Roadmap for the next decade of plant programmed cell death research.

Programmed cell death (PCD) is fundamentally important for plant development, abiotic stress responses and immunity, but our understanding of its regulation remains fragmented. Building a stronger research community is required to accelerate progress in this area through knowledge exchange and constructive debate. In this Viewpoint, we aim to initiate a collective effort to integrate data across a diverse set of experimental models to facilitate characterisation of the fundamental mechanisms underlying plant PCD and ultimately aid the development of a new plant cell death classification system in the future. We also put forward our vision for the next decade of plant PCD research stemming from discussions held during the 31st New Phytologist workshop, 'The Life and Death Decisions of Plant Cells' that took place at University College Dublin in Ireland (14-15 June 2023). We convey the key areas of significant progress and possible future research directions identified, including resolving the spatiotemporal control of cell death, isolation of its molecular and genetic regulators, and harnessing technical advances for studying PCD events in plants. Further, we review the breadth of potential impacts of plant PCD research and highlight the promising new applications of findings from this dynamically evolving field.

Growth control of Marchantia polymorpha gemmae using nonthermal plasma irradiation.

Several studies have documented that treatment by cold atmospheric pressure plasma (CAPP) on plants foster seed germination and growth in recent years. However, the molecular processes that underlie the action of CAPP on the seeds and plants remain mostly enigmatic. We here introduce gemmae of Marchantia polymorpha, a basal liverwort, as a novel model plant material suitable for CAPP research. Treating the gemmae with CAPP for a constant time interval at low power resulted in consistent growth enhancement, while growth inhibition at higher power in a dose-dependent manner. These results distinctly demonstrate that CAPP irradiation can positively and negatively regulate plant growth depending on the plasma intensity of irradiation, offering a suitable experimental system for understanding the molecular mechanisms underlying the action of CAPP in plants.

Rapid Propagation of Ca2+ Waves and Electrical Signals in the Liverwort Marchantia polymorpha.

In response to both biotic and abiotic stresses, vascular plants transmit long-distance Ca2+ and electrical signals from localized stress sites to distant tissues through their vasculature. Various models have been proposed for the mechanisms underlying the long-distance signaling, primarily centered around the presence of vascular bundles. We here demonstrate that the non-vascular liverwort Marchantia polymorpha possesses a mechanism for propagating Ca2+ waves and electrical signals in response to wounding. The propagation velocity of these signals was approximately 1-2 mm s-1, equivalent to that observed in vascular plants. Both Ca2+ waves and electrical signals were inhibited by La3+ as well as tetraethylammonium chloride, suggesting the crucial importance of both Ca2+ channel(s) and K+ channel(s) in wound-induced membrane depolarization as well as the subsequent long-distance signal propagation. Simultaneous recordings of Ca2+ and electrical signals indicated a tight coupling between the dynamics of these two signaling modalities. Furthermore, molecular genetic studies revealed that a GLUTAMATE RECEPTOR-LIKE (GLR) channel plays a central role in the propagation of both Ca2+ waves and electrical signals. Conversely, none of the three two-pore channels were implicated in either signal propagation. These findings shed light on the evolutionary conservation of rapid long-distance Ca2+ wave and electrical signal propagation involving GLRs in land plants, even in the absence of vascular tissue.

Novel in silico screening system for plant defense activators using deep learning-based prediction of reactive oxygen species accumulation.

