Jun Hidema

UV Radiation–Sensitive Norin 1 Rice Contains Defective Cyclobutane Pyrimidine Dimer Photolyase

Norin 1, a progenitor of many economically important Japanese rice strains, is highly sensitive to the damaging effects of UVB radiation (wavelengths 290 to 320 nm). Norin 1 seedlings are deficient in photorepair of cyclobutane pyrimidine dimers. However, the molecular origin of this deficiency was not known and, because rice photolyase genes have not been cloned and sequenced, could not be determined by examining photolyase structural genes or upstream regulatory elements for mutations. We therefore used a photoflash approach, which showed that the deficiency in photorepair in vivo resulted from a functionally altered photolyase. These results were confirmed by studies with extracts, which showed that the Norin 1 photolyase–dimer complex was highly thermolabile relative to the wild-type Sasanishiki photolyase. This deficiency results from a structure/function alteration of photolyase rather than of nonspecific repair, photolytic, or regulatory elements. Thus, the molecular origin of this plant DNA repair deficiency, resulting from a spontaneously occurring mutation to UV radiation sensitivity, is defective photolyase.

Effects of supplemental UV-B radiation on the growth and yield of two cultivars of Japanese lowland rice (Oryza sativa L.) under the field in a cool rice-growing region of Japan

Abstract An investigation was made of the variations in growth and grain yield in response to increased exposure to UV-B radiation of Japanese lowland rice ( Oryza sativa L.) in a cool rice-growing region. Two cultivars, UV-resistant cv. ‘Sasanishiki’ and UV-sensitive cv. ‘Norin 1’, were examined in a lowland field at Kashimadai (37°28′E, 141°06′E) in Miyagi Prefecture, Japan, for four cropping seasons from 1994 to 1997. The two cultivars were grown in a lowland field with or without supplemental UV-B radiation, which was provided by UV-B-emitting fluorescent lamps, with a 0.1-mm-thick cellulose diacetate film as a filter. In both cultivars, significant decreases in tiller number as the result of supplemental UV-B radiation were observed during the tillering stage in 1994, 1995 and 1997. Furthermore, decreases in grain size from supplemental UV-B radiation were recorded in all seasons. The trend towards small grain size was pronounced in 1996. In that year, the mean daily middle temperatures were lower throughout most of the cropping season and the mean daily hours of sunshine during the tillering stage and between the end of the panicle differentiation stage and the beginning of the ripening stage were shorter. In 1993 when the temperature and the amount of sunshine were both lower, the tiller number, the dry mass of aboveground parts and the panicle number were significantly reduced by supplemental unfiltered UV-B radiation. There was a cultivar difference in the inhibitory effects of supplemental UV-B radiation on growth between the sensitive cultivar Norin 1 and the resistant cultivar Sasanishiki. These results indicate that supplemental UV-B radiation has a positive effect on the growth and grain development of rice, which may be enhanced by unusual climatic conditions such as lower temperature and less sunshine, in cool rice-growing regions.

Autophagy Contributes to Nighttime Energy Availability for Growth in Arabidopsis

Autophagic recycling of proteins supplies nighttime energy sources such as amino acids under conditions of limited sugar availability. Autophagy is an intracellular process leading to the vacuolar degradation of cytoplasmic components. Autophagic degradation of chloroplasts is particularly activated in leaves under conditions of low sugar availability. Here, we investigated the importance of autophagy in the energy availability and growth of Arabidopsis (Arabidopsis thaliana). autophagy-deficient (atg) mutants showed reduced growth under short-day conditions. This growth inhibition was largely relieved under continuous light or under short-day conditions combined with feeding of exogenous sucrose, suggesting that autophagy is involved in energy production at night for growth. Arabidopsis accumulates starch during the day and degrades it for respiration at night. Nighttime energy availability is perturbed in starchless mutants, in which a lack of starch accumulation causes a transient sugar deficit at night. We generated starchless and atg double mutants and grew them under different photoperiods. The double mutants showed more severe phenotypes than did atg or starchless single mutants: reduced growth and early cell death in leaves were observed when plants were grown under 10-h photoperiods. Transcript analysis of dark-inducible genes revealed that the sugar starvation symptoms observed in starchless mutants became more severe in starchless atg double mutants. The contents of free amino acids (AAs) increased, and transcript levels of several genes involved in AA catabolism were elevated in starchless mutant leaves. The increases in branched-chain AA and aromatic AA contents were partially compromised in starchless atg double mutants. We conclude that autophagy can contribute to energy availability at night by providing a supply of alternative energy sources such as AAs.

EFFECTS OF LIGHT ENVIRONMENT DURING CULTURE ON UV-INDUCED CYCLOBUTYL PYRIMIDINE DIMERS AND THEIR PHOTOREPAIR IN RICE (ORYZA SATIVA L.)

