Auxin signaling is essential for organogenesis but not for cell survival in the liverwort Marchantia polymorpha.

Auxin plays pleiotropic roles in plant development via gene regulation upon its perception by the receptors TRANSPORT INHIBITOR RESPONSE 1/AUXIN SIGNALING F-BOX (TIR1/AFBs). This auxin-regulated transcriptional control mechanism originated in the common ancestor of land plants. Although the complete loss of TIR1/AFBs causes embryonic lethality in Arabidopsis thaliana, it is unclear whether the requirement for TIR1-mediated auxin perception in cell viability can be generalized. The model liverwort Marchantia polymorpha has a minimal auxin signaling system with only a single TIR1/AFB, MpTIR1. Here we show by genetic, biochemical, and transcriptomic analyses that MpTIR1 functions as an evolutionarily conserved auxin receptor. Null mutants and conditionally knocked-out mutants of MpTIR1 were viable but incapable of forming any organs and grew as cell masses. Principal component analysis performed using transcriptomes at various developmental stages indicated that MpTIR1 is involved in the developmental transition from spores to organized thalli, during which apical notches containing stem cells are established. In Mptir1 cells, stem cell- and differentiation-related genes were up- and downregulated, respectively. Our findings suggest that, in M. polymorpha, auxin signaling is dispensable for cell division but is essential for three-dimensional patterning of the plant body by establishing pluripotent stem cells for organogenesis, a derived trait of land plants.

Regulation of Photosynthetic Carbohydrate Metabolism by a Raf-Like Kinase in the Liverwort Marchantia polymorpha.

To optimize growth and development, plants monitor photosynthetic activities and appropriately regulate various cellular processes. However, signaling mechanisms that coordinate plant growth with photosynthesis remain poorly understood. To identify factors that are involved in signaling related to photosynthetic stimuli, we performed a phosphoproteomic analysis with Marchantia polymorpha, an extant bryophyte species in the basal lineage of land plants. Among proteins whose phosphorylation status changed differentially between dark-treated plants and those after light irradiation but failed to do so in the presence of a photosynthesis inhibitor, we identified a B4-group Raf-like kinase, named PHOTOSYNTHESIS-RELATED RAF (MpPRAF). Biochemical analyses confirmed photosynthesis-activity-dependent changes in the phosphorylation status of MpPRAF. Mutations in the MpPRAF gene resulted in growth retardation. Measurement of carbohydrates demonstrated both hyper-accumulation of starch and reduction of sucrose in Mppraf mutants. Neither inhibition of starch synthesis nor exogenous supply of sucrose alleviated the growth defect, suggesting serious impairment of Mppraf mutants in both the synthesis of sucrose and the repression of its catabolism. As a result of the compromised photosynthate metabolism, photosynthetic electron transport was downregulated in Mppraf mutants. A mutated MpPRAF with a common amino acid substitution for inactivating kinase activity was unable to rescue the Mppraf mutant defects. Our results provide evidence that MpPRAF is a photosynthesis signaling kinase that regulates sucrose metabolism.

A pollen coat-inducible autoinhibited Ca2+-ATPase expressed in stigmatic papilla cells is required for compatible pollination in the Brassicaceae.

In the Brassicaceae, intraspecific non-self pollen (compatible pollen) can germinate and grow into stigmatic papilla cells, while self-pollen or interspecific pollen is rejected at this stage. However, the mechanisms underlying this selective acceptance of compatible pollen remain unclear. Here, using a cell-impermeant calcium indicator, we showed that the compatible pollen coat contains signaling molecules that stimulate Ca(2+) export from the papilla cells. Transcriptome analyses of stigmas suggested that autoinhibited Ca(2+)-ATPase13 (ACA13) was induced after both compatible pollination and compatible pollen coat treatment. A complementation test using a yeast Saccharomyces cerevisiae strain lacking major Ca(2+) transport systems suggested that ACA13 indeed functions as an autoinhibited Ca(2+) transporter. ACA13 transcription increased in papilla cells and in transmitting tracts after pollination. ACA13 protein localized to the plasma membrane and to vesicles near the Golgi body and accumulated at the pollen tube penetration site after pollination. The stigma of a T-DNA insertion line of ACA13 exhibited reduced Ca(2+) export, as well as defects in compatible pollen germination and seed production. These findings suggest that stigmatic ACA13 functions in the export of Ca(2+) to the compatible pollen tube, which promotes successful fertilization.

