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The dispersal of seeds away from parent plants seems to be the underlying selective force in the evolution of fleshy fruits attractive to animals. Secondary metabolites, which are not essential compounds for plant survival, are involved in the interaction of fleshy fruits with seed dispersers and antagonists. Plant volatile organic compounds (VOCs) are secondary metabolites that play important roles in biotic interactions and in abiotic stress responses. They are usually accumulated at high levels in specific plant tissues and organs, such as fleshy fruits. The study of VOCs emitted during fruit development and after different biotic challenges may help to determine the interactions of fleshy fruits not only with legitimate vertebrate dispersers, but also with insects and microorganisms. A knowledge of fruit VOCs could be used in agriculture to generate attraction or repellency to pests and resistance to pathogens in fruits. This review provides an examination of specific fruit VOC blends as signals for either seed dispersal or predation through simple or complex trophic chains, which may also have consequences for an understanding of the importance of biodiversity in wild areas.

To recover transgenic citrus plants in the most efficient manner, the use of selection marker genes is essential. In this work, it was shown that the mutated forms of the acetolactate synthase ( ALS ) gene in combination with the herbicide selection agent imazapyr (IMZ) added to the selection medium may be used to achieve this goal. This approach enables the development of cisgenic regenerants, namely, plants without the incorporation of those bacterial genes currently employed for transgenic selection, and additionally it allows the generation of edited, non-transgenic plants with altered endogenous ALS genes leading to IMZ resistance. In this work, the citrus mutants, in which ALS has been converted into IMZ-resistant forms using a base editor system, were recovered after cocultivation of the explants with Agrobacterium tumefaciens carrying a cytidine deaminase fused to nSpCas9 in the T-DNA and selecting regenerants in the culture medium supplemented with IMZ. Analysis of transgene-free plants indicated that the transient expression of the T-DNA genes was sufficient to induce ALS mutations and thus generate IMZ-resistant shoots at 11.7% frequency. To our knowledge, this is the first report of T-DNA-free edited citrus plants. Although further optimization is required to increase edition efficiency, this methodology will allow generating new citrus varieties with improved organoleptic/agronomic features without the need to use foreign genes.

Huanglongbing (HLB) is the most destructive, yet incurable disease of citrus. Finding sources of genetic resistance to HLB-associated ‘ Candidatus Liberibacter asiaticus’ (Las) becomes strategic to warrant crop sustainability, but no resistant Citrus genotypes exist. Some Citrus relatives of the family Rutaceae, subfamily Aurantioideae, were described as full-resistant to Las, but they are phylogenetically far, thus incompatible with Citrus . Partial resistance was indicated for certain cross-compatible types. Moreover, other genotypes from subtribe Citrinae, sexually incompatible but graft-compatible with Citrus , may provide new rootstocks able to restrict bacterial titer in the canopy. Use of seedlings from monoembryonic species and inconsistencies in previous reports likely due to Las recalcitrance encouraged us to evaluate more accurately these Citrus relatives. We tested for Las resistance a diverse collection of graft-compatible Citrinae species using an aggressive and consistent challenge-inoculation and evaluation procedure. Most Citrinae species examined were either susceptible or partially resistant to Las. However, Eremocitrus glauca and Papua/New Guinea Microcitrus species as well as their hybrids and those with Citrus arose here for the first time as full-resistant, opening the way for using these underutilized genotypes as Las resistance sources in breeding programs or attempting using them directly as possible new Las-resistant Citrus rootstocks or interstocks.

