Plant and Cell Physiology - Advance Access

Global Gene Expression Responses to Waterlogging in Roots and Leaves of Cotton (Gossypium hirsutum L.)

Christianson, J. A., Llewellyn, D. J., Dennis, E. S., Wilson, I. W. — Wed, 18 Nov 2009 06:49:46 PST

Waterlogging stress causes yield reduction in cotton (Gossypium hirsutum L.). A major component of waterlogging stress is the lack of oxygen available to submerged tissues. While changes in expressed protein, gene transcription and metabolite levels have been studied in response to low-oxygen stress, little research has been done on molecular responses to waterlogging in cotton. We assessed cotton growth responses to waterlogging and assayed global gene transcription responses in root and leaf cotton tissues of partially submerged plants. Waterlogging caused significant reductions in stem elongation, shoot mass, root mass, leaf number and altered the expression of 1,012 genes (4% of genes assayed) in root tissue as early as 4 h after flooding. Many of these genes were associated with cell wall modification and growth pathways, glycolysis, fermentation, mitochondrial electron transport and nitrogen metabolism. Waterlogging of plant roots also altered global gene expression in leaf tissues, significantly changing the expression of 1,305 genes (5% of genes assayed) after 24 h of flooding. Genes affected were associated with cell wall growth and modification, tetrapyrrole synthesis, hormone response, starch metabolism and nitrogen metabolism Implications of these results for the development of waterlogging tolerant cotton are discussed.

Metabolome analysis of response to oxidative stress in rice suspension cells overexpressing cell death suppressor Bax inhibitor-1

Mon, 16 Nov 2009 22:46:29 PST

Bax inhibitor-1 (BI-1) is a cell death suppression factor widely conserved in higher plants and animals. Overexpression of Arabidopsis BI-1 (AtBI-1) in plants confers tolerance to various cell death-inducible stresses. However, apart from the cell death suppressing activity, little is known about the physiological roles of BI-1-overexpressing plants. In this study, we evaluated the effects of AtBI-1 overexpression on rice metabolome in response to oxidative stress. AtBI-1-overexpressing rice cells in suspension displayed enhanced tolerance to menadione-induced oxidative stress compared to vector control cells, whereas AtBI-1-overexpression did not influence increase of intracellular H₂O₂ concentration or inhibition of oxidative stress-sensitive aconitase activity. Capillary electrophoresis-mass spectrometry (CE-MS)-based metabolome analysis revealed dynamic metabolic changes in oxidative-stressed rice cells, e.g., depletion of the central metabolic pathway, imbalance in redox state and energy charge, and accumulation of amino acids. Furthermore, comparative metabolome analysis demonstrated that AtBI-1 overexpression did not affect primary metabolism in rice cells under normal growth condition but significantly altered metabolite composition within several distinct pathways under cell death-inducible oxidative stress. The AtBI-1-mediated metabolic alteration included recovery of redox state and energy charge, which are known as important factors for metabolic defense to oxidative stress. These observations suggest that although AtBI-1 does not affect rice metabolism directly, rather, its cell death suppression activity leads to enhanced capacity to acclimate oxidative stress.

Quantitative Analysis of ER Body Morphology in an Arabidopsis Mutant

Wed, 11 Nov 2009 07:29:41 PST

Although fluorescence microscopy screening has proven useful in the identification of genes involved in plant organelle biogenesis and integrity, the quantitative and statistical study of the geometric phenotype is highly limited. This situation could generate unconscious bias in the understanding and presentation of a mutant phenotype. Therefore, we have developed an automated quantification system for green fluorescent protein (GFP) images, which enabled us to easily obtain quantitative data on ER bodies (an ER-derived organelle). We isolated an ER body morphology mutant of Arabidopsis thaliana, leb-1 (long ER body). The leb-1 mutant had significantly fewer and larger ER bodies than the wild-type. An amino acid substitution of cysteine-29 with tyrosine (C29Y) on PYK10, a major component protein of ER bodies, was found in leb-1. Non-reducing SDS-PAGE revealed that the electrophoretic mobility of PYK10 in the leb-1 mutant was clearly different from that in the wild-type. This difference suggests that the C29Y amino acid substitution caused a tertiary structural change of the PYK10 protein. While the bglu21-1 and pyk10-1 single mutations slightly affected the number and morphology of the ER bodies, a bglu21-1 pyk10-1 double mutant had fewer and larger ER bodies than the wild-type. The quantitative ER body phenotypes of leb-1 were similar to those of bglu21-1 pyk10-1 and bglu21-1leb-1, suggesting that the leb-1 mutation allele acts dominantly to the BGLU21 wild-type allele. The leb-1 type PYK10 protein, which has an abnormal structure, may competitively inhibit interactions between the wild-type BGLU21/PYK10 protein and an unknown partner.

