Suppression of OsVPE3 Enhances Salt Tolerance by Attenuating Vacuole Rupture during Programmed Cell Death and Affects Stomata Development in Rice

Abstract

Background: Vacuolar processing enzymes (VPEs) are cysteine proteinases that act as crucial mediators of programmed cell death (PCD) in plants. In rice, however, the role of VPEs in abiotic stress-induced PCD remains largely unknown. In this study, we generated OsVPE3 overexpression and suppression transgenic lines to elucidate the function of this gene in rice.

Results: Survival rate and chlorophyll retention analyses showed that suppression of OsVPE3 clearly enhanced salt stress tolerance in transgenic rice compared with wild type. Furthermore, fragmentation of genomic DNA was inhibited in plants with down-regulated OsVPE3. Vital staining studies indicated that vacuole rupture occurred prior to plasma membrane collapse during salt-induced PCD. Notably, overexpression of OsVPE3 promoted vacuole rupture, whereas suppression of OsVPE3 attenuated or delayed the disintegration of vacuolar membranes. Moreover, we found that suppression of OsVPE3 caused decreased leaf width and guard cell length in rice.

Conclusions: Taken together, these results indicated that suppression of OsVPE3 enhances salt tolerance by attenuating vacuole rupture during PCD. Therefore, we concluded that OsVPE3 plays a crucial role in vacuole-mediated PCD and in stomatal development in rice.

Keywords: OsVPE3; Programmed cell death; Rice; Salt stress; Stomata; Vacuolar processing enzyme.

Fig. 1

Sequence analysis, subcellular localization and expression pattern of OsVPE3. a The OsVPE3 gene structure based on the genomic sequence and full-length cDNA clone (BAC41387). Black boxes represent exons. Arrows indicate the primers used to isolate OsVPE3cDNA. b OsVPE3 amino acid sequence. Amino acids necessary for caspase-like activity are indicated by coloured letters. c The subcellular localization of OsVPE1 and OsVPE3 by confocal fluorescent microscopy. Scale bar = 10 μm. d qRT-PCR analysis of OsVPE3 expression in various rice tissues. Total RNA was extracted from leaf, root, booting panicle and immature embryo samples. OsUBQ5 was used as an internal control. Values are means, and error bars represent the SD from three independent experiments

Fig. 2

Generation and analysis of transgenic lines. A diagram of the vector constructs used to generate the overexpression lines (a) and RNAi line (b). c Expression levels of OsVPE3 in WT and transgenic lines were determined by qRT-PCR. OsUBQ5 was used as an internal control. Values are means, and error bars represent the SD (n > 1) from three independent experiments. Asterisks indicate a significant difference between WT and transgenic lines (t-test; **, P < 0.01). d SDS-PAGE analysis of seed storage proteins in WT and transgenic lines. The intensity of protein bands was measured by Image J software. Arrow indicates the 57-kDa glutelin precursor

Fig. 3

Suppression of OsVPE3 improved survival rate and inhibited chlorosis under salt stress. a Plants were grown for 4 weeks, treated with 150 mMNaCl for 3 days and then allowed to recover for 7 days. b Expression levels of OsVPE3 in WT and transgenic lines as determined by qRT-PCR. Total RNA was extracted from the fourth leaves. OsUBQ5 was used as an internal control. Values are means, and error bars represent the SD from three independent experiments. Asterisks indicate a significant difference between WT and transgenic lines (t-test; **, P < 0.01). c The fourth leaves of NaCl-treated plants. Bar = 1 cm. d Chlorophyll retention in the fourth leaves of NaCl-treated plants prior to recovery. Values are means, and error bars represent the SD from three independent experiments. Asterisks indicate a significant difference between WT and transgenic lines (t-test; *, P < 0.05). e Survival rates of NaCl-treated plants 7 days after recovery. Values are means, and error bars represent the SD (n > 20) of three independent experiments. Asterisks indicate a significant difference between WT and transgenic lines (t-test; *, P < 0.05)

Fig. 4

Inhibition of the primary root growth and DNA ladder formation in WT and transgenic lines under salt stress. a Three-day-old seedlings were treated with 0, 100 and 150 mM NaCl for 5 days. The root relative elongation rates were calculated as described in the Materials and methods. Values are means, and error bars represent the SD of three independent experiments n > 30) and the different letters denote the significant difference among different lines (ANOVA, P < 0.05). b Primary root cell viability was presented using Evans Blue dye in WT and transgenic lines treated with 0 or 150 mMNaCl for 3 days. Bars = 200 μm. c DNA ladder formation after treatment with 300 mMNaCl for 8 h in root tips. d Relative expression levels of OsVPE homology genes determined by qRT-PCR. Total RNA was extracted from the roots. OsUBQ5 was used as an internal control. Values are means, and error bars represent the SD from three independent experiments

Fig. 5

Suppression of OsVPE3 alleviates vacuole rupture under salt stress. a Protoplasts from WT and transgenic lines were stained with the BCECF-AM vital dye (final concentration = 10 μM) for 2 h and then treated with 100 mM NaCl for 3 h. Before the BCECF fluorescent images were inspected, the protoplasts were stained with Trypan Blue (TB). Type 1 protoplasts were living cells with intact vacuoles. Type 2 protoplasts were living cells with disintegrated vacuolar membranes. Type 3 protoplasts were dead. b Statistical analysis of three types of protoplasts after treatment with 100 mM NaCl. Data represent the means ± SD (n > 100) for three independent replicates. Asterisks indicate a significant difference between the WT and transgenic lines (t-test; *, P < 0.05)

Fig. 6

Fourth leaf width and guard cell size in WT and transgenic lines. a Images of fourth leaves of WT and transgenic lines. Bars = 1 cm. b Measurements of the maximum fourth leaf width in WT and transgenic lines. Values indicate the mean, and error bars represent the SD (n >10). An asterisk indicates a significant difference between the WT and transgenic lines (t-test; P < 0.05). c Images of stomata in WT and transgenic lines. Bars = 5 μm. d Percentage of stomata with various guard cell lengths in WT and transgenic lines (n > 500)

Fig. 7

Effect of OsVPE3 on the expression levels of genes related to stomatal development. Expression levels of OsTMM, OsSPCH1, OsSPCH2, and OsMUTE in WT and transgenic lines as determined by qRT-PCR. Total RNA was extracted from the shoots of 5-day-old plants. OsUBQ5 was used as an internal control. Values are means, and error bars represent the SD from three independent experiments. Asterisks indicate a significant difference between WT and transgenic lines (t-test; **, P < 0.01)