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. 2016 Oct 4;172(3):2044–2056. doi: 10.1104/pp.16.01150

Phytoglobins Improve Hypoxic Root Growth by Alleviating Apical Meristem Cell Death1,[OPEN]

Mohamed M Mira 1,2, Robert D Hill 1, Claudio Stasolla 1,*
PMCID: PMC5100795  PMID: 27702845

Phytoglobin expression in hypoxic root apical meristems alleviates programmed cell death by removing NO and moderating ethylene and ROS in the maize meristematic cells.

Abstract

Hypoxic root growth in maize (Zea mays) is influenced by the expression of phytoglobins (ZmPgbs). Relative to the wild type, suppression of ZmPgb1.1 or ZmPgb1.2 inhibits the growth of roots exposed to 4% oxygen, causing structural abnormalities in the root apical meristems. These effects were accompanied by increasing levels of reactive oxygen species (ROS), possibly through the transcriptional induction of four Respiratory Burst Oxidase Homologs. TUNEL-positive nuclei in meristematic cells indicated the involvement of programmed cell death (PCD) in the process. These cells also accumulated nitric oxide and stained heavily for ethylene biosynthetic transcripts. A sharp increase in the expression level of several 1-aminocyclopropane synthase (ZmAcs2, ZmAcs6, and ZmAcs7), 1-aminocyclopropane oxidase (Aco15, Aco20, Aco31, and Aco35), and ethylene-responsive (ZmErf2 and ZmEbf1) genes was observed in hypoxic ZmPgb-suppressing roots, which overproduced ethylene. Inhibiting ROS synthesis with diphenyleneiodonium or ethylene perception with 1-methylcyclopropene suppressed PCD, increased BAX inhibitor-1, an effective attenuator of the death programs in eukaryotes, and restored root growth. Hypoxic roots overexpressing ZmPgbs had the lowest level of ethylene and showed a reduction in ROS staining and TUNEL-positive nuclei in the meristematic cells. These roots retained functional meristems and exhibited the highest growth performance when subjected to hypoxic conditions. Collectively, these results suggest a novel function of Pgbs in protecting root apical meristems from hypoxia-induced PCD through mechanisms initiated by nitric oxide and mediated by ethylene via ROS.

Oxygen deficiency (hypoxia), experienced by plants grown in poorly drained soils or subjected to flooding, impairs plant growth and results in heavy crop losses (Dennis et al., 2000). Submergence or flooding reduces oxygen availability for plant cells, inhibiting the gas exchange required for basic physiological processes (Bailey-Serres and Voesenek, 2008). Both roots and shoots are affected by hypoxia, regardless of whether the plant is submerged or only the root is exposed to the condition. The consequences to shoots of prolonged root hypoxia include reduced photosynthetic rate and stomatal conductance, decreased leaf growth and senescence, wilting of the aboveground organs, and alterations in plant water relations (Mustroph and Albrecht, 2003). Ethylene accumulates rapidly in flooded Rumex palustris root cells (Visser et al., 1996), and in some species, ethylene affects the selective death of cortical cells generating lysogenous aerenchyma (Drew et al., 1979, 2000; Drew, 1997) Precursors of ethylene have been shown to induce changes in Brassica napus growth behavior and root architecture (Patrick et al., 2009). Depending upon the concentration and species, ethylene can either stimulate or inhibit root growth (Konings and Jackson, 1979). Ethylene regulation of programmed cell death (PCD) is not restricted to hypoxia but, rather, is observed in response to many adverse growth conditions (Drew et al., 1979; Feldman, 1984; Abeles et al., 1992; Pitts et al., 1998; Clark et al., 1999; Buer et al., 2003). The execution of PCD in maize (Zea mays) roots under hypoxic conditions is triggered by a rapid increase in ethylene level resulting from the transcriptional induction of 1-aminocyclopropane-1-carboxylate synthase (ACS) and oxidase (ACO; Geisler-Lee et al., 2010) and transduced through the generation of reactive oxygen species (ROS) produced by NADPH oxidase activity (Torres and Dangl, 2005). The progression of these events in maize roots has been shown using 1-methylcyclopropene (1-MCP) as a specific inhibitor of ethylene perception or diphenyleneiodonium (DPI) to inhibit ROS production (Takahashi et al., 2015).

While considerable attention has been paid to the mechanisms underlying PCD during aerenchyma formation, no information is currently available on other death programs occurring in other regions of hypoxic roots, including the root tip. Maize root tips are very sensitive to flooding stress and die after a few hours, compromising survival upon the reestablishment of normoxic conditions (Roberts et al., 1984). The root apical meristem (RAM) harbors stem cells and performs the task of organizing centers for postembryonic morphogenesis (Jiang and Feldman, 2005). These crucial functions are evidenced by its conserved structure. The maize RAM consists of a quiescent center (QC), comprising 800 to 1,200 slowly dividing cells, surrounded by more actively dividing stem cells (Kerk and Feldman, 1995). Genetic or environmental perturbations of RAM function lead to growth inhibition or cessation (Blilou et al., 2005). Recent work identified ethylene as a central regulator of RAM function (Street et al., 2015).

