The Plant Cell recent issues

Epistasis and Genetic Regulation of Variation in the Arabidopsis Metabolome

Eckardt, N. A. — 2008-06-30

Newly Isolated Circadian Clock Components Conserved across Eukaryotes

Mach, J. — 2008-06-30

An Exocyst Vesicle Tethering Complex in Plants

Eckardt, N. A. — 2008-06-30

The Scientific Roots of Modern Plant Biotechnology

Sussex, I. M. — 2008-06-30

Biochemical Networks and Epistasis Shape the Arabidopsis thaliana Metabolome

Rowe, H. C., Hansen, B. G., Halkier, B. A., Kliebenstein, D. J. — 2008-06-30

Genomic approaches have accelerated the study of the quantitative genetics that underlie phenotypic variation. These approaches associate genome-scale analyses such as transcript profiling with targeted phenotypes such as measurements of specific metabolites. Additionally, these approaches can help identify uncharacterized networks or pathways. However, little is known about the genomic architecture underlying data sets such as metabolomics or the potential of such data sets to reveal networks. To describe the genetic regulation of variation in the Arabidopsis thaliana metabolome and test our ability to integrate unknown metabolites into biochemical networks, we conducted a replicated metabolomic analysis on 210 lines of an Arabidopsis population that was previously used for targeted metabolite quantitative trait locus (QTL) and global expression QTL analysis. Metabolic traits were less heritable than the average transcript trait, suggesting that there are differences in the power to detect QTLs between transcript and metabolite traits. We used statistical analysis to identify a large number of metabolite QTLs with moderate phenotypic effects and found frequent epistatic interactions controlling a majority of the variation. The distribution of metabolite QTLs across the genome included 11 QTL clusters; 8 of these clusters were associated in an epistatic network that regulated plant central metabolism. We also generated two de novo biochemical network models from the available data, one of unknown function and the other associated with central plant metabolism.

Signals Derived from YABBY Gene Activities in Organ Primordia Regulate Growth and Partitioning of Arabidopsis Shoot Apical Meristems

Goldshmidt, A., Alvarez, J. P., Bowman, J. L., Eshed, Y. — 2008-06-30

Shoot apical meristems (SAMs) are self-sustaining groups of cells responsible for the ordered initiation of all aerial plant tissues, including stems and lateral organs. The precise coordination of these processes argues for crosstalk between the different SAM domains. The products of YABBY (YAB) genes are limited to the organ primordium domains, which are situated at the periphery of all SAMs and which are separated by a margin of three to seven cells from the central meristem zone marked by WUSCHEL and CLAVATA3 expression. Mutations in the two related YAB1 genes, FILAMENTOUS FLOWER and YABBY3 (YAB3), cause an array of defects, including aberrant phyllotaxis. We show that peripheral YAB1 activity nonautonomously and sequentially affects the phyllotaxis and growth of subsequent primordia and coordinates the expression of SAM central zone markers. These effects support a role for YAB1 genes in short-range signaling. However, no evidence was found that YAB1 gene products are themselves mobile. A screen for suppression of a floral YAB1 overexpression phenotype revealed that the YAB1-born signals are mediated in part by the activity of LATERAL SUPPRESSOR. This GRAS protein is expressed at the boundary of organ primordia and the SAM central zone, distinct from the YAB1 expression domain. Together, these results suggest that YAB1 activity stimulates signals from the organs to the meristem via a secondary message or signal cascade, a process essential for organized growth of the SAM.

Dual Effects of miR156-Targeted SPL Genes and CYP78A5/KLUH on Plastochron Length and Organ Size in Arabidopsis thaliana

Wang, J.-W., Schwab, R., Czech, B., Mica, E., Weigel, D. — 2008-06-30

Leaves of flowering plants are produced from the shoot apical meristem at regular intervals, with the time that elapses between the formation of two successive leaf primordia defining the plastochron. We have identified two genetic axes affecting plastochron length in Arabidopsis thaliana. One involves microRNA156 (miR156), which targets a series of SQUAMOSA PROMOTER BINDING PROTEIN-LIKE (SPL) genes. In situ hybridization studies and misexpression experiments demonstrate that miR156 is a quantitative, rather than spatial, modulator of SPL expression in leaf primordia and that SPL activity nonautonomously inhibits initiation of new leaves at the shoot apical meristem. The second axis is exemplified by a redundantly acting pair of cytochrome P450 genes, CYP78A5/KLUH and CYP78A7, which are likely orthologs of PLASTOCHRON1 of rice (Oryza sativa). Inactivation of CYP78A5, which is expressed at the periphery of the shoot apical meristem, accelerates the leaf initiation rate, whereas cyp78a5 cyp78a7 double mutants often die as embryos with supernumerary cotyledon primordia. The effects of both miR156-targeted SPL genes and CYP78A5 on organ size are correlated with changes in plastochron length, suggesting a potential compensatory mechanism that links the rate at which leaves are produced to final leaf size.

XAP5 CIRCADIAN TIMEKEEPER Coordinates Light Signals for Proper Timing of Photomorphogenesis and the Circadian Clock in Arabidopsis

Martin-Tryon, E. L., Harmer, S. L. — 2008-06-30

Numerous, varied, and widespread taxa have an internal circadian clock that allows anticipation of rhythmic changes in the environment. We have identified XAP5 CIRCADIAN TIMEKEEPER (XCT), an Arabidopsis thaliana gene important for light regulation of the circadian clock and photomorphogenesis. XCT is essential for proper clock function: xct mutants display a shortened circadian period in all conditions tested. Interestingly, XCT plays opposite roles in plant responses to light depending both on trait and wavelength. The clock in xct plants is hypersensitive to red but shows normal responses to blue light. By contrast, inhibition of hypocotyl elongation in xct is hyposensitive to red light but hypersensitive to blue light. Finally, XCT is important for ribulose-1,5-bisphosphate carboxylase/oxygenase production and plant greening in response to light. This novel combination of phenotypes suggests XCT may play a global role in coordinating growth in response to the light environment. XCT contains a XAP5 domain and is well conserved across diverse taxa, suggesting it has a common function in higher eukaryotes. Downregulation of the XCT ortholog in Caenorhabditis elegans is lethal, suggesting that studies in Arabidopsis may be instrumental to understanding the biochemical activity of XCT.

SOMNUS, a CCCH-Type Zinc Finger Protein in Arabidopsis, Negatively Regulates Light-Dependent Seed Germination Downstream of PIL5

2008-06-30

Light absorbed by seed phytochromes of Arabidopsis thaliana modulates abscisic acid (ABA) and gibberellic acid (GA) signaling pathways at least partly via PHYTOCHROME-INTERACTING FACTOR3-LIKE5 (PIL5), a phytochrome-interacting basic helix-loop-helix transcription factor. Here, we report a new mutant, somnus (som), that germinates in darkness, independently of various light regimens. SOM encodes a nucleus-localized CCCH-type zinc finger protein. The som mutant has lower levels of ABA and elevated levels of GA due to expressional changes in ABA and GA metabolic genes. Unlike PIL5, however, SOM does not regulate the expression of GA-INSENSITIVE and REPRESSOR OF GA1 (RGA/RGA1), two DELLA genes encoding GA negative signaling components. Our in vivo analysis shows that PIL5 activates the expression of SOM by binding directly to its promoter, suggesting that PIL5 regulates ABA and GA metabolic genes partly through SOM. In agreement with these results, we also observed that the reduced germination frequency of a PIL5 overexpression line is rescued by the som mutation and that this rescue is accompanied by expressional changes in ABA and GA metabolic genes. Taken together, our results indicate that SOM is a component in the phytochrome signal transduction pathway that regulates hormone metabolic genes downstream of PIL5 during seed germination.

