Comprehensive integration of single-cell transcriptomic data illuminates the regulatory network architecture of plant cell fate specification

Plant morphogenesis relies on precise gene expression programs at the proper time and position which is orchestrated by transcription factors(TFs)in intricate regulatory networks in a cell-type specific manner.Here we introduced a comprehensive single-cell transcriptomic atlas of Arabidopsis seedlings.This atlas is the result of meticulous integration of 63 previously published scRNA-seq datasets,addressing batch effects and conserving biological variance.This integration spans a broad spectrum of tissues,including both below-and above-ground parts.Utilizing a rigorous approach for cell type annotation,we identified 47 distinct cell types or states,largely expanding our current view of plant cell compositions.We systematically constructed cell-type specific gene regulatory networks and uncovered key regulators that act in a coordinated manner to control cell-type specific gene expression.Taken together,our study not only offers extensive plant cell atlas exploration that serves as a valuable resource,but also provides molecular insights into gene-regulatory programs that varies from different cell types.

MORPHOLOGICAL MODEL FOR RHEOLOGICAL PROPERTIES OF PLANT CELL SUSPENSION CULTURE SYSTEM

This paper puts forward a physical and mathematical model for the rheological properties of a plant cell suspension culture system.The model can explain why the system is pseudoplastic satisfactorily,thus the rheological properties of the system as the effect of the flow behavior index on plant cell concentration are interpreted correctly and the mechanism of the rheological properties of the system is further understood.Therefore the model can be applied in the technological design and optimum conditions of the system and the reformation,evaluation and scale up of reactors.

Transient Expression of Exogenous Gene into Plant Cell Mediated by PEI Nanovector

This study was carried out to investigate the transfection effect of exogenous gene into plant protoplast cell mediated by polyethylenimine （PEI） nanovector, based on PEI gene delivery system in the field of medical science. PEI/DNA complexes were prepared by using PEI polymer to bind the plant expression plasmid, pCMl205-GFPn. The ability of PEI combining and protecting DNA was investigated by agarose gel electrophoresis retardation assay. The surface characteristics of PEI/DNA complexes were observed with transmission electron microscope. The transfection efficiency of Arabidopsis thaliana protoplasts mediated by PEI/DNA complexes at different N/P ratios was analyzed based on observation of transient expression of green fluorescent protein with confocal laser scanning microscope. PEI could bind and condense DNA, and form stable 100-200 nm PEI/DNA complexes when the proportion of PEl and DNA is in the range of 5：1-1：4. Transfection efficiency of PEI/DNA complexes increased with N/P ratios in range of N/P〈5 and reached the highest at N/P=5, and began to decrease beyond N/P〉5 as higher toxicity to cells. The transfection efficiency of PEI/DNA complexes at N/P=5 was higher than PEG. This study confirmed that PEI nanovector could effectively mediate foreign gene entering into A. thaliana protoplast cell to obtain transient expression, which may be developed as a hopeful and novel transgenic method combined with plant protoplast regeneration.

Synthesis of Fluorescent Carbon Quantum Dots and Their Application in the Plant Cell Imaging

Carbon quantum dots(CQDs) exhibit tremendous advantages for plant growth study due to its strong fluorescence and good biocompatibility. The fluorescent CQDs were synthesized by the onestep microwave method with the raw materials of citric acid(CA) and urea(UR), and expressed a unique green fluorescence with the optimal excitation wavelength of over 400 nm through adjusting the doping of N elements. It is demonstrated that CQDs can act as deliver media in plant and fluorescent probes for plant cell imaging through directly cultivated in the seedlings of melon and wheat, respectively. Based on the effects of the fluorescent CQDs on plants growth, we can further study the mechanisms of the ions transport in plants.

