Plant Physiology recent issues

[EDITORIALS - FOCUS ISSUE] Membrane Trafficking: Intracellular Highways and Country Roads

Cheung, A. Y., de Vries, S. C. — 2008-08-04

[UPDATES] Nanoscale Architecture of Endoplasmic Reticulum Export Sites and of Golgi Membranes as Determined by Electron Tomography

Staehelin, L. A., Kang, B.-H. — 2008-08-04

[UPDATES] Advances in Fluorescent Protein-Based Imaging for the Analysis of Plant Endomembranes

Held, M. A., Boulaflous, A., Brandizzi, F. — 2008-08-04

[UPDATES] The Endosomal System of Plants: Charting New and Familiar Territories

Robinson, D. G., Jiang, L., Schumacher, K. — 2008-08-04

[UPDATES] What Is Moving in the Secretory Pathway of Plants?

Rojo, E., Denecke, J. — 2008-08-04

[UPDATES] SNAREs: Cogs and Coordinators in Signaling and Development

Bassham, D. C., Blatt, M. R. — 2008-08-04

[UPDATES] The Regulatory RAB and ARF GTPases for Vesicular Trafficking

Nielsen, E., Cheung, A. Y., Ueda, T. — 2008-08-04

[UPDATES] Regulation of Membrane Trafficking, Cytoskeleton Dynamics, and Cell Polarity by ROP/RAC GTPases

Yalovsky, S., Bloch, D., Sorek, N., Kost, B. — 2008-08-04

[UPDATES] Vesicle Trafficking during Somatic Cytokinesis

Van Damme, D., Inze, D., Russinova, E. — 2008-08-04

[UPDATES] PIN Polar Targeting

Feraru, E., Friml, J. — 2008-08-04

[UPDATES] Plasma Membrane Receptor Complexes

Aker, J., de Vries, S. C. — 2008-08-04

[UPDATES] Plant Receptors Go Endosomal: A Moving View on Signal Transduction

Geldner, N., Robatzek, S. — 2008-08-04

[UPDATES] Secretory Pathways in Plant Immune Responses

Kwon, C., Bednarek, P., Schulze-Lefert, P. — 2008-08-04

[UPDATES] A Proteomics Approach to Membrane Trafficking

Groen, A. J., de Vries, S. C., Lilley, K. S. — 2008-08-04

[RESEARCH ARTICLES] Comparison of the Dynamics and Functional Redundancy of the Arabidopsis Dynamin-Related Isoforms DRP1A and DRP1C during Plant Development

Konopka, C. A., Bednarek, S. Y. — 2008-08-04

Members of the Arabidopsis (Arabidopsis thaliana) DYNAMIN-RELATED PROTEIN1 (DRP1) family are required for cytokinesis and cell expansion. Two isoforms, DRP1A and DRP1C, are required for plasma membrane maintenance during stigmatic papillae expansion and pollen development, respectively. It is unknown whether the DRP1s function interchangeably or if they have distinct roles during cell division and expansion. DRP1C was previously shown to form dynamic foci in the cell cortex, which colocalize with part of the clathrin endocytic machinery in plants. DRP1A localizes to the plasma membrane, but its cortical organization and dynamics have not been determined. Using dual color labeling with live cell imaging techniques, we showed that DRP1A also forms discreet dynamic foci in the epidermal cell cortex. Although the foci overlap with those formed by DRP1C and clathrin light chain, there are clear differences in behavior and response to pharmacological inhibitors between DRP1A and DRP1C foci. Possible functional or regulatory differences between DRP1A and DRP1C were supported by the failure of DRP1C to functionally compensate for the absence of DRP1A. Our studies indicated that the DRP1 isoforms function or are regulated differently during cell expansion.

[RESEARCH ARTICLES] Sucrose Synthase Is Associated with the Cell Wall of Tobacco Pollen Tubes

Persia, D., Cai, G., Del Casino, C., Faleri, C., Willemse, M. T.M., Cresti, M. — 2008-08-04

Sucrose synthase (Sus; EC 2.4.1.13) is a key enzyme of sucrose metabolism in plant cells, providing carbon for respiration and for the synthesis of cell wall polymers and starch. Since Sus is important for plant cell growth, insights into its structure, localization, and features are useful for defining the relationships between nutrients, growth, and cell morphogenesis. We used the pollen tube of tobacco (Nicotiana tabacum) as a cell model to characterize the main features of Sus with regard to cell growth and cell wall synthesis. Apart from its role during sexual reproduction, the pollen tube is a typical tip-growing cell, and the proper construction of its cell wall is essential for correct shaping and direction of growth. The outer cell wall layer of pollen tubes consists of pectins, but the inner layer is composed of cellulose and callose; both polymers require metabolic precursors in the form of UDP-glucose, which is synthesized by Sus. We identified an 88-kD polypeptide in the soluble, plasma membrane and Golgi fraction of pollen tubes. The protein was also found in association with the cell wall. After purification, the protein showed an enzyme activity similar to that of maize (Zea mays) Sus. Distribution of Sus was affected by brefeldin A and depended on the nutrition status of the pollen tube, because an absence of metabolic sugars in the growth medium caused Sus to distribute differently during tube elongation. Analysis by bidimensional electrophoresis indicated that Sus exists as two isoforms, one of which is phosphorylated and more abundant in the cytoplasm and cell wall and the other of which is not phosphorylated and is specific to the plasma membrane. Results indicate that the protein has a role in the construction of the extracellular matrix and thus in the morphogenesis of pollen tubes.

[RESEARCH ARTICLES] An Actin-Binding Protein, LlLIM1, Mediates Calcium and Hydrogen Regulation of Actin Dynamics in Pollen Tubes

Wang, H.-J., Wan, A.-R., Jauh, G.-Y. — 2008-08-04

Actin microfilaments are crucial for polar cell tip growth, and their configurations and dynamics are regulated by the actions of various actin-binding proteins (ABPs). We explored the function of a lily (Lilium longiflorum) pollen-enriched LIM domain-containing protein, LlLIM1, in regulating the actin dynamics in elongating pollen tube. Cytological and biochemical assays verified LlLIM1 functioning as an ABP, promoting filamentous actin (F-actin) bundle assembly and protecting F-actin against latrunculin B-mediated depolymerization. Overexpressed LlLIM1 significantly disturbed pollen tube growth and morphology, with multiple tubes protruding from one pollen grain and coaggregation of FM4-64-labeled vesicles and Golgi apparatuses at the subapex of the tube tip. Moderate expression of LlLIM1 induced an oscillatory formation of asterisk-shaped F-actin aggregates that oscillated with growth period but in different phases at the subapical region. These results suggest that the formation of LlLIM1-mediated overstabilized F-actin bundles interfered with endomembrane trafficking to result in growth retardation. Cosedimentation assays revealed that the binding affinity of LlLIM1 to F-actin was simultaneously regulated by both pH and Ca²⁺: LlLIM1 showed a preference for F-actin binding under low pH and low Ca²⁺ concentration. The potential functions of LlLIM1 as an ABP sensitive to pH and calcium in integrating endomembrane trafficking, oscillatory pH, and calcium circumstances to regulate tip-focused pollen tube growth are discussed.

