Special Issue: Gene Expressions in Response to Diseases, Abiotic Stresses, and Pest Damage of Horticultural Products

1. Introduction

Horticultural crops are continuously exposed to pathogens, pests, and abiotic stress, which collectively result in significant economic losses. Over time, plants have evolved sophisticated defense mechanisms to counteract both pathogen and insect attacks, as well as to face abiotic stresses. The identification of genes involved in defense responses and stress tolerance is fundamental for selecting resistant crops, which could enable the sustainable management of diseases and damages. Gene expression is subject to complex regulation, influenced not only by developmental and tissue-specific factors, but also by both biotic and abiotic stimuli in the environment. The fluctuation in expression levels, or the gene expression profile, provides essential insights into the biological functions of genes. Comprehensive expression profiles established under various experimental conditions could facilitate the understanding of gene functions. Similarities in expression profiles between genes are an effective index for predicting gene function and mechanisms of gene expression regulation [1]. If two genes have similar expression profiles, it is reasonable to expect that they have the same biological function, even in different plant species. In addition, similar expression profiles suggest that two genes are regulated by the same mechanism (e.g., a transcription factor (TF) and a cis-element). Therefore, genes with highly similar expression profiles are likely to have the same biological functions and regulatory mechanisms. Transcriptomics enables the simultaneous analysis of gene expression in plants, pathogens, and pest interactions (Figure 1).

2. Overview of Published Articles

Several gene families are involved in plant responses to biotic and abiotic stresses, becoming objects of research aiming to enable breeding improvement. The first two manuscripts address this issue by examining the evolutionary relationship, conserved motif composition, gene structure, chromosomal location, cis-regulatory elements within the promoter region, and primary expression patterns in various plant tissues (stem, root, leaf, tendril, and flower) and in response to both abiotic and biotic stresses.

In Contribution 1, the organ-specific expression patterns and stress-responsive gene expression patterns of 24 eggplant DIR family genes were investigated. Directing proteins (DIR) play essential roles in lignin and lignan pathway biosynthesis, protective responses, and secondary metabolism, as well as in plant disease resistance. The results identified 24 DIR genes in eggplant, divided into three subfamilies (DIR-a, DIR-b/d, and DIR-e). Among the identified cis-elements, some were involved in hormone response, stress defense, and the expression of plant tissues, suggesting a correlation with plant development and plant response to abiotic and biotic stresses. Specifically, the expression of each SmDIR gene displayed organ-specificity and typical expression (down- or upregulation) according to cultivar sensitivity. The SmDIR22 gene was among the candidates for chief association with stress response, being differentially expressed in response to abiotic and biotic stresses. The results of the study will provide a foundation for future research on the biological functions of the SmDIR22 gene, which is promising for the molecular breeding of resistant eggplant.

In Contribution 2, the authors characterized the SIMILAR TO RCD-ONE (SRO) gene family in two-leaf-stage seedlings of Chinese Long cucumber; SRO is a family of small, plant-specific proteins regulating numerous biological processes, including physiological development and stress reactions. These genes, which encode transcription factor (TF) proteins CsSRO1, CsSRO2, CsSRO3, and CsSRO4, were successfully isolated, cloned, and found to be deeply induced under drought and salt stresses, displaying different expressions in sensitive and resistant cucumber plants. Expression profiling revealed that the four CsSRO genes were differentially expressed in various tissues and under different stress conditions, indicating their involvement in plant physiological processes and defense mechanisms. The SRO family genes were relatively conservative, with the authors revealing conserved motif arrangements within each subgroup and highlighting differences between monocot- and dicot-specific patterns. Except for CsSRO2, which is located in both the nucleus and mitochondria, the other SRO genes were found in the nucleus. In the future, the four SRO genes could be considered potential candidates for breeding stress-tolerant cucumber varieties. Being related to phytohormone signaling, they could also be involved in defense responses to biotic stresses.

Plants have evolved wide phytohormone signaling pathways to improve survival in response to drought stress, such as the auxin (IAA), cytokinin (CK), abscisic acid (ABA), jasmonic acid (JA), salicylic acid (SA), ethylene (ET), and gibberellin (GA) pathways [2,3]. Genes and pathways involved in stress response were physiologically and transcriptionally studied in the Contribution 3, and Bletilla striata, a widely used medicinal plant, was found to be highly susceptible to drought stress during the planting chain. In plant tissues, biotic and abiotic stresses caused ROS accumulation and early defense relied on antioxidants such as catalase (CAT), peroxidase (POX), superoxide dismutase (SOD), glutathione sulfo-transferase (GST). Indeed, these pathways were upregulated: The upregulation of SnRK2 and SLAH2 in B. striata suggests that drought stress promotes the closure of stomata to prevent water loss. Additionally, SnRK2 can activate downstream genes, such as ABF; its overexpression in Arabidopsis resulted in ABA hypersensitivity and drought tolerance. Also, the upregulation of SnRK2 can activate different cation or anion channels, such as S-type anion channel 3 (SLAH3), involved in forcing stomata closure [4]. Enhanced O₂ production also protected the photosystem under drought stress. Furthermore, the drought-induced increase in dehydrin (DHN) gene expression proved ROS scavenging to be the activation of mechanism of drought tolerance [5]. These results indicate that B. striata leaves reduce drought-induced oxidative damage by activating the expression of SOD, CAT, and POX, which scavenge ROS, confirming this mode of action.

