Loss of nuclear flavanols during drought periods in Taxus baccata

RESEARCH PAPER

Loss of nuclear flavanols during drought periods in TaxusbaccataW. Feucht1, D. Treutter1, H. Dithmar2 & J. Polster2

1 Department for Plant Science, Unit Fruit Science, Technische Universitat Munchen, Center of Life and Food Science Weihenstephan, Freising, Germany

2 Department for Physical Biochemistry, Technische Universitat Munchen, Center of Life and Food Science Weihenstephan, Freising, Germany

INTRODUCTION

The global climate has become progressively warmer andextreme drought periods occur more frequently as, for exam-ple, in 2003, 2007 and 2010 in Southern Germany. Amongthe conifer species studied for several years in our laborato-ries, Taxus baccata was shown to have a comparatively highcontent of nuclear flavanols (Polster et al. 2006). During thegrowing season, flavanols stain blue with the p-dimethylami-nocinnamaldehyde (DMACA) reagent. However, in the dor-mant season the nuclear flavanols were much decreased oreven absent.

Plants in general have evolved adaptive strategies to ensuresurvival under high temperature and drought conditions.Special signalling molecules finally induce transcription fac-tors that activate stress-responsive genes (Xiong et al. 2002).Any epigenetic modification of histone structures due to anextreme environment is supposed to have consequences forstress-responsive gene expression (Hsieh & Fischer 2005).Loosening or compacting of histones in response to favour-able or harsh epigenetic conditions (Luger & Richmond1998) is visualised as light or dark blue flavanol staining ofnuclei or chromosomes (Feucht et al. 2007).

Phenolic compounds, once defined as waste products, havereceived more and more attention as signalling compounds(Taylor & Grotewold 2005). Catechins of tea (Camellia sinen-sis) have been shown to complex with DNA and RNA (Kuzu-

hara et al. 2006) and might modify specific genes; moreover,genes must be protected against UV radiation (Ries et al.2000). Catechin, epicatechin and epigallocatechin were identi-fied as flavanol components in nuclei of Taxus microspores(Feucht et al. 2008). The paper presented here shows thatdrought and heat stress result in a complete loss of nuclearflavanols of T. baccata. Following return to a rainy period,through rewatering of the study site, all nuclei of the currentyear growth again established nuclear flavanols.

A first indication that targets of flavanols in nuclei couldbe the histone proteins of nucleosomes was provided in Pol-ster et al. (2003), and these finding have been further con-firmed these findings (Feucht et al. 2004, 2005). It is wellknown that post-translational modifications of histone pro-teins, mainly acetylation, methylation, phosphorylation,formylation and ADP-ribosylation in the N-terminal taildomains but also in the core domains, are important mecha-nisms for the regulation of chromatin structure (Goll &Bestor 2002; Mersfelder & Parthun 2006) and gene expression(Iizuka & Smith 2003).

If histones are actually flavanol targets, then the ‘epigenetichistone code’ is equipped with a further modification factor.In order to investigate this hypothesis, modified peptideswere synthesised in analogy to the histone code, representingfragment 71–85 of the H4 protein. The results show thatthese modified peptides have different interactions with theflavanol catechin.

Keywords

Catechin; dimethylaminocinnamaldehyde;

histochemistry; histone protein; nucleus.

Correspondence

D. Treutter, Unit Fruit Science, Department

Plant Science, Duernast 2, D- 85354 Freising,

Germany.

E-mail: [email protected]

Editor

B. Piechulla

Received: 23 March 2012; Accepted: 29 June

2012

doi:10.1111/j.1438-8677.2012.00661.x

ABSTRACT

Normally, needles of Taxus baccata during the growth period prominently stainblue for nuclear flavanols with the histochemical DMACA procedure. However,under excess heat and drought conditions, nuclear flavanols of current-year needlesdecline to zero. Nevertheless, greenish-yellow-coloured flavonols (quercetin deriva-tives) were still observed in nuclei. All of these yellow nuclei were in a silencedstate and without mitosis. This link between drought and loss of nuclear flavanolswas found in 3 years, 2003, 2007 and 2010. In 2007, exceptional drought occurredin early spring, interrupted by short rains. This, in turn, led to flushing of newsprouts, a characteristic feature in which nuclei were overloaded with flavanols. Bythe end of three drought periods, all nuclei developed blue-coloured nuclear flava-nols. The flavanols seem to be associated with the histone proteins of chromatin.The oxidative degradation of catechin in Tris buffer (pH 8.0) containing MgCl2 wasstudied in the presence of the H4-core fragment TYTEHAKRKTVTAMD, modifiedaccording to the epigenetic histone code. The results show that catechin degradationcan be significantly inhibited by the non-modified peptides and the methylated pep-tides (methylation at both lysine residues). The acetylated and formylated peptidesdo not show this behaviour. These observations indicate that flavanol association atchromosomes appears to be regulated by the epigenetic histone code.

Plant Biology ISSN 1435-8603

462 Plant Biology 15 (2013) 462–470 ª 2012 German Botanical Society and The Royal Botanical Society of the Netherlands

MATERIAL AND METHODS

Study site

The sample trees of T. baccata and T. baccata DovastoniiAureovariegata (5–10 years old), 2–3 m in height, grow inthe Botanical Garden of Weihenstephan, 30 km north ofMunich (530 m a.s.l.) in deep and loamy soil with highwater-holding capacity. Mean annual rainfall in the last5 years ranged from 650 to 750 mm per year, with no dis-tinct seasonality in precipitation. In 2007, four trees locatedin close proximity, within about 50 m, were investigated.More than six trees were used for further sampling; some ofwhich are shaded beneath tall, broadleaf trees (mainly Quer-cus robur) but without major competition from grass roots.In 2003 and 2010 other groups of trees without an overstoreycanopy were investigated.

