Phytoglobins improve hypoxic root growth by alleviating apical meristem cell death

32 Pages·2016·4.57 MB·English

Plant Physiology Preview. Published on October 4, 2016, as DOI:10.1104/pp.16.01150
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2 Phytoglobins improve hypoxic root growth by alleviating apical meristem cell death
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5 Mohamed M. Mira1, Robert D. Hill2, Claudio Stasolla2*
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7 1 Permanent address: Department of Botany, Faculty of Science, Tanta University, Tanta, Egypt
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9 2Department of Plant Science, University of Manitoba, Winnipeg, Manitoba, R3T 2N2, Canada
10 *Corresponding author E-mail: [email protected]
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12 Tel. [phone]
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14 Author contributions: MMM performed most of the experiments and contributed to the
15 analysis of the data and writing of the manuscript; RDH contributed to the conception of the
16 project, the research plans the data analysis and the writing of the manuscript; CS contributed to
17 the project conception, the research plans, data analysis, some of the experimentation,
18 manuscript writing and was responsible for the overall supervision of the project.
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22 Running title: Phytoglobin influences hypoxic root growth
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24 Word count: 4790
25 Abstract, 227; Introduction, 728; Discussion, 980; Experimental Procedure, 1318, Figure
26 Legend 488
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33 ABSTRACT
34 Hypoxic root growth in maize is influenced by expression of phytoglobins (ZmPgbs). Relative
35 to WT, suppression of ZmPgb1.1 or ZmPgb1.2 inhibits growth of roots exposed to 4% oxygen
36 causing structural abnormalities in the root apical meristems. These effects were accompanied
37 by increasing levels of reactive oxygen species (ROS), possibly through the transcriptional
38 induction of four Respiratory Burst Oxidase Homologs (Rbohs). TUNEL-positive nuclei in
39 meristematic cells indicated the involvement of programmed cell death (PCD) in the process.
40 These cells also accumulated nitric oxide (NO) and stained heavily for ethylene biosynthetic
41 transcripts. A sharp increase in the expression level of several ACC synthase (ZmAcs2, 6, and
42 7), ACC oxidase (Aco15, 20, 31, and 35), and ethylene responsive (ZmErf2 and ZmEbf1) genes
43 was observed in hypoxic ZmPgb-suppressing roots, that overproduced ethylene. Inhibiting ROS
44 synthesis with diphenyleneiodonium or ethylene perception with 1-methylcyclopropene (1-MCP)
45 suppressed PCD, increased BAX inhibitor-1 (Bi-1), an effective attenuator of the death programs
46 in eukaryotes, and restored root growth. Hypoxic roots over-expressing ZmPgbs had the lowest
47 level of ethylene and showed a reduction in ROS staining and TUNEL-positive nuclei in the
48 meristematic cells. These roots retained functional meristems and exhibited the highest growth
49 performance when subjected to hypoxic conditions. Collectively these results suggest a novel
50 function of PGBs in protecting root apical meristems from hypoxia-induced PCD through
51 mechanisms initiated by NO and mediated by ethylene via ROS.
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53 Keywords: ethylene, hypoxia, maize, phytoglobins, programmed cell death, reactive oxygen
54 species, root apical meristem.
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61 INTRODUCTION
62 Oxygen deficiency (hypoxia), experienced by plants grown in poorly drained soils or subjected
63 to flooding, impairs plant growth and results in heavy crop losses (Dennis et al., 2000).