Phytoglobins (Pgbs), previously termed nonsymbiotic hemoglobins (Hill et al., 2016), are heme-containing proteins characterized mainly for their ability to remove nitric oxide (NO) under adverse conditions, including hypoxia (Hill, 2012). Pgbs are induced rapidly in cells grown under limited oxygen (Silva-Cardenas et al., 2003), and experimental changes in their expression level affect the plant response to stress. In Arabidopsis (Arabidopsis thaliana), ectopic expression of one Pgb enhanced survival to low-oxygen conditions (Hunt et al., 2002), while hypoxic alfalfa (Medicago sativa) roots and maize cells overexpressing Pgbs maintained growth and sustained a high-energy status (Dordas et al., 2003a; Igamberdiev and Hill, 2004). In culture, the suppression of Pgbs enhances ethylene synthesis (Manac’h-Little et al., 2005) and induces PCD in maize through ROS production (Huang et al., 2014). These observations, in conjunction with the root tip localization of one maize Pgb (Dordas et al., 2003b; Zhao et al., 2008), are the premises of this work, to determine whether Pgbs exercise a protective role by limiting meristematic cell death in the hypoxic RAM through the regulation of ethylene and ROS.

RESULTS

The Expression of ZmPgbs Affects Hypoxic Root Growth

The root growth of 5-d-old seedlings with altered expression of ZmPbg1.1 or ZmPgb1.2 was compared under normoxic (ambient air) or hypoxic (4% oxygen) conditions. The growth of wild-type hypoxic roots was more than 40% impaired after hypoxic treatment for 24 h, while ZmPgb1.1- and ZmPgb1.2-overexpressing roots [ZmPgb1.1(S) and ZmPgb1.2(S)] showed less than 30% reduction in growth (Fig. 1A). In the lines suppressing either ZmPgb [ZmPgb1.1(A) or ZmPgb1.2(A)], there was substantially reduced root growth of the order of 60% to 80% during the same period, with evidence of abnormalities within the root apices. Structural disorganization of the root tip (Fig. 1A) and the formation of large vacuoles within cells of the QC (Supplemental Fig. S1A), a sign of differentiation, often were observed in roots suppressing either ZmPgb1.1 or ZmPgb1.2.

Figure 1.

Figure 1.

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Root growth behavior and localization of Pgb transcripts in hypoxic roots of maize. A, Root growth and structure of the root apical meristems of wild-type (WT) roots and roots overexpressing (S) or down-regulating (A) ZmPgb1.1 and ZmPgb1.2 after 24 h of 4% oxygen treatment. Numbers on line graphs represent the relative elongation (4% oxygen/ambient air) at each day of treatment. Values are means ± se of three biological replicates each consisting of at least 20 roots. RC, Root cap. *, Significantly different at the same time point (P < 0.05). B, Serial sections used for RNA and protein localization studies. Ct, Cortex; Ep, epidermis; St, stele. C, RNA in situ localization of ZmPgb1.1 and ZmPgb1.2 transcripts in the five serial sections outlined in B of roots exposed to ambient air (Amb) or 4% oxygen. NC, Negative control in which sections were hybridized with sense riboprobes.

The expression of ZmPgbs was measured in segments (0–2, 2–5, 5–10, and 10–20 mm from the tip) of hypoxic wild-type roots. Hypoxia induced ZmPgb1.1 and ZmPgb1.2, especially in proximity to the root tip (segments 0–2 and 2–5 mm), with maximum expression occurring at 12 h (Supplemental Fig. S2). Differences in expression levels between normoxic and hypoxic conditions were attenuated in more mature regions of the root (segments 5–10 and 10–20 mm). To enhance resolution, RNA in situ localization studies of both ZmPgbs were performed on progressive transverse sections along the RAM (Fig. 1B). These sections included the root cap (section I), the QC (section II), domains with initial (section III) and advanced (section IV) regions of cellular differentiation, and mature fully differentiated tissue (section V; Fig. 1B). Hypoxic conditions increased the staining of ZmPgb1.1 and, to a lesser extent, ZmPgb1.2 in the central cells of the root cap (section I; Fig. 1C). Increased expression of ZmPgbs as a result of hypoxia was particularly evident in the QC region (section II) and in tissue undergoing early differentiation (section III). Heavy induction of ZmPgb1.1 also was observed in hypoxic cells at advanced stages of differentiation (section IV). The specificity of the signal was verified using sense riboprobes as a negative control (Fig. 1C). Longitudinal sections of hypoxic roots also displayed evidence of heavy staining for ZmPgbs (Supplemental Fig. S1B).

ZmPgb Regulation of NO, ROS, and PCD in Hypoxic RAM

The different growth behaviors of maize roots with altered expression of ZmPgbs was examined further in light of the following observations: Pgbs scavenge NO (Dordas et al., 2003a), and when Pgb expression is suppressed, NO accumulates, inducing ROS production (Huang et al., 2014) that triggers PCD (Van and Dat, 2006). Altered ZmPgb expression was achieved by the use of maize transgenic lines (Youseff et al., 2016) that constitutively expressed ZmPgb1.1 or ZmPgb1.2 in either the sense (S) or antisense (A) orientation. The relative expression of a particular ZmPgb in normoxic root lines is shown in Supplemental Figure S3B. ZmPgb(S) lines had ZmPgb levels approximately 15- to 20-fold higher than the wild-type line, while Pgb(A) lines had expression levels that were less than 10% of the wild-type line.

Examining the effect of varying Pgb expression, with the exception of the root cap (section I), there was visual evidence of an increase in staining for NO, ROS, and PCD in ZmPgb(A) lines and a decrease in ZmPgb(S) lines relative to the wild type as a result of hypoxia (Fig. 2). Consistent with the evidence of PCD in the sections, staining for transcripts of BAX inhibitor-1 (Bi-1), an attenuator of PCD (Watanabe and Lam 2006), was pronounced in sense lines and reduced in antisense lines compared with the wild type. In sections II, III, and IV, the extent of PCD as measured by TUNEL assays was significantly different from that in wild-type sections for certain cell types in both sense and antisense lines of the two class 1 Pgbs.

Figure 2.

Figure 2.

