Proline accumulation and antioxidant response are crucial for citrus tolerance to UV-B light-induced stress.

Plants face a wide range of biotic and abiotic stress conditions, which are further intensified by climate change. Among these stressors, increased irradiation in terms of intensity and wavelength range can lead to detrimental effects, such as chlorophyll degradation, destruction of the PSII reaction center, generation of ROS, alterations to plant metabolism, and even plant death. Here, we investigated the responses of two citrus genotypes, Citrus macrophylla (CM), and Troyer citrange (TC) to UV-B light-induced stress, by growing plants of both genotypes under control and UV-B stress conditions for 5 days to evaluate their tolerance mechanisms. TC seedlings had higher sensitivity to UV-B light than CM seedlings, as they showed more damage and increased levels of oxidative harm (indicated by the accumulation of MDA). In contrast, CM seedlings exhibited specific adaptive mechanisms, including accumulation of higher levels of proline under stressful conditions, and enhanced antioxidant capacity, as evidenced by increased ascorbate peroxidase activity and upregulation of the CsAPX2 gene. Phytohormone accumulation patterns were similar in both genotypes, with a decrease in ABA content in response to UV-B light. Furthermore, expression of genes involved in light perception and response was specifically affected in the tolerant CM seedlings, which exhibited higher expression of CsHYH/CsHY5 and CsRUP1-2 genes. These findings underscore the importance of the antioxidant system in citrus plants subjected to UV-B light-induced stress and suggest that CsHYH/CsHY5 and CsRUP1-2 could be considered genes associated with tolerance to such challenging conditions.

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.

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.

Regulation of magnesium and calcium homeostasis in citrus seedlings under varying magnesium supply.

Magnesium (Mg) and calcium (Ca) are two essential macronutrients in plants; however, the characteristics of Mg and Ca concentrations in organ, subcellular and chemical forms and their relationships in citrus plants, especially under varying Mg supply, are not well understood. In this study, Citrus sinensis seedlings (cv. Xuegan) were cultivated in conditions of Mg deficiency (0 mmol Mg2+ L-1) and Mg sufficiency (2 mmol Mg2+ L-1) to investigate the responses of Mg and Ca homeostasis in different organs and fractions. Compared with Mg sufficiency, Mg deficiency significantly decreased root and shoot growth, with the shoot biomass reduction of branch organs was greater than that of parent organs. In addition to increasing the Ca concentration in the parent stem and lateral root organs, Mg deficiency significantly decreased the concentrations and accumulations of Mg and Ca in citrus seedlings, further altering their distribution in different organs. More than 50% of Ca and Mg were sequestrated in the cell wall and soluble fractions, respectively, with Mg concentration decreasing by 15.4% in roots and 46.9% in leaves under Mg deficiency, while Ca concentration decreased by 27.6% in roots and increased by 23.6% in parent leaves. Approximately 90% of Mg exists in inorganic, water-soluble, and pectate and protein-bound forms, and nearly 90% of Ca exists in water-soluble, pectate and protein-bound, phosphate and oxalate acid forms. Except for the decreased inorganic Mg in roots and water-soluble Mg and Ca in leaves, Mg deficiency increased the proportions of Mg and Ca in all chemical forms. However, Mg deficiency generally increased the Ca/Mg ratio in various organs, subcellular and chemical forms, with negative relationships between Mg concentration and Ca/Mg ratio, and the variations of Mg and Ca were highly separated between Mg supply and organs. In conclusion, our results provide insights into the effects of Mg supply on Mg and Ca homeostasis in citrus plants.

Small RNAs contribute to citrus Huanglongbing tolerance by manipulating methyl salicylate signaling and exogenous methyl salicylate primes citrus groves from emerging infection.

