Nitric Oxide-Mediated Maize Root Apex Responses to Nitrate are Regulated by Auxin and Strigolactones

Abstract

Nitrate (NO3 (-)) is a key element for crop production but its levels in agricultural soils are limited. Plants have developed mechanisms to cope with these NO3 (-) fluctuations based on sensing nitrate at the root apex. Particularly, the transition zone (TZ) of root apex has been suggested as a signaling-response zone. This study dissects cellular and molecular mechanisms underlying NO3 (-) resupply effects on primary root (PR) growth in maize, confirming nitric oxide (NO) as a putative modulator. Nitrate restoration induced PR elongation within the first 2 h, corresponding to a stimulation of cell elongation at the basal border of the TZ. Xyloglucans (XGs) immunolocalization together with Brefeldin A applications demonstrated that nitrate resupply induces XG accumulation. This effect was blocked by cPTIO (NO scavenger). Transcriptional analysis of ZmXET1 confirmed the stimulatory effect of nitrate on XGs accumulation in cells of the TZ. Immunolocalization analyses revealed a positive effect of nitrate resupply on auxin and PIN1 accumulation, but a transcriptional regulation of auxin biosynthesis/transport/signaling genes was excluded. Short-term nitrate treatment repressed the transcription of genes involved in strigolactones (SLs) biosynthesis and transport, mainly in the TZ. Enhancement of carotenoid cleavage dioxygenases (CCDs) transcription in presence of cPTIO indicated endogenous NO as a negative modulator of CCDs activity. Finally, treatment with the SLs-biosynthesis inhibitor (TIS108) restored the root growth in the nitrate-starved seedlings. Present report suggests that the NO-mediated root apex responses to nitrate are accomplished in cells of the TZ via integrative actions of auxin, NO and SLs.

Keywords: auxin; nitrate; nitric oxide; root; strigolactones; transition zone.

FIGURE 1

Nitrate availability modulates Zea mays L. primary root (PR) growth. Two days maize seedlings were grown for 24 h in -NO₃^- solution and then transferred to + NO₃^-(A,C) or – NO₃^-(A,B) medium (T₀). PR length was measured at T₀ and after 2, 6, 24, and 48 h of nitrate depletion/provision. The time course of Δ length respect to the T₀ was calculated (A). Data are means ± SE of three biological replicates. Confocal images (derived from XG labeling) were analyzed to determine the cortical cell number in the transition zone (TZ) of each plant after 2 h of NO₃^- supply (C) depletion (B). (B,C) were derived from XG labeling pictures. Histograms summarizing quantification of cortical cell number (D) and calculation of the average cortex cell length (E) in the cortex of maize root TZ. Results are presented as mean ± SE from three experiments (n = 5–10). Bar = 100 μm. Asterisk indicates significant differences, P < 0.01, based on ANOVA.

FIGURE 2

Immunolocalization of xyloglucans (green staining) in cells of root TZ. The blue-emitting fluorescence is due to DAPI (4′,6-diamidino-2-phenylindole) compound which was used to better characterized nuclear staining. (B) Nitrate treatment resulted in a very abundant accumulation of XGs, especially in cross walls, in comparison with the negative control (A) and + NO₃^- roots treated with cPTIO (C). (D–F) In BFA-treated cells, almost all XGs internalized into BFA compartments. (D) Roots grown in a nitrate-depleted solution, or (E) nitrate-resupply solution, or (F) nitrate-resupply and cPTIO solution. Bars: for (A,B,D) 18 μm; for (F) 20 μm; for (E) 22 μm.

FIGURE 3

Real-time quantitative PCR analysis of mRNA encoding ZmXET1 (GRMZM2G026980). (A) Real time validation and investigation of ZmXET1 in the four root portions (M, meristem; TZ, transition zone; EZ, elongation zone; MZ, mature zone). Data are expressed as ratio of +N/-N relative expression values. Quantitative real-time PCR was performed in triplicate (three biological replicates). Asterisk indicates significant differences between the treatments, P < 0.01, based on ANOVA. (B) RNA-Seq RPKM values in maize root TZ of 2 days seedlings grown for 24 h in a NO₃^- depleted solution and then moved to a NO₃^- supplied (+N) or deprived (-N) solution for 2 h.