BACKGROUND: Plant defense activators offer advantages over pesticides by avoiding the emergence of drug-resistant pathogens. However, only a limited number of compounds have been reported. Reactive oxygen species (ROS) act as not only antimicrobial agents but also signaling molecules that trigger immune responses. They also affect various cellular processes, highlighting the potential ROS modulators as plant defense activators. Establishing a high-throughput screening system for ROS modulators holds great promise for identifying lead chemical compounds with novel modes of action (MoAs). RESULTS: We established a novel in silico screening system for plant defense activators using deep learning-based predictions of ROS accumulation combined with the chemical properties of the compounds as explanatory variables. Our screening strategy comprised four phases: (1) development of a ROS inference system based on a deep neural network that combines ROS production data in plant cells and multidimensional chemical features of chemical compounds; (2) in silico extensive-scale screening of seven million commercially available compounds using the ROS inference model; (3) secondary screening by visualization of the chemical space of compounds using the generative topographic mapping; and (4) confirmation and validation of the identified compounds as potential ROS modulators within plant cells. We further characterized the effects of selected chemical compounds on plant cells using molecular biology methods, including pathogenic signal-triggered enzymatic ROS induction and programmed cell death as immune responses. Our results indicate that deep learning-based screening systems can rapidly and effectively identify potential immune signal-inducible ROS modulators with distinct chemical characteristics compared with the actual ROS measurement system in plant cells. CONCLUSIONS: We developed a model system capable of inferring a diverse range of ROS activity control agents that activate immune responses through the assimilation of chemical features of candidate pesticide compounds. By employing this system in the prescreening phase of actual ROS measurement in plant cells, we anticipate enhanced efficiency and reduced pesticide discovery costs. The in-silico screening methods for identifying plant ROS modulators hold the potential to facilitate the development of diverse plant defense activators with novel MoAs.

Enhanced Ca2+ binding to EF-hands through phosphorylation of conserved serine residues activates MpRBOHB and chitin-triggered ROS production.

NADPH oxidases/RBOHs catalyze apoplastic ROS production and act as key signaling nodes, integrating multiple signal transduction pathways regulating plant development and stress responses. Although RBOHs have been suggested to be activated by Ca2+ binding and phosphorylation by various protein kinases, a mechanism linking Ca2+ binding and phosphorylation in the activity regulation remained elusive. Chitin-triggered ROS production required cytosolic Ca2+ elevation and Ca2+ binding to MpRBOHB in a liverwort Marchantia polymorpha. Heterologous expression analysis of truncated variants revealed that a segment of the N-terminal cytosolic region highly conserved among land plant RBOHs encompassing the two EF-hand motifs is essential for the activation of MpRBOHB. Within the conserved regulatory domain, we have identified two Ser residues whose phosphorylation is critical for the activation in planta. Isothermal titration calorimetry analyses revealed that phosphorylation of the two Ser residues increased the Ca2+ binding affinity of MpRBOHB, while Ca2+ binding is indispensable for the activation, even if the two Ser residues are phosphorylated. Our findings shed light on a mechanism through which phosphorylation potentiates the Ca2+ -dependent activation of MpRBOHB, emphasizing the pivotal role of Ca2+ binding in mediating the Ca2+ and phosphorylation-driven activation of MpRBOHB, which is likely to represent a fundamental mechanism conserved among land plant RBOHs.

Diversity and characteristics of plant immunity-activating bacteria from Brassicaceae plants.

