Examination of aquaporin (AQP) membrane channels in extremophile plants may increase our understanding of plant tolerance to high salt, drought or other conditions. Here, we cloned a tonoplast AQP gene (TsTIP1;2) from the halophyte Thellungiella salsuginea and characterized its biological functions. TsTIP1;2 transcripts accumulate to high levels in several organs, increasing in response to multiple external stimuli. Ectopic overexpression of TsTIP1;2 in Arabidopsis significantly increased plant tolerance to drought, salt and oxidative stresses. TsTIP1;2 had water channel activity when expressed in Xenopus oocytes. TsTIP1;2 was also able to conduct H2O2 molecules into yeast cells in response to oxidative stress. TsTIP1;2 was not permeable to Na+ in Xenopus oocytes, but it could facilitate the entry of Na+ ions into plant cell vacuoles by an indirect process under high-salinity conditions. Collectively, these data showed that TsTIP1;2 could mediate the conduction of both H2O and H2O2 across membranes, and may act as a multifunctional contributor to survival of T. salsuginea in highly stressful habitats.
Aquaporin; Channeling activity; Stress tolerance; Thellungiella salsuginea
Comprehensive integration of large-scale omics resources such as genomes, transcriptomes and metabolomes will provide deeper insights into broader aspects of molecular biology. For better understanding of plant biology, we aim to construct a next-generation sequencing (NGS)-derived gene expression network (GEN) repository for a broad range of plant species. So far we have incorporated information about 745 high-quality mRNA sequencing (mRNA-Seq) samples from eight plant species (Arabidopsis thaliana, Oryza sativa, Solanum lycopersicum, Sorghum bicolor, Vitis vinifera, Solanum tuberosum, Medicago truncatula and Glycine max) from the public short read archive, digitally profiled the entire set of gene expression profiles, and drawn GENs by using correspondence analysis (CA) to take advantage of gene expression similarities. In order to understand the evolutionary significance of the GENs from multiple species, they were linked according to the orthology of each node (gene) among species. In addition to other gene expression information, functional annotation of the genes will facilitate biological comprehension. Currently we are improving the given gene annotations with natural language processing (NLP) techniques and manual curation. Here we introduce the current status of our analyses and the web database, PODC (Plant Omics Data Center; http://bioinf.mind.meiji.ac.jp/podc/), now open to the public, providing GENs, functional annotations and additional comprehensive omics resources.
Correspondence analysis; Database; Gene expression network; Manual curation; Natural language processing (NLP); Omics
In transcriptome analysis, accurate annotation of each transcriptional unit and its expression profile is essential. A full-length cDNA (FL-cDNA) collection facilitates the refinement of transcriptional annotation, and accurate transcription start sites help to unravel transcriptional regulation. We constructed a normalized FL-cDNA library from eight growth stages of aerial tissues in Sorghum bicolor and isolated 37,607 clones. These clones were Sanger sequenced from the 5′ and/or 3′ ends and in total 38,981 high-quality expressed sequence tags (ESTs) were obtained. About one-third of the transcripts of known genes were captured as FL-cDNA clone resources. In addition to these, we also annotated 272 novel genes, 323 antisense transcripts and 1,672 candidate isoforms. These clones are available from the RIKEN Bioresource Center. After obtaining accurate annotation of transcriptional units, we performed expression profile analysis. We carried out spikelet-, seed- and stem-specific RNA sequencing (RNA-Seq) analysis and confirmed the expression of 70.6% of the newly identified genes. We also downloaded 23 sorghum RNA-Seq samples that are publicly available and these are shown on a genome browser together with our original FL-cDNA and RNA-Seq data. Using our original and publicly available data, we made an expression profile of each gene and identified the top 20 genes with the most similar expression. In addition, we visualized their relationships in gene co-expression networks. Users can access and compare various transcriptome data from S, bicolor at http://sorghum.riken.jp.
