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The conjugation of sterols with a Glc moiety is catalyzed by sterol glucosyltransferases (SGTs). A portion of the resulting steryl glucosides (SG) are then esterified with a long-chain fatty acid to form acyl-SG (ASG). SG and ASG are prevalent components of plant cellular membranes and influence their organization and functional properties. Mutant analysis had previously inferred that two Arabidopsis SGTs, UGT80A2 and UGT80B1/TT15, could have specialized roles in the production of SG in seeds, despite an overlap in their enzymatic activity. Here, we establish new roles for both enzymes in the accumulation of polysaccharides in seed coat epidermal cells (SCEs). The rhamnogalacturonan-I (RG-I) content of the inner layer of seed mucilage was higher in ugt80A2 , whereas RG-I accumulation was lower in mutants of UGT80B1 , with double mutant phenotypes indicating that UGT80A2 acts independently from UGT80B1. In contrast, an additive phenotype was observed in double mutants for increased galactoglucomannan (GGM) content. Double mutants also exhibited increased polymer density within the inner mucilage layer. In contrast, cell wall defects were only observed in mutants defective for UGT80B1 , while more mucilage cellulose was only observed when UGT80A2 was mutated. The generation of a range of phenotypic effects, simultaneously within a single cell type, demonstrates that the adjustment of the SG and ASG composition of cellular membranes by UGT80A2 and UGT80B1 tailors polysaccharide accumulation in Arabidopsis seeds.

Despite a general view that asparagine synthetase generates asparagine as an amino acid for long-distance transport of nitrogen to sink organs, its role in nitrogen metabolic pathways in floral organs during seed nitrogen filling has remained undefined. We demonstrate that the onset of pollination in Arabidopsis induces selected genes for asparagine metabolism, namely ASN1 (At3g47340), GLN2 (At5g35630), GLU1 (At5g04140), AapAT2 (At5g19950), ASPGA1 (At5g08100) and ASPGB1 (At3g16150), particularly at the ovule stage (stage 0), accompanied by enhanced asparagine synthetase protein, asparagine and total amino acids. Immunolocalization confined asparagine synthetase to the vascular cells of the silique cell wall and septum, but also to the outer and inner seed integuments, demonstrating the post-phloem transport of asparagine in these cells to developing embryos. In the asn1 mutant, aberrant embryo cell divisions in upper suspensor cell layers from globular to heart stages assign a role for nitrogen in differentiating embryos within the ovary. Induction of asparagine metabolic genes by light/dark and nitrate supports fine shifts of nitrogen metabolic pathways. In transgenic Arabidopsis expressing promoter Ca MV 35S ::ASN1 fusion, marked metabolomics changes at stage 0, including a several-fold increase in free asparagine, are correlated to enhanced seed nitrogen. However, specific promoter Napin2S ::ASN1 expression during seed formation and a six-fold increase in asparagine toward the desiccation stage result in wild-type seed nitrogen, underlining that delayed accumulation of asparagine impairs the timing of its use by releasing amide and amino nitrogen. Transcript and metabolite profiles in floral organs match the carbon and nitrogen partitioning to generate energy via the tricarboxylic acid cycle, GABA shunt and phosphorylated serine synthetic pathway., (© 2017 The Authors The Plant Journal © 2017 John Wiley & Sons Ltd.)

The phloem is a complex tissue composed of highly specialized cells with unique subcellular structures and a compact organization that is challenging to study in vivo at cellular resolution. We used confocal scanning laser microscopy and subcellular fluorescent markers in companion cells and sieve elements, for live imaging of the phloem in Arabidopsis leaves. This approach provided a simple framework for identifying phloem cell types unambiguously. It highlighted the compactness of the meshed network of organelles within companion cells. By contrast, within the sieve elements, unknown bodies were observed in association with the PP2-A1:GFP, GFP:RTM1 and RTM2:GFP markers at the cell periphery. The phloem lectin PP2-A1:GFP marker was found in the parietal ground matrix. Its location differed from that of the P-protein filaments, which were visualized with SEOR1:GFP and SEOR2:GFP. PP2-A1:GFP surrounded two types of bodies, one of which was identified as mitochondria. This location suggested that it was embedded within the sieve element clamps, specific structures that may fix the organelles to each another or to the plasma membrane in the sieve tubes. GFP:RTM1 was associated with a class of larger bodies, potentially corresponding to plastids. PP2-A1:GFP was soluble in the cytosol of immature sieve elements. The changes in its subcellular localization during differentiation provide an in vivo blueprint for monitoring this process. The subcellular features obtained with these companion cell and sieve element markers can be used as landmarks for exploring the organization and dynamics of phloem cells in vivo.

