Volume 98, Issue 2 pp. 291-300

Original Article

Free Access

Comparative analysis of the reactive oxygen species-producing enzymatic activity of Arabidopsis NADPH oxidases

Hidetaka Kaya,

Corresponding Author

Hidetaka Kaya

[email protected]

Department of Applied Biological Science, Tokyo University of Science, 2641 Yamazaki, Noda, Chiba, 278–8510, Japan

Graduate School of Agriculture, Ehime University, 3-5-7 Tarumi, Matsuyama, Ehime, 790-8566 Japan

These authors contributed equally to this work.For correspondence (e-mails [email protected]; [email protected]).Search for more papers by this author

Seiji Takeda,

Seiji Takeda

Graduate School of Life and Environmental Sciences, Kyoto Prefectural University, 1-5 Hangi-cho, Shimogamo, Sakyo-ku, Kyoto, 606-8522 Japan

Biotechnology Research Department, Kyoto Prefectural Agriculture Forestry and Fisheries Technology Center, Kitainayazuma Oji 74, Seika-cho, Soraku-gun, Kyoto, 619-0244 Japan

These authors contributed equally to this work.Search for more papers by this author

Masaki J. Kobayashi,

Masaki J. Kobayashi

Japan International Research Center for Agricultural Sciences, 1-1 Ohwashi, Tsukuba, Ibaraki, 305-8686 Japan

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Sachie Kimura,

Sachie Kimura

Department of Applied Biological Science, Tokyo University of Science, 2641 Yamazaki, Noda, Chiba, 278–8510, Japan

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Ayako Iizuka,

Ayako Iizuka

Department of Applied Biological Science, Tokyo University of Science, 2641 Yamazaki, Noda, Chiba, 278–8510, Japan

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Aya Imai,

Aya Imai

Department of Applied Biological Science, Tokyo University of Science, 2641 Yamazaki, Noda, Chiba, 278–8510, Japan

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Haruka Hishinuma,

Haruka Hishinuma

Department of Applied Biological Science, Tokyo University of Science, 2641 Yamazaki, Noda, Chiba, 278–8510, Japan

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Tomoko Kawarazaki,

Tomoko Kawarazaki

Department of Applied Biological Science, Tokyo University of Science, 2641 Yamazaki, Noda, Chiba, 278–8510, Japan

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Kyoichiro Mori,

Kyoichiro Mori

Department of Applied Biological Science, Tokyo University of Science, 2641 Yamazaki, Noda, Chiba, 278–8510, Japan

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Yuta Yamamoto,

Yuta Yamamoto

Department of Applied Biological Science, Tokyo University of Science, 2641 Yamazaki, Noda, Chiba, 278–8510, Japan

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Yuki Murakami,

Yuki Murakami

Department of Applied Biological Science, Tokyo University of Science, 2641 Yamazaki, Noda, Chiba, 278–8510, Japan

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Ayuko Nakauchi,

Ayuko Nakauchi

Department of Applied Biological Science, Tokyo University of Science, 2641 Yamazaki, Noda, Chiba, 278–8510, Japan

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Mitsutomo Abe,

Mitsutomo Abe

Department of Biological Science, Graduate School of Science, The University of Tokyo, Bunkyo-ku, Tokyo, 113-0033 Japan

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Kazuyuki Kuchitsu,

Corresponding Author

Kazuyuki Kuchitsu

[email protected]

Department of Applied Biological Science, Tokyo University of Science, 2641 Yamazaki, Noda, Chiba, 278–8510, Japan

For correspondence (e-mails [email protected]; [email protected]).Search for more papers by this author

Hidetaka Kaya,

Corresponding Author

Hidetaka Kaya

[email protected]

Department of Applied Biological Science, Tokyo University of Science, 2641 Yamazaki, Noda, Chiba, 278–8510, Japan

Graduate School of Agriculture, Ehime University, 3-5-7 Tarumi, Matsuyama, Ehime, 790-8566 Japan

These authors contributed equally to this work.For correspondence (e-mails [email protected]; [email protected]).Search for more papers by this author

Seiji Takeda,

Seiji Takeda

Graduate School of Life and Environmental Sciences, Kyoto Prefectural University, 1-5 Hangi-cho, Shimogamo, Sakyo-ku, Kyoto, 606-8522 Japan

