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Plant Physiology Preview. Published on October 4, 2016, as DOI:10.1104/pp.16.01150
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Phytoglobins improve hypoxic root growth by alleviating apical meristem cell death
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Mohamed M. Mira1, Robert D. Hill2, Claudio Stasolla2*
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Permanent address: Department of Botany, Faculty of Science, Tanta University, Tanta, Egypt
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Department of Plant Science, University of Manitoba, Winnipeg, Manitoba, R3T 2N2, Canada
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Corresponding author E-mail: [email protected]
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Tel. 204-474-6098
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Author contributions: MMM performed most of the experiments and contributed to the
analysis of the data and writing of the manuscript; RDH contributed to the conception of the
project, the research plans the data analysis and the writing of the manuscript; CS contributed to
the project conception, the research plans, data analysis, some of the experimentation,
manuscript writing and was responsible for the overall supervision of the project.
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Running title: Phytoglobin influences hypoxic root growth
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Word count: 4790
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Abstract, 227; Introduction, 728; Discussion, 980; Experimental Procedure, 1318,
Legend 488
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Figure
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ABSTRACT
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Hypoxic root growth in maize is influenced by expression of phytoglobins (ZmPgbs). Relative
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to WT, suppression of ZmPgb1.1 or ZmPgb1.2 inhibits growth of roots exposed to 4% oxygen
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causing structural abnormalities in the root apical meristems. These effects were accompanied
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by increasing levels of reactive oxygen species (ROS), possibly through the transcriptional
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induction of four Respiratory Burst Oxidase Homologs (Rbohs). TUNEL-positive nuclei in
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meristematic cells indicated the involvement of programmed cell death (PCD) in the process.
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These cells also accumulated nitric oxide (NO) and stained heavily for ethylene biosynthetic
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transcripts. A sharp increase in the expression level of several ACC synthase (ZmAcs2, 6, and
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7), ACC oxidase (Aco15, 20, 31, and 35), and ethylene responsive (ZmErf2 and ZmEbf1) genes
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was observed in hypoxic ZmPgb-suppressing roots, that overproduced ethylene. Inhibiting ROS
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synthesis with diphenyleneiodonium or ethylene perception with 1-methylcyclopropene (1-MCP)
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suppressed PCD, increased BAX inhibitor-1 (Bi-1), an effective attenuator of the death programs
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in eukaryotes, and restored root growth. Hypoxic roots over-expressing ZmPgbs had the lowest
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level of ethylene and showed a reduction in ROS staining and TUNEL-positive nuclei in the
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meristematic cells. These roots retained functional meristems and exhibited the highest growth
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performance when subjected to hypoxic conditions. Collectively these results suggest a novel
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function of PGBs in protecting root apical meristems from hypoxia-induced PCD through
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mechanisms initiated by NO and mediated by ethylene via ROS.
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Keywords: ethylene, hypoxia, maize, phytoglobins, programmed cell death, reactive oxygen
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species, root apical meristem.
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INTRODUCTION
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Oxygen deficiency (hypoxia), experienced by plants grown in poorly drained soils or subjected
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to flooding, impairs plant growth and results in heavy crop losses (Dennis et al., 2000).
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Submergence or flooding reduces oxygen availability for plant cells inhibiting gas exchange
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required for basic physiological processes (Bailey-Serres and Voesenek, 2008). Both roots and
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shoots are affected by hypoxia, regardless of whether the plant is submerged or only the root is
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exposed to the condition. The consequences to shoots of prolonged root hypoxia
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reduced photosynthetic rate and stomatal conductance, decreased leaf growth and senescence,
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wilting of the above ground organs and alterations in plant water relations (Mustroph and
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Albrecht, 2003).
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(Visser et al., 1996) and in some species ethylene affects the selective death of cortical cells
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generating lysogenous aerenchyma (Drew et al., 2000;Drew et al., 1979;Drew, 1997) Precursors
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of ethylene have been shown to induce changes in B. napus growth behavior and root
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architecture (Patrick et al., 2009).
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either stimulate or inhibit root growth (Konings and Jackson, 1979). Ethylene regulation of
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programmed cell death (PCD) is not restricted to hypoxia, but rather is observed in response to
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many adverse growth conditions (Abeles.et.al., 1992;Buer et al., 2003;Clark et al., 1999;Drew et
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al., 1979;Feldman, 1984;Pitts et al., 1998). Execution of PCD in maize roots under hypoxic
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conditions is triggered by a rapid increase in ethylene level resulting from the transcriptional
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induction of 1-aminocyclopropane-1-carboxylate synthase (ACS) and oxidase (ACO) (Geisler-
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Lee et al., 2010) and transduced through the generation of reactive oxygen species (ROS)
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produced by NADPH oxidase activity
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in maize roots has been shown using 1-methylcyclopropene (1-MCP), as a specific inhibitor of
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ethylene perception, or diphenyleneiodonium (DPI) to inhibit ROS production (Takahashi et al.,
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2015).
include
Ethylene accumulates rapidly in flooded Rumex palustris Sm root cells
Depending upon concentration and species, ethylene can
(Torres and Dangl, 2005).
Progression of these events
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While considerable attention has been paid to the mechanisms underlying PCD during
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aerenchyma formation, no information is currently available on other death programs occurring
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in other regions of hypoxic roots, including the root tip. Maize root tips are very sensitive to
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flooding stress and die after a few hours, compromising survival upon the reestablishment of
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normoxic conditions (Roberts et al., 1984). The root apical meristem (RAM) harbors stem cells
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and performs the task of organizing centers for post-embryonic morphogenesis (Jiang and
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Feldman, 2005). These crucial functions are evidenced by its conserved structure. The maize
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RAM consists of a
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surrounded by more actively dividing stem cells (Kerk and Feldman, 1995).
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environmental perturbations of RAM function lead to growth inhibition or cessation (Blilou et
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al., 2005). Recent work identified ethylene as a central regulator of RAM function (Street et al.,
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2015).
