De-Etiolation of Wheat Seedling Leaves: Cross Talk between Heme Oxygenase/Carbon Monoxide and Nitric Oxide

Greening of etiolated plants is predominantly stimulated by light but the complete molecular mechanism is still unknown. Multiple studies currently focus on the important physiological effects of heme oxygenase (HO)/carbon monoxide (CO) in plants. In this report, firstly, the role of HO/CO in light-induced de-etiolation process was investigated. We discovered that light could significantly increase HO activities, HO-1 gene expression, CO release, and chlorophyll accumulation, all of which were sensitive to zinc protoporphyrin (ZnPPIX), the potent inhibitor of HO-1, respectively. Both HO-1 inducer hematin (H) and CO aqueous solution were able to relieve etiolation in wheat seedling leaves under completely darkness by up-regulating endogenous HO/CO system, so as nitric oxide (NO) donor sodium nitroprusside (SNP) did. Similarly, endogenous NO production was also boost in response to light, SNP, hematin and CO aqueous solution in wheat seedling leaves. Additionally, the restoration of chlorophyll contents was blocked, when the inhibitor of mammalian nitric oxide synthase NG-nitro-L-arginine methylester hydrochloride (L-NAME) or the specific scavenger of NO 2-(4-carboxyphenyl)-4, 4, 5, 5-tetramethylimidazoline-1-oxyl-3-oxide potassium salt (cPTIO) was added, respectively. Furthermore, the inducible effects of light were different from those of SNP, hematin, and CO on Pfr accumulation and PHYA transcripts. However, all of sodium nitroprusside (SNP), hematin, and CO could accelerate NO emission, which suggested that HO/CO in wheat seedlings de-etiolation under dark-light transition may have a cross talk with NO.


Introduction
De-etiolation of etioplasts lack of chlorophyll is a complex developmental process that is triggered predominantly by light. One of the important light-dependent steps is the phototransformation of protochlorophyll (ide) to chlorophyll (ide) [1]. During the subsequent greening period, the rapid accumulation of chlorophylls occurs. In fact, plants have developed elaborate photo sensory systems to optimize its growth and development to daily and seasonal changes. However, the complete molecular mechanisms by which light regulates development are largely unknown.
Heme oxygenases (HOs, EC 1.14.99.3) were originally identified in animals [2], which catalyzes the first and rate-limiting step in the degradation of heme. Via oxidation, HO cleaves heme molecules to yield equimolar quantities of biliverdin IXa (BV), carbon monoxide (CO), and free iron. Indeed, ample evidence currently supports the notion that HO serves to provide potent cytoprotective effects in many models of oxidant-induced cellular and tissue injury both in animals and plants. For example, recent works have indicated the enhancement of HO activities and transcripts in the antioxidant defense system in soybean leaves subjected to lower levels of cadmium (Cd) stress or UV-B irradiation [3,4].
Moreover, in plants, HOs are not only involved in adaptation or defense against oxidative stress, but also identified to regulate the first step of phycobilin biosynthesis [5]. These linear tetrapyrrole molecules are precursors of the chromophores for the photoreceptor phytochromes, which are traditionally responsible for the detection of far-red light (FR) and red light (R). They are pigments consisting of a polypeptide of about 125 kD carrying a chromophore moiety, the phytochromobilin, which is a linear tetrapyrrole co-valently attached to a conserved cysteine residue in the Nterminal region [6][7][8][9]. Phytochromes are encoded by gene families in all higher plants [10], each of which uniquely contributes to assemble holo-phytochromes for regulating plant photomorphogenesis.
CO, catalyzed by HOs, was found to play various important physiological roles in animals [11][12][13]. More recently, it has been reported to be involved in various biological processes in plant kingdom. For instance, it acts as a compound with hormonal effects, including affecting the seed dormancy and germination [14], inducing lateral root formation, involving in abscisic acid (ABA)-induced stomatal closure [15,16], alleviating the cadmium-induced oxidative damage in Medicago sativa seedling root tissues [17] and even involving in the auxin-induced cucumber adventitious rooting process [18], most of which were similar to some behaviors reported by NO in plants [19,20]. Previous reports have shown that NO signal, which was mainly produced by nitric oxide synthase-like protein or nitrate reductase (NR, EC 1.6.6.1/2) in plants [20][21][22][23], participated in the course of wheat leaves de-etiolation [19]. Considering the similarities between NO and CO, we hypothesized that CO might exert the same function. On the other hand, mounting evidence has demonstrated the presence of CO release in plants [17,[24][25][26][27][28], which also provided the evidence for above hypothesis.
To reveal the role of HO/CO system in light-induced deetiolation, we analyzed the HO expression and CO release of wheat leaves in light-dark transition. In addition, we discussed the mechanism of hematin and CO on restoring the reduction of chlorophyll content in absence of light and suggested a possible link between HO/CO and NO.

