NOX enzyme, by producing ROS, is needed to sustain the normal rate of pollen tube growth and this is likely to be a general mechanism in the control of tip growth of polarized plant cells
[26,27][26][27]. NOXs generate tip-localized, pulsating amounts of H
2O
2 that functions, possibly through Ca
2+ channel activation, to maintain a steady, tip-focused Ca
2+ gradient in pollen tube tip during growth
[28]. On the other hand, pollen NOX can be activated by Ca
2+ and Ca
2+ can increase its activity in vivo
[26,29][26][29]. This process would happen especially when the pollen grains are hydrated under mild conditions, so the activity of pollen NOX could be concentrated in those insoluble fractions, which could facilitate the exposure of tissues to ROS produced by this enzyme. It is worth mentioning that the extent of this exposure could differ among the plant families according to where the NOX resides in pollen grains and during hydration in the mucosa
[30]. In addition, pollen NOX can also be activated by low abundant signaling phospholipids, such as phosphatidic acid (PA) and phosphatidylinositol 4, 5-bisphosphate in vitro and in vivo, so there is a possible synergism between Ca
2+ and phospholipid-mediated NOX activation in pollen
[29]. In plants, ROP/RAC GTPases (the subfamily of Rho-type GTPases, which belong to the Rat sarcoma superfamily of small GTP-binding proteins) are also necessary for normal pollen tube growth by regulating ROS production
[29]. Besides these items mentioned above, O
3 was also indicated to increase ragweed pollen allergenicity through stimulation of ROS-generating NADPH oxidase
[31]. Recently, two NOXs/RBOHs, AtRbohH and AtRbohJ, have been shown to localize at the plasma membrane of pollen tube tip and the ROS production by the NOXs/RBOHs presumably plays a critical role in the positive feedback regulation that maintains the pollen tube tip growth
[32,33][32][33]. Furthermore, apoplastic ROS derived from AtRbohH and AtRbohJ are involved in pollen tube elongation by affecting the cell wall metabolism
[33]. More intriguing is that ectopic expressing of AtRbohC/hair root2 (hrd2) in pollen tubes could restore
atrbohH and
atrbohJ defects in tip growth of pollen tubes
[34], which implies that AtRbohC/hrd2 also plays roles in regulating the development of pollen tubes. Moreover, AtRbohE was also suggested to be critical for programmed cell death (PCD) and pollen development in
Arabidopsis thaliana L.
[35]. Meanwhile, OeRbohH, possessing a high degree of identity with AtRbohH/J, plays an important role in pollen germination and pollen tube growth in olive
[36]. Genetic interference with the temporal ROS pattern by manipulating
NOX/RBOH genes, affected the timing of tapetal PCD (programmed cell death) and resulted in aborted male gametophytes
[35]. All in all, we still cannot figure out how many factors are related to the regulation of pollen NOX. Nevertheless, what we already know is that pollen NOXs play a significant part in the regulation of pollen germination.
3.2. Root and Root Hair Development
The elongation of roots and root hairs is essential for uptake of minerals and water from the soil. Ca
2+ influx from the extracellular store is required for cell elongation in roots
[37]. It was suggested that plasma membrane NOXs/RBOHs and H
+-ATPases (a H
+ pump by coupling with energy of ATP hydrolysis on plasma membranes) are functionally synchronized and they work cooperatively to maintain the membrane electrical balance while mediating plant cell growth through wall relaxation
[38]. Observations on maize roots indicate that the activities of plasma membrane-associated NADPH oxidase respond both to gravity and to imposed centrifugal forces
[39]. In an early study, AtRHD2, a NADPH oxidase in
Arabidopsis, was reported as controlling root development by making ROS that regulates plant cell expansion through the activation of Ca
2+ channels
[40]. Further, both AtRbohC/RHD2 and ROP (RHO of plants) GTPases were found to be required for normal root hair growth by regulating ROS production
[41]. Coincidentally, the maize (
Zea mays L.)
roothairless5 (
rth5) which encodes a monocot-specific NADPH oxidase, was found to be responsible for establishing the high levels of ROS in the tips of growing root hairs
[42]. In rice,
OsNOX3 was also reported to play critical roles in root hair initiation and elongation by regulating the content of superoxide and hydrogen peroxide in root hair tips
[27]. In addition, both AtRbohD and AtRbohF are essential for ABA (abscisic acid)-promoted ROS production in
Arabidopsis roots, and ROS subsequently activate Ca
2+ signaling as well as reduce auxin sensitivity of roots, thus positively regulating ABA-inhibited primary root growth
[43]. Moreover, AtRbohD and AtRbohF negatively modulate lateral root development by changing the peroxidase activity and increasing the local generation of ·O
2- in primary roots in an auxin-independent manner
[44]. Similar results were also acquired in the legume-rhizobia symbiosis and legumes use different RBOHs for different stages of nodulation
[45,46][45][46]. Moreover, nitric oxide (NO) can activate NADPH oxidase activity, resulting in increased generation of ·O
2-, which subsequently induces growth of adventitious roots and acts downstream of auxin action in the process of root growth and development
[47]. These results suggest a vital role of NOXs/RBOHs in root and root hair development in plants.
