OsNOA1 functions in a threshold-dependent manner to regulate chloroplast proteins in rice at lower temperatures

Abstract

Background: Although decreased protein expressions have been observed in NOA1 (Nitric Oxide Associated protein 1) deficient plants, the molecular mechanisms of how NOA1 regulates protein metabolism remain poorly understood. In this study, we have used a global comparative proteomic approach for both OsNOA1 suppression and overexpression transgenic lines under two different temperatures, in combination with physiological and biochemical analyses to explore the regulatory mechanisms of OsNOA1 in rice.

Results: In OsNOA1-silenced or highly overexpressed rice, considerably different expression patterns of both chlorophyll and Rubisco as well as distinct phenotypes were observed between the growth temperatures at 22 °C and 30 °C. These observations led us to hypothesize there appears a narrow abundance threshold for OsNOA1 to function properly at lower temperatures, while higher temperatures seem to partially compensate for the changes of OsNOA1 abundance. Quantitative proteomic analyses revealed higher temperatures could restore 90% of the suppressed proteins to normal levels, whereas almost all of the remaining suppressed proteins were chloroplast ribosomal proteins. Additionally, our data showed 90% of the suppressed proteins in both types of transgenic plants at lower temperatures were located in the chloroplast, suggesting a primary effect of OsNOA1 on chloroplast proteins. Transcript analyses, along with in vitro pull-down experiments further demonstrated OsNOA1 is associated with the function of chloroplast ribosomes.

Conclusions: Our results suggest OsNOA1 functions in a threshold-dependent manner for regulation of chloroplast proteins at lower temperatures, which may be mediated by interactions between OsNOA1 and chloroplast ribosomes.

Keywords: Chloroplast; NOA1; Quantitative proteomics; Rice; Tandem mass tag; Threshold-dependent.

Fig. 1

Phenotypes of transgenic plants (RNAi-20, Ox-2, Ox-22 and Ox-45) along with WT grown at different temperatures. Germinated seeds were grown in growth chambers at 22 °C (a) or 30 °C (b) under 80 μmol•m^− 2 s^− 1 light intensity. Transcripts of OsNOA1 were determined by real-time PCR (n = 4) under both temperatures (c), and the mean values of NOA1 protein in two biological sets grown at 22 °C were detected by TMT-LC-MS/MS (d). Contents of soluble proteins (e) and chlorophyll (f) were determined (n = 4). Plots of abundance of Rubisco and chlorophyll against OsNOA1 expression level were shown in the inset of (e and f), respectively. Data are means ±SD of n biological replicates and representative of three independent experiments. For (e and f), data significances were determined by DMRT (Duncan’s new Multiple Range Test). Differences were considered significant at the level of P < 0.01

Fig. 2

Changes of Rubisco abundance in etiolated seedlings after being exposure to light (a) or in non-etiolated seedlings after being transferred to the lower temperature (b). A: Germinated seeds were first grown in a dark chamber under 30 °C for 4d, the etiolated seedlings were then transferred to either 30 °C or 15 °C for various times (3d, 6d and 9d). The first fully expanded leaves were collected at 0d and leaves at the same leaf-position were sampled at other time points for analyses (n = 4); B: the post-germinated seeds were first grown at 30 °C for 10d, then transferred to 15 °C for various time (3d, 6d and 9d). The first fully expanded leaves were collected at 0d and leaves at the same leaf-position were sampled at other time points for analyses (n = 4). Data are means ±SD of n biological replicates and representative of three independent experiments

Fig. 3

Semi-quantitative RT-PCR analyses of chloroplast rRNAs. Semi-quantitative RT-PCR analyses were performed using plants grown at either 22 °C/22 °C or 30 °C/30 °C (day/night). Primers used for RT-PCR were listed in Table 1

Fig. 4

Comparison of log₂ TMT ratios (127/126,129/126,130/126 and 131/126) for 2290 of quantified proteins which were commonly identified in both sets of biological replicates: set1 and set2. (a) log₂ ratio of 127/126 group (b) log₂ ratio of 129/126 group (c) log₂ ratio of 130/126 group (d) log₂ ratio of 131/126 group. As the internal error for log₂ ratio of 129/126 group (0.96) was the largest among all these ratios, the correspondence 2-fold was used as a threshold in this study

Fig. 5

Venn diagram for assessment of differently expressed proteins identified in both OsNOA1-silenced and overexpressed lines. The significantly down-regulated proteins were shown in panel (a) and up-regulated proteins were shown in panel (b)

Fig. 6

The effects of temperature (22 °C versus 30 °C) on proteome changes. Significantly down-regulated proteins in the RNAi-20 line at 22 °C were compared to those proteins quantified at 30 °C. Red regions represented significantly down-regulated protein species, while blue ones represented protein species at normal levels

Fig. 7

Functional categories of differently expressed proteins in the OsNOA1-regulated rice plants: NOA1-silenced (RNAi-20) and overexpression (Ox-45). Differentially expressed proteins were classified based on biological process at a GO annotation level of 3 by web-based software Blast2go. The top ten functional categories were selected to cover the data sets for down-regulated (panel a) or up-regulated (panel b) proteins, respectively

Fig. 8

A predicted interaction network of OsNOA1 (COG1161). The interaction network (as displayed by EMBL STRING) for genetically interacting proteins possibly related in function with OsNOA1 was shown. Green lines indicated text mining (literature citations), purple lines indicated high-throughput experimental data (like Y2H), and blue lines indicated database (previous knowledge). Top candidates (score ≥ 0.999) were shown in this figure; of all these proteins, COG0244 and COG2007 were components of ribosomes, others were also involved in the process of translation. NOG45551 and COG1084 took part in the biogenesis of ribosomes, while COG5117 affected the transport of pre-ribosomes. COG1499 affected ribosome stability and mRNA decay

Fig. 9

Interaction proteins of OsNOA1 identified by in vitro pull-down. The interaction proteins of OsNOA1 were affinity pulled down from crude extracts of rice leaves. Proteins were separated by SDS-PAGE and visualized by silver staining. Lane 1, purified NOA1 only; Lane 2, crude extracts of rice leaves only; Lane 3, pull down reaction replicate 1; Lane4, pull down reaction replicate 2. Possible bands (marked by asterisks) of interaction proteins were identified by mass spectrometry