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. 2012 Sep 6;34(4):367–374. doi: 10.1007/s10059-012-0111-5

Overexpression of a LAM Domain Containing RNA-Binding Protein LARP1c Induces Precocious Leaf Senescence in Arabidopsis

Bangyue Zhang 1, Jianheng Jia 1, Min Yang 1, Chunxia Yan 1, Yuzhen Han 1,*
PMCID: PMC3887763  PMID: 22965746

Abstract

Leaf senescence is the final stage of leaf life history, and it can be regulated by multiple internal and external cues. La-related proteins (LARPs), which contain a well-conserved La motif (LAM) domain and normally a canonical RNA recognition motif (RRM) or noncanonical RRM-like motif, are widely present in eukaryotes. Six LARP genes (LARP1a-1c and LARP6a-6c) are present in Arabidopsis, but their biological functions have not been studied previously. In this study, we investigated the biological roles of LARP1c from the LARP1 family. Constitutive or inducible overexpression of LARP1c caused premature leaf senescence. Expression levels of several senescence-associated genes and defense-related genes were elevated upon overexpression of LARP1c. The LARP1c null mutant 1c-1 impaired ABA-, SA-, and MeJA-induced leaf senescence in detached leaves. Gene expression profiles of LARP1c showed age-dependent expression in rosette leaves. Taken together, our results suggest LARP1c is involved in regulation of leaf senescence.

Keywords: Arabidopsis, LARP1c, senescence, senescence-associated genes

INTRODUCTION

Leaf senescence, which is the final stage of leaf life history, is a self-regulatory developmental process influenced by growth and environmental factors. Dramatic changes in cell structure and metabolism occur during leaf senescence, including the degradation of chloroplasts, mitochondria, and nuclei, and simultaneous catabolism of chlorophyll pigments, nucleic acids, proteins, and lipids. The nutrients released from senescing leaves are transported to newly-developing leaves, fruits, or seeds (Buchanan-Wollaston et al., 2005; Lim et al., 2007a; Munné-Bosch and Alegre, 2004).

Leaf senescence can be induced by biotic or abiotic stresses in the environment, including pathogen infection, nutrient deficiency, and osmotic, light, and temperature stresses (Hopkins et al., 2007; Lim et al., 2007a; Zhou et al., 2009; 2011). Leaf senescence can also be initiated by internal signals, such as developmental cues and phytohormone balance. The internal or environmental cues that induce aging are perceived by plants, which then express senescence-associated genes (SAGs), resulting in dysfunctional developmental processes and, ultimately, the appearance of a senescence phenotype (Guo and Gan, 2005; Lim et al., 2007a). A large number of genes show enhanced or reduced expression during leaf senescence. Transcriptome analysis of senescent leaves indicated that approximately 2,500 genes (approximately 10%) in the Arabidopsis genome were expressed in senescent leaves (He et al., 2001). Microarray analysis showed that more than 800 SAGs were distinctively up-regulated during leaf senescence (Buchanan-Wollaston et al., 2005; Van Der Graaff et al., 2006).

Post-transcriptional regulation plays an essential role in the regulation of gene expression during plant growth, development, and response to external stresses. RNA-binding proteins are involved in multiple steps of post-transcriptional regulation, including pre-mRNA splicing, mRNA transport, localization, translation, and stability (Dreyfuss et al., 2002). Although many RNA-binding proteins are up- or down-regulated during leaf senescence, the biological functions of these genes are largely unknown (Buchanan-Wollaston et al., 2005; Van Der Graaff et al., 2006). Overexpression of three UBA2 genes (UBA2a, UBA2b, and UBA2c), which encode heterogeneous nuclear ribonucleoprotein (hnRNP)-type RNA-binding proteins, induce leaf senescence and hypersensitive-like cell death (Kim et al., 2008).

