Control of Rice Embryo Development,Shoot Apical Meristem Maintenance,and Grain Yield by a Novel Cytochrome P450

Angiosperm seeds usually consist of two major parts： the embryo and the endosperm. However, the molec- ular mechanism（s） underlying embryo and endosperm development remains largely unknown, particularly in rice, the model cereal. Here, we report the identification and functional characterization of the rice GIANT EMBRYO （GE） gene. Mutation of GE resulted in a large embryo in the seed, which was caused by excessive expansion of scuteUum cells. Post-embryonic growth of ge seedling was severely inhibited due to defective shoot apical meristem （SAM） mainte- nance. Map-based cloning revealed that GE encodes a CYP78A subfamily P450 monooxygenase that is localized to the endoplasmic reticulum. GE is expressed predominantly in the scutellar epithelium, the interface region between embryo and endosperm. Overexpression of GE promoted cell proliferation and enhanced rice plant growth and grain yield, but reduced embryo size, suggesting that GE is critical for coordinating rice embryo and endosperm development. Moreover, transgenic Arabidopsis plants overexpressing AtCYP78AlO, a GE homolog, also produced bigger seeds, implying a con- served role for the CYP78A subfamily of P450s in regulating seed development. Taken together, our results indicate that GE plays critical roles in regulating embryo development and SAM maintenance.     

Arabidopsis tic62 trol Mutant Lacking Thylakoid- Bound Ferredoxin-NADP＋ Oxidoreductase Shows Distinct Metabolic Phenotype

Ferredoxin-NADP＋ oxidoreductase （FNR）, functioning in the last step of the photosynthetic electron transfer chain, exists both as a soluble protein in the chloroplast stroma and tightly attached to chloroplast membranes. Surface plasmon resonance assays showed that the two FNR isoforms, LFNR1 and LFNR2, are bound to the thylakoid membrane via the C-terminal domains of Tic62 and TROL proteins in a pH-dependent manner. The tic62 trol double mutants contained a reduced level of FNR, exclusively found in the soluble stroma. Although the mutant plants showed no visual phenotype or defects in the function of photosystems under any conditions studied, a low ratio of NADPH/NADP~ was detected. Since the CO2 fixation capacity did not differ between the tic62 trol plants and wild-type, it seems that the plants are able to funnel reducing power to most crucial reactions to ensure survival and fitness of the plants. However, the activity of malate dehydrogenase was down-regulated in the mutant plants. Apparently, the plastid metabolism is able to cope with substantial changes in directing the electrons from the light reactions to stromal metabolism and thus only few differences are visible in steady-state metabolite pool sizes of the tic62 trol plants.     

Redox Regulation of Arabidopsis Mitochondrial Citrate Synthase

Citrate synthase has a key role in the tricarboxylic （TCA） cycle of mitochondria of all organisms, as it cata- lyzes the first committed step which is the fusion of a carbon-carbon bond between oxaloacetate and acetyl CoA. The regulation of TCA cycle function is especially important in plants, since mitochondrial activities have to be coordinated with photosynthesis. The posttranslational regulation of TCA cycle activity in plants is thus far almost entirely unexplored. Although several TCA cycle enzymes have been identified as thioredoxin targets in vitro, the existence of any thioredoxin-dependent regulation as known for the Calvin cycle, yet remains to be demonstrated. Here we have investigated the redox regulation of the Arabidopsis citrate synthase enzyme by site-directed mutagenesis of its six cysteine residues. Our results indicate that oxidation inhibits the enzyme activity by the formation of mixed disulfides, as the partially oxidized citrate synthase enzyme forms large redox-dependent aggregates. Furthermore, we were able to demonstrate that thioredoxin can cleave diverse intraas well as intermolecular disulfide bridges, which strongly enhances the activity of the enzyme. Activity measurements with the cysteine variants of the enzyme revealed important cysteine residues affecting total enzyme activity as well as the redox sensitivity of the enzyme.     

