植物科学

Cyanobacteria are Gram-negative oxygen-producing photosynthetic bacteria that are useful in the pharmaceutical and biofuel industries. Monitoring of oxidative stress under fluctuating environmental conditions is important for determining the fitness, survival, and growth of cyanobacteria in the laboratory as well as in large scale cultivation systems. Here, we provide a protocol developed using unicellular Synechococcus elongatus PCC 7942 and filamentous Fremyella diplosiphon BK14 cyanobacteria for high-throughput oxidative stress measurement by 2′,7′-dichlorodihydrofluorescein-diacetate (DCFH-DA) and flow cytometry (FCM). We also provide details for the optimization of cell number, dye concentration, and FCM parameters for each organism before it can be utilized to quantify reactive oxygen species (ROS). FCM-based method can be used to measure ROS in a large population of cyanobacterial cells in a high-throughput manner.

Graphical abstract:

The brown alga Ectocarpus has a haploid-diploid life cycle that involves alternation between two multicellular generations, the sporophyte and the gametophyte. Life cycle generation is not determined by ploidy but by a genetic system that includes two different three amino acid loop extension homeodomain transcription factors called OUROBOROS and SAMSARA. In addition, sporophytes have been shown to secrete a diffusible factor into the medium that can induce gametophyte initial cells to switch from the gametophyte to the sporophyte developmental program. The protocol presented here describes how to produce sporophyte-conditioned medium containing the diffusible sporophyte-inducing factor and how to assay for activity of the factor using a meio-spore-based bioassay. The protocol, which describes how several steps of these procedures can be optimised, will represent a useful tool for future work aimed at characterising the diffusible factor and investigating its mode of action.

The ability to achieve nuclear or chloroplast transformation in plants has been a long standing goal, especially in microalgae research. Over past years there has been only little success, but transient and stable nuclear transformation has been achieved in multiple species. Our newly developed method allows for relatively simple transformation of Cyanidioschizon merolae in both nuclear and chloroplast genome by means of homologous recombination between the genome and a transformation vector. The use of chloramphenicol resistance gene as the selectable marker allows for plate-based efficient selection of mutant colonies. Overall, the method allows the generation of mutant strains within 6 months.

The unicellular red alga Cyanidioschyzon merolae has been used as a eukaryotic photosynthetic model for various basic and applied studies. Although the nuclear genome of C. merolae can be modified by homologous recombination with exogenously introduced DNA, it has been difficult to modify multiple chromosome loci within the same strain because of the limited number of available positive selection markers. Recently, we reported a modified URA5.3 gene cassette (URA5.3T), which can be used repeatedly for nuclear genome transformation using the pMKT plasmid vectors for epitope tagging (3x FLAG- or 3x Myc-) of nuclear-encoded proteins. In addition, these plasmid vectors can also be used to knock out multiple genes one by one. This report describes the construction of DNA fragments for transformation and the detailed transformation procedure.

The unicellular red alga Cyanidioschyzon merolae has been used as a model photosynthetic eukaryote for various basic and applied studies, and several of these molecular genetics techniques have been reported. However, there are still improvements to be made concerning the plating method. The conventional plating method often generates diffuse colonies and single colonies cannot be easily isolated. To overcome these problems, we established a novel plating method for C. merolae, making use of melted cornstarch as the use of top agar plating in bacterial genetics. This method improved the formation of defined colonies in at least 4-fold higher efficiency than the conventional method, and made the handling procedure much easier than the previous method.

This is a protocol for quantitative determination of storage and total carbohydrates in algae and cyanobacteria. The protocol is simple, fast and sensitive and it requires only few standard chemicals. Great advantage of this protocol is that both storage and total saccharides can be determined in the cellular pellets that were already used for chlorophyll and carotenoids quantification. Since it is recommended to perform the pigments measurement in triplicates, each pigment analysis can generate samples for both total saccharide and glycogen/starch content quantification.

The protocol was applied for quantification of both storage and total carbohydrates in cyanobacteria Synechocystis sp. PCC 6803, Cyanothece sp. ATCC 51142 and Cyanobacterium sp. IPPAS B-1200. It was also applied for estimation of storage polysaccharides in Galdieria (IPPAS P-500, IPPAS P-507, IPPAS P-508, IPPAS P-513), Cyanidium caldarium IPPAS P-510, in green algae Chlorella sp. IPPAS C-1 and C-1210, Parachlorella kessleri IPPAS C-9, Nannochloris sp. C-1509, Coelastrella sp. IPPAS H-626, Haematococcus sp. IPPAS H-629 and H-239, and in Eustigmatos sp. IPPAS H-242 and IPPAS C-70.

Genome editing in diatoms has recently been established for the model species Phaeodactylum tricornutum and Thalassiosira pseudonana. The present protocol, although developed for T. pseudonana, can be modified to edit any diatom genome as we utilize the flexible, modular Golden Gate cloning system. The main steps include how to design a construct using Golden Gate cloning for targeting two sites, allowing a precise deletion to be introduced into the target gene. The transformation protocol is explained, as are the methods for screening using band shift assay and/or restriction site loss.

We analyzed the reactive oxygen species (ROS) accumulation in the colony-forming green microalga Botryococcus braunii in response to several stress inducers such as NaCl, NaHCO₃, salicylic acid (SA), methyl jasmonate, and acetic acid. A staining assay using the fluorescent dye CellROX Green was used. CellROX Green is a fluorogenic probe used for measuring oxidative stress in live cells. The dye is weakly fluorescent inside cells in a reduced state but exhibits bright green photostable fluorescence upon oxidation by ROS and subsequent binding to DNA. The large amount of liquid hydrocarbons produced and excreted by B. braunii, creates a highly hydrophobic extracellular environment that makes difficult to study short times defense responses on this microalga. The procedure developed here allowed us to detect ROS in this microalga even within a short period of time (in minutes) after treatment of cells with different stress inducers.

Glutathione is an important molecule involved in the primary and secondary metabolism of all organisms. The Glutathione redox status is an indicator of the cellular redox state. Therefore, it is important to have precise methods on hand to determine the glutathione redox status in the cell. In this protocol, we describe an improved spectrophotometric method to estimate the content of reduced (GSH) and oxidized (GSSG) forms of glutathione in the extremophilic microalga Galdieria phlegrea.

Cryopreservation is commonly used for storing viable cells, tissues, organs or organisms at ultralow temperatures, and usually involves immersion in liquid nitrogen at -196 °C. Here we provide a detailed cryopreservation protocol for C. reinhardtii based on Crutchfield’s work (Crutchfield et al., 1999), with minor changes (Yang and Li, 2016). In this study, we compared the cryoprotection effect of two common cryopreservation agents (CPAs), methanol and DMSO. Furthermore, the two-step cryopreservation process was divided into five stages to study the factors affecting the survival rate at each stage. We found that the use of methanol as the CPA, combined with the cooling process outlined here (cooling from 25 °C to -55 °C at a rate of 1 °C/min), were indispensable for cell survival after cryopreservation. The thawing process described here (thawing at 35 °C for 5 min) was also important for increasing the survival rate.