[RESEARCH ARTICLES] SCAMPs Highlight the Developing Cell Plate during Cytokinesis in Tobacco BY-2 Cells

Lam, S. K., Cai, Y., Hillmer, S., Robinson, D. G., Jiang, L. — 2008-08-04

We previously demonstrated that rice (Oryza sativa) SECRETORY CARRIER MEMBRANE PROTEIN1 (OsSCAMP1)-yellow fluorescent protein in transgenic tobacco (Nicotiana tabacum) Bright Yellow-2 cells locates to the plasma membrane and to motile punctate structures, which represent the trans-Golgi network/early endosome and are tubular-vesicular in nature. Here, we now show that SCAMPs are diverted to the cell plate during cytokinesis dividing Bright Yellow-2 cells. As cells progress from metaphase to cytokinesis, punctate OsSCAMP1-labeled structures begin to collect in the future division plane. Together with the internalized endosomal marker FM4-64, they then become incorporated into the cell plate as it forms and expands. This was confirmed by immunogold electron microscopy. We also monitored for the Golgi apparatus and the prevacuolar compartment (PVC)/multivesicular body. Golgi stacks tend to accumulate in the vicinity of the division plane, but the signals are clearly separate to the cell plate. The situation with the PVC (labeled by green fluorescent protein-BP-80) is not so clear. Punctate BP-80 signals are seen at the advancing periphery of the cell plate, which was confirmed by immunogold electron microscopy. Specific but weak labeling was observed in the cell plate, but no evidence for a fusion of the PVC/multivesicular body with the cell plate could be obtained. Our data, therefore, support the notion that cell plate formation is mainly a secretory process involving mass incorporation of domains of the trans-Golgi network/early endosome membrane. We regard the involvement of multivesicular late endosomes in this process to be equivocal.

[RESEARCH ARTICLES] Magnitude and Direction of Vesicle Dynamics in Growing Pollen Tubes Using Spatiotemporal Image Correlation Spectroscopy and Fluorescence Recovery after Photobleaching

2008-08-04

The delivery of cell wall material and membrane to growing plant cell surfaces requires the spatial and temporal coordination of secretory vesicle trafficking. Given the small size of vesicles, their dynamics is difficult to quantify. To quantitatively analyze vesicle dynamics in growing pollen tubes labeled with the styryl dye FM1-43, we applied spatiotemporal correlation spectroscopy on time-lapse series obtained with high-speed confocal laser scanning microscopy recordings. The resulting vector maps revealed that vesicles migrate toward the apex in the cell cortex and that they accumulate in an annulus-shaped region adjacent to the extreme tip and then turn back to flow rearward in the center of the tube. Fluorescence recovery after photobleaching confirmed vesicle accumulation in the shoulder of the apex, and it revealed that the extreme apex never recovers full fluorescence intensity. This is consistent with endocytotic activity occurring in this region. Fluorescence recovery after photobleaching analysis also allowed us to measure the turnover rate of the apical vesicle population, which was significantly more rapid than the theoretical rate computed based on requirements for new cell wall material. This may indicate that a significant portion of the vesicles delivered to the apex does not succeed in contacting the plasma membrane for delivery of their contents. Therefore, we propose that more than one passage into the apex may be needed for many vesicles before they fuse to the plasma membrane and deliver their contents.

[RESEARCH ARTICLES] A Class I ADP-Ribosylation Factor GTPase-Activating Protein Is Critical for Maintaining Directional Root Hair Growth in Arabidopsis

Yoo, C.-M., Wen, J., Motes, C. M., Sparks, J. A., Blancaflor, E. B. — 2008-08-04

Membrane trafficking and cytoskeletal dynamics are important cellular processes that drive tip growth in root hairs. These processes interact with a multitude of signaling pathways that allow for the efficient transfer of information to specify the direction in which tip growth occurs. Here, we show that AGD1, a class I ADP ribosylation factor GTPase-activating protein, is important for maintaining straight growth in Arabidopsis (Arabidopsis thaliana) root hairs, since mutations in the AGD1 gene resulted in wavy root hair growth. Live cell imaging of growing agd1 root hairs revealed bundles of endoplasmic microtubules and actin filaments extending into the extreme tip. The wavy phenotype and pattern of cytoskeletal distribution in root hairs of agd1 partially resembled that of mutants in an armadillo repeat-containing kinesin (ARK1). Root hairs of double agd1 ark1 mutants were more severely deformed compared with single mutants. Organelle trafficking as revealed by a fluorescent Golgi marker was slightly inhibited, and Golgi stacks frequently protruded into the extreme root hair apex of agd1 mutants. Transient expression of green fluorescent protein-AGD1 in tobacco (Nicotiana tabacum) epidermal cells labeled punctate bodies that partially colocalized with the endocytic marker FM4-64, while ARK1-yellow fluorescent protein associated with microtubules. Brefeldin A rescued the phenotype of agd1, indicating that the altered activity of an AGD1-dependent ADP ribosylation factor contributes to the defective growth, organelle trafficking, and cytoskeletal organization of agd1 root hairs. We propose that AGD1, a regulator of membrane trafficking, and ARK1, a microtubule motor, are components of converging signaling pathways that affect cytoskeletal organization to specify growth orientation in Arabidopsis root hairs.

[RESEARCH ARTICLES] A Distinct Endosomal Ca2+/Mn2+ Pump Affects Root Growth through the Secretory Process

Li, X., Chanroj, S., Wu, Z., Romanowsky, S. M., Harper, J. F., Sze, H. — 2008-08-04

Ca²⁺ is required for protein processing, sorting, and secretion in eukaryotic cells, although the particular roles of the transporters involved in the secretory system of plants are obscure. One endomembrane-type Ca-ATPase from Arabidopsis (Arabidopsis thaliana), AtECA3, diverges from AtECA1, AtECA2, and AtECA4 in protein sequence; yet, AtECA3 appears similar in transport activity to the endoplasmic reticulum (ER)-bound AtECA1. Expression of AtECA3 in a yeast (Saccharomyces cerevisiae) mutant defective in its endogenous Ca²⁺ pumps conferred the ability to grow on Ca²⁺-depleted medium and tolerance to toxic levels of Mn²⁺. A green fluorescent protein-tagged AtECA3 was functionally competent and localized to intracellular membranes of yeast, suggesting that Ca²⁺ and Mn²⁺ loading into internal compartment(s) enhanced yeast proliferation. In mesophyll protoplasts, AtECA3-green fluorescent protein associated with a subpopulation of endosome/prevacuolar compartments based on partial colocalization with the Ara7 marker. Interestingly, three independent eca3 T-DNA disruption mutants showed severe reduction in root growth normally stimulated by 3 mm Ca²⁺, indicating that AtECA3 function cannot be replaced by an ER-associated AtECA1. Furthermore, root growth of mutants is sensitive to 50 µm Mn²⁺, indicating that AtECA3 is also important for the detoxification of excess Mn²⁺. Curiously, Ateca3 mutant roots produced 65% more apoplastic protein than wild-type roots, as monitored by peroxidase activity, suggesting that the secretory process was altered. Together, these results demonstrate that the role of AtECA3 is distinct from that of the more abundant ER AtECA1. AtECA3 supports Ca²⁺-stimulated root growth and the detoxification of high Mn²⁺, possibly through activities mediated by post-Golgi compartments that coordinate membrane traffic and sorting of materials to the vacuole and the cell wall.

