Nitric oxide is involved in hydrogen gas-induced cell cycle activation during adventitious root formation in cucumber
© The Author(s). 2016
Received: 29 April 2016
Accepted: 16 June 2016
Published: 28 June 2016
Adventitious root development is a complex process regulated through a variety of signaling molecules. Hydrogen gas (H2) and nitric oxide (NO), two new signaling molecules are both involved in plant development and stress tolerance.
To investigate the mechanism of adventitious root development induced by hydrogen-rich water (HRW), a combination of fluorescence microscopy and molecular approaches was used to study cell cycle activation and cell cycle-related gene expression in cucumber (Cucumis sativus ‘Xinchun 4’) explants. The results revealed that the effect of HRW on adventitious root development was dose-dependent, with maximal biological responses at 50 % HRW. HRW treatment increased NO content in a time-dependent fashion. The results also indicated that HRW and NO promoted the G1-to-S transition and up-regulated cell cycle-related genes: CycA (A-type cyclin), CycB (B-type cyclin), CDKA (cyclin-dependent kinase A) and CDKB (cyclin-dependent kinase B) expression. Additionally, target genes related to adventitious rooting were up-regulated by HRW and NO in cucumber explants. While, the responses of HRW-induced adventitious root development and increase of NO content were partially blocked by a specific NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide potassium salt, NO synthase (NOS)-like enzyme inhibitor NG –nitro-L-arginine methylester hydrochloride, or nitrate reductase inhibitors tungstate and NaN3. These chemicals also partially reversed the effect of HRW on cell cycle activation and the transcripts of cell cycle regulatory genes and target genes related adventitious root formation.
Together, NO may emerge as a downstream signaling molecule in H2-induced adventitious root organogenesis. Additionally, H2 mediated cell cycle activation via NO pathway during adventitious root formation.
Adventitious rooting is a key step in the vegetative propagation of plants. Understanding the mechanism of adventitious rooting is of significant importance to strategize breeding efforts to maximizing its marketable yield . Adventitious root development is a complex process regulated by several lines of environmental and endogenous factors. In recent years, there has been increasing interest in the field of signal transduction during adventitious rooting. Untill now, nitric oxide (NO) [2, 3], Ca2+ ions, calmodulin (CaM) , Ca2+-dependent protein kinase activities (CDPK) , cyclic guanosinemonophosphate (cGMP) , ethylene , mitogen-activated protein kinase , carbon monoxide , polyamines , hydrogen peroxide , hydrogen sulfide  and hydrogen gas (H2)  have been suggested to be involved in adventitious rooting process. However, the complex network of signaling molecule associated with adventitious rooting remains unclear. A better understanding of the regulation of initiation of adventitious rooting by signaling molecules will advance our understanding of the molecular mechanisms regulating adventitious root development.
The gaseous compound NO is a redox-active small signaling molecule which may regulate almost all biotic and abiotic stress responses [13, 14]. Previous studies have also demonstrated the involvement of NO in plant various physiological processes such as maturation and senescence [15, 16], seed germination or dormancy [17, 18], floral transition  and stomatal movement . NO is also required for root organogenesis, including lateral root formation , root hair formation , and adventitious rooting [3, 23]. Although numerous studies have demonstrated the involvement of NO in adventitious roots formation, there is currently little information on its mechanism.
H2, a colorless, odorless and tasteless gas is the structurally simplest gas in nature and known as an inert gas. Previous studies have demonstrated that H2 is a potential therapeutic medical gas . It has received worldwide attention because it could selectively reduce hydroxyl radical and peroxynitrite in cell . Subsequently, accumulated evidence from a variety of animal experiments and clinical tests indicated that H2 may act as an anti-inflammatory, anti-apoptotic, and anti-allergic agent . More recently, there have been some reports indicating that H2 may play critical roles in plant stress response including salinity [27–29], drought [29, 30], paraquat-induced oxidative stress , cadmium toxicity , aluminum stress , mercury toxicity , and UV-A irradiation . Hydrogen-rich water also could delay postharvest ripening and senescence of kiwifruit during storage by regulating the antioxidant defence . Notably, Lin et al.  found that H2 might regulate cucumber adventitious root development in a heme oxygenase-1/carbon monoxide-dependent manner. The author suggested that exogenous HRW treatment might be a good option to induce plant root organogenesis. However, the mechanism of H2 on regulating adventitious root development needs to be fully investigated.