Results
Light-induced wheat seedlings de-etiolation is sensitive to ZnPPIX, the potent inhibitor of HO-1 In Arabidopsis, genetic analysis of the HY1 locus showed that hy1 mutant plants display long hypocotyls and decrease chlorophyll accumulation consistent with a substantial deficiency in photo-chemical active phytochromes (phys) [29]. Meanwhile, the potent HO-1 inhibitor ZnPPIX was first found to inhibit HO activity in both animal and plant tissues [18,30]. In our test, comparison of the chlorophyll content in seedling leaves of wheat plants transferred from dark to light (DRL) with dark-grown controls (DRD) showed that transferred plants (DRL) maintained relatively higher chlorophyll level after 3 days (P,0.05, Fig. 1), as found previously. Interestingly, above light-mediated effect was prevented when ZnPPIX was added (DRL+ZnPPIX), further suggesting a possible interrelationship among light, HO-1, and chlorophyll content in our experimental conditions.
The effects of ZnPPIX on chlorophyll content in the presence of light treatment were also evaluated. As shown in Fig. 1, chlorophyll content of light-grown seedling leaves (LRL) was markedly reduced by addition ZnPPIX (LRL+ZnPPIX, P,0.05). Similarly, inhibition of HO-1 activity with ZnPPIX in dark-grown sample resulted in a 33% decrease in chlorophyll content, confirming a possible physiological role for HO-1 in light-induced wheat seedlings de-etiolation.
Both HO-1 inducer hematin and CO aqueous solution were able to restore the chlorophyll decrease of wheat seedling leaves in completely darkness In further investigations, we noticed that HO-1 inducer hematin (H) or CO aqueous solution (CO), when applied to etiolated wheat seedlings, could dose-dependently restore the loss of chlorophyll content induced by darkness pretreatment. In comparison with dark-grown control plants (DRD), a maximal response at 10 mM Figure 1. Comparative effects of HO-1 inducer hematin, COsaturated aqueous solution, and light on the restoration of chlorophyll content in etiolated wheat seedling leaves. 14-dayold wheat seedlings were kept at 25uC in continuous darkness for 5 days. After that, some were transferred into light, while others were still left in darkness without (DRD) or with different contentions of hematin (1.0, 10, and 100 mM, DRD+H1.0, 10, 100), and different saturations of CO aqueous solution (0.1, 1.0, 10, and 50%, DRD+CO0.1, 1.0, 10, 50%). LRL+ZnPPIX, DRD+ZnPPIX, and DRL+ZnPPIX DRH10+Hb stand for combination with HO-1 specific inhibitor ZnPPIX (100 mM), and CO/NO scavenger hemoglobin (Hb, 0.1 gL 21 ), for additional 3 days. LRL stands for wheat seedlings were grown in normal light cycle. Bars denoted by the same letter did not significantly differ at P,0.05 according to Duncan's multiple range tests. doi:10.1371/journal.pone.0081470.g001 hematin and 1.0% CO aqueous solution was observed. Additionally, above hematin-mediated effect was prevented when the CO scavenger hemoglobin (Hb) was added. Thus, above concentration of hematin and CO aqueous solution were used to investigate the role of the HO/CO system throughout this investigation. Furthermore, the results shown in Fig. 2 also confirmed that 10 mM hematin, as an effective inducer of HO-1, was able to partially reverse the negative impact of darkness on chlorophyll content in a time-dependent manner. In a word, above results further illustrated that both hematin and CO aqueous solution could exert the same inducible function as light in continuous darkness.

