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.

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.

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.

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.

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.

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.

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.

Comparative analysis of the reactive oxygen species-producing enzymatic activity of Arabidopsis NADPH oxidases.

Reactive oxygen species (ROS) produced by NADPH oxidases, called respiratory burst oxidase homologs (Rbohs), play crucial roles in development as well as biotic and abiotic stress responses in plants. Arabidopsis has 10 Rboh genes, AtRbohA to AtRbohJ. Five AtRbohs (AtRbohC, -D, -F, -H and -J) are synergistically activated by Ca2+ -binding and protein phosphorylation to produce ROS that play various roles in planta, although the activities of the other Rbohs remain unknown. With a heterologous expression system, we found a range of ROS-producing activity among the AtRbohs with differences up to 100 times, indicating that the required amounts of ROS are different in each situation where AtRbohs act. To specify the functions of AtRbohs involved in cell growth, we focused on AtRbohC, -H and -J, which are involved in tip growth of root hairs or pollen tubes. Ectopic expression of the root hair factor AtRbohC/ROOT HAIR DEFECTIVE 2 (RHD2) in pollen tubes restored the atrbohH atrbohJ defects in tip growth of pollen tubes. However, expression of AtRbohH or -J in root hairs did not complement the tip growth defect in the atrbohC/rhd2 mutant. Our data indicate that Rbohs possess different ranges of enzymatic activity, and that some Rbohs have evolved to carry specific functions in cell growth.

Harnessing host ROS-generating machinery for the robust genome replication of a plant RNA virus.

As sessile organisms, plants have to accommodate to rapid changes in their surrounding environment. Reactive oxygen species (ROS) act as signaling molecules to transduce biotic and abiotic stimuli into plant stress adaptations. It is established that a respiratory burst oxidase homolog B of Nicotiana benthamiana (NbRBOHB) produces ROS in response to microbe-associated molecular patterns to inhibit pathogen infection. Plant viruses are also known as causative agents of ROS induction in infected plants; however, the function of ROS in plant-virus interactions remains obscure. Here, we show that the replication of red clover necrotic mosaic virus (RCNMV), a plant positive-strand RNA [(+)RNA] virus, requires NbRBOHB-mediated ROS production. The RCNMV replication protein p27 plays a pivotal role in this process, redirecting the subcellular localization of NbRBOHB and a subgroup II calcium-dependent protein kinase of N. benthamiana (NbCDPKiso2) from the plasma membrane to the p27-containing intracellular aggregate structures. p27 also induces an intracellular ROS burst in an RBOH-dependent manner. NbCDPKiso2 was shown to be an activator of the p27-triggered ROS accumulations and to be required for RCNMV replication. Importantly, this RBOH-derived ROS is essential for robust viral RNA replication. The need for RBOH-derived ROS was demonstrated for the replication of another (+)RNA virus, brome mosaic virus, suggesting that this characteristic is true for plant (+)RNA viruses. Collectively, our findings revealed a hitherto unknown viral strategy whereby the host ROS-generating machinery is diverted for robust viral RNA replication.

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.

Fine-tuning of RBOHF activity is achieved by differential phosphorylation and Ca2+ binding.

RBOHF from Arabidopsis thaliana represents a multifunctional NADPH oxidase regulating biotic and abiotic stress tolerance, developmental processes and guard cell aperture. The molecular components and mechanisms determining RBOHF activity remain to be elucidated. Here we combined protein interaction studies, biochemical and genetic approaches, and pathway reconstitution analyses to identify and characterize proteins that confer RBOHF regulation and elucidated mechanisms that adjust RBOHF activity. While the Ca2+ sensor-activated kinases CIPK11 and CIPK26 constitute alternative paths for RBOHF activation, the combined activity of CIPKs and the kinase open stomata 1 (OST1) triggers complementary activation of this NADPH oxidase, which is efficiently counteracted through dephosphorylation by the phosphatase ABI1. Within RBOHF, several distinct phosphorylation sites (p-sites) in the N-terminus of RBOHF appear to contribute individually to activity regulation. These findings identify RBOHF as a convergence point targeted by a complex regulatory network of kinases and phosphatases. We propose that this allows for fine-tuning of plant reactive oxygen species (ROS) production by RBOHF in response to different stimuli and in diverse physiological processes.

rgs-CaM Detects and Counteracts Viral RNA Silencing Suppressors in Plant Immune Priming.