64 Submergence or flooding reduces oxygen availability for plant cells inhibiting gas exchange
65 required for basic physiological processes (Bailey-Serres and Voesenek, 2008). Both roots and
66 shoots are affected by hypoxia, regardless of whether the plant is submerged or only the root is
67 exposed to the condition. The consequences to shoots of prolonged root hypoxia include
68 reduced photosynthetic rate and stomatal conductance, decreased leaf growth and senescence,
69 wilting of the above ground organs and alterations in plant water relations (Mustroph and
70 Albrecht, 2003). Ethylene accumulates rapidly in flooded Rumex palustris Sm root cells
71 (Visser et al., 1996) and in some species ethylene affects the selective death of cortical cells
72 generating lysogenous aerenchyma (Drew et al., 2000;Drew et al., 1979;Drew, 1997) Precursors
73 of ethylene have been shown to induce changes in B. napus growth behavior and root
74 architecture (Patrick et al., 2009). Depending upon concentration and species, ethylene can
75 either stimulate or inhibit root growth (Konings and Jackson, 1979). Ethylene regulation of
76 programmed cell death (PCD) is not restricted to hypoxia, but rather is observed in response to
77 many adverse growth conditions (Abeles.et.al., 1992;Buer et al., 2003;Clark et al., 1999;Drew et
78 al., 1979;Feldman, 1984;Pitts et al., 1998). Execution of PCD in maize roots under hypoxic
79 conditions is triggered by a rapid increase in ethylene level resulting from the transcriptional
80 induction of 1-aminocyclopropane-1-carboxylate synthase (ACS) and oxidase (ACO) (Geisler-
81 Lee et al., 2010) and transduced through the generation of reactive oxygen species (ROS)
82 produced by NADPH oxidase activity (Torres and Dangl, 2005). Progression of these events
83 in maize roots has been shown using 1-methylcyclopropene (1-MCP), as a specific inhibitor of
84 ethylene perception, or diphenyleneiodonium (DPI) to inhibit ROS production (Takahashi et al.,
85 2015).
86 While considerable attention has been paid to the mechanisms underlying PCD during
87 aerenchyma formation, no information is currently available on other death programs occurring
88 in other regions of hypoxic roots, including the root tip. Maize root tips are very sensitive to
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89 flooding stress and die after a few hours, compromising survival upon the reestablishment of
90 normoxic conditions (Roberts et al., 1984). The root apical meristem (RAM) harbors stem cells
91 and performs the task of organizing centers for post-embryonic morphogenesis (Jiang and
92 Feldman, 2005). These crucial functions are evidenced by its conserved structure. The maize
93 RAM consists of a quiescent center (QC), comprising 800-1200 slowly dividing cells,
94 surrounded by more actively dividing stem cells (Kerk and Feldman, 1995). Genetic or
95 environmental perturbations of RAM function lead to growth inhibition or cessation (Blilou et
96 al., 2005). Recent work identified ethylene as a central regulator of RAM function (Street et al.,
97 2015).
98 Phytoglobins (PGBs), previously termed nonsymbiotic hemoglobins (Hill et al, 2016), are
99 heme-containing proteins characterized mainly for their ability to remove nitric oxide (NO)
100 under adverse conditions, including hypoxia (Hill, 2012). Phytoglobins are rapidly induced in
101 cells grown under limited oxygen (Silva-Cardenas et al., 2003) and experimental changes in their
102 expression level affect plant response to stress. In Arabidopsis, ectopic expression of one Pgb
103 enhanced survival to low oxygen conditions (Hunt et al., 2002), while hypoxic alfalfa roots and
104 maize cells over-expressing Pgbs maintained growth and sustained a high energy status (Dordas
105 et al., 2003a;Igamberdiev and Hill, 2004). In culture, suppression of Pgbs enhances ethylene
106 synthesis (Manac'h-Little et al., 2005) and induces PCD in maize through ROS production
107 (Huang et al., 2014). These observations, in conjunction with the root tip localization of one
108 maize Pgb (Dordas et al., 2003b;Zhao et al., 2008) are the premises of the present work, to
109 determine whether Pgbs exercise a protective role by limiting meristematic cell death in the
110 hypoxic RAM through regulation of ethylene and ROS.
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113 RESULTS
114 Expression of ZmPgbs affects hypoxic root growth
115 Root growth of 5 day-old seedlings with altered expression of ZmPbg1.1 or ZmPgb1.2 was
116 compared under normoxic (ambient air) or hypoxic (4% oxygen) conditions. Growth of WT
117 hypoxic roots was more than 40 percent impaired after hypoxic treatment for 24 h, while
118 ZmPgb1.1 or ZmPgb1.2 over-expressing roots [ZmPgb1.1(S) and ZmPgb1.2(S)] showed less
119 than 30 percent reduction in growth (Fig. 1A). In the lines suppressing either of the ZmPgbs
120 [ZmPgb1.1(A) and ZmPgb1.2(A)] there was substantially reduced root growth of the order of 60
121 to 80 percent during the same period with evidence of abnormalities within the root apices.