MTA Is an Arabidopsis Messenger RNA Adenosine Methylase and Interacts with a Homolog of a Sex-Specific Splicing Factor

Zhong, S., Li, H., Bodi, Z., Button, J., Vespa, L., Herzog, M., Fray, R. G. — 2008-06-30

N⁶-Methyladenosine is a ubiquitous modification identified in the mRNA of numerous eukaryotes, where it is present within both coding and noncoding regions. However, this base modification does not alter the coding capacity, and its biological significance remains unclear. We show that Arabidopsis thaliana mRNA contains N⁶-methyladenosine at levels similar to those previously reported for animal cells. We further show that inactivation of the Arabidopsis ortholog of the yeast and human mRNA adenosine methylase (MTA) results in failure of the developing embryo to progress past the globular stage. We also demonstrate that the arrested seeds are deficient in mRNAs containing N⁶-methyladenosine. Expression of MTA is strongly associated with dividing tissues, particularly reproductive organs, shoot meristems, and emerging lateral roots. Finally, we show that MTA interacts in vitro and in vivo with At FIP37, a homolog of the Drosophila protein FEMALE LETHAL2D and of human WILMS' TUMOUR1-ASSOCIATING PROTEIN. The results reported here provide direct evidence for an essential function for N⁶-methyladenosine in a multicellular eukaryote, and the interaction with At FIP37 suggests possible RNA processing events that might be regulated or altered by this base modification.

Identification of a Xylogalacturonan Xylosyltransferase Involved in Pectin Biosynthesis in Arabidopsis

2008-06-30

Xylogalacturonan (XGA) is a class of pectic polysaccharide found in plant cell walls. The Arabidopsis thaliana locus At5g33290 encodes a predicted Type II membrane protein, and insertion mutants of the At5g33290 locus had decreased cell wall xylose. Immunological studies, enzymatic extraction of polysaccharides, monosaccharide linkage analysis, and oligosaccharide mass profiling were employed to identify the affected cell wall polymer. Pectic XGA was reduced to much lower levels in mutant than in wild-type leaves, indicating a role of At5g33290 in XGA biosynthesis. The mutated gene was designated xylogalacturonan deficient1 (xgd1). Transformation of the xgd1-1 mutant with the wild-type gene restored XGA to wild-type levels. XGD1 protein heterologously expressed in Nicotiana benthamiana catalyzed the transfer of xylose from UDP-xylose onto oligogalacturonides and endogenous acceptors. The products formed could be hydrolyzed with an XGA-specific hydrolase. These results confirm that the XGD1 protein is a XGA xylosyltransferase. The protein was shown by expression of a fluorescent fusion protein in N. benthamiana to be localized in the Golgi vesicles as expected for a glycosyltransferase involved in pectin biosynthesis.

A Mutant Impaired in the Production of Plastome-Encoded Proteins Uncovers a Mechanism for the Homeostasis of Isoprenoid Biosynthetic Enzymes in Arabidopsis Plastids

2008-06-30

The plastid-localized methylerythritol phosphate (MEP) pathway synthesizes the isoprenoid precursors for the production of essential photosynthesis-related compounds and hormones. We have identified an Arabidopsis thaliana mutant, rif1, in which posttranscriptional upregulation of MEP pathway enzyme levels is caused by the loss of function of At3g47450, a gene originally reported to encode a mitochondrial protein related to nitric oxide synthesis. However, we show that nitric oxide is not involved in the regulation of the MEP pathway and that the encoded protein is a plastid-targeted homolog of the Bacillus subtilis YqeH protein, a GTPase required for proper ribosome assembly. Consistently, in rif1 seedlings, decreased levels of plastome-encoded proteins were observed, with the exception of ClpP1, a catalytic subunit of the plastidial Clp protease complex. The unexpected accumulation of ClpP1 in plastids with reduced protein synthesis suggested a compensatory mechanism in response to decreased Clp activity levels. In agreement, a negative correlation was found between Clp protease activity and MEP pathway enzyme levels in different experiments, suggesting that Clp-mediated degradation of MEP pathway enzymes might be a mechanism used by individual plastids to finely adjust plastidial isoprenoid biosynthesis to their functional and physiological states.

Mutation of a Rice Gene Encoding a Phenylalanine Biosynthetic Enzyme Results in Accumulation of Phenylalanine and Tryptophan

2008-06-30

Two distinct biosynthetic pathways for Phe in plants have been proposed: conversion of prephenate to Phe via phenylpyruvate or arogenate. The reactions catalyzed by prephenate dehydratase (PDT) and arogenate dehydratase (ADT) contribute to these respective pathways. The Mtr1 mutant of rice (Oryza sativa) manifests accumulation of Phe, Trp, and several phenylpropanoids, suggesting a link between the synthesis of Phe and Trp. Here, we show that the Mtr1 mutant gene (mtr1-D) encodes a form of rice PDT with a point mutation in the putative allosteric regulatory region of the protein. Transformed callus lines expressing mtr1-D exhibited all the characteristics of Mtr1 callus tissue. Biochemical analysis revealed that rice PDT possesses both PDT and ADT activities, with a preference for arogenate as substrate, suggesting that it functions primarily as an ADT. The wild-type enzyme is feedback regulated by Phe, whereas the mutant enzyme showed a reduced feedback sensitivity, resulting in Phe accumulation. In addition, these observations indicate that rice PDT is critical for regulating the size of the Phe pool in plant cells. Feeding external Phe to wild-type callus tissue and seedlings resulted in Trp accumulation, demonstrating a connection between Phe accumulation and Trp pool size.

An Exocyst Complex Functions in Plant Cell Growth in Arabidopsis and Tobacco

2008-06-30

The exocyst, an octameric tethering complex and effector of Rho and Rab GTPases, facilitates polarized secretion in yeast and animals. Recent evidence implicates three plant homologs of exocyst subunits (SEC3, SEC8, and EXO70A1) in plant cell morphogenesis. Here, we provide genetic, cell biological, and biochemical evidence that these and other predicted subunits function together in vivo in Arabidopsis thaliana. Double mutants in exocyst subunits (sec5 exo70A1 and sec8 exo70A1) show a synergistic defect in etiolated hypocotyl elongation. Mutants in exocyst subunits SEC5, SEC6, SEC8, and SEC15a show defective pollen germination and pollen tube growth phenotypes. Using antibodies directed against SEC6, SEC8, and EXO70A1, we demonstrate colocalization of these proteins at the apex of growing tobacco pollen tubes. The SEC3, SEC5, SEC6, SEC8, SEC10, SEC15a, and EXO70 subunits copurify in a high molecular mass fraction of 900 kD after chromatographic fractionation of an Arabidopsis cell suspension extract. Blue native electrophoresis confirmed the presence of SEC3, SEC6, SEC8, and EXO70 in high molecular mass complexes. Finally, use of the yeast two-hybrid system revealed interaction of Arabidopsis SEC3a with EXO70A1, SEC10 with SEC15b, and SEC6 with SEC8. We conclude that the exocyst functions as a complex in plant cells, where it plays important roles in morphogenesis.

Dual Fatty Acyl Modification Determines the Localization and Plasma Membrane Targeting of CBL/CIPK Ca2+ Signaling Complexes in Arabidopsis

Batistic, O., Sorek, N., Schultke, S., Yalovsky, S., Kudla, J. — 2008-06-30

Arabidopsis thaliana calcineurin B–like proteins (CBLs) interact specifically with a group of CBL-interacting protein kinases (CIPKs). CBL/CIPK complexes phosphorylate target proteins at the plasma membrane. Here, we report that dual lipid modification is required for CBL1 function and for localization of this calcium sensor at the plasma membrane. First, myristoylation targets CBL1 to the endoplasmic reticulum. Second, S-acylation is crucial for endoplasmic reticulum-to-plasma membrane trafficking via a novel cellular targeting pathway that is insensitive to brefeldin A. We found that a 12–amino acid peptide of CBL1 is sufficient to mediate dual lipid modification and to confer plasma membrane targeting. Moreover, the lipid modification status of the calcium sensor moiety determines the cellular localization of preassembled CBL/CIPK complexes. Our findings demonstrate the importance of S-acylation for regulating the spatial accuracy of Ca²⁺-decoding proteins and suggest a novel mechanism that enables the functional specificity of calcium sensor/kinase complexes.

