Noriko Nagata

Allelopathic Effects of Volatile Monoterpenoids Produced by Salvia leucophylla: Inhibition of Cell Proliferation and DNA Synthesis in the Root Apical Meristem of Brassica campestris Seedlings

Salvia leucophylla, a shrub observed in coastal south California, produces several volatile monoterpenoids (camphor, 1,8-cineole, β-pinene, α-pinene, and camphene) that potentially act as allelochemicals. The effects of these were examined using Brassica campestris as the test plant. Camphor, 1,8-cineole, and β-pinene inhibited germination of B. campestris seeds at high concentrations, whereas α-pinene and camphene did not. Root growth was inhibited by all five monoterpenoids in a dose-dependent manner, but hypocotyl growth was largely unaffected. The monoterpenoids did not alter the sizes of matured cells in either hypocotyls or roots, indicating that cell expansion is relatively insensitive to these compounds. They did not decrease the mitotic index in the shoot apical region, but specifically lowered mitotic index in the root apical meristem. Moreover, morphological and biochemical analyses on the incorporation of 5-bromo-2′-deoxyuridine into DNA demonstrated that the monoterpenoids inhibit both cell-nuclear and organelle DNA synthesis in the root apical meristem. These results suggest that the monoterpenoids produced by S. leucophylla could interfere with the growth of other plants in its vicinity through inhibition of cell proliferation in the root apical meristem.

A Heterocomplex of Iron Superoxide Dismutases Defends Chloroplast Nucleoids against Oxidative Stress and Is Essential for Chloroplast Development in Arabidopsis

There are three iron superoxide dismutases in Arabidopsis thaliana: FE SUPEROXIDE DISMUTASE1 (FSD1), FSD2, and FSD3. Their biological roles in chloroplast development are unknown. Here, we show that FSD2 and FSD3 play essential roles in early chloroplast development, whereas FSD1, which is found in the cytoplasm, does not. An fsd2-1 fsd3-1 double mutant had a severe albino phenotype on agar plates, whereas fsd2 and fsd3 single knockout mutants had pale green phenotypes. Chloroplast development was arrested in young seedlings of the double mutant. The mutant plants were highly sensitive to oxidative stress and developed increased levels of reactive oxygen species (ROS) during extended darkness. The FSD2 and FSD3 proteins formed a heteromeric protein complex in the chloroplast nucleoids. Furthermore, transgenic Arabidopsis plants overexpressing both the FSD2 and FSD3 genes showed greater tolerance to oxidative stress induced by methyl viologen than did the wild type or single FSD2- or FSD3-overexpressing lines. We propose that heteromeric FSD2 and FSD3 act as ROS scavengers in the maintenance of early chloroplast development by protecting the chloroplast nucleoids from ROS.

Arabidopsis MALE STERILITY1 encodes a PHD-type transcription factor and regulates pollen and tapetum development.

The Arabidopsis thaliana MALE STERILITY1 (MS1) gene encodes a nuclear protein with Leu zipper–like and PHD-finger motifs and is important for postmeiotic pollen development. Here, we examined MS1 function using both cell biological and molecular biological approaches. We introduced a fusion construct of MS1 and a transcriptional repression domain (MS1-SRDX) into wild-type Arabidopsis, and the transgenic plants showed a semisterile phenotype similar to that of ms1. Since the repression domain can convert various kinds of transcriptional activators to dominant repressors, this suggested that MS1 functioned as a transcriptional activator. The Leu zipper–like region and the PHD motif were required for the MS1 function. Phenotypic analysis of the ms1 mutant and the MS1-SRDX transgenic Arabidopsis indicated that MS1 was involved in formation of pollen exine and pollen cytosolic components as well as tapetum development. Next, we searched for MS1 downstream genes by analyzing publicly available microarray data and identified 95 genes affected by MS1. Using a transgenic ms1 plant showing dexamethasone-inducible recovery of fertility, we further examined whether these genes were immediately downstream of MS1. From these results, we discuss a role of MS1 in pollen and tapetum development and the conservation of MS1 function in flowering plants.

Mevalonic acid partially restores chloroplast and etioplast development in Arabidopsis lacking the non-mevalonate pathway

Abstract. Isopentenyl diphosphate (IPP) is produced via two independent biosynthetic pathways in higher plants: the mevalonate (MVA) pathway in the cytoplasm and the non-mevalonate 2-C-methyl-D-erythritol-4-phosphate (MEP) pathway in plastids. It has been previously suggested that IPP or IPP-derived products can be exchanged between the cytoplasm and plastids. However, the issue of whether the exchanged products reflect efficient synthesis of functional isoprenoids remains unresolved. We fed exogenous mevalonic acid to the Arabidopsis thaliana (L.) Heynh. albino mutant cla1-1, a null mutant of the first-step enzyme in the MEP pathway. This resulted in the recovery of thylakoid membrane stacking in chloroplasts in the light, and the formation of prolamellar bodies and plastoglobuli in etioplasts in the dark. By contrast, exogenous lovastatin, an inhibitor of mevalonic acid biosynthesis, induced complete depigmentation and further inhibition of plastid development in both the light and the dark. These results suggest that mevalonic acid-derived products contribute to the formation of functional plastidic isoprenoids, such as the chlorophylls and carotenoids required for plastid development.