Induction of HO/CO system in response to light treatment
We analyzed the expression of HO-1 mRNA in wheat seedling leaves in response to the transition from dark to light at different times. Quantitative real-time RT-PCR revealed that seedling leaves of wheat plants transferred from dark to light (DRL) with dark-and light-grown controls (DRD, and LRL) brought about the highest induction of HO-1 gene (Fig. 3A). For example, the results shown that HO-1 mRNA level was increased more than 3 times after transition (12 h) respect to control values, while 1.5 time enhancement was observed at 6 h of transition. This fact indicated that HO-1 gene response occurred in a time-dependent manner. On the other hand, treatment either with dark or light for 12 h produced the slightly decreased expression pattern of HO-1 (Fig. 3A).
To assess whether the aforementioned different mRNA levels reflected changes in HO activities, enzyme activity determination was performed. As shown in Table 1, the observed differences in transcripts were also positively correlated with enzyme activities. Interestingly, it is noteworthy that, as expected, treatment with ZnPPIX in seedling leaves of wheat plants transferred from dark to light produced the significantly decrease of HO activity (P,0.05). Moreover, the changes of CO content under different treatments were in conformity with HO activities (Fig. 3B).
HO activities and HO-1 transcripts were increased in response to SNP, hematin, and CO aqueous solution in etiolated wheat seedling leaves The light-like effect of applying HO-1 inducer hematin or CO aqueous solution to dark-grown samples first led us to investigate whether changes in endogenous HO activities and HO-1 transcripts occurred in response to hematin or CO aqueous solution, as well as these actions of light (Fig. 3, Table 1), or a well known NO donor sodium nitroprusside (SNP). In our experimental conditions, 10 mM hematin (H), or 1.0% CO aqueous solution (CO), applied to dark-grown wheat seedlings, induced rapid enhancement of HO activity (Fig. 4). The time course test shown that in comparison with dark-grown controls (DRD), a rapidly maximum response was discovered as early as 1 day after different treatments (DRD+S, DRD+H, and DRD+CO), followed by a gradual decrease until the third day ( Fig. 4). A weaker increased level of HO activities after treatment in the transition from dark to light (DRL) was also observed (Fig. 4). Meanwhile, changes in HO-1 transcripts (Fig. 5) were found to be similar to those for HO activities under different treatments. We also noted that the induction of HO expression elicited by hematin apparently preceded light-induced de-etiolation.
The application of L-NAME and cPTIO blocked the restoration of chlorophyll contents in etiolated wheat seedling leaves induced by SNP, hematin, and CO aqueous solution The observation that de-etiolation of wheat seedling leaves displayed similar sensitivity to light, SNP, hematin, and CO aqueous solution prompted us to examine the effects of the inhibitor of mammalian NOS-like protein (L-NAME) and a specific NO scavenger cPTIO on the changes of chlorophyll contents in etiolated wheat seedling leaves with or without SNP, hematin, and CO aqueous solution. As shown in Fig. 6, similar to the dark-induced decay of chlorophyll, the ameliorating effects of SNP (S), hematin (H), and CO aqueous solution (CO) on etiolation was significantly blocked by L-NAME or cPTIO, respectively. Further, the negative effects of L-NAME and cPTIO on light- induced de-etiolation were observed. These results suggested that endogenous NO is likely to be involved in de-etiolation elicited by light, hematin and CO aqueous solution, and that mammalian NOS-like enzyme-mediated NO production plays an important role in above process.
NO production was increased in response to SNP, hematin, and CO aqueous solution in etiolated wheat seedling leaves To further confirm whether de-etiolation elicited by light, hematin, and CO aqueous solution is related to endogenous NO, Greiss reagent method (Fig. 7) or a fluorescence method with or without the specific fluorescent probe 4,5-diaminofluorescein diacetate (DAF-2 DA), the negative probe 4-amino fluorescein diacetate (AF 4-DA), and NO scavenger cPTIO (Fig. 8), was used to evaluate the changes of NO signal, respectively. As expected [31], in comparison with the dark-grown control samples (DRD), the time course experiments for additional three days illustrated that a burst of endogenous NO production appeared on the second day with light, SNP, hematin, and CO treatments, respectively, and then followed by a gradual decrease till the third day (Fig. 7).
Furthermore, a slight decrease of DAF-2 DA-dependent green fluorescence was firstly observed in the dark-grown sample (DRD, 2 d) respect to the light-grown control (LRL). The green fluorescence was increased by the addition of the NO donor SNP and reduced by the specific scavenger cPTIO, respectively, further confirming that DAF-2 DA-dependent green fluorescence is associated with endogenous NO concentration. As expected (Fig. 7), an increase in endogenous NO production in seedling leaves was found upon exposure to light, SNP, hematin, and CO, as demonstrated by 37.4, 69.2, 47.7 and 36.6% induction of   fluorescence intensity following 2 days. cPTIO, a specific scavenger of NO, also inhibited above inductions. Moreover, similar increase in fluorescence was not apparently observed when AF 4-DA, the negative control probe for DAF-2 DA, was applied, respectively. Together, these results reveal that an induction of endogenous NO production in the seedling leaves might be a critical event in above effects elicited by light, SNP, hematin, and CO aqueous solution during de-etiolation process.

