Rice TCD8 Encoding a Multi-Domain GTPase Is Crucial for Chloroplast Development of Early Leaf Stage at Low Temperatures

Abstract

The multi-domain GTPase (MnmE) is conservative from bacteria to human and participates in tRNA modified synthesis. However, our understanding of how the MnmE is involved in plant chloroplast development is scarce, let alone in rice. A novel rice mutant, thermo-sensitive chlorophyll-deficient mutant 8 (tcd8) was identified in this study, which apparently presented an albino phenotype at 20 °C but a normal green over 24 °C, coincided with chloroplast development and chlorophyll content. Map-based cloning and complementary test revealed the TCD8 encoded a multi-domain GTPase localized in chloroplasts. In addition, the disturbance of TCD8 suppressed the transcripts of certain chloroplast-related genes at low temperature, although the genes were recoverable to nearly normal levels at high temperature (32 °C), indicating that TCD8 governs chloroplast development at low temperature. The multi-domain GTPase gene in rice is first reported in this study, which endorses the importance in exploring chloroplast development in rice.

Keywords: albino phenotype; chloroplast; low temperature; multi-domain GTPase; rice.

Figure 1

Characterization of the tcd8 mutants: (a) 3-leaf-stage phenotype of (WT (left) and tcd8 mutant (right) grown at 20 °C, 24 °C, 28 °C, and 32 °C, respectively; (b) photosynthetic pigment contents of 3-leaf-stage seedlings at 20 °C, and (c) at 32 °C. Chl, total chlorophyll; Chla, chlorophyll a; Chlb, chlorophyll b; Car, carotenoid. Bars represent the mean ± SD of three biological replicates. Raw data were used to conduct Student’s t-test; asterisks indicate statistically significant differences: ** p < 0.01.

Figure 2

TEM (Transmission electron microscopic) images of chloroplasts in WT and tcd8 mutant: (a) WT cells under 20 °C; (c) The cells of tcd8 mutant under 20 °C; (b,d) Magnified views of chloroplasts in the (a,c); (e) WT cells under 32 °C; (g) tcd8 cells mutant under 32 °C; (f,h) Magnified views of chloroplasts in (e,g). c, chloroplast; g, grana lamella stacks.

Figure 3

Mapped-cloning of the TCD8 gene: (a) TCD8 was located between the OP08-19i and MM2936 on chromosome 8 (Chr.8) using 112 F₂ mutant individuals; (b) Fine-mapping of TCD8 between BAC1 (AP004636.3) and BAC2 (AP00491.2) within a 173 kb region by the markers ID13441 and ID13582 using 868 mutant individuals; (c) The target region contains fifteen predicted candidate genes (LOC_Os08g31320, LOC_Os08g31340, LOC_Os08g31360, LOC_Os08g31410, LOC_Os08g31420, LOC_Os08g31430, LOC_Os08g31440, LOC_Os08g31450, LOC_Os08g31460, LOC_Os08g31470, LOC_Os08g31510, LOC_Os08g31520, LOC_Os08g31550, LOC_Os08g31560, LOC_Os08g31569); (d) Comparison with wild-type gene sequence revealed a single nucleotide (A) deletion mutation at the eighth exon (represented by black squares from left to right). The (A) deletion mutation at the position 3214 bp from the ATG start codon in LOC_Os08g31460, and (e) Complementation of the tcd8 mutant, segregation of T₁ plants obtained from transgenic T₀ plants.

Figure 4

Subcellular localization of TCD8 gene: (a) Empty GFP vector and (b) TCD8-GFP fusion protein. The scale bar represents 5 μm.

Figure 5

Transcript levels of TCD8 in various tissues. R (young-seedling roots), S (young-seedling stems), YL (young-seedling leaves), SL (second leaf from the top), FL (flag leaf at heading), P (young panicles). OsActin was used as a control.

Figure 6

qRT-PCR analysis of related genes in WT and tcd8 mutant at the 3-leaf-stage under 20 °C: (a) Analysis of genes associated with chlorophyll biosynthesis; (b) Photosynthesis, and (c) chloroplast development. OsActin was used as an internal control to analyze the relative expression of genes in WT and tcd8. The relative expression level of each gene in WT and tcd8 was analyzed by using OsActin as an internal control. Bars represent the mean ± SD of three biological replicates. Raw data were used to conduct Student’s t-test; asterisks indicate statistically significant differences: ** p < 0.01.

Figure 7

qRT-PCR analysis of related genes in WT and tcd8 mutant at the 3-leaf-stage under 32 °C: (a) Analysis of genes associated with chlorophyll biosynthesis; (b) photosynthesis, and (c) chloroplast development. OsActin was used as an internal control to analyze the relative expression genes in WT and tcd8. Bars represent the mean ± SD of three biological replicates. Raw data were used to perform Student’s-test; asterisks indicate statistically significant differences: ** p < 0.01.