Plant and Cell Physiology - current issue

Jasmonate-Induced Nicotine Formation in Tobacco is Mediated by Tobacco COI1 and JAZ Genes

Shoji, T., Ogawa, T., Hashimoto, T. — 2008-07-11

Biosynthesis of many plant alkaloids is enhanced by endogenous accumulation and exogenous application of jasmonates, but the general and specific signaling components are not well understood. In Arabidopsis, jasmonate-induced ZIM-domain-containing (JAZ) proteins have recently been found to be critical transcriptional repressors linking CORONATINE INSENSTIVE1 (COI1)-mediated jasmonate perception and jasmonate-regulated transcriptional regulation. Insect herbivory on tobacco leaves activates the jasmonate signaling pathway, leading to up-regulation of nicotine biosynthesis genes in roots. We show here that roots of COI1-silenced tobacco plants are insensitive to growth inhibition by methyl jasmonate, and do not activate nicotine biosynthesis genes after jasmonate treatment or wounding of leaves. Tobacco JAZ proteins appeared to be rapidly degraded after jasmonate treatment, whereas a C-terminally truncated form lacking the conserved Jas motif did not. When the non-degradable JAZ forms were expressed in tobacco hairy roots, jasmonate induction of nicotine biosynthesis was strongly inhibited. Formation of tobacco alkaloids in jasmonate-elicited tobacco BY-2 cells was also effectively suppressed by the COI1 RNAi (RNA interference) construct and by the dominant-negative truncated JAZ constructs. In addition, jasmonate-mediated induction of nicotine biosynthesis genes was diminished by treatment with a proteasome inhibitor MG132. These results indicate that jasmonate-triggered, COI1-mediated degradation of JAZ repressors activates transcriptional regulation of nicotine biosynthesis genes in tobacco roots.

Loss-of-Function Mutations in the Arabidopsis Heterotrimeric G-protein {alpha} Subunit Enhance the Developmental Defects of Brassinosteroid Signaling and Biosynthesis Mutants

Gao, Y., Wang, S., Asami, T., Chen, J.-G. — 2008-07-11

Loss-of-function alleles of the sole heterotrimeric G-protein subunit in Arabidopsis, GPA1, display defects in cell proliferation throughout plant development. Previous studies indicated that GPA1 is involved in brassinosteroid (BR) response. Here we provide genetic evidence that loss-of-function mutations in GPA1, gpa1-2 and gpa1-4, enhance the developmental defects of bri1-5, a weak allele of a BR receptor mutant, and det2-1, a BR-deficient mutant in Arabidopsis. gpa1-2 bri1-5 and gpa1-4 det2-1 double mutants had shorter hypocotyls, shorter roots and fewer lateral roots, and displayed more severe dwarfism than bri1-5 and det2-1 single mutants, respectively. By using the Arabidopsis hypocotyl as a model system where the parameters of cell division and cell elongation can be simultaneously measured, we found that gpa1 can specifically enhance the cell division defects of bri1-5 and det2-1 mutants. Similarly, gpa1 specifically enhances cell division defects in the primary roots of bri1-5 and det2-1 mutants. Furthermore, an additive effect on cell division between gpa1 and bri1-5 or det2-1 mutations was observed in the hypocotyls, whereas a synergistic effect was observed in the roots. Taken together, these results provided the first genetic evidence that G-protein- and BR-mediated pathways may be converged to modulate cell proliferation in a cell/tissue-specific manner.

Domain II Mutations in CRANE/IAA18 Suppress Lateral Root Formation and Affect Shoot Development in Arabidopsis thaliana

Uehara, T., Okushima, Y., Mimura, T., Tasaka, M., Fukaki, H. — 2008-07-11

Lateral root formation is an important developmental component of root systems in vascular plants. Several regulatory genes for lateral root formation have been identified from recent studies mainly using Arabidopsis thaliana. In this study, we isolated two dominant mutant alleles, crane-1 and crane-2, which are defective in lateral root formation in Arabidopsis. The crane mutants have dramatically reduced lateral root and auxin-induced lateral root formation, indicating that the crane mutations reduce auxin sensitivity. In addition, the crane mutants have pleiotropic phenotypes in the aerial shoots, including long hypocotyls when grown in the light, up-curled leaves and reduced fertility. The crane mutant phenotypes are caused by a gain-of-function mutation in domain II of IAA18, a member of the Aux/IAA transcriptional repressor family which is expressed in almost all organs. In roots, IAA18 promoter::GUS was expressed in the early stages of lateral root development. In the yeast two-hybrid system, IAA18 interacts with AUXIN RESPONSE FACTOR 7 (ARF7) and ARF19, transcriptional activators that positively regulate lateral root formation. Taken together, our results indicate that CRANE/IAA18 is involved in lateral root formation in Arabidopsis, and suggest that it negatively regulates the activity of ARF7 and ARF19 for lateral root formation.