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.

A unique intracellular compartment formed during the oligotrophic growth of Rhodococcus erythropolis N9T-4.

Rhodococcus erythropolis N9T-4, isolated from stored crude oil, shows extremely oligotrophic features and can grow on a basal medium without any additional carbon, nitrogen, sulfur, and energy sources, but requires CO2 for its oligotrophic growth. Transmission electron microscopic observation showed that a relatively large and spherical compartment was observed in a N9T-4 cell grown under oligotrophic conditions. In most cases, only one compartment was observed per cell, but in some cases, it was localized at each pole of the cell, suggesting that it divides at cell division. We termed this unique bacterial compartment an oligobody. The oligobody was not observed or very rarely observed in small sizes under nutrient rich conditions, whereas additional carbon sources did not affect oligobody formation. Energy dispersive X-ray spectroscopy analysis revealed remarkable peaks corresponding to phosphorus and potassium in the oligobody. The oligobodies in N9T-4 cells could be stained by Toluidine blue, suggesting that the oligobody is composed of inorganic polyphosphate and is a type of acidocalcisome. Two genes-encoding polyphosphate kinases, ppk1 and ppk2, were found in the N9T-4 genome: ppk1 disruption caused a negative effect on the formation of the oligobody. Although it was suggested that the oligobody plays an important role for the oligotrophic growth, both ppk-deleted mutants showed the same level of oligotrophic growth as the wild-type strain.

Cullin1-P is an Essential Component of Non-Self Recognition System in Self-Incompatibility in Petunia.

Self-incompatibility (SI) in flowering plants is a genetic reproductive barrier to distinguish self- and non-self pollen to promote outbreeding. In Solanaceae, self-pollen is rejected by the ribonucleases expressed in the styles (S-RNases), via its cytotoxic function. On the other side, the male-determinant is the S-locus F-box proteins (SLFs) expressed in pollen. Multiple SLFs collaboratively detoxify non-self S-RNases, therefore, non-self recognition is the mode of self-/non-self discrimination in Solanaceae. It is considered that SLFs function as a substrate-recognition module of the Skp1-Cullin1-F-box (SCF) complex that inactivates non-self S-RNases via their polyubiquitination, which leads to degradation by 26S proteasome. In fact, PhSSK1 (Petunia hybrida SLF-interacting Skp1-like1) was identified as a specific component of SCFSLF and was shown to be essential for detoxification of S-RNase in Petunia However, different molecules are proposed as the candidate Cullin1, another component of SCFSLF, and there is as yet no definite conclusion. Here, we identified five Cullin1s from the expressed sequence tags (ESTs) derived from the male reproductive organ in Petunia Among them, only PhCUL1-P was co-immunoprecipitated with S7-SLF2. In vitro protein-binding assay suggested that PhSSK1 specifically forms a complex with PhCUL1-P in an SLF-dependent manner. Knockdown of PhCUL1-P suppressed fertility of transgenic pollen in cross-compatible pollination in the functional S-RNase-dependent manner. These results suggested that SCFSLF selectively uses PhCUL1-P. Phylogeny of Cullin1s indicates that CUL1-P is recruited into the SI machinery during the evolution of Solanaceae, suggesting that the SI components have evolved differently among species in Solanaceae and Rosaceae, despite both families sharing the S-RNase-based SI.

Trans-acting small RNA determines dominance relationships in Brassica self-incompatibility.

A diploid organism has two copies of each gene, one inherited from each parent. The expression of two inherited alleles is sometimes biased by the effects known as dominant/recessive relationships, which determine the final phenotype of the organism. To explore the mechanisms underlying these relationships, we have examined the monoallelic expression of S-locus protein 11 genes (SP11), which encode the male determinants of self-incompatibility in Brassica. We previously reported that SP11 expression was monoallelic in some S heterozygotes, and that the promoter regions of recessive SP11 alleles were specifically methylated in the anther tapetum. Here we show that this methylation is controlled by trans-acting small non-coding RNA (sRNA). We identified inverted genomic sequences that were similar to the recessive SP11 promoters in the flanking regions of dominant SP11 alleles. These sequences were specifically expressed in the anther tapetum and processed into 24-nucleotide sRNA, named SP11 methylation inducer (Smi). Introduction of the Smi genomic region into the recessive S homozygotes triggered the methylation of the promoter of recessive SP11 alleles and repressed their transcription. This is an example showing sRNA encoded in the flanking region of a dominant allele acts in trans to induce transcriptional silencing of the recessive allele. Our finding may provide new insights into the widespread monoallelic gene expression systems.

