Circadian changes in endogenous concentrations of indole-3-acetic acid, melatonin, serotonin, abscisic acid and jasmonic acid in Characeae (Chara australis Brown)

Abstract

Giant-celled Characeae (Chara australis Brown), grown for 4 months on 12/12 hr day/night cycle and summer/autumn temperatures, exhibited distinct concentration maxima in auxin (indole-3-acetic acid; IAA), melatonin and serotonin about 4 hr after subjective daybreak. These concentration peaks persisted after 3 day pretreatment in continuous darkness: confirming a circadian rhythm, rather than a response to “light on.” The plants pretreated for 3 d in continuous light exhibited several large IAA concentration maxima throughout the 24 hr. The melatonin and serotonin concentrations decreased and were less synchronized with IAA. Chara plants grown on 9/15 hr day/night cycle for 4 months and winter/spring temperatures contained much smaller concentrations of IAA, melatonin and serotonin. The IAA concentration maxima were observed in subjective dark phase. Serotonin concentration peaks were weakly correlated with those of IAA. Melatonin concentration was low and mostly independent of circadian cycle. The “dark” IAA concentration peaks persisted in plants treated for 3 d in the dark. The plants pretreated for 3 d in the light again developed more IAA concentration peaks. In this case the concentration maxima in melatonin and serotonin became more synchronous with those in IAA. The abscisic acid (ABA) and jasmonic acid (JA) concentrations were also measured in plants on winter regime. The ABA concentration did not exhibit circadian pattern, while JA concentration peaks were out of phase with those of IAA. The data are discussed in terms of crosstalk between metabolic pathways.

Introduction

Auxin (indole-3-acetic acid; IAA) is an essential hormone for setting spatial patterns of embryophyte growth and development, as well as for responding to environmental cues of gravity, light and water. IAA is synthesized in shoot apical meristem and transported throughout the plant to control growth. Modern genetic profiling established intimate entanglement between circadian clock- and auxin-signaling pathways. The nature of this interdependence is under intense investigation with 50% of auxin-responsive genes being rhythmically expressed (for review see Covington & Harmer).¹ While exogenous auxin has small effects on the circadian clock, the circadian clock controls plant transcriptional and growth responses to exogenous auxin. This process is called “gating.”

Endogenous IAA concentration peak was measured in the subjective day in dicotyledonous plant Chenopodium rubrum L.,² model plant Arabidopsis,³ and Solanaceae tobacco.⁴ In Arabidopsis the IAA maximum coincided with maximal growth rate of the first inflorescence node.³ In C. rubrum the IAA concentration maximum shifted to dark phase in short day/long night regime and coincided with flowering induction.² In tobacco leaves there was a secondary IAA concentration increase 2 hours after “light-off”.⁴ Cuin,⁵ reviewed the hypothesis that plants lack the central oscillator, such as the suprachiasmatic nucleus in the mammal brain. Until recently, the prevailing view was that plants have independent oscillators in different parts of roots and shoots, which are out of phase and lack extensive crosstalk. However, recent experiments suggest that there is circadian signaling between cells, tissues and organs (for review see Dodd et al.).⁶

Abscisic acid (ABA) is one of the major plant phytohormones involved in seed maturation, dormancy and senescence. It also has an important role in water stressed plant tissues. In guard cells ABA mobilizes K⁺ channels (or channel conductance) to efflux K⁺ for stomatal closure.⁷ Consequently, ABA signaling was associated with higher land plants.⁸ However, ABA turns out to be an ancient molecule detected in many types of organisms from cyanobacteria, algae, fungi to land plants.⁹ Jasmonic acid (JA) is another stress hormone discovered in land plants subjected to biotic or abiotic stress.

The hormone melatonin is involved in setting or stabilizing circadian rhythms in animal kingdom, where it signals darkness by an increase in concentration.¹⁰ Serotonin, which is an intermediate in the melatonin synthetic pathway, functions as a neurotransmitter in brains of animals. Melatonin was discovered in plants in 2 surveys of common fruits and vegetables.^11,12 Murch et al.¹³ described its synthesis from tryptophan via pathway that can also produce IAA.^14,15 It is now thought that similar to ABA, melatonin is of ancient origin, introduced into both plant and animal kingdoms through cyanobacteria and proteobacteria that became, respectively, chloroplasts and mitochondria.¹⁶ The genetic basis and regulation of the synthesis of melatonin in plants are now probed by molecular techniques.^17-22 However, animal-like melatonin receptor sequences have not been found so far in plant genomes.²³