Abstract— We examined the effects of a light environment during culture of rice plants (Oryza sativa) on the steady‐state cyclobutyl pyrimidine dimer (CPD) level, CPD induction by challenge UVB exposure and the ability to photorepair CPD. The steady‐state CPD level in plants grown under visible radiation with supplemental UVB radiation in a growth chamber was several times higher than in plants grown without supplemental UVB radiation, whereas in outdoor‐grown plants, it was not enhanced by supplemental UVB radiation. The susceptibility to CPD induction by challenge UVB exposure was highest in dark‐grown plants and decreased with increasing irradiance of visible radiation at low and high levels and outdoors. Chronic UVB radiation reduced the susceptibility to UV‐induced CPD in plants grown both indoors and outdoors. There was a significant negative correlation between CPD levels induced by challenge UVB exposure and the content of UV‐absorbing compounds. The UV‐induced CPD could be reduced by subsequent blue radiation in all samples except in dark‐grown seedlings. The higher the irradiance of visible radiation in the culture, the greater the ability to photorepair CPD. Chronic UVB radiation did not increase the ability to photorepair CPD.

UVB-induced cyclobutyl pyrimidine dimer and photorepair with progress of growth and leaf age in rice

Abstract We have examined the susceptibility to cyclobutyl pyrimidine dimer (CPD) induction by UVB radiation, the ability to photorepair cyclobutyl pyrimidine dimers (CPDs), and the levels of ultraviolet (UV)-absorbing compounds in various developing stages of leaves in UVB-resistant (Sasanishiki) and -sensitive (Norin 1) rices (Oryza sativa L.). UVB produces more CPDs in the second, third and fourth fully expanded leaves than in the fifth and sixth fully expanded leaves. In leaves in which the levels of UV-absorbing compounds are lower, more CPDs are produced. The ability to photorepair CPDs in fully expanded leaves of different stages of each cultivar remains approximately constant, although the photorepair capacity in the fourth leaf rapidly increases from leaf emergence until full expansion. The photorepair capacity is lower in Norin 1 than that in Sasanishiki in all the leaf stages or different aged leaves. These results therefore suggest that rice plants could be more susceptible to CPD induction at the stage when the second, third or fourth leaves are developing, and UVB sensitivity correlates with low CPD photorepair.

Entire Photodamaged Chloroplasts Are Transported to the Central Vacuole by Autophagy

An autophagy process termed chlorophagy is induced by photodamage and serves to eliminate entire damaged chloroplasts via transport to the vacuole in Arabidopsis leaves. Turnover of dysfunctional organelles is vital to maintain homeostasis in eukaryotic cells. As photosynthetic organelles, plant chloroplasts can suffer sunlight-induced damage. However, the process for turnover of entire damaged chloroplasts remains unclear. Here, we demonstrate that autophagy is responsible for the elimination of sunlight-damaged, collapsed chloroplasts in Arabidopsis thaliana. We found that vacuolar transport of entire chloroplasts, termed chlorophagy, was induced by UV-B damage to the chloroplast apparatus. This transport did not occur in autophagy-defective atg mutants, which exhibited UV-B-sensitive phenotypes and accumulated collapsed chloroplasts. Use of a fluorescent protein marker of the autophagosomal membrane allowed us to image autophagosome-mediated transport of entire chloroplasts to the central vacuole. In contrast to sugar starvation, which preferentially induced distinct type of chloroplast-targeted autophagy that transports a part of stroma via the Rubisco-containing body (RCB) pathway, photooxidative damage induced chlorophagy without prior activation of RCB production. We further showed that chlorophagy is induced by chloroplast damage caused by either artificial visible light or natural sunlight. Thus, this report establishes that an autophagic process eliminates entire chloroplasts in response to light-induced damage.

qUVR-10 , a Major Quantitative Trait Locus for Ultraviolet-B Resistance in Rice, Encodes Cyclobutane Pyrimidine Dimer Photolyase

Rice qUVR-10, a quantitative trait locus (QTL) for ultraviolet-B (UVB) resistance on chromosome 10, was cloned by map-based strategy. It was detected in backcross inbred lines (BILs) derived from a cross between the japonica variety Nipponbare (UV resistant) and the indica variety Kasalath (UV sensitive). Plants homozygous for the Nipponbare allele at the qUVR-10 locus were more resistant to UVB compared with the Kasalath allele. High-resolution mapping using 1850 F2 plants enabled us to delimit qUVR-10 to a <27-kb genomic region. We identified a gene encoding the cyclobutane pyrimidine dimer (CPD) photolyase in this region. Activity of CPD photorepair in Nipponbare was higher than that of Kasalath and nearly isogenic with qUVR-10 [NIL(qUVR-10)], suggesting that the CPD photolyase of Kasalath was defective. We introduced a genomic fragment containing the CPD photolyase gene of Nipponbare to NIL(qUVR-10). Transgenic plants showed the same level of resistance as Nipponbare did, indicating that the qUVR-10 encoded the CPD photolyase. Comparison of the qUVR-10 sequence in the Nipponbare and Kasalath alleles revealed one probable candidate for the functional nucleotide polymorphism. It was indicated that single-base substitution in the CPD photolyase gene caused the alteration of activity of CPD photorepair and UVB resistance. Furthermore, we were able to develop a UV-hyperresistant plant by overexpression of the photolyase gene.

