Plant Physiology current issue

[ON THE INSIDE] On the Inside

Minorsky, P. V. — 2008-04-28

[EDITORIALS] Editor's Choice Series: The Next Generation of Biotech Crops

Martino-Catt, S. J., Sachs, E. S. — 2008-04-28

[EDITOR'S CHOICE SERIES ON THE NEXT GENERATION OF BIOTECH CROPS] The Outlook for Protein Engineering in Crop Improvement

Rao, A. G. — 2008-04-28

[EDITOR'S CHOICE SERIES ON THE NEXT GENERATION OF BIOTECH CROPS] Targets for Crop Biotechnology in a Future High-CO2 and High-O3 World

Ainsworth, E. A., Rogers, A., Leakey, A. D.B. — 2008-04-28

[EDITOR'S CHOICE SERIES ON THE NEXT GENERATION OF BIOTECH CROPS] Regulating the Regulators: The Future Prospects for Transcription-Factor-Based Agricultural Biotechnology Products

Century, K., Reuber, T. L., Ratcliffe, O. J. — 2008-04-28

[BREAKTHROUGH TECHNOLOGIES] Global Characterization of Cell-Specific Gene Expression through Fluorescence-Activated Sorting of Nuclei

Zhang, C., Barthelson, R. A., Lambert, G. M., Galbraith, D. W. — 2008-04-28

We describe a simple and highly effective means for global identification of genes that are expressed within specific cell types within complex tissues. It involves transgenic expression of nuclear-targeted green fluorescent protein in a cell-type-specific manner. The fluorescent nuclei are then purified from homogenates by fluorescence-activated sorting, and the RNAs employed as targets for microarray hybridization. We demonstrate the validity of the approach through the identification of 12 genes that are selectively expressed in phloem.

[BIOINFORMATICS] Annotating Genes of Known and Unknown Function by Large-Scale Coexpression Analysis

2008-04-28

About 40% of the proteins encoded in eukaryotic genomes are proteins of unknown function (PUFs). Their functional characterization remains one of the main challenges in modern biology. In this study we identified the PUF encoding genes from Arabidopsis (Arabidopsis thaliana) using a combination of sequence similarity, domain-based, and empirical approaches. Large-scale gene expression analyses of 1,310 publicly available Affymetrix chips were performed to associate the identified PUF genes with regulatory networks and biological processes of known function. To generate quality results, the study was restricted to expression sets with replicated samples. First, genome-wide clustering and gene function enrichment analysis of clusters allowed us to associate 1,541 PUF genes with tightly coexpressed genes for proteins of known function (PKFs). Over 70% of them could be assigned to more specific biological process annotations than the ones available in the current Gene Ontology release. The most highly overrepresented functional categories in the obtained clusters were ribosome assembly, photosynthesis, and cell wall pathways. Interestingly, the majority of the PUF genes appeared to be controlled by the same regulatory networks as most PKF genes, because clusters enriched in PUF genes were extremely rare. Second, large-scale analysis of differentially expressed genes was applied to identify a comprehensive set of abiotic stress-response genes. This analysis resulted in the identification of 269 PKF and 104 PUF genes that responded to a wide variety of abiotic stresses, whereas 608 PKF and 206 PUF genes responded predominantly to specific stress treatments. The provided coexpression and differentially expressed gene data represent an important resource for guiding future functional characterization experiments of PUF and PKF genes. Finally, the public Plant Gene Expression Database (http://bioweb.ucr.edu/PED) was developed as part of this project to provide efficient access and mining tools for the vast gene expression data of this study.

[SCIENTIFIC CORRESPONDENCE] Activity Range of Arabidopsis Small RNAs Derived from Different Biogenesis Pathways

Tretter, E. M., Alvarez, J. P., Eshed, Y., Bowman, J. L. — 2008-04-28

[BIOCHEMICAL PROCESSES AND MACROMOLECULAR STRUCTURES] Cell Wall Modifications in Arabidopsis Plants with Altered {alpha}-L-Arabinofuranosidase Activity

2008-04-28

Although cell wall remodeling is an essential feature of plant growth and development, the underlying molecular mechanisms are poorly understood. This work describes the characterization of Arabidopsis (Arabidopsis thaliana) plants with altered expression of ARAF1, a bifunctional -l-arabinofuranosidase/β-d-xylosidase (At3g10740) belonging to family 51 glycosyl-hydrolases. ARAF1 was localized in several cell types in the vascular system of roots and stems, including xylem vessels and parenchyma cells surrounding the vessels, the cambium, and the phloem. araf1 T-DNA insertional mutants showed no visible phenotype, whereas transgenic plants that overexpressed ARAF1 exhibited a delay in inflorescence emergence and altered stem architecture. Although global monosaccharide analysis indicated only slight differences in cell wall composition in both mutant and overexpressing lines, immunolocalization experiments using anti-arabinan (LM6) and anti-xylan (LM10) antibodies indicated cell type-specific alterations in cell wall structure. In araf1 mutants, an increase in LM6 signal intensity was observed in the phloem, cambium, and xylem parenchyma in stems and roots, largely coinciding with ARAF1 expression sites. The ectopic overexpression of ARAF1 resulted in an increase in LM10 labeling in the secondary walls of interfascicular fibers and xylem vessels. The combined ARAF1 gene expression and immunolocalization studies suggest that arabinan-containing pectins are potential in vivo substrates of ARAF1 in Arabidopsis.

[BIOCHEMICAL PROCESSES AND MACROMOLECULAR STRUCTURES] UDP-Xylose-Stimulated Glucuronyltransferase Activity in Wheat Microsomal Membranes: Characterization and Role in Glucurono(arabino)xylan Biosynthesis

Zeng, W., Chatterjee, M., Faik, A. — 2008-04-28

Microsomal membranes from etiolated wheat (Triticum aestivum) seedlings cooperatively incorporated xylose (Xyl), arabinose, and glucuronic acid residues from their corresponding uridine 5'-diphosphosugars into an ethanol-insoluble glucurono(arabino)xylan (GAX)-like product. A glucuronyltransferase activity that is enhanced by the presence of UDP-Xyl was also identified in these microsomes. Wheat glucuronyltransferase activity was optimal at pH 7 and required manganese ions, and several lines of evidence suggest its involvement in GAX-like biosynthesis. The GAX characteristics of the ¹⁴C-product were confirmed by digestion with a purified endo-xylanase from Aspergillus awamori (endo-xylanase III) and by total acid hydrolysis, resulting in a Xyl:arabinose:glucuronic acid molar ratio of approximately 105:34:1. Endo-xylanase III released only three types of oligosaccharides in addition to free Xyl. No radiolabel was released as xylobiose, xylotriose, or xylotetraose, indicating the absence of long stretches of unbranched Xyl residues in the nascent GAX-like product. High-pH anion exchange chromatography analysis of the resulting oligosaccharides along with known arabinoxylan oligosaccharide standards suggests that a portion of the nascent GAX-like product has a relatively regular structure. The other portion of the [¹⁴C]GAX-like polymer was resistant to proteinase K, endo-polygalacturonase, and endo-xylanase III (GH11 family) but was degraded by Driselase, supporting the hypothesis that the xylan backbone in this portion of the product is most likely highly substituted. Size exclusion chromatography indicated that the nascent GAX-like polymer had an apparent molecular mass of approximately 10 to 15 kD; however, mature GAXs from wheat cell walls had larger apparent molecular masses (>66 kD).