Database; FL-cDNA; New transcript; NGS; Plant; Sorghum
With the number of sequenced plant genomes growing, the number of predicted genes and functional annotations is also increasing. The association between genes and phenotypic traits is currently of great interest. Unfortunately, the information available today is widely scattered over a number of different databases. Information retrieval (IR) has become an all-encompassing bioinformatics methodology for extracting knowledge from complex, heterogeneous and distributed databases, and therefore can be a useful tool for obtaining a comprehensive view of plant genomics, from genes to traits. Here we describe LAILAPS (http://lailaps.ipk-gatersleben.de), an IR system designed to link plant genomic data in the context of phenotypic attributes for a detailed forward genetic research. LAILAPS comprises around 65 million indexed documents, encompassing >13 major life science databases with around 80 million links to plant genomic resources. The LAILAPS search engine allows fuzzy querying for candidate genes linked to specific traits over a loosely integrated system of indexed and interlinked genome databases. Query assistance and an evidence-based annotation system enable time-efficient and comprehensive information retrieval. An artificial neural network incorporating user feedback and behavior tracking allows relevance sorting of results. We fully describe LAILAPS’s functionality and capabilities by comparing this system’s performance with other widely used systems and by reporting both a validation in maize and a knowledge discovery use-case focusing on candidate genes in barley.
Functional gene annotation; Information retrieval; Integrative search engine; Plant genomics resources; Traits
Arabidopsis thaliana is an important model species for studies of plant gene functions. Research on Arabidopsis has resulted in the generation of high-quality genome sequences, annotations and related post-genomic studies. The amount of annotation, such as gene-coding regions and structures, is steadily growing in the field of plant research. In contrast to the genomics resource of animals and microorganisms, there are still some difficulties with characterization of some gene functions in plant genomics studies. The acquisition of information on protein structure can help elucidate the corresponding gene function because proteins encoded in the genome possess highly specific structures and functions. In this study, we calculated multiple physicochemical and secondary structural parameters of protein sequences, including length, hydrophobicity, the amount of secondary structure, the number of intrinsically disordered regions (IDRs) and the predicted presence of transmembrane helices and signal peptides, using a total of 208,333 protein sequences from the genomes of six representative plant species, Arabidopsis thaliana, Glycine max (soybean), Populus trichocarpa (poplar), Oryza sativa (rice), Physcomitrella patens (moss) and Cyanidioschyzon merolae (alga). Using the PASS tool and the Rosetta Stone method, we annotated the presence of novel functional regions in 1,732 protein sequences that included unannotated sequences from the Arabidopsis and rice proteomes. These results were organized into the Plant Protein Annotation Suite database (Plant-PrAS), which can be freely accessed online at http://plant-pras.riken.jp/.
Database; Gene function; Physicochemical property; Plant protein; Protein property
Comparative genomics combined with phylogenetic reconstructions are powerful approaches to study the evolution of genes and genomes. However, the current rapid expansion of the volume of genomic information makes it increasingly difficult to interrogate, integrate and synthesize comparative genome data while taking into account the maximum breadth of information available. GenomicusPlants (http://www.genomicus.biologie.ens.fr/genomicus-plants) is an extension of the Genomicus webserver that addresses this issue by allowing users to explore flowering plant genomes in an intuitive way, across the broadest evolutionary scales. Extant genomes of 26 flowering plants can be analyzed, as well as 23 ancestral reconstructed genomes. Ancestral gene order provides a long-term chronological view of gene order evolution, greatly facilitating comparative genomics and evolutionary studies. Four main interfaces (‘views’) are available where: (i) PhyloView combines phylogenetic trees with comparisons of genomic loci across any number of genomes; (ii) AlignView projects loci of interest against all other genomes to visualize its topological conservation; (iii) MatrixView compares two genomes in a classical dotplot representation; and (iv) Karyoview visualizes chromosome karyotypes ‘painted’ with colours of another genome of interest. All four views are interconnected and benefit from many customizable features.
Ancestral reconstruction; Evolution; Flowering plants; Genomics; Synteny
Recent developments in DNA sequencing have enabled the large and complex genomes of many crop species to be determined for the first time, even those previously intractable due to their polyploid nature. Indeed, over the course of the last 2 years, the genome sequences of several commercially important cereals, notably barley and bread wheat, have become available, as well as those of related wild species. While still incomplete, comparison with other, more completely assembled species suggests that coverage of genic regions is likely to be high. Ensembl Plants (http://plants.ensembl.org) is an integrative resource organizing, analyzing and visualizing genome-scale information for important crop and model plants. Available data include reference genome sequence, variant loci, gene models and functional annotation. For variant loci, individual and population genotypes, linkage information and, where available, phenotypic information are shown. Comparative analyses are performed on DNA and protein sequence alignments. The resulting genome alignments and gene trees, representing the implied evolutionary history of the gene family, are made available for visualization and analysis. Driven by the case of bread wheat, specific extensions to the analysis pipelines and web interface have recently been developed to support polyploid genomes. Data in Ensembl Plants is accessible through a genome browser incorporating various specialist interfaces for different data types, and through a variety of additional methods for programmatic access and data mining. These interfaces are consistent with those offered through the Ensembl interface for the genomes of non-plant species, including those of plant pathogens, pests and pollinators, facilitating the study of the plant in its environment.
