Advertisement
Browse Subject Areas
?

Click through the PLOS taxonomy to find articles in your field.

For more information about PLOS Subject Areas, click here.

  • Loading metrics

NOA1 Functions in a Temperature-Dependent Manner to Regulate Chlorophyll Biosynthesis and Rubisco Formation in Rice

  • Qiaosong Yang,

    Affiliations Key Laboratory of Plant Functional Genomics and Biotechnology, Education Department of Guangdong Province, South China Agricultural University, Guangzhou, China, Institute of Fruit Tree Research, Guangdong Academy of Agricultural Sciences, Guangzhou, China

  • Han He,

    Affiliation Key Laboratory of Plant Functional Genomics and Biotechnology, Education Department of Guangdong Province, South China Agricultural University, Guangzhou, China

  • Heying Li,

    Affiliation Key Laboratory of Plant Functional Genomics and Biotechnology, Education Department of Guangdong Province, South China Agricultural University, Guangzhou, China

  • Hua Tian,

    Affiliation College of Agriculture, South China Agricultural University, Guangzhou, China

  • Jianjun Zhang,

    Affiliation Key Laboratory of Plant Functional Genomics and Biotechnology, Education Department of Guangdong Province, South China Agricultural University, Guangzhou, China

  • Liguang Zhai,

    Affiliation Key Laboratory of Plant Functional Genomics and Biotechnology, Education Department of Guangdong Province, South China Agricultural University, Guangzhou, China

  • Jiandong Chen,

    Affiliation Key Laboratory of Plant Functional Genomics and Biotechnology, Education Department of Guangdong Province, South China Agricultural University, Guangzhou, China

  • Hong Wu,

    Affiliation Key Laboratory of Plant Functional Genomics and Biotechnology, Education Department of Guangdong Province, South China Agricultural University, Guangzhou, China

  • Ganjun Yi,

    Affiliation Institute of Fruit Tree Research, Guangdong Academy of Agricultural Sciences, Guangzhou, China

  • Zheng-Hui He,

    Affiliation Department of Biology, San Francisco State University, San Francisco, California, United States of America

  • Xinxiang Peng

    xpeng@scau.edu.cn

    Affiliation Key Laboratory of Plant Functional Genomics and Biotechnology, Education Department of Guangdong Province, South China Agricultural University, Guangzhou, China

NOA1 Functions in a Temperature-Dependent Manner to Regulate Chlorophyll Biosynthesis and Rubisco Formation in Rice

  • Qiaosong Yang, 
  • Han He, 
  • Heying Li, 
  • Hua Tian, 
  • Jianjun Zhang, 
  • Liguang Zhai, 
  • Jiandong Chen, 
  • Hong Wu, 
  • Ganjun Yi, 
  • Zheng-Hui He
PLOS
x

Abstract

NITRIC OXIDE-ASSOCIATED1 (NOA1) encodes a circularly permuted GTPase (cGTPase) known to be essential for ribosome assembly in plants. While the reduced chlorophyll and Rubisco phenotypes were formerly noticed in both NOA1-supressed rice and Arabidopsis, a detailed insight is still necessary. In this study, by using RNAi transgenic rice, we further demonstrate that NOA1 functions in a temperature-dependent manner to regulate chlorophyll and Rubisco levels. When plants were grown at 30°C, the chlorophyll and Rubisco levels in OsNOA1-silenced plants were only slightly lower than those in WT. However, at 22°C, the silenced plants accumulated far less chlorophyll and Rubisco than WT. It was further revealed that the regulation of chlorophyll and Rubisco occurs at the anabolic level. Etiolated WT seedlings restored chlorophyll and Rubisco accumulations readily once returned to light, at either 30°C or 15°C. Etiolated OsNOA1-silenced plants accumulated chlorophyll and Rubisco to normal levels only at 30°C, and lost this ability at low temperature. On the other hand, de-etiolated OsNOA1-silenced seedlings maintained similar levels of chlorophyll and Rubisco as WT, even after being shifted to 15°C for various times. Further expression analyses identified several candidate genes, including OsPorA (NADPH: protochlorophyllide oxidoreductase A), OsrbcL (Rubisco large subunit), OsRALyase (Ribosomal RNA apurinic site specific lyase) and OsPuf4 (RNA-binding protein of the Puf family), which may be involved in OsNOA1-regulated chlorophyll biosynthesis and Rubisco formation. Overall, our results suggest OsNOA1 functions in a temperature-dependent manner to regulate chlorophyll biosynthesis, Rubisco formation and plastid development in rice.

Introduction

Nitric oxide (NO) functions not only in plant defense responses, but also in the regulation of various plant developmental processes, including germination, root growth, vascular differentiation, stomatal closure, and flowering [1]. The mechanism underlying nitric oxide (NO) synthesis has been well established in animals, and depends primarily on a heme-flavoprotein NO synthase (NOS). NOS catalyzes the conversion of arginine to citrulline and NO in the presence of NADPH and oxygen [2]. NOS1 in Arabidopsis (AtNOS1) was first characterized as a potential nitric oxide synthase, which shares homology to a hypothetical snail NOS or NOS partner [3]. NO levels in Atnos1-knockout mutants were significantly reduced compared to WT plants [3], [4], [5], and moreover, the yellowish phenotype of the Atnos1 mutants was partially rescued by the application of NO donor compounds [3], [6], [7]. The NOS1-deficient plants also showed decreased NO accumulation in response to ABA, salicylic acid, salinity, and elicitor treatments [4], [5], [8][10].

Studies so far have yielded conflicting data regarding the true function of NOS1. In some studies it was observed that the accumulation of NO in response to different hormones or oxidative stresses was similar in NOS1-deficient and WT plants [11][13]; and contrastingly different NO responses in the mutants to different treatments have been detected, even under similar experimental conditions [5]. It is thus possible that NOS1 is involved in NO production in response to some stimuli but not to the others [1]. In addition, a role for NOS1 unrelated to NO synthesis was suggested by the observation that not all of the phenotypes noticed in the mutants could be rescued by NO supplementation [7]. Direct evidence came about recently by using recombinant NOS1 systems, where NOS activity was not observed [14][16], leading to the later renaming of AtNOS1 to NO-associated protein 1 (AtNOA1) [14]. It is currently more accepted that NOA1 affects NO production in an indirect manner [1].

AtNOA1 was identified and annotated as an open reading frame of 1686 bp, corresponding to a 561 amino acid protein that shares no sequence homology to animal NOSs. It belongs to a circularly permuted GTPase (cGTPase) family [16], [17]. In bacteria and some eukaryotes, this family of GTP-binding proteins is associated with the RNA/ribosome binding function [16][18]. Loss of function of AtNOA1 causes a chlorotic phenotype and an up-regulation of methylerythritol phosphate (MEP) pathway enzyme levels [7]. In the rice genome, only one homologous gene was identified: Os02g0104700 (OsNOA1). OsNOA1 was recently shown to be a functional homolog of AtNOA1/RIF1 required for ribosome biogenesis [1], [7] and plastid function [19]. However, how exactly NOA1 plays a role in plastid function is still poorly understood.

While the chlorophyll and Rubisco decreasing phenotypes were formerly noticed in either NOA1-supressed rice or Arabidopsis, a detailed insight is still lacking. In this study, by using RNAi transgenic rice plants, we further demonstrate that NOA1 functions in a temperature-dependent manner to regulate chlorophyll and Rubisco levels. It was further revealed that the regulation of chlorophyll and Rubisco occurs at the anabolic level, and that reduced chlorophyll and Rubisco levels may result in defective plastids.

Materials and Methods

Plant materials

Oryza sativa (japonica cultivar-group) Zhonghua 11 was used for the physiological study and for constructing the transgenic plants.

