Materials and reagents

Biological materials

Chlamydomonas reinhardtii represents a valuable research organism with a sequenced genome, simple life cycle, and ability to grow in multiple metabolic states. It is a model organism for studies in areas like flagella structure, chloroplast biogenesis, photosynthesis, and cell–cell recognition. Its cell wall is composed of glycoproteins and polysaccharides in a multi-layered structure. Strain CC125 represents a popular wild-type (WT) alga for the species; strain cw15 [6] is a cell wall–deficient mutant that facilitates genetic manipulation and cellular process studies; and sta6 is a mutant strain deficient in starch accumulation, which is very useful for studies on carbon partitioning and lipid accumulation [7].

Scenedesmus obliquus and Chlorella sorokiniana are thoroughly used for biotechnological applications such as the production of biofuels and food/feed ingredients and wastewater treatment. They show very robust and prolific growth in cultivation facilities. In contrast to C. reinhardtii, they currently offer limited possibilities for genetic modification and reduced availability of mutant strains. While the S. obliquus cell wall comprises a resistant outer layer of algaenan and an inner layer of cellulosic material, the C. sorokiniana cell wall presents a thin trilaminate layer of hemicellulose and a glucosamine-based residue and produces mucilage [8].

A. thaliana, a model organism in plant biology, boasts features like a small genome, rapid life cycle, and easy genetic modification. Its cell wall, composed of cellulose, hemicellulose, and pectin, provides strength, flexibility, and porosity. Some cells even develop a secondary cell wall with lignin for rigidity. Structural proteins, enzymes, and glycoproteins contribute to cell signaling and growth. Remodeling occurs during growth and in response to environmental stress [9].

All of these organisms produce starch as a carbon and energy reserve, and sugars with different physiological roles.

Organisms' cultivation conditions

The microalgal strains C. reinhardtii CC125 (WT), cw15 (cell wall–deficient), and sta6 (starchless mutant) were kindly provided by C. Benning (Michigan State University), and strains C. sorokiniana RP and S. obliquus C1S were isolated [10] and characterized [11] by our research group. These algal strains belong to the INBIOTEC culture collection.

The microalgal strains were cultivated and induced for carbohydrate accumulation as previously described in Bader et al. [12] under nitrogen-deficient conditions.

The A. thaliana used in this study was a wild type of Columbia (Col-O) stored in our laboratory. Seeds were sown in pots and grown under 16/8 h light/dark (light 120 μmol photons m^-2·s^-1) at 23 °C in a growth room. Fourteen-day-old A. thaliana plants were treated with 150 mM NaCl for 14 days before leaves were collected for carbohydrate analysis.

Reagents

1. Distilled water (dH₂O)

2. Double-distilled water (ddH₂O) for anthrone solution

3. Ethanol 96% (C₂H₆O) (e.g., Alco Protect, Porta, Argentina)

4. Perchloric acid 60% (HClO₄) (Fluka, catalog number: 77232)

5. Sulfuric acid 96%–98% (H₂SO₄) (Biopack, Brand, catalog number: 0303A0036)

6. Glucose analytical grade (C₆H₁₂O₆) (Glc) (Sigma-Aldrich, catalog number: G5767)

7. Anthrone (C₁₄H₁₀O) [Fluka, catalog number: A 866 (0740)]

8. Thiourea (CH₄N₂S) (Sigma-Aldrich, catalog number: T7875-500G)

   Note: Both sulfuric acid and perchloric acid are hazardous to touch and inhalation; therefore, it is necessary to work with caution and use personal protective equipment (gloves, lab coat, closed-toe shoes, and safety goggles).

Solutions

For carbohydrate quantification, the anthrone method was used, which has been widely validated and allows for the quantification of a broad range of carbohydrates, hexoses, and aldopentoses [13]. In the presence of concentrated sulfuric acid, carbohydrates are dehydrated into furfurals (or hydroxymethylfurfurals), which condense with anthrone (10-keto-9,10-dihydroxyanthracene) (Figure 1) to produce a blue-green complex. The intensity of the color is quantified by absorbance at 620 nm.

Figure 1. Reaction of the anthrone assay. Modified from Gerwig [14]. The extinction coefficient of the complex between 5-hydroxy methyl furfural and anthrone at 620 nm is 76,000 M^-1·cm^-1.

1. 67% sulfuric acid solution (see Recipes)

2. Anthrone solution (72% sulfuric acid final concentration) (see Recipes)

3. 10 mM glucose solution (see Recipes)

1. 67% sulfuric acid solution (10 mL)

   Conduct the process under a fume hood and using personal protective equipment (gloves, lab coat, closed-toe shoes, and safety goggles). In a glass container, pour 3.3 mL of ddH₂O. Place the container on ice and then slowly add 6.7 mL of sulfuric acid to prevent acid splashes upon contact with water.




2. Anthrone solution (100 mL)

   Conduct the process under a fume hood and using personal protective equipment (gloves, lab coat, closed-toe shoes, and safety goggles). In a glass container, pour 28 mL of ddH₂O. Place the container on ice and then slowly add 35 mL of sulfuric acid to prevent acid splashes upon contact with water, while stirring with a magnetic stir bar. Add 50 mg of anthrone and 1 g of thiourea. Complete with 37 mL of sulfuric acid. Leave to stir for 16 h in darkness.




3. 10 mM Glc solution (10 mL)

   Prepare by adding 18 mg of Glc to 10 mL of ddH₂O.

Equipment

1. General lab supplies: microtubes, pipettes, tips, tube racks, etc.

2. Block heater (Thermolyne, model: Type 17600 Dri-Bath)

3. Bench-top vortex (Labnet, model: VX100)

4. Benchtop centrifuge for 2 mL tubes at room temperature and 4 °C (Thermo Scientific, model: Sorvall Legend Micro 21R)

5. Fume Hood (Esco, model: EFA-4UDRVW-8)

6. 5 mL glass test tubes

7. Analytical balance (Sartorius, model: Entris2241-1S)

8. UV/VIS spectrometer (Shimadzu Corp., serial number A114548, 05789)

9. 1 cm Pathlength quartz cuvettes; disposable or glass cuvettes can also be used

10. Gas mask (3M, model: 6899B, filters 3M 6003) for measuring samples with anthrone

11. Thermostatic water bath for laboratory (Daglef Patz, model: Hor, 18.3867)

12. Magnetic stirring bar and magnetic stirrer (Thermolyne, model: Nuova II)

Procedure