NGS transcriptomic analysis uncovers the possible resistance mechanisms of olive to Spilocea oleagina leaf spot infection.

Spilocea oleagina is a dangerous obligate fungal pathogen of olive, feared in the Mediterranean countries, causing Peacock's eye or leaf spot infection, which can lead to a serious yield loss of approximately 20% or higher depending on climatic conditions. Coping with this disease is much more problematic for organic farms. To date, knowledge on the genetic control of possible mechanisms of resistance/low susceptibility is quite limited. In this work, comparative transcriptomic analysis (RNA-seq) was conducted in leaf tissues of a low susceptible cultivar Koroneiki and a high susceptible cultivar Nocellara del Belice, both tested in the field using the NaOH test, considering two stages-"zero sign of disease" and "evident sign of infection". Cultivars showed a very large number of differentially expressed genes (DEGs) in both stages. 'Koroneiki' showed an extensive hormonal crosstalk, involving Abscisic acid (ABA) and ethylene synergistically acting with Jasmonate, with early signaling of the disease and remarkable defense responses against Spilocea through the over-expression of many resistance gene analogs or pathogenesis-related (PR) genes: non-specific lipid-transfer genes (nsLTPs), LRR receptor-like serine/threonine-protein kinase genes, GDSL esterase lipase, defensin Ec-AMP-D2-like, pathogenesis-related leaf protein 6-like, Thaumatin-like gene, Mildew resistance Locus O (MLO) gene, glycine-rich protein (GRP), MADS-box genes, STH-21-like, endochitinases, glucan endo-1,3-beta-glucosidases, and finally, many proteinases. Numerous genes involved in cell wall biogenesis, remodeling, and cell wall-based defense, including lignin synthesis, were also upregulated in the resistant cultivar, indicating the possible role of wall composition in disease resistance. It was remarkable that many transcription factors (TS), some of which involved in Induced Systemic Resistance (ISR), as well as some also involved in abiotic stress response, were found to be uniquely expressed in 'Koroneiki', while 'Nocellara del Belice' was lacking an effective system of defense, expressing genes that overlap with wounding responses, and, to a minor extent, genes related to phenylpropanoid and terpenoid pathways. Only a Thaumatin-like gene was found in both cultivars showing a similar expression. In this work, the genetic factors and mechanism underlying the putative resistance trait against this fungal pathogen were unraveled for the first time and possible target genes for breeding resistant olive genotypes were found.

Physiological and Structural Responses to Prolonged Water Deficit in Young Trees of Two Olive Cultivars.

This study aimed to characterize the physiological and structural responses of potted one-year-old olive trees belonging to two olive cultivars­'Nocellara del Belice' and 'Cerasuola'­exposed to prolonged drought under greenhouse conditions. Two irrigation treatments based on evapotranspiration (ET) were imposed for 69 days, i.e., well-watered (WW, 100% ET) and drought-stressed (DS, 10−30% ET). Leaf stomatal conductance (gs), stem water potential (Ψstem), transpiration (E), photosynthetic capacity (Amax), water use efficiency (WUE), stem (Kstem) and root (Kroot) hydraulic conductance, trunk diameter variations (TDV), and leaf patch attenuated pressure fluctuations (pp, a proxy of the inverse of leaf turgor pressure) were measured in WW and DS trees at different stages of the experiment. Leaf gs did not significantly differ between cultivars under DS, whereas differences in Ψstem only became significant at the end of prolonged drought, when 'Nocellara del Belice' experienced Ψstem < −4 MPa. 'Cerasuola' trees expressed the best WUE under drought, although they were more susceptible to photoinhibition under optimal plant water status. Both cultivars tended to increase their Kstem at the end of the drought period. A marked reduction in Kroot occurred in 'Cerasuola' plants after prolonged drought; however, a similar mechanism was not observed in 'Nocellara del Belice'. The ratio between Kstem and Kroot exponentially increased towards the end of the prolonged drought period in both cultivars, but more markedly in 'Cerasuola'. TDV and pp trends suggested that 'Cerasuola' plants keep better plant water status under severe drought compared to 'Nocellara del Belice' by maintaining high leaf turgor and reduced trunk diameter fluctuations. These responses may be related to reduced cell wall elasticity and xylem vessel size and/or wall thickness­drought avoidance mechanisms. The Kstem/Kroot ratio can serve as an indicator of drought stress avoidance mechanisms to compare genotype-specific responses to drought stress.

