Molecular Plant Advance Access published June 3, 2014 RESEARCH ARTICLE
Molecular Plant
A Novel Chloroplast-Localized Pentatricopeptide Repeat Protein Involved in Splicing Affects Chloroplast Development and Abiotic Stress Response in Rice Junjie Tana,b,2,
Zhenhua Tana,b,2,
Fuqing Wub,2,
Peike Shenga,b,
Yueqin Hengb,
Xinhua Wangc,
Yulong Renb,c, Jiulin Wangb, Xiuping Guob, Xin Zhangb, Zhijun Chengb, Ling Jiangc, Xuanming Liua, Haiyang Wangb, and Jianmin Wanb,c,1
ABSTRACT Pentatricopeptide repeat (PPR) proteins comprise a large family in higher plants and modulate organellar gene expression by participating in various aspects of organellar RNA metabolism. In rice, the family contains 477 members, and the majority of their functions remain unclear. In this study, we isolated and characterized a rice mutant, white stripe leaf (wsl), which displays chlorotic striations early in development. Map-based cloning revealed that WSL encodes a newly identified rice PPR protein which targets the chloroplasts. In wsl mutants, PEP-dependent plastid gene expression was significantly down-regulated, and plastid rRNAs and translation products accumulate to very low levels. Consistently with the observations, wsl shows a strong defect in the splicing of chloroplast transcript rpl2, resulting in aberrant transcript accumulation and its product reduction in the mutant. The wsl shows enhanced sensitivity to ABA, salinity, and sugar, and it accumulates more H2O2 than wild-type. These results suggest the reduced translation efficiency may affect the response of the mutant to abiotic stress. Key words: PPR protein; WSL; ABA; chloroplast; RNA splicing; rpl2; abiotic stress responses. Tan J. J., Tan Z. H., Wu F. Q., Sheng P. K., Heng Y. Q., Wang X. H., Ren Y. L., Wang J. L., Guo X. P., Zhang X., Cheng Z. J., Jiang L., Liu X. M., Wang H. Y., and Wan J. M. (2014). A novel chloroplast-localized pentatricopeptide repeat protein involved in splicing affects chloroplast development and abiotic stress response in rice. Mol. Plant. 00, 1–22.
Introduction The eukaryotic pentatricopeptide repeat (PPR) gene family was first discovered by bioinformatics analysis while screening Arabidopsis proteins targeted to mitochondria and chloroplasts (Lurin et al., 2004). In higher plants, the PPR family contains many members, with 450 in Arabidopsis and 477 in rice (Lurin et al., 2004; Schmitz-Linneweber and Small, 2008). PPR proteins usually contain 2–26 tandem arrays of a degenerate 35 amino acid repeat (PPR motif), which shows a structure similar to the tetratricopeptide (TPR) motif (Fisk et al., 1999; Small and Peeters, 2000; Lurin et al., 2004). PPR motifs have been classified into three types: P, L, or S, according to size and variation. Based on their motifs, PPR proteins were classified into P and PLS subfamilies (Lurin et al., 2004). The PLS subfamily was further divided into PLS, E, E+, and DYW subgroups based on
the presence or absence of C-terminal motifs (Lurin et al., 2004). The majority of these proteins are localized to chloroplasts or mitochondria (Lurin et al., 2004; Ding et al., 2006). Posttranscriptional modulation of gene expression plays a very important role in controlling gene activity in
1 To whom correspondence should be addressed. E-mail wanjm@ njau.edu.cn, [email protected], tel. +86-25-84396516, fax +86-25-84396516. 2 These authors contributed equally to this work. © The Author 2014. Published by the Molecular Plant Shanghai Editorial Office in association with Oxford University Press on behalf of CSPB and IPPE, SIBS, CAS. doi:10.1093/mp/ssu054 Received 7 January 2014; accepted 23 April 2014
Downloaded from http://mplant.oxfordjournals.org/ at North Dakota State University on October 28, 2014
a State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Biology, Hunan University, Changsha 410082, P.R. China b National Key Facility for Crop Gene Resources and Genetic Improvement, Institute of Crop Science, Chinese Academy of Agricultural Sciences, Beijing 100081, P.R. China c National Key Laboratory for Crop Genetics and Germplasm Enhancement, Nanjing Agricultural University, Nanjing 210095, P.R. China
2
Roles of WSL in Chloroplast Development and Abiotic Stress
In the current study, we isolated and characterized a rice mutant wsl, which shows white stripes on the leaves as well as altered responses to ABA, sugar, and salinity early in development. By map-based cloning, we isolated the WSL gene and demonstrated that it encodes a PPR protein. Further investigation showed wsl exhibited reduced translation efficiency which was most likely to result from abnormal splicing of rpl2 gene. Our work indicated the reduced translation efficiency may affect the response of the mutant to abiotic stress.