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quiescent center (QC), comprising 800-1200 slowly dividing cells,
Genetic or
Phytoglobins (PGBs), previously termed nonsymbiotic hemoglobins (Hill et al, 2016),
are
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heme-containing proteins characterized mainly for their ability to remove nitric oxide (NO)
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under adverse conditions, including hypoxia (Hill, 2012). Phytoglobins are rapidly induced in
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cells grown under limited oxygen (Silva-Cardenas et al., 2003) and experimental changes in their
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expression level affect plant response to stress. In Arabidopsis, ectopic expression of one Pgb
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enhanced survival to low oxygen conditions (Hunt et al., 2002), while hypoxic alfalfa roots and
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maize cells over-expressing Pgbs maintained growth and sustained a high energy status (Dordas
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et al., 2003a;Igamberdiev and Hill, 2004). In culture, suppression of Pgbs enhances ethylene
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synthesis (Manac'h-Little et al., 2005) and induces PCD in maize through ROS production
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(Huang et al., 2014). These observations, in conjunction with the root tip localization of one
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maize Pgb (Dordas et al., 2003b;Zhao et al., 2008) are the premises of the present work, to
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determine whether Pgbs exercise a protective role by limiting meristematic cell death in the
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hypoxic RAM through regulation of ethylene and ROS.
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RESULTS
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Expression of ZmPgbs affects hypoxic root growth
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Root growth of 5 day-old seedlings with altered expression of ZmPbg1.1 or ZmPgb1.2 was
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compared under normoxic (ambient air) or hypoxic (4% oxygen) conditions. Growth of WT
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hypoxic roots was more than 40 percent impaired after hypoxic treatment for 24 h, while
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ZmPgb1.1 or ZmPgb1.2 over-expressing roots [ZmPgb1.1(S) and ZmPgb1.2(S)] showed less
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than 30 percent reduction in growth (Fig. 1A). In the lines suppressing either of the ZmPgbs
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[ZmPgb1.1(A) and ZmPgb1.2(A)] there was substantially reduced root growth of the order of 60
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to 80 percent during the same period with evidence of abnormalities within the root apices.
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Structural disorganization of the root tip (Fig. 1A) and formation of large vacuoles within cells of
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the quiescent center (QC) (Supplemental Fig. 1A), a sign of differentiation, were often observed
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in roots suppressing either ZmPgb1.1 or ZmPgb1.2.
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Expression of ZmPgbs was measured in segments (0-2, 2-5, 5-10, and 10-20 mm from the tip)
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of hypoxic WT roots. Hypoxia induced ZmPgb1.1 and ZmPgb1.2, especially in proximity of the
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root tip (segments 0-2mm and 2-5mm), with maximum expression occurring at 12 h
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(Supplemental Fig. 2).
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conditions were attenuated in more mature regions of the root (segments 5-10 and 10-20mm).
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To enhance resolution, RNA in situ localization studies of both ZmPgbs were performed on
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progressive transverse sections along the RAM (Fig. 1B). These sections included the root cap
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(section I), the QC (section II), domains with initial (section III) and advanced (section IV)
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regions of cellular differentiation, and in mature fully differentiated tissue (section V) (Fig. 1B).
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Hypoxic conditions increased the staining of ZmPgb1.1, and, to a lesser extent, ZmPgb1.2, in the
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central cells of the root cap (section I, Fig. 1C). Increased expression of ZmPgbs as a result of
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hypoxia was particularly evident in the QC region (section II) and in tissue undergoing early
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differentiation (section III). Heavy induction of ZmPgb1.1 was also observed in hypoxic cells
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at advanced stages of differentiation (section IV). Specificity of the signal was verified using
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sense ribo-probes as a negative control (NC) (Fig. 1C). Longitudinal sections of hypoxic roots
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also displayed evidence of heavy staining for ZmPgbs (Supplemental Fig. 1B).
Differences in expression levels between normoxic and hypoxic
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ZmPgb regulation of NO, ROS and PCD in hypoxic RAM
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The different growth behavior of corn roots with altered expression of ZmPgbs was further
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examined in light of the following observations:
phytoglobins scavenge NO (Dordas et al.,
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2003a); when Pgb expression is suppressed, NO accumulates, inducing ROS production (Huang
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et al., 2014) that triggers PCD (Van and Dat, 2006). Altered ZmPgb expression was achieved by
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the use of maize transgenic lines (Youseff et al, 2016) that constitutively expressed ZmPgb1.1 or
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ZmPgb1.2 in either the sense (S) or antisense (A) orientations. The relative expression of a
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particular ZmPgb in normoxic root lines is shown in Supplemental Fig. 3B. ZmPgb (S) lines
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had ZmPgb levels approximately 15-20 fold higher than the WT line while Pgb(A) lines had
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expression levels that were less than 10 percent of the WT line.
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Examining the effect of varying Pgb expression, with the exception of the root cap
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(Section I), there was visual evidence of an increase in staining for NO, ROS and PCD in
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ZmPgb(A) lines and a decrease in ZmPgb(S) lines relative to WT as a result of hypoxia (Fig. 2).
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Consistent with the evidence of PCD in the sections, staining for transcripts of BAX inhibitor-1
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(Bi-1), an attenuator of PCD (Watanabe and Lam 2006), was pronounced in sense lines and
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reduced in antisense lines compared to the WT. In sections II, III and IV, the extent of PCD as
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measured by TUNEL assays was significantly different from that in WT sections for certain cell
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types in both sense and antisense lines of the two Class 1 Pgbs.
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With respect to NO, ROS and PCD in the various sections, the response to hypoxia in the
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root cap (Section I) displayed no apparent change as a result of varying Pgb expression (Fig. 2).
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Although there were some slight visual differences for NO and ROS in micrographs of the more
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mature, fully differentiated section V, there were no significant differences in the extent of PCD
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amongst the lines. Most of the effects of Pgb variation on NO, ROS and PCD appeared to be in
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the root meristem (Section II) and tissue undergoing differentiation (Sections III and IV).
