The Plant Cell recent issues

Refining Our Standards

Martin, C. — 2008-08-25

Effector Trafficking: RXLR-dEER as Extra Gear for Delivery into Plant Cells

Govers, F., Bouwmeester, K. — 2008-08-25

Effector XopD Suppresses Tissue Degeneration in Xanthomonas-Infected Tomato Leaves

Mach, J. — 2008-08-25

They All Scream for ICE1/SCRM2: Core Regulatory Units in Stomatal Development

Hofmann, N. R. — 2008-08-25

Auxin Regulation of Late Stamen Development

Eckardt, N. A. — 2008-08-25

Towards a Systematic Validation of References in Real-Time RT-PCR

Gutierrez, L., Mauriat, M., Pelloux, J., Bellini, C., Van Wuytswinkel, O. — 2008-08-25

Eleven Golden Rules of Quantitative RT-PCR

Udvardi, M. K., Czechowski, T., Scheible, W.-R. — 2008-08-25

The Evolving Complexity of the Auxin Pathway

Lau, S., Jurgens, G., De Smet, I. — 2008-08-25

Calmodulin7 Plays an Important Role as Transcriptional Regulator in Arabidopsis Seedling Development

Kushwaha, R., Singh, A., Chattopadhyay, S. — 2008-08-25

Although calmodulin (CaM) is known to play multiple regulatory roles in eukaryotes, its direct function as transcriptional regulator is unknown. Furthermore, the physiological functions of CaM are largely unknown in plants. Here, we show that one of the four Arabidopsis thaliana CaM isoforms, CAM7, is a transcriptional regulator that directly interacts with the promoters of light-inducible genes and promotes photomorphogenesis. CAM7 overexpression causes hyperphotomorphogenic growth and an increase in the expression of light-inducible genes. Mutations in CAM7 produce no visible effects on photomorphogenic growth, indicating likely redundant gene functions. However, cam7 mutants display reduced expression of light-inducible genes, and cam7 hy5 double mutants show an enhancement of the hy5 phenotype. Moreover, overexpression of CAM7 can partly suppress the hy5 phenotype, indicating that the two factors work together to control light-induced seedling development. The mutational and transgenic studies, together with physiological analyses, illustrate the concerted function of CAM7 and HY5 basic leucine zipper transcription factor in Arabidopsis seedling development.

Auxin Regulates Arabidopsis Anther Dehiscence, Pollen Maturation, and Filament Elongation

Cecchetti, V., Altamura, M. M., Falasca, G., Costantino, P., Cardarelli, M. — 2008-08-25

We provide evidence on the localization, synthesis, transport, and effects of auxin on the processes occurring late in Arabidopsis thaliana stamen development: anther dehiscence, pollen maturation, and preanthesis filament elongation. Expression of auxin-sensitive reporter constructs suggests that auxin effects begin in anthers between the end of meiosis and the bilocular stage in the somatic tissues involved in the first step of dehiscence as well as in the microspores and in the junction region between anther and filament. In situ hybridizations of the auxin biosynthetic genes YUC2 and YUC6 suggest that auxin is synthesized in anthers. In agreement with the timing of auxin effects, the TIR1, AFB1, AFB2, and AFB3 auxin receptor-encoding genes are transcribed in anthers only during late stages of development starting at the end of meiosis. We found that in tir1 afb triple and quadruple mutants, anther dehiscence and pollen maturation occur earlier than in the wild type, causing the release of mature pollen grains before the completion of filament elongation. We also assessed the contribution of auxin transport to late stamen developmental processes. Our results suggest that auxin synthesized in anthers plays a major role in coordinating anther dehiscence and pollen maturation, while auxin transport contributes to the independent regulation of preanthesis filament elongation.

SCREAM/ICE1 and SCREAM2 Specify Three Cell-State Transitional Steps Leading to Arabidopsis Stomatal Differentiation

2008-08-25

Differentiation of specialized cell types in multicellular organisms requires orchestrated actions of cell fate determinants. Stomata, valves on the plant epidermis, are formed through a series of differentiation events mediated by three closely related basic-helix-loop-helix proteins: SPEECHLESS (SPCH), MUTE, and FAMA. However, it is not known what mechanism coordinates their actions. Here, we identify two paralogous proteins, SCREAM (SCRM) and SCRM2, which directly interact with and specify the sequential actions of SPCH, MUTE, and FAMA. The gain-of-function mutation in SCRM exhibited constitutive stomatal differentiation in the epidermis. Conversely, successive loss of SCRM and SCRM2 recapitulated the phenotypes of fama, mute, and spch, indicating that SCRM and SCRM2 together determined successive initiation, proliferation, and terminal differentiation of stomatal cell lineages. Our findings identify the core regulatory units of stomatal differentiation and suggest a model strikingly similar to cell-type differentiation in animals. Surprisingly, map-based cloning revealed that SCRM is INDUCER OF CBF EXPRESSION1, a master regulator of freezing tolerance, thus implicating a potential link between the transcriptional regulation of environmental adaptation and development in plants.

Mutations in SUPPRESSOR OF VARIEGATION1, a Factor Required for Normal Chloroplast Translation, Suppress var2-Mediated Leaf Variegation in Arabidopsis

Yu, F., Liu, X., Alsheikh, M., Park, S., Rodermel, S. — 2008-08-25

The Arabidopsis thaliana yellow variegated2 (var2) mutant is variegated due to lack of a chloroplast FtsH-like metalloprotease (FtsH2/VAR2). We have generated suppressors of var2 variegation to gain insight into factors and pathways that interact with VAR2 during chloroplast biogenesis. Here, we describe two such suppressors. Suppression of variegation in the first line, TAG-FN, was caused by disruption of the nuclear gene (SUPPRESSOR OF VARIEGATION1 [SVR1]) for a chloroplast-localized homolog of pseudouridine () synthase, which isomerizes uridine to in noncoding RNAs. svr1 single mutants were epistatic to var2, and they displayed a phenotypic syndrome that included defects in chloroplast rRNA processing, reduced chloroplast translation, reduced chloroplast protein accumulation, and elevated chloroplast mRNA levels. In the second line (TAG-IE), suppression of variegation was caused by a lesion in SVR2, the gene for the ClpR1 subunit of the chloroplast ClpP/R protease. Like svr1, svr2 was epistatic to var2, and clpR1 mutants had a phenotype that resembled svr1. We propose that an impairment of chloroplast translation in TAG-FN and TAG-IE decreased the demand for VAR2 activity during chloroplast biogenesis and that this resulted in the suppression of var2 variegation. Consistent with this hypothesis, var2 variegation was repressed by chemical inhibitors of chloroplast translation. In planta mutagenesis revealed that SVR1 not only played a role in uridine isomerization but that its physical presence was necessary for proper chloroplast rRNA processing. Our data indicate that defects in chloroplast rRNA processing are a common, but not universal, molecular phenotype associated with suppression of var2 variegation.

