Plant Physiology current issue

On the Inside

Minorsky, P. V. — 2009-07-02

How Sweet It Is: Identification of Vacuolar Sucrose Transporters

Grennan, A. K., Gragg, J. — 2009-07-02

A Versatile Zero Background T-Vector System for Gene Cloning and Functional Genomics

Chen, S., Songkumarn, P., Liu, J., Wang, G.-L. — 2009-07-02

With the recent availability of complete genomic sequences of many organisms, high-throughput and cost-efficient systems for gene cloning and functional analysis are in great demand. Although site-specific recombination-based cloning systems, such as Gateway cloning technology, are extremely useful for efficient transfer of DNA fragments into multiple destination vectors, the two-step cloning process is time consuming and expensive. Here, we report a zero background TA cloning system that provides simple and high-efficiency direct cloning of PCR-amplified DNA fragments with almost no self-ligation. The improved T-vector system takes advantage of the restriction enzyme XcmI to generate a T-overhang after digestion and the negative selection marker gene ccdB to eliminate the self-ligation background after transformation. We demonstrate the feasibility and flexibility of the technology by developing a set of transient and stable transformation vectors for constitutive gene expression, gene silencing, protein tagging, protein subcellular localization detection, and promoter fragment activity analysis in plants. Because the system can be easily adapted for developing specialized expression vectors for other organisms, zero background TA provides a general, cost-efficient, and high-throughput platform that complements the Gateway cloning system for gene cloning and functional genomics.

A Visual Reporter System for Virus-Induced Gene Silencing in Tomato Fruit Based on Anthocyanin Accumulation

2009-07-02

Virus-induced gene silencing (VIGS) is a powerful tool for reverse genetics in tomato (Solanum lycopersicum). However, the irregular distribution of the effects of VIGS hampers the identification and quantification of nonvisual phenotypes. To overcome this limitation, a visually traceable VIGS system was developed for fruit, comprising two elements: (1) a transgenic tomato line (Del/Ros1) expressing Antirrhinum majus Delila and Rosea1 transcription factors under the control of the fruit-specific E8 promoter, showing a purple-fruited, anthocyanin-rich phenotype; and (2) a modified tobacco rattle virus VIGS vector incorporating partial Rosea1 and Delila sequences, which was shown to restore the red-fruited phenotype upon agroinjection in Del/Ros1 plants. Dissection of silenced areas for subsequent chemometric analysis successfully identified the relevant metabolites underlying gene function for three tomato genes, phytoene desaturase, TomloxC, and SlODO1, used for proof of concept. The C-6 aldehydes derived from lipid 13-hydroperoxidation were found to be the volatile compounds most severely affected by TomloxC silencing, whereas geranial and 6-methyl-5-hepten-2-one were identified as the volatiles most severely reduced by phytoene desaturase silencing in ripening fruit. In a third example, silencing of SlODO1, a tomato homolog of the ODORANT1 gene encoding a myb transcription factor, which regulates benzenoid metabolism in petunia (Petunia hybrida) flowers, resulted in a sharp accumulation of benzaldehyde in tomato fruit. Together, these results indicate that fruit VIGS, enhanced by anthocyanin monitoring, can be a powerful tool for reverse genetics in the study of the metabolic networks operating during fruit ripening.

TriFLDB: A Database of Clustered Full-Length Coding Sequences from Triticeae with Applications to Comparative Grass Genomics

Mochida, K., Yoshida, T., Sakurai, T., Ogihara, Y., Shinozaki, K. — 2009-07-02

The Triticeae Full-Length CDS Database (TriFLDB) contains available information regarding full-length coding sequences (CDSs) of the Triticeae crops wheat (Triticum aestivum) and barley (Hordeum vulgare) and includes functional annotations and comparative genomics features. TriFLDB provides a search interface using keywords for gene function and related Gene Ontology terms and a similarity search for DNA and deduced translated amino acid sequences to access annotations of Triticeae full-length CDS (TriFLCDS) entries. Annotations consist of similarity search results against several sequence databases and domain structure predictions by InterProScan. The deduced amino acid sequences in TriFLDB are grouped with the proteome datasets for Arabidopsis (Arabidopsis thaliana), rice (Oryza sativa), and sorghum (Sorghum bicolor) by hierarchical clustering in stepwise thresholds of sequence identity, providing hierarchical clustering results based on full-length protein sequences. The database also provides sequence similarity results based on comparative mapping of TriFLCDSs onto the rice and sorghum genome sequences, which together with current annotations can be used to predict gene structures for TriFLCDS entries. To provide the possible genetic locations of full-length CDSs, TriFLCDS entries are also assigned to the genetically mapped cDNA sequences of barley and diploid wheat, which are currently accommodated in the Triticeae Mapped EST Database. These relational data are searchable from the search interfaces of both databases. The current TriFLDB contains 15,871 full-length CDSs from barley and wheat and includes putative full-length cDNAs for barley and wheat, which are publicly accessible. This informative content provides an informatics gateway for Triticeae genomics and grass comparative genomics. TriFLDB is publicly available at http://TriFLDB.psc.riken.jp/.

The Role of Diglycosyl Lipids in Photosynthesis and Membrane Lipid Homeostasis in Arabidopsis

Holzl, G., Witt, S., Gaude, N., Melzer, M., Schottler, M. A., Dormann, P. — 2009-07-02

The galactolipid digalactosyldiacylglycerol (DGD) is an abundant thylakoid lipid in chloroplasts. The introduction of the bacterial lipid glucosylgalactosyldiacylglycerol (GGD) from Chloroflexus aurantiacus into the DGD-deficient Arabidopsis (Arabidopsis thaliana) dgd1 mutant was previously shown to result in complementation of growth, but photosynthetic efficiency was only partially restored. Here, we demonstrate that GGD accumulation in the double mutant dgd1dgd2, which is totally devoid of DGD, also complements growth at normal and high-light conditions, but photosynthetic efficiency in the GGD-containing dgd1dgd2 line remains decreased. This is attributable to an increased susceptibility of photosystem II to photodamage, resulting in reduced photosystem II accumulation already at normal light intensities. The chloroplasts of dgd1 and dgd1dgd2 show alterations in thylakoid ultrastructure, a phenotype that is restored in the GGD-containing lines. These data suggest that the strong growth retardation of the DGD-deficient lines dgd1 and dgd1dgd2 can be primarily attributed to a decreased capacity for chloroplast membrane assembly and proliferation and, to a smaller extent, to photosynthetic deficiency. During phosphate limitation, GGD increases in plastidial and extraplastidial membranes of the transgenic lines to an extent similar to that of DGD in the wild type, indicating that synthesis and transport of the bacterial lipid (GGD) and of the authentic plant lipid (DGD) are subject to the same mechanisms of regulation.

Molecular and Biochemical Characterization of the Parvulin-Type PPIases in Lotus japonicus

2009-07-02

The cis/trans isomerization of the peptide bond preceding proline is an intrinsically slow process, although important in many biological processes in both prokaryotes and eukaryotes. In vivo, this isomerization is catalyzed by peptidyl-prolyl cis/trans-isomerases (PPIases). Here, we present the molecular and biochemical characterization of parvulin-type PPIase family members of the model legume Lotus japonicus, annotated as LjPar1, LjPar2, and LjPar3. Although LjPar1 and LjPar2 were found to be homologous to PIN1 (Protein Interacting with NIMA)-type parvulins and hPar14 from human, respectively, LjPar3 represents a novel multidomain parvulin, apparently present only in plants, that contains an active carboxyl-terminal sulfurtransferase domain. All Lotus parvulins were heterologously expressed and purified from Escherichia coli, and purified protein verification measurements used a liquid chromatography-mass spectrometry-based proteomic method. The biochemical characterization of the recombinant Lotus parvulins revealed that they possess PPIase activity toward synthetic tetrapeptides, although they exhibited different substrate specificities depending on the amino acid amino terminal to proline. These differences were also studied in a structural context using molecular modeling of the encoded polypeptides. Real-time reverse transcription-polymerase chain reaction revealed that the three parvulin genes of Lotus are ubiquitously expressed in all plant organs. LjPar1 was found to be up-regulated during the later stages of nodule development. Subcellular localization of LjPar-enhanced Yellow Fluorescence Protein (eYFP) fusions expressed in Arabidopsis (Arabidopsis thaliana) leaf epidermal cells revealed that LjPar1- and LjPar2-eYFP fusions were localized in the cytoplasm and in the nucleus, in contrast to LjPar3-eYFP, which was clearly localized in plastids. Divergent substrate specificities, expression profiles, and subcellular localization indicate that plant parvulin-type PPIases are probably involved in a wide range of biochemical and physiological processes.

