Hub Genes and Pathways Related to Lemon (Citrus limon) Leaf Response to Plenodomus tracheiphilus Infection and Influenced by Pseudomonas mediterranea Biocontrol Activity.

The lemon industry in the Mediterranean basin is strongly threatened by "mal secco" disease (MSD) caused by the fungus Plenodomus tracheiphlilus. Leaf pretreatments with Pseudomonas mediterranea 3C have been proposed as innovative tools for eco-sustainable interventions aimed at controlling the disease. In this study, by exploiting the results of previously performed RNAseq analysis, WCGNA was conducted among gene expression patterns in both inoculated (Pt) and pretreated and fungus-inoculated lemon plants (Citrus limon L.) (3CPt), and two indicators of fungal infection, i.e., the amount of fungus DNA measured in planta and the disease index (DI). The aims of this work were (a) to identify gene modules significantly associated with those traits, (b) to construct co-expression networks related to mal secco disease; (c) to define the effect and action mechanisms of P. mediterranea by comparing the networks. The results led to the identification of nine hub genes in the networks, with three of them belonging to receptor-like kinases (RLK), such as HERK1, CLAVATA1 and LRR, which play crucial roles in plant-pathogen interaction. Moreover, the comparison between networks indicated that the expression of those receptors is not induced in the presence of P. mediterranea, suggesting how powerful WCGNA is in discovering crucial genes that must undergo further investigation and be eventually knocked out.

Investigating the impact of pedoclimatic conditions on the oenological performance of two red cultivars grown throughout southern Italy.

The cultivated grapevine, Vitis vinifera subsp. vinifera, possesses a rich biodiversity with numerous varieties. Each variety adapts differently to varying pedoclimatic conditions, which greatly influence the terroir expression of wine regions. These conditions impact vine growth, physiology, and berry composition, ultimately shaping the unique characteristics and typicity of the wines produced. Nowadays, the potential of the different adaptation capacities of grape varieties has not yet been thoroughly investigated. We addressed this issue by studying two grape varieties, Aglianico and Cabernet Sauvignon, in two different pedoclimatic conditions of Southern Italy. We evaluated and compared the effect of different pedoclimatic conditions on plant physiology, the microbial quality of grapes using Next-Generation Sequencing (NGS) technology, the expression trends of key genes in ripe berries and the concentration of phenolic compounds in grapes and wines by HPLC-MS, HPLC-DAD, NMR and spectrophotometric analyses. Metabolomic and microbiome data were integrated with quantitative gene expression analyses to examine varietal differences and plasticity of genes involved in important oenological pathways. The data collected showed that the phenotypic response of studied grapes in terms of vigor, production, and fruit quality is strongly influenced by the pedoclimatic conditions and, in particular, by soil physical properties. Furthermore, Aglianico grape variety was more influenced than the Cabernet Sauvignon by environmental conditions. In conclusion, the obtained findings not only reinforce the terroir concept and our comprehension of grape's ability to adapt to climate variations but can also have implications for the future usage of grape genetic resources.

Sicilian Rivet Wheat Landraces: Grain Characteristics and Technological Quality of Flour and Bread.

In recent years, the growth of tetraploid Sicilian wheat landraces has been arousing increasing interest. In this study, eighteen local genotypes of Triticum turgidum subsp. turgidum, belonging to the groups 'Bufala', 'Ciciredda', 'Bivona' and 'Paola', and two cultivars of Triticum turgidum subsp. durum (the old variety 'Bidì', and a more recent variety 'Simeto') were assessed for the characteristics of the grain and bread-making performance of their flours and doughs, as well as the quality of the loaves. The grain of the twenty genotypes came from a field trial conducted during 2018-2019 in south-eastern Sicily. The main commercial features of the grain (thousand kernel weight and hectolitre weight), including the defects (starchy, black pointed and shrunken kernels), were determined. The wholemeal flours and doughs obtained from the grain of each genotype were evaluated for the main technological quality (physico-chemical and rheological characteristics), and processed into loaves, whose main quality indices (volume, height, weight, moisture and porosity) were assessed. The results from such analyses allowed the authors to evaluate the genotypes' bread-making suitability. In particular, for the grain characteristics, hectolitre weight varied from 68.23 ('Bufala Rossa Lunga 01') to 77.43 ('Bidì 03') kg/hL, passing through the typical values for common and durum wheat. Among the grain defects, the black point defect was absent in all the grain samples, except for that of 'Bufala Nera Corta 01' (2%). Dry gluten content varied from 6.22 to 10.23 g/100 g, and sedimentation test values were low or medium-low, with values ranging from 22 to 35 mL. Amylase activity was low and highly variable among the genotypes, with the maximum value observed for 'Bufala Rossa Corta b01' (509 s). The doughs evidenced a poor quality for bread making with alveograph values of W ranging from 12 to 145 (10-4 × Joule) and thus the volume of the loaves varied from 346.25 cm3 of 'Bivona' and 'Ciciredda' to 415.00 cm3 of 'Bufala Rossa Lunga'. A Tandem Cluster Analysis was conducted on a set of all the response variables. The Hierarchical Cluster Analysis was initially run. A five-cluster solution identified three clusters further segmented and two single branches. Overall, the study highlighted the possibility of using some of these landraces alone for the production of traditional breads locally appreciated or together with other ingredients for the production of crumbly baked goods such as substitutes for bread and biscuits.

