Comparative Analyses and Phylogenetic Relationships between <i>Cryptomeria fortunei</i> and Related Species Based on Complete Chloroplast Genomes

Cryptomeria fortunei (Chinese cedar) is a highly adaptable woody species and one of the main forest plantation trees in subtropical high-altitude areas in China. However, there are few studies on its chloroplast (cp) genome. In this study, the complete cp genome of C. fortunei was sequenced and evaluated via comparative analyses with those of related species (formerly the Taxodiaceae) in Cupressaceae. The C. fortunei cp genome was 131,580 bp in length, and the GC content of the whole genome was 35.38%. It lost one relevant large inverted repeat and contained 114 unique genes, including 82 protein-coding genes, 28 tRNAs and 4 rRNAs. The relative synonymous codon usage (RSCU) of codons ending with A/U was more than twice that of codons ending with G/C. Thirty long repeat structures (LRSs) and 213 simple sequence repeat (SSR) loci were detected in the C. fortunei cp genome. Comparative analyses of 10 cp genomes revealed that substantial rearrangements occurred in the gene organization. Additionally, 6 cp hotspot regions (trnS-GGA, ycf1, trnP-GGG, trnC-GCA, psbZ and accD) were identified, and 4 genes (petL, psbM, rpl22 and psaM) had likely underwent positive selection. Phylogenetic analysis showed that Cupressaceae, Taxaceae and Cephalotaxaceae clustered to form a clade and that C. fortunei was most closely related to C. japonica (Japanese cedar), C. japonica cv. Wogon Hort and Taxodium distichum (baldcypress). These results provide references for future studies of population genetics, phylogenetic status and molecular markers among Cupressaceae species and for the cultivation of improved varieties.     

The chloroplast genome from a lycophyte (microphyllophyte), <Emphasis Type="Italic">Selaginella uncinata</Emphasis>, has a unique inversion,transpositions and many gene losses

We determined the complete nucleotide sequence of the chloroplast genome of Selaginella uncinata, a lycophyte belonging to the basal lineage of the vascular plants. The circular double-stranded DNA is 144,170 bp, with an inverted repeat of 25,578 bp separated by a large single copy region (LSC) of 77,706 bp and a small single copy region (SSC) of 40,886 bp. We assigned 81 protein-coding genes including four pseudogenes, four rRNA genes and only 12 tRNA genes. Four genes, rps15, rps16, rpl32 and ycf10, found in most chloroplast genomes in land plants were not present in S. uncinata. While gene order and arrangement of the chloroplast genome of another lycophyte, Hupertzia lucidula, are almost the same as those of bryophytes, those of S. uncinata differ considerably from the typical structure of bryophytes with respect to the presence of a unique 20 kb inversion within the LSC, transposition of two segments from the LSC to the SSC and many gene losses. Thus, the organization of the S. uncinata chloroplast genome provides a new insight into the evolution of lycophytes, which were separated from euphyllophytes approximately 400 million years ago. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

<i>Aspergillus tubingensis</i> Causes Leaf Spot of Cotton (<i>Gossypium hirsutum</i> L.) in Pakistan

Cotton (Gossypium hirsutum L.) is a key fiber crop of great commercial importance. Numerous phytopathogens decimate crop production by causing various diseases. During July-August 2018, leaf spot symptoms were recurrently observed on cotton leaves in Rahim Yar Khan, Pakistan and adjacent areas. Infected leaf samples were collected and plated on potato dextrose agar (PDA) media. Causal agent of cotton leaf spot was isolated, characterized and identified as Aspergillus tubingensis based on morphological and microscopic observations. Conclusive identification of pathogen was done on the comparative molecular analysis of CaM and β-tubulin gene sequences. BLAST analysis of both sequenced genes showed 99% similarity with A. tubingensis. Koch’s postulates were followed to confirm the pathogenicity of the isolated fungus. Healthy plants were inoculated with fungus and similar disease symptoms were observed. Fungus was re-isolated and identified to be identical to the inoculated fungus. To our knowledge, this is the first report describing the involvement of A. tubingensis in causing leaf spot disease of cotton in Pakistan and around the world.     