Plant Cell Wall, a Challenge for Its Characterisation

The plant cell wall is a complex 3D network composed of polysaccharides, lignin and proteins. The knowledge of the structure and content of each cell wall polymer is a prerequisite to understand their functions during plant development and adaptation but also to optimise their industrial applications. The analysis of cell wall compounds is complicated by their multiple molecular interactions. In this review, we present numerous methods to purify, characterise and quantify proteins, polysaccharides and lignin from the wall. Two kinds of approaches are detailed: the first presents in vitro methods which involve the breakdown of the molecular linkages between polymers thanking to chemical, physical and/or enzymatic treatments. The second approach describes in situ methods that allow the cell wall polymer characterisation thanking to many analytical techniques coupled with microscopy. If microscopy is the common point of all of them, their development is associated with improvement of analytical techniques, increasing their power of resolution.

Physiological Response of the Resurrection Fern Subjected to Environmental Stress as Shown by Plant Cell Membrane Integrity

Resurrection fern has a unique ability to maintain cell wall integrity when the plant cell is desiccated. It uses proteins such as late embryogenesis proteins and heat shock proteins to maintain their cellular functions. The purpose of this experiment is to determine the effects of environmental stressors on the physiological response of the resurrection fern (Pleopeltis polypodioides). The physiological response of resurrection fern plants was subjected to various temperatures (-50°C, 0°C, 25°C, and 50°C) for 7 days. Results indicated that there was a significant difference between hydrated and desiccated ferns based on the temperature. Additionally, electrolyte leakage measurements confirmed cell damage following exposure to temperature extremes of -50°C and 50°C.

Summary of the Frontier Introduction of Preparation of Secondary Metabolites in Plant Cell Culture

Plant cell culture technology is a technology that applies the research results of cell engineering to produce plant biological products at the cellular level.In recent years,the secondary metabolites of plants have attracted more and more attention.The use of plant cell culture technology is a fast and efficient method of producing secondary metabolites.

The First Observation on Plant Cell Fossils in China

For a long time, paleontologists have been focusing on hard parts of organisms during different geological periods while soft parts are rarely reported. Well-preserved plant cells, if found in fossils, are treated only as a rarity. Recent prowess in research on fossil cytoplasm indicates that plant cytoplasm not only has excellent ultrastructures preserved but also may be a quite commonly seen fossil in strata. However, up to now there is no report of plant cell fossils in China yet. Here plant cell fossils are reported from Huolinhe Coal Mine （the early Cretaceous）, Inner Mongolia, China. The presence of plant cytoplasm fossils in two cones on the same specimen not only provides further support for the recently proposed hypothesis on plant cytoplasm fossilization but also marks the first record of plant cytoplasm fossils in China, which suggests a great research potential in this new area.

EFFECTS OF TURBULENT SHEAR STRESS ON THE GROWTH OF SUSPENDED PLANT CELLS IN A BUBBLE COLUMN REACTOR

In this paper, effects of increasing the height of column, bubble size and gas flow rate on the growth of suspended Catharanthus roseus plant cells in a 1:5 litre bubble -column were studied. Statistical characteristics of turbulence such as auto-correlation function, and auto-power spectral density function of the fluctuating liquid velocity were measured in a bubble column employing pseudo-cell granulated agar. Calculations of turbulent shear stress were carried out. From these studies it is suggested that in plan! cell suspension culture sparged with large bubbles, turbulent shear stress is probably one of the most important reasons for the inhibited cell growth and cell damage. Larger bubbles or higher gas flow rates lead to the increase in shear stress and are more detrimental to plant cells.

A Uniaxial Loading Device for Studying Mechanoresponses of Single Plant Cell

A system which consists of a loading chamber unit, displacement sensor, data collector and processor, and a feedback control, was established for applying mechanical forces to single plant cells. The method works by compressing an agar cell-suspension block between parallel surfaces through using a force-feedback control circuit coupled to a microchip, delivering the pre-defined. The actual controlled stimulus is achieved whilst measuring the force being imposed on the cell, and its deformation. TheArabidopsisprotoplasts were utilized to test the system. It provides an experimental approach to investigate the mechanoresponses of plant cellsin vitroconditions.