An essential role of a TatC homologue of a ΔpH- dependent protein transporter in thylakoid membrane formation during chloroplast development in Arabidopsis thaliana

At least three transport systems function in targeting nuclear-encoded chloroplast proteins to the chloroplast thylakoid membrane. One of these systems requires a thylakoid pH gradient and is named the ΔpH-dependent protein transport system. A similar ΔpH export system of Escherichia coli contains four components, twin arginine translocation A (TatA), TatB, TatC, and TatE. TatC is a major component of the ΔpH-dependent protein transporter in E. coli and functions in the translocation of tightly folded proteins across membranes. We have isolated four transposon-inserted albino mutants named albino and pale green 2 (apg2) from Arabidopsis thaliana and showed that the transposons were inserted into different sites of a single gene. The APG2 gene product (named cpTatC) has sequence similarity with bacterial TatC and contains six putative transmembrane domains, including bacterial TatC proteins and a transit peptide in its N terminus. apg2 mutants showed albino phenotypes and could not grow in soil. The apg2 plastids were highly vacuolated, lacked internal membrane structures and lamellae of the thylakoid membrane, and contained many densely stained globule structures, like undifferentiated proplastids. Immunoblot analysis detected no thylakoid membrane proteins such as D1, light-harvesting complex, and OE23 in apg2 plastids, whereas soluble proteins such as rubisco large and small subunits were not decreased. These results indicate an essential role of cpTatC in chloroplast development, especially in thylakoid membrane formation.

An Arabidopsis Cell Cycle–Dependent Kinase-Related Gene, CDC2b, Plays a Role in Regulating Seedling Growth in Darkness

The Arabidopsis CDC2b gene has been defined as a plant-specific cell cycle–dependent kinase-related gene, although it lacks the conserved cyclin binding motif, and its exact function is not known. Here, we report that in etiolated seedlings, the expression of the CDC2b gene is correlated with elongation rate of the hypocotyl. Inhibition of CDC2b gene expression by using an inducible antisense construct resulted in short-hypocotyl and open-cotyledon phenotypes when transgenic seedlings were grown in the dark. The severity of these phenotypes in dark-grown seedlings could be correlated with the level of the antisense gene expression. The short hypocotyl of seedlings underexpressing CDC2b was a result of inhibition of cell elongation rather than a reduction in cell number, whereas in cotyledons, inhibition of CDC2b expression resulted in large, open cotyledons with amyloplasts rather than etioplasts. Although the nuclear DNA was less compact in the antisense hypocotyl cells, DNA content and endoreduplication were not affected. Cell division of the shoot apical meristem also was not affected by antisense expression. The short-hypocotyl phenotype of these transgenic plants was partially rescued by the addition of brassinolide. Brassinolide can only induce CDC2b expression in darkness. These results suggest a role for the CDC2b gene in seedling growth via regulation of hypocotyl cell elongation and cotyledon cell development.

INCREASED LEVEL OF POLYPLOIDY1, a Conserved Repressor of CYCLINA2 Transcription, Controls Endoreduplication in Arabidopsis

Endoreduplication is a type of cell cycle in which DNA replication continues without cell division. We have isolated several dominant mutants from Arabidopsis thaliana activation tagging lines by flow cytometry. One of the mutants, increased level of polyploidy1-1D (ilp1-1D), showed increased polyploidy in both light- and dark-grown hypocotyls. The corresponding gene of ilp1-1D encodes a protein homologous to the C-terminal region of mammalian GC binding factor. We demonstrate that this protein functions as a transcriptional repressor in vivo. The expression of all members of the CYCLINA2 (CYCA2) family was reduced in an ILP1 overexpressing line, and the mouse (Mus musculus) homolog of ILP1 repressed cyclin A2 expression in mouse NIH3T3 cells. T-DNA insertion mutants of ILP1 showed reduced polyploidy and upregulated all CYCA2 expression. Furthermore, loss of CYCA2;1 expression induces an increase in polyploidy in Arabidopsis. We demonstrate that this protein regulates endoreduplication through control of CYCA2 expression in Arabidopsis.