Chloroplast genome aberration in micropropagation-derived albino <Emphasis Type="Italic">Bambusa edulis</Emphasis> mutants, <Emphasis Type="Italic">ab1</Emphasis> and <Emphasis Type="Italic">ab2</Emphasis>

Two albino mutants (ab1 and ab2) have been derived from long-term shoot proliferation of Bambusa edulis. Based on transmission electronic microscopy data, the chloroplasts of these mutants were abnormal. To study the mutation of gene regulation in the aberrant chloroplasts, we designed 19 pairs of chloroplast-encoded gene primers for genomic and RT-PCR. Only putative NAD(P)H-quinone oxidoreductase chain 4L (ndhE; DQ908943) and ribosomal protein S7 (rps7; DQ908931) were conserved in both the mutant and wild-type plants. The deletions in the chloroplast genome of these two mutants were different: nine genes were deleted in the chloroplast genomic aberration in ab1 and 11 genes in ab2. The chloroplast genes, NAD(P)H-quinone oxidoreductase chain 4 (ndhD; DQ908944), chloroplast 50S ribosomal protein L14 (rpl14; DQ908934), and ATP synthase beta chain (atpB; DQ908948) were abnormal in both mutants. The gene expressions of 18 of these 20 genes were correlated with their DNA copy number. The two exceptions were: ATP synthase CF0 A chain (atpI; DQ908946), whose expression in both mutants was not reduced even though the copy number was reduced; ribosomal protein S19 (rps19; DQ908949), whose expression was reduced or it was not expressed at all even though there was no difference in genomic copy number between the wild-type and mutant plants. The genomic PCR results showed that chloroplast genome aberrations do occur in multiple shoot proliferation, and this phenomenon may be involved in the generation of albino mutants.     

Molecular adaptation and expression evolution following duplication of genes for organellar ribosomal protein S13 in rosids

Background  

Gene duplication has been a fundamental process in the evolution of eukaryotic genomes. After duplication one copy (or both) can undergo divergence in sequence, expression pattern, and function. Two divergent copies of the ribosomal protein S13 gene (rps13) of chloroplast origin are found in the nucleus of the rosids Arabidopsis, Gossypium, and Glycine. One encodes chloroplast-imported RPS13 (nucp rps13), while the other encodes mitochondria-imported RPS13 (numit rps13). The rps13 gene has been lost from mitochondrial DNA (mt rps13) of many rosids.     

Genome-Wide Analysis of the <i>KANADI</i> Gene Family and Its Expression Patterns under Different Nitrogen Concentrations Treatments in <i>Populus trichocarpa</i>

KANADI (KAN) is a plant-specific gene that controlled the polarity development of lateral organs. It mainly acted on the abaxial characteristics of plants to make the lateral organs asymmetrical. However, it had been less identified in woody plants. In this study, the members of the KAN gene family in Populus trichocarpa were identified and analyzed using the bioinformatics method. The results showed that a total of 8 KAN family members were screened out, and each member contained the unique GARP domain and conserved region of the family proteins. Phylogenetic analysis and their gene structures revealed that all KAN genes from P. trichocarpa, Arabidopsis thaliana, and Nicotiana benthamiana could be divided into four subgroups, while the eight genes in P. trichocarpa were classified into three subgroups, respectively. The analysis of tissue-specific expression indicated that PtKAN1 was highly expressed in young leaves, PtKAN6 was highly expressed in young leaves and mature leaves, PtKAN2, PtKAN5, and PtKAN7 were highly expressed in nodes and internodes, PtKAN8 was highly expressed in roots, and PtKAN3 and PtKAN4 showed low expression levels in all tissues. Among them, PtKAN2 and PtKAN6, and PtKAN4 and PtKAN5 might have functional redundancy. Under high nitrogen concentrations, PtKAN2 and PtKAN8 were highly expressed in mature stems and leaves, respectively, while PtKAN4, PtKAN5, and PtKAN7 were highly expressed in roots. This study laid a theoretical foundation for further study of the KAN gene-mediated nitrogen effect on root development.     