Comparative genomics; Functional genomics; Genetic variation; Genome browser; Transcriptomics; Triticeae
The clustered regularly interspaced short palindromic repeat (CRISPR)-associated endonuclease 9 (CRISPR/Cas9) system has been demonstrated to be a robust genome engineering tool in a variety of organisms including plants. However, it has been shown that the CRISPR/Cas9 system cleaves genomic DNA sequences containing mismatches to the guide RNA strand. We expected that this low specificity could be exploited to induce multihomeologous and multiparalogous gene knockouts. In the case of polyploid plants, simultaneous modification of multiple homeologous genes, i.e. genes with similar but not identical DNA sequences, is often needed to obtain a desired phenotype. Even in diploid plants, disruption of multiparalogous genes, which have functional redundancy, is often needed. To validate the applicability of the CRISPR/Cas9 system to target mutagenesis of paralogous genes in rice, we designed a single-guide RNA (sgRNA) that recognized 20 bp sequences of cyclin-dependent kinase B2 (CDKB2) as an on-target locus. These 20 bp possess similarity to other rice CDK genes (CDKA1, CDKA2 and CDKB1) with different numbers of mismatches. We analyzed mutations in these four CDK genes in plants regenerated from Cas9/sgRNA-transformed calli and revealed that single, double and triple mutants of CDKA2, CDKB1 and CDKB2 can be created by a single sgRNA.
CDK; CRISPR/Cas9; Off-target mutation; Rice
Gene targeting (GT) is a technique used to modify endogenous genes in target genomes precisely via homologous recombination (HR). Although GT plants are produced using genetic transformation techniques, if the difference between the endogenous and the modified gene is limited to point mutations, GT crops can be considered equivalent to non-genetically modified mutant crops generated by conventional mutagenesis techniques. However, it is difficult to guarantee the non-incorporation of DNA fragments from Agrobacterium in GT plants created by Agrobacterium-mediated GT despite screening with conventional Southern blot and/or PCR techniques. Here, we report a comprehensive analysis of herbicide-tolerant rice plants generated by inducing point mutations in the rice ALS gene via Agrobacterium-mediated GT. We performed genome comparative genomic hybridization (CGH) array analysis and whole-genome sequencing to evaluate the molecular composition of GT rice plants. Thus far, no integration of Agrobacterium-derived DNA fragments has been detected in GT rice plants. However, >1,000 single nucleotide polymorphisms (SNPs) and insertion/deletion (InDels) were found in GT plants. Among these mutations, 20–100 variants might have some effect on expression levels and/or protein function. Information about additive mutations should be useful in clearing out unwanted mutations by backcrossing.
Acetolactate synthase; Gene targeting; Herbicide tolerance; Rice
Brefeldin A suppresses vesicle trafficking by inhibiting exchange of GDP for GTP in ADP-ribosylation factor. We report that brefeldin A suppresses mobilization of triacylglycerols in Chlamydomonas reinhardtii, a model organism of green microalgae. Analyses revealed that brefeldin A causes Chlamydomonas to form lipid droplets in which triacylglycerols accumulate in a dose-dependent manner. Pulse labeling experiment using fluorescent fatty acids suggested that brefeldin A inhibits the cells from degrading fatty acids. The experiment also revealed that the cells transiently form novel compartments that accumulate exogenously added fatty acids in the cytoplasm, designated fatty acid-induced microbodies (FAIMs). Brefeldin A up-regulates the formation of FAIMs, whereas nitrogen deprivation that up-regulates triacylglycerol synthesis in Chlamydomonas does not cause the cells to form FAIMs. These results underscore the role of the vesicle trafficking machinery in triacylglycerol metabolism in green microalgae.
