Os03g0654600

NONYELLOW COLORING1 (NYC1) and NYC1-LIKE (NOL) are thought to encode Chl b reductases, which catalyze the conversion of Chl b to 7-hydroxymethyl Chl a (HMChl).

Annotated Information

Function

• The researchers therefore predicted that NYC1 and NOL are Chl b reductases with distinct roles in Chl b degradation.

• To examine the hypothesis thatt NYC1 and NOL directly interact with each other, the researchers performed immunoprecipitation analysis. NOL and NYC1 proteins were produced in insect cells infected with baculovirus harboring NOL–HA, NYC1–FLAG or both. The produced proteins were detected by immunoblot analysis using anti-FLAG and anti-HA antibodies (Figure 8a). NYC1– FLAG was precipitated using anti-HA only in the presence of NOL–HA, indicating that NOL and NYC1 directly interact with each other in vitro (Figure 8b).

Figure 8. Physical interaction between NOL and NYC1 in vitro. (a) Expression of NOL–HA and NYC1–FLAG proteins in insect cells infected with recombinant baculoviruses. (b) Immunoprecipitation analysis of NOL–HA and NYC1–FLAG proteins. Proteins extracted from the insect cells were immunoprecipitated using anti-HA Sepharose conjugate. .

• Because NYC1 is localized in plastids and has transmembrane domains, it is likely thatNOL and NYC1 are co-localized in thylakoid membranes facing the stromal side where they form a complex that functions as a Chl b reductase (Figure 10).

Figure 10. A model for the regulation of chlorophyll and LHCII degradation during leaf senescence in rice. .

Mutation

• As seen in the screening of stay-green mutants, nol-1, nol-2 and nol-3 showed the stay-green phenotype 7 days after dark incubation (DAD), in comparison to the wild-type, which showed full yellowing at this stage (Figure 1b). The leaves of nol-1 retained more greenness relative to the wildtype and did not turn yellow even at the very late stage of natural senescence (Figure 1c).

Figure 1. Mutation positions and the stay-green phenotype of nol mutants. .

• The change in chlorophyll content during dark-induced senescence was analyzed in nol-1, which showed prominent inhibition of Chl b degradation until 8 DAD (Figure 2a). Inhibition of Chl a degradation in nol-1 was not obvious until 4 DAD, but became significant at 6 DAD and thereafter. The ratio of Chl a to Chl b was close to 1 at 8 DAD. These characteristics were also observed in nol-2 and nol-3. As other parameters of leaf senescence, we measured the change in the Fv/Fm value (the ratio of variable to maximum fluorescence; a parameter of PSII activity) and membrane ion leakage during dark-induced senescence. The Fv/Fm value for nol-1 during dark-induced senescence decreased in a similar manner to the wild-type (Figure 2b), and membrane ion leakage also increased similarly to the wild-type (Figure 2c). These results indicate that nol shows the stay-green phenotype due to the retention of chlorophyll, but leaf functionality decreases during senescence.

Figure 2. Physiological characterization of nol-1 during senescence. (a) Change in the chlorophyll content during dark incubation. Chlorophyll was extracted with 80% acetone and its content was measured spectrophotometrically. Solid line, nol-1; dotted line, wild-type; closed circles, Chl a; open circles, Chl b. Bars indicate standard errors (n = 3). (b) Change in Fv/Fm values during dark incubation. Solid line, nol-1; dotted line, wild-type. Bars indicate standard errors (n = 3). (c) Change in membrane ion leakage during dark incubation. Solid line, nol-1; dotted line, wild-type. Bars indicate standard errors (n = 3). .

• To examine the behavior of the chlorophyll–protein complexes in nol mutants, we performed a ‘green-gel’ \ analysis (Figure 3a). The amounts of chlorophyll–protein complexes, namely the PSI reaction center, LHCII trimer, CP43/47, LHCI dimer and LHCII monomer, were similar between the wildtype and nol-1 before dark treatment. During dark-induced senescence, all chlorophyll–protein complexes were degraded in the wild-type, and the LHCII monomer and LHCII trimer were selectively retained in nol-1 (Figure 3a). Immunoblot analysis revealed that all LHCI proteins

examined, namely Lhca1, Lhca3 and Lhca4, and PsaF, a component of the PSI core complex, were degraded at the late stage of senescence in both wild-type and nol-1 (Figure 3b).

