Phenotypic characterization of the tcd7 mutant
Seedlings for the wild-type japonica rice variety Jiahua 1 and the tcd7 mutantwere grown at four distinct temperatures (20ºC, 24ºC, 28ºC and 32ºC). Seedlings from WT displayed normal green leaves regardless of the growth temperature and leaf-stages, as expected. In sharp contrast, the tcd7 mutant produced albino leaves when grown at 20ºC, up to the fourth leaf. Surprisingly, all subsequent leaves starting with the fifth leaf took on a normal appearance, similar to WT seedlings (Figure 1A). In addition, the albino phenotype seen in the tcd7 mutant was conditional, as we observed white leaves only when seedlings were grown at 20ºC, but not at 24ºC, 28ºC or 32ºC (Figure 1B-D). We failed to rescue the albino phenotype when tcd7 seedlings were transferred from 20ºC to 32°C, as third and fourth white leaves did not turn green (data not shown). These results indicated that the mutant phenotype is temperature sensitive during the early seedling stage.
In agreement with our visual phenotypic assessment, seedling contents of chlorophyll a (Chl a), chlorophyll b (Chl b) and carotenoids (Car) before the four-leaf stage were much lower in white tcd7 leaves than in WT seedlings when grown at 20°C (Figure 2A), but were similar to WT (Figure 2B) at the higher growth temperature of 32°C. This result suggested that chlorophyll accumulation in younger tcd7 seedling may be blocked under cold stress. To determine whether the observed reduction of photosynthetic pigments in tcd7 seedlings at 20ºC might be due to altered chloroplast ultrastructure, we observed chloroplasts in seedlings of both genotypes grown at 20ºC or 32ºC by transmission electron microscopy (TEM). WT mesophyll cells contained numerous uniform chloroplasts, regardless of the growth temperature (Figure 2C, 2D). By contrast, leaves from tcd7 seedlings grown at 20°C were characterized by far fewer grana and abnormal grana structure (Figure 2E), whereas chloroplasts in leaves from tcd7 mutant seedlings grown at 32ºC had normal morphology, consistent with their lack of a distinct phenotype at this temperature (Figure 2F). We therefore presume that the abnormal chloroplast structures seen in seedlings grown at 20°C may lead to reduced chlorophyll content in the mutant.
Map-based cloning of TCD7
To identify TCD7, we generated a mapping population by crossing tcd7 plants with the polymorphic variety Peiai64S (indica). Leaves of F1 hybrid seedlings were a healthy green, indicating that tcd7 is a recessive mutation. In the subsequent F2 generation, the mutant phenotype segregated as a monogenic recessive Mendelian trait, with a wild-type to mutant phenotype ratio of 3:1 (Additional file 1: Table S1, χ2 = 0.21<χ20.05 = 3.84). We then selected 94 F2 seedlings with the mutant phenotype for initial mapping, which placed TCD7 between markers MM3645 and MM3833 on chromosome 3 (Figure 3A, B). We then increased the size of the mapping population to 624 F2 seedlings with the mutant phenotype for fine mapping, allowing us to narrow the mapping interval containing TCD7 to a 123-kb region between markers ID14867 and RM15419. This interval was covered by four bacterial artificial chromosomes (BACs AC097276, AC092778, AC097276 and AC109601) and contained six predicted genes (Figure 3C). We amplified all candidate genes by PCR from tcd7 genomic DNA and sequenced all PCR amplicons to detect a polymorphism relative to the wild-type sequence: only LOC_Os03g40550, which encodesthe TAC protein most similar to Arabidopsis FLN2 (thus also named OsFLN2 in this study), carried a 9-bp (GTTG CTCTT) deletion in exon 3, 775 bp downstream from the translation start codon (Figure 3D, 3E). Because of its length, this deletion does not disrupt the open reading frame of OsFLN2/TCD7, but results in a predicted protein lacking three amino acids (Val, Ala, Leu) compared to WT.