The complete nucleotide sequence of the hornwort (Anthoceros formosae) chloroplast genome: insight into the earliest land plants

It is generally believed that bryophytes are the earliest land plants. However, the phylogenetic relationships among bryophytes, including mosses, liverworts and hornworts, are not clearly resolved. To obtain more information on the earliest land plants, we determined the complete nucleotide sequence of the chloroplast genome from the hornwort Anthoceros formosae. The circular double-stranded DNA of 161 162 bp is the largest genome ever reported among land plant chloroplasts. It contains 76 protein, 32 tRNA and 4 rRNA genes and 10 open reading frames (ORFs), which are identical with the chloroplast genome of the other green plants analyzed. The major difference is a larger inverted repeat than that of the liverwort Marchantia, Anthoceros contains an excess of ndhB and rps7 genes and the 3′ exon of rps12. The genes matK and rps15, commonly found in the chloroplast genomes of land plants, are pseudogenes. The intron of rrn23 is the first finding in the known chloroplast genomes of land plants. A striking feature of the hornwort chloroplast is that more than half of the protein-coding genes have nonsense codons, which are converted into sense codons by RNA editing. Maximum-likelihood (ML) analysis, based on 11 518 amino acid sites of 52 proteins encoded in the chloroplast genomes of the green plants, placed liverworts as the sister to all other land plants.     

Identification and Characterization of a Novel Yellow Leaf Mutant <i>yl1</i> in Rice

Leaf-color mutants play an important role in the study of chlorophyll metabolism, chloroplast development, and photosynthesis system. In this study, the yellow leaf 1 (yl1) rice mutant was identified from the ethyl methane sulfonate-treated mutant progeny of Lailong, a glutinous japonica rice landrace cultivated in Guizhou Province, China. Results showed that yl1 exhibited yellow leaves with decreased chlorophyll content throughout the growth period. Chloroplast development in the yl1 mutant was disrupted, and the grana lamellae was loosely packed and disordered. RNA sequencing and real-time quantitative polymerase chain reaction (qRT-PCR) analysis revealed that the chlorophyll synthesis-related genes OsCHLH, OsCHLM, OsCHLG, PORB, and YGL8, as well as the chloroplast development-related genes FtsZ, OsRpoTp, and RbcL, were down-regulated in the yl1 mutant. Genetic analysis revealed that the yellow leaf phenotype of yl1 was controlled by recessive nuclear gene. By employing the MutMap method, the mutation responsible for the phenotype was mapped to a 6.17 Mb region between 17.34 and 23.51 Mb on chromosome 3. Two non-synonymous single-nucleotide polymorphisms (SNPs) located in the gene locus LOC_Os03g31210 and LOC_Os03g36760 were detected in this region. The two SNPs were further confirmed by PCR and Sanger sequencing. The expression patterns of the two candidate genes indicated that LOC_Os03g36760 showed greater potential for functional verification. Subcellular protein localization revealed that the encoded product of LOC_Os03g36760 was localized in the nucleus, cytoplasm, and plasma membrane. These results will be useful for further characterization and cloning of the yl1 gene, and for research on the molecular mechanisms controlling biogenesis and chloroplast biochemical processes.     

Extensive Rearrangements in the Chloroplast Genome of <Emphasis Type="Italic">Trachelium caeruleum</Emphasis> Are Associated with Repeats and tRNA Genes

Chloroplast genome organization, gene order, and content are highly conserved among land plants. We sequenced the chloroplast genome of Trachelium caeruleum L. (Campanulaceae), a member of an angiosperm family known for highly rearranged genomes. The total genome size is 162,321 bp, with an inverted repeat (IR) of 27,273 bp, large single-copy (LSC) region of 100,114 bp, and small single-copy (SSC) region of 7,661 bp. The genome encodes 112 different genes, with 17 duplicated in the IR, a tRNA gene (trnI-cau) duplicated once in the LSC region, and a protein-coding gene (psbJ) with two duplicate copies, for a total of 132 putatively intact genes. ndhK may be a pseudogene with internal stop codons, and clpP, ycf1, and ycf2 are so highly diverged that they also may be pseudogenes. ycf15, rpl23, infA, and accD are truncated and likely nonfunctional. The most conspicuous feature of the Trachelium genome is the presence of 18 internally unrearranged blocks of genes inverted or relocated within the genome relative to the ancestral gene order of angiosperm chloroplast genomes. Recombination between repeats or tRNA genes has been suggested as a mechanism of chloroplast genome rearrangements. The Trachelium chloroplast genome shares with Pelargonium and Jasminum both a higher number of repeats and larger repeated sequences in comparison to eight other angiosperm chloroplast genomes, and these are concentrated near rearrangement endpoints. Genes for tRNAs occur at many but not all inversion endpoints, so some combination of repeats and tRNA genes may have mediated these rearrangements.     

