Programmed cell death (PCD) in maize seed– an overview of the methods for observing PCD eventsinsitu in plant tissues

Aleš Kladnik, Karen Chamusco, Marina Dermastia, Prem S. Chourey

Abstract

Abbreviations:

DAP – days after pollination, kb – kilobase-pairs, P-C – placento-chalaza, PCD – programmed cell death.

Introduction

Programmed cell death (PCD) is referred to any process by which protoplast, with or without the cell wall that encloses it, is eliminated as part of an adaptive event in the life cycle of the plant (Dangl et al., 2000). PCD is extremely important in development of multicellular organisms. Many developmental processes in animals and plants require removal of cells by PCD (Buckner et al., 1998; Vaux and Korsmeyer, 1999; Krishnamurthy et al., 2000).

PCD in animal tissues is often referred to a highly conservative cell death pathway with a very distinct sequence of morphological changes named ‘apoptosis’, after Greek word for senescence and falling of leaves.Apoptosis begins with cell shrinkage and chromatin condensation, followed by fragmentation of nucleus and cytoplasm. The cellular remains are packaged in plasma membrane vesicles named ‘apoptotic bodies’ that are later phagocytosed by neighbouring cells or macrophages. These morphological characteristics are used to distinguish death by apoptosis from death by necrosis.The latter is abrupt, there is no gradual shrinkage of cell and cellular contents are not packaged into apoptotic bodies (Kerr et al., 1972). On the molecular level, DNA degradation in necrosis occurs at more or less random sites and genomic DNA appears as a smear on the agarose gel after electrophoresis. On the other hand the genomic DNA in apoptotic cells is cleaved by endonucleasesat internucleosomal sites giving rise to fragments of multiples of approximately 180 base pairs, and such DNA appears as a ladder on the gel (Kerr and Harmon, 1991).Apoptosis in animals is mediated by a class of aspartate-specific cysteine proteases called caspases. No caspase homologues are reported thus far in plants, but there are numerous reports of involvement of caspase-like proteases (metacaspases, putative caspase related protease family) in the control of cell death activation in plants (Watanabe and Lam, 2004).

Several PCD patways were described in plants. The unique property of plant cells, the cell wall, even enables plant cells to function after their death. Plant cells die in different ways, from rapid vacuole mediated cell death to slower apoptotic-like cell death.

DNA fragmentation occurs also in plant cell death, where the size of fragments ranges from 0.14 to 50 kb.

We investigated the role of PCD in development of a novel transport system in the base of the maize (Zea mays) seed in the placento-chalazal (P-C) layer, the maternal tissue just below the basal endosperm cells and just above vascular termini in the pedicel. It is believed to play a critical role in transport of water, sugar and other nutrients into developing seed (Kiesselbach, 1949; Felker and Shannon, 1980). An additional function of the P-C layer, especially in maize, sorghum and teosinte is in the accumulation of endosperm secreted peptides with antifungal function, which may prevent the entrance of pathogens from maternal into the filial tissues (Serna et al., 2001; Cai et al., 2002).Whether the endosperm-secreted peptides along with the collapsed or crushed mass of cellular tissue in the P-C cells at seed maturity leads to the so called ‘black layer’ or abscission layer (Felker and Shannon, 1980), is not clear. Similar cellular autolytic events are also reported in the nucellar projection in barley (Linnestad et al., 1998) and in thin walled parenchymatous cells in developing cotyledons of fava bean (Weber et al., 1997). It is probable that these events are attributable to the phenomenon of programmed cell death (PCD).

In research of developmental PCD, we should analyse temporal progression of several anatomical and physiological changes.We used several light microscopy techniques to document PCD progression in maize caryopsis, however the disruption of cellular integrity was most accurately observed by transmission electron microscopy.Fragmented DNA in apoptotic-like nuclei has many free 3’-OH ends that were detected in situ with TUNEL rection (terminal deoxynucleotidyl transferase dUTP nick end labeling), where the enzyme terminal deoxynucleotidyl transferase is used to incorporate labeled nucleotides into DNA strand breaks (Gavrieli et al., 1992). Degradation of nuclei can be visualized with DAPI staining, that emits strong fluorescence when bound to DNA, thus specifically staining nuclei (Kubista et al., 1987). Crystal violet is used to stain chromosomes (Jensen, 1962) and can also be used to identify condensation of chromatin in interphase nuclei. Endoreduplication of nuclei in the P-C layer was measured with image analysis based densitometry on Feulgen stained tissue sections (Kladnik et al., 2004).