Negative feedback by IRE1ß optimizes mucin production in goblet cells.

In mammals, the prototypical endoplasmic reticulum (ER) stress sensor inositol-requiring enzyme 1 (IRE1) has diverged into two paralogs. IRE1α is broadly expressed and mediates the unconventional splicing of X-box binding protein 1 (XBP1) mRNA during ER stress. By contrast, IRE1ß is expressed selectively in the digestive tract, and its function remains unclear. Here, we report that IRE1ß plays a distinctive role in mucin-secreting goblet cells. In IRE1ß(-/-) mice, aberrant mucin 2 (MUC2) accumulated in the ER of goblet cells, accompanied by ER distension and elevated ER stress signaling such as increased XBP1 mRNA splicing. In contrast, conditional IRE1α(-/-) mice showed no such ER distension but a marked decrease in spliced XBP1 mRNA. mRNA stability assay revealed that MUC2 mRNA was greatly stabilized in IRE1ß(-/-) mice. These findings suggest that in goblet cells, IRE1ß, but not IRE1α, promotes efficient protein folding and secretion in the ER by optimizing the level of mRNA encoding their major secretory product, MUC2.

Transcriptional characteristics and differences in Arabidopsis stigmatic papilla cells pre- and post-pollination.

Pollination is an important early step in sexual plant reproduction. In Arabidopsis thaliana, sequential pollination events, from pollen adhesion onto the stigma surface to pollen tube germination and elongation, occur on the stigmatic papilla cells. Following successful completion of these events, the pollen tube penetrates the stigma and finally fertilizes a female gametophyte. The pollination events are thought to be initiated and regulated by interactions between papilla cells and pollen. Here, we report the characterization of gene expression profiles of unpollinated (UP), compatible pollinated (CP) and incompatible pollinated (IP) papilla cells in A. thaliana. Based on cell type-specific transcriptome analysis from a combination of laser microdissection and RNA sequencing, 15,475, 17,360 and 16,918 genes were identified as expressed in UP, CP and IP papilla cells, respectively, and, of these, 14,392 genes were present in all three data sets. Differentially expressed gene (DEG) analyses identified 147 and 71 genes up-regulated in CP and IP papilla cells, respectively, and 115 and 46 genes down-regulated. Gene Ontology and metabolic pathway analyses revealed that papilla cells play an active role as the female reproductive component in pollination, particularly in information exchange, signal transduction, internal physiological changes and external morphological modification. This study provides fundamental information on the molecular mechanisms involved in pollination in papilla cells, furthering our understanding of the reproductive role of papilla cells.

Cytoplasmic Ca2+ changes dynamically during the interaction of the pollen tube with synergid cells.

The directional growth of the pollen tube from the stigma to the embryo sac in the ovules is regulated by pollen-pistil interactions based on intercellular communication. Although pollen tube growth is regulated by the cytoplasmic Ca(2+) concentration ([Ca(2+)](cyt)), it is not known whether [Ca(2+)](cyt) is involved in pollen tube guidance and reception. Using Arabidopsis expressing the GFP-based Ca(2+)-sensor yellow cameleon 3.60 (YC3.60) in pollen tubes and synergid cells, we monitored Ca(2+) dynamics in these cells during pollen tube guidance and reception under semi-in vivo fertilization conditions. In the pollen tube growing towards the micropyle, pollen tubes initiated turning within 150 µm of the micropylar opening; the [Ca(2+)](cyt) in these pollen tube tips was higher than in those not growing towards an ovule in assays with myb98 mutant ovules, in which pollen tube guidance is disrupted. These results suggest that attractants secreted from the ovules affect Ca(2+) dynamics in the pollen tube. [Ca(2+)](cyt) in synergid cells did not change when the pollen tube grew towards the micropyle or entered the ovule. Upon pollen tube arrival at the synergid cell, however, [Ca(2+)](cyt) oscillation began at the micropylar pole of the synergid, spreading towards the chalazal pole. Finally, [Ca(2+)](cyt) in the synergid cell reached a maximum at pollen tube rupture. These results suggest that signals from the pollen tube induce Ca(2+) oscillations in synergid cells, and that this Ca(2+) oscillation is involved in the interaction between the pollen tube and synergid cell.

Dominance relationships between self-incompatibility alleles controlled by DNA methylation.

