Photosynthesis Research 49: 169-181, 1996. @ 1996 Kluwer Academic Publishers. Printed in the Netherlands.

Regular paper

Photosynthesis in the basal growing zone of barley leaves

M a r g a r e t e Ba i e r 1 , W o l f g a n g Bi lge r 1 , R a i n e r W o l f 2 & K a r l - J o s e f D ie t z 1,* 1Julius-von-Sachs-Institut fiir Biowissenschaften der Universitiit, Mittlerer Dallenbergweg 64, 97082 Wiirzburg, Germany; 2Biozentrum der Universitiit, Am Hubland, 97082 Wiirzburg. Germany; *Author for correspondence and~or reprints

Received 3 October 1995; accepted in revised form 15 July 1996

Key words: basiplast, carotenoids, chlorophyll, chloroplast development, gene expression, Hordeum vulgare

Abstract

Cell proliferation, elongation, determination and differentiation mainly take place in the basal 5 mm of a barley leaf, the so-called basiplast. A considerable portion of cDNAs randomly selected from a basiplast cDNA library represented photosynthetic genes such as CP29, RUBISCO-SSU and type I-LHCP II. Therefore, we became interested in the role of the basiplast in establishing photosynthesis. (1) Northern blot analysis revealed expression of photosynthetic genes in the basiplast, although at a low level. Analysis of basiplasts at different developmental stages of the leaves revealed maximal expression of photosynthetic genes during early leaf development. The activity of these genes shows that plastid differentiation involves the development of the photosynthetic apparatus even at this early state of leaf cell expansion. (2) This conclusion was supported by the fact that chlorophylls and carotenoids are synthesized in the basiplast. The qualitative pattern of pigment composition was largely similar to that of fully differentiated green leaves. (3) The transition from proplastids to chloroplasts progressed in the basal 5 mm of the leaf, so that the number of grana lamellae per thylakoid stack increased with distance from the meristem from zero to about five. (4) Photosynthetic function was studied by chlorophyll a-fluorescence measurements. In dark-adapted 8-day-old primary leaves, the fluorescence ratio (Fp - Fo)/Fp was little decreased in basiplasts as compared to leaf blades. During steady state photosynthesis, the ratio (FM' -- Fo)/FM' was high in the leaf blade (0.5), but low in the sheath (0.25) and in the basiplast (0.18), indicating the existence of functional, albeit low light-adapted chloroplasts in the basiplast. (5) Further on, chlorophyll a fluorescence analysis in relation to seedling age revealed efficient photosynthetic performance in the basiplast of 3- to 6-day-old seedlings which later-on differentiates into leaf blade as compared to the basiplast of 7- to 12-day-old seedlings which develops into leaf sheath and finally ceases to grow. The leaf age dependent changes in basiplast photosynthesis were reflected by changes in pigment contents and LHCP II expression both of which also revealed a maximum in the basiplast of 4-day-old seedlings.

Abbreviations: bas 1-basiplast-associated gene 1 encoding a peroxide reductase; cab-chlorophyll a/b binding protein; CP 29 - 29 kDa chlorophyll binding protein; DIG - digoxigenin; EMIP - epidermal major intrinsic protein; LHCP II - light harvesting complex of Photosystem II; L S U - large subunit of Rubisco; NPQ- non photochemical chlorophyll a fluorescence quenching; PS I/PS II - Photosystem I/II; P Q - photochemical chlorophyll a fluorescence quenching; Rubisco - Ribulose- 1,5-bisphosphate carboxylase; SSU - small subunit of Rubisco

Introduction

As is typical for monocots, almost all cells of barley leaves derive from a meristem located at the base of

the leaves. In subsequent zones the meristematic cells expand and develop into morphologically and physio- logically distinct cell types such as epidermis, meso- phyll and cells of the bundle sheath. Photosynthesis

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is the leaf's predominant physiological function. The development of the photosynthetic machinery depends on a dramatic specialization of cells and plastids, which is based on a complex interaction of nuclear and plas- tidic genes (Mullet 1993).

In barley primary leaves, differentiation of the mesophyll is accompanied by an increase in plastid number per cell from approximately 10 to 65 (Mullet 1988). The small proplastids present at the leaf base with a diameter of 1-2 #m are enlarged 4- to 6-fold and develop into disk-shaped chloroplasts (Robertson and Laetsch 1974; Mullet 1988). According to Mullet (1988), chloroplast development in barley leaves may be divided in five phases: (1) Plastid number remains low for approximately one day after cessation of cell division. All plastids are still similar to proplastids (Dannenhoffer and Evert 1994). (2) In the cell elon- gation zone which is 2 to 3 cm long (Baumgartner et al. 1989) the plastid number per cell increases (Baum- gartner et al. 1989; Boffey et al. 1982). Plastid volume remains rather unchanged (Mullet 1988). Plastid tran- scription is stimulated (Baumgartner et al. 1989) and carotenoids and chlorophylls accumulate (Boffey et al. 1980). (3) Plastid transcriptional and translational activity is high (Baumgartner et al. 1989), and the bulk of the thylakoid membranes is synthesized (Dannen- hoffer and Evert 1994). (4)Plastids are photosyntheti- cally fully active in the mature leaf. (5) Degradation of plastids takes place during leaf senescence (Wardley et al. 1984).

The developmental changes in plastid morphology are accompanied by the assembly of the photosynthet- ic machinery (Dannenhoffer and Evert 1994; Webber et al. 1984). This involves an enhanced and coordinat- ed expression of plastidic and nuclear genes (Mullet 1988; Rapp et al. 1992; Ougham and Davies 1990). In contrast to the model suggested for plastid develop- ment in barley (Mullet 1988), photosynthetic activity was shown to start early in leaf development of vari- ous Gramineae, for instance maize, wheat and Lolium temulentum. Measurements of gas exchange (Baker and Leech 1977; Gay and Thomas 1995), chlorophyll a fluorescence induction kinetics (Webber et al. 1984), and light induced acidification of the suspension media of thylakoids (Webber et al. 1986) demonstrated that chloroplasts are photosynthetically active even in the early phases of leaf and plastid development.

