Amino Acids Supplied through the Autophagy/Endocytosis Pathway Promote Starch Synthesis in Physcomitrella Protonemal Cells

Abstract

The physiological implications of autophagy in plant cells have not been fully elucidated. Therefore, we investigated the consequences of autophagy in the moss Physcomitrella by measuring biochemical parameters (fresh and dry weights; starch, amino acid, carbohydrate, and NH3 content) in wild-type (WT) and autophagy-deficient atg5 Physcomitrella cells. We found higher starch levels and a higher net starch synthesis rate in WT cells than in atg5 cells cultured in a glucose-containing culture medium, whereas net starch degradation was similar in the two strains cultured in a glucose-deficient culture medium. Additionally, the treatment of cells with the autophagy inhibitor 3-methyladenine suppressed starch synthesis. Loading bovine serum albumin into atg5 cells through endocytosis, i.e., supplying proteins to vacuoles in the same way as through autophagy, accelerated starch synthesis, whereas loading glutamine through the plasma membrane had no such effect, suggesting that Physcomitrella cells distinguish between different amino acid supply pathways. After net starch synthesis, NH3 levels increased in WT cells, although the change in total amino acid content did not differ between WT and atg5 cells, indicating that autophagy-produced amino acids are oxidized rapidly. We conclude that autophagy promotes starch synthesis in Physcomitrella by supplying the energy obtained by oxidizing autophagy-produced amino acids.

Keywords: Physcomitrella (Physcomitrium); amino acid; autophagy; endocytosis; starch synthesis.

Figure 1

Growth of wild-type (WT) and atg5 mutant Physcomitrella colonies. WT and atg5 mutant cells of Physcomitrella were transferred onto BCDATG agar medium and cultured under light conditions for 7 d. (A) The same colonies were photographed immediately (0 d), 3, 5, and 7 d after transfer. Scale bar: 2 mm. (B) Fresh weight (FW) of 50 WT and atg5 colonies was measured immediately (0 d) and 7 d after transfer. (C) Dry weight (DW) was also measured, and the DW to FW ratio (DW/FW) was calculated. (B,C) Data are shown as means ± standard deviation (SD) (n = 3, * p < 0.05).

Figure 2

Extracellular sugars and starch in WT and atg5 mutant Physcomitrella cells. (A) WT and atg5 cells were cultured on BCDATG agar medium for 7 d under light conditions. The cell wall components were fractionated into hot water extracted (HW), pectin, hemicellulose, and cellulose fractions, and the carbohydrate content of each fraction was measured. (B) Starch content was measured in 7-d-old WT and atg5 cells cultured on BCDATG or BCDAT agar medium. (A,B) Data are shown as means ± SD (n = 3, *** p < 0.005, * p < 0.05).

Figure 3

Starch synthesis and degradation in WT and atg5 mutant Physcomitrella colonies. (A) Seven-day-old WT and atg5 Physcomitrella colonies cultured on BCDAT agar medium were transferred to BCDATG agar medium and cultured for another 7 d. The starch content of colonies was measured for 7 d. (B) Seven-day-old WT and atg5 Physcomitrella colonies cultured on BCDATG agar medium were transferred to BCDAT agar medium and cultured for another 7 d under light conditions. After transfer, the starch content of the colonies was measured for 7 d. (A,B) Data are shown as means ± SD (n = 3).

Figure 4

Effects of the autophagy inhibitor 3-methyladenine (3-MA) on starch synthesis. (A) WT and atg5 mutant Physcomitrella cells cultured on BCDAT agar medium were transferred to BCDATG liquid medium and cultured for a further 7 d under light conditions, during which their starch content was measured. (B) WT Physcomitrella colonies grown on a BCDAT agar medium were transferred to a BCDATG liquid medium containing 5 mM of 3-MA or water (as a solvent control) and cultured under light conditions. The starch content of colonies was measured 2 d after transfer. (A,B) Data are shown as means ± SD (n = 3, *** p < 0.005).

Figure 5

Effects of bovine serum albumin (BSA) and glutamine (Gln) on starch synthesis in atg5 mutants. Physcomitrella atg5 colonies cultured on a BCDAT agar medium were transferred to BCDATG liquid medium containing 2% (w/v) BSA, 1 mM Gln, or water (as a solvent control) and cultured under light conditions. The starch content of colonies was measured immediately (0 d) and 2 d after transfer. Data are shown as means ± SD (n = 3). Different letters denote significant differences from each other, p < 0.01).

Figure 6

Changes in the amino acid levels in WT and atg5 Physcomitrella cells following their transfer from BCDAT to BCDATG medium. WT and atg5 mutant Physcomitrella cells grown on BCDAT agar medium were transferred to BCDATG liquid medium and grown under light conditions. (A) Amino acids were extracted from WT and atg5 cells immediately (0 d) and 1 d after transfer and measured. (B) Among the 15 amino acids detected and γ-aminobutyric acid (GABA), the 6 amino acids with significantly different levels in WT and atg5 cells immediately (0 d) and 1 d after transfer are presented. (C) Total amino acid levels i.e., the summation of the 16 amino acid levels immediately (0 d) and 1 d after transfer in WT and atg5 cells are shown. (A–C) Data are shown as means ± SD (n = 3, * p < 0.05, ** p < 0.01, *** p < 0.005).

Figure 7

Changes in the ammonia (NH₃) content of WT and atg5 Physcomitrella colonies following their transfer from BCDAT to BCDATG medium. Seven-day-old WT and atg5 Physcomitrella colonies grown on BCDAT agar medium were transferred to BCDATG liquid medium and grown under light conditions. NH₃ content in WT and atg5 mutants was measured immediately (0 d) and 1 d after transfer. Data are shown as means ± SD (n = 3, ** p < 0.01).

Figure 8

Effects of BSA administration on the NH₃ content of atg5 cells. Seven-day-old atg5 Physcomitrella colonies grown on a BCDAT agar medium were transferred to BCDATG liquid medium containing 2% (w/v) BSA or water (as a solvent control) and cultured for 1 d under light conditions. NH₃ ions were measured immediately (0 d) and 1 d after transfer. Data are shown as means ± SD (n = 3, *** p < 0.005).

Figure 9

Changes in the glucose and sucrose levels in WT and atg5 Physcomitrella colonies following their transfer from BCDAT to BCDATG medium. Seven-day-old WT and atg5 Physcomitrella colonies grown on BCDAT agar medium were transferred to BCDATG liquid medium and grown under light conditions. (A) Glucose and (B) sucrose levels in WT and atg5 cells were measured immediately (0 d) and 1 d after transfer. (A,B) Data are shown as means ± SD (n = 3).