Weather conditions of study site

Significant drought periods occurred in 2003, 2007 and 2010.In August 2003, temperatures were generally high, with maxi-mum daily values above 36 �C and precipitation of only12 mm. In July 2010, the first 3 weeks were extremely hot,with 8 days above 30 �C and precipitation was near zero, butby the end July, precipitation was extremely high (90 mm).

However, during spring 2007 the weather conditions wereatypical; high temperatures occurred already at the beginningof April (up to 5 �C above average) so that swelling of the budsstarted 3–4 weeks earlier than normal. In April 2007, there wasonly 11 mm of rain, resulting in a gradual decline in growth tonearly zero in May. From 1 April to 16 June, there were threeperiods of sporadic rainfall (8–9 May: 36 mm, 28–29 May:57 mm, 12 and 15 June: together 38 mm). By 28–29 May, infew hours 57 mm of rain fell, but most was subject to superfi-cial run-off because the dry soil was unable to fully absorb thefast-flowing water. These rains caused new post-rain flushes tosprout from quiescent buds about 3–4 days later.

In field-grown trees, physiological effects of high irradi-ance, evapotranspiration and water deficit can be con-founded. In April 2007, when leaves of the overstorey treeswere still not fully developed, the elevated temperatures prob-ably had the strongest influence. However in May ⁄ June, whencanopies of overstorey trees were closed, the Taxus trees werein a shaded microclimate so that mainly water-limited condi-tions were considered the dominant stress factor.

Sampling of needles

In 2007, sampling was performed with two types of needle:(i) expanded needles of the normal current-season shoots;and (ii) only in 2007, newly emerged needles from post-rainperiods. For analytical determination of flavanols, three nee-dle groups from the needle-expansion period were sampled(5–10, 10–15 and 15–20 mm in length). Small, newlyemerged needles were additionally sampled after occasionalrainfall in May and June. From the current growth, a total of15 samplings were made during the entire investigation per-iod (20, 25, 30 April, 5, 15 May, 5, 15 June, 5, 20 July). Post-rain samples were collected on 13 and 17 May and 2, 5, 18and 21 June. Each sample consisted of five needles.

In 2003, sampling was performed with fully developed nee-dles on 5, 15 and 25 August. In the post-rain period, twodates per month were chosen for further samplings in Sep-tember, October and November. In 2010, fully developedneedles were sampled on dates, 5, 12 and 19 July. Addition-ally, in the post-rain period, some further samples weretaken.

Histological techniques

Microscopic histochemical observations were made from 300to 500 cells per needle.

Fresh tissues were used to study the presence of flavanols innuclei. Usual fixation and embedding was omitted because theamount of soluble flavanols was reduced during dehydrationprocedures with alcohol. Immediately following collection,excised needle tissues were placed directly on a microscopeslide to perform selective blue flavanol staining with theDMACA reagent (1% p-dimethylaminocinnamaldehyde in1.5 N methanolic sulphuric acid). After 10–20 min, when thereagent had nearly evaporated, the tissue was rinsed in waterand the flavanols turned blue. By lightly squeezing the stainedtissues under a cover slip the individual cells separated fromone another. Diphenylboric acid-b-aminoethylester (DPBA,1% in methanol) was applied to detect compounds with yellowfluorescence, using the filter sets, G 395-440, FT 460 and LP480. Photomicrographs were made with a Zeiss Axioscop andan UV photomicroscope (Zeiss, Oberkochen, Germany). Scan-ning of the micrographs was performed with a CoolScan appa-ratus (Nikon LE 450; Nikon Corp., Tokyo, Japan).

Determination of flavanols with HPLC

Phenolic compounds were extracted from lyophilised andpowdered needles with methanol within 30 min in a soni-cated water bath (4 �C). After centrifugation, an aliquot ofthe supernatant was injected into the HPLC consisting ofan autosampler (Gilson Abimed Modell 231, Abimed, Lan-genfeld, Germany), two pumps (Kontron Modell 422, BiotekKontron, Neufahrn, Germany), and a diode array detector(BioTek Kontron 540, Biotek Kontron). Separation wasdone on a column (250 mm · 4 mm ID) prepacked withHypersil ODS (3 lm particle size). For post-column derivat-isation, a further analytical HPLC pump (Gynkotek ModellM 300 C, Abimed) and a detector 432 nm were used. Flav-ones and flavonols were detected at 280 nm. For detectingflavanols, 1% DMACA in 3 N H2SO4 ⁄ MeOH was used,resulting in a highly selective colour complex at 640 nm.Step-wise gradients were applied using mixtures of solventA (formic acid, 10% in water) and solvent B (methanol,gradient grade) from 95:5 (v ⁄ v) to 100:90 (v ⁄ v) with a flowrate of 0.5 ml min)1 (Treutter et al. 1994). The gradientused was: 0–5 min, isocratic, 5% B in A; 5–15 min, 5–10%B in A; 15–30 min, isocratic, 10% B in A; 30–50 min, 10–15% B in A; 50–70 min, isocratic, 15% B in A; 70–85 min,15–20% B in A; 85–90 min, isocratic, 20% B in A; 95–110 min, 20–25% B in A; 11–140 min, 25–30% B in A;140–160 min, 30–40% B in A; 160–175 min, 40–50% B inA; 175–190 min, 50–90% B in A. Methodological variationsin the method were below 10%.