Citrus production is severely threatened by the devastating Huanglongbing (HLB) disease globally. By studying and analyzing the defensive behaviors of an HLB-tolerant citrus cultivar 'Shatangju', we discovered that citrus can sense Candidatus Liberibacter asiaticus (CLas) infection and induce immune responses against HLB, which can be further strengthened by both endogenously produced and exogenously applied methyl salicylate (MeSA). This immune circuit is turned on by an miR2977-SAMT (encoding a citrus Salicylate [SA] O-methyltransferase) cascade, by which CLas infection leads to more in planta MeSA production and aerial emission. We provided both transgenic and multi-year trail evidences that MeSA is an effective community immune signal. Ambient MeSA accumulation and foliage application can effectively induce defense gene expression and significantly boost citrus performance. We also found that miRNAs are battle fields between citrus and CLas, and about 30% of the differential gene expression upon CLas infection are regulated by miRNAs. Furthermore, CLas hijacks host key processes by manipulating key citrus miRNAs, and citrus employs miRNAs that coordinately regulate defense-related genes. Based on our results, we proposed that miRNAs and associated components are key targets for engineering or breeding resistant citrus varieties. We anticipate that MeSA-based management, either induced expression or external application, would be a promising tool for HLB control.

Photosynthetic Characteristics and Chloroplast Ultrastructure Responses of Citrus Leaves to Copper Toxicity Induced by Bordeaux Mixture in Greenhouse.

Bordeaux mixture (Bm) is a copper (Cu)-based pesticide that has been widely used for controlling citrus scab and citrus canker. However, frequent spraying of Bm is toxic to citrus. To our knowledge, few studies are available that discuss how the photosynthetic characteristics and chloroplast ultrastructure of citrus leaves are affected by Cu toxicity induced by excessive Bm. In the study, two-year-old seedlings of Citrus grandis (C. grandis) and Citrus sinensis (C. sinensis), which were precultured in pots, were foliar-sprayed with deionized water (as control) or Bm diluted 500-fold at intervals of 7 days for 6 times (4 times as recommended by the manufacturer) to investigate the leaf Cu absorption, photosynthesis, chloroplast ultrastructure and antioxidant enzymatic activities. Bm foliar-sprayed 6 times on citrus seedlings increased the leaf Cu content, decreased the photosynthetic pigments content and destroyed the chloroplast ultrastructure, which induced leaf chlorosis and photosynthetic inhibition. A lower Cu absorption, a higher light photon-electron transfer efficiency, a relative integrity of chloroplast ultrastructure and a promoted antioxidant protection contributed to a higher photosynthetic activity of C. grandis than C. sinensis under excessive spraying of Bm. The present study provides crucial references for screening and selecting citrus species with a higher tolerance to Cu toxicity induced by excessive Bm.

Water stress signaling and hydraulic traits in three congeneric citrus species under water deficit.

Morpho-physiological strategies to deal with water deficit vary among citrus species and the chemical signaling through ABA and anatomical, hydraulic, and physiological traits were evaluated in saplings of Rangpur lime, Swingle citrumelo and Valencia sweet orange. Trunk and roots of Swingle citrumelo presented lower vessel diameter and higher vessel frequency as compared to the other species. However, relative water content at the turgor loss point (RWCTLP), the osmotic potential at full turgor (Ψ0), the osmotic potential at the turgor loss point (ΨTLP), bulk modulus of elasticity (ε) and the xylem water potential when hydraulic conductivity is reduced by 50% (Ψ50) and 88% (Ψ88) indicated similar hydraulic traits among citrus species, with Rangpur lime showing the highest hydraulic safety margin. Roots of Rangpur lime and Swingle citrumelo were more water conductive than ones of Valencia sweet orange, which was linked to higher stomatal conductance. Chemical signaling through ABA prevented shoot dehydration in Rangpur lime under water deficit, with this species showing a more conservative stomatal behavior, sensing, and responding rapidly to low soil moisture. Taken together, our results suggest that Rangpur lime - the drought tolerant species - has an improved control of leaf water status due to chemical signaling and effective stomatal regulation for reducing water loss as well as decreased root hydraulic conductivity for saving water resources under limiting conditions.

Citrus heat shock transcription factor CitHsfA7-mediated citric acid degradation in response to heat stress.