RESULTS Phenotypic Characteristics of the wsl Mutant The wsl mutant developed leaves with white stripes before the fifth leaf stage, particularly at the third leaf (L3) stage. Newly developed leaves after the fifth leaf stage displayed normal green color. The white stripes appeared to be randomly distributed on the surface of the mutant leaves, but were predominantly near the leaf margins (Figure 1A–1E). The chlorophyll and carotenoid contents in leaves of wsl mutant were much lower than in wild-type (WT) before the five-leaf stage, but were similar to the WT thereafter (Supplemental Figure 1). The white-striped leaves never reverted to normal green as the plants developed (Figure 1F). At maturity, the major agronomic traits of the mutant, including plant height and seed size, were indistinguishable from those of WT plants (Figure 1G and 1H, and Supplemental Table 1). Additionally, the degree of whitestriping was influenced by temperature and light. The phenotypes of wsl plants were more apparent in conditions of low temperature or high light intensity (Supplemental Figure 2). Previous studies suggested a close link between the formation of photosynthetic pigments and the development of chloroplasts (Su et al., 2012). Therefore, we compared the ultrastructure of chloroplasts among white and green sectors of wsl mutant leaves and WT normal leaves using transmission electron microscopy. Cells in WT leaves contained normal chloroplasts displaying well-organized lamellar structures and were equipped with normally stacked grana and thylakoid membranes (Figure 1I). However, most cells within the white sectors in wsl mutants were heteroplastidic and contained non-pigmented plastids which were severely vacuolated and lacked organized lamellar structures (Figure 1J). Some of cells had no plastids (Figure 1K) and, more seriously, their morphologies were abnormal (Figure 1L). The cells in green sectors of wsl leaves contained apparently normal chloroplasts similar to those in WT leaves (Figure 1M). These observations implied that WSL plays an important role in chloroplast development as well as maintaining normal cell activity in leaves at an early seedling stage.
Downloaded from http://mplant.oxfordjournals.org/ at North Dakota State University on October 28, 2014
organelles. PPR protein is a RNA-binding protein and fulfills an important role in regulating organelle gene expression by controlling RNA editing, splicing, stability, or translation (Lurin et al., 2004; Schmitz-Linneweber and Small, 2008). A combinatorial amino acid code for sequence-specific RNA recognition by PPR tracts was recently proposed (Barkan et al., 2012). RNA editing acts as an important step in posttranscriptional control of organelle gene expression, by modifying transcript sequences mainly by converting cytidine (C) to uridine (U) so that it differs from that encoded in the organelle genome. Besides editing, splicing of introns is also required for many organellar transcripts in plants. Chloroplast genomes in angiosperms contain approximately 20 introns, the majority of which are classified as group II introns based on their similarity to self-splicing group II intron ribozymes. However, none of them has been reported to self-splice in vitro and there has been more recent evidence for the involvement of multiple nucleus-encoded proteins in the splicing of each chloroplast intron in vivo (de Longevialle et al., 2010). Considerable progress has been made in identifying these proteins and to date approximately 15 nuclear genes have been reported to facilitate splicing of one or more chloroplast introns (de Longevialle et al., 2010; Khrouchtchova et al., 2012; Germain et al., 2013). However, no such proteins have been reported in rice. Accumulating data from previous studies reveal that PPR proteins regulate a wide variety