A transcriptional cascade involving BBX22 and HY5 finely regulates both plant height and fruit pigmentation in citrus.

Dwarfing is a pivotal agronomic trait affecting both yield and quality. Citrus species exhibit substantial variation in plant height, among which internode length is a core element. However, the molecular mechanism governing internode elongation remains unclear. Here, we unveiled that the transcriptional cascade consisting of B-BOX DOMAIN PROTEIN 22 (BBX22) and ELONGATED HYPOCOTYL 5 (HY5) finely tunes plant height and internode elongation in citrus. Loss-of-function mutations of BBX22 in an early-flowering citrus (Citrus hindsii "SJG") promoted internode elongation and reduced pigment accumulation, whereas ectopic expression of BBX22 in SJG, sweet orange (C. sinensis), pomelo (C. maxima) or heterologous expression of BBX22 in tomato (Solanum lycopersicum) significantly decreased internode length. Furthermore, exogenous application of gibberellin A3 (GA3) rescued the shortened internode and dwarf phenotype caused by BBX22 overexpression. Additional experiments revealed that BBX22 played a dual role in regulation internode elongation and pigmentation in citrus. On the one hand, it directly bound to and activated the expression of HY5, GA metabolism gene (GA2 OXIDASE 8, GA2ox8), carotenoid biosynthesis gene (PHYTOENE SYNTHASE 1, PSY1) and anthocyanin regulatory gene (Ruby1, a MYB DOMAIN PROTEIN). On the other hand, it acted as a cofactor of HY5, enhancing the ability of HY5 to regulate target genes expression. Together, our results reveal the critical role of the transcriptional cascade consisting of BBX22 and HY5 in controlling internode elongation and pigment accumulation in citrus. Unraveling the crosstalk regulatory mechanism between internode elongation and fruit pigmentation provides key genes for breeding of novel types with both dwarf and health-beneficial fortification in citrus.

Utilization of somatic fusion techniques for the development of HLB tolerant breeding resources employing the Australian finger lime (Citrus australasica).

The Australian finger lime is a unique citrus species that has gained importance due to its unique fruit characteristics and perceived tolerance to Huanglongbing (HLB), an often-fatal disease of citrus trees. In this study, we developed allotetraploid finger lime hybrids and cybrids by utilizing somatic cell fusion techniques to fuse diploid 'OLL8' sweet orange or 'Page' tangelo callus-derived protoplasts with finger lime (FL) mesophyll-derived protoplasts. Six somatic fusions were regenerated from the 'OLL8' + FL fusion, while three putative cybrids were regenerated from the 'Page' + FL fusion. Ploidy levels and nuclear-expressed sequence tag derived simple sequence repeat (EST-SSR) markers confirmed the somatic hybrid production, and mitochondrial DNA primer sets confirmed the cybrid nature. Several trees produced by the somatic fusion remained HLB negative even after 6 years of growth in an HLB-endemic environment. Pathogenesis related (PR) and other genes that are often upregulated in HLB-tolerant trees were also upregulated in our somatic fusions. These newly developed somatic fusions and cybrids could potentially be used as breeding parents to develop the next generation of improved HLB-tolerant rootstocks and scions.

Reprogramming of Stem Cell Activity to Convert Thorns into Branches.

Thorns arise from axillary shoot apical meristems that proliferate for a time and then terminally differentiate into a sharp tip. Like other meristems, thorn meristems contain stem cells but, in the case of thorns, these stem cells undergo a programmed cessation of proliferative activity. Using Citrus, we characterize a gene network necessary for thorn development. We identify two Citrus genes, THORN IDENTITY1 (TI1) and THORN IDENTITY2 (TI2), encoding TCP transcription factors, as necessary for stem cell quiescence and thorn identity. Disruption of TI1 and TI2 function results in reactivation of stem cells and concomitant conversion of thorns to branches. Expression of WUSCHEL (WUS) defines the shoot stem cell niche in the apical meristems of many angiosperm species; we show that TI1 binds to the Citrus WUS promoter and negatively regulates its expression to terminate stem cell proliferation. We propose that shifts in the timing and function of components of this gene network can account for the evolution of Citrus thorn identity. Modulating this pathway can significantly alter plant architecture and could be leveraged to improve crop yields.

Changes in Cuticle Components and Morphology of 'Satsuma' Mandarin (Citrus unshiu) during Ambient Storage and Their Potential Role on Penicillium digitatum Infection.