Heat stress is a major abiotic stress for plants, which can generate a range of biochemical and genetic responses. In 'Ponkan' mandarin fruit, hot air treatment (HAT) accelerates the degradation of citric acid. However, the transcriptional regulatory mechanisms of citrate degradation in response to HAT remain to be elucidated. Here, 17 heat shock transcription factor sequences were isolated, and dual-luciferase assays were employed to investigate whether the encoded proteins that could trans-activate the promoters of key genes in the GABA shunt, involved in citrate metabolism. We identified four heat shock transcription factors (CitHsfA7, CitHsfA3, CitHsfA4b and CitHsfA8) that showed trans-activation effects on CitAco3, CitIDH3 and CitGAD4, respectively. Transient expression of the CitHsfs in citrus fruits indicated that CitHsfA7 was the only factor that resulted in a significant lowering of the citric acid content, and these results were confirmed by a virus-induced gene silencing system (VIGS). Sub-cellar localization showed that CitHsfA7 is located in the nucleus and is capable of binding directly to a putative HSE in the CitAco3 promoter and enhance its expression. We proposed that the induction of CitHsfA7 transcript level contributes to citric acid degradation in citrus fruit, via modulation of CitAco3 in response to HAT.

Short-term warming does not affect intrinsic thermotolerance but induces strong sustaining photoprotection in tropical evergreen citrus genotypes.

Consequences of warming and postwarming events on photosynthetic thermotolerance (PT ) and photoprotective responses in tropical evergreen species remain elusive. We chose Citrus to answer some of the emerging questions related to tropical evergreen species' PT behaviour including (i) how wide is the genotypic variation in PT ? (ii) how does PT respond to short-term warming and (iii) how do photosynthesis and photoprotective functions respond over short-term warming and postwarming events? A study on 21 genotypes revealed significant genotypic differences in PT , though these were not large. We selected five genotypes with divergent PT and simulated warming events: Tmax 26/20°C (day-time highest maximum/night-time lowest maximum) (Week 1) < Tmax 33/30°C (Week 2) < Tmax 36/32°C (Week 3) followed by Tmax 26/16°C (Week 4, recovery). The PT of all genotypes remained unaltered despite strong leaf megathermy (leaf temperature > air temperature) during warming events. Though moderate warming showed genotype-specific stimulation in photosynthesis, higher warming unequivocally led to severe loss in net photosynthesis and induced higher nonphotochemical quenching. Even after a week of postwarming, photoprotective mechanisms strongly persisted. Our study points towards a conservative PT in evergreen citrus genotypes and their need for sustaining higher photoprotection during warming as well as postwarming recovery conditions.

Photodynamic control of citrus crop diseases.

Citrus are economically important fruit crops to which infectious diseases like citrus canker caused by Xanthomonas citri subs. citri, citrus variegated chlorosis caused by Xylella fastidiosa, "huanglongbing" associated with the presence of Candidatus liberibacter species, anthracnose caused by Colletotrichum gloeosporioides and citrus black spot caused by Phyllosticta citricarpa, impose significant losses. Control measures involve chemical treatment of orchards but often, eradication of infected plants is unavoidable. To circumvent the environmental impacts of pesticides and the socio-economic impacts of eradication, innovative antimicrobial approaches like photodynamic inactivation are being tested. There is evidence of the susceptibility of Xanthomonas citri subs. citri and C. gloeosporioides to photodynamic damage. However, the realistic assessment of perspectives for widespread application of photodynamic inactivation in the control of citrus diseases, necessarily implies that other microorganisms are also considered. This review intends to provide a critical summary of the current state of research on photodynamic inactivation of citrus pathogens and to identify some of the current limitations to the widespread use of photodynamic treatments in citrus crops.

Citrus transcription factor CsHB5 regulates abscisic acid biosynthetic genes and promotes senescence.

