Plant volatile-triggered defense in citrus against biotic stressors.

Plants employ sophisticated defense mechanisms, including releasing volatile organic compounds, to defend against biotic and abiotic stresses. These compounds play a crucial role in plant defense by attracting natural enemies and facilitating communication between plants to activate defense mechanisms. However, there has been no research on how exposure to these compounds activates defense mechanisms in citrus plants. To elucidate the underlying mechanisms governing citrus defensive activation, we conducted a molecular analysis of the rootstock Citrange carrizo [a hybrid of Citrus sinensis × Poncirus trifoliata] in response to defense activation by the volatile (Z)-3-hexenyl propanoate [(Z)-3-HP], utilizing a groundbreaking transcriptomic analysis involving the genomes of both parental species. Our results revealed significant gene expression changes, notably the overexpression of genes related to plant immunity, antioxidant activity, defense against herbivores, and tolerance to abiotic stress. Significantly, P. trifoliata contributed most notably to the hybrid's gene expression profile in response to (Z)-3-HP. Additionally, plants exposed to (Z)-3-HP repelled several citrus pests, attracted natural predators, and led to diminished performance of two key citrus pests. Our study emphasizes the complex molecular basis of volatile-triggered defenses in citrus and highlights the potential of plant volatiles in pest control strategies.

Silicon nanoparticles confer hypoxia tolerance in citrus rootstocks by modulating antioxidant activities and carbohydrate metabolism.

Citrus is a remarkable fruit crop, extremely sensitive to flooding conditions, which frequently trigger hypoxia stress and cause severe damage to citrus plants. Silicon nanoparticles (SiNPs) are beneficial and have the potential to overcome this problem. Therefore, the present study aimed to investigate the effect of silicon nanoparticles to overcome hypoxia stress through modulating antioxidant enzyme activity and carbohydrate metabolism. Three citrus rootstocks (Carrizo citrange, Roubidoux, and Rich 16-6) were exposed to flooding (with and without oxygen) through different SiNP treatments via foliar and root zone. SiNPs applied treatment plants showed a significant increase in photosynthesis, leaf greenness, antioxidant enzymes, and carbohydrate metabolic activities, besides the higher accumulation of proline and glycine betaine. The rate of lipid peroxidation was drastically higher in flooded plants; however, SiNPs application reduced it significantly, ultimately reducing oxidative damage. Overall, Rich16-6 rootstock showed good performance via root zone application compared to other rootstocks, possibly due to genotypical variation in silicon uptake. Our outcomes demonstrate that SiNPs significantly affect plant growth during hypoxia stress conditions, and their use is an optimal strategy to overcome this issue. This study laid the foundation for future research to use at the commercial level to overcome hypoxia stress and a potential platform for future research.

Rootstocks with Different Tolerance Grade to Citrus Tristeza Virus Induce Dissimilar Volatile Profile in Citrus sinensis and Avoidance Response in the Vector Aphis gossypii Glover.

The citrus tristeza virus (CTV) is an agent of devastating epidemics of the citrus plant grafted on Citrus aurantium, one of the main rootstocks still used in the Mediterranean area. Consequently, CTV-tolerant alternative citrus rootstocks are considered necessary to manage this disease and/or its vector; that in Mediterranean countries is the aphid Aphis gossypii. In this study, we analyzed the VOCs emitted from Citrus sinensis plants grafted on the CTV-susceptible C. aurantium and on the CTV-tolerant Volkamer lemon, Forner-Alcaide no. 5, and Carrizo citrange. Furthermore, the aphid preference/avoidance response toward these combinations was evaluated in a semi-field experiment. The VOC profiles recorded on the leaves of C. sinensis grafted on the four rootstocks listed above showed significant differences in the abundances and ratios of the compounds emitted. The behavioral experiments indicated that A. gossypii prefers to orient and establish on the C. sinensis plants grafted on C. aurantium rather than on that grafted on the three CTV-tolerant varieties. The possibility that this avoidance mechanism is triggered by the different profile of the VOC emitted by the different combinations and the consequent susceptibility/tolerance shown toward CTV is discussed.