When analyzing relative gene expression, a preliminary and foundational step is the employment of robust and reliable housekeeping genes. The ideal reference gene is continuously expressed in all tissues across various conditions, and this issue was evaluated in the Contribution 4. Considering eight frequently used housekeeping genes (EF1α, ACT, SKD1, YLS8, UBQ, GAPDH, TUB, and WDP), the authors evaluated the expression of these candidate reference genes in pear (Pyrus bretschneideri Rehd.) leaves subjected to hormonal (SA, MeJA, ETH, and ABA), biotic (Venturia nashicola), and abiotic stresses (cold, heat, water deficit). No one gene was consistently expressed across all stress conditions; however, WDP was relatively stable except for under methyl jasmonate stress, where ACT had the most stable expression. As they are robustly expressed in plant tissues under different stresses, it is suggested that only EF1α and GAPDH should be used as reference genes in treatments. These results provide valid and updated inputs for selecting housekeeping genes in pears exposed to biotic and abiotic stresses in the future.

In view of sustainable agriculture, the Contribution 5 investigated the antimicrobial effects related to mycorrhizal-induced resistance (MIR) in tropical chili pepper seedlings differing in mycorrhizal response and pathogen resistance. In their work, the impact of mycorrhizae on the relative expression of genes involved in jasmonic acid (JA)- and salicylic acid (SA)-mediated signaling pathways was studied in plants challenged by Ralstonia solanacearum. The effect of mycorrhizae on the phenotypic traits of tropical chili peppers was also studied. In ten tropical chili pepper genotypes, mycorrhiza treatment plus infection with R. solanacearum (M1R1) increased the relative gene expression of JA, which was higher in genotypes with high mycorrhizal responsiveness than in those infected with R. solanacearum (M0R1). A similar result was found for the resistant-control cultivar. Based on phytohormone balance and relative expression, the results showed a significant increase in the relative gene expression of SA and JA pathways; particularly, the latter enhanced induced systemic resistance (ISR) caused by interactions between the plant and beneficial microorganisms. Generally, the relative gene expression of SA was associated with pathogen resistance; however, JA expression was deeply related to the tropical chili peppers’ response to both mycorrhizal colonization and pathogen resistance. The rise in the JA and SA signaling pathways was influenced by the resistance properties of the tropical pepper genotypes to R. solanacearum, differences which affected plant phenotypes. The effectiveness of symbiosis depended on the mycorrhizal species and host genotype. It is probable the beneficial effects due to mycorrhizae and observed under biotic stressors could be spread to abiotic stress conditions as well.

The genus Dianthus, a member of the family Caryophyllaceae [6], is an ornamental flower with production characteristics similar to those of Chrysanthemum and Rosa. In Contribution 6, gene expression profiles were studied after the exposure of a Dianthus hybrida flower to tactile stimuli. Touch stimulation induced concentration enhancement of free cytosolic Ca²⁺ and activation of Ca²⁺-binding proteins involved in the regulation of downstream cascades through phosphorylation signaling; the concentration of JA also improved. Touch stimuli induced the upregulation of genes encoding protein kinase, xyloglucan endotransglucosylase, calmodulin, and components involved in JA biosynthesis and signaling, as well as the downregulation of photosynthetic genes, and the significant of phytohormones was highlighted. The authors applied RNA-seq methods and analyses to assess the molecular mechanisms involved in the touch stimulus responses of D. hybrida flower buds. Among the tested genes, those encoding serine/threonine kinases were only found in flower buds displaying an organ-specific response to stimulation. This knowledge can be applied in future breeding to enhance flower morphology and develop new varieties.