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Localization of NO by DAF-2DA, ROS by dihydroethidonium, cell death by TUNEL, and Bi-1 transcripts by RNA in situ hybridization in the five serial sections of hypoxic roots of wild-type (WT) seedlings and seedlings overexpressing (S) or down-regulating (A) ZmPgb1.1 and ZmPgb1.2 after 24 h of 4% oxygen treatment. Bar graphs indicate the percentage of TUNEL-positive nuclei ± se in different root domains. Ct, Cortex; Ep, epidermis; RC, root cap; St, stele. *, Significantly different from wild-type values (P < 0.05).

With respect to NO, ROS, and PCD in the various sections, the response to hypoxia in the root cap (section I) displayed no apparent change as a result of varying Pgb expression (Fig. 2). Although there were some slight visual differences for NO and ROS in micrographs of the more mature, fully differentiated section V, there were no significant differences in the extent of PCD among the lines. Most of the effects of Pgb variation on NO, ROS, and PCD appeared to be in the root meristem (section II) and tissue undergoing differentiation (sections III and IV). Evidence of increased NO and ROS and significantly increased PCD in the QC, compared with the wild type, was found in antisense lines of section II, with decreased expression of Bi-1. About 90% PCD occurred in the cells of the QC in the ZmPgb(A) lines. The situation was reversed for NO, ROS, and PCD in the sense lines, with PCD declining significantly in the QC to around 5% of the cells. In section III, where most of the cells are in the stage of early differentiation, altering Pgb expression had an effect on the staining of NO, ROS, and PCD in the cortex, epidermis, and portions of the stele as a result of hypoxia. The antisense lines had significantly increased PCD in these regions, with the extent of cell death approaching 90% in some instances. In the sense lines, PCD was depressed significantly, to around 1% in the epidermis and stele of the ZmPgb1.1 line and to around 5% in the stele of the ZmPgb1.2 line. In the region of more advanced differentiation (section IV), altering Pgb expression had an effect largely in the area of the cortex, where increased expression reduced the intensity of staining for NO and ROS and increased that of Bi-1. PCD was significantly lower in the cortex of the ZmPgb(S) lines. The reverse effect was observed in the antisense lines, with significantly increased PCD in the cortex of both lines. The level of PCD in the cortical cells of this region, even in the antisense lines, reached only 30% in comparison with the meristem and early differentiation regions of the root, where PCD approached 90%. Longitudinal sections of hypoxic roots showed similar patterns when stained for NO, ROS, and PCD (Supplemental Fig. S1C).

To further examine the relationship between Pgb expression and PCD, the expression of Respiratory Burst Oxidase Homologs (Rbohs) and Bi-1 in root sections of the lines was determined by quantitative reverse transcription-PCR over the 24 h of the hypoxic treatment. In the 0- to 2-mm region of the root tip (Fig. 3A), antisensing either one of the two Pgbs resulted in significantly increased levels of most Rboh transcripts relative to the wild type throughout the hypoxic treatment, with maximum levels occurring in the period 6 to 12 h after the initiation of the treatment. Constitutive overexpression of the Pgbs gave varying results, ranging from significant decreases in transcript abundance for RbohA to no differences for RbohB throughout the treatment. For RbohC and RbohD, there was a significant decrease in transcript levels in the sense lines at 12 h of hypoxia, largely due to increased expression of these two genes in the wild-type line at that time point. Similar results were obtained for sections 2 to 5, 5 to 10, and 10 to 20 mm back from the root tip (Supplemental Figs. S4 and S5), although the differences become less distinct and significant in the regions farthest from the tip. Significantly higher levels of Bi-1 transcripts relative to the wild type were present in the 0- to 2-mm section at the beginning of the hypoxic treatment in the sense lines and remained significantly higher throughout the treatment, with the ZmPgb1.1 line being slightly higher (Fig. 3B). The effects of Pgb variation on Bi-1 transcript abundance were similar for the 2- to 5-mm, 5- to 10-mm, and 10- to 20-mm sections (Supplemental Fig. S6), although the level of expression declined in all lines as the distance from the root tip increased.

Figure 3.

Figure 3.

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Relative expression of the four Rbohs (A–D; A) and Bi-1 (B) transcripts in root tips (0–2 mm) of maize seedlings subjected to 4% oxygen treatments. Values are means ± se of three biological replicates and are normalized to the wild-type (WT) value of day 0 (set at 1). Root tips were harvested from wild-type seedlings and seedlings overexpressing (S) or down-regulating (A) ZmPgb1.1 and ZmPgb1.2. *, Significantly different from wild-type values (P < 0.05) at the same time point.

The results of Figures 2 and 3 suggest that Pgbs are a factor in maintaining the viability of the root meristem and differentiating cells during hypoxic stress. This suggestion is supported by the results of Figure 1, which show increased expression of both Pgbs in the region of the QC of section II, increased expression of ZmPgb1.2 in portions of the stele, cortex, and epidermis of section III, and a general increase in expression of ZmPgb1.1 throughout section IV when the lines are exposed to hypoxia. Levels of Pgb double or triple in the root meristem region within 2 h of the start of the hypoxic treatment, becoming 5- to 6-fold higher for ZmPgb1.2 within 12 h (Supplemental Fig. S2).

Spatial and Temporal Regulation of Ethylene Biosynthetic and Ethylene-Responsive Genes by ZmPgb under Hypoxia

Hypoxic responses in roots are mediated by ethylene through the production of its precursor 1-aminocyclopropane and the induction of ACS and ACO (Wang and Arteca, 1992; Zhou et al., 2001). To assess if ZmPgb modulates ethylene production and response, the expression and localization patterns of ethylene biosynthetic and ethylene-responsive genes were analyzed in normoxic and hypoxic roots.

