Combined Cytological and Transcriptomic Analysis Reveals a Nitric Oxide Signaling Pathway Involved in Cold-Inhibited Camellia sinensis Pollen Tube Growth

Nitric oxide (NO) as a signaling molecule plays crucial roles in many abiotic stresses in plant development processes, including pollen tube growth. Here, the signaling networks dominated by NO during cold stress that inhibited Camellia sinensis pollen tube growth are investigated in vitro. Cytological analysis show that cold-induced NO is involved in the inhibition of pollen tube growth along with disruption of the cytoplasmic Ca2+ gradient, increase in ROS content, acidification of cytoplasmic pH and abnormalities in organelle ultrastructure and cell wall component distribution in the pollen tube tip. Furthermore, differentially expressed genes (DEGs)-related to signaling pathway, such as NO synthesis, cGMP, Ca2+, ROS, pH, actin, cell wall, and MAPK cascade signal pathways, are identified and quantified using transcriptomic analyses and qRT-PCR, which indicate a potential molecular mechanism for the above cytological results. Taken together, these findings suggest that a complex signaling network dominated by NO, including Ca2+, ROS, pH, RACs signaling and the crosstalk among them, is stimulated in the C. sinensis pollen tube in response to cold stress, which further causes secondary and tertiary alterations, such as ultrastructural abnormalities in organelles and cell wall construction, ultimately resulting in perturbed pollen tube extension.


INTRODUCTION
Low temperature is a major factor that significantly constrains the life cycle of higher plants, including germination, growth, development, flowering, and seed setting (Klemens et al., 2014;Maruyama et al., 2014). Among these processes, reproductive processes, particularly pollen tube growth, are negatively regulated by low temperatures (Hedhly, 2011). According to previous reports, cold stress significantly reduces the pollen tube growth of Cicer arietinum (Srinivasan et al., 1999) and Pyrus bretschneideri  and disrupts the morphology of the pollen tube tip zone (Srinivasan et al., 1999). Recently, the actin cytoskeleton, endocytosis and some signaling molecules, such as the calcium ion (Ca 2+ ) and reactive oxygen species (ROS), have been implicated in the cold stress-inhibited pollen tube growth in vitro (Wu et al., 2012;Gao et al., 2014). However, the underlying basis of the cellular mechanisms of pollen tube growth under cold stress remains largely unknown.
Nitric oxide (NO) is a highly active gaseous signaling molecule that plays crucial roles in many key physiological processes in plants, including seed germination, photo morphogenesis, mitochondrial activity, leaf expansion, root growth, regulation of stomatal movement, fruit maturation, senescence and iron metabolism, etc. (Lanteri et al., 2008;Neill et al., 2008;Sanz et al., 2015). Furthermore, many investigations have revealed that NO plays an important role in the reproductive processes of higher plants, including flower bud differentiation, flowering induction, fertilization and seed setting, particularly pollen tube tip growth (Prado et al., 2008;Domingos et al., 2015). For example, NO participated in self-incompatibility-induced programmed cell death (PCD) in the Papaver rhoeas pollen tube through interactions with Ca 2+ , ROS and actin signaling (Wilkins et al., 2011), and there was a suspected potential association between NO and other signaling factors, such as the MAPK cascade and cytoplasmic pH, in this process (Wilkins et al., 2014(Wilkins et al., , 2015. Similarly, NO was found to modulate both the influx of extracellular Ca 2+ and actin filament organization during cell wall construction to regulate the tip growth of Pinus bungeana pollen tubes . Moreover, NO is also involved in the tolerance of plants to various abiotic stresses (Qiao et al., 2014), such as high salt (Qiao et al., 2009), heat (Xuan et al., 2010), drought (Liao et al., 2012), heavy metals (Saxena and Shekhawat, 2013;Kováčik et al., 2014), and UV-B irradiation stress (Tossi et al., 2009), particularly in cold acclimation and freezing tolerance of plants. For example, Majláth et al. (2012) reported an increased production of NO in Triticum aestivum roots after exposure to cold stress, but a decrease of NO content was found in Capsicum annuum leaves when they were exposed to low temperatures (Airaki et al., 2012). Recently, NO has been proposed to protect plants from chilling injury by increasing their antioxidant defenses and thereby preventing ROS damage; NO stimulated the activity of S-nitrosylated proteins in Brassica juncea under cold stress (Sehrawat et al., 2013a). Additionally, NO participates in cold-triggered root growth inhibition by regulating the content of long-chain bases and the expression of cold-responsive genes (Guillas et al., 2013;Puyaubert and Baudouin, 2014). These data therefore suggest that there is a potential signal regulatory network that depends on NO in a plant's response to cold stress; further investigation is required to clarify the underlying mechanisms of this process.
Above all, NO is thought to act as a core signaling molecule in the cold stress-mediated inhibition of pollen tube growth, and this hypothesis has been supported by physiological and pharmacological findings in our previous research, which showed that NO production from NO synthase (NOS)-like activity decreased cold-responsive pollen germination, inhibited pollen tube growth and reduced proline (Pro) accumulation, partly via the cGMP signaling pathway in Camellia sinensis (Wang et al., 2012). However, the role of the NO-dependent complex signaling network, including cGMP, Ca 2+ , ROS, actin, and pH signaling and the cross-talk among them, in the process of cold stressinhibited pollen tube growth, has not yet been elucidated. In the present study, we also investigated the signal transduction roles of NO during pollen tube elongation in response to cold stress in C. sinensis. Specifically, we focused on cold-induced NO that is involved in inhibiting the tip growth of the pollen tube, in addition to several linked cellular features that are essential for the NO signaling pathway under cold tolerance, including the cytoplasmic Ca 2+ gradient, the ROS concentrations, the acidification of the cytoplasm, the tip ultrastructure, and the composition of the cell wall. Moreover, we also performed the identification of differentially expressed genes in the coldinduced NO signaling pathway in C. sinensis pollen tubes, including genes involved in NO synthesis, cGMP, Ca 2+ , ROS, pH, actin, the cell wall, and the MAPK cascade, using transcriptomic analyses, which provided insight into the molecular mechanisms that underlie the above events. These data provide further insights into the regulation of NO signaling in the pollen tube response to cold stress in C. sinensis.