In crucifers, the pollen S-determinant gene, SP11, is sporophytically expressed in the anther tapetum, and the pollen self-incompatibility phenotype is determined by the dominance relationships between the two S-haplotypes it carries. We report here that 5' promoter sequences of recessive SP11 alleles are specifically methylated in the tapetum before the initiation of SP11 transcription. These results suggest that tissue-specific monoallelic de novo DNA methylation is involved in determining the dominance interactions that determine the cruciferous self-incompatibility phenotype.

Self/non-self recognition mechanisms in sexual reproduction: new insight into the self-incompatibility system shared by flowering plants and hermaphroditic animals.

Sexual reproduction is an essential process for generating a genetic variety in the next generation. However, most flowering plants and hermaphroditic animals potentially allow self-fertilization. Approximately 60% of angiosperms possess a self-incompatibility (SI) system to avoid inbreeding. The SI system functions at a process of interaction between pollen (or pollen tube) and the pistil. These SI-responsible factors (S-determinants) in pollen and the pistil are encoded by highly polymorphic multiallelic genes in the S-locus, which are tightly linked making a single haplotype. Different taxonomic families utilize different types of S-determinant proteins. In contrast to the plant system, the mechanisms of SI in simultaneously hermaphroditic animals are largely unknown. Among them, promising candidates for SI in ascidians (primitive chordates) were recently identified. The SI system in the ascidian Cionaintestinalis was found to be very similar to those in flowering plants: The products of sperm- and egg-side multiallelic SI genes, which are tight linked and highly polymorphic, appear to be responsible for the SI system as revealed by genetic analysis. These findings led us to speculate that the SI systems in plants and animals evolved in a manner of convergent evolution. Here, we review the current understanding of the molecular mechanisms of the SI system in flowering plants, particularly Brassicacea, and in ascidians from the viewpoint of common mechanisms shared by plants and animals.

Physiological and genetic analysis of CO2-induced breakdown of self-incompatibility in Brassica rapa.

Self-incompatibility (SI) of the Brassicaceae family can be overcome by CO2 gas treatment. This method has been used for decades as an effective means to obtain a large amount of inbred seeds which can then be used for F1 hybrid seed production; however, the molecular mechanism by which CO2 alters the SI pathway has not been elucidated. In this study, to obtain new insights into the mechanism of CO2-induced SI breakdown, the focus was on two inbred lines of Brassica rapa (syn. campestris) with different CO2 sensitivity. Physiological examination using X-ray microanalysis suggested that SI breakdown in the CO2-sensitive line was accompanied by a significant accumulation of calcium at the pollen-stigma interface. Pre-treatment of pollen or pistil with CO2 gas before pollination showed no effect on the SI reaction, suggesting that some physiological process after pollination is necessary for SI to be overcome. Genetic analyses using F1 progeny of a CO2-sensitive × CO2-insensitive cross suggested that CO2 sensitivity is a semi-dominant trait in these lines. Analysis of F2 progeny suggested that CO2 sensitivity could be a quantitative trait, which is controlled by more than one gene. Quantitative trait locus (QTL) analyses identified two major loci, BrSIO1 and BrSIO2, which work additively in overcoming SI during CO2 treatment. No QTL was detected at the loci previously shown to affect SI stability, suggesting that CO2 sensitivity is determined by novel genes. The QTL data presented here should be useful for determining the responsible genes, and for the marker-assisted selection of desirable parental lines with stable but CO2-sensitive SI in F1 hybrid breeding.

Conserved CKI1-mediated signaling is required for female germline specification in Marchantia polymorpha.

In land plants, gametes derive from a small number of dedicated haploid cells.1 In angiosperms, one central cell and one egg cell are differentiated in the embryo sac as female gametes for double fertilization, while in non-flowering plants, only one egg cell is generated in the female sexual organ, called the archegonium.2,3 The central cell specification of Arabidopsis thaliana is controlled by the histidine kinase CYTOKININ-INDEPENDENT 1 (CKI1), which is a two-component signaling (TCS) activator sharing downstream regulatory components with the cytokinin signaling pathway.4,5,6,7 Our phylogenetic analysis suggested that CKI1 orthologs broadly exist in land plants. However, the role of CKI1 in non-flowering plants remains unclear. Here, we found that the sole CKI1 ortholog in the liverwort Marchantia polymorpha, MpCKI1, which functions through conserved downstream TCS components, regulates the female germline specification for egg cell development in the archegonium. In M. polymorpha, the archegonium develops three-dimensionally from a single cell accumulating MpBONOBO (MpBNB), a master regulator for germline initiation and differentiation.8 We visualized female germline specification by capturing the distribution pattern of MpBNB in discrete stages of early archegonium development, and found that MpBNB accumulation is restricted to female germline cells. MpCKI1 is required for the proper MpBNB accumulation in the female germline, and is critical for the asymmetric cell divisions that specify the female germline cells. These results suggest that CKI1-mediated TCS originated during early land plant evolution and participates in female germ cell specification in deeply diverged plant lineages.