Pepper production is affected by abiotic and biotic stress [7,8]. In particular, the fungus Verticillium dahliae poses a serious threat to global pepper production. With this in mind, Contribution 7. physiologically and transcriptomically compared detached pepper leaves at three time-points after inoculation with V. dahliae to define resistant plants in the eighth contribution to this Special Issue. Susceptible and resistant cultivars shared increases in the same signaling pathways related to the transduction of plant–pathogen interaction, MAPK, and phytohormone signaling. The resistant cultivar MS72 exhibited higher chlorophyll levels and a lower malondialdehyde content after inoculation compared to the susceptible cultivar MS66. The research indicates that infection with V. dahliae causes cell membrane lipid peroxidation in MS66. Moreover, MS72 maintained high chlorophyll levels and a low MDA content, indicating its resistance to V. dahliae. Thirty-six hub genes contribute to the resistance response, such as the transcription factor bHLH93, defense-like protein 1, and miraculin-like. Simultaneously, pathways involved in setting up the cell wall, phenylpropanoid biosynthesis, and photosynthesis (chlorophylls reduced over 70% in susceptible cultivars) were differentially expressed and regulated in resistant cultivars, improving pepper resistance. The key genes and regulatory mechanisms may serve as targets for improving pepper resistance in breeding programs and selecting improved varieties.

A different approach to controlling the oomycete Phytophthora infestans, responsible for the late blight disease of the Solanaceae family, was used in the seventh contribution by Contribution 8: virus-induced gene silencing (VIGS) was used to deliver homologous sequences. PiLLP, the ortholog of plant loricrin-like protein (LLP), was silenced, arranging the recombinant vector containing both tobacco mosaic virus and LLP. Thus, RNAinterference (RNAi) could be expressed in the A1 mating type, preventing sexual reproduction and reducing losses and management costs related to late blight disease. RNA interference (RNAi) is a powerful tool for studying gene function that can be deployed through constitutive transformation or transient expression, such as in VIGS experiments. The study provides the first evidence of downregulation of a constitutive gene in P. infestans using a recombinant vector based on a plant virus.

Good agronomic practices, such as fertilization, could reduce symptoms caused by biotic and abiotic stress. Contribution 9 provide a comprehensive study on iron deficiency chlorosis limiting the productivity of ‘Yali’ pears in alkaline soils. Studies on photosynthesis, root vitality, enzymatic activity, soil physicochemical properties, iron and citrate contents, and relative expression of genes linked to iron absorption and transport could contribute to controlling homeostasis regulation and lead to selecting iron-efficient cultivars. This study systematically investigated the physiological and molecular responses to various degrees of iron deficiency, focusing on the roles of PbFRO2 (Pyrus bretschneideri Ferric Reductase Oxidase 2), PbIRT1 (P. bretschneideri Iron-Regulated Transporter 1), and PbCS2 (P. bretschneideri Citrate Synthase 2) in iron uptake. In this paper the authors compared physiological and molecular iron deficiency and leaf/root chlorosis of different severity (moderate, normal, and severe) over time in ‘Yali’ pear. The bivalent iron content was significant and inversely related to severe chlorosis; furthermore, maximum photochemical efficiency was reduced due to iron stress. The results demonstrated that moderate iron deficiency upregulated PbFRO2, enhanced root ferric reductase (FCR) activity, and promoted Fe³⁺ reduction and Fe²⁺ transport, while severe deficiency suppressed the expression of these genes. To evaluate these metabolic pathways, the relative expression of corresponding genes was evaluated and PbFRO2 was induced by chlorosis to improve radical iron uptake. Globally, normal and moderate chlorotic leaves exhibited similar physiological and molecular features, despite the fact that severe chlorosis induces significant changes. The described mechanisms were led by the PbFRO2 gene, with the differences related to the season and displaying stage-specific responses to chlorosis according to the “gene–metabolite–phenotype” pattern. This hub gene is suitable for studying adaptive responses, for reducing chlorosis, and for defining potential aims for iron biofortification. Above all, this study proposes PbFRO2 as a potential molecular target for breeding iron-efficient pear cultivars.

3. Conclusions

Most of the papers in this Special Issue share a common scientific approach, highlighting the morphological, molecular, and metabolic processes that different plant species exhibit in response to biotic and abiotic stresses. These stresses cause a reduction in chlorophyll amounts, chiefly appreciable in leaf yellowing; simultaneously, plant defense mechanisms are stimulated. To do this, genes involved in the phenylpropanoid pathway, phytohormone signaling, antioxidant production, and pathogenesis-related proteins are up- or downregulated, all according to cultivar susceptibility and plant organ. Furthermore, the displayed results bring out functional overlap within the same gene family among different plant species during plant–pathogen interactions. According to each time-point of stress development, differential gene expression between susceptible and resistant cultivars is a key point in breeding. Thanks to cheaper high-throughput sequencing technologies, transcriptome sequencing, and the ability to obtain reliable data, key resistance genes and knowledge of regulatory mechanisms can facilitate the development of breeding programs that represent a new level of defense for controlling biotic and abiotic stresses.