Plant Material and In vitro Pollen Culture
Mature pollen was collected from "C. sinensis (L.) O. Kuntze cv.

Observation of Pollen Tube Elongation and Morphology
To measure the mean length of the pollen tubes, approximately 50 pollen tubes were detected in each of the three replicates at 0.5, 1, and 2 h after different treatments. The morphology of the pollen tubes was examined using a Leica DM2500 biological microscope, and digital images were captured with a Leica DFC290 digital color camera (Leica, Germany).

Measurement of Cytoplasmic Ca 2+ Gradient
The pollen tubes were loaded with the fluorescent Ca 2+ indicator Fluo-4/AM ester (Life Technologies, Invitrogen, USA) according to Spinelli and Gillespie (2012) with slight modifications. Briefly, after the treatments, the samples were incubated at 25 or 4 • C in the dark in culture medium containing 20 µM Fluo-4/AM ester (prepared with DMSO) for 15 min. Then, the pollen tubes were rinsed three times with the corresponding culture medium to wash out excess fluorophore. The fluorescence of at least 20 pollen tubes in each of three replicates was detected using a 488-nm argon laser attached to a Laser Scanning Confocal Microscope (LSCM, Zeiss LSM 780, Germany) with the same parameter settings, and emission signals were collected at 515 nm. Image analysis was performed with pseudo color technology (Rainbow2) in ZEN 2013 software.

Measurement of Cytoplasmic ROS
The presence of ROS in the pollen tubes was assayed and visualized with CM-H 2 DCF-DA (Invitrogen, USA) as described by Wilkins et al. (2011) with slight modifications. In brief, the samples were incubated in 5 µM CM-H 2 DCF-DA for 15 min in the dark; then, the excess fluorescent indicator was washed out. The specimens were mounted and photographed with a Zeiss LSM 780 LSCM (excitation at 488 nm and emission at 515 nm). To allow comparisons between images, identical parameter settings were used throughout each experiment. The quantification of relative fluorescence units of at least 20 pollen tubes in each of three replicates was performed using the ImageJ software package, and the mean relative fluorescence intensities were calculated.

Measurement of Cytoplasmic pH
Intracellular [pH] cyt was determined in the living pollen tubes with BCECF AM (Invitrogen, USA) as described by Wilkins et al. (2015) with slight modifications. The pollen tubes were loaded with 2.5 µM BCECF AM for 15 min followed by washing with the corresponding culture medium. The pollen tubes were only imaged within 5 to 10 min after the addition of BCECF AM because this time frame allowed for accurate reporting of [pH] cyt . The images of at least 20 pollen tubes in each of three replicates were captured using a Zeiss LSM 780 LSCM with sequential excitation at 488 nm and emission at 510-550 nm, and the image analysis was performed with pseudo color technology (Rainbow2) in ZEN 2013 software.

Ultrastructure Observation with a Transmission Electron Microscope (TEM)
A TEM analysis was performed according to Wang et al. (2014) and Sheng et al. (2006) with slight modifications. The pollen tubes were collected after treatment for 1 h and then fixed in 2.5% glutaraldehyde in 100 mM PBS (pH 7.2) at 4 • C for 4 h. Then, they were washed with 100 mM PBS and post-fixed with 2% OsO 4 for 2 h, washed again, dehydrated in an ethanol series (50, 70, 90, and 100%) and finally embedded in Spurr's epoxy resin. Sections were cut with an LKB-V ultramicrotome, stained with 2% uranyl acetate (w/v) in 70% methanol (v/v), and 0.5% lead citrate and observed using a TEM (H-7650, Hitachi Hightechnologies Corporation, Japan) at 80 kV.

Fluorescent Immunolabeling of Pectins and AGPs in the Pollen Tube Cell Wall
The immunolabeling of pectins and AGPs in pollen tube cell walls was performed with LM19, LM20 and LM2 antibodies (PlantProbes, Leeds, UK) according to Chen et al. (2009) with slight modifications. Pollen tubes that had been treated for 1 h were collected and fixed in 4% paraformaldehyde in 100 mM phosphate buffer solution (PBS, pH 7.2) for 30 min at room temperature and rinsed three times for 5 min each with PBS. Subsequently, the specimens were incubated for 2.5 h at room temperature with primary antibodies against acidic pectin (LM19), esterified pectin (LM20) and AGPs (LM2) at a dilution of 1:5. After incubation, the pollen tubes were washed with PBS three times for 10 min each, incubated with a secondary antibody, fluorescein isothiocyanate (FITC)-labeled sheep antirat IgG (KPL, Inc. USA), diluted 1:50 with PBS for at least 2 h at room temperature and then washed with PBS three times. The samples were mounted, and then at least 20 pollen tubes in each of three replicates were observed and photographed with a Zeiss LSM 780 LSCM (excitation at 488 m and emission at 522 nm).