Results

Fig. 1:? longitudinal section of maize caryopsis (with labeled tissues)

Fig. 1: negative DAPI images 2, 4, 8, 12, 20/24 DAP + graph layers count (wt).

Fig. 2: crystal violet (wt 10 DAP)

Fig. 3: TUNEL 12 DAP P-C layer (also some apoptotic bodies at 12 DAP), 4 DAP nucellus PCD, embrio+endosperm 10 DAP?; (DAPI + TUNEL all)

Fig. 4: TEM 9 DAP (vacuole fragmentation, degradation of protoplast; gradient of morphological changes)

Fig. 5: flavonoids (other stages, without alkaline?, monochrome image)

Fig. 6: endoreduplication

In sections stained with DAPI (Fig. 1) we saw cells without nuclei in the P-C region very early in seed development, already at 4 days after pollination (DAP). DAPI specifically stained DNA and cell walls were visible due to their autofluorescence with UV excitation. The number of cells without nuclei increased with time of seed development and reached a maximum of 20-25 cell layers around 24 DAP (Fig. 1). To investigate PCD events in the P-C region we used several histology-based methods in addition to DAPI staining.

Crystal violet is used to stain plant chromosomes (Jensen, 1962), however we found that it is also useful to identify the extent of chromatin condensation in nuclei. Cells of the P-C layer that were located immediately below cells that were already without nuclei, contained small nuclei with completely condensed chromatin (picnotic nuclei), whereas in nuclei below them the appearance of condensed chromatin was spotted and nuclei were larger (Fig. 2).

To determinewhether the PCD of P-C cells was apoptotic-like, we used TUNEL reaction on the tissue sections (Fig. 3). Nuclei that contained fragmented DNA, showed stronger fluorescence. Since plant tissues emit strong autofluorescence, the fluorescence of TUNEL-stained nuclei was compared to the fluorescence of DAPI-stained nuclei, where the entire DNA is stained. In the P-C layer at 12 DAP there were several layers of cells containing nuclei with fragmented DNA, just underneath the cells that were already enucleated.In some instances, the TUNEL-staining pattern appeared as foci concentrated around the periphery of cell (arrow in Fig. 3). It is possible that such pattern of staining is due to the membrane-bound apoptotic-like bodies containing fragmented DNA as observed in animal cells undergoing apoptosis (Bursch et al., 1990).TUNEL staining was observed also in nuclei of the cells in the nucellus surrouding the expanding endosperm at 4 DAP and innuclei of endosperm cells surrounding embryo at 10 DAP (Fig. 3).

To describe PCD process in greater detail we used transmission electron microscopy. Ultrastructural analysis of successive P-C layers of caryopsis at 9 DAP showed a progression of cellular change during development (Fig. 4A – D). Most importantly, the cells directly underneath the basal endosperm cells were enucleated, without organelles and only the degenerated remains of protoplasts could be seen, however the cell walls remained in place (Fig. 4A, B). The next layer (distal to basal endosperm cells) was comprised of cells with dense cytoplasm but no discernible nucleus and vacuole, whereas, large vacuoles and membranous organization was clearly seen in cells further away from the endosperm (Fig. 4C and D, respectively). The vacuole in the upper part of Fig. 4 D already started with fragmentation.

We also confirmed the different nature of two layers inside the P-C region with staining for phenolic substances. Different colour of UV-induced autofluorescence of sections in alkaline buffer revealed presence of different hydroxycinnamic acids: blue-gren coloration in the nucellar P-C layer indicated sinapic acid and bright blue in the integumentary P-C layer indicated ferulic or caffeic acid (Harborne, 1998). Moreover, we also observed strong staining for flavonoids in the integumentary P-C layer only in sections treated with Naturstoffreagenz A.

At 4 DAP nuclei in the cells forming the P-C layer were of normal ploidy levels 2 C and 4 C, typical for mitotic population of cells, whereas at 12 DAP we observed several layers of cells with endopolyploid nuclei with DNA content 8 C (Figure 6).