Biotechnology Research Department, Kyoto Prefectural Agriculture Forestry and Fisheries Technology Center, Kitainayazuma Oji 74, Seika-cho, Soraku-gun, Kyoto, 619-0244 Japan

These authors contributed equally to this work.Search for more papers by this author

Masaki J. Kobayashi,

Masaki J. Kobayashi

Japan International Research Center for Agricultural Sciences, 1-1 Ohwashi, Tsukuba, Ibaraki, 305-8686 Japan

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Sachie Kimura,

Sachie Kimura

Department of Applied Biological Science, Tokyo University of Science, 2641 Yamazaki, Noda, Chiba, 278–8510, Japan

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Ayako Iizuka,

Ayako Iizuka

Department of Applied Biological Science, Tokyo University of Science, 2641 Yamazaki, Noda, Chiba, 278–8510, Japan

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Aya Imai,

Aya Imai

Department of Applied Biological Science, Tokyo University of Science, 2641 Yamazaki, Noda, Chiba, 278–8510, Japan

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Haruka Hishinuma,

Haruka Hishinuma

Department of Applied Biological Science, Tokyo University of Science, 2641 Yamazaki, Noda, Chiba, 278–8510, Japan

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Tomoko Kawarazaki,

Tomoko Kawarazaki

Department of Applied Biological Science, Tokyo University of Science, 2641 Yamazaki, Noda, Chiba, 278–8510, Japan

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Kyoichiro Mori,

Kyoichiro Mori

Department of Applied Biological Science, Tokyo University of Science, 2641 Yamazaki, Noda, Chiba, 278–8510, Japan

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Yuta Yamamoto,

Yuta Yamamoto

Department of Applied Biological Science, Tokyo University of Science, 2641 Yamazaki, Noda, Chiba, 278–8510, Japan

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Yuki Murakami,

Yuki Murakami

Department of Applied Biological Science, Tokyo University of Science, 2641 Yamazaki, Noda, Chiba, 278–8510, Japan

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Ayuko Nakauchi,

Ayuko Nakauchi

Department of Applied Biological Science, Tokyo University of Science, 2641 Yamazaki, Noda, Chiba, 278–8510, Japan

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Mitsutomo Abe,

Mitsutomo Abe

Department of Biological Science, Graduate School of Science, The University of Tokyo, Bunkyo-ku, Tokyo, 113-0033 Japan

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Kazuyuki Kuchitsu,

Corresponding Author

Kazuyuki Kuchitsu

[email protected]

Department of Applied Biological Science, Tokyo University of Science, 2641 Yamazaki, Noda, Chiba, 278–8510, Japan

For correspondence (e-mails [email protected]; [email protected]).Search for more papers by this author

First published: 20 December 2018

https://doi.org/10.1111/tpj.14212

Citations: 56

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Summary

Reactive oxygen species (ROS) produced by NADPH oxidases, called respiratory burst oxidase homologs (Rbohs), play crucial roles in development as well as biotic and abiotic stress responses in plants. Arabidopsis has 10 Rboh genes, AtRbohA to AtRbohJ. Five AtRbohs (AtRbohC, -D, -F, -H and -J) are synergistically activated by Ca²⁺-binding and protein phosphorylation to produce ROS that play various roles in planta, although the activities of the other Rbohs remain unknown. With a heterologous expression system, we found a range of ROS-producing activity among the AtRbohs with differences up to 100 times, indicating that the required amounts of ROS are different in each situation where AtRbohs act. To specify the functions of AtRbohs involved in cell growth, we focused on AtRbohC, -H and -J, which are involved in tip growth of root hairs or pollen tubes. Ectopic expression of the root hair factor AtRbohC/ROOT HAIR DEFECTIVE 2 (RHD2) in pollen tubes restored the atrbohH atrbohJ defects in tip growth of pollen tubes. However, expression of AtRbohH or -J in root hairs did not complement the tip growth defect in the atrbohC/rhd2 mutant. Our data indicate that Rbohs possess different ranges of enzymatic activity, and that some Rbohs have evolved to carry specific functions in cell growth.