The similar structure of melatonin and serotonin molecules to that of IAA suggests a role for these substances in plant growth and development including regulation of shoot and root morphogenesis.^14,24-26 Melatonin and serotonin concentrations increase at specific stages of development of Datura metel L. flower,²⁷ and concentration levels fluctuate through the veraison (ripening) of grapes.²⁸ Melatonin also detoxifies reactive oxygen species (ROS) in plant tissues.^29-33 For more detailed description of melatonin multiple roles in growth, senescence, biotic and abiotic stress, see comprehensive review by Reiter et al.²³

Is melatonin involved in circadian rhythms outside the animal kingdom? A dark phase concentration peak was found in dinoflagellate Gonyaulax polyedra,³⁴ and in shoots of C. rubrum.³⁵ The concentration rhythm persisted without light cues in Gonyaulax,³⁶ but not in another dinoflagellate Symbiodinium.³⁷ Van Tassel et al.³⁸ observed an indistinct melatonin concentration peak in the subjective day in the shoots of Morning glory (Pharbitis nil), while Tan et al.³⁹ found 2 melatonin concentration peaks in middle of the day and at the onset of darkness in water hyacinth (Eichhornia crassipes (Mart.) Solms). Boccalandro et al.⁴⁰ measured greater melatonin concentration at night with a peak at dawn in grapes (Vitis vinifera L. cv Malbec). The green marine macroalga Ulva sp. synchronized melatonin concentration maxima with semi-lunar rhythm coinciding with low spring tides, which expose the plants to multiple environmental stresses.⁴¹ These rhythms persisted in laboratory grown plants not exposed to tidal variation. Thus, similarly to IAA concentration, the circadian rhythms are present, but vary from tissue to tissue and plant to plant (see also Fig. 5 in Arnao & Hernandez-Ruiz).⁴²

Figure 5.

Chromatographic separation of serotonin, melatonin, indole-3-acetic acid, abscisic acid, and jasmonic acid.

The multiple roles of melatonin as antioxidant and growth regulator (see Reiter et al.²³ for comprehensive review) make it difficult to separate out the involvement in circadian and infradian rhythms. One approach is to remove the photoperiodic, temperature or other cues to test whether the rhythms are endogenously sustained. Another experimental technique is application of exogenous melatonin to disrupt existing circadian rhythm or developmental stage. This approach was successful in the dinoflagellate Lingulodinium, but not in higher land plants.⁴³ The external application of melatonin might be also “gated” by the circadian signaling.

The current research compares diel endogenous concentrations of IAA, melatonin and serotonin in Characeae Chara australis Brown grown in summer and winter conditions. In the second experiment, abscisic acid (ABA) and jasmonic acid (JA) were also measured. In both experiments pre-cut explants were subjected to 3 day pretreatment in continuous dark or light. We have selected a model system that consists of small numbers of cells to reduce cell-to-cell signaling and tissue-to-tissue variation. Fresh water C. australis plants consist of large cells up to 1 mm in diameter and several cm in length (see Fig. 1). The large internodal cells contain multiple nuclei, but do not divide. Clabeaux & Bisson,⁴⁴ established that Chara plants display apical dominance (decapitated plants produce more shoots), but were unable to demonstrate exogenous IAA response. Boot et al.⁴⁵ and Raven,⁴⁶ described cell-to-cell polar IAA transport (PAT) in Characeae, demonstrating that IAA also has an ancient origin. C. australis contains melatonin and is responsive to melatonin signaling.^33,47 These studies have wide applicability to higher plants in general, since the Characeae are the sister group to the ancestors of all land plants,⁴⁸ and their use as experimental system has already established the fundamentals of plant cell electrophysiology,^49,50 cell to cell transport,^49,50 cytoplasmic streaming,⁵¹ and gravitropism,⁵²

Figure 1.