BACKGROUND: Microorganisms that activate plant immune responses are useful for application as biocontrol agents in agriculture to minimize crop losses. The present study was conducted to identify and characterize plant immunity-activating microorganisms in Brassicaceae plants. RESULTS: A total of 25 bacterial strains were isolated from the interior of a Brassicaceae plant, Raphanus sativus var. hortensis. Ten different genera of bacteria were identified: Pseudomonas, Leclercia, Enterobacter, Xanthomonas, Rhizobium, Agrobacterium, Pantoea, Rhodococcus, Microbacterium, and Plantibacter. The isolated strains were analyzed using a method to detect plant immunity-activating microorganisms that involves incubation of the microorganism with tobacco BY-2 cells, followed by treatment with cryptogein, a proteinaceous elicitor of tobacco immune responses. In this method, cryptogein-induced production of reactive oxygen species (ROS) in BY-2 cells serves as a marker of immune activation. Among the 25 strains examined, 6 strains markedly enhanced cryptogein-induced ROS production in BY-2 cells. These 6 strains colonized the interior of Arabidopsis plants, and Pseudomonas sp. RS3R-1 and Rhodococcus sp. RS1R-6 selectively enhanced plant resistance to the bacterial pathogens Pseudomonas syringae pv. tomato DC3000 and Pectobacterium carotovorum subsp. carotovorum NBRC 14082, respectively. In addition, Pseudomonas sp. RS1P-1 effectively enhanced resistance to both pathogens. We also comprehensively investigated the localization (i.e., cellular or extracellular) of the plant immunity-activating components produced by the bacteria derived from R. sativus var. hortensis and the components produced by previously isolated bacteria derived from another Brassicaceae plant species, Brassica rapa var. perviridis. Most gram-negative strains enhanced cryptogein-induced ROS production in BY-2 cells via the presence of cells themselves rather than via extracellular components, whereas many gram-positive strains enhanced ROS production via extracellular components. Comparative genomic analyses supported the hypothesis that the structure of lipopolysaccharides in the outer cell envelope plays an important role in the ROS-enhancing activity of gram-negative Pseudomonas strains. CONCLUSIONS: The assay method described here based on elicitor-induced ROS production in cultured plant cells enabled the discovery of novel plant immunity-activating bacteria from R. sativus var. hortensis. The results in this study also suggest that components involved in the ROS-enhancing activity of the bacteria may differ depending largely on genus and species.

Regulation of PaRBOH1-mediated ROS production in Norway spruce by Ca2+ binding and phosphorylation.

Plant respiratory burst oxidase homologs (RBOHs) are plasma membrane-localized NADPH oxidases that generate superoxide anion radicals, which then dismutate to H2O2, into the apoplast using cytoplasmic NADPH as an electron donor. PaRBOH1 is the most highly expressed RBOH gene in developing xylem as well as in a lignin-forming cell culture of Norway spruce (Picea abies L. Karst.). Since no previous information about regulation of gymnosperm RBOHs exist, our aim was to resolve how PaRBOH1 is regulated with a focus on phosphorylation. The N-terminal part of PaRBOH1 was found to contain several putative phosphorylation sites and a four-times repeated motif with similarities to the Botrytis-induced kinase 1 target site in Arabidopsis AtRBOHD. Phosphorylation was indicated for six of the sites in in vitro kinase assays using 15 amino-acid-long peptides for each of the predicted phosphotarget site in the presence of protein extracts of developing xylem. Serine and threonine residues showing positive response in the peptide assays were individually mutated to alanine (kinase-inactive) or to aspartate (phosphomimic), and the wild type PaRBOH1 and the mutated constructs transfected to human kidney embryogenic (HEK293T) cells with a low endogenous level of extracellular ROS production. ROS-producing assays with HEK cells showed that Ca2+ and phosphorylation synergistically activate the enzyme and identified several serine and threonine residues that are likely to be phosphorylated including a novel phosphorylation site not characterized in other plant species. These were further investigated with a phosphoproteomic study. Results of Norway spruce, the first gymnosperm species studied in relation to RBOH regulation, show that regulation of RBOH activity is conserved among seed plants.

Detection of NO3- introduced in plasma-irradiated dry lettuce seeds using liquid chromatography-electrospray ionization quantum mass spectrometry (LC-ESI QMS).

Discharge plasma irradiates seeds with reactive oxygen and nitrogen species (RONS). However, RONS introduced in seeds by plasma irradiation have not been successfully detected thus far. This study provides experimental evidence that nitrate ion NO3- is introduced in lettuce seeds as RONS upon irradiation with atmospheric-pressure air dielectric barrier discharge plasma. Plasma irradiation for 5 min promotes seed germination. The components of the plasma-irradiated seeds were examined using electrospray ionization quantum mass spectrometry (ESI QMS), which revealed that the plasma irradiation introduced an ion with a mass of 62 m/z in detectable amounts. This ion was identified as NO3- by liquid chromatography (LC), multiple wavelength detector (MWD), and LC-ESI QMS. A one-dimensional simulation at electron temperature Te = 1 eV, electron density Ne = 1013/m3, and gas temperature Tg = 300 K indicated the introduction of NO3-, involving nitric oxide NO. NO3- is one of the most important ions that trigger signal transduction for germination when introduced in seeds. The scanning electron microscopy (SEM) images revealed that there was no change on the surface of the seeds after plasma irradiation. Plasma irradiation is an effective method of introducing NO3- in seeds in a dry process without causing damage.