[BIOCHEMICAL PROCESSES AND MACROMOLECULAR STRUCTURES] Arabidopsis Sucrose Transporter AtSUC1 Is Important for Pollen Germination and Sucrose-Induced Anthocyanin Accumulation

Sivitz, A. B., Reinders, A., Ward, J. M. — 2008-04-28

The Arabidopsis (Arabidopsis thaliana) sucrose transporter AtSUC1 (At1g71880) is highly expressed in pollen; however, its function has remained unknown. Here, we show that suc1 mutant pollen is defective in vivo, as evidenced by segregation distortion, and also has low rates of germination in vitro. AtSUC1-green fluorescent protein was localized to the plasma membrane in pollen tubes. AtSUC1 is also expressed in roots and external application of sucrose increased AtSUC1 expression in roots. AtSUC1 is important for sucrose-dependent signaling leading to anthocyanin accumulation in seedlings. suc1 mutants accumulated less anthocyanins in response to exogenous sucrose or maltose and microarray analysis revealed reduced expression of many genes important for anthocyanin biosynthesis. The results indicate that AtSUC1 is important for sugar signaling in vegetative tissue and for normal male gametophyte function.

[BIOENERGETICS AND PHOTOSYNTHESIS] Decrease in Manganese Superoxide Dismutase Leads to Reduced Root Growth and Affects Tricarboxylic Acid Cycle Flux and Mitochondrial Redox Homeostasis

2008-04-28

Superoxide dismutases (SODs) are key components of the plant antioxidant defense system. While plastidic and cytosolic isoforms have been extensively studied, the importance of mitochondrial SOD at a cellular and whole-plant level has not been established. To address this, transgenic Arabidopsis (Arabidopsis thaliana) plants were generated in which expression of AtMSD1, encoding the mitochondrial manganese (Mn)SOD, was suppressed by antisense. The strongest antisense line showed retarded root growth even under control growth conditions. There was evidence for a specific disturbance of mitochondrial redox homeostasis in seedlings grown in liquid culture: a mitochondrially targeted redox-sensitive green fluorescent protein was significantly more oxidized in the MnSOD-antisense background. In contrast, there was no substantial change in oxidation of cytosolically targeted redox-sensitive green fluorescent protein, nor changes in antioxidant defense components. The consequences of altered mitochondrial redox status of seedlings were subtle with no widespread increase of mitochondrial protein carbonyls or inhibition of mitochondrial respiratory complexes. However, there were specific inhibitions of tricarboxylic acid (TCA) cycle enzymes (aconitase and isocitrate dehydrogenase) and an inhibition of TCA cycle flux in isolated mitochondria. Nevertheless, total respiratory CO₂ output of seedlings was not decreased, suggesting that the inhibited TCA cycle enzymes can be bypassed. In older, soil-grown plants, redox perturbation was more pronounced with changes in the amount and/or redox poise of ascorbate and glutathione. Overall, the results demonstrate that reduced MnSOD affects mitochondrial redox balance and plant growth. The data also highlight the flexibility of plant metabolism with TCA cycle inhibition having little effect on overall respiratory rates.

[BIOENERGETICS AND PHOTOSYNTHESIS] Mild Reductions in Mitochondrial Citrate Synthase Activity Result in a Compromised Nitrate Assimilation and Reduced Leaf Pigmentation But Have No Effect on Photosynthetic Performance or Growth

2008-04-28

Transgenic tomato (Solanum lycopersicum) plants, expressing a fragment of the mitochondrial citrate synthase gene in the antisense orientation and exhibiting mild reductions in the total cellular activity of this enzyme, displayed essentially no visible phenotypic alteration from the wild type. A more detailed physiological characterization, however, revealed that although these plants were characterized by relatively few changes in photosynthetic parameters they displayed a decreased relative flux through the tricarboxylic acid cycle and an increased rate of respiration. Furthermore, biochemical analyses revealed that the transformants exhibited considerably altered metabolism, being characterized by slight decreases in the levels of organic acids of the tricarboxylic acid cycle, photosynthetic pigments, and in a single line in protein content but increases in the levels of nitrate, several amino acids, and starch. We additionally determined the maximal catalytic activities of a wide range of enzymes of primary metabolism, performed targeted quantitative PCR analysis on all three isoforms of citrate synthase, and conducted a broader transcript profiling using the TOM1 microarray. Results from these studies confirmed that if the lines were somewhat impaired in nitrate assimilation, they were not severely affected by this, suggesting the presence of strategies by which metabolism is reprogrammed to compensate for this deficiency. The results are discussed in the context of carbon-nitrogen interaction and interorganellar coordination of metabolism.

[CELL BIOLOGY AND SIGNAL TRANSDUCTION] Arabidopsis Ribosomal Proteins RPL23aA and RPL23aB Are Differentially Targeted to the Nucleolus and Are Disparately Required for Normal Development

Degenhardt, R. F., Bonham-Smith, P. C. — 2008-04-28

Protein synthesis is catalyzed by the ribosome, a two-subunit enzyme comprised of four ribosomal RNAs and, in Arabidopsis (Arabidopsis thaliana), 81 ribosomal proteins (r-proteins). Plant r-protein genes exist as families of multiple expressed members, yet only one r-protein from each family is incorporated into any given ribosome, suggesting that many r-protein genes may be functionally redundant or development/tissue/stress specific. Here, we characterized the localization and gene-silencing phenotypes of a large subunit r-protein family, RPL23a, containing two expressed genes (RPL23aA and RPL23aB). Live cell imaging of RPL23aA and RPL23aB in tobacco with a C-terminal fluorescent-protein tag demonstrated that both isoforms accumulated in the nucleolus; however, only RPL23aA was targeted to the nucleolus with an N-terminal fluorescent protein tag, suggesting divergence in targeting efficiency of localization signals. Independent knockdowns of endogenous RPL23aA and RPL23aB transcript levels using RNA interference determined that an RPL23aB knockdown did not alter plant growth or development. Conversely, a knockdown of RPL23aA produced a pleiotropic phenotype characterized by growth retardation, irregular leaf and root morphology, abnormal phyllotaxy and vasculature, and loss of apical dominance. Comparison to other mutants suggests that the phenotype results from reduced ribosome biogenesis, and we postulate a link between biogenesis, microRNA-target degradation, and maintenance of auxin homeostasis. An additional RNA interference construct that coordinately silenced both RPL23aA and RPL23aB demonstrated that this family is essential for viability.

