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.

In-depth comparative transcriptome analysis of Purpureocillium sp. CB1 under cadmium stress.

Fungal bioremediation is a very attractive tool to cope with environmental pollution. We aimed to decipher the cadmium (Cd) response of Purpureocillium sp. CB1, isolated from polluted soil, at transcriptome level by RNA-sequencing (RNA-seq). We used 500 and 2500 mg/L of Cd2+ concentrations at two time points (t6;36). RNA-seq determined 620 genes that were co-expressed in all samples. The highest number of differentially expressed genes (DEGs) was obtained within the first six h of exposure to 2500 mg/L of Cd2+. Several genes encoding transcriptional regulators, transporters, heat shock proteins, and oxidative stress-related genes were differentially expressed under Cd2+ stress. Remarkably, the genes that encode salicylate hydroxylase, which is involved in naphthalene biodegradation pathway, were significantly overexpressed. Utilization of diesel as the sole carbon source by CB1 even in the presence of Cd2+ supported concomitant upregulation of hydrocarbon degradation pathway genes. Furthermore, leucinostatin-related gene expression levels increased under Cd2+ stress. In addition, leucinostatin extracts from Cd2+-treated CB1 cultures showed higher antifungal activity than the control. Notably, Cd2+ in CB1 was mainly found as bound to the cell wall, thus confirming its adsorption potential. Cd2+ stress slightly reduced growth and led to mycelial malformation due to Cd2+ adsorption, especially at a concentration of 2500 mg/L at t36. A strong correlation was recorded between RNA-seq and reverse-transcriptase-quantitative polymerase chain reaction (RT-qPCR) data. In conclusion, the study represents the first transcriptome analysis of Purpureocillium sp. under Cd2+ stress, providing insights into the primary targets for rational engineering to construct strains with remarkable bioremediation potency. KEY POINTS: • Upregulation of genes encoding salicylate hydroxylases under Cd2+ stress • Maximum Cd2+ adsorption at 500 mg/L at t36 as tightly bound to the cell wall • Concordant bioremediation potential of CB1 on Cd2+ and diesel.

Development of Highly Efficient Resistance to Beet Curly Top Iran Virus (Becurtovirus) in Sugar Beet (B. vulgaris) via CRISPR/Cas9 System.

Beet Curly Top Iran Virus (BCTIV, Becurtovirus) is a dominant and widespread pathogen responsible for great damage and yield reduction in sugar beet production in the Mediterranean and Middle East. CRISPR-based gene editing is a versatile tool that has been successfully used in plants to improve resistance against many viral pathogens. In this study, the efficiency of gRNA/Cas9 constructs targeting the expressed genes of BCTIV was assessed in sugar beet leaves by their transient expression. Almost all positive control sugar beets revealed systemic infection and severe disease symptoms (90%), with a great biomass reduction (68%) after BCTIV agroinoculation. On the other hand, sugar beets co-agronioculated with BCTIV and gRNA/Cas9 indicated much lower systemic infection (10-55%), disease symptoms and biomass reduction (13-45%). Viral inactivation was also verified by RCA and qPCR assays for gRNA/Cas9 treated sugar beets. PCR-RE digestion and sequencing assays confirmed the gRNA/Cas9-mediated INDEL mutations at the target sites of the BCTIV genome and represented high efficiencies (53-88%), especially for those targeting BCTIV's movement gene and its overlapping region between capsid and ssDNA regulator genes. A multiplex CRISPR approach was also tested. The most effective four gRNAs targeting all the genes of BCTIV were cloned into a Cas9-containing vector and agroinoculated into virus-infected sugar beet leaves. The results of this multiplex CRISPR system revealed almost complete viral resistance with inhibition of systemic infection and mutant escape. This is the first report of CRSIPR-mediated broad-spectrum resistance against Becurtovirus in sugar beet.

General guidelines for CRISPR/Cas-based genome editing in plants.

CRISPR (clustered regularly interspaced short palindromic repeats)/Cas (CRISPR-associated) technology is a versatile genome editing tool that has been used to improve agriculturally important plant traits. Due to its precision, CRISPR/Cas9 is more effective than either conventional plant breeding methods or standard genetic engineering approaches for the rapid development of new varieties resilient to climate change. In addition to knowledge in tissue culture-based plant transformation, effective gene-specific single guide RNA (sgRNA) design, prediction of its off-target effect and utilization of vectors, promoters, Cas proteins and terminators is required for CRISPR/Cas9. Various bioinformatics tools are available for the best sgRNA design and screening of the off-targets. Various tools are used in the delivery of CRISPR/Cas components into cells and the genome. Moreover, some recent studies proved the simultaneous silencing of different paralogs in the same family or several genes working in the same pathway by using multiple-target sgRNA designs. This review summarizes the type of promoters, Cas proteins, recognition sequences, and terminators available for the development of knock-out and overexpression plant lines. It also provides a general guideline for the development of genome-edited plants from the design of sgRNAs to the selection of non-transgenic genome-edited T2 generation.

