Mikio Nishimura

Plants continuously remodel and degrade their organelles due to damage from their metabolic activities and environmental stressors, as well as an integral part of their cell differentiation programs. Whereas certain organelles use local hydrolytic enzymes for limited remodeling, most of the pathways that control the partial or complete dismantling of organelles rely on vacuolar degradation. Specifically, selective autophagic pathways play a crucial role in recognizing and sorting plant organelle cargo for vacuolar clearance, especially under cellular stress conditions induced by factors like heat, drought, and damaging light. In these short reviews, we discuss the mechanisms that control the vacuolar degradation of chloroplasts, mitochondria, endoplasmic reticulum, Golgi, and peroxisomes, with an emphasis on autophagy, recently discovered selective autophagy receptors for plant organelles, and crosstalk with other catabolic pathways.

Light is essential for plant growth, but excessive light energy produces reactive oxygen species (ROS), which can seriously damage cells. Mutants defective in ATG (autophagy related) genes show light intensity-dependent leaf damage and ROS accumulation. We found that autophagy is one of the crucial systems in protecting plants from ROS-induced damage by removing oxidative peroxisomes. Damaged peroxisomes are targeted by the PtdIns3P marker and specifically engulfed by phagophores labeled by ATG18a-GFP. Under high-intensity light, huge peroxisome aggregates are induced and captured by vacuolar membranes. Research provides a deeper understanding of plant stress response to light irradiation.

Although light is essential for photosynthesis, it has the potential to elevate intracellular levels of reactive oxygen species (ROS). Since high ROS levels are cytotoxic, plants must alleviate such damage. However, the cellular mechanism underlying ROS-induced leaf damage alleviation in peroxisomes was not fully explored. Here, we show that autophagy plays a pivotal role in the selective removal of ROS-generating peroxisomes, which protects plants from oxidative damage during photosynthesis. We present evidence that autophagy-deficient mutants show light intensity-dependent leaf damage and excess aggregation of ROS-accumulating peroxisomes. The peroxisome aggregates are specifically engulfed by pre-autophagosomal structures and vacuolar membranes in both leaf cells and isolated vacuoles, but they are not degraded in mutants. ATG18a-GFP and GFP-2×FYVE, which bind to phosphatidylinositol 3-phosphate, preferentially target the peroxisomal membranes and pre-autophagosomal structures near peroxisomes in ROS-accumulating cells under high-intensity light. Our findings provide deeper insights into the plant stress response caused by light irradiation.

Light is essential for photosynthesis, but it has the potential to elevate intracellular levels of reactive oxygen species (ROS) during photosynthesis. Photorespiration is a metabolic pathway in photosynthesis to metabolise oxidised by products from chloroplasts, and it generates a high level of ROS in peroxisomes. Since high levels of ROS are toxic, plants must manage damage from ROS. However, the cellular mechanism to elude leaf damage from ROS in peroxisomes is not fully explored. Here we show that autophagy plays a pivotal role in the selective removal of ROS-generating peroxisomes, which protects plants from oxidative damage during photosynthesis. We found that a series of peup mutants, which is a defect in autophagy degradation of peroxisomes, showed light-intensity-dependent leaf damage and excess aggregation of ROS-accumulating peroxisomes. The peroxisome aggregates were specifically engulfed by pre-autophagosomal structures and vacuolar membranes, but they were not degraded in the mutants. ATG18a-GFP and GFP-2 × FYVE, which both bind to phosphatidylinositol 3-phosphate, preferentially targeted the peroxisomal membranes and pre-autophagosomal structures near peroxisomes in ROS-accumulated cells under high-intensity light conditions. Our findings provide new information to better understand the plant stress response caused by light irradiation.

Brassicaceae plants have a dual-cell type of chemical defense against herbivory. Here, we show a novel single-cell defense involving endoplasmic reticulum (ER)-derived organelles (ER bodies) and the vacuoles. We identify various glucosinolates as endogenous substrates of the ER-body β-glucosidases BGLU23 and BGLU21. Woodlice strongly prefer to eat seedlings of bglu23 bglu21 or a glucosinolate-deficient mutant over wild-type seedlings, confirming that the β-glucosidases have a role in chemical defense: production of toxic compounds upon organellar damage. Deficiency of the Brassicaceae-specific protein NAI2 prevents ER-body formation, which results in a loss of BGLU23 and a loss of resistance to woodlice. Hence, NAI2 that interacts with BGLU23 is essential for sequestering BGLU23 in ER bodies and preventing its degradation. Artificial expression of NAI2 and BGLU23 in non-Brassicaceae plants results in the formation of ER bodies, indicating that acquisition of NAI2 by Brassicaceae plants is a key step in developing their single-cell defense system.