Senescence is a gradual physiological process involving the integration of numerous internal and environmental signals. Abscisic acid (ABA) is a well-known inducer of senescence. However, the regulatory mechanisms underlying ABA-mediated senescence remain largely unknown. Here, we report that the citrus homeodomain leucine zipper I (HD-ZIP I) transcription factor CsHB5 functions as a regulator of ABA-triggered senescence. CsHB5 acts as a nucleus-localized transcriptional activator, the expression of which appeared to be closely associated with citrus senescence. Overexpression of CsHB5 in citrus calli upregulated the expression of ABA- and reactive oxygen species (ROS)-related genes, and significantly increased the content of ABA and hydrogen peroxide (H2 O2 ), whereas silencing CsHB5 in citrus calli downregulated the expression of ABA-related genes. Additionally, heterogenous overexpression of CsHB5 in Solanum lycopersicum (tomato) and Arabidopsis thaliana (Arabidopsis) leads to early leaf yellowing under dark-induced senescence conditions. Meanwhile, the levels of ABA and H2 O2 in transgenic tomatoes increased significantly and the lycopene content decreased. Transcriptome analysis of CsHB5-overexpressing citrus calli and tomato showed that CsHB5 was involved in multiple senescence-associated processes, including chlorophyll degradation, nutrient compound biosynthesis and transport, as well as ABA and ROS signal transduction. The results of yeast one-hybrid assays, electrophoretic mobility shift assays and dual luciferase assays indicated that CsHB5 directly binds to the promoters of ABA biosynthetic genes, including ß-carotene hydroxylase 1 (BCH1) and 9-cis-epoxycarotenoid dioxygenase 2 (NCED2), thereby activating their transcription. Our findings revealed that CsHB5 participates in senescence, at least partly, by directly controlling ABA accumulation. Our work provides insight into the regulatory mechanisms underlying ABA-mediated senescence.

ERF108 from Poncirus trifoliata (L.) Raf. functions in cold tolerance by modulating raffinose synthesis through transcriptional regulation of PtrRafS.

Ethylene-responsive factors (ERFs) are plant-specific transcription factors involved in cold stress response, and raffinose is known to accumulate in plants exposed to cold. However, it remains elusive whether ERFs function in cold tolerance by modulating raffinose synthesis. Here, we identified a cold-responsive PtrERF108 from trifoliate orange (Poncirus trifoliata (L.) Raf.), a cold-tolerant plant closely related to citrus. PtrERF108 is localized in the nucleus and has transcriptional activation activity. Overexpression of PtrERF108 conferred enhanced cold tolerance of transgenic lemon, whereas virus-induced gene silencing (VIGS)-mediated knockdown of PtrERF108 in trifoliate orange greatly elevated cold sensitivity. Transcriptome profiling showed that PtrERF108 overexpression caused extensive reprogramming of genes associated with signaling transduction, physiological processes and metabolic pathways. Among them, a raffinose synthase (RafS)-encoding gene, PtrRafS, was confirmed as a direct target of PtrERF108. RafS activity and raffinose content were significantly increased in PtrERF108-overexpressing transgenic plants, but prominently decreased in the VIGS plants under cold conditions. Meanwhile, exogenous replenishment of raffinose could recover the cold tolerance of PtrERF108-silenced plants, whereas VIGS-mediated knockdown of PtrRafS resulted in cold-sensitive phenotype. Taken together, the current results demonstrate that PtrERF108 plays a positive role in cold tolerance by modulation of raffinose synthesis via regulating PtrRafS. Our findings reveal a new transcriptional module composed of ERF108-RafS underlying cold-induced raffinose accumulation in plants.

Quantification of Flavonoids, Phenols and Antioxidant Potential from Dropped Citrus reticulata Blanco Fruits Influenced by Drying Techniques.

