The rice LATE ELONGATED HYPOCOTYL enhances salt tolerance by regulating Na+/K+ homeostasis and ABA signalling.

The circadian clock plays multiple functions in the regulation of plant growth, development and response to various abiotic stress. Here, we showed that the core oscillator component late elongated hypocotyl (LHY) was involved in rice response to salt stress. The mutations of OsLHY gene led to reduced salt tolerance in rice. Transcriptomic analyses revealed that the OsLHY gene regulates the expression of genes related to ion homeostasis and the abscisic acid (ABA) signalling pathway, including genes encoded High-affinity K+ transporters (OsHKTs) and the stress-activated protein kinases (OsSAPKs). We demonstrated that OsLHY directly binds the promoters of OsHKT1;1, OsHKT1;4 and OsSAPK9 to regulate their expression. Moreover, the ossapk9 mutants exhibited salt tolerance under salt stress. Taken together, our findings revealed that OsLHY integrates ion homeostasis and the ABA pathway to regulate salt tolerance in rice, providing insights into our understanding of how the circadian clock controls rice response to salt stress.

Effect of Crop Rotation on Fusarium Mycotoxins and Fusarium Species in Cereals in Sichuan Province (China).

The present study was performed to evaluate the effect of crop rotation on Fusarium mycotoxins and species in cereals in Sichuan Province. A total of 311 cereal samples were randomly collected and analyzed from 2018 to 2019 in Sichuan Province. The results of mycotoxin analysis showed that the major trichothecene mycotoxins in Sichuan Province were nivalenol (NIV) and deoxynivalenol (DON), and the mean concentration of total trichothecenes (including NIV, fusarenone X [4ANIV], DON, 3-acetyldeoxynivalenol [3ADON], and 15-acetyldeoxynivalenol [15ADON]) in wheat was significantly higher than that in maize and rice. The concentration of total trichothecenes in the succeeding crops was significantly higher than that in the previous crops. In addition, wheat grown after maize had reduced incidence and concentration of trichothecene mycotoxins compared with that grown after rice, and ratooning rice grown after rice had increased incidence and concentration of trichothecene mycotoxins. Our data indicated that Fusarium asiaticum with the NIV chemotype was predominant in wheat and rice samples, while the number of the NIV chemotypes of F. asiaticum and Fusarium meridionale and the 15ADON chemotype of Fusarium graminearum in maize were almost the same. Although the composition of Fusarium species was affected by crop rotations, there were no differences when comparing the same crop rotation except for the maize-wheat rotation. Moreover, the same species and chemotype of Fusarium strains originated from different crops in various rotations, but there were no significant differences in pathogenicity in wheat and rice. These results contribute to the knowledge of the effect of crop rotation on Fusarium mycotoxins and species affecting cereals in Sichuan Province, which may lead to improved strategies for control of Fusarium mycotoxins and fungal disease in China.

Molecular Research for Cereal Grain Quality.

Cereals such as wheat (Triticum aestivum L [...].

A new demethylase gene, OsDML4, is involved in high temperature-increased grain chalkiness in rice.

High temperature (HT) can affect the accumulation of seed storage materials and cause adverse effects on the yield and quality of rice. DNA methylation plays an important role in plant growth and development. Here, we identified a new demethylase gene OsDML4 and discovered its function in cytosine demethylation to affect endosperm formation. Loss of function of OsDML4 induced chalky endosperm only under HT and dramatically reduced the transcription and accumulation of glutelins and 16 kDa prolamin. The expression of two transcription factor genes RISBZ1 and RPBF was significantly decreased in the osdml4 mutants, which caused adverse effects on the formation of protein bodies (PBs) with greatly decreased PB-II number, and incomplete and abnormally shaped PB-IIs. Whole-genome bisulfite sequencing analysis of seeds at 15 d after pollination revealed much higher global methylation levels of CG, CHG, and CHH contexts in the osdml4 mutants compared with the wild type. Moreover, the RISBZ1 promoter was hypermethylated but the RPBF promoter was almost unchanged under HT. No significant difference was detected between the wild type and osdml4 mutants under normal temperature. Our study demonstrated a novel OsDML4-mediated DNA methylation involved in the formation of chalky endosperm only under HT and provided a new perspective in regulating endosperm development and the accumulation of seed storage proteins in rice.