Thus, H2 and NO have been considered as signaling modulators with multiple biological functions in plants. Little information is available, however, about the connection between H2 and NO in regulating physiological process. It was found that H2 inhibited LPS/IFNγ-induced NO production through modulation of signal transduction in macrophages and ameliorates inflammatory arthritis in mice . It provided the molecular basis for H2 effects on inflammation and a functional interaction between H2 and NO. The interaction effect between H2 and NO was also found in alfalfa. Recently, HRW was reported to alleviate aluminum-induced inhibition of root elongation via decreasing NO production . More recently, the crosstalk between H2 and NO was reported to play central roles in the ABA signaling cascade during stomatal movement .
The cell cycle consists of two major events, cell development which is the critical driving forces in completion of the ontogenic program during plants life cycle. All phases of cell cycle are regulated by the heterodimeric complexes of highly conserved proteins, cyclin-dependent kinase (CDK) and cyclin . In higher eukaryotes, the activation of CYCD and CDKA leads to produce a repressor protein kinase that phosphorylates the retinoblastoma protein at G1-to-S phase transition . In the next checkpoint, B-type CKDs and A-and B-type cyclins are involved in G2-to-M phase transition . In addition, CDK/cyclin complexes are inactivated by a class of CDK-inhibitory proteins .
It was suggested that H2 and NO have positive effects on adventitious root formation. Our previous results showed that 50 μM NO donor sodium nitroprusside (SNP) had significant effect on adventitious root . However, the crosstalk of NO and H2 in promoting adventitious rooting and its mechanism are still puzzled. In the study, molecular and pharmacological approaches were used to investigate the influence of H2 and NO on the adventitious root development in cucumber (Cucumis sativus ‘Xinchun 4’) explants, as well as cell cycle activation and cell cycle- and adventitious rooting related genes in hypocotyls. Therefore, the objectives of this study are to determine the role of NO in H2-regulated cell cycle during adventitious rooting.
HRW promoted adventitious root development in a dose-dependent manner
The HRW-induced adventitious root development was reversed by NO elimination
The production of endogenous NO was involved in HRW-induced of adventitious root formation
NO may be involved in HRW-induced cell cycle activation during adventitious root development
The highest percentage of cells in G1 phase was found in the control, which were 700.50 % and 634.20 % higher than that in HRW and SNP treatments, respectively (Fig. 4b). However, the percentage of total cell population in HRW and SNP treatments was higher than that in the control (Fig. 4b). Thus, H2 and NO promoted G1 to S transition phase, suggesting that a subpopulation of cells in G1 phase was induced to enter a new cell cycle in a synchronous manner. If cPTIO, L-NAME, or NaN3 was added to the HRW solution simultaneously, the percentage of cells in G1 phase was increased by 43.64, 25.01 and 45.33 %, but the percentage of cells in S phase was decreased by 28.48, 21.72 and 27.3 % (Fig. 4b).
NO participates in HRW-mediated transcript levels of cell cycle regulatory genes during adventitious root development
NO was responsible for HRW-induced the expression profiles of CsDNAJ-1,CsDPK1 and CsCDPK5 during adventitious root development
Adventitious roots play important roles in nutrient and water uptake in plant; and their formation is widely used for plant clonal propagation. Previous studies have shown the role and relationship between H2 and NO in plant stress response  and stomatal closure . However, there is little research in the crosstalk between H2 and NO during adventitious rooting. Here, we focus on the involvement of NO H2-induced in cell cycle activation during adventitious rooting.
H2 has aroused worldwide attention because of its selective reduction . Accumulating evidence indicates that H2 is a new signaling molecular and plays important roles in plants. In the present study, we illustrated that HRW, when applied exogenously, enhanced the number and length of adventitious root in a dose-dependent manner (Fig. 1). These findings are also consistent with a previous report showing that exogenous H2 was able to regulate cucumber adventitious root development . Much research has focused on the physiological roles of H2 in plant responses against salt stress [27, 28], cadmium toxicity , mercury toxicity , paraquat-induced oxidative stress  and aluminum stress . Ample evidences have shown that NO classified as a gasotransmitter was able to induce adventitious rooting . We have shown previously that NO, when applied exogenously, also promoted the formation of adventitious root in marigold . NO was found to promote adventitious root development in Panax ginseng by the generation of O2− . In order to ascertain whether NO signaling pathway is involved in H2-induced adventitious root, NO scavenger cPTIO and inhibitors L-NAME tungstate and NaN3 were used in the experiment. We found that cPTIO, L-NAME tungstate and NaN3 caused partial inhibition of HRW-induced adventitious root development (Fig. 2). In view of the inhibitory roles of NO-scavenger, NOS-inhibitor and NR-inhibitor in HRW-induced response, NO might be an essential gas signaling molecule in H2-mediated adventitious rooting. Until now, the relationship between H2 and NO both in animals and plants still remains to be elucidated. Although H2 could not inhibit intracellular NO production, it significantly suppressed NO-induced cytotoxicity in PC12 cells . In alfalfa, H2 alleviated aluminum stress via decreasing NO production . Xie et al.  demonstrated that NO production was contributed to H2-promoted stomatal closure in Arabidopsis. Our results also showed that when NO production was blocked, the promotive roles of HRW in adventitious rooting were reversed (Fig. 2). These results indicate that NO may act as a downstream signaling molecule involved in H2-induced adventitious root development. However, further studies are needed to determine the crosstalk between H2 and NO in different physiological processes.
