tRNA Nuclear Export in Saccharomyces cerevisiae: In Situ Hybridization Analysis

To understand the factors specifically affecting tRNA nuclear export, we adapted in situ hybridization procedures to locate endogenous levels of individual tRNA families in wild-type and mutant yeast cells. Our studies of tRNAs encoded by genes lacking introns show that nucleoporin Nup116p affects both poly(A) RNA and tRNA export, whereas Nup159p affects only poly(A) RNA export. Los1p is similar to exportin-t, which facilitates vertebrate tRNA export. A los1 deletion mutation affects tRNA but not poly(A) RNA export. The data support the notion that Los1p and exportin-t are functional homologues. Because LOS1 is nonessential, tRNA export in vertebrate and yeast cells likely involves factors in addition to exportin-t. Mutation of RNA1, which encodes RanGAP, causes nuclear accumulation of tRNAs and poly(A) RNA. Many yeast mutants, including those with the rna1-1 mutation, affect both pre-tRNA splicing and RNA export. Our studies of the location of intron-containing pre-tRNAs in the rna1-1 mutant rule out the possibility that this results from tRNA export occurring before splicing. Our results also argue against inappropriate subnuclear compartmentalization causing defects in pre-tRNA splicing. Rather, the data support “feedback” of nucleus/cytosol exchange to the pre-tRNA splicing machinery.     

Polycomb-Group Proteins and FLOWERING LOCUS T Maintain Commitment to Flowering in Arabidopsis thaliana

The switch from vegetative to reproductive growth is extremely stable even if plants are only transiently exposed to environmental stimuli that trigger flowering. In the photoperiodic pathway, a mobile signal, florigen, encoded by FLOWERING LOCUS T (FT) in Arabidopsis thaliana, induces flowering. Because FT activity in leaves is not maintained after transient photoperiodic induction, the molecular basis for stable floral commitment is unclear. Here, we show that Polycomb-group (Pc-G) proteins, which mediate epigenetic gene regulation, maintain the identity of inflorescence and floral meristems after floral induction. Thus, plants with reduced Pc-G activity show a remarkable increase of cauline leaves under noninductive conditions and floral reversion when shifted from inductive to noninductive conditions. These phenotypes are almost completely suppressed by loss of FLOWERING LOCUS C (FLC) and SHORT VEGETATIVE PHASE, which both delay flowering and promote vegetative shoot identity. Upregulation of FLC in Pc-G mutants leads to a strong decrease of FT expression in inflorescences. We find that this activity of FT is needed to prevent floral reversion. Collectively, our results reveal that floral meristem identity is at least partially maintained by a daylength-independent role of FT whose expression is indirectly sustained by Pc-G activity.     

HEN1 functions pleiotropically in Arabidopsis development and acts in C function in the flower

Four classes of floral homeotic MADS domain proteins specify the identities of the four organ types in an Arabidopsis flower. While the activities of the MADS domain proteins are essentially confined to the flower or to the inflorescence, several genes, such as APETALA2, HUA1 and HUA2, also act outside the flower in addition to their organ identity functions inside the flower. We identified a new gene, HUA ENHANCER 1 (HEN1) from a sensitized genetic screen in the hua1-1 hua2-1 background that is compromised in floral homeotic C function. We showed that HEN1, like the C function gene AGAMOUS, acts to specify reproductive organ identities and to repress A function. HEN1 also shares AG's non-homeotic function in controlling floral determinacy. HEN1 may achieve these functions by regulating the expression of AG. hen1 single mutants exhibit pleiotropic phenotypes such as reduced organ size, altered rosette leaf shape and increased number of coflorescences, during most stages of development. Therefore, HEN1, like the A function gene AP2, plays multiple roles in plant development as well as acting in organ identity specification in the flower. HEN1 codes for a novel protein and is expressed throughout the plant.     