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Evidence of increased NO, ROS and significantly increased PCD in the quiescent center,
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compared to the WT, was found in antisense lines of Section II, with decreased expression of Bi-
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1. About 90 percent PCD occurred in the cells of the quiescent center in the ZmPgb(A) lines. The
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situation was reversed for NO, ROS and PCD in the sense lines, with PCD declining
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significantly in the quiescent center to around 5 percent of the cells. In Section III, where most of
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the cells are in the stage of early differentiation, altering Pgb expression had an effect on the
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staining of NO, ROS and PCD in the cortex, epidermis and portions of the stele as a result of
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hypoxia. The antisense lines had significantly increased PCD in these regions with the extent of
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cell death approaching 90 percent in some instances. In the sense lines, PCD was significantly
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depressed to around one percent in the epidermis and stele of the ZmPgb1.1 line and to around 5
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percent in the stele of the ZmPgb1.2 line. In the region of more advanced differentiation (Section
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IV), altering Pgb expression had an effect largely in the area of the cortex, where increased
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expression reduced the intensity of staining for NO and ROS and increased that of Bi-1. PCD
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was significantly lower in the cortex of the ZmPgb(S) lines. The reverse effect was observed in
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the antisense lines, with significantly increased PCD in the cortex of both lines. The level of
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PCD in the cortical cells of this region, even in the antisense lines, reached only 30 percent in
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comparison to the meristem and early differentiation regions of the root where PCD approached
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90 percent. Longitudinal sections of hypoxic roots showed similar patterns when stained for NO,
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ROS and PCD (Supplemental Fig. 1C).
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To further examine the relationship between Pgb expression and PCD, the expression of
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Rbohs and Bi-1 in root sections of the lines was determined by qRT-PCR over the 24h of the
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hypoxic treatment. In the 0-2 mm region of the root tip (Fig. 3A), anti-sensing either one of the
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two Pgbs resulted in significantly increased levels of most Rboh transcripts relative to WT
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throughout the hypoxic treatment, with maximum levels occurring in the period 6 to 12h after the
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initiation of the treatment. Constitutive over-expression of the Pgbs gave varying results, ranging
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from significant decreases in transcript abundance for RbohA to no differences for RbohB
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throughout the treatment. For RbohC and RbohD there was a significant decrease in transcript
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levels in the sense lines at 12h hypoxia, largely due to increased expression of these two genes in
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the WT line at that time point. Similar results were obtained for sections 2-5, 5-10 and 10-20 mm
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back from the root tip (Supplemental Fig. 4 and 5), although the differences become less distinct
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and significant in the regions farthest removed from the tip. Significantly higher levels of Bi-1
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transcripts relative to WT were present in the 0-2 mm section at the beginning of the hypoxic
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treatment in the sense lines and remained significantly higher throughout the treatment, with the
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ZmPgb1.1 line being slightly higher (Fig. 3B). The effects of Pgb variation on Bi-1 transcript
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abundance were similar for the 2-5, 5-10, and 10-20 mm sections (Supplemental Fig. 6),
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although the level of expression declined in all lines as the distance from the root tip increased.
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The results of Fig. 2 and 3 suggest that PGBs are a factor in maintaining the viability of
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root meristem and differentiating cells during hypoxic stress. This suggestion is supported by the
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results of Fig. 1 that show increased expression of both Pgbs in the regions of the QC of Section
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II, increased expression of ZmPgb1.2 in portions of the stele, cortex and epidermis of section III
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and a general increase in expression of ZmPgb1.1 throughout section IV when the lines are
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exposed to hypoxia. Levels of Pgb double or triple in the root meristem region within two hours
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of the start of the hypoxic treatment, becoming 5-6 fold higher for ZmPgb1.2 within 12 hours
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(Supplemental Fig. 2).
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Spatial and temporal regulation of ethylene biosynthetic and ethylene-responsive genes by
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ZmPgb under hypoxia
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Hypoxic responses in roots are mediated by ethylene through production of its precursor ACC
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and induction of ACC synthase and ACC oxidase (Wang and Arteca, 1992;Zhou et al., 2001).
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To assess if ZmPgb modulates ethylene production and response,
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localization patterns of ethylene biosynthetic and responsive genes were analysed in normoxic
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and hypoxic roots.
the expression and
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Ethylene biosynthesis in maize is regulated by the 1-aminocyclopropane (ACC) synthase
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(ACS) gene family composed of ZmAcs2, 6, and 7, and the ACC oxidase (ACO) gene family
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including ZmAco 15, 20, 31, and 35 (Gallie and Young, 2004). Expression of the three ZmAcs
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genes was rapidly induced in the hypoxic root apex segments (0-2 mm) of lines down-regulating
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ZmPgbs (Fig. 4A), with time course profiles similar to those of the Rboh genes (Fig. 3A). Up-
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regulation of ZmPgbs resulted in the suppression of ZmAcs transcripts especially at 12h of the
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4% oxygen treatment (Fig. 4A).
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ZmAco15, 20, and 35. No consistent differences among corn lines were observed for ZmAco31
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(Fig. 4A).
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A similar ZmPgb-transcriptional regulation occurred for
Ethylene response at the root tip was assessed by measuring the expression of two genes with
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high homology to EIN3-binding F-box protein 1 (Ebf1),
and ethylene responsive factor 2
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(Erf2), known mediators of ethylene signalling in corn roots (Takahashi et al., 2015). Relative to
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wild type, the expression of both ZmErf2 and ZmEbf1 was rapidly induced in hypoxic root apices
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(0-2mm) down-regulating ZmPgb1.1 or ZmPgb1.2, and reduced in those up-regulating the two
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ZmPgbs (Fig. 4B). The observed ZmPgb transcriptional regulation of the ethylene biosynthetic
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and responsive genes also occurred in more mature hypoxic root segments (2-5, 5-10, and 10-
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20mm) but with some differences (Supplemental Fig. 7, 8). Of note, an up-regulation of several
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ethylene biosynthetic and response genes occurred in hypoxic WT segments (zone 5-10mm).
