Plant Physiology current issue

Legume Focus: Model Species Sequenced, Mutagenesis Approaches Extended, and Debut of a New Model

O'Brian, M. R., Vance, C. P., VandenBosch, K. A. — Tue, 03 Nov 2009 12:22:26 PST

Three Sequenced Legume Genomes and Many Crop Species: Rich Opportunities for Translational Genomics

Cannon, S. B., May, G. D., Jackson, S. A. — Tue, 03 Nov 2009 12:22:26 PST

Mutagenesis and Beyond! Tools for Understanding Legume Biology

Tadege, M., Wang, T. L., Wen, J., Ratet, P., Mysore, K. S. — Tue, 03 Nov 2009 12:22:26 PST

Pea Has Its Tendrils in Branching Discoveries Spanning a Century from Auxin to Strigolactones

Beveridge, C. A., Dun, E. A., Rameau, C. — Tue, 03 Nov 2009 12:22:26 PST

Legume Transcription Factor Genes: What Makes Legumes So Special?

Libault, M., Joshi, T., Benedito, V. A., Xu, D., Udvardi, M. K., Stacey, G. — Tue, 03 Nov 2009 12:22:26 PST

MicroRNAs in the Rhizobia Legume Symbiosis

Simon, S. A., Meyers, B. C., Sherrier, D. J. — Tue, 03 Nov 2009 12:22:26 PST

Will Elevated Carbon Dioxide Concentration Amplify the Benefits of Nitrogen Fixation in Legumes?

Rogers, A., Ainsworth, E. A., Leakey, A. D.B. — Tue, 03 Nov 2009 12:22:26 PST

Emerging Approaches to Broaden Resistance of Soybean to Soybean Cyst Nematode as Supported by Gene Expression Studies

Klink, V. P., Matthews, B. F. — Tue, 03 Nov 2009 12:22:26 PST

Post-Genomics Studies of Developmental Processes in Legume Seeds

Thompson, R., Burstin, J., Gallardo, K. — Tue, 03 Nov 2009 12:22:26 PST

Soybean Oil: Genetic Approaches for Modification of Functionality and Total Content

Clemente, T. E., Cahoon, E. B. — Tue, 03 Nov 2009 12:22:26 PST

Venturing Beyond Beans and Peas: What Can We Learn from Chamaecrista?

Tue, 03 Nov 2009 12:22:26 PST

A Nomadic Subtelomeric Disease Resistance Gene Cluster in Common Bean

Tue, 03 Nov 2009 12:22:26 PST

The B4 resistance (R) gene cluster is one of the largest clusters known in common bean (Phaseolus vulgaris [Pv]). It is located in a peculiar genomic environment in the subtelomeric region of the short arm of chromosome 4, adjacent to two heterochromatic blocks (knobs). We sequenced 650 kb spanning this locus and annotated 97 genes, 26 of which correspond to Coiled-Coil-Nucleotide-Binding-Site-Leucine-Rich-Repeat (CNL). Conserved microsynteny was observed between the Pv B4 locus and corresponding regions of Medicago truncatula and Lotus japonicus in chromosomes Mt6 and Lj2, respectively. The notable exception was the CNL sequences, which were completely absent in these regions. The origin of the Pv B4-CNL sequences was investigated through phylogenetic analysis, which reveals that, in the Pv genome, paralogous CNL genes are shared among nonhomologous chromosomes (4 and 11). Together, our results suggest that Pv B4-CNL was derived from CNL sequences from another cluster, the Co-2 cluster, through an ectopic recombination event. Integration of the soybean (Glycine max) genome data enables us to date more precisely this event and also to infer that a single CNL moved from the Co-2 to the B4 cluster. Moreover, we identified a new 528-bp satellite repeat, referred to as khipu, specific to the Phaseolus genus, present both between B4-CNL sequences and in the two knobs identified at the B4 R gene cluster. The khipu repeat is present on most chromosomal termini, indicating the existence of frequent ectopic recombination events in Pv subtelomeric regions. Our results highlight the importance of ectopic recombination in R gene evolution.

Dynamic Rearrangements Determine Genome Organization and Useful Traits in Soybean

Kim, K. D., Shin, J. H., Van, K., Kim, D. H., Lee, S.-H. — Tue, 03 Nov 2009 12:22:26 PST

Soybean (Glycine max) is a paleopolyploid whose genome has gone through at least two rounds of polyploidy and subsequent diploidization events. Several studies have investigated the changes in genome structure produced by the relatively recent polyploidy event, but little is known about the ancient polyploidy due to the high frequency of gene loss after duplication. Our previous study, regarding a region responsible for bacterial leaf pustule, reported two homeologous Rxp regions produced by the recent whole-genome duplication event. In this study, we identified the full set of four homeologous Rxp regions (ranging from 1.96 to 4.60 Mb) derived from both the recent and ancient polyploidy events, and this supports the quadruplicated structure of the soybean genome. Among the predicted genes on chromosome 17 (linkage group D2), 71% of them were conserved in a recently duplicated region, while 21% and 24% of duplicated genes were retained in two homeologous regions formed by the ancient polyploidy. Furthermore, comparative analysis showed a 2:1 relationship between soybean and Medicago truncatula, since M. truncatula did not undergo the recent polyploidy event that soybean did. Unlike soybean, M. truncatula homeologous regions were highly fractionated and their synteny did not exist, revealing different rates of diploidization process between the two species. Our data show that extensive synteny remained in the four homeologous regions in soybean, even though the soybean genome experienced dynamic genome rearrangements following paleopolyploidy events. Moreover, multiple Rxp quantitative trait loci on different soybean chromosomes actually comprise homeologous regions produced by two rounds of polyploidy events.

Deletion-Based Reverse Genetics in Medicago truncatula

Rogers, C., Wen, J., Chen, R., Oldroyd, G. — Tue, 03 Nov 2009 12:22:26 PST

The primary goal of reverse genetics, the identification of null mutations in targeted genes, is achieved through screening large populations of randomly mutagenized plants. T-DNA and transposon-based mutagenesis has been widely employed but is limited to species in which transformation and tissue culture are efficient. In other species, TILLING (for Targeting Induced Local Lesions IN Genomes), based on chemical mutagenesis, has provided an efficient method for the identification of single base pair mutations, only 5% of which will be null mutations. Furthermore, the efficiency of inducing point mutations, like insertion-based mutations, is dependent on target size. Here, we describe an alternative reverse genetic strategy based on physically induced genomic deletions that, independent of target size, exclusively recovers knockout mutants. Deletion TILLING (De-TILLING) employs fast neutron mutagenesis and a sensitive polymerase chain reaction-based detection. A population of 156,000 Medicago truncatula plants has been structured as 13 towers each representing 12,000 M2 plants. The De-TILLING strategy allows a single tower to be screened using just four polymerase chain reaction reactions. Dual screening and three-dimensional pooling allows efficient location of mutants from within the towers. With this method, we have demonstrated the detection of mutants from this population at a rate of 29% using five targets per gene. This De-TILLING reverse genetic strategy is independent of tissue culture and efficient plant transformation and therefore applicable to any plant species. De-TILLING mutants offer advantages for crop improvement as they possess relatively few background mutations and no exogenous DNA.

Site-Specific Integration of Transgenes in Soybean via Recombinase-Mediated DNA Cassette Exchange

Li, Z., Xing, A., Moon, B. P., McCardell, R. P., Mills, K., Falco, S. C. — Tue, 03 Nov 2009 12:22:26 PST

A targeting method to insert genes at a previously characterized genetic locus to make plant transformation and transgene expression predictable is highly desirable for plant biotechnology. We report the successful targeting of transgenes to predefined soybean (Glycine max) genome sites using the yeast FLP-FRT recombination system. First, a target DNA containing a pair of incompatible FRT sites flanking a selection gene was introduced in soybean by standard biolistic transformation. Transgenic events containing a single copy of the target were retransformed with a donor DNA, which contained the same pair of FRT sites flanking a different selection gene, and a FLP expression DNA. Precise DNA cassette exchange was achieved between the target and donor DNA via recombinase-mediated cassette exchange, so that the donor DNA was introduced at the locus previously occupied by the target DNA. The introduced donor genes expressed normally and segregated according to Mendelian laws.

Integrated Metabolite and Transcript Profiling Identify a Biosynthetic Mechanism for Hispidol in Medicago truncatula Cell Cultures

Tue, 03 Nov 2009 12:22:26 PST

Metabolic profiling of elicited barrel medic (Medicago truncatula) cell cultures using high-performance liquid chromatography coupled to photodiode and mass spectrometry detection revealed the accumulation of the aurone hispidol (6-hydroxy-2-[(4-hydroxyphenyl)methylidene]-1-benzofuran-3-one) as a major response to yeast elicitor. Parallel, large-scale transcriptome profiling indicated that three peroxidases, MtPRX1, MtPRX2, and MtPRX3, were coordinately induced with the accumulation of hispidol. MtPRX1 and MtPRX2 exhibited aurone synthase activity based upon in vitro substrate specificity and product profiles of recombinant proteins expressed in Escherichia coli. Hispidol possessed significant antifungal activity relative to other M. truncatula phenylpropanoids tested but has not been reported in this species before and was not found in differentiated roots in which high levels of the peroxidase transcripts accumulated. We propose that hispidol is formed in cell cultures by metabolic spillover when the pool of its precursor, isoliquiritigenin, builds up as a result of an imbalance between the upstream and downstream segments of the phenylpropanoid pathway, reflecting the plasticity of plant secondary metabolism. The results illustrate that integration of metabolomics and transcriptomics in genetically reprogrammed plant cell cultures is a powerful approach for the discovery of novel bioactive secondary metabolites and the mechanisms underlying their generation.

A WD40 Repeat Protein from Medicago truncatula Is Necessary for Tissue-Specific Anthocyanin and Proanthocyanidin Biosynthesis But Not for Trichome Development

Tue, 03 Nov 2009 12:22:26 PST

WD40 repeat proteins regulate biosynthesis of anthocyanins, proanthocyanidins (PAs), and mucilage in the seed and the development of trichomes and root hairs. We have cloned and characterized a WD40 repeat protein gene from Medicago truncatula (MtWD40-1) via a retrotransposon-tagging approach. Deficiency of MtWD40-1 expression blocks accumulation of mucilage and a range of phenolic compounds, including PAs, epicatechin, other flavonoids, and benzoic acids, in the seed, reduces epicatechin levels without corresponding effects on other flavonoids in flowers, reduces isoflavone levels in roots, but does not impair trichome or root hair development. MtWD40-1 is expressed constitutively, with highest expression in the seed coat, where its transcript profile temporally parallels those of PA biosynthetic genes. Transcript profile analysis revealed that many genes of flavonoid biosynthesis were down-regulated in a tissue-specific manner in M. truncatula lines harboring retrotransposon insertions in the MtWD40-1 gene. MtWD40-1 complemented the anthocyanin, PA, and trichome phenotypes of the Arabidopsis (Arabidopsis thaliana) transparent testa glabrous1 mutant. We discuss the function of MtWD40-1 in natural product formation in M. truncatula and the potential use of the gene for engineering PAs in the forage legume alfalfa (Medicago sativa).