Complementation and genome editing of TCD7
To assess whether the mutation we identified in LOC_Os03g40550might be responsible for the tcd7 mutant phenotype under cold stress, we used two complementary approaches. First, we tested for rescue of the albino phenotype upon introduction of a functional copy of LOC_Os03g40550in the tcd7 mutant, driven by the cauliflower mosaic virus (CaMV) 35S promoter. All T0 transgenic plants produced only green leaves when grown at 20ºC (Figure 4B), whereas independent T0 transgenic plants transformed with the empty vector pCAMBIA1301 retained the mutant phenotype (Figure 4A). During the subsequent T1 generation, T1 plants with green leaves also carried a copy of the transgene when grown at 20ºC, whereas none of the T1 plants with albino leaves did (Figure 4C), indicating that the overexpression of LOC_Os03g40550in the tcd7 mutant background rescued the albino phenotype characteristic of the mutant. Independently, we generated two types of homozygous and heterozygous transgenic T0 plants by CRISPR/Cas9 genome editing, respectively. One edited plant harbored the same 9-bp (CATCAGAAG) deletion detected in TCD7; we designated this line as T0-a. The other edited plant, which we designated T0-b, carried multiple deletions and mutations in TCD7 (Additional file 2: Figure S1). Importantly, both homozygous T0 plants exhibited the same albino phenotype as the tcd7 mutant when grown at 20ºC (Figure 4D). T0 plants heterozygous at these two edited sites were fully green, indicating that both mutations are recessive. Moreover, the T1 progeny of these heterozygous edited T0 transgenic plants segregated for the albino phenotype when grown at 20°C (Figure 4E, 4F). Taken together, these results confirm that LOC_Os03g40550 is TCD7.
Analysis of TCD7 expression and subcellular localization of TCD7
We next determined the expression pattern of TCD7 in various tissues in wild-type seedlings by semi-quantitative RT-PCR. TCD7 was highly expressed in leaves (second, third and flag leaves), but much less expressed in roots, stems or panicles (Figure 5A). These results were consistent with the rice gene expression atlas available in the RiceXPro database (Additional file 2: Figure S2), which showed a tissue-specific expression pattern and a vital role in leaf chloroplast development for TCD7.
As TCD7, one of the subunits of the TAC complex, we investigated its subcellular localization. The TargetP 1.1 Server predicted that TCD7 localizes to the chloroplast (http://www.cbs.dtu.dk/services/TargetP/) (Emanuelsson et al. 2000). To test this hypothesis, we fused the first 149 amino acids of TCD7, including any potential signal targeting sequence, to the N terminus of green fluorescent protein (GFP). The resulting clone was driven by the 35S promoter and transiently expressed in tobacco protoplasts. Confocal microscopy revealed co-localization of GFP fluorescence and chlorophyll auto-fluorescence (Figure5B), demonstrating that TCD7 is a chloroplast-localized protein.
Characterization of the TCD7 protein
The TCD7 locus consists of five exons and six introns (Figure 3D) and encodes a protein of 589 amino acids with a predicted molecular mass of about 65 kDa. A search of the Pfam database revealed that TCD7 belongs to the transcriptionally active chromosome (TAC) complex and contains a pfkb domain (Additional file 2: Figure S3). In addition, the tcd7 mutation results in the deletion of three conserved amino acids (Val, Ala, Leu) within the pfkb domain, which is predicted to disrupt the α-helical structure of TCD7, possibly leading to a compromised overall structure (Additional file 2: Figure S3B). TCD7 is highly conserved across land plants, and was closest to its putative ortholog from the monocot purple false brome (Brachypodium distachyon) (Figure 6A, B). Notably, TCD7/OsFLN2 shared only 55% identity with Arabidopsis FLN2 (Huang et al. 2013) (Additional file 2: Figure S4), which differed at the three amino acids deleted in tcd7 (Val, Ala, Ile instead of Val, Ala, Leu). The conservative change of a leucine to isoleucine might result in different functions in rice and Arabidopsis.
Expression of chloroplast- and photosynthesis-related Genes in tcd7
We observed ultrastructural defects in tcd7 chloroplasts when seedlings were grown at 20ºC (Figure 2C-2F), which might explain the albino phenotype. However, this possibility does not preclude an effect of the tcd7 mutation on the expression of genes involved in photosynthesis or chlorophyll biogenesis or chloroplast development. Therefore, we surveyed relative expression levels for 26 genes associated with chlorophyll biosynthesis (PORA, HEMA1, CAO1, YGL1), photosynthesis (Cab1R, RbcS, rbcL, psaA, psbA, LhcpII) and chloroplast development (RpoTp, rpoA, rpoB, rpoC1, rpoC2, FtsZ, aptA, 23S rRNA, 16S rRNA, rps7, rps20, V1, V2, OsV4, TSV3, petA)in WT and tcd7 seedlings at the third-leaf stage grown at 20ºC or 32ºC. In the tcd7 mutant grown at 20°C, we measured drastically reduced transcript levels for chlorophyll biosynthesis genes encoding glutamyl tRNA reductase (HEMA), chlorophyllide A oxygenase (CAO1), protochlorophyllide oxidoreductase (PORA) and the CHLG subunit of chlorophyll synthase (named Yellow-Green Leaf1 [YGL1] in rice) (Figure 7A), in agreement with the reduced chlorophyll contents seen earlier (Figure 2A and the albino phenotype (Figure 1A).