MOSAIC FLORAL ORGANS1, an AGL6-Like MADS Box Gene,Regulates Floral Organ Identity and Meristem Fate in Rice

Floral organ identity and meristem determinacy in plants are controlled by combinations of activities mediated by MADS box genes. AGAMOUS-LIKE6 (AGL6)-like genes are MADS box genes expressed in floral tissues, but their biological functions are mostly unknown. Here, we describe an AGL6-like gene in rice (Oryza sativa), MOSAIC FLORAL ORGANS1 (MFO1/MADS6), that regulates floral organ identity and floral meristem determinacy. In the flower of mfo1 mutants, the identities of palea and lodicule are disturbed, and mosaic organs were observed. Furthermore, the determinacy of the floral meristem was lost, and extra carpels or spikelets developed in mfo1 florets. The expression patterns of floral MADS box genes were disturbed in the mutant florets. Suppression of another rice AGL6-like gene, MADS17, caused no morphological abnormalities in the wild-type background, but it enhanced the phenotype in the mfo1 background, indicating that MADS17 has a minor but redundant function with that of MFO1. Whereas single mutants in either MFO1 or the SEPALLATA-like gene LHS1 showed moderate phenotypes, the mfo1 lhs1 double mutant showed a severe phenotype, including the loss of spikelet meristem determinacy. We propose that rice AGL6-like genes help to control floral organ identity and the establishment and determinacy of the floral meristem redundantly with LHS1.     

Phosphatidic acid is a major phospholipid class in reproductive organs of Arabidopsis thaliana

Phospholipids are the crucial components of biological membranes and signal transduction. Among different tissues, flower phospholipids are one of the least characterized features of plant lipidome. Here, we report that floral reproductive organs of Arabidopsis thaliana contain high levels of phosphatidic acid (PA), a known lipid second messenger. By using floral homeotic mutants enriched with specific floral organs, lipidomics study showed increased levels of PA species in ap3-3 mutant with enriched pistils. Accompanied gene expression study for 7 diacylglycerol kinases and 11 PA phosphatases revealed distinct floral organ specificity, suggesting an active phosphorylation/dephosphorylation between PA and diacylglycerol in flowers. Our results suggest that PA is a major phospholipid class in floral reproductive organs of A. thaliana.     

Regulation of flower development in Arabidopsis by SCF complexes

SCF complexes are the largest and best studied family of E3 ubiquitin protein ligases that facilitate the ubiquitylation of proteins targeted for degradation. The SCF core components Skp1, Cul1, and Rbx1 serve in multiple SCF complexes involving different substrate-specific F-box proteins that are involved in diverse processes including cell cycle and development. In Arabidopsis, mutations in the F-box gene UNUSUAL FLORAL ORGANS (UFO) result in a number of defects in flower development. However, functions of the core components Cul1 and Rbx1 in flower development are poorly understood. In this study we analyzed floral phenotypes caused by altering function of Cul1 or Rbx1, as well as the effects of mutations in ASK1 and ASK2. Plants homozygous for a point mutation in the AtCUL1 gene showed reduced floral organ number and several defects in each of the four whorls. Similarly, plants with reduced AtRbx1 expression due to RNA interference also exhibited floral morphological defects. In addition, compared to the ask1 mutant, plants homozygous for ask1 and heterozygous for ask2 displayed enhanced reduction of B function, as well as other novel defects of flower development, including carpelloid sepals and an inhibition of petal development. Genetic analyses demonstrate that AGAMOUS (AG) is required for the novel phenotypes observed in the first and second whorls. Furthermore, the genetic interaction between UFO and AtCUL1 supports the idea that UFO regulates multiple aspects of flower development as a part of SCF complexes. These results suggest that SCF complexes regulate several aspects of floral development in Arabidopsis.     