Growth condition and treatments

Pre-germinated seeds were grown for 10d in Kimura B complete nutrient solution, which was renewed every 3 days [20] under a greenhouse condition [average temperature of 30/25°C (day/night), relative humidity 60–80%, photosynthetically active radiation 600–1000 µmol·m−2 s−1 and photoperiod of 14 h day/10 h night] or in a phytotron chamber [for the experiments of gradient temperatures, photosynthetically active radiation 80 µmol·m−2 s−1, relative humidity 65%, photoperiod 12 h light/12 h dark, 22/22°C, 26/26°C or 30/30°C (light/dark); for the experiments of gradient light intensities, temperature 22/22°C(light/dark), relative humidity 65%, photoperiod 12 h light/12 h dark, 80, 150 or 350 µmol·m−2 s−1]. The second leaf from the top was detached at about 2 cm, and then stored at −75°C for subsequent analyses.

Construction of OsNOA1-silenced plants

To generate the OsNOA1 interference construct, a 446 bp sequence ranging from 404 to 849 bp of the ORF cDNA was amplified by RT-PCR with an upstream primer 5′- TTGTGAGCTCTGCTATGTGGGAGATGC-3′ and a downstream primer 5′- ACAGGATCCCCAACATTTGCTGAACC-3′. A skeleton RNAi vector termed pYLRNAi.5 was kindly provided by Dr. Yao-Guang Liu (College of Life Sciences, South China Agricultural University, China). The vector harbors two multi-cloning sites (MCS) so as to be able to more conveniently insert the target sequence in sense vs. antisense orientations. The cloned OsNOA1 cDNA fragment was first inserted in a sense orientation at MCS1 between SacI and BamHI. Both PCR with the specific primers and restriction enzyme cutting verified that the fragment had been correctly inserted into the vector. This first round ligated vector was then used as a template to amplify a second sequence with two unique restriction sites at the ends (RNAi-MluI: 5′-CACCCTGACGCGTGGTGTTACTTCTGAAGAGG-3′, RNAi-PstI: 5′-ACTAGAACTGCAGCCTCAGATCTACCATGGTCG-3′). The second sequence was subsequently cloned at MCS2 between PstI and MluI, thereby resulting in an opposite orientation as the sequence in MCS1. Restriction enzyme cutting showed that the second target fragment had been correctly inserted into the vector. Finally, sequencing analyses further confirmed the correct orientation and the cDNA identity [99% identical to that reported in the NCBI (Os02g0104700)]. The constructed interference vector named as pYLRNAi-Ubi-OsNOA1 was then transformed into rice callus by Agrobacterium-mediated infection (strain EHA105). T0 lines were first analyzed by Southern blot. T1 seeds from T0 lines with a single T-DNA insertion were grown to produce T2 seeds. Further screening with hygromycin-resistance identified the homozygous plants.

Measurements of photosynthesis, stomatal conductance and transpiration

Leaf net photosynthetic rates, stomatal conductance and transpiration rates were measured by a portable gas analysis system, LI-COR 6400 with a light-emitting diode light source (LI-COR Inc., Lincoln, NE). The second leaf from the top was used for the analyses. The measurement conditions were set as follows: leaf temperature 25°C, photon flux density 800 µmol·m−2 s−1, humidity 65%, and CO2 concentration 0.038%. All of the above measurements were conducted between 11:00 and 14:00 on the day of measurement.

Subcellular localization in protoplasts

The cDNA of OsNOA1 (1.7 kb) was amplified by RT-PCR from Oryza sativa (japonica cultivar-group) and confirmed by sequencing. The following primers were used in the PCR to create SalI and SacI sites: 5′-TATGTCGACCTCCTCCTGCTCCTAGT-3′ and 5′- GTCGAGCTCCAGTAATGCCATTTAGGT-3′. p35S-OsNOA1-GFP constructs were generated by cloning the 1.7 kb cDNA into the SalI-SacI site of pUC19-GFP vector. After polyethylene glycol (PEG)-mediated transformation of Arabidopsis leaf-derived and rice stem-derived protoplasts [21], the fluorescent signals of GFP and chloroplast were detected using an Olympus fluorescent microscope, and all images acquired were analyzed using Image-Pro software (Olympus, Tokyo, Japan).

Electron microscopy

Fresh leaf tissue was cut into blocks (1 mm3) with a sharp blade and prefixed in Karnovsky's fixative (3% paraformaldehyde, 4% gluteraldehyde, 0.1 M phosphate buffer, pH 7.2) for 4 h at 4°C, washed 3 times in 0.1 M phosphate buffer (pH 7.2), postfixed for 1 h in 1% OsO4, transferred back into phosphate buffer and then washed 3 times. After dehydration through an acetone series, the samples were rinsed in 50% Epon812 epoxy resin overnight, and subsequently transferred into 100% epoxy resin overnight before polymerization at 40°C for 24 h and 60°C for 24 h. Ultrathin sections (70-80 nm) were cut using a diamond knife on a LEICA EM UC6 microtome and then stained with uranyl acetate for 20 min, and counterstained with lead citrate for 10 min. The sections were examined and photographed on a Philip Fei-Tecnai 12 transmission electron microscope.

Microarray analysis

Whole leaves and stems from either WT or line 20 grown at 22°C for 6d were pooled together in liquid N2 for microarray analysis. RNA was isolated using Trizol Reagent (Invitrogen). Affymetrix GeneChip Rice Genome Arrays were used, and the genechip analyses were performed at CapitalBio Corporation (Beijing, China). The transcripts whose expressions were changed by at least 2-fold were defined to be differentially expressed. Results for gene transcripts changed by at least 2-fold can be found in Table S1 available at PLoS ONE online. All microarray data is MIAME compliant and the raw data from this article have been deposited in the ArrayExpress database (http://www.ebi.ac.uk/arrayexpress/) under accession number E-MEXP-3136.

Quantitative RT-PCR

Total RNA was isolated using TRIZOL reagents (Invitrogen). One microgram of RNA was used as a template for first-strand cDNA synthesis using ReverTra Ace (Toyobo, Osaka, Japan) with random hexamers according to the manufacturer's instructions. Primer pairs for real-time quantitative PCR (see Table S3 online) were designed using Primer Premier 5.0 (Premier Biosoft, Palo Alto, USA). The PCR reaction consisted of 10 µL of 2×SYBR Green PCR Master Mix (Toyobo), 200 nM primers, and 2 µL of 1:40-diluted template cDNA in a total volume of 20 µL. No template controls were set for each primer pair. Real-time PCR was performed employing the DNA Engine Option 2 Real-Time PCR Detection system and Opticon Monitor software (Bio-Rad, USA).

Protein extraction, detection of Rubisco abundance and western blot analyses

Protein was extracted by homogenizing 0.1 g of fresh leaves in 0.5 ml of 20 mM phosphate buffer (pH 7.5). The homogenate was centrifuged at 12,000×g for 15 min. Equally loaded proteins (10 µg) were fractionated on 12.5% SDS-PAGE gels, which were stained with CBB R-250. The gels were scanned by WinRHIZO LA 1600+ scanner (Epson, Japan) at a resolution of 300 dpi. All images were analyzed using Quantity One software (Bio-Rad, USA). For the western blot analysis of OsNOA1, proteins from equal fresh weight were loaded in each lane, separated by 10% SDS-PAGE, transferred onto a nitrocellulose membrane, and finally immunodetected using rabbit anti-OsNOA1 serum.

Measurement of chlorophyll content

Chlorophyll was extracted from leaves with mixed solution (45% acetone, 45% ethanol and 10% distilled water), and the content was then determined spectrophotometrically at 645 and 663 nm according to Lichtenthaler [22].