EFFECTS OF SHEAR STRESS ON THE VIABILITY PLANT CELLS

Plant cell lines differred greatly in the ability to withstand shear stresses. Using to-bacco cells and licorice cells as model plant cells, we studied the effects of shear stresses on the vi-ability of plant cells. Our experiments were carried out on a high shear rate Couette rheometerproviding homogeneous and constant shear stresses of laminar flow. The viability was determinedby TTC（2,3,5-Triphenyl tetrazolium chloride）. The results were as follows. （1）The viability（V）dropped exponentially with time（t）, namely V=Exp（-kt）, （k】o is a constant）. This meant thetenability of statistical homogeneity. （2）The value of k was a function of plant cells’ mechanicalproperties and the shear stress acting on the plant cells. The shear rate corresponding to k=o wasthe critical shear rate that the plant cells could withstand. It can be easily determinded by extrapo-lation. For 7-day-old tobacco cells, it was 1090 s-1 and for 9-day-old licorice cells, it was6566 s-1.（3）The plant cell suspensions were pseudoplastic fluids fitting τ=Kγn. For the tobacco cell sus-pension tested, n=O. 73, and for the licorice cellsuspension tested n=0. 7. Thus the critical shearstress for the tobacco cells was 25 dynes/cm2 and for the licorice cells it was 80 dynes/cm2. （4）One of their reasons for licorice cells to have greater tolerance to shear stresses than tobacaccocells may be the geometric features of the cells and the sizes of the cells. The licorice cells wererod-shaped, but the tobacco cells were spherical and larger than the licorice cells.

Virus-Induced Gene Silencing Offers a Functional Genomics Platform for Studying Plant Cell Wall Formation

Virus-induced gene silencing （VIGS） is a powerful genetic tool for rapid assessment of plant gene functions in the post-genomic era. Here, we successfully implemented a Tobacco Rattle Virus （TRV）-based VlGS system to study functions of genes involved in either primary or secondary cell wall formation in Nicotiana benthamiana plants. A 3-week post- VIGS time frame is sufficient to observe phenotypic alterations in the anatomical structure of stems and chemical composition of the primary and secondary cell walls. We used cell wall glycan-directed monoclonal antibodies to demonstrate that alteration of cell wall polymer synthesis during the secondary growth phase of VIGS plants has profound effects on the extractability of components from woody stem cell walls. Therefore, TRV-based VlGS together with cell wall component profiling methods provide a high-throughput gene discovery platform for studying plant cell wall formation from a bioenergy perspective.

Mechanical Response of Single Plant Cells to Cell Poking:A Numerical Simulation Model

Cell poking is an experimental technique that is widely used to study the mechanical properties of plant cells. A full understanding of the mechanical responses of plant cells to poking force Is helpful for experimental work. The aim of this study was to numerically investigate the stress distribution of the cell wall, cell turgor, and deformation of plant cells in response to applied poking force. Furthermore, the locations damaged during poking were analyzed. The model simulates cell poking, with the cell treated as a spherical, homogeneous, isotropic elastic membrane, filled with incompressible, highly viscous liquid. Equilibrium equations for the contact region and the non-contact regions were determined by using membrane theory. The boundary conditions and continuity conditions for the solution of the problem were found. The forcedeformation curve, turgor pressure and tension of the cell wall under cell poking conditions were obtained. The tension of the cell wall circumference was larger than that of the meridian. In general, maximal stress occurred at the equator around. When cell deformation increased to a certain level, the tension at the poker tip exceeded that of the equator. Breakage of the cell wall may start from the equator or the poker tip, depending on the deformation. A nonlinear model is suitable for estimating turgor, stress, and stiffness, and numerical simulation is a powerful method for determining plant cell mechanical properties.