[DEVELOPMENT AND HORMONE ACTION] Elucidating the Germination Transcriptional Program Using Small Molecules

2008-04-28

The transition from seed to seedling is mediated by germination, a complex process that starts with imbibition and completes with radicle emergence. To gain insight into the transcriptional program mediating germination, previous studies have compared the transcript profiles of dry, dormant, and germinating after-ripened Arabidopsis (Arabidopsis thaliana) seeds. While informative, these approaches did not distinguish the transcriptional responses due to imbibition, shifts in metabolism, or breaking of dormancy from those triggered by the initiation of germination. In this study, three mechanistically distinct small molecules that inhibit Arabidopsis seed germination (methotrexate, 2, 4-dinitrophenol, and cycloheximide) were identified using a small-molecule screen and used to probe the germination transcriptome. Germination-responsive transcripts were defined as those with significantly altered transcript abundance across all inhibitory treatments with respect to control germinating seeds, using data from ATH1 microarrays. This analysis identified numerous germination regulators as germination responsive, including the DELLA proteins GAI, RGA, and RGL3, the abscisic acid-insensitive proteins ABI4, ABI5, ABI8, and FRY1, and the gibberellin receptor GID1A. To help visualize these and other publicly available seed microarray data, we designed a seed mRNA expression browser using the electronic Fluorescent Pictograph platform. An overall decrease in gene expression and a 5-fold greater number of transcripts identified as statistically down-regulated in drug-inhibited seeds point to a role for mRNA degradation or turnover during seed germination. The genes identified in our study as responsive to germination define potential uncharacterized regulators of this process and provide a refined transcriptional signature for germinating Arabidopsis seeds.

[DEVELOPMENT AND HORMONE ACTION] Further Characterization of a Rice AGL12 Group MADS-Box Gene, OsMADS26

2008-04-28

Plant MADS-box genes can be divided into 11 groups. Genetic analysis has revealed that most of them function in flowering-time control, reproductive organ development, and vegetative growth. Here, we elucidated the role of OsMADS26, a member of the AGL12 group. Transcript levels of OsMADS26 were increased in an age-dependent manner in the shoots and roots. Transgenic plants of both rice (Oryza sativa) and Arabidopsis (Arabidopsis thaliana) overexpressing this gene manifested phenotypes related to stress responses, such as chlorosis, cell death, pigment accumulation, and defective root/shoot growth. In addition, apical hook development was significantly suppressed in Arabidopsis. Plants transformed with the OsMADS26-GR (glucocorticoid receptor) fusion construct displayed those stress-related phenotypes when treated with dexamethasone. Microarray analyses using this inducible system showed that biosynthesis genes for jasmonate, ethylene, and reactive oxygen species, as well as putative downstream targets involved in the stress-related process, were up-regulated in OsMADS26-overexpressing plants. These results suggest that OsMADS26 induces multiple responses that are related to various stresses.

[DEVELOPMENT AND HORMONE ACTION] SHORT HYPOCOTYL IN WHITE LIGHT1, a Serine-Arginine-Aspartate-Rich Protein in Arabidopsis, Acts as a Negative Regulator of Photomorphogenic Growth

Bhatia, S., Gangappa, S. N., Kushwaha, R., Kundu, S., Chattopadhyay, S. — 2008-04-28

Light is an important factor for plant growth and development. We have identified and functionally characterized a regulatory gene SHORT HYPOCOTYL IN WHITE LIGHT1 (SHW1) involved in Arabidopsis (Arabidopsis thaliana) seedling development. SHW1 encodes a unique serine-arginine-aspartate-rich protein, which is constitutively localized in the nucleus of hypocotyl cells. Transgenic analyses have revealed that the expression of SHW1 is developmentally regulated and is closely associated with the photosynthetically active tissues. Genetic and molecular analyses suggest that SHW1 acts as a negative regulator of light-mediated inhibition of hypocotyl elongation, however, plays a positive regulatory role in light-regulated gene expression. The shw1 mutants also display shorter hypocotyl in dark, and analyses of shw1 cop1 double mutants reveal that SHW1 acts nonredundantly with COP1 to control hypocotyl elongation in the darkness. Taken together, this study provides evidences that SHW1 is a regulatory protein that is functionally interrelated to COP1 and plays dual but opposite regulatory roles in photomorphogenesis.

[DEVELOPMENT AND HORMONE ACTION] Amino Acid Substitutions in Homologs of the STAY-GREEN Protein Are Responsible for the green-flesh and chlorophyll retainer Mutations of Tomato and Pepper

Barry, C. S., McQuinn, R. P., Chung, M.-Y., Besuden, A., Giovannoni, J. J. — 2008-04-28

Color changes often accompany the onset of ripening, leading to brightly colored fruits that serve as attractants to seed-dispersing organisms. In many fruits, including tomato (Solanum lycopersicum) and pepper (Capsicum annuum), there is a sharp decrease in chlorophyll content and a concomitant increase in the synthesis of carotenoids as a result of the conversion of chloroplasts into chromoplasts. The green-flesh (gf) and chlorophyll retainer (cl) mutations of tomato and pepper, respectively, are inhibited in their ability to degrade chlorophyll during ripening, leading to the production of ripe fruits characterized by both chlorophyll and carotenoid accumulation and are thus brown in color. Using a positional cloning approach, we have identified a point mutation at the gf locus that causes an amino acid substitution in an invariant residue of a tomato homolog of the STAY-GREEN (SGR) protein of rice (Oryza sativa). Similarly, the cl mutation also carries an amino acid substitution at an invariant residue in a pepper homolog of SGR. Both GF and CL expression are highly induced at the onset of fruit ripening, coincident with the ripening-associated decline in chlorophyll. Phylogenetic analysis indicates that there are two distinct groups of SGR proteins in plants. The SGR subfamily is required for chlorophyll degradation and operates through an unknown mechanism. A second subfamily, which we have termed SGR-like, has an as-yet undefined function.