[RESEARCH ARTICLES] Imaging of the Yellow Cameleon 3.6 Indicator Reveals That Elevations in Cytosolic Ca2+ Follow Oscillating Increases in Growth in Root Hairs of Arabidopsis

Monshausen, G. B., Messerli, M. A., Gilroy, S. — 2008-08-04

In tip-growing cells, the tip-high Ca²⁺ gradient is thought to regulate the activity of components of the growth machinery, including the cytoskeleton, Ca²⁺-dependent regulatory proteins, and the secretory apparatus. In pollen tubes, both the Ca²⁺ gradient and cell elongation show oscillatory behavior, reinforcing the link between the two. We report that in growing root hairs of Arabidopsis (Arabidopsis thaliana), an oscillating tip-focused Ca²⁺ gradient can be resolved through imaging of a cytosolically expressed Yellow Cameleon 3.6 fluorescence resonance energy transfer-based Ca²⁺ sensor. Both elongation of the root hairs and the associated tip-focused Ca²⁺ gradient show a similar dynamic character, oscillating with a frequency of 2 to 4 min^–1. Cross-correlation analysis indicates that the Ca²⁺ oscillations lag the growth oscillations by 5.3 ± 0.3 s. However, growth never completely stops, even during the slow cycle of an oscillation, and the concomitant tip Ca²⁺ level is always slightly elevated compared with the resting Ca²⁺ concentration along the distal shaft, behind the growing tip. Artificially increasing Ca²⁺ using the Ca²⁺ ionophore A23187 leads to immediate cessation of elongation and thickening of the apical cell wall. In contrast, dissipating the Ca²⁺ gradient using either the Ca²⁺ channel blocker La³⁺ or the Ca²⁺ chelator EGTA is accompanied by an increase in the rate of cell expansion and eventual bursting of the root hair tip. These observations are consistent with a model in which the maximal oscillatory increase in cytosolic Ca²⁺ is triggered by cell expansion associated with tip growth and plays a role in the subsequent restriction of growth.

[RESEARCH ARTICLES] Synthetic Lipid (DOPG) Vesicles Accumulate in the Cell Plate Region But Do Not Fuse

Esseling-Ozdoba, A., Vos, J. W., van Lammeren, A. A.M., Emons, A. M. C. — 2008-08-04

The cell plate is the new cell wall, with bordering plasma membrane, that is formed between two daughter cells in plants, and it is formed by fusion of vesicles (approximately 60 nm). To start to determine physical properties of cell plate forming vesicles for their transport through the phragmoplast, and fusion with each other, we microinjected fluorescent synthetic lipid vesicles that were made of 1,2-dioleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (DOPG) into Tradescantia virginiana stamen hair cells. During interphase, the 60-nm wide DOPG vesicles moved inside the cytoplasm comparably to organelles. During cytokinesis, they were transported through the phragmoplast and accumulated in the cell plate region together with the endogenous vesicles, even inside the central cell plate region. Because at this stage microtubules are virtually absent from that region, while actin filaments are present, actin filaments may have a role in the transport of vesicles toward the cell plate. Unlike the endogenous vesicles, the synthetic DOPG vesicles did not fuse with the developing cell plate. Instead, they redistributed into the cytoplasm of the daughter cells upon completion of cytokinesis. Because the redistribution of the vesicles occurs when actin filaments disappear from the phragmoplast, actin filaments may be involved in keeping the vesicles inside the developing cell plate region.

[RESEARCH ARTICLES] An Abscisic Acid-Induced Protein, HVA22, Inhibits Gibberellin-Mediated Programmed Cell Death in Cereal Aleurone Cells

Guo, W.-J., David Ho, T.-H. — 2008-08-04

Plant HVA22 is a unique abscisic acid (ABA)/stress-induced protein first isolated from barley (Hordeum vulgare) aleurone cells. Its yeast homolog, Yop1p, functions in vesicular trafficking and in the endoplasmic reticulum (ER) network in vivo. To examine the roles of plant HVA22, barley HVA22 was ectopically expressed in barley aleurone cells. Overexpression of HVA22 proteins inhibited gibberellin (GA)-induced formation of large digestive vacuoles, which is an important aspect of GA-induced programmed cell death in aleurone cells. The effect of HVA22 was specific, because overexpression of green fluorescent protein or another ABA-induced protein, HVA1, did not lead to the same effect. HVA22 acts downstream of the transcription factor GAMyb, which activates programmed cell death and other GA-mediated processes. Moreover, expression of HVA22:green fluorescent protein fusion proteins showed network and punctate fluorescence patterns, which were colocalized with an ER marker, BiP:RFP, and a Golgi marker, ST:mRFP, respectively. In particular, the transmembrane domain 2 was critical for protein localization and stability. Ectopic expression of the most phylogenetically similar Arabidopsis (Arabidopsis thaliana) homolog, AtHVA22D, also resulted in the inhibition of vacuolation to a similar level as HVA22, indicating function conservation between barley HVA22 and some Arabidopsis homologs. Taken together, we show that HVA22 is an ER- and Golgi-localized protein capable of negatively regulating GA-mediated vacuolation/programmed cell death in barley aleurone cells. We propose that ABA induces the accumulation of HVA22 proteins to inhibit vesicular trafficking involved in nutrient mobilization to delay coalescence of protein storage vacuoles as part of its role in regulating seed germination and seedling growth.

[RESEARCH ARTICLES] Genetic Evidence That Cellulose Synthase Activity Influences Microtubule Cortical Array Organization

Paredez, A. R., Persson, S., Ehrhardt, D. W., Somerville, C. R. — 2008-08-04

To identify factors that influence cytoskeletal organization we screened for Arabidopsis (Arabidopsis thaliana) mutants that show hypersensitivity to the microtubule destabilizing drug oryzalin. We cloned the genes corresponding to two of the 131 mutant lines obtained. The genes encoded mutant alleles of PROCUSTE1 and KORRIGAN, which both encode proteins that have previously been implicated in cellulose synthesis. Analysis of microtubules in the mutants revealed that both mutants have altered orientation of root cortical microtubules. Similarly, isoxaben, an inhibitor of cellulose synthesis, also altered the orientation of cortical microtubules while exogenous cellulose degradation did not. Thus, our results substantiate that proteins involved in cell wall biosynthesis influence cytoskeletal organization and indicate that this influence on cortical microtubule stability and orientation is correlated with cellulose synthesis rather than the integrity of the cell wall.

[RESEARCH ARTICLES] The PRA1 Gene Family in Arabidopsis

2008-08-04

Prenylated Rab acceptor 1 (PRA1) domain proteins are small transmembrane proteins that regulate vesicle trafficking as receptors of Rab GTPases and the vacuolar soluble N-ethylmaleimide-sensitive factor attachment receptor protein VAMP2. However, little is known about PRA1 family members in plants. Sequence analysis revealed that higher plants, compared with animals and primitive plants, possess an expanded family of PRA1 domain-containing proteins. The Arabidopsis (Arabidopsis thaliana) PRA1 (AtPRA1) proteins were found to homodimerize and heterodimerize in a manner corresponding to their phylogenetic distribution. Different AtPRA1 family members displayed distinct expression patterns, with a preference for vascular cells and expanding or developing tissues. AtPRA1 genes were significantly coexpressed with Rab GTPases and genes encoding vesicle transport proteins, suggesting an involvement in the vesicle trafficking process similar to that of their animal counterparts. Correspondingly, AtPRA1 proteins were localized in the endoplasmic reticulum, Golgi apparatus, and endosomes/prevacuolar compartments, hinting at a function in both secretory and endocytic intracellular trafficking pathways. Taken together, our data reveal a high functional diversity of AtPRA1 proteins, probably dealing with the various demands of the complex trafficking system.