Auxin Biosynthesis in Pea: Characterization of the Tryptamine Pathway

Tue, 03 Nov 2009 12:22:26 PST

One pathway leading to the bioactive auxin, indole-3-acetic acid (IAA), is known as the tryptamine pathway, which is suggested to proceed in the sequence: tryptophan (Trp), tryptamine, N-hydroxytryptamine, indole-3-acetaldoxime, indole-3-acetaldehyde (IAAld), IAA. Recently, this pathway has been characterized by the YUCCA genes in Arabidopsis (Arabidopsis thaliana) and their homologs in other species. YUCCA is thought to be responsible for the conversion of tryptamine to N-hydroxytryptamine. Here we complement the genetic findings with a compound-based approach in pea (Pisum sativum), detecting potential precursors by gas chromatography/tandem-mass spectrometry. In addition, we have synthesized deuterated forms of many of the intermediates involved, and have used them to quantify the endogenous compounds, and to investigate their metabolic fates. Trp, tryptamine, IAAld, indole-3-ethanol, and IAA were detected as endogenous constituents, whereas indole-3-acetaldoxime and one of its products, indole-3-acetonitrile, were not detected. Metabolism experiments indicated that the tryptamine pathway to IAA in pea roots proceeds in the sequence: Trp, tryptamine, IAAld, IAA, with indole-3-ethanol as a side-branch product of IAAld. N-hydroxytryptamine was not detected, but we cannot exclude that it is an intermediate between tryptamine and IAAld, nor can we rule out the possibility of a Trp-independent pathway operating in pea roots.

The Metabolic Role of the Legume Endosperm: A Noninvasive Imaging Study

Tue, 03 Nov 2009 12:22:26 PST

Although essential for normal seed development in the legumes, the metabolic role of the endosperm remains uncertain. We designed noninvasive nuclear magnetic resonance tools for the in vivo study of key metabolites in the transient liquid endosperm of intact pea (Pisum sativum) seeds. The steady-state levels of sucrose, glutamine, and alanine could be monitored and their distribution within the embryo sac visualized. Seed structure was digitalized as a three-dimensional model, providing volume information for distinct seed organs. The nuclear magnetic resonance method, combined with laser microdissection, isotope labeling, in situ hybridization, and electron microscopy, was used to contrast the wild-type endosperm with that of a mutant in which embryo growth is retarded. Expression of sequences encoding amino acid and sucrose transporters was up-regulated earlier in the endosperm than in the embryo, and this activity led to the accumulation of soluble metabolites in the endosperm vacuole. The endosperm provides a temporary source of nutrition, permits space for embryo growth, and acts as a buffer between the maternal organism and its offspring. The concentration of sucrose in the endosperm vacuole is developmentally controlled, while the total amount accumulated depends on the growth of the embryo. The endosperm concentration of glutamine is a limiting factor for protein storage. The properties of the endosperm ensure that the young embryo develops within a homeostatic environment, necessary to sustain embryogenesis. We argue for a degree of metabolite-mediated control exerted by the endosperm on the growth of, and assimilate storage by, the embryo.

Knockdown of CELL DIVISION CYCLE16 Reveals an Inverse Relationship between Lateral Root and Nodule Numbers and a Link to Auxin in Medicago truncatula

Tue, 03 Nov 2009 12:22:26 PST

The postembryonic development of lateral roots and nodules is a highly regulated process. Recent studies suggest the existence of cross talk and interdependency in the growth of these two organs. Although plant hormones, including auxin and cytokinin, appear to be key players in coordinating this cross talk, very few genes that cross-regulate root and nodule development have been uncovered so far. This study reports that a homolog of CELL DIVISION CYCLE16 (CDC16), a core component of the Anaphase Promoting Complex, is one of the key mediators in controlling the overall number of lateral roots and nodules. A partial suppression of this gene in Medicago truncatula leads to a decrease in number of lateral roots and a 4-fold increase in number of nodules. The roots showing lowered expression of MtCDC16 also show reduced sensitivity to phytohormone auxin, thus providing a potential function of CDC16 in auxin signaling.

Molecular and Chromosomal Evidence for Allopolyploidy in Soybean

Tue, 03 Nov 2009 12:22:26 PST

Recent studies have documented that the soybean (Glycine max) genome has undergone two rounds of large-scale genome and/or segmental duplication. To shed light on the timing and nature of these duplication events, we characterized and analyzed two subfamilies of high-copy centromeric satellite repeats, CentGm-1 and CentGm-2, using a combination of computational and molecular cytogenetic approaches. These two subfamilies of satellite repeats mark distinct subsets of soybean centromeres and, in at least one case, a pair of homologs, suggesting their origins from an allopolyploid event. The satellite monomers of each subfamily are arranged in large tandem arrays, and intermingled monomers of the two subfamilies were not detected by fluorescence in situ hybridization on extended DNA fibers nor at the sequence level. This indicates that there has been little recombination and homogenization of satellite DNA between these two sets of centromeres. These satellite repeats are also present in Glycine soja, the proposed wild progenitor of soybean, but could not be detected in any other relatives of soybean examined in this study, suggesting the rapid divergence of the centromeric satellite DNA within the Glycine genus. Together, these observations provide direct evidence, at molecular and chromosomal levels, in support of the hypothesis that the soybean genome has experienced a recent allopolyploidization event.

Conservation of Lotus and Arabidopsis Basic Helix-Loop-Helix Proteins Reveals New Players in Root Hair Development

Tue, 03 Nov 2009 12:22:26 PST

Basic helix-loop-helix (bHLH) proteins constitute a large family of transcriptional regulators in plants. Although they have been shown to play important roles in a wide variety of developmental processes, relatively few have been functionally characterized. Here, we describe the map-based cloning of the Lotus japonicus ROOTHAIRLESS1 (LjRHL1) locus. Deleterious mutations in this locus prevent root hair development, which also aborts root hair-dependent colonization of the host root by nitrogen-fixing bacteria. We show that the LjRHL1 gene encodes a presumed bHLH transcription factor that functions in a nonredundant manner to control root hair development in L. japonicus. Homology search and cross-species complementation experiments defined three members of the Arabidopsis (Arabidopsis thaliana) bHLH protein family, At2g24260, At4g30980, and At5g58010, as functionally equivalent to LjRHL1. Curiously, At2g24260 and At4g30980 mRNA species accumulate independently from the known positive regulators of root hair cell fate, while all three genes act in a partially redundant manner to regulate root hair development in Arabidopsis.

(Homo)glutathione Depletion Modulates Host Gene Expression during the Symbiotic Interaction between Medicago truncatula and Sinorhizobium meliloti

Tue, 03 Nov 2009 12:22:26 PST

Under nitrogen-limiting conditions, legumes interact with symbiotic rhizobia to produce nitrogen-fixing root nodules. We have previously shown that glutathione and homoglutathione [(h)GSH] deficiencies impaired Medicago truncatula symbiosis efficiency, showing the importance of the low M_r thiols during the nodulation process in the model legume M. truncatula. In this study, the plant transcriptomic response to Sinorhizobium meliloti infection under (h)GSH depletion was investigated using cDNA-amplified fragment length polymorphism analysis. Among 6,149 expression tags monitored, 181 genes displayed significant differential expression between inoculated control and inoculated (h)GSH depleted roots. Quantitative reverse transcription polymerase chain reaction analysis confirmed the changes in mRNA levels. This transcriptomic analysis shows a down-regulation of genes involved in meristem formation and a modulation of the expression of stress-related genes in (h)GSH-depleted plants. Promoter-β-glucuronidase histochemical analysis showed that the putative MtPIP2 aquaporin might be up-regulated during nodule meristem formation and that this up-regulation is inhibited under (h)GSH depletion. (h)GSH depletion enhances the expression of salicylic acid (SA)-regulated genes after S. meliloti infection and the expression of SA-regulated genes after exogenous SA treatment. Modification of water transport and SA signaling pathway observed under (h)GSH deficiency contribute to explain how (h)GSH depletion alters the proper development of the symbiotic interaction.

A Nuclear-Targeted Cameleon Demonstrates Intranuclear Ca2+ Spiking in Medicago truncatula Root Hairs in Response to Rhizobial Nodulation Factors

Tue, 03 Nov 2009 12:22:26 PST

Lipochitooligosaccharide nodulation factors (NFs) secreted by endosymbiotic nitrogen-fixing rhizobia trigger Ca²⁺ spiking in the cytoplasmic perinuclear region of host legume root hairs. To determine whether NFs also elicit Ca²⁺ responses within the plant cell nucleus we have made use of a nucleoplasmin-tagged cameleon (NupYC2.1). Confocal microscopy using this nuclear-specific calcium reporter has revealed sustained and regular Ca²⁺ spiking within the nuclear compartment of Medicago truncatula root hairs treated with Sinorhizobium meliloti NFs. Since the activation of Ca²⁺ oscillations is blocked in M. truncatula nfp, dmi1, and dmi2 mutants, and unaltered in a dmi3 background, it is likely that intranuclear spiking lies on the established NF-dependent signal transduction pathway, leading to cytoplasmic calcium spiking. A semiautomated mathematical procedure has been developed to identify and analyze nuclear Ca²⁺ spiking profiles, and has revealed high cell-to-cell variability in terms of both periodicity and spike duration. Time-lapse imaging of the cameleon Förster resonance energy transfer-based ratio has allowed us to visualize the nuclear spiking variability in situ and to demonstrate the absence of spiking synchrony between adjacent growing root hairs. Finally, spatio-temporal analysis of the asymmetric nuclear spike suggests that the initial rapid increase in Ca²⁺ concentration occurs principally in the vicinity of the nuclear envelope. The discovery that rhizobial NF perception leads to the activation of cell-autonomous Ca²⁺ oscillations on both sides of the nuclear envelope raises major questions about the respective roles of the cytoplasmic and nuclear compartments in transducing this key endosymbiotic signal.