Results

Generation of the OsNOA1-silenced lines

Examination of the rice genome identified a single gene (Os02g0104700) that is homologous to AtNOA1/RIF1, which we named OsNOA1. OsNOA1 has an open reading frame (ORF) of 1644 bp that encodes a protein of 547 amino acids with 62% similarity to AtNOA1/RIF1. The protein has a conserved YqeH domain (137–340aa) and a predicted GTPase domain. A sequence ranging from 404 to 849 bp of the ORF was amplified by RT-PCR to be used to construct the RNAi vectors. The constructs were then transformed into WT rice via Agrobacterium. Southern blot analyses identified several independent T0 lines with a single T-DNA insertion, including lines 20 and 40 used in this study. The T1 seeds from these two lines were grown to produce T2 seeds, from which homozygous lines were identified through a hygromycin-resistance screen. Real-time PCR analyses confirmed that OsNOA1 transcript levels are indeed significantly reduced by about 90% and 80% for lines 20 and 40, respectively (Fig. 1A). Therefore, we used these two independent homozygous lines for further functional analyses.

thumbnail
Figure 1. The phenotypes of OsNOA1-silenced lines 20 and 40 under normal natural growth conditions.

(A) Transcriptional expression of OsNOA1 (n = 6); (B) Images of rice growth; (C) Relative height (n = 24), photosynthesis rates (Pn) (n = 16), chlorophyll (n = 3) and Rubisco (n = 3) in the silenced and WT plants; (D) Relative tillering (n = 24), stomatal conductance (Gs) (n = 16) and transpiration rates (E) (n = 16). The second leaf from the top was sampled for the analyses. The data are the means ±SD of n replicates and representative of at least three independent experiments.

https://doi.org/10.1371/journal.pone.0020015.g001

Phenotypes

Under normal natural conditions, the OsNOA1-silenced plants had similar growth rates to WT (Fig. 1B, C). The contents of chlorophyll and Rubisco were slightly lower in the silenced plants than those in WT (Fig. 1C). No significant differences were detected in the photosynthetic rate (Pn) and the ultrastructure of plastids (Fig. S3). In contrast, the tiller number was reduced by 50% and 21% for lines 20 and 40, respectively, while the stomatal conductance and transpiration rate were increased (Fig. 1D). The increased stomatal conductance and transpiration may be due to reduced NO in the NOA1-suprressed plants [3], [16]. OsNOA1 fused with a GFP reporter gene was transfected into both Arabidopsis leaf protoplasts and rice stem protoplasts. The results demonstrated that OsNOA1 is localized to the plastids of rice (Fig. S1), in agreement with previous reports that the OsNOA1-EYFP fusion protein is targeted to chloroplasts in transgenic Arabidopsis plants [19].

It has been reported that Atnoa1 knockout mutants display dwarf and yellow leaf phenotypes under normal conditions [3], [7], [16]. Interestingly, as addressed above, we did not see the same phenotypes in our OsNOA1-silenced rice plants under the normal natural growth conditions. However, dwarf and yellow leaf phenotypes were observed in our OsNOA1-silenced plants when the growth temperature was decreased (Fig. 2A).

thumbnail
Figure 2. Growth, chlorophyll and Rubisco as affected by temperature and light intensities in OsNOA1-silenced lines.

Germinated seeds were grown in growth chambers at different temperatures or various light intensities for 10d. (A) The morphological phenotypes for lines 20, 40 and WT; (B, D, F) Relative content of chlorophyll and Rubisco in lines 20 and 40 at different temperatures under 80 µmol·m−2 s−1 light intensity; (C, E, G) Relative content of chlorophyll and Rubisco in lines 20 and 40 under different light intensities at 22°C. The second leaf from the top was sampled for the analyses. The data are means ±SD of 3 replicates and representative of at least three independent experiments.

https://doi.org/10.1371/journal.pone.0020015.g002

Temperature-dependent changes in chlorophyll, Rubisco and plastids

We next examined in detail the effects of various temperatures and light intensities on chlorophyll and Rubisco accumulations in the OsNOA1-silenced plants. At 30°C, only minor reductions were detected in the contents of chlorophyll and Rubisco in the silenced plants as compared to WT (Fig. 2B, D, F). Significant differences occurred when the growth temperature was decreased to 26°C; and only trace amounts of chlorophyll and Rubisco could be detected in the silenced plants at 22°C (Fig. 2B, D, F; Fig. 5C, D). In order to confirm the temperature-dependence of the NOA1 function, we also tested the Atnoa1-knockout Arabidopsis in this respect. As shown in Fig. S2, at 22°C the levels of chlorophyll and Rubisco were only slightly lower in the mutants than those in WT, and a lower temperature (12°C) markedly enhanced the chlorotic phenotype in the mutants, with Rubisco decreased to a very low level in the mutants. In contrast, different light intensities had no obvious impact on the abundance of chlorophyll and Rubisco in the silenced plants at 22°C (Fig. 2C, E, G), indicating that OsNOA1 regulates accumulation of chlorophyll and Rubisco independent of light intensity.

When the etiolated seedlings were transferred to light at 30°C, both OsNOA1-silenced and WT plants turned green readily, with chlorophyll and Rubisco continuously accumulated. Similar amounts of chlorophyll and Rubisco were detected at 10 d after transfer in the silenced plants relative to WT (Fig. 3). If the etiolated seedlings were transferred to 15°C, the silenced plants failed to accumulate chlorophyll and Rubisco, in contrast to WT that was able to accumulate appreciable amounts of chlorophyll and Rubisco at the lower temperature (Fig. 3). If the silenced seedlings were first grown at 30°C for 10d and then transferred to 15°C, those leaves that had already fully expanded could maintain similar amounts of chlorophyll and Rubisco after transfer (Fig. 4), while those newly-emerged leaves after transfer were yellowish, just like the phenotype of the silenced seedlings that were continuously grown at the lower temperature (Fig. 2A). In addition, it was found that Rubisco accumulation lagged behind chlorophyll. For instance, the chlorophyll level in the silenced seedlings recovered to about 45%, 72% and 95% relative to WT, at 3d, 6d and 10d, respectively, while Rubisco was restored to 14%, 41% and 93% under the same condition (Fig. 3).

thumbnail
Figure 3. Accumulation of chlorophyll (A) and Rubisco (B) in etiolated silenced seedlings after exposure to light.

Germinated seeds (lines 20, 40 and WT) were grown in a growth chamber at 28°C in the dark for 4d, then the etiolated seedlings were exposed to light at either at 30°C or 15°C for various time (0, 3, 6 and 10 d). The second leaf from the top was sampled for the analyses. The data are means ±SD of 3 replicates and representative of at least three independent experiments.

https://doi.org/10.1371/journal.pone.0020015.g003

thumbnail
Figure 4. Changes in the accumulated chlorophyll and Rubisco under low temperature.

Germinated seeds (lines 20, 40 and WT) were grown in a growth chamber at 30°C and under 80 µmol·m−2 s−1 light intensity for 10d, then the seedlings were transferred to 15°C under the same light intensity for various times (0, 3, 6d). Leaves that had already fully expanded before transfer were sampled for the analyses. The data are means ±SD of 3 replicates and representative of at least three independent experiments.

https://doi.org/10.1371/journal.pone.0020015.g004

As shown in Fig. 5A, obvious differences in plastid ultrastructure could be seen between the OsNOA1-silenced and WT plants grown at 30°C for 3d. WT had thickly stacked thylakoid membranes, while the silenced plants had vague thylakoid membranes. As the plants grew up at this temperature, the difference gradually became smaller and almost disappeared at 10d. Clearly, a positive correlation exists between chlorophyll and Rubsico accumulations and plastid development (Fig. 5A vs. Fig. 5C). In contrast, little differences in plastid ultrastructure could be seen between the OsNOA1-silenced and WT plants grown at 22°C for 3d (Fig. 5B). As the plants grew up at this lower temperature, the plastids in WT gradually developed and finally matured, while the plastids in the silenced plants were degraded gradually and almost totally dissolved at 10d (Fig. 5B). It was meanwhile noticed that, at 22°C, chlorophyll and Rubisco remained at a very low level throughout the time course (Fig. 5C, D).

thumbnail
Figure 5. Changes in plastid ultrastructure in relation to the accumulation of chlorophyll and Rubisco in the silenced plants.