BslA(YuaB) forms a hydrophobic layer on the surface of Bacillus subtilis biofilms.

Biofilms are surface-associated bacterial aggregates, in which bacteria are enveloped by polymeric substances known as the biofilm matrix. Bacillus subtilis biofilms display persistent resistance to liquid wetting and gas penetration, which probably explains the broad-spectrum resistance of the bacteria in these biofilms to antimicrobial agents. In this study, BslA (formerly YuaB) was identified as a major contributor to the surface repellency of B. subtilis biofilms. Disruption of bslA resulted in the loss of surface repellency and altered the biofilm surface microstructure. BslA localized to the biofilm matrix in an exopolysaccharide-dependent manner. Purified BslA exhibited amphiphilic properties and formed polymers in response to increases in the area of the air-water interface in vitro. Genetic and biochemical analyses showed that the self-polymerization activity of BslA was essential for its ability to localize to the biofilm matrix. Confocal laser scanning microscopy showed that BslA formed a layer on the biofilm surface. Taken together, we propose that BslA, standing for biofilm-surface layer protein, is responsible for the hydrophobic layer on the surface of biofilms.

Cell type-specific transcriptome of Brassicaceae stigmatic papilla cells from a combination of laser microdissection and RNA sequencing.

Pollination is an early and critical step in plant reproduction, leading to successful fertilization. It consists of many sequential processes, including adhesion of pollen grains onto the surface of stigmatic papilla cells, foot formation to strengthen pollen-stigma interaction, pollen hydration and germination, and pollen tube elongation and penetration. We have focused on an examination of the expressed genes in papilla cells, to increase understanding of the molecular systems of pollination. From three representative species of Brassicaceae (Arabidopsis thaliana, A. halleri and Brassica rapa), stigmatic papilla cells were isolated precisely by laser microdissection, and cell type-specific gene expression in papilla cells was determined by RNA sequencing. As a result, 17,240, 19,260 and 21,026 unigenes were defined in papilla cells of A. thaliana, A. halleri and B. rapa, respectively, and, among these, 12,311 genes were common to all three species. Among the17,240 genes predicted in A. thaliana, one-third were papilla specific while approximately half of the genes were detected in all tissues examined. Bioinformatics analysis revealed that genes related to a wide range of reproduction and development functions are expressed in papilla cells, particularly metabolism, transcription and membrane-mediated information exchange. These results reflect the conserved features of general cellular function and also the specific reproductive role of papilla cells, highlighting a complex cellular system regulated by a diverse range of molecules in these cells. This study provides fundamental biological knowledge to dissect the molecular mechanisms of pollination in papilla cells and will shed light on our understanding of plant reproduction mechanisms.

The transcription factor OsNAC4 is a key positive regulator of plant hypersensitive cell death.

The hypersensitive response (HR) is a common feature of plant immune responses and a type of programmed cell death. However, little is known about the induction mechanism of HR cell death. We report that overexpression of OsNAC4, which encodes a plant-specific transcription factor, leads to HR cell death accompanied by the loss of plasma membrane integrity, nuclear DNA fragmentation and typical morphological changes. In OsNAC4 knock-down lines, HR cell death is markedly decreased in response to avirulent bacterial strains. After induction by an avirulent pathogen recognition signal, OsNAC4 is translocated into the nucleus in a phosphorylation-dependent manner. A microarray analysis showed that the expression of 139 genes including OsHSP90 and IREN, encoding a Ca(2+)-dependent nuclease, were different between the OsNAC4 knock-down line and control line during HR cell death. During the induction of HR cell death, OsHSP90 is involved in the loss of plasma membrane integrity, whereas IREN causes nuclear DNA fragmentation. Overall, our results indicate that two important events occurring during HR cell death are regulated by independent pathways.

