through pathway enrichment analysis and metabolite mapping. We measured physio-biochemical responses and growth and yield characteristics. With these, the relationships between metabolic network and the variables measured above were investigated. We found that moderate-shade alleviates sink limitation by preventing excessive starch accumulation and increasing foliar sucrose levels. Increased growth and fruit yield in shaded compared with non-shaded trees were associated with increased photosystem II efficiency and leaf carbon fixation pathway metabolites. Our study also shows that, in HLB-affected trees under shade, the signaling of plant hormones (auxins and cytokinins) and nitrogen supply were downregulated with reducing new shoot production likely due to diminished needs of cell damage repair and tissue regeneration under shade. Overall, our findings provide the first glimpse of the complex dynamics between cellular metabolites and leaf physiological functions in citrus HLB pathosystem under shade, and reveal the mechanistic basis of how shade ameliorates HLB disease.

MicroRNAs and Transcripts Associated with an Early Ripening Mutant of Pomelo (Citrus grandis Osbeck).

'Liuyuezaoyou' is an early-ripening cultivar selected from a bud mutation of Citrus grandis Osbeck 'Guanximiyou'. They were designated here as MT and WT, respectively. The fruit of MT matures about 45 days earlier than WT, which was accompanied by significant changes in key phytohormones, sugar compounds and organic acids. Recent studies have showed that microRNAs (miRNAs) play an important role in regulation of fruit ripening process. The aim of this study was to compare MT fruits with WT ones to uncover if miRNAs were implicated in the ripening of C. grandis. Fruits of both WT and MT at four developmental stages were analyzed using high-throughput sequencing and RT-PCR. Several independent miRNA libraries were constructed and sequenced. A total of 747 known miRNAs were identified and 99 novel miRNAs were predicted across all libraries. The novel miRNAs were found to have hairpin structures and possess star sequences. These results showed that transcriptome and miRNAs are substantially involved in a complex and comprehensive network in regulation of fruit ripening of this species. Further analysis of the network model revealed intricate interactions of miRNAs with mRNAs during the fleshy fruit ripening process. Several identified miRNAs have potential targets. These include auxin-responsive protein IAA9, sucrose synthase 3, V-type proton ATPase, NCED1 (ABA biosynthesis) and PL1/5 (pectate lyase genes), as well as NAC100 putative coordinated regulation networks, whose interactions with respective miRNAs may contribute significantly to fruit ripening of C. grandis.

The Citrus yellow mosaic badnavirus ORFI functions as a RNA-silencing suppressor.

Citrus yellow mosaic badnavirus (CMBV) causes mosaic disease in all economically important citrus cultivars of India, with losses reaching up to 70%. CMBV belongs to the genus Badnavirus, family Caulimoviridae, possessing a circular double-stranded (ds) DNA genome with six open reading frames (ORFs I to VI), whose functions are yet to be deciphered. The RNA-silencing suppressor (RSS) activity has not been assigned to any CMBV ORF as yet. In the present study, it was found that ORFI exhibited RSS activity among all the six CMBV ORFs tested. Studies were done by employing the well-established Agrobacterium-mediated transient assay based on the transgenic Nicotiana benthamiana 16c plant line expressing the green fluorescent protein (GFP). The RSS activity of ORFI was confirmed by the analysis of the GFP visual expression in the agroinfiltrated leaves, further supported by quantification of GFP expression by RT-PCR. Based on the GFP visual expression, the CMBV ORFI was a weak RSS when compared to the p19 protein of tomato bushy stunt virus. In contrast, the ORFII, ORFIV, ORFV, ORFVI, and CP gene did not exhibit any RSS activity. Hence, ORFI is the first ORF of CMBV to be identified with RNA-silencing suppression activity.

Characterization of two closely related citrus cultivars using UPLC-ESI-MS/MS-based widely targeted metabolomics.