Germinated seeds (line 20 and WT) were grown in growth chambers at either 30°C (A) or 22°C (B) and under 80 µmol·m−2 s−1 light intensity for various times (3, 6, 10d). The newest leaf (3, 6d) or the second leaf from the top (10d) was sampled for the observation of ultrastructure of plastids (A and B) and determination of chlorophyll and Rubisco content (C and D). The ultrastructure was observed by a Philip Fei-Tecnai 12 transmission electron microscope. Bars = 0.5 µm. The data are means ±SD of 3 replicates and representative of at least three independent experiments.

https://doi.org/10.1371/journal.pone.0020015.g005

The above data collectively indicate that the decreased chlorophyll and Rubisco levels are ultimately due to their impaired biosynthesis or formation, rather than to an increase in degradation. This decrease in production may then affect plastid development.

Transcriptional responses to OsNOA1 suppression

To further understand the possible reasons behind the decreased chlorophyll biosynthesis and Rubisco formation, a cDNA microarray analysis was performed to screen for candidate genes that are affected by the OsNOA1 silencing. The assay identified a total of 2292 transcripts that showed more than a 2-fold change in expression (Table S1 and S2). Among these genes, 1362 were up-regulated and 930 were down-regulated (Table S1). Two genes, NADPH: protochlorophyllide oxidoreductase A (PorA, Os04g0678700) and chlorophyllide a oxygenase that are responsible for chlorophyll biosynthesis, were highly down-regulated in OsNOA1-silenced plants at 22°C, whereas the genes involved in chlorophyll metabolism were not significantly affected (Tables S1 and S2). RbcL, PsbC, PsbD,ferredoxin-NADP+ reductase and ATPase subunit ε were down-regulated and the other genes involved in the photosystems, carbon fixation and the TCA cycle were not altered (Table S1 and S2). A translational repressor gene OsPuf4 (Os12g048890), and OsRALyase (Os02g0493100) encoding a putative ribosomal RNA apurinic site specific lyase, were greatly up-regulated (Table S1) in the silenced plants at 22°C. Subsequent real-time PCR analyses confirmed that the expressions of OsPorA, OsrbcL, OsPuf4 and OsRALyase were indeed dramatically altered at 22°C, and restored to normal when the growth temperature was increased to 30°C (Fig. 6B–F). In addition, expression of our target gene OsNOA1 was highly suppressed at both mRNA and protein levels in WT at 22°C, such that the expression difference between the silenced and WT plants were even larger at 30°C than at 22°C (Fig. 6A), seemingly inconsistent with the phenotypes (Fig. 2A).

thumbnail
Figure 6. Expressions of some genes in response to OsNOA1 silencing at either 22°C or 30°C.

Germinated seeds (line 20 and WT) were grown at either 22°C or 30°C for 10d. The whole leaves and stems were sampled for various analyses (real-time PCR for all the genes listed; additional western-blot for OsNOA1 as in A). The data are means ±SD of 3 replicates and representative of two independent experiments. GenBank/EMBL accession numbers: OsNOA1, NM_001052149; OsrbcL, NP_039391; OsPorA, NM_001060809; OsPorB, NM_001071490; OsPuf4, NM_001073324 and OsRALyase, NM_001053416.

https://doi.org/10.1371/journal.pone.0020015.g006

Discussion

Temperature-dependent phenotypes and possible reasons behind

Most recently, Liu et al. [19] observed that OsNOA1-silenced rice plants had yellowish leaves and decreased Rubisco abundance when grown in a temperature-controlled green house (30°C day/25°C night). It was also noticed that when Arabidopsis was grown at constant 22 or 24°C (16h light/8h dark) the newly-emerged young leaves of Atnoa1-knockout mutants displayed a yellow phenotype though mature leaves had only a slight reduction in chlorophyll [6], [7]. In our current study, however, under a normal natural condition (average 30°C day/average 25°C night) or at constant 30°C in a growth chamber (12h light/12h dark), OsNOA1-silenced plants had only slightly lower levels of chlorophyll and Rubisco than WT plants, without visual chlorotic phenotypes (Figs. 1B and 2A). Clearly, quantitative differences occurred between these experiments. We consider that these inconsistencies could be caused by the ambient temperature differences. In our study, 30°C during the day is an average estimated value, but the maximum could reach as high as 35°C at noon. It has been noticed that an even higher temperature (e. g. constant 35°C) was able to completely abolish the minor differences that were observed at constant 30°C (data not shown). This temperature-dependence was subsequently examined in detail. We observed that, as growth temperature was decreased, the differences became increasingly larger, with chlorophyll and Rubisco dramatically reduced in the silenced plants at low temperature (Figs. 2 and 3). Similar phenotypes were also observed in the Atnoa1-knockout Arabidopsis (Fig. S2). To the best of our knowledge, such a temperature-dependent regulation of NOA1 function has not been addressed so far.

It is an intriguing question as to how temperature affects NOA1 function, with special regard to the regulation of chlorophyll and Rubisco. Temperature-sensitive plant mutants and variants were previously reported, but a common type is those that show normal phenotypes at low temperature and mutant phenotypes at high temperature [23][25]. The type like NOA1-silenced rice observed in this study, i. e. permissive at high temperature and restrictive at low temperature, has been also noticed more recently. For instance, a mutant designated as tds (a recessive temperature dependent shooty mutant of Nicotiana tabacum L) displays thick and narrow leaves with abnormal mesophyll cells at 21°C. A higher temperature at 30°C reverses this mutant phenotype and leads to formation of normal leaves and restoration of apical dominance [26]. A phan mutant has arrested growth at 15°C, but shifting the mutant from 15°C to 25°C allows meristem function to resume [27]. An Arabidopsis mutant, called chilling sensitive5 (chs5), contains a missense mutation in a conserved amino acid of 1-deoxy-d-xylulose 5-phosphate synthase (DXP synthase). The mutant displayed a temperature-sensitive phenotype, being normal at 22°C but chlorotic at 15°C [28]. In a pepper mutant named Sy-2, the true leaf at 20°C is irregularly shaped with a smaller area, but is almost normal when the growth temperature is above 24°C [29], [30].

