Abstract: SECONDARY WALL-ASSOCIATED NAC DOMAIN PROTEIN1 (SND1) is a master transcriptional switch activating the developmental program of secondary wall biosynthesis. Here, we demonstrate that a battery of SND1-regulated transcription factors is required for normal secondary wall biosynthesis in Arabidopsis thaliana. The expression of 11 SND1-regulated transcription factors, namely, SND2, SND3, MYB103, MYB85, MYB52, MYB54, MYB69, MYB42, MYB43, MYB20, and KNAT7 (a Knotted1-like homeodomain protein), was developmentally associated with cells undergoing secondary wall thickening. Of these, dominant repression of SND2, SND3, MYB103, MYB85, MYB52, MYB54, and KNAT7 significantly reduced secondary wall thickening in fiber cells. Overexpression of SND2, SND3, and MYB103 increased secondary wall thickening in fibers, and overexpression of MYB85 led to ectopic deposition of lignin in epidermal and cortical cells in stems. Furthermore, SND2, SND3, MYB103, MYB85, MYB52, and MYB54 were able to induce secondary wall biosynthetic genes. Direct target analysis using the estrogen-inducible system revealed that MYB46, SND3, MYB103, and KNAT7 were direct targets of SND1 and also of its close homologs, NST1, NST2, and vessel-specific VND6 and VND7. Together, these results demonstrate that a transcriptional network consisting of SND1 and its downstream targets is involved in regulating secondary wall biosynthesis in fibers and that NST1, NST2, VND6, and VND7 are functional homologs of SND1 that regulate the same downstream targets in different cell types.

Abstract: Nuclear factor Y (NF-Y) is a ubiquitous transcription factor composed of three distinct subunits (NF-YA, NF-YB, and NF-YC). We found that the Arabidopsis thaliana NFYA5 transcript is strongly induced by drought stress in an abscisic acid (ABA)-dependent manner. Promoter:β-glucuronidase analyses showed that NFYA5 was highly expressed in vascular tissues and guard cells and that part of the induction by drought was transcriptional. NFYA5 contains a target site for miR169, which targets mRNAs for cleavage or translational repression. We found that miR169 was downregulated by drought stress through an ABA-dependent pathway. Analysis of the expression of miR169 precursors showed that miR169a and miR169c were substantially downregulated by drought stress. Coexpression of miR169 and NFYA5 suggested that miR169a was more efficient than miR169c at repressing the NFYA5 mRNA level. nfya5 knockout plants and plants overexpressing miR169a showed enhanced leaf water loss and were more sensitive to drought stress than wild-type plants. By contrast, transgenic Arabidopsis plants overexpressing NFYA5 displayed reduced leaf water loss and were more resistant to drought stress than the wild type. Microarray analysis indicated that NFYA5 is crucial for the expression of a number of drought stress–responsive genes. Thus, NFYA5 is important for drought resistance, and its induction by drought stress occurs at both the transcriptional and posttranscriptional levels.

Abstract: Chitin, a polymer of N -acetyl-d-glucosamine, is found in fungal cell walls but not in plants. Plant cells can perceive chitin fragments (chitooligosaccharides) leading to gene induction and defense responses. We identified a LysM receptor-like protein (LysM RLK1) required for chitin signaling in Arabidopsis thaliana . The mutation in this gene blocked the induction of almost all chitooligosaccharide-responsive genes and led to more susceptibility to fungal pathogens but had no effect on infection by a bacterial pathogen. Additionally, exogenously applied chitooligosaccharides enhanced resistance against both fungal and bacterial pathogens in the wild-type plants but not in the mutant. Together, our data indicate that LysM RLK1 is essential for chitin signaling in plants (likely as part of the receptor complex) and is involved in chitin-mediated plant innate immunity. The LysM RLK1-mediated chitin signaling pathway is unique, but it may share a conserved downstream pathway with the FLS2/flagellin- and EFR/EF-Tu–mediated signaling pathways. Additionally, our work suggests a possible evolutionary relationship between the chitin and Nod factor perception mechanisms due to the similarities between their potential receptors and between the signal molecules perceived by them.

