Ovule-specific MADS-box proteins have conserved protein-protein interactions in monocot and dicot plants

OsMADS13 is a rice MADS-box gene that is specifically expressed in developing ovules. The amino acid sequence of OsMADS13 shows 74% similarity to those of FLORAL BINDING PROTEIN 7 (FBP7) and FBP11, the products of two MADS-box genes that are necessary and sufficient to determine ovule identity in Petunia. To assess whether OsMADS13, the putative rice ortholog of FBP7 and FBP11, has an equivalent function, several analyses were performed. Ectopic expression of FBP7 and FBP11 in Petunia results in ectopic ovule formation on sepals and petals. Here we show that ectopic expression of OsMADS13 in rice and Arabidopsis does not result in the formation of such structures. Furthermore, ectopic expression of FBP7 and FBP11 in Arabidopsis also fails to induce ectopic ovule formation. To determine whether protein-protein interactions involving putative class D MADS-box proteins have been conserved, yeast two-hybrid assays were performed. These experiments resulted in the identification of three putative partners of OsMADS13, all of them encoded by AGL2-like genes. Interestingly the Petunia FBP7 protein also interacts with AGL2-like proteins. The evolutionary conservation of the MADS-box protein partners of these ovule-specific factors was confirmed by exchange experiments which showed that the protein partners of OsMADS13 interact with FBP7 and vice versa.     

Analogues of auxin modifying growth and development of some monocot and dicot plants

The dependence of morphogenetic processes in the formation of vegetative and generative organs in spring oilseed rape and barley on exogenously applied physiological analogues of auxin: 2,4-D (2,4-dichlorphenoxyacetic acid), NAA (naphthalene-1-acetic acid), TA-12 (1-[2-chloroethoxycarbonyl-methyl]-4-naphthalenesulfonic acid calcium salt) and TA-14 (1-[2-dimethylaminoethoxicarbonylmethyl]naphtalene chlormethylate) were investigated. The experiments were performed with hypocotyl tissue cultures of oilseed rape and barley microspores in vitro. The auxin analogues applied revealed differences of morphogenetic competence in dedifferentiation-redifferentiation processes that occurred in oilseed rape cultures. TA-12 and TA-14 applied together with NAA and BA (6-benzylaminopurine) caused more intensive callus growth in comparison with 2,4-D. Rhizogenesis was induced when 2,4-D was substituted by TA-12. Compound TA-14, unlike TA-12, facilitated the appearance and development of cotyledons in callus tissues. Hower the compounds TA-12 and TA-14 have no positive effect in monocot plant — barly anther culture for callogenesis and regeneration in comparison to indole-3-acetic acid (IAA). TA-14 and TA-12 showed similar but not identical auxin properties and demonstrated high efficiency as modifiers of rape-dicot plant growth and morphogenesis.     

Siderophores of Pseudomonas putida as an iron source for dicot and monocot plants

Iron uptake from ferrated (⁵⁹Fe) pseudobactin (PSB), a Pseudomonas putida siderophore, by various plant species was studied in nutrient solution culture under short term (10 h) and long term (3 weeks) conditions. In the short term experiments, ⁵⁹Fe uptake rate from ⁵⁹FePSB by dicots (peanuts, cotton and sunflower) was relatively low when compared with ⁵⁹Fe uptake rate from ⁵⁹FeEDDHA. Iron uptake rate from ⁵⁹FePSB was pH and concentration dependent, as was the Fe uptake rate from ⁵⁹FeEDDHA. The rate was about 10 times lower than that of Fe uptake from the synthetic chelate. Results were similar for long term experiments.Monocots (sorghum) in short term experiments exhibited significantly higher uptake rate of Fe from FePSB than from FeEDDHA. In long term experiments, FePSB was less efficient than FeEDDHA as an Fe source for sorghum at pH 6, but the same levels of leaf chlorophyll concentration were obtained at pH 7.3.Fe uptake rates by dicots from the siderophore and FeEDDHA were found to correlate with Fe reduction rates and reduction potentials (E₀) of both chelates. Therefore, it is suggested that the reduction mechanism governs the Fe uptake process from PSB by dicots. Further studies will be conducted to determine the role of pH in Fe aquisition from PSB by monocots.     

