ORCID IDs: 0000-0001-5222-9163 (M.I.); 0000-0002-8682-4566 (H.I.); 0000-0002-8218-6894 (S.N.); 0000-0002-3798-8257 (J.H.)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-Bsensitive 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.

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.

The amounts of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), total chlorophyll (Chi), and total leaf nitrogen were measured in fully expanded, young leaves of wheat (Triticum aestivum L.), rice (Oryza sativa L.), spinach (Spinacia oleracea L.), bean (Phaseolus vulgaris L.), and pea (Pisum sativum L.). In addition, the activities of whole-chain electron transport and carbonic anhydrase were measured. All plants were grown hydroponically at different nitrogen concentrations. Although a greater than proportional increase in Rubisco content relative to leaf nitrogen content and Chi was found with increasing nitrogen supply for rice, spinach, bean, and pea, the ratio of Rubisco to total leaf nitrogen or Chi in wheat was essentially independent of nitrogen treatment. In addition, the ratio of Rubisco to electron transport activities remained constant only in wheat. Nevertheless, gas-exchange analysis showed that the in vivo balance between the capacities of Rubisco and electron transport in wheat, rice, and spinach remained almost constant, irrespective of nitrogen treatment. The in vitro carbonic anhydrase activity in wheat was very low and strongly responsive to increasing nitrogen content. Such a response was not found for the other C3 plants examined, which had 10-to 30-fold higher carbonic anhydrase activity than wheat at any leafnitrogen content. These distinctive responses of carbonic anhydrase activity in wheat were discussed in relation to CO2-transfer resistance and the in vivo balance between the capacities of Rubisco and electron transport. ach, clarified the relation between nitrogen nutrition and nitrogen partitioning into the various photosynthetic components and activities. They found that although nitrogen supply increased the ratio of Rubisco activity to electron transport activity, ATPase, Chl, or total leaf nitrogen, the balance between the in vivo activities of Rubisco and electron transport remained constant. They concluded that this difference was compensated for by the presence of a C02-transfer resistance between intercellular air spaces and the carboxylation sites. As a result of this resistance, the in vivo Rubisco specific activity was reduced progressively with increasing amount of enzyme because the partial pressure of CO2 at the carboxylation sites was reduced and kept in a constant balance with electron transport activity. The increase in the ratio of Rubisco to total leaf nitrogen or Chl with nitrogen supply is frequently found for other C3 species, such as tobacco (1), cotton (32), Solanum (11), bean (26), and pea (18).However, in spite of the existence of significant C02-transfer resistance in wheat (5,8,23, 30), the ratio of Rubisco to total leaf nitrogen or Chl in fully expanded young leaves seems to be independent of nitrogen nutrition (5,17,18 Plant Physiol. Vol. 100, 1992 CO2 diffusion to the carboxylation sites remains uncertain. In addition, the response of CA activity to changing nitrogen content is not known.In this study, we used fully expanded, youn...

Plant chloroplasts constantly accumulate damage caused by visible wavelengths of light during photosynthesis. Our previous study revealed that entire photodamaged chloroplasts are subjected to vacuolar digestion through an autophagy process termed chlorophagy; however, how this process is induced and executed remained poorly understood. In this study, we monitored intracellular induction of chlorophagy in Arabidopsis () leaves and found that mesophyll cells damaged by high visible light displayed abnormal chloroplasts with a swollen shape and 2.5 times the volume of normal chloroplasts. In wild-type plants, the activation of chlorophagy decreased the number of swollen chloroplasts. In the autophagy-deficient mutants, the swollen chloroplasts persisted, and dysfunctional chloroplasts that had lost chlorophyll fluorescence accumulated in the cytoplasm. Chloroplast swelling and subsequent induction of chlorophagy were suppressed by the application of exogenous mannitol to increase the osmotic pressure outside chloroplasts or by overexpression of VESICLE INDUCING PROTEIN IN PLASTID1, which maintains chloroplast envelope integrity. Microscopic observations of autophagy-related membranes showed that swollen chloroplasts were partly surrounded by autophagosomal structures and were engulfed directly by the tonoplast, as in microautophagy. Our results indicate that an elevation in osmotic potential inside the chloroplast due to high visible light-derived envelope damage results in chloroplast swelling and serves as an induction factor for chlorophagy, and this process mobilizes entire chloroplasts via tonoplast-mediated sequestering to avoid the cytosolic accumulation of dysfunctional chloroplasts.

SummaryRice cultivars vary widely in their sensitivity to ultraviolet B (UVB) and this has been correlated with cyclobutane pyrimidine dimer (CPD) photolyase mutations that alter the structure/function of this photorepair enzyme. Here, we tested whether CPD photolyase function determines the UVB sensitivity of rice (Oryza sativa) by generating transgenic rice plants bearing the CPD photolyase gene of the UV-resistant rice cultivar Sasanishiki in the sense orientation (S-B and S-C lines) or the antisense orientation (AS-D line). The S-B and S-C plants had 5.1-and 45.7-fold higher CPD photolyase activities than the wild-type, respectively, were significantly more resistant to UVB-induced growth damage, and maintained significantly lower CPD levels in their leaves during growth under elevated UVB radiation. Conversely, the AS-D plant had little photolyase activity, was severely damaged by elevated UVB radiation, and maintained higher CPD levels in its leaves during growth under UVB radiation. Notably, the S-C plant was not more resistant to UVB-induced growth inhibition than the S-B plant, even though it had much higher CPD photolyase activity. These results strongly indicate that UVB-induced CPDs are one of principal causes of UVB-induced growth inhibition in rice plants grown under supplementary UVB radiation, and that increasing CPD photolyase activity can significantly alleviate UVB-caused growth inhibition in rice. However, further protection from UVB-induced damage may require the genetic enhancement of other systems as well.

