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Drought and low temperature are two key environmental factors that induce adult citrus flowering. However, the underlying regulation mechanism is poorly understood. The bZIP transcription factor FD is a key component of the florigen activation complex (FAC) which is composed of FLOWERING LOCUS T (FT), FD, and 14-3-3 proteins. In this study, isolation and characterization of CiFD in citrus found that there was alternative splicing (AS) of CiFD, forming two different proteins (CiFDα and CiFDβ). Further investigation found that their expression patterns were similar in different tissues of citrus, but the subcellular localization and transcriptional activity were different. Overexpression of the CiFD DNA sequence (CiFD-DNA), CiFDα, or CiFDβ in tobacco and citrus showed early flowering, and CiFD-DNA transgenic plants were the earliest, followed by CiFDβ and CiFDα. Interestingly, CiFDα and CiFDβ were induced by low temperature and drought, respectively. Further analysis showed that CiFDα can form a FAC complex with CiFT, Ci14-3-3, and then bind to the citrus APETALA1 (CiAP1) promoter and promote its expression. However, CiFDβ can directly bind to the CiAP1 promoter independently of CiFT and Ci14-3-3. These results showed that CiFDβ can form a more direct and simplified pathway that is independent of the FAC complex to regulate drought-induced flowering through AS. In addition, a bHLH transcription factor (CibHLH96) binds to CiFD promoter and promotes the expression of CiFD under drought condition. Transgenic analysis found that CibHLH96 can promote flowering in transgenic tobacco. These results suggest that CiFD is involved in drought- and low-temperature-induced citrus flowering through different regulatory patterns.

In the present study, we evaluated the protective effect of exogenous heme oxygenase-1 (HO-1 EC 1.14.99.3) inducer hematin against mercury-induced oxidative damage in the roots of Alfalfa (Medicago sativa L.). Plants exposed to mercury (HgCl2) exhibited a significant increase of lipid peroxidation, as well as inhibition of root elongation. However, hematin (50 μM) supplementation to HgCl2 (100 μmol/L) treated plants effectively reduced the lipid peroxidation and partially increased the root elongation. These responses were mimicked by the application of aqueous solution of carbon monoxide (CO) with 50% saturation. Also, treatment with hematin could result in the potent induction of HO-1 transcript in the root tissues, as detected 12 h following treatment. Moreover, the activation of anti-oxidant enzyme, including glutathione reductase, monodehydroascorbate reductase and superoxide dismutase activities, and the decrease of lipoxygenase activity, were induced by hematin at 12 h or 24 h, which was further confirmed by histochemical staining for the detection of lipid peroxidation and loss of membrane integrity. Whereas, ascorbate peroxidase and guaiacol peroxidase isozyme activities or their transcripts were reduced, respectively, indicating that hydrogen peroxide might act as a signal to mediate Hg-tolerance at the beginning of treatment. The ameliorating effects of hematin were specific, since the CO scavenger hemoglobin differentially reversed the above actions. Taken together, our results suggested that hematin exhibits a vital role in protecting the plant against Hg-induced oxidative damage.

Carbon monoxide (CO), an odorless, tasteless and colorless gas, has recently proved to be an important bioactive or signal molecule in mammalian cells, with its effects mediated mainly by nitric oxide (NO). In the present report, we show that exogenous CO induces lateral root (LR) formation, an NO-dependent process. Administration of the CO donor hematin to rapeseed (Brassica napus L. Yangyou 6) seedlings for 3 days, dose-dependently promoted the total length and number of LRs. These responses were also seen following the application of gaseous CO aqueous solutions of different saturated concentrations. Furthermore, the actions of CO on seedlings were fully reversed when the CO scavenger hemoglobin (Hb) or the CO-specific synthetic inhibitor zinc protoporphyrin-IX (ZnPPIX) were added. Interestingly, depletion of endogenous NO using its specific scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide potassium salt (cPTIO) or the nitric oxide synthase (NOS) inhibitor NG-nitro-l-arginine methyl ester (l-NAME), led to the complete abolition of LR development, illustrating an important role for endogenous NO in the action of CO on LR formation. However, the induction of LR development by 200 μmol/L sodium nitroprusside (SNP), an NO donor, was not affected by the presence or absence of ZnPPIX. Furthermore, using an anatomical approach combined with laser scanning confocal microscopy with the NO-specific fluorophore 4,5-diaminofluorescein diacetate, we observed that both hematin and SNP increased NO release compared with control samples and that the NO signal was mainly distributed in the LR primordia (LRP), especially after 36 h treatment. The LRP were found to have similar morphology in control, SNP- and hematin-treated seedlings. Similarly, the enhancement of the NO signal by CO at 36 h was differentially quenched by the addition of cPTIO, l-NAME, ZnPPIX and Hb. In contrast, the induction of NO caused by SNP was not affected by the application of ZnPPIX. Therefore, we further deduced that CO induces LR formation probably mediated by the NO/NOS pathway and NO may act downstream of CO signaling, which has also been shown to occur in animals.

