WEI Kang, WANG Li-yuan, RUAN Li, ZHANG Cheng-cai, WU Li-yun, LI Hai-lin, CHENG Hao

Key Laboratory of Tea Biology and Resources Utilization, Ministry of Agriculture, National Center for Tea Improvement, Tea Research Institute Chinese Academy of Agricultural Sciences (TRICAAS), Hangzhou 310008, P.R.China

Abstract Nitric oxide (NO) and hydrogen peroxide (H2O2) are essential signaling molecules with key roles in auxin induced adventitious root formation in many plants. However, whether they are the sole determinants for adventitious root formation is worth further study. In this study, endogenous NO and H2O2 were monitored in tea cutting with or without indole-3-butyric acid(IBA) treatment by using the fluorescent probes diaminofluorescein diacetate (DAF-2DA) and 2’,7’-dichlorodihydrofluorescein diacetate (DCF-DA), respectively. The overproduction of NO and H2O2 was detected in the rooting parts of tea cuttings treated with or without IBA. But little NO and H2O2 was detected before the initiation phase of tea cuttings even with IBA treatment indicating that they might be not directly induced by IBA. Further carbon and nitrogen analysis found that the overproduction of NO and H2O2 were coincident with the consumption of soluble sugars and the assimilation of nitrogen.These results suggest that rooting phases should be taken into consideration with the hypothesis that auxin induces adventitious root formation via NO- and H2O2-dependent pathways and sink establishment might be a prerequisite for NO and H2O2 mediated adventitious root formation.

Keywords: nitric oxide, hydrogen peroxide, indole-3-butyric acid, tea cuttings

1. Introduction

The formation of adventitious roots is an essential process for the clonal propagation of many economically important horticultural and woody species. Slow or insufficient rooting will lead to heavy losses for farmers. In the pastfew decades, many root stimulators were developed to improve plant propagation efficiency. The phytohormone auxins are the most widely used in agricultural practice. The rooting efficiency of cuttings treated with auxins will increase significantly (Rout 2006). However, current understanding of the physiological and biochemical basis of auxin-induced adventitious rooting remains at a rudimentary stage(Pagnussat et al. 2003; Pacurar et al. 2014; Pop et al. 2011).

Recently, it was reported that nitric oxide (NO) and hydrogen peroxide (H2O2) mediate the auxin responses during adventitious or primary root formation (Li et al.2009; Fernández-Marcos et al. 2011). The NO and H2O2are versatile signaling molecules and are involved in many physiological processes including growth, development and defense responses (Neill et al. 2002). Exogenous application of auxin induces overproduction of NO and H2O2(Pagnussat et al. 2003; Li et al. 2009). Importantly,exogenous H2O2or NO-donor sodium nitroprusside mimic the auxin-induced adventitious root formation in cucumber(Pagnussat et al. 2003), marigold (Liao et al. 2009) and mung bean (Li et al. 2010). Furthermore, the induction of NO and H2O2always occur in parallel, which suggests they may act synergistically (Lum et al. 2002; Neill et al. 2003).

Therefore, a recent hypothesis proposes that auxin induces adventitious root formation via NO- and H2O2-dependent cyclic guanosine monophosphate (cGMP)signaling (Bai et al. 2012). The latter is closely associated with cell enlargement and division (Pagnussat et al. 2003;Li et al. 2010). This hypothesis has been proven in many herbaceous plants (Pagnussat et al. 2003; Liao et al. 2009;Li et al. 2010). However, the extent to which this hypothesis could be used in woody species is still unknown yet because the rooting processes of woody plants are much longer and more complex than herbaceous plants.

The tea plant (Camellia sinensis), a type of evergreen woody species is one of the most important beverage plants in the world. It is currently propagated primarily by nodal cuttings. However, losses always occur due to rooting difficulty. Thus, the auxin derivative indole-3-butyric acid(IBA) is widely used to stimulate adventitious root formation in tea cuttings. However, the underlying mechanism is not well studied (Rout 2006). The aim of this study was:1) to investigate whether the hypothesis of auxin-induced adventitious root formation via NO and H2O2is suitable for the tea plant and 2) to explore the underlying metabolism.