The EPIP Peptide of INFLORESCENCE DEFICIENT IN ABSCISSION Is Sufficient to Induce Abscission in Arabidopsis through the Receptor-Like Kinases HAESA and HAESA-LIKE2

2008-08-25

In Arabidopsis thaliana, the final step of floral organ abscission is regulated by INFLORESCENCE DEFICIENT IN ABSCISSION (IDA): ida mutants fail to abscise floral organs, and plants overexpressing IDA display earlier abscission. We show that five IDA-LIKE (IDL) genes are expressed in different tissues, but plants overexpressing these genes have phenotypes similar to IDA-overexpressing plants, suggesting functional redundancy. IDA/IDL proteins have N-terminal signal peptides and a C-terminal conserved motif (extended PIP [EPIP]) at the C terminus (EPIP-C). IDA can, similar to CLAVATA3, be processed by an activity from cauliflower meristems. The EPIP-C of IDA and IDL1 replaced IDA function in vivo, when the signal peptide was present. In addition, synthetic IDA and IDL1 EPIP peptides rescued ida and induced early floral abscission in wild-type flowers. The EPIP-C of the other IDL proteins could partially substitute for IDA function. Similarly to ida, a double mutant between the receptor-like kinases (RLKs) HAESA (HAE) and HAESA-LIKE2 (HSL2) displays nonabscising flowers. Neither overexpression of IDA nor synthetic EPIP or EPIP-C peptides could rescue the hae hsl2 abscission deficiency. We propose that IDA and the IDL proteins constitute a family of putative ligands that act through RLKs to regulate different events during plant development.

Arabidopsis 10-Formyl Tetrahydrofolate Deformylases Are Essential for Photorespiration

2008-08-25

In prokaryotes, PurU (10-formyl tetrahydrofolate [THF] deformylase) metabolizes 10-formyl THF to formate and THF for purine and Gly biosyntheses. The Arabidopsis thaliana genome contains two putative purU genes, At4g17360 and At5g47435. Knocking out these genes simultaneously results in plants that are smaller and paler than the wild type. These double knockout (dKO) mutant plants show a 70-fold increase in Gly levels and accumulate elevated levels of 5- and 10-formyl THF. Embryo development in dKO mutants arrests between heart and early bent cotyledon stages. Mature seeds are shriveled, accumulate low amounts of lipids, and fail to germinate. However, the dKO mutant is only conditionally lethal and is rescued by growth under nonphotorespiratory conditions. In addition, culturing dKO siliques in the presence of sucrose restores normal embryo development and seed viability, suggesting that the seed and embryo development phenotypes are a result of a maternal effect. Our findings are consistent with the involvement of At4g17360 and At5g47435 proteins in photorespiration, which is to prevent excessive accumulation of 5-formyl THF, a potent inhibitor of the Gly decarboxylase/Ser hydroxymethyltransferase complex. Supporting this role, deletion of the At2g38660 gene that encodes the bifunctional 5,10-methylene THF dehydrogenase/5,10-methenyl THF cyclohydrolase that acts upstream of 5-formyl THF formation restored the wild-type phenotype in dKO plants.

Mutation of the Plastidial {alpha}-Glucan Phosphorylase Gene in Rice Affects the Synthesis and Structure of Starch in the Endosperm

2008-08-25

Plastidial phosphorylase (Pho1) accounts for ~96% of the total phosphorylase activity in developing rice (Oryza sativa) seeds. From mutant stocks induced by N-methyl-N-nitrosourea treatment, we identified plants with mutations in the Pho1 gene that are deficient in Pho1. Strikingly, the size of mature seeds and the starch content in these mutants showed considerable variation, ranging from shrunken to pseudonormal. The loss of Pho1 caused smaller starch granules to accumulate and modified the amylopectin structure. Variation in the morphological and biochemical phenotype of individual seeds was common to all 15 pho1-independent homozygous mutant lines studied, indicating that this phenotype was caused solely by the genetic defect. The phenotype of the pho1 mutation was temperature dependent. While the mutant plants grown at 30°C produced mainly plump seeds at maturity, most of the seeds from plants grown at 20°C were shrunken, with a significant proportion showing severe reduction in starch accumulation. These results strongly suggest that Pho1 plays a crucial role in starch biosynthesis in rice endosperm at low temperatures and that one or more other factors can complement the function of Pho1 at high temperatures.

Badh2, Encoding Betaine Aldehyde Dehydrogenase, Inhibits the Biosynthesis of 2-Acetyl-1-Pyrroline, a Major Component in Rice Fragrance

2008-08-25

In rice (Oryza sativa), the presence of a dominant Badh2 allele encoding betaine aldehyde dehydrogenase (BADH2) inhibits the synthesis of 2-acetyl-1-pyrroline (2AP), a potent flavor component in rice fragrance. By contrast, its two recessive alleles, badh2-E2 and badh2-E7, induce 2AP formation. Badh2 was found to be transcribed in all tissues tested except for roots, and the transcript was detected at higher abundance in young, healthy leaves than in other tissues. Multiple Badh2 transcript lengths were detected, and the complete, full-length Badh2 transcript was much less abundant than partial Badh2 transcripts. 2AP levels were significantly reduced in cauliflower mosaic virus 35S-driven transgenic lines expressing the complete, but not the partial, Badh2 coding sequences. In accordance, the intact, full-length BADH2 protein (503 residues) appeared exclusively in nonfragrant transgenic lines and rice varieties. These results indicate that the full-length BADH2 protein encoded by Badh2 renders rice nonfragrant by inhibiting 2AP biosynthesis. The BADH2 enzyme was predicted to contain three domains: NAD binding, substrate binding, and oligomerization domains. BADH2 was distributed throughout the cytoplasm, where it is predicted to catalyze the oxidization of betaine aldehyde, 4-aminobutyraldehyde (AB-ald), and 3-aminopropionaldehyde. The presence of null badh2 alleles resulted in AB-ald accumulation and enhanced 2AP biosynthesis. In summary, these data support the hypothesis that BADH2 inhibits 2AP biosynthesis by exhausting AB-ald, a presumed 2AP precursor.