Live-imaging provides an atlas of cellular growth dynamics in the stamen

Development of multicellular organisms is a complex process involving precise coordination of growth among individual cells. Understanding organogenesis requires measurements of cellular behaviors over space and time. In plants, such a quantitative approach has been successfully used to dissect organ development in both leaves and external floral organs, such as sepals. However, the observation of floral reproductive organs is hampered as they develop inside tightly closed floral buds, and are therefore difficult to access for imaging. We developed a confocal time-lapse imaging method, applied here to Arabidopsis (Arabidopsis thaliana), which allows full quantitative characterization of the development of stamens, the male reproductive organs. Our lineage tracing reveals the early specification of the filament and the anther. Formation of the anther lobes is associated with a temporal increase of growth at the lobe surface that correlates with intensive growth of the developing locule. Filament development is very dynamic and passes through three distinct phases: (1) initial intense, anisotropic growth, and high cell proliferation; (2) restriction of growth and proliferation to the filament proximal region; and (3) resumption of intense and anisotropic growth, displaced to the distal portion of the filament, without cell proliferation. This quantitative atlas of cellular growth dynamics provides a solid framework for future studies into stamen development.     

Suppression of late-flowering and semi-dwarf phenotypes in the arabidopsis clock mutant lhy-12;cca1-101 by phyB under continuous light

Photoperiodic flowering in Arabidopsis is controlled not only by floral activators such as GI, CO and FT, but also by repressors such as SVP and FLC. Double mutations in LHY and CCA1 (lhy;cca1) accelerated flowering under short days, mainly by the GI-CO dependent pathway. In contrast, lhy;cca1 showed delayed flowering under continuous light (LL), probably due to the GI-CO independent pathway. This late-flowering phenotype was suppressed by svp, flc and elf3. However, how SVP, FLC and ELF3 mediate LHY/CCA1 and flowering time is not fully understood. We found that lhy;cca1 exhibited short hypocotyls and petioles under LL, but the molecular mechanism for these effects has not been elucidated.To address these questions, we performed a screen for mutations that suppress either or both of the lhy;cca1 phenotypes under LL, using two different approaches. We identified two novel mutations, a dominant (del1) and a recessive (phyB-2511) allele of phyB. The flowering times of single mutants of three phyB alleles, hy3-1, del1 and phyB-2511, are almost the same and earlier than those of wild-type plants. A similar level of acceleration of flowering time was observed in all three phyB mutants tested when combined with the late-flowering mutations co-2 and SVPox. However, the effect of phyB-2511 on lhy;cca1 was different from those by hy3-1 or del1. svp-3 did not strongly enhance the early-flowering phenotypes of phyB-2511 or del1. These results suggest that light signaling via PhyB may affect factors downstream of the clock proteins, controlling flowering time and organ elongation. phyB mutations with different levels of effects on lhy;cca1-dependent late flowering would be useful to determine a specific role for PHYB in the flowering pathway controlled by lhy;cca1 under LL.Key words: Arabidopsis thaliana, CCA1, circadian clock, CO, FT, LHY, organ elongation, photoperiodic flowering, PHYB, SVP     

OsPIE1, the Rice Ortholog of Arabidopsis PHOTOPERIOD-INDEPENDENT EARLY FLOWERING1, Is Essential for Embryo Development

Background

The SWR1 complex is important for the deposition of histone variant H2A.Z into chromatin necessary to robustly regulate gene expression during growth and development. In Arabidopsis thaliana, the catalytic subunit of the SWR1-like complex, encoded by PIE1 (PHOTOPERIOD-INDEPENDENT EARLY FLOWERING1), has been shown to function in multiple developmental processes including flowering time pathways and petal number regulation. However, the function of the PIE1 orthologs in monocots remains unknown.

Methodology/Findings

We report the identification of the rice (Oryza sativa) ortholog, OsPIE1. Although OsPIE1 does not exhibit a conserved exon/intron structure as Arabidopsis PIE1, its encoded protein is highly similar to PIE1, sharing 53.9% amino acid sequence identity. OsPIE1 also has a very similar expression pattern as PIE1. Furthermore, transgenic expression of OsPIE1 completely rescued both early flowering and extra petal number phenotypes of the Arabidopsis pie1-2 mutant. However, homozygous T-DNA insertional mutants of OsPIE1 in rice were embryonically lethal, in contrast to the viable mutants in the orthologous genes for yeast, Drosophila and Arabidopsis (Swr1, DOMINO and PIE1, respectively).

Conclusions/Significance

Taken together, our results suggest that OsPIE1 is the rice ortholog of Arabidopsis PIE1 and plays an essential role in rice embryo development.     