Effects of secondary structure on pre-mRNA splicing: hairpins sequestering the 5'' but not the 3'' splice site inhibit intron processing in Nicotiana plumbaginifolia.

We have performed a systematic study of the effect of artificial hairpins on pre-mRNA splicing in protoplasts of a dicot plant, Nicotiana plumbaginifolia. Hairpins with a potential to form 18 or 24 bp stems strongly inhibit splicing when they sequester the 5' splice site or are placed in the middle of short introns. However, similar 24 bp hairpins sequestering the 3' splice site do not prevent this site from being used as an acceptor. Utilization of the stem-located 3' site requires that the base of the stem is separated from the upstream 5' splice site by a minimum of approximately 45 nucleotides and that another 'helper' 3' splice site is present downstream of the stem. The results indicate that the spliceosome or factors associated with it may have a potential to unfold secondary structure present in the downstream portion of the intron, prior to or at the step of the 3' splice site selection. The finding that the helper 3' site is required for utilization of the stem-located acceptor confirms and extends previous observations, obtained with HeLa cell in vitro splicing systems, indicating that the 3' splice site may be recognized at least twice during spliceosome assembly.     

Molecular analysis of dicot and monocot small nuclear RNA populations.

Oligonucleotides directed against conserved small nuclear RNA (snRNA) sequences have been used to identify the individual U1, U2, U4, U5, and U6 snRNAs in dicot and monocot nuclei. The plant snRNA populations are significantly more heterogeneous than the mammalian or Saccharomyces cerevisiae snRNA populations. U6 snRNA exists as a single species of similar size in monocot and dicot nuclei. The abundance and molecular weights of the U1, U2, U4, and U5 snRNAs expressed in monocot and dicot nuclei are significantly different. Whereas most dicot nuclei contain one or two predominant forms of U2 snRNA and a small number of U4 snRNAs, monocot nuclei contain multiple forms of U2 snRNA ranging from 208 to 260 nucleotides and multiple forms of U4 snRNA from 159 to 176 nucleotides. Multiple forms of U1 and U5 snRNA exist in both plant groups. All prominent size variants of U1, U2, U4, and U5 snRNA identified in monocot nuclei can be immunoprecipitated with anti-trimethylguanosine antibody. We conclude that the sizes and number of snRNA molecules involved in intron excision differ considerably in dicot and monocot nuclei. In wheat nuclei, we have identified an additional U1-like RNA that is differentially expressed during development.     

A G-box motif (GCCACGTGCC) tetramer confers high-level constitutive expression in dicot and monocot plants

GUS reporter expression from 11 basal promoters (CaMV –90) with G-box cores (CACGTG) was analysed to evaluate the regulatory roles of G-box flanking sequences. While most G-box motifs exhibited some tissue preference of gene expression, a distinct tissue-specific expression was not apparent. However, one of 11 G-box sequences, the G-box 10 (GCCACGTGCC) tetramer, conferred a high-level constitutive expression in seed, root, leaf, axillary bud, almost all parts of flower buds and pollen of transgenic tobacco plants. Furthermore, the G-box 10 tetramer promoter exhibited high-level expression in transgenic dicot carrot and monocot rice. This is apparently the first report of a G-box motif conferring a high-level constitutive expression in a non-tissue-specific manner.     

A promoter from sugarcane bacilliform badnavirus drives transgene expression in banana and other monocot and dicot plants

A 1369 bp DNA fragment (Sc) was isolated from a full-length clone of sugarcane bacilliform badnavirus (ScBV) and was shown to have promoter activity in transient expression assays using monocot (banana, maize, millet and sorghum) and dicot plant species (tobacco, sunflower, canola and Nicotiana benthamiana). This promoter was also tested for stable expression in transgenic banana and tobacco plants. These experiments showed that this promoter could drive high-level expression of the -glucuronidase (GUS) reporter gene in most plant cells. The expression level was comparable to the maize ubiquitin promoter in standardised transient assays in maize. In transgenic banana plants the expression levels were variable for different transgenic lines but was generally comparable with the activities of both the maize ubiquitin promoter and the enhanced cauliflower mosaic virus (CaMV) 35S promoter. The Sc promoter appears to express in a near-constitutive manner in transgenic banana and tobacco plants. The promoter from sugarcane bacilliform virus represents a useful tool for the high-level expression of foreign genes in both monocot and dicot transgenic plants that could be used similarly to the CaMV 35S or maize polyubiquitin promoter.     