Ethylene biosynthesis in maize is regulated by the ACS gene family, composed of ZmAcs2, ZmAcs6, and ZmAcs7, and the ACO gene family, including ZmAco15, ZmAco20, ZmAco31, and ZmAco35 (Gallie and Young, 2004). Expression of the three ZmAcs genes was induced rapidly in the hypoxic root apex segments (0–2 mm) of lines down-regulating ZmPgbs (Fig. 4A), with time-course profiles similar to those of the Rboh genes (Fig. 3A). Up-regulation of ZmPgbs resulted in the suppression of ZmAcs transcripts especially at 12 h of the 4% oxygen treatment (Fig. 4A). A similar ZmPgb transcriptional regulation occurred for ZmAco15, ZmAco20, and ZmAco35. No consistent differences among maize lines were observed for ZmAco31 (Fig. 4A).

Figure 4.

Figure 4.

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Relative expression of the ethylene biosynthetic genes ACS and ACO (A) and the ethylene response genes Erf2 and Ebf1 (B) in root tips (0–2 mm) of maize seedlings subjected to 4% oxygen treatments. Values are means ± se of three biological replicates and are normalized to the wild-type (WT) value of day 0 (set at 1). Root tips were harvested from wild-type seedlings and seedlings overexpressing (S) or down-regulating (A) ZmPgb1.1 and ZmPgb1.2. *, Significantly different from wild-type values (P < 0.05) at the same time point.

The ethylene response at the root tip was assessed by measuring the expression of two genes with high homology to EIN3-BINDING F-BOX PROTEIN1 (Ebf1) and ETHYLENE-RESPONSIVE FACTOR2 (Erf2), known mediators of ethylene signaling in maize roots (Takahashi et al., 2015). Relative to the wild type, the expression of both ZmErf2 and ZmEbf1 was induced rapidly in hypoxic root apices (0–2 mm) down-regulating ZmPgb1.1 or ZmPgb1.2 and reduced in those up-regulating the two ZmPgbs (Fig. 4B). The observed ZmPgb transcriptional regulation of the ethylene biosynthetic and ethylene-responsive genes also occurred in more mature hypoxic root segments (2–5, 5–10, and 10–20 mm) but with some differences (Supplemental Figs. S7 and S8). Of note, an up-regulation of several ethylene biosynthetic and ethylene-responsive genes occurred in hypoxic wild-type segments (zone 5–10 mm).

To better understand the function of ethylene biosynthetic and ethylene-responsive genes at the root tip, RNA in situ hybridization studies were performed along the cross sections (I–V) of the RAM described in Figure 1B. Due to the high degree of similarity in nucleotide sequence between some of the genes, the localization analyses document the combined expression of ZmAco20 and ZmAco35 (probe ZmAco20/35), ZmAco15 and ZmAco31 (probe ZmAco15/31), and ZmAcs2 and ZmAcs7 (probe ZmAcs2/7). A faint ZmAco15/31 and ZmAcs2/7 signal was detected in normoxic root caps (section I) of all lines (Fig. 5). Exposure to 4% oxygen enhanced the staining pattern of all genes throughout the root cap, with the strongest signal observed in the ZmPgb down-regulating lines probed with ZmAco15/31.

Figure 5.

Figure 5.

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Localization of the ethylene biosynthetic (ZmAco20/35, ZmAco15/31, ZmAcs2/7, and ZmAcs6) and ethylene response (ZmEbf1 and ZmErf2) transcripts using RNA in situ hybridization in the serial sections (I–IV) of roots of wild-type (WT) seedlings and seedlings overexpressing (S) or down-regulating (A) ZmPgb1.1 and ZmPgb1.2 exposed for 24 h to 4% oxygen or ambient air. NC, Negative control using sense riboprobes (ACO, ACS, Ebf1, and Erf2).

Relative to normoxic conditions, hypoxic cells of the QC region (section II) and the distal cells showing early signs of differentiation (section III) were heavily labeled by ethylene biosynthetic and response riboprobes. Staining for ZmAco15/31 and ZmErf2 (section II) and ZmAco20/35, ZmAco15/31, ZmAcs2/7, and ZmAcs6 (section III) was particularly intense in roots suppressing ZmPgb1.1 or ZmPgb1.2 (Fig. 5) and with high levels of NO, ROS, and PCD (Fig. 2). In section III, hypoxic cells up-regulating ZmPgbs had the lowest signals of ZmAco15/31 and ZmAcs2/7. These cells also had reduced levels of NO and ROS and limited PCD (Fig. 2).

The expression of many ethylene biosynthetic and ethylene-responsive genes in hypoxic tissue at advanced stages of differentiation (section IV) was restricted to the outer cortical region. Of note, the expression of ZmAco15/31 and ZmEbf1 in the ZmPgb-down-regulating hypoxic roots extended through inner cortical layers, in a pattern mimicking that of NO, ROS, and PCD (Fig. 2). A fainter staining pattern was observed for ZmAco20/35 and ZmEbf1 in tissues where ZmPgbs were induced (Fig. 5). In fully mature tissue (section V) of all maize lines, the differences in staining intensity and localization patterns were attenuated for all the genes analyzed. A moderate induction, mostly unrelated to ZmPgb expression, was observed following exposure to 4% oxygen (Supplemental Fig. S9).

The general pattern of expression of ethylene biosynthetic genes coincided with that of ethylene production by hypoxic root tips, which, relative to the wild type, increased markedly in roots suppressing ZmPgbs and decreased slightly in those where the levels of either ZmPgb was up-regulated (Table I).