Application of SNP, hematin and CO saturated aqueous solution induced the accumulation of Pfr in etiolated wheat seedling leaves
Phytochromes are molecular light switches that regulate various aspects of plant growth and development. Phytochromes exist in two photointerconvertible forms, Pr and Pfr. Pfr is thought to be the active form of phytochrome, and can be converted back to Pr by far-red light. Therefore, we further detected the relative amount of Pfr to investigate whether HO/CO involving in lightmediated signals in plants depend on photoreceptors. Results shown in Fig. 9 illustrated that Pfr content in dark-grown plant (DRD) was decreased with respect to the light-grown control (LRL) (P,0.05). By comparison, the dark-induced decrease of Pfr level was markedly recovered by SNP, hematin, and CO aqueous solution treatment for additional 3 days. On the other hand, ZnPPIX or cPTIO significantly blocked hematin-induced effects. Meanwhile, dark-light transition (DRL) even led to the more serious decrease of Pfr content.
It is previously reported that in dark-grown seedlings, PHYA is the most abundant phytochrome, which is responsible for deetiolation under far-red light [8]. In the following test, we analyzed the expression profile of PHYA using quantitative real-time RT-PCR and discovered that SNP, hematin and 1.0% CO saturated solution all were able to induce PHYA gene expression as well as dark treatment (Fig. 9A). However, DRL treatment also exhibited no obvious positive effect.