Quantitative Analysis for ROS-Producing Activity and Regulation of Plant NADPH Oxidases in HEK293T Cells.

Reactive oxygen species (ROS) produced by plant NADPH oxidases, respiratory burst oxidase homologs (RBOHs), play key roles in biotic and abiotic stress responses and development in plants. While properly controlled amounts of ROS function as signaling molecules, excessive accumulation of ROS can cause undesirable side effects due to their ability to oxidize DNA, lipids, and proteins. To limit the damaging consequences of unrestricted ROS accumulation, RBOH activity is tightly controlled by post-translational modifications (PTMs) and protein-protein interactions. In order to analyze these elaborate regulatory mechanisms, it is crucial to quantitatively assess the ROS-producing activity of RBOHs. Given the high endogenous ROS generation in plants, however, it can be challenging in plant cells to measure ROS production derived from specific RBOHs and to analyze the contribution of regulatory events for their activation and inactivation. Here we describe human embryonic kidney 293T (HEK293T) cells as a heterologous expression system and a useful tool to quantitatively monitor ROS production by RBOHs. This system permits the reconstitution of regulatory events to dissect the effects of Ca2+, phosphorylation, and protein-protein interactions on RBOH-dependent ROS production.

Interactions between pH, reactive species, and cells in plasma-activated water can remove algae.

Lightning strikes cause nitrogen to dissolve in water and form reactive nitrogen and oxygen species, which form natural fertilizers that can be absorbed through plant roots. Such processes during rainstorm events can be simulated by applying plasma to a solution. Plasma-activated water (PAW) has great potential as a source of various dissolved reactive chemical species. Different mixtures of species are produced using different solution compositions. Here, basil seeds were grown in PAW to prevent blooms of Chlorella vulgaris and ion chromatography and UV-vis spectroscopy were used to quantify reactive ions. NO2 -, NO3 -, and H2O2 were found to be key to the antialgal effect. Secondary reactive ions such as peroxynitrite (ONOO-, ONOOH) were also involved. The antialgal effect was strongly related to the pH around the algal cells. Acidification was predominantly caused by the generation of NO2 - and H2O2. After two weeks monitoring basil growth, the antifungal properties were preserved, few reactive oxygen species formed in the plasma zone, and only reactive nitrogen species were transformed into reactive peroxynitrite ions. The pH around the cells was determined using an iridium oxide microelectrode. The PAW antialgal mechanism depended on acidic conditions (pH 2.2, at which peroxynitrite can be generated) under which ONOOH penetrated the algal cell membranes, destroying the cells and preventing growth. This practical and sustainable PAW process allows a surprising amount of fertilizer to be generated with an antialgal effect that could be used in various eco-friendly agricultural processes under ambient conditions.

Functional Analyses of the Two Distinctive Types of Two-Pore Channels and the Slow Vacuolar Channel in Marchantia polymorpha.

