A Single-cell transcriptome atlas reveals the trajectory of early cell fate transition during callus induction in Arabidopsis.

The acquisition of pluripotent callus from somatic cells plays an important role in plant development studies and the genetic improvement of crops. This developmental process incorporates a series of cell fate transitions and reprogramming. However, our knowledge of cell heterogeneity and of the mechanisms of cell fate transition during callus induction remains quite limited. Here, we performed a time series single-cell transcriptome experiment on Arabidopsis root explants that were induced in callus induction medium for 0 days, 1 day, and 4 days, and we constructed a detailed single-cell transcriptional atlas of the callus induction process. We identified the cell types responsible for initiating the early callus: lateral root primordia-initiating (LRPI)-like cells and quiescent center (QC)-like cells. LRPI-like cells are derived from xylem pole pericycle cells and are similar to lateral root primordia. We delineate the developmental trajectory of the dedifferentiation of LRPI-like cells into QC-like cells. QC-like cells are undifferentiated pluripotent acquired cells that appear in the early stages of callus formation and play a critical role in later callus development and organ regeneration. We further inferred the transcription factors that regulating QC-like cells and the gene expression signatures that are related to cell fate decisions. Overall, our cell-lineage transcriptome atlas for callus induction provides a distinct perspective on cell fate transition during callus formation, and significantly improves understanding of callus formation.

Chromosome-level genomes of three key Allium crops and their trait evolution.

Allium crop breeding remains severely hindered due to the lack of high-quality reference genomes. Here we report high-quality chromosome-level genome assemblies for three key Allium crops (Welsh onion, garlic and onion), which are 11.17 Gb, 15.52 Gb and 15.78 Gb in size with the highest recorded contig N50 of 507.27 Mb, 109.82 Mb and 81.66 Mb, respectively. Beyond revealing the genome evolutionary process of Allium species, our pathogen infection experiments and comparative metabolomic and genomic analyses showed that genes encoding enzymes involved in the metabolic pathway of Allium-specific flavor compounds may have evolved from an ancient uncharacterized plant defense system widely existing in many plant lineages but extensively boosted in alliums. Using in situ hybridization and spatial RNA sequencing, we obtained an overview of cell-type categorization and gene expression changes associated with spongy mesophyll cell expansion during onion bulb formation, thus indicating the functional roles of bulb formation genes.

Spatial transcriptomics reveals light-induced chlorenchyma cells involved in promoting shoot regeneration in tomato callus.

Callus is a reprogrammed cell mass involved in plant regeneration and gene transformation in crop engineering. Pluripotent callus cells develop into fertile shoots through shoot regeneration. The molecular basis of the shoot regeneration process in crop callus remains largely elusive. This study pioneers the exploration of the spatial transcriptome of tomato callus during shoot regeneration. The findings reveal the presence of highly heterogeneous cell populations within the callus, including epidermis, vascular tissue, shoot primordia, inner callus, and outgrowth shoots. By characterizing the spatially resolved molecular features of shoot primordia and surrounding cells, specific factors essential for shoot primordia formation are identified. Notably, chlorenchyma cells, enriched in photosynthesis-related processes, play a crucial role in promoting shoot primordia formation and subsequent shoot regeneration. Light is shown to promote shoot regeneration by inducing chlorenchyma cell development and coordinating sugar signaling. These findings significantly advance our understanding of the cellular and molecular aspects of shoot regeneration in tomato callus and demonstrate the immense potential of spatial transcriptomics in plant biology.

Scaling-up and proteomic analysis reveals photosynthetic and metabolic insights toward prolonged H2 photoproduction in Chlamydomonas hpm91 mutant lacking proton gradient regulation 5 (PGR5).