Establishment of Monitoring Methods for Autophagy in Rice Reveals Autophagic Recycling of Chloroplasts and Root Plastids during Energy Limitation

Autophagy is responsible for the degradation of leaf chloroplasts and root plastids in rice plants and functions during energy limitation caused by interruption of photosynthesis. Autophagy is an intracellular process leading to vacuolar or lysosomal degradation of cytoplasmic components in eukaryotes. Establishment of proper methods to monitor autophagy was a key step in uncovering its role in organisms, such as yeast (Saccharomyces cerevisiae), mammals, and Arabidopsis (Arabidopsis thaliana), in which chloroplastic proteins were found to be recycled by autophagy. Chloroplast recycling has been predicted to function in nutrient remobilization for growing organs or grain filling in cereal crops. Here, to develop our understanding of autophagy in cereals, we established monitoring methods for chloroplast autophagy in rice (Oryza sativa). We generated transgenic rice-expressing fluorescent protein (FP) OsAuTophaGy8 (OsATG8) fusions as autophagy markers. FP-ATG8 signals were delivered into the vacuolar lumen in living cells of roots and leaves mainly as vesicles corresponding to autophagic bodies. This phenomenon was not observed upon the addition of wortmannin, an inhibitor of autophagy, or in an ATG7 knockout mutant. Markers for the chloroplast stroma, stromal FP, and FP-labeled Rubisco were delivered by a type of autophagic body called the Rubisco-containing body (RCB) in the same manner. RCB production in excised leaves was suppressed by supply of external sucrose or light. The release of free FP caused by autophagy-dependent breakdown of FP-labeled Rubisco was induced during accelerated senescence in individually darkened leaves. In roots, nongreen plastids underwent both RCB-mediated and entire organelle types of autophagy. Therefore, our newly developed methods to monitor autophagy directly showed autophagic degradation of leaf chloroplasts and root plastids in rice plants and its induction during energy limitation.

UV-B induced stress responses in three rice cultivars

UV-B responses of three rice (Oryza sativa L.) cultivars (Sasanishiki, Norin 1 and Surjamkhi) with different photolyase activity were investigated. Carbon dioxide assimilation data support that Sasanishiki was less sensitive to UV-B than Norin 1 and Surjamkhi. UV-B radiation sharply decreased the content of Rubisco protein in Surjamkhi and has no effect in Sasanishiki. The photochemical activities of photosystem (PS) 1 and PS 2 was slightly affected by UV-B treatment. The content of H2O2 and the activities of antioxidant enzymes, catalase (CAT), peroxides (POX) and superoxide dismutase (SOD) were enhanced after UV-B treatment. The activities of CAT and POX isoenzymes in Sasanishiki were more enhanced by UV-B radiation than those in Norin 1 and Surjamkhi.

Cyclobutane pyrimidine dimer (CPD) photolyase repairs ultraviolet‐B‐induced CPDs in rice chloroplast and mitochondrial DNA

Plants use sunlight as energy for photosynthesis; however, plant DNA is exposed to the harmful effects of ultraviolet-B (UV-B) radiation (280-320 nm) in the process. UV-B radiation damages nuclear, chloroplast and mitochondrial DNA by the formation of cyclobutane pyrimidine dimers (CPDs), which are the primary UV-B-induced DNA lesions, and are a principal cause of UV-B-induced growth inhibition in plants. Repair of CPDs is therefore essential for plant survival while exposed to UV-B-containing sunlight. Nuclear repair of the UV-B-induced CPDs involves the photoreversal of CPDs, photoreactivation, which is mediated by CPD photolyase that monomerizes the CPDs in DNA by using the energy of near-UV and visible light (300-500 nm). To date, the CPD repair processes in plant chloroplasts and mitochondria remain poorly understood. Here, we report the photoreactivation of CPDs in chloroplast and mitochondrial DNA in rice. Biochemical and subcellular localization analyses using rice strains with different levels of CPD photolyase activity and transgenic rice strains showed that full-length CPD photolyase is encoded by a single gene, not a splice variant, and is expressed and targeted not only to nuclei but also to chloroplasts and mitochondria. The results indicate that rice may have evolved a CPD photolyase that functions in chloroplasts, mitochondria and nuclei, and that contains DNA to protect cells from the harmful effects of UV-B radiation.