La-related proteins (LARPs), which contain a conserved La motif (LAM) domain and are normally immediately followed by a canonical RNA recognition motif (RRM) or noncanonical RRM-like (RRM-L) motif, are widely present in eukaryotes (Bayfield et al., 2010; Bousquet-Antonelli and Deragon, 2009). The co-evolving LAM-RRM/RRM-L regions are suggested to cooperate functionally for RNA (e.g., SnRNA and mRNA) binding activity (Bousquet-Antonelli and Deragon, 2009). Genuine La proteins specifically recognize and bind the 3′-UUU-OH of RNA polymerase III primary transcripts (Wolin and Cedervall, 2002), but knowledge of the function of the four LARP subfamilies (LARP1, 4, 6 and 7) is still limited (Bousquet-Antonelli and Deragon, 2009). The LARP1 subfamily members contain a LAM immediately followed by a noncanonical RRM-L5, with the exception of some members that lack this motif (Bousquet-Antonelli and Deragon, 2009). A DM15 box of unknown function is also found in the C-terminal region of some members of this subfamily (Bousquet-Antonelli and Deragon, 2009; Nykamp et al., 2008).

The yeast Saccharomyces cerevisiae LARP1 proteins Sro9p and Slf1p, which have a LAM but lack an adjacent RRM or DM15 box, are mainly cytoplasmic proteins able to bind translating mRNAs (Sobel and Wolin, 1999). Sro9p inhibits the activity of the transcript activator Hap1p by assembling a repressed protein complex, and this inhibition can be released by an increase in the intracellular heme concentration (Lan et al., 2004). The Drosophila melanogaster LARP1 was reported to be associated with the poly (A)-binding protein (PABP) and was important for spermatogenesis (Blagden et al., 2009; Chauvet et al., 2000; Ichihara et al., 2007). In Caenorhabditis elegans, Ce-LARP1 is required for normal oogenesis but does not affect the viability of the worm (Nykamp et al., 2008). CeLARP1 is localized in the procession bodies (P-bodies) where mRNA decay occurs, and null-mutant worms accumulated a high level of mRNAs in the Ras-MAPK pathway, indicating that CeLARP1 functions to attenuate specific mRNA levels by promoting mRNA decay (Nykamp et al., 2008). More recently, human LARP1 was shown to form a complex with PABP and the cap-binding protein eukaryote initiation factor 4E (eIF4E) (Burrows et al., 2010). Reduced expression of LARP1 in HeLa cells led to decreased global protein synthesis rates, and resulted in cell cycle arrest and an inhibition of cell migration (Burrows et al., 2010). Drosophila LARP1 appears to participate in a RNA-binding complex in which LARP1 is associated with 7SK and Bin3 (Bicoid interacting protein 3), a RNA methyltransferase that methylates the 5′ end of 7SK RNA, to serve as a scaffold for PABP and Ago2, which bind directly to mRNAs and negatively regulate initiation of translation (Singh et al., 2011).

Bioinformatic analyses of the LAM region showed that the Arabidopsis genome harbors six LARP genes (LARP1a-1c and LARP6a-6c) (Bayfield et al., 2010; Bousquet-Antonelli and Deragon, 2009), but their biological functions have not been studied previously. Here, we report that overexpression of LARP1c led to premature leaf senescence. A number of SAGs and defense-related genes were elevated upon overexpression of LARP1c. The LARP1c null mutant 1c-1 retarded abscisic acid (ABA)-, salicylic acid (SA)-, and methyl jasmonate (MeJA)-induced leaf senescence in detached leaves. Gene expression profiles of LARP1c showed age-dependent expression in rosette leaves. These data suggest that the Arabidopsis LARP1c is involved in regulation of leaf senescence.

MATERIALS AND METHODS

Plant materials and growth conditions

Arabidopsis plants were grown in an environmentally controlled culture room at 22°C with a relative humidity of ∼60% under long-day conditions (16 h light/8 h dark photoperiod). The TDNA insertional mutant 1c-1 (FLAG_372B03, Wassilewskija [WS] background) was obtained from the French National Institute for Agricultural Research. Gene-specific primers 1c-F, 1c-R, and LB4 (T-DNA left border primer for 1c-1) were used to isolate the T-DNA insertional homozygous lines. The sequences of primers used for T-DNA insertional analysis and gene expression in the mutant lines are shown in Supplementary Table 2. All Arabidopsis transgenic plants, including 1c-GUS, 1a-OE, 1b-OE, 1c-OE, 1c-DEX, and 1c-GFP, were generated by the floral dip method in the Col-0 background (Clough and Bent, 1998). Transgenic plants were selected on plates containing 25 mg L−1 hygromycin. Homozygous T3/T4 lines were used in the study unless otherwise specified. To induce LARP1c expression, 4-week-old 1c-DEX transgenic plants were once sprayed with 15 μM dexamethasone (∼1 mL per plant).

