Label-Free and Simultaneous Mechanical and Electrical Characterization of Single Plant Cells Using Microfluidic Impedance Flow Cytometry.

Despite that single-cell-type-level analyses have been extensively conducted on animal models to gain new insights into complex biological processes; the unique biological and physiological properties of plant cells have not been widely studied at single-cell resolution. In this work, an electrical impedance flow cytometry was fabricated based on microfluidics with constriction microchannel to simultaneously characterize the mechanical and electrical properties of single plant cells. Protoplasts from two model plant species, the herbaceous Arabidopsis thaliana and the woody Populus trichocarpa, could be readily discriminated by their respective mechanical traits, but not by electrical impedance. On the contrary, overexpression of a red fluorescent protein on plasma membrane resulted in changes in cell electrical impedance instead of cell deformability. During primary cell wall (PCW) regeneration, this extracellular layer outside of protoplasts introduced dramatic variations in both mechanical and electrical properties of single plant cells. Furthermore, the effects of auxin, an essential phytohormone regulating PCW reformation, were validated on this platform. Taken together, our results revealed a novel application of microfluidic impedance flow cytometry in the field of plant science to simultaneously characterize dual biophysical properties at single-cell resolution, which could be further developed as a powerful and reliable tool for plant cell phenotyping and cell fate specification.

An impedance-coupled microfluidic device for single-cell analysis of primary cell wall regeneration.

Primary cell wall (PCW) is a rigid yet flexible cell wall surrounding plant cells and it plays key roles in plant growth, cell differentiation, intercellular communication, water movement and defence. As a technique widely used to study the characteristics of mammalian cells, electrical impedance spectroscopy (EIS) is rarely used in plant science. In this work, we designed and fabricated an EIS based biosensor coupled with microfluidic platform to investigate the formation process of PCWat the single-cell level. Arabidopsis mesophyll cells with completely regenerated PCW showed significantly higher impedance values compared to the nascent protoplasts without PCW, demonstrating that PCW formation caused a dramatic change in cell electrical properties. The device could also discriminate plant mutant cells with modified PCW compositions, thus provided a novel tool for physical phenotyping of plant cells. The dose-dependent effects of exogenously applied auxin on PCW regeneration were corroborated on this platform which revealed its potential to sensitively detect the influences of in vitro stimuli. This work not only provided one novel application of impedance-based biosensor to characterize a plant-specific developmental event, but also revealed the promises of EIS integrated microfluidic system as a sensitive, time-effective and low-cost platform to characterize single plant cells and make new scientific discoveries in plant science.

Genome-wide identification, expression and functional analysis of Populus xylogen-like genes.

As an extracellular arabinogalactan protein (AGP) containing a non-specific lipid transfer protein (nsLTP) domain, xylogen mediates the local intercellular communication required for tracheary element (TE) differentiation in Zinnia cell culture. Although XYLP (xylogen-like protein) gene families have been reported in Arabidopsis and rice, no comprehensive analysis has been performed in woody plants. In this work, 31 XYLP genes in five phylogenetic groups were identified from Populus trichocarpa genome and a comprehensive bioinformatic analysis including gene and protein structures, chromosomal locations and duplication events were conducted. In-silico data and qRT-PCR results indicated that PtXYLP1 is predominantly expressed in poplar apex, young leaves and roots, while PtXYLP2 is uniformly expressed across a variety of tissues with a low abundance. Analysis on PtXYLP1pro:GUS and PtXYLP2pro:GUS in Arabidopsis revealed their differential expression patterns during seed germination and specific inductions by exogenously applied phytohormones including auxin, cytokinin and GA. When overexpressed in Arabidopsis, PtXYLP1 but not PtXYLP2 resulted in cotyledons with defective venation patterns and interrupted secondary (2°) vein loops, which phenotype was underpinned by the down-regulation of genes indispensably required by embryonic venation development at procambium and/or vessel level.

Truncated BAM receptors interfere the apical meristematic activity in a dominant negative manner when ectopically expressed in Arabidopsis.

Small, secreted signaling peptides that are perceived by receptor-like kinases (RLKs) constitute an important regulatory mechanism in plant organ formation and stem cell maintenance. However, functional redundancy at the level of both ligand and receptor families often makes it difficult to clearly discern the role of individual members by a genetic approach. Here, we show that driven by a constitutive CaMV 35S promoter, a truncated BAM protein (BAMΔ) that lacks either the signal peptide (SP) or the cytoplasmic kinase (Ki) domain could cause defective shoot apical meristem (SAM) maintenance, which phenotypically resembled the triple bam mutant. Such a dominant-negative effect could also be achieved when the same transgene was driven by the native AtBAM1 promoter, but not by the CLV1 promoter. When introduced into a clv1-4 background, BAMΔ proteins abolished the typical clv phenotype by suppressing the transcriptional level of clv1-4. In addition to a clear reduction in root length and a decreased number of meristematic cells, the 35S:BAMΔ transgenic seedlings exhibited considerable resistance to CLE40p- but not to CLV3p-mediated root growth inhibition, implying that BAMs play key roles in the regulation of proximal meristem activity in root through CLE40 peptide. Findings present here not only provide evidence that truncated BAM proteins are strongly dominant negative in regulating apical meristem development but also propose that expression of a truncated version of plant LRR receptor kinase could potentially be used as a powerful tool to reveal its in vivo function in signal transduction.