Physiologically dropped immature Citrus reticulata Blanco fruits are regarded as waste and discarded in the citrus orchard but are a good source of bioactive compounds including flavonoids, antioxidants and total phenols. A study was undertaken to identify and quantify these bioactive compounds and to investigate the influence of different drying techniques, namely freeze drying and hot air oven drying, on flavonoids namely flavanone glycosides, antioxidant potential and total phenol content in immature dropped fruits of Citrus reticulata Blanco. Flavonoids were quantified in high-performance liquid chromatography (HPLC). The antioxidant activity were investigated with three assays azino-bis [3-ethylbenzthiazoline-6-sulfonic acid]) (ABTS), 2,2-diphenyl-1-picrylhydrazyl radical (DPPH), Ferric Reducing Ability of Plasma (FRAP) and total phenol content was determined. Freeze dried samples of 12 and 14 mm size retained maximum hesperidin flavonoid content (27.03% and 27.20%) as compared to the hot air dried samples (17.99%) and retained higher phenolic content ranged from 50.54-54.19 mg GAEL-1. The antioxidant activity in freeze dried fruits was from 12.21-13.55 mM L-1 Trolox and 15.27-16.72 mM L-1 Trolox with ABTS, DPPH assay and FRAP values ranging from 7.31-9.07 mM L-1 Trolox. Significant positive correlation was found between the flavonoid hesperidin with antioxidant assays and total phenolic content (TPC). The results showed that waste citrus fruits can act as potential source of bioflavonoids, especially hesperidin, and antioxidants for pharmaceutical as well as nutraceutical industry.

Differences in morphological and physiological features of citrus seedlings are related to Mg transport from the parent to branch organs.

BACKGROUND: In this study, we aimed to test the hypothesis that magnesium (Mg) remobilization in citrus plants is regulated by Mg supply and contributes to differences in the growth of the parent and branch organs. Citrus seedlings were grown in sand under Mg deficient (0 mmol Mg2+ L-1, -Mg) and Mg sufficient (2 mmol Mg2+ L-1, + Mg) conditions. The effects on biomass, Mg uptake and transport, gas exchange and chlorophyll fluorescence, as well as related morphological and physiological parameters were evaluated in different organs. RESULTS: Mg deficiency significantly decreased plant biomass, with a decrease in total plant biomass of 39.6%, and a greater than twofold decrease in the branch organs compared with that of the parent organs. Reduced photosynthesis capacity was caused by a decreased in pigment levels and photosynthetic electron transport chain disruption, thus affecting non-structural carbohydrate accumulation and plant growth. However, the adaptive responses of branch leaves to Mg deficiency were greater than those in parent leaves. Mg deficiency inhibited plant Mg uptake but enhanced Mg remobilization from parent to branch organs, thus changing related growth variables and physiological parameters, including protein synthesis and antioxidant enzyme activity. Moreover, in the principal components analysis, these variations were highly clustered in both the upper and lower parent leaves, but highly separated in branch leaves under the different Mg conditions. CONCLUSIONS: Mg deficiency inhibits the growth of the parent and branch organs of citrus plants, with high Mg mobility contributing to differences in physiological metabolism. These findings suggest that Mg management should be optimized for sustainable citrus production.

Phytoene desaturase-silenced citrus as a trap crop with multiple cues to attract Diaphorina citri, the vector of Huanglongbing.

Huanglongbing (HLB) is one of the most destructive diseases in citrus worldwide. Unfortunately, HLB has no cure and management relies on insecticides to reduce populations of the vector, Diaphorina citri Kuwayama (Hemiptera: Liviidae). We propose an attract-and-kill strategy using a trap crop as an alternative to vector control to reduce transmission of the pathogen, 'Candidatus Liberibacter asiaticus'. We evaluated vector response to phytoene desaturase-silenced citrus trees using virus-induced gene silencing technology. Citrus tristeza virus (CTV) was used to produce a phytoene desaturase-silenced citrus (CTV-tPDS) that expresses visual, olfactory, and gustatory cues to attract D. citri. We found that D. citri were more attracted to CTV-tPDS plants with noticeably better fecundity and overall population fitness than on control plants. Moreover, rearing D. citri on CTV-tPDS plants significantly increased their survival probability compared with those reared on control plants. CTV-tPDS plants possessed reduced content of both carotenoid and chlorophyll pigments resulting in a consistent photobleached phenotype on citrus leaves which provided a sufficient close-range visual attractant to stimulate D. citri landing. Additionally, CTV-tPDS plants exhibited an enriched profile of volatile organic compounds (VOCs), which offered adequate olfactory cues to attract psyllid from long-range. Finally, CTV-tPDS plants exhibited an enriched metabolite content of phloem sap and leaves which offered appropriate gustatory cues that influenced probing/feeding behavior. We believe that introducing CTV-tPDS plants (as a trap crop) to D. citri-infested orchards will attract and congregate psyllids to facilitate their removal from the target crop with insecticides or by other means. This new strategy could be deployed relatively quickly and economically to HLB-impacted citrus industries. Moreover, it is an eco-friendly strategy because it should partially reduce the input of chemical insecticides ameliorating the indirect cost of HLB infection.