Comparative transcriptomic analyses of citrus cold-resistant vs. sensitive rootstocks might suggest a relevant role of ABA signaling in triggering cold scion adaption.

BACKGROUND: The citrus genus comprises a number of sensitive tropical and subtropical species to cold stress, which limits global citrus distribution to certain latitudes and causes major economic loss. We used RNA-Seq technology to analyze changes in the transcriptome of Valencia delta seedless orange in response to long-term cold stress grafted on two frequently used citrus rootstocks: Carrizo citrange (CAR), considered one of the most cold-tolerant accessions; C. macrophylla (MAC), a very sensitive one. Our objectives were to identify the genetic mechanism that produce the tolerant or sensitive phenotypes in citrus, as well as to gain insights of the rootstock-scion interactions that induce the cold tolerance or sensitivity in the scion. RESULTS: Plants were kept at 1 ºC for 30 days. Samples were taken at 0, 15 and 30 days. The metabolomic analysis showed a significant increase in the concentration of free sugars and proline, which was higher for the CAR plants. Hormone quantification in roots showed a substantially increased ABA concentration during cold exposure in the CAR roots, which was not observed in MAC. Different approaches were followed to analyze gene expression. During the stress treatment, the 0-15-day comparison yielded the most DEGs. The functional characterization of DEGs showed enrichment in GO terms and KEGG pathways related to abiotic stress responses previously described in plant cold adaption. The DEGs analysis revealed that several key genes promoting cold adaption were up-regulated in the CAR plants, and those repressing it had higher expression levels in the MAC samples. CONCLUSIONS: The metabolomic and transcriptomic study herein performed indicates that the mechanisms activated in plants shortly after cold exposure remain active in the long term. Both the hormone quantification and differential expression analysis suggest that ABA signaling might play a relevant role in promoting the cold hardiness or sensitiveness of Valencia sweet orange grafted onto Carrizo citrange or Macrophylla rootstocks, respectively. Our work provides new insights into the mechanisms by which rootstocks modulate resistance to abiotic stress in the production variety grafted onto them.

In Planta Localization of Endophytic Cordyceps fumosorosea in Carrizo Citrus.

Entomopathogenic fungi can be a useful resource for controlling insect vectors of citrus plant pathogens, such as the Asian citrus psyllid (Diaphorina citri) associated with huanglongbing or the citrus root weevil (Diaprepes abbreviatus) associated with the spread of Phytophtora spp. In this study, Cordyceps fumosorosea (Cfr) was investigated in planta as a potential endophytic entomopathogenic fungus and various inoculation techniques were used to determine if it would colonize the Carrizo citrange (Citrus × insitorum) seeds and plants. The four inoculation methodologies evaluated were seed soaking, stem injection, foliar spray, and soil drench. Seed immersion trials demonstrated that the roots of the Carrizo citrange plant can be inoculated successfully with Cfr. Stem injection, foliar spray, and soil drench also provided successful inoculation of Cfr. However, this fungus was only endophytic in the plant stem. Sand cores indicated that Cfr moved down through the sand column and was able to inoculate the roots. Given the prevalence of Cfr in the soil during the drench experiment, and that the fungus was able to colonize Carrizo citrange roots through seed immersion, this finding provides evidence of the potential endophytism of this fungus when applied to citrus plant species.

Arbuscular mycorrhizal symbiosis improves tolerance of Carrizo citrange to excess boron supply by reducing leaf B concentration and toxicity in the leaves and roots.

