Plant and Cell Physiology - recent issues

Recent Progress in Plant Reproduction Research: The Story of the Male Gametophyte through to Successful Fertilization

Suzuki, G. — Wed, 11 Nov 2009 07:54:39 PST

Sexual reproduction is an important biological event not only for evolution but also for breeding in plants. It is a well known fact that Charles Darwin (1809–1882) was interested in the reproduction system of plants as part of his concept of ‘species’ and ‘evolution.’ His keen observation and speculation is timeless even in the current post-genome era. In the Darwin anniversary year of 2009, I have summarized recent molecular genetic studies of plant reproduction, focusing especially on male gametophyte development, pollination and fertilization. We are just beginning to understand the molecular mechanisms of the elaborate reproduction system in flowering plants, which have been a mystery for >100 years.

RESOPS: A Database for Analyzing the Correspondence of RNA Editing Sites to Protein Three-Dimensional Structures

Yura, K., Sulaiman, S., Hatta, Y., Shionyu, M., Go, M. — Wed, 11 Nov 2009 07:54:39 PST

Transcripts from mitochondrial and chloroplast DNA of land plants often undergo cytidine to uridine conversion-type RNA editing events. RESOPS is a newly built database that specializes in displaying RNA editing sites of land plant organelles on protein three-dimensional (3D) structures to help elucidate the mechanisms of RNA editing for gene expression regulation. RESOPS contains the following information: unedited and edited cDNA sequences with notes for the target nucleotides of RNA editing, conceptual translation from the edited cDNA sequence in pseudo-UniProt format, a list of proteins under the influence of RNA editing, multiple amino acid sequence alignments of edited proteins, the location of amino acid residues coded by codons under the influence of RNA editing in protein 3D structures and the statistics of biased distributions of the edited residues with respect to protein structures. Most of the data processing procedures are automated; hence, it is easy to keep abreast of updated genome and protein 3D structural data. In the RESOPS database, we clarified that the locations of residues switched by RNA editing are significantly biased to protein structural cores. The integration of different types of data in the database also help advance the understanding of RNA editing mechanisms. RESOPS is accessible at http://cib.cf.ocha.ac.jp/RNAEDITING/.

Differential Downward Stream of Auxin Synthesized at the Tip Has a Key Role in Gravitropic Curvature via TIR1/AFBs-Mediated Auxin Signaling Pathways

Nishimura, T., Nakano, H., Hayashi, K.-i., Niwa, C., Koshiba, T. — Wed, 11 Nov 2009 07:54:39 PST

Since the early days of Darwin, monocot coleoptiles have been used to investigate indole-3-acetic acid (IAA) production, polar transport and tropisms. Here, using maize coleoptiles, we first showed that polar transport of IAA synthesized at the tip region is regulated by ZmPIN(s). Then, the TIR/AFBs-mediated auxin signaling pathway corresponds to the asymmetric IAA flow after gravi-stimulus, which results in tropic curvature. When [¹³C₁₁¹⁵N₂]Trp was applied to coleoptile tips, substantial amounts of the stable isotope were incorporated into IAA at the tip region, and the labeled IAA was transported in a polar manner at approximately 7 mm h^–1. Immunohistochemical analyses revealed that ZmPIN1(s) was present in almost all cells. ZmPIN1(s) showed a relatively non-polar distribution at the tip, but a basal cellular localization at lower regions. Application of the IAA transport inhibitors 1-N-naphthylphthalamic acid (NPA) and brefeldin A (BFA) at the very tip region almost completely inhibited IAA movement from the tip. These inhibitors also severely suppressed gravitropic bending. PEO-IAA, an auxin antagonist that binds to TIR1/AFBs, suppressed not only the expression of an auxin-responsive ZmSAUR2 gene, but also gravitropic curvature. Expression of ZmSAUR2 was up-regulated on the lower side and down-regulated on the upper side of the coleoptile elongation zone, corresponding to the asymmetric IAA distribution. These results indicate that the asymmetric downward streams of IAA control the differential growth rate of the cells by attenuating TIR1/AFBs-mediated auxin response genes, including ZmSAUR2, and therefore result in tropic curvature.

Rice BRITTLE CULM 5 (BRITTLE NODE) is Involved in Secondary Cell Wall Formation in the Sclerenchyma Tissue of Nodes

Aohara, T., Kotake, T., Kaneko, Y., Takatsuji, H., Tsumuraya, Y., Kawasaki, S. — Wed, 11 Nov 2009 07:54:39 PST

Several brittle culm (bc) mutants known in grasses are considered excellent materials to study the process of secondary cell wall formation. The brittle phenotype of the rice bc5 (brittle node) mutant appears exclusively in the developed nodes, which is distinct from other bc mutants (bc1, 2, 3, 4, 6 and 7) that show the brittle phenotype in culms and leaves. To address the defects of the rice bc5 mutant in node-specific cell wall formation, we analyzed tissue morphology and cell wall composition. The bc5 mutation was found to affect the cell wall deposition of node sclerenchyma tissues at 1 week after heading, the stage at which the cell wall sugar content is reduced, in the bc5 nodes, compared with wild-type nodes. Moreover, decreased accumulation of lignin and thickness of cell walls in the sclerenchyma tissues were also observed in the bc5 nodes. The amounts of cellulose and hemicellulose were reduced to 53 and 65% of those in the wild-type plants, respectively. Sugar composition and glycosidic linkage analyses of the hemicellulose showed that the accumulation of glucuronosyl arabinoxylan in bc5 nodes was perturbed by the mutation. The bc5 locus was narrowed to an approximately 3.1 Mb region of chromosome 2, where none of the other bc genes is located. The bc5 mutation appeared to reduce the expression levels of the OsCesA genes in the nodes after heading. The results indicate that the BC5 gene regulates the development of secondary cell walls of node sclerenchyma tissues.

The TL29 Protein is Lumen Located, Associated with PSII and Not an Ascorbate Peroxidase

Wed, 11 Nov 2009 07:54:39 PST

The TL29 protein is one of the more abundant proteins in the thylakoid lumen of plant chloroplasts. Based on its sequence homology to ascorbate peroxidases, but without any supporting biochemical evidence, TL29 was suggested to be involved in the plant defense system against reactive oxygen species and consequently renamed to APX4. Our in vivo and in vitro analyses failed to show any peroxidase activity associated with TL29; it bound neither heme nor ascorbate. Recombinant overexpressed TL29 had no ascorbate-dependent peroxidase activity, and various mutational analyses aiming to convert TL29 into an ascorbate peroxidase failed. Furthermore, in the thylakoid lumen no such activity could be associated with TL29 and, additionally, TL29 knock-out mutants did not show any decreased peroxidase activity or increased content of radical oxygen species when grown under light stress. Instead we could show that TL29 is a lumen-located component associated with PSII.