The Arabidopsis D-Type Cyclin CYCD4 Controls Cell Division in the Stomatal Lineage of the Hypocotyl Epidermis

Cyclin D (CYCD) plays an important role in cell cycle progression and reentry in response to external signals. Here, we demonstrate that Arabidopsis thaliana CYCD4 is associated with specific cell divisions in the hypocotyl. We observed that cycd4 T-DNA insertion mutants had a reduced number of nonprotruding cells and stomata in the hypocotyl epidermis. Conversely, CYCD4 overexpression enhanced cell division in nonprotruding cell files in the upper region of the hypocotyls, where stomata are usually formed in wild-type plants. The overproliferative cells were of stomatal lineage, which is marked by the expression of the TOO MANY MOUTHS gene, but unlike the meristemoids, most of them were not triangular. Although the phytohormone gibberellin promoted stomatal differentiation in the hypocotyl, inhibition of gibberellin biosynthesis did not prevent CYCD4 from inducing cell division. These results suggested that CYCD4 has a specialized function in the proliferation of stomatal lineage progenitors rather than in stomatal differentiation. We propose that CYCD4 controls cell division in the initial step of stomata formation in the hypocotyl.

Complete blockage of the mevalonate pathway results in male gametophyte lethality

Plants have two isoprenoid biosynthetic pathways: the cytosolic mevalonate (MVA) pathway and the plastidic 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway. Since the discovery of the MEP pathway, possible metabolic cross-talk between these pathways has prompted intense research. Although many studies have shown the existence of such cross-talk using feeding experiments, it remains to be determined if native cross-talk, rather than exogenously applied metabolites, can compensate for complete blockage of the MVA pathway. Previously, Arabidopsis mutants for HMG1 and HMG2 encoding HMG-CoA reductase (HMGR) were isolated. Although it was shown that HMGR1 is a functional HMGR, the enzyme activity of HMGR2 has not been confirmed. It is demonstrated here that HMG2 encodes a functional reductase with similar activity to HMGR1, using enzyme assays and complementation experiments. To estimate the contribution of native cross-talk, an attempt was made to block the MVA pathway by making double mutants lacking both HMG1 and HMG2, but no double homozygotes were detected in the progeny of self-pollinated HMG1/hmg1 hmg2/hmg2 plants. hmg1 hmg2 male gametophytes appeared to be lethal based on crossing experiments, and microscopy indicated that ∼50% of the microspores from the HMG1/hmg1 hmg2/hmg2 plant appeared shrunken and exhibited poorly defined endoplasmic reticulum membranes. In situ hybridization showed that HMG1 transcripts were expressed in both the tapetum and microspores, while HMG2 mRNA appeared only in microspores. It is concluded that native cross-talk from the plastid cannot compensate for complete blockage of the MVA pathway, at least during male gametophyte development, because either HMG1 or HMG2 is required for male gametophyte development.

Treatment of dark-grown Arabidopsis thaliana with a brassinosteroid-biosynthesis inhibitor, brassinazole, induces some characteristics of light-grown plants.

Abstract. When a brassinosteroid biosynthesis inhibitor, brassinazole (Brz), was applied at concentrations ranging from 0.1 to 2 μM, Arabidopsis thaliana (L.) Heynh seedlings grown in the dark exhibited morphological features of light-grown plants, i.e. short hypocotyls, expanded cotyledons, and true leaves, in a dose-dependent manner. Control (non Brz-treated) seedlings grown in the dark for 40 d did not develop leaf primordia. However, treatment with the lowest concentration of Brz induced the development of leaf buds, although it hardly induced any short hypocotyls, and treatment with the highest concentration of Brz induced both short hypocotyls and leaves. Labeling experiments with the thymidine analogue 5-bromo-2′-deoxyuridine revealed that amplification of cell nuclei and organellar nucleoids is activated in the shoot apical meristems of dark-grown Brz-treated seedlings. These results suggest that Brz-treatment induces development of true leaves. Furthermore, condensation and scattering of plastid nucleoids, which is known to occur during the differentiation of etioplasts into chloroplasts, was observed in the plastids of dark-grown Brz-treated cotyledons. In addition, high levels of ribulose-1,5-bisphosphate carboxylase-oxygenase proteins accumulated in the plastids of the cotyledons. Electron microscopy showed that the plastids were etioplasts with a prolamellar body and few thylakoid membranes. These results suggest that Brz treatment in the dark induces the initial steps of plastid differentiation, which occur prior to the development of thylakoid membranes. This is a novel presumed function of brassinosteroids. These cytological changes seen in Brz-treated Arabidopsis were exactly the same as those seen in a brassinosteroid-biosynthesis-deficient mutant, det2, supporting the hypothesis that Brz has no side-effects except inhibiting brassinosteroid biosynthesis, and should prove a useful tool in clarifying the role of brassinosteroids.