Large-Scale Analysis of Putative Soybean Regulatory Gene Expression Identifies a Myb Gene Involved in Soybean Nodule Development

Tue, 03 Nov 2009 12:22:26 PST

Nodulation is the result of a symbiosis between legumes and rhizobial bacteria in soil. This symbiosis is mutually beneficial, with the bacteria providing a source of nitrogen to the host while the plant supplies carbon to the symbiont. Nodule development is a complex process that is tightly regulated in the host plant cell through networks of gene expression. In order to examine this regulation in detail, a library of quantitative reverse transcription-polymerase chain reaction primer sets was developed for a large number of soybean (Glycine max) putative regulatory genes available in the current expressed sequence tag collection. This library contained primers specific to soybean transcription factor genes as well as genes involved in chromatin modification and translational regulation. Using this library, we analyzed the expression of this gene set during nodule development. A large number of genes were found to be differentially expressed, especially at the later stages of nodule development when active nitrogen fixation was occurring. Expression of these putative regulatory genes was also analyzed in response to the addition of nitrate as a nitrogen source. This comparative analysis identified genes that may be specifically involved in nitrogen assimilation, metabolism, and the maintenance of active nodules. To address this possibility, the expression of one such candidate was studied in more detail by expressing in soybean roots promoter β-glucuronidase and green fluorescent protein fusions. This gene, named Control of Nodule Development (CND), encoded a Myb transcription factor gene. When the CND gene was silenced, nodulation was reduced. These results, associated with a strong expression of the CND gene in the vascular tissues, suggest a role for CND in controlling soybean nodulation.

Global Changes in the Transcript and Metabolic Profiles during Symbiotic Nitrogen Fixation in Phosphorus-Stressed Common Bean Plants

Tue, 03 Nov 2009 12:22:26 PST

Phosphorus (P) deficiency is widespread in regions where the common bean (Phaseolus vulgaris), the most important legume for human consumption, is produced, and it is perhaps the factor that most limits nitrogen fixation. Global gene expression and metabolome approaches were used to investigate the responses of nodules from common bean plants inoculated with Rhizobium tropici CIAT899 grown under P-deficient and P-sufficient conditions. P-deficient inoculated plants showed drastic reduction in nodulation and nitrogenase activity as determined by acetylene reduction assay. Nodule transcript profiling was performed through hybridization of nylon filter arrays spotted with cDNAs, approximately 4,000 unigene set, from the nodule and P-deficient root library. A total of 459 genes, representing different biological processes according to updated annotation using the UniProt Knowledgebase database, showed significant differential expression in response to P: 59% of these were induced in P-deficient nodules. The expression platform for transcription factor genes based in quantitative reverse transcriptase-polymerase chain reaction revealed that 37 transcription factor genes were differentially expressed in P-deficient nodules and only one gene was repressed. Data from nontargeted metabolic profiles indicated that amino acids and other nitrogen metabolites were decreased, while organic and polyhydroxy acids were accumulated, in P-deficient nodules. Bioinformatics analyses using MapMan and PathExpress software tools, customized to common bean, were utilized for the analysis of global changes in gene expression that affected overall metabolism. Glycolysis and glycerolipid metabolism, and starch and Suc metabolism, were identified among the pathways significantly induced or repressed in P-deficient nodules, respectively.

LIN, a Novel Type of U-Box/WD40 Protein, Controls Early Infection by Rhizobia in Legumes

Tue, 03 Nov 2009 12:22:26 PST

The formation of a nitrogen-fixing nodule requires the coordinated development of rhizobial colonization and nodule organogenesis. Based on its mutant phenotype, lumpy infections (lin), LIN functions at an early stage of the rhizobial symbiotic process, required for both infection thread growth in root hair cells and the further development of nodule primordia. We show that spontaneous nodulation activated by the calcium- and calmodulin-dependent protein kinase is independent of LIN; thus, LIN is not necessary for nodule organogenesis. From this, we infer that LIN predominantly functions during rhizobial colonization and that the abortion of this process in lin mutants leads to a suppression of nodule development. Here, we identify the LIN gene in Medicago truncatula and Lotus japonicus, showing that it codes for a predicted E3 ubiquitin ligase containing a highly conserved U-box and WD40 repeat domains. Ubiquitin-mediated protein degradation is a universal mechanism to regulate many biological processes by eliminating rate-limiting enzymes and key components such as transcription factors. We propose that LIN is a regulator of the component(s) of the nodulation factor signal transduction pathway and that its function is required for correct temporal and spatial activity of the target protein(s).

MERE1, a Low-Copy-Number Copia-Type Retroelement in Medicago truncatula Active during Tissue Culture

Tue, 03 Nov 2009 12:22:26 PST

We have identified an active Medicago truncatula copia-like retroelement called Medicago RetroElement1-1 (MERE1-1) as an insertion in the symbiotic NSP2 gene. MERE1-1 belongs to a low-copy-number family in the sequenced Medicago genome. These copies are highly related, but only three of them have a complete coding region and polymorphism exists between the long terminal repeats of these different copies. This retroelement family is present in all M. truncatula ecotypes tested but also in other legume species like Lotus japonicus. It is active only during tissue culture in both R108 and Jemalong Medicago accessions and inserts preferentially in genes.

The Nematode Resistance Allele at the rhg1 Locus Alters the Proteome and Primary Metabolism of Soybean Roots

Tue, 03 Nov 2009 12:22:26 PST

Heterodera glycines, the soybean cyst nematode (SCN), causes the most damaging chronic disease of soybean (Glycine max). Host resistance requires the resistance allele at rhg1. Resistance destroys the giant cells created in the plant's roots by the nematodes about 24 to 48 h after commencement of feeding. In addition, 4 to 8 d later, a systemic acquired resistance develops that discourages later infestations. The molecular mechanisms that control the rhg1-mediated resistance response appear to be multigenic and complex, as judged by transcript abundance changes, even in near isogenic lines (NILs). This study aimed to focus on key posttranscriptional changes by identifying proteins and metabolites that were increased in abundance in both resistant and susceptible NILs. Comparisons were made among NILs 10 d after SCN infestation and without SCN infestation. Two-dimensional gel electrophoresis resolved more than 1,000 protein spots on each gel. Only 30 protein spots with a significant (P < 0.05) difference in abundance of 1.5-fold or more were found among the four treatments. The proteins in these spots were picked, trypsin digested, and analyzed using quadrupole time-of-flight tandem mass spectrometry. Protein identifications could be made for 24 of the 30 spots. Four spots contained two proteins, so that 28 distinct proteins were identified. The proteins were grouped into six functional categories. Metabolite analysis by gas chromatography-mass spectrometry identified 131 metabolites, among which 58 were altered by one or more treatment; 28 were involved in primary metabolism. Taken together, the data showed that 17 pathways were altered by the rhg1 alleles. Pathways altered were associated with systemic acquired resistance-like responses, including xenobiotic, phytoalexin, ascorbate, and inositol metabolism, as well as primary metabolisms like amino acid synthesis and glycolysis. The pathways impacted by the rhg1 allelic state and SCN infestation agreed with transcript abundance analyses but identified a smaller set of key proteins. Six of the proteins lay within the same small region of the interactome identifying a key set of 159 interacting proteins involved in transcriptional control, nuclear localization, and protein degradation. Finally, two proteins (glucose-6-phosphate isomerase [EC 5.3.1.9] and isoflavone reductase [EC 1.3.1.45]) and two metabolites (maltose and an unknown) differed in resistant and susceptible NILs without SCN infestation and may form the basis of a new assay for the selection of resistance to SCN in soybean.

TILLING in Lotus japonicus Identified Large Allelic Series for Symbiosis Genes and Revealed a Bias in Functionally Defective Ethyl Methanesulfonate Alleles toward Glycine Replacements

Tue, 03 Nov 2009 12:22:27 PST

We have established tools for forward and reverse genetic analysis of the legume Lotus (Lotus japonicus). A structured population of M2 progeny of 4,904 ethyl methanesulfonate-mutagenized M1 embryos is available for single nucleotide polymorphism mutation detection, using a TILLING (for Targeting Induced Local Lesions IN Genomes) protocol. Scanning subsets of this population, we identified a mutation load of one per 502 kb of amplified fragment. Moreover, we observed a 1:10 ratio between homozygous and heterozygous mutations in the M2 progeny. This reveals a clear difference in germline genetics between Lotus and Arabidopsis (Arabidopsis thaliana). In addition, we assembled M2 siblings with obvious phenotypes in overall development, starch accumulation, or nitrogen-fixing root nodule symbiosis in three thematic subpopulations. By screening the nodulation-defective population of M2 individuals for mutations in a set of 12 genes known to be essential for nodule development, we identified large allelic series for each gene, generating a unique data set that combines genotypic and phenotypic information facilitating structure-function studies. This analysis revealed a significant bias for replacements of glycine (Gly) residues in functionally defective alleles, which may be explained by the exceptional structural features of Gly. Gly allows the peptide chain to adopt conformations that are no longer possible after amino acid replacement. This previously unrecognized vulnerability of proteins at Gly residues could be used for the improvement of algorithms that are designed to predict the deleterious nature of single nucleotide polymorphism mutations. Our results demonstrate the power, as well as the limitations, of ethyl methanesulfonate mutagenesis for forward and reverse genetic studies. (Original mutant phenotypes can be accessed at http://data.jic.bbsrc.ac.uk/cgi-bin/lotusjaponicus Access to the Lotus TILLING facility can be obtained through http://www.lotusjaponicus.org or http://revgenuk.jic.ac.uk)

On the Inside

Minorsky, P. V. — Tue, 03 Nov 2009 12:22:27 PST

Discovery and Characterization of a Novel Lachrymatory Factor Synthase in Petiveria alliacea and Its Influence on Alliinase-Mediated Formation of Biologically Active Organosulfur Compounds

Musah, R. A., He, Q., Kubec, R. — Tue, 03 Nov 2009 12:22:27 PST

A novel lachrymatory factor synthase (LFS) was isolated and purified from the roots of the Amazonian medicinal plant Petiveria alliacea. The enzyme is a heterotetrameric glycoprotein comprised of two -subunits (68.8 kD each), one -subunit (22.5 kD), and one -subunit (11.9 kD). The two -subunits are glycosylated and connected by a disulfide bridge. The LFS has an isoelectric point of 5.2. It catalyzes the formation of a sulfine lachrymator, (Z)-phenylmethanethial S-oxide, only in the presence of P. alliacea alliinase and its natural substrate, S-benzyl-l-cysteine sulfoxide (petiveriin). Depending on its concentration relative to that of P. alliacea alliinase, the LFS sequesters, to varying degrees, the sulfenic acid intermediate formed by alliinase-mediated breakdown of petiveriin. At LFS:alliinase of 5:1, LFS sequesters all of the sulfenic acid formed by alliinase action on petiveriin, and converts it entirely to (Z)-phenylmethanethial S-oxide. However, starting at LFS:alliinase of 5:2, the LFS is unable to sequester all of the sulfenic acid produced by the alliinase, with the result that sulfenic acid that escapes the action of the LFS condenses with loss of water to form S-benzyl phenylmethanethiosulfinate (petivericin). The results show that the LFS and alliinase function in tandem, with the alliinase furnishing the sulfenic acid substrate on which the LFS acts. The results also show that the LFS modulates the formation of biologically active thiosulfinates that are downstream of the alliinase in a manner dependent upon the relative concentrations of the LFS and the alliinase. These observations suggest that manipulation of LFS-to-alliinase ratios in plants displaying this system may provide a means by which to rationally modify organosulfur small molecule profiles to obtain desired flavor and/or odor signatures, or increase the presence of desirable biologically active small molecules.