Table I. Ethylene level (nmol g−1 fresh weight h−1) measured in tips (0–10 mm) and more mature regions (more than 10 mm) of maize roots after the imposition of a 24-h normoxic (ambient) or hypoxic (4% oxygen) treatment.

Values are means ± se of three biological replicates. Asterisks indicate statistically significant differences (P < 0.05) from wild-type values in the same treatment using one-way ANOVA.

Sample Ambient 4% Oxygen
Root tip (0–10 mm)
 Wild type 0.152 ± 0.018 0.563 ± 0.028
 ZmPgb1.1(S) 0.104 ± 0.019 0.269 ± 0.087
 ZmPgb1.1(A) 0.245 ± 0.117 1.518 ± 0.228*
 ZmPgb1.2(S) 0.100 ± 0.028 0.304 ± 0.113
 ZmPgb1.2(A) 0.185 ± 0.053 1.962 ± 0.259*
Mature root (more than 10 mm)
 Wild type 0.068 ± 0.013 0.571 ± 0.165
 ZmPgb1.1(S) 0.064 ± 0.025 0.531 ± 0.149
 ZmPgb1.1(A) 0.114 ± 0.031 0.579 ± 0.070
 ZmPgb1.2(S) 0.105 ± 0.015 0.614 ± 0.070
 ZmPgb1.2(A) 0.120 ± 0.006 0.641 ± 0.066
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Collectively, these results indicate that ZmPgbs contribute to the regulation of ethylene synthesis in root tips, possibly through the transcriptional modulation of biosynthetic genes, especially in proximity to the QC, and in differentiating tissue of the RAM.

Ethylene Mediates the ZmPgb Regulation of ROS, PCD, and Growth Under Hypoxic Conditions

The relationship between the transcriptional induction of ethylene in the meristematic regions, the accumulation of ROS and TUNEL-positive nuclei, as well as the stunted growth in hypoxic roots suppressing ZmPgbs was investigated using DPI and 1-MCP. DPI is used frequently as an inhibitor of NADPH oxidase, reducing ROS production (Takahashi et al., 2015), although it does inhibit other flavoprotein-containing enzymes (Wind et al., 2010). 1-MCP competes with ethylene, inhibiting its perception, and is thought to be specific in its action (Sisler and Serek, 1997).

Pretreatments of ZmPgb1.1- and ZmPgb1.2-suppressing roots with either 1-MCP or DPI restored growth under hypoxic conditions (Fig. 6A). Along with repressing the expression of the four ZmRbohs (Supplemental Figs. S10–S13) and inducing that of ZmBi-1 (Supplemental Fig. S14), applications of 1-MCP in the same roots reduced the accumulation of ROS and the number of TUNEL-positive nuclei and increased the ZmBi-1 signal in the five sections of the RAM (compare Fig. 2 with Fig. 6B and with Supplemental Fig. S15, showing localization in normoxic roots).

Figure 6.

Figure 6.

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Effects of the suppression of ROS by DPI and ethylene perception by 1-MCP on root behavior. A, Effects of DPI and 1-MCP on the root growth of wild-type (WT) seedlings and seedlings down-regulating (A) ZmPgb1.1 or ZmPgb1.2 after 24 h of 4% oxygen treatment. Values are means ± se of three biological replicates. *, Significantly different from the wild-type value (ambient). B, Localization of ROS by dihydroethidonium, PCD by TUNEL, and Bi-1 transcripts by RNA in situ hybridization in the serial sections (I–V) of hypoxic roots of ZmPgb1.1- or ZmPgb1.2-suppressing seedlings treated with DPI or 1-MCP.

Treatments with DPI phenocopied 1-MCP in reducing PCD and enhancing the expression and localization signal of ZmBi-1 (Figs. 1 and 6B; Supplemental Fig. S14). Assuming that the DPI effect is solely on Rbohs, the inhibition of root growth and the compromised structure of the RAM observed in hypoxic roots in which Pgb expression has been suppressed appear to be mediated by ethylene.

Pgbs are known to scavenge NO (Dordas et al., 2003b). To verify that the phenotypes observed by altering ZmPgbs are mediated by NO, we manipulated NO content pharmacologically using the NO donors sodium nitroprusside (SNP) and S-nitroso-N-acetylpenicillamine (SNAP) and the NO scavenger 2-4-carboxyphenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO). Increasing NO with SNP or SNAP in the ZmPgb-overexpressing lines reduced root growth, while applications of cPTIO restored root growth in lines suppressing ZmPgbs (Table II). A similar NO-mediated regulation also was observed for many ethylene and ROS biosynthetic genes influenced by ZmPgbs. The expression of these genes following applications of SNP, SNAP, or cPTIO was measured at 12 h (Supplemental Figs. S16–S19), corresponding to the most pronounced alterations observed in the transgenic roots (Figs. 3 and 4).

Table II. Root growth of maize seedlings overexpressing (S) or down-regulating (A) ZmPgb1.1 or ZmPgb1.2 exposed for 24 h to 4% oxygen.

Roots were grown in the absence (untreated) or presence of the NO donors SNP or SNAP and/or the NO scavenger cPTIO. Values are means ± se of three biological replicates Asterisks indicate statistically significant differences (P < 0.05) from the respective untreated roots using one-way ANOVA.

