Genome-wide characterization and expression profiling of E2F/DP gene family members in response to abiotic stress in tomato (Solanum lycopersicum L.).

BACKGROUND: E2F/DP (Eukaryotic 2 transcription factor/dimerization partner) family proteins play an essential function in the cell cycle development of higher organisms. E2F/DP family genes have been reported only in a few plant species. However, comprehensive genome-wide characterization analysis of the E2F/DP gene family of Solanum lycopersicum has not been reported so far. RESULTS: This study identified eight nonredundant SlE2F/DP genes that were classified into seven groups in the phylogenetic analysis. All eight genes had a single E2F-TDP domain and few genes had additional domains. Two segmental duplication gene pairs were observed within tomato, in addition to cis-regulatory elements, miRNA target sites and phosphorylation sites which play an important role in plant development and stress response in tomato. To explore the three-dimensional (3D) models and gene ontology (GO) annotations of SlE2F/DP proteins, we pointed to their putative transporter activity and their interaction with several putative ligands. The localization of SlE2F/DP-GFP fused proteins in the nucleus and endoplasmic reticulum suggested that they may act in other biological functions. Expression studies revealed the differential expression pattern of most of the SlE2F/DP genes in various organs. Moreover, the expression of E2F/DP genes against abiotic stress, particularly SlE2F/DP2 and/or SlE2F/DP7, was upregulated in response to heat, salt, cold and ABA treatment. Furthermore, the co-expression analysis of SlE2F/DP genes with multiple metabolic pathways was co-expressed with defence genes, transcription factors and so on, suggested their crucial role in various biological processes. CONCLUSIONS: Overall, our findings provide a way to understand the structure and function of SlE2F/DP genes; it might be helpful to improve fruit development and tolerance against abiotic stress through marker-assisted selection or transgenic approaches.

Complete genome sequence of a tentative novel capillovirus isolated from Gerbera jamesonii.

The currently named gerbera virus A (GeVA) has been shown to be a novel capillovirus with a complete genome of 6929 nucleotides (nt) (GenBank accession no. OM525829.1). GeVA was detected in Gerbera jamesonii using high-throughput RNA sequencing analysis. The GeVA genome is a single linear RNA with two open reading frames (ORF), similar to those of other capilloviruses. The larger ORF encodes a polyprotein containing four domains, while the smaller ORF encodes a movement protein. The complete genome had 41.0-54.9% nt sequence identity to other those of capilloviruses, while the polyprotein and the movement protein had 26.5-36.4% and 13.1-32.2% amino acid (aa) sequence identity, respectively. Two UUAGGU promoters for subgenomic RNA (sgRNA) transcription were also identified in this study. BLAST analysis demonstrated that the GeVA genome shared the highest sequence similarity with rubber tree capillovirus 1 (MN047299.1) (complete nucleotide sequence identity, 68.54%; polyprotein amino acid sequence identity, 44.53%). Phylogenetic analysis based on complete genome and replication protein sequences placed GeVA alongside other members of the genus Capillovirus in the family Betaflexiviridae. These data suggest that GeVA is a new member of the genus Capillovirus.

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.

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.

Comprehensive Genome-Wide Analysis and Expression Pattern Profiling of the SlHVA22 Gene Family Unravels Their Likely Involvement in the Abiotic Stress Adaptation of Tomato.