Studies of a Novel Cysteine Sulfoxide Lyase from Petiveria alliacea: The First Heteromeric Alliinase

Musah, R. A., He, Q., Kubec, R., Jadhav, A. — Tue, 03 Nov 2009 12:22:27 PST

A novel alliinase (EC 4.4.1.4) was detected and purified from the roots of the Amazonian medicinal plant Petiveria alliacea. The isolated enzyme is a heteropentameric glycoprotein composed of two -subunits (68.1 kD each), one β-subunit (56.0 kD), one -subunit (24.8 kD), and one -subunit (13.9 kD). The two -subunits are connected by a disulfide bridge, and both - and β-subunits are glycosylated. The enzyme has an isoelectric point of 4.78 and pH and temperature optima of 8.0 and approximately 52°C, respectively. Its activation energy with its natural substrate S-benzyl-l-cysteine sulfoxide is 64.6 kJ mol^–1. Kinetic studies showed that both K_m and V_max vary as a function of substrate structure, with the most preferred substrates being the naturally occurring P. alliacea compounds S-benzyl-l-cysteine sulfoxide and S-2-hydroxyethyl-l-cysteine sulfoxide. The alliinase reacts with these substrates to produce S-benzyl phenylmethanethiosulfinate and S-(2-hydroxyethyl) 2-hydroxyethanethiosulfinate, respectively.

Identification of an Arabidopsis Feruloyl-Coenzyme A Transferase Required for Suberin Synthesis

Molina, I., Li-Beisson, Y., Beisson, F., Ohlrogge, J. B., Pollard, M. — Tue, 03 Nov 2009 12:22:27 PST

All plants produce suberin, a lipophilic barrier of the cell wall that controls water and solute fluxes and restricts pathogen infection. It is often described as a heteropolymer comprised of polyaliphatic and polyaromatic domains. Major monomers include -hydroxy and ,-dicarboxylic fatty acids, glycerol, and ferulate. No genes have yet been identified for the aromatic suberin pathway. Here we demonstrate that Arabidopsis (Arabidopsis thaliana) gene AT5G41040, a member of the BAHD family of acyltransferases, is essential for incorporation of ferulate into suberin. In Arabidopsis plants transformed with the AT5G41040 promoter:YFP fusion, reporter expression is localized to cell layers undergoing suberization. Knockout mutants of AT5G41040 show almost complete elimination of suberin-associated ester-linked ferulate. However, the classic lamellar structure of suberin in root periderm of at5g41040 is not disrupted. The reduction in ferulate in at5g41040-knockout seeds is associated with an approximate stoichiometric decrease in aliphatic monomers containing -hydroxyl groups. Recombinant AT5G41040p catalyzed acyl transfer from feruloyl-coenzyme A to -hydroxyfatty acids and fatty alcohols, demonstrating that the gene encodes a feruloyl transferase. CYP86B1, a cytochrome P450 monooxygenase gene whose transcript levels correlate with AT5G41040 expression, was also investigated. Knockouts and overexpression confirmed CYP86B1 as an oxidase required for the biosynthesis of very-long-chain saturated ,-bifunctional aliphatic monomers in suberin. The seed suberin composition of cyp86b1 knockout was surprisingly dominated by unsubstituted fatty acids that are incapable of polymeric linkages. Together, these results challenge our current view of suberin structure by questioning both the function of ester-linked ferulate as an essential component and the existence of an extended aliphatic polyester.

Identification of the Endodermal Vacuole as the Iron Storage Compartment in the Arabidopsis Embryo

Roschzttardtz, H., Conejero, G., Curie, C., Mari, S. — Tue, 03 Nov 2009 12:22:27 PST

Deciphering how cellular iron (Fe) pools are formed, where they are localized, and which ones are remobilized represents an important challenge to better understand Fe homeostasis. The recent development of imaging techniques, adapted to plants, has helped gain insight into these events. We have analyzed the localization of Fe during embryo development in Arabidopsis (Arabidopsis thaliana) with an improved histochemical staining based on Perls coloration intensified by a second reaction with diaminobenzidine and hydrogen peroxide. The procedure, quick to set up and specific for Fe, was applied directly on histological sections, which dramatically increased its subcellular resolution. We have thus unambiguously shown that in dry seeds Fe is primarily stored in the endodermis cell layer, within the vacuoles, from which it is remobilized during germination. In the vit1-1 mutant, in which the Fe pattern is disturbed, Fe is stored in vacuoles of cortex cells of the hypocotyl/radicle axis and in a single subepidermal cell layer in the cotyledons. During the early stages of embryo development, Fe is evenly distributed in the cells of both wild-type and vit1-1 mutants. Fe eventually accumulates in endodermal cells as the vascular system develops, a process that is impaired in vit1-1. Our results have uncovered a new role for the endodermis in Fe storage in the embryo and have established that the Perls/diaminobenzidine staining is a method of choice to detect Fe in plant tissues and cells.

Coordination of Plastid Protein Import and Nuclear Gene Expression by Plastid-to-Nucleus Retrograde Signaling

Kakizaki, T., Matsumura, H., Nakayama, K., Che, F.-S., Terauchi, R., Inaba, T. — Tue, 03 Nov 2009 12:22:27 PST

Expression of nuclear-encoded plastid proteins and import of those proteins into plastids are indispensable for plastid biogenesis. One possible cellular mechanism that coordinates these two essential processes is retrograde signaling from plastids to the nucleus. However, the molecular details of how this signaling occurs remain elusive. Using the plastid protein import2 mutant of Arabidopsis (Arabidopsis thaliana), which lacks the atToc159 protein import receptor, we demonstrate that the expression of photosynthesis-related nuclear genes is tightly coordinated with their import into plastids. Down-regulation of photosynthesis-related nuclear genes is also observed in mutants lacking other components of the plastid protein import apparatus. Genetic studies indicate that the coordination of plastid protein import and nuclear gene expression is independent of proposed plastid signaling pathways such as the accumulation of Mg-protoporphyrin IX and the activity of ABA INSENSITIVE4 (ABI4). Instead, it may involve GUN1 and the transcription factor AtGLK. The expression level of AtGLK1 is tightly correlated with the expression of photosynthesis-related nuclear genes in mutants defective in plastid protein import. Furthermore, the activity of GUN1 appears to down-regulate the expression of AtGLK1 when plastids are dysfunctional. Based on these data, we suggest that defects in plastid protein import generate a signal that represses photosynthesis-related nuclear genes through repression of AtGLK1 expression but not through activation of ABI4.

Arabidopsis LON2 Is Necessary for Peroxisomal Function and Sustained Matrix Protein Import

Lingard, M. J., Bartel, B. — Tue, 03 Nov 2009 12:22:27 PST

Relatively little is known about the small subset of peroxisomal proteins with predicted protease activity. Here, we report that the peroxisomal LON2 (At5g47040) protease facilitates matrix protein import into Arabidopsis (Arabidopsis thaliana) peroxisomes. We identified T-DNA insertion alleles disrupted in five of the nine confirmed or predicted peroxisomal proteases and found only two—lon2 and deg15, a mutant defective in the previously described PTS2-processing protease (DEG15/At1g28320)—with phenotypes suggestive of peroxisome metabolism defects. Both lon2 and deg15 mutants were mildly resistant to the inhibitory effects of indole-3-butyric acid (IBA) on root elongation, but only lon2 mutants were resistant to the stimulatory effects of IBA on lateral root production or displayed Suc dependence during seedling growth. lon2 mutants displayed defects in removing the type 2 peroxisome targeting signal (PTS2) from peroxisomal malate dehydrogenase and reduced accumulation of 3-ketoacyl-CoA thiolase, another PTS2-containing protein; both defects were not apparent upon germination but appeared in 5- to 8-d-old seedlings. In lon2 cotyledon cells, matrix proteins were localized to peroxisomes in 4-d-old seedlings but mislocalized to the cytosol in 8-d-old seedlings. Moreover, a PTS2-GFP reporter sorted to peroxisomes in lon2 root tip cells but was largely cytosolic in more mature root cells. Our results indicate that LON2 is needed for sustained matrix protein import into peroxisomes. The delayed onset of matrix protein sorting defects may account for the relatively weak Suc dependence following germination, moderate IBA-resistant primary root elongation, and severe defects in IBA-induced lateral root formation observed in lon2 mutants.