Mechanisms of Progressive Water Deficit Tolerance and Growth Recovery of Chinese Maize Foundation Genotypes of Huangzao 4 and Chang 7-2, Which are Proposed on the Basis of Comparison of Physiological and Transcriptomic Responses

Li, Y., Sun, C., Huang, Z., Pan, J., Wang, L., Fan, X. — Wed, 11 Nov 2009 07:29:41 PST

Maize inbred lines of Huangzao 4 (HZ4) and Chang 7-2 (C7-2) are the foundation genotypes key to maize crossing breeding in China. C7-2 is derived from HZ-4. In this study, changes in phenotype, physiology and gene expression of three-leaf-old seedlings of HZ4 and C7-2 under the conditions of progressive water deficit (WD) and re-watering (RW) were compared to drop a hint for breeding new maize foundation genotypes with higher drought tolerance. Progressive WD was made by adding PEG (PEG 6000) at the 24-h interval in Hoagland's nutrient solution, resulting in the water potentials of -0.15, -0.3 and finally -0.5 MPa, respectively, at 24, 48 and 72 h. The seedlings treated for 24 h at -0.3 MPa were treated for RW in the solution without complementation with PEG. The results showed that C7-2 seedlings are more tolerant to progressive WD than HZ4 seedlings in part because the former has a larger stomatal resistance, a relative stronger leaf water-holding capacity, and timely and stable increase in activities of antioxidant enzymes (superoxide dismutase and peroxydase) especially in roots upon WD. Oligonucleotide probe arrays-based analysis discovered a number of WD- and RW-regulated genes in both inbred lines, and clearly indicated that fine transcriptional coordination between maize leaves and roots is one of factors constituting higher WD tolerance and stronger ability of growth recovery from WD. On the basis of the resulting data and co-regulation of responsive genes in tissues, we proposed a model of the whole maize plant tolerance to and recovery from WD.

ABA Hypersensitive Germination2-1 Causes the Activation of Both Abscisic Acid and Salicylic Acid Responses in Arabidopsis

Thu, 05 Nov 2009 07:14:46 PST

Abscisic acid (ABA) and salicylic acid (SA) are believed to act antagonistically. We previously reported that an ABA hypersensitive mutant ahg2-1, which had a reduced expression of polyA specific ribonuclease (PARN), exhibited pleiotropic phenotypes including unique enhanced ABA- and SA-sensitive phenotypes. In this study, we characterised the increased SA-sensitive phenotype of this mutant in detail and addressed its relation with ABA-related and dwarf phenotypes. We found that the ahg2-1 mutant had a high endogenous SA level and an elevated resistance to bacterial pathogen. Double mutant analyses showed that Arabidopsis plants defective in the SA-signaling pathway (npr1 and pad4 mutants and nahG transgenic plants) could suppress neither the ABA hypersensitivity nor the dwarf phenotypes. These results indicate that ABA-related, SA- related, and dwarf phenotypes of the ahg2-1 are independent each other. To obtain more insight into the molecular basis of ahg2-1 effect, microarray analyses were conducted not only for ahg2-1 but also for ahg2sid2 or ahg2abi1 so as to reduce the secondary effects of SA or ABA. The resulting data indicates that ahg2-1 has a unique gene expression profile, consistent with the novel phenotype of this mutant. Detailed comparison of the expression profiles of up- or down-regulated genes implied that ahg2-1 somehow affects mitochondrial function. Our data suggests that a partial loss of PARN activity affects ABA, SA, and mitochondrial function independently, and that the regulation of mRNA levels is deeply implicated in diverse cellular functions.

The peptidoglycan biosynthesis genes, MurA and MraY, are related to chloroplast division in the moss Physcomitrella patens.