of biological processes, such as cytoplasmic male sterility (CMS) (Bentolila et al., 2002; Brown et al., 2003; Koizuka et al., 2003; Hu et al., 2012), embryogenesis (Cushing et al., 2005; Sosso et al., 2012), chloroplast development (Chi et al., 2008; Su et al., 2012), retrograde signaling (Kobayashi et al., 2007; Laluk et al., 2011), and seed development (Liu et al., 2013). Three PPR proteins were shown to be involved in abiotic stress responses in plants. In Arabidopsis, PPR40 is localized to mitochondria and is involved in the regulation of ubiquinol-cytochrome c reductase activity of complex III in the mitochondrial electron transport chain. A ppr40 mutant showed enhanced sensitivity to ABA and salinity in addition to accumulating ROS and altered stressresponsive gene expression (Zsigmond et al., 2008). PGN is a mitochondrion-localized PPR protein that regulates ROS homeostasis and stress-induced ABA responses (Laluk et al., 2011). PGN inactivation leads to ROS accumulation and down-regulation of stress-inducible genes resulting in susceptibility to necrotrophic fungal pathogens as well as enhanced abiotic stress sensitivity (Laluk et al., 2011). So far, only one chloroplast-localized PPR protein, GUN1, is reported to be involved in abiotic responses. GUN1 modulates the expression of nuclear-encoded ABI4 and photooxidative stress responses (Kobayashi et al., 2007). Two chloroplast-localized PPR proteins have been reported in rice (Gothandam et al., 2005; Su et al., 2012), but neither is associated with abiotic stress responses.
Molecular Plant
Molecular Plant
Roles of WSL in Chloroplast Development and Abiotic Stress
3
(A–E) Phenotypes of wild-type (left) and wsl mutant (right) seedlings at the one- (A), two- (B), three- (C), four- (D), and five- (E) leaf stages. The mutant developed leaves (especially the third leaf) with white stripes predominantly along the edges. The white box above the seedlings shows enlarged views of the regions indicated in wild-type and wsl leaves, respectively. Bars = 1 mm. (F) The juvenile white-striped leaves never reverted to green, even in mature plants. (G) Phenotypes of WT (left) and wsl (right) plants at the heading stage. (H) Comparison of the WT and wsl seeds (de-hulled). (I–M) Transmission electron microscopic images of cells from wild-type, white, and green sectors of wsl mutant at the three-leaf stage. (I) Mesophyll cells in wild-type plants showed normal, well ordered chloroplasts. (J–L) Cells in white sectors of the mutants displayed some abnormalities, including vacuolated plastids and lack of organized thylakoid membranes (J), lack of most organelles (K), and abnormal cell morphology (L). (M) Chloroplasts from green sectors of wsl seedlings were indistinguishable from those of wild-type. Scale bar: 1 μm in (I, M), 500 nm in (L), 2 μm in (J, K).
The wsl Mutant Has Enhanced ABA Sensitivity Because the only visible effect of wsl was the leaf striping, we investigated responses to various plant hormones. Under normal conditions, wsl germinated slightly more slowly than the WT (Figure 2D). Abscisic acid (ABA) is a crucial phytohormone that modulates seed germination. We wondered whether wsl had altered responses to ABA. Seed germination and seedling growth tests
showed that the germination rate of wsl was significantly lower than that of the WT with the application of 2 μM of ABA (Figure 2E). Higher levels of ABA (5 μM) strongly inhibited germination of WT seeds (38%–45%) at day 5, but the inhibition was much stronger in wsl (0%–10%) (Figure 2F). Moreover, ABA inhibition of shoot and root length was dramatically more enhanced in the wsl mutant with an increase of ABA concentration from 2 to
Downloaded from http://mplant.oxfordjournals.org/ at North Dakota State University on October 28, 2014
Figure 1 Phenotypic Characteristics of the wsl Mutant.