Dynamics of Arabidopsis Dynamin-Related Protein 1C and a Clathrin Light Chain at the Plasma Membrane

Konopka, C. A., Backues, S. K., Bednarek, S. Y. — 2008-06-30

Plant morphogenesis depends on polarized exocytic and endocytic membrane trafficking. Members of the Arabidopsis thaliana dynamin-related protein 1 (DRP1) subfamily are required for polarized cell expansion and cytokinesis. Using a combination of live-cell imaging techniques, we show that a functional DRP1C green fluorescent fusion protein (DRP1C-GFP) was localized at the division plane in dividing cells and to the plasma membrane in expanding interphase cells. In both tip growing root hairs and diffuse-polar expanding epidermal cells, DRP1C-GFP organized into dynamic foci at the cell cortex, which colocalized with a clathrin light chain fluorescent fusion protein (CLC-FFP), suggesting that DRP1C may participate in clathrin-mediated membrane dynamics. DRP1C-GFP and CLC-GFP foci dynamics are dependent on cytoskeleton organization, cytoplasmic streaming, and functional clathrin-mediated endocytic traffic. Our studies provide insight into DRP1 and clathrin dynamics in the plant cell cortex and indicate that the clathrin endocytic machinery in plants has both similarities and striking differences to that in mammalian cells and yeast.

A Transporter Regulating Silicon Distribution in Rice Shoots

Yamaji, N., Mitatni, N., Ma, J. F. — 2008-06-30

Rice (Oryza sativa) accumulates very high concentrations of silicon (Si) in the shoots, and the deposition of Si as amorphous silica helps plants to overcome biotic and abiotic stresses. Here, we describe a transporter, Lsi6, which is involved in the distribution of Si in the shoots. Lsi6 belongs to the nodulin-26 intrinsic protein III subgroup of aquaporins and is permeable to silicic acid. Lsi6 is expressed in the leaf sheath and leaf blades as well as in the root tips. Cellular localization studies revealed that Lsi6 is found in the xylem parenchyma cells of the leaf sheath and leaf blades. Moreover, Lsi6 showed polar localization at the side facing toward the vessel. Knockdown of Lsi6 did not affect the uptake of Si by the roots but resulted in disordered deposition of silica in the shoots and increased excretion of Si in the guttation fluid. These results indicate that Lsi6 is a transporter responsible for the transport of Si out of the xylem and subsequently affects the distribution of Si in the leaf.

MAPK Signaling Regulates Nitric Oxide and NADPH Oxidase-Dependent Oxidative Bursts in Nicotiana benthamiana

Asai, S., Ohta, K., Yoshioka, H. — 2008-06-30

Nitric oxide (NO) and reactive oxygen species (ROS) act as signals in innate immunity in plants. The radical burst is induced by INF1 elicitin, produced by the oomycete pathogen Phytophthora infestans. NO ASSOCIATED1 (NOA1) and NADPH oxidase participate in the radical burst. Here, we show that mitogen-activated protein kinase (MAPK) cascades MEK2-SIPK/NTF4 and MEK1-NTF6 participate in the regulation of the radical burst. NO generation was induced by conditional activation of SIPK/NTF4, but not by NTF6, in Nicotiana benthamiana leaves. INF1- and SIPK/NTF4-mediated NO bursts were compromised by the knockdown of NOA1. However, ROS generation was induced by either SIPK/NTF4 or NTF6. INF1- and MAPK-mediated ROS generation was eliminated by silencing Respiratory Burst Oxidase Homolog B (RBOHB), an inducible form of the NADPH oxidase. INF1-induced expression of RBOHB was compromised in SIPK/NTF4/NTF6-silenced leaves. These results indicated that INF1 regulates NOA1-mediated NO and RBOHB-dependent ROS generation through MAPK cascades. NOA1 silencing induced high susceptibility to Colletotrichum orbiculare but not to P. infestans; conversely, RBOHB silencing decreased resistance to P. infestans but not to C. orbiculare. These results indicate that the effects of the radical burst on the defense response appear to be diverse in plant–pathogen interactions.

Prepenetration Apparatus Assembly Precedes and Predicts the Colonization Patterns of Arbuscular Mycorrhizal Fungi within the Root Cortex of Both Medicago truncatula and Daucus carota

Genre, A., Chabaud, M., Faccio, A., Barker, D. G., Bonfante, P. — 2008-06-30

Arbuscular mycorrhizas (AM) are widespread, ancient endosymbiotic associations that contribute significantly to soil nutrient uptake in plants. We have previously shown that initial fungal penetration of the host root is mediated via a specialized cytoplasmic assembly called the prepenetration apparatus (PPA), which directs AM hyphae through the epidermis (Genre et al., 2005). In vivo confocal microscopy studies performed on Medicago truncatula and Daucus carota, host plants with different patterns of AM colonization, now reveal that subsequent intracellular growth across the root outer cortex is also PPA dependent. On the other hand, inner root cortical colonization leading to arbuscule development involves more varied and complex PPA-related mechanisms. In particular, a striking alignment of polarized PPAs can be observed in adjacent inner cortical cells of D. carota, correlating with the intracellular root colonization strategy of this plant. Ultrastructural analysis of these PPA-containing cells reveals intense membrane trafficking coupled with nuclear enlargement and remodeling, typical features of arbusculated cells. Taken together, these findings imply that prepenetration responses are both conserved and modulated throughout the AM symbiosis as a function of the different stages of fungal accommodation and the host-specific pattern of root colonization. We propose a model for intracellular AM fungal accommodation integrating peri-arbuscular interface formation and the regulation of functional arbuscule development.

High-Resolution Imaging of Cortical Microtubule Arrays

Eckardt, N. A. — 2008-05-27

Surprising New Member of the KNOTTED1-Like Family of Transcriptional Regulators Lacks a Homeodomain

Mach, J. — 2008-05-27

PLP3 Proteins Function in Microtubule Assembly in Arabidopsis

Eckardt, N. A. — 2008-05-27

Probing the Role of Auxin in Wood Formation

Farquharson, K. L. — 2008-05-27

Regulation of Plastid Gene Expression in the Chloroplast-to-Chromoplast Transition

Hofmann, N. R. — 2008-05-27

The coi1-16 Mutant Harbors a Second Site Mutation Rendering PEN2 Nonfunctional

Westphal, L., Scheel, D., Rosahl, S. — 2008-05-27

Histone Acetylation and Chromatin Remodeling Are Required for UV-B-Dependent Transcriptional Activation of Regulated Genes in Maize

2008-05-27

The nuclear proteomes of maize (Zea mays) lines that differ in UV-B tolerance were compared by two-dimensional gel electrophoresis after UV light treatment. Differential accumulation of chromatin proteins, particularly histones, constituted the largest class identified by mass spectrometry. UV-B–tolerant landraces and the B73 inbred line show twice as many protein changes as the UV-B–sensitive b, pl W23 inbred line and transgenic maize expressing RNA interference constructs directed against chromatin factors. Mass spectrometic analysis of posttranslational modifications on histone proteins demonstrates that UV-B–tolerant lines exhibit greater acetylation on N-terminal tails of histones H3 and H4 after irradiation. These acetylated histones are enriched in the promoter and transcribed regions of the two UV-B–upregulated genes examined; radiation-sensitive lines lack this enrichment. DNase I and micrococcal nuclease hypersensitivity assays indicate that chromatin adopts looser structures around the selected genes in the UV-B–tolerant samples. Chromatin immunoprecipitation experiments identified additional chromatin factor changes associated with the nfc102 test gene after UV-B treatment in radiation-tolerant lines. Chromatin remodeling is thus shown to be a key process in acclimation to UV-B, and lines deficient in this process are more sensitive to UV-B.