Homi, S., Takechi, K., Tanidokoro, K., Sato, H., Takio, S., Takano, H. — Wed, 04 Nov 2009 06:01:09 PST

In the moss Physcomitrella patens, 10 Mur genes involved in peptidoglycan biosynthesis were found, and the MurE and Pbp genes are related to plastid division. Although the MraY and MurG genes were missing in our previous EST screening, they were discovered in the P. patens genome in this study, indicating that P. patens has a full set of genes capable of synthesizing peptidoglycan. In addition, a second MurA gene (PpMurA2) was found. Whereas Northern analyses indicated that PpMurA1, PpMurG, and PpMraY were expressed, transcripts of PpMurA2 were detected only when RT-PCR was employed. Whereas GFP fusion proteins with either PpMurA1 or PpMraY were detected in chloroplasts, the PpMurA2 fusion proteins were located in the cytoplasm. Protonema cells in the wild-type plants had an average of 46 chloroplasts. PpMurA1 gene-disrupted lines had fewer than 10 chloroplasts, whereas approximately 30 chloroplasts existed in the PpMurA2 knockout lines. The PpMurA1/A2 double-knockout lines had only a few macrochloroplasts, suggesting a redundant function for these two genes. Disruption of the PpMraY gene in P. patens resulted in the appearance of macrochloroplasts. The Anabaena MraY, fused with the N-terminal region of PpMraY and the A. thaliana MraY, could complement the macrochloroplast phenotype in the PpMraY knockout line. Electron microscopic observations showed no obvious differences in the shape or stacking of thylakoid membranes between all knockout transformants and wild-type plants, suggesting that these Mur genes are related only to plastid division in moss.

Metabolic Engineering of Lignan Biosynthesis in Forsythia Cell Culture

Wed, 04 Nov 2009 06:01:10 PST

Lignans are a large class of secondary metabolites in plants, with numerous biological effects in mammals, including anti-tumor and anti-oxidant activities. Sesamin, the most abundant furofuran-class lignan in sesame seeds (Sesamum plants), is produced by the cytochrome P450 enzyme CYP81Q1 from the precursor lignan, pinoresinol. In contrast, Forsythia plants produce dibenzylbutyrolactone-class lignans, such as matairesinol, from pinoresinol via the catalysis of pinoresinol/lariciresinol reductase (PLR) and secoisolariciresinol dehydrogenase. Here we present the engineering of lignan biosynthesis in Forsythia cell suspension cultures for the development of an efficient production method of beneficial lignans. A suspension cell culture prepared from leaves of Forsythia koreana produced lignans, mainly pinoresinol and matairesinol glucosides, at levels comparable to that obtained from the leaves. In an attempt to increase the pinoresinol content in Forsythia, we generated a transgenic cell line overexpressing an RNA interference (RNAi) construct of PLR (PLR-RNAi). Down-regulation of PLR expression led to a complete loss of matairesinol and an accumulation of approximately 20-fold pinoresinol in its glucoside form in comparison with the non-transformant. Moreover, the Forsythia transgenic cells co-expressing CYP81Q1 and PLR-RNAi exhibited production of sesamin as well as accumulation of pinoresinol glucoside. These data suggest Forsythia cell suspension to be a promising tool for the engineering of lignan production. To the best of our knowledge, this is the first report on transgenic production of an exogenous lignan in a plant species.

Suppression of Peroxisome Biogenesis Factor 10 Reduces Cuticular Wax Accumulation by Disrupting the ER Network in Arabidopsis thaliana

Kamigaki, A., Kondo, M., Mano, S., Hayashi, M., Nishimura, M. — Wed, 04 Nov 2009 06:01:08 PST

Peroxisome biogenesis factor 10 (PEX10) is a component of the peroxisomal matrix protein import machinery. To analyze the physiological function of PEX10, we used transgenic AtPEX10i Arabidopsis plants that had suppressed expression of the PEX10 gene due to RNA interference. AtPEX10i plants had patches of paleness on leaves, and abnormal floral organs that were typical of cuticular wax-deficient mutants. Quantitative analysis of cuticular wax revealed that the amount of wax in AtPEX10i plants was indeed lower than that in control plants. This result was confirmed by toluidine blue staining and scanning electron microscopic analysis of AtPEX10i. The CER1, CER4, WAX2 and SHN1 genes are known to be responsible for wax biosynthesis in Arabidopsis. Of these, CER1, CER4 and WAX2 were found to be localized on the ER. In AtPEX10i plants, the expression of these genes was down-regulated, and CER1, CER4 and WAX2 were mislocalized to the cytosol. We also found that AtPEX10i plants had defects in ER morphology. Based on these results, we propose that PEX10 is essential for the maintenance of ER morphology and for the expression of CER1, CER4, WAX2 and SHN1 genes, which contribute to the biosynthesis of cuticular wax.