Total RNA Extraction and Transcriptomic Analysis
Pollen was pre-incubated in standard culture medium at 25 • C in the dark for 30 min, followed by the various treatments, including the control (25 • C, CK), cold stress (4 • C, LT) and NO donor (25 µM DEA NONOate, NO), for 1 h in the dark. After incubation, the pollen tubes were collected with a nylon mesh screen (200 meshes) to exclude ungerminated pollen grains; then, they were immediately subjected to grinding in liquid nitrogen. Total RNA from the pollen tubes of three independent experiments (CK1, LT1 and NO1; CK2, LT2 and NO2; and CK3, LT3 and NO3) was extracted using RNAiso Plus (TaKaRa, Japan), and the quality of the total RNA was verified using a 2100 Bioanalyzer RNA Nano chip device (Agilent, Santa Clara, CA, USA) and a NanoDrop ND-1000 spectrophotometer (NanoDrop, Wilmington, DE). The cDNA libraries were constructed and sequenced using an Illumina HiSeq TM 2000 located at the Beijing Genomics Institute (Shenzhen, China; http://www.genomics.cn/index). To compare the differences in gene expression, a stringent cutoff (a probability > 0.7 and a |log 2 Ratio| ≥ 1.0), was used to identify differentially expressed genes (DEGs).

Quantitative Real-Time PCR (qRT-PCR) Analysis
Total RNA was isolated from pollen tubes that were subjected to the various treatments described above using RNAiso Plus (TaKaRa, Japan) and treated with DNase I to remove any genomic DNA contamination. The amplification regimen was set up as described by Ren et al. (2014), and three biological replicates were performed for each sample. Relative expression levels were calculated by including the C. sinensis 18 sRNA gene as the reference based on the 2 − CT method (Livak and Schmittgen, 2001). Primers used for the qRT-PCR are listed in Table S1.

Statistical Analysis
All data are expressed as the means ± standard deviations (SD) obtained from at least three independent replicates. Statistical significance was calculated by one-way ANOVA using Duncan's test in the SPSS 20 software, and the significant differences among various treatment groups are represented by different letters (P < 0.05).

Pollen Tube Growth and Morphological Observations
As shown in Figure 1, cold stress and NO donor DEA NONOate significantly delayed pollen tube growth after the 0.5, 1, and 2 h treatments. In addition, the NO scavenger cPTIO was used to confirm the role of NO in the cold stress-induced inhibition of pollen tube growth. Interestingly, the inhibitory effects of cold stress on pollen tube growth were markedly relieved by the simultaneous presence of cPTIO. Furthermore, under control conditions, the pollen tube showed a uniform diameter and a clear zone at the tip ( Figure S1A), whereas the pollen tube exhibited obvious abnormalities such as a swollen tip and a loss of the clear zone at the tube tip after cold stress ( Figure S1B) or DEA NONOate treatment for 1 h ( Figure S1C). Moreover, the effects of cold stress on the morphology of the pollen tube tip were reduced by cPTIO ( Figure S1D).

Effects of Cold Stress and No on the Cytoplasmic Ca 2+ Gradient in the Pollen Tube Tip
Because Ca 2+ plays a central role in the tip growth of pollen tubes, the tip-focused Ca 2+ gradient is also necessary for structural organization of the cytoskeleton in angiosperm pollen tubes (Sheng et al., 2006). Pollen tubes were loaded with Fluo-4/AM to test the possible effects of cold stress and NO treatments on the Ca 2+ distribution. The results showed that pollen tubes grown under normal conditions exhibited FIGURE 1 | Effects of cold stress or DEA NONOate on the growth of C. sinensis pollen tube. The growth of pollen tubes was significantly delayed after treatment with cold stress (4 • C) or 25 µM DEA NONOate, and the effects of cold stress were reduced by 200 µM cPTIO. The values are the means of three replicates ± SD (n ≥ 50). Different letters on bars denote significant differences at P < 0.05 according to Duncan's test.
the typical tip-to-base cytoplasmic Ca 2+ concentration gradient (Figures 2A,E,I), whereas this tip-focused Ca 2+ gradient was disrupted after cold stress treatment, and stronger fluorescence erratically filled the entire tip of the pollen tubes ( Figures 2B,F (Figures 3A,E). Similarly, the quantification analysis showed that the average fluorescence intensity of the ROS was significantly increased by cold stress or DEA NONOate by 3.07fold and 1.86-fold compared with the control, respectively, and the increase in the average fluorescence intensity induced by cold stress was decreased 2.18-fold with 200 µM cPTIO ( Figure S2). These data suggest that increases in NO can stimulate increases in ROS under cold stress in pollen tubes of C. sinensis.