Discussion
In animal research, HOs have been widely studied and well understood. Recently, an increasing number of researches concentrate on its function in plants. It is an important effecter of growth, development and defense response, but there were few reports about the involvement of HO/CO in formation of photosynthetic performance. In our experiment, we discovered that when etiolated wheat seedling induced by darkness, were transferred to light for greening, HO activities, HO-1 transcript, and CO release were obviously up-regulated (Fig. 3), which were consistent with the increase of chlorophyll content (Fig. 1). However, addition of ZnPPIX blocks this inducible effect. It is suggested that HO/CO system might participate in light-induced wheat seedling de-etiolation.  An earlier study [32] indicated that when treated with hematin, NaCl-induced chlorophyll decay of wheat seedling leaves was evidently reduced. Our results also confirmed that application of hematin, a HO-1 inducer or CO donor, could impair the reduction of chlorophyll contents in entirely darkness, but treatment with Hb (a CO scavenger) arrested this positive effects during the greening process (Fig. 1). The block action of Hb suggested that endogenous CO, may possibly from HO, be responsible for wheat seedling de-etiolation. Meanwhile, lower concentration of CO aqueous solution acted as the same function on restoring the decrease of chlorophyll contents, which also confirmed above hypothesis. Besides, hematin (H), or 1.0% CO aqueous solution (CO), could enhance HO activity in dark-grown wheat seedlings (Fig. 4), which suggested that hematin, acting as the substrate of endogenous HO and exogenous lower concentration of CO aqueous solution might regulate wheat seedling deetiolation in absence of light through activating endogenous HO/ CO system. Zhang et al. [33] reported that endogenous NO participated in light-inducing greening of etiolated barley seedlings. In this study, based on the relationship between HO/CO and NO shown previously in animals and recently in plants, we investigated the putative role of NO in light, hematin, and CO-induced responses by determining NO in vivo using fluorescence probe as well as the usage of chemicals that can manipulate the NO concentrations during greening process. Our results indicated that hematin and CO could induce the production of endogenous NO and its peak value appeared on the second day, which lag behind the increase of HO activity. Application of NOS inhibitor L-NAME (200 mM) and NO scavenger cPTIO could block the increase of chlorophyll content induced by hematin and 1.0% saturated CO aqueous solution in etiolated wheat seedlings, which suggested that NO might intermediate wheat seedling de-etiolation aroused by light or hematin/CO. Intriguingly, we also discovered that SNP treatment could also induce the elevation of HO activities in time-dependent manner in wheat seedling greening process, which was indicated by Noriega et al. [4] in soybean leaves. Based on these results, it is deduced that there exists the possible link between HO/CO and NO in wheat seedling de-etiolation in absence or presence of light. Phytochromes are traditionally known as biliprotein photoreceptors in plants and PHYA is predominantly responsible for deetiolation. To investigate the role of phytochrome in light or hematin/CO induced de-etiolation, we analyzed the changes of Pfr content and discovered that the amount of Pfr and PHYA expression were more substantive in hematin/CO treated wheat seedlings in completely darkness (Fig. 9). Furthermore, continuous dark treatment (DRD) also exhibited the same inducible effects. However, when wheat seedlings transferred from dark to light, both Pfr content and PHYA transcripts were decreased. From these data we could presume that hematin and CO accomplish wheat seedling de-etiolation process by increasing phytochrome expression through the following way: hematin and exogenous CO might activate endogenous HO activities to synthesize the precursors of the chromophores for the plant light-harvesting photobiliproteins [34,35]; another offspring, endogenous CO up-regulated the expression of phytochrome apoprotein genes. Afterwards, both of them linked together through a thiol-ether bond to form integrated phytochrome which could regulate chlorophyll accumulation through influencing the formation of 5-aminolevulinate [36]. SNP, a potent NO donor have more obvious function on increasing the content of Pfr than that hematin and CO did; whereas, adding cPTIO led to the decrease. Therefore, it is speculated that phytochrome might mediate the HO/CO signal transduction and act as a downstream regulator of HO/CO and NO. The mechanism of HO/CO in regulating wheat seedlings greening in continuous darkness might be different from light inducible effects. Chlorophyll and heme are both synthesized in the plastid from ALA and share a common pathway between ALA and Proto IX [37]. In the dark chlorophyll synthesis proceeds only as far as protochlorophyllide because the enzyme protochlorophyllide oxidoreductase (POR) has an absolute requirement for light. That etiolated seedlings transferred from dark to light activate the activity of POR, which finally leads to the chlorophyll accumulation. In the absence of light, the increase of HO activities accelerated the degradation of endogenous heme and consequently speeded up their common metabolic pathway, as a result, improving chlorophyll synthesis.
To our knowledge, the present study provided the first description of endogenous HO/CO and NO in wheat seedling de-etiolation. Both hematin and SNP displayed effects on upregulation of endogenous HO/CO system and NO releasing. The mutual induction effects suggested that there might be an inseparable relationship between HO/CO and NO during the greening process of wheat seedlings. Moreover, HO/CO system and NO might achieve the physiological function described above by modulating phytochrome transformation and synthesis.

CO-saturated aqueous solution preparation
CO gas was prepared by heating concentrated sulfuric acid (H 2 SO 4 ) with formic acid (HCOOH) at the speed of 3-5 seconds per drop. In our experiment, CO-saturated aqueous solution was freshly obtained by bubbling above CO gas gently through a glass tube into 300 ml of above Hoagland solution for about 30 min, a duration time long enough to make the solution saturated with CO. Then the saturated stock solution (100% of saturation) was immediately diluted with fresh prepared Hoagland solution to the concentration the experiment required (0.1, 1.0, 10 and 50% of saturation).