Previous animal researches showed that there is a relationship between H2 and NO in some situations [36, 43]. In plants, Xie et al.  discovered that exposure of Arabidopsis to HRW resulted in an increase in NO content. Additionally, the effect of H2 in alleviating aluminum-induced inhibition of root elongation in alfalfa may derive from the decrease of NO . In addition, an increase in H2-mediated NO production catalyzed by NOS-like protein and NR might be required and be part of the molecular events involved in H2 action. Here, our evidence supports the possibility that NOS-like- and NR-dependent NO production contributes to H2-promoted adventitious root development in cucumber (Fig. 3). Coincidently, it has been demonstrated that NO levels are apparent during the H2-induced stomatal closure . It has also been found opposite observation that H2 decreased NO production in aluminum-induced inhibition of root elongation . Therefore, present researches indicate that there is a complex interaction between H2 and NO in some physiological processes.
Previous studies have shown that cell cycle regulation in the xylem pericycle may play crucial roles in root organogenesis [44, 45]. Cell cycle regulation occurred in the xylem pericycle, in which cells proceed to G2 phase, whereas the rest of pericycle remained at G1 phase . Here, our analyses indicated that that H2- and NO-induced an accumulation of cells in the S phase during adventitious rooting, indicating that H2- and NO-induced cell cycle activation contributed to rooting (Fig. 4). It has been previously reported that cell cycle induction played a key function in adventitious root growth  and lateral root formation . This is the first report to show that H2 and NO are involved in cell cycle progression during adventitious root development. Interestingly, cPTIO, L-NAME, and NaN3 all partially reduced H2-induced accumulation of cells in S phase and then inhibited the adventitious root development (Fig. 4). It is generally accepted that plant hormones play a central role in the reactivation of the cell cycle during root development . The inducible effect of NO on cell cycle activation has been reported in tomato lateral formation . The authors noted that auxin-dependent cell cycle gene regulation might be dependent on NO. Thus, our pharmacological evidence supports the possibility that, at least in our experimental condition, H2 and NO might form a linear signaling pathway in regulating cell cycle activation during adventitious rooting.
Cell-cycle regulatory genes have been shown to be involved in regulating cell division in internode growth  and root meristem induction . To understand the mechanisms of NO involved in H2-regulated cell cycle that resulting in root initiation, detailed molecular studies were conducted in this study. The genes involved in the transition of G1 to S, CycA, CycB, CDKA and CDKB were significantly up-regulated by NO and H2 treatments (Fig. 5), which consistent with the phase change of cell cycle. Otvos et al.  reported that NO promoted cell division and embryogenic cell formation in leaf protoplast-derived cells of alfalfa. NO transiently induced CyCA2;1 and CYCD3;1 mRNA expression in cell suspensions. These results correspond well with the expression of CycA and CycB in our experimental condition. Previous study also found that NO mediated the induction of the CYCD3;1 gene at the beginning of lateral root primordial formation . Thus, these results validate the involvement of NO in regulating cell cycle-related genes during lateral and adventitious rooting. It is noteworthy that the expression of CDKA was markedly up-regulated by H2 and NO, suggesting that A-type CDKs may play a main role in G1-to-S transition. Himanen et al.  also found that the transcription level of CDKA was induced in auxin-mediated cell cycle activation during early lateral root initiation. The level of the CDKA transcript remained at high levels in NO treatment during lateral rooting indicating that pericycle cells may be competent for cell division in tomato . Additionally, our data showed that the transcript level of CDKB was less than that of CDKA after H2 treatment. It may because B-type CDKs regulates cell cycle progression to the mitotic phase, G2 to M . This study further showed that the transcript levels of CycA, CycB, CDKA and CDKB induced by H2 were partially inhibited by the NO scavenger cPTIO, NOS inhibitor L-NAME and NR inhibitor NaN3 (Fig. 5). Thus, the transcript profile presented here allows a simple model to be proposed that NO may be involved in cell cycle activation during H2-induced adventitious rooting. The evidence provided here further confirms that H2 and NO may specifically regulate cell cycle activation and adventitious root formation. Furthermore, NO may be downstream signal molecule during H2 –induced adventitious rooting, present in competent pericycle cells, which finally activates cell cycle-related genes.