Dissecting the Molecular Basis of the Regulation of Wood Formation by Auxin in Hybrid Aspen

2008-05-27

Indole acetic acid (auxin) is a key regulator of wood formation, and an observed overlap between auxin concentration gradient and developing secondary xylem cells has led to the hypothesis that auxin regulates wood formation by acting as a morphogen. We dissected the role of auxin in wood formation by identifying the auxin-responsive transcriptome in wood-forming tissues and investigating alterations in wood formation in transgenic hybrid aspen plants (Populus tremula x Populus tremuloides) with perturbed auxin signaling. We showed that auxin-responsive genes in wood-forming tissues respond dynamically to changes in cellular auxin levels. However, the expression patterns of most of the auxin-responsive genes displayed limited correlation with the auxin concentration across this developmental zone. Perturbing auxin signaling by reducing auxin responsiveness reduced the cambial cell division activity, caused spatial deregulation of cell division of the cambial initials, and led to reductions in not only radial but also axial dimensions of fibers and vessels. We propose that, instead of acting as a morphogen, changes in auxin concentration in developing secondary xylem cells may provide important regulatory cues that modulate the expression of a few key regulators; these, in turn, may control the global gene expression patterns that are essential for normal secondary xylem development.

Plastid Transcriptomics and Translatomics of Tomato Fruit Development and Chloroplast-to-Chromoplast Differentiation: Chromoplast Gene Expression Largely Serves the Production of a Single Protein

Kahlau, S., Bock, R. — 2008-05-27

Plastid genes are expressed at high levels in photosynthetically active chloroplasts but are generally believed to be drastically downregulated in nongreen plastids. The genome-wide changes in the expression patterns of plastid genes during the development of nongreen plastid types as well as the contributions of transcriptional versus translational regulation are largely unknown. We report here a systematic transcriptomics and translatomics analysis of the tomato (Solanum lycopersicum) plastid genome during fruit development and chloroplast-to-chromoplast conversion. At the level of RNA accumulation, most but not all plastid genes are strongly downregulated in fruits compared with leaves. By contrast, chloroplast-to-chromoplast differentiation during fruit ripening is surprisingly not accompanied by large changes in plastid RNA accumulation. However, most plastid genes are translationally downregulated during chromoplast development. Both transcriptional and translational downregulation are more pronounced for photosynthesis-related genes than for genes involved in gene expression, indicating that some low-level plastid gene expression must be sustained in chromoplasts. High-level expression during chromoplast development identifies accD, the only plastid-encoded gene involved in fatty acid biosynthesis, as the target gene for which gene expression activity in chromoplasts is maintained. In addition, we have determined the developmental patterns of plastid RNA polymerase activities, intron splicing, and RNA editing and report specific developmental changes in the splicing and editing patterns of plastid transcripts.

KNOX Lost the OX: The Arabidopsis KNATM Gene Defines a Novel Class of KNOX Transcriptional Regulators Missing the Homeodomain

Magnani, E., Hake, S. — 2008-05-27

Three amino acid loop extension (TALE) homeodomain transcriptional regulators play a central role in plant and animal developmental programs. Plant KNOTTED1-like homeobox (KNOX) and animal Myeloid ecotropic viral integration site (MEIS) proteins share a TALE homeodomain and a MEINOX (MEIS-KNOX) domain, suggesting that an ancestral MEINOX-TALE protein predates the divergence of plants from fungi and animals. In this study, we identify and characterize the Arabidopsis thaliana KNATM gene, which encodes a MEINOX domain but not a homeodomain. Phylogenetic analysis of the KNOX family places KNATM in a new class and shows conservation in dicotyledons. We demonstrate that KNATM selectively interacts with Arabidopsis BELL TALE proteins through the MEINOX domain. The homeodomain is known to be necessary for KNOX–KNOX interaction. On the contrary, KNATM specifically dimerizes with the KNOX protein BREVIPEDICELLUS through an acidic coiled-coil domain. KNATM is expressed in proximal-lateral domains of organ primordia and at the boundary of mature organs; in accordance, genetic analyses identify a function for KNATM in leaf proximal-distal patterning. In vivo domain analyses highlighted KNATM functional regions and revealed a role as transcriptional regulator. Taken together, our data reveal a homeodomain-independent mechanism of KNOX dimerization and transcriptional regulation.

Interaction of KNAT6 and KNAT2 with BREVIPEDICELLUS and PENNYWISE in Arabidopsis Inflorescences

Ragni, L., Belles-Boix, E., Gunl, M., Pautot, V. — 2008-05-27

The three amino acid loop extension (TALE) homeodomain superfamily, which comprises the KNOTTED-like and BEL1-like families, plays a critical role in regulating meristem activity. We previously demonstrated a function for KNAT6 (for KNOTTED-like from Arabidopsis thaliana 6) in shoot apical meristem and boundary maintenance during embryogenesis. KNAT2, the gene most closely related to KNAT6, does not play such a role. To investigate the contribution of KNAT6 and KNAT2 to inflorescence development, we examined their interactions with two TALE genes that regulate internode patterning, BREVIPEDICELLUS (BP) and PENNYWISE (PNY). Our data revealed distinct and overlapping interactions of KNAT6 and KNAT2 during inflorescence development. Removal of KNAT6 activity suppressed the pny phenotype and partially rescued the bp phenotype. Removal of KNAT2 activity had an effect only in the absence of both BP and KNAT6 or in the absence of both BP and PNY. Consistent with this, KNAT6 and KNAT2 expression patterns were enlarged in both bp and pny mutants. Thus, the defects seen in pny and bp are attributable mainly to the misexpression of KNAT6 and to a lesser extent of KNAT2. Hence, our data showed that BP and PNY restrict KNAT6 and KNAT2 expression to promote correct inflorescence development. This interaction was also revealed in the carpel.

REBELOTE, SQUINT, and ULTRAPETALA1 Function Redundantly in the Temporal Regulation of Floral Meristem Termination in Arabidopsis thaliana

2008-05-27

In Arabidopsis thaliana, flowers are determinate, showing a fixed number of whorls. Here, we report on three independent genes, a novel gene REBELOTE (RBL; protein of unknown function), SQUINT (SQN; a cyclophilin), and ULTRAPETALA1 (ULT1; a putative transcription factor) that redundantly influence floral meristem (FM) termination. Their mutations, combined with each other or with crabs claw, the genetic background in which they were isolated, trigger a strong FM indeterminacy with reiterations of extra floral whorls in the center of the flower. The range of phenotypes suggests that, in Arabidopsis, FM termination is initiated from stages 3 to 4 onwards and needs to be maintained through stage 6 and beyond, and that RBL, SQN, and ULT1 are required for this continuous regulation. We show that mutant phenotypes result from a decrease of AGAMOUS (AG) expression in an inner 4th whorl subdomain. However, the defect of AG activity alone does not explain all reported phenotypes, and our genetic data suggest that RBL, SQN, and, to a lesser extent, ULT1 also influence SUPERMAN activity. Finally, from all the molecular and genetic data presented, we argue that these genes contribute to the more stable and uniform development of flowers, termed floral developmental homeostasis.