Mechanisms underlying the temperature-dependent phenotypes, particularly where low temperature is restrictive, are poorly understood thus far. One possible explanation for such an effect may be that NOA1 itself directly serves as an effecter to maintain or even up-regulate the tolerance of plants to low temperature, and loss of its function would increase sensitivities to low temperature, thereby resulting in reduced chlorophyll and Rubisco in plants. If it is true, NOA1 would be of evolutionary significance and particularly important for the tropically-originated species, such as rice, to adapt to cold environments. It was further thought that NO may have mediated the regulation by NOA1 since NO was shown to be involved in cold tolerance [31], [32] and reduced NO production was observed in NOA1-deficient plants [3], [31]. However, it was detected that the supplementation of NO failed to restore the reduced chlorophyll and Rubisco (Fig. S4) [19], ruling out the above possibility. In addition, reactive oxygen species (ROS) were usually increased in NOA1-deficient plants [6], [8]. Persistent increase in ROS may attenuate the tolerance to low temperature leading to a faster degradation of chlorophyll and Rubisco [33], [34]. We have shown, however, that the reduced chlorophyll and Rubisco are ultimately due to the impaired chlorophyll biosynthesis and Rubisco formation, rather than to their degradation (Fig. 3 and 4). Thus, a direct involvement of ROS in the reduced chlorophyll and Rubisco seems also less likely, and whether indeed NOA1 functions in the regulation of low-temperature tolerance needs to be further investigated. A second possibility has been previously raised by other authors [26], [28] that a kind of protein exists to be able to compensate for the absence of the gene product at high temperatures but not at low temperature. This compensatory protein could be either homoeologous (same family member) or distinct. As described above, since only one gene copy is identified for OsNOA1 in the rice genome, the proposed protein is more likely to be distinct from OsNOA1. It is highly interesting to identify the proposed compensatory protein and those linking components between NOA1 and chlorophyll/Rubisco turnover. In this study, by using microarray and real-time PCR analyses, we preliminarily identified several possible linking genes, i. e. OsPorA, OsrbcL, OsRALyase and OsPuf4 which are known to be involved in chlorophyll and Rubisco anabolism. But, how NOA1 regulates these genes and what those interaction proteins are remains to be understood.

OsNOA1 expression in WT plants was found to be highly down-regulated at low temperature (Fig. 6A), and similar results were also observed in Arabidopsis [31]. This result is puzzling because the difference in OsNOA1 expression between the silenced and WT plants at 22°C was even smaller than that at 30°C (Fig. 6A), which is seemingly inconsistent with the phenotypes (Fig. 2). It is not clear as to why no significant phenotypes occur in WT at 22°C, even though OsNOA1 expression is also suppressed (Fig. 6A). We here propose a possibility that OsNOA1 expression is redundant and that there is a threshold for its function. At low temperature, this threshold for WT plants might be lower than that for silenced plants. This notion is based on the evidence that certain proteins, presumably including the compensatory one, were more suppressed in the silenced plants than in WT plants, particularly at low temperature (see more discussions below) [19].

Chlorophyll and Rubisco are regulated by OsNOA1 at the anabolic level

It was previously proposed that the yellow leaf phenotype in NOA1-suppressed plants was caused by the oxidative stress-induced degradation of chlorophyll resulting from impaired ribosome biogenesis due to loss of function of NOA1 [16], [19]. It is also possible that the oxidative stress could be directly elicited by the suboptimal growth temperature. But generally, the critical minimum temperature for rice shoot elongation ranges from 7 to 16°C and that for root elongation from 12 to 16°C [35]. Here we show several lines of evidence to indicate that OsNOA1 regulates chlorophyll biosynthesis and Rubisco formation rather than their degradation: (i) The etiolated WT plants restored chlorophyll and Rubisco accumulations readily once returned to light at either 30°C or 15°C, while the etiolated silenced seedlings could normally accumulate chlorophyll and Rubisco only at 30°C and lost the ability at the low temperature (Fig. 3); (ii) Once the silenced plants had accumulated sufficient chlorophyll and Rubisco (in the light at 30°C), their levels were not significantly reduced even after they were transferred to 15°C for various times (Fig. 4); (iii) Expression analyses further point to such a possibility. PorA and OsrbcL, a gene for chlorophyll biosynthesis [36] and for the Rubisco large subunit, respectively, were heavily suppressed at 22°C but almost normal at 30°C in the NOA1-silenced plants, and all the genes detected for chlorophyll catabolism were not significantly affected (Table S2). The other two genes, OsRALyase and OsPuf4, were highly up-regulated at 22°C but returned to normal levels at 30°C (Fig. 6E, F and Table S1). OsRALyase was identified as an rRNA apurinic site-specific lyase (RALyase) [37], [38], which cleaves phosphodiester bonds at the depurinated site produced by ribosome-inactivating protein and diminishes the residual elongation activities of depurinated ribosomes. In support, it was detected that certain rRNA species needed for ribosome assembly were indeed reduced in the silenced rice plants at 22°C (data not shown), which was also noticed by Liu et al. [19]. Pufs are able to suppress protein translation by binding their specific target to mRNAs [39]. These results further indicate that the formation of either Rubisco or some other proteins may be retarded at the translational level in the silenced plants at 22°C. There have also been studies showing that NOA1 is required for the correct formation of 70S ribosomes and stability of 30S subunits in Arabidopsis [16], [40] and in rice [19]. Overall, it appears highly likely that OsNOA1 regulates chlorophyll biosynthesis and Rubisco formation rather than their degradation in rice plants.

Plastid development and NO production in relation to chlorophyll and Rubisco

Chlorophyll, Rubisco and plastids were all affected in the silenced plants at low temperature (Figs. 2, 3 and 5). Thus, we are curious about whether the three of them are mechanistically interrelated. It was noticed that chlorophyll was accumulated faster than Rubisco (Figs. 3 and 5), so there is a possibility that Rubisco formation is subsequently regulated by chlorophyll accumulation. But, this notion seems to be negated by the data from both Paddock et al. [41] and Bevins et al. [42], who showed that, in some mutants of Arabidopsis where chlorophyll was mostly lost, Rubisco large subunit levels were still indistinguishable from that in WT plants. Thus, chlorophyll and Rubisco could be independently regulated by OsNOA1. Loss of any inclusion, including chlorophyll, Rubisco and NO, could be simply accounted for by pleiotropic effects of defective plastids [7], [31]. However, our time-course dynamic observation in this study indicated that the deficiency of chlorophyll and Rubisco might have acted as the cause, rather than the effect, of the defective plastids (Fig. 5). This notion is supported by some previous experiments. For instance, a POR mutant that was retarded in chlorophyll biosynthesis developed a seedling-lethal xantha phenotype at the cotyledon stage and possessed defective plastids [41]; a sweetclover chlorophyll-deficient mutant also showed abnormal development of plastids [42]. Indeed, our OsNOA1-silenced seedlings grown at 22°C displayed similar plastid ultrastructure as the above mutants [41], [42], where those authors held the view that the defective plastid ultrastructure in the mutants might be caused by the reduced chlorophyll biosynthesis [41][43]. In addition, a total of 28 plastid ribosomal proteins were substantially reduced in OsNOA1-silenced rice [19], such a reduction in plastome-encoded proteins was also considered to be the cause of defective plastids [7]. Collectively, the possibility exists that altered chlorophyll and Rubisco might have affected plastid development.

NOA1 was once considered as a NO synthase in plants, but it is currently more accepted that NOA1 affects NO production in an indirect manner [1]. According to Moreau et al. [16], decreased NO production in Atnoa1 knockout mutants may result from increased production of reactive oxygen species (ROS), since interaction of the elevated ROS with NO reduces the amount of NO. Increased ROS could simply be caused by the pleiotropic effect of defective plastids [6], [7], [8], [16] and/or disrupted chlorophyll biosynthesis [44][46]. It has been reported that the ‘nonphotoactive’ Pchlide (protochlorophyllide a), due to its dissociation from the POR active site, may serve as a photosensitizer for the formation of singlet oxygen [44][46]. A heavy down-regulation of PorA in our silenced rice, therefore, may likely elicit ROS production in such a similar manner. Indeed, several studies have observed increases in ROS production in NOA-suppressed plants [3], [8].

Supporting Information

Figure S1.