Among secondary metabolites, flavonoids are particularly important for the plant life cycle and could be beneficial for human health. The study of Arabidopsis thaliana transparent testa mutants showed that seed flavonoids are important for environmental adaptation, reactive oxygen species homeostasis, dormancy and longevity. Compared with Arabidopsis and maize (Zea mays L.), far less research has been conducted on rice (Oryza sativa L.) particularly for cultivars with non-pigmented seeds. In this study, we describe the localization, nature and relative abundance of flavonoids in mature and germinated non-pigmented Nipponbare seeds using a combination of confocal microscopy, mass spectrometry and gene expression analysis. The mature seed exclusively accumulates flavones mostly in the embryo and to a lesser extent in the pericarp/testa. Due to the variety of flavone conjugation patterns, 21 different flavones were identified, including sulfated flavones never mentioned before in cereals. Schaftoside (apigenin-6-C-glucoside-8-C-arabinoside) and its two isomers represent nearly 50% of all rice seed flavones and are the only flavonoids accumulated in the pericarp/testa seed compartment. These 21 conjugated flavones showed a very stable profile during rice seed germination sensu stricto, while expression of key flavone synthesis genes strongly increases before the completion of germination. We discuss the potential roles of these rice seed flavones in a seed biology context., (© The Author 2014. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

We investigated the function of ASN2, one of the three genes encoding asparagine synthetase (EC 6.3.5.4), which is the most highly expressed in vegetative leaves of Arabidopsis thaliana. Expression of ASN2 and parallel higher asparagine content in darkness suggest that leaf metabolism involves ASN2 for asparagine synthesis. In asn2-1 knockout and asn2-2 knockdown lines, ASN2 disruption caused a defective growth phenotype and ammonium accumulation. The asn2 mutant leaves displayed a depleted asparagine and an accumulation of alanine, GABA, pyruvate and fumarate, indicating an alanine formation from pyruvate through the GABA shunt to consume excess ammonium in the absence of asparagine synthesis. By contrast, asparagine did not contribute to photorespiratory nitrogen recycle as photosynthetic net CO(2) assimilation was not significantly different between lines under both 21 and 2% O(2). ASN2 was found in phloem companion cells by in situ hybridization and immunolocalization. Moreover, lack of asparagine in asn2 phloem sap and lowered (15) N flux to sinks, accompanied by the delayed yellowing (senescence) of asn2 leaves, in the absence of asparagine support a specific role of asparagine in phloem loading and nitrogen reallocation. We conclude that ASN2 is essential for nitrogen assimilation, distribution and remobilization (via the phloem) within the plant., (© 2012 Blackwell Publishing Ltd.)

Land plant cells assemble microtubule arrays without a conspicuous microtubule organizing center like a centrosome. In Arabidopsis thaliana, the TONNEAU1 (TON1) proteins, which share similarity with FOP, a human centrosomal protein, are essential for microtubule organization at the cortex. We have identified a novel superfamily of 34 proteins conserved in land plants, the TON1 Recruiting Motif (TRM) proteins, which share six short conserved motifs, including a TON1-interacting motif present in all TRMs. An archetypal member of this family, TRM1, is a microtubule-associated protein that localizes to cortical microtubules and binds microtubules in vitro. Not all TRM proteins can bind microtubules, suggesting a diversity of functions for this family. In addition, we show that TRM1 interacts in vivo with TON1 and is able to target TON1 to cortical microtubules via its C-terminal TON1 interaction motif. Interestingly, three motifs of TRMs are found in CAP350, a human centrosomal protein interacting with FOP, and the C-terminal M2 motif of CAP350 is responsible for FOP recruitment at the centrosome. Moreover, we found that TON1 can interact with the human CAP350 M2 motif in yeast. Taken together, our results suggest conservation of eukaryotic centrosomal components in plant cells.

