How Cysteine Protease Gene PtCP5 Affects Seed Germination by Mobilizing Storage Proteins in Populus trichocarpa

Abstract

In higher plants, seed storage proteins are deposited in protein storage vacuoles (PSVs) and degraded by protease, especially cysteine proteases, as a source of nitrogen for seed germination. In this study, a cathepsin B-like cysteine protease PtCP5, which is important for seed germination and pollen development, was first cloned in Populus trichocarpa. The GUS staining of the ProPtCP5-GUS reporter line showed that PtCP5 is expressed in the roots, stems, leaves, flowers, siliques and seeds of Arabidopsis. We reveal that PtCP5 is present in plasma membrane and co-localizes with the plasma membrane marker REM1.3. Both seed germination and early seedling development are slower in OX-PtCP5 transgenic Arabidopsis when compared with the wild-type. Further analysis revealed that, when stained with toluidine blue, the observed storage protein accumulation was lower in OX-PtCP5 than in the wild-type. Our results also show that the number of abnormal pollen grains is higher and the germination rate of pollen is lower in OX-PtCP5 than in the wild-type. These results indicate that PtCP5 is an important factor in mobilizing storage proteins and that the proper expression of PtCP5 is necessary for both pollen and seed maturation and germination. This study sheds further light on the biological functions of cysteine proteases and provides further reference for seed development research on woody plants.

Keywords: cysteine protease PtCP5; pollen; promoter; seed germination; tissue-specific expression.

Figure 1

PtCP5 sequence analysis.

Figure 2

Phylogenetic analysis of PtCP5 based on an NJ tree with 1000 bootstrap replicates among 70 PLCPs subfamily proteins.

Figure 3

Expression patterns of PtCP5: (A) PtCP5 in P. trichocarpa. The expression levels were normalized to that of β-actin-F and β-actin-R. Data are mean ± SE (n = 3 experiments). (B–J) proPtCP5:GUS expression patterns in Arabidopsis: seedling (B); cotyledons (C); flower (D); siliques (E); seed (F); stem (G); petiole (H); leaf (I); and root (J). (B–G) bar = 1 mm, (H–J) bar = 250 μm. (K–N) Subcellular fluorescence localization of PtCP5-GFP, bar = 30 μm.

Figure 4

Phenotype of OX-PtCP5 Arabidopsis: (A) relative expression of PtCP5 gene in OX-PtCP5. (B) Seed germination, bar = 1 cm. (C) Seed germination rate. Each medium plate contained about 30 seeds of OX-PtCP5 and wild-type respectively. Error line represents standard deviation (SD), * represents significant difference between OX-PTCP5 and wild-type (p < 0.05), ** represents extremely significant difference between OX-PTCP5 and wild-type (p < 0.01), similarly hereafter. (D) Seedlings, bar = 2 cm. (E) Plants, bar = 5 cm. (F) Stem height. The number of samples from each strain was about 30. (G) Flower, bar = 1 cm. (H) Siliques, bar = 1 cm.

Figure 5

Seeds of OX-PtCP5: (A–J) thin sections from dry seeds of OX-PtCP5 and wild-type, bar = 100 μm. (K) Seed coat thickness. (L–O) PSVs in the cells of cotyledons and hypocotyls, bar = 8 μm.

Figure 6

Pollen of OX-PtCP5: (A,B) pollen germination of OX-PtCP5 and wild-type, bar = 500 μm. (C) Pollen germination rate. Pollen samples number of OX-PTCP5 and wild-type were 200 each. ** Represents extremely significant difference between OX-PTCP5 and wild-type (p < 0.01). (D,E) Pollen of wild-type and OX-PtCP5 Arabidopsis by scanning electron microscope, bar = 100 μm.