Recently in animals, endogenous carbon monoxide (CO), like nitric oxide, was implicated as another important physiological messenger or bioactive molecule. However, little information is known about the physiological roles of CO in the whole plant. In the present study, we report that different concentrations of the CO donor hematin (0.1, 1.0 and 10.0 μmol/L) alleviated wheat (Triticum aestivum L. Yangmai 158) seed germination inhibition caused by 250 mmol/L NaCl stress in a dose-dependent manner. These responses were also proved by the addition of different gaseous CO aqueous solutions from 0.1% to 100.0% of saturation. Among these treatments, the effect of 1.0 μmol/L hematin and 1.0% saturation of CO aqueous solution were the most obvious. Furthermore, compared with non-hematin treatment, the degradation of storage reserves in wheat seeds was also accelerated. Time-course analyses showed that application of hematin dose-dependently increased the activities of superoxide dismutase, catalase, ascorbate peroxidase, and guaiacol peroxidase activities, thus decreasing the lipid peroxidation in germinating wheat seed subjected to salt stress. Meanwhile, the responses of hematin were specific for CO because the addition of the CO scavenger hemoglobin (0.2 g/L) blocked the various actions of 1.0 μmol/L hematin. Taken together, the results of the present study demonstrate that CO, at a low concentration, is able to attenuate the seed germination inhibition produced by salinity stress and counteract the lipid peroxidation in germinating wheat seeds. (Managing editor: Ping He)

Carbon monoxide (CO), a by-product released during the degradation of heme by heme oxygenases (EC 1.14.99.3) in animals, is regarded as an important physiological messenger or bioactive molecule involved in many biological events that has been recently reported as playing a major role in mediating the cytoprotection against oxidant-induced lung injury. In the present study, we first determined the protective effect of exogenous CO against salt-induced oxidative damage in wheat seedling leaves. Wheat seedlings treated with 0.01 μmol/L hematin as the CO donor demonstrated significant reversal of chlorophyll decay, dry weight, and water loss induced by 300 mmol/L NaCl stress. Interestingly, the increase in lipid peroxidation observed in salt-treated leaves was reversed by 0.01 nmol/L hematin treatment. Time-course analyses showed that application of 0.01 μmol/L hematin enhanced guaiacol peroxidase, superoxide dismutase, ascorbate peroxidase and catalase activities in wheat seedling leaves subjected to salt stress. These effects are specific for CO because the CO scavenger hemoglobin (1.2 mg/L) blocked the actions of the CO donor hematin. However, higher concentration of the CO donor (1.0 μmol/L) did not alleviate dry weight and water loss of salt-stressed wheat seedlings. These results suggest that exogenous application of low levels of a CO donor may be advantageous against salinity toxicity. (Managing editor: Ping He)

Pretreatment of lower H(2)O(2) doses (0.05, 0.5 and 5 mM) for 24 h was able to dose-dependently attenuate lipid peroxidation in wheat seedling leaves mediated by further oxidative damage elicited by higher dose of H(2)O(2) (150 mM) for 6 h, with 0.5 mM H(2)O(2) being the most effective concentrations. Further results illustrated that 0.5 mM H(2)O(2) pretreatment triggered the biphasic production of H(2)O(2) during a 24 h period. We also noticed that only peak I (0.25 h) rather than peak II (4 h) was approximately consistent with the enhancement of heme oxygenase (HO) activity, HO-1 gene expression. Meanwhile, enhanced superoxide dismutase (SOD) activity, Mn-SOD and Cu,Zn-SOD transcripts might be a potential source of peak I of endogenous H(2)O(2). Further results confirmed that 0.5 mM H(2)O(2) treatment for 0.5 h was able to upregulate HO gene expression, which was detected by enzyme activity determination, semi-quantitative reverse transcription-polymerase chain reaction and western blotting. Meanwhile, the application of N,N'-dimethylthiourea, a trap for endogenous H(2)O(2), not only blocked the upregulation of HO, but also reversed the corresponding oxidation attenuation. Together, the above results suggest that endogenous H(2)O(2) production (peak I) plays a positive role in the induction of HO by enhancing its mRNA level and protein expression, thus leading to the acclimation to oxidative stress.