Genetic Screens for Floral Mutants in Arabidopsis thaliana: Enhancers and Suppressors.

The flower is a hallmark feature that has contributed to the evolutionary success of land plants. Diverse mutagenic agents have been employed as a tool to genetically perturb flower development and identify genes involved in floral patterning and morphogenesis. Since the initial studies to identify genes governing processes such as floral organ specification, mutagenesis in sensitized backgrounds has been used to isolate enhancers and suppressors to further probe the molecular basis of floral development. Here, we first describe two commonly employed methods for mutagenesis (using ethyl methanesulfonate (EMS) or T-DNAs as mutagens), and then describe three methods for identifying a mutation that leads to phenotypic alterations: traditional map-based cloning, modified high-efficiency thermal asymmetric interlaced PCR (mhiTAIL-PCR), and deep sequencing in the plant model Arabidopsis thaliana.

In-silico, evolutionary, and functional analysis of CHUP1 and its related proteins in Bienertia sinuspersici-a comparative study across C3, C4, CAM, and SCC4 model plants.

Single-cell C4 (SCC4) plants with bienertioid anatomy carry out photosynthesis in a single cell. Chloroplast movement is the underlying phenomenon, where chloroplast unusual positioning 1 (CHUP1) plays a key role. This study aimed to characterize CHUP1 and CHUP1-like proteins in an SCC4 photosynthetic plant, Bienertia sinuspersici. Also, a comparative analysis of SCC4 CHUP1 was made with C3, C4, and CAM model plants including an extant basal angiosperm, Amborella. The CHUP1 gene exists as a single copy from the basal angiosperms to SCC4 plants. Our analysis identified that Chenopodium quinoa, a recently duplicated allotetraploid, has two copies of CHUP1. In addition, the numbers of CHUP1-like and its associated proteins such as CHUP1-like_a, CHUP1-like_b, HPR, TPR, and ABP varied between the species. Hidden Markov Model analysis showed that the gene size of CHUP1-like_a and CHUP1-like_b of SCC4 species, Bienertia, and Suaeda were enlarged than other plants. Also, we identified that CHUP1-like_a and CHUP1-like_b are absent in Arabidopsis and Amborella, respectively. Motif analysis identified several conserved and variable motifs based on the orders (monocot and dicot) as well as photosynthetic pathways. For instance, CAM plants such as pineapple and cactus shared certain motifs of CHUP1-like_a irrespective of their distant phylogenetic relationship. The free ratio model showed that CHUP1 maintained purifying selection, whereas CHUP1-like_a and CHUP1-like_b have adaptive functions between SCC4 plants and quinoa. Similarly, rice and maize branches displayed functional diversification on CHUP1-like_b. Relative gene expression data showed that during the subcellular compartmentalization process of Bienertia, CHUP1 and actin-binding proteins (ABP) genes showed a similar pattern of expression. Altogether, the results of this study provide insight into the evolutionary and functional details of CHUP1 and its associated proteins in the development of the SCC4 system in comparison with other C3, C4, and CAM model plants.

Anticancer Effects of High Glucosinolate Synthesis Lines of Brassica rapa on Colorectal Cancer Cells.

Chemoprevention is a method of health control in modern industrialized societies. Traditional breeding (hybridization) has been widely used to produce new (sub)species with beneficial phenotypes. Previously, we produced a number of doubled haploid (DH) lines of Brassica rapa with a high glucosinolate (GSL) content. In this study, we evaluated the anticancer activities of extracts from three selected high-GSL (HGSL)-containing DH lines (DHLs) of Brassica rapa in human colorectal cancer (CRC) cells. The three HGSL DHL extracts showed anti-proliferative activities in the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium assay and pro-apoptotic activities in the cell cycle or annexin V analysis with the induction of pro-apoptotic protein expression in CRC cells. Mechanistically, HGSL DHL extracts inhibited the NF-κB and ERK pathways, leading to a reduction in the nuclear localization of NF-κB p65. In addition, reactive oxygen species were induced by HGSL DHL extract treatment in CRC cells. In conclusion, our data suggest that the newly developed HGSL DHLs possess enhanced anticancer activities and are potentially helpful as a daily vegetable supplement with chemopreventive activities.

Analysis of Phenotypic Characteristics and Sucrose Metabolism in the Roots of Raphanus sativus L.

