Production of biologically active human basic fibroblast growth factor (hFGFb) using Nicotiana tabacum transplastomic plants.

MAIN CONCLUSION: We generated transplastomic tobacco lines that stably express a human Basic Fibroblast Growth Factor (hFGFb) in their chloroplasts stroma and purified a biologically active recombinant hFGFb. MAIN: The use of plants as biofactories presents as an attractive technology with the potential to efficiently produce high-value human recombinant proteins in a cost-effective manner. Plastid genome transformation stands out for its possibility to accumulate recombinant proteins at elevated levels. Of particular interest are recombinant growth factors, given their applications in animal cell culture and regenerative medicine. In this study, we produced recombinant human Fibroblast Growth Factor (rhFGFb), a crucial protein required for animal cell culture, in tobacco chloroplasts. We successfully generated two independent transplastomic lines that are homoplasmic and accumulate rhFGFb in their leaves. Furthermore, the produced rhFGFb demonstrated its biological activity by inducing proliferation in HEK293T cell lines. These results collectively underscore plastid genome transformation as a promising plant-based bioreactor for rhFGFb production.

Comprehensive identification, characterization, and expression analysis of the MORF gene family in Brassica napus.

BACKGROUND: RNA editing in chloroplast and mitochondrion transcripts of plants is an important type of post-transcriptional RNA modification in which members of the multiple organellar RNA editing factor gene family (MORF) play a crucial role. However, a systematic identification and characterization of MORF members in Brassica napus is still lacking. RESULTS: In this study, a total of 43 MORF genes were identified from the genome of the Brassica napus cultivar "Zhongshuang 11". The Brassica napus MORF (BnMORF) family members were divided into three groups through phylogenetic analysis. BnMORF genes distributed on 14 chromosomes and expanded due to segmental duplication and whole genome duplication repetitions. The majority of BnMORF proteins were predicted to be localized to mitochondria and chloroplasts. The promoter cis-regulatory element analysis, spatial-temporal expression profiling, and co-expression network of BnMORF genes indicated the involvement of BnMORF genes in stress and phytohormone responses, as well as growth and development. CONCLUSION: This study provides a comprehensive analysis of BnMORF genes and lays a foundation for further exploring their physiological functions in Brassica napus.

Comparative chloroplast genomics and phylogenetic analysis of Oreomecon nudicaulis (Papaveraceae).

Oreomecon nudicaulis, commonly known as mountain poppy, is a significant perennial herb. In 2022, the species O. nudicaulis, which was previously classified under the genus Papaver, was reclassified within the genus Oreomecon. Nevertheless, the phylogenetic status and chloroplast genome within the genus Oreomecon have not yet been reported. This study elucidates the chloroplast genome sequence and structural features of O. nudicaulis and explores its evolutionary relationships within Papaveraceae. Using Illumina sequencing technology, the chloroplast genome of O. nudicaulis was sequenced, assembled, and annotated. The results indicate that the chloroplast genome of O. nudicaulis exhibits a typical circular quadripartite structure. The chloroplast genome is 153,903 bp in length, with a GC content of 38.87%, containing 84 protein-coding genes, 8 rRNA genes, 38 tRNA genes, and 2 pseudogenes. The genome encodes 25,815 codons, with leucine (Leu) being the most abundant codon, and the most frequently used codon is AUU. Additionally, 129 microsatellite markers were identified, with mononucleotide repeats being the most abundant (53.49%). Our phylogenetic analysis revealed that O. nudicaulis has a relatively close relationship with the genus Meconopsis within the Papaveraceae family. The phylogenetic analysis supported the taxonomic status of O. nudicaulis, as it did not form a clade with other Papaver species, consistent with the revised taxonomy of Papaveraceae. This is the first report of a phylogenomic study of the complete chloroplast genome in the genus Oreomecon, which is a significant genus worldwide. This analysis of the O. nudicaulis chloroplast genome provides a theoretical basis for research on genetic diversity, molecular marker development, and species identification, enriching genetic information and supporting the evolutionary relationships among Papaveraceae.

Comparative chloroplast-specific SNP and nSCoT markers analysis and population structure study in kiwifruit plants.