Plastid Signals and the Bundle Sheath： Mesophyll Development in Reticulate Mutants

The development of a plant leaf is a meticulously orchestrated sequence of events producing a complex organ comprising diverse cell types. The reticulate class of leaf variegation mutants displays contrasting pigmentation between veins and interveinal regions due to specific aberrations in the development of mesophyll cells. Thus, the reticulate mutants offer a potent tool to investigate cell-type-specific developmental processes. The discovery that most mutants are affected in plastid-localized, metabolic pathways that are strongly expressed in vasculature-associated tis- sues implicates a crucial role for the bundle sheath and their chloroplasts in proper development of the mesophyll cells. Here, we review the reticulate mutants and their phenotypic characteristics, with a focus on those in Arabidopsis thali- ana. Two alternative models have been put forward to explain the relationship between plastid metabolism and meso- phyll cell development, which we call here the supply and the signaling hypotheses. We critically assess these proposed models and discuss their implications for leaf development and bundle sheath function in C3 species. The characteriza- tion of the reticulate mutants supports the significance of plastid retrograde signaling in cell development and highlights the significance of the bundle sheath in C3 photosynthesis.     

Organelle pH in the Arabidopsis Endomembrane System

The pH of intracellular compartments is essential for the viability of cells. Despite its relevance, little is known about the pH of these compartments. To measure pH in vivo, we have first generated two pH sensors by combining the improved-solubility feature of solubility-modified green fluorescent protein （GFP） （smGFP） with the pH-sensing capabil- ity of the pHluorins and codon optimized for expression in Arabidopsis. PEpHluorin （plant-solubility-modified ecliptic pHluorin） gradually loses fluorescence as pH is lowered with fluorescence vanishing at pH 6.2 and PRpHluorin （plant- solubility-modified ratiomatric pHluorin）, a dual-excitation sensor, allowing for precise measurements. Compartment- specific sensors were generated by further fusing specific sorting signals to PEpHluorin and PRpHluorin. Our results show that the pH of cytosol and nucleus is similar （pH 7.3 and 7.2）, while peroxisomes, mitochondrial matrix, and plastidial stroma have alkaline pH. Compartments of the secretory pathway reveal a gradual acidification, spanning from pH 7.1 in the endoplasmic reticulum （ER） to pH 5.2 in the vacuole. Surprisingly, pH in the trans-Golgi network （TGN） and mul- tivesicular body （MVB） is, with pH 6.3 and 6.2, quite similar. The inhibition of vacuolar-type H＋-ATPase （V-ATPase） with concanamycin A （ConcA） caused drastic increase in pH in TGN and vacuole. Overall, the PEpHluorin and PRpHluorin are excellent pH sensors for visualization and quantification of pH in vivo, respectively.     

A New Set of Reversibly Photoswitchable Fluorescent Proteins for Use in Transgenic Plants

Fluorescent reporter proteins that allow repeated switching between a fluorescent and a non-fluorescent state in response to specific wavelengths of light are novel tools for monitoring of protein trafficking and super-resolu- tion fluorescence microscopy in living organisms. Here, we describe variants of the reversibly photoswitchable fluores- cent proteins rsFastLime, bsDronpa, and Padron that have been codon-optimized for the use in transgenic Arabidopsis plants. The synthetic proteins, designated rsFastLIME-s, bsDRONPA-s, and PADRON C-s, showed photophysical properties and switching behavior comparable to those reported for the original proteins. By combining the ＇positively switchable＇ PADRON C-s with the ＇negatively switchable＇ rsFastLIME-s or bsDRONPA-s, two different fluorescent reporter proteins could be imaged at the same wavelength upon transient expression in Nicotiana benthamiana cells. Thus, co-localiza- tion analysis can be performed using only a single detection channel. Furthermore, the proteins were used to tag the RNA-binding protein AtGRP7 （Arabidopsis thaliana glycine-rich RNA-binding protein 7） in transgenic Arabidopsis plants. Because the new reversibly photoswitchable fluorescent proteins show an increase in signal strength during each pho- toactivation cycle, we were able to generate a large number of scans of the same region and reconstruct 3-D images of AtGRP7 expression in the root tip. Upon photoactivation of the AtGRP7：rsFastLIME-s fusion protein in a defined region of a transgenic Arabidopsis root, spreading of the fluorescence signal into adjacent regions was observed, indicating that movement from cell to cell can be monitored. Our results demonstrate that rsFastLIME-s, bsDRONPA-s, and PADRON C-s are versatile fluorescent markers in plants, Furthermore, the proteins also show strong fluorescence in mammalian cells including COS-7 and HeLa cells.     