Dramatic Acidification of the Pollen Tube Tip is Induced by Cold Stress and No
Pollen tube [pH] cyt (cytoplasmic pH) has been demonstrated to play a vital role in pollen tube growth Wilkins et al., 2015). We therefore investigated cold stress-induced [pH] cyt changes using the ratiometric pH indicator 2,7-bis-(2-carboxyethyl)-5-(and-6)-carboxy fluorescein (BCECF) acetoxymethyl ester (AM). The results showed that the [pH] cyt decreased after 1 h of cold stress treatment (Figures 4B,F,J) compared to that in the control pollen tubes (Figures 4A,E,I), implying that cold stress induced the acidification in the pollen tube tip zone. Similarly, the levels of the fluorescence signal were also decreased after treatment with 25 µM DEA NONOate (Figures 4C,G,K). To examine whether NO is involved in cold-induced cytoplasmic acidification, 200 µM cPTIO was used to treat pollen tubes under cold stress treatment, and the [pH] cyt was examined. The results showed that the degree of pollen tube cytoplasmic acidification was significantly reduced, although this reduction did not completely reverse the effects of the cold stress on the pollen

Cold Stress and No Disrupt the Distribution of Organelles and Induce Cell Wall Abnormalities in C. sinensis Pollen Tubes
TEM was performed, and the results showed that the extreme apical zone of the pollen tube was filled with numerous secretory vesicles in the control condition (Figures 5A,E). Fusion of the vesicles with the plasma membrane was frequently observed, as shown by the black arrows ( Figure 5E), indicating that the cell wall materials were actively released into the cell wall. A large number of other organelles, particularly mitochondria and smooth endoplasmic reticulum (sER), accumulated in the subapical zone (Figures 5A,E). However, substantial variation was observed in the pollen tube tips that were treated with cold stress (Figures 5B,F) or exogenous NO for 1 h (Figures 5C,G). The most obvious change was a disruption of the distribution of organelles, as shown by the sharp decline in the number of vesicles, mitochondria and sER, and the feature of other organelles, including the rough endoplasmic reticulum (rER) and vacuoles at the tip of the pollen tube ( Figures 5B,C,F,G). Moreover, the configuration of the rER was altered, and it appeared to wrap around vacuoles and other organelles when treated with cold stress ( Figure S3A) or exogenous NO ( Figure S3B). Furthermore, the use of cPTIO only partly reversed the effect of cold stress on the organelle ultrastructure of the pollen tubes (Figures 5D,H), suggesting that NO is not a unique factor in the process in which cold stress affects the organelle ultrastructure of pollen tubes.
In the control pollen tubes, thick, brown, smooth cell walls were attached at the base of the pollen tube ( Figures S4A,B) to maintain the mechanical support, whereas the cell wall at the tip region was thin, translucent and bumpy to maintain the high flexibility of the tip region (Figures 5A,E); these differences in the cell wall resulted in fast polarized growth of the pollen tube. However, the typical feature of the cell wall at the tip region was changed to that of a cell wall at the base region after the cold stress treatment (Figures 5B,F) or treatment with 25 µM DEA   NONOate (Figures 5C,G). In addition, treatment with 200 µM cPTIO effectively reduced the effects of cold stress on the cell wall ultrastructure (Figures 5D,H), indicating that the cell wall was another key factor in the process of NO involvement in coldinhibited C. sinensis pollen tube growth. Thus, we hypothesized that the cold-induced NO disrupts the organization of the cell wall, resulting in the retarded growth of the tubes and tip swelling.

Effects of Cold Stress and No Treatment on the Distribution of Pectin and AGPs
To further confirm the role of the cell wall, we studied the distribution of the cell wall components in pollen tubes. In pollen tubes grown under standard conditions, the localization of LM19-reactive pectin indicates the distribution of acidic pectin only in the basal regions of the tube, and the intensity of the antigen signal decreases gradually toward the apex of the tube (Figure 6A), whereas the localization of the LM20-reactive esterified pectin was limited to the very tip of the growing tubes ( Figure 6E). In contrast, pollen tubes treated with cold stress (Figures 6B,F) or 25 µM DEA NONOate (Figures 6C,G) showed completely different pectin distributions compared with the control pollen tubes (Figures 6A,E); for example, acidic pectins were detected across the entire surface of the pollen tubes, including the tips (Figures 6B,C), and esterified pectins were detected only in the basal region near the germinating aperture (Figures 6F,G). Notably, the fluorescence signal of acidic pectin at the pollen tube tip was decreased by 200 µM cPTIO under cold stress ( Figure 6D). In addition, treatment with cPTIO increased the distribution of esterified pectin on the shank of the pollen tubes, although the signal was still not detected at the pollen tube tip ( Figure 6H). Furthermore, pollen tubes grown under standard conditions showed a characteristic dotstrengthening with remarkable periodicity of AGPs deposition along the entire length, as revealed by immune-localization with the LM2 antibodies, and the ring-like structures based on dotstrengthening were visualized, particularly in the apical region of the pollen tubes ( Figure 7A) In contrast, the pollen tubes that were treated with cold stress or 25 µM DEA NONOate showed a completely different distribution of AGPs compared to the control. The dot-strengthening feature and the ring-like structures at the tip disappeared, and the fluorescence signal was observed only on the shank region of the pollen tubes (Figures 7B,C). However, the changes in the AGPs distribution caused by cold stress were effectively reversed by 200 µM cPTIO ( Figure 7D).