BACKGROUND: Kiwifruit (Actinidiaceae family) is an economically important fruit tree in China and New Zealand. It is a typical dioecious plant that has undergone frequent natural hybridization, along with chromosomal ploidy diversity within the genus Actinidia, resulting in higher genetic differences and horticultural diversity between interspecific and intraspecific traits. This diversity provides a rich genetic base for breeding. China is not only the original center of speciation for the Actinidia genus but also its distribution center, housing the most domesticated species: A. chinensis var. chinensis, A. chinensis var. deliciosa, A. arguta, and A. polygama. However, there have been relatively few studies on the application of DNA markers and the genetic basis of kiwifruit plants. By combining information from chloroplast-specific SNPs and nuclear SCoT (nSCoT) markers, we can uncover complementary aspects of genetic variation, population structure, and evolutionary relationships. In this study, one chloroplast DNA (cpDNA) marker pair was selected out of nine cpDNA candidate pairs. Twenty nSCoT markers were selected and used to assess the population structure and chloroplast-specific DNA haplotype diversity in 55 kiwifruit plants (Actinidia), including 20 samples of A. chinensis var. chinensis, 22 samples of A. chinensis var. deliciosa, 11 samples of A. arguta, and two samples of A. polygama, based on morphological observations collected from China. RESULTS: The average genetic distance among the 55 samples was 0.26 with chloroplast-specific SNP markers and 0.57 with nSCoT markers. The Mantel test revealed a very small correlation (r = 0.21). The 55 samples were categorized into different sub-populations using Bayesian analysis, the Unweighted Pair Group Method with the Arithmetic Mean (UPGMA), and the Principal Component Analysis (PCA) method, respectively. Based on the analysis of 205 variable sites, a total of 15 chloroplast-specific DNA haplotypes were observed, contributing to a higher level of polymorphism with an Hd of 0.78. Most of the chloroplast-specific DNA haplotype diversity was distributed among populations, but significant diversity was also observed within populations. H1 was shared by 24 samples, including 12 of A. chinensis var. chinensis and 12 of A. chinensis var. deliciosa, indicating that H1 is an ancient and dominant haplotype among the 55 chloroplast-specific sequences. H2 may not have evolved further.The remaining haplotypes were rare and unique, with some appearing to be exclusive to a particular variety and often detected in single individuals. For example, the H15 haplotype was found exclusively in A. polygama. CONCLUSION: The population genetic variation explained by chloroplast-specific SNP markers has greater power than that explained by nSCoTs, with chloroplast-specific DNA haplotypes being the most efficient. Gene flow appears to be more evident between A. chinensis var. chinensis and A. chinensis var. deliciosa, as they share chloroplast-specific DNA haplotypes, In contrast, A.arguta and A. polygama possess their own characteristic haplotypes, derived from the haplotype of A. chinensis var. chinensis. Compared with A. chinensis, the A.arguta and A. polygama showed better grouping. It also seems crucial to screen out, for each type of molecular marker, especially haplotypes, the core markers of the Actinidia genus.

Transcriptomic study of the role of MeFtsZ2-1 in pigment accumulation in cassava leaves.

MeFtsZ2-1 is a key gene for plant plastid division, but the mechanism by which MeFtsZ2-1 affects pigment accumulation in cassava (Manihot esculenta Crantz) through plastids remains unclear. We found that MeFtsZ2-1 overexpression in cassava (OE) exhibited darker colors of leaves, with increased levels of anthocyanins and carotenoids. Further observation via Transmission Electron Microscopy (TEM) revealed no apparent defects in chloroplast structure but an increase in the number of plastoglobule in OE leaves. RNA-seq results showed 1582 differentially expressed genes (DEGs) in leaves of OE. KEGG pathway analysis indicated that these DEGs were enriched in pathways related to flavonoid, anthocyanin, and carotenoid biosynthesis. This study reveals the role of MeFtsZ2-1 in cassava pigment accumulation from a physiological and transcriptomic perspective, providing a theoretical basis for improving cassava quality.

Complete Chloroplast Genome of Alternanthera sessilis and Comparative Analysis with Its Congeneric Invasive Weed Alternanthera philoxeroides.