Plasmid constructs

For histological assays, the promoter region (1,453 bp) of LARP1c was amplified from Arabidopsis DNA and cloned into the pCAMBIA 1391 vector. To generate LARP1a, 1b, or 1c overexpression plasmids (1a-OE, 1b-OE or 1c-OE), the individual full-length coding sequence (CDs) was cloned into Super Promoter 1300 vector (pSuper 1300), a binary vector carrying the Cauliflower mosaic virus (CaMV) 35S promoter for overexpression. For DEX-inducible expression of LARP1c, the fulllength LARP1c CDs without stop codon was cloned into the pTA7002 vector, a dexamethasone (DEX)-inducible expression vector. To generate the 1c-GFP construct, the full-length LARP1c CDs without stop codon was fused to the 5′-terminal of the pSuper 1300-GFP vector, a binary vector generated from pSuper 1300. All of the PCR amplifications, enzyme digestions, and plasmid transformations used in plasmid constructs were performed using standard protocols. The sequences of primers and restriction enzymes used are presented in Supplementary Table 2.

Histological assays and subcellular localization assay

For histochemical analysis of β-glucuronidase staining (GUS) activity, the 1c-GUS transgenic materials were submerged in stain solution [50 mM sodium phosphate, pH 7.2, 0.5 mM K3Fe(CN)6, 0.5 mM K4Fe(CN)6 and 2 mM 5-bromo-4-chloro-3-indolyl-β-D-glucuronide (Sigma-Aldrich, China)], and subsequently incubated at 37°C for ∼3 h. The materials were then transferred to ethanol:acetic acid (3:1, v/v) solution to remove background pigmentation. The stained tissues were examined with an Olympus SZ16 stereomicroscope and Olympus SZX16-DP72 microscope (Olympus, Japan) and digital images recorded with a Canon G12 camera (Canon, Japan).

For subcellular localization assays, live roots of 5-day-old seedlings grown on Murashige and Skoog medium were used for GFP analysis. All tissues were examined with a Zeiss 510 Meta laser scanning confocal microscope (Zeiss, Germany).

Chlorophyll content and membrane ion leakage analysis

Chlorophyll was extracted from leaves and measured according to the protocol of Grbi and Bleecker (1995). The leaves used at each sampling time point were excised from at least three separate plants. Membrane ion leakage was measured as described by Guo and Gan (2006).

Trypan blue staining and hormone treatments

To visualize dying cells in senescing leaves, Trypan blue (TB) staining was performed similar to the method of Koch et al. (1990). Leaves were submerged in lactophenol-TB solution (0.1% TB, 50% methanol, 16.6% glycerol, 16.6% lactic acid, and 16.7% water-saturated phenol) and stained in a boiling water bath for 2 min. The samples were then transferred to 2.5 g mL−1 chloral hydrate solution to remove background pigmentation.

For hormone treatments, the 5th rosette leaves were collected from 4-week-old WS and 1c-1 mutant plants and placed adaxial side up on treatment solutions containing 3 mM 2-(4-morpholino) ethanesulfonic acid (MES) and either 20 μM ABA, 200 μM MeJA, 300 μM SA, or MES only (control). The leaves were incubated at 22°C under continuous illumination. The leaves were incubated in ABA or SA for 6 days and in MeJA for 5 days.

RNA isolation, reverse-transcription PCR and quantitative real-time PCR analysis

Total RNA was isolated from plant samples following a previously described protocol (Oñate-Sánchez, L., and Vicente-Carbajosa, J., 2008). First-strand cDNA was synthesized from RNA using the Promega M-MLV Reverse Transcription system according to the manufacturer’s protocol (Promega, China). The sample cDNA was used for subsequent analyses by reverse-transcription PCR (RT-PCR) and quantitative real-time PCR (qRT-PCR). For qRT-PCR, the ABI7500 Fast Real-Time PCR system (Applied Biosystems, USA) using the SYBR Green I Master Mix (Takara, Beijing) in a volume of 20 μL was employed. AT4G34270 was used as an internal control gene. The qRT-PCR reaction was performed following the protocol recommended by the manufacturer. For RT-PCR, the first-strand cDNA was used and PCR products were detected with GoldView (Newprobe, China), a novel dye for staining of nucleic acids. Actin2 was used as an internal control. All qRTPCR and RT-PCR primers can be found in Supplementary Table 2. All RT-PCR reactions were performed for 25–28 cycles.