[DEVELOPMENT AND HORMONE ACTION] Nitric Oxide Triggers Phosphatidic Acid Accumulation via Phospholipase D during Auxin-Induced Adventitious Root Formation in Cucumber

Lanteri, M. L., Laxalt, A. M., Lamattina, L. — 2008-04-28

Auxin and nitric oxide (NO) play fundamental roles throughout plant life. NO is a second messenger in auxin signal transduction leading to root developmental processes. The mechanisms triggered by auxin and NO that direct adventitious root (AR) formation are beginning to be unraveled. The goal of this work was to study phospholipid (PL) signaling during the auxin- and NO-induced AR formation in cucumber (Cucumis sativus) explants. Explants were labeled with ³²P-inorganic phosphate and treated with the auxins indole-3-acetic acid or 1-naphthylacetic acid, or the NO donor S-nitroso N-acetyl penicillamine, in the presence or absence of the specific NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide. PLs were separated by thin-layer chromatography and quantified. We report that the signaling PLs phosphatidic acid (PA), phosphatidylinositol phosphate, and phosphatidylinositol bisphosphate accumulated within 1 min after auxin or NO treatment. Both auxin and NO evoked similar and transient time course responses, since signaling PLs returned to control levels after 20 or 30 min of treatment. The results indicate that auxin relies on NO in inducing PA, phosphatidylinositol phosphate, and phosphatidylinositol bisphosphate accumulation. Furthermore, we demonstrate that auxin and NO trigger PA formation via phospholipase D (PLD) activity. Explants treated for 10 min with auxin or NO displayed a 200% increase in AR number compared with control explants. In addition, PLD activity was required for the auxin- and NO-induced AR formation. Finally, exogenously applied PA increased up to 300% the number of ARs. Altogether, our data support the idea that PLD-derived PA is an early signaling event during AR formation induced by auxin and NO in cucumber explants.

[DEVELOPMENT AND HORMONE ACTION] The Pea DELLA Proteins LA and CRY Are Important Regulators of Gibberellin Synthesis and Root Growth

2008-04-28

The theory that bioactive gibberellins (GAs) act as inhibitors of inhibitors of plant growth was based originally on the slender pea (Pisum sativum) mutant (genotype la cry-s), but the molecular nature of this mutant has remained obscure. Here we show that the genes LA and CRY encode DELLA proteins, previously characterized in other species (Arabidopsis [Arabidopsis thaliana] and several grasses) as repressors of growth, which are destabilized by GAs. Mutations la and cry-s encode nonfunctional proteins, accounting for the fact that la cry-s plants are extremely elongated, or slender. We use the la and cry-s mutations to show that in roots, DELLA proteins effectively promote the expression of GA synthesis genes, as well as inhibit elongation. We show also that one of the DELLA-regulated genes is a second member of the pea GA 3-oxidase family, and that this gene appears to play a major role in pea roots.

[ENVIRONMENTAL STRESS AND ADAPTATION TO STRESS] Ammonia Triggers Photodamage of Photosystem II in the Cyanobacterium Synechocystis sp. Strain PCC 6803

Drath, M., Kloft, N., Batschauer, A., Marin, K., Novak, J., Forchhammer, K. — 2008-04-28

Ammonia has long been known to be toxic for many photosynthetic organisms; however, the target for its toxicity remains elusive. Here, we show that in the cyanobacterium Synechocystis sp. strain PCC 6803, ammonia triggers a rapid photodamage of photosystem II (PSII). Whereas wild-type cells can cope with this damage by turning on the FtsH2-dependent PSII repair cycle, the FtsH2-deficient mutant is highly sensitive and loses PSII activity at millimolar concentration of ammonia. Ammonia-triggered PSII destruction is light dependent and occurs already at low photon fluence rates. Experiments with monochromatic light showed that ammonia-promoted PSII photoinhibition is executed by wavebands known to directly destroy the manganese cluster in the PSII oxygen-evolving complex, suggesting that the oxygen-evolving complex may be a direct target for ammonia toxicity.

[ENVIRONMENTAL STRESS AND ADAPTATION TO STRESS] The Central Role of a SNRK2 Kinase in Sulfur Deprivation Responses

Gonzalez-Ballester, D., Pollock, S. V., Pootakham, W., Grossman, A. R. — 2008-04-28

In the absence of sulfur (S), Chlamydomonas reinhardtii increases the abundance of several transcripts encoding proteins associated with S acquisition and assimilation, conserves S amino acids, and acclimates to suboptimal growth conditions. A positive regulator, SAC1 (for sulfur acclimation protein 1), and a negative regulator, SAC3, were shown to participate in the control of these processes. In this study, we investigated two allelic mutants (ars11 and ars44) affected in a gene encoding a SNRK2 (for SNF1-related protein kinase 2) kinase designated SNRK2.1. Like the sac1 mutant, both snrk2.1 mutants were deficient in the expression of S-responsive genes. Furthermore, the mutant cells bleached more rapidly than wild-type cells during S deprivation, although the phenotypes of ars11 and ars44 were not identical: ars11 exhibited a more severe phenotype than either ars44 or sac1. The phenotypic differences between the ars11 and ars44 mutants reflected distinct alterations of SNRK2.1 mRNA splicing caused by insertion of the marker gene. The ars11 phenotype could be rescued by complementation with SNRK2.1 cDNA. In contrast to the nonepistatic relationship between SAC3 and SAC1, characterization of the sac3 ars11 double mutant showed that SNRK2.1 is epistatic to SAC3. These data reveal the crucial regulatory role of SNRK2.1 in the signaling cascade critical for eliciting S deprivation responses in Chlamydomonas. The phylogenetic relationships and structures of the eight members of the SNRK2 family in Chlamydomonas are discussed.

[ENVIRONMENTAL STRESS AND ADAPTATION TO STRESS] Isolation and Characterization of Mutants of Common Ice Plant Deficient in Crassulacean Acid Metabolism

2008-04-28

Crassulacean acid metabolism (CAM) is a specialized mode of photosynthesis that improves water use efficiency by shifting part or all of net atmospheric CO₂ uptake to the night. Genetic dissection of regulatory and metabolic attributes of CAM has been limited by the difficulty of identifying a reliable phenotype for mutant screening. We developed a novel and simple colorimetric assay to measure leaf pH to screen fast neutron-mutagenized populations of common ice plant (Mesembryanthemum crystallinum), a facultative CAM species, to detect CAM-deficient mutants with limited nocturnal acidification. The isolated CAM-deficient mutants showed negligible net dark CO₂ uptake compared with wild-type plants following the imposition of salinity stress. The mutants and wild-type plants accumulated nearly comparable levels of sodium in leaves, but the mutants grew more slowly than the wild-type plants. The mutants also had substantially reduced seed set and seed weight relative to wild type under salinity stress. Carbon-isotope ratios of seed collected from 4-month-old plants indicated that C₃ photosynthesis made a greater contribution to seed production in mutants compared to wild type. The CAM-deficient mutants were deficient in leaf starch and lacked plastidic phosphoglucomutase, an enzyme critical for gluconeogenesis and starch formation, resulting in substrate limitation of nocturnal C₄ acid formation. The restoration of nocturnal acidification by feeding detached leaves of salt-stressed mutants with glucose or sucrose supported this defect and served to illustrate the flexibility of CAM. The CAM-deficient mutants described here constitute important models for exploring regulatory features and metabolic consequences of CAM.