Transcriptomic profile of lettuce seedlings (Lactuca sativa) response to microalgae extracts used as biostimulant agents.

To reduce the use of chemical fertilizers and maximize agricultural yields, the use of microalgae extracts as biostimulants has recently attracted significant attention due to their favourable impact on both plant growth and their ability to induce tolerance towards environmental stressors. Lettuce (Lactuca sativa) is one of the most important fresh vegetables that often requires applications of chemical fertilizers to increase quality and productivity. Accordingly, the purpose of this study was to analyse the transcriptome reprogramming of lettuce (L. sativa) seedlings in response to either Chlorella vulgaris or Scenedesmus quadricauda extracts by applying an RNAseq approach. Differential gene expression analysis revealed that the core gene set that responded to microalgal treatments in a species-independent manner includes 1330 clusters, 1184 of which were down-regulated and 146 up-regulated, clearly suggesting that the repression of gene expression is the main effect of algal treatments. The deregulation of 7197 transcripts in the C. vulgaris treated seedlings compared to control samples (LsCv vs. LsCK) and 7118 transcripts in the S. quadricauda treated seedlings compared to control samples (LsSq vs. LsCK) were counted. Although the number of deregulated genes turned out to be similar between the algal treatments, the level of deregulation was higher in LsCv versus LsCK than in LsSq versus LsCK. In addition, 2439 deregulated transcripts were observed in the C. vulgaris treated seedlings compared to S. quadricauda treated samples (LsCv vs. LsSq comparison) suggesting that a specific transcriptomic profile was induced by the single algal extracts. 'Plant hormone signal transduction' category includes a very elevated number of DEGs, many of them specifically indicating that C. vulgaris actives both genes involved in the auxin biosynthesis and transduction pathways, whereas S. quadricauda up-regulates genes implicated in the cytokinin biosynthesis pathway. Finally, algal treatments induced the deregulation of genes encoding small hormone-like molecules that are known to act alone or by interacting with major plant hormones. In conclusion, this study offers the groundwork to draw up a list of putative gene targets with the aim of lettuce genetic improvement that will allow a limited or even null use of synthetic fertilizers and pesticides in the management of this crop.

Global leaf and root transcriptome in response to cadmium reveals tolerance mechanisms in Arundo donax L.

The expected increase of sustainable energy demand has shifted the attention towards bioenergy crops. Due to their know tolerance against abiotic stress and relatively low nutritional requirements, they have been proposed as election crops to be cultivated in marginal lands without disturbing the part of lands employed for agricultural purposes. Arundo donax L. is a promising bioenergy crop whose behaviour under water and salt stress has been recently studied at transcriptomic levels. As the anthropogenic activities produced in the last years a worrying increase of cadmium contamination worldwide, the aim of our work was to decipher the global transcriptomic response of A. donax leaf and root in the perspective of its cultivation in contaminated soil. In our study, RNA-seq libraries yielded a total of 416 million clean reads and 10.4 Gb per sample. De novo assembly of clean reads resulted in 378,521 transcripts and 126,668 unigenes with N50 length of 1812 bp and 1555 bp, respectively. Differential gene expression analysis revealed 5,303 deregulated transcripts (3,206 up- and 2,097 down regulated) specifically observed in the Cd-treated roots compared to Cd-treated leaves. Among them, we identified genes related to "Protein biosynthesis", "Phytohormone action", "Nutrient uptake", "Cell wall organisation", "Polyamine metabolism", "Reactive oxygen species metabolism" and "Ion membrane transport". Globally, our results indicate that ethylene biosynthesis and the downstream signal cascade are strongly induced by cadmium stress. In accordance to ethylene role in the interaction with the ROS generation and scavenging machinery, the transcription of several genes (NADPH oxidase 1, superoxide dismutase, ascorbate peroxidase, different glutathione S-transferases and catalase) devoted to cope the oxidative stress is strongly activated. Several small signal peptides belonging to ROTUNDIFOLIA, CLAVATA3, and C-TERMINALLY ENCODED PEPTIDE 1 (CEP) are also among the up-regulated genes in Cd-treated roots functioning as messenger molecules from root to shoot in order to communicate the stressful status to the upper part of the plants. Finally, the main finding of our work is that genes involved in cell wall remodelling and lignification are decisively up-regulated in giant reed roots. This probably represents a mechanism to avoid cadmium uptake which strongly supports the possibility to cultivate giant cane in contaminated soils in the perspective to reserve agricultural soil for food and feed crops.