To elucidate the role of fruit cuticle in fungal infection, changes in cuticle composition and morphology of 'Satsuma' mandarin during ambient (at 25 °C) storage and their role in Penicillium digitatum infection were investigated. Results showed that the epicuticular wax yield increased from 1.11 µg cm-2 to 4.21 µg cm-2 during storage for 20 days and then decreased to 1.35 µg cm-2 as storage time prolonged to 40 days. Intracuticular wax content of fruits stored for 20 days showed a peak value that was 1.7-fold higher than that of fruits stored for 40 days. The contents of cutin monomers of fruits showed a decreased trend during storage, while their proportions in the cutin stayed stable. Acids were identified as the most abundant components in epicuticular wax independently of the storage time, followed by alkanes and terpenoids. Terpenoids were found as the predominant components in intracuticular wax during the whole storage, followed by alkanes and acids. The flattened platelets crystals of fruits at harvest changed into small granule-like wax ones after 10 days of storage then gradually distributed across the surface of the fruits as stored for 40 days. Results of in vitro tests showed that mycelial growth of Penicillium digitatum could be promoted by epicuticular wax and conidial germination could be inhibited by cutin at different storage stages. These results shed new light on the chemical basis for cuticle involvement in fungal infection.

Genetic, morphological and chemical investigations reveal the genetic origin of Pompia (C. medica tuberosa Risso & Poiteau) - An old endemic Sardinian citrus fruit.

Citrus fruits have been introduced to the Mediterranean area from Asia for centuries and spontaneous crosses have generated several hybrid forms, some of which have had agricultural or industrial success while others have remained niche food or ornamental products, or have disappeared. Pompia (C. medica tuberosa Risso & Poiteau) is an old endemic citrus fruit from Sardinia of unknown genetic origin. Initial phenotypic and molecular characterizations revealed a high degree of similarity with lemon (C. limon (L.) Burm.) and citron (C. medica L.). To identify the ancestors of Pompia, 70 citrus species of the Citrus genus were genotyped with 36 codominant molecular markers (SSR and InDel) of nuclear and cytoplasmic genomes. Diversity analysis and allelic comparisons between each citrus species at each locus indicated that Pompia resembles lemon and limonette of Marrakech, i.e. the result of a cross between sour orange (C. aurantium L.) and citron, where citron was the pollinator. Two Italian citron varieties were identified as potential male parents, i.e. Diamante and Common Poncire. However, we were unable to differentiate varieties of sour oranges because varietal diversification in this horticultural group resulted from DNA sequence variations that SSR or InDel markers could not reveal. Rhob el Arsa and Poncire de Collioure were found to be two synonyms of Pompia. Pompia appeared to be equally distinct from citron, lemon and sour orange based on the overall analysis of the fruit, leaf and seed phenotype, and juice chemical composition. At the leaf level, the Pompia essential oil (EO) composition is close to that of citron whereas the zest is much closer to that of sour orange.

Intelligent microscopic approach for identification and recognition of citrus deformities.

Plant diseases are accountable for economic losses in an agricultural country. The manual process of plant diseases diagnosis is a key challenge from last one decade; therefore, researchers in this area introduced automated systems. In this research work, automated system is proposed for citrus fruit diseases recognition using computer vision technique. The proposed method incorporates five fundamental steps such as preprocessing, disease segmentation, feature extraction and reduction, fusion, and classification. The noise is being removed followed by a contrast stretching procedure in the very first phase. Later, watershed method is applied to excerpt the infectious regions. The shape, texture, and color features are subsequently computed from these infection regions. In the fourth step, reduced features are fused using serial-based approach followed by a final step of classification using multiclass support vector machine. For dimensionality reduction, principal component analysis is utilized, which is a statistical procedure that enforces an orthogonal transformation on a set of observations. Three different image data sets (Citrus Image Gallery, Plant Village, and self-collected) are combined in this research to achieving a classification accuracy of 95.5%. From the stats, it is quite clear that our proposed method outperforms several existing methods with greater precision and accuracy.

Accelerating multi-slice spatiotemporally encoded MRI with simultaneous echo refocusing.