Arabidopsis NAC transcription factor, ANAC078, regulates flavonoids biosynthesis under high-light

Tue, 03 Nov 2009 06:42:45 PST

We have isolated a combination of high-light and heat-shock (HL + HS) stress-inducible genes, including a NAC transcription factor designated ANAC078 (Nishizawa et al. 2006). Here we explored the physiological function of ANAC078 under HL stress. Yeast transcription activity assays showed that ANAC078 functions as a transcriptional activator. A fusion protein composed of green fluorescent protein (GFP) and the full-length ANAC078 was detected in the nucleus and cytoplasm, while fusion proteins comprising GFP and ANAC078 deleted of a putative transmembrane motif were found only in the nucleus. In ANAC078-overexpressing Arabidopsis plants (Ox-ANAC078-43), the transcription of 166 genes was up-regulated compared with the levels in wild-type plants under HL (1,200 µmol m^-2 s^-1, 30°C). These genes included some for transcription factors regulating the expression of genes related to the biosynthesis of flavonoids. Interestingly, the transcript levels of some genes related to flavonoid biosynthesis and the levels of anthocyanins were significantly increased in the Ox-ANAC0478 plants and reduced in knock-out ANAC078 plants (KO-ANAC078) compared with the wild-type plants under HL stress.

The present findings suggest that ANAC078 protein is associated with the induction of genes related to flavonoid biosynthesis, leading to the accumulation of anthocyanins, in response to HL stress.

Identification of a novel major quantitative trait locus controlling distribution of Cd between roots and shoots in rice

Ueno, D., Koyama, E., Kono, I., Ando, T., Yano, M., Ma, J. F. — Mon, 02 Nov 2009 06:58:53 PST

Accumulation of Cd in rice grain is a serious concern of food safety since rice as a staple food is a major source of Cd intake in Asian countries. However, the mechanisms controlling Cd accumulation in rice are still poorly understood. Herein, we performed both physiological and genetic analysis of two rice cultivars contrasting in Cd accumulation, which were screened from a core-collection of rice cultivars. The cultivar Anjana Dhan (Indica) accumulated much higher Cd than Nipponbare (Japonica) in the shoots and grains when grown in both soil and solution culture. A short-term uptake experiment (20 min) showed that Cd uptake by Nipponbare was higher than that by Anjana Dhan. However, the concentration of Cd in the shoot and xylem sap was much higher in Anjana Dhan than in Nipponbare. Of the Cd taken up by the roots, less than 4% was translocated to the shoots in Nipponbare, compared with 10-25% in Anjana Dhan, indicating a higher root-to-shoot translocation of Cd in the latter. A quantitative trait locus (QTL) analysis for Cd accumulation was performed using a F2 population derived from Anjana Dhan and Nipponbare. A QTL with large effect for Cd accumulation was detected on the short arm of chromosome 7, explaining 85.6% of the phenotypic variance in the shoot Cd concentration of the F2 population. High accumulation is likely to be controlled by a single recessive gene. Candidate genomic region was defined less than 1.9 Mb by mean of substitution mapping.

Exogenous Polyamines Elicit Herbivore-Induced Volatiles in Lima Bean Leaves: Involvement of Calcium, H2O2 and Jasmonic Acid

Mon, 02 Nov 2009 06:58:52 PST

We investigated the role of polyamines (PAs) in lima bean (Phaseolus lunatus) leaves on the production of herbivorous mite (Tetranuchus urticae)-induced plant volatiles that attract carnivorous natural enemies of the herbivores. To do this, we focused on the effects of the exogenous PAs [cadaverine, putrescine, spermidine and spermine (Spm)] on the production of volatiles, H₂O₂ and jasmonic acid (JA) and the levels of defensive genes, cytosolic calcium and reactive oxygen species (ROS). Among the tested PAs, Spm was most active in inducing the production of volatile terpenoids known to be induced by T. urticae. An increase in JA levels was also found after Spm treatment, indicating that Spm induces the biosynthesis of JA, which has been shown elsewhere to regulate the production of some volatile terpenoids. Further, treatment with JA and Spm together resulted in greater volatile emission than that with JA alone. In a Y-tube olfactometer, leaves treated with Spm+JA attracted more predatory mites (Phytoseiulus persimilis) than those treated with JA alone. After treatment with Spm+JA, no effects were found on enzyme activity of polyamine oxidase (PAO) and Cu-amine oxidase (CuAO). However, induction of calcium influx and ROS production, and increased enzyme activities and gene expressions for NADPH-oxidase complex, superoxide dismutase, catalase, ascorbate peroxidase, glutathione reductase and glutathione peroxidase, were found after treatment with Spm+JA. These results indicate that Spm plays an important role in the production of T. urticae-induced lima bean leaf volatiles.