Allele-specific effects of PDF2 on floral morphology in Arabidopsis thaliana

The class IV Homeodomain-leucine zipper (HD-ZIP IV) gene family includes several genes that are functionally significant in epidermal development. Our recent study revealed that double mutants of the epidermis-expressed HD-ZIP IV members, PROTODERMAL FACTOR2 (PDF2) in combination with some HOMEODOMAIN GLABROUS (HDG, pronounced “hedge”) genes, affect stamen development and specification of petal and stamen identity, possibly in a non cell-autonomous manner. However, the effect of the pdf2 mutations on the floral development was largely different depending on T-DNA insertion locations: pdf2–1 hdg flowers exhibited homeotic conversion of petals and stamens, while pdf2–2 hdg flowers had only a reduced number of stamens. Here, we used 2 additional pdf2 alleles to make double mutants and found that their floral phenotypes were rather similar to those of pdf2–2 hdg. The allele-specific effect caused by pdf2–1, which carries a T-DNA in a steroidogenic acute regulatory protein-related lipid transfer (START) domain-encoding region, suggests the importance of the START domain in proper function of HD-ZIP IV proteins.     

Arabidopsis (Brassicaceae) flower development and gynoecium patterning in wild type and Ettin mutants

Screening for mutations that alter flower development in Arabidopsis has led to the identification of two general types of genetic loci: those affecting meristem and organ identity, and those affecting growth and development independent of identity. ettin (ett) mutants belong to the latter class and exhibit pleiotropic phenotypes distinct from previously described Arabidopsis mutants. These phenotypes include increases in sepal and petal number, decreases in stamen number and anther locule number, and gross alteration of tissue patterning in the gynoecium. To determine when and how differences in ett floral meristems originate, flower development was compared between the wild type and ett mutants. ett floral meristems exhibit increases in abaxial sepal and petal primordia number without apparent increases in meristem size. Extra sepal and petal primordia develop into normal organs. In contrast, stamen and carpel primordia exhibit alterations in shape and form, subsequent to premature elongation of the terminal floral meristem. Phenotypes are allele-strength dependent. The stigma develops precociously and style differentiation is basally and abaxially misplaced in ett gynoecia. The data are discussed in the context of a model suggesting that two concentric boundaries specify the apical-basal pattern of gynoecium differentiation.     

On the origin of class B floral homeotic genes: functional substitution and dominant inhibition in Arabidopsis by expression of an orthologue from the gymnosperm Gnetum

Class B floral homeotic genes are involved in specifying stamen and petal identity in angiosperms (flowering plants). Here we report that gymnosperms, the closest relatives of the angiosperms, contain at least two different clades representing putative orthologues of class B genes, termed GGM2-like and DAL12-like genes. To obtain information about the functional conservation of the class B genes in seed plants, the representative of one of these clades from Gnetum, termed GGM2, was expressed under the control of the CaMV 35S promoter in Arabidopsis wild-type plants and in different class B mutants. In wild-type plants and in a conditional mutant grown at a permissive temperature, gain-of-function phenotypes were obtained in whorls 1 and 4, where class B genes are usually not expressed. In contrast, loss-of-function phenotypes were observed in whorls 2 and 3, where class B genes are expressed. In different class B gene null mutants of Arabidopsis, and in the conditional B mutant grown at the non-permissive temperature, a partial complementation of the mutant phenotype was obtained. In situ hybridization studies and class B gene promoter test fusion experiments demonstrated that the gain-of-function phenotypes are not due to an upregulation of the endogenous B genes from Arabidopsis, and hence probably involve interactions between GGM2 protein homodimers and class B protein target genes other than the Arabidopsis class B genes itself. To our knowledge, this is the first time that partial complementation of a homeotic mutant by an orthologous gene from a distantly related species has been reported. These data suggest that GGM2 has a function in the gymnosperm Gnetum which is related to that of class B floral organ identity genes of angiosperms. That function may be in the specification of male reproductive organ identity, and in distinguishing male from female reproductive organs.     