Chara australis plant (male, identified by orange antheridia). Scale bar = 50 mm. The circle shows the top part of the plant used in sampling (adapted from Beilby & Casanova).⁵⁰

Results

Experiment 1: Summer plants

In plants, grown for 4 months on 12/12 light/dark cycle and in summer/atumn temperatures, IAA, melatonin and serotonin exhibited sharp distinct maxima in the middle of the subjective day. Serotonin (blue) and melatonin (red) reached their first concentration peak at 11 o‘clock and a secondary smaller peak at 13 o'clock (See Fig. 2a and Table 1a for peak magnitudes). By 15 o‘clock both substances returned to low background levels: melatonin at 4 ± 3 ng/g of tissue and serotonin at 19 ± 18 ng/g of tissue. IAA concentration (green) started to increase with a slight delay, reaching a high peak at 14 o’clock and decreasing sharply to the background level (9 ± 11 ng/g of tissue) in the next hour. The background levels were computed by averaging the “flat” part of the cycle for each substance. Note that the background serotonin level is the highest, followed by IAA and melatonin background levels. The only large concentration peak in the dark phase belonged to melatonin at 20 o'clock. Melatonin remained slightly elevated (∼10 ng/g of tissue) until 23 o’clock. IAA and serotonin did exhibit several smaller concentration peaks in the dark phase (see Fig. 2a and Table 1a for peak magnitudes).

Figure 2.

Diurnal concentration of IAA (green), melatonin (red) and serotonin (blue) in characean plants acclimated to different photoperiodic regimes: (A) 12/12 light/dark cycle, the gray rectangles indicate darkness (B) Explants pretreated 3 d in darkness, the dashed line rectangles indicate where plants experienced dark in the 12/12 hour regime, (C) Explants pretreated 3 d in light, the dashed line rectangles indicate where plants experienced dark in the 12/12 hour regime. Bars indicate standard error of the triplicate analysis of each sample.

 .

(B) Winter Data

Time of day	9/15 hr LIGHT/DARK				DARK				LIGHT
	IAA	JA	Ser	Mel	IAA	JA	Ser	Mel	IAA	JA	Ser	Mel
0.5					213 ?	14
1.5		11							39	19
2.5	21		0.38		50		1
3.5									35			0.35
4.5						19
5.5					52
6.5										22
7.5								1.6	20		0.2	0.07
8.5		15			12
9.5						14				14
10.5					14				16
11.5		10									0.07	0.12
12.5
13.5						10
14.5										9
15.5
16.5
17.5					15				33		0.12
18.5										46		17?
19.5						25			34		0.09
20.5	34	12	0.15
21.5					35					4
22.5							0.4
23.5									23		0.035

The explants pretreated in the dark for 3 d exhibited concentration maxima in all the indoleamines at the similar phase of the cycle as the day/night plants (see Fig. 2b and Table 1a). IAA concentration peaked at 11 o‘clock with subsequent smaller peaks, while serotonin and melatonin concentration peaks aligned with some of the IAA concentration maxima (see Fig. 2b and Table 1a). There was a small concentration peak in melatonin at 20 o'clock, below the resolution of the figure, but noticable above the background values similar to those described in Fig. 2a.

The explants pretreated in continuous light for 3 d showed rather different indoleamine concentration profiles (see Fig. 2c). IAA retained a smaller concentration peak at 11 o'clock. However, there were other large concentration peaks (see Fig. 2c and Table 1a). Serotonin and melatonin concentrations remained low with smaller concentration peaks (see Fig. 2c and Table 1a). The background levels for all 3 substances were again similar to data described in Fig. 2a.

Experiment 2: Winter plants

The plants were exposed to short day/long night for 4 months, so they experienced a prolonged winter (at the same time as Australian winter, but with shorter day length). The concentration of IAA (green) decreased by an order of magnitude, serotonin (blue) by 2 orders of magnitude and melatonin (red) by 3 orders of magnitude compared to Summer plants (see Fig. 3, keeping in mind that concentrations of melatonin and serotonin were multiplied by similar factors to resolve the data on the graph). For magnitudes of concentration maxima see Table 1b. The IAA concentration still displayed circadian variation, but the peaks appeared in the dark phase (Fig. 3a and Table 1b). The background level was 0.15 ± 0.2 ng/g of tissue. Serotonin background concentration was 0.027 ± 0.03 ng/g of tissue and concentration peaks were synchronised with the IAA concentration peaks (Fig. 3a and Table 1b). Melatonin concentration varied between pg/g to 1.6 ng/g of tissue (background of 0.016 ± 0.02 ng/g of tissue) and seemed independent of day/night cycle, although some of the small concentration peaks did coincide with those of IAA (Fig. 3a).

Figure 3.