The Function of DNA Demethylase Gene ROS1a Null Mutant on Seed Development in Rice (Oryza Sativa) Using the CRISPR/CAS9 System.

The endosperm is the main nutrient source in cereals for humans, as it is a highly specialized storage organ for starch, lipids, and proteins, and plays an essential role in seed growth and development. Active DNA demethylation regulates plant developmental processes and is ensured by cytosine methylation (5-meC) DNA glycosylase enzymes. To find out the role of OsROS1a in seed development, the null mutant of OsROS1a was generated using the CRISPR/Cas9 system. The null mutant of OsROS1a was stable and heritable, which affects the major agronomic traits, particularly in rice seeds. The null mutant of OsROS1a showed longer and narrower grains, and seeds were deformed containing an underdeveloped and less-starch-producing endosperm with slightly irregularly shaped embryos. In contrast to the transparent grains of the wild type, the grains of the null mutant of OsROS1a were slightly opaque and rounded starch granules, with uneven shapes, sizes, and surfaces. A total of 723 differential expression genes (DEGs) were detected in the null mutant of OsROS1a by RNA-Seq, of which 290 were downregulated and 433 were upregulated. The gene ontology (GO) terms with the top 20 enrichment factors were visualized for cellular components, biological processes, and molecular functions. The key genes that are enriched for these GO terms include starch synthesis genes (OsSSIIa and OsSSIIIa) and cellulose synthesis genes (CESA2, CESA3, CESA6, and CESA8). Genes encoding polysaccharides and glutelin were found to be downregulated in the mutant endosperm. The glutelins were further verified by SDS-PAGE, suggesting that glutelin genes could be involved in the null mutant of OsROS1a seed phenotype and OsROS1a could have the key role in the regulation of glutelins. Furthermore, 378 differentially alternative splicing (AS) genes were identified in the null mutant of OsROS1a, suggesting that the OsROS1a gene has an impact on AS events. Our findings indicated that the function on rice endosperm development in the null mutant of OsROS1a could be influenced through regulating gene expression and AS, which could provide the base to properly understand the molecular mechanism related to the OsROS1a gene in the regulation of rice seed development.

Loss of Function of the RRMF Domain in OsROS1a Causes Sterility in Rice (Oryza sativa L.).

For crop seed production, the development of anthers and male fertility are the main agronomic traits and key biological processes for flowering plants. Active DNA demethylation regulates many plant developmental processes and is ensured by 5-meC DNA glycosylase enzymes. To find out the role of OsROS1a, OsROS1a gene editing mutants were generated using the CRISPR/Cas9 system. The osros1a mutants had shrink spikelets, smaller anthers and pollen grains, and were not stained by iodine staining showing a significant reduction in total soluble sugar and starch contents as compared to wildtype (WT), which caused complete male sterility. Similarly, the expression of genes involved in pollen and anther development was decreased in osros1a mutants as compared to WT. Furthermore, bisulfite sequencing showed that the CG and CHG methylation of the OsPKS2 gene promoter was significantly increased in the osros1a mutant, which caused a reduced expression of OsPKS2 in osros1a mutants. DNA methylation of the TDR gene promoter was similar between WT and osros1a mutants, indicating that the DNA methylation effect by OsROS1a was gene specific. The expression of OsROS1a in the mutants was not changed, but it produced a frame-shift mutation to truncate the Pem-CXXC and RRMF domains. Combined with previous studies, our findings suggested that the RRMF domain in OsROS1a is the functional domain and loss of RRMF for OsROS1a causes sterility in rice.

Analysis of the Fusarium graminearum Species Complex from Gramineous Weeds Near Wheat Fields in Jiangsu Province, China.

Several weed species are known as alternative hosts of the Fusarium graminearum species complex (FGSC), and their epidemiological significance in Fusarium head blight (FHB) has been investigated; however, scant information is available regarding FGSC occurrence in weeds near Chinese wheat fields. To evaluate the potential role of gramineous weeds surrounding wheat fields in FHB, 306 FGSC isolates were obtained from 210 gramineous weed samples in 2018 in Jiangsu Province. Among them, 289 were Fusarium asiaticum, and the remainder were F. graminearum. Trichothecene genotype and mycotoxin analyses revealed that 74.3% of the F. asiaticum isolates were the 3-acetyldeoxynivalenol (3ADON) chemotype, and the remainder were the nivalenol (NIV) chemotype. Additionally, 82.4% of F. graminearum isolates were the 15-acetyldeoxynivalenol (15ADON) chemotype, and the remainder were the NIV chemotype. FHB severity and trichothecene analysis indicated that F. asiaticum isolates with the 3ADON chemotype were more aggressive than those with the NIV chemotype in wheat. 3ADON and NIV chemotypes of F. asiaticum isolated from weeds and wheat showed no significant differences in pathogenicity in wheat. All selected F. asiaticum isolates produced perithecia, with little difference between the 3ADON and NIV chemotypes. These results highlight the epidemiology of the FGSC isolated from weeds near wheat fields, with implications for reducing FHB inoculum in China.