Plant Inner Membrane Anion Channel (PIMAC) Function in Plant Mitochondria

Laus, M. N., Soccio, M., Trono, D., Cattivelli, L., Pastore, D. — 2008-07-11

To date, the existence of the plant inner membrane anion channel (PIMAC) has been shown only in potato mitochondria, but its physiological role remains unclear. In this study, by means of swelling experiments in K⁺ and ammonium salts, we characterize a PIMAC-like anion-conducting pathway in mitochondria from durum wheat (DWM), a monocotyledonous species phylogenetically far from potato. DWM were investigated since they possess a very active potassium channel (PmitoK_ATP), so implying a very active matching anion uniport pathway and, possibly, a coordinated function. As in potato mitochondria, the electrophoretic uptake of chloride and succinate was inhibited by matrix [H⁺], propranolol, and tributyltin, and was insensitive to Mg²⁺, N,N'-dicyclohexylcarbodiimide (DCCD) and mercurials, thus showing PIMAC's existence in DWM. PIMAC actively transports dicarboxylates, oxodicarboxylates, tricarboxylates and Pi. Interestingly, a novel mechanism of swelling in ammonium salts of isolated plant mitochondria is reported, based on electrophoretic anion uptake via PIMAC and ammonium uniport via PmitoK_ATP. PIMAC is inhibited by physiological compounds, such as ATP and free fatty acids, by high electrical membrane potential (), but not by acyl-CoAs or reactive oxygen species. PIMAC was found to cooperate with dicarboxylate carrier by allowing succinate uptake that triggers succinate/malate exchange in isolated DWM. Similar results were obtained using mitochondria from the dicotyledonous species topinambur, so suggesting generalization of results. We propose that PIMAC is normally inactive in vivo due to ATP and inhibition, but activation may occur in mitochondria de-energized by PmitoK_ATP (or other dissipative systems) to replace or integrate the operation of classical anion carriers.

Enzymatic and Metabolic Diagnostic of Nitrogen Deficiency in Arabidopsis thaliana Wassileskija Accession

2008-07-11

Adaptation to steady-state low-nutrient availability was investigated by comparing the Wassileskija (WS) accession of Arabidopsis thaliana grown on 2 or 10 mM nitrate. Low nitrogen conditions led to a limited rosette biomass and seed yield. The latter was mainly due to reduced seed number, while seed weight was less affected. However, harvest index was lower in high nitrate compared with limited nitrate conditions. Under nitrogen-limiting conditions, nitrate reductase activity was decreased while glutamine synthetase activity was increased due to a higher accumulation of the cytosolic enzyme. The level of nitrogen remobilization to the seeds was higher under low nitrogen, and the vegetative parts of the plants remaining after seed production stored very low residual nitrogen. Through promoting nitrogen remobilization and recycling pathways, nitrogen limitation modified plant and seed compositions. Rosette leaves contained more sugars and less free amino acids when grown under nitrogen-limiting conditions. Compared with high nitrogen, the levels of proline, asparagine and glutamine were decreased. The seed amino acid composition reflected that of the rosette leaves, thus suggesting that phloem loading for seed filling was poorly selective. The major finding of this report was that together with decreasing biomass and yield, nitrogen limitation triggers large modifications in vegetative products and seed quality.