Diurnal concentration of IAA (green), melatonin (red) and serotonin (blue) in characean plants acclimated to different photoperiodic regimes: (A) 9/15 light/dark cycle, the gray rectangles indicate darkness. The melatonin and serotonin concentrations were low compared to IAA concentration and were multiplied by 100 and 20, respectively, to be visible on the graph (see Table 1b for the values of the concentration maxima). (B) Explants pretreated 3 d in darkness, the dashed line rectangles indicate where plants experienced dark in the 9/15 hour regime. Melatonin and serotonin concentrations were multiplied by 10 and 20, respectively, to be visible on the graph (see Table 1b for the values of the concentration maxima). (C) Explants pretreated 3 d in light, the dashed line rectangles indicate where plants experienced dark in the 9/12 hour regime. Melatonin and serotonin concentrations were multiplied by 50 and 50, respectively, to be visible on the graph (see Table 1b for the values of the concentration maxima).

Table 1:

Diel peak magnitudes in ng/g of tissue. The 24 hr clock is used with 0 o’clock designating midnight. In the Summer data samples were collected on the hour, in the Winter data at 30 min past each hour. The shaded portion of the time column signifies darkness in the day/night regime. The data from plants pretreated for three days in the dark are designated “dark”, for three days in the light are designated “light” (see Methods section). Summer data background concentrations: 9 ± 11 (IAA), 19 ± 18 (serotonin), 4 ± 3 (melatonin), all in ng/g of tissue. Winter data background concentrations: 0.15 ± 0.2 (IAA), 0.027 ± 0.03 (serotonin), 0.016 ± 0.02 (melatonin), all in ng/g of tissue. The two spurious data points in Winter data are marked with question marks. These concentrations in IAA and melatonin are much higher than all the other Winter data and there are no concentration peaks at the same phase in the other data (although the latter condition alone would not make them dubious). (A) Summer Data

Time of day	12/12 hr LIGHT/DARK			DARK			LIGHT
	IAA	Ser	Mel	IAA	Ser	Mel	IAA	Ser	Mel
0
1
2
3	17	41
4							201	61
5
6
7							733
8
9
10
11		380	137	637	515	428	384	12
12									69
13		101	106	260		551
14	824						109	47	14
15				211	50	39
16
17
18				101					18
19							44	19
20	42	67	410			14
21
22	43			43			786
23					58

The explants pretreated in the dark for 3 d exhibited concentration maxima in IAA and serotonin at similar times as the day/night plants (see Fig. 3b and Table 1b), with smaller concentration peaks in “day time” and early “night-fall” (Fig. 3b and Table 1b). There was a high concentration data point at 0.30 o'clock. This result was judged spurious, mainly because the value was much higher than all other winter measurments and, less importantly, no maxima were observed at that phase in the other data (see Table 2b). Serotonin concentration peaks were synchronised with 2 of the IAA concentration peaks. The melatonin concentration remained low with one peak, which was not synchronised with the other indoleamines.

Table 2.

Mass Spectrometer Parameters

Compound	Formula	Parent Ion (m/z)	Daughter Ion (m/z)	Cone (V)	Collision Cell (eV)
Indole-3-Acetic Acid	C10H9NO2	176.07	103	30	25
Indole-3-Acetic Acid	C10H9NO2	176.07	130	30	13
Serotonin	C10H12N2O	177.10	115	45	27
Serotonin	C10H12N2O	177.10	160	45	10
Jasmonic Acid	C12H18O3	211.13	133	22	14
Jasmonic Acid	C12H18O3	211.13	151	22	10
Melatonin	C13H16N2O2	233.13	159	30	23
Melatonin	C13H16N2O2	233.13	174	30	15
Abscisic Acid	C15H20O4	265.14	135	20	22
Abscisic Acid	C15H20O4	265.14	247	20	6

The explants pretreated in continuous light for 3 d showed more IAA concentration peaks (Fig. 3c and Table 1b). The serotonin concentration peaks remained approximately synchronised with the IAA concentration peaks. The melatonin concentration remained low, but did show some synchronisation with the other indoleamines (Fig. 3c and Table 1b). There was a high concentration data point at 18.30 o’clock, again judged spurious because of much higher magnitude than all other Winter data.

The concentration of abscissic acid (ABA, red) and jasmonic acid (JA, blue) were also measured in the second experiment to evaluate whether stress responses correlate with indoleamine and auxin metabolism (Fig. 4). ABA showed 3 indistinct peaks in concentration, but only one of these concentration peaks aligned with a concentration peak in IAA (green). JA exhibited 4 main increases in concentration (Fig. 4a and Table 1b).

Figure 4.