RESULTS

Overexpression of LARP1c causes premature leaf senescence

To investigate the functions of LARP1c, we first used the T-DNA insertional mutant 1c-1 (FLAG_372B03, WS background), which has a T-DNA insertion in the second exon (Supplementary Fig. 1A). RT-PCR analysis showed that 1c-1 is a T-DNA knockout line (Supplementary Fig. 1B). Under the experimental growth conditions, the 1c-1 mutant plants showed normal growth and development almost identical to that of WS (Supplementary Fig. 1C). Because the LARP1c knockout line showed no obvious developmental defects, we employed gain-of-function approaches to investigate the roles of LARP1c. We obtained CaMV 35S promoter-driven overexpression lines of LARP1c. Fifty-seven of 85 T1 transformants with LARP1c overexpression (1c-OE) exhibited precocious leaf yellowing.

Six homozygous 1c-OE transgenic lines were generated, and their LARP1c transcript levels were detected by RT-PCR (Fig. 1A). Among them, four lines with higher expression levels of LARP1c displayed a leaf-yellowing phenotype 5 weeks after germination; the remaining two lines with expression levels similar to Col-0 plants showed no obvious leaf yellowing. The relative expression levels of LARP1c in lines 3 and 4 were examined by qRT-PCR (Fig. 1B), and these two lines were selected for further analysis.

Fig. 1.

Fig. 1.

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Screening of LARP1c overexpression (1c-OE) lines. (A) Seven-day-old seedlings of Col-0 and each of six homozygous lines were used for RNA extraction. The yellowing phenotype was observed by 5 weeks after germination. –, yellowing phenotype absent; +, yellowing phenotype present. (B) Relative expression of LARP1c in Col-0 and 1c-OE transgenic lines 3 and 4. Data represent the mean ± SE of three replicates.

Rosette leaves and cotyledons from 5- and 6-week-old Col-0 and 1c-OE plants are shown in Figs. 2A and 2B. The 1c-OE plants displayed premature leaf senescence compared with Col-0. The senescence phenotype was not observed in rosette leaves of 5-week-old Col-0 plants, but appeared in 2nd to 7th rosette leaves of 1c-OE transgenic plants (Fig. 2A); a more severe senescence phenotype was observed in 6-week-old 1c-OE plants (Fig. 2B). Decreased chlorophyll content (Fig. 2C) and increased membrane ion leakage (Fig. 2D) were also detected in 1c-OE plants.

Fig. 2.

Fig. 2.

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Accelerated leaf senescence in LARP1c overexpression (1c-OE) lines. Rosette leaves and cotyledons detached from (A) 5- and (B) 6-week-old Col-0 and 1c-OE transgenic lines #3 and #4. (C, D) Chlorophyll content (C) and membrane ion leakage (D) in 5th to 8th rosette leaves from plants in (A, B). Data represent mean ± SE of three replicates. Asterisks indicate significant differences between Col-0 and the 1c-OE lines (Student’s t-test, P < 0.01).

Cell death in the 4th rosette leaves of 5-week-old Col-0 and 1c-OE plants was examined by TB staining. Leaves of 1c-OE plants contained more dying cells than the wild type (Fig. 3A). RBCS1A, which encodes the small subunit of ribulose bisphosphate carboxylase, a key enzyme for CO2 assimilation, had decreased expression in 1c-OE plants (Fig. 3B), indicating that the leaves of transgenic lines had reduced photosynthetic activity.

Fig. 3.

Fig. 3.

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Cell death and expression of RBCS1A in leaves of Col-0 and LARP1c overexpression (1c-OE) transgenic lines 3 and 4. (A) Cell death of 4th rosette leaves from 5-week-old plants was examined by Trypan blue staining. Bar = 0.5 mm. (B) QRT-PCR analysis of RBCS1A expression levels. Relative transcript levels were normalized to AT4G34270. Data represent mean ± SE of three replicates. Asterisks indicate significant differences between Col-0 and the 1c-OE lines (Student’s t-test, P < 0.01).