Figure 3. Protein degradation during senescence in nol-1, nyc1-2 and nol-1 nyc1-2. .

• To examine the behavior of photosynthetic pigments during senescence, the researchers performed HPLC analysis of extracts from detached leaves of nol-1 and the wild-type (Figure 4). The chromatograms were very similar before dark incubation, but at 7 DAD nol-1 showed apparent retention of not only Chl a and Chl b but also neoxanthin and lutein, all of which are LHCII-bound pigments. The stability of violaxanthin, a weakly LHCII-bound pigment, was less affected in nol-1. This result suggests that the inhibition of photosynthetic pigment breakdown in nol is due to the stability of LHCII during senescence, as is the case for nyc1.

Figure 4. HPLC elution profiles of photosynthetic pigments extracted from non-senescent (0 DAD) and fully senescent (7 DAD) leaves of the wild-type and nol-1. Absorption was measured at 440 nm. The same weight of leaves was extracted with the same volume of 80% acetone. N, neoxanthin; V, violaxanthin; L, lutein; La, lutein 3-acetate; b, Chl b; a, Chl a; C, b-carotene. .

• The researchers therefore examined the ultrastructure of chloroplasts during senescence in the wild-type and nol-1 (Figure 5). There was no obvious difference at 0 DAD, but at 4 DAD chloroplasts became round in shape and plastoglobules became obvious in both the wild-type and nol-1, and initial degradation of grana stacks was observed in the wild-type. At 7 DAD, only a small volume of thylakoid membranes remained in the wildtype, but very thick and wide grana stacks were observed in nol-1. These observations indicate that grana stacks are not properly degenerated during senescence in nol.

Figure 5. Ultrastructures of chloroplasts during leaf senescence in the wild-type and nol-1. (a)–(c) wild-type; (d)–(f) nol-1. (a,d), 0 DAD; (b,e), 4 DAD; (c,f), 7 DAD. g, grana; p, plastoglobule. The arrowhead indicates residual grana stacks. Scale bars = 500 nm. .

• The observations described above indicate that nol and nyc1 show a very similar phenotype, suggesting that they act in the same process in chlorophyll degradation. To examine the genetic interaction between NOL and NYC1, the researchers raised a double mutant of the probable null mutations nol-1 and nyc1-2. In both the nol-1 and nyc1-2 mutants, Chl b was slightly degraded at the late stage of senescence. The chlorophyll contents at 0 and 6 DAD were also measured in the wild-type, nol-1, nyc1-2 and nol-1 nyc1-2 (Figure 6). The degree of inhibition of chlorophyll degradation was broadly similar among the nol-1, nyc1-2 and nol-1 nyc1-2 mutants, with a slight enhancement of Chl b retention in nol-1 nyc1-2.

Figure 6. Chlorophyll degradation during leaf senescence in the wild-type, nol-1, nyc1-2 and nol-1 nyc1-2. Chl a and Chl b contents at 0 and 6 DAD in the wild-type, nol-1, nyc1-2 and nol-1 nyc1-2 were measured spectrophotometrically. Bars indicate standard errors (n = 6). .

Subcellular localization

• To examine the intracellular localization of NOL, we fused the region encoding the putative transit peptide of NOL to the green fluorescent protein (GFP) gene and introduced the fusion into the epidermal cells of onion (Allium cepa) by particle bombardment. The GFP signal co-localized with that of red fluorescent protein fused to the transit peptide of plastid-localizing rice S9 ribosomal protein. This result indicates that NOL is localized in the plastids.

Expression

• Expression of both NYC1 and NOL is induced during senescence in rice.

Evolution

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Labs working on this gene

• Graduate School of Agricultural and Life Sciences, University of Tokyo, Tokyo, 113-8657, Japan,
• Institute of Radiation Breeding, National Institute of Agrobiological Sciences, Hitachi-ohmiya, 219-2293, Japan,
• The Institute of Low Temperature Science, Hokkaido University, Sapporo 060-0819, Japan, and
• Graduate School of Science, Hiroshima University, Higashi-hiroshima, 739-8526, Japan