Primary infection of a plant with a pathogen that causes high accumulation of salicylic acid in the plant typically via a hypersensitive response confers enhanced resistance against secondary infection with a broad spectrum of pathogens, including viruses. This phenomenon is called systemic acquired resistance (SAR), which is a plant priming for adaption to repeated biotic stress. However, the molecular mechanisms of SAR-mediated enhanced inhibition, especially of virus infection, remain unclear. Here, we show that SAR against cucumber mosaic virus (CMV) in tobacco plants (Nicotiana tabacum) involves a calmodulin-like protein, rgs-CaM. We previously reported the antiviral function of rgs-CaM, which binds to and directs degradation of viral RNA silencing suppressors (RSSs), including CMV 2b, via autophagy. We found that rgs-CaM-mediated immunity is ineffective against CMV infection in normally growing tobacco plants but is activated as a result of SAR induction via salicylic acid signaling. We then analyzed the effect of overexpression of rgs-CaM on salicylic acid signaling. Overexpressed and ectopically expressed rgs-CaM induced defense reactions, including cell death, generation of reactive oxygen species, and salicylic acid signaling. Further analysis using a combination of the salicylic acid analogue benzo-(1,2,3)-thiadiazole-7-carbothioic acid S-methyl ester (BTH) and the Ca2+ ionophore A23187 revealed that rgs-CaM functions as an immune receptor that induces salicylic acid signaling by simultaneously perceiving both viral RSS and Ca2+ influx as infection cues, implying its autoactivation. Thus, secondary infection of SAR-induced tobacco plants with CMV seems to be effectively inhibited through 2b recognition and degradation by rgs-CaM, leading to reinforcement of antiviral RNA silencing and other salicylic acid-mediated antiviral responses.IMPORTANCE Even without an acquired immune system like that in vertebrates, plants show enhanced whole-plant resistance against secondary infection with pathogens; this so-called systemic acquired resistance (SAR) has been known for more than half a century and continues to be extensively studied. SAR-induced plants strongly and rapidly express a number of antibiotics and pathogenesis-related proteins targeted against secondary infection, which can account for enhanced resistance against bacterial and fungal pathogens but are not thought to control viral infection. This study showed that enhanced resistance against cucumber mosaic virus is caused by a tobacco calmodulin-like protein, rgs-CaM, which detects and counteracts the major viral virulence factor (RNA silencing suppressor) after SAR induction. rgs-CaM-mediated SAR illustrates the growth versus defense trade-off in plants, as it targets the major virulence factor only under specific biotic stress conditions, thus avoiding the cost of constitutive activation while reducing the damage from virus infection.

Ice Nucleation Activity in Plants: The Distribution, Characterization, and Their Roles in Cold Hardiness Mechanisms.

Control of freezing in plant tissues is a key issue in cold hardiness mechanisms. Yet freeze-regulation mechanisms remain mostly unexplored. Among them, ice nucleation activity (INA) is a primary factor involved in the initiation and regulation of freezing events in plant tissues, yet the details remain poorly understood. To address this, we developed a highly reproducible assay for determining plant tissue INA and noninvasive freeze visualization tools using MRI and infrared thermography. The results of visualization studies on plant freezing behaviors and INA survey of over 600 species tissues show that (1) freezing-sensitive plants tend to have low INA in their tissues (thus tend to transiently supercool), while wintering cold-hardy species have high INA in some specialized tissues; and (2) the high INA in cold-hardy tissues likely functions as a freezing sensor to initiate freezing at warm subzero temperatures at appropriate locations and timing, resulting in the induction of tissue-/species-specific freezing behaviors (e.g., extracellular freezing, extraorgan freezing) and the freezing order among tissues: from the primary freeze to the last tissue remaining unfrozen (likely INA level dependent). The spatiotemporal distributions of tissue INA, their characterization, and functional roles are detailed. INA assay principles, anti-nucleation activity (ANA), and freeze visualization tools are also described.

Ca2+-activated reactive oxygen species production by Arabidopsis RbohH and RbohJ is essential for proper pollen tube tip growth.