Promotion of cyclic electron transport around photosystem I during the evolution of NADP-malic enzyme-type C4 photosynthesis in the genus Flaveria.

C4 plants display higher cyclic electron transport activity than C3 plants. This activity is suggested to be important for the production of ATPs required for C4 metabolism. To understand the process by which photosystem I (PSI) cyclic electron transport was promoted during C4 evolution, we conducted comparative analyses of the functionality of PSI cyclic electron transport among members of the genus Flaveria, which contains several C3, C3-C4 intermediate, C4-like and C4 species. The abundance of NDH-H, a subunit of NADH dehydrogenase-like complex, increased markedly in bundle sheath cells with the activity of the C4 cycle. By contrast, PROTON GRADIENT REGULATION5 (PGR5) and PGR5-LIKE1 increased in both mesophyll and bundle sheath cells in C4-like Flaveria palmeri and C4 species. Grana stacks were drastically reduced in bundle sheath chloroplasts of C4-like F. palmeri and C4 species; these species showed a marked increase in PSI cyclic electron transport activity. These results suggest that both the expression of proteins involved in PSI cyclic electron transport and changes in thylakoid structure contribute to the high activity of cyclic electron flow in NADP-malic enzyme-type C4 photosynthesis. We propose that these changes were important for the establishment of C4 photosynthesis from C3-C4 intermediate photosynthesis in Flaveria.

FZL, a dynamin-like protein localized to curved grana edges, is required for efficient photosynthetic electron transfer in Arabidopsis.

Photosynthetic electron transfer and its regulation processes take place on thylakoid membranes, and the thylakoid of vascular plants exhibits particularly intricate structure consisting of stacked grana and flat stroma lamellae. It is known that several membrane remodeling proteins contribute to maintain the thylakoid structure, and one putative example is FUZZY ONION LIKE (FZL). In this study, we re-evaluated the controversial function of FZL in thylakoid membrane remodeling and in photosynthesis. We investigated the sub-membrane localization of FZL and found that it is enriched on curved grana edges of thylakoid membranes, consistent with the previously proposed model that FZL mediates fusion of grana and stroma lamellae at the interfaces. The mature fzl thylakoid morphology characterized with the staggered and less connected grana seems to agree with this model as well. In the photosynthetic analysis, the fzl knockout mutants in Arabidopsis displayed reduced electron flow, likely resulting in higher oxidative levels of Photosystem I (PSI) and smaller proton motive force (pmf). However, nonphotochemical quenching (NPQ) of chlorophyll fluorescence was excessively enhanced considering the pmf levels in fzl, and we found that introducing kea3-1 mutation, lowering pH in thylakoid lumen, synergistically reinforced the photosynthetic disorder in the fzl mutant background. We also showed that state transitions normally occurred in fzl, and that they were not involved in the photosynthetic disorders in fzl. We discuss the possible mechanisms by which the altered thylakoid morphology in fzl leads to the photosynthetic modifications.

Glycosylation regulates specific induction of rice immune responses by Acidovorax avenae flagellin.

Plants have a sensitive system that detects various pathogen-derived molecules to protect against infection. Flagellin, a main component of the bacterial flagellum, from the rice avirulent N1141 strain of the Gram-negative phytopathogenic bacterium Acidovorax avenae induces plant immune responses including H2O generation, whereas flagellin from the rice virulent K1 strain of A. avenae does not induce these immune responses. To clarify the molecular mechanism that leads to these differing responses between the K1 and N1141 flagellins, recombinant K1 and N1141 flagellins were generated using an Escherichia coli expression system. When cultured rice cells were treated with recombinant K1 or N1141 flagellin, both flagellins equally induced H2O2 generation, suggesting that post-translational modifications of the flagellins are involved in the specific induction of immune responses. Mass spectrometry analyses using glycosyltransferase-deficient mutants showed that 1,600- and 2,150-Da glycans were present on the flagellins from N1141 and K1, respectively. A deglycosylated K1 flagellin induced immune responses in the same manner as N1141 flagellin. Site-directed mutagenesis revealed that glycans were attached to four amino acid residues (Ser¹78, Ser¹8³, Ser²¹², and Thr³5¹) in K1 flagellin. Among mutant K1 flagellins in which each glycan-attached amino acid residue was changed to alanine, S178A and S183A, K1 flagellin induced a strong immune response in cultured rice cells, indicating that the glycans at Ser¹78 and Ser¹8³ in K1 flagellin prevent epitope recognition in rice.