[RESEARCH ARTICLES] Tracking Monolignols during Wood Development in Lodgepole Pine

2008-08-04

Secondary xylem (wood) formation in gymnosperms requires that the tracheid protoplasts first build an elaborate secondary cell wall from an array of polysaccharides and then reinforce it with lignin, an amorphous, three-dimensional product of the random radical coupling of monolignols. The objective of this study was to track the spatial distribution of monolignols during development as they move from symplasm to apoplasm. This was done by feeding [³H]phenylalanine ([³H]Phe) to dissected cambium/developing wood from lodgepole pine (Pinus contorta var latifolia) seedlings, allowing uptake and metabolism, then rapidly freezing the cells and performing autoradiography to detect the locations of the monolignols responsible for lignification. Parallel experiments showed that radioactivity was incorporated into polymeric lignin and a methanol-soluble pool that was characterized by high-performance liquid chromatography. [³H]Phe was incorporated into expected lignin precursors, such as coniferyl alcohol and p-coumaryl alcohol, as well as pinoresinol. Coniferin, the glucoside of coniferyl alcohol, was detected by high-performance liquid chromatography but was not radioactively labeled. With light microscopy, radiolabeled phenylpropanoids were detected in the rays as well as the tracheids, with the two cell types showing differential sensitivity to inhibitors of protein translation and phenylpropanoid metabolism. Secondary cell walls of developing tracheids were heavily labeled when incubated with [³H]Phe. Inside the cell, cytoplasm was most strongly labeled followed by Golgi and low-vacuole label. Inhibitor studies suggest that the Golgi signal could be attributed to protein, rather than phenylpropanoid, origins. These data, produced with the best microscopy tools that are available today, support a model in which unknown membrane transporters, rather than Golgi vesicles, export monolignols.

[ON THE INSIDE] On the Inside

Minorsky, P. V. — 2008-08-04

[GENOME ANALYSIS] Comparative Analysis of Divergent and Convergent Gene Pairs and Their Expression Patterns in Rice, Arabidopsis, and Populus

Krom, N., Ramakrishna, W. — 2008-08-04

Comparative analysis of the organization and expression patterns of divergent and convergent gene pairs in multiple plant genomes can identify patterns that are shared by more than one species or are unique to a particular species. Here, we study the coexpression and interspecies conservation of divergent and convergent gene pairs in three plant species: rice (Oryza sativa), Arabidopsis (Arabidopsis thaliana), and black cottonwood (Populus trichocarpa). Strongly correlated expression levels between divergent and convergent genes were found to be quite common in all three species, and the frequency of strong correlation appears to be independent of intergenic distance. Conservation of divergent or convergent arrangement among these species appears to be quite rare. However, conserved arrangement is significantly more frequent when the genes display strongly correlated expression levels or have one or more Gene Ontology (GO) classes in common. A correlation between intergenic distance in divergent and convergent gene pairs and shared GO classes was observed, in varying degrees, in rice and Populus but not in Arabidopsis. Furthermore, multiple GO classes were either overrepresented or underrepresented in Arabidopsis and Populus gene pairs, while only two GO classes were underrepresented in rice divergent gene pairs. Three cis-regulatory elements common to both Arabidopsis and rice were overrepresented in the intergenic regions of strongly correlated divergent gene pairs compared to those of noncorrelated pairs. Our results suggest that shared as well as unique mechanisms operate in shaping the organization and function of divergent and convergent gene pairs in different plant species.

[BREAKTHROUGH TECHNOLOGIES] Imaging Nutrient Distributions in Plant Tissue Using Time-of-Flight Secondary Ion Mass Spectrometry and Scanning Electron Microscopy

Metzner, R., Schneider, H. U., Breuer, U., Schroeder, W. H. — 2008-08-04

A new approach to trace the transport routes of macronutrients in plants at the level of cells and tissues and to measure their elemental distributions was developed for investigating the dynamics and structure-function relationships of transport processes. Stem samples from Phaseolus vulgaris were used as a test system. Shock freezing and cryo-preparation were combined in a cryogenic chain with cryo-time-of-flight secondary ion mass spectrometry (cryo-ToF-SIMS) for element and isotope-specific imaging. Cryo-scanning electron microscopy (cryo-SEM) was integrated into the cryogenic workflow to assess the quality of structural preservation. We evaluated the capability of these techniques to monitor transport pathways and processes in xylem and associated tissues using supplementary sodium (Na) and tracers for potassium (K), rubidium (Rb), and ⁴¹K added to the transpiration stream. Cryo-ToF-SIMS imaging produced detailed mappings of water, K, calcium, magnesium, the K tracers, and Na without quantification. Lateral resolutions ranged from 10 µm in survey mappings and at high mass resolution to approximately 1 µm in high lateral resolution imaging in reduced areas and at lower mass resolution. The tracers Rb and ⁴¹K, as well as Na, were imaged with high sensitivity in xylem vessels and surrounding tissues. The isotope signature of the stable isotope tracer was utilized for relative quantification of the ⁴¹K tracer as a fraction of total K at the single pixel level. Cryo-SEM confirmed that tissue structures had been preserved with subcellular detail throughout all procedures. Overlays of cryo-ToF-SIMS images onto the corresponding SEM images allowed detailed correlation of nutrient images with subcellular structures.

[BIOINFORMATICS] A Community-Based Annotation Framework for Linking Solanaceae Genomes with Phenomes

Menda, N., Buels, R. M., Tecle, I., Mueller, L. A. — 2008-08-04

The amount of biological data available in the public domain is growing exponentially, and there is an increasing need for infrastructural and human resources to organize, store, and present the data in a proper context. Model organism databases (MODs) invest great efforts to functionally annotate genomes and phenomes by in-house curators. The SOL Genomics Network (SGN; http://www.sgn.cornell.edu) is a clade-oriented database (COD), which provides a more scalable and comparative framework for biological information. SGN has recently spearheaded a new approach by developing community annotation tools to expand its curational capacity. These tools effectively allow some curation to be delegated to qualified researchers, while, at the same time, preserving the in-house curators' full editorial control. Here we describe the background, features, implementation, results, and development road map of SGN's community annotation tools for curating genotypes and phenotypes. Since the inception of this project in late 2006, interest and participation from the Solanaceae research community has been strong and growing continuously to the extent that we plan to expand the framework to accommodate more plant taxa. All data, tools, and code developed at SGN are freely available to download and adapt.

[SCIENTIFIC CORRESPONDENCE] Single-Stranded DNA-Binding Protein Whirly1 in Barley Leaves Is Located in Plastids and the Nucleus of the Same Cell

Grabowski, E., Miao, Y., Mulisch, M., Krupinska, K. — 2008-08-04

[BIOCHEMICAL PROCESSES AND MACROMOLECULAR STRUCTURES] Quantitative 1H Nuclear Magnetic Resonance Metabolite Profiling as a Functional Genomics Platform to Investigate Alkaloid Biosynthesis in Opium Poppy

Hagel, J. M., Weljie, A. M., Vogel, H. J., Facchini, P. J. — 2008-08-04

Opium poppy (Papaver somniferum) produces a diverse array of bioactive benzylisoquinoline alkaloids and has emerged as a versatile model system to study plant alkaloid metabolism. The plant is widely cultivated as the only commercial source of the narcotic analgesics morphine and codeine. Variations in plant secondary metabolism as a result of genetic diversity are often associated with perturbations in other metabolic pathways. As part of a functional genomics platform, we used ¹H nuclear magnetic resonance (NMR) metabolite profiling for the analysis of primary and secondary metabolism in opium poppy. Aqueous and chloroform extracts of six different opium poppy cultivars were subjected to chemometric analysis. Principle component analysis of the ¹H NMR spectra for latex extracts clearly distinguished two varieties, including a low-alkaloid variety and a high-thebaine, low-morphine cultivar. Distinction was also made between pharmaceutical-grade opium poppy cultivars and a condiment variety. Such phenotypic differences were not observed in root extracts. Loading plots confirmed that morphinan alkaloids contributed predominantly to the variance in latex extracts. Quantification of 34 root and 21 latex metabolites, performed using Chenomx NMR Suite version 4.6, showed major differences in the accumulation of specific alkaloids in the latex of the low-alkaloid and high-thebaine, low-morphine varieties. Relatively few differences were found in the levels of other metabolites, indicating that the variation was specific for alkaloid metabolism. Exceptions in the low-alkaloid cultivar included an increased accumulation of the alkaloid precursor tyramine and reduced levels of sucrose, some amino acids, and malate. Real-time polymerase chain reaction analysis of 42 genes involved in primary and secondary metabolism showed differential gene expression mainly associated with alkaloid biosynthesis. Reduced alkaloid levels in the condiment variety were associated with the reduced abundance of transcripts encoding several alkaloid biosynthetic enzymes.