Diurnal concentration of IAA (green), JA (blue) and ABA (red) in characean plants acclimated to different photoperiodic regimes: (A) 9/15 light/dark cycle, the gray rectangles indicate darkness. (B) Explants pretreated 3 d in darkness, the dashed line rectangles indicate where plants experienced dark in the 9/15 hour regime. (C) Explants pretreated 3 d in light, the dashed line rectangles indicate where plants experienced dark in the 9/15 hour regime.

The explants pretreated in 3 d of darkness exhibited greater increases in concentration of both ABA and JA. ABA showed 5 concentration peaks (Fig. 4b). JA concentration peaks occured out of phase with those of IAA (Fig. 4b and Table 1b). The explants pretreated in 3 d of light also showed higher concentrations of JA. ABA concentration exhibited less peaks than day/night and dark data (Fig. 4c). The JA concentration peaks were again out of phase with those of IAA (Fig. 4c and Table 1b).

Discussion

Circadian synchronisation of IAA, melatonin and serotonin in Summer plants

In the summer months the growing cells at the top of Chara thallus exhibit large IAA concentration maximum in the middle of the subjective day (see Fig. 2a and Table 1a), similar to Chenopodium,² Arabidopsis,³ and tobacco leaves.⁴ A much smaller “dark” concentration peak was also recorded in Chara and in tobacco leaves.⁴ The midday melatonin concentration peak (Fig. 2a and Table 1a) is similar to that observed in water hyacinth,³⁹ and fruits of the sweet cherry.⁵³ The synchronous melatonin concentration peak with that of IAA supports suggestion of Arnao and Hernandez-Ruiz,⁴² that melatonin acts as parallel growth regulator. Another possible melatonin role is to modulate auxin signaling pathway to senescence through indole-3-acetic acid inducible 17 (AtIAA17) in the growing summer plants.⁵⁴ The second melatonin concentration peak at the begining of the dark phase (Fig. 2a and Table 1a) is similar to that observed in water hyacinth,³⁹ while in sweet cherry secondary concentration peak was observed in early morning.⁵³

In the explants exposed to 3 d of total darkness the “mid-day” concentration peaks persisted, with the magnitude of melatonin and serotonin peaks increasing and the magnitude of IAA peak decreasing slightly (see Fig. 2a and Table 1a). The persistence of the “mid-day” IAA, melatonin and serotonin concentration peaks in absence of day/night cue confirms the self-sustaining circadian nature of the variation: the concentrations changes were not a response to “light on.” The secondary “dark” melatonin concentration peak at 20 o'clock (Fig. 2a) was replaced by a very small melatonin concentration peak and a small broad concentration increase in serotonin, suggesting that it was not a spurious data point (compare Figs. 2a and b). In the 12/12 photoperiod plants this melatonin concentration increase could be a response to onset of darkness, when it is no longer needed as a strong antioxidant for byproducts of photosynthesis (Lazar et al.³³ and references there in). Tan et al.³⁹ observed AFMK (N¹-acetyl-N²-formyl-5-methoxykynuramine) concentration peaks synchronised with those of melatonin in water hyacinth. As AFMK is part of melatonin antioxidant cascade, melatonin was performing its antioxidant role.

The explants pretreated in 3 d of continuous light exhibited very different pattern: the IAA concentration became disconnected from the circadian cycle and only weakly connected to concentration changes of melatonin and serotonin. The concentrations of these 2 substances were close to background, except for some indistinct maxima (see Fig. 2c and Table 1a). Melatonin could be consumed by reactive oxygen species (ROS) produced by photosynthesis. The concentration of IAA exhibited 4 larger peaks and some smaller peaks (Fig. 2c and Table 1a) with approximate period of 4 hrs. This behavior is reminiscent of underdamped negative feedback circuit “ringing” (oscilating) after a change in the input (in this case lack of expected darkness). Thus, in case of constant darkness, the system maintains status quo, while in constant light a change in IAA production is initiated. This increased rate of IAA production explains greater rhizoid formation in explants exposed to continuous light, as IAA stimulates rhizoid development.⁵⁵

