LMPA Regulates Lesion Mimic Leaf and Panicle Development Through ROS-Induced PCD in Rice

Abstract

Leaf and panicle are important nutrient and yield organs in rice, respectively. Although several genes controlling lesion mimic leaf and panicle abortion have been identified, a few studies have reported the involvement of a single gene in the production of both the traits. In this study, we characterized a panicle abortion mutant, lesion mimic leaf and panicle apical abortion (lmpa), which exhibits lesions on the leaf and causes degeneration of apical spikelets. Molecular cloning revealed that LMPA encodes a proton pump ATPase protein that is localized in the plasma membrane and is highly expressed in leaves and panicles. The analysis of promoter activity showed that the insertion of a fragment in the promoter of lmpa caused a decrease in the transcription level. Cellular and histochemistry analysis indicated that the ROS accumulated and cell death occurred in lmpa. Moreover, physiological experiments revealed that lmpa was more sensitive to high temperatures and salt stress conditions. These results provide a better understanding of the role of LMPA in panicle development and lesion mimic formation by regulating ROS homeostasis.

Keywords: cell death; lesion mimic; panicle apical abortion; plasma membrane H+-ATPase; reactive oxygen species; rice.

Figure 1

Phenotypic characterization of the lmpa mutant. (A) Phenotypes of the mature wild-type (WT) plant and lmpa mutant. The arrow indicates the degenerated spikelets. (B) Flag leaf phenotype in WT and lmpa mutant. (C) Phenotypic comparison of mature panicles between WT and lmpa mutant. (D) Magnified view of the white boxed areas in (C). (E–I) Comparison of spikelet abortion rate (E), panicle length (F), plant height (G), number of grains per panicle (H), and 1,000-grain weight (I) between the WT and lmpa mutant. Error bars represent standard deviation (SD) (n = 10); **Significant difference at p < 0.01 compared with the WT by Student's t-test. NDS, no degenerated spikelet. Bars =10 cm in (A), 5 cm in (B), 2 cm in (C), and 1 cm in (D).

Figure 2

Histological characterization of WT and lmpa mutant. (A–C) Cross-sections of WT apical spikelet hulls. (D–F) Cross-sections of lmpa apical spikelet hulls. (G) Cross-sections of lmpa leaves. (H) Normal part of lmpa leaves, magnified view of the white boxed areas in (G). (I) Spotted part of lmpa leaves, magnified view of the white boxed areas in (G). le, lemma; pa, palea; st, stamen; sc, silicified cells; nc, non-silicified cells; fs, fibrous sclerenchyma; spc, spongy parenchymatous cells. SC(ab), abaxial sclerenchyma; BC, bulliform cells; P, phloem; X, xylem; VBS, vascular bundle sheath; MC, mesophyll cells; SC(ad), adaxial sclerenchyma. Bars= 400 μm in (A,D,G), 50 μm in (B,C,E,F,H,I).

Figure 3

Detection of DNA fragmentation by the TUNEL assay. (A–F) TUNEL assay of leaves in WT (A–C) and lmpa mutant (D–F). (G–L) TUNEL assay of apical spikelet hull cells in WT (G–I) and in lmpa (J–L). (M–R) TUNEL assay of apical spikelet anthers in WT (M–O) and in lmpa (P–R). TUNEL-positive signals are indicated by the green fluorescence of fluorescein, and nuclei fluoresce deep red signals due to counterstaining with DAPI. le, lemma; pa, palea; C, connective tissue; V, vascular bundle cells; T, tapetum. Bars = 20 μm in (A–L), 50 μm in (M–R).

Figure 4

Determination of ROS content in the wild type. (A) DAB straining in leaves of WT (left) and lmpa (right). (B) NBT staining in leaves of WT (left) and lmpa (right). (C–H) Measurement of H₂O₂ content (C), MDA content (D), APX activity (E), POD activity (F), SOD activity (G), and CAT activity (H) in leaves. (I–M) Expression levels of OsVPE2 (I), OsVPE3 (J), CATA (K), CATB (L), and CATC (M) in leaves. Error bars represent standard deviation (SD) (n = 3); **Significant difference at p < 0.01 compared with the WT by Student's t-test. Bars = 1 cm in (A,B).

Figure 5

Map-based cloning of lmpa. (A) Fine mapping of lmpa. (B) Gene structure of the candidate gene. (C) Agarose electrophoresis confirmation of the mutation. DNA fragments were amplified using pF and pR primers indicated in (B). (D) Deletion mutation at the target site in two representative knockout lines generated by the CRISPR/Cas9 technology. (E) Expression levels of lmpa in WT, lmpa mutant, and two knockout mutant lines. (F–H) Phenotypes of WT and knockout mutant plants. Error bars represent standard deviation (SD) (n = 3); **Significant difference at p < 0.01 compared with the WT by Student's t-test. Bars = 10 cm in (F), 1 cm in (G,H).

Figure 6

Promoter activity analysis. (A,B) GUS staining of Pro^WT-GUS and Pro^lmpa–GUS transgenic plants. (C) The GUS activity in WT and lmpa roots at the seedling stage. (D,E) Transient expression assays of the WT promoter and lmpa promoter. Error bars represent standard deviation (SD) (n = 3); **Significant difference at p < 0.01 compared with the WT by Student's t-test. Bars = 1 cm in (A,B).

Figure 7

Expression pattern and subcellular localization of the LMPA protein. (A) Relative expression of LMPA in various tissues. (B) Tissue-specific expression of the GUS gene driven by the LMPA promoter. (C–J) Plasma membrane localization of LMPA in LMPA-GFP transgenic plants (C–F) and leaf cells of N. benthamiana (G–J). FM4-64 was used as a membrane marker. Error bars represent standard deviation (SD) (n = 3). Bars = 1 cm in (B), 50 μm in (C–J).

Figure 8

Temperature treatment, and DAB and NBT staining. (A,B) Phenotypes of WT and lmpa mutant under different temperatures. (C,D) DAB staining of leaves grown at 30°C (C) and 20°C (D). (E,F) NBT staining of leaves grown at 30°C (E) and 20°C (F). Bars = 2 cm in (A,B).