Editing of the ethylene biosynthesis gene in carnation using CRISPR-Cas9 ribonucleoprotein complex.

The study aimed to edit ethylene (ET) biosynthesis genes [1-aminocyclopropane-1-carboxylic acid (ACC) synthetase 1 (ACS1) and ACC oxidase 1 (ACO1)] in carnation using the CRISPR/Cas9 ribonucleoprotein (RNP) complex system. Initially, the conserved regions of the target genes (ACS1 and ACO1) were validated for the generation of different single guide RNAs (sgRNAs), followed by the use of an in vitro cleavage assay to confirm the ability of the sgRNAs to cleave the target genes specifically. The in vitro cleavage assay revealed that the sgRNAs were highly effective in cleaving their respective target regions. The complex of sgRNA: Cas9 was directly delivered into the carnation protoplast, and the target genes in the protoplast were deep-sequenced. The results revealed that the sgRNAs were applicable for editing the ET biosynthesis genes, as the mutation frequency ranged from 8.8 to 10.8% for ACO1 and 0.2-58.5% for ACS1. When sequencing the target genes in the callus derived from the protoplasts transformed with sgRNA: Cas9, different indel patterns (+ 1, - 1, and - 8 bp) in ACO1 and (- 1, + 1, and + 11) in ACS1 were identified. This study highlighted the potential application of CRISPR/Cas9 RNP complex system in facilitating precise gene editing for ET biosynthesis in carnation.

Loss of ACO4 in petunia improves abiotic stress tolerance by reducing the deleterious effects of stress-induced ethylene.

To investigate the role of ethylene (ET) in abiotic stress tolerance in petunia cv. 'Mirage Rose', petunia plants in which the ET biosynthesis gene 1-aminocyclopropane-1-carboxylic acid oxidase 4 (ACO4) was knocked out (phaco4 mutants) and wild-type (WT) plants were exposed to heat and drought conditions. Loss of function of ACO4 significantly delayed leaf senescence and chlorosis under heat and drought stress by maintaining the SPAD values and the relative water content, indicating a greater stress tolerance of phaco4 mutants than that of WT plants. This tolerance was related to the lower ET and reactive oxygen species levels in the mutants than in WT plants. Furthermore, the stress-induced expression of genes related to ET signal transduction, antioxidant and proline activities, heat response, and biosynthesis of abscisic acid was higher in the mutants than in WT plants, indicating a greater stress tolerance in the former than in the latter. These results demonstrate the deleterious effects of stress-induced ET on plant growth and provide a better physiological and molecular understanding of the role of stress ET in the abiotic stress response of petunia. Because the loss of function of ACO4 in petunia improved stress tolerance, we suggest that ACO4 plays a vital role in stress-induced leaf senescence and acts as a negative regulator of abiotic stress tolerance.

Removal of heavy metals using Iris species: A potential approach for reclamation of heavy metal-polluted sites and environmental beautification.

Globally, the number of heavy metal (HM)-polluted sites has increased rapidly in recent years, posing a serious threat to agricultural productivity, human health, and environmental safety. Hence, it is necessary to remediate HM-polluted sites to increase cultivatable lands for agricultural productivity, prevent hazardous effects to human health, and promote environmental safety. Removal of HMs using plants (phytoremediation) is a promising method as it is eco-friendly. Recently, ornamental plants have been widely used in phytoremediation programs as they can simultaneously eliminate HMs and are aesthetically pleasing. Among the ornamental plants, Iris species are frequently used; however, their role in HM remediation has not been reviewed yet. Here, the importance of Iris species in the ornamental industry and their different commercial aspects are briefly described. Additionally, the mechanisms of how the plant species absorb and transport the HMs to the above-ground tissues and tolerate HM stress are highlighted. The variation in HM remediation efficiency depending on the plant species, HM type and concentration, use of certain supplements, and experimental conditions are also discussed. Iris species are able to remove other hazards as well, such as pesticides, pharmaceutical compounds, and industrial wastes, from polluted soils or waste-water. Owing to the valuable information presented in this review, we expect more applications of the species in reclaiming polluted sites and beautifying the environment.

Protoplast isolation and transient gene expression in different petunia cultivars.