Single-shot spatiotemporally encoded (SPEN) ultrafast magnetic resonance imaging (MRI) is of great value to both scientific research and clinical application owing to its capability for delivering MR images with greater robustness to magnetic field inhomogeneity and chemical-shift displacement effects than conventional methods like EPI due to high effective phase-encoded bandwidth. Many SPEN MRI methods have been developed, among which multi-slice SPEN MRI arises as a promising supplement to ultrafast multi-slice sampling. In this work, we propose a new multi-slice SPEN MRI method, termed multi-echo segmented SPEN (ME-SeSPEN) method, which produces multiple images within a single train of echoes and successively samples widely separated slices. The resulting images were reconstructed using de-convolution super-resolved algorithm. The robustness and efficiency of the proposed method were demonstrated by phantom, lemon and in vivo experiments in comparison with spin-echo EPI, spin-echo simultaneous echo refocusing (SER), and segmented SPEN (SeSPEN) MRI. The results indicate that the new method effectively shortens the sampling time (20% reduction practically in comparison with SeSPEN when two slices are simultaneously sampled). ME-SeSPEN also reduces eddy current effects while maintaining the benefits of SPEN MRI, such as similar robustness to field inhomogeneity, spatial resolution and signal-to-noise ratio to SeSPEN MRI. The new method will promote the versatility of multi-slice MRI in practical applications.

Dissection of the Mechanism for Compatible and Incompatible Graft Combinations of Citrus grandis (L.) Osbeck ('Hongmian Miyou').

'Hongmian miyou' (Citrus grandis L. Osbeck) is mutated from 'Guanxi miyou', with a different spongy layer coloration. Trifoliate orange (Poncirus trifoliata) is widely used as rootstocks in 'Guanxi miyou' grafting, whereas 'Hongmian miyou' is incompatible with available trifoliate orange rootstocks. To explore the reasons for the etiolation of leaves of 'Hongmian miyou'/trifoliate orange, anatomical differences among different graft unions, gene expression profiles, and auxin levels of scion were investigated in this study. A histological assay indicated that there was no significant difference in anatomical structure between the compatible and incompatible combinations. A total of 1950 significant differentially-expressed genes (DEGs) were identified and analyzed. The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis revealed that genes involved in carbohydrate metabolism, energy metabolism, amino acid metabolism, and plant hormone signal transduction were significantly enriched. Moreover, the expression of nine genes in the auxin pathway were upregulated and three were downregulated in compatible combinations compared with those in the incompatible group. Further experiments verified that indole-3-acetic acid (IAA) content increases in the compatible graft combination, which suggests that IAA might promote graft compatibility.

Physiologic, Anatomic, and Gene Expression Changes in Citrus sunki, Poncirus trifoliata, and Their Hybrids After 'Candidatus Liberibacter asiaticus' Infection.

Huanglongbing (HLB) is a destructive disease of citrus caused by phloem-limited bacteria, namely 'Candidatus Liberibacter asiaticus' (Las), 'Candidatus Liberibacter africanus', and 'Candidatus Liberibacter americanus'. Although there are no known HLB-resistant citrus species, studies have reported Poncirus trifoliata as being more tolerant. Assuming that callose deposition in the phloem of infected plants can inhibit translocation of photosynthetic products and cause starch accumulation, we compared callose deposition in petioles and starch accumulation in infected leaves of three genotypes (Citrus sinensis, C. sunki, and P. trifoliata) and 15 hybrids (C. sunki × P. trifoliata). Compared with the mock-inoculated plants, higher bacterial counts and greater accumulation of callose and starch were found in C. sinensis, C. sunki, and 10 of the hybrid plants. Lower titer and fewer metabolic changes due to Las infection were observed in P. trifoliata and in two Las-positive hybrids while three hybrids were Las-negative. Callose accumulation was linked to and correlated with genes involved in phloem functionality and starch accumulation was linked to up-regulation of genes involved in starch biosynthesis and repression of those related to starch breakdown. Lower expression of genes involved in phloem functionality in resistant and tolerant plants can partially explain the absence of distinct disease symptoms associated with starch accumulation that are usually observed in HLB-susceptible genotypes.

Protective effects of peel and seed extracts of Citrus aurantium on glutamate-induced cytotoxicity in PC12 cell line.

Oxidative stress and apoptosis contribute to neuronal degeneration in many neurodegenerative diseases such as Alzheimer's disease. Glutamate is a major excitatory neurotransmitter in the central nervous system (CNS) and is considered responsible for the pathogenesis of many neurological disorders. Reactive oxygen species (ROS) production is thought to be involved in glutamate-induced apoptosis process. In this study, the neuroprotective effects of Citrus aurantium in the glutamate-induced rat's adrenal pheochromocytoma cell line (PC12 cells) were investigated. The cell viability and apoptotic cell death were measured using MTT and propidium iodine (PI)-staining methods, respectively. In addition, intracellular ROS and malondialdehyde (MDA) levels were determined by fluorometric methods. The results showed that glutamate cytotoxicity in PC12 cells was accompanied by an increment of MDA content, ROS generation, and apoptotic induction. However, pretreatment with peel and seed extracts of C. aurantium significantly reduced MDA content, ROS generation, and apoptotic cells. All these findings indicated that C. aurantium protected PC12 cells against glutamate-induced apoptosis by inhibiting ROS production. Therefore, the present study supports that C. aurantium extracts possess neuroprotective effects against glutamate-induced toxicity in PC12 cell line. The protective effect of C. aurantium might be attributed to its antioxidant properties.