Treatments
Wheat seedlings were grown in growth chamber until the second leaves were fully expanded nearly for 14 days. The further experimental design thus consisted of the following treatment groups: LRL: stands for wheat seedlings were grown in normal condition. Other seedlings were kept in complete darkness for five days. After that, they were treated with different concentrations of hematin (1.0, 10, and 100 mM) and CO-saturated aqueous solution (0.1, 1.0, 10, and 50%) for a few additional days in the dark (abbreviated to DRD+H1.0, 10, and 100 or DRD+CO0.1%, 1.0%, 10%, and 50%). Among them, 10 mM hematin and 1.0% CO-saturated aqueous solution were chosen as the suitable treatment because of its better positive results, termed as DRD+H and DRD+CO in the following experiments. 100 mM SNP treatment was brought into comparison (abbreviated to DRD+S). DRD stands for these five-day dark-treated seedlings were continuous kept in the darkness. When these etiolated seedlings were transferred to light again (300 mmol m 22 s 21 ), it was abbreviated to DRL. NOS inhibitor L-NAME (200 mM), NO scavenger cPTIO (100 mM), NO/CO scavenger Hb (0.1 gL 21 ), and HO-1 inhibitor ZnPPIX (100 mM) were used respectively. All groups were arranged in plastic beakers with at least three replicates. Additionally, all the containers were separated to avoid the CO gas escaping or any possible CO gas interference, and above solutions were renewed each day. All tests were carried out at least three independent sets of experiments with similar results.

Chlorophyll content determination
Chlorophyll quantification was performed by the method described by Sa et al. [41].

Heme oxygenase preparation and activity determination
Preparation of crude membrane fractions and detection of HO activity were determined by the method of Liu et al. [42]. HO activity was calculated by measuring the formation of biliverdin-IXa (BV). The concentration of BV was estimated using a molar absorption coefficient at 650 nm of 6.25 mM 21 cm 21 in 0.1 M HEPES-NaOH buffer (pH 7.2). One unit of activity (U) was calculated by taking the quantity of the enzyme to produce 1 nmol BV at 37uC per 30 min.s

Phytochrome content determination
Phytochrome content was determined by the method of Lane et al. [43]. The phytochrome in wheat seedlings was assayed with a dual-wavelength difference photometer. The instrument measured the optical-density difference, DOD, between 730 and 800 mp, rather than the difference between 660 and 730 my, in order to eliminate the optical-density changes due to protochlorophyll transformations. The relative amounts of Pfr could be determined. After a given treatment, measurement of the sample would give a certain DOD reading, R1, on the instrument. The sample would then be irradiated with the actinic source of far-red light to convert any Pfr present to Pr and decrease the DOD reading to R2. The amount of Pfr would be equal to k (R1-R2), where k is a constant of proportionality. The difference between DOD readings is referred to as D(DOD).

Protein content determination
Protein concentration was determined by the method of Bradford [44] using BSA as the standard.
NO content determination by using Greiss reagent NO content from wheat leaves was determined using the method described by Zhou et al. [45]. Absorbance was assayed at 540 nm; NO content was calculated by comparison to a standard curve of NaNO 2 .

Imaging of endogenous NO by LSCM
In our experiment, 2.0-2.5 mm transversal sections from the center of leaves, was further treated in the presence or absence of 1 mM cPTIO for 50 min, and then incubated with the specific fluorescent probe DAF-2 DA in 20 mM BES-KCL buffer (pH6.2) in darkness for 1 h. The extra probes were removed by washing the sections at 20 mM BES-KCL buffer three times for 15 min and then images were taken using a TCS-SP2 laser scanning confocal microscope (Leica lasertechnik Gmbh, Heidelberg, exciting with the 488 nm, and emission using 490-530 nm for NO analysis). The negative probe AF 4-DA was also used as control. Experiments were repeated six times and similar results obtained. All manipulations were performed at 25uC61uC.

CO content determination
The determination of CO content by gas chromatography and mass spectrometry (GC/MS) were carried out according to the method described by our previous report [42].

Statistical analyses
Each experiment was repeated at least three times with similar results. Values were expressed as means 6 SE at least three replicates obtained from three independent experiments. Different measurements were subjected to analysis of variance (ANOVA) using SPSS with significances of P,0.05 and P,0.01, respectively.