Transcriptome analysis of the thermosensitive genic male-sterile line provides new insights into fertility alteration in rice (Oryza sativa).

Thermosensitive genic male-sterile (TGMS) lines have been widely used for rice breeding to produce hybrid rice. Here, RNA-Seq was performed to investigate the expression profiles of young panicle from rice TGMS line at the microspore mother cell and meiosis stages under sterile and fertile conditions. A total of 1070 differentially expressed genes (DEGs) were identified, which were enriched in protein folding, protein binding, regulation of transcription, transcription factor activity and metabolic related processes. Further analyses showed hub genes UbL40s were predicted to interact with proteolysis-related genes and DNA-directed RNA polymerase subunit, and HSPs interacted with kinases to play important roles in regulating fertility alteration. It suggested that besides UbL40s, DNA-directed RNA polymerase subunit, kinases and HSPs might be involved in fertility alteration in rice TGMS lines. These findings provided new insights into fertility alteration in rice TGMS line, which will benefit for further application of TGMS in rice breeding.

Identification, characterization, and functional prediction of circular RNAs in maize.

Circular RNAs (circRNAs) are a new type of intracellular regulator that have been widely identified in animals and plants by high-throughput sequencing. However, there are still few functional studies on circRNAs in plants. To better understand maize circRNAs and their potential functions, we identified 1199 circRNAs in maize from RiboMinus RNA-Seq transcriptome data, and found distinct features of splicing site selection bias, longer flanking introns, and miniature inverted-repeat transposable element (MITE) insertions in flanking introns in maize circRNAs compared to other plant circRNAs. In total, 31 and 36 orthologous circRNAs were identified in rice and maize, respectively, but the orthologous parental genes could not produce orthologous circRNAs, mostly because of long-sequence insertions/deletions at flanking introns and approximately 24.3% of them contained MITE sequences. The majority of maize circRNAs showed high diversity of expression under different treatments and/or in different genetic backgrounds, implying that circRNAs could be involved in various regulatory networks. Twenty-six ecircRNAs were predicted to contain one or more target mimics, and 229 circRNAs had high coding potential, indicating that circRNAs could perform peptide-encoding functions in plants. These results will broaden understanding of the roles of circRNAs in plants and support further functional work on maize.

Interactions between CO2-Responsive Switchable Emulsion Droplets Determined by Using Optical Tweezers.

CO2-responsive switchable emulsions have been of great interest in some industrial processes where the stability of the emulsion is only required temporarily, such as oil transport, drug delivery, and fossil fuel production. The good understanding of the stability and instability mechanism is vital to the switchable behavior between emulsification and demulsification. Herein, a novel approach was developed to determine the interactions between two switchable emulsion droplets directly by a dual-laser optical tweezers instrument. The repulsive force between a couple of tetradecane droplets occurs to increase progressively with the increasing concentration of switchable surfactant in solutions. However, the repulsive force appears to decrease progressively in turn when the switchable surfactant concentration is far higher than the critical micelle concentration (CMC). Moreover, the depletion effect starts to emerge in the higher surfactant concentration which is attributed to the switchable surfactant micelles generated in solutions. In addition, according to the measurements of interaction forces, a mechanism of the switchable behavior is well proposed, which is established by the principle of self-assembly/detachment of the switchable surfactant, resulting in the weakening and re-enhancing of the electrostatic double-layer (EDL) repulsive forces between tetradecane droplets, upon selective introduction and removal of CO2. Based on this work, a novel perspective was provided to study the switchable emulsion, which can contribute instructive messages for the understanding of stability and instability mechanisms of switchable emulsions.

In Situ Measurements of Interactions between Switchable Surface-Active Colloid Particles Using Optical Tweezers.