Citrus cultivars are widely spread worldwide, and some of them only differ by specific mutations along the genome. It is difficult to distinguish them by traditional morphological identification. To accurately identify such similar cultivars, the subtle differences between them must be detected. In this study, UPLC-ESI-MS/MS-based widely targeted metabolomics analysis was conducted to study the chemical differences between two closely related citrus cultivars, Citrus reticulata 'DHP' and C. reticulata 'BZH'. Totally 352 metabolites including 11 terpenoids, 35 alkaloids, 80 phenolic acids, 25 coumarins, 7 lignans, 184 flavonoids and 10 other compounds were detected and identified; Among them, 15 metabolites are unique to DHP and 16 metabolites are unique to BZH. Hierarchical cluster analysis (HCA), principal component analysis (PCA), and orthogonal signal correction and partial least squares-discriminant analysis (OPLS-DA) can be used to clearly discriminate between DHP and BZH. 93 metabolites including 36 down-regulated and 57 up-regulated are significantly different in DHP and BZH. They are mainly involved in the biosynthesis of flavonoids, flavones, flavonols, and isoflavonoids. In addition, the relative content levels of flavonoids, alkaloids, and terpenoids are much higher in the peel of DHP than that of BZH, the presence of which may correlate with the quality difference of the peels. The results reported herein indicate that metabolite analysis based on UPLC-ESI-MS/MS is an effective means of identifying cultivars with different genotypes, especially those that cannot be distinguished based on traditional identification methods.

A MADS-Box Gene CiMADS43 Is Involved in Citrus Flowering and Leaf Development through Interaction with CiAGL9.

MADS-box genes are involved in various developmental processes including vegetative development, flower architecture, flowering, pollen formation, seed and fruit development. However, the function of most MADS-box genes and their regulation mechanism are still unclear in woody plants compared with model plants. In this study, a MADS-box gene (CiMADS43) was identified in citrus. Phylogenetic and sequence analysis showed that CiMADS43 is a GOA-like Bsister MADS-box gene. It was localized in the nucleus and as a transcriptional activator. Overexpression of CiMADS43 promoted early flowering and leaves curling in transgenic Arabidopsis. Besides, overexpression or knockout of CiMADS43 also showed leaf curl phenotype in citrus similar to that of CiMADS43 overexpressed in Arabidopsis. Protein-protein interaction found that a SEPALLATA (SEP)-like protein (CiAGL9) interacted with CiMADS43 protein. Interestingly, CiAGL9 also can bind to the CiMADS43 promoter and promote its transcription. Expression analysis also showed that these two genes were closely related to seasonal flowering and the development of the leaf in citrus. Our findings revealed the multifunctional roles of CiMADS43 in the vegetative and reproductive development of citrus. These results will facilitate our understanding of the evolution and molecular mechanisms of MADS-box genes in citrus.

The transcriptome landscapes of citrus leaf in different developmental stages.

KEY MESSAGE: The temporal expression profiles of citrus leaves explain the sink-source transition of immature leaves to mature leaves and provide knowledge regarding the differential responses of mature and immature leaves to biotic stress such as citrus canker and Asian citrus psyllid (Diaphorina citri). Citrus is an important fruit crop worldwide. Different developmental stages of citrus leaves are associated with distinct features, such as differences in susceptibilities to pathogens and insects, as well as photosynthetic capacity. Here, we investigated the mechanisms underlying these distinctions by comparing the gene expression profiles of mature and immature citrus leaves. Immature (stages V3 and V4), transition (stage V5), and mature (stage V6) Citrus sinensis leaves were chosen for RNA-seq analyses. Carbohydrate biosynthesis, photosynthesis, starch biosynthesis, and disaccharide metabolic processes were enriched among the upregulated differentially expressed genes (DEGs) in the V5 and V6 stages compared with that in the V3 and V4 stages. Glucose level was found to be higher in V5 and V6 than in V3 and V4. Among the four stages, the largest number of DEGs between contiguous stages were identified between V5 and V4, consistent with a change from sink to source, as well as with the sucrose and starch quantification data. The differential expression profiles related to cell wall synthesis, secondary metabolites such as flavonoids and terpenoids, amino acid biosynthesis, and immunity between immature and mature leaves may contribute to their different responses to Asian citrus psyllid infestation. The expression data suggested that both the constitutive and induced gene expression of immunity-related genes plays important roles in the greater resistance of mature leaves against Xanthomonas citri compared with immature leaves. The gene expression profiles in the different stages can help identify stage-specific promoters for the manipulation of the expression of citrus traits according to the stage. The temporal expression profiles explain the sink-source transition of immature leaves to mature leaves and provide knowledge regarding the differential responses to biotic stress.