The protocol optimized for Petunia hybrida cv. Mirage Rose produced high protoplast yields in 3 out of other 11 cultivars (Damask White, Dreams White, and Opera Supreme White). Factors optimized in the protoplast transfection process showed that the best transfection efficiency (80%) was obtained using 2.5 × 105 protoplast density, 40% polyethylene glycol (PEG) concentration, 10 µg plasmid DNA, and 15 min of transfection time. Assessing the usability of the protocol for other cultivars (Damask White, Dreams White, and Opera Supreme White), a reasonable protoplast transfection efficiency (⁓50%) was observed in the cultivars Dreams White and Opera Supreme White, with lower efficiency (⁓50%) observed in the cv. Damask White. The transient expression of enhanced green fluorescent protein (eGFP) in the nucleus of the transfected protoplasts of all cultivars was confirmed using PCR. This system could be valuable for genome editing of unwanted genes in petunias using the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9) technology. Furthermore, it could contribute to other studies on protein subcellular localization, protein-protein interactions, and functional gene expression in the petunias.

Comprehensive Genome-Wide Analysis and Expression Pattern Profiling of PLATZ Gene Family Members in Solanum Lycopersicum L. under Multiple Abiotic Stresses.

PLATZ (plant AT-rich sequence and zinc-binding) family proteins with two conserved zinc-dependent DNA-binding motifs are transcription factors specific to the plant kingdom. The functions of PLATZ proteins in growth, development, and adaptation to multiple abiotic stresses have been investigated in various plant species, but their role in tomato has not been explored yet. In the present work, 20 non-redundant Solanum lycopersicum PLATZ (SlPLATZ) genes with three segmentally duplicated gene pairs and four tandemly duplicated gene pairs were identified on eight tomato chromosomes. The comparative modeling and gene ontology (GO) annotations of tomato PLATZ proteins indicated their probable roles in defense response, transcriptional regulation, and protein metabolic processes as well as their binding affinity for various ligands, including nucleic acids, peptides, and zinc. SlPLATZ10 and SlPLATZ17 were only expressed in 1 cm fruits and flowers, respectively, indicating their preferential involvement in the development of these organs. The expression of SlPLATZ1, SlPLATZ12, and SlPLATZ19 was up- or down-regulated following exposure to various abiotic stresses, whereas that of SlPLATZ11 was induced under temperature stresses (i.e., cold and heat stress), revealing their probable function in the abiotic stress tolerance of tomato. Weighted gene co-expression network analysis corroborated the aforementioned findings by spotlighting the co-expression of several stress-associated genes with SlPLATZ genes. Confocal fluorescence microscopy revealed the localization of SlPLATZ−GFP fusion proteins in the nucleus, hinting at their functions as transcription factors. These findings provide a foundation for a better understanding of the structure and function of PLATZ genes and should assist in the selection of potential candidate genes involved in the development and abiotic stress adaptation in tomato.

Overexpression of acdS in Petunia hybrida Improved Flower Longevity and Cadmium-Stress Tolerance by Reducing Ethylene Production in Floral and Vegetative Tissues.

The role of acdS, which encodes the 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase enzyme, in extending flower longevity and improving tolerance to cadmium (Cd) stress was assessed using transgenic Petunia hybrida cv. 'Mirage Rose' overexpressing acdS and wild-type (WT) plants. The overexpression of acdS reduced ethylene production in floral tissue via suppression of ethylene-related genes and improved flower longevity, approximately 2 to 4 days longer than WT flowers. Under Cd stress, acdS significantly reduced Cd-induced ethylene production in vegetable tissues of transgenic plants through suppression of ethylene-related genes. This resulted in a lower accumulation of ethylene-induced reactive oxygen species (ROS) in the transgenic plants than in WT plants. In addition, expression of the genes involved in the activities of antioxidant and proline synthesis as well as the metal chelation process was also higher in the former than in the latter. Moreover, Cd accumulation was significantly higher in WT plants than in the transgenic plants. These results are linked to the greater tolerance of transgenic plants to Cd stress than the WT plants, which was determined based on plant growth and physiological performance. These results highlight the potential applicability of using acdS to extend flower longevity of ornamental bedding plants and also reveal the mechanism by which acdS improves Cd-stress tolerance. We suggest that acdS overexpression in plants can extend flower longevity and also help reduce the negative impact of Cd-induced ethylene on plant growth when the plants are unavoidably cultivated in Cd-contaminated soil.