The taproot of radish (Raphanus sativus L.) is an important sink organ; it is morphologically diverse and contains large amounts of secondary metabolites. Sucrose metabolism is believed to be important in the development of sink organs. We measured the amounts of glucose, fructose, and sucrose in the roots of sixty three radish accessions and analyzed the association between the sugar content and the root phenotype. Fructose content correlated with the root color and length characteristics, glucose was the most abundant sugar in the roots, and the sucrose content was very low, compared to that of the hexoses in most of the accessions. Expression analysis of the genes involved in sucrose metabolism, transportation, starch synthesis, and cell wall synthesis was performed through RNA sequencing. The genes encoding sucrose synthases (SUSY) and the enzymes involved in the synthesis of cellulose were highly expressed, indicating that SUSY is involved in cell wall synthesis in radish roots. The positive correlation coefficient (R) between the sucrose content and the expression of cell wall invertase and sugar transporter proteins suggest that hexose accumulation could occur through the apoplastic pathway in radish roots. A positive R score was also obtained when comparing the expression of genes encoding SUSY and fructokinase (FK), suggesting that the fructose produced by SUSY is mostly phosphorylated by FK. In addition, we concluded that sucrose was the most metabolized sugar in radish roots.

Systemic Expression of Genes Involved in the Plant Defense Response Induced by Wounding in Senna tora.

Wounds in tissues provide a pathway of entry for pathogenic fungi and bacteria in plants. Plants respond to wounding by regulating the expression of genes involved in their defense mechanisms. To analyze this response, we investigated the defense-related genes induced by wounding in the leaves of Senna tora using RNA sequencing. The genes involved in jasmonate and ethylene biosynthesis were strongly induced by wounding, as were a large number of genes encoding transcription factors such as ERFs, WRKYs, MYBs, bHLHs, and NACs. Wounding induced the expression of genes encoding pathogenesis-related (PR) proteins, such as PR-1, chitinase, thaumatin-like protein, cysteine proteinase inhibitor, PR-10, and plant defensin. Furthermore, wounding led to the induction of genes involved in flavonoid biosynthesis and the accumulation of kaempferol and quercetin in S. tora leaves. All these genes were expressed systemically in leaves distant from the wound site. These results demonstrate that mechanical wounding can lead to a systemic defense response in the Caesalpinioideae, a subfamily of the Leguminosae. In addition, a co-expression analysis of genes induced by wounding provides important information about the interactions between genes involved in plant defense responses.

Influence of Genotype on High Glucosinolate Synthesis Lines of Brassica rapa.

This study was conducted to investigate doubled haploid (DH) lines produced between high GSL (HGSL) Brassica rapa ssp. trilocularis (yellow sarson) and low GSL (LGSL) B. rapa ssp. chinensis (pak choi) parents. In total, 161 DH lines were generated. GSL content of HGSL DH lines ranged from 44.12 to 57.04 µmol·g-1·dry weight (dw), which is within the level of high GSL B. rapa ssp. trilocularis (47.46 to 59.56 µmol g-1 dw). We resequenced five of the HGSL DH lines and three of the LGSL DH lines. Recombination blocks were formed between the parental and DH lines with 108,328 single-nucleotide polymorphisms in all chromosomes. In the measured GSL, gluconapin occurred as the major substrate in HGSL DH lines. Among the HGSL DH lines, BrYSP_DH005 had glucoraphanin levels approximately 12-fold higher than those of the HGSL mother plant. The hydrolysis capacity of GSL was analyzed in HGSL DH lines with a Korean pak choi cultivar as a control. Bioactive compounds, such as 3-butenyl isothiocyanate, 4-pentenyl isothiocyanate, 2-phenethyl isothiocyanate, and sulforaphane, were present in the HGSL DH lines at 3-fold to 6.3-fold higher levels compared to the commercial cultivar. The selected HGSL DH lines, resequencing data, and SNP identification were utilized for genome-assisted selection to develop elite GSL-enriched cultivars and the industrial production of potential anti-cancerous metabolites such as gluconapin and glucoraphanin.

Genome-wide analysis of the MADS-Box gene family in Chrysanthemum.