Because the disruption of housekeeping or homeostatic genes could lead to early senescence symptoms, the premature leaf senescence phenotype observed in 1c-OE transgenic plants may be due to indirect disturbances of developmental processes. Inducible overexpression experiments could avoid potential complications in interpreting the phenotype (Woo et al., 2010). We applied a glucocorticoid-mediated transcriptional induction system (Aoyama and Chua, 1997) to generate conditional LARP1c transgenic plants. LARP1c was cloned into the pTA7002 vector, which harbors a DEX (a synthetic glucocorticoid)-inducible promoter. After DEX induction for 15 h, the relative expression of LARP1c in 4-week-old T2 transgenic lines was examined by qRT-PCR, and the leaf yellowing phenotype was recorded at later stages (Fig. 4). Only the inducible-lines with higher LARP1c expression levels displayed subsequent leaf yellowing phenotypes (Fig. 4), suggesting that overexpression of LARP1c was the cause of the phenotype.

Fig. 4.

Fig. 4.

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Quantitative RT-PCR analysis of LARP1c expression in dexamethasone (DEX)-inducible lines revealed a correlation between LARP1c overexpression and leaf yellowing. Four-week-old transgenic plants (T2 generation) were sprayed with DEX (15 μM), and aerial parts were collected 15 h later. The leaf yellowing phenotype was observed 4 days after application of DEX. –, yellowing phenotype absent; +, yellowing phenotype present. Data represent mean ± SE of three replicates.

Several 1c-DEX homozygous lines were generated for further analysis. After 3–4 days of DEX treatment, the 4-week-old 1c-DEX transgenic lines showed leaf yellowing in rosette leaves, but the transgenic plants harboring the empty pTA7002 vector displayed no yellowing after DEX induction (Fig. 5A). Gene expression analysis showed that LARP1c was induced in 1c-DEX transgenic plants (Fig. 5B). Consistent with the yellowing phenotype in the leaves of 1c-DEX transgenic lines (Fig. 5A), the chlorophyll content decreased rapidly (Fig. 5D) and the frequency of cell death increased markedly (Fig. 5C) after DEX treatment for 4 days. Based on these results and the senescence phenotype of 1c-OE lines, we conclude that LARP1c overexpression was responsible for the premature leaf senescence.

Fig. 5.

Fig. 5.

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Inducible overexpression of LARP1c led to precocious leaf senescence. (A) Phenotypes of transgenic lines after dexamethasone (DEX) induction. The image was taken 4 days after 4-week-old plants were sprayed with 15 μM DEX. (B) RT-PCR analysis of the expression of LARP1c in 5th to 8th rosette leaves of plants pictured in (A). (C) Cell death in 8th rosette leaves of plants pictured in (A). Bar = 0.1 mm. (D) Chlorophyll content of 5th to 8th rosette leaves after DEX treatment. Data represent mean ± SE of three replicates. Asterisks indicate significant differences between Col-0 and the 1c-DEX transgenic lines (Student’s t-test, P < 0.01).

Inducible overexpression of LARP1c elevates transcript levels of SAGs and defense-related genes

To confirm that LARP1c plays an important role in regulating leaf senescence, we analyzed the expression of several SAGs in the 1c-DEX transgenic lines after DEX induction for 24 and 48 h, before the transgenic plants showed signs of chlorosis. In parallel with LARP1c overexpression, increased expression of several SAGs, including SAG13 (Lohman et al., 1994), SEN4 (Park et al., 1998), and WRKY6 (Robatzek and Somssich, 2001), were observed (Fig. 6). SAG12 (cysteine proteinase), a highly senescence-specific marker gene (Noh and Amasino, 1999; Pontier et al., 1999), was also detected 72 h after treatment, when the transgenic plants showed signs of yellowing (data not shown). The regulatory pathway of leaf senescence has substantial cross-talk with plant defense signaling pathways (Quirino et al., 2000). Thus, we examined the expression levels of several defense-related genes by RT-PCR. Pathogenesis-related (PR) genes PR1 and PR2 are SA pathway defense genes (Mang et al., 2009). Phytoalexin deficient 4 (PAD4) and Enhanced Disease Resistance 1 (EDS1) act upstream from SA biosynthesis but are also induced by SA (Rustérucci et al., 2001). Plant defensin gene 1.2 (PDF1.2) is a marker gene frequently used to monitor jasmonate response (Pieterse et al., 2009). Arabidopsis aldehyde oxidase 3 (AAO3) and 9-cisepoxycarotenoid dioxygenase 3 (NCED3) encode key enzymes in the biosynthesis of ABA (Seo and Koshiba, 2002). All of these defense-related genes examined were upregulated in 1c-DEX transgenic lines after DEX treatment for 24 and 48 h (Fig. 6).