It has been observed that the target genes of adventitious root development, CsDNAJ-1, CsCDPK1 and CsCDPK5 could be induced by auxin, CO , and hydrogen sulfide . All DnaJ-like proteins characterized by a J domain regulated interactions with Hsp70 in protein folding and the assembly and disassembly of protein complexes. It has illustrated that the genes of DnaJ-like protein (s) and calcium-dependent protein kinases (CDPKs) were involved in the initiation and development of adventitious root [8, 52]. Additionally, IAA and NO induced the activity of CDPK that associated with cell differentiation, division, and /or differentiation during the formation of adventitious root . In this study, the molecular evidence showed that H2 and NO treatments both induced high expression of the CsDNAJ-1, CsCDPK1 and CsCDPK5 genes (Fig. 6), which were consistent with the number of adventitious roots observed. Bai et al.  found that IAA, 3-O-C10-HL and H2O2 increased the expression of CDC2, ARC2, and CDPK as well as the Aux/IAA gene family members AUX22c, AUX22d, and AUX22e in mung bean. Recently, Lin et al.  also reported that H2 induced the expression of target genes related to adventitious root formation in a heme oxygenase-1/carbon monoxide-dependent manner during cucumber adventitious rooting. Application of NO scavenger cPTIO, NOS inhibitor L-NAME and NR inhibitor NaN3 were able to down-regulate H2-induced transcription levels of CsDNAJ-1, CsCDPK1 and CsCDPK5 (Fig. 6). Thus, we deduced that H2-induced CsDNAJ-1, CsCDPK1 and CsCDPK5 expression might be regulated by NO production. These findings might suggest that CsDNAJ-1, CsCDPK1 and CsCDPK5 expression at the earlier state might be required for H2-induced adventitious root development in a NO-independent manner.
Plant material and growth conditions
Cucumber (Cucumis sativus ‘Xinchun 4’) seeds were kindly supplied by Gansu Academy of Agricultural Sciences, Lanzhou, China. Selected identical seeds were germinated in Petri dishes on filter papers soaked in distilled water, then transferred to an illuminating incubator and maintained at 25 ± 1 °C for 5d with a 14-h photoperiod (photosynthetically active radiation = 200 μmol m−2 s−1). Primary roots of 5-d-old seedlings were removed and the cucumber explants were then maintained under the same temperature and photoperiod conditions described above for another 5 d in the presence of different media as indicated below. Then, records were taken of root number per explants, root length. Meanwhile, corresponding photographs were taken.
Treatments of explants, chemicals
After primary roots were removed, every ten cucumber explants were put into a Petri dish containing 6 ml of distilled water, different concentration of hydrogen-rich water (HRW) as indicated in Fig. 1. 30 μM N-nitro-L-arginine methyl ester (L-NAME, Sigma, USA) or 200 μM 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO, Sigma, USA), or 100 μM tungstate or 10 μM NaN3 were added together with optimum concentration of HRW. 200 μM cPTIO was added together with optimum concentration of SNP. Inactivated SNP (50 μM) had previously been exposed to light to drive off NO about 24 h. The concentration of these chemicals was selected based on the results of preliminary experiment and previous experiment . The solutions were prepared in complete darkness, and immediately diluted to the demanded concentrations at pH values of 6.5. Unless stated otherwise, the remaining chemicals were of analytical grade from Chinese companies.
Preparation of hydrogen-rich water (HRW)
Purified H2 gas (99.99 %, v/v) generated from a hydrogen gas generator (QL-300, Saikesaisi Hydrogen Energy Co., Ltd., China) was bubbled into 2 l distilled water at a rate of 300 ml min−1 for 30 min. Then, the corresponding HRW was rapidly diluted to required saturations (1, 10, 50 %, [v/v]). In our experimental conditions, H2 concentration in freshly prepared HRW determined with a “Dissolved hydrogen portable meter” (Trustlex Co., Led, ENH-1000, Japan) was 0.45 mM, and maintained at a relative constant level in 25 °C for at least 12 h.