In flowering plants, pollen germinates on the stigma and pollen tubes grow through the style to fertilize the ovules. Enzymatic production of reactive oxygen species (ROS) has been suggested to be involved in pollen tube tip growth. Here, we characterized the function and regulation of the NADPH oxidases RbohH and RbohJ (Respiratory burst oxidase homolog H and J) in pollen tubes in Arabidopsis thaliana. In the rbohH and rbohJ single mutants, pollen tube tip growth was comparable to that of the wild type; however, tip growth was severely impaired in the double mutant. In vivo imaging showed that ROS accumulation in the pollen tube was impaired in the double mutant. Both RbohH and RbohJ, which contain Ca(2+) binding EF-hand motifs, possessed Ca(2+)-induced ROS-producing activity and localized at the plasma membrane of the pollen tube tip. Point mutations in the EF-hand motifs impaired Ca(2+)-induced ROS production and complementation of the double mutant phenotype. We also showed that a protein phosphatase inhibitor enhanced the Ca(2+)-induced ROS-producing activity of RbohH and RbohJ, suggesting their synergistic activation by protein phosphorylation and Ca(2+). Our results suggest that ROS production by RbohH and RbohJ is essential for proper pollen tube tip growth, and furthermore, that Ca(2+)-induced ROS positive feedback regulation is conserved in the polarized cell growth to shape the long tubular cell.

Autophagy, programmed cell death and reactive oxygen species in sexual reproduction in plants.

Autophagy is one of the major cellular processes of recycling of proteins, metabolites and intracellular organelles, and plays crucial roles in the regulation of innate immunity, stress responses and programmed cell death (PCD) in many eukaryotes. It is also essential in development and sexual reproduction in many animals. In plants, although autophagy-deficient mutants of Arabidopsis thaliana show phenotypes in abiotic and biotic stress responses, their life cycle seems normal and thus little had been known until recently about the roles of autophagy in development and reproduction. Rice mutants defective in autophagy show sporophytic male sterility and immature pollens, indicating crucial roles of autophagy during pollen maturation. Enzymatic production of reactive oxygen species (ROS) by respiratory burst oxidase homologues (Rbohs) play multiple roles in regulating anther development, pollen tube elongation and fertilization. Significance of autophagy and ROS in the regulation of PCD of transient cells during plant sexual reproduction is discussed in comparison with animals.

Stomatal closure induced by phytosphingosine-1-phosphate and sphingosine-1-phosphate depends on nitric oxide and pH of guard cells in Pisum sativum.

MAIN CONCLUSION: Phyto-S1P and S1P induced stomatal closure in epidermis of pea ( Pisum sativum ) by raising the levels of NO and pH in guard cells. Phosphosphingolipids, such as phytosphingosine-1-phosphate (phyto-S1P) and sphingosine-1-phosphate (S1P), are important signaling components during drought stress. The biosynthesis of phyto-S1P or S1P is mediated by sphingosine kinases (SPHKs). Although phyto-S1P and S1P are known to be signaling components in higher plants, their ability to induce stomatal closure has been ambiguous. We evaluated in detail the effects of phyto-S1P, S1P and SPHK inhibitors on signaling events leading to stomatal closure in the epidermis of Pisum sativum. Phyto-S1P or S1P induced stomatal closure, along with a marked rise in nitric oxide (NO) and cytoplasmic pH of guard cells, as in case of ABA. Two SPHK inhibitors, DL-threo dihydrosphingosine and N',N'-dimethylsphingosine, restricted ABA-induced stomatal closure and prevented the increase of NO or pH by ABA. Modulators of NO or pH impaired both stomatal closure and increase in NO or pH by phyto-S1P/S1P. The stomatal closure by phyto-S1P/S1P was mediated by phospholipase D and phosphatidic acid (PA). When present, PA elevated the levels of pH, but not NO of guard cells. Our results demonstrate that stomatal closure induced by phyto-S1P and S1P depends on rise in pH as well as NO of guard cells. A scheme of signaling events initiated by phyto-S1P/S1P, and converging to cause stomatal closure, is proposed.

Establishment of monitoring methods for autophagy in rice reveals autophagic recycling of chloroplasts and root plastids during energy limitation.

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.

Autophagy supports biomass production and nitrogen use efficiency at the vegetative stage in rice.