Abstract: The Guard Model for disease resistance postulates that plant resistance proteins act by monitoring (guarding) the target of their corresponding pathogen effector. We posit, however, that guarded effector targets are evolutionarily unstable in plant populations polymorphic for resistance (R) genes. Depending on the absence or presence of the R gene, guarded effector targets are subject to opposing selection forces (1) to evade manipulation by effectors (weaker interaction) and (2) to improve perception of effectors (stronger interaction). Duplication of the effector target gene or independent evolution of a target mimic could relax evolutionary constraints and result in a decoy that would be solely involved in effector perception. There is growing support for this Decoy Model from four diverse cases of effector perception involving Pto, Bs3, RCR3, and RIN4. We discuss the differences between the Guard and Decoy Models and their variants, hypothesize how decoys might have evolved, and suggest ways to challenge the Decoy Model.

Abstract: Plants have evolved robust mechanisms to respond and adapt to unfavorable environmental conditions, such as low temperature. The C-repeat/drought-responsive element binding factor CBF1/DREB1b gene encodes a transcriptional activator transiently induced by cold that controls the expression of a set of genes responding to low temperature (the CBF regulon). Constitutive expression of CBF1 confers freezing tolerance but also slows growth. Here, we propose that low temperature-induced CBF1 expression restrains growth at least in part by allowing the accumulation of DELLAs, a family of nuclear growth-repressing proteins, the degradation of which is stimulated by gibberellin (GA). We show that cold/CBF1 enhances the accumulation of a green fluorescent protein (GFP)-tagged DELLA protein (GFP-RGA) by reducing GA content through stimulating expression of GA-inactivating GA 2-oxidase genes. Accordingly, transgenic plants that constitutively express CBF1 accumulate less bioactive GA and as a consequence exhibit dwarfism and late flowering. Both phenotypes are suppressed when CBF1 is expressed in a line lacking two DELLA proteins, GA-INSENSITIVE and REPRESSOR OF GA1-3. In addition, we show that DELLAs contribute significantly to CBF1-induced cold acclimation and freezing tolerance by a mechanism that is distinct from the CBF regulon. We conclude that DELLAs are components of the CBF1-mediated cold stress response.

Abstract: Reverse transcription followed by quantitative polymerase chain reaction analysis, or qRT-PCR, is an extremely sensitive, cost-effective method for quantifying gene transcripts from plant cells. The availability of nonspecific double-stranded DNA (dsDNA) binding fluorophors, such as SYBR Green, and

Abstract: In Arabidopsis thaliana, lateral roots are formed from root pericycle cells adjacent to the xylem poles. Lateral root development is regulated antagonistically by the plant hormones auxin and cytokinin. While a great deal is known about how auxin promotes lateral root development, the mechanism of cytokinin repression is still unclear. Elevating cytokinin levels was observed to disrupt lateral root initiation and the regular pattern of divisions that characterizes lateral root development in Arabidopsis. To identify the stage of lateral root development that is sensitive to cytokinins, we targeted the expression of the Agrobacterium tumefaciens cytokinin biosynthesis enzyme isopentenyltransferase to either xylem-pole pericycle cells or young lateral root primordia using GAL4-GFP enhancer trap lines. Transactivation experiments revealed that xylem-pole pericycle cells are sensitive to cytokinins, whereas young lateral root primordia are not. This effect is physiologically significant because transactivation of the Arabidopsis cytokinin degrading enzyme cytokinin oxidase 1 in lateral root founder cells results in increased lateral root formation. We observed that cytokinins perturb the expression of PIN genes in lateral root founder cells and prevent the formation of an auxin gradient that is required to pattern lateral root primordia.

Abstract: 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.