Alternanthera sessilis is considered the closest relative to the invasive weed Alternanthera philoxeroides in China, making it an important native species for studying the invasive mechanisms and adaptations of A. philoxeroides. Chloroplasts play a crucial role in a plant's environmental adaptation, with their genomes being pivotal in the evolution and adaptation of both invasive and related species. However, the chloroplast genome of A. sessilis has remained unknown until now. In this study, we sequenced and assembled the complete chloroplast genome of A. sessilis using high-throughput sequencing. The A. sessilis chloroplast genome is 151,935 base pairs long, comprising two inverted repeat regions, a large single copy region, and a small single copy region. This chloroplast genome contains 128 genes, including 8 rRNA-coding genes, 37 tRNA-coding genes, 4 pseudogenes, and 83 protein-coding genes. When compared to the chloroplast genome of the invasive weed A. philoxeroides and other Amaranthaceae species, we observed significant variations in the ccsA, ycf1, and ycf2 regions in the A. sessilis chloroplast genome. Moreover, two genes, ccsA and accD, were found to be undergoing rapid evolution due to positive selection pressure. The phylogenetic trees were constructed for the Amaranthaceae family, estimating the time of independent species formation between A. philoxeroides and A. sessilis to be approximately 3.5186-8.8242 million years ago. These findings provide a foundation for understanding the population variation within invasive species among the Alternanthera genus.

Complete Chloroplast Genome of Krascheninnikovia ewersmanniana: Comparative and Phylogenetic Analysis.

Krascheninnikovia ewersmanniana is a dominant desert shrub in Xinjiang, China, with high economic and ecological value. However, molecular systematics research on K. ewersmanniana is lacking. To resolve the genetic composition of K. ewersmanniana within Amaranthaceae and its systematic relationship with related genera, we used a second-generation Illumina sequencing system to detect the chloroplast genome of K. ewersmanniana and analyze its assembly, annotation, and phylogenetics. Total length of the chloroplast genome of K. ewersmanniana reached 152,287 bp, with 84 protein-coding genes, 36 tRNAs, and eight rRNAs. Codon usage analysis showed the majority of codons ending with base A/U. Mononucleotide repeats were the most common (85.42%) of the four identified simple sequence repeats. A comparison with chloroplast genomes of six other Amaranthaceae species indicated contraction and expansion of the inverted repeat boundary region in K. ewersmanniana, with some genes (rps19, ndhF, ycf1) differing in length and distribution. Among the seven species, the variation in non-coding regions was greater. Phylogenetic analysis revealed Krascheninnikovia ceratoides, Dysphania ambrosioides, Dysphania pumilio, and Dysphania botrys to have a close monophyletic relationship. By sequencing the K. ewersmanniana chloroplast genome, this research resolves the relatedness among 35 Amaranthaceae species, providing molecular insights for germplasm utilization, and theoretical support for studying evolutionary relationships.

Complete Chloroplast Genome Sequence Structure and Phylogenetic Analysis of Kohlrabi (Brassica oleracea var. gongylodes L.).

Kohlrabi is an important swollen-stem cabbage variety belonging to the Brassicaceae family. However, few complete chloroplast genome sequences of this genus have been reported. Here, a complete chloroplast genome with a quadripartite cycle of 153,364 bp was obtained. A total of 132 genes were identified, including 87 protein-coding genes, 37 transfer RNA genes and eight ribosomal RNA genes. The base composition analysis showed that the overall GC content was 36.36% of the complete chloroplast genome sequence. Relative synonymous codon usage frequency (RSCU) analysis showed that most codons with values greater than 1 ended with A or U, while most codons with values less than 1 ended with C or G. Thirty-five scattered repeats were identified and most of them were distributed in the large single-copy (LSC) region. A total of 290 simple sequence repeats (SSRs) were found and 188 of them were distributed in the LSC region. Phylogenetic relationship analysis showed that five Brassica oleracea subspecies were clustered into one group and the kohlrabi chloroplast genome was closely related to that of B. oleracea var. botrytis. Our results provide a basis for understanding chloroplast-dependent metabolic studies and provide new insight for understanding the polyploidization of Brassicaceae species.

Comparative chloroplast genomics, phylogenetic relationships and molecular markers development of Aglaonema commutatum and seven green cultivars of Aglaonema.