Switchable surface-active colloid particles are critical to the preparation of switchable Pickering emulsions, which are widely involved in multitudinous fundamental and practical fields, such as biomedical, food products, and spinning cosmetics. The stability of switchable surface-active particles relies on the full understanding of interaction forces between individual colloid particles quantitatively. In this work, a dual-laser optical tweezers instrument was applied to measure the interaction forces between silica particles coated with a common cationic surfactant (cetyltrimethylammonium bromide, CTAB) in water, and all of the measured forces can be well fitted with the theoretical model derived from the Derjaguin-Landau-Verwey-Overbeek (DLVO) theory. It was revealed that the minimum surface distance to engender the interaction forces between silica particles was closer progressively with the increase of CTAB concentrations, suggesting that the introduction of CTAB molecules in the solution thinned the electric double layer. In addition, the minimum surface distance between surface-inactive silica particles further decreased compared to surface-active states, although the ζ-potential has returned to the initial value of bare silica in pure water when the molecular ratio of 1:1 anionic surfactant (sodium dodecyl sulfate, SDS) was added into the solution to switch the surface-active silica particles to surface-inactive states. Our results provide a considerate methodology for quantifying the interaction forces and investigating the switchable behaviors of CTAB molecules from the adsorption to desorption at the particle-water interfaces, which provide vital foresights into the stabilization mechanism of switchable surface-active colloid particles and the further development of switchable Pickering emulsions.

Cumulibacter soli sp. nov., isolated from farmland soil.

A Gram-stain-positive, strictly aerobic, non-motile, non-spore-forming and rod-shaped bacterium, designated as strain G-1T, was isolated from farmland soil sampled in in Fuyang, Anhui Province, PR China. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain G-1T was closely related to Cumulibacter manganitolerans 2-36T (97.7 % similarity). Strain G-1T contained iso-C16 : 0, C17 : 1ω6c, iso-C15 : 0 and iso-C14 : 0 as the predominant fatty acids. The polar lipids of strain G-1T were diphosphatidylglycerol, phosphatidylethanolamine, an unidentified phospholipid, an unidentified lipid and two unidentified glycolipids. The predominant respiratory quinone of strain G-1T was MK-9(H4). The cell wall contained meso-diaminopimelic acid as the diagnostic diamino acid. The G+C content of the genomic DNA based on genome calculations was 64.2 mol%. Average nucleotide identity and the digital DNA-DNA hybridization values for the draft genomes between strain G-1T and strain 2-36T were 75.7 and 20.2 %, respectively. On the basis of phenotypic and phylogenetic data, strain G-1T is considered to represent a novel species of the genus Cumulibacter, for which the name Cumulibacter soli sp. nov. is proposed. The type strain is G-1T (=CCTCC AB2019021T=KCTC 49258T).

Analysis of Fusarium graminearum Species Complex from Freshly Harvested Rice in Jiangsu Province (China).

Members of Fusarium graminearum species complex (FGSC) are the major pathogens that cause Fusarium head blight (FHB) in cereals worldwide. Symptoms of FHB on rice, including dark staining or browning of rice glumes, were recently observed in Jiangsu Province, China. To improve our understanding of the pathogens involved, 201 FGSC isolates were obtained from freshly harvested rice samples and identified by phylogenetic analyses. Among the 201 FGSC isolates, 196 were F. asiaticum and the remaining 5 were F. graminearum. Trichothecene chemotype and chemical analyses showed that 68.4% of the F. asiaticum isolates were the 3-acetyldeoxynivalenol (3ADON) chemotype and the remainder were the nivalenol (NIV) chemotype. All of the F. graminearum isolates were the 15-acetyldeoxynivalenol chemotype. Pathogenicity assays showed that both the 3ADON and NIV chemotypes of F. asiaticum could infect wheat and rice spikes. FHB severity and trichothecene toxin analysis revealed that F. asiaticum with the NIV chemotype was less aggressive than that with the 3ADON chemotype in wheat, while the NIV-producing strains were more virulent than the 3ADON-producing strains in rice. F. asiaticum isolates with different chemotypes did not show significant differences in mycelial growth, sporulation, conidial dimensions, or perithecial production. These findings would provide useful information for developing management strategies for the control of FHB in China.

Dynamic gene amplification and function diversification of grass-specific O-methyltransferase gene family.