Genotype Treatment Growth
mm
ZmPgb1.1(S) Untreated 12.17 ± 0.44
SNP 5.33 ± 1.25*
SNAP 5.50 ± 0.79*
ZmPgb1.1(A) Untreated 3.67 ± 0.37
cPTIO 12.00 ± 1.67*
ZmPgb1.2(S) Untreated 12.17 ± 0.44
SNP 5.17 ± 0.66*
SNAP 5.83 ± 0.59*
ZmPgb1.2(A) Untreated 4.53 ± 0.34
cPTIO 14.20 ± 0.49*
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DISCUSSION

In a natural growth environment, plants exposed to excess water have a limited availability of oxygen necessary to perform many energy-consuming metabolic processes (Dennis et al., 2000). As a result, morphological and physiological processes are affected and growth compromised (Jackson and Colmer, 2005). Plant root growth is sustained by the continuous supply of cells provided by the RAM, which is composed of a cluster of less mitotically active cells, the QC, surrounded by cells with higher proliferative activity (Dinneny and Benfey, 2008). The RAM harbors stem cells; therefore, its preservation during adverse conditions is paramount to survival. Oxygen deprivation causes root growth arrest and root tip death in a variety of species (Subbaiah et al., 2000), an observation consistent with the behavior of maize seedlings exposed to 4% oxygen (Fig. 1A).

The expression of ZmPgb1.1 and ZmPgb1.2 in the root tip and their up-regulation under hypoxic conditions appear to be a requirement for protecting the meristematic cells and the architecture of the RAM, resulting in limits to the inhibitory effect of oxygen deprivation on root growth. The suppression of ZmPgbs induces PCD, with cells of the QC (section II) and those at the initial stages of differentiation (section III) being the most affected (Fig. 2). The pluripotent cells of the QC are the source of stem cells and integrators of several processes required for normal root growth (Jiang and Feldman, 2005). A key function of the QC is to maintain the root initials in an undifferentiated state through non-cell-autonomous signals (van den Berg et al., 1997; Scheres, 2007). Therefore, it is not surprising that dismantling of the QC cells and their distal derivatives by PCD, as observed extensively in hypoxic roots down-regulating ZmPgbs, compromises root growth (Fig. 1A). This is in contrast to ZmPgb-up-regulating lines, which show limited PCD as well as the greatest root growth rate under hypoxic conditions. Execution of the death program might be due, at least in part, to the suppression of the maize homolog Bi-1, an attenuator of the death programs in eukaryotes (Watanabe and Lam, 2006). In addition to being induced in cells up-regulating ZmPgbs (Fig. 3B), the expression of this gene occurred in domains where PCD was limited (Fig. 2). The effect of ZmPgbs in regulating the cell death/survival decision was obvious in immature tissue of the root proper (sections I–IV) but less apparent in the proximal domains (section I) or the distal domains (section V) of the RAM, where the frequency of PCD is unrelated to the expression of ZmPgbs (Fig. 2). This observation suggests that cell responsiveness to ZmPgbs might vary along the root profile, with the meristematic cells (sections II–IV) of the root proper being the most sensitive. While a definitive explanation for this different behavior cannot be made, future research might focus on the concept that ZmPgb action is critical in those regions of the root that are most susceptible to reduced oxygen diffusion and availability. These regions are likely to include the tightly packed meristematic cells and their immediate derivatives (sections II–IV) that experience oxygen limitation even under normoxic conditions (Armstrong et al., 1994). The protective role of ZmPgbs, therefore, might be essential to alleviate stress and reduce death in these meristematic regions.

The results suggest that the ZmPgb control of cell fate in hypoxic root tips is mediated by NO. The effect of Pgb on hypoxic growth likely occurs upstream of ROS and ethylene production, since the alleviation of hypoxia on maize root growth can be achieved by either constitutive expression of Pgbs (Fig. 1A) or inhibiting ethylene perception or ROS production in antisense Pgb lines (Fig. 6). In agreement with the NO-scavenging properties of ZmPgbs (Dordas et al., 2003b), NO staining appears to increase in meristematic and differentiating cells (sections II–IV) of lines suppressing ZmPgb and decreases in those where the genes are up-regulated (Fig. 2). Regions staining for NO correspond to those accumulating ROS and TUNEL-positive cells (Fig. 2). In maize culture cells, the level of ROS is modulated by ZmPgbs through NO (Huang et al., 2014). Hypoxic roots suppressing ZmPgbs have increased expression of Rbohs (Fig. 3A). The expression of these genes is reduced in cells where ZmPgbs are up-regulated. DPI, an inhibitor of flavoprotein-containing enzymes including NADPH oxidase, decreased the number of cells undergoing PCD (Fig. 6B) and increased the expression of Bi-1 (Supplemental Fig. S14) and growth (Fig. 6A) in ZmPgb-suppressing roots under hypoxic conditions, supporting the contention that ROS are required for the induction of the death program. ROS are implicated in several PCD-inducing responses in maize, including the formation of aerenchyma in hypoxic roots (Takahashi et al., 2015) and shaping the body of developing somatic embryos (Huang et al., 2014).

The production of ROS requires ethylene, as the inhibition of ethylene perception by 1-MCP reduces the transcript levels of Rbohs, ROS signal, and the number of TUNEL-positive nuclei and increases the expression of Bi-1 (Fig. 6; Supplemental Figs. S10–S14). This regulation is consistent with previous studies demonstrating a ROS dependence of several ethylene responses, some of which trigger the death program (Yamauchi et al., 2011). Synthesized during adverse growing conditions, ethylene accumulates preferentially in hypoxic tissue, and its production is regulated by the activity of ACS and ACO, encoded by multigene families (Gallie and Young, 2004). A rapid transcriptional induction of several members of ACS and ACO multigene families, as well as the ethylene signaling-related genes Ebf1 and Erf2, follows exposure to low oxygen (Geisler-Lee et al., 2010; Takahashi et al., 2015).

