Nitrogen deficiency hinders etioplast development in stems of dark-grown pea (Pisum sativum) shoot cultures Annamária Kósa, Éva Preininger and Béla Böddi* Eötvös University, Institute of Biology, Department of Plant Anatomy, H-1117 Budapest, Pázmány P. s. 1/c, Hungary *Corresponding author, e-mail: [email protected]

The effects of nitrogen (N)-deprivation were studied in etiolated pea plants (Pisum sativum cv. Zsuzsi) grown in shoot cultures. The average shoot lengths decreased and the stems significantly altered considering their pigment contents, 77 K fluorescence spectra and ultrastructural properties. The protochlorophyllide content and the relative contribution of the 654–655 nm emitting flash photoactive protochlorophyllide form significantly decreased. The etioplast inner membrane structure characteristically changed: N-deprivation correlated with a decrease in the size and number of prolamellar bodies. These results show that N-deficiency directly hinders the pigment production, as well as the synthesis of other etioplast inner membrane components in etiolated pea stems. Abbreviations – BAP, 6-benzylaminopurine; Chl, chlorophyll; IBA, indole-3-butyric acid; MS, Murashige-Skoog; N, nitrogen; NAA, 1-naphthaleneacetic acid; Pchl, protochlorophyll; Pchlide, protochlorophyllide; PLB, prolamellar body; POR, NADPH:protochlorophyllide oxidoreductase; PT, prothylakoid.

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Introduction Chlorosis and reduced size of leaves are characteristic symptoms of plants grown under nitrogen (N) limitation (Diaz et al. 2006, Zhao et al. 2005). The former phenomenon indicates the disordered development of the photosynthetic apparatus, due to the decreased chlorophyll (Chl) and carotenoid contents (Huang et al. 2004). Ultrastructural changes of chloroplasts can also be observed; the thylakoid system is reduced and plastoglobuli and starch grains accumulate in the stroma (Doncheva et al. 2001). Senescence symptoms were also reported as a result of remobilization of N from elder to developing tissues (Distelfeld et al. 2014, Masclaux et al. 2000, Masclaux-Daubresse et al. 2010). During this ageing the main source of N is the degradation of chloroplast proteins (Guiboileau et al. 2012). The effect of N deficiency is well-documented in green plants, but it is lesser-known in etiolated ones, as the seeds of higher plants provide sufficient amounts of carbon and nitrogen for the early development of seedlings. Therefore, the effect of nitrogen supply on etioplast development has received only little attention in the literature (El Amrani et al. 1994). There are suggestions that N limitation represses the differentiation of etioplasts into chloroplasts in cotyledons of young Arabidopsis seedlings, however, the process may be affected more by the C:N ratio than by the N status (Vidal et al. 2014). Studies on Arabidopsis plantlets grown on synthetic medium with deprived N content showed a strong effect on the galactolipid composition (Gaude et al. 2007); these lipids are characteristic in etioplast inner membranes, prolamellar bodies (PLB-s) and prothylakoids (PT-s) (Selstam and Sandelius 1984). The biosynthesis of the NADPH:protochlorophyllide oxidoreductase (POR; EC enzyme, the main protein component of PLB-s (Blomqvist et al. 2008, Ikeuchi and Murakami 1982, Selstam and Sandelius 1984) is also affected, however, the down-regulation of POR gene in response to chronically low N condition is controversial (Bi et al. 2007, Wu et al. 2011). The POR apoprotein, NADPH and protochlorophyllide (Pchlide) form ternary complexes (Griffiths 1978), which exist in different spectral forms in the etioplasts (Amirjani 2010, Böddi et al. 1989, Schoefs et al. 2000). PLB-s contain the flash photoactive forms with fluorescence emission maxima at 644 and 655–657 nm (Ryberg and Sundqvist 1982), which were described as dimeric or oligomeric complexes, respectively (Amirjani 2010, Böddi et al. 1989, Schoefs et al. 2000). The PT membranes were characterized with the presence of Pchlide and/or protochlorophyll (Pchl) monomers, showing fluorescence emission maxima at 633 nm in etiolated leaves (Amirjani 2010, Böddi et al. 1989, Schoefs et al. 2000) or at 630 and 636 nm in pea epicotyls (Böddi et al 1994, 1998). Remarkable effects can be expected on the etioplast development in case of N-deficiency because of reduced Chl biosynthesis (Gaude et al 2007) and the disturbed development of etioplast inner membranes. Etiolated pea stems were deemed to be promising experimental materials to study the effects of N-deficiency because of their presumed similarity to pea epicotyls, which show an apical-basal gradient in Pchlide content and in the organization of PLB-s (Böddi et al. 1994). However, in case of etiolated seedlings, sufficient amounts of N are supplied from the cotyledons This article is protected by copyright. All rights reserved