AM Fungi and <i>Piriformospora indica</i> Improve Plant Growth of <i>Pinus elliottii</i> Seedlings

Pinus elliottii is an exotic afforestation pine extensively distributed in southern parts of China. In order to understand whether endophytic fungi can affect seedling growth of P. elliottii, Piriformospora indica (Pi), Funnelifcrmis mosseae (Fm), and Diversispora tortuosa (Dt) were inoculated respectively, and the non-inoculated group was set as control. The growth indexes, the contents of soluble sugar and soluble protein, and plant endogenous hormone levels in the leaves of P. elliottii, were analyzed. The results showed that Fm, Dt and Pi colonized the P. elliottii roots to form mycorrhizal structure and chlamydospores arranged in beads respectively. Three fungal inoculants exhibited the stimulated growth responses, whilst Dt illustrated the most positive effect on plant height, single fresh weight, trunk diameter and root system structure, compared with the control. On the other hand, the soluble sugar and soluble protein contents were increased distinctively in mycorrhizal plants. The endogenous IAA, GA3, ZR contents were increased, while the ABA contents were reduced in mycorrhizal plants versus non-mycorrhizal plants. The fungi-induced endogenous hormone changes triggered plant growth improvement of P. elliottii seedlings. This research unraveled the positive effect of AM fungi and P. indica on growth of pine seedlings, while, more application of endophytic fungi to fields needs to be explored.     

First Report of <i>Fusarium striatum</i> Causing Root Rot Disease of <i>Panax notoginseng</i> in Yunnan,China

Panax notoginseng is a traditional Chinese medicinal plant. Root rot of P. notoginseng is one of the most serious diseases affecting P. notoginseng growth and causes wilted leaves, fewer lateral roots and rotten roots. Root rot is a soil-borne disease, and mainly occurs from June to August in Yunnan Province when the temperatures are high and the air is humid. In this study, the endophytic fungal genus Fusarium isolate E-2018.1.22-#3.2 was obtained from a P. notoginseng embryo. Fusarium isolate E-2018.1.22-#3.2 was identified as Fusarium striatum based on morphological characteristics and molecular analysis. The fungus was found to have conidiophores and macroconidia, and its ITS, LSU and TEF-1α genes shared 100%, 99.2% and 99% identities with the homologous genes of Fusarium striatum, respectively. Isolate F. striatum E-2018.1.22-#3.2 can cause root rot symptoms, including black, soft roots, fewer lateral roots and leaf wilt, in 93% of the experimental P. notoginseng plants, and could be re-isolated, fulfilling Koch’s postulates. When the P. notoginseng plants were treated with the fungicide pyraclostrobin, isolate F. striatum E-2018.1.22-#3.2 was unable to cause root rot. We have therefore demonstrated that F. striatum E-2018.1.22-#3.2 is able to cause root rot disease in P. notoginseng. This is the first report of root rot disease caused by F. striatum on P. notoginseng in China.     

Development of a New Cold-Tolerant Maize (<i>Zea mays</i> L.) Germplasm Using the <i>ICE1</i> Gene from <i>Arabidopsis thaliana</i>

To develop cold-tolerant maize germplasms and identify the activation of INDUCER OF CRT/DRE-BINDING FACTOR EXPRESSION (ICE1) expression in response to cold stress, RT-PCR was used to amplify the complete open reading frame sequence of the ICE1 gene and construct the plant expression vector pCAMBIA3301-ICE1-Bar. Immature maize embryos and calli were transformed with the recombinant vector using Agrobacterium tumefaciens-mediated transformations. From the regenerated plantlets, three T1 lines were screened and identified by PCR. A Southern blot analysis showed that a single copy of the ICE1 gene was integrated into the maize (Zea mays L.) genomes of the three T1 generations. Under low temperature-stress conditions (4°C), the relative conductivity levels decreased by 27.51%–31.44%, the proline concentrations increased by 12.50%–17.50%, the malondialdehyde concentrations decreased by 16.78%–18.37%, and the peroxidase activities increased by 19.60%–22.89% in the T1 lines compared with those of the control. A real-time quantitative PCR analysis showed that the ICE1 gene was ectopically expressed in the roots, stems, and leaves of the T1 lines. ICE1 positively regulates the expression of the CBF genes in response to cold stress. Thus, this study showed the successful transformation of maize with the ICE1 gene, resulting in the generation of a new maize germplasm that had increased tolerance to cold stress.     