Triacylglycerol mobilization; Lipid droplets; microalgae; brefeldin A; peroxisome precursor; ADP-ribosylation factor
In plants, genes involved in photosynthesis are encoded separately in nuclei and plastids, and tight cooperation between these two genomes is therefore required for the development of functional chloroplasts. Golden2-like (GLK) transcription factors are involved in chloroplast development, directly targeting photosynthesis-associated nuclear genes for up-regulation. Although overexpression of GLKs leads to chloroplast development in non-photosynthetic organs, the mechanisms of coordination between the nuclear gene expression influenced by GLKs and the photosynthetic processes inside chloroplasts are largely unknown. To elucidate the impact of GLK-induced expression of photosynthesis-associated nuclear genes on the construction of photosynthetic systems, chloroplast morphology and photosynthetic characteristics in greenish roots of Arabidopsis thaliana lines overexpressing GLKs were compared with those in wild-type roots and leaves. Overexpression of GLKs caused up-regulation of not only their direct targets but also non-target nuclear and plastid genes, leading to global induction of chloroplast biogenesis in the root. Large antennae relative to reaction centers were observed in wild-type roots and were further enhanced by GLK overexpression due to the increased expression of target genes associated with peripheral light-harvesting antennae. Photochemical efficiency was lower in the root chloroplasts than in leaf chloroplasts, suggesting that the imbalance in the photosynthetic machinery decreases the efficiency of light utilization in root chloroplasts. Despite the low photochemical efficiency, root photosynthesis contributed to carbon assimilation in Arabidopsis. Moreover, GLK overexpression increased CO2 fixation and promoted phototrophic performance of the root, showing the potential of root photosynthesis to improve effective carbon utilization in plants.
Arabidopsis root; Chloroplast development; Construction of photosynthetic systems; GLK; Photosynthesis
Among signaling components downstream of phytochrome A (phyA), HY5, HFR1 and LAF1 are transcription factors that regulate expression of phyA-responsive genes. Previous work has shown that FHY1/FHL distribute phyA signals directly to HFR1 and LAF1, both of which regulate largely independent pathways, but the relationship of HY5 to these two factors was unclear. Here, we investigated the genetic relationship among the genes encoding these three transcription factors, HY5, HFR1 and LAF1. Analyses of double and triple mutants showed that HY5, a basic leucine zipper (bZIP) factor, HFR1, a basic helix–loop–helix (bHLH) factor, and LAF1, a Myb factor, independently transmit phyA signals downstream. We showed that HY5 but not its homolog, HYH, could interact with HFR1 and LAF1; on the other hand, FHY1 and its homolog, FHL did not interact with HY5 or HYH. Together, our results suggest that HY5 transmits phyA signals through an FHY1/FHL-independent pathway but it may also modulate FHY1/FHL signal through its interaction with HFR1 and LAF1.
Double and triple mutants; Light signaling; Phytochrome A; Protein interaction; Signaling cascade; Transcription factors
The Rubiaceae species, Ophiorrhiza pumila, accumulates camptothecin, an anti-cancer alkaloid with a potent DNA topoisomerase I inhibitory activity, as well as anthraquinones that are derived from the combination of the isochorismate and hemiterpenoid pathways. The biosynthesis of these secondary products is active in O. pumila hairy roots yet very low in cell suspension culture. Deep transcriptome analysis was conducted in O. pumila hairy roots and cell suspension cultures using the Illumina platform, yielding a total of 2 Gb of sequence for each sample. We generated a hybrid transcriptome assembly of O. pumila using the Illumina-derived short read sequences and conventional Sanger-derived expressed sequence tag clones derived from a full-length cDNA library constructed using RNA from hairy roots. Among 35,608 non-redundant unigenes, 3,649 were preferentially expressed in hairy roots compared with cell suspension culture. Candidate genes involved in the biosynthetic pathway for the monoterpenoid indole alkaloid camptothecin were identified; specifically, genes involved in post-strictosamide biosynthetic events and genes involved in the biosynthesis of anthraquinones and chlorogenic acid. Untargeted metabolomic analysis by Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS) indicated that most of the proposed intermediates in the camptothecin biosynthetic pathway accumulated in hairy roots in a preferential manner compared with cell suspension culture. In addition, a number of anthraquinones and chlorogenic acid preferentially accumulated in hairy roots compared with cell suspension culture. These results suggest that deep transcriptome and metabolome data sets can facilitate the identification of genes and intermediates involved in the biosynthesis of secondary products including camptothecin in O. pumila.