3.3. Seed Germination
NOXs/RBOHs also play important roles in seed germination. The functional mechanism has been proposed in many plant species, such as
Arabidopsis thaliana L. rice (
Oryza sativa L.) and barley (
Hordeum vulgare L.). AtRbohB is a major producer of ·O
2- in germinating seeds, and inhibition of the ·O
2- production by diphenylene iodonium (DPI) leads to a delay in seed germination of
Arabidopsis and cress
[48]. In rice, OsNOX5, 7 and 9 might play crucial roles in radicle and root elongation during seed germination by regulating ROS production
[49]. Similarly, ·O
2- produced by NADPH oxidase also regulates seed germination and seedling growth in barley
[50,51][50][51]. Moreover, NOX/RBOH-mediated ROS production promotes gibberellic acid (GA) biosynthesis in barley embryos through regulation of HvKAO1 and HvGA3ox1 proteins, while GA induces and activates NOXs/RBOHs for ROS production in aleurone cells to induce α-amylase activity of the cells and therefore increases seed germination
[5,52,53][5][52][53].
3.4. Plant-Microorganism Ineractions
Phaseolus vulgaris NADPH oxidase is crucial for successful rhizobial colonization and probably maintains proper infection thread growth and shape
[54]. Moreover, it also has critical roles in reducing arbuscular mycorrhizal fungal (AMF) colonization. Overexpression of PvRbohB augments nodule efficiency by enhancing nitrogen fixation and delaying nodule senescence but impairs AMF colonization
[55]. A
Medicago truncatula NADPH oxidase, MtRbohA, has similar effects. It is significantly upregulated in
Sinorhizobium meliloti-induced symbiotic nodules, while hypoxia prevailing in the nodule-fixing zone may stimulate MtRbohA expression, which would, in turn, lead to the regulation of nodule functioning
[56]. Moreover, NoxA, a NADPH oxidase isoform in the grass endosymbiont
Epichloë festucae, was identified as essential for the establishment of systemic compatible infections in host plants
[50]. ROS produced by NoxA or NoxB (from
Fusarium oxysporum) regulate hyphal growth of a fungal pathogen towards roots of the host plants to maintain a mutualistic and symbiotic interaction
[57,58][57][58]. Recently, four GmNOXs from soybean genome (
Glycine max) also showed strong expression in nodules, pointing to their probable involvement in nodulation
[59]. All these results suggest that, NOXs/RBOHs, regulated by several different factors, play a significant role in mutualistic and symbiotic processes between plant and microorganism.
3.5. Fungal Development
When it comes to fungi, NOXs/RBOHs participate in a wide range of biological processes from their growth, differentiation and reproduction, to rhizobial colonization. In
Aspergillus nidulans, NoxA plays crucial roles in fungal physiology and differentiation by generating ROS
[60]. Moreover, genetic analysis of
Δnox2 (lacking the NADPH oxidase 2 gene),
Δnox1 (lacking the NADPH oxidase 1 gene) and a transcription factor deletion mutant
Δste12 in
Sordaria macrospora, reveals that the mutation of NOXs could lead to ascospore germination defect
[61]. In yeast, NoxA, NoxB and their associated regulators (BemA, Cdc24 and NoxR) have distinct or overlapping functions for the regulation of different hyphal morphogenesis processes
[57]. Furthermore, in
Neurospora crassa, NOX-1 elimination results in complete female sterility, decreased asexual development and reduced hyphal growth; whereas, a lack of NOX-2 does not affect any of these processes but led to the production of sexual spores that failed to germinate
[62]. In this study, the function of NOX-generated ROS acting as critical cell differentiation signals highlights a novel role for ROS in the regulation of fungal growth
[62]. The function of NOX-generated ROS in regulating the reproductive process was also found in other fungi. In
Botrytis cinerea, NOX complexes are essential for conidial anastomosis tubes’ formation and fusion
[63].
3.6. Other Aspects
Besides functioning in the regulation of pollen germination, root development and seed germination, as well as fungal development, NOXs/RBOHs have other effects as well. It was suggested that solar ultraviolet irradiation regulates anthocyanin synthesis in apple peel by modulating the production of ROS via NADPH oxidase
[64]. Moreover, chloroplastic NADPH oxidase-like activity mediates perpetual H
2O
2 generation, which probably induces apoptotic-like cell death of
B. napus leaf protoplasts
[65]. Furthermore, it was reported that among the seven homologues of NADPH oxidases in potato, the expression of
StRbohA and
StRbohB was detected in particular when dormancy break
[66]. These results suggest very extensive roles of NOXs/RBOHs in the plant kingdom, participating in various important biological processes.