Much of the nitrogen in leaves is distributed to chloroplasts, mainly in photosynthetic proteins. During leaf senescence, chloroplastic proteins, including Rubisco, are rapidly degraded, and the released nitrogen is remobilized and reused in newly developing tissues. Autophagy facilitates the degradation of intracellular components for nutrient recycling in all eukaryotes, and recent studies have revealed critical roles for autophagy in Rubisco degradation and nitrogen remobilization into seeds in Arabidopsis (Arabidopsis thaliana). Here, we examined the function of autophagy in vegetative growth and nitrogen usage in a cereal plant, rice (Oryza sativa). An autophagy-disrupted rice mutant, Osatg7-1, showed reduced biomass production and nitrogen use efficiency compared with the wild type. While Osatg7-1 showed early visible leaf senescence, the nitrogen concentration remained high in the senescent leaves. (15)N pulse chase analysis revealed suppression of nitrogen remobilization during leaf senescence in Osatg7-1. Accordingly, the reduction of nitrogen available for newly developing tissues in Osatg7-1 likely led its reduced leaf area and tillers. The limited leaf growth in Osatg7-1 decreased the photosynthetic capacity of the plant. Much of the nitrogen remaining in senescent leaves of Osatg7-1 was in soluble proteins, and the Rubisco concentration in senescing leaves of Osatg7-1 was about 2.5 times higher than in the wild type. Transmission electron micrographs showed a cytosolic fraction rich with organelles in senescent leaves of Osatg7-1. Our results suggest that autophagy contributes to efficient nitrogen remobilization at the whole-plant level by facilitating protein degradation for nitrogen recycling in senescent leaves.

Freezing behaviours in wintering Cornus florida flower bud tissues revisited using MRI.

How plant tissues control their water behaviours (phase and movement) under subfreezing temperatures through adaptative strategies (freezing behaviours) is important for their survival. However, the fine details of freezing behaviours in complex organs and their regulation mechanisms are poorly understood, and non-invasive visualization/analysis is required. The localization/density of unfrozen water in wintering Cornus florida flower buds at subfreezing temperatures was visualized with high-resolution magnetic resonance imaging (MRI). This allowed tissue-specific freezing behaviours to be determined. MRI images revealed that individual anthers and ovules remained stably supercooled to -14 to -21 °C or lower. The signal from other floral tissues decreased during cooling to -7 °C, which likely indicates their extracellular freezing. Microscopic observation and differential thermal analyses revealed that the abrupt breakdown of supercooled individual ovules and anthers resulted in their all-or-nothing type of injuries. The distribution of ice nucleation activity in flower buds determined using a test tube-based assay corroborated which tissues primarily froze. MRI is a powerful tool for non-invasively visualizing unfrozen tissues. Freezing events and/or dehydration events can be located by digital comparison of MRI images acquired at different temperatures. Only anthers and ovules preferentially remaining unfrozen are a novel freezing behaviour in flower buds. Physicochemical and biological mechanisms/implications are discussed.

Mitogen-activated protein kinase 4-like carrying an MEY motif instead of a TXY motif is involved in ozone tolerance and regulation of stomatal closure in tobacco.

The mitogen-activated protein kinases (MAPKs/MPKs) are important factors in the regulation of signal transduction in response to biotic and abiotic stresses. Previously, we characterized a MAPK from tobacco, Nicotiana tabacum MPK4 (NtMPK4). Here, we found a highly homologous gene, NtMPK4-like (NtMPK4L), in tobacco as well as other species in Solanaceae and Gramineae. Deduced amino acid sequences of their translation products carried MEY motifs instead of conserved TXY motifs of the MAPK family. We isolated the full length NtMPK4L gene and examined the physiological functions of NtMPK4L. We revealed that NtMPK4L was activated by wounding, like NtMPK4. However, a constitutively active salicylic acid-induced protein kinase kinase (SIPKK(EE)), which phosphorylates NtMPK4, did not phosphorylate NtMPK4L. Moreover, a tyrosine residue in the MEY motif was not involved in NtMPK4L activation. We also found that NtMPK4L-silenced plants showed rapid transpiration caused by remarkably open stomata. In addition, NtMPK4L-silenced plants completely lost the ability to close stomata upon ozone treatment and were highly sensitive to ozone, suggesting that this atypical MAPK plays a role in ozone tolerance through stomatal regulation.