Feucht, Treutter, Dithmar & Polster Loss of nuclear flavanols during drought

Plant Biology 15 (2013) 462–470 ª 2012 German Botanical Society and The Royal Botanical Society of the Netherlands 463

Identification of flavonoids

The flavonoid peaks were identified according to their UVabsorbance, chromatographic behaviour on HPLC, develop-ment of blue colour using post-column derivatisation andTLC. Catechin, epicatechin and epigallocatechin were used asauthentic standards (Roth, Karlsruhe, Germany). Identifica-tion of flavones and flavonols was performed with authenticstandards (quercetin, myricetin, apigenin, kaempferol, luteo-lin) by comparison of chromatographic behaviour on HPLC,TLC and UV absorbance spectra.

Thin layer chromatography

Cellulose plates (Merck, Darmstadt, Germany) were used fortwo-dimensional flavanol separation. The solvents were: firstdirection, butanol ⁄ acetic acid ⁄ water (BAW, 4:1:2.2); seconddirection, 10% formic acid. The flavanol spots were visualisedby spraying with the DMACA reagent. The two dominatingspots, catechin and epicatechin, as well as epigallocatechin,were compared with authentic standards from Roth. TLCwas repeated four times (once per week) per species to con-firm the data obtained with HPLC.

Chemicals for kinetic measurements

Peptides (lyophilisates) were prepared from Peptides and Ele-phants GmbH (Nuthetal, Germany; see Table 3); (+)-catechin(puriss.; article no: 6200) and Tris = Tris-(hydroxymethyl)amino methane (PUFFERAN�, ‡99.9%, p.a., Roth, Karlsruhe,Germany) were obtained from Roth; ethanol (for spectros-copy; ‘‘Uvasol’’) from Merck; oxygen 4.5 (content 12 l; Mini-can; 12 bar) from Linde (Unterschleißheim, Germany);hydrochloric acid (min. 25%; puriss. p.a.) from Riedel-deHaen (Sigma Aldrich, Seelze, Germany); and magnesiumchloride hexahydrate (AnalaR Normapur, 99.0–102.0%) fromVWR (Leuven, Belgium).

Buffer and solutions for kinetic measurements

Catechin was dissolved in ethanol to a concentration of10 mg ml)1 (34.5 mm) as stock solution. Buffer peptide solu-tion: the pH of a dilute solution containing 0.1 m Tris and10 mm MgCl was adjusted to 8.0 with HCl (pH meter:WTW inoLab pH Level 1). About 3.3% ethanol was added tothe buffer peptide solution and dissolved in ethanolic bufferto a concentration of about 1 mg ml)1 (see Table 3). Thesolution was carefully gassed with oxygen for 3 min in awater bath at 20 �C.

Kinetic measurement of degradation of catechin

A total of 500 ll of the prepared ethanolic buffer peptidesolution (or pure ethanolic buffer solution without peptidebut gassed with oxygen) were placed in a quartz cuvette(1 cm path length, total volume 600 ll) and the cuvette waskept at 20.0 �C for 5 min. Then, kinetic measurements werestarted after addition of 10 ll catechin stock solution (at20.0 �C). Reaction spectra were recorded with a diode arrayspectrometer (Hewlett Packard, model 8453; Agilent Technol-ogies, Waldbronn, Germany). Kinetics were followed for

about 15 h (parameters of kinetic measurement: integrationtime 0.1 s, run time 54,000 s, cycle time 600 s, incrementcycle time: 2% after 7200 s). Approximately 60 spectra wererecorded in each run. Time-dependent curves were plottedfor the wavelength 434 nm.

RESULTS

Loss of nuclear flavanols during spring drought in 2007

In 2007, bud break of Taxus started during the first days ofApril, with a medium degree of blue staining in nuclei ofnewly sprouting needles. However, by the end of April theblue colour had slowly disappeared, and about 10 days later(first days of May), most nuclei were pale yellowish in col-our. Occasionally, nuclear structures appeared green, being amixture of yellow flavonoids and blue flavanols, which wasmore pronounced around newly formed tracheids (Fig. 1a).Later in May, during progression of the drought, in a groupof five to ten cells, only one had a greenish nucleus (Fig. 1b).A severely affected nucleus contained some filiform streakswith a pale greenish tint (Fig. 1c), which on magnificationrevealed about ten greenish, chromosome-like structures(Fig. 1d). To confirm the presence of yellow flavonoids,nuclei were stained with Naturstoff reagent (Fig. 1e). Up tolate June, the overall yellow appearance of nuclei corre-sponded to those shown in Fig. 1a–e.

Low to medium levels of nuclear flavanols in post-rain flushes

About 3–5 days after the short rain periods (8 and 29 May,12 and 15 June), new, sprouting flushes about 2 cm in finallength appeared, in which trans-differentiation of small cellswas seen. A limited number of helical tracheary elementsformed close to sieve cells of the vascular bundles, with char-acteristic helical winding of secondary non-lignified wallthickening. The nuclei of these cells slowly disappeared dur-ing thickening of the helical windings. However, most strik-ing point was that around the helical tracheids, small,rounded parenchyma-like cells formed, which developed bluestaining nuclei (Fig. 1f). Occasionally, two prominent nucle-oli were present and the surrounding nucleoplasm consistedof fine-grained euchromatin (Fig. 1g, left). The neighbouringmetaphase chromosomes showed little to moderate intensityof blue staining flavanols (Fig. 1g, right).