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To better understand the function of ethylene biosynthetic and responsive genes at the root
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tip, RNA in situ hybridization studies were performed along the cross sections (I-V) of the RAM
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described in Fig. 1B. Due to the high degree of similarity in nucleotide sequence between some
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of the genes, the localization analyses document the combined expression of ZmAco20 and
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ZmAco 35 (probe ZmAco20/35), ZmAco15 and ZmAco31 (probe ZmAco15/31), and ZmAcs2 and
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ZmAcs7 (probe ZmAcs2/7). A faint ZmAco15/31 and ZmAcs2/7 signal was detected in normoxic
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root caps (section I) of all lines (Fig. 5). Exposure to 4% oxygen enhanced the staining pattern
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of all genes throughout the root cap with the strongest signal observed in the ZmPgb down-
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regulating lines probed with ZmAco15/31.
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Relative to normoxic conditions, hypoxic cells of the QC region (section II) and the distal cells
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showing early signs of differentiation (section III) were heavily labeled by ethylene biosynthetic
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and response ribo-probes. Staining for ZmAco15/31 and ZmErf2 (section II) and ZmAco20/35,
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ZmAco15/31, ZmAcs2/7, and ZmAcs6, (section III) was particularly intense in roots suppressing
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ZmPgb1.1 or ZmPgb1.2 (Fig. 5) and high levels of NO, ROS and PCD (Fig. 2). In section III,
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hypoxic cells up-regulating ZmPgbs had the lowest signal of ZmAco15/31, and ZmAcs2/7. These
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cells also had reduced levels of NO, ROS, and limited PCD (Fig. 2).
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Expression of many ethylene biosynthetic and responsive genes in hypoxic tissue at
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advanced stages of differentiation (section IV) was restricted to the outer cortical region. Of
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note, expression of ZmAco15/31 and ZmEbf1 in the ZmPgb down-regulating hypoxic roots
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extended through inner cortical layers, in a pattern mimicking that of NO, ROS and PCD (Fig.
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2). A fainter staining pattern was observed for ZmAco20/35 and ZmEbf1 in tissues where
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ZmPgbs were induced (Fig. 5). In fully mature tissue (section V) of all corn lines the differences
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in staining intensity and localization patterns were attenuated for all the genes analysed. A
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moderate induction, mostly unrelated to ZmPgb expression, was observed following exposure to
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4% oxygen (Supplemental Fig. 9).
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The general pattern of expression of ethylene biosynthetic genes coincided with that of ethylene
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production by hypoxic root tips, which relative to the WT increased markedly in roots
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suppressing ZmPgbs and decreased slightly in those where the levels of either ZmPgb was up-
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regulated (Table 1).
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Collectively, these results indicate that ZmPGBs contribute to the regulation of ethylene
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synthesis in root tips, possibly through the transcriptional modulation of biosynthetic genes
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especially in proximity of the QC and in differentiating tissue of the RAM.
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Ethylene mediates the ZmPgb regulation of ROS, PCD, and growth under hypoxic
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conditions
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The relationship between the transcriptional induction of ethylene in the meristematic regions,
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the accumulation of ROS and TUNEL-positive nuclei, as well as the stunted growth in hypoxic
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roots suppressing ZmPgbs was investigated using diphenyleneiodonium (DPI) and 1-
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methylcyclopropene (1-MCP). DPI is frequently used as an inhibitor of NADPH oxidase,
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reducing ROS production (Takahashi et al., 2015), although it does inhibit other flavoprotein-
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containing enzymes (Wind et al., 2010). 1-MCP competes with ethylene, inhibiting its
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perception and is thought to be specific in its action (Sisler and Serek, 1997).
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Pre-treatments of ZmPgb1.1- and ZmPgb1.2-suppressing roots with either 1-MCP or DPI
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restored growth under hypoxic conditions (Fig. 6A).
Along with repressing the expression of
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the four ZmRbohs (Supplemental Fig. 10-13) and inducing that of ZmBi-1 (Supplemental Fig.
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14), applications of 1-MCP in the same roots reduced the accumulation of ROS, the number of
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TUNEL-positive nuclei, and increased the ZmBi-1 signal in the five sections of the RAM
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(compare Fig. 2 with Fig. 6B and with Supplemental Fig. 15 showing localization in normoxic
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roots).
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Treatments with DPI phenocopied 1-MCP in reducing PCD and enhancing the expression and
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localization signal of ZmBi-1 (Fig. 1, 6B, and Supplemental Fig. 14). Assuming the DPI effect is
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solely on RBOHs, the inhibition of root growth and the compromised structure of the RAM
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observed in hypoxic roots in which Pgb expression has been suppressed appear to be mediated
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by ethylene.
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Phytoglobins are known to scavenge NO (Dordas et al., 2003b). To verify that the phenotypes
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observed by altering ZmPgbs are mediated by NO, we manipulated NO content
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pharmacologically using the nitric oxide donors, sodium nitroprusside (SNP) and S-nitroso-N-
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acetylpenicillamine (SNAP), and the nitric oxide scavenger, 2-4-carboxyphenyl-4,4,5,5-
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tetramethylimidazoline-1-oxyl-3-oxide (cPTIO).
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ZmPgbs over-expressing lines reduced root growth, while applications of cPTIO restored root
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growth in lines suppressing ZmPgbs (Table 2).
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observed for many ethylene and ROS biosynthetic genes influenced by ZmPgbs.
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expression of these genes following applications of SNP, SNAP or cPTIO was measured at 12h
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(Supplemental Fig. 16-19), corresponding to the most pronounced alterations observed in the
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transgenic roots (Fig. 3,4).
Increasing NO with SNP or SNAP in the
A similar NO mediated regulation was also
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DISCUSSION
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In a natural growth environment, plants exposed to excess water have a limited availability of
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oxygen necessary to perform many energy-consuming metabolic processes (Dennis et al., 2000).
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As a result, morphological and physiological processes are affected and growth compromised
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(Jackson and Colmer, 2005).