MADS-box family transcription factors play key roles in various developmental processes in plants. Here, we identified 108 MADS-box genes in the genome of chrysanthemum (Chrysanthemum nankingense). We classified these genes based on their phylogenetic relationships with MADS-box genes in Arabidopsis thaliana and lettuce (Lactuca sativa). Type I genes were subdivided into classes Mα (19 genes), Mß (12 genes), and Mγ (10 genes), and type II genes were subdivided into classes MIKCC (64 genes) and MIKC* (3 genes). The MIKCC class genes were further divided into 16 subclasses that included genes described in the ABCDE flower development model. Each group of MADS-box genes showed a specific pattern of conserved protein motifs and exon-intron structure. We analyzed the expression levels of each MADS-box gene in root, stem, leaf, flower bud, disc floret, and ray floret tissues. Subfamilies AGL18, FLC, and SVP contained more members in chrysanthemum. The asterid-specific TM8 subfamily and eleven Asteraceae Specific-MADS CnMADS genes were present in chrysanthemum. Chrysanthemum is the lacking members of the AGL15 subfamily. Among the genes responsible for the ABCDE model, B-class genes were expanded in chrysanthemum with three AP3 and four PI genes. One AP3 homolog functions in marginal ray floret development, whereas the two other AP3 homologs function in the development of the central disc floret. Two of the four PI genes are expressed in chrysanthemum, specifically in both types of florets. The results of this study lay the foundation for further studies of the roles of MADS-box genes in flower development in chrysanthemum and of the evolution of MADS-box genes in plants.

Engineering disease resistant plants through CRISPR-Cas9 technology.

Plants are susceptible to phytopathogens, including bacteria, fungi, and viruses, which cause colossal financial shortfalls (pre- and post-harvest) and threaten global food safety. To combat with these phytopathogens, plant possesses two-layer of defense in the form of PAMP-triggered immunity (PTI), or Effectors-triggered immunity (ETI). The understanding of plant-molecular interactions and revolution of high-throughput molecular techniques have opened the door for innovations in developing pathogen-resistant plants. In this context, Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) has transformed genome editing (GE) technology and being harnessed for altering the traits. Here we have summarized the complexities of plant immune system and the use of CRISPR-Cas9 to edit the various components of plant immune system to acquire long-lasting resistance in plants against phytopathogens. This review also sheds the light on the limitations of CRISPR-Cas9 system, regulation of CRISPR-Cas9 edited crops and future prospective of this technology.

Genome-enabled discovery of anthraquinone biosynthesis in Senna tora.

Senna tora is a widely used medicinal plant. Its health benefits have been attributed to the large quantity of anthraquinones, but how they are made in plants remains a mystery. To identify the genes responsible for plant anthraquinone biosynthesis, we reveal the genome sequence of S. tora at the chromosome level with 526 Mb (96%) assembled into 13 chromosomes. Comparison among related plant species shows that a chalcone synthase-like (CHS-L) gene family has lineage-specifically and rapidly expanded in S. tora. Combining genomics, transcriptomics, metabolomics, and biochemistry, we identify a CHS-L gene contributing to the biosynthesis of anthraquinones. The S. tora reference genome will accelerate the discovery of biologically active anthraquinone biosynthesis pathways in medicinal plants.

Induction of Glucoraphasatin Biosynthesis Genes by MYB29 in Radish (Raphanus sativus L.) Roots.

Glucoraphasatin (GRH) is a specific aliphatic glucosinolate (GSL) that is only abundant in radish (Raphanus sativus L.). The gene expression regulating GRH biosynthesis in radish is still poorly understood. We employed a total of 59 radish accessions to analyze GSL profiles and showed that GRH was specific and predominant among the aliphatic GSLs in radish roots. We selected five accessions roots with high, moderate and low GSL biosynthesis, respectively, to conduct a comparative transcriptome analysis and the qRT-PCR of the biosynthesis genes for aliphatic GSLs. In this study, among all the accessions tested, roots with the accession RA157-74 had a high GRH content and showed a significant expression of the aliphatic GSL biosynthesis genes. We defined the genes involved in the GRH biosynthesis process and found that they were regulated by a transcription factor (RSG00789) at the MYB29 locus in radish roots. We found 13 aliphatic GSL biosynthesis genes regulated by the RSG00789 gene in the GRH biosynthesis pathway.

A comparative analysis of the complete chloroplast genomes of three Chrysanthemum boreale strains.