Tetraploidy enhances the ability to exclude chloride from leaves in carrizo citrange seedlings.

Tetraploid citrus seedlings are more tolerant to salt stress than diploid genotypes. To provide insight into the causes of differences in salt tolerance due to ploidy and thus to better understand Cl- exclusion mechanisms in citrus, diploid and tetraploid seedlings of Carrizo citrange (CC) were grown at 0 (control) and 40mM NaCl (salt-treated) medium for 20 days. Chloride uptake and root-to-shoot translocation rates were on average 1.4-fold higher in diploid than in tetraploid salt-treated plants, which resulted in a greater (1.6-fold) Cl- build up in the leaves of the former. Root hydraulic conductance and leaf transpiration rate were 58% and 17% lower, respectively, in tetraploid than in diploid control plants. Differences remained after salt treatment which reduced these parameters by 30-40% in both genotypes. Morphology of the root system was significantly influenced by ploidy. Tetraploid roots were less branched and with lower number of root tips than those of diploid plants. The cross-section diameter and area were lower in the diploid, and consequently specific root length was higher (1.7-fold) than in tetraploid plants. The exodermis in sections close to the root apex was broader and with higher deposition of suberin in cell walls in the tetraploid than in the diploid genotype. Net CO2 assimilation rate in tetraploid salt-treated seedlings was 1.5-fold higher than in diploid salt-treated plants, likely due to the loss of photosynthetic capacity of diploid plants induced by Cl- toxicity. Leaf damage was much higher, in terms of burnt area and defoliation, in diploid than in tetraploid salt-treated plants (8- and 6-fold, respectively). Salt treatment significantly reduced (37%) the dry weight of the diploid plants, but did not affect the tetraploids. In conclusion, tetraploid CC plants appear more tolerant to salinization and this effect seems mainly due to differences in morphological and histological traits of roots affecting hydraulic conductance and transpiration rate. These results may suggest that tetraploid CC used as rootstock could improve salt tolerance in citrus trees.

An Integrated View of Whole-Tree Hydraulic Architecture. Does Stomatal or Hydraulic Conductance Determine Whole Tree Transpiration?

Hydraulic conductance exerts a strong influence on many aspects of plant physiology, namely: transpiration, CO2 assimilation, growth, productivity or stress response. However we lack full understanding of the contribution of root or shoot water transport capacity to the total water balance, something which is difficult to study in trees. Here we tested the hypothesis that whole plant hydraulic conductance modulates plant transpiration using two different seedlings of citrus rootstocks, Poncirus trifoliata (L.) Raf. and Cleopatra mandarin (Citrus reshni Hort ex Tan.). The two genotypes presented important differences in their root or shoot hydraulic conductance contribution to whole plant hydraulic conductance but, even so, water balance proved highly dependent on whole plant conductance. Further, we propose there is a possible equilibrium between root and shoot hydraulic conductance, similar to that between shoot and root biomass production, which could be related with xylem anatomy.

Resistance to pathogens in terpene down-regulated orange fruits inversely correlates with the accumulation of D-limonene in peel oil glands.

Volatile organic compounds (VOCs) are secondary metabolites acting as a language for the communication of plants with the environment. In orange fruits, the monoterpene D-limonene accumulates at very high levels in oil glands from the peel. Drastic down-regulation of D-limonene synthase gene expression in the peel of transgenic oranges harboring a D-limonene synthase transgene in antisense (AS) configuration altered the monoterpene profile in oil glands, mainly resulting in reduced accumulation of D-limonene. This led to fruit resistance against Penicillium digitatum (Pd), Xanthomonas citri subsp. citri (Xcc) and other specialized pathogens. Here, we analyze resistance to pathogens in independent AS and empty vector (EV) lines, which have low, medium or high D-limonene concentrations and show that the level of resistance is inversely related to the accumulation of D-limonene in orange peels, thus explaining the need of high D-limonene accumulation in mature oranges in nature for the efficient attraction of specialized microorganism frugivores.