High Temperatures Cause Male Sterility in Rice Plants with Transcriptional Alterations During Pollen Development

Wed, 11 Nov 2009 07:54:39 PST

Plant male reproductive development is highly organized and sensitive to various environmental stressors, including high temperature. We have established an experimental procedure to evaluate high temperature injury in japonica rice plants. High temperature treatment (39°C/30°C) starting at the microspore stage repeatedly reduced spikelet fertility in our system. Morphological observations revealed that pollen viability in plants exposed to high temperatures was lower than that in control plants. Most pollen grains in high temperature-treated plants displayed a normal round shape and stained reddish purple with Alexander’s reagent; however, the pollen grains were very poorly attached and displayed limited germination on the stigma. To investigate gene regulatory mechanisms in the anther in high temperature environments, DNA microarray analysis was performed by comparing non-treated samples with samples treated with 2–4 d of high heat. Genes responsive to high temperatures were identified from clustering of microarray data. Among these, at least 13 were designated as high temperature-repressed genes in the anther. Expression analyses revealed that these genes were expressed specifically in the immature anther mainly in the tapetum at the microspore stage and down-regulated after 1 d of high temperature. The expression levels of Osc6, OsRAFTIN and TDR, which are tapetum-specific genes, were unaffected by high temperatures. These results suggest that not all tapetal genes are inhibited by increased temperatures and the tapetum itself is not degraded in such an environment. However, high temperatures may disrupt some of the tapetum functions required for pollen adhesion and germination on the stigma.

Arabidopsis OPT6 is an Oligopeptide Transporter with Exceptionally Broad Substrate Specificity

Pike, S., Patel, A., Stacey, G., Gassmann, W. — Wed, 11 Nov 2009 07:54:39 PST

Oligopeptide transporters (OPTs) are found in fungi, bacteria and plants. The nine Arabidopsis thaliana OPT genes are expressed mainly in the vasculature and are thought to transport tetra- and pentapeptides, and peptide-like substrates such as glutathione. Expression of AtOPT6 in Xenopus laevis oocytes demonstrated that AtOPT6 transports many tetra- and pentapeptides. In addition, AtOPT6 transported reduced glutathione (GSH), a tripeptide, but not oxidized glutathione (GSSG). Recent data showed that Candida albicans OPTs can transport peptides up to eight amino acids in length. AtOPT6 transported mammalian signaling peptides up to 10 amino acids in length and, in addition, known plant development- and nematode pathogenesis-associated peptides up to 13 amino acids long. AtOPT6 displayed high affinity for penta- and dodecapeptides, but low affinity for GSH. In comparison the Saccharomyces cerevisiae ScOPT1 was incapable of transporting any of the longer peptides tested. These data demonstrate the necessity of experimentally determining substrate specificity of individual OPTs, and lay a foundation for structure/function studies. Characterization of the AtOPT6 substrate range provides a basis for investigating the possible physiological function of AtOPT6 in peptide signaling and thiol transport in response to stress.

Ectopic Overexpression of The Transcription Factor OsGLK1 Induces Chloroplast Development in Non-Green Rice Cells

Wed, 11 Nov 2009 07:54:39 PST

For systematic and genome-wide analyses of rice gene functions, we took advantage of the full-length cDNA overexpresser (FOX) gene-hunting system and generated >12 000 independent FOX-rice lines from >25 000 rice calli treated with the rice-FOX Agrobacterium library. We found two FOX-rice lines generating green calli on a callus-inducing medium containing 2,4-D, on which wild-type rice calli became ivory yellow. In both lines, OsGLK1 cDNA encoding a GARP transcription factor was ectopically overexpressed. Using rice expression-microarray and northern blot analyses, we found that a large number of nucleus-encoded genes involved in chloroplast functions were highly expressed and transcripts of plastid-encoded genes, psaA, psbA and rbcL, increased in the OsGLK1-FOX calli. Transmission electron microscopy showed the existence of differentiated chloroplasts with grana stacks in OsGLK1-FOX calli cells. However, in darkness, OsGLK1-FOX calli did not show a green color or develop grana stacks. Furthermore, we found developed chloroplasts in vascular bundle and bundle sheath cells of coleoptiles and leaves from OsGLK1-FOX seedlings. The OsGLK1-FOX calli exhibited high photosynthetic activity and were able to grow on sucrose-depleted media, indicating that developed chloroplasts in OsGLK1-FOX rice calli are functional and active. We also observed that the endogenous OsGLK1 mRNA level increased synchronously with the greening of wild-type calli after transfer to plantlet regeneration medium. These results strongly suggest that OsGLK1 regulates chloroplast development under the control of light and phytohormones, and that it is a key regulator of chloroplast development.

MYB83 Is a Direct Target of SND1 and Acts Redundantly with MYB46 in the Regulation of Secondary Cell Wall Biosynthesis in Arabidopsis

McCarthy, R. L., Zhong, R., Ye, Z.-H. — Wed, 11 Nov 2009 07:54:39 PST

It has been proposed that the transcriptional regulation of secondary wall biosynthesis in Arabidopsis is controlled by a transcriptional network mediated by SND1 and its close homologs. Uncovering all the transcription factors and deciphering their interrelationships in the network are essential for our understanding of the molecular mechanisms underlying the transcriptional regulation of biosynthesis of secondary walls, the major constituent of wood and fibers. Here, we present functional evidence that the MYB83 transcription factor is another molecular switch in the SND1-mediated transcriptional network regulating secondary wall biosynthesis. MYB83 is specifically expressed in fibers and vessels where secondary wall thickening occurs. Its expression is directly activated by SND1 and its close homologs, including NST1, NST2, VND6 and VND7, indicating that MYB83 is their direct target. MYB83 overexpression is able to activate a number of the biosynthetic genes of cellulose, xylan and lignin and concomitantly induce ectopic secondary wall deposition. In addition, its overexpression upregulates the expression of several transcription factors involved in regulation of secondary wall biosynthesis. Dominant repression of MYB83 functions or simultaneous RNAi inhibition of MYB83 and MYB46 results in a reduction in secondary wall thickening in fibers and vessels and a deformation of vessels. Furthermore, double T-DNA knockout mutations of MYB83 and MYB46 cause a lack of secondary walls in vessels and an arrest in plant growth. Together, these results demonstrate that MYB83 and MYB46, both of which are SND1 direct targets, function redundantly in the transcriptional regulatory cascade leading to secondary wall formation in fibers and vessels.