Discussion

The PCD in the P-C cells described here may actually be causal to the activation of the transport function of these cells. Unlike the cells in the nucellus that are autolysed, the dead P-C layer cells were present throughout the duration of seed development as a structural bridge for post-phloem transport of water, sugars and other nutrients from vascular tissue in the pedicel to a developing seed. The P-C cells are thus similar to the transport cells in xylem, the tracheary elements (TEs), which also undergo a rapid PCD prior to becoming functionally mature (reviewed by McCann, 1997). Xylogenesis is best analyzed in the TEs of Zinnia elegans; PCD in these cells occurs by vacuolar-lysis which in turn leads to rapid nuclear degradation through an S1 type nuclease, ZEN1 (Obara et al. 2001; Ito and Fukuda, 2002). It is likely that the PCD in P-C layer is executed through a similar pathway. Despite the functional similarities between the P-C cells and the TEs, the former lacked the characteristic structural features of the mature TEs, which include secondary cell walls of annular, spiral reticulate or pitted wall thickenings. Endoreduplication in P-C layer was not as extensive as in tracheary elements (TE), reaching only 8 C in contrast to 64 C of TEs. Nevertheless, endoreduplication is well spread in the PC-layer alone, indicating that this is a terminally differentiated tissue. The difference in ploidy level between two cell types most likely reflects their different need for cell size. Number of studies have shown the correlation between endoreduplication and cell size, thus the high endopolyploidy level of TEs may be related to theirlarge volume.

The P-C layer thus appears to be a novel transport system between phloem termini in pedicel and basal endosperm. Overall, the PCD in P-C layer may be an adaptive strategy to allow a clear passage of water with solutes through the dead cells into developing filial tissues.

Cells in the PC-layer also entered a special variant of the cell cycle, endoreduplication, doubling of DNA without condensation of chromosomes, mitosis and cell division. Cells often enter endoreduplication cycle when switching from proliferation to differentiation. Endopolyploid cells do not divide anymore and are usually larger and metabolically active. Some cell types, for example tracheary elements, undergo several cycles of endoreduplication, before they execute the PCD programme.

Evans Blue is a dye that is excluded from living cells with intact plasma membrane, thereby staining only the cytoplasm of nonviable cells. It was succesfully used to detect progression of PCD in maize endosperm (Young and Gallie, 1997). Staining of fresh hand-cut sections of caryopsis with Evans blue did not prove useful for documenting PCD progression in the P-C layer, since no characteristic pattern of staining was seen (data not shown).

With analysis of extracted DNA we lose positional information about PCD processes in the tissue. Moreover, if the PCD process occurs in only few cells at a time, the DNA fragments would be present in a relatively small concentration, compared to DNA from the whole tissue. TUNEL reaction combines molecular and anatomical approach in PCD study.

Since the tissue of interest represents only few layers of cells in the maize caryopsis, genomic DNA was not checked for laddering. However the positive TUNEL result in the nuclei, condensation of chromatin, the ultrastructural analyses and the exact temporal progression of cell death support the theory that the cell death in P-C layer is in fact developmental programmed cell death. Cells in the P-C layer perform their first important function very early in the caryopsis development, almost immediately after fertilization they synthesize large amount of flavonoids, that may serve as an antimicrobial barrier on the way from the phloem in the maternal tissue of pedicel to the filial endosperm together with endosperm secreted antimicrobial peptides (Serna et al., 2001; Cai et al., 2002). Furthermore, the cells in the P-C that are dying, continue functioning also after their death, serving as a transport tissue.

With the advance of imaging techniques there are also great opportunities to monitor living cells, especially the production of reactive oxygen species, mitochondrial activity and calcium activity (Chaerle and Van Der Straeten, 2001). See also the largecollection of apoptosis assays at the web site of Molecular Probes (URL address although majority of the assays are developed for human cells, many procedures can be also used on plant tissues.

Materials and methods

Preparation of plant material for light microscopy

Maize (Zea mays L.) plants of the W22 inbred line were grown in the greenhouse. Female flowers were hand pollinated, either with the pollen from the same plant or from other plants of the same genotype. Developing caryopses were harvested at 0 – 28 DAP (days after pollination) and immediately fixed in cold FAA fixative (3.7% formaldehyde, 5% acetic acid, 50% ethanol) for 24 hours, followed by dehydration in series of ethanol and tertiary butyl alcohol and embedding in Paraplast Plus (Fisher Scientific). Paraffin embedded caryopses were sectioned to 8 – 12 micrometers thick sections with a rotary microtome (Microm 325, Carl Zeiss).