Miniature1-Encoded Cell Wall Invertase Is Essential for Assembly and Function of Wall-in-Growth in the Maize Endosperm Transfer Cell

Kang, B.-H., Xiong, Y., Williams, D. S., Pozueta-Romero, D., Chourey, P. S. — Tue, 03 Nov 2009 12:22:27 PST

The miniature1 (mn1) seed phenotype in maize (Zea mays) is due to a loss-of-function mutation at the Mn1 locus that encodes a cell wall invertase (INCW2) that localizes exclusively to the basal endosperm transfer cells (BETCs) of developing seeds. A common feature of all transfer cells is the labyrinth-like wall-in-growth (WIG) that increases the plasma membrane area, thereby enhancing transport capacity in these cells. To better understand WIG formation and roles of INCW2 in the BETC development, we examined wild-type and mn1 mutant developing kernels by cryofixation and electron microscopy. In Mn1 seeds, WIGs developed uniformly in the BETC layer during 7 to 17 d after pollination, and the secretory/endocytic organelles proliferated in the BETCs. Mitochondria accumulated in the vicinity of WIGs, suggesting a functional link between them. In the mn1 BETCs, WIGs were stunted and their endoplasmic reticulum was swollen; Golgi density in the mutant BETCs was 51% of the Mn1 Golgi density. However, the polarized distribution of mitochondria was not affected. INCW2-specific immunogold particles were detected in WIGs, the endoplasmic reticulum, Golgi stacks, and the trans-Golgi network in the Mn1 BETCs, while immunogold particles were extremely rare in the mutant BETCs. Levels of WIG development in the empty pericarp4 mutant was heterogeneous among BETCs, and INCW2 immunogold particles were approximately four times more abundant in the larger WIGs than in the stunted WIGs. These results indicate that polarized secretion is activated during WIG formation and that INCW2 is required for normal development of WIGs to which INCW2 is localized.

LBD18/ASL20 Regulates Lateral Root Formation in Combination with LBD16/ASL18 Downstream of ARF7 and ARF19 in Arabidopsis

Lee, H. W., Kim, N. Y., Lee, D. J., Kim, J. — Tue, 03 Nov 2009 12:22:27 PST

The LATERAL ORGAN BOUNDARIES DOMAIN/ASYMMETRIC LEAVES2-LIKE (LBD/ASL) genes encode proteins harboring a conserved amino acid domain, referred to as the LOB (for lateral organ boundaries) domain. While recent studies have revealed developmental functions of some LBD genes in Arabidopsis (Arabidopsis thaliana) and in crop plants, the biological functions of many other LBD genes remain to be determined. In this study, we have demonstrated that the lbd18 mutant evidenced a reduced number of lateral roots and that lbd16 lbd18 double mutants exhibited a dramatic reduction in the number of lateral roots compared with lbd16 or lbd18. Consistent with this observation, significant β-glucuronidase (GUS) expression in Pro_LBD18:GUS seedlings was detected in lateral root primordia as well as in the emerged lateral roots. Whereas the numbers of primordia of lbd16, lbd18, and lbd16 lbd18 mutants were similar to those observed in the wild type, the numbers of emerged lateral roots of lbd16 and lbd18 single mutants were reduced significantly. lbd16 lbd18 double mutants exhibited additively reduced numbers of emerged lateral roots compared with single mutants. This finding indicates that LBD16 and LBD18 may function in the initiation and emergence of lateral root formation via a different pathway. LBD18 was shown to be localized into the nucleus. We determined whether LBD18 functions in the nucleus using a steroid regulator-inducible system in which the nuclear translocation of LBD18 can be regulated by dexamethasone in the wild-type, lbd18, and lbd16 lbd18 backgrounds. Whereas LBD18 overexpression in the wild-type background induced lateral root formation to some degree, other lines manifested the growth-inhibition phenotype. However, LBD18 overexpression rescued lateral root formation in lbd18 and lbd16 lbd18 mutants without inducing any other phenotypes. Furthermore, we demonstrated that LBD18 overexpression can stimulate lateral root formation in auxin response factor7/19 (arf7 arf19) mutants with blocked lateral root formation. Taken together, our results suggest that LBD18 functions in the initiation and emergence of lateral roots, in conjunction with LBD16, downstream of ARF7 and ARF19.

Expressing the Diphtheria Toxin A Subunit from the HAP2(GCS1) Promoter Blocks Sperm Maturation and Produces Single Sperm-Like Cells Capable of Fertilization

Frank, A. C., Johnson, M. A. — Tue, 03 Nov 2009 12:22:27 PST

After meiosis, the male germline of flowering plants undergoes two mitoses, producing two sperm that are carried within a pollen tube to an ovule. One sperm fuses with the egg to form the zygote and the other fuses with the central cell to form the primary endosperm. The mechanisms that control male germline development and gene expression, and ensure that sperm properly fuse with female gametes are just beginning to be understood. Expression of the potent translation inhibitor, diphtheria toxin A subunit, from the Arabidopsis (Arabidopsis thaliana) HAP2(GCS1) promoter blocked sperm development before the final cell division, resulting in pollen tubes that carried a single sperm-like cell rather than two sperm. These pollen tubes targeted ovules and fertilized either the egg or the central cell, producing seeds with either endosperm or an embryo, but not both. Endosperm-only seeds significantly outnumbered embryo-only seeds, suggesting that single sperm-like cells preferentially fuse with the central cell. These experiments show that de novo translation is required for completion of sperm development, that the HAP2(GCS1) promoter is very tightly controlled, and that disruption of gene expression can result in male germ cells with a bias for gamete fusion.

The SPOROCYTELESS/NOZZLE Gene Is Involved in Controlling Stamen Identity in Arabidopsis

Tue, 03 Nov 2009 12:22:27 PST

The stamen, which consists of an anther and a filament, is the male reproductive organ in a flower. The specification of stamen identity in Arabidopsis (Arabidopsis thaliana) is controlled by a combination of the B genes APETALA3 (AP3) and PISTILLATA, the C gene AGAMOUS (AG), and the E genes SEPALLATA1 (SEP1) to SEP4. The "floral organ-building" gene SPOROCYTELESS/NOZZLE (SPL/NZZ) plays a central role in regulating anther cell differentiation. However, much less is known about how "floral organ identity" and floral organ-building genes interact to control floral organ development. In this study, we report that ectopic expression of SPL/NZZ not only affects flower development in the wild-type background but also leads to the transformation of petal-like organs into stamen-like organs in flowers of ap2-1, a weak ap2 mutant allele. Moreover, our loss-of-function analysis indicates that the spl/nzz mutant enhances the phenotype of the ag weak allele ag-4. Furthermore, ectopic expression and overexpression of SPL/NZZ altered expression of AG, SEP3, and AP2 in rosette leaves and flowers, while ectopic expression of SPL/NZZ resulted in ectopic expression of AG and SEP3 in the outer whorls of flowers. Our results indicate that the SPL/NZZ gene is engaged in controlling stamen identity via interacting with genes required for stamen identity in Arabidopsis.

A Leaky Mutation in DWARF4 Reveals an Antagonistic Role of Brassinosteroid in the Inhibition of Root Growth by Jasmonate in Arabidopsis

Tue, 03 Nov 2009 12:22:27 PST

The F-box protein CORONATINE INSENSITIVE1 (COI1) plays a central role in jasmonate (JA) signaling and is required for all JA responses in Arabidopsis (Arabidopsis thaliana). To dissect JA signal transduction, we isolated the partially suppressing coi1 (psc1) mutant, which partially suppressed coi1 insensitivity to JA inhibition of root growth. The psc1 mutant partially restored JA sensitivity in coi1-2 background and displayed JA hypersensitivity in wild-type COI1 background. Genetic mapping, sequence analysis, and complementation tests revealed that psc1 is a leaky mutation of DWARF4 (DWF4) that encodes a key enzyme in brassinosteroid (BR) biosynthesis. Physiological analysis showed that an application of exogenous BR eliminated the partial restoration of JA sensitivity by psc1 in coi1-2 background and the JA hypersensitivity of psc1 in wild-type COI1 background. Exogenous BR also attenuated JA inhibition of root growth in the wild type. In addition, the expression of DWF4 was inhibited by JA, and this inhibition was dependent on COI1. These results indicate that (1) BR is involved in JA signaling and negatively regulates JA inhibition of root growth, and (2) the DWF4 is down-regulated by JA and is located downstream of COI1 in the JA-signaling pathway.

Functional Analysis of {alpha}-DOX2, an Active {alpha}-Dioxygenase Critical for Normal Development in Tomato Plants

Tue, 03 Nov 2009 12:22:27 PST

Plant -dioxygenases initiate the synthesis of oxylipins by catalyzing the incorporation of molecular oxygen at the -methylene carbon atom of fatty acids. Previously, -DOX1 has been shown to display -dioxygenase activity and to be implicated in plant defense. In this study, we investigated the function of a second -dioxygenase isoform, -DOX2, in tomato (Solanum lycopersicum) and Arabidopsis (Arabidopsis thaliana). Recombinant Sl-DOX2 and At-DOX2 proteins catalyzed the conversion of a wide range of fatty acids into 2(R)-hydroperoxy derivatives. Expression of Sl-DOX2 and At-DOX2 was found in seedlings and increased during senescence induced by detachment of leaves. In contrast, microbial infection, earlier known to increase the expression of -DOX1, did not alter the expression of Sl-DOX2 or At-DOX2. The tomato mutant divaricata, characterized by early dwarfing and anthocyanin accumulation, carries a mutation at the Sl-DOX2 locus and was chosen for functional studies of -DOX2. Transcriptional changes in such mutants showed the up-regulation of genes playing roles in lipid and phenylpropanoid metabolism, the latter being in consonance with the anthocyanin accumulation. Transgenic expression of At-DOX2 and Sl-DOX2 in divaricata partially complemented the compromised phenotype in mature plants and fully complemented it in seedlings, thus indicating the functional exchangeability between -DOX2 from tomato and Arabidopsis. However, deletion of At-DOX2 in Arabidopsis plants did not provoke any visible phenotypic alteration indicating that the relative importance of -DOX2 in plant physiology is species specific.

A Nuclear Factor Regulates Abscisic Acid Responses in Arabidopsis

Kim, M. J., Shin, R., Schachtman, D. P. — Tue, 03 Nov 2009 12:22:27 PST

Abscisic acid (ABA) is a plant hormone that regulates plant growth as well as stress responses. In this study, we identified and characterized a new Arabidopsis (Arabidopsis thaliana) protein, Nuclear Protein X1 (NPX1), which was up-regulated by stress and treatment with exogenous ABA. Stomatal closure, seed germination, and primary root growth are well-known ABA responses that were less sensitive to ABA in NPX1-overexpressing plants. NPX1-overexpressing plants were more drought sensitive, and the changes in response to drought were due to the altered guard cell sensitivity to ABA in transgenic plants and not to a lack of ABA production. The nuclear localization of NPX1 correlated with changes in the expression of genes involved in ABA biosynthesis and ABA signal transduction. To understand the function of NPX1, we searched for interacting proteins and found that an ABA-inducible NAC transcription factor, TIP, interacted with NPX1. Based on the whole plant phenotypes, we hypothesized that NPX1 acts as a transcriptional repressor, and this was demonstrated in yeast, where we showed that TIP was repressed by NPX1. Our results indicate that the previously unknown protein NPX1 acts as a negative regulator in plant response to changes in environmental conditions through the control of ABA-regulated gene expression. The characterization of this factor enhances our understanding of guard cell function and the mechanisms that plants use to modulate water loss from leaves under drought conditions.