122 Structural disorganization of the root tip (Fig. 1A) and formation of large vacuoles within cells of
123 the quiescent center (QC) (Supplemental Fig. 1A), a sign of differentiation, were often observed
124 in roots suppressing either ZmPgb1.1 or ZmPgb1.2.
125 Expression of ZmPgbs was measured in segments (0-2, 2-5, 5-10, and 10-20 mm from the tip)
126 of hypoxic WT roots. Hypoxia induced ZmPgb1.1 and ZmPgb1.2, especially in proximity of the
127 root tip (segments 0-2mm and 2-5mm), with maximum expression occurring at 12 h
128 (Supplemental Fig. 2). Differences in expression levels between normoxic and hypoxic
129 conditions were attenuated in more mature regions of the root (segments 5-10 and 10-20mm).
130 To enhance resolution, RNA in situ localization studies of both ZmPgbs were performed on
131 progressive transverse sections along the RAM (Fig. 1B). These sections included the root cap
132 (section I), the QC (section II), domains with initial (section III) and advanced (section IV)
133 regions of cellular differentiation, and in mature fully differentiated tissue (section V) (Fig. 1B).
134 Hypoxic conditions increased the staining of ZmPgb1.1, and, to a lesser extent, ZmPgb1.2, in the
135 central cells of the root cap (section I, Fig. 1C). Increased expression of ZmPgbs as a result of
136 hypoxia was particularly evident in the QC region (section II) and in tissue undergoing early
137 differentiation (section III). Heavy induction of ZmPgb1.1 was also observed in hypoxic cells
138 at advanced stages of differentiation (section IV). Specificity of the signal was verified using
139 sense ribo-probes as a negative control (NC) (Fig. 1C). Longitudinal sections of hypoxic roots
140 also displayed evidence of heavy staining for ZmPgbs (Supplemental Fig. 1B).
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142 ZmPgb regulation of NO, ROS and PCD in hypoxic RAM
143 The different growth behavior of corn roots with altered expression of ZmPgbs was further
144 examined in light of the following observations: phytoglobins scavenge NO (Dordas et al.,
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145 2003a); when Pgb expression is suppressed, NO accumulates, inducing ROS production (Huang
146 et al., 2014) that triggers PCD (Van and Dat, 2006). Altered ZmPgb expression was achieved by
147 the use of maize transgenic lines (Youseff et al, 2016) that constitutively expressed ZmPgb1.1 or
148 ZmPgb1.2 in either the sense (S) or antisense (A) orientations. The relative expression of a
149 particular ZmPgb in normoxic root lines is shown in Supplemental Fig. 3B. ZmPgb (S) lines
150 had ZmPgb levels approximately 15-20 fold higher than the WT line while Pgb(A) lines had
151 expression levels that were less than 10 percent of the WT line.
152 Examining the effect of varying Pgb expression, with the exception of the root cap
153 (Section I), there was visual evidence of an increase in staining for NO, ROS and PCD in
154 ZmPgb(A) lines and a decrease in ZmPgb(S) lines relative to WT as a result of hypoxia (Fig. 2).
155 Consistent with the evidence of PCD in the sections, staining for transcripts of BAX inhibitor-1
156 (Bi-1), an attenuator of PCD (Watanabe and Lam 2006), was pronounced in sense lines and
157 reduced in antisense lines compared to the WT. In sections II, III and IV, the extent of PCD as
158 measured by TUNEL assays was significantly different from that in WT sections for certain cell
159 types in both sense and antisense lines of the two Class 1 Pgbs.
160 With respect to NO, ROS and PCD in the various sections, the response to hypoxia in the
161 root cap (Section I) displayed no apparent change as a result of varying Pgb expression (Fig. 2).