NDF6: A Thylakoid Protein Specific to Terrestrial Plants is Essential for Activity of Chloroplastic NAD(P)H Dehydrogenase in Arabidopsis

Ishikawa, N., Takabayashi, A., Ishida, S., Hano, Y., Endo, T., Sato, F. — 2008-07-11

NAD(P)H dehydrogenase (NDH) is a homolog of respiratory complex I and mediates one of the two pathways of cyclic electron flow around PSI (CEF I). Although 15 ndh subunits have been identified in the chloroplastic and nuclear genomes of higher plants, no electron accepter subunits have been identified to date. To identify the missing chloroplastic NDH subunits, we undertook an in silico approach based on co-expression analysis. In this report, we characterized the novel gene NDF6 (NDH-dependent flow 6; At1g18730) which encodes a protein that is essential for NDH activity. NDF6 has one transmembrane domain and is localized in the thylakoid membrane fraction. Homologous proteins of NDF6 were identified in the genomes of terrestrial plants; however, no homologs have been found in cyanobacteria, which are thought to be the origin of chloroplasts and have a minimal NDH complex unit. NDF6 is unstable in ndhB-impaired or disrupted mutants of higher plants in which the chloroplastic NDH complex is thought to be degraded. These results suggest that NDF6 is a novel subunit of chloroplastic NDH that was added to terrestrial plants during evolution.

Mitochondrial Dynamics in Plant Male Gametophyte Visualized by Fluorescent Live Imaging

2008-07-11

Visualization of organelles in living cells is a powerful method for studying their dynamic behavior. Here we attempted to visualize mitochondria in angiosperm male gametophyte (pollen grain from Arabidopsis thaliana) that are composed of one vegetative cell (VC) and two sperm cells (SCs). Combination of mitochondria-targeted fluorescent proteins with VC- or SC-specific expression allowed us to observe the precise number and dynamic behavior of mitochondria in the respective cell types. Furthermore, live imaging of SC mitochondria during double fertilization confirmed previous observations, demonstrated by electron microscopy in other species, that sperm mitochondria enter into the egg and central cells. We also attempted to visualize mutant mitochondria that were elongated due to a defect in mitochondrial division. This mutant phenotype was indeed detectable in VC mitochondria of a heterozygous F₁ plant, suggesting active mitochondrial division in male gametophyte. Finally, we performed mutant screening and isolated a putative mitochondrial protein transport mutant whose phenotype was detectable only in haploid cells. The transgenic materials presented in this work are useful not only for live imaging but also for studying mitochondrial functions by mutant analysis.

Agrobacterium-Mediated Transformation of the Haploid Liverwort Marchantia polymorpha L., an Emerging Model for Plant Biology

Ishizaki, K., Chiyoda, S., Yamato, K. T., Kohchi, T. — 2008-07-11

Agrobacterium-mediated transformation has not been practical in pteridophytes, bryophytes and algae to date, although it is commonly used in model plants including Arabidopsis and rice. Here we present a rapid Agrobacterium-mediated transformation system for the haploid liverwort Marchantia polymorpha L. using immature thalli developed from spores. Hundreds of hygromycin-resistant plants per sporangium were obtained by co-cultivation of immature thalli with Agrobacterium carrying the binary vector that contains a reporter, the β-glucuronidase (GUS) gene with an intron, and a selection marker, the hygromycin phosphotransferase (hpt) gene. In this system, individual gemmae, which arise asexually from single initial cells, were analyzed as isogenic transformants. GUS activity staining showed that all hygromycin-resistant plants examined expressed the GUS transgene in planta. DNA analyses verified random integration of 1–5 copies of the intact T-DNA between the right and the left borders into the M. polymorpha genome. The efficient and rapid Agrobacterium-mediated transformation of M. polymorpha should provide molecular techniques to facilitate comparative genomics, taking advantage of this unique model plant that retains many features of the common ancestor of land plants.

Methyl Jasmonate Induces Production of Reactive Oxygen Species and Alterations in Mitochondrial Dynamics that Precede Photosynthetic Dysfunction and Subsequent Cell Death