Increased tRNA level in yeast cells with mutant translation termination factors eRF1 and eRF3

We have earlier characterized Saccharomyces cerevisiae strains with mutations of essential SUP45 and SUP35, which code for translation termination factors eRF1 and eRF3, respectively. In this work, the sup45 and sup35 nonsense mutants were compared with respect to the levels of eight tRNAs: tRNA^Tyr, tRNA^Gln, tRNA^Trp, tRNA^Leu, tRNA^Arg (described as potential suppressor tRNAs), tRNA^Pro, tRNA^His, and tRNA^Gly. The mutants did not display a selective increase in tRNAs, capable of a noncanonical read-through at stop codons. Most of the mutations increased the level of all tRNAs under study. The mechanisms providing for the viability of the sup45 and sup35 nonsense mutants are discussed.     

Mutations associated with floral organ number in rice

How floral organ number is specified is an interesting subject and has been intensively studied in Arabidopsis thaliana. In rice (Oryza sativa L.), mutations associated with floral organ number have been identified. In three mutants of rice, floral organ number 1 (fon1) and the two alleles, floral organ number 2-1 (fon2-1) and floral organ number 2-2 (fon2-2), the floral organs were increased in number centripetally. Lodicules, homologous to petals, were rarely affected, and stamens were frequently increased from six to seven or eight. Of all the floral organs the number of pistils was the most frequently increased. Among the mutants, fon1 showed a different spectrum of organ number from fon2 -1 and fon2 -2. Lodicules were the most frequently affected in fon1, but pistils of more than half of fon1 flowers were unaffected; in contrast, the pistils of most flowers were increased in fon2 -1 and fon2-2. Homeotic conversion of organ identity was also detected at a low frequency in ectopically formed lodicules and stamens. Lodicules and stamens were partially converted into anthers and stigmas, respectively. Concomitant with the increased number of floral organs, each mutant had an enlarged apical meristem. Although meristem size was comparable among the three mutants and wild type in the early phase of flower development, a significant difference became apparent after the lemma primordium had differentiated. In these mutants, the size of the shoot apical meristem in the embryo and in the vegetative phase was not affected, and no phenotypic abnormalities were detected. These results do not coincide with those for Arabidopsis in which clavatal affects the sizes of both shoot and floral meristems, leading to abnormal phyllotaxis, inflorescence fasciation and increased floral organs. Accordingly, it is considered that FON1 and FON2 function exclusively in the regulation of the floral meristem, not of the vegetative meristem.Abbreviation DIC differential interference contrast This work was supported in part by Grant-in-Aid for Scientific Research on Priority Areas from the Ministry of Education, Science and Culture of Japan.     

PORPHOBILINOGEN DEAMINASE Deficiency Alters Vegetative and Reproductive Development and Causes Lesions in Arabidopsis

The Arabidopsis rugosa1 (rug1) mutant has irregularly shaped leaves and reduced growth. In the absence of pathogens, leaves of rug1 plants have spontaneous lesions reminiscent of those seen in lesion-mimic mutants; rug1 plants also express cytological and molecular markers associated with defence against pathogens. These rug1 phenotypes are made stronger by dark/light transitions. The rug1 mutant also has delayed flowering time, upregulation of the floral repressor FLOWERING LOCUS C (FLC) and downregulation of the flowering promoters FT and SOC1/AGL20. Vernalization suppresses the late flowering phenotype of rug1 by repressing FLC. Microarray analysis revealed that 280 nuclear genes are differentially expressed between rug1 and wild type; almost a quarter of these genes are involved in plant defence. In rug1, the auxin response is also affected and several auxin-responsive genes are downregulated. We identified the RUG1 gene by map-based cloning and found that it encodes porphobilinogen deaminase (PBGD), also known as hydroxymethylbilane synthase, an enzyme of the tetrapyrrole biosynthesis pathway, which produces chlorophyll, heme, siroheme and phytochromobilin in plants. PBGD activity is reduced in rug1 plants, which accumulate porphobilinogen. Our results indicate that Arabidopsis PBGD deficiency impairs the porphyrin pathway and triggers constitutive activation of plant defence mechanisms leading to leaf lesions and affecting vegetative and reproductive development.