Overexpression of acdS gene encoding 1-aminocyclopropane-1-carboxylic acid deaminase enzyme in petunia negatively affects seed germination.

KEY MESSAGE: Overexpression of acdS in petunia negatively affects seed germination by suppression of ethylene biosynthesis and signaling genes and induction of abscisic acid biosynthesis genes in the seeds. The acdS gene, which encodes 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase, has been overexpressed in horticultural crops to improve their tolerance to abiotic stress. However, the role of acdS in the germination of crop seeds has not been investigated, despite its suppression of ethylene production. In this study, acdS overexpression significantly reduced seed weight and germination rate in transgenic petunia cv. Merage Rose (T5, T7, and T12) relative to wild type via the suppression of ethylene biosynthesis and signaling genes and induction of abscisic acid (ABA) biosynthesis genes. The germination rate of T7 was significantly lower than those of T5 and T12, which was linked to higher expression of acdS in the former than the latter. The addition of exogenous ACC and gibberellic acid (GA3) to the germination medium improved the germination rate of T5 seeds and GA3 promoted the germination rate of T12 seeds. However, neither ACC nor GA3 promoted the germination rate of T7 seeds. The improved germination rates in T5 and T12 were associated with the transcriptional regulation of ethylene biosynthesis genes, particularly that of the ACO1 gene, signaling genes, and ABA biosynthesis genes. In this study, we discovered a negative role of acdS in seed germination in petunia. Thus, we highlight the need to consider the negative effect of acdS on seed germination when overexpressing the gene in horticultural crops to improve tolerance to abiotic stress.

Role of Ethylene Biosynthesis Genes in the Regulation of Salt Stress and Drought Stress Tolerance in Petunia.

Ethylene plays a critical signaling role in the abiotic stress tolerance mechanism. However, the role of ethylene in regulating abiotic stress tolerance in petunia has not been well-investigated, and the underlying molecular mechanism by which ethylene regulates abiotic stress tolerance is still unknown. Therefore, we examined the involvement of ethylene in salt and drought stress tolerance of petunia using the petunia wild type cv. "Merage Rose" and the ethylene biosynthesis genes (PhACO1 and PhACO3)-edited mutants (phaco1 and phaco3). Here, we discovered that editing PhACO1 and PhACO3 reduced ethylene production in the mutants, and mutants were more sensitive to salt and drought stress than the wild type (WT). This was proven by the better outcomes of plant growth and physiological parameters and ion homeostasis in WT over the mutants. Molecular analysis revealed that the expression levels of the genes associated with antioxidant, proline synthesis, ABA synthesis and signaling, and ethylene signaling differed significantly between the WT and mutants, indicating the role of ethylene in the transcriptional regulation of the genes associated with abiotic stress tolerance. This study highlights the involvement of ethylene in abiotic stress adaptation and provides a physiological and molecular understanding of the role of ethylene in abiotic stress response in petunia. Furthermore, the finding alerts researchers to consider the negative effects of ethylene reduction on abiotic stress tolerance when editing the ethylene biosynthesis genes to improve the postharvest quality of horticultural crops.

Characterizing the effects of different chemicals on stem bending of cut snapdragon flower.

BACKGROUND: This study investigated the effects of ethylene release compounds (ethephon), ethylene-action inhibitors (silver thiosulfate: STS), and nitric oxide donor (sodium nitroprusside: SNP) on stem bending of snapdragon flowers. Moreover, the effects of plant growth supplements [6-benzyladenine (BA), gibberellic acid 3 (GA3), and calcium chloride (CaCl2)] on the stem bending were also extensively investigated. RESULTS: Ethephon completely prevented stem bending until 9 days after treatment (9 DAT). STS exhibited the highest bending rate, while SNP did not significantly affect the bending compared to the controls. The bending results were associated with the results of stem curvature, relative shoot elongation, ethylene production, and lignin content, that are involved in the stem bending mechanism. This was proven by the expression analysis of genes involved in ethylene and lignin biosynthetic pathways. The addition of plant growth supplements slightly or significantly delayed stem bending in the treatments (control, SNP, and STS) and significantly reduced petal senescence in ethephon at 9 DAT. CONCLUSION: These results show the preventive role of ethephon in the stem bending of cut snapdragon. Moreover, the combination of ethephon with supplements also provided information that could guide the development of strategies to delay stem bending in other cut flowers that undergo serious bending during a short vase life.