BACKGROUND: Chrysanthemum boreale Makino (Anthemideae, Asteraceae) is a plant of economic, ornamental and medicinal importance. We characterized and compared the chloroplast genomes of three C. boreale strains. These were collected from different geographic regions of Korea and varied in floral morphology. METHODS: The chloroplast genomes were obtained by next-generation sequencing techniques, assembled de novo, annotated, and compared with one another. Phylogenetic analysis placed them within the Anthemideae tribe. RESULTS: The sizes of the complete chloroplast genomes of the C. boreale strains were 151,012 bp (strain 121002), 151,098 bp (strain IT232531) and 151,010 bp (strain IT301358). Each genome contained 80 unique protein-coding genes, 4 rRNA genes and 29 tRNA genes. Comparative analyses revealed a high degree of conservation in the overall sequence, gene content, gene order and GC content among the strains. We identified 298 single nucleotide polymorphisms (SNPs) and 106 insertions/deletions (indels) in the chloroplast genomes. These variations were more abundant in non-coding regions than in coding regions. Long dispersed repeats and simple sequence repeats were present in both coding and noncoding regions, with greater frequency in the latter. Regardless of their location, these repeats can be used for molecular marker development. Phylogenetic analysis revealed the evolutionary relationship of the species in the Anthemideae tribe. The three complete chloroplast genomes will be valuable genetic resources for studying the population genetics and evolutionary relationships of Asteraceae species.

Comparative Analysis of the Complete Chloroplast Genome of Mainland Aster spathulifolius and Other Aster Species.

Aster spathulifolius, a common ornamental and medicinal plant, is widely distributed in Korea and Japan, and is genetically classified into mainland and island types. Here, we sequenced the whole chloroplast genome of mainland A. spathulifolius and compared it with those of the island type and other Aster species. The chloroplast genome of mainland A. spathulifolius is 152,732 bp with a conserved quadripartite structure, has 37.28% guanine-cytosine (GC) content, and contains 114 non-redundant genes. Comparison of the chloroplast genomes between the two A. spathulifolius lines and the other Aster species revealed that their sequences, GC contents, gene contents and orders, and exon-intron structure were well conserved; however, differences were observed in their lengths, repeat sequences, and the contraction and expansion of the inverted repeats. The variations were mostly in the single-copy regions and non-coding regions, which, together with the detected simple sequence repeats, could be used for the development of molecular markers to distinguish between these plants. All Aster species clustered into a monophyletic group, but the chloroplast genome of mainland A. spathulifolius was more similar to the other Aster species than to that of the island A. spathulifolius. The accD and ndhF genes were detected to be under positive selection within the Aster lineage compared to other related taxa. The complete chloroplast genome of mainland A. spathulifolius presented in this study will be helpful for species identification and the analysis of the genetic diversity, evolution, and phylogenetic relationships in the Aster genus and the Asteraceae.

De novo transcriptome sequence of Senna tora provides insights into anthraquinone biosynthesis.

Senna tora is an annual herb with rich source of anthraquinones that have tremendous pharmacological properties. However, there is little mention of genetic information for this species, especially regarding the biosynthetic pathways of anthraquinones. To understand the key genes and regulatory mechanism of anthraquinone biosynthesis pathways, we performed spatial and temporal transcriptome sequencing of S. tora using short RNA sequencing (RNA-Seq) and long-read isoform sequencing (Iso-Seq) technologies, and generated two unigene sets composed of 118,635 and 39,364, respectively. A comprehensive functional annotation and classification with multiple public databases identified array of genes involved in major secondary metabolite biosynthesis pathways and important transcription factor (TF) families (MYB, MYB-related, AP2/ERF, C2C2-YABBY, and bHLH). Differential expression analysis indicated that the expression level of genes involved in anthraquinone biosynthetic pathway regulates differently depending on the degree of tissues and seeds development. Furthermore, we identified that the amount of anthraquinone compounds were greater in late seeds than early ones. In conclusion, these results provide a rich resource for understanding the anthraquinone metabolism in S. tora.

CRISPR-Cas9 system: A genome-editing tool with endless possibilities.

The discovery of CRISPR: Cas9 and its application as a powerful gene-editing tool has transformed the world of basic and applied science, especially the molecular biology dome. Also, the smooth, quick, flexible, and very efficient nature of this technology has enabled the biologists to alter the genome of prokaryotes to complex eukaryotic systems, including plants and animals. Using CRISPR and associated tools, investigation, control, and modification of significant biological events have been more accessible than before. These biological scissors are now being used to accelerate breeding programs of crop and livestock, engineer new antimicrobials, and control disease-carrying pathogens. However, like other techniques, these cutters emerged as a double-edged sword and put several challenges to the scientific society. Here in this review article, we summarized the beneficial application of the CRISPR: Cas9 system and unsafe perception to the society if handled carelessly. We also discussed the limitations and ethical issues related to CRISPR: Cas9 technology.

Comparative Analysis of the YABBY Gene Family of Bienertia sinuspersici, a Single-Cell C4 Plant.