Enlarged, rounded mesophyll cells located outside the bun-dle sheath displayed moderate blue-stained nuclei with fine-grained euchromatin (Fig. 1h). Close examination showed amosaic-like distribution of the chromatin (Fig. 1i magnifica-tion of 1 h), which is characteristic of activated nuclei. Whencomparing Fig. 1i with 1j, it is of interest that the mesophyllnuclei of sprouting needles often showed a unique nuclearflavanol pattern, even within the same cell cluster. Thesecould be related to differences in diffuseness of the flavanolsas well as expression of darker blue spots of heterochromatin.

High levels of nuclear flavanols in post-rain flushes

A few days after the rain (8–9 May) diffused flavanols wereobserved in and around the water conducting xylem vesselsof the newly emerging leaf flushes. The site of flavanol syn-

Loss of nuclear flavanols during drought Feucht, Treutter, Dithmar & Polster


thesis might be in the roots or in the new flush itself. Darkblue nuclei were found only around these xylem vessels, whileless blue nuclei developed some distance away (Fig. 1k). Theentire scenario points to excessive leaching of flavanols withinand directly around the tracheary elements. Even the cellwalls displayed some affinity for flavanols (Fig. 1l*). Twoadjacent post-rain nuclei are shown in Fig. 1m because oftheir different nuclear staining pattern. The small and com-pacted dark blue nucleus (top) was only 6 lm in diameter,and apart from the nucleolus, no further structures wereseen. The second nucleus (bottom, Fig. 1m), about 8 lm indiameter, displayed a looser, open configuration, with somefine-grained flavanol structures.

The two examples of interphase nuclei had many aggrega-tions of darker heterochromatin that was randomly distrib-uted over the nucleus (Fig. 1n and o). Both nuclei wereclearly different with regard to the amounts of diffuseeuchromatin and patchy heterochromatin. At late metaphase,extremely dense chromosome arms were aligned in parallel(Fig. 1p). However, one chromosome arm was cast (marked

by an asterisk) outside the bulk of the chromosomes, perhapsdue to centromeric malfunction.

Excess levels of nuclear flavanols in post-rain flushes

A number of nuclei in the post-rain needles had pro-nounced accumulation of flavanols. Assembly of the meta-phase chromosomes (Fig. 1q, polar view) was less orderlyand the flavanols appeared to be rather variable, as seen inboth pale and dark blue domains along the chromosomes,and nuclei were even more compacted, distorted and irregu-lar (Fig. 1r), suggesting a mismatch. A further intensificationof chromosomal perturbance, combined with an excess offlavanols (Fig. 1s), indicated maximum clumping of flava-nols and loss of any regular mitotic structures. Finally, adrastic example of chaotic and overloaded nuclear structuresis shown in the telophase stage (Fig. 1t). The newly develop-ing daughter nuclei were overloaded with flavanols and theregion between the daughter cells was stained a faint blue,which is most unusual in our experience. Also, the nearby

a b c d e

jihgf

k l m o p

u

n

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Fig. 1. (a–u) Chromatin state maps for 2007 resulting from blue staining of flavanols. Diameter of nucleus (n) in lm. (a–e) Severe drought. (a) Helical tra-

cheary xylem element (* very pale blue) and adjacent greenish chromosomal structures becoming diffuse. The green colour results from a mixture of yel-

low flavonoids and light blue staining flavanols (see also Figs b, c, d). a–d: DMACA reagent. (b) Mesophyll cells with only one cell showing a greenish, ill-

defined nucleus (*). n, 7 lm. (c) Magnified cell with very pale green nucleus (*). n, 7 lm. (d) Magnified nucleus (shown in c) with some green chromo-

somal structures. n, 7 lm. (e) Nuclei with a yellow fluorescence after DPBA reagent. n, 8 lm. (f–j) Post-rain flush with moderate nuclear flavanol binding.

(f) Fairly blue nuclei positioned close to single tracheary elements. Helical windings (*) stain light blue. n, 8 lm. (g) Moderately stained nucleus (n, 10 lm)

with two large nucleoli and metaphase with pale staining chromosomes. (h) Pale blue nucleus (*) of a large mesophyll cell. n, 8 lm. (i) Magnified nucleus

(shown in g) exhibiting fine-grained, moderate blue euchromatin spots. The interchromatin space apparently lacks flavanols. n, 9 lm. (j) Nucleus showing

differential staining pattern, i.e. rather pale staining of euchromatin and a few spots of darker blue heterochromatin. n, 9 lm. (k–u) Post-rain period with

dense to extreme nuclear flavanol binding. (k) Needle sector with some elongated single tracheary elements and dark stained nuclei located nearby. Diffus-

ing flavanols coincide with regions of tracheary elements (*). n, 7–9 lm. (l) To compare with Fig. k, a moderately stained nucleus near a single tracheary

element during normal weather conditions in June 2006. n, 9 lm. (m) Two nuclei (7 and 10 lm in diameter) with different chromatin structures. (n)

Nucleus with dark blue euchromatin and a number of condensed spots of heterochromatin. (o) Loosely structured nucleus, with some dark patches of het-

erochromatin. n, 8 lm. (p) Metaphase with normal flavanol binding but with a tendency to a diffuse appearance. One chromosome is lagging beyond the

regularly aligned bulk of chromosomes. (q) Distorted chromosome structures at metaphase with some dense heterochromatin spots. n, 12 lm. (r) Severely

disturbed mitotic state with abnormal and dark blue chromatin structures. n, 11 lm. (s) Nuclear chromatin excessively overloaded with flavanols, impeding

any normal organisation of mitosis. n, 12 lm. (t) Excess flavanols bound to segregating telophases causes some flavanols to be retained in the cytoplasmic

space between the telophases. (u) Rather pale flavanol staining of telophase in late July, with correct segregation of the haploid chromosome clusters.



interphase nucleus was stained extremely dark blue (Fig. 1t).For comparison, a pale blue telophase configuration, sam-pled 3 weeks after the drought period (20 July) is shown(Fig. 1u).