4
Roles of WSL in Chloroplast Development and Abiotic Stress
Molecular Plant
(A) Five-day-old wild-type and wsl seedlings on media supplemented with increasing concentrations of ABA. Shoot (B) and root (C) lengths of wild-type and wsl seedlings in (A). The error bar represents ±SE of approximately 30 plants. Asterisks indicate statistically significant differences compared with the control (Student’s t-test: * P
Molecular Plant
A Novel Chloroplast-Localized Pentatricopeptide Repeat Protein Involved in Splicing Affects Chloroplast Development and Abiotic Stress Response in Rice Junjie Tana,b,2,
Zhenhua Tana,b,2,
Fuqing Wub,2,
Peike Shenga,b,
Yueqin Hengb,
Xinhua Wangc,
Yulong Renb,c, Jiulin Wangb, Xiuping Guob, Xin Zhangb, Zhijun Chengb, Ling Jiangc, Xuanming Liua, Haiyang Wangb, and Jianmin Wanb,c,1
ABSTRACT Pentatricopeptide repeat (PPR) proteins comprise a large family in higher plants and modulate organellar gene expression by participating in various aspects of organellar RNA metabolism. In rice, the family contains 477 members, and the majority of their functions remain unclear. In this study, we isolated and characterized a rice mutant, white stripe leaf (wsl), which displays chlorotic striations early in development. Map-based cloning revealed that WSL encodes a newly identified rice PPR protein which targets the chloroplasts. In wsl mutants, PEP-dependent plastid gene expression was significantly down-regulated, and plastid rRNAs and translation products accumulate to very low levels. Consistently with the observations, wsl shows a strong defect in the splicing of chloroplast transcript rpl2, resulting in aberrant transcript accumulation and its product reduction in the mutant. The wsl shows enhanced sensitivity to ABA, salinity, and sugar, and it accumulates more H2O2 than wild-type. These results suggest the reduced translation efficiency may affect the response of the mutant to abiotic stress. Key words: PPR protein; WSL; ABA; chloroplast; RNA splicing; rpl2; abiotic stress responses. Tan J. J., Tan Z. H., Wu F. Q., Sheng P. K., Heng Y. Q., Wang X. H., Ren Y. L., Wang J. L., Guo X. P., Zhang X., Cheng Z. J., Jiang L., Liu X. M., Wang H. Y., and Wan J. M. (2014). A novel chloroplast-localized pentatricopeptide repeat protein involved in splicing affects chloroplast development and abiotic stress response in rice. Mol. Plant. 00, 1–22.
Introduction The eukaryotic pentatricopeptide repeat (PPR) gene family was first discovered by bioinformatics analysis while screening Arabidopsis proteins targeted to mitochondria and chloroplasts (Lurin et al., 2004). In higher plants, the PPR family contains many members, with 450 in Arabidopsis and 477 in rice (Lurin et al., 2004; Schmitz-Linneweber and Small, 2008). PPR proteins usually contain 2–26 tandem arrays of a degenerate 35 amino acid repeat (PPR motif), which shows a structure similar to the tetratricopeptide (TPR) motif (Fisk et al., 1999; Small and Peeters, 2000; Lurin et al., 2004). PPR motifs have been classified into three types: P, L, or S, according to size and variation. Based on their motifs, PPR proteins were classified into P and PLS subfamilies (Lurin et al., 2004). The PLS subfamily was further divided into PLS, E, E+, and DYW subgroups based on
the presence or absence of C-terminal motifs (Lurin et al., 2004). The majority of these proteins are localized to chloroplasts or mitochondria (Lurin et al., 2004; Ding et al., 2006). Posttranscriptional modulation of gene expression plays a very important role in controlling gene activity in
1 To whom correspondence should be addressed. E-mail wanjm@ njau.edu.cn, [email protected], tel. +86-25-84396516, fax +86-25-84396516. 2 These authors contributed equally to this work. © The Author 2014. Published by the Molecular Plant Shanghai Editorial Office in association with Oxford University Press on behalf of CSPB and IPPE, SIBS, CAS. doi:10.1093/mp/ssu054 Received 7 January 2014; accepted 23 April 2014
Downloaded from http://mplant.oxfordjournals.org/ at North Dakota State University on October 28, 2014
a State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Biology, Hunan University, Changsha 410082, P.R. China b National Key Facility for Crop Gene Resources and Genetic Improvement, Institute of Crop Science, Chinese Academy of Agricultural Sciences, Beijing 100081, P.R. China c National Key Laboratory for Crop Genetics and Germplasm Enhancement, Nanjing Agricultural University, Nanjing 210095, P.R. China
2
Roles of WSL in Chloroplast Development and Abiotic Stress
In the current study, we isolated and characterized a rice mutant wsl, which shows white stripes on the leaves as well as altered responses to ABA, sugar, and salinity early in development. By map-based cloning, we isolated the WSL gene and demonstrated that it encodes a PPR protein. Further investigation showed wsl exhibited reduced translation efficiency which was most likely to result from abnormal splicing of rpl2 gene. Our work indicated the reduced translation efficiency may affect the response of the mutant to abiotic stress.