Functional Characterization of the Arabidopsis {beta}-Ketoacyl-Coenzyme A Reductase Candidates of the Fatty Acid Elongase

2009-07-02

In plants, very-long-chain fatty acids (VLCFAs; >18 carbon) are precursors of sphingolipids, triacylglycerols, cuticular waxes, and suberin. VLCFAs are synthesized by a multiprotein membrane-bound fatty acid elongation system that catalyzes four successive enzymatic reactions: condensation, reduction, dehydration, and a second reduction. A bioinformatics survey of the Arabidopsis (Arabidopsis thaliana) genome has revealed two sequences homologous to YBR159w encoding a Saccharomyces cerevisiae β-ketoacyl reductase (KCR), which catalyzes the first reduction during VLCFA elongation. Expression analyses showed that both AtKCR1 and AtKCR2 genes were transcribed in siliques, flowers, inflorescence stems, leaves, as well as developing embryos, but only AtKCR1 transcript was detected in roots. Fluorescent protein-tagged AtKCR1 and AtKCR2 were localized to the endoplasmic reticulum, the site of fatty acid elongation. Complementation of the yeast ybr159 mutant demonstrated that the two KCR proteins are divergent and that only AtKCR1 can restore heterologous elongase activity similar to the native yeast KCR gene. Analyses of insertional mutants in AtKCR1 and AtKCR2 revealed that loss of AtKCR1 function results in embryo lethality, which cannot be rescued by AtKCR2 expression using the AtKCR1 promoter. In contrast, a disruption of the AtKCR2 gene had no obvious phenotypic effect. Taken together, these results indicate that only AtKCR1 is a functional KCR isoform involved in microsomal fatty acid elongation. To investigate the roles of AtKCR1 in postembryonic development, transgenic lines expressing RNA interference and overexpression constructs targeted against AtKCR1 were generated. Morphological and biochemical characterization of these lines confirmed that suppressed KCR activity results in a reduction of cuticular wax load and affects VLCFA composition of sphingolipids, seed triacylglycerols, and root glycerolipids, demonstrating in planta that KCR is involved in elongation reactions supplying VLCFA for all these diverse classes of lipids.

Oil Bodies and Oleosins in Physcomitrella Possess Characteristics Representative of Early Trends in Evolution

Huang, C.-Y., Chung, C.-I, Lin, Y.-C., Hsing, Y.-I. C., Huang, A. H.C. — 2009-07-02

Searches of sequenced genomes of diverse organisms revealed that the moss Physcomitrella patens is the most primitive organism possessing oleosin genes. Microscopy examination of Physcomitrella revealed that oil bodies (OBs) were abundant in the photosynthetic vegetative gametophyte and the reproductive spore. Chromatography illustrated the neutral lipids in OBs isolated from the gametophyte to be largely steryl esters and triacylglycerols, and SDS-PAGE showed the major proteins to be oleosins. Reverse transcription-PCR revealed the expression of all three oleosin genes to be tissue specific. This tissue specificity was greatly altered via alternative splicing, a control mechanism of oleosin gene expression unknown in higher plants. During the production of sex organs at the tips of gametophyte branches, the number of OBs in the top gametophyte tissue decreased concomitant with increases in the number of peroxisomes and level of transcripts encoding the glyoxylate cycle enzymes; thus, the OBs are food reserves for gluconeogenesis. In spores during germination, peroxisomes adjacent to OBs, along with transcripts encoding the glyoxylate cycle enzymes, appeared; thus, the spore OBs are food reserves for gluconeogenesis and equivalent to seed OBs. The one-cell-layer gametophyte could be observed easily with confocal microscopy for the subcellular OBs and other structures. Transient expression of various gene constructs transformed into gametophyte cells revealed that all OBs were linked to the endoplasmic reticulum (ER), that oleosins were synthesized in extended regions of the ER, and that two different oleosins were colocated in all OBs.

RNA Interference of LIN5 in Tomato Confirms Its Role in Controlling Brix Content, Uncovers the Influence of Sugars on the Levels of Fruit Hormones, and Demonstrates the Importance of Sucrose Cleavage for Normal Fruit Development and Fertility

2009-07-02

It has been previously demonstrated, utilizing intraspecific introgression lines, that Lycopersicum Invertase5 (LIN5), which encodes a cell wall invertase, controls total soluble solids content in tomato (Solanum lycopersicum). The physiological role of this protein, however, has not yet been directly studied, since evaluation of data obtained from the introgression lines is complicated by the fact that they additionally harbor many other wild species alleles. To allow a more precise comparison, we generated transgenic tomato in which we silenced the expression of LIN5 using the RNA interference approach. The transformants were characterized by an altered flower and fruit morphology, displaying increased numbers of petals and sepals per flower, an increased rate of fruit abortion, and a reduction in fruit size. Evaluation of the mature fruit revealed that the transformants were characterized by a reduction of seed number per plant. Furthermore, detailed physiological analysis revealed that the transformants displayed aberrant pollen morphology and a reduction in the rate of pollen tube elongation. Metabolite profiling of ovaries and green and red fruit revealed that metabolic changes in the transformants were largely confined to sugar metabolism, whereas transcript and hormone profiling revealed broad changes both in the hormones themselves and in transcripts encoding their biosynthetic enzymes and response elements. These results are discussed in the context of current understanding of the role of sugar during the development of tomato fruit, with particular focus given to its impact on hormone levels and organ morphology.

AtBXL1 Encodes a Bifunctional {beta}-D-Xylosidase/{alpha}-L-Arabinofuranosidase Required for Pectic Arabinan Modification in Arabidopsis Mucilage Secretory Cells

2009-07-02

Following pollination, the epidermal cells of the Arabidopsis (Arabidopsis thaliana) ovule undergo a complex differentiation process that includes the synthesis and polar secretion of pectinaceous mucilage followed by the production of a secondary cell wall. Wetting of mature seeds leads to the rapid bursting of these mucilage secretory cells to release a hydrophilic gel that surrounds the seed and is believed to aid in seed hydration and germination. A novel mutant is identified where mucilage release is both patchy and slow and whose seeds display delayed germination. While developmental analysis of mutant seeds reveals no change in mucilage secretory cell morphology, changes in monosaccharide quantities are detected, suggesting the mucilage release defect results from altered mucilage composition. Plasmid rescue and cloning of the mutant locus revealed a T-DNA insertion in AtBXL1, which encodes a putative bifunctional β-d-xylosidase/-l-arabinofuranosidase that has been implicated as a β-d-xylosidase acting during vascular development. Chemical and immunological analyses of mucilage extracted from bxl1 mutant seeds and antibody staining of developing seed coats reveal an increase in (1->5)-linked arabinans, suggesting that BXL1 is acting as an -l-arabinofuranosidase in the seed coat. This implication is supported by the ability to rescue mucilage release through treatment of bxl1 seeds with exogenous -l-arabinofuranosidases. Together, these results suggest that trimming of rhamnogalacturonan I arabinan side chains is required for correct mucilage release and reveal a new role for BXL1 as an -l-arabinofuranosidase acting in seed coat development.

The RNA Hydrolysis and the Cytokinin Binding Activities of PR-10 Proteins Are Differently Performed by Two Isoforms of the Pru p 1 Peach Major Allergen and Are Possibly Functionally Related

Zubini, P., Zambelli, B., Musiani, F., Ciurli, S., Bertolini, P., Baraldi, E. — 2009-07-02

PR-10 proteins are a family of pathogenesis-related (PR) allergenic proteins playing multifunctional roles. The peach (Prunus persica) major allergen, Pru p 1.01, and its isoform, Pru p 1.06D, were found highly expressed in the fruit skin at the pit hardening stage, when fruits transiently lose their susceptibility to the fungal pathogen Monilinia spp. To investigate the possible role of the two Pru p 1 isoforms in plant defense, the recombinant proteins were expressed in Escherichia coli and purified. Light scattering experiments and circular dichroism spectroscopy showed that both proteins are monomers in solution with secondary structures typical of PR-10 proteins. Even though the proteins do not display direct antimicrobial activity, they both act as RNases, a function possibly related to defense. The RNase activity is different for the two proteins, and only that of Pru p 1.01 is affected in the presence of the cytokinin zeatin, suggesting a physiological correlation between Pru p 1.01 ligand binding and enzymatic activity. The binding of zeatin to Pru p 1.01 was evaluated using isothermal titration calorimetry, which provided information on the stoichiometry and on the thermodynamic parameters of the interaction. The structural architecture of Pru p 1.01 and Pru p 1.06D was obtained by homology modeling, and the differences in the binding pockets, possibly accounting for the observed difference in binding activity, were evaluated.