2. Materials and methods

2.1. Plant material

Freshly growing twigs of Camellia sinensis var. Longjing 43 were collected from the tea garden of the Tea Research Institute Chinese Academy of Agricultural Sciences,Hangzhou, China and brought to the laboratory in a water cooled box. Each twig was further segmented into smaller pieces consisting of one node with a single leaf in each segment. The total length of the stem of the cuttings was about 3.5 cm. The single node cuttings of uniform size were then selected and put in 1 000-mL glass beakers containing 200 mL basic nutrient solution (1/10 Hoagland nutrient solution). The basal region of the nodal cuttings was dipped into the solution. The pH of the nutrient solution was maintained at 5.8 (Brinker et al. 2004). The IBA was added to the corresponding beakers to form 2 treatments:(1) the control contained only the basic solution; and (2)IBA (0.4 mmol L–1) for 24 h, which was modified from our previous experiment design (Wei et al. 2013). After that, the nodal cuttings were transplanted into 30-L containers filled with potting medium (Pindstrup Peat moss) and placed in a greenhouse for further cultivation.

Five tea cuttings of each treatment were collected 21 days after treatment. The basal region of the tea cuttings was washed with distilled water 3 times and used for NO and H2O2detection. The roots of tea cuttings under control treatment were also collected after 3 months’ cultivation for NO and H2O2detection. Meanwhile, the remainder of the cutting samples was collected 21, 28 and 35 days after treatment for carbon and nitrogen analysis.

2.2. Confocal laser scanning microscopy

NO was visualized using the specific NO dye DAF-2DA(Calbiochem, Nottingham, UK), using the method described by Desikan et al. (2002). The stems of control, the stems,stem-root bases and roots of IBA treated cuttings were dyed with 10 µmol L–1DAF-2DA dissolved in MES/KCl buffer for 1 h at 25°C in darkness. After labeling, the samples were washed 4 times in 20 min with MES buffer (10-3mol L–1,pH 6.15) and were prepared on microscope slides. A Zeiss Axiowert 200M-type Fluorescent Microscope (Carl Zeiss,Germany) was used to detect fluorescence intensity. Digital photographs were collected with a high-resolution digital camera (Axiocam HR, Carl Zeiss, Germany). To measure the fluorescence intensity, the IMAGEJ software (NIH,Bethesda, MD, USA) was applied according to Bonales et al. (2013). At least 5 samples were measured for each treatment. For each of them, the relative fluorescence values of 20 cells (technical replicates) were averaged as described by Wang et al. (2017).

The H2O2production was monitored using the H2O2-sensitive fluorescent probe DCF-DA (Molecular Probes,Leiden, the Netherlands) using the method described by Lee et al. (2008). The stems and roots of the tea cuttings were incubated in 20 µmol L–1DCF-DA for 1 h, then washed with 0.1 mmol L–1KCl and 0.1 mmol L–1CaCl2(pH 6.0). The samples were then incubated for a further 60 min before images were visualized. All samples were also imaged with a Zeiss fluorescent microscope. Images were analyzed using IMAGEJ software (NIH, Bethesda, MD, USA). Data are presented as the mean pixel intensities. At least 5 samples were measured for each treatment. The relative fluorescence values of 20 cells (technical replicates) were averaged as described by Wang et al. (2017).

2.3. Carbon and nitrogen analysis

The basal region of the cuttings (about 2.5 cm) were cut and used for carbon and nitrogen analysis. The basal region of the IBA-treated cuttings also included adventitious roots. The total carbon and total nitrogen contents were determined successively using a VarioMAX CN-analyzer(Elementar, Hanau, Germany). The soluble protein and total soluble sugars were quantified in potassium phosphate buffer (KPB) (50 mmol L–1, pH 7.5) extracts of fresh samples(0.3 g). These extracts were filtered through four cheese cloth layers and centrifuged at 15 500 r min–1for 15 min at 4°C. The supernatant was collected and stored at 4°C for the determination of soluble protein and sugars. The soluble protein was measured by the protein dye-binding method of Bradford (1976) using bovine serum albumin asstandard. Quantitative determination of total soluble sugars was carried out according to a colorimetric method (Yemm and Willis 1954). The total carbon, total nitrogen, soluble protein and soluble sugars were analyzed with 3 replicates per treatment.

3. Results

3.1. Effect of lBA treatment on rooting

The effect of IBA on adventitious root formation was obvious in the tea cuttings after cultivation in potting medium for 3 weeks (Fig. 1). Adventitious roots were initiated in the IBA treated cuttings, but no root was observed in the tea cuttings without IBA treatment (the control) even 35 days after cultivation (data not shown). These results are consistent with the finding of Rout (2006). It is also worth noting that the adventitious roots were mainly present in the middle region of the IBA treated cuttings rather than the basal region,which might be correlated with the oxygen levels in the microenvironment of tea cuttings during cultivation (Fig. 1).

