Loss of the circadian clock-associated protein 1 in Arabidopsis results in altered clock-regulated gene expression

Little is known about plant circadian oscillators, in spite of how important they are to sessile plants, which require accurate timekeepers that enable the plants to respond to their environment. Previously, we identified a circadian clock-associated (CCA1) gene that encodes an Myb-related protein that is associated with phytochrome control and circadian regulation in plants. To understand the role CCA1 plays in phytochrome and circadian regulation, we have isolated an Arabidopsis line with a T DNA insertion that results in the loss of CCA1 RNA, of CCA1 protein, and of an Lhcb-promoter binding activity. This mutation affects the circadian expression of all four clock-controlled genes that we examined. The results show that, despite their similarity, CCA1 and LHY are only partially redundant. The lack of CCA1 also affects the phytochrome regulation of gene expression, suggesting that CCA1 has an additional role in a signal transduction pathway from light, possibly acting at the point of integration between phytochrome and the clock. Our results indicate that CCA1 is an important clock-associated protein involved in circadian regulation of gene expression.

Control of fertilization-independent endosperm development by the MEDEA polycomb gene in Arabidopsis

Higher plant reproduction is unique because two cells are fertilized in the haploid female gametophyte. Egg and sperm nuclei fuse to form the embryo. A second sperm nucleus fuses with the central cell nucleus that replicates to generate the endosperm, a tissue that supports embryo development. To understand mechanisms that initiate reproduction, we isolated a mutation in Arabidopsis, f644, that allows for replication of the central cell and subsequent endosperm development without fertilization. When mutant f644 egg and central cells are fertilized by wild-type sperm, embryo development is inhibited, and endosperm is overproduced. By using a map-based strategy, we cloned and sequenced the F644 gene and showed that it encodes a SET-domain polycomb protein. Subsequently, we found that F644 is identical to MEDEA (MEA), a gene whose maternal-derived allele is required for embryogenesis [Grossniklaus, U., Vielle-Calzada, J.-P., Hoeppner, M. A. & Gagliano, W. B. (1998) Science 280, 446–450]. Together, these results reveal functions for plant polycomb proteins in the suppression of central cell proliferation and endosperm development. We discuss models to explain how polycomb proteins function to suppress endosperm and promote embryo development.

Recessive and dominant mutations in the ethylene biosynthetic gene ACS5 of Arabidopsis confer cytokinin insensitivity and ethylene overproduction, respectively

We identified a set of cytokinin-insensitive mutants by using a screen based on the ethylene-mediated triple response observed after treatment with low levels of cytokinins. One group of these mutants disrupts ACS5, a member of the Arabidopsis gene family that encodes 1-aminocyclopropane-1-carboxylate synthase, the first enzyme in ethylene biosynthesis. The ACS5 isoform is mainly responsible for the sustained rise in ethylene biosynthesis observed in response to low levels of cytokinin and appears to be regulated primarily by a posttranscriptional mechanism. Furthermore, the dominant ethylene-overproducing mutant eto2 was found to be the result of an alteration of the carboxy terminus of ACS5, suggesting that this domain acts as a negative regulator of ACS5 function.

Lignin monomer composition is determined by the expression of a cytochrome P450-dependent monooxygenase in Arabidopsis

The phenylpropanoid pathway provides precursors for the biosynthesis of soluble secondary metabolites and lignin in plants. Ferulate-5-hydroxylase (F5H) catalyzes an irreversible hydroxylation step in this pathway that diverts ferulic acid away from guaiacyl lignin biosynthesis and toward sinapic acid and syringyl lignin. This fact led us to postulate that F5H was a potential regulatory step in the determination of lignin monomer composition. To test this hypothesis, we have used Arabidopsis to examine the impact of F5H overexpression. Arabidopsis is a useful model system in which to study lignification because in wild-type plants, guaiacyl and syringyl lignins are deposited in a tissue-specific fashion, while the F5H-deficient fah1 mutant accumulates only guaiacyl lignin. Here we show that ectopic overexpression of F5H in Arabidopsis abolishes tissue-specific lignin monomer accumulation. Surprisingly, overexpression of F5H under the control of the lignification-associated cinnamate-4-hydroxylase promoter, but not the commonly employed cauliflower mosaic virus 35S promoter, generates a lignin that is almost entirely comprised of syringylpropane units. These experiments demonstrate that modification of F5H expression may enable engineering of lignin monomer composition in agronomically important plant species.