Citrus species are among the most important fruit tree crops grown worldwide. Their long juvenile period joined with their complex genetic and reproductive characteristics severely hampers genomic studies and the improvement of traits of interest. Among these, seedlessness represents a major fruit quality trait. Genetic engineering is the fastest way to unequivocally characterize the function of citrus genes and to develop better varieties. In this study, two genes from Citrus clementina Hort. ex Tan., CcGLT1 and CcRBP1 , that putatively encode a glycosyltransferase-like (GLT) protein and an RNA binding (RBP) family protein, respectively, were characterized as highly expressed in male and female reproductive tissues and then evaluated as candidate genes involved in male and/or female gametic development by silencing them using RNA interference (RNAi) in Carrizo citrange, used as model citrus type easy to transform. Concurrently, the early flowering and fruiting phenotype was induced by ectopic overexpression of the citrus ortholog of the floral integrator FLOWERING LOCUS T gene ( FT ) which enabled flower and fruit production less than six months after transformation. Histological observations of flower tissues from genetically modified plants showed that silencing CcGLT1 affects pollen performance by reducing pollen germinability and viability which results in an increased rate of ovule abortion resulting in fewer seeds in self-pollinated fruits. Conversely, the silencing of CcRBP1 led to severe alterations in plant growth and development in the transgenic RBP lines preventing the characterization of its role in fertility, which therefore remains unresolved. These results provide useful insights into male and female sterility in citrus for the genetic improvement of commercial varieties aimed to obtain seedless fruits.

Citrus aroma and flavor, chief traits of fruit quality, are derived from their high content in essential oils of most plant tissues, including leaves, stems, flowers, and fruits. Accumulated in secretory cavities, most components of these oils are volatile terpenes. They contribute to defense against herbivores and pathogens, and perhaps also protect tissues against abiotic stress. In spite of their importance, our understanding of the physiological, biochemical, and genetic regulation of citrus terpene volatiles is still limited. The availability of the sweet orange ( L. Osbeck) genome sequence allowed us to characterize for the first time the terpene synthase (TPS) family in a citrus type. is one of the largest angiosperm TPS families characterized so far, formed by 95 loci from which just 55 encode for putative functional TPSs. All TPS angiosperm families, TPS-a, TPS-b, TPS-c, TPS-e/f, and TPS-g were represented in the sweet orange genome, with 28, 18, 2, 2, and 5 putative full length genes each. Additionally, sweet orange β-farnesene synthase, ( )-β-cubebene/α-copaene synthase, two β-caryophyllene synthases, and three multiproduct enzymes yielding β-cadinene/α-copaene, β-elemene, and β-cadinene/ledene/allo-aromandendrene as major products were identified, and functionally characterized via recombinant assays.

Carotenoid-rich foods such as citrus fruits have a wide range of functions in human health. They primarily exert antioxidant effects, but individual carotenoids may also act through other health-promoting mechanisms such as β-carotene as pro-vitamin A. Here, we show that red-fleshed sweet oranges grown in tropical climates are 4–9 times richer in carotenoids than their orange-fleshed counterparts, regardless of their maturation stage. The most significant difference observed between both varieties was the presence of lycopene at moderate concentrations (around 8 µg/g FW) in the mature pulp of the red varieties, which was absent in the blond ones. This is because the red-fleshed sweet oranges grown in tropical climates with high temperatures increase lycopene and β-carotene concentrations in their pulp during fruit maturation. Due to lycopene accumulation, red orange juice offers a promising addition to popular blond-orange, with the new varieties Carrancas and Pinhal being perfectly suitable for blending to enhance juice colour. Sao Paulo, one of the world’s leading citrus orange juice producers, as well as other tropical citrus regions could benefit from cultivating using such lycopene-rich cultivars and industrially.

The Asian Citrus Psyllid (ACP), Diaphorina citri , severely threatens citrus production worldwide by transmitting the greening (= Huanglongbing)—causing bacterium Candidatus Liberibacter asiaticus. There is growing evidence that the push-pull strategy is suitable to partially mitigate HLB by repelling ACP with transgenic plants engineered to produce repellents and attracting the vector to plants with a minimal disease transmission rate. Species that pull ACP away from commercial citrus plants have been identified, and transgenic plants that repel ACP have been developed. The concept of a repellent-producing plant was first demonstrated with an Arabidopsis line engineered to overexpress a gene controlling the synthesis of β -caryophyllene and other sesquiterpenes. We have analyzed the volatile organic compounds released by this Arabidopsis line and identified α -humulene, α -copaene, and trace amounts of β -elemene, in addition to β -caryophyllene. Behavioral measurements demonstrated that α -copaene repels ACP at doses ca. 100× lower than those needed for β -caryophyllene repellence. In contrast, α -humulene is innocuous at the level emitted by the transgenic plant. We confirmed that a mixture of the three sesquiterpenes in the ratio 1:100:10 repels ACP. Likewise, a commercial sample of copaiba oil containing the three sesquiterpenes, in a proportion similar to that in the transgenic plant, repelled ACP.