In this work we analyse gene expression, pigment composition, chloroplast ultra structure, and chloro- phyll a fluorescence kinetics in order to gain addi- tional and refined information on photosynthesis in

the basal growing zone of barley leaves. We think that it is necessary to determine all the parameters simultaneously in one type of identical leaf material in order to avoid a comparison of data which are at least sometimes incompatible for reasons of species specificity or with respect to variation in growth con- ditions. The questions addressed in the paper are: (1) Is there a correlation between expression of various nuclear and plastome encoded photosynthetic genes and the functionality of the photosynthetic apparatus? (2) Is the composition of photosynthetic pigments sub- jected to only quantitative or also major qualitative changes during early plastid development? How are these changes related to photosynthetic activity? (3) During leaf development, the basiplast develops into leaf blade and later on into leaf sheath. Is this devel- opmental transition reflected by differences in plastid development in the basiplast?

Materials and methods

Plant growth and preparation of lea f fractions

Barley (Hordeum vulgare L. vat. Gerbel) seeds were germinated in vermiculite saturated with distilled water under controlled environmental conditions (14 h: 20 °C; 10 h 18 °C) for 2 days in darkness. Seedlings were transferred to hydroponic culture with a modi- fied nutrient solution after Hoagland and Arnon (1938) containing 1.25 mM KNO3, 1 mM KC1, 1.5 mM Ca(NO3)2, 32 #M Fe-EDTA, 750 #M MgSO4, 375 #M KH2PO4, 68.9 #M H3BO2, 13.7 #M MnCI2, 1.5 #M ZnSO4, 0.5 #M CuC12, and 0.2 #M Na2MoO4 and illuminated under a controlled light/dark cycle (light: 14 h, 170 #mol quanta m -2 s -1, 20°C; dark: 10 h, 18 °C) (Brune et al. 1994). The residual caryopsis, the coleoptile and the secondary leaf were gently removed from 2 to 14 day old seedlings. The basal 5 mm of the primary leaf were taken as one section and will be referred to as basiplast in the following. The upper part was cut into 1 cm sections; or fractionated into basi- plast (BP), lower sheath (PC1), upper sheath (PC2), lower part of the blade (PB), middle part of the blade (PM) and leaf tip (PS).

Isolation of nucleic acids from plant material

Plant tissue was ground to a fine powder in liquid N2 and extracted in a buffer (2.5 ml buffer / g tissue) containing 25 mM EDTA, 25 mM EGTA, 100 mM

Tris-HC1 (pH 8.5), 100 mM 13-mercaptoethanol and 2% (v/v) SDS in the presence of phenol (2 ml / g tissue) and chloroform (1 ml / g tissue). After repeated extrac- tion of the aqueous phase with equal volumes of phe- nol/chloroform (1:1) followed by chloroform, nucleic acids were precipitated by addition of one volume iso- propanol. After dissolving the pellet in water, nucleic acids were precipitated with 2.5 volumes ethanol and 1/10 volume 3 M sodium acetate and resuspended in water. Nucleic acids were quantified spectrophotomet- rically.

For cDNA synthesis, ribonucleic acids were puri- fied by differential precipitation with 2-butoxyethanol as described by Manning (1991). Polyadenylated mRNA, was then enriched using the 'PolyATtract mRNA Isolation System' (Promega, Madison, USA) according to the supplier's protocol.

Synthesis of DIG-labeled RNA and radiolabeled DNA probes

cDNA harbouring plasmid (CP29, LHCP, SSU) equiv- alent to 1 #g was cut downstream of the cDNA- insert by restriction endonucleases and then used as template to synthesize digoxigenin-labeled RNA probes. Strand-specific transcription was performed in 40 mM Tris-HC1 (pH 8.0), 8 mM MgC12, 50 mM NaC1, 2 mM spermidine, and 30 mM DTT with T3- RNA polymerase at 37 °C for 1 h. Nucleotides were added as 'DIG RNA labeling mixture' (2 #1 / 25 #1) (Boehringer, Mannheim, Germany). Synthesized RNA was stabilized with 10 U RNasin (Promega, Madison, USA). Following transcription, the DNA-template was removed by digestion with RNase-free DNase. Ran- dom primed labeling was performed with DNA repre- senting fragments of the maize plastome (DI: pZmc 427, Larrinua et al. 1983, LSU: pZmc 460, Rodermel and Bogorad 1985; cytochrome b559:850 bp-Bam HI fragment, Larrinua et al. 1983) and with a cDNA-clone of triosephosphate isomerase (Marchionni and Gilbert 1986). DNA equivalent to 200 ng was denatured at 95 °C for 10 rain followed by addition of 2 #1 hexanu- cleotide mixture (Boehringer, Mannheim, Germany), dATP, dTTP, and dGTP (Pharmacia, Heidelberg, Ger- many) to final concentrations of 0.5 mM each, 30 #Ci [a32p]-dCTP (Amersham, Braunschweig, Germany), and 3 U Klenow fragment (Boehringer, Mannheim, Germany). The labeling reaction was terminated by passage of a 'Sephacryl S-300 HR MicroSpin' col- umn for nucleotide removal (Pharmacia, Heidelberg, Germany).

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Northern blot analysis

After separation of 20 #g RNA/lane in 1% formamide containing agarose gels (Sambrook et al. 1989) nucleic acids were blotted onto Nylon membranes (Hybond- N, Amersham, Braunschweig, Germany) by capillary transfer using 20 x SSC (Sambrook et al. 1989). 0.5- 1 #g DIG-labeled RNA probes were hybridized to the blots in 50% (v/v) formamide, 6 x SSC, 2 x Denhardt's solution, 0.1% (w/v) SDS and 2.5 mg torula-yeast RNA (Sigma, Deisenhofen, Germany) at 60°C for 18 h. Non-specifically bound probe was removed by three repetitive washes in 0.2 x SSC, 0.1% (v/v) SDS at 60 °C for 30 min each. Detection was performed with the 'DIG nucleic acid detection kit' (Boehringer, Mannheim, Germany) according to the supplier's pro- tocol.

Northern blot hybridization with radiolabeled probes was performed in 4x SSC, 3 x Denhardt's solu- tion and 0.5% (w/v) SDS and 0.5 mg salmon sperm DNA (Boehringer, Mannheim, Germany) at 65 °C for 18 h. Following hybridization, the blots were washed in 2xSSC, lx SSC, and 0.5x SSC containing 0.5% (w/v) SDS at 65 °C for 30 min each. Kodak XOmat AR films were developed after exposure to the membranes for 2 and 18 h.