FIGURE 4

Nitrate does not affect accumulation of auxin related transcripts in root TZ. (A) RNA-Seq analysis of transcript levels of transcripts involved in auxin biosynthesis, signaling and transport in the roots of 2-day-old plants grown in a nitrate-depleted solution for 24 h and treated without (-N) or with (+N) 1 mM NO₃^- for 2 h. Colors indicate the range of each gene expression, with least expression shown in black and highest expression shown in red. Transcripts abundance represented by the heat map are the average of transcript abundance values from three independent. Data shown are expressed as log₁₀ of RPKM values of the RNA-seq analysis (Trevisan et al., 2015) and are means of two independent experiments. (B) Quantitative RT-PCR validation of RNA-seq expression profiling of six PINs transcripts (ZmPIN1a, ZmPIN1b, ZmPIN1c, ZmPIN2, ZmPIN5c, ZmPIN9) in various root portions. The levels of PINs transcripts were measured in total RNAs from: meristem-enriched zone (<3 mm from the root tip); TZ-enriched portion (the next 0.8 cm); elongation zone-enriched portion (the next 0.8 cm); and maturation zone (the residual portion). Expression levels are expressed as +N/-N fold change in the four zones of nitrate-supplied seedlings (+N) relative to nitrate deprived seedlings (-N).

FIGURE 5

Immunolocalization of auxin (A,B) and PIN1 (C,D) in cells of maize root TZ. (A) The localization of auxin (IAA) in -NO₃^- roots showed that a prominent IAA signal was visible at the cytoplasm. (B) Exposure of root apices to NO₃^- resulted in a strong immunofluorescence at the cross walls (end-poles). (C) A prominent PIN1 signal was scored within nuclei in -NO₃^- root cells. (D) In the NO₃^- treated roots, PIN1 labeling within nuclei slightly vanished while end-poles was clearly enriched with PIN1. Bar in (C): for (A,C) 20 μm; for (D) 18 μm; for (B) 16 μm.

FIGURE 6

In situ mRNA hybridization of maize root with DIG-labeled antisense probes of GRMZM2G145008. All images represent longitudinal sections of B73 inbred line root apexes of 2 days maize seedlings grown for 24 h in a NO₃^- depleted solution and then moved to a NO₃^- supplied solution. The hybridization signal is represented by the red-purple staining. The figure shows sections hybridized with antisense probes (A–F) and with sense probes (G) (negative controls). Longitudinal sections through a primary maize root hybridized with GRMZM2G145008 antisense transcript showing the hybridization signal in root TZ (A,B,E), meristem (C–F), elongation zone (E). (C) Portrays a magnification of the root cap and (D) represents a higher magnification of (C) showing the purple staining in the cells of the root meristem. Bars, 200 μm.

FIGURE 7

Nitrate modulates SL-related gene expression. Expression patterns of strigolactone biosynthesis, signaling and transport genes in maize root zones (M, meristem; TZ, transition zone; EZ, elongation zone; MZ, mature zone) expressed in response to the short term nitrate provision. Relative mRNA levels were normalized for individual genes relative to MEP (Manoli et al., 2012). Quantitative real-time PCR was performed in triplicate (three biological replicates) and mean values are shown as ratio (+NO₃^-/-NO₃^-).

FIGURE 8

Nitric oxide affects SL-related gene expression. Expression patterns of strigolactone biosynthesis, signaling and transport genes in maize root TZ expressed in response to the short term nitrate provision (+N), and to nitrate in combination to cPTIO (+N/+cPTIO) (A). Expression patterns of strigolactone biosynthesis and transport genes in maize root TZ expressed in response to the short term nitrate provision (+N), and to nitrate starvation in combination to SNP (-N/+SNP) (B). For each gene, the expression level in the control plants (-N) was equal to 1 (red lines).

FIGURE 9

Strigolactones and nitrate modulate Zea mays L. PR growth. Two days maize seedlings were grown for 24 h in -NO₃^- solution and then transferred to +NO₃^- (gray bar), – NO₃^- (white bar) or – NO₃^- in combination to TIS108 (black bar). PR length was collected at T₀ and after 2 h of nitrate depletion/provision. The time course of Δ length respect to the T₀ was calculated. Data are means ± SE of three biological replicates. Asterisk indicates significant differences between the treatments, P < 0.01, based on ANOVA.

FIGURE 10

Hypothetical model of how nitrate signaling might control PR growth, through triggering TZ enlargement. 2 h nitrate provision would produce a NO boost which inhibits SL biosynthesis, leading to an auxin (IAA) and PIN1 re-distribution and to a reduction of cell division in favor of cell growth, also affecting xyloglucans deposition and cytoskeleton.