Changes of phenolic compounds during the growth phase ofneedles

Growing needles were compared with fully expanded needleswith respect to amounts of different flavanol types (Table 1):catechin was several-fold higher compared with epicatechinand epigallocatechin. Few other flavanols, probably dimers,could not be completely identified. Comparing needle groupsof different length, catechin, and epicatechin were quite simi-lar in amount in the shorter needle groups (5–10 and 10–15 mm in length), reaching roughly twice the concentrationsof the longer needles (15–20 mm in length). A similar ten-dency was found for the unidentified flavanols. Notably, inthe case of epigallocatechin, the younger needles 5–15 mm inlength reached four-fold the values in the expanded olderneedle group. The hydroxycinnamic acids followed the flava-nol pattern (Table 2); they decreased in amount from theyounger to the mature needle group. The same was true forflavonols with an absorption maximum at 350 nm, however,the flavones showed the reverse trend. Overall, these resultssuggest an overall tendency to increasing amounts of pheno-lic compounds in the faster-growing younger needles.

With regard to phenols in the shoot axis (Table 1), it was evi-dent that catechin followed the trend found for the youngerneedles (5–15 mm); moreover, the absolute value was as high asthat found in these needles, whereas epigallocatechin and epi-catechin amounts were very low. Unidentified flavanols reachedvalues close to those of the younger needles. The flavone levelsof the shoot axis were rather low compared with those of theneedles, and this was paralleled by the flavonols. Regardinghydroxycinnamic acids, the amounts in the shoot axis were ashigh as in both of the younger needle groups (Table 2).

Drought period July 2010: Active growth

In July 2010 there were three short heatwaves after the expo-nential growth period had finished so that further rewateringwith rain did not result in new growth flushes. During Mayand June, weather conditions were within the normal range,and nuclear staining in T. baccata cells reached standard levels.A potentially meristematic cell cluster in May 2010 clearly haslight blue nuclei, indicating a low level of flavanols; however,this cluster was surrounded by non-meristematic cells staineddark blue, with flavanol-rich nuclei (Fig. 2a); the cytoplasmaround the light blue nuclei is completely white, and thereforecannot be distinguished. Magnification of a group of meriste-matic cell clusters demonstrates the distinctly differentiatedsubnuclear flavanol structures (Fig. 2b). The paler blue euchro-matic domains are massively overloaded with large patches andsmall spots of dark blue heterochromatin. The prominentnucleoli point to increased nuclear activity in five of the tencells, two of which are relative large.

As a further example of mosaic-like flavanol structures, acell lineage with four cells is shown (Fig. 2c). Compared withthe individual (post-lineage) cells of Fig. 1b, the lineage cellsare relatively similar in their nuclear flavanol expression. Thesize of the nuclei, relative proportions of cells and nuclei,staining density, and pattern of euchromatin and heterochro-matin distribution point to a clonal feature. In addition, asingle cell (Fig. 2c*) located outside the lineage clearly showsdistinct individual morphological characteristics that differfrom the lineage cells, i.e. larger cell size with a small nucleushaving dense and patchy flavanols. In the following example(Fig. 2d), two single, loosened nuclei have a similar appear-ance with regard to euchromatic structures, with moderateflavanols; denser chromatin spots are low in number.

Silencing of nuclei through drought-induced loss of flavanols

Upon drought stress in July, the nuclear flavanol patternchanged to a homogenous pale blue (marked with an aster-isk). This can be seen in elongated, mature palisade cells,containing densely packed starch grains (Fig. 2e); when mag-nified (Fig. 2f), the pale diffuseness is easily recognised. Manyof the mesophyll cells were yellow in colour, with a lightgreenish nucleus (Fig. 2g). Other needle sectors lack yellowcompounds and nuclei had already dissolved; the flavanols ofthese are diffusely distributed throughout the cells (Fig. 2h).In one case, diffuse blue nuclear remnants were more dis-tinct, indicating the original position of the nucleus (Fig. 2h*). Cells with dense vacuolar flavanols indicate that the con-tent of stored flavanols was not affected by drought, althoughthe nucleus had become pale blue (Fig. 2i*). Other cell types,such as starch storing cells, had the same pale blue nucleiwith a diffuse appearance (Fig. 2j*) and this was more pro-nounced in the following cells (Fig. 2k*): one of these nucleihas lost its rounded structure and the previously blue flava-nols had changed into reddish compounds (Fig. 2k*).

By late July, young fruits (arils) appeared as green-col-oured organs. Within the seed, the embryo still could not beobserved, but the developing integuments (Fig. 2l) showedactivated nuclei that were moderate to dark blue in colour,and the cytoplasm contained a number of fine-grained flava-nol spots with minimum space between them. From newly

Table 1. Concentrations (mg g)1 DW) of flavanols from post-rain flushes

(5–15-mm long) and mature needles (15–20-mm long). C, catechin, EGC,

epigallocatechin, EC epicatechin, UF, unknown flavanols. C, EGC and EC

were calculated using identical standards; UF was calculated as procyani-

din B2.

needle (mm) C EGC EC UF

5–10 8.8 1.5 1.9 9.9

10–15 8.3 1.3 2.4 10.2

15–20 4.0 0.4 1.1 4.7

Shoot axis (flush) 8.0 0.3 0.9 9.2

Table 2. Concentrations (mg g)1 DW) of hydroxycinnamic acids, flavones

and flavonols (needles as in Table 1). Hydroxycinnamic acids were calcu-

lated as chlorogenic acid, flavones as apigenin, and flavonols as rutin.

needle

(mm)

hydroxycinnamic

acids flavones flavonols

5–10 22.6 0.7 14.6

10–15 16.3 1.7 10.1

15–20 4.8 2.1 3.0

Axis 17.8 0.4 5.6



developing axillary buds to the youngest leaflets about 1 mmin length, dark blue nuclei appeared that were occasionallyenclosed in red-coloured cells (Fig. 2m). Red pigmentation ofthese leaflets is not the rule but can be more or less fre-quently observed.