ZIP Genes Encode Proteins Involved In Membrane Trafficking Of The TGN-PVC/vacuoles.

Niihama, M., Takemoto, N., Hashiguchi, Y., Tasaka, M., Morita, M. T. — Mon, 02 Nov 2009 06:58:54 PST

The Arabidopsis zigzgag (zig) is a loss-of-function mutant of Qb-SNARE VTI11 which is involved in vesicle trafficking between the trans-Golgi network (TGN) and vacuoles. zig-1 exhibits abnormality both in shoot gravitropism and morphology. To elucidate the molecular network of the post-Golgi membrane trafficking in plant cells, we have isolated the suppressor mutants of zig. Here we report zig suppressor 2 (zip2) and zip4 that are recessive mutants and partially suppress abnormality both in gravitropism and morphology. The ZIP2 encodes AtVPS41/AtVAM2 protein that is thought to be an Arabidopsis ortholog of yeast Vps41p/Vam2p, which is involved in protein sorting to vacuoles as a subunit of tethering complex HOPS. Yeast Vps41p is also proposed to function in budding of AP-3 coated vesicles from the Golgi. The zip2 mutation is a missense mutation in a conserved amino acid of a putative clathrin heavy chain repeat (CHCR) domain. AtVPS41 is a single-copy gene in Arabidopsis genome and the T-DNA insertion mutant appears to be lethal, whereas zip2 single mutant showed no obvious phenotype. On the other hand, the zip4 is a loss-of-function mutant of a putative ortholog of yeast AP-3 µ subunit. In addition, loss-of-function mutant of other subunits of AP-3, ap-3β and ap-3, also exhibit suppressive effect on zig-1 phenotype. Although these genes are also single-copy genes in the genome, the loss-of-function mutants of AP-3 grow normally. Our results suggest that AtVPS41 and AP-3 play roles in proper function of the post-Golgi trafficking network and support membrane trafficking to vacuoles.

Identification of Zinc-Responsive Proteins in the Roots of Arabidopsis thaliana using a Highly Improved Method of Two-Dimensional Electrophoresis

Fukao, Y., Ferjani, A., Fujiwara, M., Nishimori, Y., Ohtsu, I. — Fri, 30 Oct 2009 02:20:57 PDT

Zinc (Zn) is an essential micronutrient for various physiological and metabolic processes in plants, although it is toxic in excess. To better understand Zn-responsive proteins, we developed a highly improved method of isoelectric focusing (IEF) in which whole lysate from Arabidopsis roots is subjected to IEF without any desalting steps. In this method, samples extracted with lysis buffer containing 1.5% SDS can also be directly applied to IEF. By applying this method to Zn-treated Arabidopsis roots, 10 upregulated and 17 downregulated proteins were identified, 15 of which showed a significant correlation with previously reported transcriptomic data.

Raffinose in Chloroplasts is Synthesized in the Cytosol and Transported across the Chloroplast Envelope

Schneider, T., Keller, F. — Fri, 30 Oct 2009 00:38:30 PDT

In chloroplasts, several water-soluble carbohydrates have been suggested to act as stress protectants. The trisaccharide raffinose (-1,6-galactosyl sucrose) is such a carbohydrate but has received little attention. We here demonstrate by compartmentation analysis of leaf mesophyll protoplasts that raffinose is clearly (to about 20%) present in chloroplasts of cold-treated common bugle (Ajuga reptans L.), spinach (Spinacia oleracea L.), and Arabidopsis [Arabidopsis thaliana (L.) Heynh.] plants. The two dedicated enzymes needed for raffinose synthesis, galactinol synthase and raffinose synthase, were found to be extrachloroplastic (probably cytosolic) in location, suggesting that the chloroplast envelope contains a raffinose transporter. Uptake experiments with isolated Ajuga and Arabidopsis chloroplasts clearly demonstrated that raffinose is indeed transported across the chloroplast envelope by a raffinose transporter, probably actively. Raffinose uptake into Ajuga chloroplasts was a saturable process with apparent K_m and v_max values of 27.8 mM and 3.3 µmol mg^-1 Chl min^-1, respectively.