Clean and sustainable H2 production is crucial to a carbon-neutral world. H2 generation by Chlamydomonas reinhardtii is an attractive approach for solar-H2 from H2O. However, it is currently not large-scalable because of lacking desirable strains with both optimal H2 productivity and sufficient knowledge of underlying molecular mechanism. We hereby carried out extensive and in-depth investigations of H2 photoproduction of hpm91 mutant lacking PGR5 (Proton Gradient Regulation 5) toward its up-scaling and fundamental mechanism issues. We show that hpm91 is at least 100-fold scalable (up to 10 L) with continuous H2 collection of 7287 ml H2/10L-HPBR in averagely 26 days under sulfur deprivation. Also, we show that hpm91 is robust and active during sustained H2 photoproduction, most likely due to decreased intracellular ROS relative to wild type. Moreover, we obtained quantitative proteomic profiles of wild type and hpm91 at four representing time points of H2 evolution, leading to 2229 and 1350 differentially expressed proteins, respectively. Compared to wild type, major proteome alterations of hpm91 include not only core subunits of photosystems and those related to anti-oxidative responses but also essential proteins in photosynthetic antenna, C/N metabolic balance, and sulfur assimilation toward both cysteine biosynthesis and sulfation of metabolites during sulfur-deprived H2 production. These results reveal not only new insights of cellular and molecular basis of enhanced H2 production in hpm91 but also provide additional candidate gene targets and modules for further genetic modifications and/or in artificial photosynthesis mimics toward basic and applied research aiming at advancing solar-H2 technology.

Protoplast technology enables the identification of efficient multiplex genome editing tools in Phalaenopsis.

Phalaenopsis orchids are popular ornamental plants worldwide. The application and optimization of efficient CRISPR-Cas genome editing toolkits in Phalaenopsis greatly accelerate the development of orchid gene function and breeding research. However, these methods are greatly hindered by the deficiency of a rapid screening system. In this study, we established a fast and convenient Phalaenopsis protoplast technology for the identification of functional genome editing tools. Two multiplex genome editing tools, PTG-Cas9-HPG (PTG, polycistronic tRNA-gRNA) system and RMC-Cpf1-HPG (RMC, ribozyme-based multi-crRNA) system, were developed for Phalaenopsis genome editing and further evaluated by established protoplast technology. We successfully detected various editing events comprising substitution and indel at designed target sites of the PDS gene and MADS gene, showing that both PTG-Cas9-HPG and RMC-Cpf1-HPG multiplex genome editing systems are functional in Phalaenopsis. Additionally, by optimizing the promoter that drives Cpf1 expression, we found that Super promoter can significantly improve the editing efficiency of the RMC-Cpf1-HPG system. Altogether, we successfully developed two efficient multiplex genome editing systems, PTG-Cas9-HPG and RMC-Cpf1-HPG, for Phalaenopsis, and the established protoplast-based screening technology provides a valuable foundation for developing more diverse and efficient genome editing toolkits and facilitating the development of orchid precision breeding.

Whole-cell biotransformation for large scale production of carcinine in Escherichia coli.

Carcinine is a natural imidazole-containing peptide derivative. It is widely used in the cosmetics industry as anti-aging supplement with antioxidant, anti-glycation and glycation reversal functions, and it also has a notable pharmacological effect as anti-tumor drug and in protection against retinopathy. However, a technological method for synthesis and production of carcinine has not been established. In this study, a whole-cell transformation system converting ß-alanine and histamine to carcinine by the enzymes Ebony and phosphopantetheine transferase (Sfp) has been developed. The results revealed that the catalytic efficiency of the strain containing the fusion protein of Ebony and Sfp (Sfp-glycine-serine-glycine-Ebony, SGE) in Escherichia coli W3110 (WSGE strain) is significantly higher (7.45 mM) than the combinatorial strain of pET28a-ebony and pACYCDuet-sfp in E. coli BL21(DE3) (BSE strain) (2.17 mM). Under the optimal reaction conditions (25 â„ƒ, pH 7.0, 12.5 g/L wet cells, 20 mM ß-alanine and 40 mM histamine), the carcinine can be quickly synthesized within 24 h up to a concentration of 22.63 mM. To achieve a continuous and efficient conversion of the precursors, a batch-feeding catalysis was designed. With this system, ß-alanine (40 mM) and histamine (40 mM) could be completely transformed to carcinine (40.34 mM) in 36 h with a productivity of 0.204 g/L h reaching a titer of 7.34 g/L. Hence, the batch-feeding whole-cell biocatalysis is a promising technology for the high yield production of carcinine which can promote the industrial production of carcinine.