ZmPgbs regulate the transcription and localization of ethylene biosynthetic and ethylene-responsive genes in hypoxic roots. With the exception of ZmAco31, the suppression of ZmPgbs induces the expression of all ethylene biosynthetic genes analyzed, while the overexpression of ZmPgbs has a repressive effect (Fig. 4). This pattern, closely reflecting the root tip-specific ZmPgb regulation of ethylene accumulation (Table I), confirms the transcriptional induction of ethylene, modulated by NO, in alfalfa cells limited in Pgb1 protein (Manac’h-Little et al., 2005). The regulation of many ethylene biosynthetic genes by ZmPgbs was more apparent in tissues of the root proper harboring meristematic cells (sections II and III in Fig. 5) and was consistent with the accumulation of NO and ROS and the induction of PCD (Fig. 2).

Collectively, this study demonstrates a novel function of Pgbs in protecting hypoxic root apical meristems from the NO-mediated accumulation of ethylene leading to the overproduction of ROS and death. The up-regulation of Pgbs in meristematic cells during hypoxia may be indicative of a root acclimation to hypoxia, with the suppression of Pgb expression being associated with avoidance mechanisms, such as aerenchyma formation, that accompany flooding responses. The Pgb protective role of meristematic cells might be a universal response to other forms of stress, including salt and drought, known to modulate Pgbs and reduce root growth by compromising meristem function.

MATERIALS AND METHODS

Plant Material and Hypoxic Treatment Conditions

The generation and characterization of maize (Zea mays) plants overexpressing or down-regulating ZmPgb1.1 or ZmPgb1.2 was described in previous studies (Youseff et al., 2016; Supplemental Fig. S3). Lines constitutively expressing class 1 Pgbs are designated with an (S) following the gene abbreviation, while those expressing the Pgb in an antisense configuration are designated with an (A). Hypoxic conditions were created exactly as described (Geisler-Lee et al., 2010). Briefly, the roots of 5-d-old seedlings were immersed into a liquid solution (one-half-strength Murashige and Skoog medium) through which air (normoxic conditions) or 4% (v/v) oxygen/96% nitrogen (hypoxic conditions) was bubbled at a rate of 350 mL min−1 for 24 h. Experiments were performed at 22°C under a light intensity of 15 µmol s−1 m−2.

Histological Analyses and Localization of NO, ROS, and PCD

For histological examinations, roots were fixed in 2.5% glutaraldehyde and 1.6% paraformaldehyde in 0.05 m phosphate buffer (pH 6.9), dehydrated with methyl cellosolve followed by three washings with absolute ethanol, and then infiltrated and embedded in Historesin (Leica). Sections (3 µm) were stained with Toluidine Blue (Yeung, 1990).

For NO and ROS localization, maize roots were sectioned using a compresstome and stained with DAF-2DA (Elhiti et al., 2013) or dihydroethidonium (Tsukagoshi et al., 2010), respectively.

Nuclear DNA fragmentation was detected with the In Situ Cell Death Detection Kit-Fluorescein (Roche; as described by Huang et al. [2014]). Tissue was fixed in 4% paraformaldehyde, dehydrated in an ethanol series, and embedded in wax. Sections (10 μm) were dewaxed in xylene and labeled with the TUNEL kit (Roche) according to the manufacturer’s protocol, with the exclusion of the permeabilization step by proteinase K. Omission of TdT was used for negative controls.

RNA in Situ Hybridization

RNA in situ hybridization studies were performed following the procedure described by Elhiti et al. (2010). Roots were fixed in 4% (w/v) freshly prepared paraformaldehyde in phosphate-buffered saline, pH 7.4. Following a 15-min vacuum infiltration, the samples were incubated for 3 h at room temperature, dehydrated in an ethanol series (30%, 50%, 70%, 95%, 100%, and 100%) for 45 min each at 4°C, and left overnight in 100% ethanol. The roots were then treated with increasing levels of xylene and incubated overnight at 42°C in xylene containing a few pellets of paraffin. After incubation at 60°C, xylene was replaced gradually with paraffin, and the samples were embedded into blocks that were sectioned at a thickness of 10 μm using disposable blades in a Leica (RM 2145) microtome. Before hybridization, paraffin was removed from the slides with two changes in xylene and gradual rehydration.

For hybridization, cDNAs encoding ZmPgbs, ZmAcs, ZmAco, ZmEbf1, and ZmErf2 were amplified and used for the preparation of digoxigenin-labeled sense and antisense riboprobes, following the procedure described in the DIG Application Manual (Roche Diagnostics). Tissue treatments and prehybridization steps were performed as described by Canton et al. (1999).

Sections were hybridized with sense or antisense probe in 1× Denhardt’s, 1 mg mL−1 tRNA, 10% dextran sulfate, 50% formamide, and 1× salts (Regan et al., 1999). Hybridization was conducted at 50°C for 16 h. Posthybridization washes and antibody treatments were performed as described by Regan et al. (1999). Detection of digoxigenin-labeled probes was carried out using a Western Blue-stabilized substrate for alkaline phosphatase (Promega).

Chemical Treatments

The NO scavenger cPTIO and the NO donors SNP and SNAP were applied at concentrations of 200 µm (cPTIO) and 100 µm (SNP and SNAP) to one-half-strength Murashige and Skoog medium during hypoxia conditions. Pretreatments with 40 μm DPI or 1 ppm 1-MCP (a gift from AgroFresh) were performed exactly as described previously (Rajhi et al., 2011; Yamauchi et al., 2011).