<Emphasis Type="Italic">SulP</Emphasis>, a nuclear gene encoding a putative chloroplast-targeted sulfate permease in<Emphasis Type="Italic"> Chlamydomonas reinhardtii</Emphasis>

Genomic, proteomic, phylogenetic and evolutionary aspects of a novel gene encoding a putative chloroplast-targeted sulfate permease of prokaryotic origin in the green alga Chlamydomonas reinhardtii are described. This nuclear-encoded sulfate permease gene (SulP) contains four introns, whereas all other known chloroplast sulfate permease genes lack introns and are encoded by the chloroplast genome. The deduced amino acid sequence of the protein showed an extended N-terminus, which includes a putative chloroplast transit peptide. The mature protein contains seven transmembrane domains and two large hydrophilic loops. This novel prokaryotic-origin gene probably migrated from the chloroplast to the nuclear genome during evolution of C. reinhardtii. The SulP gene, or any of its homologues, has not been retained in vascular plants, e.g. Arabidopsis thaliana, although it is encountered in the chloroplast genome of a liverwort (Marchantia polymorpha). A comparative structural analysis and phylogenetic origin of chloroplast sulfate permeases in a variety of species is presented.     

Cloning and Characterization of <i>EuGID1</i> in <i>Eucommia ulmoides</i> Oliver

Gibberellic acid controlled the key developmental processes of the life cycle of landing plants, and regulated the growth and development of plants. In this study, a novel gibberellin receptor gene EuGID1 was obtained from Eucommia ulmoides Oliver. The cDNA of EuGID1 was 1556 bp, and the open reading frame was 1029 bp, which encoded 343 amino acids. EuGID1 had the homology sequence with the hormone-sensitive lipase family. Amino acid sequence alignment confirmed EuGID1 protein had the highest homology with the GID1 protein of Manihot esculenta. EuGID1 was located in the nucleus and cell membrane and had expression in four plant organs. Overexpression of EuGID1 in transgenic Arabidopsis plants promoted plant elongation and increased siliques yield.     

Regeneration of somatic hybrid plants formed between Lycopersicon esculentum and L. pennellii

Summary Somatic hybrid plants have been regenerated following polyethylene glycol mediated fusion of leaf mesophyll protoplasts from tomato and protoplasts from Lycopersicon pennellii callus. Three different cultivars of tomato were used as sources of protoplasts: Early Girl, Manapal, and UC82B. Fusions were performed between protoplasts of these tomato cultivars and protoplasts of L. pennellii, and between protoplasts of the cultivars and protoplasts of L. pennellii that had been exposed to 3 or 6 krads of gamma radiation. Somatic hybrid plants were identified on the basis of heterozygous isozyme banding patterns, and leaf and flower morphology. Somatic hybrid plants were regenerated following fusion of tomato protoplasts with either untreated or irradiated L. pennellii protoplasts. All were heterozygous for isozyme loci on five different chromosomes. Regenerated somatic hybrids showed inheritance of either or both parental chloroplast genomes, but predominantly the L. pennellii mitochondrial genome. The regenerated somatic hybrid plants exhibited reduced fertility, less than 20% viable pollen. A total of 34 somatic hybrid calli were identified. Of these, 21 regenerated shoots, and 7 produced seed following manual pollinations.     