Space travel has advanced significantly over the last six decades with astronauts spending up to 6 months at the International Space Station. Nonetheless, the living environment while in outer space is extremely challenging to astronauts. In particular, exposure to space radiation represents a serious potential long-term threat to the health of astronauts because the amount of radiation exposure accumulates during their time in space. Therefore, health risks associated with exposure to space radiation are an important topic in space travel, and characterizing space radiation in detail is essential for improving the safety of space missions. In the first part of this review, we provide an overview of the space radiation environment and briefly present current and future endeavors that monitor different space radiation environments. We then present research evaluating adverse biological effects caused by exposure to various space radiation environments and how these can be reduced. We especially consider the deleterious effects on cellular DNA and how cells activate DNA repair mechanisms. The latest technologies being developed, e.g., a fluorescent ubiquitination-based cell cycle indicator, to measure real-time cell cycle progression and DNA damage caused by exposure to ultraviolet radiation are presented. Progress in examining the combined effects of microgravity and radiation to animals and plants are summarized, and our current understanding of the relationship between psychological stress and radiation is presented. Finally, we provide details about protective agents and the study of organisms that are highly resistant to radiation and how their biological mechanisms may aid developing novel technologies that alleviate biological damage caused by radiation. Future research that furthers our understanding of the effects of space radiation on human health will facilitate risk-mitigating strategies to enable long-term space and planetary exploration.

There is a cultivar difference in the response to ultraviolet-B (UVB: 280-320 nm) in rice (Oryza sativa L.). Among Japanese lowland rice cultivars, Sasanishiki, a leading Japanese rice cultivar, is resistant to the damaging effects of UVB while Norin 1, a close relative, is less resistant. We found previously that Norin 1 was deficient in cyclobutane pyrimidine dimer (CPD) photorepair ability and suggested that the UVB sensitivity in rice depends largely on CPD photorepair ability. In order to verify that suggestion, we examined the correlation between UVB sensitivity and CPD photolyase activity in 17 rice cultivars of progenitors and relatives in breeding of UV-resistant Sasanishiki and UV-sensitive Norin 1. The amino acid at position 126 of the deduced amino acid sequence of CPD photolyase in cultivars including such as Norin 1 was found to be arginine, the CPD photolyase activities of which were lower. The amino acid at that position in cultivars including such as Sasanishiki was glutamine. Furthermore, cultivars more resistant to UVB were found to exhibit higher photolyase activities than less resistant cultivars. These results emphasize that single amino acid alteration from glutamine to arginine leads to a deficit of CPD photolyase activity and that CPD photolyase activity is one of the main factors determining UVB sensitivity in rice.

Effects of irrpdiance on photosynthetic characteristics were examined in senescent leaves of rice (Oryza sativa L.). Two irradiance treatments (100 and 20% natural sunlight) were imposed after the full expansion of the 13th leaf through senescence. The photosynthetic rate was measured as a function of intercellular C02 pressure with a gas-exchange system. The amounts of cytochrome f, coupling factor 1, ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), and chlorophyll were determined. The coupling factor 1 and cytochrome f contents decreased rapidly during senescence, and their rates of decrease were much faster from the 20% sunlight treatment than from the full sunlight treatment. These changes were well correlated with those in the photosynthetic rate at C02 pressure = 600 microbars, but not with those under the ambient air condition (350 microbars C02) and 200 microbars CO2. This suggested that the amounts of coupling factor I and cytochrome f from the full sunlight treatment cannot be limiting factors for the photosynthetic rate at ambient air conditions. The Rubisco content also decreased during senescence, but its decrease from the 20% sunlight treatment was appreciably retarded. However, this difference was not reflected in the photosynthetic rates at the ambient and 200 microbars C02. This implied that in vivo Rubisco activity may be regulated in the senescent leaves from the 20% sunlight treatment. The chlorophyll content decreased most slowly. In the 20% sunlight treatment, it remained apparentdy constant with a decline in chlorophyll a/b ratio. These photosynthetic characteristics of the senescent rice leaves under low irradiance were discussed in relation to acclimation of shade plants.thesis during senescence is mainly caused by a decrease in a functional unit of the photosynthetic system.When plants are transferred to a different irradiance, they show acclimation of the photosynthetic system. The characteristics of plants acclimated to low irradiance have been reviewed in detail (1, 2, 7). These plants generally have relatively more Chl b, low capacities of electron transport per unit of Chl, and a reduction in soluble protein relative to Chl. However, the photosynthetic characteristics of leaves aged under low irradiance after full expansion are not known. In addition, it is unclear whether the senescent leaves show the regulatory light acclimation.In this study, we examined the effects of irradiance on the in vivo and in vitro photosynthetic characteristics of senescent leaves of rice. Two irradiance treatments (100 and 20% sunlight) were imposed after the full expansion of the 13th leaf through senescence. To deduce the in vivo balance between Rubisco and electron transport limitation, we first examined the rate of CO2 assimilation as a function of C,2 according to the photosynthetic model of Farquhar and Caemmerer (9). Second, we determined the amounts of Rubisco, CF,, and Cyt f proteins in relation to the gas-exchange data. The limitation of CFI(ATP-ase) and Cyt f contents for ...