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.

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.

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.

Overexpression of snapdragon Delila (Del) gene in tobacco enhances anthocyanin accumulation and abiotic stress tolerance.

BACKGROUND: Rosea1 (Ros1) and Delila (Del) co-expression controls anthocyanin accumulation in snapdragon flowers, while their overexpression in tomato strongly induces anthocyanin accumulation. However, little data exist on how Del expression alone influences anthocyanin accumulation. RESULTS: In tobacco (Nicotiana tabacum 'Xanthi'), Del expression enhanced leaf and flower anthocyanin production through regulating NtCHS, NtCHI, NtF3H, NtDFR, and NtANS transcript levels. Transgenic lines displayed different anthocyanin colors (e.g., pale red: T0-P, red: T0-R, and strong red: T0-S), resulting from varying levels of biosynthetic gene transcripts. Under salt stress, the T2 generation had higher total polyphenol content, radical (DPPH, ABTS) scavenging activities, antioxidant-related gene expression, as well as overall greater salt and drought tolerance than wild type (WT). CONCLUSION: We propose that Del overexpression elevates transcript levels of anthocyanin biosynthetic and antioxidant-related genes, leading to enhanced anthocyanin production and antioxidant activity. The resultant increase of anthocyanin and antioxidant activity improves abiotic stress tolerance.

Genome-wide identification, characterization and expression profiling of LIM family genes in Solanum lycopersicum L.

LIM domain proteins, some of which have been shown to be actin binding proteins, are involved in various developmental activities and cellular processes in plants. To date, the molecular defense-related functions of LIM family genes have not been investigated in any solanaceous vegetable crop species. In this study, we identified 15 LIM family genes in tomato (Solanum lycopersicum L.) through genome-wide analysis and performed expression profiling in different organs of tomato, including fruits at six different developmental stages. We also performed expression profiling of selected tomato LIM genes in plants under ABA, drought, cold, NaCl and heat stress treatment. The encoded proteins of the 15 tomato LIM genes were classified into two main groups, i.e., proteins similar to cysteine-rich proteins and plant-specific DAR proteins, based on differences in functional domains and variability in their C-terminal regions. The DAR proteins contain a so far poorly characterized zinc-finger-like motif that we propose to call DAR-ZF. Six of the 15 LIM genes were expressed only in flowers, indicating that they play flower-specific roles in plants. The other nine genes were expressed in all organs and at various stages of fruit development. SlßLIM1b was expressed relatively highly at the later stage of fruit development, but three other genes, SlWLIM2a, SlDAR2 and SlDAR4, were expressed at the early stage of fruit development. Seven genes were induced by ABA, five by cold, seven by drought, eight by NaCl and seven by heat treatment respectively, indicating their possible roles in abiotic stress tolerance. Our results will be useful for functional analysis of LIM genes during fruit development in tomato plants under different abiotic stresses.

Production of polyploids and unreduced gametes in Lilium auratum × L. henryi hybrid.

Intergenomic F1 hybrids between L. auratum x L. henryi and their BC1 progeny were investigated through genomic in situ hybridization technique (GISH) to determine their potential value in lily breeding. We confirmed that F1 intergenomic hybrids possessed a set of chromosomes (x=12) from both parents and that flowers of the F1 auratum × henryi hybrid showed an intermediate morphological phenotype. Pollen size, viability and germination ability were measured through microscopic observations. F1 intergenomic hybrids produced a relevant frequency of 2n-gametes, which were successfully used to perform crosses with Oriental hybrids, resulting in the triploid Oriental Auratum Henryi (OAuH) hybrid. Twenty BC1 plants were generated by crossing between four different Oriental hybrid cultivars and F1 AuH hybrids using an in vitro embryo rescue technique, after which the genome constitution and chromosome composition were analyzed by GISH. All plants were triploid, showing 12 from female parents (diploid Oriental hybrid) and 24 from male parents (diploid F1 AuH hybrid). Overall, 16 out of 20 BC1 progeny possessed recombinant chromosomes with 1-5 crossover sites per plant. Cytological analysis of 20 BC1 plants by GISH verified that the occurrence of 2n pollen formation in all F1 AuH hybrids was derived from the FDR (first division restitution) mechanism, in which the genome composition of all BC1 plants possess 12 Oriental + 12 L. auratum + 12 L. henryi chromosomes. Allotriploids derived from the AuH hybrid were used as female for crossing with the diploid Oriental hybrid cultivar 'Sorbonne' and considerable numbers of plants (0-6.5 plants per ovary) were only obtained when female OAuH (BC1) triploids were used. Taken together, the results of this study indicate that production and analysis of F1 AuH hybrids and their progeny through sexual polyploidization can be useful for efficient creation of important horticultural traits.