Hormone- and Light-Mediated Regulation of Heat-Induced Differential Petiole Growth in Arabidopsis

van Zanten, M., Voesenek, L. A.C.J., Peeters, A. J.M., Millenaar, F. F. — Tue, 03 Nov 2009 12:22:27 PST

Plants react quickly and profoundly to changes in their environment. A sudden increase in temperature, for example, induces differential petiole growth-driven upward leaf movement (hyponastic growth) in Arabidopsis (Arabidopsis thaliana). We show that accessions that face the strongest fluctuations in diurnal temperature in their natural habitat are least sensitive for heat-induced hyponastic growth. This indicates that heat-induced hyponastic growth is a trait subject to natural selection. The response is induced with kinetics remarkably similar to ethylene- and low light-induced hyponasty in several accessions. Using pharmacological assays, transcript analysis, and mutant analyses, we demonstrate that ethylene and the photoreceptor protein phytochrome B are negative regulators of heat-induced hyponastic growth and that low light, phytochrome A, auxin, polar auxin transport, and abscisic acid are positive regulators of heat-induced hyponastic growth. Furthermore, auxin, auxin polar transport, phytochrome A, phytochrome B, and cryptochromes are required for a fast induction of heat-induced hyponastic growth.

Dual Roles of Reactive Oxygen Species and NADPH Oxidase RBOHD in an Arabidopsis-Alternaria Pathosystem

Tue, 03 Nov 2009 12:22:27 PST

Arabidopsis (Arabidopsis thaliana) NADPH oxidases have been reported to suppress the spread of pathogen- and salicylic acid-induced cell death. Here, we present dual roles of RBOHD (for respiratory burst oxidase homolog D) in an Arabidopsis-Alternaria pathosystem, suggesting either initiation or prevention of cell death dependent on the distance from pathogen attack. Our data demonstrate that a rbohD knockout mutant exhibits increased spread of cell death at the macroscopic level upon inoculation with the fungus Alternaria brassicicola. However, the cellular patterns of reactive oxygen species accumulation and cell death are fundamentally different in the AtrbohD mutant compared with the wild type. Functional RBOHD causes marked extracellular hydrogen peroxide accumulation as well as cell death in distinct, single cells of A. brassicicola-infected wild-type plants. This single cell response is missing in the AtrbohD mutant, where infection triggers spreading-type necrosis preceded by less distinct chloroplastic hydrogen peroxide accumulation in large clusters of cells. While the salicylic acid analog benzothiadiazole induces the action of RBOHD and the development of cell death in infected tissues, the ethylene inhibitor aminoethoxyvinylglycine inhibits cell death, indicating that both salicylic acid and ethylene positively regulate RBOHD and cell death. Moreover, A. brassicicola-infected AtrbohD plants hyperaccumulate ethylene and free salicylic acid compared with the wild type, suggesting negative feedback regulation of salicylic acid and ethylene by RBOHD. We propose that functional RBOHD triggers death in cells that are damaged by fungal infection but simultaneously inhibits death in neighboring cells through the suppression of free salicylic acid and ethylene levels.

SET DOMAIN GROUP25 Encodes a Histone Methyltransferase and Is Involved in FLOWERING LOCUS C Activation and Repression of Flowering

Berr, A., Xu, L., Gao, J., Cognat, V., Steinmetz, A., Dong, A., Shen, W.-H. — Tue, 03 Nov 2009 12:22:27 PST

Covalent modifications of histone lysine residues by methylation play key roles in the regulation of chromatin structure and function. In contrast to H3K9 and H3K27 methylations that mark repressive states of transcription and are absent in some lower eukaryotes, H3K4 and H3K36 methylations are considered as active marks of transcription and are highly conserved in all eukaryotes from yeast (Saccharomyces cerevisiae) to Homo sapiens. Paradoxically, protein complexes catalyzing H3K4 and H3K36 methylations are less-extensively characterized in higher eukaryotes, particularly in plants. Arabidopsis (Arabidopsis thaliana) contains 12 SET DOMAIN GROUP (SDG) proteins phylogenetic classified to Trithorax Group (TrxG) and thus potentially involved in H3K4 and H3K36 methylations. So far only some genes of this family had been functionally characterized. Here we report on the genetic and molecular characterization of SDG25, a previously uncharacterized member of the Arabidopsis TrxG family. We show that the loss-of-function mutant sdg25-1 has an early flowering phenotype associated with suppression of FLOWERING LOCUS C (FLC) expression. Recombinant SDG25 proteins could methylate histone H3 from oligonucleosomes and mutant sdg25-1 plants showed weakly reduced levels of H3K36 dimethylation at FLC chromatin. Interestingly, sdg25-1 transcriptome shared a highly significant number of differentially expressed genes with that of sdg26-1, a previously characterized mutant exhibiting late-flowering phenotype and elevated FLC expression. Taken together, our results provide, to our knowledge, the first demonstration for a biological function of SDG25 and reveal additional layers of complexity of overlap and nonoverlap functions of the TrxG family genes in Arabidopsis.

SLOW WALKER2, a NOC1/MAK21 Homologue, Is Essential for Coordinated Cell Cycle Progression during Female Gametophyte Development in Arabidopsis

Tue, 03 Nov 2009 12:22:27 PST

Morphogenesis requires the coordination of cell growth, division, and cell differentiation. Female gametogenesis in flowering plants, where a single haploid spore undergoes continuous growth and nuclear division without cytokinesis to form an eight-nucleate coenocytic embryo sac before cellularization, provides a good system to study the genetic control of such processes in multicellular organisms. Here, we report the characterization of an Arabidopsis (Arabidopsis thaliana) female gametophyte mutant, slow walker2 (swa2), in which the progression of the mitotic cycles and the synchrony of female gametophyte development were impaired, causing an arrest of female gametophytes at the two-, four-, or eight-nucleate stage. Delayed pollination test showed that a portion of the mutant ovules were able to develop into functional embryo sacs and could be fertilized. SWA2 encodes a nucleolar protein homologous to yeast NUCLEOLAR COMPLEX ASSOCIATED PROTEIN1 (NOC1)/MAINTENANCE OF KILLER21 that, together with NOC2, is involved in preribosome export from the nucleus to the cytoplasm. Similarly, SWA2 can physically interact with a putative Arabidopsis NOC2 homologue. SWA2 is expressed ubiquitously throughout the plant, at high levels in actively dividing tissues and gametophytes. Therefore, we conclude that SWA2 most likely plays a role in ribosome biogenesis that is essential for the coordinated mitotic progression of the female gametophyte.

Plant SMU-1 and SMU-2 Homologues Regulate Pre-mRNA Splicing and Multiple Aspects of Development

Chung, T., Wang, D., Kim, C.-S., Yadegari, R., Larkins, B. A. — Tue, 03 Nov 2009 12:22:27 PST

In eukaryotes, alternative splicing of pre-mRNAs contributes significantly to the proper expression of the genome. However, the functions of many auxiliary spliceosomal proteins are still unknown. Here, we functionally characterized plant homologues of nematode suppressors of mec-8 and unc-52 (smu). We compared transcript profiles of maize (Zea mays) smu2 endosperm with those of wild-type plants and identified pre-mRNA splicing events that depend on the maize SMU2 protein. Consistent with a conserved role of plant SMU-2 homologues, Arabidopsis (Arabidopsis thaliana) smu2 mutants also show altered splicing of similar target pre-mRNAs. The Atsmu2 mutants occasionally show developmental phenotypes, including abnormal cotyledon numbers and higher seed weights. We identified AtSMU1 as one of the SMU2-interacting proteins, and Atsmu1 mutations cause similar developmental phenotypes with higher penetrance than Atsmu2. The AtSMU2 and AtSMU1 proteins are localized to the nucleus and highly prevalent in actively dividing tissues. Taken together, our data indicated that the plant SMU-1 and SMU-2 homologues appear to be involved in splicing of specific pre-mRNAs that affect multiple aspects of development.

The Grapevine R2R3-MYB Transcription Factor VvMYBF1 Regulates Flavonol Synthesis in Developing Grape Berries

Tue, 03 Nov 2009 12:22:27 PST

Flavonols are important ultraviolet light protectants in many plants and contribute substantially to the quality and health-promoting effects of fruits and derived plant products. To study the regulation of flavonol synthesis in fruit, we isolated and characterized the grapevine (Vitis vinifera ‘Shiraz’) R2R3-MYB transcription factor VvMYBF1. Transient reporter assays established VvMYBF1 to be a specific activator of flavonol synthase1 (VvFLS1) and several other promoters of grapevine and Arabidopsis (Arabidopsis thaliana) genes involved in flavonol synthesis. Expression of VvMYBF1 in the Arabidopsis mutant myb12 resulted in complementation of its flavonol-deficient phenotype and confirmed the function of VvMYBF1 as a transcriptional regulator of flavonol synthesis. Transcript analysis of VvMYBF1 throughout grape berry development revealed its expression during flowering and in skins of ripening berries, which correlates with the accumulation of flavonols and expression of VvFLS1. In addition to its developmental regulation, VvMYBF1 expression was light inducible, implicating VvMYBF1 in the control of VvFLS1 transcription. Sequence analysis of VvMYBF1 and VvFLS1 indicated conserved putative light regulatory units in promoters of both genes from different cultivars. By analysis of the VvMYBF1 amino acid sequence, we identified the previously described SG7 domain and an additional sequence motif conserved in several plant MYB factors. The described motifs have been used to identify MYB transcription factors from other plant species putatively involved in the regulation of flavonol biosynthesis. To our knowledge, this is the first functional characterization of a light-inducible MYB transcription factor controlling flavonol synthesis in fruit.

