preparation-intact-tissue-for-microscopic-analysis-endosperm-cell

Preparation of Intact Tissue for Microscopic Analysis of the Endosperm Cell Layer in Developing and Mature <em>Arabidopsis</em> Seeds

In Arabidopsis seeds, the endosperm, a single layer of living cells located between the embryo and the testa, plays a critical role in regulating seed ...

Meiji University

the university of tokyo

Nutrient recycling and mobilization from organ to organ all along the plant lifespan is essential for plant survival under changing environments. Nutrient remobilization to the seeds is also essential for good seed production. In this review, we summarize the recent advances made to understand how plants manage nutrient remobilization from senescing organs to sink tissues and what is the contribution of autophagy in this process. Plant engineering manipulating autophagy for better yield and plant tolerance to stresses will be presented.

Autophagy and Nutrients Management in Plants.

Zinc (Zn) is an essential micronutrient for plant growth. Accordingly, Zn deficiency (-Zn) in agricultural fields is a serious problem, especially in developing regions. Autophagy, a major intracellular degradation system in eukaryotes, plays important roles in nutrient recycling under nitrogen and carbon starvation. However, the relationship between autophagy and deficiencies of other essential elements remains poorly understood, especially in plants. In this study, we focused on Zn due to the property that within cells most Zn is tightly bound to proteins, which can be targets of autophagy. We found that autophagy plays a critical role during -Zn in Arabidopsis (). Autophagy-defective plants ( mutants) failed to grow and developed accelerated chlorosis under -Zn. As expected, -Zn induced autophagy in wild-type plants, whereas in  mutants, various organelle proteins accumulated to high levels. Additionally, the amount of free Zn was lower in  mutants than in control plants. Interestingly, -Zn symptoms in  mutants recovered under low-light, iron-limited conditions. The levels of hydroxyl radicals in chloroplasts were elevated, and the levels of superoxide were reduced in -Zn  mutants. These results imply that the photosynthesis-mediated Fenton-like reaction, which is responsible for the chlorotic symptom of -Zn, is accelerated in  mutants. Together, our data indicate that autophagic degradation plays important functions in maintaining Zn pools to increase Zn bioavailability and maintain reactive oxygen species homeostasis under -Zn in plants.

Autophagy Increases Zinc Bioavailability to Avoid Light-Mediated Reactive Oxygen Species Production under Zinc Deficiency.

Autophagy, which is one of the self-degradation systems, promotes intracellular zinc (Zn) recycling under Zn deficiency (-Zn) in plants. Therefore, autophagy defective plants show severe chlorosis under -Zn. Root is the plant organ which directly exposed to Zn deficient environment, however, in our recent study, -Zn symptom was prominently observed in leaves as chlorosis. Here, we conducted micrografting to determine which organ's autophagic activities are important to suppress the -Zn induced chlorosis. Grafted plants that have autophagic activities only in roots or leaves were grown under -Zn and then compared chlorotic phenotypes among them. As a result, regardless of the autophagic activities in rootstocks, -Zn induced-chlorosis in leaves was occurred only when autophagy in scion was defective. This data indicates that Zn resupplied by autophagic degradation in root cells could not contribute to suppress the chlorosis in leaves. Thus, autophagy in the aerial part is critical for controlling -Zn induced-chlorosis in leaves. Taken together, along with our recently reported data, we conclude that the mechanism of Zn resupply by autophagic degradation is not systemic throughout the plant but rather a local system.

Importance of non-systemic leaf autophagy for suppression of zinc starvation induced-chlorosis.

