Figure 1.
Localization of nuclear DNA fragmentation detected by the TUNEL assay at 6 (A–C), 8 (D–F), 10 (G–I), 16 (J, K), and 18 DAF (L–N).
TUNEL-positive nuclei are visualized as green signals and indicated by red arrows. Upper panel demonstrates positions of histological sections used for TUNEL assay at the reconstructed cross and longitudal views of a barley grain. al, aleurone; cl, chlorenchyma, em, embryo; es, endosperm; esr, embryo surrounding region; mvb, main vascular bundle; np, nucellar projection; nu, nucellus; p, pericarp, tc, transfer cells. Bars = 200 µm.
Figure 2.
Caspase-like activities and effect of specific caspase inhibitors on corresponding caspase-like activity in barley pericarp.
Data are means ± SD, n = 4, values followed by the same letter do not differ significantly at p>0.05.
Figure 3.
Caspase-like activities and effect of specific caspase inhibitors on corresponding caspase-like activity in the developing endosperm.
Data are means ± SD, n = 4, values followed by the same letter do not differ significantly at p>0.05.
Figure 4.
Phylogenetic trees of proteasome subunits PBA and PBB (A) and phytaspases (B) drawn with the ClustalW software.
The horizontal scale represents the evolutionary distance expressed as a number of substitutions per amino acid. The putative phytaspases and proteasome subunits PBA1 of barley are shown in bolt. (A) Putative barley (Hv) proteasome subunits PBA and PBB are similar to the corresponding genes from Arabidopsis thaliana (At), Populus tomentosa (Pt) and Oryza sativa (Os). (B) Putative barley (Hv) phytaspases together with phytaspases from Nicotiana tabacum (Nt) and Oryza sativa (Os) belong to the subgroup 1 of subtilase-like proteases. The phytaspases with proven caspase-6 activity are shown in italic. Only one Arabidopsis subtilase-like protease per subgroup is shown (Rautengarten et al., 2008) in order to simplify the figure.
Figure 5.
Transcript profiling of the proteasome subunits PBA and PBB (A) and phytaspase (PhS) genes (B) in pericarp (left) and endosperm fractions (right) of the developing barley grains determined by real-time quantitative RT-PCR analysis.
Data are means ± SD, n = 3, values followed by the same letter do not differ significantly at p>0.05.
Figure 6.
Expression profiles of the vacuolar processing enzymes VPE2a-VPE2d (A), proteasome subunits PBA and PBB (B) and phytaspase (PhS) genes (C) in the different tissues micro-dissected from the developing barley grains.
Figure 7.
Scheme illustrating programmed cell death processes together with potentially involved activities and genes (in brackets) in distinct tissues of the developing barley grains.
Activities: cas1, caspase-1-like; cas3, caspase-3-like; cas4, caspase-4-like; cas6, caspase-6-like; cas8, caspase-8-like; cas9, caspase-9-like. Genes: VPE, vacuolar processing enzyme; PhS, phytaspase.