eskimo1 mutants of Arabidopsis are constitutively freezing-tolerant

Temperate plants develop a greater ability to withstand freezing in response to a period of low but nonfreezing temperatures through a complex, adaptive process of cold acclimation. Very little is known about the signaling processes by which plants perceive the low temperature stimulus and transduce it into the nucleus to activate genes needed for increased freezing tolerance. To help understand the signaling processes, we have isolated mutants of Arabidopsis that are constitutively freezing-tolerant in the absence of cold acclimation. Freezing tolerance of wild-type Arabidopsis was increased from −5.5°C to −12.6°C by cold acclimation whereas the freezing tolerance of 26 mutant lines ranged from −6.8°C to −10.6°C in the absence of acclimation. Plants with mutations at the eskimo1 (esk1) locus accumulated high levels of proline, a compatible osmolyte, but did not exhibit constitutively increased expression of several cold-regulated genes involved in freezing tolerance. RNA gel blot analysis suggested that proline accumulation in esk1 plants was mediated by regulation of transcript levels of genes involved in proline synthesis and degradation. The characterization of esk1 mutants and results from other mutants suggest that distinct signaling pathways activate different aspects of cold acclimation and that activation of one pathway can result in considerable freezing tolerance without activation of other pathways.

Histidine kinase activity of the ETR1 ethylene receptor from Arabidopsis

ETR1 represents a prototypical ethylene receptor. Homologues of ETR1 have been identified in Arabidopsis as well as in other plant species, indicating that ethylene perception involves a family of receptors and that the mechanism of ethylene perception is conserved in plants. The amino-terminal half of ETR1 contains a hydrophobic domain responsible for ethylene binding and membrane localization. The carboxyl-terminal half of the polypeptide contains domains with homology to histidine kinases and response regulators, signaling motifs originally identified in bacteria. The putative histidine kinase domain of ETR1 was expressed in yeast as a fusion protein with glutathione S-transferase and affinity purified. Autophosphorylation of the purified fusion protein was observed on incubation with radiolabeled ATP. The incorporated phosphate was resistant to treatment with 3 M NaOH, but was sensitive to 1 M HCl, consistent with phosphorylation of histidine. Autophosphorylation was abolished by mutations that eliminated either the presumptive site of phosphorylation (His-353) or putative catalytic residues within the kinase domain. Truncations were used to delineate the region required for histidine kinase activity. An examination of cation requirements indicated that ETR1 requires Mn2+ for autophosphorylation. These results demonstrate that higher plants contain proteins with histidine kinase activity. Furthermore, these results indicate that aspects of ethylene signaling may be regulated by changes in histidine kinase activity of the receptor.

A role for jasmonate in pathogen defense of Arabidopsis

To investigate the role of jasmonate in the defense of plants against fungal pathogens, we have studied a mutant of Arabidopsis, fad3–2 fad7–2 fad8, that cannot accumulate jasmonate. Mutant plants were extremely susceptible to root rot caused by the fungal root pathogen Pythium mastophorum (Drechs.), even though neighboring wild-type plants were largely unaffected by this fungus. Application of exogenous methyl jasmonate substantially protected mutant plants, reducing the incidence of disease to a level close to that of wild-type controls. A similar treatment with methyl jasmonate did not protect the jasmonate-insensitive mutant coi1 from infection, showing that protective action of applied jasmonate against P. mastophorum was mediated by the induction of plant defense mechanisms rather than by a direct antifungal action. Transcripts of three jasmonate-responsive defense genes are induced by Pythium challenge in the wild-type but not in the jasmonate-deficient mutant. Pythium species are ubiquitous in soil and root habitats world-wide, but most (including P. mastophorum) are considered to be minor pathogens. Our results indicate that jasmonate is essential for plant defense against Pythium and, because of the high exposure of plant roots to Pythium inoculum in soil, may well be fundamental to survival of plants in nature. Our results further indicate that the fad3–2 fad7–2 fad8 mutant is an appropriate genetic model for studying the role of this important signaling molecule in pathogen defense.

NMR characterization of lignins in Arabidopsis altered in the activity of ferulate 5-hydroxylase