Aglaonema commutatum is a famous species in the Aglaonema genus, which has important ornamental and economic value. However, its chloroplast genome information and phylogenetic relationships among popular green cultivars of Aglaonema in southern China have not been reported. Herein, chloroplast genomes of one variety of A. commutatum and seven green cultivars of Aglaonema, namely, A. commutatum 'San Remo', 'Kai Sa', 'Pattaya Beauty', 'Sapphire', 'Silver Queen', 'Snow White', 'White Gem', and 'White Horse Prince', were sequenced and assembled for comparative analysis and phylogeny. These eight genomes possessed a typical quadripartite structure that consisted of a LSC region (90,799-91,486 bp), an SSC region (20,508-21,137 bp) and a pair of IR regions (26,661-26,750 bp). Each genome contained 112 different genes, comprising 79 protein-coding genes, 29 tRNA genes and 4 rRNA genes. The gene orders, GC contents, codon usage frequency, and IR/SC boundaries were highly conserved among these eight genomes. Long repeats, SSRs, SNPs and indels were analyzed among these eight genomes. Comparative analysis of 15 Aglaonema chloroplast genomes identified 7 highly variable regions, including trnH-GUG-exon1-psbA, trnS-GCU-trnG-UCC-exon1, trnY-GUA-trnE-UUC, psbC-trnS-UGA, trnF-GAA-ndhJ, ccsA-ndhD, and rps15-ycf1-D2. Reconstruction of the phylogenetic trees based on chloroplast genomes, strongly supported that Aglaonema was a sister to Anchomanes, and that the Aglaonema genus was classified into two sister clades including clade I and clade II, which corresponded to two sections, Aglaonema and Chamaecaulon, respectively. One variety and five cultivars, including A. commutatum 'San Remo', 'Kai Sa', 'Pattaya Beauty', 'Silver Queen', 'Snow White', and 'White Horse Prince', were classified into clade I; and the rest of the two cultivars, including 'Sapphire' and 'White Gem', were classified into clade II. Positive selection was observed in 34 protein-coding genes at the level of the amino acid sites among 77 chloroplast genomes of the Araceae family. Based on the highly variable regions and SSRs, 4 DNA markers were developed to differentiate the clade I and clade II in Aglaonema. In conclusion, this study provided chloroplast genomic resources for Aglaonema, which were useful for its classification and phylogeny.

Differential Transcription Profiling Reveals the MicroRNAs Involved in Alleviating Damage to Photosynthesis under Drought Stress during the Grain Filling Stage in Wheat.

To explore the possible novel microRNA (miRNA) regulatory pathways in Zhengmai 1860, a newly cultivated drought-tolerant wheat (Triticum aestivum L.) cultivar, miRNA transcriptome sequencing of the flag leaves of Zhengmai 1860, drought-sensitive variety Zhoumai 18, and drought-resistant variety Bainong 207 was performed during the grain filling stage. We also observed changes in the chloroplast ultrastructure, phytohormone levels, and antioxidant- and photosynthesis-related physiological indicators in three wheat varieties. The results showed that the flag leaves of the drought-tolerant variety Zhengmai 1860 had higher chlorophyll contents and net photosynthetic rates than those of Zhoumai 18 under drought stress during the grain filling stage; in addition, the chloroplast structure was more complete. However, there was no significant difference between Zhengmai 1860 and Bainong 207. MiRNA transcriptome analysis revealed that the differential expression of the miRNAs and mRNAs exhibited variable specificity. The KEGG pathway enrichment results indicated that most of the genes were enriched in the MAPK signaling pathway, plant hormone signal transduction, photosynthetic antennae protein, and amino acid and carbohydrate metabolism. In the drought-tolerant cultivar Zhengmai 1860, tae-miR408 was targeted to regulate the allene oxide synthase (AOS) gene, inhibit its expression, reduce the AOS content, and decrease the synthesis of jasmonic acid (JA) and abscisic acid (ABA). The results of this study suggest that Zhengmai 1860 could improve the photosynthetic performance of flag leaves by inhibiting the expression of genes involved in the JA pathway through miRNAs under drought conditions. Moreover, multiple miRNAs may target chlorophyll, antioxidant enzymes, phytohormone signal transduction, and other related pathways; thus, it is possible to provide a more theoretical basis for wheat molecular breeding.