Background Information: The idea that GPCRs (G-protein-coupled receptors) may exist as homo- or hetero-oligomers, although still controversial, is now widely accepted. Nevertheless, the functional roles of oligomerization are still unclear and gaining greater insight into the mechanisms underlying the dynamics of GPCR assembly and, in particular, assessing the effect of ligands on this process seems important. We chose to focus our present study on the effect of MT7 (muscarinic toxin 7), a highly selective allosteric peptide ligand, on the oligomerization state of the hM1 (human M1 muscarinic acetylcholine receptor subtype)., Results: We analysed the hM1 oligomerization state in membrane preparations or in live cells and observed the effect of MT7 via four complementary techniques: native-PAGE electrophoresis analysed by both Western blotting and autoradiography on solubilized membrane preparations of CHO-M1 cells (Chinese-hamster ovary cells expressing muscarinic M1 receptors); FRET (fluorescence resonance energy transfer) experiments on cells expressing differently tagged M1 receptors using either an acceptor photobleaching approach or a novel fluorescence emission anisotropy technique; and, finally, by BRET (bioluminescence resonance energy transfer) assays. Our results reveal that MT7 seems to protect the M1 receptor from the dissociating effect of the detergent and induces an increase in the FRET and BRET signals, highlighting its ability to affect the dimeric form of the receptor., Conclusions: Our results suggest that MT7 binds to a dimeric form of hM1 receptor, favouring the stability of this receptor state at the cellular level, probably by inducing some conformational rearrangements of the pre-existing muscarinic receptor homodimers.

This study was aimed at investigating the physiological role of ferredoxin-glutamate synthases (EC 1.4.1.7), NADH-glutamate synthase (EC 1.4.1.14) and carbamoylphosphate synthetase (EC 6.3.5.5) in Arabidopsis. Phenotypic analysis revealed a high level of photorespiratory ammonium, glutamine/glutamate and asparagine/aspartate in the GLU1 mutant lacking the major ferredoxin-glutamate synthase, indicating that excess photorespiratory ammonium was detoxified into amino acids for transport out of the veins. Consistent with these results, promoter analysis and in situ hybridization demonstrated that GLU1 and GLU2 were expressed in the mesophyll and phloem companion cell-sieve element complex. However, these phenotypic changes were not detected in the GLU2 mutant defective in the second ferredoxin-glutamate synthase gene. The impairment in primary ammonium assimilation in the GLT mutant under nonphotorespiratory high-CO(2) conditions underlined the importance of NADH-glutamate synthase for amino acid trafficking, given that this gene only accounted for 3% of total glutamate synthase activity. The excess ammonium from either endogenous photorespiration or the exogenous medium was shifted to arginine. The promoter analysis and slight effects on overall arginine synthesis in the T-DNA insertion mutant in the single carbamoylphosphate synthetase large subunit gene indicated that carbamoylphosphate synthetase located in the chloroplasts was not limiting for ammonium assimilation into arginine. The data provided evidence that ferredoxin-glutamate synthases, NADH-glutamate synthase and carbamoylphosphate synthetase play specific physiological roles in ammonium assimilation in the mesophyll and phloem for the synthesis and transport of glutamine, glutamate, arginine, and derived amino acids.

We investigated the role of glutamine synthetases (cytosolic GS1 and chloroplast GS2) and glutamate synthases (ferredoxin-GOGAT and NADH-GOGAT) in the inorganic nitrogen assimilation and reassimilation into amino acids between bundle sheath cells and mesophyll cells for the remobilization of amino acids during the early phase of grain filling in Zea mays L. The plants responded to a light/dark cycle at the level of nitrate, ammonium and amino acids in the second leaf, upward from the primary ear, which acted as the source organ. The assimilation of ammonium issued from distinct pathways and amino acid synthesis were evaluated from the diurnal rhythms of the transcripts and the encoded enzyme activities of nitrate reductase, nitrite reductase, GS1, GS2, ferredoxin-GOGAT, NADH-GOGAT, NADH-glutamate dehydrogenase and asparagine synthetase. We discerned the specific role of the isoproteins of ferredoxin and ferredoxin:NADP(+) oxidoreductase in providing ferredoxin-GOGAT with photoreduced or enzymatically reduced ferredoxin as the electron donor. The spatial distribution of ferredoxin-GOGAT supported its role in the nitrogen (re)assimilation and reallocation in bundle sheath cells and mesophyll cells of the source leaf. The diurnal nitrogen recycling within the plants took place via the specific amino acids in the phloem and xylem exudates. Taken together, we conclude that the GS1/ferredoxin-GOGAT cycle is the main pathway of inorganic nitrogen assimilation and recycling into glutamine and glutamate, and preconditions amino acid interconversion and remobilization.