Characterization of the Entire Cystatin Gene Family in Barley and Their Target Cathepsin L-Like Cysteine-Proteases, Partners in the Hordein Mobilization during Seed Germination

Martinez, M., Cambra, I., Carrillo, L., Diaz-Mendoza, M., Diaz, I. — Tue, 03 Nov 2009 12:22:27 PST

Plant cystatins are inhibitors of cysteine-proteases of the papain C1A and legumain C13 families. Cystatin data from multiple plant species have suggested that these inhibitors act as defense proteins against pests and pathogens and as regulators of protein turnover. In this study, we characterize the entire cystatin gene family from barley (Hordeum vulgare), which contain 13 nonredundant genes, and identify and characterize their target enzymes, the barley cathepsin L-like proteases. Cystatins and proteases were expressed and purified from Escherichia coli cultures. Each cystatin was found to have different inhibitory capability against barley cysteine-proteases in in vitro inhibitory assays using specific substrates. Real-time reverse transcription-polymerase chain reaction revealed that inhibitors and enzymes present a wide variation in their messenger RNA expression patterns. Their transcripts were mainly detected in developing and germinating seeds, and some of them were also expressed in leaves and roots. Subcellular localization of cystatins and cathepsin L-like proteases fused to green fluorescent protein demonstrated the presence of both protein families throughout the endoplasmic reticulum and the Golgi complex. Proteases and cystatins not only colocalized but also interacted in vivo in the plant cell, as revealed by bimolecular fluorescence complementation. The functional relationship between cystatins and cathepsin L-like proteases was inferred from their common implication as counterparts of mobilization of storage proteins upon barley seed germination. The opposite pattern of transcription expression in gibberellin-treated aleurones presented by inhibitors and enzymes allowed proteases to specifically degrade B, C, and D hordeins stored in the endosperm of barley seeds.

Unique Features of Plant Cleavage and Polyadenylation Specificity Factor Revealed by Proteomic Studies

Zhao, H., Xing, D., Li, Q. Q. — Tue, 03 Nov 2009 12:22:27 PST

Cleavage and polyadenylation of precursor mRNA is an essential process for mRNA maturation. Among the 15 to 20 protein factors required for this process, a subgroup of proteins is needed for both cleavage and polyadenylation in plants and animals. This subgroup of proteins is known as the cleavage and polyadenylation specificity factor (CPSF). To explore the in vivo structural features of plant CPSF, we used tandem affinity purification methods to isolate the interacting protein complexes for each component of the CPSF subunits using Arabidopsis (Arabidopsis thaliana ecotype Landsberg erecta) suspension culture cells. The proteins in these complexes were identified by mass spectrometry and western immunoblots. By compiling the in vivo interaction data from tandem affinity purification tagging as well as other available yeast two-hybrid data, we propose an in vivo plant CPSF model in which the Arabidopsis CPSF possesses AtCPSF30, AtCPSF73-I, AtCPSF73-II, AtCPSF100, AtCPSF160, AtFY, and AtFIPS5. Among them, AtCPSF100 serves as a core with which all other factors, except AtFIPS5, are associated. These results show that plant CPSF possesses distinct features, such as AtCPSF73-II and AtFY, while sharing other ortholog components with its yeast and mammalian counterparts. Interestingly, these two unique plant CPSF components have been associated with embryo development and flowering time controls, both of which involve plant-specific biological processes.

Stable Transcription Activities Dependent on an Orientation of Tam3 Transposon Insertions into Antirrhinum and Yeast Promoters Occur Only within Chromatin

Tue, 03 Nov 2009 12:22:27 PST

Transposon insertions occasionally occur in the promoter regions of plant genes, many of which are still capable of being transcribed. However, it remains unclear how transcription of such promoters is able to occur. Insertion of the Tam3 transposon into various genes of Antirrhinum majus can confer leaky phenotypes without its excision. These genes, named Tam3-permissible alleles, often contain Tam3 in their promoter regions. Two alleles at different anthocyanin biosynthesis loci, nivea^{recurrens::Tam3} (niv^rec) and pallida^{recurrens::Tam3} (pal^rec), both contain Tam3 at a similar position immediately upstream of the promoter TATA-box; however, these insertions had different phenotypic consequences. Under conditions where the inserted Tam3 is immobilized, the niv^rec line produces pale red petals, whereas the pal^rec line produces no pigment. These pigmentation patterns are correlated with the level of transcripts from the niv^rec or pal^rec alleles, and these transcriptional activities are independent of DNA methylation in their promoter regions. In niv^rec, Tam3 is inserted in an orientation that results in the 3' end of Tam3 adjacent to the 5' region of the gene coding sequence. In contrast, the pal^rec allele contains a Tam3 insertion in the opposite orientation. Four of five different nonrelated genes that are also Tam3-permissible alleles and contain Tam3 within the promoter region share the same Tam3 orientation as niv^rec. The different transcriptional activities dependent on Tam3 orientation in the Antirrhinum promoters were consistent with expression of luciferase reporter constructs introduced into yeast chromosomes but not with transient expression of these constructs in Antirrhinum cells. These results suggest that for Tam3 to sustain stable transcriptional activity in various promoters it must be embedded in chromatin.

A Genome-Scale Metabolic Model of Arabidopsis and Some of Its Properties

Poolman, M. G., Miguet, L., Sweetlove, L. J., Fell, D. A. — Tue, 03 Nov 2009 12:22:27 PST

We describe the construction and analysis of a genome-scale metabolic model of Arabidopsis (Arabidopsis thaliana) primarily derived from the annotations in the Aracyc database. We used techniques based on linear programming to demonstrate the following: (1) that the model is capable of producing biomass components (amino acids, nucleotides, lipid, starch, and cellulose) in the proportions observed experimentally in a heterotrophic suspension culture; (2) that approximately only 15% of the available reactions are needed for this purpose and that the size of this network is comparable to estimates of minimal network size for other organisms; (3) that reactions may be grouped according to the changes in flux resulting from a hypothetical stimulus (in this case demand for ATP) and that this allows the identification of potential metabolic modules; and (4) that total ATP demand for growth and maintenance can be inferred and that this is consistent with previous estimates in prokaryotes and yeast.

Starch Synthesis in Arabidopsis Is Achieved by Spatial Cotranscription of Core Starch Metabolism Genes

Tsai, H.-L., Lue, W.-L., Lu, K.-J., Hsieh, M.-H., Wang, S.-M., Chen, J. — Tue, 03 Nov 2009 12:22:27 PST

Starch synthesis and degradation require the participation of many enzymes, occur in both photosynthetic and nonphotosynthetic tissues, and are subject to environmental and developmental regulation. We examine the distribution of starch in vegetative tissues of Arabidopsis (Arabidopsis thaliana) and the expression of genes encoding core enzymes for starch synthesis. Starch is accumulated in plastids of epidermal, mesophyll, vascular, and root cap cells but not in root proper cells. We also identify cells that can synthesize starch heterotrophically in albino mutants. Starch synthesis in leaves is regulated by developmental stage and light. Expression of gene promoter-β-glucuronidase fusion constructs in transgenic seedlings shows that starch synthesis genes are transcriptionally active in cells with starch synthesis and are inactive in root proper cells except the plastidial phosphoglucose isomerase. In addition, ADG2 (for ADPG PYROPHOSPHORYLASE2) is not required for starch synthesis in root cap cells. Expression profile analysis reveals that starch metabolism genes can be clustered into two sets based on their tissue-specific expression patterns. Starch distribution and expression pattern of core starch synthesis genes are common in Arabidopsis and rice (Oryza sativa), suggesting that the regulatory mechanism for starch metabolism genes may be conserved evolutionarily. We conclude that starch synthesis in Arabidopsis is achieved by spatial coexpression of core starch metabolism genes regulated by their promoter activities and is fine-tuned by cell-specific endogenous and environmental controls.

A Systems-Level Analysis of the Effects of Light Quality on the Metabolism of a Cyanobacterium

Tue, 03 Nov 2009 12:22:28 PST

Photosynthetic organisms experience changes in light quantity and light quality in their natural habitat. In response to changes in light quality, these organisms redistribute excitation energy and adjust photosystem stoichiometry to maximize the utilization of available light energy. However, the response of other cellular processes to changes in light quality is mostly unknown. Here, we report a systematic investigation into the adaptation of cellular processes in Synechocystis species PCC 6803 to light that preferentially excites either photosystem II or photosystem I. We find that preferential excitation of photosystem II and photosystem I induces massive reprogramming of the Synechocystis transcriptome. The rewiring of cellular processes begins as soon as Synechocystis senses the imbalance in the excitation of reaction centers. We find that Synechocystis utilizes the cyclic photosynthetic electron transport chain for ATP generation and a major part of the respiratory pathway to generate reducing equivalents and carbon skeletons during preferential excitation of photosystem I. In contrast, cytochrome c oxidase and photosystem I act as terminal components of the photosynthetic electron transport chain to produce sufficient ATP and limited amounts of NADPH and reduced ferredoxin during preferential excitation of photosystem II. To overcome the shortage of NADPH and reduced ferredoxin, Synechocystis preferentially activates transporters and acquisition pathways to assimilate ammonia, urea, and arginine over nitrate as a nitrogen source. This study provides a systematic analysis of cellular processes in cyanobacteria in response to preferential excitation and shows that the cyanobacterial cell undergoes significant adjustment of cellular processes, many of which were previously unknown.

The Cyclization of the 3,6-Anhydro-Galactose Ring of {iota}-Carrageenan Is Catalyzed by Two D-Galactose-2,6-Sulfurylases in the Red Alga Chondrus crispus

Tue, 03 Nov 2009 12:22:28 PST

Carrageenans are sulfated galactans found in the cell walls of numerous red seaweeds (Rhodophyta). They are classified according to the number and the position of sulfate ester groups and the occurrence of 3,6-anhydro-galactose. Although the carrageenan biosynthesis pathway is not fully understood, it is usually accepted that the last step consists of the formation of a 3,6-anhydro ring found in - and -carrageenans through the enzymatic conversion of d-galactose-6-sulfate or d-galactose-2,6-disulfate occurring in µ- and -carrageenan, respectively. We purified two enzymes, sulfurylase I (65 kD) and sulfurylase II (32 kD), that are able to catalyze the conversion of - into -carrageenan. We compared their sulfate release rates (i.e. arising from the formation of the anhydro ring) with the viscosity of the solution and demonstrated two distinct modes of action. In addition, we found that some mixtures of sulfurylase I and II lead to the formation of carrageenan solutions with unexpectedly low viscosities. We discuss the implication of these findings for the assembly of a densely aggregated matrix in red algal cell walls.