Variation for Resistance to <i>Alternaria tenuissima</i> and Potential Structural Mechanism among Different Cultivars of <i>Chrysanthemum morifolium</i>

Black spot disease, caused by the necrotrophic fungus Alternaria tenuissima (Fr.) Wiltsh (A. tenuissima), is considered a highly destructive disease of Chrysanthemum (Chrysanthemum morifolium Ramat.). A set of 17 accessions of commercial chrysanthemum cultivars were evaluated for resistance to A. tenuissima by seedling artificial inoculation. It was found that the reaction of the accessions to artificial inoculation ranged from resistant to highly susceptible. Five varieties of chrysanthemum (‘Zhongshan Taogui’, ‘Jinba’, ‘Zhongshan Jinguan’, ‘Jinling Wanhuang’ and ‘Jinling Yangguang’) were resistant; two varieties of chrysanthemum (‘Zhongshan Xinggui’ and ‘Zhongshan Jinkui’) were moderately resistant; and others were susceptible to various degrees, four varieties of chrysanthemum (‘Zhongshan Zihe’, ‘Zhongshan Jiuhong’, ‘Zaoyihong’ and ‘Jinling Jiaohuang’) were highly susceptible, especially. Some leaf morphological features of two resistant and two highly susceptible cultivars were further researched. Trichome density, length, height, gland size and stomata density were found to be associated with plant passive resistance. Resistant varieties that were identified in present study will be promising germplasm for exploitation of breeding programmes aimed at developing A. tenuissima-resistant cultivars and increasing genetic diversity.     

Effects of Barium Stress in <i>Brassica juncea</i> and <i>Cakile maritima</i>: The Indicator Role of Some Antioxidant Enzymes and Secondary Metabolites

Soil contamination by toxic trace metal elements, like barium (Ba), may stimulate various undesirable changes in the metabolic activity of plants. The plant responses are fast and with, direct or indirect, generation of reactive oxygen species (ROS). To cope with the stress imposed by the ROS production, plants developed a dual cellular system composed of enzymatic and non-enzymatic players that convert ROS, and their by-products, into stable nontoxic molecules. To assess the Ba stress response of two Brassicaceae species (Brassica juncea, a glycophyte, and Cakile maritime, a halophyte), plants were exposure to different Ba concentrations (0, 100, 200, 300 and 500 μM). The plants response was evaluated through their morphology and development, the determination of plant leaves antioxidant enzymatic activities and by the production of plants secondary metabolites. Results indicated that the two Brassicaceae species have the ability to survive in an environment containing Ba (even at 500 μM). The biomass production of C. maritima was slightly affected whereas an increase in biomass B. juncea was noticed. The stress imposed by Ba activated the antioxidant defense system in the two species, noticed by the changes in the leaves activity of catalase (CAT), ascorbate peroxidase (APX) and guaicol peroxidase (GPX), and of the secondary metabolites, through the production of total phenols and flavonoids. The enzymatic response was not similar within the two plant species: CAT and APX seem to have a more important role against the oxidative stress in C. maritima while in B. juncea is GPX. Overall, total phenols and flavonoids production was more significant in the plants aerial part than in the roots, of the both species. Although the two Brassicaceae species response was different, in both plants catalytic and non-catalytic transformation of ROS occurs, and both were able to overcome the Ba toxicity and prevent the cell damage.     

The chloroplast and mitochondrial genomes of two Gomphonema parvulum (Bacillariophyta) environmental isolates from South Carolina (United States) and Virginia (United States)

Gomphonema parvulum is a cosmopolitan freshwater diatom that is used as an indicator in water quality biomonitoring. In this study, we report the culturing of two geographically separated isolates from southeastern North America, their morphology, and the sequencing and assembly of their mitochondrial and chloroplast genomes. Morphologically, both strains fit G. parvulum sensu lato, but the frustules from a protected habitat in South Carolina were smaller than those cited in the historic data of this species from the same location as well as a second culture from Virginia. Phylogenetic analyses using the rbcL gene placed both within a clade with G. parvulum. Genetic markers, including full chloroplast and mitochondrial genomes and the nuclear small subunit rRNA gene region were assembled from each isolate. The organellar genomes of the two strains varied slightly in size due to small differences in intergenic regions with chloroplast genomes of 121,035 bp and 121,482 bp and mitochondrial genomes of 34,639 bp and 34,654 bp. The intraspecific pairwise identities of the chloroplast and mitochondrial genomes of these two isolates were 97.9% and 95.4%, respectively. Multigene phylogenetic analysis demonstrated a close relationship between G. parvulum, Gomphoneis minuta, and Didymosphenia geminata.