Open and Closed： The Roles of Linker Histones in Plants and Animals

Histones package DNA in all eukaryotes and play key roles in regulating gene expression. Approximately 150 base pairs of DNA wraps around an octamer of core histones to form the nucleosome, the basic unit of chromatin. Linker histones compact chromatin further by binding to and neutralizing the charge of the DNA between nucleosomes. It is well established that chromatin packing is regulated by a complex pattern of posttranslational modifications （PTMs） to core histones, but linker histone function is less well understood. In this review, we describe the current understand- ing of the many roles that linker histones play in cellular processes, including gene regulation, cell division, and devel- opment, while putting the linker histone in the context of other nuclear proteins. Although intriguing roles for plant linker histones are beginning to emerge, much of our current understanding comes from work in animal systems. Many unanswered questions remain and additional work is required to fully elucidate the complex processes mediated by linker histones in plants.     

Natural Variation in OsPRR37 Regulates Heading Date and Contributes to Rice Cultivation at a Wide Range of Latitudes

Heading date and photoperiod sensitivity are fundamental traits that determine rice adaptation to a wide range of geographic environments. By quantitative trait locus （QTL） mapping and candidate gene analysis using whole- genome re-sequencing, we found that Oryza sativa Pseudo-Response Regulator37 （OsPRR37; hereafter PRR37） is respon- sible for the Early heading7-2 （EH7-2）/Heading date2 （Hd2） QTL which was identified from a cross of late-heading rice ＇Milyang23 （M23）＇ and early-heading rice ＇H143＇. H143 contains a missense mutation of an invariantly conserved amino acid in the CCT （CONSTANS, CO-like, and TOC1） domain of PRR37 protein. In the world rice collection, different types of nonfunctional PRR37 alleles were found in many European and Asian rice cultivars. Notably, the japonica varieties harboring nonfunctional alleles of both Ghd7/Hd4 and PRR37/Hd2 flower extremely early under natural long-day condi- tions, and are adapted to the northernmost regions of rice cultivation, up to 53~ N latitude. Genetic analysis revealed that the effects of PRR37 and Ghd7 alleles on heading date are additive, and PRR37 down-regulates Hd3a expression to suppress flowering under long-day conditions. Our results demonstrate that natural variations in PRR37/Hd2 and GhdT/ Hd4 have contributed to the expansion of rice cultivation to temperate and cooler regions.     

Heteromeric and Homomeric Geranyl Diphosphate Synthases from Catharanthus roseus and Their Role in Monoterpene Indole Alkaloid Biosynthesis