A Redox-Mediated Modulation of Stem Bolting in Transgenic Nicotiana sylvestris Differentially Expressing the External Mitochondrial NADPH Dehydrogenase

2009-07-02

Cytosolic NADPH can be directly oxidized by a calcium-dependent NADPH dehydrogenase, NDB1, present in the plant mitochondrial electron transport chain. However, little is known regarding the impact of modified cytosolic NADPH reduction levels on growth and metabolism. Nicotiana sylvestris plants overexpressing potato (Solanum tuberosum) NDB1 displayed early bolting, whereas sense suppression of the same gene led to delayed bolting, with consequential changes in flowering time. The phenotype was dependent on light irradiance but not linked to any change in biomass accumulation. Whereas the leaf NADPH/NADP⁺ ratio was unaffected, the stem NADPH/NADP⁺ ratio was altered following the genetic modification and strongly correlated with the bolting phenotype. Metabolic profiling of the stem showed that the NADP(H) change affected relatively few, albeit central, metabolites, including 2-oxoglutarate, glutamate, ascorbate, sugars, and hexose-phosphates. Consistent with the phenotype, the modified NDB1 level also affected the expression of putative floral meristem identity genes of the SQUAMOSA and LEAFY types. Further evidence for involvement of the NADPH redox in stem development was seen in the distinct decrease in the stem apex NADPH/NADP⁺ ratio during bolting. Additionally, the potato NDB1 protein was specifically detected in mitochondria, and a survey of its abundance in major organs revealed that the highest levels are found in green stems. These results thus strongly suggest that NDB1 in the mitochondrial electron transport chain can, by modifying cell redox levels, specifically affect developmental processes.

LPA66 Is Required for Editing psbF Chloroplast Transcripts in Arabidopsis

Cai, W., Ji, D., Peng, L., Guo, J., Ma, J., Zou, M., Lu, C., Zhang, L. — 2009-07-02

To gain insight into the molecular mechanism of RNA editing, we have characterized the low psii accumulation66 (lpa66) Arabidopsis (Arabidopsis thaliana) mutant, which displays a high chlorophyll fluorescence phenotype. Its perturbed chlorophyll fluorescence is reflected in reduced levels of photosystem II (PSII) proteins. In vivo protein labeling showed that synthesis rates of the PSII reaction center protein D1/D2 were lower, and turnover rates of PSII core proteins higher, than in wild-type counterparts. The assembly of newly synthesized proteins into PSII occurs in the lpa66 mutant but with reduced efficiency compared with the wild type. LPA66 encodes a chloroplast protein of the pentatricopeptide repeat family. In lpa66 mutants, editing of psbF that converts serine to phenylalanine is specifically impaired. Thus, LPA66 is specifically required for editing the psbF transcripts in Arabidopsis, and the amino acid alternation due to lack of editing strongly affects the efficiency of the assembly of PSII complexes.

Refining the Definition of Plant Mitochondrial Presequences through Analysis of Sorting Signals, N-Terminal Modifications, and Cleavage Motifs

Huang, S., Taylor, N. L., Whelan, J., Millar, A. H. — 2009-07-02

Mitochondrial protein import is a complex multistep process from synthesis of proteins in the cytosol, recognition by receptors on the organelle surface, to translocation across one or both mitochondrial membranes and assembly after removal of the targeting signal, referred to as a presequence. In plants, import has to further discriminate between mitochondria and chloroplasts. In this study, we determined the precise cleavage sites in the presequences for Arabidopsis (Arabidopsis thaliana) and rice (Oryza sativa) mitochondrial proteins using mass spectrometry by comparing the precursor sequences with experimental evidence of the amino-terminal peptide from mature proteins. We validated this method by assessments of false-positive rates and comparisons with previous available data using Edman degradation. In total, the cleavable presequences of 62 proteins from Arabidopsis and 52 proteins from rice mitochondria were determined. None of these proteins contained amino-terminal acetylation, in contrast to recent findings for chloroplast stromal proteins. Furthermore, the classical matrix glutamate dehydrogenase was detected with intact and amino-terminal acetylated sequences, indicating that it is imported into mitochondria without a cleavable targeting signal. Arabidopsis and rice mitochondrial presequences had similar isoelectric points, hydrophobicity, and the predicted ability to form an amphiphilic -helix at the amino-terminal region of the presequence, but variations in length, amino acid composition, and cleavage motifs for mitochondrial processing peptidase were observed. A combination of lower hydrophobicity and start point of the amino-terminal -helix in mitochondrial presequences in both Arabidopsis and rice distinguished them (98%) from Arabidopsis chloroplast stroma transit peptides. Both Arabidopsis and rice mitochondrial cleavage sites could be grouped into three classes, with conserved –3R (class II) and –2R (class I) or without any conserved (class III) arginines. Class II was dominant in both Arabidopsis and rice (55%–58%), but in rice sequences there was much less frequently a phenylalanine (F) in the –1 position of the cleavage site than in Arabidopsis sequences. Our data also suggest a novel cleavage motif of (F/Y)(S/A) in plant class III sequences.

The Transcription Factor ABI4 Is a Regulator of Mitochondrial Retrograde Expression of ALTERNATIVE OXIDASE1a

Giraud, E., Van Aken, O., Ho, L. H.M., Whelan, J. — 2009-07-02

Plant cells integrate signals from external sources and from organelles to regulate gene expression, referred to as anterograde and retrograde signaling, respectively. Functional characterization of the promoter of ALTERNATIVE OXIDASE1a (AOX1a) from Arabidopsis (Arabidopsis thaliana), a marker for mitochondrial retrograde response, was carried out by testing the ability of the AOX1a promoter to drive expression of the reporter gene GUS. This approach identified a strong repressor element, designated the B element, that was necessary for an increased promoter activity in response to the mitochondrial complex I inhibitor rotenone. This element overlaps with a previously identified potential binding site for the transcription factor ABSCISIC ACID INSENSITIVE4 (ABI4). AOX1a promoter activity was fully derepressed in abi4 mutants and was unresponsive to rotenone. Furthermore, deletion of the B element of the AOX1a promoter resulted in increased GUS staining activity compared to the wild-type promoter in transgenic plants. Binding of the ABI4 transcription factor to this region of the AOX1a promoter was demonstrated by electromobility shift and yeast one-hybrid assays. Analysis of transcript abundance for AOX1a in abi4 mutant lines revealed significantly increased levels of AOX1a mRNA that could not be further induced by rotenone, consistent with the role of ABI4 as a repressor that is derepressed in response to rotenone. These results show that ABI4 plays a central role in mediating mitochondrial retrograde signals to induce the expression of AOX1a. Furthermore, they provide a molecular link between mitochondrial and chloroplast retrograde signaling, as ABI4 has been previously shown to act downstream of at least two chloroplast retrograde signaling pathways.

The Histidine Kinase-Related Domain of Arabidopsis Phytochrome A Controls the Spectral Sensitivity and the Subcellular Distribution of the Photoreceptor

Muller, R., Fernandez, A. P., Hiltbrunner, A., Schafer, E., Kretsch, T. — 2009-07-02

Phytochrome A (phyA) is the primary photoreceptor for sensing extremely low amounts of light and for mediating various far-red light-induced responses in higher plants. Translocation from the cytosol to the nucleus is an essential step in phyA signal transduction. EID1 (for EMPFINDLICHER IM DUNKELROTEN LICHT1) is an F-box protein that functions as a negative regulator in far-red light signaling downstream of the phyA in Arabidopsis (Arabidopsis thaliana). To identify factors involved in EID1-dependent light signal transduction, pools of ethylmethylsulfonate-treated eid1-3 seeds were screened for seedlings that suppress the hypersensitive phenotype of the mutant. The phenotype of the suppressor mutant presented here is caused by a missense mutation in the PHYA gene that leads to an amino acid transition in its histidine kinase-related domain. The novel phyA-402 allele alters the spectral sensitivity and the persistence of far-red light-induced high-irradiance responses. The strong eid1-3 suppressor phenotype of phyA-402 contrasts with the moderate phenotype observed when phyA-402 is introgressed into the wild-type background, which indicates that the mutation mainly alters functions in an EID1-dependent signaling cascade. The mutation specifically inhibits nuclear accumulation of the photoreceptor molecule upon red light irradiation, even though it still interacts with FHY1 (for far-red long hypocotyl 1) and FHL (for FHY1-like protein), two factors that are essential for nuclear accumulation of phyA. Degradation of the mutated phyA is unaltered even under light conditions that inhibit its nuclear accumulation, indicating that phyA degradation may occur mostly in the cytoplasm.