Zhang, L., Xing, D. — 2008-07-11

Methyl jasmonate (MeJa) is a well-known plant stress hormone. Upon exposure to stress, MeJa is produced and causes activation of programmed cell death (PCD) and defense mechanisms in plants. However, the early events and the signaling mechanisms of MeJa-induced cell death have yet to be fully elucidated. To obtain some insights into the early events of this cell death process, we investigated mitochondrial dynamics, chloroplast morphology and function, production and localization of reactive oxygen species (ROS) at the single-cell level as well as photosynthetic capacity at the whole-seedling level under MeJa stimulation. Our results demonstrated that MeJa induction of ROS production, which first occurred in mitochondria after 1 h of MeJa treatment and subsequently in chloroplasts by 3 h of treatment, caused a series of alterations in mitochondrial dynamics including the cessation of mitochondrial movement, the loss of mitochondrial transmembrane potential (MPT), and the morphological transition and aberrant distribution of mitochondria. Thereafter, photochemical efficiency dramatically declined before obvious distortion in chloroplast morphology, which is prior to MeJa-induced cell death in protoplasts or intact seedlings. Moreover, treatment of protoplasts with ascorbic acid or catalase prevented ROS production, organelle change, photosynthetic dysfunction and subsequent cell death. The permeability transition pore inhibitor cyclosporin A gave significant protection against MPT loss, mitochondrial swelling and subsequent cell death. These results suggested that MeJa induces ROS production and alterations of mitochondrial dynamics as well as subsequent photosynthetic collapse, which occur upstream of cell death and are necessary components of the cell death process.

Floral Transition and Nitric Oxide Emission During Flower Development in Arabidopsis thaliana is Affected in Nitrate Reductase-Deficient Plants

2008-07-11

The nitrate reductase (NR)-defective double mutant of Arabidopsis thaliana (nia1 nia2) has previously been shown to present a low endogenous content of NO in its leaves compared with the wild-type plants. In the present study, we analyzed the effect of NR mutation on floral induction and development of A. thaliana, as NO was recently described as one of the signals involved in the flowering process. The NO fluorescent probes diaminofluorescein-2 diacetate (DAF-2DA) and 1,2-diaminoanthraquinone (1,2-DAA) were used to localize NO production in situ by fluorescence microscopy in the floral structures of A. thaliana during floral development. Data were validated by incubating the intact tissues with DAF-2 and quantifying the DAF-2 triazole by fluorescence spectrometry. The results showed that NO is synthesized in specific cells and tissues in the floral structure and its production increases with floral development until anthesis. In the gynoecium, NO synthesis occurs only in differentiated stigmatic papillae of the floral bud, and, in the stamen, only anthers that are producing pollen grains synthesize NO. Sepals and petals do not show NO production. NR-deficient plants emitted less NO, although they showed the same pattern of NO emission in their floral organs. This mutant blossomed precociously when compared with wild-type plants, as measured by the increased caulinar/rosette leaf number and the decrease in the number of days to bolting and anthesis, and this phenotype seems to result from the markedly reduced NO levels in roots and leaves during vegetative growth. Overall, the results reveal a role for NR in the flowering process.

RNAi-Mediated Knockdown of the XIP-Type Endoxylanase Inhibitor Gene, OsXIP, Has No Effect on Grain Development and Germination in Rice

Tokunaga, T., Miyata, Y., Fujikawa, Y., Esaka, M. — 2008-07-11

OsXIP (Oryza sativa xylanase inhibitor protein) is a XIP-type xylanase inhibitor which was identified as a protein encoded by a wound stress-responsive gene in rice. Although the OsXIP gene was specifically expressed in mature grains under basal conditions, recombinant OsXIP had no effect on rice endogenous xylanases, and OsXIP-suppressed transgenic rice plants did not exhibit any change in grain development and germination, suggesting that rice development may be independent of OsXIP. Analysis using an OsXIP-specific antibody revealed that OsXIP is markedly accumulated in apoplast in rice root cells by wounding. These results reinforced the possibility that OsXIP is involved in plant defense mechanisms against phytopathogens.

Effects of Polylinker uATGs on the Function of Grass HKT1 Transporters Expressed in Yeast Cells

Banuelos, M. A., Haro, R., Fraile-Escanciano, A., Rodriguez-Navarro, A. — 2008-07-11

HvHKT1 mediates K⁺ or Na⁺ uniport in yeast cells if the expression promoter is joined directly to the HvHKT1 cDNA, and Na⁺–K⁺ symport if a 59 nucleotide polylinker is inserted. Our results show that three ATG triplets in the polylinker decreased the synthesis of the transporter and that the lower amount of transporter caused the functional change. With the rice HKT1 cDNA, the 59 nt polylinker changed the mode of Na⁺ uptake from K⁺-insensitive to K⁺-inhibitable. These two modes of Na⁺ uptake also occurred in rice plants.

Targeting of proConA to the Plant Vacuole depends on its Nine Amino-acid C-terminal Propeptide

2008-07-11