Anthraquinone; Camptothecin; Hairy root; Metabolome; Ophiorrhiza pumila; Transcriptome
The grana thylakoids of higher plant chloroplasts are crowded with PSII and the associated light-harvesting complexes (LHCIIs). They constitute supercomplexes, and often form semi-crystalline arrays in the grana. The crowded condition of the grana may be necessary for efficient trapping of excitation energy by LHCII under weak light, but it might hinder proper movement of LHCII necessary for reversible aggregation of LHCII in the energy-dependent quenching of Chl fluorescence under moderate high light. When the thylakoids are illuminated with extreme high light, the reaction center-binding D1 protein of PSII is photodamaged, and the damaged protein migrates to the grana margins for degradation and subsequent repair. In both moderate and extreme high-light conditions, fluidity of the thylakoid membrane is crucial. In this review, we first provide an overview of photoprotective processes, then discuss changes in membrane fluidity and mobility of the protein complexes in the grana under excessive light, which are closely associated with photoprotection of PSII. We hypothesize that reversible aggregation of LHCII, which is necessary to avoid light stress under moderate high light, and swift turnover of the photodamaged D1 protein under extreme high light are threatened by irreversible protein aggregation induced by reactive oxygen species in photochemical reactions.
Light stress; Membrane crowdedness; Non-photochemical quenching; Photoinhibition; Photosystem II; Protein aggregation
In this review, I outline the indirect evidence for the formation of singlet oxygen (1O2) obtained from experiments with the isolated PSII reaction center complex. I also review the methods we used to measure singlet oxygen directly, including luminescence at 1,270 nm, both steady state and time resolved. Other methods we used were histidine-catalyzed molecular oxygen uptake (enabling 1O2 yield measurements), and dye bleaching and difference absorption spectroscopy to identify where quenchers of 1O2 can access this toxic species. We also demonstrated the protective behavior of carotenoids bound within Chl–protein complexes which bring about a substantial amount of 1O2 quenching within the reaction center complex. Finally, I describe how these techniques have been used and expanded in research on photoinhibition and on the role of 1O2 as a signaling molecule in instigating cellular responses to various stress factors. I also discuss the current views on the role of 1O2 as a signaling molecule and the distance it might be able to travel within cells.
Chlorophyll; Photosynthesis; Reactive oxygen species; Triplet states
Plant vacuoles play critical roles in development, growth and stress responses. In mature cells, vacuolar membranes (VMs) display several types of structures, which are formed by invagination and folding of VMs into the lumenal side and can gradually move and change shape. Although such VM structures are observed in a broad range of tissue types and plant species, the molecular mechanism underlying their formation and maintenance remains unclear. Here, we report that a novel HEAT-repeat protein, SHOOT GRAVITROPISM6 (SGR6), of Arabidopsis is involved in the control of morphological changes and dynamics of VM structures in endodermal cells, which are the gravity-sensing cells in shoots. SGR6 is a membrane-associated protein that is mainly localized to the VM in stem endodermal cells. The sgr6 mutant stem exhibits a reduced gravitropic response. Higher plants utilize amyloplast sedimentation as a means to sense gravity direction. Amyloplasts are surrounded by VMs in Arabidopsis endodermal cells, and the flexible and dynamic structure of VMs is important for amyloplast sedimentation. We demonstrated that such dynamic features of VMs are gradually lost in sgr6 endodermal cells during a 30 min observation period. Histological analysis revealed that amyloplast sedimentation was impaired in sgr6. Detailed live-cell imaging analyses revealed that the VM structures in sgr6 had severe defects in morphological changes and dynamics. Our results suggest that SGR6 is a novel protein involved in the formation and/or maintenance of invaginated VM structures in gravity-sensing cells.