Network analysis of postharvest senescence process in citrus fruits revealed by transcriptomic and metabolomic profiling.

Citrus (Citrus spp.), a nonclimacteric fruit, is one of the most important fruit crops in global fruit industry. However, the biological behavior of citrus fruit ripening and postharvest senescence remains unclear. To better understand the senescence process of citrus fruit, we analyzed data sets from commercial microarrays, gas chromatography-mass spectrometry, and liquid chromatography-mass spectrometry and validated physiological quality detection of four main varieties in the genus Citrus. Network-based approaches of data mining and modeling were used to investigate complex molecular processes in citrus. The Citrus Metabolic Pathway Network and correlation networks were constructed to explore the modules and relationships of the functional genes/metabolites. We found that the different flesh-rind transport of nutrients and water due to the anatomic structural differences among citrus varieties might be an important factor that influences fruit senescence behavior. We then modeled and verified the citrus senescence process. As fruit rind is exposed directly to the environment, which results in energy expenditure in response to biotic and abiotic stresses, nutrients are exported from flesh to rind to maintain the activity of the whole fruit. The depletion of internal substances causes abiotic stresses, which further induces phytohormone reactions, transcription factor regulation, and a series of physiological and biochemical reactions.

Reference-free unwarping of single-shot spatiotemporally encoded MRI using asymmetric self-refocused echoes acquisition.

This paper presents a phase evolution rewinding algorithm for correcting the geometric and intensity distortions in single-shot spatiotemporally encoded (SPEN) MRI with acquisition of asymmetric self-refocused echo trains. Using the field map calculated from the phase distribution of the source image, the off-resonance induced phase errors are successfully rewound through deconvolution. The alias-free partial Fourier transform reconstruction helps improve the signal-to-noise ratio of the field maps and the output images. The effectiveness of the proposed algorithm was validated through 7 T MRI experiments on a lemon, a water phantom, and in vivo rat head. SPEN imaging was evaluated using rapid acquisition by sequential excitation and refocusing (RASER) which produces uniform T2 weighting. The results indicate that the new technique can more robustly deal with the cases in which the images obtained with conventional single-shot spin-echo EPI are difficult to be restored due to serious field variations.

Optimization of culture conditions (sucrose, pH, and photoperiod) for in vitro regeneration and early detection of somaclonal variation in ginger lime (Citrus assamensis).

Various explants (stem, leaf, and root) of Citrus assamensis were cultured on MS media supplemented with various combinations and concentrations (0.5-2.0 mg L(-1)) of NAA and BAP. Optimum shoot and root regeneration were obtained from stem cultures supplemented with 1.5 mg L(-1) NAA and 2.0 mg L(-1) BAP, respectively. Explant type affects the success of tissue culture of this species, whereby stem explants were observed to be the most responsive. Addition of 30 gL(-1) sucrose and pH of 5.8 was most optimum for in vitro regeneration of this species. Photoperiod of 16 hours of light and 8 hours of darkness was most optimum for shoot regeneration, but photoperiod of 24 hours of darkness was beneficial for production of callus. The morphology (macro and micro) and anatomy of in vivo and in vitro/ex vitro Citrus assamensis were also observed to elucidate any irregularities (or somaclonal variation) that may arise due to tissue culture protocols. Several minor micromorphological and anatomical differences were observed, possibly due to stress of tissue culture, but in vitro plantlets are expected to revert back to normal phenotype following full adaptation to the natural environment.

Surface barriers of mandarin 'okitsu' leaves make a major contribution to canker disease resistance.

Field evaluations have shown that Satsuma mandarin (Citrus unshiu) 'Okitsu' is one of the mandarin cultivars that shows substantial resistance to Xanthomonas citri subsp. citri (X. citri), the causal agent of citrus bacterial canker disease. However, the mechanisms underlying this resistance are not well understood. In this study, we have shown that 'Okitsu' leaves are nevertheless susceptible to X. citri infection during a period of their development; however, this period is shorter than that seen in the susceptible mandarin 'Clemenules' (C. clementina). Under controlled growth conditions, the resistance of 'Okitsu' to X. citri was associated with the age of the leaf and was evident in spray-inoculated plants but not in those inoculated by infiltration. Furthermore, X. citri showed reduced attachment and biofilm formation in 'Okitsu' leaves compared with 'Clemenules'. Taken together, our data suggest that structural features of the 'Okitsu' leaf surface, such as the physical properties of the cuticle, are involved in the resistance to X. citri.