CENTRORADIALIS maintains shoot meristem indeterminacy by antagonizing THORN IDENTITY1 in Citrus.

Differential regulation of stem cell activity in shoot meristems contributes to the wide variation in shoot architecture.1-3 In most Citrus species, a thorn meristem and a dormant axillary meristem co-localize at each leaf base, offset from each other in a spiral phyllotactic pattern. We recently identified THORN IDENTITY1 (TI1) and THORN IDENTITY2 (TI2), encoding TEOSINTE BRANCHED1/CYCLOIDEA/PCF (TCP) transcription factors, as necessary for the termination of meristem proliferation and concomitant thorn production in Citrus.4 However, how the dormant axillary meristem at the same leaf axil maintains stem cell activity is still unknown. The phosphatidylethanolamine-binding protein (PEBP)-type transcription factors CENTRORADIALIS (CEN) and TERMINAL FLOWER1 (TFL1) maintain inflorescence meristem indeterminacy in many plant species by antagonizing floral meristem identity regulators.5-9 Here, we show that, in Citrus, Citrus CEN (CsCEN) maintains vegetative axillary meristem indeterminacy by antagonizing TI1. CsCEN is expressed in the axillary meristem, but not in the thorn meristem. Disruption of CsCEN function results in termination of the stem cell activity and conversion of dormant axillary meristems into thorns, although ectopic overexpression of CsCEN represses TI1 expression and converts thorns into dormant buds, a phenotype similar to the ti1 mutant. We further show that CsCEN interacts with Citrus FD (CsFD) to repress TI1 expression. CsCEN activity depends on the function of TI1 and TI2, as mutations in TI1 and TI2 rescue the cscen mutant phenotype. We suggest that the antagonistic roles of CsCEN and TI1 define the pattern of axillary meristem determinacy, which shapes vegetative Citrus tree shoot architecture.

The endophytic microbiota of Citrus limon is transmitted from seed to shoot highlighting differences of bacterial and fungal community structures.

Citrus limon (L.) Burm. F. is an important evergreen fruit crop whose rhizosphere and phyllosphere microbiota  have been characterized, while seed microbiota is still unknown. Bacterial and fungal endophytes were isolated from C. limon surface-sterilized seeds. The isolated fungi-belonging to Aspergillus, Quambalaria and Bjerkandera genera-and bacteria-belonging to Staphylococcus genus-were characterized for indoleacetic acid production and phosphate solubilization. Next Generation Sequencing based approaches were then used to characterize the endophytic bacterial and fungal microbiota structures of surface-sterilized C. limon seeds and of shoots obtained under aseptic conditions from in vitro growing seedlings regenerated from surface-sterilized seeds. This analysis highlighted that Cutibacterium and Acinetobacter were the most abundant bacterial genera in both seeds and shoots, while Cladosporium and Debaryomyces were the most abundant fungal genera in seeds and shoots, respectively. The localization of bacterial endophytes in seed and shoot tissues was revealed by Fluorescence In Situ Hybridization coupled with Confocal Laser Scanning Microscopy revealing vascular bundle colonization. Thus, these results highlighted for the first time the structures of endophytic microbiota of C. limon seeds and the transmission to shoots, corroborating the idea of a vertical transmission of plant microbiota and suggesting its crucial role in seed germination and plant development.