Establishing a Reference Baseline for Midday Stem Water Potential in Olive and Its Use for Plant-Based Irrigation Management.

Midday stem water potential (SWP) is rapidly becoming adopted as a standard tool for plant-based irrigation management in many woody perennial crops. A reference or "baseline" SWP has been used in some crops (almond, prune, grape, and walnut) to account for the climatic influence of air vapor pressure deficit (VPD) on SWP under non-limiting soil moisture conditions. The baseline can be determined empirically for field trees maintained under such non-limiting conditions, but such conditions are difficult to achieve for an entire season. We present the results of an alternative survey-based approach, using a large set of SWP and VPD data collected over multiple years, from irrigation experiments in olive orchards located in multiple countries [Spain, United States (California), Italy, and Argentina]. The relation of SWP to midday VPD across the entire data set was consistent with an upper limit SWP which declined with VPD, with the upper limit being similar to that found in Prunus. A best fit linear regression estimate for this upper limit (baseline) was found by selecting the maximum R 2 and minimum probability for various upper fractions of the SWP/VPD relation. In addition to being surprisingly similar to the Prunus baseline, the olive baseline was also similar (within 0.1 MPa) to a recently published mechanistic olive soil-plant-atmosphere-continuum (SPAC) model for "super high density" orchard systems. Despite similarities in the baseline, the overall physiological range of SWP exhibited by olive extends to about -8 MPa, compared to about -4 MPa for economically producing almond. This may indicate that, despite species differences in physiological responses to low water availability (drought), there may be convergent adaptations/acclimations across species to high levels of water availability. Similar to its use in other crops, the olive baseline will enable more accurate and reproducible plant-based irrigation management for both full and deficit irrigation practices, and we present tentative SWP guidelines for this purpose.

Algerian Olive Germplasm and Its Relationships with the Central-Western Mediterranean Varieties Contributes to Clarify Cultivated Olive Diversification.

Olive tree with its main final product, olive oil, is an important element of Mediterranean history, considered the emblematic fruit of a civilization. Despite its wide diffusion and economic and cultural importance, its evolutionary and phylogenetic history is still difficult to clarify. As part of the Mediterranean basin, Algeria was indicated as a secondary diversification center. However, genetic characterization studies from Maghreb area, are currently underrepresented. In this context, we characterized 119 endemic Algerian accessions by using 12 microsatellite markers with the main goal to evaluate the genetic diversity and population structure. In order to provide new insights about the history of olive diversification events in the Central-Western Mediterranean basin, we included and analyzed a sample of 103 Italian accessions from Sicily and, a set of molecular profiles of cultivars from the Central-Western Mediterranean area. The phylogenetic investigation let us to evaluate genetic relationships among Central-Mediterranean basin olive germplasm, highlight new synonymy cases to support the importance of vegetative propagation in the cultivated olive diffusion and consolidate the hypothesis of more recent admixture events occurrence. This work provided new information about Algerian germplasm biodiversity and contributed to clarify olive diversification process.

Detecting Mild Water Stress in Olive with Multiple Plant-Based Continuous Sensors.

A comprehensive characterization of water stress is needed for the development of automated irrigation protocols aiming to increase olive orchard environmental and economical sustainability. The main aim of this study is to determine whether a combination of continuous leaf turgor, fruit growth, and sap flow responses improves the detection of mild water stress in two olive cultivars characterized by different responses to water stress. The sensitivity of the tested indicators to mild stress depended on the main mechanisms that each cultivar uses to cope with water deficit. One cultivar showed pronounced day to day changes in leaf turgor and fruit relative growth rate in response to water withholding. The other cultivar reduced daily sap flows and showed a pronounced tendency to reach very low values of leaf turgor. Based on these responses, the sensitivity of the selected indicators is discussed in relation to drought response mechanisms, such as stomatal closure, osmotic adjustment, and tissue elasticity. The analysis of the daily dynamics of the monitored parameters highlights the limitation of using non-continuous measurements in drought stress studies, suggesting that the time of the day when data is collected has a great influence on the results and consequent interpretations, particularly when different genotypes are compared. Overall, the results highlight the need to tailor plant-based water management protocols on genotype-specific physiological responses to water deficit and encourage the use of combinations of plant-based continuously monitoring sensors to establish a solid base for irrigation management.