[ENVIRONMENTAL STRESS AND ADAPTATION TO STRESS] The Arabidopsis Putative Selenium-Binding Protein Family: Expression Study and Characterization of SBP1 as a Potential New Player in Cadmium Detoxification Processes

Dutilleul, C., Jourdain, A., Bourguignon, J., Hugouvieux, V. — 2008-04-28

In Arabidopsis (Arabidopsis thaliana), the putative selenium-binding protein (SBP) gene family is composed of three members (SBP1–SBP3). Reverse transcription-polymerase chain reaction analyses showed that SBP1 expression was ubiquitous. SBP2 was expressed at a lower level in flowers and roots, whereas SBP3 transcripts were only detected in young seedling tissues. In cadmium (Cd)-treated seedlings, SBP1 level of expression was rapidly increased in roots. In shoots, SBP1 transcripts accumulated later and for higher Cd doses. SBP2 and SBP3 expression showed delayed or no responsiveness to Cd. In addition, luciferase (LUC) activity recorded on Arabidopsis lines expressing the LUC gene under the control of the SBP1 promoter further showed dynamic regulation of SBP1 expression during development and in response to Cd stress. Western-blot analysis using polyclonal antibodies raised against SBP1 showed that SBP1 protein accumulated in Cd-exposed tissues in correlation with SBP1 transcript amount. The sbp1 null mutant displayed no visible phenotype under normal and stress conditions that was explained by the up-regulation of SBP2 expression. SBP1 overexpression enhanced Cd accumulation in roots and reduced sensitivity to Cd in wild type and, more significantly, in Cd-hypersensitive cad mutants that lack phytochelatins. Similarly, in Saccharomyces cerevisiae, SBP1 expression led to increased Cd tolerance of the Cd-hypersensitive ycf1 mutant. In vitro experiments showed that SBP1 has the ability to bind Cd. These data highlight the importance of maintaining the adequate SBP protein level under healthy and stress conditions and suggest that, during Cd stress, SBP1 accumulation efficiently helps to detoxify Cd potentially through direct binding.

[ENVIRONMENTAL STRESS AND ADAPTATION TO STRESS] The Ionic Environment Controls the Contribution of the Barley HvHAK1 Transporter to Potassium Acquisition

2008-04-28

The control of potassium (K⁺) acquisition is a critical requirement for plant growth. Although HAK1 (high affinity K⁺ 1) transporters provide a pathway for K⁺ acquisition, the effect exerted by the ionic environment on their contribution to K⁺ capture remains essentially unknown. Here, the influence of the ionic environment on the accumulation of transcripts coding for the barley (Hordeum vulgare) HvHAK1 transporter as well as on HvHAK1-mediated K⁺ capture has been examined. In situ mRNA hybridization studies show that HvHAK1 expression occurs in most root cells, being augmented at the outermost cell layers. Accumulation of HvHAK1 transcripts is enhanced by K⁺ deprivation and transiently by exposure to high salt concentrations. In addition, studies on the accumulation of transcripts coding for HvHAK1 and its close homolog HvHAK1b revealed the presence of two K⁺-responsive pathways, one repressed and the other insensitive to ammonium. Experiments with Arabidopsis (Arabidopsis thaliana) HvHAK1-expressing transgenic plants showed that K⁺ deprivation enhances the capture of K⁺ mediated by HvHAK1. A detailed study with HvHAK1-expressing Saccharomyces cerevisiae cells also revealed an increase of K⁺ uptake after K⁺ starvation. This increase did not occur in cells grown at high Na⁺ concentrations but took place for cells grown in the presence of NH₄⁺. 3,3'-Dihexyloxacarbocyanine iodide accumulation measurements indicate that the increased capture of K⁺ in HvHAK1-expressing yeast cells cannot be explained only by changes in the membrane potential. It is shown that the yeast protein phosphatase PPZ1 as well as the halotolerance HAL4/HAL5 kinases negatively regulate the HvHAK1-mediated K⁺ transport.

[ENVIRONMENTAL STRESS AND ADAPTATION TO STRESS] Disruption of the Arabidopsis Circadian Clock Is Responsible for Extensive Variation in the Cold-Responsive Transcriptome

2008-04-28

In plants, low temperature causes massive transcriptional changes, many of which are presumed to be involved in the process of cold acclimation. Given the diversity of developmental and environmental factors between experiments, it is surprising that their influence on the identification of cold-responsive genes is largely unknown. A systematic investigation of genes responding to 1 d of cold treatment revealed that diurnal- and circadian-regulated genes are responsible for the majority of the substantial variation between experiments. This is contrary to the widespread assumption that these effects are eliminated using paired diurnal controls. To identify the molecular basis for this variation, we performed targeted expression analyses of diurnal and circadian time courses in Arabidopsis (Arabidopsis thaliana). We show that, after a short initial cold response, in diurnal conditions cold reduces the amplitude of cycles for clock components and dampens or disrupts the cycles of output genes, while in continuous light all cycles become arrhythmic. This means that genes identified as cold-responsive are dependent on the time of day the experiment was performed and that a control at normal temperature will not correct for this effect, as was postulated up to now. Time of day also affects the number and strength of expression changes for a large number of transcription factors, and this likely further contributes to experimental differences. This reveals that interactions between cold and diurnal regulation are major factors in shaping the cold-responsive transcriptome and thus will be an important consideration in future experiments to dissect transcriptional regulatory networks controlling cold acclimation. In addition, our data revealed differential effects of cold on circadian output genes and a unique regulation of an oscillator component, suggesting that cold treatment could also be an important tool to probe circadian and diurnal regulatory mechanisms.

[GENETICS, GENOMICS, AND MOLECULAR EVOLUTION] Tobacco Transcription Factors: Novel Insights into Transcriptional Regulation in the Solanaceae

2008-04-28

Tobacco (Nicotiana tabacum) is a member of the Solanaceae, one of the agronomically most important groups of flowering plants. We have performed an in silico analysis of 1.15 million gene-space sequence reads from the tobacco nuclear genome and report the detailed analysis of more than 2,500 tobacco transcription factors (TFs). The tobacco genome contains at least one member of each of the 64 well-characterized TF families identified in sequenced vascular plant genomes, indicating that evolution of the Solanaceae was not associated with the gain or loss of TF families. However, we found notable differences between tobacco and non-Solanaceae species in TF family size and evidence for both tobacco- and Solanaceae-specific subfamily expansions. Compared with TF families from sequenced plant genomes, tobacco has a higher proportion of ERF/AP2, C2H2 zinc finger, homeodomain, GRF, TCP, zinc finger homeodomain, BES, and STERILE APETALA (SAP) genes and novel subfamilies of BES, C2H2 zinc finger, SAP, and NAC genes. The novel NAC subfamily, termed TNACS, appears restricted to the Solanaceae, as they are absent from currently sequenced plant genomes but present in tomato (Solanum lycopersicum), pepper (Capsicum annuum), and potato (Solanum tuberosum). They constitute approximately 25% of NAC genes in tobacco. Based on our phylogenetic studies, we predict that many of the more than 50 tobacco group IX ERF genes are involved in jasmonate responses. Consistent with this, over two-thirds of group IX ERF genes tested showed increased mRNA levels following jasmonate treatment. Our data are a major resource for the Solanaceae and fill a void in studies of TF families across the plant kingdom.