Effects of phosphorus deficiency on the absorption of mineral nutrients, photosynthetic system performance and antioxidant metabolism in Citrus grandis.

Phosphorus (P) is an essential macronutrient for plant growth, development and production. However, little is known about the effects of P deficiency on nutrient absorption, photosynthetic apparatus performance and antioxidant metabolism in citrus. Seedlings of 'sour pummelo' (Citrus grandis) were irrigated with a nutrient solution containing 0.2 mM (Control) or 0 mM (P deficiency) KH2PO4 until saturated every other day for 16 weeks. P deficiency significantly decreased the dry weight (DW) of leaves and stems, and increased the root/shoot ratio in C. grandis but did not affect the DW of roots. The decreased DW of leaves and stems might be induced by the decreased chlorophyll (Chl) contents and CO2 assimilation in P deficient seedlings. P deficiency heterogeneously affected the nutrient contents of leaves, stems and roots. The analysis of Chl a fluorescence transients showed that P deficiency impaired electron transport from the donor side of photosystem II (PSII) to the end acceptor side of PSI, which showed a greater impact on the performance of the donor side of PSII than that of the acceptor side of PSII and photosystem I (PSI). P deficiency increased the contents of ascorbate (ASC), H2O2 and malondialdehyde (MDA) as well as the activities of superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX), dehydroascorbate reductase (DHAR) and glutathione reductase (GR) in leaves. In contrast, P deficiency increased the ASC content, reduced the glutathione (GSH) content and the activities of SOD, CAT, APX and monodehydroascorbate reductase (MDHAR), but did not increase H2O2 production, anthocyanins and MDA content in roots. Taking these results together, we conclude that P deficiency affects nutrient absorption and lowers photosynthetic performance, leading to ROS production, which might be a crucial cause of the inhibited growth of C. grandis.

High efficient de novo root-to-shoot organogenesis in Citrus jambhiri Lush.: Gene expression, genetic stability and virus indexing.

A protocol for high-frequency direct organogenesis from root explants of Kachai lemon (Citrus jambhiri Lush.) was developed. Full-length roots (~3 cm) were isolated from the in vitro grown seedlings and cultured on Murashige and Skoog basal medium supplemented with Nitsch vitamin (MSN) with different concentrations of cytokinin [6-benzylaminopurine, (BAP)] and gibberellic acid (GA3). The frequency of multiple shoot proliferation was very high, with an average of 34.3 shoots per root explant when inoculated on the MSN medium supplemented with BAP (1.0 mg L-1) and GA3 (1.0 mg L-1). Optimal rooting was induced in the plantlets under half strength MSN medium supplemented with indole-3-acetic acid (IAA, 0.5-1.0 mg L-1). IAA induced better root structure than 1-naphthaleneacetic acid (NAA), which was evident from the scanning electron microscopy (SEM). The expressions of growth regulating factor genes (GRF1 and GRF5) and GA3 signaling genes (GA2OX1 and KO1) were elevated in the regenerants obtained from MSN+BAP (1.0 mg L-1)+GA3 (1.0 mg L-1). The expressions of auxin regulating genes were high in roots obtained in ½ MSN+IAA 1.0 mg L-1. Furthermore, indexing of the regenerants confirmed that there was no amplicons detected for Huanglongbing bacterium and Citrus tristeza virus. Random amplified polymorphic DNA (RAPD) and inter simple sequence repeat (ISSR) markers detected no polymorphic bands amongst the regenerated plants. This is the first report that describes direct organogenesis from the root explant of Citrus jambhiri Lush. The high-frequency direct regeneration protocol in the present study provides an enormous significance in Citrus organogenesis, its commercial cultivation and genetic conservation.

Exogenous Application of ABA and NAA Alleviates the Delayed Coloring Caused by Puffing Inhibitor in Citrus Fruit.