Fig. 6.

Fig. 6.

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Expression of SAGs and defense-related genes in control and 1c-DEX transgenic plants. Four-week-old Arabidopsis plants were sprayed with 15 μM dexamethasone (DEX), and 5th to 8th rosette leaves were collected at 0, 24, and 48 h after treatment. RTPCR was done for 25–28 cycles using gene-specific primers. Actin2 served as an internal control.

The LARP1c mutant 1c-1 impairs ABA-, SA-, and MeJA-induced leaf senescence

We showed above that inducible overexpression of LARP1c elevated transcript levels of SAGs and defense-related genes (Fig. 6). Thus, we tested the effects of senescence- and defense-related plant hormones ABA, SA, and MeJA on mutant 1c-1. Fifth rosette leaves were detached from 4-week-old plants and treated with ABA, SA, or MeJA. As shown in Fig. 7, ABA, SA and MeJA prompted the senescence of detached leaves in WS and 1c-1. However, compared with WS, the leaves from 1c-1 mutant displayed delayed yellowing (Fig. 7A) and retained more chlorophyll content (Fig. 7B) after ABA, SA and MeJA treatment for 6 or 5 days. These results suggested that LARP1c mediated ABA-, SA-, and MeJA-induced senescence of detached leaves.

Fig. 7.

Fig. 7.

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Leaf senescence responses of the WS and 1c-1 mutant to methyl jasmonate (MeJA), salicylic acid (SA), and abscisic acid (ABA) treatments. The 5th rosette leaves from 4-week-old plants were detached and incubated under continuous light in 2-(4-morpholino) ethanesulfonic acid (MES) buffer containing hormone or MES only as mock. Chlorophyll content was examined 0, 5, and 6 days after treatments. Data represent mean ± SE of four replicates. Asterisks indicate significant differences between WS and the 1c-1 mutant (Student’s t-test, 0.01≤ P < 0.05). Bar = 5 mm.

LARP1c expression in rosette leaves is age-dependent

To investigate the expression pattern of LARP1c during leaf development, qRT-PCR analyses at different leaf developmental stages were carried out. As shown in Fig. 8, transcript levels of LARP1c increased as leaves developed and senesced. The relative expression level of LARP1c in early-senescent leaves was double that in young leaves, and more was detected in late senescent leaves (Fig. 8).

Fig. 8.

Fig. 8.

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Quantitative RT-PCR analysis of LARP1c expression during leaf development in Arabidopsis. YL, young leaves, half the size of fully-expanded leaves; NS, non-senescent, fully-expanded leaves; ES, early-senescent leaves with < 25% leaf-area yellowing; LS, late-senescent leaves, with > 50% leaf-area yellowing. Relative transcript levels were normalized to AT4G34270. Data represent mean ± SE of three replicates.

To further confirm the expression pattern of LARP1c, a ∼1.5 kb promoter region upstream of the start codon was cloned and inserted into the pCAMBIA-1391 vector. Transgenic plants were generated and the GUS activity was analyzed in 4- to 7-week-old plants. As shown in Fig. 9, GUS activity was detected in the older rosette leaves of these plants, such as the 1st-5th rosette leaves of 5-week-old plants. Older plants had more rosette leaves with GUS activity, increasing from four in 4-week-old plants to eight in 7-week-old plants. Interestingly, some blue spots were also detectable at the leaf margins of younger rosette leaves, such as the 8th rosette leaves of 5-week-old plants (Supplementary Fig. 2A), which might be the initial sites of LARP1c expression. We also searched for LARP1c for gene expression information in AtGenExpress (http://jsp.weigelworld.org/expviz/expviz.jsp). As shown in Supplementary Table 1, LARP1c expression increased in senescing leaves compared with other rosette leaves examined, which is consistent with our expression data. These results indicated that LARP1c expression in rosette leaves was age-dependent. GUS activity was also detected in other tissues, such as seedlings and inflorescences (Supplementary Figs. 2B–2E), implying that LARP1c plays diverse roles in Arabidopsis.