For four decades, an influential hypothesis has posited that competition for food resources between microbes and vertebrates selects for microbes to alter these resources in ways that make them unpalatable to vertebrates. We chose an understudied cross kingdom interaction to experimentally evaluate the effect of fruit infection by fungi on both vertebrate (mammals and birds) fruit preferences and on ecologically relevant fruit traits (volatile compounds, toughness, etc). Our well-replicated field experiments revealed that, in contrast to previous studies, frugivorous mammals and birds consistently preferred infested over intact fruits. This was concordant with the higher level of attractive volatiles (esters, ethanol) in infested fruits. This investigation suggests that vertebrate frugivores, fleshy-fruited plants, and microbes form a tripartite interaction in which each part could interact positively with the other two (e.g. both orange seeds and fungal spores are likely dispersed by mammals). Such a mutualistic view of these complex interactions is opposed to the generalized idea of competition between frugivorous vertebrates and microorganisms. Thus, this research provides a new perspective on the widely accepted plant evolutionary dilemma to make fruits attractive to mutualistic frugivores while unattractive to presumed antagonistic microbes that constrain seed dispersal.

Novel, suitable and sustainable alternative control tactics that have the potential to reduce migration of Diaphorina citri into commercial citrus orchards are essential to improve management of huanglongbing (HLB). In this study, the effect of orange jasmine ( Murraya paniculata ) as a border trap crop on psyllid settlement and dispersal was assessed in citrus orchards. Furthermore, volatile emission profiles and relative attractiveness of both orange jasmine and sweet orange ( Citrus  ×  aurantium L., syn. Citrus sinensis (L.) Osbeck) nursery flushes to D. citri were investigated. In newly established citrus orchards, the trap crop reduced the capture of psyllids in yellow sticky traps and the number of psyllids that settled on citrus trees compared to fallow mowed grass fields by 40% and 83%, respectively. Psyllids were attracted and killed by thiamethoxam-treated orange jasmine suggesting that the trap crop could act as a ‘sink’ for D. citri . Additionally, the presence of the trap crop reduced HLB incidence by 43%. Olfactometer experiments showed that orange jasmine plays an attractive role on psyllid behavior and that this attractiveness may be associated with differences in the volatile profiles emitted by orange jasmine in comparison with sweet orange. Results indicated that insecticide-treated M. paniculata may act as a trap crop to attract and kill D. citri before they settled on the edges of citrus orchards, which significantly contributes to the reduction of HLB primary spread.

Production of citrus, the main fruit tree crop worldwide, is severely threatened by Huanglongbing (HLB), for which as yet a cure is not available. Spread of this bacterial disease in America and Asia is intimately connected with dispersal and feeding of the insect vector Diaphorina citri , oligophagous on rutaceous host plants. Effective control of this psyllid is an important component in successful HLB management programs. Volatiles released from the non-host guava have been shown to be repellent to the psyllid and to inhibit its response to citrus odour. By analysing VOC emission from guava we identified one volatile compound, ( E )-β-caryophyllene, which at certain doses exerts a repellent effect on D . citri . Non-host plant rejection mediated by ( E )-β-caryophyllene is demonstrated here by using Arabidopsis over-expression and knock-out lines. For the first time, results indicate that genetically engineered Arabidopsis plants with modified emission of VOCs can alter the behaviour of D . citri . This study shows that transgenic plants with an inherent ability to release ( E )-β-caryophyllene can potentially be used in new protection strategies of citrus trees against HLB.