The two-pore channel (TPC) family is widely conserved in eukaryotes. Many vascular plants, including Arabidopsis and rice, possess a single TPC gene which functions as a slow vacuolar (SV) channel-voltage-dependent cation-permeable channel located in the vacuolar membrane (tonoplast). On the other hand, a liverwort Marchantia polymorpha genome encodes three TPC homologs: MpTPC1 is similar to TPCs in vascular plants (type 1 TPC), while MpTPC2 and MpTPC3 are classified into a distinctive group (type 2 TPC). Phylogenetic analysis suggested that the type 2 TPC emerged before the land colonization in plant evolution and was lost in vascular plants and hornworts. All of the three MpTPCs were shown to be localized at the tonoplast. We generated knockout mutants of tpc1, tpc2, tpc3 and tpc2 tpc3 double mutant by clustered regularly interspaced short palindromic repeats/Cas9 genome editing and performed patch-clamp analyses of isolated vacuoles. The SV channel activity was abolished in the Mptpc1 loss-of-function mutant (Mptpc1-1KO), while Mptpc2-1KO, Mptpc3-1KO and Mptpc2-2/tpc3-2KO double mutant exhibited similar activity to the wild type, indicating that MpTPC1 (type 1) is solely responsible for the SV channel activity. Activators of mammalian TPCs, phosphatidylinositol-3,5-bisphosphate and nicotinic acid adenine dinucleotide phosphate, did not affect the ion channel activity of any MpTPCs. These results indicate that the type 1 TPCs, which are well conserved in all land plant species, encode the SV channel, while the type 2 TPCs likely encode other tonoplast cation channel(s) distinct from the SV channel and animal TPCs.

Identification of the sex-determining factor in the liverwort Marchantia polymorpha reveals unique evolution of sex chromosomes in a haploid system.

Sex determination is a central process for sexual reproduction and is often regulated by a sex determinant encoded on a sex chromosome. Rules that govern the evolution of sex chromosomes via specialization and degeneration following the evolution of a sex determinant have been well studied in diploid organisms. However, distinct predictions apply to sex chromosomes in organisms where sex is determined in the haploid phase of the life cycle: both sex chromosomes, female U and male V, are expected to maintain their gene functions, even though both are non-recombining. This is in contrast to the X-Y (or Z-W) asymmetry and Y (W) chromosome degeneration in XY (ZW) systems of diploids. Here, we provide evidence that sex chromosomes diverged early during the evolution of haploid liverworts and identify the sex determinant on the Marchantia polymorpha U chromosome. This gene, Feminizer, encodes a member of the plant-specific BASIC PENTACYSTEINE transcription factor family. It triggers female differentiation via regulation of the autosomal sex-determining locus of FEMALE GAMETOPHYTE MYB and SUPPRESSOR OF FEMINIZATION. Phylogenetic analyses of Feminizer and other sex chromosome genes indicate dimorphic sex chromosomes had already been established 430 mya in the ancestral liverwort. Feminizer also plays a role in reproductive induction that is shared with its gametolog on the V chromosome, suggesting an ancestral function, distinct from sex determination, was retained by the gametologs. This implies ancestral functions can be preserved after the acquisition of a sex determination mechanism during the evolution of a dominant haploid sex chromosome system.

Ultrafine bubble water mitigates plant growth in damaged soil.

Water containing ultrafine/nano bubbles (UFBs) promoted the growth of tomato (Solanum lycopersicum) in soil damaged by cultivation of tomato in the previous year or bacterial wilt-like disease and also promoted the growth of lettuce (Lactuca sativa) when lettuce was grown in the soil damaged by repeated cultivation of lettuce. On the other hand, UFB supply did not affect plant growth in rock wool or healthy soil. Furthermore, the growth of lettuce was not affected by UFB water treatment in the soil damaged by the cultivation of tomato. UFB water partly suppressed the growth of the pathogen of bacteria wilt disease, Ralstonia solanacearum in vitro. These data suggest that UFB water is effective to recover the plant growth from soil damage.

Impact of Mammalian Two-Pore Channel Inhibitors on Long-Distance Electrical Signals in the Characean Macroalga Nitellopsis obtusa and the Early Terrestrial Liverwort Marchantia polymorpha.