HD-ZIP III Activity Is Modulated by Competitive Inhibitors via a Feedback Loop in Arabidopsis Shoot Apical Meristem Development

2008-05-27

Shoot apical meristem (SAM) development is coordinately regulated by two interdependent signaling events: one maintaining stem cell identity and the other governing the initiation of lateral organs from the flanks of the SAM. The signaling networks involved in this process are interconnected and are regulated by multiple molecular mechanisms. Class III homeodomain-leucine zipper (HD-ZIP III) proteins are the most extensively studied transcription factors involved in this regulation. However, how different signals are integrated to maintain stem cell identity and to pattern lateral organ polarity remains unclear. Here, we demonstrated that a small ZIP protein, ZPR3, and its functionally redundant homolog, ZPR4, negatively regulate the HD-ZIP III activity in SAM development. ZPR3 directly interacts with PHABULOSA (PHB) and other HD-ZIP III proteins via the ZIP motifs and forms nonfunctional heterodimers. Accordingly, a double mutant, zpr3-2 zpr4-2, exhibits an altered SAM activity with abnormal stem cell maintenance. However, the mutant displays normal patterning of leaf polarity. In addition, we show that PHB positively regulates ZPR3 expression. We therefore propose that HD-ZIP III activity in regulating SAM development is modulated by, among other things, a feedback loop involving the competitive inhibitors ZPR3 and ZPR4.

The Receptor Kinase CORYNE of Arabidopsis Transmits the Stem Cell-Limiting Signal CLAVATA3 Independently of CLAVATA1

Muller, R., Bleckmann, A., Simon, R. — 2008-05-27

Stem cells in shoot and floral meristems of Arabidopsis thaliana secrete the signaling peptide CLAVATA3 (CLV3) that restricts stem cell proliferation and promotes differentiation. The CLV3 signaling pathway is proposed to comprise the receptor kinase CLV1 and the receptor-like protein CLV2. We show here that the novel receptor kinase CORYNE (CRN) and CLV2 act together, and in parallel with CLV1, to perceive the CLV3 signal. Mutations in CRN cause stem cell proliferation, similar to clv1, clv2, and clv3 mutants. CRN has additional functions during plant development, including floral organ development, that are shared with CLV2. The CRN protein lacks a distinct extracellular domain, and we propose that CRN and CLV2 interact via their transmembrane domains to establish a functional receptor.

Distinct Light-Initiated Gene Expression and Cell Cycle Programs in the Shoot Apex and Cotyledons of Arabidopsis

2008-05-27

In darkness, shoot apex growth is repressed, but it becomes rapidly activated by light. We show that phytochromes and cryptochromes play largely redundant roles in this derepression in Arabidopsis thaliana. We examined the light activation of transcriptional changes in a finely resolved time course, comparing the shoot apex (meristem and leaf primordia) and the cotyledon and found >5700 differentially expressed genes. Early events specific to the shoot apices included the repression of genes for Really Interesting New Gene finger proteins and basic domain/leucine zipper and basic helix-loop-helix transcription factors. The downregulation of auxin and ethylene and the upregulation of cytokinin and gibberellin hormonal responses were also characteristic of shoot apices. In the apex, genes involved in ribosome biogenesis and protein translation were rapidly and synchronously induced, simultaneously with cell proliferation genes, preceding visible organ growth. Subsequently, the activation of signaling genes and transcriptional signatures of cell wall expansion, turgor generation, and plastid biogenesis were apparent. Furthermore, light regulates the forms and protein levels of two transcription factors with opposing functions in cell proliferation, E2FB and E2FC, through the Constitutively Photomorphogenic1 (COP1), COP9-Signalosome5, and Deetiolated1 light signaling molecules. These data provide the basis for reconstruction of the regulatory networks for light-regulated meristem, leaf, and cotyledon development.

Phosducin-Like Protein 3 Is Required for Microtubule-Dependent Steps of Cell Division but Not for Meristem Growth in Arabidopsis

Castellano, M. M., Sablowski, R. — 2008-05-27

Given the central role of cell division in meristems, one might expect meristem growth to be regulated by mitotic checkpoints, including checkpoints for correct microtubule function. Here, we studied the role of two close Phosducin-Like Protein 3 homologs from Arabidopsis thaliana (PLP3a and PLP3b) in the microtubule assembly pathway and determined the consequences of inhibiting PLP3a and PLP3b expression in the meristem. PLP3 function is essential in Arabidopsis: impairing PLP3a and PLP3b expression disrupted microtubule arrays and caused polyploidy, aneuploidy, defective cytokinesis, and disoriented cell growth. Consistent with a role in microtubule formation, PLP3a interacted with β-tubulin in the yeast two-hybrid assay and, when overexpressed, increased resistance to drugs that inhibit tubulin polymerization. Inhibition of PLP3 function targeted to the meristem caused severe mitotic defects, but the cells carried on cycling through DNA replication and abortive cytokinesis. Thus, we showed that PLP3 is involved in microtubule formation in Arabidopsis and provided genetic evidence that cell viability and growth in the meristem are not subordinate to successful completion of microtubule-dependent steps of cell division.

Analysis of Cortical Arrays from Tradescantia virginiana at High Resolution Reveals Discrete Microtubule Subpopulations and Demonstrates That Confocal Images of Arrays Can Be Misleading

Barton, D. A., Vantard, M., Overall, R. L. — 2008-05-27

Cortical microtubule arrays are highly organized networks involved in directing cellulose microfibril deposition within the cell wall. Their organization results from complex interactions between individual microtubules and microtubule-associated proteins. The precise details of these interactions are often not evident using optical microscopy. Using high-resolution scanning electron microscopy, we analyzed extensive regions of cortical arrays and identified two spatially discrete microtubule subpopulations that exhibited different stabilities. Microtubules that lay adjacent to the plasma membrane were often bundled and more stable than the randomly aligned, discordant microtubules that lay deeper in the cytoplasm. Immunolabeling revealed katanin at microtubule ends, on curves, or at sites along microtubules in line with neighboring microtubule ends. End binding 1 protein also localized along microtubules, at microtubule ends or junctions between microtubules, and on the plasma membrane in direct line with microtubule ends. We show fine bands in vivo that traverse and may encircle microtubules. Comparing confocal and electron microscope images of fluorescently tagged arrays, we demonstrate that optical images are misleading, highlighting the fundamental importance of studying cortical microtubule arrays at high resolution.

Arabidopsis SCARs Function Interchangeably to Meet Actin-Related Protein 2/3 Activation Thresholds during Morphogenesis

2008-05-27

During polarized growth and tissue morphogenesis, cells must reorganize their cytoplasm and change shape in response to growth signals. Dynamic polymerization of actin filaments is one cellular component of polarized growth, and the actin-related protein 2/3 (ARP2/3) complex is an important actin filament nucleator in plants. ARP2/3 alone is inactive, and the Arabidopsis thaliana WAVE complex translates Rho-family small GTPase signals into an ARP2/3 activation response. The SCAR subunit of the WAVE complex is the primary activator of ARP2/3, and plant and vertebrate SCARs are encoded by a small gene family. However, it is unclear if SCAR isoforms function interchangeably or if they have unique properties that customize WAVE complex functions. We used the Arabidopsis distorted group mutants and an integrated analysis of SCAR gene and protein functions to address this question directly. Genetic results indicate that each of the four SCARs functions in the context of the WAVE-ARP2/3 pathway and together they define the lone mechanism for ARP2/3 activation. Genetic interactions among the scar mutants and transgene complementation studies show that the activators function interchangeably to meet the threshold for ARP2/3 activation in the cell. Interestingly, double, triple, and quadruple mutant analyses indicate that individual SCAR genes vary in their relative importance depending on the cell type, tissue, or organ that is analyzed. Differences among SCARs in mRNA levels and the biochemical efficiency of ARP2/3 activation may explain the functional contributions of individual genes.