Post-transcriptional Gene Silencing Induced by Short Interfering RNAs in Cultured Transgenic Plant Cells

Short interfering RNA (siRNA) is widely used for studyingpost-transcriptional gene silencing and holds great promise as a tool for both identifying functionof novel genes and validating drug targets. Two siRNA fragments (siRNA-a and -b), which weredesigned against different specific areas of coding region of the same target green fluorescentprotein (GFP) gene, were used to silence GFP expression in cultured gfp transgenic cells of rice(Oryza sativa L.; OS), cotton (Gossypium hirsutum L.; GH), Eraser fir [Abies fraseri (Pursh) Poir;AF], and Virginia pine (Pinus virginiana Mill.; PV). Differential gene silencing was observed in thebombarded transgenic cells between two siRNAs, and these results were consistent with theinactivation of GFP confirmed by laser scanning microscopy, Northern blot, and siRNA analysis intested transgenic cell cultures. These data suggest that siRNA-mediated gene inactivation can be thesiRNA specific in different plant species. These results indicate that siRNA is a highly specifictool for targeted gene knockdown and for establishing siRNA-mediated gene silencing, which could bea reliable approach for large-scale screening of gene function and drug target validation.

Tanned or Sunburned:How Excessive Light Triggers Plant Cell Death

Plants often encounter light intensities exceeding the capacity of photosynthesis(excessive light)mainly due to biotic and abiotic factors,which lower C02 fixation and reduce light energy sinks.Under excessive light,the photosynthetic electron transport chain generates damaging molecules,hence leading to photooxidative stress and eventually to cell death.In this review,we summarize the mechanisms linking the excessive absorption of light energy in chloroplasts to programmed cell death in plant leaves.We highlight the importance of reactive carbonyl species generated by lipid photooxidation,their detoxification,and the integrating role of the endoplasmic reticulum in the adoption of phototolerance or cell-death pathways.Finally,we invite the scientific community to standardize the conditions of excessive light treatments.

Plant cell totipotency: Insights into cellular reprogramming

Plant cells have a powerful capacity in their propagation to adapt to environmental change, given that a single plant cell can give rise to a whole plant via somatic embryogenesis without the need for fertilization. The reprogramming of somatic cells into totipotent cells is a critical step in somatic embryogenesis. This process can be induced by stimuli such as plant hormones, transcriptional regulators and stress. Here, we review current knowledge on how the identity of totipotent cells is determined and the stimuli required for reprogramming of somatic cells into totipotent cells. We highlight key molecular regulators and associated networks that control cell fate transition from somatic to totipotent cells. Finally,we pose several outstanding questions that should be addressed to enhance our understanding of the mechanisms underlying plant cell totipotency.

Biomass accumulation of Panax vietnamensis in cell suspension cultures varies with addition of plant growth regulators and organic additives

Objective: To evaluate the impact of plant growth regulators including kinetin(KN),benzyl adenine and naphthalene acetic acid, yeast extract and casein hydrolyzate on biomass accumulation of Vietnamese ginseng Panax vietnamensis(P. vietnamensis) in cell suspension culture.Methods: Cell suspension cultures were established from friable calluses derived from leaves and petioles of 3-year-old in-vitro P. vietnamensis plants. The cell suspension cultures were grown in Murashige and Skoog basal media supplemented with various concentrations of KN, benzyl adenine, naphthalene acetic acid, and yeast extract and casein hydrolyzate.Results: All tested factors generated an increase in the cell biomass of P. vietnamensis in suspension culture, but the impact of each varies depended on the factor type, concentration, and incubation period. Addition of 2.0 mg/L KN resulted in the largest biomass increase after 24 d,(57.0 ± 0.9) and(3.1 ± 0.1) mg/m L fresh and dry weight, respectively,whereas addition of benzyl adenine or naphthalene acetic acid produced optimum levels of Panax cell biomass at 1.0 and 1.5 mg/L, respectively. Addition of the elicitor yeast extract led to a 1.4–2.4 fold increase in biomass of P. vietnamensis, while addition of casein hydrolyzate enhanced biomass accumulation 1.8–2.6 fold.Conclusions: The addition of each factor causes significant changes in biomass accumulation of P. vietnamensis. The largest biomass accumulation is from cultures grown in MS media containing 2.0 mg/L KN for 24 d. The outcome of the present study provides new insights into the optimal suspension culture conditions for studies on the in vitro cell biomass production of P. vietnamensis.