The Tryptophan Conjugates of Jasmonic and Indole-3-Acetic Acids Are Endogenous Auxin Inhibitors

Staswick, P. E. — 2009-07-02

Most conjugates of plant hormones are inactive, and some function to reduce the active hormone pool. This study characterized the activity of the tryptophan (Trp) conjugate of jasmonic acid (JA-Trp) in Arabidopsis (Arabidopsis thaliana). Unexpectedly, JA-Trp caused agravitropic root growth in seedlings, unlike JA or nine other JA-amino acid conjugates. The response was dose dependent from 1 to100 µm, was independent of the COI1 jasmonate signaling locus, and unlike the jasmonate signal JA-isoleucine, JA-Trp minimally inhibited root growth. The Trp conjugate with indole-3-acetic acid (IAA-Trp) produced a similar response, while Trp alone and conjugates with benzoic and cinnamic acids did not. JA-Trp and IAA-Trp at 25 µm nearly eliminated seedling root inhibition caused by 2 µm IAA. The TIR1 auxin receptor is required for activity because roots of tir1-1 grew only approximately 60% of wild-type length on IAA plus JA-Trp, even though tir1-1 is auxin resistant. However, neither JA-Trp nor IAA-Trp interfered with IAA-dependent interaction between TIR1 and Aux/IAA7 in cell-free assays. Trp conjugates inhibited IAA-stimulated lateral root production and DR5-β-glucuronidase gene expression. JA-deficient mutants were hypersensitive to IAA and a Trp-overaccumulating mutant was less sensitive, suggesting endogenous conjugates affect auxin sensitivity. Conjugates were present at 5.8 pmol g^–1 fresh weight or less in roots, seedlings, leaves, and flowers, and the values increased approximately 10-fold in roots incubated in 25 µm Trp and IAA or JA at 2 µm. These results show that JA-Trp and IAA-Trp constitute a previously unrecognized mechanism to regulate auxin action.

Fine-Tuning of the Cytoplasmic Ca2+ Concentration Is Essential for Pollen Tube Growth

2009-07-02

Pollen tube growth is crucial for the delivery of sperm cells to the ovule during flowering plant reproduction. Previous in vitro imaging of Lilium longiflorum and Nicotiana tabacum has shown that growing pollen tubes exhibit a tip-focused Ca²⁺ concentration ([Ca²⁺]) gradient and regular oscillations of the cytosolic [Ca²⁺] ([Ca²⁺]_cyt) in the tip region. Whether this [Ca²⁺] gradient and/or [Ca²⁺]_cyt oscillations are present as the tube grows through the stigma (in vivo condition), however, is still not clear. We monitored [Ca²⁺]_cyt dynamics in pollen tubes under various conditions using Arabidopsis (Arabidopsis thaliana) and N. tabacum expressing yellow cameleon 3.60, a fluorescent calcium indicator with a large dynamic range. The tip-focused [Ca²⁺]_cyt gradient was always observed in growing pollen tubes. Regular oscillations of the [Ca²⁺]_cyt, however, were rarely identified in Arabidopsis or N. tabacum pollen tubes grown under the in vivo condition or in those placed in germination medium just after they had grown through a style (semi-in vivo condition). On the other hand, regular oscillations were observed in vitro in both growing and nongrowing pollen tubes, although the oscillation amplitude was 5-fold greater in the nongrowing pollen tubes compared with growing pollen tubes. These results suggested that a submicromolar [Ca²⁺]_cyt in the tip region is essential for pollen tube growth, whereas a regular [Ca²⁺] oscillation is not. Next, we monitored [Ca²⁺] dynamics in the endoplasmic reticulum ([Ca²⁺]_ER) in relation to Arabidopsis pollen tube growth using yellow cameleon 4.60, which has a lower affinity for Ca²⁺ compared with yellow cameleon 3.60. The [Ca²⁺]_ER in pollen tubes grown under the semi-in vivo condition was between 100 and 500 µm. In addition, cyclopiazonic acid, an inhibitor of ER-type Ca²⁺-ATPases, inhibited growth and decreased the [Ca²⁺]_ER. Our observations suggest that the ER serves as one of the Ca²⁺ stores in the pollen tube and cyclopiazonic acid-sensitive Ca²⁺-ATPases in the ER are required for pollen tube growth.

Evidence for a Role of Gibberellins in Salicylic Acid-Modulated Early Plant Responses to Abiotic Stress in Arabidopsis Seeds

2009-07-02

Exogenous application of gibberellic acid (GA₃) was able to reverse the inhibitory effect of salt, oxidative, and heat stresses in the germination and seedling establishment of Arabidopsis (Arabidopsis thaliana), this effect being accompanied by an increase in salicylic acid (SA) levels, a hormone that in recent years has been implicated in plant responses to abiotic stress. Furthermore, this treatment induced an increase in the expression levels of the isochorismate synthase1 and nonexpressor of PR1 genes, involved in SA biosynthesis and action, respectively. In addition, we proved that transgenic plants overexpressing a gibberellin (GA)-responsive gene from beechnut (Fagus sylvatica), coding for a member of the GA₃ stimulated in Arabidopsis (GASA) family (FsGASA4), showed a reduced GA dependence for growth and improved responses to salt, oxidative, and heat stress at the level of seed germination and seedling establishment. In 35S:FsGASA4 seeds, the improved behavior under abiotic stress was accompanied by an increase in SA endogenous levels. All these data taken together suggest that this GA-responsive gene and exogenous addition of GAs are able to counteract the inhibitory effects of these adverse environmental conditions in seed germination and seedling growth through modulation of SA biosynthesis. Furthermore, this hypothesis is supported by the fact that sid2 mutants, impaired in SA biosynthesis, are more sensitive to salt stress than wild type and are not affected by exogenous application of GA₃.

Triple Loss of Function of Protein Phosphatases Type 2C Leads to Partial Constitutive Response to Endogenous Abscisic Acid

2009-07-02

The phytohormone abscisic acid (ABA) is a key regulator of plant growth and development as well as plant responses to situations of decreased water availability. Protein phosphatases type 2C (PP2Cs) from group A, which includes the ABI1/HAB1 and PP2CA branches, are key negative regulators of ABA signaling. Specifically, HAB1, ABI1, ABI2, and PP2CA have been shown to affect both seed and vegetative responses to ABA. To further understand their contribution to ABA signaling and to unravel possible genetic interactions and functional redundancy among them, we have generated different combinations of double and triple mutants impaired in these PP2Cs. Interestingly, hab1-1pp2ca-1 and abi1-2pp2ca-1 double mutants showed reduced water loss and enhanced resistance to drought stress, which further supports the role of PP2CA in vegetative responses to ABA. Two triple hab1-1abi1-2abi2-2 and hab1-1abi1-2pp2ca-1 mutants were generated, which showed an extreme response to exogenous ABA, impaired growth, and partial constitutive response to endogenous ABA. Thus, transcriptomic analysis revealed a partial up-regulation/down-regulation of a subset of ABA-responsive genes in both triple mutants in the absence of exogenous ABA. Comparison of ABA responses in the different pp2c mutants showed that a progressive increase in ABA sensitivity could be obtained through combined inactivation of these PP2Cs. These results indicate that ABA response is finely tuned by the integrated action of these genes, which is required to prevent a constitutive response to endogenous ABA that might have a deleterious effect on growth and development in the absence of environmental stress.