Transcriptome Analysis of Plenodomus tracheiphilus Infecting Rough Lemon (Citrus jambhiri Lush.) Indicates a Multifaceted Strategy during Host Pathogenesis.

The causal agent of mal secco disease is the fungus Plenodomus tracheiphilus, mainly affecting lemon tree survival in the Mediterranean area. Using a fully compatible host-pathogen interaction, the aim of our work was to retrieve the fungus transcriptome by an RNA seq approach during infection of rough lemon (Citrus jambhiri Lush.) to identify crucial transcripts for pathogenesis establishment and progression. A total of 2438 clusters belonging to P. tracheiphilus were retrieved and classified into the GO and KEGG categories. Transcripts were categorized mainly within the "membrane", "catalytic activity", and "primary metabolic process" GO terms. Moreover, most of the transcripts are included in the "ribosome", "carbon metabolism", and "oxidative phosphorylation" KEGG categories. By focusing our attention on transcripts with FPKM values higher than the median, we were able to identify four main transcript groups functioning in (a) fungus cell wall remodeling and protection, (b) destroying plant defensive secondary metabolites, (c) optimizing fungus development and pathogenesis, and (d) toxin biosynthesis, thus indicating that a multifaceted strategy to subdue the host was executed.

Genetic and Morpho-Agronomic Characterization of Sicilian Tetraploid Wheat Germplasm.

Cereal landraces are a very valuable resource in contemporary agriculture. A renewed focus for breeding purposes could ameliorate some negative consequences of modern agriculture and conventional breeding, such as the loss of genetic diversity. One strategy combining molecular genotyping and characterization of morpho-agronomic traits related to productivity is proposed to assess a group of tetraploid wheat landraces named Bufala, historically cultivated in the mountain areas of Sicily and characterized by adaptability in terms of cold tolerance, ability to grow in marginal soils, weed competitiveness and resistance to diseases. A total of 55 SSR molecular markers were used to detect patterns of diversity in 30 rivet and durum wheat genotypes. Furthermore, phenotyping was then conducted for 8 morpho-agronomic traits. Discriminant analysis of principal components (DAPC), STRUCTURE and phylogenetical analysis allowed to identify three groups, two of them genetically close and including both Bufala and Bufala-related rivet landraces. To the third group, old and more recent durum wheat varieties, constituting the outgroup, were assigned. Clustering was confirmed by Principal Component Analysis (PCA). Finally, a correlation analysis showed that Bufala genotypes are characterized by lower ear density, major ear length and later earing time compared with the other studied genotypes. The levels of diversity and population structure could be an important contribution to parent selection in tetraploid wheat breeding programs, as well as to germplasm conservation and management.

De Novo Transcriptome Sequencing of Rough Lemon Leaves (Citrus jambhiri Lush.) in Response to Plenodomus tracheiphilus Infection.

Mal secco is one of the most severe diseases of citrus, caused by the necrotrophic fungus Plenodomus tracheiphilus. With the main aim of identifying candidate genes involved in the response of citrus plants to "Mal secco", we performed a de novo transcriptome analysis of rough lemon seedlings subjected to inoculation of P. tracheiphilus. The analysis of differential expressed genes (DEGs) highlighted a sharp response triggered by the pathogen as a total of 4986 significant DEGs (2865 genes up-regulated and 2121 down-regulated) have been revealed. The analysis of the most significantly enriched KEGG pathways indicated that a crucial role is played by genes involved in "Plant hormone signal transduction", "Phenylpropanoid biosynthesis", and "Carbon metabolism". The main findings of this work are that under fungus challenge, the rough lemon genes involved both in the light harvesting and the photosynthetic electron flow were significantly down-regulated, thus probably inducing a shortage of energy for cellular functions. Moreover, the systemic acquired resistance (SAR) was activated through the induced salicylic acid cascade. Interestingly, RPM1 interacting protein 4, an essential positive regulator of plant defense, and BIR2, which is a negative regulator of basal level of immunity, have been identified thus representing useful targets for molecular breeding.

Fungal Infection Induces Anthocyanin Biosynthesis and Changes in DNA Methylation Configuration of Blood Orange [Citrus sinensis L. (Osbeck)].