Introduction

Aerobic organisms need to deal with the unavoidable generation of reactive oxygen species (ROS) by aerobic respiration, including the superoxide anion radical (O₂⁻), hydrogen peroxide (H₂O₂) and the hydroxyl radical (^•OH), because they can damage DNA, protein and lipids and thus are highly toxic in cells (Kehrer, 2000). This situation is particularly severe in plants due to the generation of ROS through photosynthesis. Recent findings have revealed that most organisms produce ROS by several kinds of enzymes and use them as signaling molecules for environmental responses and development (Kärkönen and Kuchitsu, 2015). In plants, ROS are involved in numerous processes such as disease resistance responses (Levine et al., 1994), salinity stress responses (Kurusu et al., 2015; Hossain and Dietz, 2016), cell growth by scission or cross-linking of cell walls (Everdeen et al., 1988; Fry, 1998; Schweikert et al., 2000; Schopfer, 2001; Rodríguez et al., 2002; Schopfer et al., 2002), root gravitropism (Joo et al., 2001), lateral root emergence (Orman-Ligeza et al., 2016), cell-fate transition in roots (Tsukagoshi et al., 2010), the development of male and female gametophytes (Jiménez-Quesada et al., 2016), mutualistic symbiosis with fungi (Tanaka et al., 2006), long-distance signal transduction (Miller et al., 2009), the development of Casparian strips (Lee et al., 2013) and organ abscission (Lee et al., 2018).

The NADPH oxidases (Nox) are ROS-producing enzymes that are conserved in many eukaryotes and play roles in signal transduction in defense and developmental processes. The Nox proteins carry six transmembrane domains and a conserved C-terminal region containing the NADPH-binding and FAD-binding motifs, which are crucial for transferring electrons to generate superoxide anions (Sumimoto, 2008). The mammalian gp91^phox/Nox2, acting together with p22^phox, requires other protein components such as p47^phox, p67^phox and Rac small GTPase for its activation, but plants do not appear to carry the clear homologs of p47^phox or p67^phox (Sumimoto, 2008). Generally, plant Nox proteins, called respiratory burst oxidase homologs (Rbohs), carry two EF-hand motifs and phosphorylation sites at the cytological N-terminal region, in addition to the transmembrane domains and C-terminal region (Figure 1a; Sumimoto, 2008; Oda et al., 2010; Kärkönen and Kuchitsu, 2015). This indicates that during evolution organisms have established their own regulatory system to activate the Nox enzymes.

Details are in the caption following the image — **Figure 1**
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Comparison of ionomycin-induced reactive oxygen species (ROS)-producing activities among Arabidopsis respiratory burst oxidase homologs (AtRbohs).(a) Schematic representation of the construct for AtRboh expression. The AtRbohs carry N-terminal EF-hand motifs and C-terminal FAD- and NADPH-binding domains, connected by six transmembrane (TM) helices. (b) Western blotting analysis of the expression levels of N-terminal 3×FLAG-tagged AtRbohA–AtRbohJ proteins in transfected human embryonic kidney (HEK) 293T cells. β-actin was used as a loading control. (c)–(e) Ionomycin-induced production of reactive oxygen species (ROS) in *AtRbohA–AtRbohJ* transfected HEK293T cells. Empty vector-transfected cells were used to measure an internal ROS-producing activity in HEK293T. After baseline measurements of ROS production for 5 min, 1 μm ionomycin was added to the culture medium. The ROS production was measured by chemiluminescence and is shown as relative luminescence units (RLU) per second. Results given are means ± standard error (n = 3). (f) Comparison of maximum ROS-producing activities between *AtRbohA–AtRbohJ* transfected HEK293T cells. The P-values were determined using Student's t-test (*P < 0.05; **P > 0.05).

The Rboh genes were first isolated from rice and then from Arabidopsis thaliana as homologs of mammalian gp91^phox/Nox2 (Groom et al., 1996; Torres et al., 1998). So far nine and ten Rbohs have been identified in rice and Arabidopsis, respectively (Kaur et al., 2014; Kaur and Pati, 2016). They have also been identified in other plants including tobacco, potato, tomato, Medicago and maize, where they regulate pathogen defense, abiotic stress responses, symbiosis or cell growth (Yoshioka et al., 2001, 2003; Simon-Plas et al., 2002; Liszkay et al., 2004; Sagi et al., 2004; Marino et al., 2011; Nestler et al., 2014). The 10 Rboh genes in Arabidopsis are referred to as AtRbohA to AtRbohJ, and genetic studies have suggested that they have distinct roles (Table 1).