[BIOCHEMICAL PROCESSES AND MACROMOLECULAR STRUCTURES] The Role of Acetyl-Coenzyme A Synthetase in Arabidopsis

Lin, M., Oliver, D. J. — 2008-08-04

The acs1 knockout mutant that has a disruption in the plastidic acetyl-coenzyme A (CoA) synthetase (ACS; At5g36880) gene was used to explore the role of this protein and plastidic acetate metabolism in Arabidopsis (Arabidopsis thaliana). Disruption of the ACS gene decreased ACS activity by 90% and largely blocked the incorporation of exogenous ¹⁴C-acetate and ¹⁴C-ethanol into fatty acids. Whereas the disruption had no significant effect on the synthesis of bulk seed triacylglycerols, the acs1 plants were smaller and flowered later. This suggests that the pyruvate dehydrogenase bypass provided by the aerobic fermentation pathway that converts pyruvate to acetate and probably on to fatty acids is important to the plants during normal growth. The role of ACS in destroying fermentative intermediates is supported by the increased sensitivity of the acs1 mutant to exogenous acetate, ethanol, and acetaldehyde compared to wild-type plants. Whereas these observations suggest that flux through the aerobic fermentation pathway is important, the reason for this flux is unclear. Interestingly, acetate is able to support high rates of plant growth on medium and this growth is blocked in the acs1 mutant.

[BIOCHEMICAL PROCESSES AND MACROMOLECULAR STRUCTURES] A Proteomic Profiling Approach to Reveal a Novel Role of Brassica napus Drought 22 kD/Water-Soluble Chlorophyll-Binding Protein in Young Leaves during Nitrogen Remobilization Induced by Stressful Conditions

2008-08-04

Despite its water-soluble chlorophyll-binding protein (WSCP) function, the putative trypsin inhibitor (TI) activity of the Brassica napus drought 22 kD (BnD22) protein and its physiological function in young leaves during leaf nitrogen (N) remobilization promoted by stressful conditions remains an enigma. Therefore, our objectives were to determine (1) if BnD22 is related to the 19-kD TI previously detected in B. napus young leaves, and (2) if the levels of BnD22 transcripts, BnD22 protein, and TI activity in young leaves are associated with plant responses to stress conditions (N starvation and methyl jasmonate [MeJA] treatments) that are able to modulate leaf senescence. Compared to control, N starvation delayed initiation of senescence and induced 19-kD TI activity in the young leaves. After 3 d with MeJA, the 19-kD TI activity was 7-fold higher than the control. Using two-dimensional electrophoresis gel, TI activity, and electrospray ionization liquid chromatography tandem mass spectrometry analysis, it was demonstrated that two 19-kD proteins with isoelectric points 5.0 and 5.1 harboring TI activity correspond to BnD22 perfectly. BnD22 gene expression, TI activities, and BnD22 protein presented similar patterns. Using polyclonal anti-WSCP antibodies of Brassica oleracea, six polypeptides separated by two-dimensional electrophoresis were detected in young leaves treated with MeJA. Electrospray ionization liquid chromatography tandem mass spectrometry analysis of six polypeptides confirms their homologies with WSCP. Results suggest that BnD22 possesses dual functions (WSCP and TI) that lead to the protection of younger tissues from adverse conditions by maintaining metabolism (protein integrity and photosynthesis). By sustaining sink growth of stressed plants, BnD22 may contribute to a better utilization of recycling N from sources, a physiological trait that improves N-use efficiency.

[BIOCHEMICAL PROCESSES AND MACROMOLECULAR STRUCTURES] Bridging the Gap between Plant and Mammalian Polyamine Catabolism: A Novel Peroxisomal Polyamine Oxidase Responsible for a Full Back-Conversion Pathway in Arabidopsis

2008-08-04

In contrast to animals, where polyamine (PA) catabolism efficiently converts spermine (Spm) to putrescine (Put), plants have been considered to possess a PA catabolic pathway producing 1,3-diaminopropane, ¹-pyrroline, the corresponding aldehyde, and hydrogen peroxide but unable to back-convert Spm to Put. Arabidopsis (Arabidopsis thaliana) genome contains at least five putative PA oxidase (PAO) members with yet-unknown localization and physiological role(s). AtPAO1 was recently identified as an enzyme similar to the mammalian Spm oxidase, which converts Spm to spermidine (Spd). In this work, we have performed in silico analysis of the five Arabidopsis genes and have identified PAO3 (AtPAO3) as a nontypical PAO, in terms of homology, compared to other known PAOs. We have expressed the gene AtPAO3 and have purified a protein corresponding to it using the inducible heterologous expression system of Escherichia coli. AtPAO3 catalyzed the sequential conversion/oxidation of Spm to Spd, and of Spd to Put, thus exhibiting functional homology to the mammalian PAOs. The best substrate for this pathway was Spd, whereas the N¹-acetyl-derivatives of Spm and Spd were oxidized less efficiently. On the other hand, no activity was detected when diamines (agmatine, cadaverine, and Put) were used as substrates. Moreover, although AtPAO3 does not exhibit significant similarity to the other known PAOs, it is efficiently inhibited by guazatine, a potent PAO inhibitor. AtPAO3 contains a peroxisomal targeting motif at the C terminus, and it targets green fluorescence protein to peroxisomes when fused at the N terminus but not at the C terminus. These results reveal that AtPAO3 is a peroxisomal protein and that the C terminus of the protein contains the sorting information. The overall data reinforce the view that plants and mammals possess a similar PA oxidation system, concerning both the subcellular localization and the mode of its action.

[BIOENERGETICS AND PHOTOSYNTHESIS] Identification of Regulatory Pathways Controlling Gene Expression of Stress-Responsive Mitochondrial Proteins in Arabidopsis

2008-08-04

In this study we analyzed transcript abundance and promoters of genes encoding mitochondrial proteins to identify signaling pathways that regulate stress-induced gene expression. We used Arabidopsis (Arabidopsis thaliana) alternative oxidase AOX1a, external NADP H-dehydrogenase NDB2, and two additional highly stress-responsive genes, At2g21640 and BCS1. As a starting point, the promoter region of AOX1a was analyzed and functional analysis identified 10 cis-acting regulatory elements (CAREs), which played a role in response to treatment with H₂O₂, rotenone, or both. Six of these elements were also functional in the NDB2 promoter. The promoter region of At2g21640, previously defined as a hallmark of oxidative stress, shared two functional CAREs with AOX1a and was responsive to treatment with H₂O₂ but not rotenone. Microarray analysis further supported that signaling pathways induced by H₂O₂ and rotenone are not identical. The promoter of BCS1 was not responsive to H₂O₂ or rotenone, but highly responsive to salicylic acid (SA), whereas the promoters of AOX1a and NDB2 were unresponsive to SA. Analysis of transcript abundance of these genes in a variety of defense signaling mutants confirmed that BCS1 expression is regulated in a different manner compared to AOX1a, NDB2, and At2g21640. These mutants also revealed a pathway associated with programmed cell death that regulated AOX1a in a manner distinct from the other genes. Thus, at least three distinctive pathways regulate mitochondrial stress response at a transcriptional level, an SA-dependent pathway represented by BCS1, a second pathway that represents a convergence point for signals generated by H₂O₂ and rotenone on multiple CAREs, some of which are shared between responsive genes, and a third pathway that acts via EDS1 and PAD4 regulating only AOX1a. Furthermore, posttranscriptional regulation accounts for changes in transcript abundance by SA treatment for some genes.