Editing of 1-aminocyclopropane-1-carboxylate oxidase genes negatively affects petunia seed germination.

KEY MESSAGE: Editing of ACO genes involved in ethylene biosynthesis pathway reduces ethylene production in petunia seeds and inhibits seed germination. Ethylene production in the seeds of Petunia hybrida cv. 'Mirage Rose' was associated with expression of 1-aminocyclopropane-1-carboxylic acid (ACC) oxidase (ACO) genes (PhACO1, PhACO3, and PhACO4). Suppression of their expression by ethylene inhibitor silver thiosulphate (STS) significantly reduced ethylene production and inhibited seed germination. When it was combined with ethylene precursor ACC, ethylene production was re-promoted via activation of the genes and higher seed germination was restored. This was confirmed using the mutants editing the genes and WT. In the present study, compared with wild type plants, three different mutants (phaco1, phaco3, and phaco4) showed significantly decreased germination percentages as well as delayed germination time and seedling growth. These reductions were associated with lighter seed weight, lower ACO transcript levels, and lower ethylene production in mutants. Inhibited seed germination owing to reduced ethylene production was further verified by the supplementation of exogenous ACC and gibberellic acid (GA3) to growth medium, which restored high seed germination activity in all mutants via enhanced ethylene production. In this study, we reported a key regulatory role of ethylene in seed germination mechanisms in petunia. Further, we highlighted on need to consider the negative effects of ethylene reduction in seed germination and plant growth when editing genes in the ethylene biosynthesis pathway for the maintenance of postharvest fruit, vegetable, and flower quality.

Ethylene Acts as a Negative Regulator of the Stem-Bending Mechanism of Different Cut Snapdragon Cultivars.

This study investigated whether ethylene is involved in the stem-bending mechanism of three different snapdragon cultivars 'Asrit Red', 'Asrit Yellow', and 'Merryred Pink', by treating their cut stems with an ethylene-releasing compound (ethephon), an ethylene-action inhibitor [silver thiosulfate (STS)], and distilled water (as the control). Ethephon completely prevented stem bending in all cultivars, whereas STS exhibited a higher bending rate compared with the control. The bending rates were influenced by several factors, such as the degree of stem curvature, relative shoot elongation, ethylene production, and lignin content, indicating their involvement in the stem-bending mechanism of the cultivars. The analysis of the expression of genes involved in the ethylene and lignin biosynthetic pathways also supported the importance of lignin and ethylene in the stem-bending mechanism. Taken together, as ethephon completely prevented stem bending of the three snapdragon cultivars, this study suggested that ethylene acts as a negative regulator of the stem-bending mechanism of snapdragon cultivars, and the information will be valuable for the prevention of stem bending in other commercially important ornamental flowers.

Overexpression of 1-Aminocyclopropane-1-Carboxylic Acid Deaminase (acdS) Gene in Petunia hybrida Improves Tolerance to Abiotic Stresses.

Abiotic stress induces the ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC) in plants, which consequently enhances ethylene production and inhibits plant growth. The bacterial ACC deaminase enzyme encoded by the acdS gene reduces stress-induced ethylene production and improves plant growth in response to stress. In this study, overexpression of acdS in Petunia hybrida ('Mirage Rose') significantly reduced expression of the ethylene biosynthesis gene ACC oxidase 1 (ACO1) and ethylene production relative to those in wild type (WT) under various abiotic stresses (cold, drought, and salt). The higher reduction of stress-induced ethylene in the transgenic plants, which was due to the overexpression of acdS, led to a greater tolerance to the stresses compared to that in the WT plants. The greater stress tolerances were proven based on better plant growth and physiological performance, which were linked to stress tolerance. Moreover, expression analysis of the genes involved in stress tolerance also supported the increased tolerance of transgenics relative to that with the WT. These results suggest the possibility that acdS is overexpressed in ornamental plants, particularly in bedding plants normally growing outside the environment, to overcome the deleterious effect of ethylene on plant growth under different abiotic stresses. The development of stress-tolerant plants will be helpful to advance the floricultural industry.