The mature needles from the current-year growth sampledfrom August to October (2010) showed only moderate flava-nol staining (Fig. 2n) as compared with previous years, prob-ably due to the frequently cool weather conditions. The evenand diffuse nuclear flavanol pattern represents a metabolicallyrather silenced state.

In T. baccata Aureovariegata, a variety with prominent yel-low needles, fairly flavanol-rich nuclei developed duringspring in May 2010, as shown by the metaphase and telo-phase or the two interphase nuclei (Fig. 2o). Here, the paleand dark blue mosaic configuration is indicative of an active

meristematic cell cluster. However, during drought stress inJuly 2010, some needles showed an extreme yellow colour-ation and greenish nuclei, being a mixture of yellow andblue, and had nearly lost the flavanols (Fig. 2p*).

European drought period 2003

The extreme heat shock period in August 2003 caused severedamage symptoms in all Taxus trees. Data from three sampledates (2, 8 and 16 August) are documented in Fig. 2q,rand t.

The four green nuclei (Fig. 2q) are rather conspicuous,each having a large nucleolus, suggesting that the transcrip-tional state is perfectly synchronised. Under extreme droughtstress the short greenish interim state was replaced by a palegreenish-brown (Fig. 2r). During this dry period, two trees

a b c d

e

j

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Fig. 2. (a–t) Chromatin state maps for 2010 and 2003 resulting from blue staining of flavanols. n, as indicated in Fig. 1. (a–d) Active growth phase. (a)

Cluster of potentially meristematic cells (light blue) surrounded by dark blue non-meristematic cells. Dark interphase cells n, 7–8 lm. (b) Cluster of mitotic

cells showing variable staining of euchromatic and heterochromatic domains. Interphase cells n, 8 lm. (c) Lineage with four cells indicating a highly active

nuclear flavanol pattern and one single cell with a very compact cluster in the centre of the nucleus. Single cell n, 7 lm. (d) Pale blue interphase mesophyll

cells with different blue subnuclear clusters. n, 8 lm. (e–k) Drought period. (e) Palisade cells with starch and pale blue, diffuse nuclei. n, 8 lm. (f) Magnifi-

cation of such a pale blue nucleus reveals a very diffuse type of chromatin. n, 8 lm. (g) Distinct greenish nucleus in a yellow cellular environment. n,

7 lm. (h) Cells from spongy parenchyma with diffuse blue remnants of chromatin, somewhat more intense in one cell (*). (i) Large vacuole with flavanols

surrounding a pale nucleus. *n, 7 lm. (j) Starch cells with dissolving nuclei (*). (k) Three nearly dissolved nuclei (*) and blue-reddish patchy remnants of a

prior nucleus. (l–t) Controls and drought damaged nuclei. (l) Cells of an integument from a developing young fruit sampled in late August. Cytoplasmic

flavanols and four nuclei. n, 7 lm. (m) Young leaflets from an axillary bud sampled in late September. In the red-pigmented leaflets, are dark blue nuclei.

n, 6 lm. (n) Cells of the spongy parenchyma sampled in late September (staining pale to moderate blue). n, 8 lm. (o) Dark blue mesophyll nuclei from

T. baccata Aureovariegata sampled in early June. Rounded nuclei, n, 8 lm. (p) Yellow mesophyll cells of T. baccata Aureovariegata sampled on 15 July,

with greenish remnants of dissolved nuclei (*). (q) Greenish, but still intact, nuclei with large nucleoli sampled at the beginning of the drought period in

August in 2003. Nucleoli 4 lm in diameter. (r) Greenish remnants of nuclei (*) sampled on 28 August 2003. n, 7 and 8 lm. (s) Irrigation of trees during

the drought period of August led to bright blue nuclei. Most elongated nucleus = 20 lm. (t) Examples of a green nucleus (*) and of severely damaged,

oxidised nuclei, still with nucleoli, sampled 28 August 2003. Small, rounded nucleus = 6 lm.



were irrigated, which resulted in bright blue stained, flavanol-rich nuclei (Fig. 2s).

As a final symptom of drought stress in 2003 (Fig. 2t), fewdays after extreme temperatures of up to 36 �C (16 August)brown-coloured nuclei appeared in sun-exposed needles,however, these still each had a large nucleolus. One nucleusremained green (Fig. 2t below*), indicating significant differ-ences in individual nuclei, even if located directly adjacent.

Different interactions between catechin and modified H4-corepeptides

Peptides were synthesised that represent the fragment 71–85 ofthe H4-core protein (T71YTEHAKRKTVTAMD85). The lysineamino acids of this fragment were acetylated (ac), methylated(me) and formylated (form) at the e-positions (Table 3). Inorder to detect possible interactions between these peptidesand catechin, the method of Feucht et al. (2009) was applied.In this paper, the degradation behaviour of catechin was inves-tigated with UV-VIS spectroscopy in dependence on time inthe presence of (complete) histone proteins. In analogy to thisprocedure, the prepared H4-core fragments were dissolved insolutions that contained 0.1 m Tris buffer (pH 8.0; 20.0 �C;3.3% ethanol) and 10 mm MgCl2 gassed with oxygen and,thereafter, the reaction spectra were recorded.