Signaling Pathway Related DEGs Quantified and Identified from Cold Stress or No Treatments
Differentially expressed genes (DEGs) were identified according to Zhao et al. (2015), with a probability >0.7 and an estimated absolute |log 2 Ratio| ≥ 1.0. Comparing the CK library (CK1, CK2, and CK3) with the LT library (LT1, LT2, and LT3), scilicet CK-VS-LT, there were 278 signaling pathway-related DEGs (130 genes up-regulated and 148 genes down-regulated, 130/148), among which there were 12 genes associated with NO synthesis and 42, 80, 11, 25, 46, 28, and 25 genes related to cGMP, Ca 2+ , ROS, pH, actin, the cell wall, and the MAPK cascade, respectively ( Figure 8A, Table S2). Similarly, 221 signaling pathway-related  DEGs (117/104) were detected in CK-VS-NO, and the numbers of DEGs involved in NO synthesis, cGMP, Ca 2+ , ROS, pH, actin, the cell wall, and the MAPK cascade were as follows: 15,36,64,6,17,48,18, and 17, respectively ( Figure 8B, Table S3). In addition, more DEGs were involved in Ca 2+ signaling than other signaling-related DEGs that were identified in the treatments, indicating the importance of Ca 2+ in the process of the pollen tube response to cold stress and NO treatments.
In our previous study, proline (Pro) accumulation played an important role in the process of NO involvement in coldinhibited C. sinensis pollen tube growth (Wang et al., 2012), but the mechanism of Pro accumulation remains unclear. Here, the expression of three rate-limiting enzyme genes in Pro metabolism, including 1 -pyrroline-5-carboxylate synthetase (P5CS), ornithine-δ-aminotransferase (δ-OAT) and proline dehydrogenase (ProDH), were detected by qRT-PCR (Figure 10). The results showed that the expression of CsP5CS increased after cold stress treatment but did not change after treatment with 25 µM DEA NONOate. Additionally, the expression of Csδ-OAT was induced by cold stress or exogenous NO, and the induction that resulted from cold stress was significantly reduced by 200 µM cPTIO. Interestingly, exogenous NO decreased the expression of CsProDH, and cPTIO caused a greater increase in CsProDH expression under cold stress compared with cold stresstreated pollen tubes. These results suggest that NO regulated Pro accumulation by increasing the expression of Csδ-OAT instead of CsP5CS and by reducing the expression of CsProDH in C. sinensis pollen tubes responding to cold stress.