The biosynthesis of sweet orange anthocyanins is triggered by several environmental factors such as low temperature. Much less is known about the effect of biotic stress on anthocyanin production in sweet orange, although in other species anthocyanins are often indicated as "defense molecules". In this work, citrus fruits were inoculated with Penicillium digitatum, the causal agent of green mold, and the amount of anthocyanins and the expression of genes related to their biosynthesis was monitored by RT-real time PCR after 3 and 5 days from inoculation (DPI). Moreover, the status of cytosine methylation of DFR and RUBY promoter regions was investigated by McrBC digestion followed in real-time. Our results highlight that fungal infection induces anthocyanin production by activating the expression of several genes in the biosynthetic pathway. The induction of gene expression is accompanied by maintenance of high levels of methylation at the DFR and RUBY promoters in the inoculated fruits, thus suggesting that DNA methylation is not a repressive mark of anthocyanin related gene expression in sweet orange subjected to biotic stress. Finally, by measuring the expression levels of the Citrus DNA demethylase genes, we found that none of them is up-regulated in response to fungal infection, this result being in accordance with the observed maintenance of high-level DFR and Ruby promoter regions methylation.

Transcriptional response of giant reed (Arundo donax L.) low ecotype to long-term salt stress by unigene-based RNAseq.

The giant reed is a fast growing herbaceous non-food crop considered as eligible alternative energy source to reduce the usage of fossil fuels. Tolerance of this plant to abiotic stress has been demonstrated across a range of stressful conditions, thus allowing cultivation in marginal or poorly cultivated land in order not to compromise food security and to overcome land use controversies. In this work, we de novo sequenced, assembled and analyzed the A. donax low G34 ecotype leaf transcriptome (RNAseq analysis) subjected to severe long-term salt stress (256.67 mM NaCl corresponding to 32 dS m-1 electric conductibility). In order to shed light upon the response to high salinity of this non model plant, we analyzed clusters related to salt sensory and signaling transduction, transcription factors, hormone regulation, Reactive Oxygen Species (ROS) scavenging and osmolyte biosynthesis, all of them showing different regulation compared to untreated plants. The analysis of clusters related to ethylene biosynthesis and signaling indicated that gene transcription is modulated towards the minimization of ethylene negative effects upon plant growth. Certainly, the photosynthesis is strongly affected since genes involved in Rubisco biosynthesis and assembly are down-regulated. However, a shift towards C4 photosynthesis is likely to occur as gene regulation is aimed to activate the primary CO2 fixation to PEP (phosphoenolpyruvate). The analysis of "carbon metabolism" category revealed that G34 ecotype under salt stress induces the expression of glycolysis and Krebs cycle related genes, this being consistent with the hypothesis that some sort of salt avoidance might be occurred in A. donax G34 low ecotype. By comparing our results with findings obtained with other giant reed ecotype, we identified several differences in the response to salt that are in accordance with the possibility that heritable phenotypic differences among clones of A. donax might be accumulated especially in ecotypes originating from distant geographical areas, despite their asexual reproduction modality. Additionally, 26,838 simple sequence repeat (SSR) markers were identified and validated. This SSR dataset definitely expands the marker catalogue of A. donax facilitating the genotypic characterization of this species.

RNASeq analysis of giant cane reveals the leaf transcriptome dynamics under long-term salt stress.

BACKGROUND: To compensate for the lack of information about the molecular mechanism involved in Arundo donax L. response to salt stress, we de novo sequenced, assembled and analyzed the A. donax leaf transcriptome subjected to two levels of long-term salt stress (namely, S3 severe and S4 extreme). RESULTS: The picture that emerges from the identification of differentially expressed genes is consistent with a salt dose-dependent response. Hence, a deeper re-programming of the gene expression occurs in those plants grew at extreme salt level than in those subjected to severe salt stress, probably representing for them an "emergency" state. In particular, we analyzed clusters related to salt sensory and signaling, transcription factors, hormone regulation, Reactive Oxygen Species (ROS) scavenging, osmolyte biosynthesis and biomass production, all of them showing different regulation either versus untreated plants or between the two treatments. Importantly, the photosynthesis is strongly impaired in samples treated with both levels of salinity stress. However, in extreme salt conditions, a dramatic switch from C3 Calvin cycle to C4 photosynthesis is likely to occur, this probably being the more impressive finding of our work. CONCLUSIONS: Considered the distinct response to salt doses, genes either involved in severe or in extreme salt response could constitute useful markers of the physiological status of A. donax to deepen our understanding of its biology and productivity in salinized soil. Finally, many of the unigenes identified in the present study have the potential to be used for the development of A. donax varieties with improved productivity and stress tolerance, in particular the knock out of the GTL1 gene acting as negative regulator of water use efficiency has been proposed as good target for genome editing.