Arabidopsis Replication Protein A 70a is Required for DNA Damage Response and Telomere Length Homeostasis

Takashi, Y., Kobayashi, Y., Tanaka, K., Tamura, K. — Wed, 11 Nov 2009 07:54:39 PST

Replication protein A1 (RPA1/RPA70) forms a heterotrimeric complex together with RPA2/RPA32 and RPA3/RPA14 subunits which plays essential roles in various aspects of DNA metabolism including replication, repair, recombination and telomere maintenance. Compared with RPA70 in yeast and mammals, limited information is available about the factor in plants. In this study, we analyzed the functions of AtRPA70a, which is most similar to human RPA70 among four paralogs in Arabidopsis thaliana. RNA blot analysis showed that AtRPA70a is expressed ubiquitously in plant organs containing differentiated and meristematic tissues, while its expression was up-regulated in response to DNA damage stress. Yeast two-hybrid and co-immunoprecipitation analyses showed that AtRPA70a interacted preferentially with Arabidopsis RPA32a, one of two paralogs. Inactivation of AtRPA70a by T-DNA insertion did not affect growth under normal conditions, but resulted in increased sensitivity to genotoxic agents such as methylmethane sulfonate, bleomycin and hydroxyurea. Terminal restriction fragment analysis revealed that telomere lengths in an AtRPA70a-deficient line were significantly larger than in the wild type, whereas those in the mutant expressing antisense AtTERT (telomerase catalytic subunit gene) were shortened during successive generations. These results demonstrate that AtRPA70a is involved in repair of double-strand DNA breaks and possibly contributes to telomerase-dependent telomere length regulation.

Arabidopsis NIP1;1 Transports Antimonite and Determines Antimonite Sensitivity

Kamiya, T., Fujiwara, T. — Wed, 11 Nov 2009 07:54:39 PST

Antimony (Sb) is toxic to organisms including plants. Although it is not essential to organisms, plants take up Sb from the environment. In this study, we identified an antimonite [Sb(III)] transporter from Arabidopsis thaliana. We examined the Sb(III) tolerance of the disruption mutant plants of arsenite [As(III)] transporters, nodulin 26-like intrinsic proteins (NIPs), since Sb(III) is similar to As(III) in structure. One of the mutants, nip1;1, showed Sb(III) tolerance and accumulated less Sb. Furthermore, yeast expressing NIP1;1 accumulated twice as much Sb as control. These data indicate that NIP1;1 transports Sb(III) and determines the Sb(III) sensitivity of A. thaliana.

The Poplar GT8E and GT8F Glycosyltransferases are Functional Orthologs of Arabidopsis PARVUS Involved in Glucuronoxylan Biosynthesis

Lee, C., Teng, Q., Huang, W., Zhong, R., Ye, Z.-H. — Wed, 11 Nov 2009 07:54:39 PST

The poplar GT8E and GT8F glycosyltransferases have previously been shown to be associated with wood formation, but their roles in the biosynthesis of wood components are not known. Here, we show that PoGT8E and PoGT8F are expressed in vessels and fibers during wood formation and their encoded proteins are predominantly located in the endoplasmic reticulum. We demonstrate that expression of PoGT8E and PoGT8F in the Arabidopsis parvus mutant rescues the defects in the content and structure of glucuronoxylan conferred by the parvus mutation. These findings suggest that PoGT8E and PoGT8F are involved in glucuronoxylan biosynthesis during wood formation in poplar.

A Highly Sensitive, Quick and Simple Quantification Method for Nicotianamine and 2'-Deoxymugineic Acid from Minimum Samples Using LC/ESI-TOF-MS Achieves Functional Analysis of These Components in Plants

Wed, 11 Nov 2009 07:54:39 PST

A highly sensitive quantitative method for assaying nicotianamine (NA) and 2'-deoxymugineic acid (DMA) using liquid chromatography/electrospray ionization time-of-flight mass spectrometry (LC/ESI-TOF-MS) was developed. The amino and hydroxyl groups of NA and DMA were derivatized using 9-fluorenylmethoxycarbonyl chloride. The amounts of NA and DMA in 10 µl of xylem sap from rice cultivated under iron (Fe)-sufficient and Fe-deficient conditions were quantified without concentration. In Fe-sufficient plants, the concentrations of NA and DMA were almost equal to that of Fe. In Fe-deficient plants, the concentration of NA did not change significantly, whereas that of DMA increased markedly.

Resolving the Role of Plant Glutamate Dehydrogenase. I. in vivo Real Time Nuclear Magnetic Resonance Spectroscopy Experiments

Wed, 11 Nov 2009 07:54:39 PST

Isozyme-Specific Modes of Activation of CTP:Phosphorylcholine Cytidylyltransferase in Arabidopsis thaliana at Low Temperature

Thu, 08 Oct 2009 08:39:56 PDT

Arabidopsis thaliana increases cellular phosphatidylcholine (PC) content during cold acclimation by up-regulating PC biosynthesis. The A. thaliana genes CCT1 and CCT2 encode CTP:phosphorylcholine cytidylyltransferases (CCTs; EC 2.7.7.15), which regulate PC biosynthesis via the CDP–choline pathway. We isolated the T-DNA-tagged knockout mutants cct1 and cct2 of A. thaliana (Wassilevskaja; WS). CCT activity in cct1 and cct2 plants accounted for 29 and 79% to the cellular CCT activity of WS plants, respectively. When plants were exposed to 2°C for 7 d, CCT activity increased in both cct1 and cct2 plants, and immunoblot analyses revealed that cct1 contained an increased level of CCT2 protein whereas cct2 exhibited little increase in CCT1 level. For each mutant grown at 23°C, CCT activity was mainly enriched in the particulate (15,000 x g pellet) and microsomal (150,000 x g pellet) fractions from rosette leaf homogenates. After exposure to cold, the particulate and microsomal fractions of cct1 plants had higher total CCT activity due to increased levels of CCT2; in contrast, the levels of CCT1 in cct2 plants remained unchanged in particulate and microsomal fractions despite a significant increase in the total CCT activity. We conclude that the CDP–choline pathway of A. thaliana is up-regulated at low temperature via isogene-specific modes: enhanced expression of CCT2 and post-translational activation/inactivation of CCT1 in membranes. PC levels were similarly maintained in both mutants and WS plants after 14 d at 2°C, suggesting that either of the CCT genes is sufficient for PC biosynthesis at low temperature.

Differential Positioning of C4 Mesophyll and Bundle Sheath Chloroplasts: Aggregative Movement of C4 Mesophyll Chloroplasts in Response to Environmental Stresses

Yamada, M., Kawasaki, M., Sugiyama, T., Miyake, H., Taniguchi, M. — Thu, 08 Oct 2009 08:39:56 PDT

In C₄ plants, mesophyll (M) chloroplasts are randomly distributed along the cell walls, while bundle sheath (BS) chloroplasts are typically located in either a centripetal or centrifugal position. We investigated whether these intracellular positions are affected by environmental stresses. When mature leaves of finger millet (Eleusine coracana) were exposed to extremely high intensity light, most M chloroplasts aggregatively re-distributed to the BS side, whereas the intracellular arrangement of BS chloroplasts was unaffected. Compared with the homologous light-avoidance movement of M chloroplasts in C₃ plants, it requires extremely high light (3,000–4,000 µmol m^–2 s^–1) and responds more slowly (distinctive movement observed in 1 h). The high light-induced movement of M chloroplasts was also observed in maize (Zea mays), another C₄ species, but with a distinct pattern of redistribution along the sides of anticlinal walls, analogous to C₃ plants. The aggregative movement of M chloroplasts occurred at normal light intensities (250–500 µmol m^–2 s^–1) in response to environmental stresses, such as drought, salinity and hyperosmosis. Moreover, the re-arrangement of M chloroplasts was observed in field-grown C₄ plants when exposed to mid-day sunlight, but also under midsummer drought conditions. The migration of M chloroplasts was controlled by actin filaments and also induced in a light-dependent fashion upon incubation with ABA, which may be the physiological signal transducer. Together these results suggest that M and BS cells of C₄ plants have different mechanisms controlling intracellular chloroplast positioning, and that the aggregative movement of C₄ M chloroplasts is thought to be a protective response under environmental stress conditions.