Isolation and Characterization of AaWRKY1, an Artemisia annua Transcription Factor that Regulates the Amorpha-4,11-diene Synthase Gene, a Key Gene of Artemisinin Biosynthesis

Fri, 30 Oct 2009 00:38:29 PDT

Amorpha-4,11-diene synthase (ADS) of Artemisia annua catalyzes the conversion of farnesyl diphosphate into amorpha-4,11-diene, the first committed step in the biosynthesis of the antimalarial drug artemisinin. The promoters of ADS contain two reverse-oriented TTGACC W-box cis-acting elements, which are the proposed binding sites of WRKY transcription factors. A full-length cDNA (AaWRKY1) was isolated from a cDNA library of the glandular secretory trichomes (GSTs) in which artemisinin is synthesized and sequestered. AaWRKY1 encodes a 311 amino acid protein containing a single WRKY domain. AaWRKY1 and ADS genes were highly expressed in GSTs and both were strongly induced by methyl jasmonate and chitosan. Transient expression analysis of the AaWRKY1-GFP reporter revealed that AaWRKY1 was targeted to nuclei. Biochemical analysis demonstrated that the AaWRKY1 protein was capable of binding to the W-box cis-acting elements of the ADS promoters, and it demonstrated transactivation activity in yeast. Coexpression of the effector construct 35S::AaWRKY1 with a reporter construct ADSpro1::GUS highly activated expression of the GUS gene in stably transformed tobacco. Furthermore, transient expression experiments in agroinfiltrated N. benthamiana and A. annua leaves showed that AaWRKY1 protein transactivated the ADSpro2 promoter activity by binding to the W-box of the promoter, disruption of the W-box abolished the activation. Transient expression of AaWRKY1 cDNA in A. annua leaves clearly activated the expression of majority of artemisinin biosynthetic genes. These results strongly suggest the involvement of the AaWRKY1 transcription factor in the regulation of artemisinin biosynthesis, and indicate that ADS is a target gene of AaWRKY1 in A. annua.

Three SnRK2 Protein Kinases are the Main Positive Regulators of Abscisic Acid Signaling in Response to Water Stress in Arabidopsis

Fri, 30 Oct 2009 00:38:28 PDT

Responses to water stress are thought to be mediated by transcriptional regulation of gene expression via reversible protein phosphorylation events. Previously, we reported that bZIP-type AREB transcription factors are involved in abscisic acid (ABA) signaling under water-stress conditions in Arabidopsis. The AREB1 protein is phosphorylated in vitro by ABA-activated SNF1-related protein kinase 2s (SnRK2s) such as SRK2D/SnRK2.2, SRK2E/SnRK2.6 and SRK2I/SnRK2.3 (SRK2D/E/I). Consistent with this, we now show that SRK2D/E/I and AREB1 colocalize and interact in nuclei in planta. Our results show that unlike srk2d, srk2e and srk2i single and double mutants, srk2d srk2e srk2i triple mutants exhibit greatly reduced tolerance to drought stress and highly enhanced insensitivity to ABA. Under water-stress conditions, ABA- and water stress-dependent gene expressions, including those of transcription factors, are globally and drastically impaired and jasmonic acid (JA)-responsive and flowering genes are upregulated in srk2d/e/i triple mutants, but not in other single and double mutants. The downregulated genes in srk2d/e/i and areb triple mutants largely overlap in ABA-dependent expression, supporting the view that SRK2D/E/I regulate AREBs in ABA signaling in response to water stress. Almost all dehydration-responsive LEA protein genes and group-A PP2C genes are strongly downregulated in the srk2d/e/i triple mutants. Further, our data show that these group-A PP2Cs, such as HAI1 and ABI1, interact with SRK2D. Together, our results indicate that SRK2D/E/I function as main positive regulators, and suggest that ABA signaling is controlled by the dual modulation of SRK2D/E/I and group-A PP2Cs.

Overexpression of a Brassica rapa NGATHA Gene in Arabidopsis thaliana Negatively Affects Cell Proliferation during Lateral Organ and Root Growth.

