Exploring the role of FAT genes in Solanaceae species through genome-wide analysis and genome editing.

Plants produce numerous fatty acid derivatives, and some of these compounds have significant regulatory functions, such as governing effector-induced resistance, systemic resistance, and other defense pathways. This study systematically identified and characterized eight FAT genes (Acyl-acyl carrier protein thioesterases), four in the Solanum lycopersicum and four in the Solanum tuberosum genome. Phylogenetic analysis classified these genes into four distinct groups, exhibiting conserved domain structures across different plant species. Promoter analysis revealed various cis-acting elements, most of which are associated with stress responsiveness and growth and development. Micro-RNA (miRNA) analysis identified specific miRNAs, notably miRNA166, targeting different FAT genes in both species. Utilizing clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9)-mediated knockout, mutant lines for SlFATB1 and SlFATB3 were successfully generated and exhibited diverse mutation types. Biochemical evaluation of selected mutant lines revealed significant changes in fatty acid composition, with linoleic and linolenic acid content variations. The study also explored the impact of FAT gene knockout on tomato leaf architecture through scanning electron microscopy, providing insights into potential morphological alterations. Knocking out of FAT genes resulted in a significant reduction in both trichome and stoma density. These findings contribute to a comprehensive understanding of FAT genes in Solanaceous species, encompassing genetic, functional, and phenotypic aspects.

CRISPR/Cas9-mediated mutagenesis of FT/TFL1 in petunia improves plant architecture and early flowering.

Petunias are renowned ornamental species widely cultivated as pot plants for their aesthetic appeal both indoors and outdoors. The preference for pot plants depends on their compact growth habit and abundant flowering. While genome editing has gained significant popularity in many crop plants in addressing growth and development and abiotic and biotic stress factors, relatively less emphasis has been placed on its application in ornamental plant species. Genome editing in ornamental plants opens up possibilities for enhancing their aesthetic qualities, offering innovative opportunities for manipulating plant architecture and visual appeal through precise genetic modifications. In this study, we aimed to optimize the procedure for an efficient genome editing system in petunia plants using the highly efficient multiplexed CRISPR/Cas9 system. Specifically, we targeted a total of six genes in Petunia which are associated with plant architecture traits, two paralogous of FLOWERING LOCUS T (PhFT) and four TERMINAL FLOWER-LIKE1 (PhTFL1) paralogous genes separately in two constructs. We successfully induced homogeneous and heterogeneous indels in the targeted genes through precise genome editing, resulting in significant phenotypic alterations in petunia. Notably, the plants harboring edited PhTFL1 and PhFT exhibited a conspicuously early flowering time in comparison to the wild-type counterparts. Furthermore, mutants with alterations in the PhTFL1 demonstrated shorter internodes than wild-type, likely by downregulating the gibberellic acid pathway genes PhGAI, creating a more compact and aesthetically appealing phenotype. This study represents the first successful endeavor to produce compact petunia plants with increased flower abundance through genome editing. Our approach holds immense promise to improve economically important potting plants like petunia and serve as a potential foundation for further improvements in similar ornamental plant species.

GATA transcription factor in common bean: A comprehensive genome-wide functional characterization, identification, and abiotic stress response evaluation.

The GATA transcription factors (TFs) have been extensively studied for its regulatory role in various biological processes in many plant species. The functional and molecular mechanism of GATA TFs in regulating tolerance to abiotic stress has not yet been studied in the common bean. This study analyzed the functional identity of the GATA gene family in the P. vulgaris genome under different abiotic and phytohormonal stress. The GATA gene family was systematically investigated in the P. vulgaris genome, and 31 PvGATA TFs were identified. The study found that 18 out of 31 PvGATA genes had undergone duplication events, emphasizing the role of gene duplication in GATA gene expansion. All the PvGATA genes were classified into four significant subfamilies, with 8, 3, 6, and 13 members in each subfamily (subfamilies I, II, III, and IV), respectively. All PvGATA protein sequences contained a single GATA domain, but subfamily II members had additional domains such as CCT and tify. A total of 799 promoter cis-regulatory elements (CREs) were predicted in the PvGATAs. Additionally, we used qRT-PCR to investigate the expression profiles of five PvGATA genes in the common bean roots under abiotic conditions. The results suggest that PvGATA01/10/25/28 may play crucial roles in regulating plant resistance against salt and drought stress and may be involved in phytohormone-mediated stress signaling pathways. PvGATA28 was selected for overexpression and cloned into N. benthamiana using Agrobacterium-mediated transformation. Transgenic lines were subjected to abiotic stress, and results showed a significant tolerance of transgenic lines to stress conditions compared to wild-type counterparts. The seed germination assay suggested an extended dormancy of transgenic lines compared to wild-type lines. This study provides a comprehensive analysis of the PvGATA gene family, which can serve as a foundation for future research on the function of GATA TFs in abiotic stress tolerance in common bean plants.

Genome-wide analysis of PvMADS in common bean and functional characterization of PvMADS31 in Arabidopsis thaliana as a player in abiotic stress responses.