Abstract: New evidence suggests a role for the plant growth hormone auxin in pathogenesis and disease resistance. Bacterial infection induces the accumulation of indole-3-acetic acid (IAA), the major type of auxin, in rice (Oryza sativa). IAA induces the expression of expansins, proteins that loosen the cell wall. Loosening the cell wall is key for plant growth but may also make the plant vulnerable to biotic intruders. Here, we report that rice GH3-8, an auxin-responsive gene functioning in auxin-dependent development, activates disease resistance in a salicylic acid signaling- and jasmonic acid signaling-independent pathway. GH3-8 encodes an IAA-amino synthetase that prevents free IAA accumulation. Overexpression of GH3-8 results in enhanced disease resistance to the rice pathogen Xanthomonas oryzae pv oryzae. This resistance is independent of jasmonic acid and salicylic acid signaling. Overexpression of GH3-8 also causes abnormal plant morphology and retarded growth and development. Both enhanced resistance and abnormal development may be caused by inhibition of the expression of expansins via suppressed auxin signaling.

Abstract: Arabidopsis thaliana WRKY38 and WRKY62, encoding two structurally similar type III WRKY transcription factors, are induced in a Nonexpressor of PR Gene1 (NPR1)-dependent manner by salicylic acid (SA) or by virulent Pseudomonas syringae. Disease resistance and SA-regulated Pathogenesis-Related1 (PR1) gene expression are enhanced in the wrky38 and wrky62 single mutants and, to a greater extent, in the double mutants. Overexpression of WRKY38 or WRKY62 reduces disease resistance and PR1 expression. Thus, WRKY38 and WRKY62 function additively as negative regulators of plant basal defense. WRKY38 and WRKY62 interact with Histone Deacetylase 19 (HDA19). Expression of HDA19 is also induced by P. syringae, and the stability of its induced transcripts depends on SA and NPR1 in infected plants. Disruption of HDA19 leads to compromised resistance, whereas its overexpression results in enhanced resistance to P. syringae. Thus, HDA19 has a role opposite from those of WRKY38 and WRKY62 in basal resistance to the bacterial pathogen. Both WRKY38 and WRKY62 are transcriptional activators in plant cells, but their activation activities are abolished by overexpressed HDA19. Interaction of WRKY38 and WRKY62 with HDA19 may act to fine-tune plant basal defense responses.

Abstract: 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.

Abstract: 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.

Abstract: 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.

Abstract: 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.

Abstract: 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.

Abstract: Little is known about the molecular and regulatory mechanisms of long-distance nitrate transport in higher plants. NRT1.5 is one of the 53 Arabidopsis thaliana nitrate transporter NRT1 (Peptide Transporter PTR) genes, of which two members, NRT1.1 (CHL1 for Chlorate resistant 1) and NRT1.2, have been shown to be involved in nitrate uptake. Functional analysis of cRNA-injected Xenopus laevis oocytes showed that NRT1.5 is a low-affinity, pH-dependent bidirectional nitrate transporter. Subcellular localization in plant protoplasts and in planta promoter-beta-glucuronidase analysis, as well as in situ hybridization, showed that NRT1.5 is located in the plasma membrane and is expressed in root pericycle cells close to the xylem. Knockdown or knockout mutations of NRT1.5 reduced the amount of nitrate transported from the root to the shoot, suggesting that NRT1.5 participates in root xylem loading of nitrate. However, root-to-shoot nitrate transport was not completely eliminated in the NRT1.5 knockout mutant, and reduction of NRT1.5 in the nrt1.1 background did not affect root-to-shoot nitrate transport. These data suggest that, in addition to that involving NRT1.5, another mechanism is responsible for xylem loading of nitrate. Further analyses of the nrt1.5 mutants revealed a regulatory loop between nitrate and potassium at the xylem transport step.