This study explores the possibility of using mycorrhization as a novel technique for diminishing the negative effects of boron (B) in the nutrient solution on seedlings of Carrizo citrange rootstock plants. For this, an experiment was planned for studying the physiological (gas exchange and chlorophyll fluorescence parameters), morphological (vegetative growth parameters), nutritional (organic solutes, carbohydrates) and oxidative stress responses of seedlings that were either mycorrhized (+AM, Rhizophagus irregularis; previously known as Glomus intraradices) or not mycorrhized (-AM), and irrigated with water containing different concentrations of B (0.5, 5 and 10 mg L-1). It was observed that an excess of B in the nutrient solution decreased the vegetative growth in both +AM and -AM plants, but this decrease was greater in -AM plants. Mycorrhized plants (+AM) under high B concentration accumulated less B in the leaves, and had a smaller reduction of net assimilation rate of CO2 and lower MDA concentration than non-mycorrhized plants. Thus, it can be concluded that mycorrhization increased the tolerance to high boron concentration in the irrigation water of citrange Carrizo seedlings by reducing both the B concentration in the plant tissue and the B toxicity in the physiological processes. The study of organic solutes and carbohydrates also pointed to a different response model between +AM and -AM plants that could be related to the different tolerance observed between these plants.

Involvement of ascorbate peroxidase and heat shock proteins on citrus tolerance to combined conditions of drought and high temperatures.

Usually several environmental stresses occur in nature simultaneously causing a unique plant response. However, most of the studies until now have focused in individually-applied abiotic stress conditions. Carrizo citrange (Poncirus trifoliata L. Raf. X Citrus sinensis L. Osb.) and Cleopatra mandarin (Citrus reshni Hort. ex Tan.) are two citrus rootstocks with contrasting tolerance to drought and heat stress and have been used in this work as a model for the study of plant tolerance to the combination of drought and high temperatures. According to our results, leaf integrity and photosynthetic machinery are less affected in Carrizo than in Cleopatra under combined conditions of drought and heat stress. The pattern of accumulation of three proteins (APX, HSP101 and HSP17.6) involved in abiotic stress tolerance shows that they do not accumulate under water stress conditions individually applied. However, contents of APX and HSP101 are higher in Carrizo than in Cleopatra under stress combination whereas HSP17.6 has a similar behavior in both types of plants. This, together with a better stomatal control and a higher APX activity of Carrizo, contributes to the higher tolerance of Carrizo plants to the combination of stresses and point to it as a better rootstock than Cleopatra (traditionally used in areas with scare water supplies) under the predictable future climatic conditions with frequent periods of drought combined with high temperatures. This work also provides the basis for testing the tolerance of different citrus varieties grafted on these rootstocks and growing under different field conditions.

Modulation of Antioxidant Defense System Is Associated with Combined Drought and Heat Stress Tolerance in Citrus.

Drought and high temperatures are two major abiotic stress factors that often occur simultaneously in nature, affecting negatively crop performance and yield. Moreover, these environmental challenges induce oxidative stress in plants through the production of reactive oxygen species (ROS). Carrizo citrange and Cleopatra mandarin are two citrus genotypes with contrasting ability to cope with the combination of drought and heat stress. In this work, a direct relationship between an increased antioxidant activity and stress tolerance is reported. According to our results, the ability of Carrizo plants to efficiently coordinate superoxide dismutase (SOD), ascorbate peroxidase (APX), catalase (CAT), and glutathione reductase (GR) activities involved in ROS detoxification along with the maintenance of a favorable GSH/GSSG ratio could be related to their relative tolerance to this stress combination. On the other hand, the increment of SOD activity and the inefficient GR activation along with the lack of CAT and APX activities in Cleopatra plants in response to the combination of drought and heat stress, could contribute to an increased oxidative stress and the higher sensibility of this citrus genotype to this stress combination.

Biolistic transformation of Carrizo citrange (Citrus sinensis Osb. × Poncirus trifoliata L. Raf.).