Catharanthus roseus is the sole source of two most important monoterpene indole alkaloid （MIA） anti- cancer agents： vinblastine and vincristine. MIAs possess a terpene and an indole moiety derived from terpenoid and shikimate pathways, respectively. Geranyl diphosphate （GPP）, the entry point to the formation of terpene moiety, is a product of the condensation of isopentenyl diphosphate （IPP） and dimethylallyl diphosphate （DMAPP） by GPP synthase （GPPS）. Here, we report three genes encoding proteins with sequence similarity to large subunit （CrGPPS.LSU） and small subunit （CrGPPS.SSU） of heteromeric GPPSs, and a homomeric GPPSs. CrGPPS.LSU is a bifunctional enzyme producing both GPP and geranyl geranyl diphosphate （GGPP）, CrGPPS.SSU is inactive, whereas CrGPPS is a homomeric enzyme forming GPP. Co-expression of both subunits in Escherichia coil resulted in heteromeric enzyme with enhanced activity producing only GPR While CrGPPS.LSU and CrGPPS showed higher expression in older and younger leaves, respectively, CrGPPS.SSU showed an increasing trend and decreased gradually. Methyl jasmonate （MelA） treatment of leaves sig- nificantly induced the expression of only CrGPPS.SSU. GFP localization indicated that CrGPPS.SSU is plastidial whereas CrGPPS is mitochondrial. Transient overexpression of AmGPPS.SSU in C. roseus leaves resulted in increased vindoline, immediate monomeric precursor of vinblastine and vincristine. Although C. roseus has both heteromeric and homomeric GPPS enzymes, our results implicate the involvement of only heteromeric GPPS with CrGPPS.SSU regulating the GPP flux for MIA biosynthesis.     

Knights in Action： Lectin Receptor-Like Kinases in Plant Development and Stress Responses

The Receptor-Like Kinase （RLK） is a vast protein family with over 600 genes in Arabidopsis and 1100 in rice. The Lectin RLK （LecRLK） family is believed to play crucial roles in saccharide signaling as well as stress perception. All the LecRLKs possess three domains： an N-terminal lectin domain, an intermediate transmembrane domain, and a C-terminal kinase domain. On the basis of lectin domain variability, LecRLKs have been subgrouped into three subclasses： L-, G-, and C-type LecRLKs. While the previous studies on LecRLKs were dedicated to classification, comparative structural analysis and expression analysis by promoter-based studies, most of the recent studies on LecRLKs have laid special emphasis on the potential of this gene family in regulating biotic/abiotic stress and developmental pathways in plants, thus mak- ing the prospects of studying the LecRLK-mediated regulatory mechanism exceptionally promising. In this review, we have described in detail the LecRLK gene family with respect to a historical, evolutionary, and structural point of view. Furthermore, we have laid emphasis on the LecRLKs roles in development, stress conditions, and hormonal response. We have also discussed the exciting research prospects offered by the current knowledge on the LecRLK gene family. The multitude of the LecRLK gene family members and their functional diversity mark these genes as both interesting and worthy candidates for further analysis, especially in the field of crop improvement.     

Diversification of SUMO-Activating Enzyme in Arabidopsis： Implications in SUMO Conjugation

Sumoylation is an essential posttranslational modification that participates in many biological processes including stress responses. However, little is known about the mechanisms that control Small Ubiquitin-like MOdifier （SUMO） conjugation in vivo. We have evaluated the regulatory role of the heterodimeric E1 activating enzyme, which catalyzes the first step in SUMO conjugation. We have established that the E1 large SAE2 and small SAE1 subunits are encoded by one and three genes, respectively, in the Arabidopsis genome. The three paralogs genes SAEla, SAElbl, and SAElb2 are the result of two independent duplication events. Since SAElbl and SAElb2 correspond to two identical cop- ies, only two E1 small subunit isoforms are present in vivo： SAEla and SAElb. The E1 heterodimer nuclear localization is modulated by the C-terminal tail of the SAE2 subunit. In vitro, SUMO conjugation rate is dependent on the SAE1 isoform contained in the E1 holoenzyme and our results suggest that downstream steps to SUMO-E1 thioester bond formation are affected. In vivo, SAEla isoform deletion in T-DNA insertion mutant plants conferred sumoylation defects upon abi- otic stress, consistent with a sumoylation defective phenotype. Our results support previous data pointing to a regula- tory role of the E1 activating enzyme during SUMO conjugation and provide a novel mechanism to control sumoylation in vivo by diversification of the E1 small subunit.     