Arabidopsis; Gravitropism; HEAT-repeat protein; Vacuolar membrane
Biomass allocation between shoots and roots is an important strategy used by plants to optimize growth in various environments. Root to shoot mass ratios typically increase in response to high CO2, a trend particularly evident under abiotic stress. We investigated this preferential root growth (PRG) in Arabidopsis thaliana plants cultivated under low pH/high CO2 or low nitrogen (N)/high CO2 conditions. Previous studies have suggested that changes in plant hormone, carbon (C) and N status may be related to PRG. We therefore examined the mechanisms underlying PRG by genetically modifying cytokinin (CK) levels, C and N status, and sugar signaling, performing sugar application experiments and determining primary metabolites, plant hormones and expression of related genes. Both low pH/high CO2 and low N/high CO2 stresses induced increases in lateral root (LR) number and led to high C/N ratios; however, under low pH/high CO2 conditions, large quantities of C were accumulated, whereas under low N/high CO2 conditions, N was severely depleted. Analyses of a CK-deficient mutant and a starchless mutant, in conjunction with sugar application experiments, revealed that these stresses induce PRG via different mechanisms. Metabolite and hormone profile analysis indicated that under low pH/high CO2 conditions, excess C accumulation may enhance LR number through the dual actions of increased auxin and decreased CKs.
Auxin; Cytokinin; High CO2; Low nitrogen; Low pH; Root to shoot ratio
Carbon (C) and nitrogen (N) are essential elements for metabolism, and their availability, called the C/N balance, must be tightly coordinated for optimal growth in plants. Previously, we have identified the ubiquitin ligase CNI1/ATL31 as a novel C/N regulator by screening plants grown on C/N stress medium containing excess sugar and limited N. To elucidate further the effect of C/N balance on plant growth and to determine the physiological function of ATL31, we performed C/N response analysis using an atmospheric CO2 manipulation system. Under conditions of elevated CO2 and sufficient N, plant biomass and total sugar and starch dramatically increased. In contrast, elevated CO2 with limited N did not increase plant biomass but promoted leaf chlorosis, with anthocyanin accumulation and increased senescence-associated gene expression. Similar results were obtained with plants grown in medium containing excess sugar and limited N, suggesting that disruption of the C/N balance affects senescence progression. In ATL31-overexpressing plants, promotion of senescence under disrupted CO2/N conditions was repressed, whereas in the loss-of-function mutant it was enhanced. The ATL31 gene was transcriptionally up-regulated under N deficiency and in senescent leaves, and ATL31 expression was highly correlated with WRKY53 expression, a key regulator of senescence. Furthermore, transient protoplast analysis implicated the direct activation of ATL31 expression by WRKY53, which was in accordance with the results of WRKY53 overexpression experiments. Together, these results demonstrate the importance of C/N balance in leaf senescence and the involvement of ubiquitin ligase ATL31 in the process of senescence in Arabidopsis.
Biomass; C/N balance; CO2; Senescence; Ubiquitin ligase
CO2 permeability of plasma membrane intrinsic protein 2 (PIP2) aquaporins of Hordeum vulgare L. was investigated. Five PIP2 members were heterologously expressed in Xenopus laevis oocytes. CO2 permeability was determined by decrease of cytosolic pH in CO2-enriched buffer using a hydrogen ion-selective microelectrode. HvPIP2;1, HvPIP2;2, HvPIP2;3 and HvPIP2;5 facilitated CO2 transport across the oocyte cell membrane. However, HvPIP2;4 that is highly homologous to HvPIP2;3 did not. The isoleucine residue at position 254 of HvPIP2;3 was conserved in PIP2 aquaporins of barley, except HvPIP2;4, which possesses methionine instead. CO2 permeability was lost by the substitution of the Ile254 of HvPIP2;3 by methionine, while water permeability was not affected. These results suggest that PIP2 aquaporins are permeable to CO2. and the conserved isoleucine at the end of the E-loop is crucial for CO2 selectivity.