RESULTS Phenotypic Characteristics of the wsl Mutant The wsl mutant developed leaves with white stripes before the fifth leaf stage, particularly at the third leaf (L3) stage. Newly developed leaves after the fifth leaf stage displayed normal green color. The white stripes appeared to be randomly distributed on the surface of the mutant leaves, but were predominantly near the leaf margins (Figure 1A–1E). The chlorophyll and carotenoid contents in leaves of wsl mutant were much lower than in wild-type (WT) before the five-leaf stage, but were similar to the WT thereafter (Supplemental Figure 1). The white-striped leaves never reverted to normal green as the plants developed (Figure 1F). At maturity, the major agronomic traits of the mutant, including plant height and seed size, were indistinguishable from those of WT plants (Figure 1G and 1H, and Supplemental Table 1). Additionally, the degree of whitestriping was influenced by temperature and light. The phenotypes of wsl plants were more apparent in conditions of low temperature or high light intensity (Supplemental Figure 2). Previous studies suggested a close link between the formation of photosynthetic pigments and the development of chloroplasts (Su et al., 2012). Therefore, we compared the ultrastructure of chloroplasts among white and green sectors of wsl mutant leaves and WT normal leaves using transmission electron microscopy. Cells in WT leaves contained normal chloroplasts displaying well-organized lamellar structures and were equipped with normally stacked grana and thylakoid membranes (Figure 1I). However, most cells within the white sectors in wsl mutants were heteroplastidic and contained non-pigmented plastids which were severely vacuolated and lacked organized lamellar structures (Figure 1J). Some of cells had no plastids (Figure 1K) and, more seriously, their morphologies were abnormal (Figure 1L). The cells in green sectors of wsl leaves contained apparently normal chloroplasts similar to those in WT leaves (Figure 1M). These observations implied that WSL plays an important role in chloroplast development as well as maintaining normal cell activity in leaves at an early seedling stage.
Downloaded from http://mplant.oxfordjournals.org/ at North Dakota State University on October 28, 2014
organelles. PPR protein is a RNA-binding protein and fulfills an important role in regulating organelle gene expression by controlling RNA editing, splicing, stability, or translation (Lurin et al., 2004; Schmitz-Linneweber and Small, 2008). A combinatorial amino acid code for sequence-specific RNA recognition by PPR tracts was recently proposed (Barkan et al., 2012). RNA editing acts as an important step in posttranscriptional control of organelle gene expression, by modifying transcript sequences mainly by converting cytidine (C) to uridine (U) so that it differs from that encoded in the organelle genome. Besides editing, splicing of introns is also required for many organellar transcripts in plants. Chloroplast genomes in angiosperms contain approximately 20 introns, the majority of which are classified as group II introns based on their similarity to self-splicing group II intron ribozymes. However, none of them has been reported to self-splice in vitro and there has been more recent evidence for the involvement of multiple nucleus-encoded proteins in the splicing of each chloroplast intron in vivo (de Longevialle et al., 2010). Considerable progress has been made in identifying these proteins and to date approximately 15 nuclear genes have been reported to facilitate splicing of one or more chloroplast introns (de Longevialle et al., 2010; Khrouchtchova et al., 2012; Germain et al., 2013). However, no such proteins have been reported in rice. Accumulating data from previous studies reveal that PPR proteins regulate a wide variety of biological processes, such as cytoplasmic male sterility (CMS) (Bentolila et al., 2002; Brown et al., 2003; Koizuka et al., 2003; Hu et al., 2012), embryogenesis (Cushing et al., 2005; Sosso et al., 