DISCUSSION
Research over the last few decades has identified NO as an important signaling molecule with diverse biological functions in plants. NO plays a crucial role in growth and development, from germination to senescence, and is also involved in plant responses to biotic and abiotic stresses, including cold stress (Sehrawat et al., 2013b). In addition, previous investigations have demonstrated that NO is involved in the regulation of pollen tube growth, particularly in the polarized tip (Prado et al., 2004). Our data show that both NO and cold stress inhibit C. sinensis pollen tube growth and lead to tip morphological abnormalities and that the NO scavenger cPTIO is able to effectively mitigate the effects of cold stress on pollen tubes, implying that NO participates in the process of cold stress-inhibited C. sinensis pollen tube growth (Wang et al., 2012). This is consistent with the results of (Prado et al., 2004(Prado et al., , 2008 who reported NO as a negative regulator of pollen tube growth in Lilium longiorum and Arabidopsis thaliana. It is well known that NO production is mainly mediated through three NO synthases (NOS) with different localizations and functions in animals, which catalyze the conversion of L-Arg to L-citrulline and NO (Qiao and Fan, 2008). However, the pathways for producing NO in plant tissues are complicated, diverse and undefined and remain a matter of discussion. Current studies have revealed that NOS activity has also been detected in higher plants, although no direct gene coding for a canonical NOS protein has been found in the genomes of Arabidopsis or any other higher plants (Domingos et al., 2015). For example, Zhao et al. (2007) reported that NOS-dependent NO production was associated with salt tolerance in Arabidopsis, and NOS-like activity-dependent endogenous NO production enhanced the tolerance to cold stress in Chorispora bungeana suspension culture cells (Liu et al., 2010). In our previous report,  NOS-like activity was confirmed to participate in the coldinduced NO production in C. sinensis pollen tubes (Wang et al., 2012). In the present study, the expression of the NO-associated protein 1 gene (NOA1, Unigene17147_All) was induced by cold stress, and cPTIO increased the effects of cold stress (Figure 9), which further supports the conclusion about cold stress-induced NO production partly from NOS-like activity in our previous studies. In addition to NOS-mediated NO production, several other NO biosynthetic enzymes may function in plant cells, including the NAD(P)H-dependent nitrate reductase (NR), xanthine oxidase/dehydrogenase and cytochrome P450 (Zhao et al., 2009;Sanz et al., 2015). Many potential NO productionrelated genes were also identified by transcriptomic analyses in the process of the C. sinensis pollen tube response to cold stress, such as the cytochrome P450 family genes and arginine metabolism-related genes (Table S3). Interestingly, NR genes were not among the differentially expressed genes after treatment with cold stress, although the role of NR in the cold-induced NO accumulation in C. sinensis pollen tubes cannot be excluded.
These results indicate that the sources of NO production under cold stress may be the result of the synergism of several pathways in C. sinensis pollen tubes.
As the most ubiquitous second messenger, Ca 2+ dependent signaling networks can respond to many physiological processes in plant cells, and it has been shown that there is a close coupling between the intracellular tip-focused Ca 2+ gradient and the polarized growth of the pollen tube (Holdaway- Clarke and Hepler, 2003). Currently, increasing evidence confirms that there is complicated crosstalk among NO and Ca 2+ signaling pathways. Prado et al. (2004) reported that a putative NO-cGMP signaling pathway induced pollen tube reorientation through effects on cytoplasmic Ca 2+ concentrations in lily. Similarly, NO was also found to modulate the cytoplasmic Ca 2+ gradient to regulate Pinus bungeana pollen tube development largely by mediating Ca 2+ influx, which is most likely dependent on cGMPactivated channels in pollen tubes ). In the present study, we found that the Ca 2+ gradient was disrupted by both cold stress and NO, and the disruption was relieved by cPTIO. Combined with our previous reports (Wang et al., 2012), it is reasonable to speculate that cold-induced NO inhibits the polarized growth of C. sinensis pollen tubes dependent on the damage to the cytoplasmic Ca 2+ gradient at the pollen tube tip zone. Additionally, pharmacological and biochemical studies have shown that NO signaling in plants is mediated by cGMP (Prado et al., 2004) and cyclic nucleotide-gated ion channels (CNGCs), which are permeable to both monovalent and divalent cations (typically K + , Na + , and Ca 2+ ) and are activated by cGMP and/or cAMP (Wang et al., 2013). Our data indicate that the expression of multiple CNGC genes is up-regulated after NO or cold stress treatment and that the expression of a large number of genes that encode proteins that activate CNGCs is induced, such as protein tyrosine kinases (PTKs) and protein tyrosine phosphatases (PTPs), which regulate the sensitivity of CNGCs on cGMP by catalytic phosphorylation (Chae et al., 2007). Recently, glutamate receptor-like channels (GLRs), a putative group of pollen Ca 2+ channels, were identified, and their Ca 2+ transport activities in pollen tubes have been confirmed based on direct electrophysiological, pharmacological and genetic evidence (Konrad et al., 2011;Michard et al., 2011). Interestingly, two GLR genes were identified from DEGs of CK-Vs-LT and CK-Vs-NO, implying that GRLs participate in the process in which NO results in a Ca 2+ gradient disruption at the pollen tube tip after treatment with cold stress. Furthermore, the expression of another class of Ca 2+ channel protein family genes, two pore calcium channel protein genes (TPC, Unigene1219_All), was shown to be up-regulated after NO or cold stress treatment, and the induction of cold stress was inhibited by cPTIO, indicating that TPC was also involved in the regulatory process of cold-induced NO disruption of the Ca 2+ gradient at the pollen tube tip. These data suggest that NO regulates the cytoplasmic Ca 2+ gradient largely by mediating Ca 2+ fluxes under cold stress, which is most likely dependent on various Ca 2+ channels in C. sinensis pollen tubes, such as CNGCs, GLRs, and TPCs.