Monitoring of Transcript Abundance

RNA extraction was carried out using TRI Reagent Solution according to the manufacturer’s protocol (Invitrogen). Total RNA was treated with DNase I recombinant, RNase-free (Roche), and the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems) was used for cDNA synthesis. Quantitative reverse transcription-PCR was performed as described by Elhiti et al. (2010). All primers used for gene expression studies are listed in Supplemental Table S1. The relative gene expression level was analyzed with the 2−∆∆CT method (Livak and Schmittgen, 2001) using actin as the reference gene.

Measurements of Ethylene Production

Ethylene measurements in normoxic or hypoxic maize roots harvested from 5-d-old seedlings were performed according to Geisler-Lee et al. (2010). Measurements were conducted on two segments: root tips (0–10 mm) and more mature root regions (more than 10 mm from the tip). To minimize the amount of ethylene produced by wounding, the root segments (about 15 per replicate) were first preincubated in an unsealed 3-mL syringe for 30 min. The syringe was then sealed and incubated in the dark for 2 h at 22°C, and 1 mL of the gas accumulated in the headspace was analyzed with a Bruker 450-GC gas chromatograph. Data analysis was carried out using the Bruker Compass Data analysis 3.0 software. All experiments were repeated in triplicate.

Statistical Analysis

Data were analyzed by one-way ANOVA using the SPSS program (SPSS Statistics for Windows, version 19.0; IBM). Treatment means were compared by Tukey’s test (α = 0.05) to differentiate the significance of differences. Structural and localization studies were performed on at least 15 roots, while growth elongation assays, ethylene measurements, and gene expression and pharmacological studies were performed using at least three biological replicates each consisting of a minimum of 20 roots.

Supplemental Data

The following supplemental materials are available.

  • Supplemental Figure S1. Features of cells from the QC of roots of wild-type seedlings and seedlings overexpressing or suppressing ZmPgb1.1 or ZmPgb1.2 after 24 h of hypoxic treatment, transcript localization of ZmPgb1.1 and ZmPgb1.2 in longitudinal sections of wild-type roots following 24 h of ambient or 4% oxygen treatment, and localization of NO, ROS, PCD, and Bi-1 transcripts in roots subjected to hypoxic treatment for 24 h.

  • Supplemental Figure S2. Relative expression of ZmPgb1.1 and ZmPgb1.2 in segments of wild-type roots exposed to 4% oxygen for 24 h.

  • Supplemental Figure S3. Characterization of transgenic lines with altered expression of ZmPgb1.1 or ZmPgb1.2.

  • Supplemental Figure S4. Relative expression of RbohA and RbohB in segments of roots exposed to 4% oxygen for 24 h.

  • Supplemental Figure S5. Relative expression of RbohC and RbohD in segments of roots exposed to 4% oxygen for 24 h.

  • Supplemental Figure S6. Relative expression of Bi-1 in segments of roots exposed to 4% oxygen for 24 h.

  • Supplemental Figure S7. Relative expression of ACS and ACO in segments of roots exposed to 4% oxygen for 24 h.

  • Supplemental Figure S8. Relative expression of Erf2 and Ebf1 in segments of roots exposed to 4% oxygen for 24 h.

  • Supplemental Figure S9. Localization of ethylene biosynthetic and response transcripts using RNA in situ hybridization in hypoxic roots after 24 h of 4% oxygen treatment.

  • Supplemental Figure S10. Relative expression of ZmRboh(A) in segments of roots pretreated with 1-MCP and exposed to 4% oxygen for 24 h.

  • Supplemental Figure S11. Relative expression of ZmRboh(B) in segments of roots pretreated with 1-MCP and exposed to 4% oxygen for 24 h.

  • Supplemental Figure S12. Relative expression of ZmRboh(D) in segments of roots pretreated with 1-MCP and exposed to 4% oxygen for 24 h.

  • Supplemental Figure S13. Relative expression of ZmBi-1 in segments of roots pretreated with DPI or 1-MCP and exposed to 4% oxygen for 24 h.

  • Supplemental Figure S14. Localization of ROS, PCD, and Bi-1 in roots of seedlings subjected to ambient oxygen treatments for 24 h.

  • Supplemental Figure S15. Root growth of maize seedlings exposed for 24 h to ambient air or 4% oxygen in the presence of SNP or SNAP and/or cPTIO.

  • Supplemental Figure S16. Relative expression of ACS in root tips exposed to 4% oxygen for 12 h.

  • Supplemental Figure S17. Relative expression of ACO in root tips exposed to 4% oxygen for 12 h.

  • Supplemental Figure S18. Relative expression of Erf2 and Ebf1 in root tips exposed to 4% oxygen for 12 h.

  • Supplemental Figure S19. Relative expression of Rbohs in root tips exposed to 4% oxygen for 12 h.

  • Supplemental Table S1. List of primers.

Supplementary Material

Supplemental Data
supp_172_3_2044__index.html (1.1KB, html)

Acknowledgments

We thank Mr. Durnin and Dr. John Markham for technical support and AgroFresh for providing 1-MCP.

Glossary

PCD

programmed cell death

ROS

reactive oxygen species

1-MCP

1-methylcyclopropene

DPI

diphenyleneiodonium

RAM

root apical meristem

QC

quiescent center

NO

nitric oxide

SNP

sodium nitroprusside

SNAP

S-nitroso-N-acetylpenicillamine

cPTIO

2-4-carboxyphenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide

Footnotes

1

This work was supported by the Manitoba Corn Growers Association (grant to C.S.).

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Associated Data

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Supplementary Materials

Supplemental Data
supp_172_3_2044__index.html (1.1KB, html)
supp_pp.16.01150_PP2016-01150DR1_Supplemental_Figures_1_19.pdf (2.5MB, pdf)
supp_pp.16.01150_PP2016-01150DR1_Supplemental_Table_1.pdf (70.2KB, pdf)

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