Table 1. Rboh genes in Arabidopsis thaliana

	Clade	AGI	Molecular weighta	Functions
Subfamilies I and II
AtRbohA	ACG	At5g07390	102934.6
AtRbohB	B	At1g09090	96389.3	Seed after-ripening1
AtRbohC	ACG	At5g51060	102517.4	Root hair tip growth2,3 , root hydrotropism4, mechanosensing5
AtRbohD	D	At5g47910	103907.7	Pathogen defense6,7, stomatal closure8 , osmosensitive metabolic changes induced by cellulose biosynthesis inhibitor9, rapid systemic signaling in response to exogenous stimuli10 , lignin deposition caused by cell wall biosynthesis inhibitor11,12, salinity stress response13 , systemic acquired acclimation by abiotic stress14, jasmonic acid-induced ROS production15, abscission of floral organs16
AtRbohG	ACG	At4g25090	96861.7
Subfamily III & IV
AtRbohE	E	At1g19230	107701.6	Programmed cell death in tapetum16
AtRbohF	F	At1g64060	108417.3	Pathogen defense6,7, stomatal closure8,osmosensitive metabolic changes induced by cellulose biosynthesis inhibitor9, lignin deposition caused by cell wall biosynthesis inhibitor11,12, salinity-stress response13, jasmonic acid-induced ROS production15, Casparian strip formation16, abscission of floral organs17
AtRbohI	I	At4g11230	106951.0	Drought stress response in seeds and roots17
Subfamily V
AtRbohH	HJ	At5g60010	100626.7	Pollen tube tip growth18,19,20, Polar root hair growth21
AtRbohJ	HJ	At3g45810	102936.2	Pollen tube tip growth18, Polar root hair growth21

^aMolecular weight information from the TAIR website.
¹Müller et al. (2009); ²Foreman et al. (2003); ³Takeda et al. (2008); ⁴Krieger et al. (2016); ⁵Monshausen et al., 2009; ⁶Torres et al., 2002; ⁷Torres et al., 2005; ⁸Kwak et al., 2003; ⁹Wormit et al., 2012; ¹⁰Miller et al., 2009; ¹¹Hamann et al., 2009; ¹²Denness et al., 2011; ¹³Ma et al., 2012; ¹⁴Suzuki et al., 2013; ¹⁵Maruta et al. 2011; ¹⁶Lee et al., 2018; ¹⁷Lee et al., 2018; He et al., 2017; ¹⁸Kaya et al., 2014; ¹⁹Boisson-Dernier et al., 2013; ²⁰Lassig et al., 2014; ²¹Mangano et al., 2017.

While the spatio-temporal control of AtRboh expression is important for their functions (Morales et al., 2016; Orman-Ligeza et al., 2016), control of the enzymatic activity of AtRboh proteins is also crucial at a cellular level, as high levels of ROS can be toxic for cells (Yun et al., 2011; Kadota et al., 2014). Their activities can be successfully analyzed by heterologous expression in the human embryonic kidney (HEK) 293T cell line (Ogasawara et al., 2008), which lacks expression of endogenous NADPH oxidases Nox1 and Nox2 and does not produce significant amounts of extracellular ROS (Shiose et al., 2001). Transient expression in HEK293T cells has shown that Ca²⁺ binding to the EF-hand and protein phosphorylation synergistically activates the ROS-producing activity of AtRbohC/ROOT HAIR DEFECTIVE 2 (RHD2), AtRbohD, AtRbohF, AtRbohH and AtRbohJ (Ogasawara et al., 2008; Takeda et al., 2008; Kimura et al., 2012; Kaya et al., 2014). Co-expression analyses of AtRbohs and their putative regulators such as AtCIPK26 or AtSRC2 have also suggested regulatory mechanisms for the activity of AtRboh proteins (Kawarazaki et al., 2013; Kimura et al., 2013). However, the ROS-producing activity of other AtRbohs remains unclear.

Here we examined the enzymatic activity of all 10 AtRbohs in the HEK293T cell line to compare their ROS-producing ability. Our data revealed striking differences in the ROS-producing activity of these enzymes. Moreover, we performed complementary analysis of AtRbohs involved in tip growth of pollen tubes or root hairs, indicating the functional diversification of AtRbohs during evolution.