[CELL BIOLOGY AND SIGNAL TRANSDUCTION] Restoration of Mature Etiolated Cucumber Hypocotyl Cell Wall Susceptibility to Expansin by Pretreatment with Fungal Pectinases and EGTA in Vitro

Zhao, Q., Yuan, S., Wang, X., Zhang, Y., Zhu, H., Lu, C. — 2008-08-04

Mature plant cell walls lose their ability to expand and become unresponsive to expansin. This phenomenon is believed to be due to cross-linking of hemicellulose, pectin, or phenolic groups in the wall. By screening various hydrolytic enzymes, we found that pretreatment of nongrowing, heat-inactivated, basal cucumber (Cucumis sativus) hypocotyls with pectin lyase (Pel1) from Aspergillus japonicus could restore reconstituted exogenous expansin-induced extension in mature cell walls in vitro. Recombinant pectate lyase A (PelA) and polygalacturonase (PG) from Aspergillus spp. exhibited similar capacity to Pel1. Pel1, PelA, and PG also enhanced the reconstituted expansin-induced extension of the apical (elongating) segments of cucumber hypocotyls. However, the effective concentrations of PelA and PG for enhancing the reconstituted expansin-induced extension were greater in the apical segments than in the basal segments, whereas Pel1 behaved in the opposite manner. These data are consistent with distribution of more methyl-esterified pectin in cell walls of the apical segments and less esterified pectin in the basal segments. Associated with the degree of esterification of pectin, more calcium was found in cell walls of basal segments compared to apical segments. Pretreatment of the calcium chelator EGTA could also restore mature cell walls' susceptibility to expansin by removing calcium from mature cell walls. Because recombinant pectinases do not hydrolyze other wall polysaccharides, and endoglucanase, xylanase, and protease cannot restore the mature wall's extensibility, we can conclude that the pectin network, especially calcium-pectate bridges, may be the primary factor that determines cucumber hypocotyl mature cell walls' unresponsiveness to expansin.

[DEVELOPMENT AND HORMONE ACTION] The Arabidopsis Phosphatidylinositol 3-Kinase Is Important for Pollen Development

Lee, Y., Kim, E.-S., Choi, Y., Hwang, I., Staiger, C. J., Chung, Y.-Y., Lee, Y. — 2008-08-04

Phosphatidylinositol 3-kinase has been reported to be important for normal plant growth. To characterize the role of the enzyme further, we attempted to isolate Arabidopsis (Arabidopsis thaliana) plants that do not express the gene, but we could not recover homozygous mutant plants. The progeny of VPS34/vps34 heterozygous plants, harboring a T-DNA insertion, showed a segregation ratio of 1:1:0 for wild-type, heterozygous, and homozygous mutant plants, indicating a gametophytic defect. Genetic transmission analysis showed that the abnormal segregation ratio was due to failure to transmit the mutant allele through the male gametophyte. Microscopic observation revealed that 2-fold higher proportions of pollen grains in heterozygous plants than wild-type plants were dead or showed reduced numbers of nuclei. Many mature pollen grains from the heterozygous plants contained large vacuoles even until the mature pollen stage, whereas pollen from wild-type plants contained many small vacuoles beginning from the vacuolated pollen stage, which indicated that vacuoles in many of the heterozygous mutant pollen did not undergo normal fission after the first mitotic division. Taken together, our results suggest that phosphatidylinositol 3-kinase is essential for vacuole reorganization and nuclear division during pollen development.

[DEVELOPMENT AND HORMONE ACTION] A Comparison of Leaf and Petal Senescence in Wallflower Reveals Common and Distinct Patterns of Gene Expression and Physiology

2008-08-04

Petals and leaves share common evolutionary origins but perform very different functions. However, few studies have compared leaf and petal senescence within the same species. Wallflower (Erysimum linifolium), an ornamental species closely related to Arabidopsis (Arabidopsis thaliana), provide a good species in which to study these processes. Physiological parameters were used to define stages of development and senescence in leaves and petals and to align these stages in the two organs. Treatment with silver thiosulfate confirmed that petal senescence in wallflower is ethylene dependent, and treatment with exogenous cytokinin and 6-methyl purine, an inhibitor of cytokinin oxidase, suggests a role for cytokinins in this process. Subtractive libraries were created, enriched for wallflower genes whose expression is up-regulated during leaf or petal senescence, and used to create a microarray, together with 91 senescence-related Arabidopsis probes. Several microarray hybridization classes were observed demonstrating similarities and differences in gene expression profiles of these two organs. Putative functions were ascribed to 170 sequenced DNA fragments from the libraries. Notable similarities between leaf and petal senescence include a large proportion of remobilization-related genes, such as the cysteine protease gene SENESCENCE-ASSOCIATED GENE12 that was up-regulated in both tissues with age. Interesting differences included the up-regulation of chitinase and glutathione S-transferase genes in senescing petals while their expression remained constant or fell with age in leaves. Semiquantitative reverse transcription-polymerase chain reaction of selected genes from the suppression subtractive hybridization libraries revealed more complex patterns of expression compared with the array data.

[DEVELOPMENT AND HORMONE ACTION] The Relationship between Auxin Transport and Maize Branching

Gallavotti, A., Yang, Y., Schmidt, R. J., Jackson, D. — 2008-08-04

Maize (Zea mays) plants make different types of vegetative or reproductive branches during development. Branches develop from axillary meristems produced on the flanks of the vegetative or inflorescence shoot apical meristem. Among these branches are the spikelets, short grass-specific structures, produced by determinate axillary spikelet-pair and spikelet meristems. We investigated the mechanism of branching in maize by making transgenic plants expressing a native expressed endogenous auxin efflux transporter (ZmPIN1a) fused to yellow fluorescent protein and a synthetic auxin-responsive promoter (DR5rev) driving red fluorescent protein. By imaging these plants, we found that all maize branching events during vegetative and reproductive development appear to be regulated by the creation of auxin response maxima through the activity of polar auxin transporters. We also found that the auxin transporter ZmPIN1a is functional, as it can rescue the polar auxin transport defects of the Arabidopsis (Arabidopsis thaliana) pin1-3 mutant. Based on this and on the groundbreaking analysis in Arabidopsis and other species, we conclude that branching mechanisms are conserved and can, in addition, explain the formation of axillary meristems (spikelet-pair and spikelet meristems) that are unique to grasses. We also found that BARREN STALK1 is required for the creation of auxin response maxima at the flanks of the inflorescence meristem, suggesting a role in the initiation of polar auxin transport for axillary meristem formation. Based on our results, we propose a general model for branching during maize inflorescence development.

[DEVELOPMENT AND HORMONE ACTION] RETARDED GROWTH OF EMBRYO1, a New Basic Helix-Loop-Helix Protein, Expresses in Endosperm to Control Embryo Growth

2008-08-04

We have isolated two dominant mutants from screening approximately 50,000 RIKEN activation-tagging lines that have short inflorescence internodes. The activation T-DNAs were inserted near a putative basic helix-loop-helix (bHLH) gene and expression of this gene was increased in the mutant lines. Overexpression of this bHLH gene produced the original mutant phenotype, indicating it was responsible for the mutants. Specific expression was observed during seed development. The loss-of-function mutation of the RETARDED GROWTH OF EMBRYO1 (RGE1) gene caused small and shriveled seeds. The embryo of the loss-of-function mutant showed retarded growth after the heart stage although abnormal morphogenesis and pattern formation of the embryo and endosperm was not observed. We named this bHLH gene RGE1. RGE1 expression was determined in endosperm cells using the β-glucuronidase reporter gene and reverse transcription-polymerase chain reaction. Microarray and real-time reverse transcription-polymerase chain reaction analysis showed specific down-regulation of putative GDSL motif lipase genes in the rge1-1 mutant, indicating possible involvement of these genes in seed morphology. These data suggest that RGE1 expression in the endosperm at the heart stage of embryo development plays an important role in controlling embryo growth.