Sucrose Control of Translation Mediated by an Upstream Open Reading Frame-Encoded Peptide

2009-07-02

Regulation of gene expression through translational control is common in many organisms. The Arabidopsis (Arabidopsis thaliana) transcription factor bZIP11 is translational repressed in response to sucrose (Suc), resulting in Suc-regulated changes in amino acid metabolism. The 5' leader of the bZIP11 mRNA harbors several upstream open reading frames (uORFs), of which the second uORF is well conserved among bZIP11 homologous genes. The uORF2 element encodes a Suc control peptide (SC-peptide) of 28 residues that is sufficient for imposing Suc-induced repression of translation (SIRT) on a heterologous mRNA. Detailed analysis of the SC-peptide suggests that it functions as an attenuator peptide. Results suggest that the SC-peptide inhibits bZIP11 translation in response to high Suc levels by stalling the ribosome on the mRNA. The conserved noncanonical AUG contexts of bZIP11 uORFs allow inefficient translational initiation of the uORF, resulting in translation initiation of the scanning ribosome at the AUG codon of the bZIP11 main ORF. The results presented show that Suc-dependent signaling mediates differential translation of mRNAs containing SC-peptides encoding uORFs.

Overexpression of the Transcription Factor AP37 in Rice Improves Grain Yield under Drought Conditions

Oh, S.-J., Kim, Y. S., Kwon, C.-W., Park, H. K., Jeong, J. S., Kim, J.-K. — 2009-07-02

Transcription factors with an APETELA2 (AP2) domain have been implicated in various cellular processes involved in plant development and stress responses. Of the 139 AP2 genes predicted in rice (Oryza sativa), we identified 42 genes in our current study that are induced by one or more stress conditions, including drought, high salinity, low temperature, and abscisic acid. Phylogenic analysis of these 42 stress-inducible AP2 genes revealed the presence of six subgroups (I–VI) with distinct signature motifs. Two genes, AP37 and AP59, representing subgroups I and II, respectively, were functionally characterized. Both genes were found to be induced upon 2 h of exposure to drought and high-salinity conditions but to differ in their expression profile upon exposure to low temperature and abscisic acid. The overexpression of AP37 and AP59 in rice under the control of the constitutive promoter OsCc1 increased the tolerance to drought and high salinity at the vegetative stage. Increased tolerance to low temperatures was observed only in OsCc1:AP37 plants. More importantly, the OsCc1:AP37 plants showed significantly enhanced drought tolerance in the field, which increased grain yield by 16% to 57% over controls under severe drought conditions, yet exhibited no significant difference under normal growth conditions. In contrast, grain yield in OsCc1:AP59 plants in the field was reduced by 23% to 43% compared with controls under both normal and drought stress conditions. Microarray experiments identified 10 and 38 genes that are up-regulated by AP37 and AP59, respectively, in addition to 37 genes that are commonly induced by both factors. Our results suggest that the AP37 gene has the potential to improve drought tolerance in rice without causing undesirable growth phenotypes.

Senescence-Induced Serotonin Biosynthesis and Its Role in Delaying Senescence in Rice Leaves

Kang, K., Kim, Y.-S., Park, S., Back, K. — 2009-07-02

Serotonin, which is well known as a pineal hormone in mammals, plays a key role in conditions such as mood, eating disorders, and alcoholism. In plants, although serotonin has been suggested to be involved in several physiological roles, including flowering, morphogenesis, and adaptation to environmental changes, its regulation and functional roles are as yet not characterized at the molecular level. In this study, we found that serotonin is greatly accumulated in rice (Oryza sativa) leaves undergoing senescence induced by either nutrient deprivation or detachment, and its synthesis is closely coupled with transcriptional and enzymatic induction of the tryptophan biosynthetic genes as well as tryptophan decarboxylase (TDC). Transgenic rice plants that overexpressed TDC accumulated higher levels of serotonin than the wild type and showed delayed senescence of rice leaves. However, transgenic rice plants, in which expression of TDC was suppressed through an RNA interference (RNAi) system, produced less serotonin and senesced faster than the wild type, suggesting that serotonin is involved in attenuating leaf senescence. The senescence-retarding activity of serotonin is associated with its high antioxidant activity compared to either tryptophan or chlorogenic acid. Results of TDC overexpression and TDC RNAi plants suggest that TDC plays a rate-limiting role for serotonin accumulation, but the synthesis of serotonin depends on an absolute amount of tryptophan accumulation by the coordinate induction of the tryptophan biosynthetic genes. In addition, immunolocalization analysis revealed that serotonin was abundant in the vascular parenchyma cells, including companion cells and xylem-parenchyma cells, suggestive of its involvement in maintaining the cellular integrity of these cells for facilitating efficient nutrient recycling from senescing leaves to sink tissues during senescence.

The Role of Annexin 1 in Drought Stress in Arabidopsis

2009-07-02

Annexins act as targets of calcium signals in eukaryotic cells, and recent results suggest that they play an important role in plant stress responses. We found that in Arabidopsis (Arabidopsis thaliana), AnnAt1 (for annexin 1) mRNA levels were up-regulated in leaves by most of the stress treatments applied. Plants overexpressing AnnAt1 protein were more drought tolerant and knockout plants were more drought sensitive than ecotype Columbia plants. We also observed that hydrogen peroxide accumulation in guard cells was reduced in overexpressing plants and increased in knockout plants both before and after treatment with abscisic acid. Oxidative protection resulting from AnnAt1 overexpression could be due to the low level of intrinsic peroxidase activity exhibited by this protein in vitro, previously linked to a conserved histidine residue found in a peroxidase-like motif. However, analyses of a mutant H40A AnnAt1 protein in a bacterial complementation test and in peroxidase activity assays indicate that this residue is not critical to the ability of AnnAt1 to confer oxidative protection. To further examine the mechanism(s) linking AnnAt1 expression to stress resistance, we analyzed the reactive S3 cluster to determine if it plays a role in AnnAt1 oligomerization and/or is the site for posttranslational modification. We found that the two cysteine residues in this cluster do not form intramolecular or intermolecular bonds but are highly susceptible to oxidation-driven S-glutathionylation, which decreases the Ca²⁺ affinity of AnnAt1 in vitro. Moreover, S-glutathionylation of AnnAt1 occurs in planta after abscisic acid treatment, which suggests that this modification could be important in regulating the cellular function of AnnAt1 during stress responses.

The Organization Pattern of Root Border-Like Cells of Arabidopsis Is Dependent on Cell Wall Homogalacturonan

2009-07-02

Border-like cells are released by Arabidopsis (Arabidopsis thaliana) root tips as organized layers of several cells that remain attached to each other rather than completely detached from each other, as is usually observed in border cells of many species. Unlike border cells, cell attachment between border-like cells is maintained after their release into the external environment. To investigate the role of cell wall polysaccharides in the attachment and organization of border-like cells, we have examined their release in several well-characterized mutants defective in the biosynthesis of xyloglucan, cellulose, or pectin. Our data show that among all mutants examined, only quasimodo mutants (qua1-1 and qua2-1), which have been characterized as producing less homogalacturonan, had an altered border-like cell phenotype as compared with the wild type. Border-like cells in both lines were released as isolated cells separated from each other, with the phenotype being much more pronounced in qua1-1 than in qua2-1. Further analysis of border-like cells in the qua1-1 mutant using immunocytochemistry and a set of anti-cell wall polysaccharide antibodies showed that the loss of the wild-type phenotype was accompanied by (1) a reduction in homogalacturonan-JIM5 epitope in the cell wall of border-like cells, confirmed by Fourier transform infrared microspectrometry, and (2) the secretion of an abundant mucilage that is enriched in xylogalacturonan and arabinogalactan-protein epitopes, in which the cells are trapped in the vicinity of the root tip.

Isolation and Characterization of Hydroxyproline-Rich Glycopeptide Signals in Black Nightshade Leaves

2009-07-02

A gene encoding a preprohydroxyproline-rich systemin, SnpreproHypSys, was identified from the leaves of black nightshade (Solanum nigrum), which is a member of a small gene family of at least three genes that have orthologs in tobacco (Nicotiana tabacum; NtpreproHypSys), tomato (Solanum lycopersicum; SlpreproHypSys), petunia (Petunia hybrida; PhpreproHypSys), potato (Solanum tuberosum; PhpreproHypSys), and sweet potato (Ipomoea batatas; IbpreproHypSys). SnpreproHypSys was induced by wounding and by treatment with methyl jasmonate. The encoded precursor protein contained a signal sequence and was posttranslationally modified to produce three hydroxyproline-rich glycopeptide signals (HypSys peptides). The three HypSys peptides isolated from nightshade leaf extracts were called SnHypSys I (19 amino acids with six pentoses), SnHypSys II (20 amino acids with six pentoses), and SnHypSys III (20 amino acids with either six or nine pentoses) by their sequential appearance in SnpreproHypSys. The three SnHypSys peptides were synthesized and tested for their abilities to alkalinize suspension culture medium, with synthetic SnHypSys I demonstrating the highest activity. Synthetic SnHypSys I was capable of inducing alkalinization in other Solanaceae cell types (or species), indicating that structural conformations within the peptides are recognized by the different cells/species to initiate signal transduction pathways, apparently through recognition by homologous receptor(s). To further demonstrate the biological relevance of the SnHypSys peptides, the early defense gene lipoxygenase D was shown to be induced by all three synthetic peptides when supplied to excised nightshade plants.