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.

During Arabidopsis embryogenesis, procambial cells undergo coordinated, asymmetric cell divisions, giving rise to vascular precursor cells (protophloem and protoxylem precursors). After germination, these cells terminally differentiate into specialized conducting cells, referred to as protophloem and protoxylem cells. Few readily identifiable markers of the onset of specification and differentiation are available, hampering the molecular genetic analysis of protophloem development. Confocal microscopy was used to investigate the patterning and differentiation of phloem cells during early plant development. Longitudinal divisions of phloem initials allowed the identification of protophloem precursor cells and adjacent metaphloem initials along the length of the plant. During germination, protophloem differentiation was observed at two independent locations, in the cotyledons and the hypocotyl. In both locations, differentiation was concomitant with cell elongation. We identified five gene-trap lines (PD1-PD5) with marker gene expression in immature protophloem elements. The spatio-temporal marker expression pattern of the lines divides them into two groups. The early specification markers PD4 and PD5 were expressed in developing organs before procambium formation and then became restricted to phloem initial cells. The protophloem precursor markers PD1-PD3 were expressed in differentiating protophloem cells at different stages of their development. All markers were expressed transiently and iteratively during the differentiation of protophloem in newly formed organs. Flanking genes were identified for four out of five gene-trap insertion lines. The possible function of these genes with respect to phloem differentiation is discussed.

Mitochondria are involved in the production of various vitamins, such as biotin, in plants. It is unclear why these biosynthetic pathways have been maintained partly or entirely within the mitochondria throughout evolution. The last step in biotin biosynthesis occurs within the mitochondria and is catalyzed by the biotin synthase complex containing the BIO2 gene product. We investigated whether the Arabidopsis Bio2 enzyme could function outside mitochondria, by trying to complement a bio2 mutant with a truncated version of BIO2 lacking the region encoding the mitochondrial targeting sequence. We describe the characterization of a new T-DNA allele of bio2, with the sole phenotype of an absence of biotin production, in contrast to the previously characterized EMS bio2 allele (Patton et al. 1998, Plant Physiol 116(3):935-946). We found that a cytosolic version of the Bio2 protein could not complement this mutant. Supplementation with the substrate dethiobiotin (DTB) also failed to rescue the mutant phenotype. Thus, the lack of availability of DTB in the cytosol is not the only factor preventing this reaction from occurring outside mitochondria. Bio2 requires mitochondrial targeting for activity, enabling it to fulfill its role in biotin synthesis. The reaction catalyzed by Bio2 may be subject to biochemical constraints, and the apparent close connection with the mitochondrial Fe-S machinery may account for the reaction being retained within the organelle.

PASTICCINO1 (PAS1) is a high molecular weight FK506-binding protein (FKBP) involved in the control of cell proliferation and differentiation during plant development. Mutations in the C-terminal region of PAS1 result in severe developmental defects. We show here that the C-terminal domain of PAS1 controls the subcellular distribution of this protein. We also demonstrated in vitro and in vivo, by Forster resonance energy transfer, that this C-terminal region is required for interaction with FAN (FKBP-associated NAC), a new member of the plant-specific family of NAC transcription factors. PAS1 and FAN are translocated into the nucleus upon auxin treatment in plant seedlings. The nuclear translocation of PAS1 is dependent on the presence of the C terminus of the protein. Finally, we showed that FAN is involved in PAS1-regulated processes because FAN overproduction partly complemented the pas1 phenotype. We suggest that PAS1 regulates the function of this NAC-like transcription factor by controlling its targeting to the nucleus upon plant cell division.