The emergence and expression of the YABBY gene family (YGF) coincided with the evolution of leaves in seed plants, and was integral to the early evidence of lamina followed by reproductive development. YGF contains six subclasses, i.e., CRC, INO, FIL, YAB2, YAB3, and YAB5. This study aims to extract the genome sequences of the YGF in Bienertia sinuspersici, an important model plant for single-cell C4 (SCC4), non-Kranz photosynthesis. A comparative genomic analysis was undertaken with Vitis vinefera, Arabidopsis thaliana, Brassica rapa, and Chenopodium quinoa. Six copies of YGF were present in B. sinuspersici and A. thaliana with a single copy of each YGF subgroup. V. vinefera possessed seven copies of YGF with duplicates in FIL and YAB2 subgroups, but no YAB3. B. rapa and C. quinoa after whole genome duplication contained additional copies of YGF. The gene structure and conserved motifs were analyzed among the YGF. In addition, the relative quantification of YGF was analyzed in the leaves, reproductive developmental stages such as the bud, and the pre-anthesis and anthesis stages in B. sinuspersici, A. thaliana, and B. rapa. CRC and INO possessed conserved floral-specific expression. Temporal and perpetual changes in the expression of YGF orthologs were observed in the leaves and reproductive developmental stages. The results of this study provide an overview of YGF evolution, copy number, and its differential expression in B. sinuspersici. Further studies are required to shed light on the roles of YABBY genes in the evolution of SCC4 plants and their distinct physiologies.

Insight on Rosaceae Family with Genome Sequencing and Functional Genomics Perspective.

Rosaceae is one of the important families possessing a variety of diversified plant species. It includes many economically valuable crops that provide nutritional and health benefits for the human. Whole genome sequences of valuable crop plants were released in recent years. Understanding of genomics helps to decipher the plant physiology and developmental process. With the information of cultivating species and its wild relative genomes, genome sequence-based molecular markers and mapping loci for economically important traits can be used to accelerate the genome assisted breeding. Identification and characterization of disease resistant capacities and abiotic stress tolerance related genes are feasible to study across species with genome information. Further breeding studies based on the identification of gene loci for aesthetic values, flowering molecular circuit controls, fruit firmness, nonacid fruits, etc. is required for producing new cultivars with valuable traits. This review discusses the whole genome sequencing reports of Malus, Pyrus, Fragaria, Prunus, and Rosa and status of functional genomics of representative traits in individual crops.

The complete chloroplast genome of an economic plant, Chrysanthemum morifolium 'Baekma'.

Chrysanthemum morifolium (Dendranthema grandiflorum), known as florist's daisy is an important ornamental and medicinal plant of the Asteraceae family. The complete chloroplast genome sequence of one economic cultivar 'Baekma' was 151,060 bp in length with a large single copy (LSC) region (82,862 bp), a small single copy (SSC) region (18,294 bp) and two inverted repeats (IRs) (24,952 bp). It contained 130 genes, including 85 protein-coding genes, 8 rRNAs and 37 tRNAs. The overall GC content was 37%. Phylogenetic analysis showed that C. morifolium 'Baekma' was grouped together with other Chrysanthemum species.

The complete mitochondrial genome sequence of Chrysanthemum boreale (Asteraceae).

Chrysanthemum is an important ornamental, herbal, and medicinal plant. We report the complete mitochondrial genome (mitogenome) sequence of Chrysanthemum boreale. The mitogenome is 211,002 bp in length, has a GC content of 45.36%, and contains 58 genes, including 35 protein-coding genes, three ribosomal RNA genes, and 20 transfer RNA genes. A phylogenetic analysis based on mitogenome protein sequences from various plants confirmed that C. boreale belongs to the Asteraceae family. This mitogenome will be useful in evolutionary and phylogenetic studies of Chrysanthemum and Asteraceae.

The complete chloroplast genome of Chrysanthemum boreale (Asteraceae).

Chrysanthemum boreale is a perennial plant in the Asteraceae family that is native to eastern Asia and has both ornamental and herbal uses. Here, we determined the complete chloroplast genome sequence for C. boreale using long-read sequencing. The chloroplast genome was 151,012 bp and consisted of a large single copy (LSC) region (82,817 bp), a small single copy (SSC) region (18,281 bp) and two inverted repeats (IRs) (24,957 bp). It was predicted to contain 131 genes, including 87 protein-coding genes, eight rRNAs and 46 tRNAs. Phylogenetic analysis of chloroplast genomes clustered C. boreale with other Chrysanthemum and Asteraceae species.