Results and Discussion

Comparison of the ROS-producing enzymatic activity of AtRbohs

To compare the ROS-producing enzymatic activity of AtRbohs we used a heterologous expression system with HEK293T cells. Coding sequences of AtRboh were fused to a 3×FLAG epitope tag (Figure 1a) to detect expressed proteins by Western blot analysis, confirming comparable expression in HEK293T cells (Figure 1b). After baseline measurements of ROS production for 5 min, 1 μm of ionomycin, a Ca²⁺ ionophore that induces influx of Ca²⁺ into the cells, was added to the medium. Ionomycin triggered rapid and transient production of ROS in all AtRboh-transfected cells, but not in empty vector-transfected controls (Figure 1c–e), suggesting that Ca²⁺ influx induces the ROS-producing enzymatic activity of AtRbohs. The highest activity was carried by AtRbohH, followed by AtRbohJ, -C and -A (Figure 1c). AtRbohA, -D, -E and -G produced around 10 times less ROS, and AtRbohG, -F, -I and -B produced even less than this (Figure 1d,e). The specific ROS-producing activity varied 100-fold between different AtRbohs in HEK293T cells (Figure 1f).

Ca²⁺ regulation in ROS production is conserved among AtRbohs

The cytosolic N-terminal region of AtRbohs contains two EF-hand motifs, where the conformation changes upon Ca²⁺-binding, leading to the activation of ROS production (Ogasawara et al., 2008; Oda et al., 2010). Point mutation in the conserved glutamic acid (E at the position –Z) of the first EF-hand Ca²⁺-binding loop reduces Ca²⁺ affinity and significantly abolishes the ROS-producing activity of AtRbohC/RHD2, AtRbohD, -F, -H and -J (Ogasawara et al., 2008; Takeda et al., 2008; Kimura et al., 2012; Kaya et al., 2014). To examine whether the importance of this amino acid residue is conserved among AtRbohs, we analyzed the ROS-producing activity of mutated AtRbohs where the E at the position –Z was converted to glutamine. Mutated proteins of AtRbohA, -B, -E and -G showed reduced ionomycin-triggered ROS-producing activity without affecting the level of expression (Figure 2a–d). The E residue at the –Z position of the first EF-hand is predicted to bind Ca²⁺, inducing a conformational change in the EF-hand region (Ogasawara et al., 2008; Oda et al., 2010). Together with the previous data, our results suggest that binding of Ca²⁺ to the EF-hand motifs is a common regulatory mechanism for production of ROS by AtRbohs.

Alignment of the two EF-hand motifs of AtRbohs revealed that AtRbohI carries unique amino acid residues and lacks the conserved aspartic acid (D) in the first EF-hand motif (Figure 3a), which is involved in Ca²⁺-binding (Oda et al., 2010). This may cause the low ionomycin-induced ROS production activity of AtRbohI in HEK293T cells (Figure 1e). Point mutation of this conserved aspartic acid (D) residue in the other AtRbohs resulted in a decrease in Ca²⁺ affinity and ROS-producing activity, indicating the importance of this residue in Ca²⁺-binding and Ca²⁺-triggered activation (Ogasawara et al., 2008; Kimura et al., 2012; Kaya et al., 2014). We generated the AtRbohI_ins268D mutant, which has an additional D to mimic the sequence of the conserved EF-hand motif. The insertional mutation did not affect the level of expression (Figure 3b) and, contrary to our expectation, the mutated protein showed lower enzymatic activity rather than improving ionomycin-triggered ROS production (Figure 3c).

In the second EF-hand motif of AtRbohI, the conserved glycine (G318) is converted to glutamine, and two additional amino acids (alanine and proline) are inserted (Figure 3a). To examine whether these variations in EF-hand motifs of AtRbohI contribute to the ionomycin-triggered ROS-producing activity, we generated AtRbohI_Q318G and AtRbohI_ins268D_Q318G mutated proteins (Figure 3a). These mutations did not affect the level of protein expression (Figure 3b). AtRbohI_Q318G mutation increased the overall activity and did not affect the response to ionomycin (Figure 3c), suggesting that the conserved G318 is not critical in the Ca²⁺ response. The AtRbohI_ins268D_Q318G mutants showed reduced ROS-producing activity similar to AtRbohI_ins268D (Figure 3c), indicating that the insertion of the conserved D to mimic the conserved EF-hand motif is rather inhibitory to the ROS-producing activity of AtRbohI. Taken together, AtRbohI has unique EF-hand structure and carries lower ionomycin-triggered ROS-producing activity.