Isolation of cDNAs from a AUni-ZAP-library

A AUni-ZAP-cDNA library was constructed from polyadenylated mRNA of barley basiplasts of 6-8-day- old seedlings using the 'ZAP cDNA Synthesis Kit' (Stratagene, La Jolla, USA). 100 phages were ran- domly selected. The cDNA harbouring pBluescript- SK plasmids were excised in vivo as described in the 'ZAP-cDNA Synthesis Kit' protocol (Stratagene, La Jolla, USA). They were amplified in E. coli TG 1 host cells and purified by alkaline lysis. Restriction diges- tion was performed with Eco RI and Xho I (Boehringer, Mannheim, Germany). DNA-fragments were seperat- ed in 1%; (w/v) agarose gels in Tris-acetat buffer (40 mM Tris-Acetat (pH 7.5), i mM EDTA) (Sambrook et al. 1988).

Sequence analysis

Dideoxy sequencing was performed with the 'T7 Sequencing Kit' (Pharmacia, Uppsala Sweden) using 35S-dATP as radiolabel (Amersham, Braunschweig, Germany) as described in the supplier's protocol. The oligonucleotides used as primers were synthesized by

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Figure 1. mRNA levels of nuclear (LHCP II, CP29, SSU, triosephos- phate isomerase) and plastid (D1, Cyt559, LSU) encoded photosyn- thetic genes in leaf blade (PM), leaf sheath (PC), basiplast (BP) and roots (R) in comparison to basl. Northern blot analysis was performed with 20 #g total RNA per lane.

Roth (Karlsruhe, Germany) or MWG (Ebersberg, Ger- many). Sequence comparisons were conducted using the online services offered by EBI Hinxton (UK) and the National Institute of Health (USA).

Pigment pattern

Chloroplast pigments were determined by HPLC (Bil- ger et al. 1995) after modification of a method pub- lished by Gilmore and Yamamoto (1991). Leaf tissue was frozen in liquid N2 and stored at - 8 0 ° C until extraction. 0.5 mg of tissue was extracted by grind- ing in a mortar or with a potter at room temperature after addition of 0.3 ml 87% acetone. The mortar was subsequently rinsed with 100% acetone essentially as described in Thayer and Bj 6rkman (1990).

Chlorophyll a-fluorescence measurements

Chl a-fluorescence of leaves was measured using a PAM 101 (Walz, Effeltrich, Germany). To increase

sensitivity and decrease fluorescence background which was not originating from leaf chlorophyll, quartz glass extension rods were placed between sample and glass fibers. The fiber for excitation light was separat- ed from the fiber connected to the detection unit (see Figure 1 in: Schreiber et al. 1995; Schreiber 1994). Both excitation and detection were performed at an angle of 45 ° in respect to the plant tissue surface, and at an angle of 90 ° between excitation and measur- ing pathway. For fluorescence analysis, dark-adapted plant material was illuminated at a photon fluence rate of 1150 #mol quanta m -2 s -1 (Fp). An additional pulse of 1200 #mol quanta m -2 s -1 of 1 s duration was applied immediately after the beginning of illu- mination. The fluorescence yield during the pulse did not exceed the initial fluorescence yield Fp suggesting that all reaction centers had been closed at Fp. The CO2 concentration was about 400 #1 1-1. The fluo- rescence parameters were also measured during steady state photosynthesis after illuminating the samples for 8 min with actinic light. Photosynthesis was activated at 1150 #tool quanta m -2 s -1 for 2.5 min. Measure- ments were then performed at 170 #mol quanta m -2 s -1 after illumination times of more than 5 min. Light pulses of an intensity of 1200 #tool quanta m -2 s -~ and of 1 s duration were applied every 60 s to transient- ly determine maximal fluorescence FM'. Calculations were performed as described by Schneiber and Bilger (1993).

Electron microscopy

Plant material was fixed for one hour in 50% Karnowsky solution containing 2% formaldehyde and 2.5% glutaraldehyde in 50 mM sodium cacodylate buffer (pH 7.2) and postfixed in 50 mM sodium cacody- late buffer (pH 7.2) supplemented with 2% OsO4 for 3 h. Before and after the subsequent overnight incuba- tion in 2% aqueous uranyl acetate the leaf sections were washed five times in water for 3 min each. Dehydration of the tissue in ethanol was followed by transfer into the intermedium propylenoxide (3 x 30 min). For embed- ding, the tissue was first incubated in propylenoxide / epon (1:1) overnight and then in epon (2 × 2 h + 1 h). Epon was prepared by mixing 4 volumes of solu- tion A (97.1 g Epon 812 + 130.8 g dodecenyl succinic anhydride) with 3 volumes solution B (90 g Epon 812 + 81.37 g methylnadic anhydride) and adding 1.5-2% (v/v) dimethyl amino methyl phenol.

Ultrathin cross sections were stained in 2% uranyl acetate in methanol for about 15 rain followed by 50%

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Reynolds' lead citrate solution (Reynolds 1963) for 10 rain. Sections were partially destained by consecutive rinsing the grids in methanol, methanol /H20 (1:1) and H20. Electron microscopy was performed using a Zeiss EM 900 at 80 kV.

Results

Genes involved in photosynthesis are expressed in the basiplast

To characterize gene expression in the basal grow- ing zone of monocotyledoneous leaves, we randomly selected 100 cDNA clones of a basiplast cDNA library, 29 of which were partially sequenced. Three cDNAs were found to encode proteins involved in photosyn- thesis as deduced from sequence homology or iden- tity. The results suggested that photosynthetic genes are expressed in the basiplast. A clone named HvB069 (Hordeum vulgare basiplast, clone 69) contained exons 4 to 6 of the gene for the 29 kD chlorophyll-binding protein (CP29) reported by Sorensen et al. (1992) (data not shown). HvB036 coded for a yet unknown member of the gene family of the small subunit of ribulose-1,5- bisphosphate carboxylase (data not shown). Nucleic acid and deduced amino acid sequences gave evidence that HvB055 (EMBL Ac. Nr. X89023) encodes the yet unknown barley homologue of a wheat cab gene pub- lished by Lamppa and coworkers in 1985. Based on this observation, it seemed interesting to understand the function of photosynthetic genes in the basiplast.