Minor Antenna Proteins CP24 and CP26 Affect the Interactions between Photosystem II Subunits and the Electron Transport Rate in Grana Membranes of Arabidopsis

de Bianchi, S., Dall'Osto, L., Tognon, G., Morosinotto, T., Bassi, R. — 2008-05-27

We investigated the function of chlorophyll a/b binding antenna proteins Chlorophyll Protein 26 (CP26) and CP24 in light harvesting and regulation of photosynthesis by isolating Arabidopsis thaliana knockout lines that completely lacked one or both of these proteins. All three mutant lines had a decreased efficiency of energy transfer from trimeric light-harvesting complex II (LHCII) to the reaction center of photosystem II (PSII) due to the physical disconnection of LHCII from PSII and formation of PSII reaction center depleted domains in grana partitions. Photosynthesis was affected in plants lacking CP24 but not in plants lacking CP26: the former mutant had decreased electron transport rates, a lower pH gradient across the grana membranes, reduced capacity for nonphotochemical quenching, and limited growth. Furthermore, the PSII particles of these plants were organized in unusual two-dimensional arrays in the grana membranes. Surprisingly, overall electron transport, nonphotochemical quenching, and growth of the double mutant were restored to wild type. Fluorescence induction kinetics and electron transport measurements at selected steps of the photosynthetic chain suggested that limitation in electron transport was due to restricted electron transport between Q_A and Q_B, which retards plastoquinone diffusion. We conclude that CP24 absence alters PSII organization and consequently limits plastoquinone diffusion.

Thylakoid Membrane Remodeling during State Transitions in Arabidopsis

2008-05-27

Adaptability of oxygenic photosynthetic organisms to fluctuations in light spectral composition and intensity is conferred by state transitions, short-term regulatory processes that enable the photosynthetic apparatus to rapidly adjust to variations in light quality. In green algae and higher plants, these processes are accompanied by reversible structural rearrangements in the thylakoid membranes. We studied these structural changes in the thylakoid membranes of Arabidopsis thaliana chloroplasts using atomic force microscopy, scanning and transmission electron microscopy, and confocal imaging. Based on our results and on the recently determined three-dimensional structure of higher-plant thylakoids trapped in one of the two major light-adapted states, we propose a model for the transitions in membrane architecture. The model suggests that reorganization of the membranes involves fission and fusion events that occur at the interface between the appressed (granal) and nonappressed (stroma lamellar) domains of the thylakoid membranes. Vertical and lateral displacements of the grana layers presumably follow these localized events, eventually leading to macroscopic rearrangements of the entire membrane network.

{beta}-AMYLASE4, a Noncatalytic Protein Required for Starch Breakdown, Acts Upstream of Three Active {beta}-Amylases in Arabidopsis Chloroplasts

2008-05-27

This work investigated the roles of β-amylases in the breakdown of leaf starch. Of the nine β-amylase (BAM)–like proteins encoded in the Arabidopsis thaliana genome, at least four (BAM1, -2, -3, and -4) are chloroplastic. When expressed as recombinant proteins in Escherichia coli, BAM1, BAM2, and BAM3 had measurable β-amylase activity but BAM4 did not. BAM4 has multiple amino acid substitutions relative to characterized β-amylases, including one of the two catalytic residues. Modeling predicts major differences between the glucan binding site of BAM4 and those of active β-amylases. Thus, BAM4 probably lost its catalytic capacity during evolution. Total β-amylase activity was reduced in leaves of bam1 and bam3 mutants but not in bam2 and bam4 mutants. The bam3 mutant had elevated starch levels and lower nighttime maltose levels than the wild type, whereas bam1 did not. However, the bam1 bam3 double mutant had a more severe phenotype than bam3, suggesting functional overlap between the two proteins. Surprisingly, bam4 mutants had elevated starch levels. Introduction of the bam4 mutation into the bam3 and bam1 bam3 backgrounds further elevated the starch levels in both cases. These data suggest that BAM4 facilitates or regulates starch breakdown and operates independently of BAM1 and BAM3. Together, our findings are consistent with the proposal that β-amylase is a major enzyme of starch breakdown in leaves, but they reveal unexpected complexity in terms of the specialization of protein function.

The Structure of Sucrose Phosphate Synthase from Halothermothrix orenii Reveals Its Mechanism of Action and Binding Mode

Chua, T. K., Bujnicki, J. M., Tan, T.-C., Huynh, F., Patel, B. K., Sivaraman, J. — 2008-05-27

Sucrose phosphate synthase (SPS) catalyzes the transfer of a glycosyl group from an activated donor sugar, such as uridine diphosphate glucose (UDP-Glc), to a saccharide acceptor d-fructose 6-phosphate (F6P), resulting in the formation of UDP and d-sucrose-6'-phosphate (S6P). This is a central regulatory process in the production of sucrose in plants, cyanobacteria, and proteobacteria. Here, we report the crystal structure of SPS from the nonphotosynthetic bacterium Halothermothrix orenii and its complexes with the substrate F6P and the product S6P. SPS has two distinct Rossmann-fold domains with a large substrate binding cleft at the interdomain interface. Structures of two complexes show that both the substrate F6P and the product S6P bind to the A-domain of SPS. Based on comparative analysis of the SPS structure with other related enzymes, the donor substrate, nucleotide diphosphate glucose, binds to the B-domain of SPS. Furthermore, we propose a mechanism of catalysis by H. orenii SPS. Our findings indicate that SPS from H. orenii may represent a valid model for the catalytic domain of plant SPSs and thus may provide useful insight into the reaction mechanism of the plant enzyme.

Functional and Physiological Characterization of Arabidopsis INOSITOL TRANSPORTER1, a Novel Tonoplast-Localized Transporter for myo-Inositol

Schneider, S., Beyhl, D., Hedrich, R., Sauer, N. — 2008-05-27

Arabidopsis thaliana INOSITOL TRANSPORTER1 (INT1) is a member of a small gene family with only three more genes (INT2 to INT4). INT2 and INT4 were shown to encode plasma membrane–localized transporters for different inositol epimers, and INT3 was characterized as a pseudogene. Here, we present the functional and physiological characterization of the INT1 protein, analyses of the tissue-specific expression of the INT1 gene, and analyses of phenotypic differences observed between wild-type plants and mutant lines carrying the int1.1 and int1.2 alleles. INT1 is a ubiquitously expressed gene, and Arabidopsis lines with T-DNA insertions in INT1 showed increased intracellular myo-inositol concentrations and reduced root growth. In Arabidopsis, tobacco (Nicotiana tabacum), and Saccharomyces cerevisiae, fusions of the green fluorescent protein to the C terminus of INT1 were targeted to the tonoplast membranes. Finally, patch-clamp analyses were performed on vacuoles from wild-type plants and from both int1 mutant lines to study the transport properties of INT1 at the tonoplast. In summary, the presented molecular, physiological, and functional studies demonstrate that INT1 is a tonoplast-localized H⁺/inositol symporter that mediates the efflux of inositol that is generated during the degradation of inositol-containing compounds in the vacuolar lumen.

Reduced V-ATPase Activity in the trans-Golgi Network Causes Oxylipin-Dependent Hypocotyl Growth Inhibition in Arabidopsis

2008-05-27

Regulated cell expansion allows plants to adapt their morphogenesis to prevailing environmental conditions. Cell expansion is driven by turgor pressure created by osmotic water uptake and is restricted by the extensibility of the cell wall, which in turn is regulated by the synthesis, incorporation, and cross-linking of new cell wall components. The vacuolar H⁺-ATPase (V-ATPase) could provide a way to coordinately regulate turgor pressure and cell wall synthesis, as it energizes the secondary active transport of solutes across the tonoplast and also has an important function in the trans-Golgi network (TGN), which affects synthesis and trafficking of cell wall components. We have previously shown that det3, a mutant with reduced V-ATPase activity, has a severe defect in cell expansion. However, it was not clear if this is caused by a defect in turgor pressure or in cell wall synthesis. Here, we show that inhibition of the tonoplast-localized V-ATPase subunit isoform VHA-a3 does not impair cell expansion. By contrast, inhibition of the TGN-localized isoform VHA-a1 is sufficient to restrict cell expansion. Furthermore, we provide evidence that the reduced hypocotyl cell expansion in det3 is conditional and due to active, hormone-mediated growth inhibition caused by a cell wall defect.

