Os07g0558500

The rice Os03g0149100 was reported as the stay-green mutant non-yellow coloring 4 (nyc4) by researchers from Japan.

Annotated Information

Function

• Os07g0558500 encodes a rice protein that is similar to THYLAKOID FORMATION1 (THF1), a chloroplast-targeted protein in A. thaliana. We introduced a genomic fragment harboring the entire Os07g0558500 gene into nyc4-1 to complement the stay-green phenotype. The resultant transformant showed yellowing but nyc4-1 retained green at 7 DAD, suggesting that Os07g0558500 is the NYC4 gene.
• NYC4 encodes the ortholog of Arabidopsis THF1. nyc4 retained chlorophyll during senescence, although changes in other senescence parameters indicated that senescence proceeded, suggesting that nyc4 is a non-functional stay-green mutant.
• In nyc4, the Fv/Fm value, which reflects PSII activity, remains at a high level during senescence. This is a very different characteristic from those observed in the non-functional stay-green mutants sgr and nyc3

Mutation

• The nyc4-1 mutation was induced by a translocation-related chromosomal rearrangement
• The nyc4-1 was obtained from a rice (Oryza sativa L. cv. Nipponbare) M2 population derived from seeds irradiated with carbon ion beams (200 MeV). For dark-induced senescence, detached leaves were incubated in water at 28ºC.
• During screening of rice M2 plants mutagenized using carbon ion beams, the reseachers isolated non-yellow coloring 4-1 (nyc4-1), the detached leaves of which retain green at 7 days after dark incubation (DAD) at 28ºC. Before dark incubation, the levels of cholorophyll were 3.09 nmol mg-1 FW (Chl a) and 0.85 nmol mg-1 FW (Chl b) in the wild-type cultivar Nipponbare, and 3.61 nmol mg-1 FW (Chl a) and 0.87 nmol mg-1 FW (Chl b) in nyc4-1 (Figure 1b). The Chl a content in nyc4-1 was slightly higher than that in the wild-type (Student’s t test, P < 0.05). During the 8 days of dark incubation, the Chl a and Chl b contents decreased drastically in Nipponbare, but 3.6- and 2.3-fold higher levels of Chl a and Chl b, respectively, were observed in nyc4-1.

• Figure 1 A comparison between Nipponbare and nyc4-1 mRNAs in pre-senescent (0 DAD) and senescent (3, 5 and 8 DAD) leaves revealed that expression of Os07g0558500, which is located within the mapped region of chromosome 7, was drastically reduced in nyc4-1.

Expression

NYC4 is expressed in various tissues, but is expressed more strongly in green tissues such as the leaf, stem, lemma and palea . Expression of NYC4 was down-regulated within 24 h of dark incubation, and then gradually increased during the extended dark incubation, and finally reached the level similar to that observed before dark incubation. This expression pattern is consistent with the function of NYC4: acclimation to high light and promotion of chlorophyll degradation in senescence.

Knowledge Extension

• Ionizing radiation often induces chromosomal rearrangements such as inversions and translocations, which cause gene disruptions or, in some cases, gene fusions. In most cases, these mutations are loss-of-function and recessive; however, both loss-of-function and gain-of-function mutations are possible for gene fusions. Theoretically, these mutations may involve two gene mutations associated with two chromosomal breakpoints.Reports of map-based cloning of mutant genes caused by such mutations are rare, probably because of the difficulty in applying map-based cloning procedures to such a situation. For example, for an inversion, it is very difficult to narrow down the candidate region because recombination is suppressed in the inverted region.
• Arabidopsis THF1 is reportedly involved in PSII activity, particularly under photo-inhibitory light conditions. In fact, light-dependent D1 degradation, which is mediated by FtsH protease in the PSII repair cycle, was shown to be impaired in the thf1 mutant. Degradation of photo-damaged D1 involves several proteases, among which FtsH plays a central role as a processive enzyme. Interestingly, lack of THF1 leads to a concomitant decrease in FtsH in chloroplasts, suggesting connectivity between THF1 and FtsH. Although physical interaction of the two proteins was not reported, these data, together with the fact that THF1 is associated with PSII, suggest that THF1 is involved in PSII maintenance through protein degradation.
• SGR is a regulator of chlorophylldegrading activity in vivo. Degradation of LHCII during senescence requires the function of the Chl b reductases NYC1 and NOL, suggesting that Chl b breakdown is necessary for LHCII degradation. Similarly, PSI and Lhca proteins were retained at high levels in sgr-2, suggesting that degradation of PSI and Lhca proteins depends on breakdown of chlorophyll, probably Chl a, because both PSI and Lhca proteins are degraded in Chl b reductase mutants during senescence.
• Detection of ROS was performed as described by Kato et al (2009) with slight modifications. In situ detection of hydrogen peroxide was performed by diaminobenzidine tetrahydrochloride staining. Leaves were vacuum-infiltrated with 0.1% diaminobenzidine tetrahydrochloride in 60 mM Tris (pH 7.5) for 1 min and incubated at room temperature under light. Chlorophyll was removed by boiling in 90% ethanol solution. In situ detection of superoxide was performed by nitroblue tetrazolium staining. Leaves were vacuum-infiltrated with 10 mM NaN3 in 10 mM potassium phosphate buffer (pH 7.8) for 1 min, and then incubated in 0.1% nitroblue tetrazolium solution at room temperature under light. Chlorophyll was removed by boiling in a solution composed of acetic acid, glycerol and ethanol (1:1:3).

Labs working on this gene

• Graduate School of Science, Hiroshima University, Higashi-Hiroshima 739–8526, Japan
• Genome Resource Unit, National Institute of Agrobiological Sciences, 2-1-2 Kannondai, Tsukuba, Ibaraki 305-8602, Japan
• Institute of Plant Science and Resources, Okayama University, Kurashiki 710-0046, Japan
• Institute of Radiation Breeding, National Institute of Agrobiological Sciences, Hitachi-Ohmiya 219-2293, Japan
• Faculty of Life and Environmental Sciences, Prefectural University of Hiroshima, Shobara 727-0023, Japan
• Institute of Low Temperature Science, Hokkaido University, Sapporo 060-0819, Japan

References

1 Yamatani, H. et al. NYC4, the rice ortholog of Arabidopsis THF1, is involved in the degradation of chlorophyll - protein complexes during leaf senescence. The Plant journal : for cell and molecular biology 74, 652-662, doi:10.1111/tpj.12154 (2013). 