HVA22 family proteins with a conserved TB2/DP1/HVA22 domain are ubiquitous in eukaryotes. HVA22 family genes have been identified in a variety of plant species. However, there has been no comprehensive genome-wide analysis of HVA22 family genes in tomato (Solanum lycopersicum L.). Here, we identified 15 non-redundant SlHVA22 genes with three segmentally duplicated gene pairs on 8 of the 12 tomato chromosomes. The predicted three-dimensional (3D) models and gene ontology (GO) annotations of SlHVA22 proteins pointed to their putative transporter activity and ability to bind to diverse ligands. The co-expression of SlHVA22 genes with various genes implicated in multiple metabolic pathways and the localization of SlHVA22-GFP fused proteins to the endoplasmic reticulum suggested that they might have a variety of biological functions, including vesicular transport in stressed cells. Comprehensive expression analysis revealed that SlHVA22 genes were differentially expressed in various organs and in response to abiotic stress conditions. The predominant expression of SlHVA22i at the ripening stage and that of SlHVA22g, SlHVA22k, and SlHVA22l in fruits at most developmental stages suggested their probable involvement in tomato fruit development and ripening. Moreover, the transcript expression of most tomato HVA22 genes, particularly SlHVA22b, SlHVA22i, SlHVA22k, SlHVA22l, SlHVA22m, and SlHVA22n, was affected by abscisic acid (ABA) and diverse abiotic stress treatments, indicating the likely involvement of these genes in tomato abiotic stress responses in an ABA-dependent manner. Overall, our findings provide a foundation to better understand the structures and functional roles of SlHVA22 genes, many of which might be useful to improve the abiotic stress tolerance and fruit quality of tomato through marker-assisted backcrossing or transgenic approaches.

Genome-wide identification, genomic organization, and expression profiling of the CONSTANS-like (COL) gene family in petunia under multiple stresses.

BACKGROUND: CONSTANS-like (CO-like, COL) are putative zinc-finger transcription factors known to play vital role in various plant biological processes such as control of flowering time, regulation of plant growth and development and responses to stresses. However, no systematic analysis of COL family gene regarding the plant development and stress response has been previously performed in any solanaceous crop. In the present study, a comprehensive genome-wide analysis of COL family genes in petunia has been conducted to figure out their roles in development of organs and stress response. RESULTS: A total of 33 COL genes, 15 PaCOL genes in P. axillaris and 18 PiCOL genes in P. inflata, were identified in petunia. Subsequently, a genome-wide systematic analysis was performed in 15 PaCOL genes. Considering the domain composition and sequence similarity the 15 PaCOL and 18 PiCOL genes were phylogenetically classified into three groups those are conserved among the flowering plants. Moreover, all of the 15 PaCOL proteins were localized in nucleus. Furthermore, differential expression patterns of PaCOL genes were observed at different developmental stages of petunia. Additionally, transcript expression of 15 PaCOL genes under various abiotic and phytohormone treatments showed their response against stresses. Moreover, several cis-elements related to stress, light-responsive, hormone signaling were also detected in different PaCOL genes. CONCLUSION: The phylogenetic clustering, organ specific expression pattern and stress responsive expression profile of conserved petunia COL genes indicating their involvement in plant growth and development and stress response mechanism. This work provide a significant foundation for understanding the biological roles of petunia COL genes in plant growth, development and in stress response.

Genome-wide identification and expression profiling of Alba gene family members in response to abiotic stress in tomato (Solanum lycopersicum L.).

BACKGROUND: Alba (Acetylation lowers binding affinity) proteins are an ancient family of nucleic acid-binding proteins that function in gene regulation, RNA metabolism, mRNA translatability, developmental processes, and stress adaptation. However, comprehensive bioinformatics analysis on the Alba gene family of Solanum lycopersicum has not been reported previously. RESULTS: In the present study, we undertook the first comprehensive genome-wide characterization of the Alba gene family in tomato (Solanum lycopersicum L.). We identified eight tomato Alba genes, which were classified into two groups: genes containing a single Alba domain and genes with a generic Alba domain and RGG/RG repeat motifs. Cis-regulatory elements and target sites for miRNAs, which function in plant development and stress responses, were prevalent in SlAlba genes. To explore the structure-function relationships of tomato Alba proteins, we predicted their 3D structures, highlighting their likely interactions with several putative ligands. Confocal microscopy revealed that SlAlba-GFP fusion proteins were localized to the nucleus and cytoplasm, consistent with putative roles in various signalling cascades. Expression profiling revealed the differential expression patterns of most SlAlba genes across diverse organs. SlAlba1 and SlAlba2 were predominantly expressed in flowers, whereas SlAlba5 expression peaked in 1 cm-diameter fruits. The SlAlba genes were differentially expressed (up- or downregulated) in response to different abiotic stresses. All but one of these genes were induced by abscisic acid treatment, pointing to their possible regulatory roles in stress tolerance via an abscisic acid-dependent pathway. Furthermore, co-expression of SlAlba genes with multiple genes related to several metabolic pathways spotlighted their crucial roles in various biological processes and signalling. CONCLUSIONS: Our characterization of SlAlba genes should facilitate the discovery of additional genes associated with organ and fruit development as well as abiotic stress adaptation in tomato.