The plot A434 nm versus time, indicating the degradationbehaviour of catechin in the presence of different H4-corefragments is shown in Fig. 3. Six curves are presented: twobelong to the non-modified H4-core fragment (15 aminoacids) and to the modified H4-core fragment (15 aminoacids, me) and closely coincide, while the other four curvesare close together. When no peptide is dissolved in the buffersolution a relatively rapid degradation of catechin can beobserved, as shown in Fig. 3 by the points belonging to ‘nopeptide’, which represent two measurements. Here, nearly allpoints lie on the same curve (barely distinguishable inFig. 3). In the presence of the ‘pure’ H4-core fragment andthe methylated peptide, however, the degradation is clearlydelayed. In contrast to this behaviour, catechin is degradedmore quickly when the H4 peptide is acetylated or formylat-ed. Obviously, catechin degradation is dependent on themodification pattern of the amino acids, which is characteris-tic for the epigenetic histone code.

DISCUSSION

Growth period 2007: Loss of nuclear flavanols and meristematicactivity as a result of drought

In spring 2007, there was a complete loss of the bluestaining nuclear flavanols because of extended dry condi-

tions. However, the nuclei still contained yellow fluorescingflavonoids of the quercetin type (Feucht et al. 2008), a fla-vonol that associates weakly with histones (Polster et al.2006). Drought-induced loss of photosynthesis affectsreprogramming of gene expression (Zhan et al. 2006), anddown-regulation of the photosynthetic machinery is appar-ently linked with a loss of nuclear flavanols. The carbohy-drate depletion caused by drought (Choinski & Johnson1993) can be experimentally overcome by supplyingsucrose, resulting in a re-establishment of flavanol synthesis(Lux-Endrich et al. 2000). Sucrose also acts specifically as asignalling molecule (Rolland et al. 2002; Koch 2004) and isinvolved in expressing gene-encoded enzymes for the syn-thesis of flavonoids and anthocyanin (Solfanelli et al.2006).

Moderate to medium levels of nuclear flavanols are involved inproliferative activity

Following Alvarez et al. (2008), the concentration of root cyt-okinins is increased in maize under drought conditions.Hence, after sporadic rain root-stored cytokinins moveupwards via the xylem (Itai & Vaadia 1971), and in the caseof Taxus, probably into the new leaf flushes that form singletracheary elements. This exceptional type of differentiation isthought to be dependent on cytokinin (0.2 lm benzylade-nine) and auxin (0.5 lm), as documented for Zinnia (Fukuda& Komamine 1980).

Epigenetic factors induced by drought or heat lead to spe-cific gene expression patterns through transcriptional activa-

Fig. 3. UV-VIS spectroscopic kinetics of catechin reaction in the presence

of H4-core peptides. Absorbance–time (A434 versus t) curves of catechin

degradation (0.2 mg catechin m)1, 0.69 mM) in the presence of H4-core

peptides (about 1 mg ml)1) with different modified lysine residues

(20.0 �C, 0.1 M Tris buffer pH 8.0, 3.3% ethanol, 10 mM MgCl2, gassed

with O2). me = both lysine residues monomethylated, ac = both lysine res-

idues acetylated, form = both lysine residues formylated. The non-modi-

fied and methylated peptides inhibit catechin degradation more effectively

than the acetylated and formylated peptides.

Table 3. H4-core peptides with different modified lysine residues used for

catechin kinetics.

sequence M (g mol)1)

weight

(mg ml)1)

TYTEHAKRKTVTAMD-NH2 1752.61 1.0

TYTEHAK(me)RK(me)TVTAMD-NH2 1779.46 1.0

TYTEHAK(ac)RK(ac)TVTAMD-NH2 1835.84 1.1

TYTEHAK(form)RK(form)TVTAMD-NH2 1807.45 1.0



tion of genes (Hwang & Sheen 2001). Apparently, after rewa-tering and subsequent shoot flushing, a set of genes producenew metabolic conditions leading to flavanol loading ofnuclei and renewed mitosis. Such a link between cytokininand catechin has already been described in Taxus (Polster etal. 2006; Feucht et al. 2008). The key aspect is that onlymoderate to medium levels of nuclear flavanols induce thecorrect conditions for cell proliferation, as indicated by thehistology. Callus cultures of grapevine display growth promo-tion by catechin even on auxin-free media (Feucht et al.1998).

Experimentally, acetylation of isolated histones leads to alowered affinity for flavanols resulting in less blue colour inthe nuclei (Feucht et al. 2008). Acetylation results in structur-ally open, transcriptionally active histones (Grewal & Moazed2003), and multi-acetylation of histones induces resumptionof rapid growth and release from dormancy (Law & Suttle2004). Using HPLC, it was shown that the flavanol concen-trations were relatively high in the young Taxus needles of 5–15 mm in length. The values for catechin and epicatechinwere twice those of fully expanded needles, and in the case ofepicatechingallate, the difference was nearly four-fold. Thusthere is a relationship between high growth activity and ele-vated levels of flavanols.