Inhibitors of human two-pore channels (TPC1 and TPC2), i.e., verapamil, tetrandrine, and NED-19, are promising medicines used in treatment of serious diseases. In the present study, the impact of these substances on action potentials (APs) and vacuolar channel activity was examined in the aquatic characean algae Nitellopsis obtusa and in the terrestrial liverwort Marchantia polymorpha. In both plant species, verapamil (20-300 µM) caused reduction of AP amplitudes, indicating impaired Ca2+ transport. In N. obtusa, it depolarized the AP excitation threshold and resting potential and prolonged AP duration. In isolated vacuoles of M. polymorpha, verapamil caused a reduction of the open probability of slow vacuolar SV/TPC channels but had almost no effect on K+ channels in the tonoplast of N. obtusa. In both species, tetrandrine (20-100 µM) evoked a pleiotropic effect: reduction of resting potential and AP amplitudes and prolongation of AP repolarization phases, especially in M. polymorpha, but it did not alter vacuolar SV/TPC activity. NED-19 (75 µM) caused both specific and unspecific effects on N. obtusa APs. In M. polymorpha, NED-19 increased the duration of repolarization. However, no inhibition of SV/TPC channels was observed in Marchantia vacuoles, but an increase in open probability and channel flickering. The results indicate an effect on Ca2+ -permeable channels governing plant excitation.

An efficient direct screening system for microorganisms that activate plant immune responses based on plant-microbe interactions using cultured plant cells.

Microorganisms that activate plant immune responses have attracted considerable attention as potential biocontrol agents in agriculture because they could reduce agrochemical use. However, conventional methods to screen for such microorganisms using whole plants and pathogens are generally laborious and time consuming. Here, we describe a general strategy using cultured plant cells to identify microorganisms that activate plant defense responses based on plant-microbe interactions. Microbial cells were incubated with tobacco BY-2 cells, followed by treatment with cryptogein, a proteinaceous elicitor of tobacco immune responses secreted by an oomycete. Cryptogein-induced production of reactive oxygen species (ROS) in BY-2 cells served as a marker to evaluate the potential of microorganisms to activate plant defense responses. Twenty-nine bacterial strains isolated from the interior of Brassica rapa var. perviridis plants were screened, and 8 strains that enhanced cryptogein-induced ROS production in BY-2 cells were selected. Following application of these strains to the root tip of Arabidopsis seedlings, two strains, Delftia sp. BR1R-2 and Arthrobacter sp. BR2S-6, were found to induce whole-plant resistance to bacterial pathogens (Pseudomonas syringae pv. tomato DC3000 and Pectobacterium carotovora subsp. carotovora NBRC 14082). Pathogen-induced expression of plant defense-related genes (PR-1, PR-5, and PDF1.2) was enhanced by the pretreatment with strain BR1R-2. This cell-cell interaction-based platform is readily applicable to large-scale screening for microorganisms that enhance plant defense responses under various environmental conditions.

Ultrastructural characterization of microlipophagy induced by the interaction of vacuoles and lipid bodies around generative and sperm cells in Arabidopsis pollen.

During pollen maturation, various organelles change their distribution and function during development as male gametophytes. We analyzed the behavior of lipid bodies and vacuoles involved in lipophagy in Arabidopsis pollen using serial section SEM and conventional TEM. At the bicellular pollen stage, lipid bodies in the vegetative cells lined up at the surface of the generative cell. Vacuoles then tightly attached, drew in, and degraded the lipid bodies and eventually occupied the space of the lipid bodies. Degradation of lipid began before transfer of the entire contents of the lipid body. At the tricellular stage, vacuoles instead of lipid bodies surrounded the sperm cells. The degradation of lipid bodies is morphologically considered microautophagy. The atg2-1 Arabidopsis mutant is deficient in one autophagy-related gene (ATG). In this mutant, the assembly of vacuoles around sperm cells was sparser than that in wild-type pollen. The deficiency of ATG2 likely prevents or slows lipid degradation, although it does not prevent contact between organelles. These results demonstrate the involvement of microlipophagy in the pollen development of Arabidopsis.

Multicolour chemical imaging of plant tissues with hyperspectral stimulated Raman scattering microscopy.