The downstream proteins that can bind Ca 2+ and act upon changes in Ca 2+ concentrations to perform specific functions play important roles in the process of pollen tube tip growth (Konrad et al., 2011), particularly Ca 2+ sensor and relay proteins, including calmodulin (CaM), CaM-like proteins (CMLs), Ca 2+dependent protein kinases (CDPKs), calcineurin B-like proteins (CBLs), and CBL-interacting protein kinases (CIPKs; Gutermuth et al., 2013;Steinhorst and Kudla, 2013;Zhou et al., 2014). Recently, Zhou et al. (2015) reported that Arabidopsis CIPK19 is required for pollen tube growth and polarity by participating in Ca 2+ homeostasis dependent on the modulation of Ca 2+ influx. In our investigation, we also found that multiple sensors and relay protein genes were up/down-regulated in C. sinensis pollen tubes in response to NO or cold stress treatment. Combined with previous results, we infer that Ca 2+ binging proteins are involved in cold-induced NO-inhibited pollen tube tip growth, which is dependent on the regulation of NO on the cytoplasmic Ca 2+ gradient (Domingos et al., 2015). In addition, accumulating evidence indicates that cytoskeleton elements (actin/myosin cables) control cytoplasmic streaming, the distribution of the endoplasmic reticulum (ER) and the transport of secretory vesicles and that actin polymerization itself also contributes to pollen tube growth (Chen et al., 2007). Concurrently, the dynamic state of the cytoskeleton elements is controlled via numerous regulatory factors, including several actin-binding proteins activated in response to Ca 2+ (Cárdenas et al., 2008). Moreover, Wang et al. (2009) reported that Factin organization in the tip region of pollen tubes sensitive to NO is partly dependent on the Ca 2+ gradient during NO signaling in P. bungeana pollen tubes. Our results show that cold-induced NO stimulation caused a sharp decline in the number of vesicles and ER distribution abnormality in the C. sinensis pollen tube tip region accompanied by a sharper Ca 2+ gradient disruption. In addition, a large number of cytoskeletonrelated genes were up/down-regulated in this process, such as actin, formin-like protein genes, myosin-like genes, kinesin genes, actin-depolymerizing factor, and the caltractin gene, which encodes a type of Ca 2+ binding protein. This suggests that vesicular trafficking and ER distribution were perturbed by the cold-induced NO accumulation, which may be partly dependent on the regulation of the NO-induced Ca 2+ gradient change in cytoskeleton elements, particularly the dynamic polymerization status of F-actin . Furthermore, mitogenactivated protein kinases (MAPKs), which are another type of important downstream target of the Ca 2+ signal, have been confirmed to mediate the guidance response in pollen tubes (Guan et al., 2014). In addition, accumulating evidence indicates that MAPKs function as intracellular targets for NO and participate in the developmental processes of plants, including pollen tube tip growth (Arasimowicz and Floryszak-Wieczorek, 2007;Domingos et al., 2015;Sanz et al., 2015). As expected, some MAPK genes and downstream genes were induced by colddependent NO in C. sinensis pollen tubes, which is consistent with a previous report showing that NO activates a potential MAPK during NO-induced PCD in A. thaliana suspension cultures (Clarke et al., 2000).
It is generally accepted that there is crosstalk among NO, Ca 2+ , and ROS signaling pathways in plants, particularly in the process of pollen tube growth and the response to environmental stress (Domingos et al., 2015). For example, NADPH oxidases (NOX) are expressed in pollen tubes and localize to the plasma membrane (PM) where they produce ROS, namely H 2 O 2 , which promotes NO synthesis through NR and/or NOA1. In contrast, NO activates protein kinases (PK), enabling NOX to bind Ca 2+ , triggering more ROS production, and NO-dependent cGMP activates Ca 2+ channels at the PM to provide adequate Ca 2+ for NOX Wilkins et al., 2011;Wudick and Feijó, 2014;Domingos et al., 2015). In addition, the activation of PM NOX also can be triggered by the CDPK in a Ca 2+dependent manner to produce ROS in pollen tubes (Kobayashi et al., 2007;Potocký et al., 2007;Kaya et al., 2014). Our results show that NO increases the accumulation of ROS accompanied by a significantly up-regulated expression of the NOX gene and Ca 2+ gradient disruption in the C. sinensis pollen tube in response to cold stress. Regardless of the reactivity of ROS and NO, our results suggest that cold-induced NO regulates the production of ROS partly through the activation of PM NOX triggered by Ca 2+ , which further supports the results of previous studies (Kaya et al., 2014;Domingos et al., 2015). Furthermore, increasing evidence suggests that small GTPases in plants called RAC/ROPs (RACs are used in this study) function as molecular switches in the polarized cell growth of pollen tubes and root hairs and in defense-related responses (Zou et al., 2011;Huang et al., 2014). Kost et al. (1999) reported that the overexpression of RAC1 enlarged the apical regions of the PM containing RAC1, which is correlated with the severity of the depolarized growth of pollen tubes. In the present study, the up-regulation of RAC1 and the down-regulation of RAC5 were detected after treatment with NO or cold stress, and the effects of cold stress were relieved by cPTIO, implying that RACs play important roles in coldinduced NO-inhibited tip growth in C. sinensis pollen tubes, which supports the above results of Kost et al. (1999). In addition, recent studies have shown that RACs affect the actin dynamics mediated by Ca 2+ and/or ADF, thereby controlling the polarized growth of pollen tubes (Chen et al., 2003;Fu, 2010;Wu et al., 2011). Moreover, RACs are found to be involved in stimulating the ROS accumulation in the polarized growth of pollen tubes (Potocký et al., 2012;Kaya et al., 2014). Given the crosstalk among NO, Ca 2+ and the ROS signaling pathway, we speculate that RACs are involved in the regulation of NO on Ca 2+ and the ROS signaling pathway in the process of cold stress inhibiting the C. sinensis pollen tube tip growth.