Effects of Site-Directed Mutations in the Chloroplast-Encoded ycf4 Gene on PSI Complex Assembly in the Green Alga Chlamydomonas reinhardtii

Onishi, T., Takahashi, Y. — Thu, 08 Oct 2009 08:39:56 PDT

The chloroplast-encoded Ycf4 plays an essential role in PSI complex assembly in the green alga Chlamydomonas reinhardtii. To gain insight into how Ycf4 functions, we generated several mutants in which residues R120, E179 and/or E181, which are conserved among oxygenic photosynthetic organisms, were changed to A or Q. Although the single mutants R120A and R120Q accumulated 80% less Ycf4 than the wild type, they assembled a functional PSI complex and grew photosynthetically like the wild type. Thus we inferred that under laboratory growth conditions, wild-type cells accumulate a superfluous amount of Ycf4. Single mutants E179A, E179Q and E181Q assembled a functional PSI complex like the wild type, whereas the single mutant E181A and double mutant E179/181A accumulated a functional PSI complex to significantly reduced levels. Double mutant E179/181Q, in contrast, accumulated Ycf4 at the wild-type level but did not assemble any mature PSI complex, suggesting that the two glutamic acid residues play crucial roles in the functionality of Ycf4. Interestingly, sucrose density gradient centrifugation of the thylakoid extracts separated a small amount of PSI subcomplex. The apparent size of the subcomplex (150–170 kDa), its composition and pulse–chase protein labeling indicate that it was an unstable subcomplex consisting of a PsaA–PsaB heterodimer. We inferred that the subcomplex was a PSI complex assembly intermediate that was detected because subsequent assembly steps were blocked by the E179/181Q mutation. We concluded that Ycf4 is involved in early processes of PSI complex assembly.

Resolving the Role of Plant Glutamate Dehydrogenase. I. in vivo Real Time Nuclear Magnetic Resonance Spectroscopy Experiments

Thu, 08 Oct 2009 08:39:56 PDT

In higher plants the glutamate dehydrogenase (GDH) enzyme catalyzes the reversible amination of 2-oxoglutarate to form glutamate, using ammonium as a substrate. For a better understanding of the physiological function of GDH either in ammonium assimilation or in the supply of 2-oxoglutarate, we used transgenic tobacco (Nicotiana tabacum L.) plants overexpressing the two genes encoding the enzyme. An in vivo real time ¹⁵N-nuclear magnetic resonance (NMR) spectroscopy approach allowed the demonstration that, when the two GDH genes were overexpressed individually or simultaneously, the transgenic plant leaves did not synthesize glutamate in the presence of ammonium when glutamine synthetase (GS) was inhibited. In contrast we confirmed that the primary function of GDH is to deaminate Glu. When the two GDH unlabeled substrates ammonium and Glu were provided simultaneously with either [¹⁵N]Glu or ¹⁵NH₄⁺ respectively, we found that the ammonium released from the deamination of Glu was reassimilated by the enzyme GS, suggesting the occurrence of a futile cycle recycling both ammonium and Glu. Taken together, these results strongly suggest that the GDH enzyme, in conjunction with NADH-GOGAT, contributes to the control of leaf Glu homeostasis, an amino acid that plays a central signaling and metabolic role at the interface of the carbon and nitrogen assimilatory pathways. Thus, in vivo NMR spectroscopy appears to be an attractive technique to follow the flux of metabolites in both normal and genetically modified plants.

Blue Light-Induced Phototropism of Inflorescence Stems and Petioles is Mediated by Phototropin Family Members phot1 and phot2

Kagawa, T., Kimura, M., Wada, M. — Thu, 08 Oct 2009 08:39:56 PDT

Phototropin family photoreceptors, phot1 and phot2, in Arabidopsis thaliana control the blue light (BL)-mediated phototropic responses of the hypocotyl, chloroplast relocation movement and stomatal opening. Phototropic responses in dark-grown tissues have been well studied but those in de-etiolated green plants are not well understood. Here, we analyzed phototropic responses of inflorescence stems and petioles of wild-type and phototropin mutant plants of A. thaliana. Similar to the results obtained from dark-grown seedlings, inflorescence stems and petioles in wild-type and phot2 mutant plants showed phototropic bending towards low fluence BL, while in phot1 mutant plants, a high fluence rate of BL was required. phot1 phot2 double mutant plants did not show any phototropic responses even under very high fluence rates of BL. We further studied the photoreceptive sites for phototropic responses of stems and petioles by partial tissue irradiation. The whole part of the inflorescence stem is sensitive to BL and shows phototropism, but in the petiole only the irradiated abaxial side is sensitive. Similar to dark-grown etiolated seedlings, phot1 plays a major role in phototropic responses under weak light, but phot2 functions under high fluence rate conditions in green plants.

Temporal Expression Patterns of Hormone Metabolism Genes during Imbibition of Arabidopsis thaliana Seeds: A Comparative Study on Dormant and Non-Dormant Accessions

Thu, 08 Oct 2009 08:39:56 PDT

Seed imbibition is a prerequisite for subsequent dormancy and germination control. Here, we investigated imbibition responses of Arabidopsis seeds by transcriptomic and hormone profile analyses using dormant [Cape Verde Islands (Cvi)] and non-dormant [Columbia (Col)] accessions. Once imbibed, seeds of both accessions swelled most up to 3 h, reflecting water uptake. Microarray analysis showed that in both accessions, seeds imbibed for 15 min, 30 min and 1 h were less active in gene expression than at 3 h. More than 2,000 genes were either up-regulated or down-regulated in seeds imbibed for 3 h. Some genes up-regulated at 3 h were already induced in seeds imbibed for 1 h, suggestive of genome reprogramming early after the onset of imbibition. Imbibition-induced genes in seeds imbibed for 3 h included those up-regulated in both Col and Cvi (common) or unique to either accession (accession specific). Up-regulated genes that were both common and Cvi-specific were over-represented for sugar metabolism and the pentose phosphate pathway, whereas Col-specific genes were over-represented for ribosomal protein genes. Quantification of plant hormones showed that ABA and salicylic acid (SA) contents were higher, but gibberellin A₄ (GA₄), N⁶-(²-isopentenyl)adenine (iP), jasmonic acid (JA), JA–isoleucine (JA-Ile) and IAA were lower in imbibed seeds of Cvi compared with Col. In addition, changes in IAA and JA were initiated before 1 h, whereas ABA and JA-Ile declined 3 h after the onset of imbibition. An increase in GA₄ and iP appeared to be correlated temporally with the initiation of secondary water uptake, which marks the completion of germination.