Recent development of stimulated Raman scattering (SRS) microscopy allows for label-free biological imaging with chemical specificity based on molecular-vibrational signatures. In particular, hyperspectral SRS imaging can acquire a molecular-vibrational spectrum at each pixel, allowing us not only to investigate the spectral difference of various biological molecules but also to discriminate different constituents based on their spectral difference. However, the number of constituents discriminated in previous label-free SRS imaging was limited to four because of the subtleness of spectral difference. Here, we report hyperspectral SRS imaging of plant tissues including leaves of Camellia japonica, roots of Arabidopsis thaliana, and thalli of a liverwort Marchantia polymorpha L. We show that SRS can discriminate as many as six components in Marchantia polymorpha L. without labeling. Our results demonstrate the effectiveness of hyperspectral SRS imaging as a tool for label-free multicolour imaging analysis of various biomolecules in plant tissues.

Cell Cycle-Dependence of Autophagic Activity and Inhibition of Autophagosome Formation at M Phase in Tobacco BY-2 Cells.

Autophagy is ubiquitous in eukaryotic cells and plays an essential role in stress adaptation and development by recycling nutrients and maintaining cellular homeostasis. However, the dynamics and regulatory mechanisms of autophagosome formation during the cell cycle in plant cells remain poorly elucidated. We here analyzed the number of autophagosomes during cell cycle progression in synchronized tobacco BY-2 cells expressing YFP-NtATG8a as a marker for the autophagosomes. Autophagosomes were abundant in the G2 and G1 phases of interphase, though they were much less abundant in the M and S phases. Autophagosomes drastically decreased during the G2/M transition, and the CDK inhibitor roscovitine inhibited the G2/M transition and the decrease in autophagosomes. Autophagosomes were rapidly increased by a proteasome inhibitor, MG-132. MG-132-induced autophagosome formation was also markedly lower in the M phases than during interphase. These results indicate that the activity of autophagosome formation is differently regulated at each cell cycle stage, which is strongly suppressed during mitosis.

Impact of Autophagy on Gene Expression and Tapetal Programmed Cell Death During Pollen Development in Rice.

Autophagy has recently been shown to be required for tapetal programmed cell death (PCD) and pollen maturation in rice. A transcriptional regulatory network is also known to play a key role in the progression of tapetal PCD. However, the relationship between the gene regulatory network and autophagy in rice anther development is mostly unknown. Here, we comprehensively analyzed the effect of autophagy disruption on gene expression profile during the tapetal PCD in rice anther development using high-throughput RNA sequencing. Expression of thousands of genes, including specific transcription factors and several proteases required for tapetal degradation, fluctuated synchronously at specific stages during tapetal PCD progression in the wild-type anthers, while this fluctuation showed significant delay in the autophagy-deficient mutant Osatg7-1. Moreover, gene ontology enrichment analysis in combination with self-organizing map clustering as well as pathway analysis revealed that the expression patterns of a variety of organelle-related genes as well as genes involved in carbohydrate/lipid metabolism were affected in the Osatg7-1 mutant during pollen maturation. These results suggest that autophagy is required for proper regulation of gene expression and quality control of organelles and timely progression of tapetal PCD during rice pollen development.

Essential roles of autophagy in metabolic regulation in endosperm development during rice seed maturation.

Autophagy plays crucial roles in the recycling of metabolites, and is involved in many developmental processes. Rice mutants defective in autophagy are male sterile due to immature pollens, indicating its critical role in pollen development. However, physiological roles of autophagy during seed maturation had remained unknown. We here found that seeds of the rice autophagy-deficient mutant Osatg7-1, that produces seeds at a very low frequency in paddy fields, are smaller and show chalky appearance and lower starch content in the endosperm at the mature stage under normal growth condition. We comprehensively analyzed the effects of disruption of autophagy on biochemical properties, proteome and seed quality, and found an abnormal activation of starch degradation pathways including accumulation of α-amylases in the endosperm during seed maturation in Osatg7-1. These results indicate critical involvement of autophagy in metabolic regulation in the endosperm of rice, and provide insights into novel autophagy-mediated regulation of starch metabolism during seed maturation.