The expressional pattern of these and other photo- synthetic genes (psbA: D1 protein; psbE cytochrome- b559; rbcL: RUBISCO large subunit; triosephosphate isomerase) was analyzed by Northern blot hybridiza- tion. Expression in the basiplast was detected for all probes (Figure 1). However, as expected, transcript amounts were much higher in leaf blades. RNA lev- els were on an intermediate level in the leaf sheath and not detectable in roots. The distribution of mRNA among tissues was similar for LHCP II and CP29. As compared to leaf blade and sheath expression of SSU was particularly low in the basiplast. Expres- sion of the plastome-encoded genes psbA, rbcL and psbF, followed the same pattern, mRNA-levels of the triosephosphate isomerase gene were maximal in roots, still high in the leaf blade and low in the sheath and basiplast. As a control we performed Northern- experiments with probes not related to photosynthe- sis. Basl encodes a 2-Cys peroxiredoxin (Baier and

Dietz 1996) and was maximally expressed in the basi- plast (Figure 1). Similar results were observed for subunit E of the vacuolar H+-ATPase (Dietz et al. 1995) and for emip which encodes a plasma membrane water channel (Hollenbach and Dietz 1995) (results not shown). The latter results demonstrate that the basi- plast is characterized by high transcriptional activity of non-photosynthetic genes.

The expression of photosynthesis-related genes was also studied during ageing of the basiplast (Figure 2). Highest amounts of all transcripts were found in the basiplast of 4-day-old plants. During leaf develop- ment, tissue derived from the basiplast until day 6 to 7 after germination differentiates into leaf blade. LHCP II expression or RNA stability decreased after day 6. This coincides with the transition from formation of leaf blade to formation of leaf sheath. The leaf sheath is characterized by low chlorophyll contents and low photosynthetic activity. Expression of LHCP II was low at day 2 due to etiolation of the seedlings which were germinated in the dark for two days before being transferred to the light (Figure 2). The mRNA level detected at day 2 corresponds to the light-independent background expression of cab genes which was similar to that in leaf blades of 8-day-old etiolated plants (data not shown). In contrast to the increased expression of LHCP II, mRNA amounts were high from the begin- ning for cyt b559, Dl-protein and Rubisco subunits and only slightly lower than at day 4. It is interest- ing that mRNA levels of D 1-protein and Rubisco-SSU remained detectable even after the primary leaf had ceased growing 10 days after sowing.

Pigment composition of the basiplast as compared to older leaf segments

Detection of gene expression of chlorophyll binding polypeptides prompted us to analyze the pigment com- position of the basiplast. Chlorophylls and carotenoids were determined in various fractions of the leaf. Total pigment contents of the basiplast as related to leaf fresh weight was only 1.2% of the leaf blade (Figure 3A). However, the qualitative composition of pigments was very similar in the basiplast, leaf sheath and leaf blade (Figure 3B).

Pigment composition in basiplasts during organogenesis

As the leaf develops, the basiplast first forms cells which differentiate into leaf blade (day 1-6) and later-

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Figure 2. mRNA levels of photosynthetic genes in relation to seedling age. The basal 5 mm zone of barley seedlings was harvested between day 2 and day 12; isolated total RNA equivalent to 20/zg was electrophoretically separated and probed with DIG-labeled antisense RNA of HvB055 (LHCP) or basl, or with radiolabelled DNA-probes (all other genes).

on leaf sheath (day 7-12) before it ceases growing. We analyzed the pigment composition during leaf organo- genesis. Chlorophyll a and b revealed highest levels in the basiplast of 4-day-old seedlings (Figure 4A). Of the carotenoids, only neoxanthin followed the same pattern of age-dependent change as the chlorophylls, whereas a- and /3-carotene were rather unchanged between day 2 and 4 and then decreased as the leaf developed. In contrast, lutein and the xanthoplhyll cycle pigments (V+A+Z) were highest in the basiplast of very young tissue and then continuously declined during aging of the seedling.

Six days after sowing, levels of all analysed pig- ments were low in the basiplast. From that time on, cells derived from the basiplast form the leaf sheath. Figure 4B depicts the relative pigment composition of basiplasts in dependence of seedling age. In 2-day-old seedlings carotenoids made up almost 70% of the pig- ments. This relative portion declined to less than 20% in the basiplast of 8-day-old plants.

The development of chloroplasts in the basiplast

The results presented in the previous sections demon- strate that at least part of the molecular basis for chloro- plast development is realized in the basiplast. There- fore, we analyzed the structural differentiation of the plastids within the basiplast. Ultrathin sections of basi- plasts from 5-day-old barley seedlings were examined in 1 mm intervals. Analysis was focused on plastid size and thylakoid formation (Figure 5). Within the 5 mm region defined as basiplast, longitudinal extensions of plastids increased about twofold from 0.87 4- 0.05 #m to 1.76 4- 0.04 #m in cross sections. Plastids initial- ly contained up to 4 stroma thylakoids but no grana stacks. Grana stacking started 3 mm above the leaf base. At the upper end of the basiplast stacks of upto 4 grana thylakoid layers were observed. Plastid divi- sion started within the 2nd and 3rd millimeter. Thus, plastid division followed cell division in close spatial and temporal sequence. The largest number of dividing plastids was counted in the 3rd mm of the basiplast.

(a) t500

1000 m O

E 500

0

~. 20o-

o 100- E r-

0- 100

o 5 0 E e-

Chlorophyll a Chlorophyll

L u t e i n c~ + I~ - C a r o t e n e

PS PM PB PC2PC 1BP PS PM PB PCzPCIBP

(a) 80

~ 6O

~ 40 C

20

0

30

m 20

E e-

l 0

175

V + A ~ Z

L u t e l n

z + e - C | r o t e n e Neoxanthln

2 4 6 8

Figure 3. Absolute (A) and relative (B) pigment contents in various leaf fractions of barley seedlings.