Thu, 29 Oct 2009 23:01:18 PDT

In an effort to elucidate biological functions of transcription factors of Brassica rapa L. (ssp. pekinensis), an NGATHA homologue, BrNGA1, that belongs to the B3-type transcription factor superfamily was identified and expressed in Arabidopsis thaliana under the control of the CaMV 35S promoter. Arabidopsis plants overexpressing BrNGA1, named BrNGA1ox, displayed markedly reduced organ growth compared to the wild type: lateral organs, such as leaves, flowers, and cotyledons, were small and distinctively narrow, and their root growth was severely retarded as well. Reduced sizes of BrNGA1ox organs were mainly due to reduction in cell numbers. Kinematic analysis on leaf growth revealed that both the rate and duration of cell proliferation declined during organogenesis, which was consistent with the reduced expression of cyclin genes. Reduction in organ growth was strongly correlated with the small size of meristematic cell pools in the shoot and root meristems. Taken together, these data indicate that BrNGA1 acts as a negative regulator of cell proliferation and may do so, in part, by regulating the size of the meristematic cell pool.

Arabidopsis SBP-box Genes SPL10, SPL11 and SPL2 Control Morphological Change in Association with Shoot Maturation in Reproductive Phase

Shikata, M., Koyama, T., Mitsuda, N., Ohme-Takagi, M. — Thu, 29 Oct 2009 23:01:17 PDT

Lateral organ traits in higher plants, such as lamina shape and trichome distribution, change gradually in association with shoot maturation. Regulation of this shoot maturation process in the vegetative phase has been extensively investigated, and members of SQUAMOSA PROMOTER BINDING PROTEIN (SBP)-box family of transcription factors have been shown to be involved in this process. However, little is known about the regulation of shoot maturation in the reproductive phase. We analyzed SPL10, SPL11 and SPL2, which are closely related members of SBP-box family in Arabidopsis. While cauline leaves had oblong lamina and few trichomes emerged on cauline leaves and flowers in wild-type plants, transgenic plants expressing a dominant repressor version of SPL10/11/2 had wide cauline leaves and many trichomes on their cauline leaves and flowers. These traits were similar to those observed at an earlier reproductive phase in wild-type plants. Loss-of-function mutants for spl10/11/2 showed similar phenotypes, indicating that SPL10, SPL11 and SPL2 redundantly control proper development of lateral organs in association with shoot maturation in the reproductive phase. In the vegetative phase, lamina shape was affected in SPL10 transgenic plants, while trichome distribution was not altered. This suggests partial regulation of shoot development in the vegetative phase by SPL10. Meanwhile, the wide cauline leaves observed in the transgenic plants and the mutants were similar to those of fruitfull (ful) mutants. We found that FUL expression in leaves increased as shoot maturation and changed in SPL10 transgenic plants. FUL may function in shoot maturation under the control of SBP-box proteins.

Genetically Modified Arabidopsis Thaliana Cells Reveal The Involvement Of Mitochondrial Fatty Acid Composition In Membrane Basal And Uncoupling Protein Mediated Proton Leaks.

Thu, 29 Oct 2009 12:04:26 PDT

We investigated the role of membrane fatty acids on basal proton leaks and uncoupling protein (UCP)-dependent proton conductance in Arabidopsis mitochondria. Using wild type cells, cold sensitive fad2 mutant cells, deficient in -6-oleate desaturase, and cold-tolerant FAD3⁺ transformant cells, over-expressing -3-linoleate desaturase, we showed that basal proton leak in non-phosphorylating state was dependent on lipid composition. The extent of membrane proton leak was drastically reduced in fad2 mutant, containing low amounts of polyunsaturated fatty acids. Conversely, this proton leak was higher in FAD3⁺ mitochondria that exhibit a higher polyunsaturated fatty acid content and high protein to lipid ratio. The dependency of membrane leaks upon membrane potential was higher in FAD3⁺ and lower in fad2. UCP content was higher in both fad2 mutant and FAD3⁺ transgenic lines compared to wild type cells and so was the UCP-activity, assayed by the reduction of phosphorylation yield (ADP/O) triggered by palmitate as UCP activator. This UCP assay was validated by measurements of UCP-proton leak in non-phosphorylating state (flux-force relationships between proton flux and membrane potential). The potential uncoupling capacity of UCP protein was high enough to allow the lost of respiratory control in the three genotypes. Taking together, data reported here suggest that the cold-tolerance of FAD3⁺ cells and the cold-sensitivity of fad2 cells are associated to changes in their mitochondrial membrane basal proton leaks, whereas, differences in functional expression of UCP are not simply related to cold adaptation in Arabidopsis cells.