Sphingolipid Long-Chain Base Hydroxylation Is Important for Growth and Regulation of Sphingolipid Content and Composition in Arabidopsis

Chen, M., Markham, J. E., Dietrich, C. R., Jaworski, J. G., Cahoon, E. B. — 2008-08-25

Sphingolipids are structural components of endomembranes and function through their metabolites as bioactive regulators of cellular processes such as programmed cell death. A characteristic feature of plant sphingolipids is their high content of trihydroxy long-chain bases (LCBs) that are produced by the LCB C-4 hydroxylase. To determine the functional significance of trihydroxy LCBs in plants, T-DNA double mutants and RNA interference suppression lines were generated for the two Arabidopsis thaliana LCB C-4 hydroxylase genes Sphingoid Base Hydroxylase1 (SBH1) and SBH2. These plants displayed reductions in growth that were dependent on the content of trihydroxy LCBs in sphingolipids. Double sbh1 sbh2 mutants, which completely lacked trihydroxy LCBs, were severely dwarfed, did not progress from vegetative to reproductive growth, and had enhanced expression of programmed cell death associated–genes. Furthermore, the total content of sphingolipids on a dry weight basis increased as the relative amounts of trihydroxy LCBs decreased. In trihydroxy LCB–null mutants, sphingolipid content was ~2.5-fold higher than that in wild-type plants. Increases in sphingolipid content resulted from the accumulation of molecular species with C16 fatty acids rather than with very-long-chain fatty acids, which are more commonly enriched in plant sphingolipids, and were accompanied by decreases in amounts of C16-containing species of chloroplast lipids. Overall, these results indicate that trihydroxy LCB synthesis plays a central role in maintaining growth and mediating the total content and fatty acid composition of sphingolipids in plants.

Dolichol Biosynthesis and Its Effects on the Unfolded Protein Response and Abiotic Stress Resistance in Arabidopsis

2008-08-25

Dolichols are long-chain unsaturated polyisoprenoids with multiple cellular functions, such as serving as lipid carriers of sugars used for protein glycosylation, which affects protein trafficking in the endoplasmic reticulum. The biological functions of dolichols in plants are largely unknown. We isolated an Arabidopsis thaliana mutant, lew1 (for leaf wilting1), that showed a leaf-wilting phenotype under normal growth conditions. LEW1 encoded a cis-prenyltransferase, which when expressed in Escherichia coli catalyzed the formation of dolichol with a chain length around C₈₀ in an in vitro assay. The lew1 mutation reduced the total plant content of main dolichols by ~85% and caused protein glycosylation defects. The mutation also impaired plasma membrane integrity, causing electrolyte leakage, lower turgor, reduced stomatal conductance, and increased drought resistance. Interestingly, drought stress in the lew1 mutant induced higher expression of the unfolded protein response pathway genes BINDING PROTEIN and BASIC DOMAIN/LEUCINE ZIPPER60 as well as earlier expression of the stress-responsive genes RD29A and COR47. The lew1 mutant was more sensitive to dark treatment, but this dark sensitivity was suppressed by drought treatment. Our data suggest that LEW1 catalyzes dolichol biosynthesis and that dolichol is important for plant responses to endoplasmic reticulum stress, drought, and dark-induced senescence in Arabidopsis.

Arabidopsis PUB22 and PUB23 Are Homologous U-Box E3 Ubiquitin Ligases That Play Combinatory Roles in Response to Drought Stress

Cho, S. K., Ryu, M. Y., Song, C., Kwak, J. M., Kim, W. T. — 2008-08-25

Ubiquitination is involved in diverse cellular processes in higher plants. In this report, we describe Arabidopsis thaliana PUB22 and PUB23, two homologous U-box–containing E3 ubiquitin (Ub) ligases. The PUB22 and PUB23 genes were rapidly and coordinately induced by abiotic stresses but not by abscisic acid. PUB22- and PUB23-overexpressing transgenic plants were hypersensitive to drought stress. By contrast, loss-of-function pub22 and pub23 mutant plants were significantly more drought-tolerant, and a pub22 pub23 double mutant displayed even greater drought tolerance. These results indicate that PUB22 and PUB23 function as negative regulators in the water stress response. Yeast two-hybrid, in vitro pull-down, and in vivo coimmunoprecipitation experiments revealed that PUB22 and PUB23 physically interacted with RPN12a, a subunit of the 19S regulatory particle (RP) in the 26S proteasome. Bacterially expressed RPN12a was effectively ubiquitinated in a PUB-dependent fashion. RPN12a was highly ubiquitinated in 35S:PUB22 plants, but not in pub22 pub23 double mutant plants, consistent with RPN12a being a substrate of PUB22 and PUB23 in vivo. In water-stressed wild-type and PUB-overexpressing plants, a significant amount of RPN12a was dissociated from the 19S RP and appeared to be associated with small-molecular-mass protein complexes in cytosolic fractions, where PUB22 and PUB23 are localized. Overall, our results suggest that PUB22 and PUB23 coordinately control a drought signaling pathway by ubiquitinating cytosolic RPN12a in Arabidopsis.

XopD SUMO Protease Affects Host Transcription, Promotes Pathogen Growth, and Delays Symptom Development in Xanthomonas-Infected Tomato Leaves

2008-08-25

We demonstrate that XopD, a type III effector from Xanthomonas campestris pathovar vesicatoria (Xcv), suppresses symptom production during the late stages of infection in susceptible tomato (Solanum lycopersicum) leaves. XopD-dependent delay of tissue degeneration correlates with reduced chlorophyll loss, reduced salicylic acid levels, and changes in the mRNA abundance of senescence- and defense-associated genes despite high pathogen titers. Subsequent structure-function analyses led to the discovery that XopD is a DNA binding protein that alters host transcription. XopD contains a putative helix-loop-helix domain required for DNA binding and two conserved ERF-associated amphiphilic motifs required to repress salicylic acid– and jasmonic acid–induced gene transcription in planta. Taken together, these data reveal that XopD is a unique virulence factor in Xcv that alters host transcription, promotes pathogen multiplication, and delays the onset of leaf chlorosis and necrosis.

RXLR-Mediated Entry of Phytophthora sojae Effector Avr1b into Soybean Cells Does Not Require Pathogen-Encoded Machinery

2008-08-25

Effector proteins secreted by oomycete and fungal pathogens have been inferred to enter host cells, where they interact with host resistance gene products. Using the effector protein Avr1b of Phytophthora sojae, an oomycete pathogen of soybean (Glycine max), we show that a pair of sequence motifs, RXLR and dEER, plus surrounding sequences, are both necessary and sufficient to deliver the protein into plant cells. Particle bombardment experiments demonstrate that these motifs function in the absence of the pathogen, indicating that no additional pathogen-encoded machinery is required for effector protein entry into host cells. Furthermore, fusion of the Avr1b RXLR-dEER domain to green fluorescent protein (GFP) allows GFP to enter soybean root cells autonomously. The conclusion that RXLR and dEER serve to transduce oomycete effectors into host cells indicates that the >370 RXLR-dEER–containing proteins encoded in the genome sequence of P. sojae are candidate effectors. We further show that the RXLR and dEER motifs can be replaced by the closely related erythrocyte targeting signals found in effector proteins of Plasmodium, the protozoan that causes malaria in humans. Mutational analysis of the RXLR motif shows that the required residues are very similar in the motifs of Plasmodium and Phytophthora. Thus, the machinery of the hosts (soybean and human) targeted by the effectors may be very ancient.