5 mm above leaf base, cells contained already a full set of chloroplasts (55-75 chloroplasts/cell: Klein and Mullet 1986; Robertson and Laetsch 1974; Baumgart- ner et al. 1989) with a very homogeneous thylakoid system. 21 plastids were distributed in a typical cross section of a subepidermal mesophyll cell at 5 mm dis- tance from the leaf meristem (data not shown). Plastid size varied from 1.2 #m to 2.4 #m. They contained grana stacks of 2 to 4 thylakoids. This compares to fully differentiated chloroplasts in the leaf blade with a size of 6 to 8 #m (Mullet 1988;) and with complex organisation of stroma lamellae and many grana thy- lakoids per stack (Eriksson et al. 1961).

Figure 4. Absolute (A) and relative (B) pigment contents in basiplasts of 2- to 8-day-old barley seedlings.

Photosynthetic activity in the basiplast

In order to complement the molecular and structural analysis we investigated photosynthetic activity in the basiplast by means of sensitive chlorophyll a fluores- cence measurements and calculation of PS II quantum efficiency. For comparison measurements were repeat- ed along the axis of complete 8-day-old primary leaves. It should be noted that fluorescence measurements in the basiplast where pigment contents were less than 2% of the leaf blade require a highly sensitive setup

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Figure 5. Ultrastructural development and mean sizes of plastids in the basiplast of 5-day-old seedlings. The size bar corresponds to 1 ttm. The basiplast of 5-day-old primary leaves was fractionated in 1 mm segments. These segments were prepared for electron microscopy as described in the methods. Dimensions of plastids were determined in cross sections in each segment. (G=grana thylakoid; ST=stroma thylakoid; Div=variable).

with insignificant background fluorescence. The flu- orescence ratio (Fp - Fo/Fp) of dark adapted tissues did not vary from the tip to the middle of the sheath of the primary leaf being close to 0.8 (Figure 6). In fact the fluorescence ratio of 0.8 also indicates that the initial light intensity of 1150 #mol quanta/m 2 s was saturating or close to saturation and that Fp was close or equal to the maximal fluorescence yield FM. Webber et al. (1984) determined the same fluorescence param- eters at 77 K and found a ratio of less than 0.8. In the lower part of the sheath it decreased considerably to about 0.65 in the basiplast which is represented by the very first data point. There was a small decrease also in

the leaf tip which may indicate beginning senescence (Wardley et al. 1984).

Following determination of quantum yield of dark adapted tissues, illumination was continued at 170 #mol quanta m -2 s -1 which corresponds to the light intensity incident on top of the canopy during growth. Following application of light pulses of high fluence rate, the fluorescence ratio (FM ~ - Fo/FM') was cal- culated which corresponds to the effective quantum yield (Genty et al. 1989). It represents PS II activi- ty when it is limited by electron consuming reactions of photosynthesis. Light titration of the photochem- ical fluorescence quenching indicated that the pulse intensity of 1200 #mol quanta/m 2 s was sufficient to

o

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0.8

0.6

0.4

0.2 T~j_j.I_j.j " F m "

0.0 I i I i I i I i I I I i I i

0 2 4 6 8 10 12

Distance from leaf base [cm]

Figure 6. Quantum yield of dark-adapted leaf segments (upper curve) and effective (lower curve) quantum yield of Photosystem II from the base to the tip of a 8-day-old primary leaf. The basiplast is defined as the zone in 0-0.5 cm distance to the base; the leaf sheath corresponds to the tissue up to 7 cm and the leaf blade to the tissue between 8 and 13 cm.

177

o . n

o t-

o 91

o

0.6

0.4 Fp

Frn" - F o

F m"

0 I l I I I

4 6 8 10 12

age [days]

Figure 7. Quantum yield of Photosystem II of basiplasts from dark- adapted primary leaves (upper curve) and effective (lower curve) quantum yield of Photosystem II of the basiplast in relation to the age of the seedling.

transiently reduce most or all reaction centers. The flu- orescence ratio was highest in the fully expanded part of the leaf blade (8-13.5 cm) while it declined in its youngest not completely expanded part (5.5-8 cm) as well as in the leaf sheath (0-5.5 cm). Minimal values were obtained in the basiplast (0-0.5 cm). However, it is important to note that a fluorescence ratio of 0.17 still indicates photosynthetic activity in the basiplast. Obviously the photosynthetic apparatus is at least to a certain extent functional and active. We also detected strong light induced non-photochemical fluorescence quenching which was rapidly reversible in the dark, indicating a build-up of a proton gradient across thy- lakoid membranes

PS H fluorescence in the basiplast in dependence of leafage

As last step of our analysis, the fluorescence parame- ters were determined in the basiplast in dependence of seedling age. The fluorescence parameter (Fp - Fo)/Fp of dark-adapted segments was not much different in the basiplast of young versus old seedlings (Figure 7). The ratio of (FM' -- Fo)/FM' during steady state pho- tosynthesis was about 0.3 in basiplasts of 3- to 6-day- old seedlings and dropped to values between 0.19 and

0.12 in the basiplast of 8- to 12-day-old seedlings. This decrease coincides with the transition of leaf blade to leaf sheath formation.

Discussion

In typical monocatyledonous plants, such as barley, leaf cells derive from a meristem at the leaf base. Therefore, monocot leaves have a well defined gra- dient of young tissue at the base and old tissue at the tip (Sharman 1942). Accordingly, monocot leaves have become a model system for studying leaf devel- opment (for instance: Baker and Leech 1977; Baum- gartner et al. 1989; Boffey and Leech 1982; Dean and Leech 1982). However, despite a large number of detailed investigations during the last years, several aspects of plastid development in the basal zone still need clarification, some of which are addressed in this paper. In fact, the main problem seems to lie in some species and growth specific variation of plastid devel- opment. Therefore, the data compiled from different plants and laboratories are often not directly compara- ble and hence, do not sum up to a complete picture of plastid development inthe basiplast. In this investiga- tion we have aimed at resolving this problem using a wide range of techniques for studying the same object.