Change of circadian rhythms in Winter plants

The concentrations of IAA, melatonin and serotonin were much lower than in the Summer plants (see Fig. 3a and Table 1b). These are large changes for melatonin and serotonin, but surveys of various fruits and whole plants show huge range of concentrations from μg/g to pg/g of tissue.¹⁵ In the same tissue a substancial drop in melatonin and serotonin levels were observed in developing flower buds of D. metel, especially if the plants were exposed to cold stress.²⁷ Large changes in endogenous IAA levels (680 to 6 ng/g of tissue) were measured in Lemna gibba L. during 45 day growth cycle.⁵⁶ The IAA concentration maxima are now found in the dark phase of the cycle (Fig. 3a and Table 1b). Further, if the Winter and Summer data are compared, it becomes clear that the dark phase IAA concentration maxima are of similar amplitudes and positions in the cycle. Thus the Winter plants simply lost the large mid day production of IAA, serotonin and melatonin. The serotonin concentration maxima were weakly syncronised with the IAA, while melatonin concentration did not exhibit circadian patterns (Fig. 3a and Table 1b). The shift of IAA concentration maximum to the dark period in long night/short day regimes was observed in short day dicotyledonous plant C. rubrum, where it induced flowering.² This does not seem to be the case in C. australis, which exhibits maximal growth and production of reproductive structures in spring and summer, although other conditions, such as water level, influence the timing.⁵⁷

In darkness pre-treated explants, the largest IAA concentration peaks remained in the “dark” part the cycle and increased in magnitude (Fig. 3b and Table 1b). Again, serotonin concentration maxima appear correlated to IAA concentration maxima, while melatonin displayed a single small uncorrelated concentration peak (Fig. 3b). Similarly to Summer plants, the light pre-treated explants exhibited more IAA concentration maxima, but of smaller amplitudes (Fig. 3c and Table 1b). In this case both serotonin and melatonin concentrations exhibited more correlated concentration peaks. Thus, in the winter plants, both constant darkness and constant light produced some “ringing” (oscillations) in IAA concentration.

Stress hormones JA and ABA in winter cells

Hackenberg and Pandey,⁵⁸ found heterotrimeric G proteins and ABA, but not JA, in closely related species of Characeae Chara braunii Gmelin. Thus Characeae contain ABA and the G proteins that modulate the plant hormone signal transduction pathways. The diel concentrations of ABA and JA are contrasted against the concentration of IAA (Fig. 4). The ABA concentration did not display obvious circadian trends in the plants on 9/15 hr cycle or after pretreatment in dark or light (Fig. 4a–c).

Concentration changes in IAA and JA in opposite direction are interesting (Fig. 4 a-c, Table 1b). Rice seedlings exposed to drought stress exhibited decrease in IAA concentration, while JA concentration increased.⁵⁹ Heat stress, on the other hand, increased IAA concentration, while JA concentration diminished. Only cold stress stimulated concentration increases in both IAA and JA.⁵⁹ During Arabidopsis seed development, IAA concentration in the silique peaked, while JA concentration went through a minimum.⁶⁰ Thus higher plants can produce increased concentrations of both IAA and JA in response to some types of environmental stress, but often the concentration changes of the 2 hormones are out of phase. Liu and Wang,⁶¹ found that auxin downregulates genes for JA biosynthesis.

Metabolic pathways interaction

The concentration peaks are very sharply defined, as each sample is dominated by contents of small number of large internodal cells with no substancial differentiation between axial and leaf internodes (see Fig. 1 and Methods section). In sampling higher plants, the concentration changes may be averaged over many cells of similar size and different types of tissue.

The amplitude and rapidity of concentration changes in all the measured hormones and indoleamines demonstrates the dynamic range of the metabolic pathways (Figs. 2–4). The IAA concentration increased 2 orders of magnitude in 2 hours in the Summer plants, with equally sudden decrease between 13 and 15 o'clock (Fig. 2a and Table 1a). Future experiments are needed to distinguish among changes in rate of production due to either more substrate or greater enzyme activity, export or further metabolism of IAA by conjugation and degradation.^62,63 Since rapid decreases in IAA concentration have been observed in explants (e.g. Figs. 2b and c), where IAA could not be transported into older parts of the thallus and the rhizoids, metabolism of IAA is more likely. IAA conjugates are used as a storage or disposal mechanism for IAA in land plants.⁶⁴ Sztein et al.⁶⁵ found IAA-ester and IAA-amide conjugates in the genus Nitella (Characeae). It will be interesting to see if Chara contains appropriate hydrolases to recover free IAA from conjugates.