The Cladosporium fulvum Virulence Protein Avr2 Inhibits Host Proteases Required for Basal Defense

2008-08-25

Cladosporium fulvum (syn. Passalora fulva) is a biotrophic fungal pathogen that causes leaf mold of tomato (Solanum lycopersicum). During growth in the apoplast, the fungus establishes disease by secreting effector proteins, 10 of which have been characterized. We have previously shown that the Avr2 effector interacts with the apoplastic tomato Cys protease Rcr3, which is required for Cf-2–mediated immunity. We now show that Avr2 is a genuine virulence factor of C. fulvum. Heterologous expression of Avr2 in Arabidopsis thaliana causes enhanced susceptibility toward extracellular fungal pathogens, including Botrytis cinerea and Verticillium dahliae, and microarray analysis showed that Avr2 expression triggers a global transcriptome reflecting pathogen challenge. Cys protease activity profiling showed that Avr2 inhibits multiple extracellular Arabidopsis Cys proteases. In tomato, Avr2 expression caused enhanced susceptibility toward Avr2-defective C. fulvum strains and also toward B. cinerea and V. dahliae. Cys protease activity profiling in tomato revealed that, in this plant also, Avr2 inhibits multiple extracellular Cys proteases, including Rcr3 and its close relative Pip1. Finally, silencing of Avr2 significantly compromised C. fulvum virulence on tomato. We conclude that Avr2 is a genuine virulence factor of C. fulvum that inhibits several Cys proteases required for plant basal defense.

Tomato Protein Kinase 1b Mediates Signaling of Plant Responses to Necrotrophic Fungi and Insect Herbivory

AbuQamar, S., Chai, M.-F., Luo, H., Song, F., Mengiste, T. — 2008-08-25

The tomato protein kinase 1 (TPK1b) gene encodes a receptor-like cytoplasmic kinase localized to the plasma membrane. Pathogen infection, mechanical wounding, and oxidative stress induce expression of TPK1b, and reducing TPK1b gene expression through RNA interference (RNAi) increases tomato susceptibility to the necrotrophic fungus Botrytis cinerea and to feeding by larvae of tobacco hornworm (Manduca sexta) but not to the bacterial pathogen Pseudomonas syringae. TPK1b RNAi seedlings are also impaired in ethylene (ET) responses. Notably, susceptibility to Botrytis and insect feeding is correlated with reduced expression of the proteinase inhibitor II gene in response to Botrytis and 1-aminocyclopropane-1-carboxylic acid, the natural precursor of ET, but wild-type expression in response to mechanical wounding and methyl-jasmonate. TPK1b functions independent of JA biosynthesis and response genes required for resistance to Botrytis. TPK1b is a functional kinase with autophosphorylation and Myelin Basis Protein phosphorylation activities. Three residues in the activation segment play a critical role in the kinase activity and in vivo signaling function of TPK1b. In sum, our findings establish a signaling role for TPK1b in an ET-mediated shared defense mechanism for resistance to necrotrophic fungi and herbivorous insects.

Induced Plant Defenses in the Natural Environment: Nicotiana attenuata WRKY3 and WRKY6 Coordinate Responses to Herbivory

Skibbe, M., Qu, N., Galis, I., Baldwin, I. T. — 2008-08-25

A plant-specific family of WRKY transcription factors regulates plant responses to pathogens and abiotic stresses. Here, we identify two insect-responsive WRKY genes in the native tobacco Nicotiana attenuata: WRKY3, whose transcripts accumulate in response to wounding, and WRKY6, whose wound responses are significantly amplified when fatty acid–amino acid conjugates (FACs) in larval oral secretions are introduced into wounds during feeding. WRKY3 is required for WRKY6 elicitation, yet neither is elicited by treatment with the phytohormone wound signal jasmonic acid. Silencing either WRKY3 or WRKY6, or both, by stable transformation makes plants highly vulnerable to herbivores under glasshouse conditions and in their native habitat in the Great Basin Desert, Utah, as shown in three field seasons. This susceptibility is associated with impaired jasmonate (JA) accumulation and impairment of the direct (trypsin proteinase inhibitors) and indirect (volatiles) defenses that JA signaling mediates. The response to wounding and herbivore-specific signals (FACs) demonstrates that these WRKYs help plants to differentiate mechanical wounding from herbivore attack, mediating a plant's herbivore-specific defenses. Differences in responses to single and multiple elicitations indicate an important role of WRKY3 and WRKY6 in potentiating and/or sustaining active JA levels during continuous insect attack.

Correction

2008-08-25

Role of Xyloglucan in Primary Cell Walls

Eckardt, N. A. — 2008-07-30

High-Resolution Three-Dimensional Imaging of Plant Tissues

Eckardt, N. A. — 2008-07-30

Ubiquitin Ligation RINGs Twice: Redundant Control of Plant Processes by E3 Ubiquitin Ligases

Mach, J. — 2008-07-30

Abscisic Acid-Mediated Suppression of Systemic Acquired Resistance Signaling

Hofmann, N. — 2008-07-30

Arabidopsis Reactome: A Foundation Knowledgebase for Plant Systems Biology

2008-07-30

Arabidopsis DDB1-CUL4 ASSOCIATED FACTOR1 Forms a Nuclear E3 Ubiquitin Ligase with DDB1 and CUL4 That Is Involved in Multiple Plant Developmental Processes

2008-07-30

The human DDB1-CUL4 ASSOCIATED FACTOR (DCAF) proteins have been reported to interact directly with UV-DAMAGED DNA BINDING PROTEIN1 (DDB1) through the WDxR motif in their WD40 domain and function as substrate-recognition receptors for CULLIN4-based E3 ubiquitin ligases. Here, we identified and characterized a homolog of human DCAF1/VprBP in Arabidopsis thaliana. Yeast two-hybrid analysis demonstrated the physical interaction between DCAF1 and DDB1 from Arabidopsis, which is likely mediated via the WD40 domain of DCAF1 that contains two WDxR motifs. Moreover, coimmunoprecipitation assays showed that DCAF1 associates with DDB1, RELATED TO UBIQUITIN–modified CUL4, and the COP9 signalosome in vivo but not with CULLIN-ASSOCIATED and NEDDYLATION-DISSOCIATED1, CONSTITUTIVE PHOTOMORPHOGENIC1 (COP1), or the COP10-DET1-DDB1 complex, supporting the existence of a distinct Arabidopsis CUL4 E3 ubiquitin ligase, the CUL4-DDB1-DCAF1 complex. Transient expression of fluorescently tagged DCAF1, DDB1, and CUL4 in onion epidermal cells showed their colocalization in the nucleus, consistent with the notion that the CUL4-DDB1-DCAF1 complex functions as a nuclear E3 ubiquitin ligase. Genetic and phenotypic analysis of two T-DNA insertion mutants of DCAF1 showed that embryonic development of the dcaf1 homozygote is arrested at the globular stage, indicating that DCAF1 is essential for plant embryogenesis. Reducing the levels of DCAF1 leads to diverse developmental defects, implying that DCAF1 might be involved in multiple developmental pathways.