during early developmental stages. To get rid of this N supply, nodal segments developed on culture medium were studied and the N source was varied experimentally. Early observations on plant tissue cultures showed that the transfer of cultures to N-free medium causes the appearance of serious deficiency symptoms and full degradation in two months (Gautheret 1955). In vitro organ cultures have recently been used to study the effect of N amount on plant growth and differentiation processes (Smolik 2013). In this work, we studied the effect of N deficiency on the ultrastructure of etioplasts and the ratio of Pchlide forms in leaves and stems grown in etiolated pea shoot cultures. The aim was to find correlation between the N-supply, the etioplast differentiation and the appearance of different Pchlide forms.

Materials and methods Shoot cultures Pea (Pisum sativum cv. Zsuzsi) seeds were surface sterilized with 0.5% mercuric chloride for 4 min, then rinsed 3 times with distilled water. The embryos were excised from the seeds, i.e. most parts of the cotyledons were removed. The dissected samples were then transferred onto Murashige-Skoog (MS) culture media (Murashige and Skoog 1962), supplemented with the following hormones: 6benzylaminopurine (BAP): 1.0 mg l–1 (4.4 μM); indole-3-butyric acid (IBA): 0.1 mg l–1 (0.5 μM); 1naphthaleneacetic acid (NAA): 0.1 mg l–1 (0.5 μM) (Sigma-Aldrich Kft., Budapest, Hungary). Shoot cultures were grown under 16 h light and 8 h dark cycles at low light intensities (the photon flux density varied between 100 and 200 μmol photons m–2 s–1). Under these conditions, green shoots developed during 4–5 weeks. Shoots of 5–8 cm were cut into nodal segments, which were transferred onto new (secondary) MS culture media and kept in darkness for 3 months. The secondary MS media contained different concentrations of N (Table 1.).

Electron microscopy Tissue pieces were fixed in 2% (v/v) glutaraldehyde for 2 h and postfixed in 1% (w/v) OsO4 for 2 h in 70 mM K-Na phosphate buffer (pH 7.2). The fixed samples were dehydrated through a graded ethanol series and embedded in Durcupan ACM epoxy resin (Fluka Chemie AG, Buchs, Switzerland). Ultrathin sections were stained with uranyl acetate and lead citrate and observed with a Hitachi 7100 electron microscope (Hitachi Corp., Tokyo, Japan). Samples were taken from 3 plantlets and 20 images were studied in case of each sample version.

Pigment content determination The pigments were extracted with 80% acetone. To determine the Pchl(ide) contents, a Pchl solution was prepared in 80% acetone from exploding cucumber (Cyclanthera explodens) seed coat and its absorption spectrum was measured with Shimadzu UV-2101 PC (Shimadzu Corp., Kyoto, Japan) This article is protected by copyright. All rights reserved

spectrophotometer in the 550–800 nm region. On the basis of the absorption maximum value at 624 nm, the pigment concentrations were calculated according to Brouers and Michel-Wolwertz (1983). A dilution series was prepared from this solution and fluorescence spectra were measured to create a calibration curve showing the relation between the fluorescence intensity and the absorbance values. As the presence or absence of the phytol has no remarkable effect on the absorbance or fluorescence emission values (Brouers and Michel-Wolwertz 1983, Mysliwa-Kurdziel et al. 2008), the Pchlide concentrations of the stem samples were determined on the basis of the linear part of this curve. Each pigment determination experiment was repeated twice