Aquaporin; Barley; Carbon dioxide; Plasma membrane intrinsic protein 2
Using 18-day-old Arabidopsis thaliana seedlings grown under increased (780 p.p.m., experimental plants) or ambient (390 p.p.m., control plants) CO2 conditions, we evaluated 14CO2 photoassimilation in and translocation from representative source leaves. The total 14CO2 photoassimilation amounts increased in the third leaves of the experimental plants in comparison with that found for the third leaves of the control plants, but the rates were comparable for the first leaves of the two groups. In contrast, translocation of labeled assimilates doubled in the first leaves of the experimental group, whereas translocation was, at best, passively enhanced even though photoassimilation increased in their third leaves. The transcript levels of the companion cell-specific sucrose:H+ symporter gene SUC2 were not significantly affected in the two groups of plants, whereas those of the sucrose effluxer gene SWEET12 and the sieve element-targeted sucrose:H+ symporter gene SUT4 were up-regulated in the experimental plants, suggesting up-regulation of SUT4-dependent apoplastic phloem loading. Compared with SUC2, SUT4 is a minor component that is expressed in companion cells but functions in sieve elements after transfer through plasmodesmata. The number of aniline blue-stained spots for plasmodesma-associated callose in the midrib wall increased in the first leaf of the experimental plants but was comparable in the third leaf between the experimental and control plants. These results suggest that A. thaliana responds to greater than normal concentrations of CO2 differentially in the first and third leaves in regards to photoassimilation, assimilate translocation and plasmodesmal biogenesis.
Arabidopsis thaliana; CO2 atmospheric levels; Photoassimilate translocation; Plasmodesma formation
To enable prediction of future rice production in a changing climate, we need to understand the interactive effects of temperature and elevated [CO2] (E[CO2]). We therefore examined if the effect of E[CO2] on the light-saturated leaf photosynthetic rate (Asat) was affected by soil and water temperature (NT, normal; ET, elevated) under open-field conditions at the rice free-air CO2 enrichment (FACE) facility in Shizukuishi, Japan, in 2007 and 2008. Season-long E[CO2] (+200 µmol mol−1) increased Asat by 26%, when averaged over two years, temperature regimes and growth stages. The effect of ET (+2°C) on Asat was not significant at active tillering and heading, but became negative and significant at mid-grain filling; Asat in E[CO2]–ET was higher than in ambient [CO2] (A[CO2])–NT by only 4%. Photosynthetic down-regulation at E[CO2] also became apparent at mid-grain filling; Asat compared at the same [CO2] in the leaf cuvette was significantly lower in plants grown in E[CO2] than in those grown in A[CO2]. The additive effects of E[CO2] and ET decreased Asat by 23% compared with that of A[CO2]–NT plants. Although total crop nitrogen (N) uptake was increased by ET, N allocation to the leaves and to Rubisco was reduced under ET and E[CO2] at mid-grain filling, which resulted in a significant decrease (32%) in the maximum rate of ribulose-1,5-bisphosphate carboxylation on a leaf area basis. Because the change in N allocation was associated with the accelerated phenology in E[CO2]–ET plants, we conclude that soil and water warming accelerates photosynthetic down-regulation at E[CO2].
Ecosystem warming; FACE (free-air CO2 enrichment); Oryza sativa; Photosynthetic down-regulation
Elevated CO2 concentrations (eCO2) trigger various plant responses. Despite intensive studies of these responses, the underlying mechanisms remain obscure. In this work, we investigated when and how leaf physiology and anatomy are affected by eCO2 in rice plants. We analyzed the most recently fully expanded leaves that developed successively after transfer of the plant to eCO2. To discriminate between the effects of eCO2 and those of nitrogen deficiency, we used three different levels of N application. We found that a decline in the leaf soluble protein content (on a leaf area basis) at eCO2 was only observed under N deficiency. The length and width of the leaf blade were reduced by both eCO2 and N deficiency, whereas the blade thickness was increased by eCO2 but was not affected by N deficiency. The change in length by eCO2 became detectable in the secondly fully expanded leaf, and those in width and thickness in the thirdly fully expanded leaf, which were at the leaf developmental stages P4 and P3, respectively, at the onset of the eCO2 treatment. The decreased blade length at eCO2 was associated with a decrease in the epidermal cell number on the adaxial side and a reduction in cell length on the abaxial side. The decreased width resulted from decreased numbers of small vascular bundles and epidermal cell files. The increased thickness was ascribed mainly to enhanced development of bundle sheath extensions at the ridges of vascular bundles. These observations enable us to identify the sites of action of eCO2 on rice leaf development.