Fig. 9.

Fig. 9.

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LARP1c promoter: GUS activity in rosette leaves of 4- to 7-week-old transgenic plants. The rosette leaves are pictured in order. Bar = 5 mm.

LARP1c is a cytoplasmic protein

To determine the subcellular localization of LARP1c in Arabidopsis, we generated the 1c-GFP construct that carried the CaMV 35S promoter for constitutive expression and obtained 1c-GFP transformants. The 1c-GFP transgenic lines displayed a premature leaf yellowing phenotype (Fig. 10A), which implied that GFP fusion did not interfere with the biological function of LARP1c. Thus, the 1c-GFP localization pattern was biologically relevant. Subcellular localization of the GFP fusion protein in the transgenic lines was analyzed by confocal laser scanning microscopy. In roots of 5-day-old seedlings, 1c-GFP fluorescence was largely localized to distinct cytoplasmic spots (Fig. 10B), in contrast with the cytoplasmic localization of 35S:GFP (Fig. 10C). This suggests that LARP1c is a cytoplasmic protein with foci distribution.

Fig. 10.

Fig. 10.

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Localization of LARP1c in the cytoplasm. (A) Phenotype of 30-day-old LARP1c-GFP transgenic (left) and 35S:GFP control (right) plants. The LARP1c-GFP transgenic line displayed a premature leaf senescence phenotype. (B) Confocal micrographs of 1c-GFP transgenic plant; GFP signal formed aggregates within the cytoplasm. (C) Confocal micrographs of 35S:GFP transgenic plant; GFP signal was evenly dispersed in the cytoplasm. Both GFP images were taken from mature root zones of 5-day-old transgenic plants. Bar = 10 μm.

Overexpression of LARP1b also causes a premature leaf yellowing phenotype

Do LARP1 members have similar functions in leaf senescence? To investigate this possibility, we also generated LARP1a and LARP1b overexpression lines. None of 12 transgenic T1 plants for LARP1a overexpression (1a-OE), regardless of increased LARP1a transcript levels, displayed premature leaf yellowing compared with wild type (data not shown). Fourteen of 74 transgenic T1 plants for LARP1b overexpression (1b-OE) displayed premature leaf yellowing (Fig. 11A) 6 weeks after germination. As expected, the transcript levels of SAGs and LARP1b were induced in 1b-OE transgenic lines (Fig. 11B). These findings suggested that LARP1b, similar to LARP1c, is involved in regulation of leaf senescence.

Fig. 11.

Fig. 11.

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Overexpression of LARP1b caused premature leaf senescence. (A) Leaf senescence phenotypes of Col-0 and LARP1b overexpression (1b-OE) transgenic lines. Compared with Col-0, 1b-OE transgenic lines showed premature leaf senescence. Rosette leaves and cotyledons were excised from soil-grown 7-week-old plants. Fourteen of 74 transgenic T1 plants displayed premature leaf senescence. (B) RT-PCR analysis of gene expression in leaves of Col-0 and 1b-OE lines. The 7th and 8th rosette leaves from 7-week-old plants of 1b-OE lines and Col-0 were used for RNA extraction. For all genes, 25 PCR cycles were employed. Actin2 was used as an internal control. The sequences of primers used for 1b-OE construct and RT-PCR analysis are presented in Supplementary Table 2.

DISCUSSION

Leaf senescence is accompanied by the activation of a subset of genes (e.g., SAGs) and the inactivation of another subset that represses the senescence program. Microarray data showed that expression of many genes that encode RNA-binding proteins is altered during leaf senescence (Buchanan-Wollaston et al., 2005), but functional information for RNA-binding proteins involved in leaf senescence is very limited. Kim et al. (2008) reported that overexpression of three hnRNP-type RNA-binding proteins (UBA2a, UBA2b, and UBA2c) induced leaf senescence and hypersensitive-like cell death. In the present study, we obtained evidence that a RNA-binding protein, LARP1c, in Arabidopsis is involved in regulating leaf senescence.