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Expression of photosynthetic and nonphotosynthet- ic genes in the basiplast. A defined portion of the cDNAs randomly selected from our basiplast cDNA library coded for proteins involved in photosynthesis. This demonstrated that a considerable part of the tran- scriptional and presumably translational activity of the basiplast is already directed towards development of the plastids. Our approach to describe the develop- mental processes in the basal growing zone of bar- ley leaves is not restricted to photosynthesis. We have demonstrated high levels of expression of structural and metabolic proteins, for instance a water channel (Hollenbach and Dietz 1995), a subunit of the tono- plast ATPase (Dietz et al. 1995) and a peroxide reduc- tase (Baier and Dietz 1996). On a fresh weight basis, the basiplast has 2-3 times the nucleic acid contents of mature leaf tissue (Ougham and Davies 1990; Baier and Dietz, unpublished). This may partly be due to the smaller cell size, but also reflects the strong expression of nonphotosynthetic genes. Therefore, in the light of an enhanced overall expression, the observed expres- sion of genes for photosynthetic components may be an underestimate of the absolute investment in the devel- opment of photosynthesis in the basal zone of leaves as compared with the expanded leaf blade. Obviously, tis- sue specific factors required as signals for chloroplast development were sufficiently active to initiate plastid development although the volume of the chloroplast compartment per cell was only small as compared to that in mature mesophyll cells where chloroplasts rep- resent a large volume fraction of the cytoplasm, for instance 28% in barley (Winter et al. 1993).

Variations in photosynthetic gene expression in the basiplast among species. 3 mm apical from the base, the meristematic cells differentiate into the cells of the epidermis, the mesophyll and the leaf bundles (Dan- nenhoffer and Evert 1994; Baier and Dietz, unpub- lished). Simultaneously, the development of the photo- synthetic machinery takes place. Based on the results of other groups (Webber et al. 1984; Mullet 1988; Ougham and Davies 1990; Gay and Thomas 1995) we attempted a more refined temporal and in part spa- tial analysis of the changes in the basiplast during the phases of leaf expansion. Up to now, usually either sections of fully expanded leaves were analyzed (Bak- er and Leech 1977; Ougham and Davies 1990), or two stages of leaf development were compared (Web- bere t al. 1984; Krupinska 1992). Already in meris- tematic cells generating mesophyll cells, plastid size is larger than in non-leaf meristems (Miyamura et al.

1986). Analyses of leaf sections from the base to the tip have shown expression of various photosynthetic genes beginning in the second and third section cor- responding to a distance of 0.5 to 1.5 cm from the scutellum (Ougham and Davies 1990). In our system, expression of photosynthetic genes was detected even in the lowest section of expanded leaves of 8-d-old seedlings, corresponding to a distance of 0 to 0.5 cm to the scutellum. In fact all probes tested, i.e. genes for nuclear and plastome encoded proteins such as CP29, LHCP II, D1, cytochrome-b559, and RUBISCO-SSU and LSU, showed a significant transcript level in the basal five millimeters of the leaves where the transi- tion of blade to sheath formation had already occurred. Expression of cab and rbcS genes was also observed in the basal centimeter of the expanding fourth foliage leaf of maize (Martineau and Taylor 1985). However at that stage of development the maize leaf meristem still forms leaf blade and is therefore comparable with basiplasts of 4 to 6-day-old barley seedlings. There seems to exist a species-specific variation of plastid development which is further modulated by environ- mental factors, particularly light. In our experiments, the photon fluence density at the top of the canopy was about 170 #mol m -2 s -1 and the seedling density high which means that the photon fluence was very low at the base of the leaves. Nevertheless, plastid develop- ment was faster than in other species such as Lolium grown at the higher irradiance of 350 #mol m -2 s -1 (Ougham and Davies 1990). The changes in expres- sion during ageing of the basiplast were similar for the nuclear encoded Rubisco-SSU and for the plastome encoded D1 protein.

Variation in photosynthetic activity and pigment com- position of the basiplast. Webber et al. (1984, 1986) reported a detailed analysis of wheat leaf development. When comparing their and our study, chlorophyll con- tents per unit leaf area changed similarly along the axis of an 8-day-old leaf of wheat and barley (Figure 4). In wheat, the chlorophyll a to b-ratio was 3.4 at the base and 2.9 at the tip. In our experiment the corre- sponding data were 3.5 and 3.4, respectively, showing that both approaches were highly comparable. How- ever there was a major difference between 4-day-old wheat and barley leaves. Usually, illumination with actinic light causes PS II-dependent chlorophyll a flu- orescence to increase to an initial maximum followed by a decrease to a lower steady state level. At high light intensities, the decrease in chlorophyll a fluorescence is mainly due to non-photochemical quenching (NPQ).

Tissue from the base of 4-day-old wheat leaves showed no NPQ and was not able to perform state 1/state 2- transitions (Webber et al. 1984). Conversely, 4-day-old barley basiplast displayed both photochemical quench- ing (PQ) and NPQ, suggesting normal photosynthetic competence of the chloroplasts in young barley basi- plast. Webber et al. (1986) found that the thylakoid membranes of 4-day-old wheat leaves had a function- al electron transport but were incapable to maintain a proton gradient whereas the development of NPQ in barley basiplast clearly demonstrates the ability to form a transthylakoid proton gradient. The data on ini- tial quantum yield as calculated from (Fp - Fo)]Fp of dark-adapted barley leaves (Figure 6) were similar to those previously reported for wheat leaves (Web- beret al. 1984). Our additional results on fluorescence during conditions of steady state photosynthesis show that photochemistry is coordinated with energy con- suming reactions in the basiplast. From this it is then quite obvious that expression of photosynthetic genes is related to functionality of the photosynthetic appara- tus at this early stage of leaf development. Further on, the two sets of data on chlorophyll a fluorescence, i.e. on the different tissue sections from the tip to the base on the one hand, and in the basiplast in dependence of leaf age on the other hand, clearly demonstrate for the first time that the differentiation of leaf tissue to the leaf blade or leaf sheath is characterized by clear- ly distinguishable properties of the chloroplasts in the basiplast.

Baker and Leech (1977) observed a delay in CO2 fixation as compared to 02 evolution when analysing photosynthetic activity along the acro-petal axis of maize leaves. The apparently higher relative expres- sion in the basiplast of genes for components of the thylakoid membrane than for Rubisco SSU may fit to this picture of a slightly delayed development of Calvin cycle reactions. Expression of nuclear and plas- tidic encoded photosynthetic genes seems not yet well coordinated at this early stage of plastid development. Additional evidence for this conclusion comes from run-on-transcription experiments (Krupinska 1992) which revealed that regulation of gene expression of proplastids and young chloroplasts is only partly reg- ulated on the level of transcription.