Protein phosphorylation-induced activation is conserved among all AtRbohs

Previous studies have shown that protein phosphorylation of several AtRbohs stimulates their ROS-producing activity. Calyculin A (CA), a protein phosphatase inhibitor, induces ROS production in HEK293T cells transfected with AtRbohC/RHD2, AtRbohD, -F, -H or -J (Ogasawara et al., 2008; Takeda et al., 2008; Kimura et al., 2012; Kaya et al., 2014), suggesting that AtRbohC, -D, -F, -H and -J are activated by protein phosphorylation. Several putative phosphorylation sites, including serine residues in the N-terminal cytosolic region, have been proposed to be critical for activation (Takeda et al., 2008; Dubiella et al., 2013; Kadota et al., 2014; Li et al., 2014; Chen et al., 2017). To investigate whether phosphorylation-dependent activation is a common regulatory mechanism in all AtRbohs, we compared the CA-induced production of ROS in HEK293T cells transfected with each AtRboh. As shown in Figure 4, all AtRbohs showed CA-induced ROS-producing activity but with striking differences, although the protein expression level did not differ much in the HEK293T cells (Figure 1b). AtRbohA showed the highest activity and AtRbohB the lowest. Calyculin A induced rapid and transient production of ROS that decreased within a couple of minutes in AtRbohA, whereas the other AtRbohs showed sustained production of ROS for at least 30 min after application of CA (Figure 4). Together with the results in response to ionomycin, our data suggest that each AtRboh possesses a different activity, indicating different responses to Ca²⁺ and protein phosphorylation.

Functional conservation of AtRbohs involved in tip growth

To investigate the functional conservation of AtRbohs in plant development, we compared AtRbohC, -H and -J, which are involved in tip growth of plant cells. AtRbohC/RHD2 locates to the root hair tip, where it maintains tip growth (Foreman et al., 2003; Takeda et al., 2008), and AtRbohH and -J, localized to the tip of pollen tubes, regulate their tip growth in a similar manner (Kaya et al., 2014). Root hairs and pollen tubes develop into long tubular structures, allowing them to play critical roles for plants: root hairs absorb water and nutrients from soil and are also involved in plant–microbe interactions, while pollen tubes deliver sperm nuclei to the female gametophyte. Tip growth in these two types of cells involves numerous common factors including Rboh proteins and ROS, Ca²⁺ and ROP GTPase (Mangano et al., 2016).

We expressed AtRbohC, -H and -J genes under the control of cis-regulatory sequences (promoter and terminator) of AtRbohH or AtRbohJ in the atrbohH-3 atrbohJ-2 double mutant, where the tip growth of pollen tubes is impaired resulting in reduced seed production (Figure 5) (Kaya et al., 2014). Expression of either AtRbohH or AtRbohJ under the control of the AtRbohH or AtRbohJ cis-regulatory sequences complemented the defects of the double mutant in pollen tube elongation and seed number (Figure 5). Intriguingly the expression of AtRbohC/RHD2 under the control of the AtRbohH or AtRbohJ cis-regulatory sequences restored pollen tube growth and seed number in the atrbohH-3 atrbohJ-2 double mutant (Figure 5). This indicates that AtRbohC/RHD2 can compensate for the AtRbohH/-J function.

Next we examined functional replacement in root hairs. In the atrbohC/rhd2-5 mutant, root hairs are initiated but elongation by tip growth is impaired (Figure 6 and Figure S1 in the online Supporting Information). Expression of AtRbohC/RHD2 under the control of the AtRbohC/RHD2 cis-regulatory sequences restored tip growth (Figures 6 and S1). However, the root hair defects were not fully complemented with the expression of AtRbohH or -J under the control of the same AtRbohC/RHD2 cis-regulatory sequences (Figures 6 and S1). We examined the accumulation of ROS at the root hair tip. Nitro blue tetrazolium staining was observed in the wild type and AtRbohC-expressing line but not in the lines expressing AtRbohH and -J (Figures S2 and S3). Alignment of AtRbohC, AtRbohH and AtRbohJ shows some specific insertions/deletions (Figure S4) that may be involved in the different activation mechanisms in root hairs and pollen tubes.

Pollen tubes deliver male nuclei to the female gametophyte, so tip growth of pollen tubes is critical for plant reproduction, while root hairs are more primitive somatic cells that absorb water and nutrients from soil. Phylogenetic analysis shows that AtRbohC and AtRbohH/-J belong to different subfamilies (Figure S5). AtRbohC/RHD2 may be a primitive type so that it can function in the other cell types, while AtRbohH and AtRbohJ cannot function in root hairs because they have acquired specific functions in later-arisen reproductive cells. This can be explained by different regulators of AtRboh protein, such as ROP (Rho in plants) GTPases, Ca²⁺-dependent protein kinases or other unknown factors, but this needs to be analyzed further in future.