The plant O-methyltransferases are dependent on S-Adenosyl-l-methionine, which can catalyze a variety of secondary metabolites. Here we identified different number of OMT genes from the respective grass genomes. Phylogenetic analysis showed that this OMT gene family is a grass-specific gene family that is different from COMT. Most of genes were expanded by tandem and segment duplication after the species split from their progenitor. Furthermore, genes from Group I and two clusters from group II are only present in Panicoideae, which included Bx10 and Bx7 involved in the benzoxazinoids pathway, suggesting these genes could participate in insect resistance in Panicoideae. Gene expression profiles showed that OMT genes were preferentially expressed in vegetative stages, especially in roots. These results revealed that this grass-specific OMT gene family could affect the development of vegetative stages, and be involved in the benzoxazinoids pathway or suberin biosynthesis that was different from COMT.

Genetic diversity and evolution of reduced sulfur storage during domestication of maize.

The domestication of maize has spanned a period of over 9000 years, during which time its wild relative teosinte underwent natural and artificial selection. We hypothesize that environmental conditions could have played a major role in this process. One factor of environmental variation is soil composition, which includes sulfur availability. Sulfur is reduced during photosynthesis and is used to synthesize cysteine and methionine, which drive the accumulation of δ10 (Zm00001d045937), δ18 (Zm00001d037436), ß15 (Zm00001d035760), γ16 (Zm00001d005793), γ27 (Zm00001d020592), and γ50 (Zm00001d020591) zeins, representing the zein2 fraction (z2) of storage proteins in maize seeds. In this study, polymorphisms and haplotypes were detected based on six z2 genes in 60 maize and teosintes lines. Haplotypes were unevenly distributed, and abundant genetic diversity was found in teosintes. Polymorphism was highest in z2δ18, whereas for z2ß15 single nucleotide polymorphism (SNP) density and insertion/deletion (indel) abundance were the lowest, indicating differential roles in seed evolution. Indels showed a clustered distribution, and most of these derived from teosintes. The indels not only led to tandem repeat polymorphisms, but also to frameshift mutations, which could also be used as null variants. In addition, neutral evolutionary tests, phylogenetic analyses, and population structures indicated that z2δ10 and z2γ50 had undergone natural selection. Indeed, a natural selection imprint could also be found with z2γ27 and z2γ16, whereas z2δ18 and z2ß15 tended to be under neutral evolution. These results suggested that genetic diversity and evolution of a subset of sulfur-rich zeins could be under environmental adaptation during maize domestication.

Smart Amphiphilic Janus Microparticles: One-Step Synthesis and Self-Assembly.

Self-assembly is the process to form ordered compound structures. Theories and experiments involving nanosized Janus particles have proved that the assembled cluster structures are related to the unit number. Micrometer-sized amphiphilic Janus particles could also act as surfactants to stabilize droplets and aggregate to form clusters. When the scale order increases to the millimeter size, particles are usually connected under shape-match mechanism, which means that the assembled structure is related to the particular particle morphology rather than the particle number. Similar to millimeter-sized particles, sub-millimeter-sized particles are larger and heavier such that their gravity cannot be ignored, whereas their Brownian motion could be neglected. To investigate the self-assembly behavior of sub-millimeter-sized Janus particles, we synthesize smart amphiphilic Janus microparticles directly from water-oil Janus droplets in one step by using a double-core capillary device. We find that the amphiphilic Janus particles could also be distributed directionally between the sides of the water-oil interface. When in oil solutions with several water droplets, the particles self-assemble into micelle-like structures to cover the water droplets with the hydrophilic phase inside. After evaporation, structures with a hydrophilic concave and a hydrophobic convex are formed. This paper demonstrates that sub-millimeter-sized amphiphilic Janus particles exhibit similar ability to nano-sized Janus particles to aggregate into clusters with ordered structures.

MiR529a modulates panicle architecture through regulating SQUAMOSA PROMOTER BINDING-LIKE genes in rice (Oryza sativa).