Ripening-Regulated Susceptibility of Tomato Fruit to Botrytis cinerea Requires NOR But Not RIN or Ethylene

2009-07-02

Fruit ripening is a developmental process that is associated with increased susceptibility to the necrotrophic pathogen Botrytis cinerea. Histochemical observations demonstrate that unripe tomato (Solanum lycopersicum) fruit activate pathogen defense responses, but these responses are attenuated in ripe fruit infected by B. cinerea. Tomato fruit ripening is regulated independently and cooperatively by ethylene and transcription factors, including NON-RIPENING (NOR) and RIPENING-INHIBITOR (RIN). Mutations in NOR or RIN or interference with ethylene perception prevent fruit from ripening and, thereby, would be expected to influence susceptibility. We show, however, that the susceptibility of ripe fruit is dependent on NOR but not on RIN and only partially on ethylene perception, leading to the conclusion that not all of the pathways and events that constitute ripening render fruit susceptible. Additionally, on unripe fruit, B. cinerea induces the expression of genes also expressed as uninfected fruit ripen. Among the ripening-associated genes induced by B. cinerea are LePG (for polygalacturonase) and LeExp1 (for expansin), which encode cell wall-modifying proteins and have been shown to facilitate susceptibility. LePG and LeExp1 are induced only in susceptible rin fruit and not in resistant nor fruit. Thus, to infect fruit, B. cinerea relies on some of the processes and events that occur during ripening, and the fungus induces these pathways in unripe fruit, suggesting that the pathogen itself can initiate the induction of susceptibility by exploiting endogenous developmental programs. These results demonstrate the developmental plasticity of plant responses to the fungus and indicate how known regulators of fruit ripening participate in regulating ripening-associated pathogen susceptibility.

Two Alternatively Spliced Isoforms of the Arabidopsis SR45 Protein Have Distinct Roles during Normal Plant Development

Zhang, X.-N., Mount, S. M. — 2009-07-02

The serine-arginine-rich (SR) proteins constitute a conserved family of pre-mRNA splicing factors. In Arabidopsis (Arabidopsis thaliana), they are encoded by 19 genes, most of which are themselves alternatively spliced. In the case of SR45, the use of alternative 3' splice sites 21 nucleotides apart generates two alternatively spliced isoforms. Isoform 1 (SR45.1) has an insertion relative to isoform 2 (SR45.2) that replaces a single arginine with eight amino acids (TSPQRKTG). The biological implications of SR45 alternative splicing have been unclear. A previously described loss-of-function mutant affecting both isoforms, sr45-1, shows several developmental defects, including defects in petal development and root growth. We found that the SR45 promoter is highly active in regions with actively growing and dividing cells. We also tested the ability of each SR45 isoform to complement the sr45-1 mutant by overexpression of isoform-specific green fluorescent protein (GFP) fusion proteins. As expected, transgenic plants overexpressing either isoform displayed both nuclear speckles and GFP fluorescence throughout the nucleoplasm. We found that SR45.1-GFP complements the flower petal phenotype, but not the root growth phenotype. Conversely, SR45.2-GFP complements root growth but not floral morphology. Mutation of a predicted phosphorylation site within the alternatively spliced segment, SR45.1-S219A-GFP, does not affect complementation. However, a double mutation affecting both serine-219 and the adjacent threonine-218 (SR45.1-T218A + S219A-GFP) behaves like isoform 2, complementing the root but not the floral phenotype. In conclusion, our study provides evidence that the two alternatively spliced isoforms of SR45 have distinct biological functions.

cis-Element- and Transcriptome-Based Screening of Root Hair-Specific Genes and Their Functional Characterization in Arabidopsis

Won, S.-K., Lee, Y.-J., Lee, H.-Y., Heo, Y.-K., Cho, M., Cho, H.-T. — 2009-07-02

Understanding the cellular differentiation of multicellular organisms requires the characterization of genes whose expression is modulated in a cell type-specific manner. The Arabidopsis (Arabidopsis thaliana) root hair cell is one model for studying cellular differentiation. In this study, root hair cell-specific genes were screened by a series of in silico and experimental filtration procedures. This process included genome-wide screening for genes with a root hair-specific cis-element in their promoters, filtering root-specific genes from the root hair-specific cis-element-containing genes, further filtering of genes that were suppressed in root hair-defective plant lines, and experimental confirmation by promoter assay. These procedures revealed 19 root hair-specific genes, including many protein kinases and cell wall-related genes, most of which have not been characterized thus far. Functional analyses of these root hair-specific genes with loss-of-function mutants and overexpressing transformants revealed that they play roles in hair growth and morphogenesis. This study demonstrates that a defined cis-element can serve as a filter to screen certain cell type-specific genes and implicates many new root hair-specific genes in root hair development.

Plant Physiological Adaptations to the Massive Foreign Protein Synthesis Occurring in Recombinant Chloroplasts

Bally, J., Nadai, M., Vitel, M., Rolland, A., Dumain, R., Dubald, M. — 2009-07-02

Genetically engineered chloroplasts have an extraordinary capacity to accumulate recombinant proteins. We have investigated in tobacco (Nicotiana tabacum) the possible consequences of such additional products on several parameters of plant development and composition. Plastid transformants were analyzed that express abundantly either bacterial enzymes, alkaline phosphatase (PhoA-S and PhoA-L) and 4-hydroxyphenyl pyruvate dioxygenase (HPPD), or a green fluorescent protein (GFP). In leaves, the HPPD and GFP recombinant proteins are the major polypeptides and accumulate to higher levels than Rubisco. Nevertheless, these engineered metabolic sinks do not cause a measurable difference in growth rate or photosynthetic parameters. The total amino acid content of transgenic leaves is also not significantly affected, showing that plant cells have a limited protein biosynthetic capacity. Recombinant products are made at the expense of resident proteins. Rubisco, which constitutes the major leaf amino acid store, is the most clearly and strongly down-regulated plant protein. This reduction is even more dramatic under conditions of limited nitrogen supply, whereas recombinant proteins accumulate to even higher relative levels. These changes are regulated posttranscriptionally since transcript levels of resident plastid genes are not affected. Our results show that plants are able to produce massive amounts of recombinant proteins in chloroplasts without profound metabolic perturbation and that Rubisco, acting as a nitrogen buffer, is a key player in maintaining homeostasis and limiting pleiotropic effects.

CDKB1;1 Forms a Functional Complex with CYCA2;3 to Suppress Endocycle Onset

2009-07-02

The mitosis-to-endocycle transition requires the controlled inactivation of M phase-associated cyclin-dependent kinase (CDK) activity. Previously, the B-type CDKB1;1 was identified as an important negative regulator of endocycle onset. Here, we demonstrate that CDKB1;1 copurifies and associates with the A2-type cyclin CYCA2;3. Coexpression of CYCA2;3 with CDKB1;1 triggered ectopic cell divisions and inhibited endoreduplication. Moreover, the enhanced endoreduplication phenotype observed after overexpression of a dominant-negative allele of CDKB1;1 could be partially complemented by CYCA2;3 co-overexpression, illustrating that both subunits unite in vivo to form a functional complex. CYCA2;3 protein stability was found to be controlled by CCS52A1, an activator of the anaphase-promoting complex. We conclude that CCS52A1 participates in endocycle onset by down-regulating CDKB1;1 activity through the destruction of CYCA2;3.

