Nitric oxide causes root apical meristem defects and growth inhibition while reducing PIN-FORMED 1 (PIN1)-dependent acropetal auxin transport

Abstract

Nitric oxide (NO) is considered a key regulator of plant developmental processes and defense, although the mechanism and direct targets of NO action remain largely unknown. We used phenotypic, cellular, and genetic analyses in Arabidopsis thaliana to explore the role of NO in regulating primary root growth and auxin transport. Treatment with the NO donors S-nitroso-N-acetylpenicillamine, sodium nitroprusside, and S-nitrosoglutathione reduces cell division, affecting the distribution of mitotic cells and meristem size by reducing cell size and number compared with NO depletion by 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO). Interestingly, genetic backgrounds in which the endogenous NO levels are enhanced [chlorophyll a/b binding protein underexpressed 1/NO overproducer 1 (cue1/nox1) mirror this response, together with an increased cell differentiation phenotype. Because of the importance of auxin distribution in regulating primary root growth, we analyzed auxin-dependent response after altering NO levels. Both elevated NO supply and the NO-overproducing Arabidopsis mutant cue1/nox1 exhibit reduced expression of the auxin reporter markers DR5pro:GUS/GFP. These effects were accompanied by a reduction in auxin transport in primary roots. NO application and the cue1/nox1 mutation caused decreased PIN-FORMED 1 (PIN1)-GFP fluorescence in a proteasome-independent manner. Remarkably, the cue1/nox1-mutant root phenotypes resemble those of pin1 mutants. The use of both chemical treatments and mutants with altered NO levels demonstrates that high levels of NO reduce auxin transport and response by a PIN1-dependent mechanism, and root meristem activity is reduced concomitantly.

Fig. 1.

Effect of NO in the regulation of Arabidopsis primary root growth. (A) (Upper) Photograph showing the length of the primary root of WT (Col-0) seedlings grown for 7 d on Murashige and Skoog (MS) agar plates that were untreated (Control) or supplemented with 10 μM, 100 μM, 200 μM, 500 μM, or 1 mM of the NO donor SNAP or with 1 mM SNAP plus 1 mM of the NO scavenger cPTIO. (Lower) Measurements were obtained 4 d after the treatment of 3-d-old seedlings. Values represent the mean of 30 measurements ± SD. Asterisks indicate significant differences compared with the untreated control (P < 0.05) (a) and with 1 mM SNAP (P < 0.05) (b). (B) Inhibition of primary root growth after delivery of NO gas (300 ppm), by treatment with the NO donors SNP (100 mM), SNAP (1 mM), or GSNO (1 mM) and in mutants with high levels of endogenous NO (cue1/nox1). Measurements were taken 4 d after the treatment of 3-d-old seedlings (n = 25). (C) Detection of endogenous NO production using DAF-2DA. Plants were grown for 2 d (Upper and Lower Left) or 7 d (Right) on agar plates and then subjected to DAF-2DA incubation. (D) (Upper) Detection of endogenous NO production using DAF-2DA in WT (Col-0) and cue1/nox1 seedlings in control conditions and after NO scavenging by cPTIO. (Lower) Measurement of NO levels in WT (Col-0) seedlings and the cue1/nox1 mutant. Asterisk indicates a statistically significant difference from the WT.

Fig. 2.