Dynamic Aspects of Ion Accumulation by Vesicle Traffic under Salt Stress in Arabidopsis

Thu, 29 Oct 2009 23:01:16 PDT

The intracellular membrane dynamics of Arabidopsis cells under high salt treatment were investigated. When Arabidopsis was treated with high levels of NaCl in hydroponic culture, root tip cells showed rapid changes in the vacuolar volume, decrease in the number of small acid compartments, active movement of vesicles and accumulation of Na⁺ both in the central vacuole and in the vesicles around the main vacuole observed with the Na⁺-dependent fluorescence of Sodium Green. Detailed observation of Arabidopsis suspension-cultured cells under high salt treatment showed a similar pattern of response to that observed in root tip cells. Immunostaining of suspension-cultured cells with antibodies against AtNHX1 clearly showed the occurrence of dotted fluorescence in the cytoplasm only under salt treatment. We also confirmed the existence of AtNHX1 in the vacuolar membrane isolated from suspension-cultured cells with immunofluorescence.

Knockout of vacuolar Q_a-SNARE protein; VAM3/SYP22 caused an increase in salt tolerance. In mutant plants, the distribution of Na+ between roots and shoots differed from that of wild type plants, with Na+ accumulating more in roots and less in the shoots of the mutant plants. The role of vesicle traffic under salt stress is discussed.

Seeing Is Believing: On the Use of Image Databases for Visually Exploring Plant Organelle Dynamics

Mano, S., Miwa, T., Nishikawa, S.-i., Mimura, T., Nishimura, M. — Mon, 14 Sep 2009 22:12:57 PDT

Organelle dynamics vary dramatically depending on cell type, developmental stage, and environmental stimuli, so that various parameters, such as size, number, and behavior, are required for the description of the dynamics of each organelle. Imaging techniques are superior to other techniques for describing organelle dynamics because these parameters are visually exhibited. Therefore, as the results can be seen immediately, investigators can more easily grasp organelle dynamics. At present, imaging techniques are emerging as fundamental tools in plant organelle research, and the development of new methodologies to visualize organelles and the improvement of analytical tools and equipment have allowed the large-scale generation of image and movie data. Accordingly, image databases that accumulate information on organelle dynamics are an increasingly indispensable part of modern plant organelle research. In addition, image databases are potentially rich data sources for computational analyses, as image and movie data reposited in the databases contain valuable and significant information, such as size, number, length, and velocity. Computational analytical tools support image-based data mining, such as segmentation, quantification, and statistical analyses, to extract biologically meaningful information from each database and combine them to construct models. In this review, we outline the image databases that are dedicated to plant organelle research and present their potential as resources for image-based computational analyses.

Arrested differentiation of proplastids into chloroplasts in variegated leaves characterized by plastid ultrastructure and nucleoid morphology

Sakamoto, W., Uno, Y., Zhang, Q., Miura, E., Kato, Y., Sodmergen, — Mon, 14 Sep 2009 22:12:56 PDT

Leaf variegation is seen in many ornamental plants and is often caused by a cell lineage-type formation of white sectors lacking functional chloroplasts. A mutant showing such leaf variegation is viable and is therefore suitable for studying chloroplast development. In this study, the formation of white sectors was temporally investigated in the Arabidopsis leaf-variegated mutant var2. Green sectors were found to emerge from white sectors after the formation of the first true leaf. Transmission electron microscopic examination of plastid ultrastructures confirmed that the peripheral zone in the var2 shoot meristem contained proplastids but lacked developing chloroplasts that were normally detected in wild type. These data suggest that chloroplast development proceeds very slowly in var2 variegated leaves. A notable feature in var2 is that the plastids in white sectors contain remarkable globular vacuolated membranes and prolamellar body-like structures. Although defective plastids were hardly observed in shoot meristems, they began to accumulate during early leaf development. Consistent with these observations, large plastid nucleoids detected in white sectors by DNA-specific fluorescent dyes were characteristic to those found in proplastids and were clearly distinguished from those in chloroplasts. These results strongly imply that in white sectors, differentiation of plastids into chloroplasts is arrested at the early stage of thylakoid development. Interestingly, large plastid nucleoids were detected in variegated sectors from species other than Arabidopsis. Thus, plastids in variegated leaves appear to share a common feature and represent a novel plastid type