[DEVELOPMENT AND HORMONE ACTION] Arginase-Negative Mutants of Arabidopsis Exhibit Increased Nitric Oxide Signaling in Root Development

2008-08-04

Mutation of either arginase structural gene (ARGAH1 or ARGAH2 encoding arginine [Arg] amidohydrolase-1 and -2, respectively) resulted in increased formation of lateral and adventitious roots in Arabidopsis (Arabidopsis thaliana) seedlings and increased nitric oxide (NO) accumulation and efflux, detected by the fluorogenic traps 3-amino,4-aminomethyl-2',7'-difluorofluorescein diacetate and diamino-rhodamine-4M, respectively. Upon seedling exposure to the synthetic auxin naphthaleneacetic acid, NO accumulation was differentially enhanced in argah1-1 and argah2-1 compared with the wild type. In all genotypes, much 3-amino,4-aminomethyl-2',7'-difluorofluorescein diacetate fluorescence originated from mitochondria. The arginases are both localized to the mitochondrial matrix and closely related. However, their expression levels and patterns differ: ARGAH1 encoded the minor activity, and ARGAH1-driven β-glucuronidase (GUS) was expressed throughout the seedling; the ARGAH2::GUS expression pattern was more localized. Naphthaleneacetic acid increased seedling lateral root numbers (total lateral roots per primary root) in the mutants to twice the number in the wild type, consistent with increased internal NO leading to enhanced auxin signaling in roots. In agreement, argah1-1 and argah2-1 showed increased expression of the auxin-responsive reporter DR5::GUS in root tips, emerging lateral roots, and hypocotyls. We propose that Arg, or an Arg derivative, is a potential NO source and that reduced arginase activity in the mutants results in greater conversion of Arg to NO, thereby potentiating auxin action in roots. This model is supported by supplemental Arg induction of adventitious roots and increased NO accumulation in argah1-1 and argah2-1 versus the wild type.

[DEVELOPMENT AND HORMONE ACTION] Mutation of the Rice Narrow leaf1 Gene, Which Encodes a Novel Protein, Affects Vein Patterning and Polar Auxin Transport

2008-08-04

The size and shape of the plant leaf is an important agronomic trait. To understand the molecular mechanism governing plant leaf shape, we characterized a classic rice (Oryza sativa) dwarf mutant named narrow leaf1 (nal1), which exhibits a characteristic phenotype of narrow leaves. In accordance with reduced leaf blade width, leaves of nal1 contain a decreased number of longitudinal veins. Anatomical investigations revealed that the culms of nal1 also show a defective vascular system, in which the number and distribution pattern of vascular bundles are altered. Map-based cloning and genetic complementation analyses demonstrated that Nal1 encodes a plant-specific protein with unknown biochemical function. We provide evidence showing that Nal1 is richly expressed in vascular tissues and that mutation of this gene leads to significantly reduced polar auxin transport capacity. These results indicate that Nal1 affects polar auxin transport as well as the vascular patterns of rice plants and plays an important role in the control of lateral leaf growth.

[DEVELOPMENT AND HORMONE ACTION] Developmental Role and Auxin Responsiveness of Class III Homeodomain Leucine Zipper Gene Family Members in Rice

Itoh, J.-I., Hibara, K.-I., Sato, Y., Nagato, Y. — 2008-08-04

Members of the Class III homeodomain leucine zipper (Class III HD-Zip) gene family are central regulators of crucial aspects of plant development. To better understand the roles of five Class III HD-Zip genes in rice (Oryza sativa) development, we investigated their expression patterns, ectopic expression phenotypes, and auxin responsiveness. Four genes, OSHB1 to OSHB4, were expressed in a localized domain of the shoot apical meristem (SAM), the adaxial cells of leaf primordia, the leaf margins, and the xylem tissue of vascular bundles. In contrast, expression of OSHB5 was observed only in phloem tissue. Plants ectopically expressing microRNA166-resistant versions of the OSHB3 gene exhibited severe defects, including the ectopic production of leaf margins, shoots, and radialized leaves. The treatment of seedlings with auxin quickly induced ectopic OSHB3 expression in the entire region of the SAM, but not in other tissues. Furthermore, this ectopic expression of OSHB3 was correlated with leaf initiation defects. Our findings suggest that rice Class III HD-Zip genes have conserved functions with their homologs in Arabidopsis (Arabidopsis thaliana), but have also acquired specific developmental roles in grasses or monocots. In addition, some Class III HD-Zip genes may regulate the leaf initiation process in the SAM in an auxin-dependent manner.

[ENVIRONMENTAL STRESS AND ADAPTATION TO STRESS] Selectable Tolerance to Herbicides by Mutated Acetolactate Synthase Genes Integrated into the Chloroplast Genome of Tobacco

2008-08-04

Strategies employed for the production of genetically modified (GM) crops are premised on (1) the avoidance of gene transfer in the field; (2) the use of genes derived from edible organisms such as plants; (3) preventing the appearance of herbicide-resistant weeds; and (4) maintaining transgenes without obstructing plant cell propagation. To this end, we developed a novel vector system for chloroplast transformation with acetolactate synthase (ALS). ALS catalyzes the first step in the biosynthesis of the branched amino acids, and its enzymatic activity is inhibited by certain classes of herbicides. We generated a series of Arabidopsis (Arabidopsis thaliana) mutated ALS (mALS) genes and introduced constructs with mALS and the aminoglycoside 3'-adenyltransferase gene (aadA) into the tobacco (Nicotiana tabacum) chloroplast genome by particle bombardment. Transplastomic plants were selected using their resistance to spectinomycin. The effects of herbicides on transplastomic mALS activity were examined by a colorimetric assay using the leaves of transplastomic plants. We found that transplastomic G121A, A122V, and P197S plants were specifically tolerant to pyrimidinylcarboxylate, imidazolinon, and sulfonylurea/pyrimidinylcarboxylate herbicides, respectively. Transplastomic plants possessing mALSs were able to grow in the presence of various herbicides, thus affirming the relationship between mALSs and the associated resistance to herbicides. Our results show that mALS genes integrated into the chloroplast genome are useful sustainable markers that function to exclude plants other than those that are GM while maintaining transplastomic crops. This investigation suggests that the resistance management of weeds in the field amid growing GM crops is possible using (1) a series of mALSs that confer specific resistance to herbicides and (2) a strategy that employs herbicide rotation.