Zinc (Zn) is nutritionally an essential metal element, but excess Zn in the environment is toxic to plants. Autophagy is a major pathway responsible for intracellular degradation. Here, we demonstrate the important role of autophagy in adaptation to excess Zn stress. We found that autophagy-defective Arabidopsis thaliana (atg2 and atg5) exhibited marked excess Zn-induced chlorosis and growth defects relative to wild-type (WT). Imaging and biochemical analyses revealed that autophagic activity was elevated under excess Zn. Interestingly, the excess Zn symptoms of atg5 were alleviated by supplementation of high levels of iron (Fe) to the media. Under excess Zn, in atg5, Fe starvation was especially severe in juvenile true leaves. Consistent with this, accumulation levels of Fe3+ near the shoot apical meristem remarkably reduced in atg5. Furthermore, excision of cotyledons induced severe excess Zn symptoms in WT, similar to those observed in atg5.Our data suggest that Fe3+ supplied from source leaves (cotyledons) via autophagy is distributed to sink leaves (true leaves) to promote healthy growth under excess Zn, revealing a new dimension, the importance of heavy-metal stress responses by the intracellular recycling.

Optimal Distribution of Iron to Sink Organs via Autophagy Is Important for Tolerance to Excess Zinc in Arabidopsis.

Inorganic phosphate (Pi) and nitrogen (N) are essential nutrients for plant growth. We found that a five-fold oversupply of nitrate rescues Arabidopsis (Arabidopsis thaliana) plants from Pi-starvation stress. Analyses of transgenic plants that overexpressed GFP-AUTOPHAGY8 showed that an oversupply of nitrate induced autophagy flux under Pi-depleted conditions. Expression of DIN6 and DIN10, the carbon (C) starvation-responsive genes, was upregulated when nitrate was oversupplied under Pi starvation, which suggested that the plants recognized the oversupply of nitrate as C starvation stress because of the reduction in the C/N ratio. Indeed, formation of Rubisco-containing bodies (RCBs), which contain chloroplast stroma and are induced by C starvation, was enhanced when nitrate was oversupplied under Pi starvation. Moreover, autophagy-deficient mutants did not release Pi (unlike wild-type plants), exhibited no RCB accumulation inside vacuoles, and were hypersensitive to Pi starvation, indicating that RCB-mediated chlorophagy is involved in Pi starvation tolerance. Thus, our results showed that the Arabidopsis response to Pi starvation is closely linked with N and C availability and that autophagy is a key factor that controls plant growth under Pi starvation.

RCB-mediated chlorophagy caused by oversupply of nitrogen suppresses phosphate-starvation stress in plants.

Under zinc (Zn) deficiency, plants take up excess iron (Fe), but the uptake is inhibited under Zn excess. Coordination between intracellular recycling, transport, and sensing is essential for Zn-Fe homeostasis. A new study shows that autophagy resupplies Zn and Fe to correct intracellular Zn-Fe imbalances.

Autophagy balances the zinc-iron seesaw caused by Zn-stress.

Inorganic phosphate (Pi) is essential for plant growth. However, Pi is often limiting in soil. Hence, plants have established several mechanisms of response to Pi starvation. One of the important mechanisms is Pi recycling, which includes membrane lipid remodeling and plastid DNA degradation via catabolic enzymes. However, the involvement of other degradation systems in Pi recycling remains unclear. Autophagy, a system for degradation of intracellular components, contributes to recycling of some nutrients, such as nitrogen, carbon, and zinc, under starvation. In the present study, we found that autophagy-deficient mutants depleted Pi early and exhibited severe leaf growth defects under Pi starvation. The main cargo of autophagy induced by early Pi depleted conditions was the endoplasmic reticulum (ER), indicating that ER-phagy, a type of autophagy that selectively degrades the ER, is involved in the response to the early phase of Pi starvation for contribution to Pi recycling. This ER-phagy was suppressed in an INOSITOL-REQUIRING ENZYME 1 double mutant, ire1a ire1b, in which ER stress responses are defective, suggesting that the early Pi starvation induced ER-phagy is induced by ER stress. Furthermore, iron limitation and inhibition of lipid-reactive oxygen species accumulation suppressed the ER-phagy. Interestingly, membrane lipid remodeling, a response to late Pi starvation, was accelerated in the ire1a ire1b under early Pi-depleted conditions. Our findings reveal the existence of two different phases of responses to Pi starvation (i.e. early and late) and indicate that ER stress-mediated ER-phagy is involved in Pi recycling in the early phase to suppress acceleration of the late phase.