Unveiling the Role of RNA Recognition Motif Proteins in Orchestrating Nucleotide-Binding Site and Leucine-Rich Repeat Protein Gene Pairs and Chloroplast Immunity Pathways: Insights into Plant Defense Mechanisms.

In plants, nucleotide-binding site and leucine-rich repeat proteins (NLRs) play pivotal roles in effector-triggered immunity (ETI). However, the precise mechanisms underlying NLR-mediated disease resistance remain elusive. Previous studies have demonstrated that the NLR gene pair Pik-H4 confers resistance to rice blast disease by interacting with the transcription factor OsBIHD1, consequently leading to the upregulation of hormone pathways. In the present study, we identified an RNA recognition motif (RRM) protein, OsRRM2, which interacted with Pik1-H4 and Pik2-H4 in vesicles and chloroplasts. OsRRM2 exhibited a modest influence on Pik-H4-mediated rice blast resistance by upregulating resistance genes and genes associated with chloroplast immunity. Moreover, the RNA-binding sequence of OsRRM2 was elucidated using systematic evolution of ligands by exponential enrichment. Transcriptome analysis further indicated that OsRRM2 promoted RNA editing of the chloroplastic gene ndhB. Collectively, our findings uncovered a chloroplastic RRM protein that facilitated the translocation of the NLR gene pair and modulated chloroplast immunity, thereby bridging the gap between ETI and chloroplast immunity.

Cloning and functional characterization of the peptide deformylase encoding gene EuPDF1B from Eucommia ulmoides Oliv.

Peptide deformylase can catalyse the removal of formyl groups from the N-terminal formyl methionine of the primary polypeptide chain. The peptide deformylase genes of a few herbaceous plants have been studied to some extent, but the peptide deformylase genes of woody plants have not been studied. In this study, we isolated EuPDF1B from Eucommia ulmoides Oliv. The full-length sequence of EuPDF1B is 1176 bp long with a poly-A tail and contains an open reading frame of 831 bp that encodes a protein of 276 amino acids. EuPDF1B was localized to the chloroplast. qRT‒PCR analysis revealed that this gene was expressed in almost all tissues tested but mainly in mature leaves. Moreover, the expression of EuPDF1B was enhanced by ABA, MeJA and GA and inhibited by shading treatment. The expression pattern of EuPDF1B was further confirmed in EuPDF1Bp: GUS transgenic tobacco plants. Among all the transgenic tobacco plants, EuPDF1Bp-3 showed the highest GUS histochemical staining and activity in different tissues. This difference may be related to the presence of enhancer elements in the region from - 891 bp to - 236 bp of the EuPDF1B promoter. In addition, the expression of the chloroplast gene psbA and the net photosynthetic rate, fresh weight and height of tobacco plants overexpressing EuPDF1B were greater than those of the wild-type tobacco plants, suggesting that EuPDF1B may promote the growth of transgenic tobacco plants. This is the first time that PDF and its promoter have been cloned from woody plants, laying a foundation for further analysis of the function of PDF and the regulation of its expression.

Comparative Analysis of Chloroplast Genomes for the Genus Manglietia Blume (Magnoliaceae): Molecular Structure and Phylogenetic Evolution.

Manglietia Blume, belonging to the Magnoliaceae family and mainly distributed in tropical and subtropical regions of Asia, has great scientific and economic value. In this study, we employed next-generation sequencing followed by de novo assembly to investigate the adaptive evolution of Manglietia using plastid genetic information. We newly sequenced the complete or nearly complete plastomes of four Manglietia species (Manglietia aromatica, Manglietia calcarea, Manglietia kwangtungensis, and Manglietia glauca) and conducted comparative analysis with seventeen published plastomes to examine the evolutionary pattern within this genus. The plastomes of these five newly sequenced Manglietia species range from 157,093 bp (M. calcarea2) to 160,493 bp (M. kwangtungensis), all exhibiting circular structures when mapped. Nucleotide diversity was observed across the plastomes, leading us to identify 13 mutational hotspot regions, comprising eight intergenic spacer regions and five gene regions. Our phylogenetic analyses based on 77 protein-coding genes generated phylogenetic relationships with high support and resolution for Manglietia. This genus can be divided into three clades, and the previously proposed infrageneric classifications are not supported by our studies. Furthermore, the close affinity between M. aromatica and M. calcarea is supported by the present work, and further studies are necessary to conclude the taxonomic treatment for the latter. These results provide resources for the comparative plastome, breeding, and plastid genetic engineering of Magnoliaceae and flowering plants.