Subcellular localization of OsNOA1-GFP in rice and Arabidopsis protoplasts. Rice stem and Arabidopsis leaf protoplasts transfected with p35S-OsNOA1-GFP constructs were observed under fluorescent microscopy. The green color image reflects the green fluorescence of OsNOA1-GFP and the red indicates the autofluorescence of chlorophyll in chloroplasts. Images were then merged to show overlapping green and red fluorescence in yellow. Bars = 5μm.

https://doi.org/10.1371/journal.pone.0020015.s001

(TIF)

Figure S2.

The phenotypes (A) and SDS-PAGE (B) of Atnoa1 mutants and WT grown at either 22°C or 12°C under 80 μmol·m-2 s-1 light intensity for 16d.

https://doi.org/10.1371/journal.pone.0020015.s002

(TIF)

Figure S3.

The plastid ultrastructure of line 20 and WT grown under normal natural growth conditions. The plants were grown under normal natural growth conditions until the booting stage, then the second leaf from the top was sampled for plastid observation. The ultrastructure was observed by a Philip Fei-Tecnai 12 transmission electron microscope. Bars noted in the bottom of each image.

https://doi.org/10.1371/journal.pone.0020015.s003

(TIF)

Figure S4.

Effects of the NO donor SNP on the decreased chlorophyll and Rubisco in OsNOA1 -silenced rice. Germinated seeds (lines 20, 40 and WT) were grown on Kimura B complete nutrient agar supplemented with different concentrations of SNP (10, 50, 100μM) in a growth chamber at 22°C and under 80 μmol·m-2 s-1. The SNP solution was renewed every 3 days. The whole leaves and stems of 6-old-day seedlings were sampled for the analyses. The data are means ±SD of 3 replicates and representative of two independent experiments.

https://doi.org/10.1371/journal.pone.0020015.s004

(TIF)

Table S1.

Transcripts at least 2-fold changed in OsNOA1-silenced line 20 compared with WT.

https://doi.org/10.1371/journal.pone.0020015.s005

(XLS)

Table S2.

Microarray analysis of gene expressions in response to OsNOA1 silencing.

https://doi.org/10.1371/journal.pone.0020015.s006

(XLS)

Table S3.

Primer pairs for real-time quantitative PCR.

https://doi.org/10.1371/journal.pone.0020015.s007

(XLS)

Acknowledgments

We thank Dr. Colin Leasure (Department of Biology, San Francisco State University) for reading this manuscript, and thank N. Crawford and colleagues (University of California) for the Atnoa1 mutant seeds.

Author Contributions

Conceived and designed the experiments: XP. Performed the experiments: QY HH HL JZ LZ JC. Analyzed the data: QY HH HW ZH-H. Contributed reagents/materials/analysis tools: XP GY. Wrote the paper: XP QY HH.