Prostaglandin E2 modulates F-actin stress fiber in FSS-stimulated MC3T3-E1 cells in a PKA-dependent manner

The effect of prostaglandin E2（PGE2） on bone mass has been well-established in vivo. Previous studies have showed that PGE2 increases differentiation, proliferation, and regu- lates cell morphology through F-actin stress fiber in statically cultured osteoblasts. However, the effect of PGE2 on osteo- blasts in the presence of fluid shear stress （FSS）, which could better uncover the anabolic effect of PGEz in vivo, has yet to be examined. Here, we hypothesized that PGE2 modulates F-actin stress fiber in FSS-stimulated MC3T3-E1 osteoblastic cells through protein kinase A （PKA） pathway. Furthermore, this PGE2-induced F-actin remodeling was associated with the recovery of cellular mechanosensitivity. Our data showed that treatment with 10 nM dmPGE2 for 15 rain significantly suppressed the F-actin stress fiber intensity in FSS-stimulated cells in a PKA-dependent manner. In addition, dmPGE2 treatment enhanced the cells＇ calcium peak magnitude and the percentage of responding cells in the second FSS stimulation, though these effects were abolished and attenuated by co-treatment with phalloidin. Our results demonstrated that 10 nM dmPGE2 was able to accelerate the ＇reset＇ process of F-actin stress fiber to its pre-stimulated level partially through PKA pathway, and thus promoted the recovery of cellular mechanosensitivity. Our finding provided a novel cellular mechanism by which PGE2 increased bone forma- tion as shown in vivo, suggesting that PGE2 could be a potential target for treatments of bone formation-related diseases.     

An XA21-Associated Kinase （OsSERK2） Regulates Immunity Mediated by the XA21 and XA3 Immune Receptors

The rice XA21 immune receptor kinase and the structurally related XA3 receptor confer immunity to Xanthomonas oryzae pv. oryzae （Xoo）, the causal agent of bacterial leaf blight. Here we report the isolation of OsSERK2 （rice somatic embryogenesis receptor kinase 2） and demonstrate that OsSERK2 positively regulates immunity mediated by XA21 and XA3 as well as the rice immune receptor FLS2 （OsFLS2）. Rice plants silenced for OsSerk2 display altered morphology and reduced sensitivity to the hormone brassinolide. OsSERK2 interacts with the intracellular domains of each immune receptor in the yeast two-hybrid system in a kinase activity-dependent manner. OsSERK2 undergoes bidi- rectional transphosphorylation with XA21 in vitro and forms a constitutive complex with XA21 in vivo. These results demonstrate an essential role for OsSERK2 in the function of three rice immune receptors and suggest that direct interaction with the rice immune receptors is critical for their function. Taken together, our findings suggest that the mechanism of OsSERK2-meditated regulation of rice XA21, XA3, and FLS2 differs from that of AtSERK3/BAK1-mediated regulation of Arabidopsis FLS2 and EFR.     

MiRNA-218, a new regulator of HMGB1, suppresses cell migration and invasion in non-small cell lung cancer

MieroRNAs （miRNAs） function as negative regulators of gene expression involved in cancer metastasis. The aim of this study is to investigate the potential roles of miR-218 in non-small cell lung cancer and validate its regulation mech- anism. Functional studies showed that miR-218 overexpres- sion inhibited cell migration and invasion, but had no effect on cell viability. Enhanced green fluorescent protein reporter assay, real-time polymerase chain reaction and western blot analysis confirmed that miR-218 suppressed the expression of high mobility group box-1 （HMGB1） by directly targeting its 31-untranslated region. Accordingly, silencing of HMGBI accorded with the effects of miR-218 on cell migration and invasion, and overexpression of HMGB1 can restore cell migration and invasion which were reduced by miR-218. In conclusion, these findings demon- strate that miR-218 functions as a tumor suppressor in lung cancer. Furthermore, miR-218 may act as a potential thera- peutic biomarker for metastatic lung cancer patients.     