Glutamate (Glu) metabolism and amino acid translocation were investigated in the young and old leaves of tobacco (Nicotiana tabacum L. cv Xanthi) using [15N]ammonium and [2-15N]Glu tracers. Regardless of leaf age, [15N]ammonium assimilation occurred via glutamine synthetase (GS; EC 6.1.1.3) and Glu synthase (ferredoxin [Fd]-GOGAT; EC 1.4.7.1; NADH-GOGAT; EC 1.4.1.14), both in the light and darkness, and it did not depend on Glu dehydrogenase (GDH; EC 1.4.1.2). The [15N]ammonium and ammonium accumulation patterns support the role of GDH in the deamination of [2-15N]Glu to provide 2-oxoglutarate and [15N]ammonium. In the dark, excess [15N]ammonium was incorporated into asparagine that served as an additional detoxification molecule. The constant Glu levels in the phloem sap suggested that Glu was continuously synthesized and supplied into the phloem regardless of leaf age. Further study using transgenic tobacco lines, harboring the promoter of the GLU1 gene (encoding Arabidopsis [Arabidopsis thaliana] Fd-GOGAT) fused to a GUS reporter gene, revealed that the expression of Fd-GOGAT remained higher in young leaves compared to old leaves, and higher in the veins compared to the mesophyll. Confocal laser-scanning microscopy localized the Fd-GOGAT protein to the phloem companion cells-sieve element complex in the leaf veins. The results are consistent with a role of Fd-GOGAT in supplying Glu for the synthesis and transport of amino acids. Taken together, the data provide evidence that the GS-GOGAT pathway and GDH play distinct roles in the source-sink nitrogen cycle of tobacco leaves.

The synthesis of cellulose microfibrils requires the presence of a membrane-bound endo-1,4-beta-D-glucanase, KORRIGAN1 (KOR1). Although the exact biochemical role of KOR1 in cellulose synthesis is unknown, we used the protein as a marker to explore the potential involvement of subcellular transport processes in cellulose synthesis. Using immunofluorescence and a green fluorescent protein (GFP)-KOR1 fusion that complemented the phenotype conferred by the kor1-1 mutant, we investigated the distribution of KOR1 in epidermal cells in the root meristem. KOR1 was localized in intracellular compartments corresponding to a heterogeneous population of organelles, which comprised the Golgi apparatus, FM4-64-labeled compartments referred to as early endosomes, and, in the case of GFP-KOR1, the tonoplast. Inhibition of cellulose synthesis by isoxaben promoted a net redistribution of GFP-KOR1 toward a homogeneous population of compartments, distinct from early endosomes, which were concentrated close to the plasma membrane facing the root surface. A redistribution of GFP-KOR1 away from early endosomes was also observed in the same cells at later stages of cell elongation. A subpopulation of GFP-KOR1-containing compartments followed trajectories along the plasma membrane, and this motility required intact microtubules. These observations demonstrate that the deposition of cellulose, like chitin synthesis in yeast, involves the regulated intracellular cycling of at least one enzyme required for its synthesis.

The aerial parts of the plant are generated by groups of rapidly dividing cells called shoot apical meristems. To analyze cell behavior in these structures, we developed a technique to visualize living shoot apical meristems using the confocal microscope. This method, combined with green fluorescent protein marker lines and vital stains, allows us to follow the dynamics of cell proliferation, cell expansion, and cell differentiation at the shoot apex. Using this approach, the effects of several mitotic drugs on meristem development were studied. Oryzalin (depolymerizing microtubules) very rapidly caused cell division arrest. Nevertheless, both cell expansion and cell differentiation proceeded in the treated meristems. Interestingly, DNA synthesis was not blocked, and the meristematic cells went through several rounds of endoreduplication in the presence of the drug. We next treated the meristems with two inhibitors of DNA synthesis, aphidicolin and hydroxyurea. In this case, cell growth and, later, cell differentiation were inhibited, suggesting an important role for DNA synthesis in growth and patterning.

Transcriptional regulation of cell cycle regulatory genes, such as B-type cyclins, is tightly linked with the mitotic activity in the meristems. To study the regulation of a B-type cyclin gene, a targeted genetic approach was undertaken. An Arabidopsis line containing a fusion construct between the CYCB1;1 promoter and a bacterial beta-glucuronidase marker gene (uidA) was used in ethyl methanesulfonate mutagenesis. The mutants were screened for altered CYCB1;1::uidA expression patterns. In a reduced CYCB1;1 expression mutant (rcb), the CYCB1;1::uidA expression was severely affected, being excluded from the shoot and root apical meristems and leaf primordia and shifted to cells associated with root cap and stomata. In addition to the overall reduction of the endogenous CYCB1;1 transcript levels, other G2-to-M phase-specific genes were also down-regulated by the mutation. In the mutant plants, the inflorescence stem growth was reduced, indicating low meristem activity. Based on the altered CYCB1;1::uidA expression patterns in rcb root meristem, a model is proposed for RCB that mediates the tissue specificity of CYCB1;1 promoter activity.