Seeds of soybean (Glycine max L.) are a major source of plant-derived oils. In the past, improvements have been made in the quantity and quality of seed oil. Triacylglycerols (TAGs) are the principal components of soybean seed oil, and understanding the metabolic regulation of TAGs in soybean seeds is essential. Here, we identified four soybean genes encoding TAG lipases, designated as SUGAR DEPENDENT1-1 (GmSDP1-1), GmSDP1-2, GmSDP1-3 and GmSDP1-4; these are homologous to Arabidopsis thaliana SDP1 (AtSDP1). To characterize the function of these genes during grain filling, transgenic lines of soybean were generated via RNA interference to knockdown the expression of all four GmSDP1 genes. The seed oil content of the transgenic soybean lines was significantly increased compared with the wild type (WT). Additionally, fatty acid profiles of the WT and transgenic soybean lines were altered; the content of linoleic acid, a major fatty acid in soybean seeds, was significantly reduced, whereas that of oleic acid was increased in transgenic soybean seeds compared with the WT. Substrate specificity experiments showed that TAG lipase preferentially cleaved oleic acid than linoleic acid in the oil body membrane in WT soybean. This study demonstrates that the GmSDP1 proteins regulate both the TAG content and fatty acid composition of soybean seeds during grain filling. These results provide a novel strategy for improving both the quantity and quality of soybean seed oil.

The dynamic behavior of organelles is essential for plant survival under various environmental conditions. Plant organelles, with various functions, migrate along actin filaments and contact other types of organelles, leading to physical interactions at a specific site called the membrane contact site. Recent studies have revealed the importance of physical interactions in maintaining efficient metabolite flow between organelles. In this review, we first summarize peroxisome function under different environmental conditions and growth stages to understand organelle interactions. We then discuss current knowledge regarding the interactions between peroxisome and other organelles, i.e., the oil bodies, chloroplast, and mitochondria from the perspective of metabolic and physiological regulation, with reference to various organelle interactions and techniques for estimating organelle interactions occurring in plant cells.

Plant immunity to avirulent bacterial pathogens is associated with subcellular membrane dynamics including fusion between the vacuolar and plasma membranes, resulting in hypersensitive cell death. Here, we report that ADAPTOR PROTEIN COMPLEX-4 (AP-4) subunits are involved in plant immunity associated with hypersensitive cell death. We isolated a mutant with a defect in resistance to an avirulent strain of Pseudomonas syringae pv. tomato (Pto) DC3000 avrRpm1 from a vacuolar protein sorting mutant library of Arabidopsis (Arabidopsis thaliana). The mutant was identical to gfs4-1, which has a mutation in the gene encoding the AP-4 subunit AP4B. Thus, we focused on AP4B and another subunit, AP4E. All of the mutants (ap4b-3, ap4b-4, ap4e-1, and ap4e-2) were defective in hypersensitive cell death and resistance to Pto DC3000 with the type III effector AvrRpm1 or AvrRpt2, both of which are recognized on the plasma membrane, while they showed slightly enhanced susceptibility to the type-III-secretion-deficient P. syringae strain hrcC. On the other hand, both ap4b-3 and ap4b-4 showed no defect in resistance to Pto DC3000 with the type III effector AvrRps4, which is recognized in the cytosol and does not induce hypersensitive cell death. Upon infection with Pto DC3000 avrRpt2, the ap4b-3 and ap4b-4 leaf cells did not show fusion between vacuolar and plasma membranes, whereas the wild-type leaf cells did. These results suggest that AP-4 contributes to cell death-associated immunity, possibly via membrane fusion, after type III effector-recognition on the plasma membrane.

Plant seeds accumulate large amounts of storage reserves comprising biodegradable organic matter. Humans rely on seed storage reserves for food and as industrial materials. Gene expression profiles are powerful tools for investigating metabolic regulation in plant cells. Therefore, detailed, accurate gene expression profiles during seed development are required for crop breeding. Acquiring highly purified RNA is essential for producing these profiles. Efficient methods are needed to isolate highly purified RNA from seeds. Here, we describe a method for isolating RNA from seeds containing large amounts of oils, proteins, and polyphenols, which have inhibitory effects on high-purity RNA isolation. Our method enables highly purified RNA to be obtained from seeds without the use of phenol, chloroform, or additional processes for RNA purification. This method is applicable to Arabidopsis, rapeseed, and soybean seeds. Our method will be useful for monitoring the expression patterns of low level transcripts in developing and mature seeds.

Seeds are storage organ in plants and main resource of plant oils to human civilization and the demand of plant oils are increasing yearly and expansion of the production capacity is an urgent issue worldwide. Thus, it is necessary to improve oil yields per unit area and generation of crops with high-oil content is needed. Arabidopsis thaliana plays a vital role in advancement of genetics and molecular biology in plant sciences. The forward and reverse genetic approaches with Arabidopsis have provided an overview of triacylglycerol metabolism. The elucidation of the overview contributes to understanding of spatiotemporal regulation of a metabolic flow of triacylglycerol metabolism in plant cell. This understanding sheds light on bottlenecks in triacylglycerol biosynthesis and provides novel clues for increasing seed triacylglycerol content. Recent advance in metabolic engineering approaches demonstrate several evidences that triacylglycerol metabolism is coordinated with other metabolisms. Most notably, triacylglycerol biosynthesis competes with biosynthesis of starch or seed storage proteins. These studies indicate that alterations of the metabolic pathways to avoid the competitions could be a novel concept for increasing seed oil content.