Nuclear magnetic resonance (NMR) of isolated lignins from an Arabidopsis mutant deficient in ferulate 5-hydroxylase (F5H) and transgenic plants derived from the mutant by overexpressing the F5H gene has provided detailed insight into the compositional and structural differences between these lignins. Wild-type Arabidopsis has a guaiacyl-rich, syringyl-guaiacyl lignin typical of other dicots, with prominent β-aryl ether (β–O–4), phenylcoumaran (β–5), resinol (β–β), biphenyl/dibenzodioxocin (5–5), and cinnamyl alcohol end-group structures. The lignin isolated from the F5H-deficient fah1–2 mutant contained only traces of syringyl units and consequently enhanced phenylcoumaran and dibenzodioxocin levels. In fah1–2 transgenics in which the F5H gene was overexpressed under the control of the cauliflower mosaic virus 35S promoter, a guaiacyl-rich, syringyl/guaiacyl lignin similar to the wild type was produced. In contrast, the isolated lignin from the fah1–2 transgenics in which F5H expression was driven by the cinnamate 4-hydroxylase promoter was almost entirely syringyl in nature. This simple lignin contained predominantly β-aryl ether units, mainly with erythro-stereochemistry, with some resinol structures. No phenylcoumaran or dibenzodioxocin structures (which require guaiacyl units) were detectable. The overexpression of syringyl units in this transgenic resulted in a lignin with a higher syringyl content than that in any other plant we have seen reported.

Interactions among enzymes of the Arabidopsis flavonoid biosynthetic pathway

Flavonoids are secondary metabolites derived from phenylalanine and acetate metabolism that perform a variety of essential functions in higher plants. Studies over the past 30 years have supported a model in which flavonoid metabolism is catalyzed by an enzyme complex localized to the endoplasmic reticulum [Hrazdina, G. & Wagner, G. J. (1985) Arch. Biochem. Biophys. 237, 88–100]. To test this model further we assayed for direct interactions between several key flavonoid biosynthetic enzymes in developing Arabidopsis seedlings. Two-hybrid assays indicated that chalcone synthase, chalcone isomerase (CHI), and dihydroflavonol 4-reductase interact in an orientation-dependent manner. Affinity chromatography and immunoprecipitation assays further demonstrated interactions between chalcone synthase, CHI, and flavonol 3-hydroxylase in lysates from Arabidopsis seedlings. These results support the hypothesis that the flavonoid enzymes assemble as a macromolecular complex with contacts between multiple proteins. Evidence was also found for posttranslational modification of CHI. The importance of understanding the subcellular organization of elaborate enzyme systems is discussed in the context of metabolic engineering.

Genetic ablation of root cap cells in Arabidopsis

The root cap is increasingly appreciated as a complex and dynamic plant organ. Root caps sense and transmit environmental signals, synthesize and secrete small molecules and macromolecules, and in some species shed metabolically active cells. However, it is not known whether root caps are essential for normal shoot and root development. We report the identification of a root cap-specific promoter and describe its use to genetically ablate root caps by directing root cap-specific expression of a diphtheria toxin A-chain gene. Transgenic toxin-expressing plants are viable and have normal aerial parts but agravitropic roots, implying loss of root cap function. Several cell layers are missing from the transgenic root caps, and the remaining cells are abnormal. Although the radial organization of the roots is normal in toxin-expressing plants, the root tips have fewer cytoplasmically dense cells than do wild-type root tips, suggesting that root meristematic activity is lower in transgenic than in wild-type plants. The roots of transgenic plants have more lateral roots and these are, in turn, more highly branched than those of wild-type plants. Thus, root cap ablation alters root architecture both by inhibiting root meristematic activity and by stimulating lateral root initiation. These observations imply that the root caps contain essential components of the signaling system that determines root architecture.

A possible role for kinase-associated protein phosphatase in the Arabidopsis CLAVATA1 signaling pathway

Continuous growth and development in plants are accomplished by meristems, groups of undifferentiated cells that persist as stem cells and initiate organs. While the structures of the apical and floral meristems in dicotyledonous plants have been well described, little is known about the underlying molecular mechanisms controlling cell proliferation and differentiation in these structures. We have shown previously that the CLAVATA1 (CLV1) gene in Arabidopsis encodes a receptor kinase-like protein that controls the size of the apical and floral meristems. Here, we show that KAPP, a gene encoding a kinase-associated protein phosphatase, is expressed in apical and young floral meristems, along with CLV1. Overexpression of KAPP mimics the clv1 mutant phenotype. Furthermore, CLV1 has kinase activity: it phosphorylates both itself and KAPP. Finally, KAPP binds and dephosphorylates CLV1. We present a model where KAPP functions as a negative regulator of the CLAVATA1 signal transduction pathway.