Although Cab1R (encoding the light harvesting Chla/b-binding protein) and RbcS (encoding the small subunit of Rubisco) showed 50-70% higher expression levels in the tcd7 mutant relative to WT, other photosynthesis-related genes (rbcL, psaA, psbA and LhcpII ) were expressed at levels 30% of WT or below in the tcd7 mutant, suggesting that tcd7 should also hinder photosynthesis (Figure 7B). Of 16 genes involved in chloroplast development, eight (rpoA, ropB, rps7, rps20, V1, V2, OsV4 and petA)were expressed at low levels at low temperatures (Figure 7C), which may lead to malformed chloroplasts (Figure 2E). The PEP subunits rpoA and ropB (Kusumi et al. 2011) were especially downregulated in tcd7, with expression levels only reaching about 20% of WT levels. Notably, the downregulation of gene expression noted above in tcd7 seedlings grown at 20ºC was largely abrogated when the seedlings were grown at 32ºC, with gene expression levels now more comparable between the WT and the mutant (Figure 8).
To explore the molecular mechanism behind the stage specificity of the albino phenotype exhibited by the tcd7 mutant, we next determined the relative transcript levels of PORA, HEMA1, CAO1, rbcL, psaA, psbA and rpoA in WT and mutant seedlings grown at 32ºC until the fourth- or fifth-leaf stage. Although all genes were significantly downregulated in the fourth leaves (Figure 9A, 9B), their relative expression returned to nearly normal levels in the fifth leaves (Figure 9C, 9D). We then turned to the PSI protein PsaA and the PSII protein D1, encoded by psaA and psbA, respectively. Indeed, we had established that expression of psaA and psbA was low in the third (Figure 7B) and fourth leaves (Figure 9B) of the tcd7 mutant at 20ºC, but was largely normal in seedlings grown at 32ºC (Figure 8B) and in the fifth mutant leaf from seedlings grown at 20ºC (Figure 9D). The accumulation patterns of PsaA and D1 were largely congruent with the levels of their corresponding transcripts, with very little protein detected in the fourth leaves of seedlings grown at 20ºC (Figure 9E), but comparable accumulation in the fifth leaves of WT and tcd7 seedlings grown at 32ºC. In conclusion, these results suggest that the altered expression of chlorophyll biosynthesis and chloroplast biogenesis genes may contribute to the albino phenotype seen in the mutant until the four-leaf stage under cold stress.
TCD7 interacts with OsTRXz in yeast
Arabidopsis FLN2, which shares 55% identity with TCD7(OsFLN2), interacts with TRXz and FLN1 (Arsova et al. 2010, Huang et al. 2013 and 2015). The putative rice ortholog to Arabidopsis TRXz is OsTRXz, encoded by LOC_Os08g29110, while AtFLN1 is most similar to OsFLN1, also named WPL2, which is encoded by LOC_Os01g63220 (Lv et al. 2017). We therefore tested whether TCD7might interact with OsTRXz and OsFLN1 in a yeast-two hybrid assay. We removed the chloroplast targeting sequences from all proteins during cloning. Unexpectedly, TCD7, interacted only with OsTRXz, and not with OsFLN1, suggesting that TCD7/OsFLN2 and ArabidopsisFLN2 may function via distinct mechanisms (Figure 10).
A possible biochemical basis for the tcd7 mutant phenotype may stem from a disrupted interaction between TCD7 and its partner OsTRXz. However, the loss of the three amino acids in FLN2tcd7 did not affect the TCD7-OsTRXz interaction (Figure 10). Structurally, the mutated TCD7, lacking three amino acids in tcd7 mutants, could still interact with OsCITRXz (Figure 10A), indicating that the tcd7 mutation may affect the function much more than the structure of rice FLN2.
The tcd7 mutant phenotype is not due to insufficient energy supply
Arabidopsis fln2 mutants display an albino and seedling-lethal phenotype at any growth temperature; however, chlorophyll accumulation can be partially rescued by exogenous supplementation with 2% sucrose, as evidenced by delayed greening (Huang et al. 2013 and 2015). We therefore wondered whether insufficient sucrose or energy supply might underlie the albino phenotype of tcd7 mutant seedlings exposed to cold stress before the fifth-leaf stage. However, no amount of exogenous sucrose from 2% to 8% succeeded in turning tcd7 albino seedlings green at the third- or fourth-leaf stage when grown at 22ºC (Figure 11). On the contrary, higher sucrose concentrations appeared to inhibit seedling growth. We conclude that thealbino phenotype is not the result of insufficient energy (sucrose) supply in tcd7 mutant seedlings.