The ACC deaminase-producing plant growth-promoting bacteria: Influences of bacterial strains and ACC deaminase activities in plant tolerance to abiotic stress.

Global climate change results in frequent occurrences and/or long durations of abiotic stress. Field grown plants are affected by abiotic stress, and they modulate ethylene in response to abiotic stress exposure and use it as a signaling molecule in stress tolerance mechanisms. However, frequent occurrences and/or long durations of stress conditions can cause plants to induce ethylene levels higher than their thresholds, resulting in a reduction of plant growth and crop productivity. The use of plant growth-promoting bacteria (PGPB) that produce 1-aminocyclopropane-1-carboxylate (ACC) deaminase has increased in various plant species to ameliorate the deleterious effects of stress-induced ethylene and promote plant growth despite abiotic stress conditions. Unfortunately, there are restrictions that limit the use of ACC deaminase-producing PGPB to protect plants from abiotic stresses. This review describes how abiotic stress induces ethylene and how stress-induced ethylene adversely affects plant growth. In addition, this review emphasizes the importance of the compatibility of PGPB strains and specific host plants and ACC deaminase activities in the reduction of stress ethylene and the promotion of plant growth, based on the research published in the last 10 years. Moreover, due to the restrictions in PGPB use, this review highlights the potential generation of transgenic plants expressing the AcdS gene that encodes the ACC deaminase enzyme as a substitute for PGPB in the future to support and uplift agricultural sustainability and food security globally.

Abiotic stress-induced anthocyanins in plants: Their role in tolerance to abiotic stresses.

Abiotic stresses, such as heat, drought, salinity, low temperature, and heavy metals, inhibit plant growth and reduce crop productivity. Abiotic stresses are becoming increasingly extreme worldwide due to the ongoing deterioration of the global climate and the increase in agrochemical utilization and industrialization. Plants grown in fields are affected by one or more abiotic stresses. The consequent stress response of plants induces reactive oxygen species (ROS), which are then used as signaling molecules to activate stress-tolerance mechanism. However, under extreme stress conditions, ROS are overproduced and cause oxidative damage to plants. In such conditions, plants produce anthocyanins after ROS signaling via the transcription of anthocyanin biosynthesis genes. These anthocyanins are then utilized in antioxidant activities by scavenging excess ROS for their sustainability. In this review, we discuss the physiological, biochemical, and molecular mechanisms underlying abiotic stress-induced anthocyanins in plants and their role in abiotic stress tolerance. In addition, we highlight the current progress in the development of anthocyanin-enriched transgenic plants and their ability to increase abiotic stress tolerance. Overall, this review provides valuable information that increases our understanding of the mechanisms by which anthocyanins respond to abiotic stress and protect plants against it. This review also provides practical guidance for plant biologists who are engineering stress-tolerant crops using anthocyanin biosynthesis or regulatory genes.

Protoplast Isolation and Shoot Regeneration from Protoplast-Derived Callus of Petunia hybrida Cv. Mirage Rose.

Despite the increasing use of protoplasts in plant biotechnology research, shoot regeneration from protoplasts remains challenging. In this study, we investigated the factors involved in protoplast isolation, callus induction, and shoot regeneration in Petunia hybrida cv. Mirage Rose. The following conditions were found to be most optimal for protoplast yield and viability: 0.6 M mannitol, 2.0% cellulase, and 6 h digestion time. A plating density of 10 × 104 protoplasts/mL under osmoticum condition (0.58 M mannitol) showed high microcolony viability in liquid culture. The Kao and Michayluk medium was found to be appropriate for callus proliferation from microcalli under a 16-h light photoperiod. Calli cultured in Murashige and Skoog medium containing 1.0 mg/L 6-benzylaminopurine and 0.2 mg/L 3-indole butyric acid showed the highest shoot regeneration frequency and number of shoots obtained per explant. Random amplification of polymorphic DNA analysis showed that the protoplast-derived shoots exhibited the same banding patterns as those of donor plants. Collectively, these findings can contribute to solving problems encountered in protoplast isolation and shoot regeneration in other petunia cultivars and related species. As the protocol developed by us is highly reproducible, it can be applied in biotechnology research on P. hybrida cv. Mirage Rose.