Peptidyl-Prolyl Isomerase Activity in Chloroplast Thylakoid Lumen is a Dispensable Function of Immunophilins in Arabidopsis thaliana

Ingelsson, B., Shapiguzov, A., Kieselbach, T., Vener, A. V. — Thu, 08 Oct 2009 08:39:56 PDT

Chloroplast thylakoid lumen of Arabidopsis thaliana contains 16 immunophilins, five cyclophilins and 11 FK506-binding proteins (FKBPs), which are considered protein folding catalysts, although only two of them, AtFKBP13 and AtCYP20-2, possess peptidyl–prolyl cis/trans isomerase (PPIase) activity. To address the question of the physiological significance of this activity, we obtained and characterized Arabidopsis mutants deficient in the most active PPIase, AtFKBP13, and a double mutant deficient in both AtFKBP13 and AtCYP20-2. Two-dimensional gel electrophoresis of isolated thylakoid lumen, as well as immunoblotting analyses of major photosynthetic membrane protein complexes did not reveal differences in protein composition between the mutants and the wild type. No changes in the relative content of photosynthetic proteins were found by differential stable isotope labeling and liquid chromatography–mass spectrometry (LC-MS) analyses. PPIase activity was measured in vitro in isolated thylakoid lumen samples using two different synthetic peptide substrates. Depending on the peptide substrate used for the assay, the PPIase activity in the thylakoid lumen of the mutants lacking either AtFKBP13 or both AtFKBP13 and AtCYP20-2 was as low as 10 or 2% of that in the wild type. Residual PPIase activity detected in the double mutant originated from AtCYP20-3, a cyclophilin from chloroplast stroma contaminating thylakoid lumen preparations. None of the mutants differed from the wild-type plants when grown under normal, cold stress or high light conditions. It is concluded that cellular functions of immunophilins in the thylakoid lumen of chloroplasts are not related to their PPIase capacity and should be investigated beyond this enzymatic activity.

The Involvement of Dual Mechanisms of Photoinactivation of Photosystem II in Capsicum annuum L. Plants

Oguchi, R., Terashima, I., Chow, W. S. — Thu, 08 Oct 2009 08:39:57 PDT

For plants, light is an indispensable resource. However, it also causes a loss of photosynthetic activity associated with photoinactivation of photosystem II (PSII). In studies of the mechanism of this photoinactivation, there are two conflicting hypotheses at present. One is that excess energy received by leaves, being neither utilized by photosynthesis nor dissipated safely in non-photochemical quenching, causes the photoinactivation. The other involves a two-step mechanism in which excitation of Mn by photons is the primary cause. In the former hypothesis, photoinactivation of PSII should not occur in low light that provides little excess energy, but in the latter hypothesis it should. Therefore, we tested these two hypotheses in different irradiances. We used a system that can measure the fraction of functional PSII complexes under natural conditions and over a long period in intact leaves, which were attached to a plant treated with lincomycin taken up via the roots. The leaves were photoinactivated in low, medium or high light (30, 60 or 950 µmol m^–2 s^–1) with white, blue, green or red light-emitting diode arrays. Our results showed that the extent of photoinactivation per photon exposure was higher in high light than in low light, consistent with the abundance of excess energy. However, photoinactivation did occur in low light with little excess energy, and blue light caused the greatest extent of photoinactivation followed by white, green and red light in this order, an order that can be predicted from the Mn absorbance spectrum. These results suggest that both mechanisms occur in the photoinactivation process.

New Dynamics in an Old Friend: Dynamic Tubular Vacuoles Radiate Through the Cortical Cytoplasm of Red Onion Epidermal Cells

Wiltshire, E. J., Collings, D. A. — Thu, 08 Oct 2009 08:39:57 PDT

The textbook image of the plant vacuole sitting passively in the centre of the cell is not always correct. We observed vacuole dynamics in the epidermal cells of red onion (Allium cepa) bulbs, using confocal microscopy to detect autofluorescence from the pigment anthocyanin. The central vacuole was penetrated by highly mobile transvacuolar strands of cytoplasm, which were also visible in concurrent transmitted light images. Tubular vacuoles also extended from the large central vacuole and radiated through the cortical cytoplasm. These tubules were thin, having a diameter of about 1.5 µm, and were connected to the central vacuole as shown by fluorescence recovery after photobleaching (FRAP) experiments. The tubules were bounded by the tonoplast, as revealed by transient expression of green fluorescent protein (GFP) targeted to the vacuolar membrane and through labeling with the dye MDY-64. Expression of endoplasmic reticulum-targeted GFP demonstrated that the vacuolar tubules were distinct from the cortical endoplasmic reticulum. Movement of the tubular vacuoles depended on actin microfilaments, as microfilament disruption blocked tubule movement and caused their collapse into minivacuoles. The close association of the tubules with GFP-tagged actin microfilaments suggests that the tubules are associated with myosin, and that tubules likely move along microfilaments. Tubular vacuoles do not require anthocyanin for their formation, as tubules were also present in white onion cells that lack anthocyanin. The function of these tubular vacuoles remains unknown, but as they greatly increase the surface area of the tonoplast, they might increase transport rates between the cytoplasm and vacuole.

Antisense Expression of Mitochondrial ATP Synthase Subunits OSCP (ATP5) and {gamma} (ATP3) Alters Leaf Morphology, Metabolism and Gene Expression in Arabidopsis

Robison, M. M., Ling, X., Smid, M. P. L., Zarei, A., Wolyn, D. J. — Thu, 08 Oct 2009 08:39:57 PDT

Determination of the role of mitochondrial (mt) ATP synthesis in plant metabolism is complicated by chloroplastic ATP synthesis. To differentiate ATP synthesis from these two organelles, we created transgenic Arabidopsis plants in which two different subunits of the mt ATP synthase, the oligomycin sensitivity-conferring protein (OSCP) (=) (ATP5) and the (ATP3) subunit, were expressed individually in antisense orientation under the control of a dexamethasone-inducible promoter. The phenotypic effects of antisense expression were identical for both atp5 and atp3. Seedling lethality resulted from induction during germination in the light, demonstrating the essentiality of both gene products. Reduced expression of either gene resulted in stunting of dark-grown (etiolated) seedlings, downward curling or wavy-edged leaf margins of light-grown plants and ball-shaped unexpanded flowers. Antisense induction reduced total ATP levels in dark-grown (etiolated) seedlings germinated on media lacking sucrose, but increased total ATP levels in induced light-grown plants and in induced dark-grown seedlings germinated on media containing sucrose. Induction reduced transcript levels for two transcription factors (TCP3 and TCP4) whose decreased expression is associated with a similar wavy-edged leaf phenotype in Arabidopsis, and increased transcript levels for dynamin-related proteins whose increased expression is associated with increased mt division. Reduced expression of these subunits of the mt ATP synthase is proposed to disturb cellular redox states, which ultimately manifest downstream as diverse and seemingly unrelated phenotypes.