[PLANTS INTERACTING WITH OTHER ORGANISMS] Biosynthesis and Accumulation of Ergoline Alkaloids in a Mutualistic Association between Ipomoea asarifolia (Convolvulaceae) and a Clavicipitalean Fungus

2008-04-28

Ergoline alkaloids occur in taxonomically unrelated taxa, such as fungi, belonging to the phylum Ascomycetes and higher plants of the family Convolvulaceae. The disjointed occurrence can be explained by the observation that plant-associated epibiotic clavicipitalean fungi capable of synthesizing ergoline alkaloids colonize the adaxial leaf surface of certain Convolvulaceae plant species. The fungi are seed transmitted. Their capacity to synthesize ergoline alkaloids depends on the presence of an intact differentiated host plant (e.g. Ipomoea asarifolia or Turbina corymbosa [Convolvulaceae]). Here, we present independent proof that these fungi are equipped with genetic material responsible for ergoline alkaloid biosynthesis. The gene (dmaW) for the determinant step in ergoline alkaloid biosynthesis was shown to be part of a cluster involved in ergoline alkaloid formation. The dmaW gene was overexpressed in Saccharomyces cerevisiae, the encoded DmaW protein purified to homogeneity, and characterized. Neither the gene nor the biosynthetic capacity, however, was detectable in the intact I. asarifolia or the taxonomically related T. corymbosa host plants. Both plants, however, contained the ergoline alkaloids almost exclusively, whereas alkaloids are not detectable in the associated epibiotic fungi. This indicates that a transport system may exist translocating the alkaloids from the epibiotic fungus into the plant. The association between the fungus and the plant very likely is a symbiotum in which ergoline alkaloids play an essential role.

[SYSTEMS BIOLOGY, MOLECULAR BIOLOGY, AND GENE REGULATION] Independence and Interaction of Regions of the INNER NO OUTER Protein in Growth Control during Ovule Development

Gallagher, T. L., Gasser, C. S. — 2008-04-28

The outer integument of the Arabidopsis (Arabidopsis thaliana) ovule develops asymmetrically, with growth and cell division occurring primarily along the region of the ovule facing the base of the gynoecium (gynobasal). This process is altered in the mutants inner no outer (ino) and superman (sup), which lead to absent or symmetrical growth of the outer integument, respectively. INO encodes a member of the YABBY family of putative transcription factors, and its expression is restricted to the gynobasal side of developing ovules via negative regulation by the transcription factor SUP. Other YABBY proteins (e.g. CRABS CLAW [CRC] and YABBY3 [YAB3]) can substitute for INO in promotion of integument growth, but do not respond to SUP regulation. In contrast, YAB5 fails to promote integument growth. To separately investigate the growth-promotive effects of INO and its inhibition by SUP, domain swaps between INO and YAB3, YAB5, or CRC were assembled. The ability of chimeric YABBY proteins to respond to SUP restriction showed a quantitative response proportional to the amount of INO protein and was more dependent on C-terminal regions of INO. A different response was seen when examining growth promotion where the number and identity of regions of INO in chimeric YABBY proteins were not the primary influence on promotion of outer integument growth. Instead, promotion of growth required a coordination of features along the entire length of the INO protein, suggesting that intramolecular interactions between regions of INO may coordinately facilitate the intermolecular interactions necessary to promote formation of the outer integument.

[SYSTEMS BIOLOGY, MOLECULAR BIOLOGY, AND GENE REGULATION] Principal Transcriptional Programs Regulating Plant Amino Acid Metabolism in Response to Abiotic Stresses

Less, H., Galili, G. — 2008-04-28

Using a bioinformatics analysis of public Arabidopsis (Arabidopsis thaliana) microarray data, we propose here a novel regulatory program, combining transcriptional and posttranslational controls, which participate in modulating fluxes of amino acid metabolism in response to abiotic stresses. The program includes the following two components: (1) the terminal enzyme of the module, responsible for the first catabolic step of the amino acid, whose level is stimulated or repressed in response to stress cues, just-in-time when the cues arrive, principally via transcriptional regulation of its gene; and (2) the initiator enzyme of the module, whose activity is principally modulated via posttranslational allosteric feedback inhibition in response to changes in the level of the amino acid, just-in-case when it occurs in response to alteration in its catabolism or sequestration into different intracellular compartments. Our proposed regulatory program is based on bioinformatics dissection of the response of all biosynthetic and catabolic genes of seven different pathways, involved in the metabolism of 11 amino acids, to eight different abiotic stresses, as judged from modulations of their mRNA levels. Our results imply that the transcription of the catabolic genes is principally more sensitive than that of the biosynthetic genes to fluctuations in stress-associated signals. Notably, the only exception to this program is the metabolic pathway of Pro, an amino acid that distinctively accumulates to significantly high levels under abiotic stresses. Examples of the biological significance of our proposed regulatory program are discussed.

[BIOCHEMICAL PROCESSES AND MACROMOLECULAR STRUCTURES] Construction of a Functional CMP-Sialic Acid Biosynthesis Pathway in Arabidopsis

2008-04-28

Previous studies have reported that plants contain negligible amounts of free or protein-bound N-acetylneuraminic acid (Neu5Ac). This is a major disadvantage for the use of plants as a biopharmaceutical expression system, since N-glycans with terminal Neu5Ac residues are important for the biological activities and half-lives of recombinant therapeutic glycoproteins in humans. For the synthesis of Neu5Ac-containing N-glycans, plants have to acquire the ability to synthesize Neu5Ac and its nucleotide-activated derivative, cytidine monophospho-N-acetylneuraminic acid. In this study, we have generated transgenic Arabidopsis (Arabidopsis thaliana) plants expressing three key enzymes of the mammalian Neu5Ac biosynthesis pathway: UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase, N-acetylneuraminic acid phosphate synthase, and CMP-N-acetylneuraminic acid synthetase. Simultaneous expression of UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase and N-acetylneuraminic acid phosphate synthase resulted in the generation of significant Neu5Ac amounts (1,275 nmol g^–1 fresh weight in leaves) in planta, which could be further converted to cytidine monophospho-N-acetylneuraminic acid (2.4 nmol g^–1 fresh weight in leaves) by coexpression of CMP-N-acetylneuraminic acid synthetase. These findings are a major step toward the production of Neu5Ac-containing glycoproteins in plants.