The close synchronisation between IAA and serotonin concentration maxima in both Summer and Winter plants suggests IAA biosynthesis by the tryptamine pathway,^62,66 which intersects with serotonin/melatonin synthesis pathway.¹⁵ Tryptamine is only found in low concentrations in Arabidopsis, compared to IAA and tryptophan and this pathway is not well defined in higher plants,⁶² but may be important in Characeae. The Summer explants exposed to continuous light lost syncronisation between concentrations of IAA and serotonin (Fig. 2c), suggesting that in this case IAA might be synthesized by different pathway or the serotonin/melatonin production is diminished. The latter is more likely, as in Winter plants, IAA and serotonin concentrations maxima are synchronised, but serotonin concentrations are low and melatonin concentrations are very low and assynchronous.

The metabolic pathway from tryptophan produces serotonin before melatonin.^14,15 If the cells were sampled more frequently, we would expect to observe a serotonin concentration peak in the Summer plants data before melatonin concentration peak. However, the changes happen too fast to resolve the sequence within the hourly sampling.

Conclusions and perspectives

To our knowledge, this is the first observation of synchronous circadian concentration changes in IAA, serotonin and melatonin in the growing plants. The connection might arise from crosstalk of IAA and serotonin/melatonin biosynthesis pathways. If melatonin acted purely as antioxidant, we would expect persistent concentration maximum in the middle of the day in the Winter plants, which received same type of illumination as the Summer/autumn plants (just for a shorter period). The large differences between Summer and Winter plants also imply infradian rhythm or a role in seasonal development. Our findings also highlight the importance of noting the time and season when collecting plants for assays of IAA, melatonin, serotonin and JA.

Charophytes, that contain the Characeae family, are the closest living relatives to ancestor of all land plants.^48,67 While recent experiments place another charophyte group, Zygnematales, as closer to land plants,^68,69 Characeae are still in strategic position near their origin. Unless the modern Characeae have undergone parallel evolution with land plants, our measurements indicate that the circadian nature of IAA and IAA conjugation pre-dates the emergence of plants onto land. Also the hormones ABA and JA were already present, supporting recent multispecies genome-wide analysis revealing components of their signaling pathways.⁷⁰ Both the circadian and seasonal rhythms in concentrations of IAA, melatonin and serotonin validate Characeae as a useful experimental system, which is both simple (small number of large cells in each sample and lack of axial and leaf internode differenciation) and closely related to higher plants. Imminent sequencing of closely related C. braunii,⁷¹ will provide genetic information on constituents of metabolic pathways.

The data described here provide a baseline to future experiments. In Summer plants the synchronisation of diel growth rate and proton pump activation with the IAA concentration maximum can be explored,^72,73 as well as concentrations of other products in the metabolic pathways (such as IAA conjugates or AFMK). The role of serotonin/melatonin pathway can be explored by inhibiting serotonin or melatonin production,²⁷ or perturbing the rhythms by fast transition from the summer into winter conditions. A separate investigation concentrating on the antheridia will continue the work of Kwiatowska et al,⁷⁴ who found circadian rhythms in protein production in these structures in related Chara vulgaris L. The differences in biochemistry of Summer and Winter plants explain inconsistent results in growth experiments using Chara nodal complexes (Beilby, unpublished results). The growth promoters can now be supplied in the appropriate part of the day to avoid gating. The functions of JA and ABA in Chara can be further assesed under range of abiotic stress conditions to probe the functions of these hormones before plants came to land.

Materials and Methods

Chara cultures

Tanks of perennial male plants of Chara australis R. Brown (see chapter 1 of Beilby and Casanova,⁵⁰ for Characeae systematics) were set up in January 2013 (experiment 1: Summer plants) and January 2014 (experiment 2: Winter plants). Plants originally came from a golf course pond on outskirts of Sydney (latitude 33.865°S), Australia, and were cultured in the laboratory for several years. Cuttings were planted into autoclaved garden soil with layer of clean sand to preserve water clarity. Tanks were illuminated by Gro-Lux fluorescent tubes, providing photosynthetically active radiation of ˜80 μmol.m⁻².s⁻¹ on a day/night cycle of 12/12 hours (experiment 1). Sydney day length varies from 14.5 to 10 hr over the year. However, tanks cultured under 14/10 day/night cycle over longer periods exhibited high plant mortality, so 12/12 photoperiod was chosen for the average “summer” photoperiod. The plants grew vigorously to about 1 m height and orange antheridia (male reproductive organs) could be observed. In experiment 2 plants were changed to day/night cycle of 9/15 hours in mid May 2014. The plants tolerated short day/longer night quite well, but grew slowly and only few antheridia still remained. The temperatures were not controlled, varying from high 20 s to mid 30s in January (southern summer) to 12–25 °C in April and September (autumn and spring) and 6–18 °C in the winter months June to August. Experiment 1 sampling was performed in April 2013 (Australian autumn) after plants experienced 4 months of 12/12 photoperoid and summer/early autumn temperatures. Experiment 2 sampling was performed in September 2014 (Australian spring) after plants experienced 4 months of 9/15 photoperiod and winter/early spring temperatures.