Abstract: Gibberellin 2-oxidases (GA2oxs) regulate plant growth by inactivating endogenous bioactive gibberellins (GAs). Two classes of GA2oxs inactivate GAs through 2β-hydroxylation: a larger class of C19 GA2oxs and a smaller class of C20 GA2oxs. In this study, we show that members of the rice (Oryza sativa) GA2ox family are differentially regulated and act in concert or individually to control GA levels during flowering, tillering, and seed germination. Using mutant and transgenic analysis, C20 GA2oxs were shown to play pleiotropic roles regulating rice growth and architecture. In particular, rice overexpressing these GA2oxs exhibited early and increased tillering and adventitious root growth. GA negatively regulated expression of two transcription factors, O. sativa homeobox 1 and TEOSINTE BRANCHED1, which control meristem initiation and axillary bud outgrowth, respectively, and that in turn inhibited tillering. One of three conserved motifs unique to the C20 GA2oxs (motif III) was found to be important for activity of these GA2oxs. Moreover, C20 GA2oxs were found to cause less severe GA-defective phenotypes than C19 GA2oxs. Our studies demonstrate that improvements in plant architecture, such as semidwarfism, increased root systems and higher tiller numbers, could be induced by overexpression of wild-type or modified C20 GA2oxs.

Abstract: Seed germination is antagonistically controlled by the phytohormones gibberellic acid (GA) and abscisic acid (ABA). GA promotes seed germination by enhancing the proteasome-mediated destruction of RGL2 (for RGA-LIKE2), a key DELLA factor repressing germination. By contrast, ABA blocks germination by inducing ABI5 (for ABA-INSENSITIVE5), a basic domain/leucine zipper transcription factor repressing germination. Decreased GA synthesis leads to an increase in endogenous ABA levels through a stabilized RGL2, a process that may involve XERICO, a RING-H2 zinc finger factor promoting ABA synthesis. In turn, increased endogenous ABA synthesis is necessary to elevate not only ABI5 RNA and protein levels but also, critically, those of RGL2. Increased ABI5 protein is ultimately responsible for preventing seed germination when GA levels are reduced. However, overexpression of ABI5 was not sufficient to repress germination, as ABI5 activity requires phosphorylation. The endogenous ABI5 phosphorylation and inhibition of germination could be recapitulated by the addition of a SnRK2 protein kinase to the ABI5 overexpression line. In sleepy1 mutant seeds, RGL2 overaccumulates; germination of these seeds can occur under conditions that produce low ABI5 expression. These data support the notion that ABI5 acts as the final common repressor of germination in response to changes in ABA and GA levels.

Abstract: 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.

Abstract: The type B Arabidopsis Response Regulators (ARRs) of Arabidopsis thaliana are transcription factors that act as positive regulators in the two-component cytokinin signaling pathway. We employed a mutant-based approach to perform a detailed characterization of the roles of ARR1, ARR10, and ARR12 in plant growth and development. The most pronounced phenotype was found in the arr1-3 arr10-5 arr12-1 triple loss-of-function mutant, which showed almost complete insensitivity to high levels of exogenously applied cytokinins. The triple mutant exhibited reduced stature due to decreased cell division in the shoot, enhanced seed size, increased sensitivity to light, altered chlorophyll and anthocyanin concentrations, and an aborted primary root with protoxylem but no metaxylem. Microarray analysis revealed that expression of the majority of cytokinin-regulated genes requires the function of ARR1, ARR10, and ARR12. Characterization of double mutants revealed differing contributions of the type B ARRs to mutant phenotypes. Our results support a model in which cytokinin regulates a wide array of downstream responses through the action of a multistep phosphorelay that culminates in transcriptional regulation by ARR1, ARR10, and ARR12.

Abstract: Reactive oxygen species (ROS) produced by NADPH oxidase play critical roles in various cellular activities, including plant innate immunity response. In contrast with the large multiprotein NADPH oxidase complex of phagocytes, in plants, only the homologs of the catalytic subunit gp91phox and the cytosolic regulator small GTPase Rac are found. Plant homologs of the gp91phox subunit are known as Rboh (for respiratory burst oxidase homolog). Although numerous Rboh have been isolated in plants, the regulation of enzymatic activity remains unknown. All rboh genes identified to date possess a conserved N-terminal extension that contains two Ca2+ binding EF-hand motifs. Previously, we ascertained that a small GTPase Rac (Os Rac1) enhanced pathogen-associated molecular pattern–induced ROS production and resistance to pathogens in rice (Oryza sativa). In this study, using yeast two-hybrid assay, we found that interaction between Rac GTPases and the N-terminal extension is ubiquitous and that a substantial part of the N-terminal region of Rboh, including the two EF-hand motifs, is required for the interaction. The direct Rac–Rboh interaction was supported by further studies using in vitro pull-down assay, a nuclear magnetic resonance titration experiment, and in vivo fluorescence resonance energy transfer (FRET) microscopy. The FRET analysis also suggests that cytosolic Ca2+ concentration may regulate Rac–Rboh interaction in a dynamic manner. Furthermore, transient coexpression of Os Rac1 and rbohB enhanced ROS production in Nicotiana benthamiana, suggesting that direct Rac–Rboh interaction may activate NADPH oxidase activity in plants. Taken together, the results suggest that cytosolic Ca2+ concentration may modulate NADPH oxidase activity by regulating the interaction between Rac GTPase and Rboh.