Differences in Expression of the RBCS Multigene Family and Rubisco Protein Content in Various Rice Plant Tissues at Different Growth Stages

Suzuki, Y., Nakabayashi, K., Yoshizawa, R., Mae, T., Makino, A. — Thu, 08 Oct 2009 08:39:57 PDT

Four out of five members of the RBCS multigene family (OsRBCS2–OsRBCS5) were highly expressed in leaf blades of rice (Oryza sativa L.) irrespective of plant growth stage, whereas accumulation of all RBCS mRNAs in leaf sheaths, roots and developing spikelets was quite low. A highly positive correlation was observed between total RBCS and RBCL mRNA levels and Rubisco content at their maxima, irrespective of tissue and growth stage. The results indicate that the total RBCS mRNA level may be a primary determinant for maximal Rubisco protein content and that Rubisco gene expression is well coordinated through the whole life of rice.

Alterations of Lysine Modifications on the Histone H3 N-Tail under Drought Stress Conditions in Arabidopsis thaliana

Thu, 08 Oct 2009 08:39:57 PDT

Arabidopsis Bile Acid:Sodium Symporter Family Protein 5 is Involved in Methionine-Derived Glucosinolate Biosynthesis

Tue, 08 Sep 2009 08:06:04 PDT

Glucosinolates (GSLs) are a group of plant secondary metabolites that have repellent activity against herbivore insects and pathogens, and anti-carcinogenic activity in humans. They are produced in plants of the Brassicaceae and other related families. Biosynthesis of GSLs from precursor amino acids takes place in two subcellular compartments; amino acid biosynthesis and side chain elongation occur mainly in the chloroplast, whereas the following core structure synthesis takes place in the cytosol. Although the genes encoding biosynthetic enzymes of GSLs are well known in Arabidopsis thaliana, the transporter genes responsible for translocation of biosynthetic intermediates between the chloroplast and cytosol are as yet unidentified. In this study, we identified the bile acid:sodium symporter family protein 5 (BASS5) gene in Arabidopsis as a candidate transporter gene involved in methionine-derived GSL (Met-GSL) biosynthesis by means of transcriptome co-expression analysis. Knocking out BASS5 resulted in a decrease of Met-GSLs and concomitant increase of methionine. A transient assay using fluorescence fusion proteins indicated a chloroplastic localization of BASS5. These results supported the idea that BASS5 plays a role in translocation across the chloroplast membranes of the biosynthetic intermediates of Met-GSLs.

Auxin Amidohydrolases from Brassica rapa Cleave the Alanine Conjugate of Indolepropionic Acid as a Preferable Substrate: A Biochemical and Modeling Approach

Tue, 08 Sep 2009 08:06:04 PDT

Two auxin amidohydrolases, BrIAR3 and BrILL2, from Chinese cabbage [Brassica rapa L. ssp. pekinensis (Lour.) Hanelt] were produced by heterologous expression in Escherichia coli, purified, and screened for activity towards N-(indol-3-ylacetyl)–l-alanine (IAA-Ala) and the long-chain auxin–amino acid conjugates, N-[3-(indol-3-yl)propionyl]–l-alanine (IPA-Ala) and N-[4-(indol-3-yl)butyryl]–l-alanine (IBA-Ala). IPA-Ala was shown to be the favored substrate of both enzymes, but BrILL2 was approximately 15 times more active than BrIAR3. Both enzymes cleaved IBA-Ala and IAA-Ala to a lesser extent. The enzyme kinetics were measured for BrILL2 and the obtained parameters suggested similar binding affinities for the long-chain auxin–amino acid conjugates (IPA-Ala and IBA-Ala). The velocity of the hydrolyzing reaction decreased in the order IPA-Ala > IBA-Ala > IAA-Ala. In a root growth bioassay, higher growth inhibition was caused by IPA-Ala and IBA-Ala in comparison with IAA-Ala. Neither these conjugates nor the corresponding free auxins affected the expression of the BrILL2 gene. A modeling study revealed several possible modes of IPA-Ala binding to BrILL2. Based on these results, two possible scenarios for substrate hydrolysis are proposed. In one the metal binding water is activated by the carboxyl group of the substrate itself, and in the other by a glutamate residue from the active site of the enzyme.

Estimation of the Relative Sizes of Rate Constants for Chlorophyll De-excitation Processes Through Comparison of Inverse Fluorescence Intensities

Kasajima, I., Takahara, K., Kawai-Yamada, M., Uchimiya, H. — Tue, 08 Sep 2009 08:06:04 PDT

The paper derives a simple way to calculate the linear relationships between all separable groups of rate constants for de-excitation of Chl a excitation energy. This is done by comparison of the inverse values of chlorophyll fluorescence intensities and is based on the matrix model of Kitajima and Butler and on the lake model of energy exchange among PSII centers. Compared with the outputs of earlier, similar calculations, the results presented here add some linear comparisons of the relative sizes of rate constants without the need for F₀' measurement. This enables us to regenerate the same alternative formula to calculate q_L as presented previously, in a different and simple form. The same former equation to calculate F₀' value from F_m, F_m' and F₀ values is also regenerated in our calculation system in a simple form. We also apply relaxation analysis to separate the rate constant for non-photochemical quenching (k_NPQ) into the rate constant for a fast-relaxing non-photochemical quenching (k_fast) and the rate constant for slow-relaxing non-photochemical quenching (k_slow). Changes in the sizes of rate constants were measured in Arabidopsis thaliana and in rice.

Amyloplast Division Progresses Simultaneously at Multiple Sites in the Endosperm of Rice

Yun, M.-S., Kawagoe, Y. — Tue, 08 Sep 2009 08:06:04 PDT

The amyloplast, a form of differentiated plastid, proliferates in sink tissues, where it synthesizes and stores starch granules. Little is known about the molecular mechanism for amyloplast division and development. The rice (Oryza sativa) endosperm provides an excellent model system for studying molecular mechanisms involved in amyloplast division and starch synthesis. We compared amyloplast division processes in the endosperm of wild type and a mutant of ARC5, a member of the dynamin superfamily. Plant growth and fertility of arc5 were not significantly different from the wild type. Unlike binary fission of chloroplast in the leaf, small amyloplasts in the endosperm of wild type divide simultaneously at multiple sites, generating a beads-on-a-string structure. In addition, large amyloplasts divide by budding-type division, giving rise to small amyloplasts attached to their surfaces. ARC5 and FtsZ2-1 fused to fluorescent proteins were targeted to the constriction sites in dividing amyloplasts. Both the loss of function of ARC5 and overexpression of ARC5 fusion proteins in the endosperm did not produce spherical amyloplasts with increased diameter, but produced either fused amyloplasts with thick connections or pleomorphic types, suggesting that proper stoichiometry between ARC5 and other components in the amyloplast division machinery is necessary for the completion of the late stage of amyloplast division. The size distribution of starch granules purified from arc5 was shifted to small and the starch gelatinization peak temperature was significantly higher than for wild-type starch, suggesting that amyloplast division processes have a significant effect on starch synthesis.