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.

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.

Ectopic expression of miRNA172 in tomato (Solanum lycopersicum) reveals novel function in fruit development through regulation of an AP2 transcription factor.

BACKGROUND: MicroRNAs (miRNAs) are short non-coding RNAs that can influence gene expression via diverse mechanisms. Tomato is a fruit widely consumed for its flavor, culinary attributes, and high nutritional quality. Tomato fruit are climacteric and fleshy, and their ripening is regulated by endogenous and exogenous signals operating through a coordinated genetic network. Much research has been conducted on mechanisms of tomato fruit ripening, but the roles of miRNA-regulated repression/expression of specific regulatory genes are not well documented. RESULTS: In this study, we demonstrate that miR172 specifically targets four SlAP2 transcription factor genes in tomato. Among them, SlAP2a was repressed by the overexpression of SlmiR172, manifesting in altered flower morphology, development and accelerated ripening. miR172 over-expression lines specifically repressed SlAP2a, enhancing ethylene biosynthesis, fruit color and additional ripening characteristics. Most previously described ripening-regulatory genes, including RIN-MADS, NR, TAGL1 and LeHB-1 were not influenced by miR172 while CNR showed altered expression. CONCLUSIONS: Tomato fruit ripening is directly influenced by miR172 targeting of the APETALA2 transcription factor, SlAP2a, with minimal influence over additional known ripening-regulatory genes. miR172a-guided SlAP2a expression provides insight into another layer of genetic control of ripening and a target for modifying the quality and nutritional value of tomato and possibly other fleshy fruit crops.

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.

Roles of R2R3-MYB transcription factors in transcriptional regulation of anthocyanin biosynthesis in horticultural plants.

KEY MESSAGE: This review contains functional roles of MYB transcription factors in the transcriptional regulation of anthocyanin biosynthesis in horticultural plants. This review describes potential uses of MYB TFs as tools for metabolic engineering for anthocyanin production. Anthocyanins (ranging from red to blue) are controlled by specific branches of the anthocyanin biosynthetic pathway and are mostly visible in ornamentals, fruits, and vegetables. In the present review, we describe which R2R3-MYB transcription factors (TFs) control the transcriptional regulation of anthocyanin structural genes involved in the specific branches of the anthocyanin biosynthetic pathway in various horticultural plants (e.g., ornamentals, fruits, and vegetables). In addition, some MYBs responsible for anthocyanin accumulation in specific tissues are described. Moreover, we highlight the phylogenetic relationships of the MYBs that suppress or promote anthocyanin synthesis in horticultural crops. Enhancement of anthocyanin synthesis via metabolic genetic engineering of anthocyanin MYBs, which is described in the review, is indicative of the potential use of the mentioned anthocyanin-related MYBs as tools for anthocyanin production. Therefore, the MYBs would be suitable for metabolic genetic engineering for improvement of flower colors, fruit quality, and vegetable nutrients.

Anti-freezing-protein type III strongly influences the expression of relevant genes in cryopreserved potato shoot tips.