Arabidopsis Encodes Four tRNase Z Enzymes

2009-07-02

Functional transfer RNA (tRNA) molecules are a prerequisite for protein biosynthesis. Several processing steps are required to generate the mature functional tRNA from precursor molecules. Two of the early processing steps involve cleavage at the tRNA 5' end and the tRNA 3' end. While processing at the tRNA 5' end is performed by RNase P, cleavage at the 3' end is catalyzed by the endonuclease tRNase Z. In eukaryotes, tRNase Z enzymes are found in two versions: a short form of about 250 to 300 amino acids and a long form of about 700 to 900 amino acids. All eukaryotic genomes analyzed to date encode at least one long tRNase Z protein. Of those, Arabidopsis (Arabidopsis thaliana) is the only organism that encodes four tRNase Z proteins, two short forms and two long forms. We show here that the four proteins are distributed to different subcellular compartments in the plant cell: the nucleus, the cytoplasm, the mitochondrion, and the chloroplast. One tRNase Z is present only in the cytoplasm, one protein is found exclusively in mitochondria, while the third one has dual locations: nucleus and mitochondria. None of these three tRNase Z proteins is essential. The fourth tRNase Z protein is present in chloroplasts, and deletion of its gene results in an embryo-lethal phenotype. In vitro analysis with the recombinant proteins showed that all four tRNase Z enzymes have tRNA 3' processing activity. In addition, the mitochondrial tRNase Z proteins cleave tRNA-like elements that serve as processing signals in mitochondrial mRNA maturation.

The K-Segment of Maize DHN1 Mediates Binding to Anionic Phospholipid Vesicles and Concomitant Structural Changes

Koag, M.-C., Wilkens, S., Fenton, R. D., Resnik, J., Vo, E., Close, T. J. — 2009-07-02

Dehydrins (DHNs; late embryogenesis abundant D11 family) are a family of intrinsically unstructured plant proteins that accumulate in the late stages of seed development and in vegetative tissues subjected to water deficit, salinity, low temperature, or abscisic acid treatment. We demonstrated previously that maize (Zea mays) DHNs bind preferentially to anionic phospholipid vesicles; this binding is accompanied by an increase in -helicity of the protein, and adoption of -helicity can be induced by sodium dodecyl sulfate. All DHNs contain at least one "K-segment," a lysine-rich 15-amino acid consensus sequence. The K-segment is predicted to form a class A2 amphipathic -helix, a structural element known to interact with membranes and proteins. Here, three K-segment deletion proteins of maize DHN1 were produced. Lipid vesicle-binding assays revealed that the K-segment is required for binding to anionic phospholipid vesicles, and adoption of -helicity of the K-segment accounts for most of the conformational change of DHNs upon binding to anionic phospholipid vesicles or sodium dodecyl sulfate. The adoption of structure may help stabilize cellular components, including membranes, under stress conditions.

Loss of the Transit Peptide and an Increase in Gene Expression of an Ancestral Chloroplastic Carbonic Anhydrase Were Instrumental in the Evolution of the Cytosolic C4 Carbonic Anhydrase in Flaveria

Tanz, S. K., Tetu, S. G., Vella, N. G.F., Ludwig, M. — 2009-07-02

C₄ photosynthesis has evolved multiple times from ancestral C₃ species. Carbonic anhydrase (CA) catalyzes the reversible hydration of CO₂ and is involved in both C₃ and C₄ photosynthesis; however, its roles and the intercellular and intracellular locations of the majority of its activity differ between C₃ and C₄ plants. To understand the molecular changes underlying the evolution of the C₄ pathway, three cDNAs encoding distinct β-CAs (CA1, CA2, and CA3) were isolated from the leaves of the C₃ plant Flaveria pringlei. The phylogenetic relationship of the F. pringlei proteins with other embryophyte β-CAs was reconstructed. Gene expression and protein localization patterns showed that CA1 and CA3 demonstrate high expression in leaves and their products localize to the chloroplast, while CA2 expression is low in all organs examined and encodes a cytosolic enzyme. The roles of the F. pringlei enzymes were considered in light of these results, other angiosperm β-CAs, and Arabidopsis (Arabidopsis thaliana) "omics" data. All three F. pringlei CAs have orthologs in the closely related C₄ plant Flaveria bidentis, and comparisons of ortholog sequences, expression patterns, and intracellular locations of their products indicated that CA1 and CA2 have maintained their ancestral role in C₄ plants, whereas modifications to the C₃ CA3 gene led to the evolution of the CA isoform that catalyzes the first step in the C₄ photosynthetic pathway. These changes included the loss of the chloroplast transit peptide and an increase in gene expression, which resulted in the high levels of CA activity seen in the cytosol of C₄ mesophyll cells.

Cytokinin-Dependent Photorespiration and the Protection of Photosynthesis during Water Deficit

Rivero, R. M., Shulaev, V., Blumwald, E. — 2009-07-02

We investigated the effects of P_SARK::IPT (for Senescence-Associated Receptor Kinase::Isopentenyltransferase) expression and cytokinin production on several aspects of photosynthesis in transgenic tobacco (Nicotiana tabacum cv SR1) plants grown under optimal or restricted (30% of optimal) watering regimes. There were no significant differences in stomatal conductance between leaves from wild-type and transgenic P_SARK-IPT plants grown under optimal or restricted watering. On the other hand, there was a significant reduction in the maximum rate of electron transport as well as the use of triose-phosphates only in wild-type plants during growth under restricted watering, indicating a biochemical control of photosynthesis during growth under water deficit. During water deficit conditions, the transgenic plants displayed an increase in catalase inside peroxisomes, maintained a physical association among chloroplasts, peroxisomes, and mitochondria, and increased the CO₂ compensation point, indicating the cytokinin-mediated occurrence of photorespiration in the transgenic plants. The contribution of photorespiration to the tolerance of transgenic plants to water deficit was also supported by the increase in transcripts coding for enzymes involved in the conversion of glycolate to ribulose-1,5-bisphosphate. Moreover, the increase in transcripts indicated a cytokinin-induced elevation in photorespiration, suggesting the contribution of photorespiration in the protection of photosynthetic processes and its beneficial role during water stress.

Identification of Nutrient-Responsive Arabidopsis and Rapeseed MicroRNAs by Comprehensive Real-Time Polymerase Chain Reaction Profiling and Small RNA Sequencing

2009-07-02

Comprehensive expression profiles of Arabidopsis (Arabidopsis thaliana) MIRNA genes and mature microRNAs (miRs) are currently not available. We established a quantitative real-time polymerase chain reaction platform that allows rapid and sensitive quantification of 177 Arabidopsis primary miR transcripts (pri-miRs). The platform was used to detect phosphorus (P) or nitrogen (N) status-responsive pri-miR species. Several pri-miR169 species as well as pri-miR398a were found to be repressed during N limitation, whereas during P limitation, pri-miR778, pri-miR827, and pri-miR399 species were induced and pri-miR398a was repressed. The corresponding responses of the biologically active, mature miRs were confirmed using specific stem-loop reverse transcription primer quantitative polymerase chain reaction assays and small RNA sequencing. Interestingly, the latter approach also revealed high abundance of some miR star strands. Bioinformatic analysis of small RNA sequences with a modified miRDeep algorithm led to the identification of the novel P limitation-induced miR2111, which is encoded by two loci in the Arabidopsis genome. Furthermore, miR2111, miR169, a miR827-like sequence, and the abundances of several miR star strands were found to be strongly dependent on P or N status in rapeseed (Brassica napus) phloem sap, flagging them as candidate systemic signals. Taken together, these results reveal the existence of complex small RNA-based regulatory networks mediating plant adaptation to mineral nutrient availability.

Abscisic Acid Negatively Regulates Elicitor-Induced Synthesis of Capsidiol in Wild Tobacco

Mialoundama, A. S., Heintz, D., Debayle, D., Rahier, A., Camara, B., Bouvier, F. — 2009-07-02

In the Solanaceae, biotic and abiotic elicitors induce de novo synthesis of sesquiterpenoid stress metabolites known as phytoalexins. Because plant hormones play critical roles in the induction of defense-responsive genes, we have explored the effect of abscisic acid (ABA) on the synthesis of capsidiol, the major wild tobacco (Nicotiana plumbaginifolia) sesquiterpenoid phytoalexin, using wild-type plants versus nonallelic mutants Npaba2 and Npaba1 that are deficient in ABA synthesis. Npaba2 and Npaba1 mutants exhibited a 2-fold higher synthesis of capsidiol than wild-type plants when elicited with either cellulase or arachidonic acid or when infected by Botrytis cinerea. The same trend was observed for the expression of the capsidiol biosynthetic genes 5-epi-aristolochene synthase and 5-epi-aristolochene hydroxylase. Treatment of wild-type plants with fluridone, an inhibitor of the upstream ABA pathway, recapitulated the behavior of Npaba2 and Npaba1 mutants, while the application of exogenous ABA reversed the enhanced synthesis of capsidiol in Npaba2 and Npaba1 mutants. Concomitant with the production of capsidiol, we observed the induction of ABA 8'-hydroxylase in elicited plants. In wild-type plants, the induction of ABA 8'-hydroxylase coincided with a decrease in ABA content and with the accumulation of ABA catabolic products such as phaseic acid and dihydrophaseic acid, suggesting a negative regulation exerted by ABA on capsidiol synthesis. Collectively, our data indicate that ABA is not required per se for the induction of capsidiol synthesis but is essentially implicated in a stress-response checkpoint to fine-tune the amplification of capsidiol synthesis in challenged plants.