Pigmentation above the constitutive level: an indicator of excimer laser radiation-induced erythema in Koreans.

Ultraviolet (UV) irradiation induces skin erythema, but it is not clear which factors have the greatest effects on UV sensitivity. Six healthy Korean adult men were enrolled and their melanin index (MI) and increment of erythema index (ΔEI) were measured. In each individual, 12 different sites were selected and 36 spots were irradiated with a single shot of monochromatic excimer laser with a dose of 350 mJ/cm(2) . The sites were categorized into three groups based on the cumulative sun exposure: UZ, unexposed zones; FEZ, frequently exposed zones; and IEZ, intermittently exposed zones. The sun exposure indexes (SEI) were also calculated based on previously described methods. ΔEI, MI and SEI were measured and calculated. The ΔEI of UZ was significantly higher than that of FEZ, but lower than that of IEZ. In general, there was a significant relationship between ΔEI and MI (R(2) = 0.135). However, IEZ did not show significant results. In contrast, there was a stronger relationship between ΔEI and SEI (R(2) = 0.344). Overall, the values were significantly higher for the SEI (0.541 [UZ], 0.281 [IEZ] and 0.228 [FEZ]) than for MI (0.311 [UZ], 0.011 [IEZ] and 0.073 [FEZ]). There were significant site variations in UV sensitivity along with skin pigmentation. In addition, significant differences were observed according to the exposure frequency. The SEI was found to be strongly correlated with UV sensitivity. These results suggest that the induced level of pigmentation above the constitutive level will be a better indicator for UV sensitivity than baseline MI.

Combined transcriptome, genetic diversity and metabolite profiling in tomato fruit reveals that the ethylene response factor SlERF6 plays an important role in ripening and carotenoid accumulation.

Solanum lycopersicum (tomato) and its wild relatives harbor genetic diversity that yields heritable variation in fruit chemistry that could be exploited to identify genes regulating their synthesis and accumulation. Carotenoids, for example, are essential in plant and animal nutrition, and are the visual indicators of ripening for many fruits, including tomato. Whereas carotenoid synthesis is well characterized, factors regulating flux through the pathway are poorly understood at the molecular level. To exploit the impact of tomato genetic diversity on carotenoids, Solanum pennellii introgression lines were used as a source of defined natural variation and as a resource for the identification of candidate regulatory genes. Ripe fruits were analyzed for numerous fruit metabolites and transcriptome profiles generated using a 12,000 unigene oligoarray. Correlation analysis between carotenoid content and gene expression profiles revealed 953 carotenoid-correlated genes. To narrow the pool, subnetwork analysis of carotenoid-correlated transcription revealed 38 candidates. One candidate for impact on trans-lycopene and ß-carotene accumulation was functionally charaterized, SlERF6, revealing that it indeed influences carotenoid biosynthesis and additional ripening phenotypes. Reduced expression of SlERF6 by RNAi enhanced both carotenoid and ethylene levels during fruit ripening, demonstrating an important role for SlERF6 in ripening, integrating the ethylene and carotenoid synthesis pathways.