Fluorescence spectroscopy The fluorescence emission spectra were recorded with a Jobin Yvon Horiba Fluoromax-3 (Paris, France) spectrofluorometer. The samples were immersed into liquid nitrogen during the measurements. The excitation wavelength was 440 nm. All spectra were measured applying 0.1 s integration time and 0.5 nm data collection frequency. The excitation and emission bandwidths were set to 2 and 5 nm, respectively. The average of 3 spectra was automatically calculated in each measurement. All spectra were corrected for the wavelength dependent sensitivity variations of the detector. The measurements were repeated with 3–12 different plantlets. Where the effects of Ndeficiency were studied, mean spectra of three samples were calculated and are shown. After recording the 77 K fluorescence spectra, several etiolated samples were thawed to 0°C, illuminated with low intensity white light (5 μmol photons m–2 s–1) for 5 min and were refrozen in liquid nitrogen to study the photoactivity of the Pchlide forms via recording the 77 K fluorescence spectra again. The acetonic pigment extracts were measured at room temperature with 430 nm excitation wavelength.

Computer analysis of the spectra Five point linear smoothing, baseline correction and deconvolution into Gaussian components were carried out with the software SPSERV V.11 (copyright Bagyinka, Cs., Biological Research Centre of Hungarian Academy of Sciences, Szeged, Hungary). The deconvolutions were performed in wavenumber function and the error of the fit was smaller than 1% at each resolution. The integral values of each component were calculated.

Results The pea shoots developed from the nodal segments in the dark showed etiolation symptoms, i.e. had elongated stems and small leaves. Often two or more shoots grew from a single nodal segment (Fig. 1). The length of the shoots had great variability even if they developed from one nodal segment, regardless of growing on complete (control) or on N-deprived media. The average length of shoots This article is protected by copyright. All rights reserved

was 8.2 ± 6.6 cm at control (n = 12) (Fig. 1c), while the plantlets grown on 1/3 or 2/3 N media were similar with average length of 5.6 ± 3.8 cm (n = 20) (Fig.1b). The plantlets on N-free medium were short: 3.9 ± 2.6 cm (n = 8); often only their shoot tips were fully etiolated (Fig. 1a). The original green nodal segments showed strong senescence symptoms in all cases; they were not studied in this work. Spectral characteristics of the dark-developed shoots were observed by measuring the 77 K fluorescence emission spectra of uppermost, middle and lowermost stem segments. The spectra of a 10 cm long shoot grown on complete MS medium are shown in Fig. 2. They showed Pchlide emission maxima at 632.5 and 654.5 nm; however their amplitude ratios significantly varied. In the spectrum of the uppermost segment, the 632.5 nm band dominated and only a small emission peak was found at 654.5 nm (Fig. 2, spectrum A). The spectrum of the middle segment had well observable independent emission band at 654.5 nm (Fig. 2, spectrum B). In the spectrum of the lowermost segment, the 632.5 nm band dominated, the amplitude of the 654.5 nm peak was relatively low and an additional maximum appeared at 680.5 nm (Fig. 2, spectrum C). The pigment contents of the segments were in good agreement with these spectral data. The basal segments of the control etiolated shoots usually contained low amounts of Chl in addition of Pchl(ide) pigments. The upper segments especially at the tip, however, contained only Pchl(ide). The mean Pchl(ide) content in the middle segments was 0.5 µg g–1 fresh mass, while the mean Chl content in the lowest segments was 0.3 µg g–1 fresh mass. To study the effect of various N levels, the spectra of uppermost, middle and lowermost segments of stems grown on media supplemented with different amounts of N were deconvoluted into Gaussian bands with maxima at around 628, 636, 643.5, 654.5 and 669 nm. The relative amplitude change of the 654.5 nm band was the most characteristic; it varied with the amount of N and with the studied regions of the stems. It was calculated as follows: the integral value of the 654.5 nm band was divided by the sum of the integral values of the above-listed five Gaussian components and multiplied by 100 (Table 2). Stems on N-free medium contained relatively low amounts of the 654.5 nm emitting Pchlide complexes independently of the studied regions. Maximum contribution of the 654.5 nm band was observed in the uppermost or in the middle stem regions depending on N content of the culture medium (Table 2). In the middle stem segment the relative contribution of the 654.5 nm band in the spectra showed positive trend with N supply (Table 2 and Fig. 3). The mean pigment content of control stems was around 0.5 µg Pchlide g–1 fresh mass (see above), which value decreased to 0.1 µg Pchlide g–1 fresh mass at 2/3 N medium and was merely 0.02 µg Pchlide g–1 fresh mass in the case of 1/3 and zero added N to the medium. To test if the 654.0–654.5 nm bands belonged to the photoactive oligomeric Pchlide complexes, some samples were thawed to 0°C after measuring their 77 K spectra and were illuminated with white light of 5 μmol photons m–2 s–1 photon flux density for 5 min. Subsequently, the samples were refrozen in liquid nitrogen and their spectra were measured again. The illumination resulted in full phototransformation of the 654.0–654.5 nm emitting Pchlide forms and in parallel the appearance of