AtRbohC, -H and -J possessed higher ROS-producing activity, while AtRbohD and AtRbohF showed rather lower enzymatic activity. AtRbohC is a root-specific AtRboh and AtRbohH and -J are pollen specific AtRbohs. AtRbohC, -H and -J are required continuously at the tip of growing root hair or pollen tube, where they may be involved in Ca²⁺-dependent positive feedback regulation and/or cell-wall regulation for tubular morphogenesis. AtRbohD and AtRbohF are expressed in many tissues throughout the plant and are important for for responses to abiotic and biotic stresses (e.g. AtRbohD is the main component of pathogen-associated molecular pattern-induced transient ROS production) (Torres et al., 2005; Kadota et al., 2015). We suppose that the amount of ROS required for endogenous development and stress responses may be different, so that the activities of AtRbohs are different depending on their biological function.

Conclusions and perspectives

The Rbohs are involved in the production of ROS in numerous aspects of plant life. Our results show that ROS-producing activity among ten AtRbohs is dramatically different in their regulation of Ca²⁺ in HEK293T cells, and suggest that all AtRbohs are activated by protein phosphorylation. We propose that each AtRboh is involved in tuning of ROS production at different sites. Point mutation in the regulatory motifs may at least partially account for the functional differentiation of AtRbohs during evolution. Complementation analysis revealed the functional conservation and distinction of the AtRbohs, suggesting that they have been modified to have specific roles in different cell types. Our data propose the functional diversification of Rboh proteins, and thus it would be interesting to compare the activity and functions in other plants. Heterologous expression in HEK293T cells would be a powerful tool for these comparative studies.

Experimental Procedures

Plasmid construction and transgenic lines

To express AtRboh in HEK293T cells with a 3×FLAG epitope tag, a synthetic DNA fragment of the Kozak (GCCGCCACC)-3×FLAG epitope tag (Sigma-Aldrich, http://www.sigmaaldrich.com/), -BamHI-EcoRV-stop(TAATAG), was inserted into NheI and KpnI sites in the pcDNA3.1(-) vector (Invitrogen, http://www.invitrogen.com/), resulting in a pcDNA3.1-Kozak-3×FLAG vector. The coding sequence (CDS) of AtRboh genes (except for AtRbohG) was PCR-amplified from a cDNA library and cloned into the BamHI or EcoRV site of pcDNA3.1-kozak-3×FLAG vector. For AtRbohG, a synthetic gene for Homo sapiens was used. EF-hand mutants were generated by point-mutation primers and the mega-primer PCR method. Oligonucleotide primer sequences for all constructions are shown in Table S1. AtRbohH-pro::AtRbohC::AtRbohH-ter and AtRbohJ-pro::AtRbohC::AtRbohJ-ter constructs were generated by replacing the CDS of AtRbohH-pro::AtRbohH::AtRbohH-ter or AtRbohJ-pro::AtRbohJ::AtRbohJ-ter (Kaya et al., 2014) with the CDS of AtRbohC, and were expressed in the atrbohH3 atrbohJ-2 double mutant. AtRbohC-pro::AtRbohC/AtRbohH/AtRbohJ::AtRbohC-ter constructs were generated by cloning the promoter, terminator of AtbohC and CDS (AtRbohC, AtRbohH or AtRbohH) into the pZP2H-lac binary vector for expression in atrbohC/rhd2-5 mutant. These plasmids were introduced into plants by Agrobacterium (GV3101::pMP90)-mediated transformation.

Cell culture and transfection

HEK293T cells were maintained at 37°C in 5% CO₂ in Dulbecco's Modified Eagle's Medium nutrient mixture Ham's F-12 (WAKO, http://ffwk.fujifilm.co.jp) supplemented with 10% fetal bovine serum (HyClone, www.gelifesciences.com). HEK293T cells were transiently transfected with pcDNA3.1-3×FLAG-AtRbohs or empty vector by GeneJuice transfection regent (Novagen, http://www.merckmillipore.com) according to the manufacturer's instructions.