Involvement of C-22-Hydroxylated Brassinosteroids in Auxin-Induced Lamina Joint Bending in Rice

Tue, 08 Sep 2009 08:06:04 PDT

The rice lamina joint is ideal material for investigating the activity of brassinosteroids (BRs) and auxin because of its high sensitivity to these compounds. Using a series of rice BR biosynthetic and receptor mutants, we conducted lamina joint tests to elucidate the mechanism of cross-talk between BR and auxin signaling in lamina joint bending. In BR biosynthetic mutants d2 and brd1, which are defective in C-23 hydroxylase and C-6 oxidase, respectively, the lamina joint response to auxin was significantly higher than that of wild-type plants. The other BR-biosynthetic mutants, brd2, osdwarf4 and d11, which are defective in C-22-hydroxylated BRs, showed less or no response to auxin. These results suggest that C-22-hydroxylated BRs are involved in auxin-induced lamina joint bending. The results were supported by the observation that inhibition of the hyper-response to auxin in d2 was reduced by treatment with brassinazole, which inhibits the function of DWARF4, the C-22 hydroxylase. In d61, which is defective in OsBRI1, a possible BR receptor in rice, the bending angle of the lamina joint in response to auxin and C-22-hydroxylated 6-deoxoBRs was nearly the same as that in wild-type plants. This implies that C-22-hydroxylated BRs function in auxin signaling independently of OsBRI1. From these observations, we propose that C-22-hydroxylated BRs participate in auxin signaling via a novel OsBRI1-independent signaling pathway.

Effects of Tobacco Ethylene Receptor Mutations on Receptor Kinase Activity, Plant Growth and Stress Responses

Tue, 08 Sep 2009 08:06:04 PDT

Ethylene receptor is the first component of ethylene signaling that regulates plant growth, development and stress responses. Previously, we have demonstrated that tobacco subfamily 2 ethylene receptor NTHK1 had Ser/Thr kinase activity, and overexpression of NTHK1 caused large rosette, reduced ethylene sensitivity, and increased salt sensitivity in transgenic Arabidopsis plants. Here we found that N-box mutation in the NTHK1 kinase domain abolished the kinase activity and led to disruption of NTHK1 roles in conferring reduced ethylene sensitivity and salt sensitive response in transgenic Arabidopsis plants. However, N-box mutation had partial effects on NTHK1 regulation of rosette growth and expression of salt- and ethylene-responsive genes AtNAC2, AtERF1 and AtCor6.6. Mutation of conserved residues in the H box did not affect kinase activity, seedling growth, ethylene sensitivity or salt-induced epinasty in transgenic plants but did influence NTHK1 function in control of specific salt- and ethylene-responsive gene expression. Compared with NTHK1, the tobacco subfamily 1 ethylene receptor NtETR1 had His kinase activity and played a weak role in regulation of rosette growth, triple response and salt response. Mutation of the conserved His residue in the NtETR1 H box eliminated phosphorylation and altered the effect of Ntetr1-1 on reporter gene activity. These results imply that the Ser/Thr kinase activity of NTHK1 is differentially required for various responses, and NTHK1 plays a larger role than NtETR1.

Enhancing Sucrose Synthase Activity in Transgenic Potato (Solanum tuberosum L.) Tubers Results in Increased Levels of Starch, ADPglucose and UDPglucose and Total Yield

Tue, 08 Sep 2009 08:06:04 PDT

Sucrose synthase (SuSy) is a highly regulated cytosolic enzyme that catalyzes the conversion of sucrose and a nucleoside diphosphate into the corresponding nucleoside diphosphate glucose and fructose. To determine the impact of SuSy activity in starch metabolism and yield in potato (Solanum tuberosum L.) tubers we measured sugar levels and enzyme activities in tubers of SuSy-overexpressing potato plants grown in greenhouse and open field conditions. We also transcriptionally characterized tubers of SuSy-overexpressing and -antisensed potato plants. SuSy-overexpressing tubers exhibited a substantial increase in starch, UDPglucose and ADPglucose content when compared with controls. Tuber dry weight, starch content per plant and total yield of SuSy-overexpressing tubers increased significantly over those of control plants. In contrast, activities of enzymes directly involved in starch metabolism in SuSy-overexpressing tubers were normal when compared with controls. Transcriptomic analyses using POCI arrays and the MapMan software revealed that changes in SuSy activity affect the expression of genes involved in multiple biological processes, but not that of genes directly involved in starch metabolism. These analyses also revealed a reverse correlation between the expressions of acid invertase and SuSy-encoding genes, indicating that the balance between SuSy- and acid invertase-mediated sucrolytic pathways is a major determinant of starch accumulation in potato tubers. Results presented in this work show that SuSy strongly determines the intracellular levels of UDPglucose, ADPglucose and starch, and total yield in potato tubers. We also show that enhancement of SuSy activity represents a useful strategy for increasing starch accumulation and yield in potato tubers.

Oxygen Sensitivity of a Nitrogenase-like Protochlorophyllide Reductase from the Cyanobacterium Leptolyngbya boryana

Yamamoto, H., Kurumiya, S., Ohashi, R., Fujita, Y. — Tue, 08 Sep 2009 08:06:04 PDT

Dark-operative protochlorophyllide (Pchlide) oxido-reductase (DPOR) is a nitrogenase-like enzyme that catalyzes Pchlide reduction, the penultimate step of chlorophyll a biosynthesis. DPOR is distributed widely among oxygenic phototrophs such as cyanobacteria, green algae and gymnosperms. To determine how DPOR operates in oxygenic photosynthetic cells, we constructed two shuttle vectors for overexpression of Strep-tagged L-protein (ChlL) and Strep-tagged NB-protein (ChlN–ChlB) in Leptolyngbya boryana (formerly Plectonema boryanum) and introduced them into mutants lacking chlL and chlB. Both transformants restored the ability to produce chlorophyll in the dark. The DPOR activity was reconstituted by L-protein and NB-protein purified from the transformants under anaerobic conditions. L-protein activity disappeared within 5 min of exposure to air while NB-protein activity persisted for >30 min in an aerobic condition, indicating that the L-protein of DPOR components is the primary target of oxygen in cyanobacterial cells. These results suggested that the DPOR from an oxygenic photosynthetic organism did not acquire oxygen tolerance during evolution; but that the cyanobacterial cell developed a mechanism to protect DPOR from oxygen.