Abstract: 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.

Abstract: The sesquiterpene (E)-b-caryophyllene is emitted by maize (Zea mays) leaves in response to attack by lepidopteran larvae like Spodoptera littoralis and released from roots after damage by larvae of the coleopteran Diabrotica virgifera virgifera. We identified a maize terpene synthase, Terpene Synthase 23 (TPS23), that produces (E)-b-caryophyllene from farnesyl diphosphate. The expression of TPS23 is controlled at the transcript level and induced independently by D. v. virgifera damage in roots and S. littoralis damage in leaves. We demonstrate that (E)-b-caryophyllene can attract natural enemies of both herbivores: entomopathogenic nematodes below ground and parasitic wasps, after an initial learning experience, above ground. The biochemical properties of TPS23 are similar to those of (E)-b-caryophyllene synthases from dicotyledons but are the result of repeated evolution. The sequence of TPS23 is maintained by positive selection in maize and its closest wild relatives, teosinte (Zea sp) species. The gene encoding TPS23 is active in teosinte species and European maize lines, but decreased transcription in most North American lines resulted in the loss of (E)-b-caryophyllene production. We argue that the (E)-b-caryophyllene defense signal was lost during breeding of the North American lines and that its restoration might help to increase the resistance of these lines against agronomically important pests.

Abstract: Stresses leading to the accumulation of misfolded proteins in the endoplasmic reticulum (ER) elicit a highly conserved ER stress response in plants called the unfolded protein response (UPR). While the response itself is well documented in plants, the components of the signaling pathway are less well known. We have identified three membrane-associated basic domain/leucine zipper (bZIP) factors in Arabidopsis thaliana that are candidates for ER stress sensors/transducers. One of these factors, bZIP28, an ER-resident transcription factor, is activated in response to treatment by tunicamycin (TM), an agent that blocks N-linked protein glycosylation. Following TM treatment, bZIP28 is processed, releasing its N-terminal, cytoplasm-facing domain, which is translocated to the nucleus. Expression of a truncated form of bZIP28, containing only the cytoplasmic domain of the protein, upregulated the expression of ER stress response genes in the absence of stress conditions. Thus, bZIP28 serves as a sensor/transducer in Arabidopsis to mediate ER stress responses related to UPR.

Abstract: 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.

Abstract: In Arabidopsis thaliana, the blue light photoreceptor cryptochromes (CRY) act to promote photomorphogenic development and the transition from vegetative to floral development in long days (LDs). We previously proposed that one of the mechanisms by which CRY regulates light responses is via its physical interaction with COP1, a RING motif-containing E3 ligase. Under LDs, the transcription of FLOWERING LOCUS T (FT) is activated by CONSTANS (CO) in leaf, and the FT protein moves to the shoot apex to induce flowering. CO protein is degraded in darkness, whereas it is stabilized by the CRY-mediated signal. However, the mechanism underlying this process is unknown. We show in this report that CO acts genetically downstream of COP1 and CRY to regulate flowering time. In addition, COP1 physically interacts with CO and functions as an E3 ligase, ubiquitinating CO in vitro and reducing CO levels in vivo. These results suggest that COP1 acts as a repressor of flowering by promoting the ubiquitin-mediated proteolysis of CO in darkness and that CRY-mediated signal may negatively regulate COP1, thereby stabilizing CO, activating FT transcription, and inducing flowering.