Analysis of the Arabidopsis Histidine Kinase ATHK1 Reveals a Connection between Vegetative Osmotic Stress Sensing and Seed Maturation

Wohlbach, D. J., Quirino, B. F., Sussman, M. R. — 2008-05-27

To cope with water stress, plants must be able to effectively sense, respond to, and adapt to changes in water availability. The Arabidopsis thaliana plasma membrane His kinase ATHK1 has been suggested to act as an osmosensor that detects water stress and initiates downstream responses. Here, we provide direct genetic evidence that ATHK1 not only is involved in the water stress response during early vegetative stages of plant growth but also plays a unique role in the regulation of desiccation processes during seed formation. To more comprehensively identify genes involved in the downstream pathways affected by the ATHK1-mediated response to water stress, we created a large-scale summary of expression data, termed the AtMegaCluster. In the AtMegaCluster, hierarchical clustering techniques were used to compare whole-genome expression levels in athk1 mutants with the expression levels reported in publicly available data sets of Arabidopsis tissues grown under a wide variety of conditions. These experiments revealed that ATHK1 is cotranscriptionally regulated with several Arabidopsis response regulators, together with two proteins containing novel sequences. Since overexpression of ATHK1 results in increased water stress tolerance, our observations suggest a new top-down route to increasing drought resistance via receptor-mediated increases in sensing water status, rather than through genetically engineered changes in downstream transcription factors or specific osmolytes.

Conserved C-Terminal Motifs Required for Avirulence and Suppression of Cell Death by Phytophthora sojae effector Avr1b

2008-05-27

The sequenced genomes of oomycete plant pathogens contain large superfamilies of effector proteins containing the protein translocation motif RXLR-dEER. However, the contributions of these effectors to pathogenicity remain poorly understood. Here, we show that the Phytophthora sojae effector protein Avr1b can contribute positively to virulence and can suppress programmed cell death (PCD) triggered by the mouse BAX protein in yeast, soybean (Glycine max), and Nicotiana benthamiana cells. We identify three conserved motifs (K, W, and Y) in the C terminus of the Avr1b protein and show that mutations in the conserved residues of the W and Y motifs reduce or abolish the ability of Avr1b to suppress PCD and also abolish the avirulence interaction of Avr1b with the Rps1b resistance gene in soybean. W and Y motifs are present in at least half of the identified oomycete RXLR-dEER effector candidates, and we show that three of these candidates also suppress PCD in soybean. Together, these results indicate that the W and Y motifs are critical for the interaction of Avr1b with host plant target proteins and support the hypothesis that these motifs are critical for the functions of the very large number of predicted oomycete effectors that contain them.

Activated Expression of an Arabidopsis HD-START Protein Confers Drought Tolerance with Improved Root System and Reduced Stomatal Density

2008-05-27

Drought is one of the most important environmental constraints limiting plant growth and agricultural productivity. To understand the underlying mechanism of drought tolerance and to identify genes for improving this important trait, we conducted a gain-of-function genetic screen for improved drought tolerance in Arabidopsis thaliana. One mutant with improved drought tolerance was isolated and designated as enhanced drought tolerance1. The mutant has a more extensive root system than the wild type, with deeper roots and more lateral roots, and shows a reduced leaf stomatal density. The mutant had higher levels of abscisic acid and Pro than the wild type and demonstrated an increased resistance to oxidative stress and high levels of superoxide dismutase. Molecular genetic analysis and recapitulation experiments showed that the enhanced drought tolerance is caused by the activated expression of a T-DNA tagged gene that encodes a putative homeodomain-START transcription factor. Moreover, overexpressing the cDNA of the transcription factor in transgenic tobacco also conferred drought tolerance associated with improved root architecture and reduced leaf stomatal density. Therefore, we have revealed functions of the homeodomain-START factor that were gained upon altering its expression pattern by activation tagging and provide a key regulator that may be used to improve drought tolerance in plants.

Identification and Regulation of TPS04/GES, an Arabidopsis Geranyllinalool Synthase Catalyzing the First Step in the Formation of the Insect-Induced Volatile C16-Homoterpene TMTT

2008-05-27

Volatile secondary metabolites emitted by plants contribute to plant–plant, plant–fungus, and plant–insect interactions. The C₁₆-homoterpene TMTT (for 4,8,12-trimethyltrideca-1,3,7,11-tetraene) is emitted after herbivore attack by a wide variety of plant species, including Arabidopsis thaliana, and is assumed to play a role in attracting predators or parasitoids of herbivores. TMTT has been suggested to be formed as a degradation product of the diterpene alcohol (E,E)-geranyllinalool. Here, we report the identification of Terpene Synthase 04 (TPS04; At1g61120) as a geranyllinalool synthase (GES). Recombinant TPS04/GES protein expressed in Escherichia coli catalyzes the formation of (E,E)-geranyllinalool from the substrate geranylgeranyl diphosphate. Transgenic Arabidopsis lines carrying T-DNA insertions in the TPS04 locus are deficient in (E,E)-geranyllinalool and TMTT synthesis, a phenotype that can be complemented by expressing the GES gene under the control of a heterologous promoter. GES transcription is upregulated under conditions that induce (E,E)-geranyllinalool and TMTT synthesis, including infestation of plants with larvae of the moth Plutella xylostella and treatment with the fungal peptide alamethicin or the octadecanoid mimic coronalon. Induction requires jasmonic acid but is independent from salicylic acid or ethylene. This study paves the ground to address the contribution of TMTT in ecological interactions and to elucidate the signaling network that regulates TMTT synthesis.

Fungal Effector Protein AVR2 Targets Diversifying Defense-Related Cys Proteases of Tomato

2008-05-27

The interaction between the fungal pathogen Cladosporium fulvum and its host tomato (Solanum lycopersicum) is an ideal model to study suppression of extracellular host defenses by pathogens. Secretion of protease inhibitor AVR2 by C. fulvum during infection suggests that tomato papain-like cysteine proteases (PLCPs) are part of the tomato defense response. We show that the tomato apoplast contains a remarkable diversity of PLCP activities with seven PLCPs that fall into four different subfamilies. Of these PLCPs, transcription of only PIP1 and RCR3 is induced by treatment with benzothiadiazole, which triggers the salicylic acid–regulated defense pathway. Sequencing of PLCP alleles of tomato relatives revealed that only PIP1 and RCR3 are under strong diversifying selection, resulting in variant residues around the substrate binding groove. The doubled number of variant residues in RCR3 suggests that RCR3 is under additional adaptive selection, probably to prevent autoimmune responses. AVR2 selectively inhibits only PIP1 and RCR3, and one of the naturally occurring variant residues in RCR3 affects AVR2 inhibition. The higher accumulation of PIP1 protein levels compared with RCR3 indicates that PIP1 might be the real virulence target of AVR2 and that RCR3 acts as a decoy for AVR2 perception in plants carrying the Cf-2 resistance gene.

Oxylipin Signaling in Plant Stress Responses

Eckardt, N. A. — 2008-04-28

The IMEter Predicts an Intron's Ability to Boost Gene Expression

Farquharson, K. L. — 2008-04-28

Aquaporins and Chloroplast Membrane Permeability

Eckardt, N. A. — 2008-04-28

Cellulose Synthesis in Phytophthora infestans Pathogenesis

Mach, J. — 2008-04-28

Heritability of the Tomato Fruit Metabolome

Eckardt, N. A. — 2008-04-28

Pond Scum Genomics: The Genomes of Chlamydomonas and Ostreococcus

Peers, G., Niyogi, K. K. — 2008-04-28

Mode of Inheritance of Primary Metabolic Traits in Tomato

2008-04-28

To evaluate components of fruit metabolic composition, we have previously metabolically phenotyped tomato (Solanum lycopersicum) introgression lines containing segmental substitutions of wild species chromosome in the genetic background of a cultivated variety. Here, we studied the hereditability of the fruit metabolome by analyzing an additional year's harvest and evaluating the metabolite profiles of lines heterozygous for the introgression (ILHs), allowing the evaluation of putative quantitative trait locus (QTL) mode of inheritance. These studies revealed that most of the metabolic QTL (174 of 332) were dominantly inherited, with relatively high proportions of additively (61 of 332) or recessively (80 of 332) inherited QTL and a negligible number displaying the characteristics of overdominant inheritance. Comparison of the mode of inheritance of QTL revealed that several metabolite pairs displayed a similar mode of inheritance of QTL at the same chromosomal loci. Evaluation of the association between morphological and metabolic traits in the ILHs revealed that this correlation was far less prominent, due to a reduced variance in the harvest index within this population. These data are discussed in the context of genomics-assisted breeding for crop improvement, with particular focus on the exploitation of wide biodiversity.