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new bands at 688–689 nm, belonging to Chlide forms. In Fig. 3 inset the emission spectra of a “dark” (continuous line) and then “illuminated” (dashed line) stem segment developed on 1/3 N medium are shown. Stems grown on different media showed remarkable differences in the organization of their etioplast inner membranes (Fig. 4 and Table 3). Almost half of the etioplasts contained PLB-s in the control stems (Fig. 4a and Table 3). N-deprivation resulted in the disturbance of the PLB membrane organization and the appearance of numerous plastoglobuli (Fig. 4b–d). In etioplasts of plantlets grown on 2/3 N medium the average size and number of PLB-s reduced (Fig. 4b and Table 3). In the case of 1/3 N medium-grown stems only one of twenty etioplasts contained small PLB, rather the presence of circularly arranged etioplast inner membranes was characteristic (Fig. 4c and Table 3). In stems grown on media containing zero added N, the etioplasts were similar to proplastids; no PLB-s but a few, PT-like simple membranes and the appearance of lipid droplets were typical (Fig. 4d and Table 3). Interestingly, the dark-developed leaves did not show the above-described symptoms of N deficiency. Even in the case of plantlets grown on N-free medium, the 654 nm band dominated in their 77 K fluorescence emission spectra (Fig. 5, right panel, solid line) and their etioplasts contained well-developed PLB-s (Fig. 5, left panel). Illumination resulted in full phototransformation of the 654 nm emitting Pchlide form into a Chlide form emitting at 689.5 nm (Fig. 5, right panel, dashed line).

Discussion The main factors required for the formation of PLB-s in etioplasts have already been identified (Solymosi and Schoefs 2010), however, the influence of nutrient starvation on the process is much less investigated. Pea was expected to react sensitively to N limitation, since its optimal N supply is ensured by N fixing bacteria under natural conditions (Newcomb 1976). All culture media applied in our work, even the N-free were suitable to produce etiolated shoots allowing to observe correlations between the N concentration of the medium and the appearance of N deprivation symptoms. On the other hand, the precise regulation of N availability for new-developing shoots cannot be achieved since the degrading green nodal segments act as N sources for the new tissues (Distelfeld et al. 2014, Drew and Sisworo 1977, Mae and Ohira 1981). The spectral properties of the dark-grown shoots studied in this work were similar to those of epicotyls of 8–10 days old etiolated pea seedlings considering the dominance of the short-wavelength (631–632 nm) emitting Pchlide form all along the axis (Fig. 2). The 655 nm form however was found in highest amounts in the hook of epicotyls (Böddi et al. 1994) but in the middle segments of the in vitro grown stems in cases of 2/3 and total N contents in the media (Fig. 2, spectrum B and Table 2). The lowermost segments of both the epicotyls (Böddi et al. 1999) and the stems (Fig 2, spectrum C) contained Chl-s with emission bands at around 680–680.5 nm. The suggested origin of these Chl forms was the translocation of chloroplasts (and Chl-s) from cotyledons in case of epicotyls (Böddi et This article is protected by copyright. All rights reserved