2012), chloroplast development (Chi et al., 2008; Su et al., 2012), retrograde signaling (Kobayashi et al., 2007; Laluk et al., 2011), and seed development (Liu et al., 2013). Three PPR proteins were shown to be involved in abiotic stress responses in plants. In Arabidopsis, PPR40 is localized to mitochondria and is involved in the regulation of ubiquinol-cytochrome c reductase activity of complex III in the mitochondrial electron transport chain. A ppr40 mutant showed enhanced sensitivity to ABA and salinity in addition to accumulating ROS and altered stressresponsive gene expression (Zsigmond et al., 2008). PGN is a mitochondrion-localized PPR protein that regulates ROS homeostasis and stress-induced ABA responses (Laluk et al., 2011). PGN inactivation leads to ROS accumulation and down-regulation of stress-inducible genes resulting in susceptibility to necrotrophic fungal pathogens as well as enhanced abiotic stress sensitivity (Laluk et al., 2011). So far, only one chloroplast-localized PPR protein, GUN1, is reported to be involved in abiotic responses. GUN1 modulates the expression of nuclear-encoded ABI4 and photooxidative stress responses (Kobayashi et al., 2007). Two chloroplast-localized PPR proteins have been reported in rice (Gothandam et al., 2005; Su et al., 2012), but neither is associated with abiotic stress responses.
Molecular Plant
Molecular Plant
Roles of WSL in Chloroplast Development and Abiotic Stress
3
(A–E) Phenotypes of wild-type (left) and wsl mutant (right) seedlings at the one- (A), two- (B), three- (C), four- (D), and five- (E) leaf stages. The mutant developed leaves (especially the third leaf) with white stripes predominantly along the edges. The white box above the seedlings shows enlarged views of the regions indicated in wild-type and wsl leaves, respectively. Bars = 1 mm. (F) The juvenile white-striped leaves never reverted to green, even in mature plants. (G) Phenotypes of WT (left) and wsl (right) plants at the heading stage. (H) Comparison of the WT and wsl seeds (de-hulled). (I–M) Transmission electron microscopic images of cells from wild-type, white, and green sectors of wsl mutant at the three-leaf stage. (I) Mesophyll cells in wild-type plants showed normal, well ordered chloroplasts. (J–L) Cells in white sectors of the mutants displayed some abnormalities, including vacuolated plastids and lack of organized thylakoid membranes (J), lack of most organelles (K), and abnormal cell morphology (L). (M) Chloroplasts from green sectors of wsl seedlings were indistinguishable from those of wild-type. Scale bar: 1 μm in (I, M), 500 nm in (L), 2 μm in (J, K).
The wsl Mutant Has Enhanced ABA Sensitivity Because the only visible effect of wsl was the leaf striping, we investigated responses to various plant hormones. Under normal conditions, wsl germinated slightly more slowly than the WT (Figure 2D). Abscisic acid (ABA) is a crucial phytohormone that modulates seed germination. We wondered whether wsl had altered responses to ABA. Seed germination and seedling growth tests
showed that the germination rate of wsl was significantly lower than that of the WT with the application of 2 μM of ABA (Figure 2E). Higher levels of ABA (5 μM) strongly inhibited germination of WT seeds (38%–45%) at day 5, but the inhibition was much stronger in wsl (0%–10%) (Figure 2F). Moreover, ABA inhibition of shoot and root length was dramatically more enhanced in the wsl mutant with an increase of ABA concentration from 2 to
Downloaded from http://mplant.oxfordjournals.org/ at North Dakota State University on October 28, 2014
Figure 1 Phenotypic Characteristics of the wsl Mutant.
4
Roles of WSL in Chloroplast Development and Abiotic Stress
Molecular Plant
(A) Five-day-old wild-type and wsl seedlings on media supplemented with increasing concentrations of ABA. Shoot (B) and root (C) lengths of wild-type and wsl seedlings in (A). The error bar represents ±SE of approximately 30 plants. Asterisks indicate statistically significant differences compared with the control (Student’s t-test: * P