Overloading of nuclear flavanols

When excessive amounts of flavanols occupied nuclei ofpost-rain flushes, the chromosomal structures of Taxus werebadly affected, showing severe distortion and compaction.Higher-order chromatin domains underwent a dramaticclumping of nucleosomes, and such a state has functionalconsequences for most processes that involve DNA (Mazum-dar & Misteli 2005). Obviously, the massive and patchy flava-nol clumps exceeded the nuclear properties to organise anorderly pattern of the chromosomes during mitosis (Fig. 1sand t). Mis-segregation of chromosomes was linked to for-mation of heterochromatins (Richards & Elgin 2002) andnucleosome clumping prevented correct transcription (Fran-cis & Halford 2006). The space between the two daughtercells (Fig. 1u) was occupied by diffuse flavanols, which is anunusual feature, and it is doubtful whether a new cell platecan form in such a situation.

Growth periods 2003 and 2010

Needle expansion during spring in both 2003 and 2010 wasregular due to normal environmental conditions. An over-view clearly reveals two groups of nuclei, one dark blue andone pale blue (Fig. 2a). Different levels of chromatin packag-ing indicate different accessibility of DNA to proteins (Rich-ards & Elgin 2002). Dense packaging leads to a silencedconformation in the dark-coloured nuclei, which means thatthe histones are hypo-acetylated and at the same time highlymethylated (Richards & Elgin 2002). The drought periods in2003 and 2010 led to a loss of nuclear flavanols, as anotherform of gene silencing.

When the blue staining, meristematic cell groups are exam-ined more in detail (Fig. 2b,c and d), a subnuclear differenti-ation of the flavanols was evident. The most prominentfeatures of spatial patterning between euchromatin and het-

erochromatin confirm that meristematic activity is character-ised by a mosaic-like differentiation of the chromatinstructures. The opening of chromatin is related to a weaken-ing of the histone–DNA interaction, which facilitates tran-scription (Kouzarides 2007). In the white space, between theblue chromatin, there are obviously non-histone proteins, e.g.high mobility proteins (HMG), which play a prominent rolein nuclear activities.

As a fundamental difference, drought in 2010 and 2003occurred in midsummer, as compared with the springdrought of 2007. Hence, post-rain flushes and formation ofsingle tracheary elements, typical for 2007, did not develop inthe former years. Somewhat intriguing is the appearance ofnucleoli in the already dark brown ‘damaged’ nucleoli (Fig. 2t). A large nucleolus is commonly related with high ribo-somal activity towards protein synthesis. Probably, there isstill high nucleolar activity because in such a stress situationthere is an enforced mobilisation and recycling of molecules(Baena-Gonzalez & Sheen 2008).

Stability of catechin in the presence of modified H4-corepeptides

Magnesium ions play an important role in condensing chro-matin and magnesium concentration can vary from 3.0 to17.0 mm in mammalian nuclei (Strick et al. 2001; Treme-thick 2007). Therefore, MgCl2 was added to the experimen-tal buffer solution at a concentration of 10 mm (see Fig. 3).The oxidation of catechin is complex and leads to a prod-uct with maximum absorbance near 440 nm (0.01 m phos-phate buffer, pH 7.5), indicating an o-quinoic catechinstructure (Jimenez-Atienzar et al. 2004). Similar absorptionbehaviour was also found under the experimental conditionsused in Fig. 3. Thus, an o-quinoic product might also beproduced here. An oxidative degradation of catechin is pre-vented to a large extent (during a tested time period ofabout 15 h) when no fragments except the complete histoneproteins are added to the buffer solution (Feucht et al.2009). Fragments of histones obviously also can potentiallydefend catechin from oxidation, although to a less degreethan complete histone proteins (see Fig. 3). It is well docu-mented that the H4-core protein can be acetylated at thelysine position K77 and K79 (Zhang et al. 2003; Hyland etal. 2005) and methylated at position K79 (Hyland et al.2005; Robin et al. 2007) inside the sequence TY-TEHAK(77)RK(79)TVTAMD; formylation can also takeplace at position K77 (Wisniewski et al. 2008). Thus, asmall range within the H4-core protein is subject to differ-ent biochemical modifications.

Complex chromatin functions are substantially regulatedthrough epigenetic mechanisms. Even the associationbehaviour of relatively small molecules, as for example cat-echin, to histone proteins seems to be dependent on theepigenetic histone code, as can be postulated from theresults in Fig. 3. Smaller segments of the H4-core proteinalso support this hypothesis, e.g. the peptide HAKRKT (H4fragment 75–80) also significantly inhibits the oxidativedegradation of catechin under the experimental conditionsof Fig. 3, whereas the peptide HAK(ac)RK(ac)T shows lessdistinct inhibition (results not shown). These kinetic resultsseem to be connected with those of Fig. 1, showing differ-



ent flavanol charges in nuclei. Not only the amount offlavanols seems to determine the flavanol accumulationon chromosomes, but also the epigenetic histone code.It can be speculated that the varying association of flava-nols (and perhaps flavonols) to the histone octamer com-plexes around which the DNA is rewound may alsoinfluence the epigenetic code of DNA (methylation patternof DNA) and thus the transcription (including RNA inter-ference) and, furthermore, the regulation of chromatinremodelling.

Final remarks

This paper demonstrates that excessive accumulation ofnuclear flavanols resulted in a chaotic, repressive state ofnuclear structures, which block further mitosis. In a broaderbiological context, epigallocatechin-gallate (EGCG) was foundto inhibit cell cycle progression in the G1 phase in humanhepatoma cells (Huang et al. 2009). In Taxus baccata such afundamental action is obviously also performed by themonomeric catechins.

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Loss of nuclear flavanols during drought periods in Taxus baccata