Nuclear localization signal binding protein from Arabidopsis mediates nuclear import of Agrobacterium VirD2 protein

T-DNA nuclear import is a central event in genetic transformation of plant cells by Agrobacterium. Presumably, the T-DNA transport intermediate is a single-stranded DNA molecule associated with two bacterial proteins, VirD2 and VirE2, which most likely mediate the transport process. While VirE2 cooperatively coats the transported single-stranded DNA, VirD2 is covalently attached to its 5′ end. To better understand the mechanism of VirD2 action, a cellular receptor for VirD2 was identified and its encoding gene cloned from Arabidopsis. The identified protein, designated AtKAPα, specifically bound VirD2 in vivo and in vitro. VirD2–AtKAPα interaction was absolutely dependent on the carboxyl-terminal bipartite nuclear localization signal sequence of VirD2. The deduced amino acid sequence of AtKAPα was homologous to yeast and animal nuclear localization signal-binding proteins belonging to the karyopherin α family. Indeed, AtKAPα efficiently rescued a yeast mutant defective for nuclear import. Furthermore, AtKAPα specifically mediated transport of VirD2 into the nuclei of permeabilized yeast cells.

Altered xanthophyll compositions adversely affect chlorophyll accumulation and nonphotochemical quenching in Arabidopsis mutants

Collectively, the xanthophyll class of carotenoids perform a variety of critical roles in light harvesting antenna assembly and function. The xanthophyll composition of higher plant photosystems (lutein, violaxanthin, and neoxanthin) is remarkably conserved, suggesting important functional roles for each. We have taken a molecular genetic approach in Arabidopsis toward defining the respective roles of individual xanthophylls in vivo by using a series of mutant lines that selectively eliminate and substitute a range of xanthophylls. The mutations, lut1 and lut2 (lut = lutein deficient), disrupt lutein biosynthesis. In lut2, lutein is replaced mainly by a stoichiometric increase in violaxanthin and antheraxanthin. A third mutant, aba1, accumulates normal levels of lutein and substitutes zeaxanthin for violaxanthin and neoxanthin. The lut2aba1 double mutant completely lacks lutein, violaxanthin, and neoxanthin and instead accumulates zeaxanthin. All mutants were viable in soil and had chlorophyll a/b ratios ranging from 2.9 to 3.5 and near wild-type rates of photosynthesis. However, mutants accumulating zeaxanthin exhibited a delayed greening virescent phenotype, which was most severe and often lethal when zeaxanthin was the only xanthophyll present. Chlorophyll fluorescence quenching kinetics indicated that both zeaxanthin and lutein contribute to nonphotochemical quenching; specifically, lutein contributes, directly or indirectly, to the rapid rise of nonphotochemical quenching. The results suggest that the normal complement of xanthophylls, while not essential, is required for optimal assembly and function of the light harvesting antenna in higher plants.

Suspensor-derived polyembryony caused by altered expression of valyl-tRNA synthetase in the twn2 mutant of Arabidopsis

The twn2 mutant of Arabidopsis exhibits a defect in early embryogenesis where, following one or two divisions of the zygote, the decendents of the apical cell arrest. The basal cells that normally give rise to the suspensor proliferate abnormally, giving rise to multiple embryos. A high proportion of the seeds fail to develop viable embryos, and those that do, contain a high proportion of partially or completely duplicated embryos. The adult plants are smaller and less vigorous than the wild type and have a severely stunted root. The twn2-1 mutation, which is the only known allele, was caused by a T-DNA insertion in the 5′ untranslated region of a putative valyl-tRNA synthetase gene, valRS. The insertion causes reduced transcription of the valRS gene in reproductive tissues and developing seeds but increased expression in leaves. Analysis of transcript initiation sites and the expression of promoter–reporter fusions in transgenic plants indicated that enhancer elements inside the first two introns interact with the border of the T-DNA to cause the altered pattern of expression of the valRS gene in the twn2 mutant. The phenotypic consequences of this unique mutation are interpreted in the context of a model, suggested by Vernon and Meinke [Vernon, D. M. & Meinke, D. W. (1994) Dev. Biol. 165, 566–573], in which the apical cell and its decendents normally suppress the embryogenic potential of the basal cell and its decendents during early embryo development.

PICKLE is a CHD3 chromatin-remodeling factor that regulates the transition from embryonic to vegetative development in Arabidopsis

The life cycle of angiosperms is punctuated by a dormant phase that separates embryonic and postembryonic development of the sporophyte. In the pickle (pkl) mutant of Arabidopsis, embryonic traits are expressed after germination. The penetrance of the pkl phenotype is strongly enhanced by inhibitors of gibberellin biosynthesis. Map-based cloning of the PKL locus revealed that it encodes a CHD3 protein. CHD3 proteins have been implicated as chromatin-remodeling factors involved in repression of transcription. PKL is necessary for repression of LEC1, a gene implicated as a critical activator of embryo development. We propose that PKL is a component of a gibberellin-modulated developmental switch that functions during germination to prevent reexpression of the embryonic developmental state.