Determination endogenous NO content
NO content was determined using the Greiss reagent method  with some modifications. Samples of cucumber hypocotyl (0.2 g) were frozen in liquid nitrogen, then ground in a mortar and pestle in 4 mL of 50 mM ice-cold acetic acid buffer, pH 3.6, containing 4 % (w/v) zinc diacetate. The homogenates were centrifuged at 10,000 × g for 15 min at 4 °C, and the supernatants were collected. For each sample, 0.1 g charcoal (Shanghai Chemical Reagent Co. Ltd.) was added. After vortex mixing and filtration, the filtrate was leached and collected. A mixture of 1 mL of filtrate and 1 mL of Greiss reagent was incubated at room temperature for 30 min to concert nitrite into a purple azo-dye. The absorbance was then assayed at 540 nm. NO content was calculated by comparison to a standard curve of NaNO2.
Hypocotyl cell nucleus extracts
Cucumber hypocotyls were excised about 0.5 cm and fixed in 4 % formaldehyde (0.1 M phosphate buffer, pH 7.4) for 15 min. Then the hypocotyls were cut up with blade in Galbraith butter (45 mM MgCl2, 30 mM sodium citrate, 20 mM 4-morpholinopropanesulfonic acid). Finally, cell nucleus suspension was collected with 30 μm mesh screen in 5 ml tube.
Fluorescence microscopic image analysis of the cell cycle
Microscopy was conducted as described by  with modifications. Cell nucleus suspension added fluorochrome DAPI (1:100000, Molecular probe), 0.1 % Triton-X 100 and 50 μg mL−1 Rnase1 (DNase-free, Qiagen) was observed and taken photos with fluorescence microscope (Leica 400×, Planapo, Wetzlar, Germany). Microscopic photography conditions setting value was fixed and no excessive exposure of the nucleus. Each sample was used to analysis at least 1000 the fluorescence microscopic images of nucleus. The fluorescence intensity of nucleus (grey value) was measured with the help of Image Pro software (Media Cyberntics, USA).
Total RNA was extracted from about 200 mg (fresh-weight) excised cucumber hypocotyl (5 mm) 6 h and 24 h after treatment using TRIZOL reagent (Sangon, China) according to the manufacturer’s instructions.
Transcript level estimation with qRT-PCR
Primers used for qPT-PCR assays
F: 5′- GCCTCTGCTGTAACAACACTCAT-3′
R: 5′- TGTGCTGGCTGTATTTTTCTCTG -3′
F: 5′- AATGAGGGCTATTTTGGTGGA-3′
R: 5′- TATCCGAAAGGCACACAAAGTC-3′
F: 5′- ATCTAAAACCCCAAAATCTGCT-3′
R: 5′- CAAATGCTCTTGCCAGTCC -3′
R: 5′- CAAATGCTCTTGCCAGTCC-3′
F: 5′- GACCACTCTCCACGATGTCAAC-3′
R: 5′- ATCAATGTGTTATGGCGGTAGC-3′
F: 5′- GGAGTTGGAAGGAGGACGATG-3′
R: 5′- TGAGATTTAGCAGTAAGGACGC-3′
F: 5′- ATGAGGAAAGGCAATCAGGAAT-3′
R: 5′- AAAGAAGCACATAAAATCAAGCAGA
Where indicated, results were expressed as the mean values ± SE of at least three independent experiments. Statistical analysis was performed using the Statistical Package for Social Sciences for Windows (version 13.00; SPSS, Inc., Chicago, IC, USA). For statistical analysis, Duncan’s multiple test (P < 0.05) was chose as appropriate.
CDKA, cyclin-dependent kinase A; CDKB, cyclin-dependent kinase B; CycA, A-type cyclin; CycB, B-type cyclin; DAPI, 4′, 6-diamidino-2-phenylindole; H2, hydrogen gas; H2O2, hydrogen peroxide; HRW, hydrogen-rich water; LI SNP, Light-inactivated SNP; L-NAME, NO synthase (NOS)-like enzyme inhibitor NG –nitro-L-arginine methylester hydrochloride; NO, nitric oxide; SNP, souium nitroprusside; cPTIO, 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide potassium salt
The authors are grateful to the editors and the anonymous reviewers for their valuable comments and help.
This research was supported by the National Natural Science Foundation of China (no. 31160398, 31560563), the Post Doctoral Foundation of China (nos. 20100470887, 2012t50828), the Key Project of Chinese Ministry of Education (no. 211182), the Research Fund for the Doctoral Program of Higher Education (no. 20116202120005), and the Natural Science Foundation of Gansu Province, China (no. 1308RJZA179, 1308RJZA262).
Availability of data and materials
Raw data could be obtained by request to the corresponding author.
YZ and WL designed research; YZ, MW and LN performed research; YZ, WL and ZM contributed analyzed data; and YZ and WL wrote the paper. All authors read and approved the final manuscript.
The authors declare that they have no competing interest.
Consent for publication
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