SPIN1, a K Homology Domain Protein Negatively Regulated and Ubiquitinated by the E3 Ubiquitin Ligase SPL11, Is Involved in Flowering Time Control in Rice

Vega-Sanchez, M. E., Zeng, L., Chen, S., Leung, H., Wang, G.-L. — 2008-07-30

The rice (Oryza sativa) E3 ligase SPOTTED LEAF11 (SPL11) negatively regulates programmed cell death and disease resistance. We demonstrate here that SPL11 also regulates flowering via interaction with SPIN1 (for SPL11-interacting protein1), a Signal Transduction and Activation of RNA family member. SPIN1 binds RNA and DNA in vitro and interacts with SPL11 in the nucleus. Spl11 mutants have delayed flowering under long-day conditions. Spin1 overexpression causes late flowering independently of daylength; expression analyses of flowering marker genes in these lines suggested that SPIN1 represses flowering by downregulating the flowering promoter gene Heading date3a (Hd3a) via Hd1-dependent mechanisms in short days and by targeting Hd1-independent factors in long days. Both Spin1 and Spl11 are regulated diurnally in opposing phases. SPL11 negatively regulates Spin1 transcript levels, while SPIN1 also affects Spl11 expression. Moreover, we show that coincidence of high accumulation of Spin1 mRNA with the light in the morning and early evening is needed to repress flowering. SPIN1 is monoubiquitinated by SPL11, suggesting that it is not targeted for degradation. Our data are consistent with a model in which SPIN1 acts as a negative regulator of flowering that itself is negatively regulated by SPL11, possibly via ubiquitination.

The Modified Flavonol Glycosylation Profile in the Arabidopsis rol1 Mutants Results in Alterations in Plant Growth and Cell Shape Formation

2008-07-30

Flavonoids are secondary metabolites known to modulate plant growth and development. A primary function of flavonols, a subgroup of flavonoids, is thought to be the modification of auxin fluxes in the plant. Flavonols in the cell are glycosylated, and the repressor of lrx1 (rol1) mutants of Arabidopsis thaliana, affected in rhamnose biosynthesis, have a modified flavonol glycosylation profile. A detailed analysis of the rol1-2 allele revealed hyponastic growth, aberrant pavement cell and stomatal morphology in cotyledons, and defective trichome formation. Blocking flavonoid biosynthesis suppresses the rol1-2 shoot phenotype, suggesting that it is induced by the modified flavonol profile. The hyponastic cotyledons of rol1-2 are likely to be the result of a flavonol-induced increase in auxin concentration. By contrast, the pavement cell, stomata, and trichome formation phenotypes appear not to be induced by the modified auxin distribution. Together, these results suggest that changes in the composition of flavonols can have a tremendous impact on plant development through both auxin-induced and auxin-independent processes.

Mutation of Arabidopsis BARD1 Causes Meristem Defects by Failing to Confine WUSCHEL Expression to the Organizing Center

Han, P., Li, Q., Zhu, Y.-X. — 2008-07-30

Stem cell fate in the Arabidopsis thaliana shoot apical meristem (SAM) is controlled by WUSCHEL (WUS) and CLAVATA. Here, we examine BARD1 (for BRCA1-associated RING domain 1), which had previously been implicated in DNA repair functions; we find that it also regulates WUS expression. We observed severe SAM defects in the knockout mutant bard1-3. WUS transcripts accumulated >238-fold in bard1-3 compared with the wild type and were located mainly in the outermost cell layers instead of the usual organizing center. A specific WUS promoter region was recognized by nuclear protein extracts obtained from wild-type plants, and this protein-DNA complex was recognized by antibodies against BARD1. The double mutant (wus-1 bard1-3) showed prematurely terminated SAM structures identical to those of wus-1, indicating that BARD1 functions through regulation of WUS. BARD1 overexpression resulted in reduced WUS transcript levels, giving a wus-1–like phenotype. Either full-length BARD1 or a clone that encoded the C-terminal domain (BARD1:C-ter;bard1-3) was sufficient to complement the bard1-3 phenotype, indicating that BARD1 functions through its C-terminal domain. Our data suggest that BARD1 regulates SAM organization and maintenance by limiting WUS expression to the organizing center.

High-Resolution Whole-Mount Imaging of Three-Dimensional Tissue Organization and Gene Expression Enables the Study of Phloem Development and Structure in Arabidopsis

2008-07-30

Currently, examination of the cellular structure of plant organs and the gene expression therein largely relies on the production of tissue sections. Here, we present a staining technique that can be used to image entire plant organs using confocal laser scanning microscopy. This technique produces high-resolution images that allow three-dimensional reconstruction of the cellular organization of plant organs. Importantly, three-dimensional domains of gene expression can be analyzed with single-cell precision. We used this technique for a detailed examination of phloem cells in the wild type and mutants. We were also able to recognize phloem sieve elements and their differentiation state in any tissue type and visualize the structure of sieve plates. We show that in the altered phloem development mutant, a hybrid cell type with phloem and xylem characteristics develops from initially normally differentiated protophloem cells. The simplicity of sieve element data collection allows for the statistical analysis of structural parameters of sieve plates, essential for the calculation of phloem conductivity. Taken together, this technique significantly improves the speed and accuracy of the investigation of plant growth and development.

Peeking into Pit Fields: A Multiple Twinning Model of Secondary Plasmodesmata Formation in Tobacco

Faulkner, C., Akman, O. E., Bell, K., Jeffree, C., Oparka, K. — 2008-07-30

In higher plants, plasmodesmata (PD) are major conduits for cell–cell communication. Primary PD are laid down at cytokinesis, while secondary PD arise during wall extension. During leaf development, the basal cell walls of trichomes extend radially without division, providing a convenient system for studying the origin of secondary PD. We devised a simple freeze-fracture protocol for examining large numbers of PD in surface view. In the postcytokinetic wall, simple PD were distributed randomly. As the wall extended, PD became twinned at the cell periphery. Additional secondary pores were inserted at right angles to these, giving rise to pit fields composed of several paired PD. During wall extension, the number of PD increased fivefold due to the insertion of secondary PD. Our data are consistent with a model in which a subset of the original primary PD pores function as templates for the insertion of new secondary PD, spatially fixing the position of future pit fields. Many of the new PD shared the same wall collar as the original PD pore, suggesting that new PD pores may arise by fissions of existing PD progenitors. Different models of secondary PD formation are discussed. Our data are supported by a computational model, Plasmodesmap, which accurately simulates the formation of radial pit fields during cell wall extension based on the occurrence of multiple PD twinning events in the cell wall. The model predicts PD distributions with striking resemblance to those seen on fractured wall faces.