Transcriptomic Analysis of the Pistacia vera (L.) Fruits Enable the Identification of Genes and Hormone-Related Gene Linked to Inflorescence Bud Abscission.

Pistacia vera (L.) is an alternate bearing species. The tree produces axillary inflorescence buds every year. Still, they abscise in "ON" overloaded shoots, causing a limited production in the following "OFF" year, causing a significant and unfavorable production fluctuation. In this work, we carried out de novo discovery and transcriptomic analysis in fruits of "ON" and "OFF" shoots of the cultivar Bianca. We also investigated whether the fruit signaling pathway and hormone biosynthesis directly or indirectly linked to the premature fall of the inflorescence buds causing alternate bearing. We identified 1536 differentially expressed genes (DEGs) in fruits of "ON" vs. "OFF" shoots, which are involved primarily in sugar metabolism, plant hormone pathways and transcription factors. The premature bud abscission linked to the phenomenon is attributable to a lack of nutrients (primarily sugar) and the possible competition between the same branches' sinks (fruits vs. inflorescence buds). Hormone pathways are involved as a response to signals degradation and remobilization of carbon and nutrients due to the strengthening of the developing embryos. Genes of the secondary metabolism and transcription factors are also involved in tailoring the individual branches response to the nutritional stress and sink competition. Crosstalk among sugar and various hormone-related genes, e.g., ethylene, auxin, ABA and cytokinin, were determined. The discovery of putative biomarkers like callose synthase 5, trehalose-6-phosphate synthase, NAD(P)-linked oxidoreductase and MIOX2, Jasmonate, and salicylic acid-related genes can help to design precision farming practices to mitigate the alternate bearing phenomenon to increase farming profitability. The aim of the analysis is to provide insight into the gene expression profiling of the fate of "ON" and "OFF" fruits associated with the alternate bearing in the pistachio.

Gaining Insight into Exclusive and Common Transcriptomic Features Linked to Drought and Salinity Responses across Fruit Tree Crops.

The present study aimed at identifying and mapping key genes expressed in root tissues involved in drought and salinity tolerance/resistance conserved among different fruit tree species. Twenty-six RNA-Seq samples were analyzed from six published studies in five plant species (Olea europaea, Vitis riparia Michx, Prunus mahaleb, Prunus persica, Phoenix dactylifera). This meta-analysis used a bioinformatic pipeline identifying 750 genes that were commonly modulated in three salinity studies and 683 genes that were commonly regulated among three drought studies, implying their conserved role in resistance/tolerance/response to these environmental stresses. A comparison was done on the genes that were in common among both salinity and drought resulted in 82 genes, of which 39 were commonly regulated with the same trend of expression (23 were upregulated and 16 were downregulated). Gene set enrichment and pathway analysis pointed out that pathways encoding regulation of defense response, drug transmembrane transport, and metal ion binding are general key molecular responses to these two abiotic stress responses. Furthermore, hormonal molecular crosstalk plays an essential role in the fine-tuning of plant responses to drought and salinity. Drought and salinity induced a different molecular "hormonal fingerprint". Dehydration stress specifically enhanced multiple genes responsive to abscisic acid, gibberellin, brassinosteroids, and the ethylene-activated signaling pathway. Salt stress mostly repressed genes encoding for key enzymes in signaling proteins in auxin-, gibberellin-(gibberellin 2 oxidase 8), and abscisic acid-related pathways (aldehyde oxidase 4, abscisic acid-responsive element-binding protein 3). Abiotic stress-related genes were mapped into the chromosome to identify molecular markers usable for the improvement of these complex quantitative traits. This meta-analysis identified genes that serve as potential targets to develop cultivars with enhanced drought and salinity resistance and/or tolerance across different fruit tree crops in a biotechnological sustainable way.

Transcriptome Analysis of Pistacia vera Inflorescence Buds in Bearing and Non-Bearing Shoots Reveals the Molecular Mechanism Causing Premature Flower Bud Abscission.