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provided by the root apical meristem (RAM) which is comprised of a cluster of less mitotically-
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active cells, the quiescent center (QC), surrounded by cells with higher proliferative activity
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(Dinneny and Benfey, 2008). The RAM harbors stem cells and, therefore, its preservation during
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adverse conditions is paramount to survival. Oxygen deprivation causes root growth arrest and
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root tip death in a variety of species (Subbaiah et al., 2000), an observation consistent with the
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behaviour of maize seedlings exposed to 4% oxygen (Fig. 1A).
Plant root growth is sustained by the continuous supply of cells
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The expression of ZmPgb1.1 and ZmPgb1.2 in the root tip and their up-regulation under
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hypoxic conditions appear to be a requirement for protecting the meristematic cells and the
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architecture of the RAM, resulting in limits to the inhibitory effect of oxygen deprivation on root
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growth. Suppression of ZmPgbs induces PCD, with cells of the QC (section II) and those at the
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initial stages of differentiation (section III) being the most affected (Fig. 2). The pluripotent cells
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of the QC are the source of stem cells and “integrators” of several processes required for normal
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root growth (Jiang and Feldman, 2005). A key function of the QC is to maintain the root initials
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in an undifferentiated state through non-cell autonomous signals (Scheres, 2007;van den Berg et
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al., 1997). It is, therefore, not surprising that dismantling of the QC cells and their distal
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derivatives by PCD, as observed extensively in hypoxic roots down-regulating ZmPgbs,
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compromises root growth (Fig. 1A).
324
show limited PCD, as well as the greatest root growth rate under hypoxic conditions. Execution
325
of the death program might be, at least in part, due to the suppression of the maize homologue
326
BAX inhibitor-1 (Bi-1), an attenuator of the death programs in eukaryotes (Watanabe and Lam,
327
2006). In addition to being induced in cells up-regulating ZmPgbs (Fig. 3B), the expression of
328
this gene occurred in domains where PCD was limited (Fig. 2). The effect of ZmPgbs in
329
regulating the cell death/survival decision was obvious in immature tissue of the root proper
330
(sections I-IV), but less apparent in the proximal domains (section I), or the distal domains
331
(section V) of the RAM, where the frequency of PCD is unrelated to the expression of ZmPgbs
332
(Fig. 2). This observation suggests that cell responsiveness to ZmPgbs might vary along the root
333
profile, with the meristematic cells (sections II-IV) of the root proper being the most sensitive.
334
While a definitive explanation for this different behaviour cannot be made, future research
335
might focus on the concept that ZmPgb action is critical in those regions of the root that are
This is in contrast to ZmPgbs up-regulating lines that
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336
most susceptible to reduced oxygen diffusion and availability. These regions are likely to
337
include the tightly packed meristematic cells and their immediate derivatives (sections II-IV) that
338
experience oxygen limitation even under normoxic conditions (Armstrong et al., 1994). The
339
protective role of ZmPgbs might, therefore, be essential to alleviate stress and reduce death in
340
these meristematic regions.
341
The results suggest that the ZmPgb control of cell fate in hypoxic root tips is mediated by NO.
342
The effect of PGB on hypoxic growth likely occurs upstream of ROS and ethylene production,
343
since the alleviation of hypoxia on maize root growth can be achieved by either constitutive
344
expression of Pgbs (Fig. 1A) or inhibiting ethylene perception or ROS production in antisense
345
Pgb lines (Fig. 6). In agreement with the NO scavenging properties of ZmPgbs (Dordas et al.,
346
2003b), NO staining appears to increase in meristematic and differentiating cells (sections II-IV)
347
of lines suppressing ZmPgb and decreases in those where the genes are up-regulated (Fig. 2).
348
Regions staining for NO correspond to those accumulating ROS and TUNEL-positive cells (Fig.
349
2). In maize culture cells, the level of ROS is modulated by ZmPgbs through NO (Huang et al.,
350
2014). Hypoxic roots suppressing ZmPgbs, have increased expression of Rbohs (Fig. 3A). The
351
expression of these genes is reduced in cells where ZmPgbs are up-regulated. DPI, an inhibitor
352
of flavoprotein-containing enzymes including NADPH oxidase, decreased the number of cells
353
undergoing PCD (Fig. 6B) and increased expression of BAX inhibitor-1 (Bi-1) (Supplemental
354
Fig. 14) and growth (Fig. 6A) in ZmPgb-suppressing roots under hypoxic conditions, supporting
355
the contention that ROS are required for the induction of the death program.
356
species are implicated in several PCD-inducing responses in maize, including formation of
357
aerenchyma in hypoxic roots (Takahashi et al., 2015) and shaping the body of developing
358
somatic embryos (Huang et al., 2014).
Reactive oxygen
359
Production of ROS requires ethylene, as inhibition of ethylene perception by 1-MCP reduces
360
the transcript levels of Rbohs, ROS signal, the number of TUNEL-positive nuclei, and increases
361
the expression of BAX inhibitor-1 (Bi-1) (Fig. 6 and Supplemental Fig. 10-14). This regulation is
362
consistent with previous studies demonstrating a ROS dependence of several ethylene responses,
363
some of which trigger the death program (Yamauchi et al., 2011). Synthesised during adverse
364
growing conditions, ethylene accumulates preferentially in hypoxic tissue and its production is
365
regulated by the activity of ACC synthase and ACC oxidase, encoded by multigene families
18
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366
(Gallie and Young, 2004).
A rapid transcriptional induction of several members of ACC
367
synthase (ACS) and oxidase (ACO) multigene families, as well as the ethylene signaling related
368
genes Ebf1 and Erf2 follows exposure to low oxygen (Geisler-Lee et al., 2010;Takahashi et al.,
369
2015).
370
ZmPgbs regulate the transcription and localization of ethylene biosynthetic and responsive
371
genes in hypoxic roots. With the exception of ZmAco31, suppression of ZmPgbs induces the
372
expression of all ethylene biosynthetic genes analysed while over-expression of ZmPgbs has a
373
repressive effect (Fig. 4). This pattern, closely reflecting the root tip-specific ZmPgb regulation
374
of ethylene accumulation (Table 1), confirms the transcriptional induction of ethylene,
375
modulated by NO, in alfalfa cells limited in Pgb1 protein (Manac’h-Little et al., 2005).