3.2. Overproduction of endogenous NO and H2O2 occurred in rooting parts but not stems even after lBA treatment

To assess the possible involvement of NO and H2O2in IBA-induced adventitious root formation in tea cuttings,the fluorescent probe DAF-2DA and DCF-DA were used for in situ detection of NO and H2O2. Fig. 2 shows that NO was mainly present in the roots and stem-root bases of tea cuttings and present bright green fluorescence (Fig. 2-C–E).In contrast, except for the slight green auto-fluorescence detected in stem epidermises, NO was not present in thestems regardless of IBA treatment (Fig. 2-A and B). In terms of relative fluorescence intensity, NO in the roots and stemroot bases of IBA treated cuttings were 108.8 and 21.1 times higher than that in the stems of control (Fig. 3). Interestingly,roots of control also exhibited strong NO signals. However,there was no significant difference in the stems of control and IBA treated cuttings. Additional studies showed that the NO fluorescence in the stems remained hard to detect regardless of cultivation time or IBA treatment (data not shown). These results provide evidence that NO emission is coincident with adventitious root formation, but not directly induced by IBA in stems.

Fig. 1 The phenotypes of tea cuttings treated with or without(control) indole-3-butyric acid (IBA). The tea cuttings were cultivated in potting medium for 3 weeks.

H2O2exhibited a similar distribution in different parts of the tea cuttings (Figs. 4 and 5). The rooting parts including newly formed adventitious roots, and the stem-root bases had H2O2levels that were 21.0 and 23.5 times higher than that in the stems of control (Fig. 5). There was little H2O2fluorescence in the stems regardless of IBA treatment(Fig. 4-A and B), which suggests that H2O2might play the same role as NO in adventitious root formation.

3.3. NO and H2O2 emission might largely affect carbon/nitrogen metabolisms in tea cuttings

NO and H2O2emissions were coincident with the formation of adventitious roots in the tea cuttings, which may markedly affect the carbon/nitrogen (C/N) status. Therefore, the time courses of the C/N response for IBA-induced adventitious root formation were also studied (Fig. 6). The carbon levels in the basal parts of the IBA-treated cuttings are slightly lower than those of control, although a significant difference(P=0.020) was only observed 21 days after treatment.However, significantly higher nitrogen levels were identified in IBA-treated cuttings versus control in all the time courses.In terms of the C/N ratio, IBA treated cuttings were also significantly lower than those of control, which suggests that the formation of adventitious root facilitates nitrogen assimilation.

Fig. 2 DAF-2DA fluorescence and merged microscopic visualization of different tissues of tea cuttings with or without (control)indole-3-butyric acid (IBA) treatment (NO analysis). A, stem of control. B, stem of IBA treated cuttings. C, stem-root base of IBA treated cuttings. D, root of IBA treated cuttings. E. root of the control cuttings. Samples were prepared and investigated as described in Materials and methods. The selected images represent the same results from 5 replicates per treatment. Bar=100 µm.

Fig. 3 DAF-2DA fluorescence in different tissues of tea cuttings treated with or without (control) indole-3-butyric acid (IBA)treatment. Vertical bars are standard errors (n=5).

On the other hand, the soluble protein contents were significantly lower in IBA treated cuttings, except for those 28 days after treatments (Fig. 7), which is contradicted by the trend in nitrogen contents. Furthermore, the soluble sugar contents were also significantly decreased when NO and H2O2became elevated, suggesting that the growth of roots may be activated by NO and H2O2. This correlates to the consumption of soluble sugars and proteins.

4. Discussion

The formation of adventitious roots can be divided into three successive but interdependent phases, namely: induction,initiation and expression (Gaspar et al. 1992). The induction phase is defined as the time period preceding the initiation of cell division (Moncousin 1991). Although no visible changes could be observed, many molecular and biochemical events occur in the induction phase that are the prerequisites for latter phases. For most herbaceous plants, the induction phase appears to be very short, generally less than 24 hours(Pagnussat et al. 2003; Liao et al. 2009; Li et al. 2010). In contrast, this phase may last for many weeks in woody species (Schwambach et al. 2005). This indicates woody species may need more preparation prior to adventitious root formation.

Fig. 4 DCF-DA fluorescence and merged microscopic visualization of different tissues of tea cuttings with or without indole-3-butyric acid (IBA) treatment (H2O2 analysis). A, stem of control. B, stem of IBA treated cuttings. C, stem-root base of IBA treated cuttings. D, root of IBA treated cuttings. E, root of the control cuttings. Samples were prepared and investigated as described in Materials and methods. The selected images represent the same results from 5 replicates per treatment. Bar=100 µm.

Fig. 5 DCF-DA fluorescence in different tissues of tea cuttings treated with or without (control) indole-3-butyric acid (IBA)treatment. Vertical bars are standard errors (n=5).