KEY MESSAGE: AFP improved cryopreservation efficiency of potato (Solanum tuberosum cv. Superior) by regulating transcript levels of CBF1 and DHN1. However, the optimal AFP concentration required for strong induction of the genes was dependent on the type of vitrification solution to which the AFP was added: This finding suggests that AFP increased cryopreservation efficiency by transcriptional regulation of these genes, which might protect plant cell membranes from cold stress during cryopreservation. Despite the availability of many studies reporting the benefits of anti-freeze protein III (AFP III) as a cryoprotectant, the role of AFP III in this process has not been well demonstrated using molecular analysis. In addition, AFP III has not been exploited in the cryopreservation of potato thus far. Therefore, we studied the effects of AFP III on the cryopreservation of potato (Solanum tuberosum cv. Superior). We found that CBF1 and DHN1 genes are low temperature-inducible in potato leaves (S. tuberosum cv. Superior). Transcript levels of these genes expressed in shoot tips cryopreserved with AFP III were higher than those of the controls. However, the optimal AFP III concentration required for strong induction of the genes was dependent on the type of cryoprotection solution to which the AFP III was added: 500 ng/mL worked best for PVS2, while 1500 ng/mL was optimal for LS. Interestingly, the involvement of AFP III in the cryoprotection solutions improved cryopreservation efficiency as compared to the control, and expression levels of the detected genes were associated with cryopreservation efficiency. This finding suggests that AFP III increased cryopreservation efficiency by transcriptional regulation of these genes, which might protect plant cell membranes from cold stress during cryopreservation. Therefore, we expect that our findings will lead to the successful application of AFP III as a potent cryoprotectant in the cryopreservation of rare and commercially important plant germplasms.

Genome-wide analysis and expression profiling of zinc finger homeodomain (ZHD) family genes reveal likely roles in organ development and stress responses in tomato.

BACKGROUND: Zinc finger homeodomain proteins (ZHD) constitute a plant-specific transcription factor family with a conserved DNA binding homeodomain and a zinc finger motif. Members of the ZHD protein family play important roles in plant growth, development, and stress responses. Genome-wide characterization of ZHD genes has been carried out in several model plants, including Arabidopsis thaliana and Oryza sativa, but not yet in tomato (Solanum lycopersicum). RESULTS: In this study, we performed the first comprehensive genome-wide characterization and expression profiling of the ZHD gene family in tomato (Solanum lycopersicum). We identified 22 SlZHD genes and classified them into six subfamilies based on phylogeny. The SlZHD genes were generally conserved in each subfamily, with minor variations in gene structure and motif distribution. The 22 SlZHD genes were distributed on six of the 12 tomato chromosomes, with segmental duplication detected in four genes. Analysis of Ka/Ks ratios revealed that the duplicated genes are under negative or purifying selection. Comprehensive expression analysis revealed that the SlZHD genes are widely expressed in various tissues, with most genes preferentially expressed in flower buds compared to other tissues. Moreover, many of the genes are responsive to abiotic stress and phytohormone treatment. CONCLUSION: Systematic analysis revealed structural diversity among tomato ZHD proteins, which indicates the possibility for diverse roles of SlZHD genes in different developmental stages as well as in response to abiotic stresses. Our expression analysis of SlZHD genes in various tissues/organs and under various abiotic stress and phytohormone treatments sheds light on their functional divergence. Our findings represent a valuable resource for further analysis to explore the biological functions of tomato ZHD genes.

Characterization of the role of sodium nitroprusside (SNP) involved in long vase life of different carnation cultivars.

BACKGROUND: Sodium nitroprusside (SNP) has been previously shown to extend the vase life of various cut flowers; however, its positive effect on extending vase life of carnations has not been well documented. Moreover, the role of SNP in the mechanisms underlying determination of vase life of cut carnations has also not been well addressed. RESULTS: SNP increased vase life of Tico Viola carnations along with their relative fresh weight (RFW). Among the treatments, the flowers treated with 10 mg L-1 SNP had the longest vase life and maximum relative fresh weight (RFW). This was achieved through significant suppression of ethylene production via downregulation of ethylene biosynthesis and petal senescence-related genes, and through an increase in the scavenging mechanism of reactive oxygen species (ROS) by antioxidant activity during flower vase life. In addition, the positive efficacy of SNP could also be confirmed using 1-aminocyclopropane-1-carboxylic acid (ACC) and different cultivars, resulting in similar trends for both experiments. CONCLUSION: Taken together, these results suggest that SNP plays a crucial role in multiple modes of action that are associated with the longevity of cut carnation flowers.

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.

