A Rice PECTATE LYASE-LIKE Gene Is Required for Plant Growth and Leaf Senescence

Abstract

To better understand the molecular mechanisms behind plant growth and leaf senescence in monocot plants, we identified a mutant exhibiting dwarfism and an early-senescence leaf phenotype, termed dwarf and early-senescence leaf1 (del1). Histological analysis showed that the abnormal growth was caused by a reduction in cell number. Further investigation revealed that the decline in cell number in del1 was affected by the cell cycle. Physiological analysis, transmission electron microscopy, and TUNEL assays showed that leaf senescence was triggered by the accumulation of reactive oxygen species. The DEL1 gene was cloned using a map-based approach. It was shown to encode a pectate lyase (PEL) precursor that contains a PelC domain. DEL1 contains all the conserved residues of PEL and has strong similarity with plant PelC. DEL1 is expressed in all tissues but predominantly in elongating tissues. Functional analysis revealed that mutation of DEL1 decreased the total PEL enzymatic activity, increased the degree of methylesterified homogalacturonan, and altered the cell wall composition and structure. In addition, transcriptome assay revealed that a set of cell wall function- and senescence-related gene expression was altered in del1 plants. Our research indicates that DEL1 is involved in both the maintenance of normal cell division and the induction of leaf senescence. These findings reveal a new molecular mechanism for plant growth and leaf senescence mediated by PECTATE LYASE-LIKE genes.

Figure 1.

Comparison of phenotype between wild-type and del1 plants. A, Wild-type (Nipponbare, left) and del1 plants (right) 5 d after sowing. Scale bar = 2 cm. B, Root length of wild-type (left) and del1 plants (right) 5 d after sowing. Scale bar = 1 cm. C and D, Statistical analysis of root length and lateral root number between wild-type and del1 plants. Twenty plants were measured. Error bars indicate sd; **P < 0.01 (Student’s t test). E, Wild-type (left) and del1 plants (right) at maturity. Scale bar = 10 cm. F and G, Statistical analysis of plant height and tiller number between wild-type and del1 plants. Twenty plants were measured. Error bars indicate sd; **P < 0.01 (Student’s t test). H, Phenotype of panicle between wild-type (left) and del1 (right) plants. Scale bar = 5 cm. I, Floret with the lemma removed, wild-type (left) and del1 (right). Scale bar = 0.5 cm. J, Mature seed and brown rice of wild type (left) and del1 (right). Scale bar = 0.5 cm. K and L, Statistical analysis of panicle length and thousand grain weight between wild-type and del1 plants. Twenty panicles were measured. Error bars indicate sd; **P < 0.01 (Student’s t test).

Figure 2.

Histological characterization of culms in wild-type and del1 plants. A to D, Cross sections of internode II of wild type (A) and del1 (B). Scale bar = 500 μm. C, Magnification of A; D, magnification of B; white rectangle shows a magnification of the sclerenchyma cell layer. Scale bar = 50 μm. E and F, Statistical analysis of cell number and sclerenchyma cell layer number between wild-type and del1 plants, means ± sd of five independent replicates. G and H, Longitudinal sections of internode II of wild type (G) and del1 (H). Scale bar = 50 μm. I and J, Statistical analysis of the cell length and cell width between wild-type and del1 plants, mean ± sd of 30 cells. **P < 0.01 (Student’s t test). K, Number of parenchyma cells (PCs) for internode II of wild-type and del1 plants, mean ± sd of five independent replicates.

Figure 3.

Cell cycle analysis of wild-type and del1 plants. A and B, Flow karyotype histogram of wild-type (A) and del1 (B) leaves. C, Quantification of the DNA profiles of wild-type and del1 plants. D, Relative expression levels of cell cycle-related genes in wild-type and del1 plants, mean ± sd of three independent replicates. *P < 0.05, **P < 0.01 (Student’s t test).

Figure 4.

Leaf phenotype and identification of leaf senescence in DEL1. A to D, Leaf phenotype from young (A), tillering (B and C), and heading (D) stages. Scale bars = 1, 5, 5, and 1 cm in A, B, C, and D, respectively. E, Statistical analysis of chlorophyll content between wild-type and del1 plants, mean ± sd of five independent replicates. **P < 0.01 (Student’s t test). F and G, Transmission electron microscopy analysis of senescence leaves of wild-type (F) and del1 plants (G). Scale bar = 0.5 μm. H, Statistical analysis of photosynthesis rate between wild-type and del1 plants, mean ± sd of five independent replicates. **P < 0.01 (Student’s t test). I, Relative expression levels of senescence-related genes and transcription factors in wild-type and del1 plants, mean ± sd of three independent replicates. *P < 0.05, **P < 0.01 (Student’s t test).

Figure 5.