Identification of Chlorogenic Acid as a Resistance Factor for Thrips in Chrysanthemum

Leiss, K. A., Maltese, F., Choi, Y. H., Verpoorte, R., Klinkhamer, P. G.L. — 2009-07-02

Western flower thrips (Frankliniella occidentalis) has become a key insect pest of agricultural and horticultural crops worldwide. Little is known about host plant resistance to thrips. In this study, we investigated thrips resistance in chrysanthemum (Dendranthema grandiflora). We identified thrips-resistant chrysanthemums applying bioassays. Subsequently, nuclear magnetic resonance (NMR)-based metabolomics was applied to compare the metabolome of thrips-resistant and -susceptible chrysanthemums. NMR facilitates wide-range coverage of the metabolome. We show that thrips-resistant and -susceptible chrysanthemums can be discriminated on basis of their metabolomic profiles. Thrips-resistant chrysanthemums contained higher amounts of the phenylpropanoids chlorogenic acid and feruloyl quinic acid. Both phenylpropanoids are known for their inhibitory effect on herbivores as well as pathogens. Thus, chlorogenic and feruloyl quinic acid are the compounds of choice to improve host plants resistance to thrips in ornamentals and crops. The effect of chlorogenic acid on thrips was further studied in bioassays with artificial diets. These experiments confirmed the negative effects on thrips. Our results prove NMR to be an important tool to identify different metabolites involved in herbivore resistance. It constitutes a significant advance in the study of plant-insect relationships, providing key information on the implementation of herbivore resistance breeding strategies in plants.

Different Lepidopteran Elicitors Account for Cross-Talk in Herbivory-Induced Phytohormone Signaling

Diezel, C., von Dahl, C. C., Gaquerel, E., Baldwin, I. T. — 2009-07-02

Salicylic acid (SA), jasmonic acid (JA), ethylene (ET), and their interactions mediate plant responses to pathogen and herbivore attack. JA-SA and JA-ET cross-signaling are well studied, but little is known about SA-ET cross-signaling in plant-herbivore interactions. When the specialist herbivore tobacco hornworm (Manduca sexta) attacks Nicotiana attenuata, rapid and transient JA and ET bursts are elicited without significantly altering wound-induced SA levels. In contrast, attack from the generalist beet armyworm (Spodoptera exigua) results in comparatively lower JA and ET bursts, but amplified SA bursts. These phytohormone responses are mimicked when the species' larval oral secretions (OS_Se and OS_Ms) are added to puncture wounds. Fatty acid-amino acid conjugates elicit the JA and ET bursts, but not the SA burst. OS_Se had enhanced glucose oxidase activity (but not β-glucosidase activity), which was sufficient to elicit the SA burst and attenuate the JA and ET levels. It is known that SA antagonizes JA; glucose oxidase activity and associated hydrogen peroxide also antagonizes the ET burst. We examined the OS_Ms-elicited SA burst in plants impaired in their ability to elicit JA (antisense [as]-lox3) and ET (inverted repeat [ir]-aco) bursts and perceive ET (35s-etr1b) after fatty acid-amino acid conjugate elicitation, which revealed that both ET and JA bursts antagonize the SA burst. Treating wild-type plants with ethephone and 1-methylcyclopropane confirmed these results and demonstrated the central role of the ET burst in suppressing the OS_Ms-elicited SA burst. By suppressing the SA burst, the ET burst likely facilitates unfettered JA-mediated defense activation in response to herbivores that otherwise would elicit SA.

Giant Flowers of Southern Magnolia Are Hydrated by the Xylem

Feild, T. S., Chatelet, D. S., Brodribb, T. J. — 2009-07-02

Flowering depends upon long-distance transport to supply water for reproductive mechanisms to function. Previous physiological studies suggested that flowers operated uncoupled from stem xylem transport and received water primarily from the phloem. We demonstrate that the water balance of Southern magnolia (Magnolia grandiflora) flowers is regulated in a manner opposite from that of previously examined flowers. We show that flowers of Southern magnolia rely upon relatively efficient xylem hydraulic transport to support high water demand during anthesis. We measured rapid rates of perianth transpiration ranging from twice to 100 times greater than previous studies. We found that relatively efficient xylem pathways existed between the xylem and flower. Perianth hydraulic conductance and the amount of xylem to transpirational surface area ratios of flowers were both approximately one-third those measured for leafy shoots. Furthermore, we observed that perianth tissues underwent significant diurnal depressions in water status during transpiring conditions. Decreases in water potential observed between flowers and vegetative tissues were consistent with water moving from the stem xylem into the flower during anthesis. Xylem hydraulic coupling of flowers to the stem was supported by experiments showing that transpiring flowers were unaffected by bark girdling. With Southern magnolia being a member of a nearly basal evolutionary lineage, our results suggest that flower water balance represents an important functional dimension that influenced early flower evolution.

Processing of a Dicistronic tRNA-snoRNA Precursor: Combined Analysis in Vitro and in Vivo Reveals Alternate Pathways and Coupling to Assembly of snoRNP

2009-07-02

The C/D box small nucleolar RNAs (snoRNAs) represent an essential class of small nucleolar RNAs that guide 2'-O-Rib methylation of ribosomal RNAs and other RNAs in eukaryotes. In Arabidopsis (Arabidopsis thaliana), >100 C/D snoRNAs have been identified, most of them encoded by polycistronic gene clusters, but little is known on the factors controlling their biogenesis. Here, we focus on the identification of factors controlling the processing of tRNA-snoRNA dicistronic precursors (pre-tsnoRNA) synthesized by RNA polymerase III and producing tRNA^Gly and C/D snoR43. We produced radiolabeled RNA probes corresponding to different pre-tsnoRNA mutants to test their impact on processing in vitro by a recombinant tRNAse Z, the Arabidopsis endonuclease that processes the 3'end of tRNAs, and by nuclear extracts from cauliflower (Brassica oleracea) inflorescences that accurately process the pre-tsnoRNA. This was coupled to an in vivo analysis of the processing of tagged pre-tsnoRNA mutants expressed in Arabidopsis. Our results strongly implicate tRNase Z in endonucleolytic cleavage of the pre-tsnoRNA. In addition, they reveal an alternate pathway that could depend on a tRNA decay surveillance mechanism. Finally, we provide arguments showing that processing of pre-tsnoRNA, both in planta and by nuclear extracts, is coupled to the assembly of snoRNA with core proteins forming the functional snoRNP (for small nucleolar ribonucleoprotein complex).

FRIGIDA Delays Flowering in Arabidopsis via a Cotranscriptional Mechanism Involving Direct Interaction with the Nuclear Cap-Binding Complex

Geraldo, N., Baurle, I., Kidou, S.-i., Hu, X., Dean, C. — 2009-07-02

A major determinant of flowering time in natural Arabidopsis (Arabidopsis thaliana) variants is FRIGIDA (FRI). FRI up-regulates expression of the floral repressor FLOWERING LOCUS C (FLC), thereby conferring a vernalization requirement and a winter annual habit. FRI encodes a novel nuclear protein with no conserved domains except for two coiled-coil regions. A mutation in the large subunit of the nuclear cap-binding complex (CBC) suppresses FRI activity, so we have explored the connection between FRI and the nuclear CBC in order to gain further insight into FRI biochemical activity. Mutations in the small subunit of the CBC (CBP20) also suppress FRI up-regulation of FLC. CBP20 interacted directly with FRI in yeast and in planta, and this association of FRI with the 5' cap was reinforced by an RNA ligase-mediated rapid amplification of cDNA ends assay that showed FRI decreased the proportion of FLC transcripts lacking a 5' cap. Loss of CBP20 resulted in very low FLC mRNA levels and an increased proportion of unspliced FLC transcripts. FRI compensated for CBP20 loss, partially restoring FLC levels and normalizing the unspliced-spliced transcript ratio. Our data suggest that FRI up-regulates FLC expression through a cotranscriptional mechanism involving direct physical interaction with the nuclear CBC with concomitant effects on FLC transcription and splicing.

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2009-07-02