Cell-Type-Specific Length and Cytosolic pH Response of Superficial Cells of Arabidopsis Root to Chronic Salinity

Abstract

Soil salinity negatively affects the growth, development and yield of plants. Acidification of the cytosol in cells of glycophytes was reported under salinity, while various types of plant cells can have a specific reaction under the same conditions. Transgenic Arabidopsis plants expressing the pH sensor Pt-GFP in the cytosol were used in this work for determination of morphometric changes and cytosolic pH changes in the superficial cells of Arabidopsis roots under chronic salinity in vitro. We did not find changes in the length of the root cap cells, while there was a decrease in the length of the differentiation zone under 50, 75 mM NaCl and the size of the epidermal cells of the differentiation zone under 75 mM NaCl. The most significant changes of cytosolic pH to chronic salinity was noted in columella (decrease by 1 pH unit at 75 mM NaCl) and epidermal cells of the differentiation zone (decrease by 0.6 and 0.4 pH units at 50 and 75 mM NaCl, respectively). In developed lateral root cap cells, acidification of cytosol by 0.4 units occurred only under 75 mM NaCl in the medium. In poorly differentiated lateral cells of the root cap, there were no changes in pH under chronic salinity.

Keywords: Arabidopsis thaliana L.; Pt-GFP; differentiation zone; epidermal cells; pH; pH indicator; root; root cap; salinity.

Figure 1

Fluorescence of root cells of Arabidopsis transgenic plant expressing Pt-GFP and Arabidopsis plant ecotype Columbia. (A) Confocal images of root cells. Transmitted light images, fluorescent images (λem 500–550 nm) with excitation at λex 405 nm and λex 488 nm, and images with merged fluorescent channels. Scale bar, 20 μm. (B) Spectra of fluorescence of root cells of Arabidopsis transgenic plant and Arabidopsis plant ecotype Columbia with excitation at λex 405 nm and λex 488 nm.

Figure 2

Analysis of Pt-GFP localization in the epidermal root cells of Arabidopsis transgenic plants expressing Pt-GFP. (A) Cap plasmolysis. 7-day old transgenic plants were incubated with 1 M KNO₃. (B) Plasmalemma was stained with 17 μg/mL CellMask™ Orange Plasma membrane Stain (orange fluorescence). (C) Nucleus was stained with 1 μg/mL DAPI (blue fluorescence). Fluorescence of Pt-GFP was registered at λex 488 nm, λem 505–525 nm; fluorescence of the CellMask ™ Orange, at λex 543 nm, λem 565–615; fluorescence of the DAPI, at λex 405 nm, λem 415–455 nm. Bars, 20 μm. Arrow-end line in LSM images indicates the direction along which the fluorescence profile is estimated.

Figure 3

Organization of superficial cells of Arabidopsis root. (Left) layout of regions of superficial root cells. (Right) LSM-images of regions of superficial root cells. Fluorescent images (λem 505–525 nm) with excitation at λex 405 nm, λex 488 nm and images with merged fluorescent channels are represented. Scale bar, 20 μm. LTC—lateral root cap tip cells; PDLC—pure differentiated lateral root cap cells; DLC -differentiated lateral root cap cells.

Figure 4

The root length of Arabidopsis transgenic plants grown on MS medium with 0 mM, 50 mM and 75 mM NaCl for 7 days (n = 15). Scale bar, 10 mm. Values are mean ± SEM. *, statistically significant difference between treatment options (ANOVA, Tukey’s test, p < 0.05).

Figure 5

The root zone length of Arabidopsis transgenic plants grown on MS medium with NaCl for 7 days (n = 15). LTC—lateral root cap tip cells; PDLC—pure differentiated lateral root cap cells; DLC—differentiated lateral root cap cells. Values are mean ± SEM. *, statistically significant difference between treatment options (ANOVA, Tukey’s test p < 0.05).

Figure 6

The root cells length of Arabidopsis transgenic plants grown on MS medium with NaCl for 7 days (n = 10–55). LTC—lateral root cap tip cells; PDLC—pure differentiated lateral root cap cells; DLC—differentiated lateral root cap cells. Values are mean ± SEM. *, statistically significant difference between treatment options (ANOVA, Tukey’s test, p < 0.05).

Figure 7

In vivo calibration of the Pt-GFP of the various regions of root cap and the epidermal cells of differentiation zone of transgenic Arabidopsis plants (n = 15–80). F488/F405 is the ratio of the fluorescence intensity of the Pt-GFP sensor when excited at wavelengths of 488 nm and 405 nm, with a registration of fluorescence at 505–525 nm. 7-days-old transgenic Arabidopsis plants were incubated in a solution with an appropriate pH value in the presence of the CCCP protonophore. Values are mean ± SEM.

Figure 8

Effect of NaCl in growth medium on cytosolic pH of superficial root cells of transgenic Arabidopsis plants grown on MS medium with NaCl for 7 days. LTC—lateral root cap tip cells; PDLC—pure differentiated lateral root cap cells; DLC—differentiated lateral root cap cells. (A) Pseudocolored ratio image of Pt-GFP after a pixel-by-pixel calculation. The images were obtained by dividing the fluorescence image λem 505–525 nm, λex 488 nm, by the fluorescence image λem 505–525 nm, λex 405 nm, taking the background into account. Bars, 20 μm. (B) Calculated cytosolic pH level (n = 10–110). Values are mean ± SEM. *, statistically significant difference between treatment options (ANOVA, Tukey’s test, p < 0.05).