KEY MESSAGE: The development of transgenic citrus plants by the biolistic method. A protocol for the biolistic transformation of epicotyl explants and transgenic shoot regeneration of immature citrange rootstock, cv. Carrizo (Citrus sinensis Osb. × Poncirus trifoliata L. Raf.) and plant regeneration is described. Immature epicotyl explants were bombarded with a vector containing the nptII selectable marker and the gfp reporter. The number of independent, stably transformed tissues/total number of explants, recorded by monitoring GFP fluorescence 4 weeks after bombardment was substantial at 18.4 %, and some fluorescing tissues regenerated into shoots. Fluorescing GFP, putative transgenic shoots were micro-grafted onto immature Carrizo rootstocks in vitro, confirmed by PCR amplification of nptII and gfp coding regions, followed by secondary grafting onto older rootstocks grown in soil. Southern blot analysis indicated that all the fluorescing shoots were transgenic. Multiple and single copies of nptII integrations were confirmed in five regenerated transgenic lines. There is potential to develop a higher throughput biolistics transformation system by optimizing the tissue culture medium to improve shoot regeneration and narrowing the window for plant sampling. This system will be appropriate for transformation with minimal cassettes.

Tolerance of citrus plants to the combination of high temperatures and drought is associated to the increase in transpiration modulated by a reduction in abscisic acid levels.

BACKGROUND: In natural environments, several adverse environmental conditions occur simultaneously constituting a unique stress factor. In this work, physiological parameters and the hormonal regulation of Carrizo citrange and Cleopatra mandarin, two citrus genotypes, in response to the combined action of high temperatures and water deprivation were studied. The objective was to characterize particular responses to the stress combination. RESULTS: Experiments indicated that Carrizo citrange is more tolerant to the stress combination than Cleopatra mandarin. Furthermore, an experimental design spanning 24 h stress duration, heat stress applied alone induced higher stomatal conductance and transpiration in both genotypes whereas combined water deprivation partially counteracted this response. Comparing both genotypes, Carrizo citrange showed higher phostosystem-II efficiency and lower oxidative damage than Cleopatra mandarin. Hormonal profiling in leaves revealed that salicylic acid (SA) accumulated in response to individual stresses but to a higher extent in samples subjected to the combination of heat and drought (showing an additive response). SA accumulation correlated with the up-regulation of pathogenesis-related gene 2 (CsPR2), as a downstream response. On the contrary, abscisic acid (ABA) accumulation was higher in water-stressed plants followed by that observed in plants under stress combination. ABA signaling in these plants was confirmed by the expression of responsive to ABA-related gene 18 (CsRAB18). Modulation of ABA levels was likely carried out by the induction of 9-neoxanthin cis-epoxicarotenoid dioxygenase (CsNCED) and ABA 8'-hydroxylase (CsCYP707A) while conversion to ABA-glycosyl ester (ABAGE) was a less prominent process despite the strong induction of ABA O-glycosyl transferase (CsAOG). CONCLUSIONS: Cleopatra mandarin is more susceptible to the combination of high temperatures and water deprivation than Carrizo citrange. This is likely a result of a higher transpiration rate in Carrizo that could allow a more efficient cooling of leaf surface ensuring optimal CO2 intake. Hence, SA induction in Cleopatra was not sufficient to protect PSII from photoinhibition, resulting in higher malondialdehyde (MDA) build-up. Inhibition of ABA accumulation during heat stress and combined stresses was achieved primarily through the up-regulation of CsCYP707A leading to phaseic acid (PA) and dehydrophaseic acid (DPA) production. To sum up, data indicate that specific physiological responses to the combination of heat and drought exist in citrus. In addition, these responses are differently modulated depending on the particular stress tolerance of citrus genotypes.

Activation of Secondary Metabolism in Citrus Plants Is Associated to Sensitivity to Combined Drought and High Temperatures.