Measurement of ROS production in HEK293T cells

The ROS-producing activity of AtRbohs was assayed as described previously (Ogasawara et al., 2008). Detection of ROS production was by a peroxidase-dependent luminol-amplified chemiluminescence technique. Chemiluminescence was measured for 1 sec every 60 sec at 37°C using a microplate luminometer Centro LB960 (Berthold Technologies, https://www.berthold.com/). Production of ROS was expressed in relative luminescence units (RLU) per second. Data are presented as the average of three samples in a representative experiment. We independently replicated this experiment more than five times with similar results.

Aniline blue staining

Aniline blue staining was performed as described previously (Kaya et al., 2014). Briefly, pistils were collected 12 h after hand pollination and fixed in acetic acid:ethanol solution (25:75). The fixed pistils were softened in 1 N NaOH for 30 min at 60°C and then stained with 0.01% aniline blue in 2% potassium phosphate buffer (K₃PO₄).

Author contributions

HK designed the experiments. KK supervised this study. AI, AI, HH and TK performed the measurement of ROS production. TK, KM, YY, YM and AN contributed to the functional analysis of AtRbohs in plants. MK performed the phylogenetic tree analysis. HK, ST, MK, SK, MA and KK wrote the manuscript.

Acknowledgements

We thank Dr Michael Wrzaczek and Ms Kerri Hunter for critical reading and commenting on the manuscript. This work was supported in part by the Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan [Grants-in-Aid for Young Scientist (B) to HK (no. 21770054), for Scientific Research on Innovative Areas to HK (no. 21200068) and to K.K. (Nos. 21117516 and 23117718) and for Scientific Research B to KK (nos 19370023 and 23380027)].

Conflict of interest

The authors declare no conflict of interest.

Supporting Information

Filename	Description
tpj14212-sup-0001-FigS1.pdfPDF document, 4.7 MB	Table S1. Oligonucleotide primer sequences used in this work.
tpj14212-sup-0002-FigS2.pdfPDF document, 1.3 MB	Figure S1. Complementation analysis of the atrbohC/rhd2 mutant with AtRbohH and -J.
tpj14212-sup-0003-FigS3.pdfPDF document, 699.1 KB	Figure S2. Detection of reactive oxygen species in root hairs.
tpj14212-sup-0004-FigS4.pdfPDF document, 615.8 KB	Figure S3. Expression analysis by RT-PCR in roots.
tpj14212-sup-0005-FigS5.pdfPDF document, 92.5 KB	Figure S4. Alignment of AtRbohC, AtRbohH and AtRbohJ.
tpj14212-sup-0006-TableS1.pdfPDF document, 35.7 KB	Figure S5. Phylogenetic tree of respiratory burst oxidase homologs in plants.
tpj14212-sup-0007-Experimental.pdfPDF document, 88.7 KB
tpj14212-sup-0008-legends.docxWord document, 15.8 KB

Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

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Citing Literature

Volume98, Issue2

April 2019

Pages 291-300

Comparative analysis of the reactive oxygen species-producing enzymatic activity of Arabidopsis NADPH oxidases

Summary

Introduction

Results and Discussion

Comparison of the ROS-producing enzymatic activity of AtRbohs

Ca²⁺ regulation in ROS production is conserved among AtRbohs

Protein phosphorylation-induced activation is conserved among all AtRbohs

Functional conservation of AtRbohs involved in tip growth

Conclusions and perspectives

Experimental Procedures

Plasmid construction and transgenic lines

Cell culture and transfection

Measurement of ROS production in HEK293T cells

Aniline blue staining

Author contributions

Acknowledgements

Conflict of interest

Supporting Information

References

Citing Literature

Figures

References

Information

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Comparative analysis of the reactive oxygen species-producing enzymatic activity of Arabidopsis NADPH oxidases

Summary

Introduction

Results and Discussion

Comparison of the ROS-producing enzymatic activity of AtRbohs

Ca2+ regulation in ROS production is conserved among AtRbohs

Protein phosphorylation-induced activation is conserved among all AtRbohs

Functional conservation of AtRbohs involved in tip growth

Conclusions and perspectives

Experimental Procedures

Plasmid construction and transgenic lines

Cell culture and transfection

Measurement of ROS production in HEK293T cells

Aniline blue staining

Author contributions

Acknowledgements

Conflict of interest

Supporting Information

References

Citing Literature

Figures

References

Related

Information

Ca²⁺ regulation in ROS production is conserved among AtRbohs