Wasabi Gone Wild? Origin and Characterization of the Complete Plastomes of Ulleung Island Wasabi (Eutrema japonicum; Brassicaceae) and Other Cultivars in Korea.

Korean wasabi occurs naturally on the young oceanic, volcanic Ulleung Island off the east coast of the Korean Peninsula. Although the Ulleung Island wasabi is reported as Eutrema japonicum and has been suggested to be morphologically identical to cultivars in Korea, very little is known about its taxonomic identity and relationship with other cultivars. In this study, we sequenced the complete chloroplast DNA sequences of three naturally occurring Ulleung Island wasabi plants and six cultivars ('Daewang', 'Daruma', 'Micado', 'Orochi', 'Green Thumb', and 'Shogun') from continental Korea and determined the taxonomic identity of Korean wasabi on Ulleung Island. The size and organization of the complete chloroplast genomes of the nine accessions were nearly identical to those of previously reported wasabi cultivars. In addition, phylogenetic analysis based on the complete plastomes suggested that Ulleung Island wasabi most likely comprises various wasabi cultivars with three chlorotypes ('Shogun', 'Green Thumb', and a unique Chusan type). Based on the complete plastomes, we identified eight chlorotypes for the major wasabi cultivars and the Ulleung Island wasabi. Two major groups (1-'Mazuma' and 'Daruma', and 2-'Fujidaruma'/'Shimane No. 3'/Ulleung Island wasabi/five cultivars in Korea) were also identified based on mother line genealogical history. Furthermore, different types of variations (mutations, insertions/deletions (indels), mononucleotide repeats, and inversions) in plastomes were identified to distinguish different cultivar lines and five highly divergent hotspots. The nine newly obtained complete plastomes are valuable organelle genomic resources for species identification and infraspecific phylogeographic studies on wild and cultivated wasabi.

Comparative analysis of chloroplast genomes of Pulsatilla species reveals evolutionary and taxonomic status of newly discovered endangered species Pulsatilla saxatilis.

BACKGROUND: Pulsatilla saxatilis, a new species of the genus Pulsatilla has been discovered. The morphological information of this species has been well described, but its chloroplast genome characteristics and comparison with species of the same genus remain to be reported. RESULTS: Our results showed that the total length of chloroplast (cp.) genome of P. saxatilis is 162,659 bp, with a GC content of 37.5%. The cp. genome contains 134 genes, including 90 known protein-coding genes, 36 tRNA genes, and 8 rRNA genes. P. saxatilis demonstrated similar characteristics to other species of genus Pulsatilla. Herein, we compared cp. genomes of 10 species, including P. saxatilis, and found that the cp. genomes of the genus Pulsatilla are extremely similar, with a length of 162,322-163,851 bp. Furthermore, The SSRs of Pulsatilla ranged from 10 to 22 bp in length. Among the four structural regions of the cp. genome, most long repeats and SSRs were detected in the LSC region, followed by that in the SSC region, and least in IRA/ IRB regions. The most common types of long repeats were forward and palindromic repeats, followed by reverse repeats, and only a few complementary repeats were found in 10 cp. genomes. We also analyzed nucleotide diversity and identified ccsA_ndhD, rps16_trnK-UUU, ccsA, and rbcL, which could be used as potential molecular markers for identification of Pulsatilla species. The results of the phylogenetic tree constructed by connecting the sequences of high variation regions were consistent with those of the cp. gene phylogenetic tree, and the species more closely related to P. saxatilis was identified as the P. campanella. CONCLUSION: It was determined that the closest species to P. saxatilis is P. campanella, which is the same as the conclusion based on pollen grain characteristics, but different from the P. chinensis determined based on morphological characteristics. By revealing information on the chloroplast characteristics, development, and evolution of the cp. genome and the potential molecular markers, this study provides effective molecular data regarding the evolution, genetic diversity, and species identification of the genus Pulsatilla.