376
regulation of many ethylene biosynthetic genes by ZmPgbs was more apparent in tissues of the
377
root proper harboring meristematic cells (section II and III in Fig. 5), and was consistent with the
378
accumulation of NO, ROS and the induction of PCD (Fig. 2).
379
Collectively, this study demonstrates a novel function of Pgbs in protecting hypoxic root apical
380
meristems from the NO mediated accumulation of ethylene leading to the over-production of
381
ROS and death. Upregulation of Pgbs in meristematic cells during hypoxia may be indicative of
382
a root acclimation to hypoxia, with the suppression of Pgb expression being associated with
383
avoidance mechanisms, such as aerenchyma formation, that accompany flooding responses.
384
The Pgb protective role of meristematic cells might be a universal response to other forms of
385
stress, including salt and drought, known to modulate Pgbs and reduce root growth by
386
compromising meristem function.
The
387
388
EXPERIMENTAL PROCEDURE
389
Plant material and hypoxic treatment conditions
390
The generation and characterization of maize plants over-expressing or down-regulating
391
ZmPgb1.1 or ZmPgb1.2 was described in previous studies (Youssef et al., 2016 and
392
Supplemental Fig. 3).
393
following the gene abbreviation, while those expressing the Pgb in an anti-sense configuration
Lines constitutively expressing Class 1 Pgbs are designated with an (S)
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Copyright © 2016 American Society of Plant Biologists. All rights reserved.
394
are designated (A). Hypoxic conditions were created exactly as described (Geisler-Lee et al.,
395
2010). Briefly, the roots of 5 day-old seedlings were immersed into a liquid solution (1/2 MS
396
medium) through which air (normoxic conditions) or 4% (v/v) oxygen/96% nitrogen (hypoxic
397
conditions) were bubbled at a rate of 350 ml/min for 24h. Experiments were performed at 22oC
398
under a light intensity of 15 µmol s-1 m-2.
399
Histological analyses, and localization of NO, ROS and PCD
400
For histological examinations, roots were fixed in 2.5% glutaraldehyde and 1.6%
401
paraformaldehyde in 0.05 M phosphate buffer (pH 6.9), dehydrated with methyl cellosolve
402
followed by three washings with absolute ethanol, and then infiltrated and embedded in
403
Historesin (Leica, Concord, Ontario, Canada). Sections (3 µm) were stained with toluidine blue
404
(TBO) (Yeung, 1990).
405
For nitric oxide and reactive oxygen species localization, maize roots were sectioned using a
406
compresstome and respectively stained with DAF-2DA (Elhiti et al., 2013) or dihydroethidium
407
(Tsukagoshi et al., 2010).
408
Nuclear DNA fragmentation was detected with the In Situ Cell Death Detection Kit-
409
Fluorescein (Roche)(as described in Huang et al., 2014).
Tissue was fixed in 4%
410
paraformaldehyde, dehydrated in ethanol series and embedded in wax. Sections (10 μm) were
411
de-waxed in xylene and labeled with the TUNEL kit (Roche) according to the manufacturer’s
412
protocol, with the exclusion of the permeabilization step by proteinase K. Omission of TdT was
413
used for negative controls.
414
415
RNA in situ hybridization
416
417
RNA in situ hybridization studies were performed following the procedure described in Elhiti et
418
al., (2010).
419
Following a 15 min vacuum-infiltration, the samples were incubated for 3h at room temperature
420
and dehydrated in an ethanol series (30, 50, 70, 95, 100, and 100%) for 45 min each at 4o C and
421
left overnight in 100% ethanol. The roots were then treated with increasing levels of xylene and
422
incubated overnight at 42o C in xylene containing a few pellets of paraffin. After incubation at
423
60o C, xylene was replaced gradually with paraffin and the samples were embedded into blocks
Roots were fixed in 4% (w/v) freshly prepared paraformaldehyde in PBS pH 7.4.
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424
that were sectioned at a thickness of 10 μm using disposable blades in a Leica (RM 2145)
425
microtome.
426
xylene and gradual rehydration.
Before hybridization, paraffin was removed from the slides with two changes in
427
For hybridization, cDNAs encoding ZmPgbs, ZmAcs, ZmAco, ZmEbf1 and ZmErf2 were
428
amplified and used for the preparation of digoxigenin (DIG)-labelled sense and antisense
429
riboprobes, following the procedure described in the DIG Application Manual (Roche
430
Diagnostics). Tissue treatments and pre-hybridization steps were performed as described by
431
Canton et al., (1999).
432
Sections were hybridized with sense or antisense probe in 1X Denhardt’s, 1 mg ml-1 tRNA,
433
10% dextran sulfate, 50% formamide, and 1X salts (Regan et al., 1999). Hybridization was
434
conducted at 50oC for 16 h. Post-hybridization washes and antibody treatments were performed
435
as described by Regan et al., (1999). Detection of DIG-labeled probes was carried out using a
436
Western Blue stabilized substrate for Alkaline Phosphatase (Promega, Madison, WI).
437
438
439
Chemical treatments
440
The NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO)
441
and the NO donors sodium nitroprusside (SNP) and S-nitroso-N-acetylpenicillamine (SNAP)
442
were applied at a concentration of 200µM (cPTIO) and 100µM (SNP and SNAP) to ½ MS liquid
443
media during hypoxia conditions. Pre-treatments with 40 μM diphenyleneiodonium chloride
444
(DPI) or 1ppm 1-methylcyclopropene (1-MCP; gift from AgroFresh Inc.) were performed
445
exactly as previously described (Rajhi et al., 2011;Yamauchi et al., 2011).
446
Monitoring of Transcript abundance
447
RNA extraction was carried out using TRI Reagent Solution according to the manufacturer’s
448
protocol (Invitrogen). The total RNA was treated with DNase I recombinant, RNase-free (Roche)
449
and the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems) was used for
450
cDNA synthesis. Quantitative RT-PCR was performed as described in Elhiti et al. (2010). All
451
primers used for gene expression studies are listed in Supplementary Table 1. The relative gene
21
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Copyright © 2016 American Society of Plant Biologists. All rights reserved.