Disrupting Two Arabidopsis thaliana Xylosyltransferase Genes Results in Plants Deficient in Xyloglucan, a Major Primary Cell Wall Component

2008-07-30

Xyloglucans are the main hemicellulosic polysaccharides found in the primary cell walls of dicots and nongraminaceous monocots, where they are thought to interact with cellulose to form a three-dimensional network that functions as the principal load-bearing structure of the primary cell wall. To determine whether two Arabidopsis thaliana genes that encode xylosyltransferases, XXT1 and XXT2, are involved in xyloglucan biosynthesis in vivo and to determine how the plant cell wall is affected by the lack of expression of XXT1, XXT2, or both, we isolated and characterized xxt1 and xxt2 single and xxt1 xxt2 double T-DNA insertion mutants. Although the xxt1 and xxt2 mutants did not have a gross morphological phenotype, they did have a slight decrease in xyloglucan content and showed slightly altered distribution patterns for xyloglucan epitopes. More interestingly, the xxt1 xxt2 double mutant had aberrant root hairs and lacked detectable xyloglucan. The reduction of xyloglucan in the xxt2 mutant and the lack of detectable xyloglucan in the xxt1 xxt2 double mutant resulted in significant changes in the mechanical properties of these plants. We conclude that XXT1 and XXT2 encode xylosyltransferases that are required for xyloglucan biosynthesis. Moreover, the lack of detectable xyloglucan in the xxt1 xxt2 double mutant challenges conventional models of the plant primary cell wall.

Targeted Degradation of the Cyclin-Dependent Kinase Inhibitor ICK4/KRP6 by RING-Type E3 Ligases Is Essential for Mitotic Cell Cycle Progression during Arabidopsis Gametogenesis

2008-07-30

Following meiosis, plant gametophytes develop through two or three rounds of mitosis. Although the ontogeny of gametophyte development has been defined in Arabidopsis thaliana, the molecular mechanisms regulating mitotic cell cycle progression are not well understood. Here, we report that RING-H2 group F 1a (RHF1a) and RHF2a, two RING-finger E3 ligases, play an important role in Arabidopsis gametogenesis. The rhf1a rhf2a double mutants are defective in the formation of male and female gametophytes due to interphase arrest of the mitotic cell cycle at the microspore stage of pollen development and at female gametophyte stage 1 of embryo sac development. We demonstrate that RHF1a directly interacts with and targets a cyclin-dependent kinase inhibitor ICK4/KRP6 (for Interactors of Cdc2 Kinase 4/Kip-related protein 6) for proteasome-mediated degradation. Inactivation of the two redundant RHF genes leads to the accumulation of ICK4/KRP6, and reduction of ICK4/KRP6 expression largely rescues the gametophytic defects in rhf1a rhf2a double mutants, indicating that ICK4/KRP6 is a substrate of the RHF E3 ligases. Interestingly, in situ hybridization showed that ICK4/KRP6 was predominantly expressed in sporophytes during meiosis. Our findings indicate that RHF1a/2a-mediated degradation of the meiosis-accumulated ICK4/KRP6 is essential to ensure the progression of subsequent mitoses to form gametophytes in Arabidopsis.

Arabidopsis ELONGATED MITOCHONDRIA1 Is Required for Localization of DYNAMIN-RELATED PROTEIN3A to Mitochondrial Fission Sites

2008-07-30

Mitochondrial fission is achieved partially by the activity of self-assembling dynamin-related proteins (DRPs) in diverse organisms. Mitochondrial fission in Arabidopsis thaliana is mediated by DRP3A and DRP3B, but the other genes and molecular mechanisms involved have yet to be elucidated. To identify these genes, we screened and analyzed Arabidopsis mutants with longer and fewer mitochondria than those of the wild type. ELM1 was found to be responsible for the phenotype of elongated mitochondria. This phenotype was also observed in drp3a plants. EST and genomic sequences similar to ELM1 were found in seed plants but not in other eukaryotes. ELM1:green fluorescent protein (GFP) was found to surround mitochondria, and ELM1 interacts with both DPR3A and DRP3B. In the elm1 mutant, DRP3A:GFP was observed in the cytosol, whereas in wild-type Arabidopsis, DRP3A:GFP localized to the ends and constricted sites of mitochondria. These results collectively suggest that mitochondrial fission in Arabidopsis is mediated by the plant-specific factor ELM1, which is required for the relocalization of DRP3A (and possibly also DRP3B) from the cytosol to mitochondrial fission sites.

Arabidopsis PEROXIN11c-e, FISSION1b, and DYNAMIN-RELATED PROTEIN3A Cooperate in Cell Cycle-Associated Replication of Peroxisomes

2008-07-30

Although participation of PEROXIN11 (PEX11), FISSION1 (FISl), and DYNAMIN-RELATED PROTEIN (DRP) has been well established during induced peroxisome proliferation in response to external stimuli, their roles in cell cycle–associated constitutive replication/duplication have not been fully explored. Herein, bimolecular fluorescence complementation experiments with Arabidopsis thaliana suspension cells revealed homooligomerization of all five PEX11 isoforms (PEX11a-e) and heterooligomerizations of all five PEX11 isoforms with FIS1b, but not FIS1a nor DRP3A. Intracellular protein targeting experiments demonstrated that FIS1b, but not FIS1a nor DRP3A, targeted to peroxisomes only when coexpressed with PEX11d or PEX11e. Simultaneous silencing of PEX11c-e or individual silencing of DRP3A, but not FIS1a nor FIS1b, resulted in ~40% reductions in peroxisome number. During G2 in synchronized cell cultures, peroxisomes sequentially enlarged, elongated, and then doubled in number, which correlated with peaks in PEX11, FIS1, and DRP3A expression. Overall, these data support a model for the replication of preexisting peroxisomes wherein PEX11c, PEX11d, and PEX11e act cooperatively during G2 to promote peroxisome elongation and recruitment of FIS1b to the peroxisome membrane, where DRP3A stimulates fission of elongated peroxisomes into daughter peroxisomes, which are then distributed between daughter cells.