References

  1. 1. Gas E, Flores-Pérez , Sauret-Güeto S, Rodríguez-Concepción M (2009) Hunting for plant nitric oxide synthase provides new evidence of a central role for plastids in nitric oxide metabolism. Plant Cell 21: 18–23.E. GasFlores-PérezS. Sauret-GüetoM. Rodríguez-Concepción2009Hunting for plant nitric oxide synthase provides new evidence of a central role for plastids in nitric oxide metabolism.Plant Cell211823
  2. 2. Alderton WK, Cooper CE, Knowles RG (2001) Nitric oxide synthases: structure, function and inhibition. Biochem J 357: 593–615.WK AldertonCE CooperRG Knowles2001Nitric oxide synthases: structure, function and inhibition.Biochem J357593615
  3. 3. Guo FQ, Okamoto M, Crawford NM (2003) Identification of a plant nitric oxide synthase gene involved in hormonal signaling. Science 302: 100–103.FQ GuoM. OkamotoNM Crawford2003Identification of a plant nitric oxide synthase gene involved in hormonal signaling.Science302100103
  4. 4. Zeidler D, Zahringer U, Gerber I, Dubery I, Hartung T, et al. (2004) Innate immunity in Arabidopsis thaliana: Lipopolysaccharides activate nitric oxide synthase (NOS) and induce defense genes. Proc Natl Acad Sci U S A 101: 15811–15816.D. ZeidlerU. ZahringerI. GerberI. DuberyT. Hartung2004Innate immunity in Arabidopsis thaliana: Lipopolysaccharides activate nitric oxide synthase (NOS) and induce defense genes.Proc Natl Acad Sci U S A1011581115816
  5. 5. Bright J, Desikan R, Hancock JT, Weir IS, Neill SJ (2006) ABA-induced NO generation and stomatal closure in Arabidopsis are dependent on H2O2 synthesis. Plant J 45: 113–122.J. BrightR. DesikanJT HancockIS WeirSJ Neill2006ABA-induced NO generation and stomatal closure in Arabidopsis are dependent on H2O2 synthesis.Plant J45113122
  6. 6. Guo FQ, Crawford NM (2005) Arabidopsis nitric oxide synthase1 is targeted to mitochondria and protects against oxidative damage and dark-induced senescence. Plant Cell 17: 3436–3450.FQ GuoNM Crawford2005Arabidopsis nitric oxide synthase1 is targeted to mitochondria and protects against oxidative damage and dark-induced senescence.Plant Cell1734363450
  7. 7. Flores-Pérez U, Sauret-Güeto S, Gas E, Jarvis P, Rodríguez-Concepción M (2008) A mutant impaired in the production of plastome-encoded proteins uncovers a mechanism for the homeostasis of isoprenoid biosynthetic enzymes in Arabidopsis plastids. Plant Cell 20: 1303–1315.U. Flores-PérezS. Sauret-GüetoE. GasP. JarvisM. Rodríguez-Concepción2008A mutant impaired in the production of plastome-encoded proteins uncovers a mechanism for the homeostasis of isoprenoid biosynthetic enzymes in Arabidopsis plastids.Plant Cell2013031315
  8. 8. Zhao MG, Tian QY, Zhang WH (2007) Nitric oxide synthase dependent nitric oxide production is associated with salt tolerance in Arabidopsis. Plant Physiol 144: 206–217.MG ZhaoQY TianWH Zhang2007Nitric oxide synthase dependent nitric oxide production is associated with salt tolerance in Arabidopsis.Plant Physiol144206217
  9. 9. Zottini M, Costa A, De Michele R, Ruzzene M, Carimi F, et al. (2007) Salicylic acid activates nitric oxide synthesis in Arabidopsis. J Exp Bot 58: 1397–1405.M. ZottiniA. CostaR. De MicheleM. RuzzeneF. Carimi2007Salicylic acid activates nitric oxide synthesis in Arabidopsis.J Exp Bot5813971405
  10. 10. Asai S, Ohta K, Yoshioka H (2008) MAPK signaling regulates nitric oxide and NADPH oxidase-dependent oxidative bursts in Nicotiana benthamiana. Plant Cell 20: 1390–1406.S. AsaiK. OhtaH. Yoshioka2008MAPK signaling regulates nitric oxide and NADPH oxidase-dependent oxidative bursts in Nicotiana benthamiana.Plant Cell2013901406
  11. 11. Arnaud N, Murgia I, Boucherez J, Briat JF, Cellier F, et al. (2006) An iron-induced nitric oxide burst precedes ubiquitin-dependent protein degradation for Arabidopsis AtFer1 ferritin gene expression. J Biol Chem 281: 23579–23588.N. ArnaudI. MurgiaJ. BoucherezJF BriatF. Cellier2006An iron-induced nitric oxide burst precedes ubiquitin-dependent protein degradation for Arabidopsis AtFer1 ferritin gene expression.J Biol Chem2812357923588
  12. 12. Kolbert Z, Bartha B, Erdei L (2008) Exogenous auxin-induced NO synthesis is nitrate reductase-associated in Arabidopsis thaliana root primordia. J Plant Physiol 165: 967–975.Z. KolbertB. BarthaL. Erdei2008Exogenous auxin-induced NO synthesis is nitrate reductase-associated in Arabidopsis thaliana root primordia.J Plant Physiol165967975
  13. 13. Tun NN, Livaja M, Kieber JJ, Scherer GF (2008) Zeatin induced nitric oxide (NO) biosynthesis in Arabidopsis thaliana mutants of NO biosynthesis and of two-component signaling genes. New Phytol 178: 515–531.NN TunM. LivajaJJ KieberGF Scherer2008Zeatin induced nitric oxide (NO) biosynthesis in Arabidopsis thaliana mutants of NO biosynthesis and of two-component signaling genes.New Phytol178515531
  14. 14. Crawford NM, Gally M, Tischner R, Heimer YM, Okamoto M, et al. (2006) Plant nitric oxide synthase: back to square one. Trends Plant Sci 11: 526–527.NM CrawfordM. GallyR. TischnerYM HeimerM. Okamoto2006Plant nitric oxide synthase: back to square one.Trends Plant Sci11526527
  15. 15. Zemojtel T, Fröhlich A, Palmieri MC, Kolanczyk M, Mikula I, et al. (2006) Plant nitric oxide synthase: a never-ending story? Trends Plant Sci 11: 524–525.T. ZemojtelA. FröhlichMC PalmieriM. KolanczykI. Mikula2006Plant nitric oxide synthase: a never-ending story?Trends Plant Sci11524525
  16. 16. Moreau M, Lee GI, Wang Y, Crane BR, Klessig DF (2008) AtNOS/A1 is a functional Arabidopsis thaliana cGTPase and not a nitric oxide synthase. J Biol Chem 283: 32957–32967.M. MoreauGI LeeY. WangBR CraneDF Klessig2008AtNOS/A1 is a functional Arabidopsis thaliana cGTPase and not a nitric oxide synthase.J Biol Chem2833295732967
  17. 17. Anand B, Verma SK, Prakash B (2006) Structural stabilization of GTP-binding domains in circularly permuted GTPases: implications for RNA binding. Nucleic Acids Res 34: 2196–2205.B. AnandSK VermaB. Prakash2006Structural stabilization of GTP-binding domains in circularly permuted GTPases: implications for RNA binding.Nucleic Acids Res3421962205
  18. 18. Matsuo Y, Morimoto T, Kuwano M, Loh PC, Oshima T, et al. (2006) The GTP-binding protein YlqF participates in the late step of 50S ribosomal subunit assembly in Bacillus subtilis. J Biol Chem 281: 8110–8117.Y. MatsuoT. MorimotoM. KuwanoPC LohT. Oshima2006The GTP-binding protein YlqF participates in the late step of 50S ribosomal subunit assembly in Bacillus subtilis.J Biol Chem28181108117
  19. 19. Liu H, Lau E, Lam MP, Chu H, Li S, et al. (2010) OsNOA1/RIF1 is a functional homolog of AtNOA1/RIF1: implication for a highly conserved plant cGTPase essential for chloroplast function. New Phytol 187(1): 83–105.H. LiuE. LauMP LamH. ChuS. Li2010OsNOA1/RIF1 is a functional homolog of AtNOA1/RIF1: implication for a highly conserved plant cGTPase essential for chloroplast function.New Phytol187183105
  20. 20. Yoshida S, Forno DA, Cock JH, Gomez KA (1976) Laboratory Manual for Physiological Studies of Rice. Manila: International Rice Research Institute. S. YoshidaDA FornoJH CockKA Gomez1976Laboratory Manual for Physiological Studies of Rice.ManilaInternational Rice Research Institute
  21. 21. Bart R, Chern M, Park CJ, Bartley L, Ronald PC (2006) A novel system for gene silencing using siRNAs in rice leaf and stem-derived protoplasts. Plant Methods 2: 13.R. BartM. ChernCJ ParkL. BartleyPC Ronald2006A novel system for gene silencing using siRNAs in rice leaf and stem-derived protoplasts.Plant Methods213
  22. 22. Lichtenthaler HK (1987) Chlorophylls and carotenoids-pigments of photosynthetic biomembranes. Methods Enzymol 148: 350–382.HK Lichtenthaler1987Chlorophylls and carotenoids-pigments of photosynthetic biomembranes.Methods Enzymol148350382
  23. 23. Ohtani M, Sugiyama M (2005) Involvement of SRD2-mediated activation of snRNA transcription in the control of cell proliferation competence in Arabidopsis. Plant J 43: 479–490.M. OhtaniM. Sugiyama2005Involvement of SRD2-mediated activation of snRNA transcription in the control of cell proliferation competence in Arabidopsis.Plant J43479490
  24. 24. Liu W, Fu Y, Hu G, Si H, Zhu L, et al. (2007) Identification and fine mapping of a thermo-sensitive chlorophyll deficient mutant in rice (Oryza sativa L.). Planta 226: 785–795.W. LiuY. FuG. HuH. SiL. Zhu2007Identification and fine mapping of a thermo-sensitive chlorophyll deficient mutant in rice (Oryza sativa L.).Planta226785795