DAPI staining

Paraplast embedded sections were dewaxed in xylene and rehydrated in ethanol series, equilibrated in freshly prepared McIvaine’s buffer pH 7.0 (0.02 M citric acid and 0.16 M Na2HPO4, mixed before use), followed by staining in 600 nM DAPI (4’,6’ diamino-2-phenylindole ∙ 2 HCl; Molecular Probes) in McIlvaine’s buffer for 15 minutes at room temperature in dark, washed with distilled water, covered with coverslips and observed with UV excitation (365 nm band-pass). Cold blue fluorescent nuclei were photographed with AxioCam MRc color digital camera (Carl Zeiss Vision).

TUNEL staining

TUNEL (terminal deoxynucleotidyl transferase mediated X-dUTP nick end labeling) was performed using In situ Cell Death Detection Kit (Roche Diagnostics), essentially following the manufacturers protocol. Briefly, sections were dewaxed in xylene and rehydrated in ethanol series, treated with 20 g/ml Proteinase K (Gibco BRL) in 10 mM Tris 7.5 and 5 mM EDTA for 15 minutes at room temperature, followed by incubation in mixture of fluorescein labeled deoxynucleotides and terminal deoxynucleotidyl transferase (TUNEL mix) for 60 minutes at 37ºC. After washing the slides with PBS, the coverslips were mounted in aqueous mounting media GelMount (Sigma-Aldrich) containing 600 nM DAPI. The green fluorescein fluorescence of nuclei with fragmented DNA was observed with a microscope with blue light excitation (450 – 490 nm band-pass), and DAPI fluorescence of all nuclei was observed with UV excitation. Images were taken with AxioCam MRc color digital camera (Carl Zeiss Vision). In addition we found that the ommision of Proteinase K step does not affect the TUNEL staining intensity, so it was skipped in later experiments. But please note that the optimal staining procedure has to be adapted for each tissue and fixation type separately (see also note regarding TUNEL in materials and methods section in Wang et al., 1996).

Iodine – crystal violet staining

Staining was performed as described by Jensen (1962). Briefly, hydrated sections were stained with 1% aqueous crystal violet for 15 minutes, rinsed in distilled water, placed in 1% iodine in 2% KI, rinsed in water, dehydrated quickly in 70%, 96% and absolute ethanol, cleared in xylene and mounted with Permount (Electron Microscopy Sciences).

Transmission electron microscopy

All materials for electron microscopy processing were purchased from Electron Microscopy Sciences. Tissues were prepared for transmission electron microscopy (TEM) by fixation in 4% glutaraldehyde (v/v) and 1% paraformaldehyde (v/v) in 0.1 M phosphate buffer (pH 7.2). Fresh kernels were harvested and immediately processed for fixation on site by placing a small aliquot of chilled fixative on a glass plate and hand sectioning a sagittal section of the kernel approximately 2 mm wide with a double edged razor blade. The tissue was then put into a vial of ice cold fixative, placed under vacuum for 5 to 10 minutes to aid fixative infiltration. After vacuuming the samples were placed on a rotating plate overnight, approximately 12 hours, at 4°C.

After fixation the tissue was rinsed 3 times for 30 minutes each in chilled phosphate buffered saline (PBS) then placed in 2% aqueous osmium tetroxide overnight at 4°C. Once the osmication was completed the samples were rinsed 3 times one hour each in distilled water then dehydrated in a chilled acetone series at 20% increments for 1 hour each. After 100% acetone the samples were sent through propylene oxide as a transition solvent, infiltrated and embedded with Spurr’s resin then polymerized at 56°C for 24 hours.

Samples were sectioned on a Sorvall IIB Ultracut ultramicrotome. The sections were cut to approximately 60 nm thickness and picked up on formvar coated copper grids (50 or 100 mesh). The sections were post stained 20 minutes in filtered 2% aqueous uranyl acetate (UA), rinsed 3 times, 1 minute each in distilled water then were allowed to dry. The sections were then stained 6 minutes in Reynold’s lead citrate, rinsed once in 0.02 M NaOH for 1 minute, then three times more, 1 minute each, in distilled water. The sections were examined and photographed in a Zeiss 109 or a Zeiss 110 transmission electron microscope (TEM).

Cell walls autofluorescence shift in alkaline medium

Paraffin sections were dewaxed in xylene and rehydrated in ethanol series to water. Sections were then either treated with ammonia vapors by holding them briefly over the ammonia solution (Sigma-Aldrich) or covered with 0.15 M K2HPO4 (pH 8.2). Control sections were incubated in distilled water only. Autofluorescence of cell walls was observed with UV excitation and photographed with AxioCam MRc color digital camera (Carl Zeiss, Germany).