Light-Induced Phosphorylation and Degradation of the Negative Regulator PHYTOCHROME-INTERACTING FACTOR1 from Arabidopsis Depend upon Its Direct Physical Interactions with Photoactivated Phytochromes

Shen, H., Zhu, L., Castillon, A., Majee, M., Downie, B., Huq, E. — 2008-07-30

The phytochrome (phy) family of photoreceptors regulates changes in gene expression in response to red/far-red light signals in part by physically interacting with constitutively nucleus-localized phy-interacting basic helix-loop-helix transcription factors (PIFs). Here, we show that PIF1, the member with the highest affinity for phys, is strongly sensitive to the quality and quantity of light. phyA plays a dominant role in regulating the degradation of PIF1 following initial light exposure, while phyB and phyD and possibly other phys also influence PIF1 degradation after prolonged illumination. PIF1 is rapidly phosphorylated and ubiquitinated under red and far-red light before being degraded with a half-life of ~1 to 2 min under red light. Although PIF1 interacts with phyB through a conserved active phyB binding motif, it interacts with phyA through a novel active phyA binding motif. phy interaction is necessary but not sufficient for the light-induced phosphorylation and degradation of PIF1. Domain-mapping studies reveal that the phy interaction, light-induced degradation, and transcriptional activation domains are located at the N-terminal 150–amino acid region of PIF1. Unlike PIF3, PIF1 does not interact with the two halves of either phyA or phyB separately. Moreover, overexpression of a light-stable truncated form of PIF1 causes constitutively photomorphogenic phenotypes in the dark. Taken together, these data suggest that removal of the negative regulators (e.g., PIFs) by light-induced proteolytic degradation might be sufficient to promote photomorphogenesis.

Arabidopsis Nuclear-Encoded Plastid Transit Peptides Contain Multiple Sequence Subgroups with Distinctive Chloroplast-Targeting Sequence Motifs

Lee, D. W., Kim, J. K., Lee, S., Choi, S., Kim, S., Hwang, I. — 2008-07-30

The N-terminal transit peptides of nuclear-encoded plastid proteins are necessary and sufficient for their import into plastids, but the information encoded by these transit peptides remains elusive, as they have a high sequence diversity and lack consensus sequences or common sequence motifs. Here, we investigated the sequence information contained in transit peptides. Hierarchical clustering on transit peptides of 208 plastid proteins showed that the transit peptide sequences are grouped to multiple sequence subgroups. We selected representative proteins from seven of these multiple subgroups and confirmed that their transit peptide sequences are highly dissimilar. Protein import experiments revealed that each protein contained transit peptide–specific sequence motifs critical for protein import into chloroplasts. Bioinformatics analysis identified sequence motifs that were conserved among members of the identified subgroups. The sequence motifs identified by the two independent approaches were nearly identical or significantly overlapped. Furthermore, the accuracy of predicting a chloroplast protein was greatly increased by grouping the transit peptides into multiple sequence subgroups. Based on these data, we propose that the transit peptides are composed of multiple sequence subgroups that contain distinctive sequence motifs for chloroplast targeting.

Analysis of the Golgi Apparatus in Arabidopsis Seed Coat Cells during Polarized Secretion of Pectin-Rich Mucilage

2008-07-30

Differentiation of the Arabidopsis thaliana seed coat cells includes a secretory phase where large amounts of pectinaceous mucilage are deposited to a specific domain of the cell wall. During this phase, Golgi stacks had cisternae with swollen margins and trans-Golgi networks consisting of interconnected vesicular clusters. The proportion of Golgi stacks producing mucilage was determined by immunogold labeling and transmission electron microscopy using an antimucilage antibody, CCRC-M36. The large percentage of stacks found to contain mucilage supports a model where all Golgi stacks produce mucilage synchronously, rather than having a subset of specialist Golgi producing pectin product. Initiation of mucilage biosynthesis was also correlated with an increase in the number of Golgi stacks per cell. Interestingly, though the morphology of individual Golgi stacks was dependent on the volume of mucilage produced, the number was not, suggesting that proliferation of Golgi stacks is developmentally programmed. Mapping the position of mucilage-producing Golgi stacks within developing seed coat cells and live-cell imaging of cells labeled with a trans-Golgi marker showed that stacks were randomly distributed throughout the cytoplasm rather than clustered at the site of secretion. These data indicate that the destination of cargo has little effect on the location of the Golgi stack within the cell.

Two Distinct Interacting Classes of Nuclear Envelope-Associated Coiled-Coil Proteins Are Required for the Tissue-Specific Nuclear Envelope Targeting of Arabidopsis RanGAP

Zhao, Q., Brkljacic, J., Meier, I. — 2008-07-30

Ran GTPase plays essential roles in multiple cellular processes, including nucleocytoplasmic transport, spindle formation, and postmitotic nuclear envelope (NE) reassembly. The cytoplasmic Ran GTPase activating protein RanGAP is critical to establish a functional RanGTP/RanGDP gradient across the NE and is associated with the outer surface of the NE in metazoan and higher plant cells. Arabidopsis thaliana RanGAP association with the root tip NE requires a family of likely plant-specific nucleoporins combining coiled-coil and transmembrane domains (CC-TMD) and WPP domain–interacting proteins (WIPs). We have now identified, by tandem affinity purification coupled with mass spectrometry, a second family of CC-TMD proteins, structurally similar, yet clearly distinct from the WIP family, that is required for RanGAP NE association in root tip cells. A combination of loss-of-function mutant analysis and protein interaction data indicates that at least one member of each NE-associated CC-TMD protein family is required for RanGAP targeting in root tip cells, while both families are dispensable in other plant tissues. This suggests an unanticipated complexity of RanGAP NE targeting in higher plant cells, contrasting both the single nucleoporin anchor in metazoans and the lack of targeting in fungi and proposes an early evolutionary divergence of the underlying plant and animal mechanisms.

Identification of the Gene Encoding the {alpha}1,3-Mannosyltransferase (ALG3) in Arabidopsis and Characterization of Downstream N-Glycan Processing

2008-07-30

Glycosyltransferases are involved in the biosynthesis of lipid-linked N-glycans. Here, we identify and characterize a mannosyltransferase gene from Arabidopsis thaliana, which is the functional homolog of the ALG3 (Dol-P-Man:Man₅GlcNAc₂-PP-Dol 1,3-mannosyl transferase) gene in yeast. The At ALG3 protein can complement a alg3 yeast mutant and is localized to the endoplasmic reticulum in yeast and in plants. A homozygous T-DNA insertion mutant, alg3-2, was identified in Arabidopsis with residual levels of wild-type ALG3, derived from incidental splicing of the 11th intron carrying the T-DNAs. N-glycan analysis of alg3-2 and alg3-2 in the complex-glycan-less mutant background, which lacks N-acetylglucosaminyl-transferase I activity, reveals that when ALG3 activity is strongly reduced, almost all N-glycans transferred to proteins are aberrant, indicating that the Arabidopsis oligosaccharide transferase complex is remarkably substrate tolerant. In alg3-2 plants, the aberrant glycans on glycoproteins are recognized by endogenous mannosidase I and N-acetylglucosaminyltransferase I and efficiently processed into complex-type glycans. Although no high-mannose-type glycoproteins are detected in alg3-2 plants, these plants do not show a growth phenotype under normal growth conditions. However, the glycosylation abnormalities result in activation of marker genes diagnostic of the unfolded protein response.