Characterization of FM2382 from Fulvimarina manganoxydans sp. Nov. 8047 with potential enzymatic decontamination of sulfur mustard.

Sulfur mustard (SM) can be hydrolyzed by haloalkane dehalogenases such as DhaA, LinB and DmbA. However, the low resistance to the elevated temperatures limited the practical application of haloalkane dehalogenases. Here we reported a new thermotolerant dehalogenase FM2382 from Fulvimarina manganoxydans sp. nov. 8047. The specific activity of FM2382 to SM is 0.6 U/mg. FM2382 possessed high heat stability (45 °C) in slight alkali environment (pH 7.5) and retained approximately 50% activity after incubation at 70 °C for 40 min. The catalytic activity of FM2382 was activated by Co2+ and Mg2+, and inhibited by Zn2+, Cu2+ and Fe3+. Furthermore, site-specific mutagenesis proved that D34, K207 D232, D237 were amino acid residues related to the catalytic activity of SM. In conclusion, we found a thermostable haloacid dehalogenases (HAD) family dehalogenase showing SM-degradation activity, which may be useful for practical application in the future.

Negative Regulation of Ectoine Uptake and Catabolism in Sinorhizobium meliloti: Characterization of the EhuR Gene.

Ectoine has osmoprotective effects on Sinorhizobium meliloti that differ from its effects in other bacteria. Ectoine does not accumulate in S. meliloti cells; instead, it is degraded. The products of the ehuABCD-eutABCDE operon were previously discovered to be responsible for the uptake and catabolism of ectoine in S. meliloti However, the mechanism by which ectoine is involved in the regulation of the ehuABCD-eutABCDE operon remains unclear. The ehuR gene, which is upstream of and oriented in the same direction as the ehuABCD-eutABCDE operon, encodes a member of the MocR/GntR family of transcriptional regulators. Quantitative reverse transcription-PCR and promoter-lacZ reporter fusion experiments revealed that EhuR represses transcription of the ehuABCD-eutABCDE operon, but this repression is inhibited in the presence of ectoine. Electrophoretic mobility shift assays and DNase I footprinting assays revealed that EhuR bound specifically to the DNA regions overlapping the -35 region of the ehuA promoter and the +1 region of the ehuR promoter. Surface plasmon resonance assays further demonstrated direct interactions between EhuR and the two promoters, although EhuR was found to have higher affinity for the ehuA promoter than for the ehuR promoter. In vitro, DNA binding by EhuR could be directly inhibited by a degradation product of ectoine. Our work demonstrates that EhuR is an important negative transcriptional regulator involved in the regulation of ectoine uptake and catabolism and is likely regulated by one or more end products of ectoine catabolism. IMPORTANCE: Sinorhizobium meliloti is an important soil bacterium that displays symbiotic interactions with legume hosts. Ectoine serves as a key osmoprotectant for S. meliloti However, ectoine does not accumulate in the cells; rather, it is degraded. In this study, we characterized the transcriptional regulation of the operon responsible for ectoine uptake and catabolism in S. meliloti We identified and characterized the transcription repressor EhuR, which is the first MocR/GntR family member found to be involved in the regulation of compatible solute uptake and catabolism. More importantly, we demonstrated for the first time that an ectoine catabolic end product could modulate EhuR DNA-binding activity. Therefore, this work provides new insights into the unique mechanism of ectoine-induced osmoprotection in S. meliloti.

Design of an ectoine-responsive AraC mutant and its application in metabolic engineering of ectoine biosynthesis.

Advanced high-throughput screening methods for small molecules may have important applications in the metabolic engineering of the biosynthetic pathways of these molecules. Ectoine is an excellent osmoprotectant that has been widely used in cosmetics. In this study, the Escherichia coli regulatory protein AraC was engineered to recognize ectoine as its non-natural effector and to activate transcription upon ectoine binding. As an endogenous reporter of ectoine, the mutated AraC protein was successfully incorporated into high-throughput screening of ectoine hyper-producing strains. The ectoine biosynthetic cluster from Halomonas elongata was cloned into E. coli. By engineering the rate-limiting enzyme L-2,4-diaminobutyric acid (DABA) aminotransferase (EctB), ectoine production and the specific activity of the EctB mutant were increased. Thus, these results demonstrated the effectiveness of engineering regulatory proteins into sensitive and rapid screening tools for small molecules and highlighted the importance and efficacy of directed evolution strategies applied to the engineering of genetic components for yield improvement in the biosynthesis of small molecules.