452
expression level was analyzed with the 2−∆∆CT method (Livak and Schmittgen, 2001) using actin
453
as the reference gene.
454
Measurements of ethylene production
455
Ethylene measurements in normoxic or hypoxic corn roots harvested from 5 day old seedlings
456
were performed according to Geisler-Lee et al. (2010). Measurements were conducted on two
457
segments: root tips (0-10mm) and more mature root regions (>10mm from tip). To minimize the
458
amount of ethylene produced by wounding, the root segments (about 15 per replicate) were first
459
pre-incubated in an unsealed 3 mL syringe for 30 min. The syringe was then sealed, incubated in
460
the dark for 2h at 22oC, and 1 mL of the gas accumulated in the headspace was analysed with a
461
Bruker 450-GC Gas chromatograph. Data analysis was carried out using the Bruker Compass
462
Data analysis 3.0. software. All experiments were repeated in triplicates.
463
464
Statistical analysis
465
Data were analyzed by one way ANOVA using the SPSS program (IBM Corp. Released 2010.
466
IBM SPSS Statistics for Windows, Version 19.0. Armonk, NY: IBM Corp.). Treatments means
467
were compared by Tukey test (α = 0.05) to differentiate the significance of differences.
468
Structural and localization studies were performed on at least 15 roots while growth elongation
469
assays, ethylene measurements, and gene expression and pharmacological studies were
470
performed using at least 3 biological replicates each consisting of a minimum of 20 roots.
471
Acknowledgements
472
This work was supported by a grant to CS from the Manitoba Corn Growers Association. The
473
technical support of Mr. Durnin and Dr. John Markham are also acknowledged. The authors
474
thank AgroFresh Inc for providing 1-MCP.
475
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476
477
478
479
480
Table 1. Ethylene level (nmol g-1 FW h-1) measured in tips (0-10 mm) and more mature regions
(>10mm) of corn roots after the imposition of a 24h normoxic (ambient) or hypoxic (4%
oxygen) treatment. Values + SE are means of three biological replicates. * indicate statistical
differences (p<0.05) from WT values in the same treatment using one way ANOVA.
481
_____________________________________________________________________
482
Root tip (0-10mm)
Ambient
WT
ZmPgb1.1(S)
ZmPgb1.1(A)
ZmPgb1.2(S)
ZmPgb1.2(A)
4% oxygen
0.152 ± 0.018
0.104 ± 0.019
0.245 ± 0.117
0.100 ± 0.028
0.185 ± 0.053
0.563 ± 0.028
0.269 ± 0.087
1.518 ± 0.228 *
0.304 ± 0.113
1.962 ± 0.259 *
483
--------------------------------------------------------------------------------------------------------------------
484
Mature root (>10mm)
WT
ZmPgb1.1(S)
ZmPgb1.1(A)
ZmPgb1.2(S)
ZmPgb1.2(A)
485
Ambient
4% oxygen
0.068± 0.013
0.064 ± 0.025
0.114 ± 0.031
0.105± 0.015
0.120± 0.006
0.571 ± 0.165
0.531 ± 0.149
0.579 ± 0.070
0.614 ± 0.070
0.641 ± 0.066
____________________________________________________________________
486
487
488
489
490
491
492
493
494
495
496
497
498
499
500
501
502
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503
504
505
506
507
508
Table 2. Root growth of corn seedling over-expressing (S) or down-regulating (A) ZmPgb1.1
or ZmPgb1.2 exposed for 24h to 4% oxygen. Roots were grown in the absence (untreated) or
presence of the nitric oxide donors SNP or SNAP, or/and the nitric oxide scavanger cPTIO.
Values + SE are means of three biological replicates * Indicate statistically significant
differences (P <0.05) from the respective untreated roots using one way ANOVA.
Genotype
Treatment
Growth (mm)
ZmPgb1.1(S)
untreated
SNP
SNAP
untreated
cPTIO
untreated
SNP
SNAP
untreated
cPTIO
12.17±0.44
5.33±1.25*
5.50±0.79*
3.67±0.37
12.00±1.67*
12.17±0.44
5.17±0.66*
5.83±0.59*
4.53±0.34
14.20±0.49*
ZmPgb1.1(A)
ZmPgb1.2(S)
ZmPgb1.2(A)
509
510
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511
SUPPLEMENTAL MATERIAL
512
Figure S1 (A) Features of cells from the QC of roots of WT seedlings and seedlings over-
513
expressing or suppressing ZmPgb1.1 or ZmPgb1.2 after 24h of hypoxic treatment. (B) Transcript
514
localization of ZmPgb1.1 and ZmPgb1.2 in longitudinal sections of WT roots following 24h of
515
ambient or 4% oxygen treatment. (C) Localization of NO, ROS, PCD, and BaxI-1 transcripts in
516
roots subjected to hypoxic treatment for 24h.
517
Figure S2 Relative expression of ZmPgb1.1 and ZmPgb1.2 in segments of WT roots exposed to
518
4% oxygen for 24 h.
519
Figure S3 Characterization of transgenic lines with altered expression of ZmPgb1.1 or
520
ZmPgb1.2.
521
Figure S4 Relative expression of Respiratory Burst Oxidase Homologs A and B (RbohA, B) in
522
segments of roots exposed to 4% oxygen for 24 h.
523
Figure S5 Relative expression of Respiratory Burst Oxidase Homologs C and D (RbohC, D) in
524
segments of roots exposed to 4% oxygen for 24 h.
525
Figure S6 Relative expression of Bi-1 in segments of roots exposed to 4% oxygen for 24 h.
526
Figure S7 Relative expression of ACC synthase and ACC oxidase in segments of roots exposed
527
to 4% oxygen for 24 h.
528
Figure S8 Relative expression of Erf2 and Ebf1 in segments of roots exposed to 4% oxygen for
529
24 h.
530
Figure S9 Localization of ethylene biosynthetic and response transcripts using RNA in situ
531
hybridization in hypoxic roots after 24h of 4% oxygen for treatment.