Similarity between relative pigment composition in basiplast and other leaf segments. The basiplast con- tained the whole set of pigments required in the photo- synthetic apparatus. The large quantitative difference in pigment contents between leaf blade and basiplast

179

has frequently been described (e.g. Boffey et al. 1980; Webber et al. 1984). However, it was striking to see the similarity in relative pigment composition of basi- plast and leaf blade. Assembly and stabilization of LHC II and PS II require chlorophyll a, chlorophyll b, lutein, violaxanthin and neoxanthin (Paulsen et al. 1990; Ktihlbrandt et al. 1994). The chlorophyll a/chlorophyll b-ratio was constant, similarly to oth- er studies (Baker and Leech 1977). But, in addition, we show that all pigments essential for the formation of functional antennas are present in barley basiplasts. Lutein and V+A+Z are present in relative excess in respect to chlorophyll comparable to etiolated tissues (PfiJndel and Strasser 1988).

Spatial pattern of structural plastid differentiation. In many studies, the differentiation of plastids was investigated in large leaf sections of 1 cm length or more which only provides a coarse picture of the devel- opmental processes in the basiplast (Baker and Leech 1977; Webber et al. 1984). We show that develop- ment proceeds within a well-defined program so that each millimeter is characterized by a specific structural state of plastids. In primary leaves of 5-day-old barley seedlings the zone of plastid division was restricted to the 2nd and 3rd millimeter of the leaf. In 5 mm distance from the leaf base the plastids were already very homogeneous as far as thylakoid organisation is concerned, although their size still varied by a factor of up to 2. Grana stacking is not yet completed. During further development of the leaf the average appression of the thylakoids will increase by a factor of 2 (Robert- son and Laetsch 1974). Plastid size and plastid volume increase during leaf maturation, the latter by a factor of about four. Both processes contribute to the higher pigment contents of the leaf blade.

Changes in the basiplast as indication of leaf devel- opment. Chloroplast development in the basiplast changes during leaf development. Thylakoids are less stacked in the basiplast of young leaves (Webber et al. 1984). PS I- and LHC-PS II-complexes are more ran- domly distributed in thylakoid membranes. LHCP II transcript amounts were highest in basiplasts of 4-day- old seedlings and decreased in the basiplast of old- er leaves. An almost identical role was observed for the chlorophyll content. All other pigments were also present to build up functional photosystems. Chloro- phyll fluorescence kinetics indicated that the chloro- plasts in the basiplast of 4-day-old seedlings are capa- ble to efficiently perform the photochemical reactions

180

of photosynthesis even at elevated light intensities. According to the model proposed by Webber et al. (1984), the excitation energy is partitioned between P S I and PS II in order to protect PS II. In conclusion, three phases of plastid development in the basiplast are to be distinguished on the basis of photosynthetic parameters:

(1) day 3-6: Growth rate and expression of photosyn- thetic genes is highest. Effective quantum yield is between 0.25 and 0.27. The basiplast forms cells

of the later photosynthetically highly active leaf

blade.

(2) day 8-11: The basiplast is still active and forms leaf sheath, however at a decreasing rate. Effective quantum yield is between 0.14 and 0.19.

(3) day 12 and older: The basiplast ceases to grow and becomes part of the leaf sheath only. Effective quantum yield drops below 0.13.

In a model previously suggested by Mullet (1993), distinct zones of DNA, replication RNA transcrip- tion, photosynthetic protein synthesis and light induced transcription are distinguished. It is obvious from our and previous results that this model only gives the rough picture of plastid development in the basiplast and needs some refinement. The developmental activi- ties attributed to distinct zones in the Mullet model take in fact place in close temporal and spatial vicinity with- in the basiplast. As a consequence, the basiplast con- tains always functional chloroplasts, even after cessa- tion of leaf growth. In relation to the questions posed in the introduction, we conclude for barley that (i) expres- sion of photosynthetic genes in the basiplast is direct- ly related to photosynthetic activity, (ii) the basiplast contains the full complement of pigments required for photosynthesis in relative composit ion similar to the leaf blade, and (iii) chloroplast development in the basiplast indeed reflects the developmental transitions

of the leaf tissue.

Acknowledgment

We are grateful to Claudia Gehrig and Prof. Georg Krohne (both from Biozentrum, Wiirzburg) for assis- tance in electron microscopy. This work was supported within the Sonderforschungsbereiche 176 and 251. M. B. acknowledges support by the Graduiertenkolle (Ka 456/5-1).

References

Baler M and Dietz K-J (1996) Primary structure and expression of plant homologues of animal and fungal thioredoxin-dependent peroxide reductases and bacterial alkyl hydroperoxide reductases. Plant Mol Biol 31:553-564

Baker NR and Leech RM (1977) Development of Photosystem I and Photosystem II activities in leaves of light-grown maize (Zea mays). Plant Physiol 60:640-644

Banmgartner B J, Rapp JC and Mullet JE (1989) Plastid transcrip- tion activity and DNA copy number increases early in barley chloroplast development. Plant Physiol 89:1011-1018

Bilger W, Fisahn J, Brummet W, KoBmann J and Willmitzer L (1995) Violaxanthin cycle pigment contents in potato and tobacco plants with genetically reduced photosynthetic capacity. Plant Physiol 108:1479-1486

Boffey SA and Leech RM (1982) Chloroplast DNA levels and the control of chloroplast division in light-grown wheat leaves. Plant Physiol 69:1387-1391

Boffey SA, Selld6n G, Leech RM (1980) Influence of cell age on chlorophyll formation in light-grown and etiolated wheat seedlings. Plant Physiol 65:680-684

Brune A, Urbach W and Dietz K-J (1994) Compat~aentation and transport of zinc in barley primary leaves as basic mechanisms involved in zinc tolerance. Plant Cell Environ. 17:153-162

Dannenhoffer JM and Evert RF (1994) Development of the vascular system in the leaf of barley (Hordeum vulgare L.). Int J Plant Sci 155:143-157

Dean C and Leech RM (1982) Genome expression during normal leaf development. I. Cellular and chloroplast numbers and DNA, RNA, and protein levels in tissues of different ages within a seven-day-old wheat leaf. Plant Physiol 69:904-910

Dietz K-J, Rudloff S, Ageorges A, Eckerskorn C, Fischer K and Arbinger B (1995) Subunit E of the vacuolar H+-ATPase of Hordeum vulgare L.: cDNA cloning, expression and immuno- logical analysis. The Plant J 8:521-529