Most mitochondrial proteins are encoded by the nucleus, translated in the cytosol, and imported. Mitochondrial precursors generally contain their targeting information in a cleavable N-terminal presequence, which is rich in hydroxylated and positively charged residues and can form amphiphilic alpha-helices. We report the in vivo targeting of green fluorescent protein (GFP) by the FDH presequence, as well as several truncated or mutated variants. Some of these mutations modify the amphiphilicity of the predicted alpha-helix. The removal of the first two residues abolishes import and some single amino acid mutations strongly inhibit import. Such strong effects on import had not been observed in similar studies on other plant mitochondrial presequences, suggesting that the FDH presequence is a particularly good model for functional studies.

Anthocyanidin reductase encoded by the BANYULS (BAN) gene is the core enzyme in proanthocyanidin (PA) biosynthesis. Here, we analyzed the developmental mechanisms that regulate the spatiotemporal expression of BAN in the developing Arabidopsis seed coat. PA-accumulating cells were localized histochemically in the inner integument (seed body and micropyle) and pigment strand (chalaza). BAN promoter activity was detected specifically in these cells. Gain-of-function experiments showed that an 86-bp promoter fragment functioned as an enhancer specific for PA-accumulating cells. Mutations in regulatory genes of PA biosynthesis abolished BAN promoter activity (transparent testa2 [tt2], tt8, and transparent testa glabra1 [ttg1]), modified its spatial pattern (tt1 and tt16), or had no influence (ttg2), thus revealing complex regulatory interactions at several developmental levels. Genetic ablation of PA-accumulating cells targeted by the BAN promoter fused to BARNASE led to the formation of normal plants that produced viable yellow seeds. Importantly, these seeds had no obvious defects in endosperm and embryo development.

The struwwelpeter (swp) mutant in Arabidopsis shows reduced cell numbers in all aerial organs. In certain cases, this defect is partially compensated by an increase in final cell size. Although the mutation does not affect cell cycle duration in the young primordia, it does influence the window of cell proliferation, as cell number is reduced during the very early stages of primordium initiation and a precocious arrest of cell proliferation occurs. In addition, the mutation also perturbs the shoot apical meristem (SAM), which becomes gradually disorganized. SWP encodes a protein with similarities to subunits of the Mediator complex, required for RNA polymerase II recruitment at target promoters in response to specific activators. To gain further insight into its function, we overexpressed the gene under the control of a constitutive promoter. This interfered again with the moment of cell cycle arrest in the young leaf. Our results suggest that the levels of SWP, besides their role in pattern formation at the meristem, play an important role in defining the duration of cell proliferation.

A seedling lethal mutant of Nicotiana plumbaginifolia (sdl-1) was isolated by transposon tagging using a maize Dissociation (Ds) element. The insertion mutation was produced by direct co-transformation of protoplasts with two plasmids: one containing Ds and a second with an Ac transposase gene. sdl-1 seedlings exhibit several phenotypes: swollen organs, short hypocotyls in light and dark conditions, and enlarged and multinucleated cells, that altogether suggest cell growth defects. Mutant cells are able to proliferate under in vitro culture conditions. Genomic DNA sequences bordering the transposon were used to recover cDNA from the normal allele. Complementation of the mutant phenotype with the cDNA confirmed that the transposon had caused the mutation. The Ds element was inserted into the first exon of the open reading frame and the homozygous mutant lacked detectable transcript. Phenocopies of the mutant were obtained by an antisense approach. SDL-1 encodes a novel protein found in several plant genomes but apparently missingfrom animal and fungal genomes; the protein is highly conserved and has a potential plastid targeting motif.