Extraction and analysis of high-quality chloroplast DNA with reduced nuclear DNA for medicinal plants.

BACKGROUND: Obtaining high-quality chloroplast genome sequences requires chloroplast DNA (cpDNA) samples that meet the sequencing requirements. The quality of extracted cpDNA directly impacts the efficiency and accuracy of sequencing analysis. Currently, there are no reported methods for extracting cpDNA from Erigeron breviscapus. Therefore, we developed a suitable method for extracting cpDNA from E. breviscapus and further verified its applicability to other medicinal plants. RESULTS: We conducted a comparative analysis of chloroplast isolation and cpDNA extraction using modified high-salt low-pH method, the high-salt method, and the NaOH low-salt method, respectively. Subsequently, the number of cpDNA copies relative to the nuclear DNA (nDNA ) was quantified via qPCR. As anticipated, chloroplasts isolated from E. breviscapus using the modified high-salt low-pH method exhibited intact structures with minimal cell debris. Moreover, the concentration, purity, and quality of E. breviscapus cpDNA extracted through this method surpassed those obtained from the other two methods. Furthermore, qPCR analysis confirmed that the modified high-salt low-pH method effectively minimized nDNA contamination in the extracted cpDNA. We then applied the developed modified high-salt low-pH method to other medicinal plant species, including Mentha haplocalyx, Taraxacum mongolicum, and Portulaca oleracea. The resultant effect on chloroplast isolation and cpDNA extraction further validated the generalizability and efficacy of this method across different plant species. CONCLUSIONS: The modified high-salt low-pH method represents a reliable approach for obtaining high-quality cpDNA from E. breviscapus. Its universal applicability establishes a solid foundation for chloroplast genome sequencing and analysis of this species. Moreover, it serves as a benchmark for developing similar methods to extract chloroplast genomes from other medicinal plants.

Knocking Out Chloroplastic Aldolases/Rubisco Lysine Methyltransferase Enhances Biomass Accumulation in Nannochloropsis oceanica under High-Light Stress.

Rubisco large-subunit methyltransferase (LSMT), a SET-domain protein lysine methyltransferase, catalyzes the formation of trimethyl-lysine in the large subunit of Rubisco or in fructose-1,6-bisphosphate aldolases (FBAs). Rubisco and FBAs are both vital proteins involved in CO2 fixation in chloroplasts; however, the physiological effect of their trimethylation remains unknown. In Nannochloropsis oceanica, a homolog of LSMT (NoLSMT) is found. Phylogenetic analysis indicates that NoLSMT and other algae LSMTs are clustered in a basal position, suggesting that algal species are the origin of LSMT. As NoLSMT lacks the His-Ala/ProTrp triad, it is predicted to have FBAs as its substrate instead of Rubisco. The 18-20% reduced abundance of FBA methylation in NoLSMT-defective mutants further confirms this observation. Moreover, this gene (nolsmt) can be induced by low-CO2 conditions. Intriguingly, NoLSMT-knockout N. oceanica mutants exhibit a 9.7-13.8% increase in dry weight and enhanced growth, which is attributed to the alleviation of photoinhibition under high-light stress. This suggests that the elimination of FBA trimethylation facilitates carbon fixation under high-light stress conditions. These findings have implications in engineering carbon fixation to improve microalgae biomass production.

Mutation mapping of a variegated EMS tomato reveals an FtsH-like protein precursor potentially causing patches of four phenotype classes in the leaves with distinctive internal morphology.