The growing top of the plant was cut for each sample (see the red circle in Fig. 1). Less than 50 large internodal cells were contained in each sample. Each sample also contained small cells from the nodal complexes and, mainly in the Summer plants, antheridia with their supporting structures. However, the contents of the internodal cells constituted the major fraction of each sample. The day/night cells were sampled directly from the tank. Pre-cut plants (referred to as explants) were stored in continuous light or continuous dark for 3 d prior to sampling. As the single cells recover overnight from being cut off the plant and survive indefinitely regenerating new plants, the explants were not expected to exhibit stress responses to cutting after 3 d pretreatment (MA Bisson, pers. com.). Samples were taken every hour. During the day the sampling was done in an unlit laboratory with curtains drawn. At night the illumination was provided by red “safe” light bulb. Cells were carefully manipulated into a pre-weighed labeled Eppendorf tubes and weighed to record the amount of wet tissue. The sample tube was then snap frozen by plunging it into liquid nitrogen and stored in dry ice and later in −80 °C freezer. The samples were couriered to Canada on dry ice and stored at −80 °C until further analysis. Samples were then extracted and analyzed using a previously published protocol for IAA, melatonin and serotonin analysis.⁷⁵

Detection and quantification

IAA, melatonin, serotonin, ABA and JA were detected and quantified using a modified version of the previously published method.⁷⁵ In brief, samples were homogenized in dark (Kontes Pellet Pestle disposable tissue grinder; Fisher Scientific) for 30s in 200 µL of 80:20 v/v methanol:0.1 N TCA in water. Samples were centrifuged (16 000 × g) for 3 min and filtered (0.2 µm, Ultrafree-MC filtered centrifuge tubes; Millipore) prior to chromatography. Serotonin (RT 1.67), melatonin (RT 3.67), IAA (RT 3.85), ABA (RT 3.98) and JA (RT 4.44) were separated from a 10 µL aliquot, at 30 °C, on a reverse phase column (150 × 2.1 mm, 1.7 μm C18 BEH, Waters Inc., Mississauga, ON) using a Waters Acquity I-Class UPLC (see Fig. 5). A gradient of 0.1% formic acid (Eluent A) and acetonitrile (Eluent B) [(A%:B%)]:0.0–05 min, 90:10; 0.5–3.5 min, 40:60; 3.5–4.2 min, 5:95; 4.2–6.5 min, 5:95; 6.5–7.0 min, 90:10] separated IAA, melatonin, serotonin, ABA and JA with a flow rate of 0.3 mL/min. Analytes were quantified with a tandem mass spectrometer (Xevo TQ-S triple quadrupole Mass Spectrometer, Waters). The capillary voltage was 3500, desolvation gas rate was 800 L/hr, cone gas rate was 150 L/hr, desolvation temperature was 550 °C, and the source temperature was 150 °C for all analyses with a dwell time of 0.02 s. Parent and daughter ions were detected using the appropriate MRM transitions (see Table 2). Standards were prepared at the following concentrations for each compound: 0, 1.6, 3.1, 6.25, 12.5, 25, 50, 100, and 200 ng/mL. For Summer data, replicate analyses were possible because of higher concentrations of most compounds. The Limit of Detection (LOD) was less than 3.1 ng/mL for all compounds in each extract and all compounds were quantified within the linear range between of 3.1–200 ng/mL. For the Winter samples, the lower concentrations required modified methods at the lower ranges. The LODs were 34 pg/mL (serotonin), 32 pg/mL (melatonin), 65 pg/mL (auxin), 58 pg/mL (abscisic acid), and 15 pg/mL (jasmonic acid). All were quantified within the individual linear range for the standards. The acquired data was processed with TargetLynx V4.1 (Waters Inc., Mississauga, ON). The chromatograms were smoothed with a 3 × 2 Mean smooth.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Acknowledgments

MJB thanks Patrick McMillan for help with organizing dry ice and liquid nitrogen at the time of sampling. We thank the referees for their instructive comments.