The vascular targeted citrus FLOWERING LOCUS T3 gene promotes non-inductive early flowering in transgenic Carrizo rootstocks and grafted juvenile scions.

Shortening the juvenile stage in citrus and inducing early flowering has been the focus of several citrus genetic improvement programs. FLOWERING LOCUS T (FT) is a small phloem-translocated protein that regulates precocious flowering. In this study, two populations of transgenic Carrizo citrange rootstocks expressing either Citrus clementina FT1 or FT3 genes under the control of the Arabidopsis thaliana phloem specific SUCROSE SYNTHASE 2 (AtSUC2) promoter were developed. The transgenic plants were morphologically similar to the non-transgenic controls (non-transgenic Carrizo citrange), however, only AtSUC2-CcFT3 was capable of inducing precocious flowers. The transgenic lines produced flowers 16 months after transformation and flower buds appeared 30-40 days on juvenile immature scions grafted onto transgenic rootstock. Gene expression analysis revealed that the expression of SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1 (SOC1) and APETALA1 (AP1) were enhanced in the transgenics. Transcriptome profiling of a selected transgenic line showed the induction of genes in different groups including: genes from the flowering induction pathway, APETALA2/ETHYLENE RESPONSE FACTOR (AP2/ERF) family genes, and jasmonic acid (JA) pathway genes. Altogether, our results suggested that ectopic expression of CcFT3 in phloem tissues of Carrizo citrange triggered the expression of several genes to mediate early flowering.

Development on Citrus medica infected with 'Candidatus Liberibacter asiaticus' has sex-specific and -nonspecific impacts on adult Diaphorina citri and its endosymbionts.

Huanglongbing (HLB) is a deadly, incurable citrus disease putatively caused by the unculturable bacterium, 'Candidatus Liberibacter asiaticus' (CLas), and transmitted by Diaphorina citri. Prior studies suggest D. citri transmits CLas in a circulative and propagative manner; however, the precise interactions necessary for CLas transmission remain unknown, and the impact of insect sex on D. citri-CLas interactions is poorly understood despite reports of sex-dependent susceptibilities to CLas. We analyzed the transcriptome, proteome, metabolome, and microbiome of male and female adult D. citri reared on healthy or CLas-infected Citrus medica to determine shared and sex-specific responses of D. citri and its endosymbionts to CLas exposure. More sex-specific than shared D. citri responses to CLas were observed, despite there being no difference between males and females in CLas density or relative abundance. CLas exposure altered the abundance of proteins involved in immunity and cellular and oxidative stress in a sex-dependent manner. CLas exposure impacted cuticular proteins and enzymes involved in chitin degradation, as well as energy metabolism and abundance of the endosymbiont 'Candidatus Profftella armatura' in both sexes similarly. Notably, diaphorin, a toxic Profftella-derived metabolite, was more abundant in both sexes with CLas exposure. The responses reported here resulted from a combination of CLas colonization of D. citri as well as the effect of CLas infection on C. medica. Elucidating these impacts on D. citri and their endosymbionts contributes to our understanding of the HLB pathosystem and identifies the responses potentially critical to limiting or promoting CLas acquisition and propagation in both sexes.

Root samples provide early and improved detection of Candidatus Liberibacter asiaticus in Citrus.