Drought and heat stresses are two of the most frequent environmental factors that take place simultaneously in the field constraining global crop productivity. Metabolism reconfiguration is often behind the adaptation of plants to adverse environmental conditions. Carrizo citrange and Cleopatra mandarin, two citrus genotypes with contrasting ability to tolerate combined heat and drought conditions, showed different metabolite patterns. Increased levels of phenylpropanoid metabolites were observed in Cleopatra in response to stress, including scopolin, a metabolite involved in defense mechanisms. Tolerant Carrizo accumulated sinapic acid and sinapoyl aldehyde, direct precursors of lignins. Finally, Cleopatra showed an accumulation of flavonols and glycosylated and polymethoxylated flavones such as tangeritin. The activation of flavonoid biosynthesis in Cleopatra could be aimed to mitigate the higher oxidative damage observed in this genotype. In general, limonoids were more severely altered in Cleopatra than in Carrizo in response to stress imposition. To conclude, all metabolite changes observed in Cleopatra suggest the activation of energy metabolism along with metabolic pathways leading to the accumulation of photoprotective and antioxidant secondary metabolites, oriented to mitigate the damaging effects of stress. Conversely, the higher ability of Carrizo to retain a high photosynthetic activity and to cope with oxidative stress allowed the maintenance of the metabolic activity and prevented the accumulation of antioxidant metabolites.

Factors affecting Agrobacterium-mediated transformation in Citrus and production of sour orange (Citrus aurantium L.) plants expressing the coat protein gene of citrus tristeza virus.

Factors influencing transformation frequencies using the Agrobacterium-mediated protocol developed for Citrus seedling internodal stem segments in this laboratory were evaluated, with particular emphasis on decreasing the numbers of ``escape'' shoots produced. Although the use of a wild-type ``shooty'' Agrobacterium strain allowed relatively high frequencies of ß-glucuronidase positive (GUS+) shoots to be produced, none of the shoots were free of wild-type T-DNA and would not root. Both use of a liquid medium/kanamycin overlay and horizontal placement of stem segments increased the efficiency of kanamycin selection. Wounding via particle bombardment prior to Agrobacterium inoculation did not increase transformation frequencies. The concentration of benzyladenine (BA) in the regeneration/selection medium inversely influenced the numbers of shoots that regenerated and the subsequent ability of the shoots to root. Regeneration in the presence of kanamycin also influenced the ability of shoots to root. Many of the shoots that regenerated on selection medium were chimeric for GUS expression, and plants established from such shoots ranged from non-staining to solidly staining for GUS. However, solidly transformed plants with integrated T-DNA were obtained, and these plants have maintained the expression of transgenes over several years. The transgenic plants include ones of sour orange (C. aurantium L.) and Key lime (C. aurantifolia (Christm.) Swing.), two species not previously transformed, and have integrated and express the coat protein gene of citrus tristeza virus. This is the first report of a potentially agriculturally important transgene being expressed in Citrus.

ACCUMULATION OF PHOSPHORUS, DRY MATTER AND BETAINE DURING NaCl STRESS OF SPLIT-ROOT CITRUS SEEDLINGS COLONIZED WITH VESICULAR-ARBUSCULAR MYCORRHIZAL FUNGI ON ZERO, ONE OR TWO HALVES.

Sodium chloride tolerance and phosphorus content were examined in split-root Carrizo citrange seedlings [Poncirus trifoliata (L.) Raf. ×Citrus sinensis (L.) Osbeck] colonized with a vesicular-arbuscular mycorrhizal fungus (Glomus intraradices Schenck & Smith) on zero, one or two root halves. Plants were treated with NaCl at 0, 25, 50 or 100 mM, and the degree of stress was measured as reduction of dry matter accumulation and rise in level of leaf proline-betaine (stachydrine). Shoot and root dry weight production during this period decreased with increasing levels of salt. Absolute reductions were similar for plants inoculated on one vs two half-root systems, but percentage decreases were less in the latter due to greater overall growth in all treatments. Betaine levels in leaf tissues were positively related to soil salt levels for each mycorrhizal treatment. Significant differences in betaine levels were also detected in plants with and without mycorrhizal fungi, and mean levels tended to be higher for those colonized on one vs two halves of their root system. In contrast, a half-root system and its fungal symbiont supplied enough phosphorus to allow concentrations of leaf P to equal those of fully infected root systems, yet the two groups did not show equal growth under control conditions or percentage reductions with NaCl stress.