The alteration of heavy ("ON/bearing") and light ("OFF/non-bearing") yield in pistachio (Pistacia vera L.) has been reported to result from the abscission of inflorescence buds on high yielding trees during the summer, but the regulatory mechanisms involved in this bud abscission remain unclear. The analysis provides insights into the transcript changes between inflorescence buds on bearing and non-bearing shoots, that we indicated as "ON" and "OFF", and shed light on the molecular mechanisms causing premature inflorescence bud abscission in the pistachio cultivar "Bianca" which can be related to the alternate bearing behavior. In this study, a transcriptome analysis was performed in inflorescence buds of "ON" and "OFF" shoots. A total of 14,330 differentially expressed genes (DEGs), most of which are involved in sugar metabolism, plant hormone pathways, secondary metabolism and oxidative stress pathway, were identified. Our results shed light on the molecular mechanisms underlying inflorescence bud abscission in pistachio and we proposed a hypothetical model behind the molecular mechanism causing this abscission in "ON" shoots. Results highlighted how changes in genes expressed in nutrient pathways (carbohydrates and mineral elements) in pistachio "ON" vs. "OFF" inflorescence buds triggers a cascade of events involving trehalose-6-phosphate and target of rapamycin (TOR) signaling, SnRK1 complex, hormones, polyamines and ROS which end, through programmed cell death and autophagy phenomena, with the abscission of inflorescence buds. This is the first study reporting gene expression profiling of the fate of "ON" and "OFF" inflorescence buds associated with the alternate bearing in the pistachio.

A Cultivar-Sensitive Approach for the Continuous Monitoring of Olive (Olea europaea L.) Tree Water Status by Fruit and Leaf Sensing.

Sustainable irrigation is crucial to reduce water use and management costs in modern orchard systems. Continuous plant-based sensing is an innovative approach for the continuous monitoring of plant water status. Olive (Olea europaea L.) genotypes can respond to drought using different leaf and fruit physiological and morphological mechanisms. This study aimed to identify whether fruit and leaf water dynamics of two different olive cultivars were differently affected by water deficit and their response to changes of midday stem water potential (Ψstem), the most common indicator of plant water status. Plant water status indicators such as leaf stomatal conductance (gs) and Ψstem were measured in the Sicilian olive cultivars Nocellara del Belice (NB) and Olivo di Mandanici (MN), in stage II and III of fruit development. Fruit gauges and leaf patch clamp pressure probes were mounted on trees and their raw data were converted in relative rates of fruit diameter change (RRfruit) and leaf pressure change (RRleaf), sensitive indicators of tissue water exchanges. The analysis of diel, diurnal and nocturnal fluctuations of RRfruit and RRleaf highlighted differences, often opposite, between the two cultivars under water deficit. A combination of statistical parameters extrapolated from RRfruit and RRleaf diurnal and nocturnal curves were successfully used to obtain significant multiple linear models for the estimation of midday Ψstem. Fruit and leaf water exchanges suggest that olive cultivar can either privilege fruit or leaf water status, with MN likely preserving leaf water status and NB increasing fruit tissue elasticity under severe water deficit. The results highlight the advantages of the integration of fruit and leaf water dynamics to estimate plant water status and the need for genotype-specific models in olive.

Transcriptomic responses to biotic stresses in Malus x domestica: a meta-analysis study.

RNA-Seq analysis is a strong tool to gain insight into the molecular responses to biotic stresses in plants. The objective of this work is to identify specific and common molecular responses between different transcriptomic data related to fungi, virus and bacteria attacks in Malus x domestica. We analyzed seven transcriptomic datasets in Malus x domestica divided in responses to fungal pathogens, virus (Apple Stem Grooving Virus) and bacteria (Erwinia amylovora). Data were dissected using an integrated approach of pathway- and gene- set enrichment analysis, Mapman visualization tool, gene ontology analysis and inferred protein-protein interaction network. Our meta-analysis revealed that the bacterial infection enhanced specifically genes involved in sugar alcohol metabolism. Brassinosteroids were upregulated by fungal pathogens while ethylene was highly affected by Erwinia amylovora. Gibberellins and jasmonates were strongly repressed by fungal and viral infections. The protein-protein interaction network highlighted the role of WRKYs in responses to the studied pathogens. In summary, our meta-analysis provides a better understanding of the Malus X domestica transcriptome responses to different biotic stress conditions; we anticipate that these insights will assist in the development of genetic resistance and acute therapeutic strategies. This work would be an example for next meta-analysis works aiming at identifying specific common molecular features linked with biotic stress responses in other specialty crops.