Huanglongbing (HLB), or Citrus Greening, is one of the most devastating diseases affecting agriculture today. Widespread throughout Citrus growing regions of the world, it has had severe economic consequences in all areas it has invaded. With no treatment available, management strategies focus on suppression and containment. Effective use of these costly control strategies relies on rapid and accurate identification of infected plants. Unfortunately, symptoms of the disease are slow to develop and indistinct from symptoms of other biotic/abiotic stressors. As a result, diagnosticians have focused on detecting the pathogen, Candidatus Liberibacter asiaticus, by DNA-based detection strategies utilizing leaf midribs for sampling. Recent work has shown that fibrous root decline occurs in HLB-affected trees before symptom development among leaves. Moreover, the pathogen, Ca. Liberibacter asiaticus, has been shown to be more evenly distributed within roots than within the canopy. Motivated by these observations, a longitudinal study of young asymptomatic trees was established to observe the spread of disease through time and test the relative effectiveness of leaf- and root-based detection strategies. Detection of the pathogen occurred earlier, more consistently, and more often in root samples than in leaf samples. Moreover, little influence of geography or host variety was found on the probability of detection.

Mutants of Citrus macrophylla rootstock obtained by gamma radiation improve salt resistance through toxic ion exclusion.

Salinity is one of the biggest challenges that need to be faced in crop production. Citrus is highly sensitive to salt stress and obtaining rootstocks with improved resistance to salinity is key for the citrus growing industry. In this study, five mutants of Citrus macrophylla rootstock, obtained through gamma radiation and in vitro pre-selected for their resistance to salinity, were irrigated with a solution containing 100 mM of NaCl. After 8 weeks of exposure, the mutants were evaluated for their performance (growth, visual leaf damage) and chlorophyll, proline, starch, soluble sugars and ion contents to determine their degree of resistance to this salinity level. In the saline conditions assayed, all the mutants showed better performance and less leaf damage than Citrus macrophylla. Our data suggest that this improved resistance to salinity was based on their capacity to accumulate less Na (MM4B and MMN1) or Cl- (MM1A, MM4A and MM3B). Besides having the lowest Cl- content, the mutants MM1A, MM4A and MM3B, had the highest NO3- concentrations in salinity. Furthermore, mutants did not show chlorophyll degradation and showed less leaf damage and acceptable plant growth. Other parameters including proline and soluble sugars, did not prove decisive in the salinity resistance of these genotypes.

The miR399-CsUBC24 Module Regulates Reproductive Development and Male Fertility in Citrus.

MicroRNA399 (miR399) regulates phosphate homeostasis in plants by down-regulating the expression of PHOSPHATE2 (PHO2, or UBC24 encoding the ubiquitin-conjugating E2 enzyme). We previously identified CsmiR399a.1 in a small RNA sequencing screen of a male-sterile somatic cytoplasmic hybrid (or cybrid) of pummelo (Citrus grandis). Here, we report that miR399 affects reproductive development and male fertility in citrus. Down-regulation of CsmiR399a.1 using a short tandem target mimic (STTM) led to abnormal floral development, inhibition of anther dehiscence, and decreased pollen fertility. When grown in inorganic phosphate (Pi)-sufficient conditions, CsmiR399a.1-STTM plants had lower total phosphorus content in their leaves than the wild type and showed typical symptoms of Pi deficiency. In CsmiR399a.1-STTM plants, the expression of genes involved in starch metabolism and Pi homeostasis was significantly different than in the wild type. Thus, we conclude that miR399-STTM mimicked Pi deficiency, disturbed starch metabolism, and was responsible for pollen grain collapse in the transgenic lines. We identified CsUBC24, a citrus homolog of Arabidopsis (Arabidopsis thaliana) AtUBC24 (PHO2), as a target of CsmiR399a.1 that physically interacts with the floral development regulators SEPALLATA family (CsSEP1.1, CsSEP1.2, and CsSEP3) and the anther dehiscence regulator INDUCER OF CBF EXPRESSION1 (CsICE1). We hypothesize that CsUBC24 downregulates the CsSEPs, which disrupts the floral meristem identity regulatory network and leads to developmental abnormalities in flowers. By interacting with CsICE1, CsUBC24 disturbs stomate function on the anther surface, which inhibits anther dehiscence. These findings indicate that a miR399-based mechanism influences both reproductive development and male fertility in citrus.