Recently, Wilkins et al. (2015) reported that cytosolic pH ([pH] cyt ) acidification was necessary and sufficient for triggering several key hallmark features of the self-incompatibility-induced PCD signaling pathway. Our data also reveal that coldinduced NO inhibits the tip growth of C. sinensis pollen tubes accompanied by a significant [pH] cyt acidification, which confirms the role of pH signaling in plant pollen tube growth (Michard et al., 2009). In addition, previous studies have suggested that the changes in [pH] cyt in the pollen tube tip are dependent on H + fluxes mediated by the regulation of Ca 2+ on PM H + -ATPase and Na + /H + antiporter (SOS1) activities (Certal et al., 2008;Michard et al., 2008;Guo et al., 2009). Interestingly, whereas cold-induced NO results in C. sinensis pollen tube tip acidification, the expression of PM H + -ATPase and the Na + /H + antiporter gene are found to be significantly upregulated, suggesting that potential H + fluxes participate in the process of pollen tube tip acidification, which is similar to reports by Sun et al. (2009) that the synergistic effect between PM H + -ATPase and the Na + /H + antiporter increased H + influx in root protoplasts under salt stress. Of course, the contribution of Ca 2+ cannot be neglected in the above process (Michard et al., 2009). Furthermore, pH as a regulator of PME activity was involved in the regulation of PME on pectin status and distribution at the pollen tube cell wall (Li et al., 1994). Increasing evidence confirms that the dynamic balance between cell wall extensibility and rigidity is another key factor that regulates tip growth in pollen tubes (Chen et al., 2007). In the present study, we also found that two PME genes were up-regulated after treatment with NO or cold stress. In addition, immunolabeling with LM19 and LM20 showed that cold-induced NO changed the distribution of acidic and esterified pectins at the cell wall of C. sinensis pollen tubes compared to that in the controls; this is similar to data showing that an increase in the degree of cell wall rigidity and a decrease in visco-elasticity influence pollen tube growth and architecture (Parre and Geitmann, 2005;Wang et al., 2009). The characteristic dot-strengthened and ring-like structures of AGPs at pollen tubes cell wall disappeared after treatment with NO or cold stress, which is similar to the results of Chen et al. (2007). In addition, ultrastructure observation showed that cold-induced NO causes cell wall thickening, smoothing and color deepening, which is likely dependent on the above changes in cell wall construction. Taking these findings together, we speculate that cold-induced NO stimulates changes in cell wall component distributions, leading to excess wall rigidity at the tip of the pollen tube, thereby inhibiting the polarized growth of the C. sinensis pollen tube tip, which may partly account for the synergistic effect of pH and PME mediated by NO.
Furthermore, it is well known that higher plants accumulate free Pro in response to a number of abiotic stresses, such as drought, salinity and freezing (Zhao et al., 2009). Nevertheless, only a few reports have indicated that NO and Pro cross-talk in cold acclimation and freezing tolerance. For example, Zhao et al. (2009) showed that cold-induced NO acts as a signal to evoke Pro accumulation via enhanced synthesis and reduced degradation by regulating related genes of the Pro biosynthetic pathway in Arabidopsis, which may be a function of NO in freezing tolerance. Similarly, our previous study showed that NO participated in stimulating Pro accumulation in C. sinensis pollen tube responses to cold stress (Wang et al., 2012). In addition, Ruan et al. (2004) reported that NO promoted the activity of P5CS1 and decreased the activity of ProDH. Interestingly, our present study showed that cold-induced NO increased the expression of Csδ-OAT and reduced the expression of CsProDH but had no effect on the expression of CsP5CS (Figure 10). These results reveal that NO regulated Pro accumulation by increasing the expression of Csδ-OAT instead of CsP5CS and by reducing the expression of CsProDH in C. sinensis pollen tubes during the response to cold stress, although the contribution of CsP5CS cannot be ignored in cold stress-induced Pro accumulation ( Figure S6).
In summary, our cytological and transcriptomic analyses provide a more global picture of the role of NO in cold stress to inhibit the polarized tip growth of C. sinensis pollen tubes. A complex signaling network dominated by NO, including Ca 2+ , ROS, pH, RACs signaling, and the crosstalk among them, was investigated in the C. sinensis pollen tube response to cold stress, which is summarized in Figure 11. This study provided two novel findings. First, cold-induced NO causes Ca 2+ gradient disruption in C. sinensis pollen tubes, most likely through Ca 2+ fluxes mediated by various Ca 2+ channels and through subsequently triggered secondary and tertiary regulatory networks, including Ca 2+ sensor and relay proteins, the MAPK cascade, ROS and pH signaling. Second, [pH] cyt acidification interacted with PMEs, leading to changes in the cell wall structure and component distributions, thereby inhibiting the polarized growth of the C. sinensis pollen tube tips after treatment with cold stress. Furthermore, RAC signaling is involved in the process of cold-induced NO-inhibited C. sinensis pollen tube polarized growth, possibly through regulating the Ca 2+ and ROS signaling FIGURE 11 | Hypothetical model summarizing the potential signaling pathway of nitric oxide (NO) involved in cold-inhibited C. sinensis pollen tube growth. This simplified model was based on the pollen tube models proposed by Wang et al. (2009 and, (Wudick and Feijó, 2014), and Domingos et al. (2015). Cold stress induces an increase in NO through the synergism of several pathways in C. sinensis pollen tubes, such as the accumulation of NOS-like activity and cytochrome P450 activity. Consequently, the cytoplasmic Ca 2+ gradient was regulated largely by mediating the Ca 2+ flux, which is dependent on various Ca 2+ channels, such as CNGCs (cGMP-activated channels), GLRs and TPCs, and this subsequently triggered secondary and tertiary regulatory networks, including Ca 2+ sensor and relay proteins, the MAPK cascade, ROS, actin, vesicles and pH signaling. In addition, Ca 2+ -dependent [pH] cyt acidification interacted with PMEs, leading to changes in the cell wall structure and component distribution. Furthermore, RAC signaling involved in the process of cold-induced NO inhibited C. sinensis pollen tube polarized growth by regulating the Ca 2+ and ROS signaling pathways. Together, the complex signaling network dominated by NO mediates the cold-inhibited C. sinensis pollen tube growth. NOS, nitric oxide synthase; GC, guanylyl cyclase; PTPs, protein tyrosine phosphatases; cPTIO, 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide; PTKs, protein tyrosine kinases; CNGCs, cyclic nucleotide-gated ion channels; GLRs, glutamate receptor-like channels; CPK, calcium-dependent protein kinase; NOX, NADPH oxidase; ROS, reactive oxygen species; ADF, actin-depolymerizing factor; PME, pectin methylesterase; RACs, Rac-like GTP-binding proteins. pathways, which is also an interesting result. Taken together, our study provides new insights into the multifaceted mechanistic framework for the functions of NO in cold-inhibited C. sinensis pollen tube growth.

AUTHOR CONTRIBUTIONS
WW, YW, and XS designed research; WW, XS, ZS, DL, and YW performed research; WW, JP, XY, PC, and YW analyzed data; WW and YW wrote the paper; XL and YW revised this paper.