It has long been suggested that NO and H2O2are the key messengers in the auxin-induced adventitious rooting process, which could mimic the effect of auxins in many herbaceous plants (Rugini et al. 1997; Pagnussat et al.2004). However, few if any woody plants were tested for the suitability of the hypothesis, although clonal propagation is even more important in these plants. In our previousstudy, both H2O2and NO-donor sodium nitroprusside pretreatments caused plant death, but did not show any positive effect on adventitious root formation in nodal tea cuttings (data not shown), which seems to be inconsistent with this hypothesis. In fact, both H2O2and NO are also signals involved in programmed cell death (Wang et al.2013). Therefore, this study was carried out for in vivo detection of NO and H2O2in the tea cuttings with or without IBA treatment. The results illustrate that NO and H2O2participate in adventitious root formation, but they might not be directly induced by IBA in tea cuttings (Figs. 2 and 4). IAA-oxidase (IAAO) activity, which was negatively correlated with endogenous auxin content, could be used as an indicator to monitor endogenous auxin changes (Nag et al. 2001). Rout (2006) studied the dynamic change of IAAO activity in tea cuttings treated with or without IBA and found the IAAO activity decreased during the induction and initiation phases but increased during expression phase in IBA-treated cuttings, which reveals endogenous auxin level was induced in IBA-treated cuttings during the firsttwo phases. Similar results were also identified in other plant species (Yan et al. 2017). However, that expression pattern was not consistent with the NO and H2O2emissions..Taking into account the NO and H2O2expression patterns in the tea cuttings, it seems that the factor of rooting phase should not be ignored in the rooting hypothesis. NO and H2O2are essential for the initial and expression phases,but at least not for the induction phase in the nodal tea cuttings. Furthermore, the promotive effect of exogenous NO and H2O2on adventitious rooting might be due to the short induction phase in herbaceous plants. However, the induction phase of wood species like tea plants is much longer. Thus, neither exogenous NO nor H2O2is able to promote adventitious root formation.

Fig. 6 The dynamic changes of carbon, nitrogen contents and carbon/nitrogen ratio in the basal region of tea cuttings with or without indole-3-butyric acid (IBA) treatment. Data are the means±standard errors (n=3). ＊ represents a significant difference (P＜0.05) between the control and IBA treatment.

Fig. 7 The dynamic changes of soluble protein and sugar contents in the basal region of tea cuttings with or without indole-3-butyric acid (IBA) treatment. Data are the means±standard errors (n=3). ＊ represents a significant difference (P＜0.05)between the control and IBA treatment.

Our results also suggest that there should be some prerequisite factors involved in the NO and H2O2mediated adventitious root formation (Figs. 2–5). These factors are mainly formed in the induction phase and able to be induced by auxins. Ahkami et al. (2009) reported that the sink establishment characterized by apoplastic unloading of sucrose and being probably mediated by jasmonates is an essential process prior to adventitious root formation.High levels of energy and carbon skeletons are needed for cell division and growth during adventitious root formation.Moreover, increasing studies found that sugars play key regulatory roles during adventitious root formation(Takahashi et al. 2003; Corrêa et al. 2005; Gibson 2005).Soluble sugars generally increase after excision and reach a maximum when roots emerge (Husen and Husen 2001;Ahkami et al. 2009). Thus, the effects of NO and H2O2on carbon and nitrogen statuses were also measured in thisstudy. Lower levels of soluble sugars were detected in the rooting tea cuttings, indicating NO and H2O2mediated pathways might largely dependent on the consumption of sugars (Fig. 7). Furthermore, higher nitrogen content and lower C/N ratio identified in the rooting tea cuttings suggests NO- and H2O2-mediated pathways may facilitate nitrogen assimilation (Fig. 6). These results are consistent with previous findings (Ahkami et al. 2009) and reveal that the sink establishment process might be the prerequisite factor for NO and H2O2promoted adventitious root formation.

5. Conclusion

Our results indicate that NO and H2O2are essential but not the sole determinants of adventitious root formation in tea cuttings. The overproduction of NO and H2O2was only detected in the rooting parts of tea cuttings. Little NO and H2O2was detected in the induction phase of tea cuttings even with IBA treatment, suggesting they might not be directly induced by IBA. Furthermore, carbon and nitrogen analysis found that the overproduction of NO and H2O2was coincident with the consumption of soluble sugars and the assimilation of nitrogen. This indicates that sink establishment during the induction phase might be a prerequisite for NO and H2O2mediated adventitious root formation.

Acknowledgements

We are greatly indebted to the earmarked fund for China Agriculture Research System (CARS-19) and the Innovation Project for Agricultural Sciences and Technology from the Chinese Academy of Agricultural Sciences (CAAS-ASTIP-2017-TRICAAS) for their financial supports.