Changing climatic conditions with rising temperatures and altered precipitation patterns pose significant challenges to agricultural productivity, particularly for common bean crops. Transcription factors (TFs) are crucial regulators that can mitigate the impact of biotic and abiotic stresses on crop production. The MADS-box TFs family has been implicated in various plant physiological processes, including stress-responsive mechanisms. However, their role in common bean and their response to stressful conditions remain poorly understood. Here, we identified 35 MADS-box gene family members in common bean, with conserved MADS-box domains and other functional domains. Gene duplication events were observed, suggesting the significance of duplication in the evolutionary development of gene families. The analysis of promoter regions revealed diverse elements, including stress-responsive elements, indicating their potential involvement in stress responses. Notably, PvMADS31, a member of the PvMADS-box gene family, demonstrated rapid upregulation under various abiotic stress conditions, including NaCl, polyethylene glycol, drought, and abscisic acid (ABA) treatments. Transgenic plants overexpressing PvMADS31 displayed enhanced lateral root development, root elongation, and seed germination under stress conditions. Furthermore, PvMADS31 overexpression in Arabidopsis resulted in improved drought tolerance, likely attributed to the enhanced scavenging of ROS and increased proline accumulation. These findings suggest that PvMADS31 might play a crucial role in modulating seed germination, root development, and stress responses, potentially through its involvement in auxin and ABA signaling pathways. Overall, this study provides valuable insights into the potential roles of PvMADS-box genes in abiotic stress responses in common bean, offering prospects for crop improvement strategies to enhance resilience under changing environmental conditions.

Investigation and Expression Analysis of R2R3-MYBs and Anthocyanin Biosynthesis-Related Genes during Seed Color Development of Common Bean (Phaseolus vulgaris).

Anthocyanins are responsible for the coloration of common bean seeds, and their accumulation is positively correlated with the expression level of anthocyanin biosynthetic genes. The MBW (MYB-bHLH-WD40) complex is thought to regulate the expression of these genes, and MYB proteins, which are a key factor in activating anthocyanin pathway genes, have been identified in several plants. This study demonstrated gene structures, chromosomal placements, gene duplications of R2R3-MYBs, miRNAs associated with R2R3-MYBs, and the interaction of these genes with other flavonoid regulatory genes. qRT-PCR was used to investigate the role of specific R2R3-MYBs and flavonoid genes in common bean seed color development. As a result of a comprehensive analysis with the help of in silico tools, we identified 160 R2R3-MYB genes in the common bean genome. We divided these genes into 16 classes on the basis of their intron-exon and motif structures. Except for three, the rest of the common bean R2R3-MYB members were distributed to all chromosomes with different densities, primarily located on chromosomes 3 and 8. We identified a total of 44 duplicated gene pairs dispersed across 11 chromosomes and evolved under purifying selection (Ka/Ks < 1), 19 of which were derived from a whole-genome duplication. Our research uncovered 25 putative repressor PvMYB proteins that contain the EAR motif. Additionally, fifty different cis-regulatory elements regulated by light, stress, and hormone were identified. Within the genome of the common bean, we discovered a total of 36 microRNAs that target a total of 72 R2R3-MYB transcripts. The effect of 16 R2R3-MYB genes and 16 phenylpropanoid pathway genes, selected on the basis of their interaction in the protein-protein interaction map, playing role in the regulation of seed coat color development was evaluated using qRT-PCR in 5 different tissues at different developmental stages. The results revealed that these specific genes have different expression levels during different developmental periods, with higher levels in the pod filling and early pod stages than in the rest of the developmental periods. Furthermore, it was shown that PvTT8 (bHLH), PvTT2 (PvMYB42), PvMYB113, PvTTG1, and PvWD68 genes have effects on the regulation of seed coat color. The findings of this study, which is the first to use whole-genome analysis to identify and characterize the R2R3-MYB genes in common bean, may serve as a reference for future functional research in the legume.

Genome-Wide Identification of the Aconitase Gene Family in Tomato (Solanum lycopersicum) and CRISPR-Based Functional Characterization of SlACO2 on Male-Sterility.

Tomato (Solanum lycopersicum) is one of the most cultivated vegetables in the world due to its consumption in a large variety of raw, cooked, or processed foods. Tomato breeding and productivity highly depend on the use of hybrid seeds and their higher yield, environmental adaption, and disease tolerance. However, the emasculation procedure during hybridization raises tomato seed production costs and labor expenses. Using male sterility is an effective way to reduce the cost of hybrid seeds and ensure cultivar purity. Recent developments in CRISPR genome editing technology enabled tomato breeders to investigate the male sterility genes and to develop male-sterile tomato lines. In the current study, the tomato Acotinase (SlACO) gene family was investigated via in silico tools and functionally characterized with CRISPR/Cas9-mediated gene disruption. Genome-wide blast and HMM search represented two SlACO genes located on different tomato chromosomes. Both genes were estimated to have a segmental duplication in the tomato genome due to their identical motif and domain structure. One of these genes, SlACO2, showed a high expression profile in all generative cells of tomato. Therefore, the SlACO2 gene was targeted with two different gRNA/Cas9 constructs to identify their functional role in tomatoes. The gene was mutated in a total of six genome-edited tomato lines, two of which were homozygous. Surprisingly, pollen viability was found to be extremely low in mutant plants compared to their wild-type (WT) counterparts. Likewise, the number of seeds per fruit also sharply decreased more than fivefold in mutant lines (10-12 seeds) compared to that in WT (67 seeds). The pollen shape, anther structures, and flower colors/shapes were not significantly varied between the mutant and WT tomatoes. The mutated lines were also subjected to salt and mannitol-mediated drought stress to test the effect of SlACO2 on abiotic stress tolerance. The results of the study indicated that mutant tomatoes have higher tolerance with significantly lower MDA content under stress conditions. This is the first CRISPR-mediated characterization of ACO genes on pollen viability, seed formation, and abiotic stress tolerance in tomatoes.