The Leaf Epidermome of Catharanthus roseus Reveals Its Biochemical Specialization

Murata, J., Roepke, J., Gordon, H., De Luca, V. — 2008-04-28

Catharanthus roseus is the sole commercial source of the monoterpenoid indole alkaloids (MIAs), vindoline and catharanthine, components of the commercially important anticancer dimers, vinblastine and vincristine. Carborundum abrasion technique was used to extract leaf epidermis–enriched mRNA, thus sampling the epidermome, or complement, of proteins expressed in the leaf epidermis. Random sequencing of the derived cDNA library established 3655 unique ESTs, composed of 1142 clusters and 2513 singletons. Virtually all known MIA pathway genes were found in this remarkable set of ESTs, while only four known genes were found in the publicly available Catharanthus EST data set. Several novel MIA pathway candidate genes were identified, as demonstrated by the cloning and functional characterization of loganic acid O-methyltransferase involved in secologanin biosynthesis. The pathways for triterpene biosynthesis were also identified, and metabolite analysis showed that oleanane-type triterpenes were localized exclusively to the cuticular wax layer. The pathways for flavonoid and very-long-chain fatty acid biosynthesis were also located in this cell type. The results illuminate the biochemical specialization of Catharanthus leaf epidermis for the production of multiple classes of metabolites. The value and versatility of this EST data set for biochemical and biological analysis of leaf epidermal cells is also discussed.

Promoter-Proximal Introns in Arabidopsis thaliana Are Enriched in Dispersed Signals that Elevate Gene Expression

Rose, A. B., Elfersi, T., Parra, G., Korf, I. — 2008-04-28

Introns that elevate mRNA accumulation have been found in a wide range of eukaryotes. However, not all introns affect gene expression, and direct testing is currently the only way to identify stimulatory introns. Our genome-wide analysis in Arabidopsis thaliana revealed that promoter-proximal introns as a group are compositionally distinct from distal introns and that the degree to which an individual intron matches the promoter-proximal intron profile is a strong predictor of its ability to increase expression. We found that the sequences responsible for elevating expression are dispersed throughout an enhancing intron, as is a candidate motif that is overrepresented in first introns and whose occurrence in tested introns is proportional to its effect on expression. The signals responsible for intron-mediated enhancement are apparently conserved between Arabidopsis and rice (Oryza sativa) despite the large evolutionary distance separating these plants.

Heme, a Plastid-Derived Regulator of Nuclear Gene Expression in Chlamydomonas

von Gromoff, E. D., Alawady, A., Meinecke, L., Grimm, B., Beck, C. F. — 2008-04-28

To gain insight into the chloroplast-to-nucleus signaling role of tetrapyrroles, Chlamydomonas reinhardtii mutants in the Mg-chelatase that catalyzes the insertion of magnesium into protoporphyrin IX were isolated and characterized. The four mutants lack chlorophyll and show reduced levels of Mg-tetrapyrroles but increased levels of soluble heme. In the mutants, light induction of HSP70A was preserved, although Mg-protoporphyrin IX has been implicated in this induction. In wild-type cells, a shift from dark to light resulted in a transient reduction in heme levels, while the levels of Mg-protoporphyrin IX, its methyl ester, and protoporphyrin IX increased. Hemin feeding to cultures in the dark activated HSP70A. This induction was mediated by the same plastid response element (PRE) in the HSP70A promoter that has been shown to mediate induction by Mg-protoporphyrin IX and light. Other nuclear genes that harbor a PRE in their promoters also were inducible by hemin feeding. Extended incubation with hemin abrogated the competence to induce HSP70A by light or Mg-protoporphyrin IX, indicating that these signals converge on the same pathway. We propose that Mg-protoporphyrin IX and heme may serve as plastid signals that regulate the expression of nuclear genes.

The Highly Similar Arabidopsis Homologs of Trithorax ATX1 and ATX2 Encode Proteins with Divergent Biochemical Functions

2008-04-28

Gene duplication followed by functional specialization is a potent force in the evolution of biological diversity. A comparative study of two highly conserved duplicated genes, ARABIDOPSIS TRITHORAX-LIKE PROTEIN1 (ATX1) and ATX2, revealed features of both partial redundancy and of functional divergence. Although structurally similar, their regulatory sequences have diverged, resulting in distinct temporal and spatial patterns of expression of the ATX1 and ATX2 genes. We found that ATX2 methylates only a limited fraction of nucleosomes and that ATX1 and ATX2 influence the expression of largely nonoverlapping gene sets. Even when coregulating shared targets, ATX1 and ATX2 may employ different mechanisms. Most remarkable is the divergence of their biochemical activities: both proteins methylate K4 of histone H3, but while ATX1 trimethylates it, ATX2 dimethylates it. ATX2 and ATX1 provide an example of separated K4 di from K4 trimethyltransferase activity.

ARABIDOPSIS TRITHORAX1 Dynamically Regulates FLOWERING LOCUS C Activation via Histone 3 Lysine 4 Trimethylation

2008-04-28

Trithorax function is essential for epigenetic maintenance of gene expression in animals, but little is known about trithorax homologs in plants. ARABIDOPSIS TRITHORAX1 (ATX1) was shown to be required for the expression of homeotic genes involved in flower organogenesis. Here, we report a novel function of ATX1, namely, the epigenetic regulation of the floral repressor FLOWERING LOCUS C (FLC). Downregulation of FLC accelerates the transition from vegetative to reproductive development in Arabidopsis thaliana. In the atx1 mutant, FLC levels are reduced and the FLC chromatin is depleted of trimethylated, but not dimethylated, histone 3 lysine 4, suggesting a specific trimethylation function of ATX1. In addition, we found that ATX1 directly binds the active FLC locus before flowering and that this interaction is released upon the transition to flowering. This dynamic process stands in contrast with the stable maintenance of homeotic gene expression mediated by trithorax group proteins in animals but resembles the dynamics of plant Polycomb group function.

EMB2473/MIRO1, an Arabidopsis Miro GTPase, Is Required for Embryogenesis and Influences Mitochondrial Morphology in Pollen

Yamaoka, S., Leaver, C. J. — 2008-04-28

The regulation of mitochondrial biogenesis, subcellular distribution, morphology, and metabolism are essential for all aspects of plant growth and development. However, the molecular mechanisms involved are still unclear. Here, we describe an analysis of the three Arabidopsis thaliana orthologs of the evolutionarily conserved Miro GTPases. Two of the genes, MIRO1 and MIRO2, are transcribed ubiquitously throughout the plant tissues, and their gene products localize to mitochondria via their C-terminal transmembrane domains. While insertional mutations in the MIRO2 gene do not have any visible impact on plant development, an insertional mutation in the MIRO1 gene is lethal during embryogenesis at the zygote to four-terminal-cell embryo stage. It also substantially impairs pollen germination and tube growth. Laser confocal and transmission electron microscopy revealed that the miro1 mutant pollen exhibits abnormally enlarged or tube-like mitochondrial morphology, leading to the disruption of continuous streaming of mitochondria in the growing pollen tube. Our findings suggest that mitochondrial morphology is influenced by MIRO1 and plays a vital role during embryogenesis and pollen tube growth.