ROS accumulation and enhancement of PCD in wild-type and del1 leaves. A and B, NBT and DAB staining of leaves between the wild-type (left) and del1 plants (right). C to E, Statistical analysis of electrolyte leakage (C), SOD activity (D), and POD activity (E) in leaves between wild-type and del1 plants, mean ± sd of five independent replicates. **P < 0.01 (Student’s t test). F, Relative expression levels of ROS detoxification-related genes in wild-type and del1 plants, mean ± sd of three independent replicates. **P < 0.01 (Student’s t test). G, Trypan blue staining of leaves in wild-type (left) and del1 plants (right). H to K, TUNEL assay of leaves. DAPI staining of wild-type (H) and del1 plants (J). Positive results of wild-type (I) and del1 plants (K). Scale bar = 50 μm.

Figure 6.

Map-based cloning and identification of DEL1. A, Fine mapping of DEL1. The del1 locus was mapped to a 45-kb region on chromosome 10. B, Schematic diagram of DEL1. Black rectangles represent exons. Black inverted triangle represents mutant site. C, Sequencing analysis of the DEL1 transcripts in T₀ transgenic lines. D and E, Phenotype of the complementation transgenic line: Wild type (left), complementation transgenic line (middle), and empty vector control (right). Scale bars = 10 cm and 4 cm, respectively. F, Expression levels of DEL1 detected by qRT-PCR in wild-type and transgenic plants, mean ± sd of three independent replicates. G to I, Statistical analysis of plant height (G), tiller number (H), and flag leaf length (I) in wild-type and transgenic plants, mean ± sd of 10 independent replicates.

Figure 7.

Prediction of the primary sequence and phylogenetic analysis of DEL1. A, The deduced amino acid sequence of DEL1. Numbers on the left refer to the positions of amino acid residues. The signal peptide is indicated with an underline; the PelC domain is shown by a dotted line; the conserved residues involved in Ca²⁺ binding (red background), disulfide bonds (orange background), catalysis (blue background), and substrate binding (purple background). B, Phylogenetic tree of PEL in Arabidopsis, rice, and other plants. The numbers at each node represent the bootstrap support (percentage), and scale bar is an indicator of genetic distance based on branch length.

Figure 8.

Expression analysis of DEL1. A, Transcription level of DEL1 in various organs, mean ± sd of three independent replicates. YR, Young root; YL, young leaf; YS, young sheath; MR, mature root; C, culm; ML, mature leaf; MS, mature sheath; P, panicle; S, spikelet. B to J, GUS analysis of DEL1 expression: B and C, Four and seven days after germination of the young plant, scale bar = 1 cm; D, root, scale bar = 500 μm; E, lateral root, scale bar = 250 μm; F, mature sheath, scale bar = 1 cm; G, mature leaf, scale bar = 1 cm; H, culm, scale bar = 1 cm; I, spikelet, scale bar = 1 cm; J, lemma and palea were removed in I, scale bar = 500 μm.

Figure 9.

The levels of PEL activity and cell wall composition and structure in wild-type and del1 plants. A, Analysis of PEL activity in wild-type and del1 plants, mean ± sd of five independent replicates. **P < 0.01 (Student’s t test). B, Comparison of cell wall composition between wild-type and del1 plants, mean ± sd of five independent replicates. **P < 0.01 (Student’s t test). HC 1, Hemicellulose 1; HC 2l hemicellulose 2. C, Neutral monosaccharide composition between wild-type and del1 plants, mean ± se of five independent replicates, **P < 0.01 (Student’s t test). D and E, Transmission electron microscopy micrographs of the bundle sheath fiber cells of wild-type (D) and del1 (E) plants, scale bar = 1 μm. F, Magnification in D, and G. magnification in E, scale bar = 0.2 μm. ml, Middle lamella; pw, primary cell wall; sw, secondary cell wall; pm, plasma membrane; c, cytoplasm. H to J, Statistical analysis of the middle lamella (H), primary cell wall (I), and secondary cell wall (J) thicknesses of bundle sheath fiber cells between the wild-type and del1 plants, mean ± sd of 30 cells, **P < 0.01 (Student’s t test).

Figure 10.

Immunohistochemical localization of HG in culm sections of wild-type and del1 plants. A to T, Immunolocalization of HG of wild-type (A and C) and del1 plants (B and D) with JIM7, wild-type (E and G) and del1 plants (F and H) with JIM5, wild-type (I and K) and del1 plants (J and L) with LM18, wild-type (M and O) and del1 plants (N and P) with LM19, and wild-type (Q and S) and del1 plants (R and T) with 2F4. Scale bar = 50 μm.

Figure 11.

A schematic model of DEL1 function in rice. Homogalacturonan is secreted in a highly methylesterified form and selectively demethylesterified by PME. The demethylesterified HG might be cleaved by DEL1 and other PELs or PGs. The alternative of the cell wall regulated the cell cycle/expansion and ROS, enabling normal rice growth and leaf senescence process.