Dataset Information

Preparation and applications of guard cell protoplasts from the leaf epidermis of Solanum lycopersicum.

ABSTRACT:

Background

Guard cell protoplasts (GCPs) isolated from various plants have proven to be especially useful for studies of signal transduction pathways and plant development. But it is not easy to isolate highly purified preparations of large numbers of GCPs from plants. In this research, our focus is on a method to isolate large numbers of guard cells from tomato leaves. The protocols described yield millions of highly purified, viable GCPs, which are also suitable for studies on guard cell physiology.

Results

We developed an efficient method for isolating GCPs from epidermal fragments of tomato leaves. The protocol requires a two-step digestion to isolate high-quality tomato GCPs. In this procedure, cellulysin (in method L) was replaced by cellulose "Onozuka" RS (in method S) in the first digestion step, which indicated that cellulase RS was more effective than cellulysin. Method S dramatically shortened the time required for obtaining high yields and high-quality GCPs. Moreover, according to the GCP yields, hydroponic plants were more effective than substrate-cultured plants.

Conclusions

In this paper, protocols for large-scale preparation of GCPs and mesophyll cell protoplasts were described, followed by some success examples of their use in biochemical and molecular approaches such as reverse-transcription polymerase chain reaction, real-time polymerase chain reaction and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The method was proved to be a more efficient GCP-isolating method, capable of providing high yields with better quality in less time.

SUBMITTER: Yao X

PROVIDER: S-EPMC5866509 | biostudies-literature | 2018

REPOSITORIES: biostudies-literature

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Publications

Preparation and applications of guard cell protoplasts from the leaf epidermis of <i>Solanum lycopersicum</i>.

Yao Xuehui X Zhao Wenchao W Yang Rui R Wang Jianli J Zhao Fukuan F Wang Shaohui S

Plant methods 20180324

<h4>Background</h4>Guard cell protoplasts (GCPs) isolated from various plants have proven to be especially useful for studies of signal transduction pathways and plant development. But it is not easy to isolate highly purified preparations of large numbers of GCPs from plants. In this research, our focus is on a method to isolate large numbers of guard cells from tomato leaves. The protocols described yield millions of highly purified, viable GCPs, which are also suitable for studies on guard ce ...[more]

PMID: 29593827

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Project description:Recently, a recessive Arabidopsis thaliana mutant with abundant stromules in leaf epidermal pavement cells was visually screened and isolated. The gene responsible for this mutant phenotype was identified as PARC6, a chloroplast division site regulator gene. The mutant allele parc6-5 carried two point mutations (G62R and W700stop) at the N- and C-terminal ends of the coding sequence, respectively. Here, we further characterized parc6-5 and other parc6 mutant alleles, and showed that PARC6 plays a critical role in plastid morphogenesis in all cell types of the leaf epidermis: pavement cells, trichome cells, and guard cells. Transient expression of PARC6 transit peptide (TP) fused to the green fluorescent protein (GFP) in plant cells showed that the G62R mutation has no or little effect on the TP activity of the PARC6 N-terminal region. Then, plastid morphology was microscopically analyzed in the leaf epidermis of wild-type (WT) and parc6 mutants (parc6-1, parc6-3, parc6-4 and parc6-5) with the aid of stroma-targeted fluorescent proteins. In parc6 pavement cells, plastids often assumed aberrant grape-like morphology, similar to those in severe plastid division mutants, atminE1, and arc6. In parc6 trichome cells, plastids exhibited extreme grape-like aggregations, without the production of giant plastids (>6 µm diameter), as a general phenotype. In parc6 guard cells, plastids exhibited a variety of abnormal phenotypes, including reduced number, enlarged size, and activated stromules, similar to those in atminE1 and arc6 guard cells. Nevertheless, unlike atminE1 and arc6, parc6 exhibited a low number of mini-chloroplasts (< 2 µm diameter) and rarely produced chloroplast-deficient guard cells. Importantly, unlike parc6, the chloroplast division site mutant arc11 exhibited WT-like plastid phenotypes in trichome and guard cells. Finally, observation of parc6 complementation lines expressing a functional PARC6-GFP protein indicated that PARC6-GFP formed a ring-like structure in both constricting and non-constricting chloroplasts, and that PARC6 dynamically changes its configuration during the process of chloroplast division.

Project description:Chloroplasts, or photosynthetic plastids, multiply by binary fission, forming a homogeneous population in plant cells. In Arabidopsis thaliana, the division apparatus (or division ring) of mesophyll chloroplasts includes an inner envelope transmembrane protein ARC6, a cytoplasmic dynamin-related protein ARC5 (DRP5B), and members of the FtsZ1 and FtsZ2 families of proteins, which co-assemble in the stromal mid-plastid division ring (FtsZ ring). FtsZ ring placement is controlled by several proteins, including a stromal factor MinE (AtMinE1). During leaf mesophyll development, ARC6 and AtMinE1 are necessary for FtsZ ring formation and thus plastid division initiation, while ARC5 is essential for a later stage of plastid division. Here, we examined plastid morphology in leaf epidermal pavement cells (PCs) and stomatal guard cells (GCs) in the arc5 and arc6 mutants using stroma-targeted fluorescent proteins. The arc5 PC plastids were generally a bit larger than those of the wild type, but most had normal shapes and were division-competent, unlike mutant mesophyll chloroplasts. The arc6 PC plastids were heterogeneous in size and shape, including the formation of giant and mini-plastids, plastids with highly developed stromules, and grape-like plastid clusters, which varied on a cell-by-cell basis. Moreover, unique plastid phenotypes for stomatal GCs were observed in both mutants. The arc5 GCs rarely lacked chlorophyll-bearing plastids (chloroplasts), while they accumulated minute chlorophyll-less plastids, whereas most GCs developed wild type-like chloroplasts. The arc6 GCs produced large chloroplasts and/or chlorophyll-less plastids, as previously observed, but unexpectedly, their chloroplasts/plastids exhibited marked morphological variations. We quantitatively analyzed plastid morphology and partitioning in paired GCs from wild-type, arc5, arc6, and atminE1 plants. Collectively, our results support the notion that ARC5 is dispensable in the process of equal division of epidermal plastids, and indicate that dysfunctions in ARC5 and ARC6 differentially affect plastid replication among mesophyll cells, PCs, and GCs within a single leaf.