al 1999). In this work, the green nodal segments could be the sources of Chl-s. The quantity of Pchlide as well as the average size of PLB-s (Table 3) and etioplasts (Fig. 4a) were similar in stems of the control cultures (Fig. 4a) and in epicotyls of etiolated pea seedlings (Böddi et al. 1994). N deprivation provoked the reduction of shoot lengths (Fig. 1) similarly to the results of Zhao et al. 2005. The N starvation and long dark incubation induced senescence process involved the evident decrease of Pchlide content (at 1/3 or at 0 added N it was only around 4% of that of the control) similarly to the results of Hukmani and Tripathy (1994). To study the effects of N-deficiency on fluorescence spectral properties, the middle stem segments were suitable, because of the positive trend between N supply and relative contribution of the 654.5 nm emitting Pchlide in the spectra (Table 2 and Fig. 3). The alteration of the etioplast inner membrane organization was observed as well (Fig. 4 and Table 3), stems developed on N-free media possessed proplastid-like plastids with single inner membranes (Fig 4d). Apart from stems grown on complete MS medium, plastoglobuli accumulated in the stroma matrix of etioplasts (Fig. 4), presumably as a result of senescence (Hopkins et al. 2007) and nitrogen deficiency (Gaude et al. 2007). Although the number and size of PLB-s decreased with the extent of N deficiency (Table 3), they are distinct from plastoglobuli on the basis of comparing their protein content (Grennan 2008). There are contradictory data in the literature regarding the correlation among Pchlide content, the amount of the 654–655 nm emitting form and the appearance of PLB-s in etioplasts. In our work, the total amounts of Pchlide and the relative contribution of the 654–655 nm form changed in parallel. This confirmed earlier observations about the positive correlation between Pchlide content and the high ratio of the 654–655 nm emitting Pchlide to other Pchlide forms (Skribanek et al. 2000). In addition, the contribution of the 654–655 nm emitting Pchlide form in fluorescence emission spectra (Fig. 3) showed strong positive correlation with the appearance of regular PLB structure (Fig. 4) confirming earlier data (Selstam et al. 2011, Sperling et al. 1998) and was in good agreement with the presumable PLB localization of these Pchlide complexes (Ryberg and Sundqvist 1982). On the other hand, stems developed on N-free media possessed plastids with single inner membranes (Fig 4d), while their 77 K fluorescence emission spectra still showed the presence of the 654–655 nm band with low amplitude (Fig. 3, spectrum D). This result suggested no strict correlation between the presence of oligomeric Pchlide and well-developed PLB-s similarly to earlier suggestions (Selstam et al. 2007). Contrary to stems, leaves of etiolated shoots grown on N-free MS media contained welldeveloped PLB-s in their etioplasts and the main maxima appeared at 654 nm in their fluorescence emission spectra (Fig. 5). These results reveal the possibility that the gradually degrading green shoot, the lateral shoot of which was examined, functioned as N source and developing leaves must be preferential sinks for the N-transport (Distelfeld et al. 2014, Drew and Sisworo 1977). The results of this work confirm the organ specific regulation of the N-distribution in case of Ndeficiency, the N-supply of young tissues (leaves and uppermost stem segment) are preferred sinks. This article is protected by copyright. All rights reserved

As a consequence, the plastid development of older stem segments is hindered, the formation of PLBs and 654–655 nm emitting flash-photoactive Pchlide complexes as well as Pchlide accumulation are reduced.