[BIOENERGETICS AND PHOTOSYNTHESIS] Expression Analysis of Genes Associated with the Induction of the Carbon-Concentrating Mechanism in Chlamydomonas reinhardtii

Yamano, T., Miura, K., Fukuzawa, H. — 2008-04-28

Acclimation to varying CO₂ concentrations and light intensities is associated with the monitoring of environmental changes by controlling genetic and physiological responses through CO₂ and light signal transduction. While CO₂ and light signals are indispensable for photosynthesis, and these environmental factors have been proposed as strongly associated with each other, studies linking these components are largely limited to work on higher plants. In this study, we examined the physiological characteristics of a green alga, Chlamydomonas reinhardtii, exposed to various light intensities or CO₂ concentrations. Acclimation to CO₂-limiting conditions by Chlamydomonas requires the induction of a carbon-concentrating mechanism (CCM) to allow the uptake of inorganic carbon (Ci) and increase the affinity for Ci. We revealed that the induction of the CCM is not solely dependent on absolute environmental Ci concentrations but is also affected by light intensity. Using a cDNA array containing 10,368 expressed sequence tags, we also obtained global expression profiles related to the physiological responses. The induction of several CCM-associated genes was strongly affected by high light as well as CO₂ concentrations. We identified novel candidates for Ci transporters and CO₂-responsive regulatory factors whose expression levels were significantly increased during the induction of the CCM.

[DEVELOPMENT AND HORMONE ACTION] Low-Temperature and Daylength Cues Are Integrated to Regulate FLOWERING LOCUS T in Barley

Hemming, M. N., Peacock, W. J., Dennis, E. S., Trevaskis, B. — 2008-04-28

Interactions between flowering time genes were examined in a doubled haploid barley (Hordeum vulgare) population segregating for H. vulgare VERNALIZATION1 (HvVRN1), HvVRN2, and PHOTOPERIOD1 (PPD-H1). A deletion allele of HvVRN2 was associated with rapid inflorescence initiation and early flowering, but only in lines with an active allele of PPD-H1. In these lines, the floral promoter FLOWERING LOCUS T (HvFT1) was expressed at high levels without vernalization, and this preceded induction of HvVRN1. Lines with the deletion allele of HvVRN2 and the inactive ppd-H1 allele did not undergo rapid inflorescence initiation and were late flowering. These data suggest that HvVRN2 counteracts PPD-H1 to prevent flowering prior to vernalization. An allele of HvVRN1 that is expressed at high basal levels (HvVRN1-1) was associated with rapid inflorescence initiation regardless of HvVRN2 or PPD-H1 genotype. HvFT1 was expressed without vernalization in lines with the HvVRN1-1 allele and HvFT1 transcript levels were highest in lines with the active PPD-H1 allele; this correlated with rapid apex development postinflorescence initiation. Thus, expression of HvVRN1 promotes inflorescence initiation and up-regulates HvFT1. Analysis of HvVRN1 expression in different genetic backgrounds postvernalization showed that HvVRN2, HvFT1, and PPD-H1 are unlikely to play a role in low-temperature induction of HvVRN1. In a vernalization responsive barley, HvFT1 is not induced by low temperatures alone, but can be induced by long days following prolonged low-temperature treatment. We conclude that low-temperature and daylength flowering-response pathways are integrated to control expression of HvFT1 in barley, and that this might occur through regulation of HvVRN2 activity.

[ENVIRONMENTAL STRESS AND ADAPTATION TO STRESS] A Third Phytoene Synthase Is Devoted to Abiotic Stress-Induced Abscisic Acid Formation in Rice and Defines Functional Diversification of Phytoene Synthase Genes

Welsch, R., Wust, F., Bar, C., Al-Babili, S., Beyer, P. — 2008-04-28

We here report on the characterization of a novel third phytoene synthase gene (PSY) in rice (Oryza sativa), OsPSY3, and on the differences among all three PSY genes with respect to the tissue-specific expression and regulation upon various environmental stimuli. The two already known PSYs are under phytochrome control and involved in carotenoid biosynthesis in photosynthetically active tissues and exhibit different expression patterns during chloroplast development. In contrast, OsPSY3 transcript levels are not affected by light and show almost no tissue-specific differences. Rather, OsPSY3 transcripts are up-regulated during increased abscisic acid (ABA) formation upon salt treatment and drought, especially in roots. The simultaneous induction of genes encoding 9-cis-epoxycarotenoid dioxygenases (NCEDs), involved in the initial steps of ABA biosynthesis, indicate that decreased xanthophyll levels are compensated by the induction of the third PSY gene. Furthermore, OsPSY3 and the OsNCEDs investigated were also induced by the application of ABA, indicating positive feedback regulation. The regulatory differences are mirrored by cis-acting elements in the corresponding promoter regions, with light-responsive elements for OsPSY1 and OsPSY2 and an ABA-response element as well as a coupling element for OsPSY3. The investigation of the gene structures and 5' untranslated regions revealed that OsPSY1 represents a descendant of an ancient PSY gene present in the common ancestor of monocots and dicots. Since the genomic structures of OsPSY2 and OsPSY3 are comparable, we conclude that they originated from the most recent common ancestor, OsPSY1.

[ENVIRONMENTAL STRESS AND ADAPTATION TO STRESS] Chaperone Activity of ERD10 and ERD14, Two Disordered Stress-Related Plant Proteins

Kovacs, D., Kalmar, E., Torok, Z., Tompa, P. — 2008-04-28

ERD10 and ERD14 (for early response to dehydration) proteins are members of the dehydrin family that accumulate in response to abiotic environmental stresses, such as high salinity, drought, and low temperature, in Arabidopsis (Arabidopsis thaliana). Whereas these proteins protect cells against the consequences of dehydration, the exact mode(s) of their action remains poorly understood. Here, detailed evidence is provided that ERD10 and ERD14 belong to the family of intrinsically disordered proteins, and it is shown in various assays that they act as chaperones in vitro. ERD10 and ERD14 are able to prevent the heat-induced aggregation and/or inactivation of various substrates, such as lysozyme, alcohol dehydrogenase, firefly luciferase, and citrate synthase. It is also demonstrated that ERD10 and ERD14 bind to acidic phospholipid vesicles without significantly affecting membrane fluidity. Membrane binding is strongly influenced by ionic strength. Our results show that these intrinsically disordered proteins have chaperone activity of rather wide substrate specificity and that they interact with phospholipid vesicles through electrostatic forces. We suggest that these findings provide the rationale for the mechanism of how these proteins avert the adverse effects of dehydration stresses.