Autophagy triggered by iron-mediated ER stress is an important stress response to the early phase of Pi starvation in plants.

To preserve germination ability, plant seeds must be protected from environmental stresses during the storage period. Here, we demonstrate that autophagy, an intracellular degradation system, maintains seed germination ability in . The germination ability of long-term (>5 years) stored dry seeds of autophagy-defective () mutant and wild-type (WT) plants was compared. Long-term stored (old) seeds of  mutants showed lower germination ability than WT seeds, although short-term stored (new) seeds of  mutants did not show such a phenotype. After removal of the seed coat and endosperm from old  mutant seeds, the embryos developed into seedlings. Autophagic flux was maintained in endosperm cells during the storage period, and autophagy defect resulted in the accumulation of oxidized proteins and accelerated endosperm cell death. Consistent with these findings, the transcripts of genes,  and , which are responsible for degradation/remodeling of the endosperm cell wall during germination, were reduced in old  mutant seeds. We conclude that autophagy maintains endosperm quality during seed storage by suppressing aging-dependent oxidative damage and cell death, which allows the endosperm to perform optimal functions during germination, i.e., cell wall degradation/remodeling, even after long-term storage.

Autophagy maintains endosperm quality during seed storage to preserve germination ability in Arabidopsis.

Grafting is an agricultural technique that joins tissues from different plants to obtain useful rootstock traits. However, cellular processes involved in joint tissue repair remain poorly understood. We analyzed Nicotiana benthamiana (Nb) and Arabidopsis thaliana (At) interfamily heterografting as a high-stress model and At homografting as a low-stress model. Transmission electron micrographs reveal the formation of autophagic structures in cells near the graft boundary over a long period in Nb/At interfamily grafts and in a short period of a few days in At homografts. Using a GFP-ATG8 marker line, the autophagosomes were observed in the cells near the graft boundary, especially on the scion side, where nutrient depletion occurred. Grafting of At autophagy-defective mutants decreases grafting success rates and post-grafting growth. NbATG5 knockdown suppresses graft establishment in Nb/At interfamily heterografts. Moreover, At autophagy-defective mutants show reduced callus formation directed to wounds under the nutrient-deficient conditions. These results suggest that autophagy is induced during grafting, promoting callus formation and contributing to tissue connectivity.

Article	Year
Autophagy and Nutrients Management in Plants. Cells\| PubMed ID: 31726766	2019
Autophagy Increases Zinc Bioavailability to Avoid Light-Mediated Reactive Oxygen Species Production under Zinc Deficiency. Plant physiology\| PubMed ID: 31941669	2020
Importance of non-systemic leaf autophagy for suppression of zinc starvation induced-chlorosis. Plant signaling & behavior\| PubMed ID: 32233726	2020
Optimal Distribution of Iron to Sink Organs via Autophagy Is Important for Tolerance to Excess Zinc in Arabidopsis. Plant & cell physiology\| PubMed ID: 33528512	2021
RCB-mediated chlorophagy caused by oversupply of nitrogen suppresses phosphate-starvation stress in plants. Plant physiology\| PubMed ID: 33721901	2021
Autophagy balances the zinc-iron seesaw caused by Zn-stress. Trends in plant science\| PubMed ID: 34330656	2021
Autophagy triggered by iron-mediated ER stress is an important stress response to the early phase of Pi starvation in plants. The Plant journal : for cell and molecular biology\| PubMed ID: 35306710	2022
Autophagy maintains endosperm quality during seed storage to preserve germination ability in Arabidopsis. Proceedings of the National Academy of Sciences of the United States of America\| PubMed ID: 38530890	2024
Autophagy is induced during plant grafting to promote wound healing. Nature communications\| PubMed ID: 40216774	2025

Daiki Shinozaki

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