Dietary fiber in plant cell walls-the healthy carbohydrates

Dietary fiber(DF)is one of the major classes of nutrients for humans.It is widely distributed in the edible parts of natural plants,with the cell wall being the main DF-containing structure.DF content varies significantly in different plant species and organs,and the processing procedure can have a dramatic effect on the DF composition of plant-based foods.Given the considerable nutritional value of DF,a deeper understanding of DF in food plants,including its composition and biosynthesis,is fundamental to the establishment of a daily intake reference of DF and is also critical to molecular breeding programs for modifying DF content.In the past decades,plant cell wall biology has seen dramatic progress,and such knowledge is of great potential to be translated into DF-related food science research and may provide future research directions for improving the health benefits of food crops.In this review,to spark interdisciplinary discussions between food science researchers and plant cell wall biologists,we focus on a specific category of DF--cell wall carbohydrates.We first summarize the content and composition of carbohydrate DF in various plant-based foods,and then discuss the structure and biosynthesis mechanism of each carbohydrate DF category,in particular the respective biosynthetic enzymes.Health impacts of DF are highlighted,and finally,future directions of DF research are also briefly outlined.

A general method for assaying homo-and hetero-transglycanase activities that act on plant cell-wall polysaccharides

Transglycanases（endotransglycosylases） cleave a polysaccharide（donor-substrate） in mid-chain, and then transfer a portion onto another poly-or oligosaccharide（acceptor-substrate）. Such enzymes contribute to plant cellwall assembly and/or re-structuring. We sought a general method for revealing novel homo- and hetero-transglycanases, applicable to diverse polysaccharides and oligosaccharides, separating transglycanase-generated3 Hpolysaccharides from unreacted3H-oligosaccharides—the former immobilized（on filter-paper, silica-gel or glassfiber）,the latter eluted. On filter-paper, certain polysaccharides [e.g.（1!3, 1!4）-b-D-glucans] remained satisfactorily adsorbed when water-washed; others（e.g. pectins） were partially lost. Many oligosaccharides（e.g. arabinan-, galactan-, xyloglucan-based） were successfully eluted in appropriate solvents, but others（e.g. [3H]xylohexaitol, [3H]mannohexaitol[3H]cellohexaitol） remained immobile. On silica-gel, all3 Holigosaccharides left an immobile ‘ghost’ spot（contaminating any3H-polysaccharides）, which was diminished but not prevented by additives e.g. sucrose or Triton X-100. The best stratum was glassfiber（GF）, onto which the reactionmixture was dried then washed in 75% ethanol. Washing led to minimal loss or lateral migration of3H-polysaccharides if conducted by slow percolation of acidified ethanol. The effectiveness of GF-blotting was well demonstrated for Chara vulgaris transb-mannanase. In conclusion, our novel GF-blotting technique ef ficiently frees transglycanase-generated3H-polysaccharides from unreacted3H-oligosaccharides,enabling high-throughput screening of multiple postulated transglycanase activities utilising chemically diverse donorand acceptor-substrates.

Plant cell cultures as heterologous bio-factories for secondary metabolite production

Synthetic biology has been developing rapidly in the last decade and is attracting increasing attention from many plant biologists.The production of high-value plant-specific secondary metabolites is,however,limited mostly to microbes.This is potentially problematic because of incorrect post-translational modification of proteins and differences in protein micro-compartmentalization,substrate availability,chaperone availability,product toxicity,and cytochrome p450 reductase enzymes.Unlike other heterologous systems,plant cells may be a promising alternative for the production of high-value metabolites.Several commercial plant suspension cell cultures from different plant species have been used successfully to produce valuable metabolites in a safe,low cost,and environmentally friendly manner.However,few metabolites are currently being biosynthesized using plant platforms,with the exception of the natural pigment anthocyanin.Both Arabidopsis thaliana and Nicotiana tabacum cell cultures can be developed by multiple gene transformations and CRISPR-Cas9 genome editing.Given that the introduction of heterologous biosynthetic pathways into Arabidopsis and N.tabacum is not widely used,the biosynthesis of foreign metabolites is currently limited;however,therein lies great potential.Here,we discuss the exemplary use of plant cell cultures and prospects for using A.thaliana and N.tabacum cell cultures to produce valuable plant-specific metabolites.