In animals, LARP1s are mainly cytoplasmic proteins that function in metabolism of specific mRNAs. LARP1 formed a complex with PABP and eIF4E and was localized to P-bodies, where mRNA decay occurs (Blagden et al., 2009; Burrows et al., 2010; Chauvet et al., 2000; Ichihara et al., 2007; Nykamp et al., 2008; Singh et al., 2011). Furthermore, LARP1 is involved in cellular remodeling, migration, and apoptosis (Burrows et al., 2010). Deletion of LARP1 leads to developmental defects during spermatogenesis in D. melanogaster (Blagden et al., 2009; Ichihara et al., 2007) and oogenesis in C. elegans (Nykamp et al., 2008). Thus, LARP1 members appear to bind to and regulate the stability and translational activities of target mRNAs.

In Arabidopsis, three members are classified into LARP1 subfamily (Bousquet-Antonelli and Deragon, 2009). No functional data for these LARP1 proteins has been reported previously. In this study, we employed gain-of-function approaches to investigate the function of LARP1s. Our results suggested that LARP1c has an important role in regulating leaf senescence. The first evidence came from constitutive overexpression of LARP1c under the control of the 35S promoter; plants with high LARP1c transcript levels displayed precocious leaf senescence (Fig. 1). The second line of evidence came from experiments in which LARP1c overexpression was induced. The inducible accumulation of LARP1c transcripts also caused premature leaf senescence (Fig. 4), as well as elevated transcript levels of SAGs, such as SAG13, SEN4, WRKY6 (Fig. 6), and SAG12. Furthermore, both RT-PCR and LARP1c promoter: GUS analyses confirmed that LARP1c expression was age-dependent (Figs. 8 and 9). More importantly, although the 1c-1 mutant showed no obvious developmental defects under normal condition, detached leaves of 1c-1 impaired ABA-, SA- and MeJA-mediated leaf senescence (Fig. 7). These observations suggested that LARP1c positively regulates leaf senescence.

The signaling pathway governing leaf senescence has substantial overlap with plant defense signaling pathways (Kim et al., 2008; Quirino et al., 2000). A number of defense-related genes are expressed during leaf senescence, and defense-related mutants also showed altered SAG expression (Quirino et al., 2000). Furthermore, ABA, SA, and MeJA have been shown to play important roles in both leaf senescence and defense signaling pathways (Adie et al., 2007; He et al., 2001; 2002; Lim et al., 2007b). We showed that several defense-related genes were upregulated upon overexpression of LARP1c (Fig. 6). The 1c-1 mutant impaired ABA-, SA- and MeJA-mediated leaf senescence (Fig. 7). These results suggest that LARP1c functioned not only in leaf senescence but also in defense signaling pathways.

Subcellular localization of 1c–GFP showed that LARP1c is a cytoplasmic protein with foci distribution (Fig. 10B). In C. elegans, CeLARP1 was reported to be a cytoplasmic protein that localizes to processing bodies (P-bodies) (Nykamp et al., 2008), which are sites of mRNA decay and storage. Interestingly, we found that 1c-GFP foci were sensitive to cycloheximide treatment, an inhibitor that can disrupt P-body formation (Goeres et al., 2007; Sheth and Parker, 2003) (data not shown). Does LARP1c localize to P-bodies? Colocalization analysis of LARP1c and P-bodies marker protein (e.g., DCP1 or DCP2) will be required to answer this question. However, one may reasonably assume that LARP1c assembles into P-bodies and regulates the dynamics of certain mRNAs that are required for initiation or facilitation of senescence in leaves.

In addition to LARP1c, there are two other LARP1 members, LARP1a and LARP1b, in Arabidopsis. Overexpression of LARP1b but not LARP1a caused premature leaf yellowing (Figs. 11A and 11B), but with minor effects compared with LARP1c overexpression. Thus, we inferred that LARP1b may have a similar role to LARP1c in regulating leaf senescence and that functional redundancy between LARP1b and LARP1c may exist. Further investigations of the effects of LARP1b on leaf senescence and on the phenotype of larp1b larp1c double mutants will reveal more about the roles of LARP1b and LARP1c in leaf senescence. For LARP1a, which might have a divergent role from other LARP1s during leaf senescence, carefully testing the LARP1a mutant may yield more information.

Supplementary Material

Acknowledgments

We thank Prof. Ying Fu for helpful discussions, Liwen Bianji (Edanz Group China) for manuscript correcting and editing. This work was supported by grants from the National Natural Science Foundation of China (Nos. 30670192 and 31070259).

Note:

Supplementary information is available on the Molecules and Cells website (www.molcells.org).

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