Elevated CO2; Leaf anatomy; Leaf development; Nitrogen deficiency; Photosynthesis; Rice
Sugar-derived reactive carbonyls (RCs), including methylglyoxal (MG), are aggressive by-products of oxidative stress known to impair the functions of multiple proteins. These advanced glycation end-products accumulate in patients with diabetes mellitus and cause major complications, including arteriosclerosis and cardiac insufficiency. In the glycolytic pathway, the equilibration reactions between dihydroxyacetone phosphate and glyceraldehyde 3-phosphate (GAP) have recently been shown to generate MG as a by-product. Because plants produce vast amounts of sugars and support the same reaction in the Calvin cycle, we hypothesized that MG also accumulates in chloroplasts. Incubating isolated chloroplasts with excess 3-phosphoglycerate (3-PGA) as the GAP precursor drove the equilibration reaction toward MG production. The rate of oxygen (O2) evolution was used as an index of 3-PGA-mediated photosynthesis. The 3-PGA- and time-dependent accumulation of MG in chloroplasts was confirmed by HPLC. In addition, MG production increased with an increase in light intensity. We also observed a positive linear relationship between the rates of MG production and O2 evolution (R = 0.88; P < 0.0001). These data provide evidence that MG is produced by the Calvin cycle and that sugar-derived RC production is inevitable during photosynthesis. Furthermore, we found that MG production is enhanced under high-CO2 conditions in illuminated wheat leaves.
Advanced glycation end-products; Methylglyoxal; Photosynthesis; Triose phosphate isomerase
The development of crops which are well suited to growth under future environmental conditions such as higher atmospheric CO2 concentrations ([CO2]) is essential to meeting the challenge of ensuring food security in the face of the growing human population and changing climate. A high-yielding indica rice variety (Oryza sativa L. cv. Takanari) has been recently identified as a potential candidate for such breeding, due to its high productivity in present [CO2]. To test if it could further increase its productivity under elevated [CO2] (eCO2), Takanari was grown in the paddy field under season-long free-air CO2 enrichment (FACE, approximately 200 µmol mol−1 above ambient [CO2]) and its leaf physiology was compared with the representative japonica variety ‘Koshihikari’. Takanari showed consistently higher midday photosynthesis and stomatal conductance than Koshihikari under both ambient and FACE growth conditions over 2 years. Maximum ribulose-1,5-bisphosphate carboxylation and electron transport rates were higher for Takanari at the mid-grain filling stage in both years. Mesophyll conductance was higher in Takanari than in Koshihikari at the late grain-filling stage. In contrast to Koshihikari, Takanari grown under FACE conditions showed no decrease in total leaf nitrogen on an area basis relative to ambient-grown plants. Chl content was higher in Takanari than in Koshihikari at the same leaf nitrogen level. These results indicate that Takanari maintains its superiority over Koshihikari in regards to its leaf-level productivity when grown in elevated [CO2] and it may be a valuable resource for rice breeding programs which seek to increase crop productivity under current and future [CO2].
Ecophysiology; FACE; Global change; Photosynthesis
Correct positioning of membrane proteins is an essential process in eukaryotic organisms. The plant hormone auxin is distributed through intercellular transport and triggers various cellular responses. Auxin transporters of the PIN-FORMED (PIN) family localize asymmetrically at the plasma membrane (PM) and mediate the directional transport of auxin between cells. A fungal toxin, brefeldin A (BFA), inhibits a subset of guanine nucleotide exchange factors for ADP-ribosylation factor small GTPases (ARF GEFs) including GNOM, which plays a major role in localization of PIN1 predominantly to the basal side of the PM. The Arabidopsis genome encodes 19 ARF-related putative GTPases. However, ARF components involved in PIN1 localization have been genetically poorly defined. Using a fluorescence imaging-based forward genetic approach, we identified an Arabidopsis mutant, bfa-visualized exocytic trafficking defective1 (bex1), in which PM localization of PIN1–green fluorescent protein (GFP) as well as development is hypersensitive to BFA. We found that in bex1 a member of the ARF1 gene family, ARF1A1C, was mutated. ARF1A1C localizes to the trans-Golgi network/early endosome and Golgi apparatus, acts synergistically to BEN1/MIN7 ARF GEF and is important for PIN recycling to the PM. Consistent with the developmental importance of PIN proteins, functional interference with ARF1 resulted in an impaired auxin response gradient and various developmental defects including embryonic patterning defects and growth arrest. Our results show that ARF1A1C is essential for recycling of PIN auxin transporters and for various auxin-dependent developmental processes.
Arabidopsis thaliana; Auxin; Embryogenesis; Exocytosis; PIN-FORMED