The single-cell stereo-seq reveals region-specific cell subtypes and transcriptome profiling in Arabidopsis leaves.

Understanding the complex functions of plant leaves requires a thorough characterization of discrete cell features. Although single-cell gene expression profiling technologies have been developed, their application in characterizing cell subtypes has not been achieved yet. Here, we present scStereo-seq (single-cell spatial enhanced resolution omics sequencing) that enabled us to show the bona fide single-cell spatial transcriptome profiles of Arabidopsis leaves. Subtle but significant transcriptomic differences between upper and lower epidermal cells have been successfully distinguished. Furthermore, we discovered cell-type-specific gene expression gradients from the main vein to the leaf edge, which led to the finding of distinct spatial developmental trajectories of vascular cells and guard cells. Our study showcases the importance of physical locations of individual cells for exerting complex biological functions in plants and demonstrates that scStereo-seq is a powerful tool to integrate single-cell location and transcriptome information for plant biology study.

The complete chloroplast genome sequence of Phalaenopsis wilsoniii (Orchidaceae).

In the present study, we reported and characterized the complete chloroplast genome of a moth orchid, Phalaenopsis wilsonii, which is endemic to South China. Its plastid genome size is 145,373 bp, consisting of a large single copy (LSC) region (84,996 bp), a small single-copy region (10,668 bp), and two inverted repeats (IRs) regions (24,855 bp). A total of 122 plastid genes were annotated, comprising 76 protein-coding genes, 38 tRNA genes, and 8 rRNA genes. The phylogenetic tree further revealed that P. wilsonii showed a sister relationship with P. lowii within subgenus Parishianae.

Arabidopsis phosphoinositide-specific phospholipase C 4 negatively regulates seedling salt tolerance.

Previous physiological and pharmacological studies have suggested that the activity of phosphoinositide-specific phospholipase C (PI-PLC) plays an important role in regulating plant salt stress responses by altering the intracellular Ca2+ concentration. However, the individual members of plant PLCs involved in this process need to be identified. Here, the function of AtPLC4 in the salt stress response of Arabidopsis seedlings was analysed. plc4 mutant seedlings showed hyposensitivity to salt stress compared with Col-0 wild-type seedlings, and the salt hyposensitive phenotype could be complemented by the expression of native promoter-controlled AtPLC4. Transgenic seedlings with AtPLC4 overexpression (AtPLC4 OE) exhibited a salt-hypersensitive phenotype, while transgenic seedlings with its inactive mutant expression (AtPLC4m OE) did not exhibit this phenotype. Using aequorin as a Ca2+ indicator in plc4 mutant and AtPLC4 OE seedlings, AtPLC4 was shown to positively regulate the salt-induced Ca2+ increase. The salt-hypersensitive phenotype of AtPLC4 OE seedlings was partially rescued by EGTA. An analysis of salt-responsive genes revealed that the transcription of RD29B, MYB15 and ZAT10 was inversely regulated in plc4 mutant and AtPLC4 OE seedlings. Our findings suggest that AtPLC4 negatively regulates the salt tolerance of Arabidopsis seedlings, and Ca2+ may be involved in regulating this process.

Interspecies competition and inhibition within the oral microbial flora: environmental factors influence the inhibition of Streptococcus mutans by Streptococcus oligofermentans.

Dental caries is a bacterial infectious disease. Streptococcus mutans is the primary pathogen that causes dental caries. Streptococcus oligofermentans is a new oral streptococcal species that can inhibit the growth of S. mutans specifically. The study aimed to assess the inhibition of S. mutans by S. oligofermentans under different oral environmental conditions. The inhibition under different carbohydrate and oxygen conditions was investigated in vitro using an interspecies interaction assay. The 4-aminoantipyine (4-ATTP) method was used to measure the yield and the initial production rate of H(2) O(2) in S. oligofermentans. The inhibitory effect was enhanced when the bacteria were cultured with carbohydrates and under aerobic conditions, or when S. oligofermentans was inoculated earlier than S. mutans. The initial synthesis rates of H(2) O(2) by S. oligofermentans were higher in the presence of carbohydrates and in aerobic culture conditions. In terms of the total H(2) O(2) yield, the effect of the environmental conditions was as follows: no carbohydrates > sucrose> glucose, and aerobic conditions > anaerobic conditions. We conclude that the presence of carbohydrate and oxygen significantly affect the ability of S. oligofermentans to inhibit the growth of S. mutans. The difference in inhibitory effect may be attributed to changes in the capacity of S. oligofermentans to produce H(2) O(2) .