  25. 25. Tamaki H, Konishi M, Daimon Y, Aida M, Tasaka M, et al. (2009) Identification of novel meristem factors involved in shoot regeneration through the analysis of temperature-sensitive mutants of Arabidopsis. Plant J 57: 1027–1039.H. TamakiM. KonishiY. DaimonM. AidaM. Tasaka2009Identification of novel meristem factors involved in shoot regeneration through the analysis of temperature-sensitive mutants of Arabidopsis.Plant J5710271039
  26. 26. Samuelsen AI, Rickson FR, Mok DW, Mok MC (1997) A temperature-dependent morphological mutant of tobacco. Planta 201(3): 303–310.AI SamuelsenFR RicksonDW MokMC Mok1997A temperature-dependent morphological mutant of tobacco.Planta2013303310
  27. 27. Waites R, Selvadurai HR, Oliver IR, Hudson A (1998) The PHANTASTICA gene encodes a MYB transcription factor involved in growth and dorsoventrality of lateral organs in Antirrhinum. Cell 93(5): 779–789.R. WaitesHR SelvaduraiIR OliverA. Hudson1998The PHANTASTICA gene encodes a MYB transcription factor involved in growth and dorsoventrality of lateral organs in Antirrhinum.Cell935779789
  28. 28. Araki N, Kusumi K, Masamoto K, Niwa Y, Iba K (2000) Temperature-sensitive Arabidopsis mutant defective in 1-deoxy-d-xylulose 5-phosphate synthase within the plastid non-mevalonate pathway of isoprenoid biosynthesis. Physiologia Plantarum 108: 19–24.N. ArakiK. KusumiK. MasamotoY. NiwaK. Iba2000Temperature-sensitive Arabidopsis mutant defective in 1-deoxy-d-xylulose 5-phosphate synthase within the plastid non-mevalonate pathway of isoprenoid biosynthesis.Physiologia Plantarum1081924
  29. 29. Koeda S, Hosokawa M, Kang BC, Yazawa S (2009) Dramatic changes in leaf development of the native Capsicum chinense from the Seychelles at temperatures below 24°C. J Plant Res 122(6): 623–631.S. KoedaM. HosokawaBC KangS. Yazawa2009Dramatic changes in leaf development of the native Capsicum chinense from the Seychelles at temperatures below 24°C.J Plant Res1226623631
  30. 30. An SJ, Pandeya D, Park SW, Li J, Kwon JK, et al. (2011) Characterization and genetic analysis of a low-temperature-sensitive mutant, sy-2, in Capsicum chinense. Theor Appl Genet 122: 459–470.SJ AnD. PandeyaSW ParkJ. LiJK Kwon2011Characterization and genetic analysis of a low-temperature-sensitive mutant, sy-2, in Capsicum chinense.Theor Appl Genet122459470
  31. 31. Zhao MG, Chen L, Zhang LL, Zhang WH (2009) Nitric reductase-dependent nitric oxide production is involved in cold acclimation and freezing tolerance in Arabidopsis. Plant Physiol 151: 755–767.MG ZhaoL. ChenLL ZhangWH Zhang2009Nitric reductase-dependent nitric oxide production is involved in cold acclimation and freezing tolerance in Arabidopsis.Plant Physiol151755767
  32. 32. Cui JX, Zhou YH, Ding JG, Xia XJ, Shi K, et al. (2011) Role of nitric oxide in hydrogen peroxide-dependent induction of abiotic stress tolerance by brassinosteroids in cucumber. Plant Cell Environ 34: 347–358.JX CuiYH ZhouJG DingXJ XiaK. Shi2011Role of nitric oxide in hydrogen peroxide-dependent induction of abiotic stress tolerance by brassinosteroids in cucumber.Plant Cell Environ34347358
  33. 33. Nakano R, Ishida H, Makino A, Mae T (2006) In vivo fragmentation of the large subunit of ribulose-1,5-bisphosphate carboxylase by reactive oxygen species in an intact leaf of cucumber under chilling-light conditions. Plant Cell Physiol 47: 270–276.R. NakanoH. IshidaA. MakinoT. Mae2006In vivo fragmentation of the large subunit of ribulose-1,5-bisphosphate carboxylase by reactive oxygen species in an intact leaf of cucumber under chilling-light conditions.Plant Cell Physiol47270276
  34. 34. Zhou Y, Huang L, Zhang Y, Shi K, Yu J, et al. (2007) Chill-induced decrease in capacity of RuBP carboxylation and associated H2O2 accumulation in cucumber leaves are alleviated by grafting onto figleaf gourd. Ann Bot 100: 839–848.Y. ZhouL. HuangY. ZhangK. ShiJ. Yu2007Chill-induced decrease in capacity of RuBP carboxylation and associated H2O2 accumulation in cucumber leaves are alleviated by grafting onto figleaf gourd.Ann Bot100839848
  35. 35. Nishiyama I (1977) Physiology of cold injury in direct-seeded rice with special reference to germination and seedling growth. Agric Hortic 52: 1353–1357, 1480-1482; 53, 30-34, 263-268.I. Nishiyama1977Physiology of cold injury in direct-seeded rice with special reference to germination and seedling growth.Agric Hortic5213531357, 1480-1482; 53, 30-34, 263-268
  36. 36. Schoefs B (1999) The light-dependent and light-independent reduction of protochlorophyllide a to chlorophyllide a. Photosynthetica 36: 481–496.B. Schoefs1999The light-dependent and light-independent reduction of protochlorophyllide a to chlorophyllide a.Photosynthetica36481496
  37. 37. Ogasawara T, Sawasaki T, Morishita R, Ozawa A, Madin K, et al. (1999) A new class of enzyme acting on damaged ribosomes: ribosomal RNA apurinic site specific lyase found in wheat germ. EMBO J 18: 6522–6531.T. OgasawaraT. SawasakiR. MorishitaA. OzawaK. Madin1999A new class of enzyme acting on damaged ribosomes: ribosomal RNA apurinic site specific lyase found in wheat germ.EMBO J1865226531
  38. 38. Ito Y, Ozawa A, Sawasaki T, Endo Y, Ochi K, et al. (2002) OsRALyase1, a putative F-box protein identified in rice, Oryza sativa, with enzyme activity identical to that of wheat RALyase. Biosci Biotechnol Biochem 66: 2727–2731.Y. ItoA. OzawaT. SawasakiY. EndoK. Ochi2002OsRALyase1, a putative F-box protein identified in rice, Oryza sativa, with enzyme activity identical to that of wheat RALyase.Biosci Biotechnol Biochem6627272731
  39. 39. Gerber AP, Herschlag D, Brown PO (2004) Extensive association of functionally and cytotopically related mRNAs with Puf family RNA-binding proteins in Yeast. PLoS Biology 2(3): 0342–0354.AP GerberD. HerschlagPO Brown2004Extensive association of functionally and cytotopically related mRNAs with Puf family RNA-binding proteins in Yeast.PLoS Biology2303420354
  40. 40. Sudhamsu J, Lee GI, Klessig DF, Crane BR (2008) The structure of YqeH. An AtNOS1/AtNOA1 ortholog that couples GTP hydrolysis to molecular recognition. J Biol Chem 283(47): 32968–32976.J. SudhamsuGI LeeDF KlessigBR Crane2008The structure of YqeH. An AtNOS1/AtNOA1 ortholog that couples GTP hydrolysis to molecular recognition.J Biol Chem283473296832976
  41. 41. Paddock TN, Mason ME, Lima DF, Armstrong GA (2010) Arabidopsis protochlorophyllide oxidoreductase A (PORA) restores bulk chlorophyll synthesis and normal development to a porB porC double mutant. Plant Mol Biol 72(4-5): 445–57.TN PaddockME MasonDF LimaGA Armstrong2010Arabidopsis protochlorophyllide oxidoreductase A (PORA) restores bulk chlorophyll synthesis and normal development to a porB porC double mutant.Plant Mol Biol724-544557
  42. 42. Bevins MA, Madhavan S, Markwell J (1993) Two Sweetclover (Melilotus alba Desr.) Mutants Temperature Sensitive for Chlorophyll Expression. Plant Physiol 103: 1123–1131.MA BevinsS. MadhavanJ. Markwell1993Two Sweetclover (Melilotus alba Desr.) Mutants Temperature Sensitive for Chlorophyll Expression.Plant Physiol10311231131
  43. 43. Philippar K, Geis T, Ilkavets I, Oster U, Schwenkert S, et al. (2007) Chloroplast biogenesis: the use of mutants to study the etioplast-chloroplast transition. Proc Natl Acad Sci U S A 104: 678–683.K. PhilipparT. GeisI. IlkavetsU. OsterS. Schwenkert2007Chloroplast biogenesis: the use of mutants to study the etioplast-chloroplast transition.Proc Natl Acad Sci U S A104678683
  44. 44. Reinbothe S, Reinbothe C, Apel K, Lebedev N (1996) Evolution of chlorophyll biosynthesis-the challenge to survive photooxidation. Cell 86: 703–705.S. ReinbotheC. ReinbotheK. ApelN. Lebedev1996Evolution of chlorophyll biosynthesis-the challenge to survive photooxidation.Cell86703705
  45. 45. Sperling U, van Cleve B, Frick G, Apel K, Armstrong GA (1997) Overexpression of light-dependent PORA or PORB in plants depleted of endogenous POR by far-red light enhances seedling survival in white light and protects against photooxidative damage. Plant J 12: 649–658.U. SperlingB. van CleveG. FrickK. ApelGA Armstrong1997Overexpression of light-dependent PORA or PORB in plants depleted of endogenous POR by far-red light enhances seedling survival in white light and protects against photooxidative damage.Plant J12649658
  46. 46. Sperling U, Franck F, van Cleve B, Frick G, Apel K, et al. (1998) Etioplast differentiation in Arabidopsis: both PORA and PORB restore the prolamellar body and photoactive protochlorophyllide-F655 to the cop1 photomorphogenic mutant. Plant Cell 10: 283–296.U. SperlingF. FranckB. van CleveG. FrickK. Apel1998Etioplast differentiation in Arabidopsis: both PORA and PORB restore the prolamellar body and photoactive protochlorophyllide-F655 to the cop1 photomorphogenic mutant.Plant Cell10283296