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.

Genome-wide identification of the BURP domain-containing genes in Phaseolus vulgaris.

Plant-specific BURP domain-containing proteins have an essential role in the plant's development and stress responses. Although BURP domain-containing proteins have been identified in several plant species, genome-wide analysis of the BURP gene family has not been investigated in the common bean. In the present study, we identified 11 BURP family members in the common bean (Phaseolus vulgaris) genome with a comprehensive in silico analysis. Pairwise alignment and phylogenetic analyses grouped PvBURP members into four subfamilies [RD-22 like (3), PG1ß-like (4), BNM2-like (3), and USP-like (1)] according to their amino acid motifs, protein domains and intron-exon structure. The physical and biochemical characteristics of amino acids, motif and intron-exon structure, and cis-regulatory elements of BURPs members were determined. Promoter regions of BURP members included stress, light, and hormone response-related cis-elements. Therefore, expression profiles of PvBURP genes were identified with in silico tools and qRT-PCR analyses under stress (salt and drought) and hormone treatment (ABA, IAA) in the current study. While significant activity changes were not observed in BURP genes in RNA-seq data sets related to salt stress, it was determined that some BURP genes were expressed differently in those with drought stress. We identified 12 different miRNA, including miRNA395, miRNA156, miRNA169, miRNA171, miRNA319, and miRNA390, targeting the nine PvBURP genes using two different in silico tools based on perfect or near-perfect complementarity to their targets. Here we present the first study to identify and characterize the BURP genes in common bean using whole-genome analysis, and the findings may serve as a reference for future functional research in common bean. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s12298-021-01052-9.

Overexpression of Ks-type dehydrins gene OeSRC1 from Olea europaea increases salt and drought tolerance in tobacco plants.

Agricultural production is greatly affected by environmental stresses, such as cold, drought and high-salinity. It is possible to produce tolerant genotypes by transferring genes encoding protective proteins or enzymes from other organisms. In this regard, the current study was aimed to clone a novel OeSRC1 gene identified during the transcriptome profiling of olives (Olea europaea L.) and to investigate the function of this gene in tobacco plants. Functional evaluation of OeSRC1 gene in putative transgenic tobacco plants were carried out under drought, cold and salt stress conditions by using molecular and biochemical tools. It was observed that the transgenic tobacco plants exhibited higher seed germination and survival rates, better root and shoot growth under cold, salt and drought stress treatments compared to wild type plants. Our results also demonstrated that, under stress conditions, transgenic plants accumulated more free proline while no significant changes were observed regarding electrolyte leakage. Ascorbate peroxidase activity of OeSRC1-overexpressing plants was higher than those of the WT plants under different stress conditions. The overall results demonstrate the explicit role of OeSRC1 gene in conferring multiple abiotic stress tolerance at the whole-plant level. The multifunctional role of olive OeSRC1 gene looks good to enhance environmental stress tolerance in diverse plants.

Gene expression analysis of bud burst process in European hazelnut (Corylus avellana L.) using RNA-Seq.

The control of bud burst process depending on temperature is crucial factor in woody perennial plants to survive in unfavorable ecological conditions. Although it has important economic and agronomic values, little information is available on the molecular mechanism of the bud burst process in Corylus avellana. Here for the first time, we conducted a de novo transcriptome-based experiment using eco-dormant leaf bud tissues. Four transcriptome libraries were constructed from the leaf bud tissues and sequenced via Illumina platform. Transcriptome analysis revealed 86,394 unigenes with a mean length of 1189 nt and an N50 of 1916 nt. Among these unigenes, 63,854 (73.78%) of them were annotated by at least one database. De novo assembled transcripts were enriched in phenylpropanoid metabolism, phytohormone biosynthesis and signal transduction pathways. Analyses of phytohormone-associated genes revealed important changes during bud burst, in response to gibberellic acid, auxin, and brassinosteroids. Approximately 2163 putative transcription factors were predicted, of which the largest number of unique transcripts belonged to the MYB transcription factor family. These results contribute to a better understanding of the regulation of bud burst genes in perennial plants.

Genome-wide characterization and expression analysis of common bean bHLH transcription factors in response to excess salt concentration.