Using a transgenic line that overexpresses a fusion of the KNAT2 (KNOTTED-like Arabidopsis) homeodomain protein and the hormone-binding domain of the glucocorticoid receptor (GR), we have investigated the possible relations between KNAT2 and various hormones. Upon activation of the KNAT2-GR fusion, we observed a delayed senescence of the leaves and a higher rate of shoot initiation, two processes that are also induced by cytokinins and inhibited by ethylene. Furthermore, the activation of the KNAT2-GR fusion induced lobing of the leaves. This feature was partially suppressed by treatment with the ethylene precursor 1-aminocyclopropane-1-carboxylic acid, or by the constitutive ethylene response ctr1 mutation. Conversely, some phenotypic traits of the ctr1 mutant were suppressed by the activation of the KNAT2-GR fusion. These data suggest that KNAT2 acts synergistically with cytokinins and antagonistically with ethylene. In the shoot apical meristem, the KNAT2 gene is expressed in the L3 layer and the rib zone. 1-Aminocyclopropane-1-carboxylic acid treatment restricted the KNAT2 expression domain in the shoot apical meristem and reduced the number of cells in the L3. The latter effect was suppressed by the activation of the KNAT2-GR construct. Conversely, the KNAT2 gene expression domain was enlarged in the ethylene-resistant etr1-1 mutant or in response to cytokinin treatment. These data suggest that ethylene and cytokinins act antagonistically in the meristem via KNAT2 to regulate the meristem activity.

In Arabidopsis ton2 mutants, abnormalities of the cortical microtubular cytoskeleton, such as disorganization of the interphase microtubule array and lack of the preprophase band before mitosis, markedly affect cell shape and arrangement as well as overall plant morphology. We present the molecular isolation of the TON2 gene, which is highly conserved in higher plants and has a vertebrate homolog of unknown function. It encodes a protein similar in its C-terminal part to B" regulatory subunits of type 2A protein phosphatases (PP2As). We show that the TON2 protein interacts with an Arabidopsis type A subunit of PP2A in the yeast two-hybrid system and thus likely defines a novel subclass of PP2A subunits that are possibly involved in the control of cytoskeletal structures in plants.

The KNAT2 (for KNOTTED-like from Arabidopsis thaliana 2) homeobox gene is expressed in the vegetative apical meristem. It is also active during flower development, suggesting a function in the structuring of flowers. To investigate its role, we used a DEXAMETHASONE (DEX)-inducible system to generate transgenic plants that overexpressed a fusion of KNAT2 with the hormone binding domain of the glucocorticoid receptor. DEX-induced plants were similar to plants overexpressing the closely related KNAT1 gene, indicating overlapping functions, although we observed differences as well. In particular, KNAT2-GR activation induced ectopic carpel features. First, KNAT2 induced the homeotic conversion of nucellus into carpel-like structures. Second, KNAT2 induced stigmatic papillae on rosette leaves in the ap2-5 background. Third, ectopic expression of the carpel identity gene AGAMOUS (AG) was observed in carpels and ovules. Interestingly, the homeotic conversion was not dependent on AG activity, because it was maintained in the ag-1 ap2-5 double mutant. Therefore, our data indicate that KNAT2 also must activate other carpel regulators. Together, these results suggest that KNAT2 plays a role in carpel development.

Mutants at the BOTERO1 locus are affected in anisotropic growth in all non-tip-growing cell types examined. Mutant cells are shorter and broader than those of the wild type. Mutant inflorescence stems show a dramatically reduced bending modulus and maximum stress at yield. Our observations of root epidermis cells show that the cell expansion defect in bot1 is correlated with a defect in the orientation of the cortical microtubules. We found that in cells within the apical portion of the root, which roughly corresponds to the meristem, microtubules were loosely organized and became much more highly aligned in transverse arrays with increasing distance from the tip. Such a transition was not observed in bot1. No defect in microtubule organization was observed in kor-1, another mutant with a radial cell expansion defect. We also found that in wild-type root epidermal cells, cessation of radial expansion precedes the increased alignment of cortical microtubules into transverse arrays. Bot1 roots still show a gravitropic response, which indicates that ordered cortical microtubules are not required for differential growth during gravitropism. Interestingly, the fact that in the mutant, these major changes in microtubule organization cause relatively subtle changes in cell morphology, suggest that other levels of control of growth anisotropy remain to be discovered. Together, these observations suggest that BOT1 is required for organizing cortical microtubules into transverse arrays in interphase cells, and that this organization is required for consolidating, rather than initiating, changes in the direction of cell expansion.