Effect of NO on the Arabidopsis root meristem. (A) Confocal images of roots from seedlings grown for 5 d on unsupplemented MS agar plates (Control), cue1-mutant seedlings, or WT seedlings supplemented with 100 μM of the NO donor SNP, 1 mM of the NO scavenger cPTIO, 1 mM SNAP, and 1 mM SNAP plus 1 mM cPTIO. Vertical lines indicate apical (AM) and basal regions (BM) of the primary meristem region (PM). Cells 1 and 15 from the QC are highlighted in green. Note that meristematic cells are enlarged in the presence of NO and that the start of net elongation is further from the QC in cPTIO-treated seedlings than in untreated controls. (B and C) Cell sizes in the cortical layer of the root. (B) Average cell size in the cortical cell layer (cells 1–40 from QC) of untreated WT seedlings, WT seedlings treated with SNP or cPTIO, and untreated cue1-mutant seedlings. (C) Average cell size in the cortical layer (cells 1–40 from QC) of WT (Col-0) seedlings grown as described above and seedlings that were untreated (control) or treated with SNAP or with SNAP plus cPTIO. Measurements were taken 2 d after the treatment of 3-d-old seedlings. (D) Average cortical cell sizes are shown for cells 1–10 and 11–20 (counted from the QC) of the seedlings in A. (E) Size of root meristem in WT (Col-0) seedlings grown for 5 d on unsupplemented MS agar plates (Control) or on medium supplemented with 1 mM of the NO scavenger cPTIO, 100 μM of the NO donor SNP, 1 mM of the NO donor SNAP, or 1 mM SNAP plus 1 mM of the NO scavenger cPTIO. A minimum of five roots per treatment was analyzed. Asterisks indicate significant differences compared with untreated control (P < 0.05). (F) Quantification of the distance between the root tip and the first root hair formed in WT (Col-0) and cue1-mutant seedlings. A minimum of 8–10 roots per genotype was analyzed. Asterisks indicate significant differences compared with untreated WT (P < 0.05). (G) Representative images of the root tip and the first root hair formed in WT Col-0) and cue1-mutant seedlings. (H and I) Representative images of the root meristem of 7-d-old WT (Col-0) and cue1-mutant seedlings stained with modified pseudo-Schiff propidium iodide (mPS-PI) (Upper) or Lugol's solution (Lower) highlighting vacuolization (H) and QC/CSC disorganization and starch accumulation (I). Red and green arrowheads indicate QCs and CSCs, respectively.

Fig. 3.

The effect of NO on cell-division activity was monitored using the CycB1;1_pro:GUS-DB reporter marking cells in the G2 stage of the cell cycle. (A) Five-day-old untreated seedlings (Control) and seedlings treated with 1 mM cPTIO, 100 μM SNP, 1 mM SNAP, 100 μM SNP plus 1 mM cPTIO, or 1 mM SNAP plus 1 mM cPTIO are shown. Pictures were taken 2 d after the treatment of 3-d-old seedlings. (B) Expression level and localization of CycB1;1_pro:GUS-DB in the cue1/nox1 background.

Fig. 4.

Pattern of DR5_pro:GUS/GFP expression in root tip tissue. (A) Confocal images of the DR5_pro:GFP reporter line in untreated (Control) seedlings and in seedlings treated with 1 mM cPTIO or with 100 μM SNP for 3, 24, and 48 h. (B) Representative close-up views of GUS staining in DR5_pro:GUS 5-d-old untreated (Control) seedlings and in seedlings treated with 100 μM SNP, 1 mM cPTIO, or 100 μM SNP plus 1 mM cPTIO. Seedlings were grown on MS plates for 3 d and then were subjected to donor/scavenger treatments for 2 d. (C) Confocal images of the DR5_pro:GFP reporter line in the cue1 background and after treatment with 1 μM NPA. (D) Acropetal auxin transport measured in roots of WT (Col-0) and cue1-mutant 7-d-old seedlings. A minimum of 15 roots per genotype was analyzed. Asterisk indicates significant difference compared with untreated WT seedlings (P < 0.05).

Fig. 5.

Disappearance of PIN1 after NO treatment and comparison of cue1 and pin1 root phenotypes. (A) Distribution of PIN1_pro:GFP-PIN1 protein is shown in untreated control plants (C), in plants treated with the NO scavenger cPTIO (1 mM; 8 h), and in plants treated with the NO donors SNP (100 μM; 3, 8, and 24 h), SNAP (1 mM; 24 h), or GSNO (1 mM; 24 h), with or without the proteasome inhibitor MG132 (100 μM; 24 h). Root tissues were stained with propidium iodide. (B) Immunoblot analysis with anti-GFP antiserum of in vivo levels of PIN1 protein in root extracts of PIN1_pro:GFP-PIN1 seedlings, in the absence or presence of NO donors and scavengers together with MG132. Actin protein levels also were determined as a loading control. (C) Confocal images of the PIN1_pro:GFP-PIN1 line in the cue1 background. (D) Confocal images after mPS-PI staining. (E and F) Root meristem size (E) and primary root length (n = 25) (F) of roots from WT (Col-0) and cue1- and pin1^−/−-mutant seedlings grown for 7 d on MS agar plates. A minimum of 8–10 roots per genotype was analyzed. Asterisks indicate significant differences compared with WT (P < 0.05).