Members of basic helix-loop-helix (bHLH) gene family found in all eukaryotes play crucial roles in response to stress. Though, most eukaryotes carry the proteins of this family, biological functions of the most bHLH family members are not deeply evaluated in plants. In this study, we conducted a comprehensive genome-wide analysis of bHLH transcription factors in salt tolerant common bean. We identified 155 bHLH protein-encoding genes (PvbHLH) by using in silico comparative genomics tools. Based on the phylogenetic tree, PvbHLH genes were classified into 8 main groups with 21 subfamilies. Exon-intron analysis indicated that proteins belonging to same main groups exhibited a closely related gene structure. While, the PvbHLH gene family has been mainly expanded through segmental duplications, a total of 11 tandem duplication were detected. Genome-wide expression analysis of bHLH genes showed that 63 PvbHLH genes were differentially expressed in at least one tissue. Three of them displayed higher expression values in both leaf and root tissues. The in silico micro-RNA target transcript analyses revealed that totally 100 PvHLH genes targeted by 86 plant miRNAs. The most abundant transcripts, which were targeted by all 18 plant miRNA, were belonging to PvHLH-22 and PvHLH-44 genes. The expression of 16 PvbHLH genes in the root and leaf tissues of salt-stressed common bean was evaluated using qRT-PCR. Among them, two of PvbHLHs, PvbHLH-54, PvbHLH-148, were found to be up-regulated in both tissues in correlation with RNA-seq measurements. The results of this study could help improve understanding of biological functions of common bean bHLH family under salt stress. Additionally, it may provide basic resources for analyzing bHLH protein function for improving economic, agronomic and ecological benefit in common bean and other species.

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.

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.

Genome editing for healthy crops: traits, tools and impacts.

Crop cultivars in commercial use have often been selected because they show high levels of resistance to pathogens. However, widespread cultivation of these crops for many years in the environments favorable to a pathogen requires durable forms of resistance to maintain "healthy crops". Breeding of new varieties tolerant/resistant to biotic stresses by incorporating genetic components related to durable resistance, developing new breeding methods and new active molecules, and improving the Integrated Pest Management strategies have been of great value, but their effectiveness is being challenged by the newly emerging diseases and the rapid change of pathogens due to climatic changes. Genome editing has provided new tools and methods to characterize defense-related genes in crops and improve crop resilience to disease pathogens providing improved food security and future sustainable agricultural systems. In this review, we discuss the principal traits, tools and impacts of utilizing genome editing techniques for achieving of durable resilience and a "healthy plants" concept.

CRISPR/Cas-mediated plant genome editing: outstanding challenges a decade after implementation.

The discovery of the CRISPR/Cas genome-editing system has revolutionized our understanding of the plant genome. CRISPR/Cas has been used for over a decade to modify plant genomes for the study of specific genes and biosynthetic pathways as well as to speed up breeding in many plant species, including both model and non-model crops. Although the CRISPR/Cas system is very efficient for genome editing, many bottlenecks and challenges slow down further improvement and applications. In this review we discuss the challenges that can occur during tissue culture, transformation, regeneration, and mutant detection. We also review the opportunities provided by new CRISPR platforms and specific applications related to gene regulation, abiotic and biotic stress response improvement, and de novo domestication of plants.

Genome-based identification of beet curly top Iran virus infecting sugar beet in Turkey and investigation of its pathogenicity by agroinfection.

Beet curly top disease (BCTD) is a yield-limiting viral infection of sugar beet (Beta vulgaris) throughout the arid and semi-arid regions of the world. Two virus species, belonging to two different genera of the family Geminiviridae (Curtovirus and Becurtovirus) had been described as the disease's causative agents on sugar beet. Despite the detection of the BCTD in some sugar beet fields of Turkey sixty years ago, the genome based characterization of BCTD-associated viruses have not been studied previously. In this study, 628 sugar beet plants exhibiting BCTD symptoms were collected from fourteen cities in central Anatolia, the major sugar beet production areas in Turkey. PCR assays of these samples using the respective Curtovirus and Becurtovirus genus-specific primers indicated that the Turkish sugar beet samples' viral sequences belong only to the genus Becurtovirus. The results of sequencing and phylogenetic analysis of the partial genome of the virus obtained from fourteen cities confirmed that BCTD-associated virus in Turkish sugar beet fields is beet curly top Iran virus (BCTIV-Becurtovirus) species. The whole genome of the collected viruses from fourteen cities were amplified by the rolling circle amplification (RCA) and the five most phylogenetically diverse viruses obtained from Afyon, Ankara, Adapazari, Yozgat and Aksaray were sequenced. The results of whole genome sequence analysis indicated >98 % sequence identities with that of a BCTIV variants reported from Urmia province (bordering Turkey) of Iran. A virus genome from Yozgat city had a genomic sequence identity of >97 % with those of BCTIV isolated from cowpea, tomato, pepper and sugar beet in the northern part of Iran. These results suggested that the spread of BCTIV through the region could create a significant threat to the production of sugar beet as well as other agricultural crops. A tandem dimer of a BCTIV-Turkish variant isolated from Ankara city was cloned into Agrobacterium plasmid to be used for agro-infection studies. Agroinoculation of this construct on sugar beet leaves generated severe BCTD symptoms (84 %) which were also confirmed by RCA and qPCR analysis. These results constituted the first genome based characterization of BCTIV Turkish variants and the first report of BCTIV spreading out of Iran.