KEY MESSAGE: MiR529a affects rice panicle architecture by targeting OsSPL2,OsSPL14 and OsSPL17 genes that could regulate their downstream panicle related genes. The panicle architecture determines the grain yield and quality of rice, which could be regulated by many transcriptional factors. The SQUAMOSA PROMOTER BINDING-LIKE (SPL) transcription factors are involved in the regulation of panicle development, which are targeted by miR156 and miR529. The expression profile demonstrated that miR529a is preferentially expressed in the early panicle of rice and it might regulate panicle development in rice. However, the regulation mechanism of miR529-SPL is still not clear. In this study, we predicted five miR529a putative target genes, OsSPL2, OsSPL14, OsSPL16, OsSPL17 and OsSPL18, while only the expression of OsSPL2, OsSPL14, and OsSPL17 was regulated by miR529a in the rice panicle. Overexpression of miR529a dramatically affected panicle architecture, which was regulated by OsSPL2, OsSPL14, and OsSPL17. Furthermore, the 117, 35, and 25 pathway genes associated with OsSPL2, OsSPL14 and OsSPL17, respectively, were predicted, and they shared 20 putative pathway genes. Our results revealed that miR529a could play a vital role in the regulation of panicle architecture through regulating OsSPL2, OsSPL14, OsSPL17 and the complex networks formed by their pathway and downstream genes. These findings will provide new genetic resources for reshaping ideal plant architecture and breeding high yield rice varieties.

Overexpression of miR529a confers enhanced resistance to oxidative stress in rice (Oryza sativa L.).

KEY MESSAGE: Overexpressing miR529a can enhance oxidative stress resistance by targeting OsSPL2 and OsSPL14 genes that can regulate the expression of their downstream SOD and POD related genes. MicroRNAs are involved in the regulation of plant developmental and physiological processes, and their expression can be altered when plants suffered environment stresses, including salt, oxidative, drought and Cadmium. The expression of microRNA529 (miR529) can be induced under oxidative stress. However, its biological function under abiotic stress responses is still unclear. In this study, miR529a was overexpressed to investigate the function of miR529a under oxidative stress in rice. Our results demonstrated that the expression of miR529a can be induced by exogenous H2O2, and overexpressing miR529a can increase plant tolerance to high level of H2O2, resulting in increased seed germination rate, root tip cell viability, reduced leaf rolling rate and chlorophyll retention. The expression of oxidative stress responsive genes and the activities of superoxide dismutase (SOD) and peroxidase (POD) were increased in miR529a overexpression plant, which could help to reduce redundant reactive oxygen species (ROS). Furthermore, only OsSPL2 and OsSPL14 were targeted by miR529a in rice seedlings, repressing their expression in miR529aOE plants could lead to strengthen plant tolerance to oxidation stress. Our study provided the evidence that overexpression of miR529a could strengthen oxidation resistance, and its target genes OsSPL2 and OsSPL14 were responsible for oxidative tolerance, implied the manipulation of miR529a and its target genes regulation on H2O2 related response genes could improve oxidative stress tolerance in rice.

Water-oil Janus emulsions: microfluidic synthesis and morphology design.

In this work we developed a facile method to prepare water-oil Janus emulsions in situ with tunable morphologies by using a double-bore capillary microfluidic device. In addition, by combining the theory model and our liquids' properties, we propose a method to design the morphology of water-oil Janus emulsions. To systematically research Janus morphologies we combined the theory model and the fluids' properties. Under the model guidance, we carefully selected the liquids system where only the interfacial tension between the water phase and the continuous phase changed while keeping the other two interfacial tensions unchanged. Thus we could adjust the Janus morphology by changing the surfactant mass fraction in the continuous phase. In addition, with the double-bore capillary, we prepared water-oil Janus emulsions with a large flow ratio range. By adjusting the flow ratio and the surfactant mass fraction, we successfully prepared Janus emulsions with gradual morphology changes, which would be meaningful in fields that have a high demand for morphology designing of amphiphilic Janus particles.

Dynamics of chloroplast genomes in green plants.

Chloroplasts are essential organelles, in which genes have widely been used in the phylogenetic analysis of green plants. Here, we took advantage of the breadth of plastid genomes (cpDNAs) sequenced species to investigate their dynamic changes. Our study showed that gene rearrangements occurred more frequently in the cpDNAs of green algae than in land plants. Phylogenetic trees were generated using 55 conserved protein-coding genes including 33 genes for photosynthesis, 16 ribosomal protein genes and 6 other genes, which supported the monophyletic evolution of vascular plants, land plants, seed plants, and angiosperms. Moreover, we could show that seed plants were more closely related to bryophytes rather than pteridophytes. Furthermore, the substitution rate for cpDNA genes was calculated to be 3.3×10(-10), which was almost 10 times lower than genes of nuclear genomes, probably because of the plastid homologous recombination machinery.