Nitric Oxide and Brassinosteroids Mediated Fungal Endophyte-lnduced Volatile Oil Production Through Protein Phosphorylation Pathways in Atractylodes lancea Plantlets

Fungal endophytes have been isolated from almost every plant, infecting their hosts without causing visible disease symptoms, and yet have still proved to be involved in plant secondary metabolites accumulation. To decipher the possible physiological mechanisms of the endophytic fungus-host interaction, the role of protein phosphorylation and the relationship between endophytic fungus-induced kinase activity and nitric oxide （NO） and brassinolide （BL） in endophyte-enhanced volatile oil accumulation in Atractylodes lancea plantlets were investigated using pharmacological and biochemical approaches. Inoculation with the endophytic fungus Gilmaniella sp. ALl2 enhanced the activities of total protein phosphorylation, Ca2~-dependent protein kinase, and volatile oil accumulation in A. lancea plantlets. The upregulation of protein kinase activity could be blocked by the BL inhibitor brassinazole. Furthermore, pretreatments with the NO-specific scavenger cPTIO significantly reduced the increased activities of protein kinases in A. lancea plantlets inoculated with endophytic fungus. Pretreatments with different protein kinase inhibitors also reduced fungus-induced NO production and volatile oil accumulation, but had barely no effect on the BL level. These data suggest that protein phosphorylation is required for endophyte- induced volatile oil production in A. lancea plantlets, and that crosstalk between protein phosphorylation and the NO pathway may occur and act as a downstream signaling event of the BL pathway.     

Cell Wall,Cytoskeleton, and Cell Expansion in Higher Plants

To accommodate two seemingly contradictory biological roles in plant physiology, providing both the rigid structural support of plant cells and the adjustable elasticity needed for cell expansion, the composition of the plant cell wall has evolved to become an intricate network of cellulosic, hemicellulosic, and pectic polysaccharides and protein. Due to its complexity, many aspects of the cell wall influence plant cell expansion, and many new and insightful observations and technologies are forthcoming. The biosynthesis of cell wall polymers and the roles of the variety of proteins involved in polysaccharide synthesis continue to be characterized. The interactions within the cell wall polymer network and the modification of these interactions provide insight into how the plant cell wall provides its dual function. The complex cell wall architecture is controlled and organized in part by the dynamic intracellular cytoskeleton and by diverse trafficking pathways of the cell wall polymers and cell wall-related machinery. Meanwhile, the cell wall is continually influenced by hormonal and integrity sensing stimuli that are perceived by the cell. These many processes cooperate to construct, maintain, and manipulate the intricate plant cell wall--an essential structure for the sustaining of the plant stature, growth, and life.     

Dissecting the Heat Stress Response in Chlamydomonas by Pharmaceutical and RNAi Approaches Reveals Conserved and Novel Aspects

To study how conserved fundamental concepts of the heat stress response （HSR） are in photosynthetic eukaryotes, we applied pharmaceutical and antisense/amiRNA approaches to the unicellular green alga Chlamydomonas reinhardtii. The Chlamydomonas HSR appears to be triggered by the accumulation of unfolded proteins, as it was induced at ambient temperatures by feeding cells with the arginine analog canavanine. The protein kinase inhibitor staurosporine strongly retarded the HSR, demonstrating the importance of phosphorylation during activation of the HSR also in Chlamydomonas. While the removal of extracellular calcium by the application of EGTA and BAPTA inhibited the HSR in moss and higher plants, only the addition of BAPTA, but not of EGTA, retarded the HSR and impaired thermotoler- ance in Chlamydomonas. The addition of cycloheximide, an inhibitor of cytosolic protein synthesis, abolished the attenu- ation of the HSR, indicating that protein synthesis is necessary to restore proteostasis. HSP90 inhibitors induced a stress response when added at ambient conditions and retarded attenuation of the HSR at elevated temperatures. In addition, we detected a direct physical interaction between cytosolic HSP90A/HSP70A and heat shock factor 1, but surprisingly this interaction persisted after the onset of stress. Finally, the expression of antisense constructs targeting chloroplast HSP70B resulted in a delay of the cell＇s entire HSR, thus suggesting the existence of a retrograde stress signaling cascade that is desensitized in HSP7OB-antisense strains.