Author contributions B. Böddi conceptualized the study, directed its implementation and did the final revision of the manuscript. B. Böddi and A. Kósa performed and analyzed spectroscopic measurements and drafted the manuscript. É. Preininger performed electron microscopy and established the organ cultures of pea. A. Kósa did the literature search and designed the figures. All authors discussed the results, read and approved the final manuscript. Acknowledgements – We are grateful to Csilla Jónás for the technical assistance in electron microscopy.

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Edited by A. Krieger-Liszkay

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Figure legends

Dark-grown pea (Pisum sativum) shhoots developped from green nodal seegments duriing three Fig. 1. D months oon MS cultu ure media conntaining zeroo (a), one thirrd (b) or totaal (c) amountt of nitrogen..

most (C) Fig. 2. 77 K fluoreescence emisssion spectraa of the upppermost (A),, middle (B)) and lowerm m developed on control MS M culture medium. m Thee spectra regions of a 10 cm long etiolateed pea stem w normalized to their maxima m were meeasured with 440 nm exciitation and were

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. Fig. 3. 777 K fluorescence emisssion spectraa of etiolatedd pea stems grown in sh hoot culturess on MS media containing c zeero (D), onee third (C), two thirds (B) or total (A) amounnt of nitrogen. Every spectrum m is the averrage of three different measurement m ts. Inset: speectrum C beefore (solid line) l and after (daashed line) illlumination with w white light of 5 µm mol photons m–2 s–1 photton flux denssity for 5 minutes. The spectraa were measuured with 440 nm excitattion and weree normalizedd to their maxxima.

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Fig. 4. E Electron microscopic imaages of etiopplasts from ettiolated pea stems grownn in shoot cuultures on control M MS media (a), ( on MS media m contaiining two thhirds (b) or one o third (c)) amount of nitrogen content oof the contro ol and on nitrrogen-free medium m (d). (Bars: 1 µm)

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Fig. 5. L Left panel: Electron E micrroscopic imaage (bar: 1 µm m) of an etiooplast from the t uppermost leaf of emission an etiolaated pea shooot cultured on nitrogen-frree MS mediium. Right panel: p 77 K fluorescence fl spectra of o a similar leaf before (solid ( line) and a after (daashed line) illlumination with w white light l of 5 – µmol phhotons m–2 s–1 photon fluxx density forr 5 minutes. The T excitatio on wavelengtth was 440 nm. n

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Table 1 Nitrogen contents of the different MS culture media




Complete MS MS with two thirds nitrogen (2/3 N) MS with one third nitrogen (1/3 N) MS without added nitrogen

(mg dm )

(mg dm-3)









Table 2 Relative contribution of the 654.5 nm emitting protochlorophyllide form in the 77 K fluorescence emission spectra of uppermost, middle and lowermost pea stem segments grown on MS culture media supplied with various amounts of nitrogen

Nitrogen content in

Relative contribution of Pchlide 654.5 (%) in uppermost stem

middle stem segment

lowermost stem

segment (mean ± SD)

(mean ± SD)

segment (mean ± SD)


13.2 ± 1.3

12.3 ± 0.3

10.3 ± 0.6

1/3 N

27.3 ± 6.6

19.5 ± 0.1

17.7 ± 0.4

2/3 N

13.9 ± 0.5

26.9 ± 1.1

17.9 ± 1.1


15.1 ± 2.7

27.3 ± 8.3

18.4 ± 1.9

culture medium

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Table 3 Size and number of prolamellar bodies in middle segments of pea stems grown on MS culture media supplied with various amounts of nitrogen. Twenty images from each sample type were analyzed

Nitrogen content in

Number of plastid sections with

Average size of prolamellar

circularly arranged


inner membranes






1/3 N




2/3 N



713.2 ± 278.9




750.7 ± 177.4

culture medium

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bodies in nm (mean ± SD)

Nitrogen deficiency hinders etioplast development in stems of dark-grown pea (Pisum sativum) shoot cultures.

The effects of nitrogen (N) deprivation were studied in etiolated pea plants (Pisum sativum cv. Zsuzsi) grown in shoot cultures. The average shoot len...
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