[ENVIRONMENTAL STRESS AND ADAPTATION TO STRESS] Drought Induction of Arabidopsis 9-cis-Epoxycarotenoid Dioxygenase Occurs in Vascular Parenchyma Cells

2008-08-04

The regulation of abscisic acid (ABA) biosynthesis is essential for plant responses to drought stress. In this study, we examined the tissue-specific localization of ABA biosynthetic enzymes in turgid and dehydrated Arabidopsis (Arabidopsis thaliana) plants using specific antibodies against 9-cis-epoxycarotenoid dioxygenase 3 (AtNCED3), AtABA2, and Arabidopsis aldehyde oxidase 3 (AAO3). Immunohistochemical analysis revealed that in turgid plants, AtABA2 and AAO3 proteins were localized in vascular parenchyma cells most abundantly at the boundary between xylem and phloem bundles, but the AtNCED3 protein was undetectable in these tissues. In water-stressed plants, AtNCED3 was detected exclusively in the vascular parenchyma cells together with AtABA2 and AAO3. In situ hybridization using the antisense probe for AtNCED3 showed that the drought-induced expression of AtNCED3 was also restricted to the vascular tissues. Expression analysis of laser-microdissected cells revealed that, among nine drought-inducible genes examined, the early induction of most genes was spatially restricted to vascular cells at 1 h and then some spread to mesophyll cells at 3 h. The spatial constraint of AtNCED3 expression in vascular tissues provides a novel insight into plant systemic response to drought stresses.

[ENVIRONMENTAL STRESS AND ADAPTATION TO STRESS] Characterization of Single and Double Inactivation Strains Reveals New Physiological Roles for Group 2 {sigma} Factors in the Cyanobacterium Synechocystis sp. PCC 6803

2008-08-04

Cyanobacteria are eubacteria that perform oxygenic photosynthesis like plants. The initiation of transcription, mediated by the RNA polymerase holoenzyme, is the main determinant of gene regulation in eubacteria. The factor of the RNA polymerase holoenzyme is responsible for the recognition of a promoter sequence. In the cyanobacterium Synechocystis sp. PCC 6803, the primary factor, SigA, is essential for cell viability. The SigB, SigC, SigD, and SigE factors show significant sequence similarity with the SigA factor but are nonessential. In this study, we have used homology modeling to construct a three-dimensional model of Synechocystis RNA polymerase holoenzyme and all group 1 and 2 factors. According to the models, the overall three-dimensional structures of group 1 and 2 factors are similar, the SigB and SigD factors being the most similar ones. In addition, we have constructed a complete set of group 2 factor double inactivation strains, sigBC, sigBD, sigBE, sigCD, sigCE, and sigDE. All double mutants grow well under standard conditions, but differences are observed in stress conditions. The transition from lag phase to exponential growth is slow in the sigBD strain, and all strains lacking the SigD factor were found to be sensitive to bright light. Furthermore, all group 2 factors were found to be involved in acclimation to salt- or sorbitol-induced osmotic stresses.

[ENVIRONMENTAL STRESS AND ADAPTATION TO STRESS] Investigation of Heavy Metal Hyperaccumulation at the Cellular Level: Development and Characterization of Thlaspi caerulescens Suspension Cell Lines

Klein, M. A., Sekimoto, H., Milner, M. J., Kochian, L. V. — 2008-08-04

The ability of Thlaspi caerulescens, a zinc (Zn)/cadmium (Cd) hyperaccumulator, to accumulate extremely high foliar concentrations of toxic heavy metals requires coordination of uptake, transport, and sequestration to avoid damage to the photosynthetic machinery. The study of these metal hyperaccumulation processes at the cellular level in T. caerulescens has been hampered by the lack of a cellular system that mimics the whole plant, is easily transformable, and competent for longer term studies. Therefore, to better understand the contribution of the cellular physiology and molecular biology to Zn/Cd hyperaccumulation in the intact plant, T. caerulescens suspension cell lines were developed. Differences in cellular metal tolerance and accumulation between the cell lines of T. caerulescens and the related nonhyperaccumulator, Arabidopsis (Arabidopsis thaliana), were examined. A number of Zn/Cd transport-related differences between T. caerulescens and Arabidopsis cell lines were identified that also are seen in the whole plant. T. caerulescens suspension cell lines exhibited: (1) higher growth requirements for Zn; (2) much greater Zn and Cd tolerance; (3) enhanced expression of specific metal transport-related genes; and (4) significant differences in metal fluxes compared with Arabidopsis. One interesting feature exhibited by the T. caerulescens cell lines was that they accumulated less Zn and Cd than the Arabidopsis cell lines, most likely due to a greater metal efflux. This finding suggests that the T. caerulescens suspension cells represent cells of the Zn/Cd transport pathway between the root epidermis and leaf. We also show it is possible to stably transform T. caerulescens suspension cells, which will allow us to alter the expression of candidate hyperaccumulation genes and thus dissect the molecular and physiological processes underlying metal hyperaccumulation in T. caerulescens.

[PLANTS INTERACTING WITH OTHER ORGANISMS] Glycerol-3-Phosphate Levels Are Associated with Basal Resistance to the Hemibiotrophic Fungus Colletotrichum higginsianum in Arabidopsis

2008-08-04

Glycerol-3-phosphate (G3P) is an important component of carbohydrate and lipid metabolic processes. In this article, we provide evidence that G3P levels in plants are associated with defense to a hemibiotrophic fungal pathogen Colletotrichum higginsianum. Inoculation of Arabidopsis (Arabidopsis thaliana) with C. higginsianum was correlated with an increase in G3P levels and a concomitant decrease in glycerol levels in the host. Plants impaired in utilization of plastidial G3P (act1) accumulated elevated levels of pathogen-induced G3P and displayed enhanced resistance. Furthermore, overexpression of the host GLY1 gene, which encodes a G3P dehydrogenase (G3Pdh), conferred enhanced resistance. In contrast, the gly1 mutant accumulated reduced levels of G3P after pathogen inoculation and showed enhanced susceptibility to C. higginsianum. Unlike gly1, a mutation in a cytosolic isoform of G3Pdh did not alter basal resistance to C. higginsianum. Furthermore, act1 gly1 double-mutant plants were as susceptible as the gly1 plants. Increased resistance or susceptibility of act1 and gly1 plants to C. higginsianum, respectively, was not due to effects of these mutations on salicylic acid- or ethylene-mediated defense pathways. The act1 mutation restored a wild-type-like response in camalexin-deficient pad3 plants, which were hypersusceptible to C. higginsianum. These data suggest that G3P-associated resistance to C. higginsianum occurs independently or downstream of the camalexin pathway. Together, these results suggest a novel and specific link between G3P metabolism and plant defense.

[PLANTS INTERACTING WITH OTHER ORGANISMS] A Positive Regulatory Role for LjERF1 in the Nodulation Process Is Revealed by Systematic Analysis of Nodule-Associated Transcription Factors of Lotus japonicus

Asamizu, E., Shimoda, Y., Kouchi, H., Tabata, S., Sato, S. — 2008-08-04

We have used reverse genetics to identify genes involved in legume-rhizobium symbiosis in Lotus japonicus. We obtained the sequences of 20 putative transcription factors from previously reported large-scale transcriptome data. The transcription factors were classified according to their DNA binding domains and patterns of expression during the nodulation process. We identified two homologues of Medicago truncatula MtHAP2-1, which encodes a CCAAT-binding protein and has been shown to play a role in nodulation. The functions of the remaining genes in the nodulation process have not been reported. Seven genes were found to encode proteins with AP2-EREBP domains, six of which were similar to proteins that have been implicated in ethylene and/or jasmonic acid signal transduction and defense gene regulation in Arabidopsis (Arabidopsis thaliana). We identified a gene, LjERF1, that is most similar to Arabidopsis ERF1, which is up-regulated by ethylene and jasmonic acid and activates downstream defense genes. LjERF1 showed the same pattern of up-regulation in roots as Arabidopsis ERF1. The nodulation phenotype of roots that overexpressed LjERF1 or inhibited LjERF1 expression using an RNA interference construct indicated that this gene functions as a positive regulator of nodulation. We propose that LjERF1 functions as a key regulator of successful infection of L. japonicus by Mesorhizobium loti.