Eriksson G, Kahn A, Walles B and v. Wettstein D (1961) Zur makromolekularen Physiologie der Chloroplasten IlL Berichte der deutschen botanischen Gesellschaft 74:211-232

Gay AP and Thomas H (1995) Leaf development in Lolium temu- lentum L.: Photosynthesis in relation to growth and senescence. New Phytol 130:159-168

Genty B, Briantais J-M, Baker NR (1989) The relationship between the quantum yield of photosynthetic electron transport and pho- tochemical quenching chlorophyll a fluorescence. Biochim Bio- phys Acta 990:87-92

Gilmore AM and Yamamoto HY (1991) Resolution of lutein and zeaxanthin using a non-encapped, lightly carbon loaded C 18 high performance liquid chromatographic column. J Chromatogr 543:137-145

Hoagland DR and Arnon DI (1938) The water culture method for growing plants without soil. University of California Agricultural Experiment Station Circular 347

Hollenbach B and Dietz K-J (1995) Molecular cloning of emip, a member of the major intrinsic protein (MIP) gene family, pref- erentially expressed in epidermal cells of barley leaves. Bot Acta 108:425-431

Klein RR and Mullet JE (1986) Regulation of chloroplast-encoded chlorophyll binding protein translation during higher plant chloroplast biogenesis. J Biol Chem 261: 11138-11145

Krupinska K (1992) Transcriptional control of plastid gene expres- sion during development of primary foliage leaves of barley grown under a dally light-dark regime. Planta 186:294-303

Ktihlbrandt W, Wang DN and Fujiyoshi Y (1994) Atomic model of plant light-harvesting complex by electron crystallography. Nature 367:614q521

Lamppa GK, Morelli G and Chua N-H. (1985) Structure and devel- opmental regulation of a wheat gene encoding the major chloro- phyll a/b-binding polypeptide. Mol Cell Biol 5:1370-1378

Larrinua JM, Muskavitch KMT, Gubbins EJ and Bogorad L (1983) A detailed restriction endonuclease site map of the Zea mays plastid genome. Plant Mol Biol 2: 129-140.

Manning K (1991) Isolation of nucleic acids from plants by differ- ential solvent precipitation. Analyt Biochem 195:45-50

Marchionni M and Gilbert W (1986) The triosephosphate isomerase gene from maize: Introns antedate the plant animal divergence. Cell 46:133-141

Martineau B and Taylor WC (1985) Photosynthetic gene expres- sion and cellular differentiation in developing maize leaves. Plant Physiol 78:399--404

Miyamura S, Nagata T and Kuroiwa T (1986) Quantitative fluo- rescence microscopy on dynamic changes of plastid nucleotides during wheat development. Protoplasma 133:66-72

Mullet JE (1988) Chloroplast development and gene expression. Ann Rev Plant Physiol Plant Mol Biol 39:475-502

Mullet JE (1993) Dynamic regulation of chloroplast transcription. Plant Physiol 103:309-313

Ougham HJ and Davies TGE (1990) Leaf development in Lolium temulentum: Gradients of RNA complement and plastid and non- plastid transcripts. Physiol Plant 79:331-338

Paulsen H, Rtimler U and Riidiger W (1990) Reconstitution of pig- ment containing complexes from light-harvesting chlorophyll a/b-binding protein overexpressed in Escherichia coIi. Planta 181:204-211

Pftindel E and Strasser RJ (1988) Violaxanthin de-epoxidase in eti- olated leaves. Photoynth Res 15:67-73

Rapp JC, Baumgartner BJ and Mullet J (1992) Quantitative analysis of transcription and RNA levels of 15 barley chloroplast genes: Transcription rates and mRNA levels vary over 300-fold; pre- dicted mRNA stabilities vary 30-fold. J Biol Chem 267: 21404- 21411

Reynolds ES (1963) The use of phibium citrate at high pH as an electron opaque stain in electron microscopy. J Cell Biol 17: 208-211

Robertson D and Laetsch WM (1974) Structure and function of developing barley plastids. Plant Physiol 54:148-159

181

Rodermel SR and Bogorad L (1985) Maize plastid photogenes: Map- ping and photoregulation of transcript levels during light induced development. J Cell Biol 100:463-476

Sambrook J, Fritsch EF and Maniatis T (1988) Molecular Cloning: A Laboratory Manual, 2nd ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY

S chreiber U (1994) New emitter-detector cuvette assembly for mea- suring modulated chlorophyll fluorescence of highly diluted sus- pensions in conjunction with the standard PAM fluorometer. Z Naturforsch 49c: 646-656

Schreiber U and Bilger W (1993) Progress in chlorophyll fluores- cence research: Major developments during the past years in retrospect. Prog Bot 54:151-173

Schreiber U, Hormann H, Neubaner C and Khighammer C (1995) Assessment of Photosystem II photochemical quantum yield by chlorophyll fluorescence quenching analysis. Aust J Plant Physiol 22:209-220

Sharman BC (1942) Developmental anatomy of the shoot of Zea mays L. Ann Bot 1: 245-282

Sorensen AB, Jensen BF and Gausing K (1992) Barley (Hordeum vulgare) gene for CP29, a core chlorophyll a/b binding protein of PS II. Plant Physiol 98:1538-1540

Thayer SS and Bj~Jrkman O (1990) Leaf xanthophyll content and composition in sun and shade determined by HPLC. Photosynth Res 23:331-343

Wardley TM, Bhalla PL and Dalling JM (1984) Changes in the number and composition of chloroplasts during senescence of mesophyll cells of attached and deattached primary leaves of wheat (Triticum aestivum L.). Plant Physiol 75:421-424

Webber AN, Baker NR, Platt-Aloia K and Thomson K (1984) Appearance of a State 1-State 2 transition during chloroplast development in the wheat leaf: Energetic and structural consid- erations. Physiol Plant 60:171-179

Webber AN, Baker NR, Paige CD and Hipkins MF (1986) Photo- synthetic electron transport and establishment of an associated trans-thylakoid proton electrochemical gradient during develop- ment of the wheat leaf. Plant Cell Environ 9:203-208

Winter H, Robinson DG and Heldt HW (1993) Subcellular volumes and metabolite concentrations in barley leaves. Planta 191: 180- 190