Is the Photosystem II Complex a Monomer or a Dimer?

Watanabe, M., Iwai, M., Narikawa, R., Ikeuchi, M. — Tue, 08 Sep 2009 08:06:04 PDT

It is widely believed that the photosystem II (PSII) complex may function as a dimer in the thylakoid membrane. Here, we report experimental conversion from the monomeric PSII to the dimeric form by treatment with high concentrations of n-dodecyl-β-d-maltopyranoside (DM). The content of the PSII monomer in a PsbTc deletion mutant was much higher than in the wild type when solubilized with low concentrations of DM. However, upon treatment with higher concentrations of DM, the PSII dimer was also recovered in the PsbTc deletion mutant. These results suggest that there are at least two distinct processes of dimerization: (i) PsbTc dependent and (ii) DM induced. We discuss the results with regard to the native assembly form(s) of PSII.

Dissection of the Relationship Between RACK1 and Heterotrimeric G-Proteins in Arabidopsis

Guo, J., Wang, S., Wang, J., Huang, W.-D., Liang, J., Chen, J.-G. — Tue, 08 Sep 2009 08:06:04 PDT

Mammalian receptor for activated C kinase 1 (RACK1) is a versatile scaffold protein, playing regulatory roles in multiple signal transduction pathways. Moreover, RACK1 interacts with the heterotrimeric G-proteins (G-proteins) and regulates some specific functions of Gβ. Although the protein sequences of both RACK1 and G-proteins are highly conserved in Arabidopsis, their relationship remains elusive. Here we provide genetic and biochemical evidence that Arabidopsis RACK1 and G-proteins may act through a mechanism that is distinct from their counterparts in mammals. Loss-of-function alleles of RACK1A (the most abundantly expressed RACK1 gene in Arabidopsis) do not appear to share morphological and developmental phenotypes with loss-of-function alleles of GPA1 (encoding the sole G in Arabidopsis) or AGB1 (encoding the sole Gβ in Arabidopsis). The analysis of gpa1 rack1a and agb1 rack1a double mutants suggested that the effect of RACK1A on morphological and developmental traits may occur independently of the presence or absence of the G-protein subunits. Although both RACK1A and G-protein subunits are negative regulators of ABA responses in the ABA inhibition of early seedling development, an additive ABA hypersensitivity was observed in gpa1 rack1a and agb1 rack1a double mutants. Biochemical analysis suggested that unlike their counterparts in mammals, RACK1 may not physically interact with AGB1. Taken together, these findings revealed some fundamental differences in the relationship of RACK1 and G-proteins between Arabidopsis and mammals.

Overexpression of the Lily p70s6k Gene in Arabidopsis Affects Elongation of Flower Organs and Indicates TOR-Dependent Regulation of AP3, PI and SUP Translation

Tzeng, T.-Y., Kong, L.-R., Chen, C.-H., Shaw, C.-C., Yang, C.-H. — Tue, 08 Sep 2009 08:06:04 PDT

The p70 ribosomal S6 kinase (p70^s6k) signaling pathway plays a key role in regulating the cell cycle via translational regulation of specific 5'TOP mRNAs. However, the function of this signaling pathway is still poorly understood in plants. Ectopic expression of the lily putative p70^s6k gene, LS6K1, resulted in up-regulation of NAP (NAC-LIKE, ACTIVATED BY AP3/PI) and PISTILLATA (PI) expression, and significantly inhibited cell expansion for petals and stamens, resulting in the male sterility phenotype in transgenic Arabidopsis. Sequence analysis revealed that the genes involved in petal and stamen development, such as APETALA3 (AP3), PI and SUPERMAN (SUP), probably encode 5'TOP mRNAs. Green fluorescent protein (GFP), fused to oligopyrimidine tract sequences that were identified in the 5'-untranslated region (UTR) of AP3, PI and SUP, was translationally regulated in human cells in response to mitogen stimulation and inhibition by the macrolide antibiotic rapamycin. Furthermore, 35S::LS6K1 significantly up-regulated β-glucuronidase (GUS) activity in the flower buds of transgenic plants carrying the GUS transgene fused to the AP3 promoter and the 5' UTR. These results have identified a novel role for the p70^s6k gene in regulating cell division and the expansion of petals and stamens by translational regulation of the 5'TOP mRNAs once ectopically expressed in Arabidopsis.

Identification of cis-Localization Elements that Target Glutelin RNAs to a Specific Subdomain of the Cortical Endoplasmic Reticulum in Rice Endosperm Cells

Washida, H., Kaneko, S., Crofts, N., Sugino, A., Wang, C., Okita, T. W — Tue, 08 Sep 2009 08:06:04 PDT

Rice glutelin RNAs are localized to the cisternal endoplasmic reticulum (ER) by a regulated RNA transport process requiring specific cis-localization elements. We set out to identify these glutelin sequences by their dominant character of being able to re-direct the normal protein body ER localization of a maize 10 kDa -zein RNA to the cisternal ER. In situ RNA localization analysis showed that the glutelin RNA contains multiple cis-localization elements; two located at the 5' and 3' ends of the coding sequences and a third located within the 3'-untranslated region. These three regions contain two conserved sequences, suggesting that these RNA recognition signals may be sequence based.

Transcriptome Analyses Revealed Diverse Expression Changes in ago1 and hyl1 Arabidopsis Mutants

Tue, 08 Sep 2009 08:06:04 PDT

MicroRNAs (miRNAs) are 20–24 nucleotide endogenous regulatory molecules conserved in higher eukaryotes. In Arabidopsis, miRNAs are produced through step-wise cleavages of primary miRNA precursors (pri-miRNAs) by DICER-LIKE1 (DCL1). This cleavage step is also supported by a double-stranded RNA-binding protein, HYPONASTIC LEAVES1 (HYL1). In many cases, mature miRNA is predominantly incorporated into an endonuclease, ARGONAUTE1 (AGO1), which degrades miRNA-targeted mRNAs. Here, we examined and revealed whole genome transcriptomes in ago1-25 and hyl1-2 mutants using tiling arrays. The data in this paper are valuable for understanding the relationship between the miRNA pathway and its effect on transcriptomes.

The Arabidopsis 26S Proteasome Subunit RPN1a is Required for Optimal Plant Growth and Stress Responses

Wang, S., Kurepa, J., Smalle, J. A. — Tue, 08 Sep 2009 08:06:05 PDT

The current literature offers contradictory results regarding the role of the proteasome subunit RPN1a in Arabidopsis development. Here we show that plants lacking RPN1a are viable and have increased cell sizes, decreased heat shock tolerance, increased oxidative stress tolerance and other phenotypes characteristic for 26S proteasome subunit mutants. These results strengthen our contention that most of the phenotypes of 26S proteasome mutants in Arabidopsis described to date reflect a general impairment in 26S proteasome function rather than a specific defect of a single subunit, and suggest that the role of the RPN1a subunit during embryogenesis needs to be reconsidered.