Regeneration of Genetically Stable Plants from in Vitro Vitrified Leaves of Different Carnation Cultivars.

This study was conducted to investigate the efficacy of shoot regeneration from different leaf types (normal leaves and vitrified leaves) from three different carnation cultivars 'Kumbuyl', 'Denev', and 'Jinju' using different combinations of 3-indole butyric acid (IBA) and thidiazuron (TDZ) concentrations. The shoot tips cultured on Murashige and Skoog (MS) basal media (Type 1 media) produced normal leaves, while those cultured-on media supplemented with plant growth regulators and/or vitamin (Type 2 media and Type 3 media) produced vitrified leaves for all cultivars. Culture of normal leaf segments on MS medium containing different combinations of IBA and TDZ concentrations induced callus in all treatments; however, the callus was unable to induce shoots and finally became necrotic. In contrast, no callus induction was observed in the control (hormone-free treatment). When vitrified leaf segments underwent the same treatments, shoots were induced from the vitrified leaves (derived from Type 2 media) but were unhealthy and gradually died, whereas those induced from Type 3 media were vitrified and healthy. The optimal combination for the best shoot regeneration and number of shoots per explants varied depending on the genotypes used. The vitrified shoots induced from the leaves of Type 3 media transformed into normal shoots and survived well under greenhouse conditions. According to the results of random amplified polymorphic DNA (RAPD) analysis, the banding patterns of twelve primers that were detected in vitrified leaf-induced normalized shoots were identical to those of normal in vitro grown plants, indicating that no genetic variation had occurred during the procedure. Taken together, this study indicates that vitrified leaves can be used for shoot regeneration of recalcitrant carnation cultivars, regardless of the genotypes and types of vitrified leaves. However, as the number of shoots per explants was still low, further investigation is warranted to obtain a more efficient shoot regeneration protocol for genetic transformation of the cultivars.

CRISPR/Cas9-mediated editing of 1-aminocyclopropane-1-carboxylate oxidase1 enhances Petunia flower longevity.

The genes that encode the ethylene biosynthesis enzyme 1-aminocyclopropane-1-carboxylate oxidase (ACO) are thought to be involved in flower senescence. Hence, we investigated whether the transcript levels of PhACO genes (PhACO1, PhACO3 and PhACO4) in Petunia cv. Mirage Rose are associated with ethylene production at different flowering stages. High transcript levels were detected in the late flowering stage and linked to high ethylene levels. PhACO1 was subsequently edited using the CRISPR/Cas9 system, and its role in ethylene production was investigated. PhACO1-edited T0 mutant lines, regardless of mutant type (homozygous or monoallelic), exhibited significantly reduced ethylene production and enhanced flower longevity compared with wild-type. Flower longevity and the reduction in ethylene production were observed to be stronger in homozygous plants than in their monoallelic counterparts. Additionally, the transmission of the edited gene to the T1 (lines 6 and 36) generation was also confirmed, with the results for flower longevity and ethylene production proving to be identical to those of the T0 mutant lines. Overall, this study increases the understanding of the role of PhACO1 in petunia flower longevity and also points to the CRISPR/Cas9 system being a powerful tool in the improvement of floricultural quality.

Silencing of the phytoene desaturase (PDS) gene affects the expression of fruit-ripening genes in tomatoes.