Abstract: Plant immune responses to pathogen attack include the hypersensitive response (HR), a form of programmed cell death occurring at invasion sites. We previously reported on Arabidopsis thaliana MYB30, a transcription factor that acts as a positive regulator of a cell death pathway conditioning the HR. Here, we show by microarray analyses of Arabidopsis plants misexpressing MYB30 that the genes encoding the four enzymes forming the acyl-coA elongase complex are putative MYB30 targets. The acyl-coA elongase complex synthesizes very-long-chain fatty acids (VLCFAs), and the accumulation of extracellular VLCFA-derived metabolites (leaf epidermal wax components) was affected in MYB30 knockout mutant and overexpressing lines. In the same lines, a lipid extraction procedure allowing high recovery of sphingolipids revealed changes in VLCFA contents that were amplified in response to inoculation. Finally, the exacerbated HR phenotype of MYB30-overexpressing lines was altered by the loss of function of the acyl-ACP thioesterase FATB, which causes severe defects in the supply of fatty acids for VLCFA biosynthesis. Based on these findings, we propose a model in which MYB30 modulates HR via VLCFAs by themselves, or VLCFA derivatives, as cell death messengers in plants.

Abstract: We show that a previously uncharacterized Arabidopsis thaliana basic helix-loop-helix (bHLH) phytochrome interacting factor (PIF), designated PIF7, interacts specifically with the far-red light-absorbing Pfr form of phyB through a conserved domain called the active phyB binding motif. Similar to PIF3, upon light exposure, PIF7 rapidly migrates to intranuclear speckles, where it colocalizes with phyB. However, in striking contrast to PIF3, this process is not accompanied by detectable light-induced phosphorylation or degradation of PIF7, suggesting that the consequences of interaction with photoactivated phyB may differ among PIFs. Nevertheless, PIF7 acts similarly to PIF3 in prolonged red light as a weak negative regulator of phyB-mediated seedling deetiolation. Examination of pif3, pif4, and pif7 double mutant combinations shows that their moderate hypersensitivity to extended red light is additive. We provide evidence that the mechanism by which these PIFs operate on the phyB signaling pathway under prolonged red light is through maintaining low phyB protein levels, in an additive or synergistic manner, via a process likely involving the proteasome pathway. These data suggest that the role of these phyB-interacting bHLH factors in modulating seedling deetiolation in prolonged red light may not be as phy-activated signaling intermediates, as proposed previously, but as direct modulators of the abundance of the photoreceptor.

Abstract: To complete the metabolic map for an entire class of compounds, it is essential to identify gene–metabolite correlations of a metabolic pathway. We used liquid chromatography–mass spectrometry (LC-MS) to identify the flavonoids produced by Arabidopsis thaliana wild-type and flavonoid biosynthetic mutant lines. The structures of 15 newly identified and eight known flavonols were deduced by LC-MS profiling of these mutants. Candidate genes presumably involved in the flavonoid pathway were delimited by transcriptome coexpression network analysis using public databases, leading to the detailed analysis of two flavonoid pathway genes, UGT78D3 (At5g17030) and RHM1 (At1g78570). The levels of flavonol 3-O-arabinosides were reduced in ugt78d3 knockdown mutants, suggesting that UGT78D3 is a flavonol arabinosyltransferase. Recombinant UGT78D3 protein could convert quercetin to quercetin 3-O-arabinoside. The strict substrate specificity of UGT78D3 for flavonol aglycones and UDP-arabinose indicate that UGT78D3 is a flavonol arabinosyltransferase. A comparison of flavonol profile in RHM knockout mutants indicated that RHM1 plays a major role in supplying UDP-rhamnose for flavonol modification. The rate of flavonol 3-O-glycosylation is more affected than those of 7-O-glycosylation by the supply of UDP-rhamnose. The precise identification of flavonoids in conjunction with transcriptomics thus led to the identification of a gene function and a more complete understanding of a plant metabolic network.