[ENVIRONMENTAL STRESS AND ADAPTATION TO STRESS] Characterization of Cold-Responsive Extracellular Chitinase in Bromegrass Cell Cultures and Its Relationship to Antifreeze Activity

Nakamura, T., Ishikawa, M., Nakatani, H., Oda, A. — 2008-04-28

A cold-responsive chitinase gene, BiCHT1, was isolated from bromegrass (Bromus inermis) ‘Manchar’ suspension cells. BiCHT1 messenger RNA was detected at low levels in nonstressed bromegrass cells, whereas its accumulation was induced by incubation at 10°C and 4°C as detected by northern- and western-blot analyses. BiCHT1 was highly homologous to rye CHT9, known to encode an antifreeze protein. BiCHT1 was overexpressed in Escherichia coli and bromegrass cells using genetic transformation procedures. BiCHT1 products expressed in both systems had chitinase activity, but the expressed proteins did not affect the growth of ice crystals in any conditions tested. Besides cold stress, the expression of the BiCHT1 gene was up-regulated by exposure to 35°C, but not by salt or osmotic stress, abscisic acid, or ethephon. BiCHT1 messenger RNA did not accumulate in response to methyl jasmonate and salicylic acid, but was slightly increased by prolonged culture at 25°C and only transiently by chitin. Antifreeze activity detected in the culture medium was induced at 4°C but only slightly at 10°C. It was also induced by ethephon treatment, but not by abscisic acid, chitin, or prolonged incubation at 25°C. The results of transgenics and expression analyses suggest that the BiCHT1 product is a major protein with chitinase activity secreted in the medium of cold-treated cells and is unlikely to be responsible for the antifreeze activity detected in the culture medium.

[ENVIRONMENTAL STRESS AND ADAPTATION TO STRESS] Increased Air Temperature during Simulated Autumn Conditions Impairs Photosynthetic Electron Transport between Photosystem II and Photosystem I

Busch, F., Huner, N. P.A., Ensminger, I. — 2008-04-28

Changes in temperature and daylength trigger physiological and seasonal developmental processes that enable evergreen trees of the boreal forest to withstand severe winter conditions. Climate change is expected to increase the autumn air temperature in the northern latitudes, while the natural decreasing photoperiod remains unaffected. As shown previously, an increase in autumn air temperature inhibits CO₂ assimilation, with a concomitant increased capacity for zeaxanthin-independent dissipation of energy exceeding the photochemical capacity in Pinus banksiana. In this study, we tested our previous model of antenna quenching and tested a limitation in intersystem electron transport in plants exposed to elevated autumn air temperatures. Using a factorial design, we dissected the effects of temperature and photoperiod on the function as well as the stoichiometry of the major components of the photosynthetic electron transport chain in P. banksiana. Natural summer conditions (16-h photoperiod/22°C) and late autumn conditions (8-h photoperiod/7°C) were compared with a treatment of autumn photoperiod with increased air temperature (SD/HT: 8-h photoperiod/22°C) and a treatment with summer photoperiod and autumn temperature (16-h photoperiod/7°C). Exposure to SD/HT resulted in an inhibition of the effective quantum yield associated with a decreased photosystem II/photosystem I stoichiometry coupled with decreased levels of Rubisco. Our data indicate that a greater capacity to keep the primary electron donor of photosystem I (P700) oxidized in plants exposed to SD/HT compared with the summer control may be attributed to a reduced rate of electron transport from the cytochrome b₆f complex to photosystem I. Photoprotection under increased autumn air temperature conditions appears to be consistent with zeaxanthin-independent antenna quenching through light-harvesting complex II aggregation and a decreased efficiency in energy transfer from the antenna to the photosystem II core. We suggest that models that predict the effect of climate change on the productivity of boreal forests must take into account the interactive effects of photoperiod and elevated temperatures.

[GENETICS, GENOMICS, AND MOLECULAR EVOLUTION] Genotype, Age, Tissue, and Environment Regulate the Structural Outcome of Glucosinolate Activation

Wentzell, A. M., Kliebenstein, D. J. — 2008-04-28

Glucosinolates are the inert storage form of a two-part phytochemical defense system in which the enzyme myrosinase generates an unstable intermediate that rapidly rearranges into the biologically active product. This rearrangement step generates simple nitriles, epithionitriles, or isothiocyanates, depending on the structure of the parent glucosinolate and the presence of proteins that promote specific structural outcomes. Glucosinolate accumulation and myrosinase activity differ by plant age and tissue type and respond to environmental stimuli such as planting density and herbivory; however, the influence of these factors on the structural outcome of the rearrangement step remains unknown. We show that the structural outcome of glucosinolate activation is controlled by interactions among plant age, planting density, and natural genetic variation in Arabidopsis (Arabidopsis thaliana) rosette leaves using six well-studied accessions. We identified a similarly complex interaction between tissue type and the natural genetic variation present within these accessions. This raises questions about the relative importance of these novel levels of regulation in the evolution of plant defense. Using mutants in the structural specifier and glucosinolate activation genes identified previously in Arabidopsis rosette leaves, we demonstrate the requirement for additional myrosinases and structural specifiers controlling these processes in the roots and seedlings. Finally, we present evidence for a novel EPITHIOSPECIFIER PROTEIN-independent, simple nitrile-specifying activity that promotes the formation of simple nitriles but not epithionitriles from all glucosinolates tested.

[PLANTS INTERACTING WITH OTHER ORGANISMS] Characterization of an Amino Acid Permease from the Endomycorrhizal Fungus Glomus mosseae

Cappellazzo, G., Lanfranco, L., Fitz, M., Wipf, D., Bonfante, P. — 2008-04-28

Arbuscular mycorrhizal (AM) fungi are capable of exploiting organic nitrogen sources, but the molecular mechanisms that control such an uptake are still unknown. Polymerase chain reaction-based approaches, bioinformatic tools, and a heterologous expression system have been used to characterize a sequence coding for an amino acid permease (GmosAAP1) from the AM fungus Glomus mosseae. The GmosAAP1 shows primary and secondary structures that are similar to those of other fungal amino acid permeases. Functional complementation and uptake experiments in a yeast mutant that was defective in the multiple amino acid uptake system demonstrated that GmosAAP1 is able to transport proline through a proton-coupled, pH- and energy-dependent process. A competitive test showed that GmosAAP1 binds nonpolar and hydrophobic amino acids, thus indicating a relatively specific substrate spectrum. GmosAAP1 mRNAs were detected in the extraradical fungal structures. Transcript abundance was increased upon exposure to organic nitrogen, in particular when supplied at 2 mm concentrations. These findings suggest that GmosAAP1 plays a role in the first steps of amino acid acquisition, allowing direct amino acid uptake from the soil and extending the molecular tools by which AM fungi exploit soil resources.

[CORRECTION] CORRECTIONS

2008-04-28