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.

Production of hyperthermostable GH10 xylanase Xyl10B from Thermotoga maritima in transplastomic plants enables complete hydrolysis of methylglucuronoxylan to fermentable sugars for biofuel production.

Overcoming the recalcitrance in lignocellulosic biomass for efficient hydrolysis of the polysaccharides cellulose and hemicellulose to fermentable sugars is a research priority for the transition from a fossilfuel-based economy to a renewable carbohydrate economy. Methylglucuronoxylans (MeGXn) are the major components of hemicellulose in woody biofuel crops. Here, we describe efficient production of the GH10 xylanase Xyl10B from Thermotoga maritima in transplastomic plants and demonstrate exceptional stability and catalytic activities of the in planta produced enzyme. Fully expanded leaves from homotransplastomic plants contained enzymatically active Xyl10B at a level of 11-15% of their total soluble protein. Transplastomic plants and their seed progeny were morphologically indistinguishable from non-transgenic plants. Catalytic activity of in planta produced Xyl10B was detected with poplar, sweetgum and birchwood xylan substrates following incubation between 40 and 90 °C and was also stable in dry and stored leaves. Optimal yields of Xyl10B were obtained from dry leaves if crude protein extraction was performed at 85 °C. The transplastomic plant derived Xyl10B showed exceptional catalytic activity and enabled the complete hydrolysis of MeGXn to fermentable sugars with the help of a single accessory enzyme (α-glucuronidase) as revealed by the sugar release assay. Even without this accessory enzyme, the majority of MeGXn was hydrolyzed by the transplastomic plant-derived Xyl10B to fermentable xylose and xylobiose.

Comparative genome-wide phylogenetic and expression analysis of SBP genes from potato (Solanum tuberosum).

Flowering time is a very important phase in transition to reproductive stage of life in higher plants. SQUAMOSA promoter-binding protein (SBP) gene family encodes plant-specific transcription factors that are involved in regulation of several developmental processes, especially flowering. Although SBP-box genes have been identified in different plants, there have been no study indicating the regulatory effect of SBP box in potato flowering. Here, we report for the first time the identification and characterization of SBP-box transcription factors as well as determination of expression level of SBP-box genes in Solanum tuberosum L. an important crop worldwide. Fifteen different SBP-box transcription factor genes were identified in potato genome. They were found to be distributed in nine chromosomes and eight of them included miR156 and miRNA157 target sites. Characterization of amino acid sequences were performed and protein interactions were predicted. In addition, expression levels of five S. tuberosum SBP-box genes were analysed by both in silico and qRT-PCR. All these results provide a better understanding of functional role of SBP-box gene family members in flowering time in potato.

Genome-wide investigation and expression analysis of AP2-ERF gene family in salt tolerant common bean.

Apetala2-ethylene-responsive element binding factor (AP2-ERF) superfamily with common AP2-DNA binding domain have developmentally and physiologically important roles in plants. Since common bean genome project has been completed recently, it is possible to identify all of the AP2-ERF genes in the common bean genome. In this study, a comprehensive genome-wide in silico analysis identified 180 AP2-ERF superfamily genes in common bean (Phaseolus vulgaris). Based on the amino acid alignment and phylogenetic analyses, superfamily members were classified into four subfamilies: DREB (54), ERF (95), AP2 (27) and RAV (3), as well as one soloist. The physical and chemical characteristics of amino acids, interaction between AP2-ERF proteins, cis elements of promoter region of AP2-ERF genes and phylogenetic trees were predicted and analyzed. Additionally, expression levels of AP2-ERF genes were evaluated by in silico and qRT-PCR analyses. In silico micro-RNA target transcript analyses identified nearly all PvAP2-ERF genes as targets of by 44 different plant species' miRNAs were identified in this study. The most abundant target genes were PvAP2/ERF-20-25-62-78-113-173. miR156, miR172 and miR838 were the most important miRNAs found in targeting and BLAST analyses. Interactome analysis revealed that the transcription factor PvAP2-ERF78, an ortholog of Arabidopsis At2G28550, was potentially interacted with at least 15 proteins, indicating that it was very important in transcriptional regulation. Here we present the first study to identify and characterize the AP2-ERF transcription factors in common bean using whole-genome analysis, and the findings may serve as a references for future functional research on the transcription factors in common bean.