Channelrhodopsin-1 Initiates Phototaxis and Photophobic Responses in Chlamydomonas by Immediate Light-Induced Depolarization

2008-07-30

Channelrhodopsins (CHR1 and CHR2) are light-gated ion channels acting as sensory photoreceptors in Chlamydomonas reinhardtii. In neuroscience, they are used to trigger action potentials by light in neuronal cells, tissues, or living animals. Here, we demonstrate that Chlamydomonas cells with low CHR2 content exhibit photophobic and phototactic responses that strictly depend on the availability of CHR1. Since CHR1 was described as a H⁺-channel, the ion specificity of CHR1 was reinvestigated in Xenopus laevis oocytes. Our experiments show that, in addition to H⁺, CHR1 also conducts Na⁺, K⁺, and Ca²⁺. The kinetic selectivity analysis demonstrates that H⁺ selectivity is not due to specific translocation but due to selective ion binding. Purified recombinant CHR1 consists of two isoforms with different absorption maxima, CHR1₅₀₅ and CHR1₄₆₃, that are in pH-dependent equilibrium. Thus, CHR1 is a photochromic and protochromic sensory photoreceptor that functions as a light-activated cation channel mediating phototactic and photophobic responses via depolarizing currents in a wide range of ionic conditions.

Antagonistic Interaction between Systemic Acquired Resistance and the Abscisic Acid-Mediated Abiotic Stress Response in Arabidopsis

2008-07-30

Systemic acquired resistance (SAR) is a potent innate immunity system in plants that is effective against a broad range of pathogens. SAR development in dicotyledonous plants, such as tobacco (Nicotiana tabacum) and Arabidopsis thaliana, is mediated by salicylic acid (SA). Here, using two types of SAR-inducing chemicals, 1,2-benzisothiazol-3(2H)-one1,1-dioxide and benzo(1,2,3)thiadiazole-7-carbothioic acid S-methyl ester, which act upstream and downstream of SA in the SAR signaling pathway, respectively, we show that treatment with abscisic acid (ABA) suppresses the induction of SAR in Arabidopsis. In an analysis using several mutants in combination with these chemicals, treatment with ABA suppressed SAR induction by inhibiting the pathway both upstream and downstream of SA, independently of the jasmonic acid/ethylene-mediated signaling pathway. Suppression of SAR induction by the NaCl-activated environmental stress response proved to be ABA dependent. Conversely, the activation of SAR suppressed the expression of ABA biosynthesis–related and ABA-responsive genes, in which the NPR1 protein or signaling downstream of NPR1 appears to contribute. Therefore, our data have revealed that antagonistic crosstalk occurs at multiple steps between the SA-mediated signaling of SAR induction and the ABA-mediated signaling of environmental stress responses.

Arabidopsis DREB2A-Interacting Proteins Function as RING E3 Ligases and Negatively Regulate Plant Drought Stress-Responsive Gene Expression

2008-07-30

The DEHYDRATION-RESPONSIVE ELEMENT BINDING PROTEIN2A (DREB2A) transcription factor controls water deficit–inducible gene expression and requires posttranslational modification for its activation. The activation mechanism is not well understood; however, the stability of this protein in the nucleus was recently found to be important for its activation. Here, we report the isolation of Arabidopsis thaliana DREB2A-INTERACTING PROTEIN1 (DRIP1) and DRIP2, C3HC4 RING domain–containing proteins that interact with the DREB2A protein in the nucleus. An in vitro ubiquitination assay showed that they function as E3 ubiquitin ligases and are capable of mediating DREB2A ubiquitination. Overexpression of DRIP1 in Arabidopsis delayed the expression of DREB2A-regulated drought-responsive genes. Drought-inducible gene expression was slightly enhanced in the single T-DNA mutants of drip1-1 and drip2-1. By contrast, significantly enhanced gene expression was revealed in the drip1 drip2 double mutant under dehydration stress. Collectively, these data imply that DRIP1 and DRIP2 function negatively in the response of plants to drought stress. Moreover, overexpression of full-length DREB2A protein was more stable in drip1-1 than in the wild-type background. These results suggest that DRIP1 and DRIP2 act as novel negative regulators in drought-responsive gene expression by targeting DREB2A to 26S proteasome proteolysis.

Spermidine Exodus and Oxidation in the Apoplast Induced by Abiotic Stress Is Responsible for H2O2 Signatures That Direct Tolerance Responses in Tobacco

2008-07-30

Polyamines (PAs) exert a protective effect against stress challenges, but their molecular role in this remains speculative. In order to detect the signaling role of apoplastic PA-derived hydrogen peroxide (H₂O₂) under abiotic stress, we developed a series of tobacco (Nicotiana tabacum cv Xanthi) transgenic plants overexpressing or downregulating apoplastic polyamine oxidase (PAO; S-pao and A-pao plants, respectively) or downregulating S-adenosyl-l-methionine decarboxylase (samdc plants). Upon salt stress, plants secreted spermidine (Spd) into the apoplast, where it was oxidized by the apoplastic PAO, generating H₂O₂. A-pao plants accumulated less H₂O₂ and exhibited less programmed cell death (PCD) than did wild-type plants, in contrast with S-pao and samdc downregulating plants. Induction of either stress-responsive genes or PCD was dependent on the level of Spd-derived apoplastic H₂O₂. Thus, in wild-type and A-pao plants, stress-responsive genes were efficiently induced, although in the latter at a lower rate, while S-pao plants, with higher H₂O₂ levels, failed to accumulate stress-responsive mRNAs, inducing PCD instead. Furthermore, decreasing intracellular PAs, while keeping normal apoplastic Spd oxidation, as in samdc downregulating transgenic plants, caused enhanced salinity-induced PCD. These results reveal that salinity induces the exodus of Spd into the apoplast, where it is catabolized by PAO, producing H₂O₂. The accumulated H₂O₂ results in the induction of either tolerance responses or PCD, depending also on the levels of intracellular PAs.

Correction

2008-07-30

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.