Analysis of Metabolic Flux Phenotypes for Two Arabidopsis Mutants with Severe Impairment in Seed Storage Lipid Synthesis

Lonien, J., Schwender, J. — Tue, 03 Nov 2009 12:22:28 PST

Major storage reserves of Arabidopsis (Arabidopsis thaliana) seeds are triacylglycerols (seed oils) and proteins. Seed oil content is severely reduced for the regulatory mutant wrinkled1 (wri1-1; At3g54320) and for a double mutant in two isoforms of plastidic pyruvate kinase (pkpβ₁pkp; At5g52920 and At3g22960). Both already biochemically well-characterized mutants were now studied by ¹³C metabolic flux analysis of cultured developing embryos based on comparison with their respective genetic wild-type backgrounds. For both mutations, in seeds as well as in cultured embryos, the oil fraction was strongly reduced while the fractions of proteins and free metabolites increased. Flux analysis in cultured embryos revealed changes in nutrient uptakes and fluxes into biomass as well as an increase in tricarboxylic acid cycle activity for both mutations. While in both wild types plastidic pyruvate kinase (PK_p) provides most of the pyruvate for plastidic fatty acid synthesis, the flux through PK_p is reduced in pkpβ₁pkp by 43% of the wild-type value. In wri1-1, PK_p flux is even more reduced (by 82%), although the genes PKpβ₁ and PKp are still expressed. Along a common paradigm of metabolic control theory, it is hypothesized that a large reduction in PK_p enzyme activity in pkpβ₁pkp has less effect on PK_p flux than multiple smaller reductions in glycolytic enzymes in wri1-1. In addition, only in the wri1-1 mutant is the large reduction in PK_p flux compensated in part by an increased import of cytosolic pyruvate and by plastidic malic enzyme. No such limited compensatory bypass could be observed in pkpβ₁pkp.

Metabolite Sorting of a Germplasm Collection Reveals the Hydroxylase3 Locus as a New Target for Maize Provitamin A Biofortification

Tue, 03 Nov 2009 12:22:28 PST

Vitamin A deficiency, a global health burden, can be alleviated through provitamin A carotenoid biofortification of major crop staples such as maize (Zea mays) and other grasses in the Poaceae. If regulation of carotenoid biosynthesis was better understood, enhancement could be controlled by limiting β-carotene hydroxylation to compounds with lower or no nonprovitamin A activity. Natural maize genetic diversity enabled identification of hydroxylation genes associated with reduced endosperm provitamin A content. A novel approach was used to capture the genetic and biochemical diversity of a large germplasm collection, representing 80% of maize genetic diversity, without having to sample the entire collection. Metabolite data sorting was applied to select a 10-line genetically diverse subset representing biochemical extremes for maize kernel carotenoids. Transcript profiling led to discovery of the Hydroxylase3 locus that coincidently mapped to a carotene quantitative trait locus, thereby prompting investigation of allelic variation in a broader collection. Three natural alleles in 51 maize lines explained 78% of variation and approximately 11-fold difference in β-carotene relative to β-cryptoxanthin and 36% of the variation and 4-fold difference in absolute levels of β-carotene. A simple PCR assay to track and identify Hydroxylase3 alleles will be valuable for predicting nutritional content in genetically diverse cultivars found worldwide.

Phosphate (Pi) Starvation Effect on the Cytosolic Pi Concentration and Pi Exchanges across the Tonoplast in Plant Cells: An in Vivo 31P-Nuclear Magnetic Resonance Study Using Methylphosphonate as a Pi Analog

Pratt, J., Boisson, A.-M., Gout, E., Bligny, R., Douce, R., Aubert, S. — Tue, 03 Nov 2009 12:22:28 PST

In vivo ³¹P-NMR analyses showed that the phosphate (Pi) concentration in the cytosol of sycamore (Acer pseudoplatanus) and Arabidopsis (Arabidopsis thaliana) cells was much lower than the cytoplasmic Pi concentrations usually considered (60–80 µm instead of >1 mm) and that it dropped very rapidly following the onset of Pi starvation. The Pi efflux from the vacuole was insufficient to compensate for the absence of external Pi supply, suggesting that the drop of cytosolic Pi might be the first endogenous signal triggering the Pi starvation rescue metabolism. Successive short sequences of Pi supply and deprivation showed that added Pi transiently accumulated in the cytosol, then in the stroma and matrix of organelles bounded by two membranes (plastids and mitochondria, respectively), and subsequently in the vacuole. The Pi analog methylphosphonate (MeP) was used to analyze Pi exchanges across the tonoplast. MeP incorporated into cells via the Pi carrier of the plasma membrane; it accumulated massively in the cytosol and prevented Pi efflux from the vacuole. This blocking of vacuolar Pi efflux was confirmed by in vitro assays with purified vacuoles. Subsequent incorporation of Pi into the cells triggered a massive transfer of MeP from the cytosol to the vacuole. Mechanisms for Pi exchanges across the tonoplast are discussed in the light of the low cytosolic Pi level, the cell response to Pi starvation, and the Pi/MeP interactive effects.

Biochemical Characterization of AtRECQ3 Reveals Significant Differences Relative to Other RecQ Helicases

Kobbe, D., Blanck, S., Focke, M., Puchta, H. — Tue, 03 Nov 2009 12:22:28 PST

Members of the conserved RecQ helicase family are important for the preservation of genomic stability. Multiple RecQ homologs within one organism raise the question of functional specialization. Whereas five different homologs are present in humans, the model plant Arabidopsis (Arabidopsis thaliana) carries seven RecQ homologs in its genome. We performed biochemical analysis of AtRECQ3, expanded upon a previous analysis of AtRECQ2, and compared their properties. Both proteins differ in their domain composition. Our analysis demonstrates that they are 3' to 5' helicases with similar activities on partial duplex DNA. However, they promote different outcomes with synthetic DNA structures that mimic Holliday junctions or a replication fork. AtRECQ2 catalyzes Holliday junction branch migration and replication fork regression, while AtRECQ3 cannot act on intact Holliday junctions. The observed reaction of AtRECQ3 on the replication fork is in line with unwinding the lagging strand. On nicked Holliday junctions, which have not been intensively studied with RecQ helicases before, AtRECQ3, but not AtRECQ2, shows a clear preference for one unwinding mechanism. In addition, AtRECQ3 is much more efficient at catalyzing DNA strand annealing. Thus, AtRECQ2 and AtRECQ3 are likely to perform different tasks in the cell, and AtRECQ3 differs in its biochemical properties from all other eukaryotic RECQ helicases characterized so far.

Plant {delta}15N Correlates with the Transpiration Efficiency of Nitrogen Acquisition in Tropical Trees

Cernusak, L. A., Winter, K., Turner, B. L. — Tue, 03 Nov 2009 12:22:28 PST

Based upon considerations of a theoretical model of ¹⁵N/¹⁴N fractionation during steady-state nitrate uptake from soil, we hypothesized that, for plants grown in a common soil environment, whole-plant ¹⁵N (_P) should vary as a function of the transpiration efficiency of nitrogen acquisition (F_N/v) and the difference between _P and root ¹⁵N (_P – _R). We tested these hypotheses with measurements of several tropical tree and liana species. Consistent with theoretical expectations, both F_N/v and _P – _R were significant sources of variation in _P, and the relationship between _P and F_N/v differed between non-N₂-fixing and N₂-fixing species. We interpret the correlation between _P and F_N/v as resulting from variation in mineral nitrogen efflux-to-influx ratios across plasma membranes of root cells. These results provide a simple explanation of variation in ¹⁵N of terrestrial plants and have implications for understanding nitrogen cycling in ecosystems.

Vascular Function in Grape Berries across Development and Its Relevance to Apparent Hydraulic Isolation

Choat, B., Gambetta, G. A., Shackel, K. A., Matthews, M. A. — Tue, 03 Nov 2009 12:22:28 PST

During the latter stages of development in fleshy fruit, water flow through the xylem declines markedly and the requirements of transpiration and further expansion are fulfilled primarily by the phloem. We evaluated the hypothesis that cessation of water transport through the xylem results from disruption or occlusion of pedicel and berry xylem conduits (hydraulic isolation). Xylem hydraulic resistance (R_h) was measured in developing fruit of grape (Vitis vinifera ‘Chardonnay’) 20 to 100 d after anthesis (DAA) and compared with observations of xylem anatomy by light and cryo-scanning electron microscopy and expression of six plasma membrane intrinsic protein (PIP) aquaporin genes (VvPIP1;1, VvPIP1;2, VvPIP1;3, VvPIP2;1, VvPIP2;2, VvPIP2;3). There was a significant increase in whole berry R_h and receptacle R_h in the latter stages of ripening (80–100 DAA), which was associated with deposition of gels or solutes in many receptacle xylem conduits. Peaks in the expression of some aquaporin isoforms corresponded to lower whole berry R_h 60 to 80 DAA, and the increase in R_h beginning at 80 DAA correlated with decreases in the expression of the two most predominantly expressed PIP genes. Although significant, the increase in berry R_h was not great enough, and occurred too late in development, to explain the decline in xylem flow that occurs at 60 to 75 DAA. The evidence suggests that the fruit is not hydraulically isolated from the parent plant by xylem occlusion but, rather, is "hydraulically buffered" by water delivered via the phloem.

A Single Amino Acid Change in the Enhancer of Zeste Ortholog CURLY LEAF Results in Vernalization-Independent, Rapid Flowering in Arabidopsis

Doyle, M. R., Amasino, R. M. — Tue, 03 Nov 2009 12:22:28 PST

Many strains of Arabidopsis (Arabidopsis thaliana) require exposure to prolonged cold for rapid flowering, a process known as vernalization. Vernalization in Arabidopsis results in the suppression of FLOWERING LOCUS C (FLC), a repressor of flowering. In a screen for mutants that no longer require vernalization for rapid flowering, we identified a dominant allele of the Enhancer of Zeste E(z) ortholog CURLY LEAF (CLF), clf-59. CLF is a Polycomb Group gene, and the clf-59 mutant protein contains a proline-to-serine transition in a cysteine-rich region that precedes the SET domain. Mutant plants are early flowering and have reduced FLC expression, but, unlike clf loss-of-function mutants, clf-59 mutants do not display additional pleiotropic phenotypes. clf-59 mutants have elevated levels of trimethylation on lysine 27 of histone H3 (H3K27me3) at FLC. Thus, clf-59 appears to be a gain-of-function allele, and this allele represses FLC without some of the components required for vernalization-mediated repression. In the course of this work, we also identified a marked difference in H3K27me3 levels at FLC between plants that contain and those that lack the FRIGIDA (FRI) gene. Furthermore, FRI appears to affect CLF occupancy at FLC; thus, our work provides insight into the molecular role that FRI plays in delaying the onset of flowering.