Combined spraying of gibberellin (GA) and prohydrojasmon (PDJ) was an effective method to reduce peel puffing in Satsuma mandarins. However, in the GA-and-PDJ combined treatment, fruit color development was delayed during the ripening process. In the present study, to improve the coloration of the GA and PDJ-treated fruit, the effects of exogenous application of 1-naphthaleneacetic acid (NAA) and abscisic acid (ABA) on chlorophyll and carotenoid accumulation were investigated. The results showed that both ABA and NAA treatments accelerated the color changes from green to orange in the GA and PDJ-treated fruit during the ripening process. With the NAA and ABA treatments, chlorophylls contents were decreased rapidly, and the contents of ß,ß-xanthophylls were significantly enhanced in the GA and PDJ-treated fruit. In addition, gene expression results showed that the changes of the chlorophyll and carotenoid metabolisms in the NAA and ABA treatments were highly regulated at the transcriptional level. The results presented in this study suggested that the application of NAA and ABA could potentially be used for improving the coloration of the GA and PDJ-treated fruit.

Genomic insights into citrus domestication and its important agronomic traits.

Citrus originated in Southeast Asia, and it has become one of the most important fruit crops worldwide. Citrus has a long and obscure domestication history due to its clonal propagation, long life cycle, wide sexual compatibility, and complex genetic background. As the genomic information of both wild and cultivated citrus becomes available, their domestication history and underlying traits or genes are becoming clear. This review outlines the genomic features of wild and cultivated species. We propose that the reduction of citric acid is a critical trait for citrus domestication. The genetic model representing the change during domestication may be associated with a regulatory complex known as WD-repeat-MYB-bHLH-WRKY (WMBW), which is involved in acidification and anthocyanin accumulation. The reduction in or loss of anthocyanins may be due to a hitchhiking effect of fruit acidity selection, in which mutation occurs in the common regulator of these two pathways in some domesticated types. Moreover, we have summarized the domestication traits and candidate genes for breeding purposes. This review represents a comprehensive summary of the genes controlling key traits of interest, such as acidity, metabolism, and disease resistance. It also sheds light on recent advances in early flowering from transgenic studies and provides a new perspective for fast breeding of citrus. Our review lays a foundation for future research on fruit acidity, flavor, and disease resistance in citrus.

Natural variations of TFIIAγ gene and LOB1 promoter contribute to citrus canker disease resistance in Atalantia buxifolia.

Citrus canker caused by Xanthomonas citri subsp. citri (Xcc) is one of the most devastating diseases in citrus industry worldwide. Most citrus cultivars such as sweet orange are susceptible to canker disease. Here, we utilized wild citrus to identify canker-resistant germplasms, and found that Atalantia buxifolia, a primitive (distant-wild) citrus, exhibited remarkable resistance to canker disease. Although the susceptibility gene LATERAL ORGAN BOUNDARIES 1 (LOB1) could also be induced in Atalantia after canker infection, the induction extent was far lower than that in sweet orange. In addition, three of amino acids encoded by transcription factor TFIIAγ in Atalantia (AbTFIIAγ) exhibited difference from those in sweet orange (CsTFIIAγ) which could stabilize the interaction between effector PthA4 and effector binding element (EBE) of LOB1 promoter. The mutation of AbTFIIAγ did not change its interaction with transcription factor binding motifs (TFBs). However, the AbTFIIAγ could hardly support the LOB1 expression induced by the PthA4. In addition, the activity of AbLOB1 promoter was significantly lower than that of CsLOB1 under the induction by PthA4. Our results demonstrate that natural variations of AbTFIIAγ and effector binding element (EBE) in the AbLOB1 promoter are crucial for the canker disease resistance of Atalantia. The natural mutations of AbTFIIAγ gene and AbLOB1 promoter in Atalantia provide candidate targets for improving the resistance to citrus canker disease.