BACKGROUND: Leaf variegation is an intriguing phenomenon observed in many plant species. However, questions remain on its mechanisms causing patterns of different colours. In this study, we describe a tomato plant detected in an M2 population of EMS mutagenised seeds, showing variegated leaves with sectors of dark green (DG), medium green (MG), light green (LG) hues, and white (WH). Cells and tissues of these classes, along with wild-type tomato plants, were studied by light, fluorescence, and transmission electron microscopy. We also measured chlorophyll a/b and carotene and quantified the variegation patterns with a machine-learning image analysis tool. We compared the genomes of pooled plants with wild-type-like and mutant phenotypes in a segregating F2 population to reveal candidate genes responsible for the variegation. RESULTS: A genetic test demonstrated a recessive nuclear mutation caused the variegated phenotype. Cross-sections displayed distinct anatomy of four-leaf phenotypes, suggesting a stepwise mesophyll degradation. DG sectors showed large spongy layers, MG presented intercellular spaces in palisade layers, and LG displayed deformed palisade cells. Electron photomicrographs of those mesophyll cells demonstrated a gradual breakdown of the chloroplasts. Chlorophyll a/b and carotene were proportionally reduced in the sectors with reduced green pigments, whereas white sectors have hardly any of these pigments. The colour segmentation system based on machine-learning image analysis was able to convert leaf variegation patterns into binary images for quantitative measurements. The bulk segregant analysis of pooled wild-type-like and variegated progeny enabled the identification of SNP and InDels via bioinformatic analysis. The mutation mapping bioinformatic pipeline revealed a region with three candidate genes in chromosome 4, of which the FtsH-like protein precursor (LOC100037730) carries an SNP that we consider the causal variegated phenotype mutation. Phylogenetic analysis shows the candidate is evolutionary closest to the Arabidopsis VAR1. The synonymous mutation created by the SNP generated a miRNA binding site, potentially disrupting the photoprotection mechanism and thylakoid development, resulting in leaf variegation. CONCLUSION: We described the histology, anatomy, physiology, and image analysis of four classes of cell layers and chloroplast degradation in a tomato plant with a variegated phenotype. The genomics and bioinformatics pipeline revealed a VAR1-related FtsH mutant, the first of its kind in tomato variegation phenotypes. The miRNA binding site of the mutated SNP opens the way to future studies on its epigenetic mechanism underlying the variegation.

Chloroplast genome analysis and evolutionary insights in the versatile medicinal plant Calendula officinalis L.

Calendula officinalis L.is a versatile medicinal plant with numerous applications in various fields. However, its chloroplast genome structure, features, phylogeny, and patterns of evolution and mutation remain largely unexplored. This study examines the chloroplast genome, phylogeny, codon usage bias, and divergence time of C. officinalis, enhancing our understanding of its evolution and adaptation. The chloroplast genome of C. officinalis is a 150,465 bp circular molecule with a G + C content of 37.75% and comprises 131 genes. Phylogenetic analysis revealed a close relationship between C. officinalis, C. arvensis, and Osteospermum ecklonis. A key finding is the similarity in codon usage bias among these species, which, coupled with the divergence time analysis, supports their close phylogenetic proximity. This similarity in codon preference and divergence times underscores a parallel evolutionary adaptation journey for these species, highlighting the intricate interplay between genetic evolution and environmental adaptation in the Asteraceae family. Moreover unique evolutionary features in C. officinalis, possibly associated with certain genes were identified, laying a foundation for future research into the genetic diversity and medicinal value of C. officinalis.

The size diversity of the Pteridaceae family chloroplast genome is caused by overlong intergenic spacers.

BACKGROUND: While the size of chloroplast genomes (cpDNAs) is often influenced by the expansion and contraction of inverted repeat regions and the enrichment of repeats, it is the intergenic spacers (IGSs) that appear to play a pivotal role in determining the size of Pteridaceae cpDNAs. This provides an opportunity to delve into the evolution of chloroplast genomic structures of the Pteridaceae family. This study added five Pteridaceae species, comparing them with 36 published counterparts. RESULTS: Poor alignment in the non-coding regions of the Pteridaceae family was observed, and this was attributed to the widespread presence of overlong IGSs in Pteridaceae cpDNAs. These overlong IGSs were identified as a major factor influencing variations in cpDNA size. In comparison to non-expanded IGSs, overlong IGSs exhibited significantly higher GC content and were rich in repetitive sequences. Species divergence time estimations suggest that these overlong IGSs may have already existed during the early radiation of the Pteridaceae family. CONCLUSIONS: This study reveals new insights into the genetic variation, evolutionary history, and dynamic changes in the cpDNA structure of the Pteridaceae family, providing a fundamental resource for further exploring its evolutionary research.