532
Figure S10 Relative expression of ZmRboh(A) in segments of roots pre-treated with 1-MCP and
533
exposed to 4% oxygen for 24 h.
534
Figure S11 Relative expression of ZmRboh(B) in segments of roots pre-treated with 1-MCP and
535
exposed to 4% oxygen for 24 h.
25
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Copyright © 2016 American Society of Plant Biologists. All rights reserved.
536
Figure S12 Relative expression of ZmRboh(D) in segments of roots pre-treated with 1-MCP and
537
exposed to 4% oxygen for 24 h.
538
Figure S13 Relative expression of ZmBi-1 in segments of roots pre-treated with DPI or 1-MCP
539
and exposed to 4% oxygen for 24 h.
540
Figure S14 Localization of ROS, PCD, and Bi-1 in roots of seedlings subjected to ambient
541
oxygen treatments for 24h.
542
Figure S15 Root growth of corn seedlings exposed for 24h to ambient air or 4% oxygen in the
543
presence of SNP or SNAP, or/and cPTIO.
544
Figure S16 Relative expression of ACC synthase root tips exposed to 4% oxygen for 12 h.
545
Figure S17 Relative expression of ACC oxidase in root tips exposed to 4% oxygen for 12 h.
546
Figure S18 Relative expression of Erf2 and Ebf1 in root tips exposed to 4% oxygen for 12 h.
547
Figure S19 Relative expression of Rbohs in root tips exposed to 4% oxygen for 12 h.
548
549
550
FIGURE LEGENDS
551
Fig. 1. Root growth behavior and localization of Pgb transcripts in hypoxic roots of maize. (A)
552
Root growth and structure of the root apical meristems of WT roots and roots over-expressing
553
(S) or down-regulating (A) ZmPgb1.1 and ZmPgb1.2 after 24h of 4% oxygen for treatment.
554
Numbers on line graphs represent the relative elongation (4% oxygen/ambient air) at each day of
555
treatment. Values are means + SE of three biological replicates each consisting of at least 20
556
roots. QC, quiescent center; RC, root cap. *significantly different at the same time point (P
557
<0.05). (B) Serial sections used for RNA and protein localization studies. QC, quiescent center;
558
RC, root cap; Ep, epidermis; Ct, cortex; St, stele. (C) RNA in situ localization of ZmPgb1.1 and
559
ZmPgb1.2 transcripts in the 5 serial sections outlined in (B) of roots exposed to ambient air
560
(Amb) or 4% oxygen. NC, negative control in which sections were hybridized with sense ribo-
561
probes.
562
26
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Copyright © 2016 American Society of Plant Biologists. All rights reserved.
563
Fig. 2.
Localization of nitric oxide (NO) by DAF-2DA, reactive oxygen species (ROS) by
564
dihydroethidonium, cell death by TUNEL, and BaxI-1 transcripts by RNA in situ hybridization,
565
in the 5 serial sections of hypoxic roots of WT seedlings and seedlings over-expressing (S) or
566
down-regulating (A) ZmPgb1.1 and ZmPgb1.2 after 24h of 4% oxygen treatment. Bar graphs
567
indicate the % of TUNEL-positive nuclei + SE in different root domains. QC, quiescent center;
568
RC, root cap; Ep, epidermis; Ct, cortex; St, stele. * Significantly different from WT values (P
569
<0.05).
570
571
Fig. 3. Relative expression of the four (A-D) Respiratory Burst Oxidase Homologs (Rbohs) (A)
572
and BAX inhibitor-1 (Bi-1) (B) transcripts in root tips (0-2mm) of maize seedlings subjected to
573
4% oxygen treatments. Values + SE are means of three biological replicates and are normalized
574
to the WT value of day 0 (set at 1). Root tips were harvested from WT seedlings and seedlings
575
over-expressing (S) or down-regulating (A) ZmPgb1.1 and ZmPgb1.2. * Significantly different
576
from WT values (P <0.05) at the same time point.
577
578
Fig. 4. Relative expression of the ethylene biosynthetic Acs and Aco (A) and response Erf2 and
579
Ebf1 (B) genes in root tips (0-2mm) of maize seedlings subjected to 4% oxygen treatments.
580
Values + SE are means of three biological replicates and are normalized to the WT value of day
581
0 (set at 1). Root tips were harvested from WT seedlings and seedlings over-expressing (S) or
582
down-regulating (A) ZmPgb1.1 and ZmPgb1.2. *Significantly different from WT values (P
583
<0.05) at the same time point.
584
Fig. 5.
585
ZmAcs6) and response (ZmEbf1 and ZmErf2) transcripts using RNA in situ hybridization in the
586
serial sections (I-IV) of roots of WT seedlings and seedlings over-expressing (S) or down-
587
regulating (A) ZmPgb1.1 and ZmPgb1.2 exposed for 24h to 4% oxygen or ambient air. NC,
588
negative control using sense riboprobes (Aco, Acs, Ebf1 and Erf2).
589
Fig. 6. Effects of suppression of ROS by diphenyleneiodonium (DPI) and ethylene perception
590
by 1-methylcyclopropene (1-MCP) on root behaviour. (A) Effects of DPI and 1-MCP on root
Localization of the ethylene biosynthetic (ZmAco20/35, ZmAco15/31, ZmAcs2/7,
27
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591
growth of WT seedlings and seedlings down-regulating (A) ZmPgb1.1 or ZmPgb1.2 after 24h of
592
4% oxygen treatment. Values + SE are means of three biological replicates.
593
statistically significant values from WT value (ambient). (B)
594
dihydroethidonium, PCD by TUNEL, and BAX inhibitor-1 transcripts by RNA in situ
595
hybridization in the serial sections (I-V) of hypoxic roots of ZmPgb1.1 or ZmPgb1.2 suppressing
596
seedlings treated with DPI or 1-MCP.
* indicates
Localization of ROS by
597
598
599
600
28
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Copyright © 2016 American Society of Plant Biologists. All rights reserved.
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