[Influence of glucose concentration on the inhibition of Streptococcus oligofermentans on Streptococcus mutans].

OBJECTIVE: To investigate the inhibition of Streptococcus oligofermentans (So) on Streptococcus mutans (Sm) and the producibility of hydrogen peroxide by So under the influence of glucose concentration environment. METHODS: The inhibition between So and Sm was observed by plating method under the different glucose concentration environment. The initial synthesis rates and production of hydrogen peroxide by So were determined under the different glucose concentration environment by 4-aminoantipyine-horseradish peroxidase method at A(510). RESULTS: Under 0, 10 and 50 mmol/L glucose environment, the inhibition of So on Sm was evident. When both Sm and So were inoculated at the same time, the ratio of inhibition area by bacterial membrane area was 0.202 ± 0.005, 0.467 ± 0.025, 0.468 ± 0.028 under 0, 10, 50 mmol/L glucose environment. When So was cultivated first and then Sm applied, the ratio was 0.394 ± 0.004, 0.811 ± 0.075 and 0.816 ± 0.007 under 0, 10 and 50 mmol/L glucose environment respectively. The inhibition under 10 and 50 mmol/L glucose environment were more significant than that under non-glucose environment. There was no significant difference between these two glucose concentrations (P > 0.05). The initial synthesis rates of H2O2 by So under the 10 mmol/L [(23.573 ± 0.263) µmo×L(-1)×min(-1)] and 50 mmol/L [(23.337 ± 0.473) µmol×L(-1)×min(-1)] glucose were higher than without glucose[(10.513 ± 0.516) µmol×L(-1)×min(-1)], P < 0.05. H2O2 was not detected in 1000 mmol/L glucose. However, the production of H2O2 by So under 0 mmol/L glucose was higher than other glucose concentrations (P < 0.05). CONCLUSIONS: The capability of the inhibition of So on Sm was affected by glucose environment and was much stronger under certain glucose concentrations (10, 50 mmol/L).

[The influence of oxygen on the inhibition between Streptococcus oligofermentans and Streptococcus mutans].

OBJECTIVE: To investigate the effect of environmental oxygen on the inhibition between Streptococcus oligofermentans (So) and Streptococcus mutans (Sm) and the producibilities of hydrogen peroxide by So. METHODS: The aerobic and anaerobic environment was established by the carbon dioxide cultivation. The inhibition between So and Sm was observed by plating method. The production and synthesis rates of hydrogen peroxide by So were determined in both aerobic and anaerobic environment by 4-ATTP-horseradish peroxidase method at A(510). RESULTS: When both Sm and So were inoculated at the same time, Sm was not inhibited under the anaerobic environment, vice versa. Sm was slightly inhibited by So under the aerobic environment, the inhibition area was 1/5 of all bacterial membrane. When So was cultivated first and then Sm applied, So could inhibite Sm growth under both anaerobic and aerobic conditions. The inhibition area was 1/5 of bacterial membrane under the anaerobic environment, and 4/5 under the aerobic environment. When Sm was cultivated first and then So applied, So was unable to proliferate under both conditions. During the logarithmic phase, the production of H2O2 by So under the aerobic environment was higher than under the anaerobic environment (P < 0.05). The initial synthesis rate of H2O2 by So during growth cycle under the anaerobic condition was (11.84 ± 3.97) µmol/L per minute, which was only 49% of that under the aerobic environment [(24.13 ± 4.46) µmol/L per minute]. CONCLUSIONS: The oxygen has the effect on the inhibition between So and Sm, and the inhibition in the aerobic environment is much stronger than in the anaerobic environment. The synthesis ability of hydrogen peroxide by So under the aerobic environment is higher than under the anaerobic environment.