Molecular Characterization and Expression Profiling of Tomato GRF Transcription Factor Family Genes in Response to Abiotic Stresses and Phytohormones.

Growth regulating factors (GRFs) are plant-specific transcription factors that are involved in diverse biological and physiological processes, such as growth, development and stress and hormone responses. However, the roles of GRFs in vegetative and reproductive growth, development and stress responses in tomato (Solanum lycopersicum) have not been extensively explored. In this study, we characterized the 13 SlGRF genes. In silico analysis of protein motif organization, intron-exon distribution, and phylogenetic classification confirmed the presence of GRF proteins in tomato. The tissue-specific expression analysis revealed that most of the SlGRF genes were preferentially expressed in young and growing tissues such as flower buds and meristems, suggesting that SlGRFs are important during growth and development of these tissues. Some of the SlGRF genes were preferentially expressed in fruits at distinct developmental stages suggesting their involvement in fruit development and the ripening process. The strong and differential expression of different SlGRFs under NaCl, drought, heat, cold, abscisic acid (ABA), and jasmonic acid (JA) treatment, predict possible functions for these genes in stress responses in addition to their growth regulatory functions. Further, differential expression of SlGRF genes upon gibberellic acid (GA3) treatment indicates their probable function in flower development and stress responses through a gibberellic acid (GA)-mediated pathway. The results of this study provide a basis for further functional analysis and characterization of this important gene family in tomato.

Inferring the Genetic Determinants of Fruit Colors in Tomato by Carotenoid Profiling.

Carotenoids are essential for plant and animal nutrition, and are important factors in the variation of pigmentation in fruits, leaves, and flowers. Tomato is a model crop for studying the biology and biotechnology of fleshy fruits, particularly for understanding carotenoid biosynthesis. In commercial tomato cultivars and germplasms, visual phenotyping of the colors of ripe fruits can be done easily. However, subsequent analysis of metabolic profiling is necessary for hypothesizing genetic factors prior to performing time-consuming genetic analysis. We used high performance liquid chromatography (HPLC), employing a C30 reverse-phase column, to efficiently resolve nine carotenoids and isomers of several carotenoids in yellow, orange, and red colored ripe tomatoes. High content of lycopene was detected in red tomatoes. The orange tomatoes contained three dominant carotenoids, namely δ-carotene, ß-carotene, and prolycopene. The yellow tomatoes showed low levels of carotenoids compared to red or orange tomatoes. Based on the HPLC profiles, genes responsible for overproducing δ-carotene and prolycopene were described as lycopene ε-cyclase and carotenoid isomerase, respectively. Subsequent genetic analysis using DNA markers for segregating population and germplasms were conducted to confirm the hypothesis. This study establishes the usefulness of metabolic profiling for inferring the genetic determinants of fruit color.

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.

Production of genetically stable and Odontoglossum ringspot virus-free Cymbidium orchid 'New True' plants via meristem-derived protocorm-like body (PLB) subcultures.

BACKGROUND: This study aimed to produce Odontoglossum ringspot virus (ORSV)-free Cymbidium orchid 'New True' plants from ORSV-infected mother plants by culturing their meristems and successively repeating subcultures of protocorm-like bodies (PLBs) derived from the meristems. RESULTS: Initially, ORSV was confirmed as the causative agent of viral symptoms in orchid leaves via reverse transcription-polymerase chain reaction (RT-PCR) analysis. Meristems from infected plants were cultured to generate PLBs, which in sequence were repeatedly subcultured up to four times. RT-PCR and quantitative RT-PCR analyses revealed that while ORSV was undetectable in shoots derived from the first subculture, complete elimination of the virus required at least a second subculture. Genetic analysis using inter-simple sequence repeat markers indicated no somaclonal variation between regenerated plants and the mother plant, suggesting that genetic consistency was maintained. CONCLUSION: Overall, our findings demonstrate that subculturing PLBs for a second time is ideal for producing genetically stable, ORSV-free Cymbidium orchids, thus offering a practical means of generating genetically stable, virus-free plants and enhancing plant health and quality in the orchid industry.