162 Although there were some slight visual differences for NO and ROS in micrographs of the more
163 mature, fully differentiated section V, there were no significant differences in the extent of PCD
164 amongst the lines. Most of the effects of Pgb variation on NO, ROS and PCD appeared to be in
165 the root meristem (Section II) and tissue undergoing differentiation (Sections III and IV).
166 Evidence of increased NO, ROS and significantly increased PCD in the quiescent center,
167 compared to the WT, was found in antisense lines of Section II, with decreased expression of Bi-
168 1. About 90 percent PCD occurred in the cells of the quiescent center in the ZmPgb(A) lines. The
169 situation was reversed for NO, ROS and PCD in the sense lines, with PCD declining
170 significantly in the quiescent center to around 5 percent of the cells. In Section III, where most of
171 the cells are in the stage of early differentiation, altering Pgb expression had an effect on the
172 staining of NO, ROS and PCD in the cortex, epidermis and portions of the stele as a result of
173 hypoxia. The antisense lines had significantly increased PCD in these regions with the extent of
174 cell death approaching 90 percent in some instances. In the sense lines, PCD was significantly
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175 depressed to around one percent in the epidermis and stele of the ZmPgb1.1 line and to around 5
176 percent in the stele of the ZmPgb1.2 line. In the region of more advanced differentiation (Section
177 IV), altering Pgb expression had an effect largely in the area of the cortex, where increased
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178 expression reduced the intensity of staining for NO and ROS and increased that of Bi-1. PCD
179 was significantly lower in the cortex of the ZmPgb(S) lines. The reverse effect was observed in
180 the antisense lines, with significantly increased PCD in the cortex of both lines. The level of
181 PCD in the cortical cells of this region, even in the antisense lines, reached only 30 percent in
182 comparison to the meristem and early differentiation regions of the root where PCD approached
183 90 percent. Longitudinal sections of hypoxic roots showed similar patterns when stained for NO,
184 ROS and PCD (Supplemental Fig. 1C).
185 To further examine the relationship between Pgb expression and PCD, the expression of
186 Rbohs and Bi-1 in root sections of the lines was determined by qRT-PCR over the 24h of the
187 hypoxic treatment. In the 0-2 mm region of the root tip (Fig. 3A), anti-sensing either one of the
188 two Pgbs resulted in significantly increased levels of most Rboh transcripts relative to WT
189 throughout the hypoxic treatment, with maximum levels occurring in the period 6 to 12h after the
190 initiation of the treatment. Constitutive over-expression of the Pgbs gave varying results, ranging
191 from significant decreases in transcript abundance for RbohA to no differences for RbohB
192 throughout the treatment. For RbohC and RbohD there was a significant decrease in transcript
193 levels in the sense lines at 12h hypoxia, largely due to increased expression of these two genes in
194 the WT line at that time point. Similar results were obtained for sections 2-5, 5-10 and 10-20 mm
195 back from the root tip (Supplemental Fig. 4 and 5), although the differences become less distinct
196 and significant in the regions farthest removed from the tip. Significantly higher levels of Bi-1
197 transcripts relative to WT were present in the 0-2 mm section at the beginning of the hypoxic
198 treatment in the sense lines and remained significantly higher throughout the treatment, with the
199 ZmPgb1.1 line being slightly higher (Fig. 3B). The effects of Pgb variation on Bi-1 transcript
200 abundance were similar for the 2-5, 5-10, and 10-20 mm sections (Supplemental Fig. 6),
201 although the level of expression declined in all lines as the distance from the root tip increased.
202 The results of Fig. 2 and 3 suggest that PGBs are a factor in maintaining the viability of
203 root meristem and differentiating cells during hypoxic stress. This suggestion is supported by the
204 results of Fig. 1 that show increased expression of both Pgbs in the regions of the QC of Section
205 II, increased expression of ZmPgb1.2 in portions of the stele, cortex and epidermis of section III
206 and a general increase in expression of ZmPgb1.1 throughout section IV when the lines are
207 exposed to hypoxia. Levels of Pgb double or triple in the root meristem region within two hours
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208 of the start of the hypoxic treatment, becoming 5-6 fold higher for ZmPgb1.2 within 12 hours
209 (Supplemental Fig. 2).
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