BACKGROUND: Past research has shown that virus-induced phytoene desaturase (PDS) gene silencing via agroinjection in the attached and detached fruit of tomato plants results in a pale-yellow fruit phenotype. Although the PDS gene is often used as a marker for gene silencing in tomatoes, little is known about the role of PDS in fruit ripening. In this study, we investigated whether the pepper PDS gene silenced endogenous PDS genes in the fruit of two tomato cultivars, Dotaerang Plus and Legend Summer. RESULTS: We found that the pepper PDS gene successfully silenced endogenous PDS in tomato fruit at a silencing frequency of 100% for both cultivars. A pale-yellow silenced area was observed over virtually the entire surface of individual fruit due to the transcriptional reduction in phytoene desaturase (PDS), zeta-carotene (ZDS), prolycopene isomerase (CrtlSO), and beta-carotene hydroxylase (CrtR-b2), which are the carotenoid biosynthesis genes responsible for the red coloration in tomatoes. PDS silencing also affected the expression levels of the fruit-ripening genes Tomato AGAMOUS-LIKE1 (TAGL1), RIPENING INHIBITOR (RIN), pectin esterase gene (PE), lipoxygenase (LOX), FRUITFULL1/FRUITFUL2 (FUL1/FUL2), and the ethylene biosynthesis and response genes 1-aminocyclopropane-1-carboxylate oxidase 1 and 3 (ACO1 and ACO3) and ethylene-responsive genes (E4 and E8). CONCLUSION: These results suggest that PDS is a positive regulator of ripening in tomato fruit, which must be considered when using it as a marker for virus-induced gene silencing (VIGS) experiments in order to avoid fruit-ripening side effects.

Tomato seeds pretreated with Antifreeze protein type I (AFP I) promotes the germination under cold stress by regulating the genes involved in germination process.

This study was conducted to investigate the involvement of antifreeze proteins (AFPs; type I and III) in the germination mechanism of tomato seeds under low temperature stress. Germination of the seeds grown at a room temperature (25°C) was observed on 5 days after sowing (DAS), while all seeds exposed to a low temperature started to germinate at 16 days after sowing (DAS). However, in comparison with control seeds (0 µg/l), seeds treated with AFP I (100, 300, or 500 µg/l) germinated earlier and at a higher percentage until 20 DAS, and seeds treated with 100 µg/l AFP I showed the highest percentage of germination. Surprisingly, AFP III did not significantly increase germination, and the rate was lower among 500 µg/l AFP III-treated seeds compared with control seeds (0 µg/l). The transcription levels of the plasma membrane-associated H+-ATPase gene and antioxidant-related superoxide dismutase (SOD) and catalase 1 (CAT1) genes were analyzed, and the transcription levels of the genes in the seeds grown at 25°C were relatively low. For low temperature-treated seeds, H+-ATPase in control seeds (0 µg/l) was higher compared with that in AFP I-treated seeds and was lower compared with that in AFP III-treated seeds. The expression levels of the antioxidant-related genes (SOD and CAT1) were lower in AFP I-treated seeds than in control seeds (0 µg/l); however, they were higher in AFP III-treated seeds than in control seeds (0 µg/l). Overall, compared with AFP III, AFP I may potentially function as a cold-protective agent by modulating the genes associated with seed germination.

The role of antifreeze proteins in the regulation of genes involved in the response of Hosta capitata to cold.

Cold temperatures are a major source of stress for plants and negatively impact crop yield. A possible way to protect plants is to treat them with antifreeze proteins (AFPs). Here, we investigated whether fish AFPs can shield the rare ornamental species Hosta capitata from low-temperature stress. We elucidated the expression patterns of the cold-inducible genes C-repeat binding factor 1 (CBF1) and dehydrin 1 (DHN1), as well as the antioxidant genes superoxide dismutase (SOD) and catalase (CAT). All were upregulated at low temperature (4 °C). With increasing exposure time, CBF1 and DHN1 expression generally rose (except CBF1 at 48 h). In contrast, SOD and CAT expression gradually declined from 6 to 48 h. Depending on exposure duration, AFP regulation of gene transcription varied with concentration. However, compared with other concentrations, 100 µg/L AFP reduced CBF1 and DHN1 expression and increased SOD and CAT expression in plants, regardless of exposure time. Both AFP I and III were likely to be most effective at protecting plants against cold stress at concentrations of 100 µg/L. Their involvement in H. capitata cold-stress treatment occurred through regulating the expression of important stress-response genes.