Identification and molecular characterization of a Brachypodium distachyon GIGANTEA gene: functional conservation in monocot and dicot plants

Developmental phase change and flowering transition are emerging as potential targets for biomass agriculture in recent years. The GIGANTEA (GI) gene is one of the central regulators that direct flowering promotion and phase transition. In this work, we isolated a GI gene orthologue from the small annual grass Brachypodium distachyon inbred line Bd21 (Brachypodium), which is perceived as a potential model monocot for studies on bioenergy grass species. A partial GI gene sequence was identified from a Brachypodium expressed sequence tag library, and a full-size gene (BdGI) was amplified from a Brachypodium cDNA library using specific primer sets designed through analysis of monocot GI gene sequences. The BdGI gene was up-regulated by light and cold. A circadian rhythm set by light–dark transition also regulated the expression of the BdGI gene. The deduced amino acid sequence of the BdGI protein shares higher than 70% of sequence identity with the GI proteins in monocots and Arabidopsis. In addition, the BdGI protein is constitutively targeted to the nucleus and physically interacts with the ZEITLUPE (ZTL) and CONSTITUTIVE PHOTOMORPHOGENIC 1 (COP1) proteins, like the Arabidopsis GI protein. Interestingly, heterologous expression of the BdGI gene in a GI-deficient Arabidopsis mutant rescued efficiently the late flowering phenotype. Together, our data indicate that the role of the GI gene in flowering induction is conserved in Arabidopsis and Brachypodium. It is envisioned that the GI genes of bioenergy grasses as well as Brachypodium could be manipulated to improve biomass by engineering developmental timing of phase transitions.     

Nuclear bodies and compartmentalization of pre-mRNA splicing factors in higher plants

We studied the fine structural organization of nuclear bodies in the root meristem during germination of maize and Arabidopsis thaliana using electron microscopy (EM). Cajal bodies (CBs) were observed in quiescent embryos and germinating cells in both species. The number and distribution of CBs were investigated. To characterize the nuclear splicing domains, immunofluorescence labelling with antibodies against splicing factors (U2B and m3G-snRNAs) and in situ hybridisation (with U1/U6 antisense probes) were performed combined with confocal microscopy. Antibodies specific to the Arabidopsis SR splicing factor atRSp31 were produced. AtRSp31 was detected in quiescent nuclei and in germinating cells. This study revealed an unexpected speckled nuclear organization of atRSp31 in root epidermal cells where micro-clusters of interchromatin granules were also observed by EM. Therefore, we examined the distribution of green fluorescent protein (GFP)-tagged atRSp31 in living cells after Agrobacterium -mediated transient expression. When expressed transiently, atRSp31-GFP exhibited a speckled distribution in leaf cells. Treatments with -amanitin, okadaic acid, staurosporine or heat shock induced the speckles to reorganize. Furthermore, we generated stable Arabidopsis transgenics expressing atRSp31-GFP. The distribution of the fusion protein was identical to that of endogenous atRSp31. Three-dimensional time-lapse confocal microscopy showed that speckles were highly dynamic domains over time.Communicated by P. ShawS. Docquier and P. Motte contributed equally to this work     

Stepwise assembly of a pre-mRNA splicing complex requires U-snRNPs and specific intron sequences

We have investigated the early events of pre-mRNA splicing in vitro by sucrose gradient sedimentation analysis. Time course experiments revealed the assembly, in two steps, of a large (50S) pre-mRNA splicing complex, preceded by formation of two other complexes that sediment at approximately 22S and 35S. Pre-mRNA and the intermediates and products of the in vitro splicing reaction cosediment with the 50S complex, while only pre-mRNA is associated with the 22S and 35S complexes. No splicing is observed in the absence of a 50S complex. Formation of the 50S complex requires ATP, whereas formation of the 22S and 35S complexes does not. U-snRNPs are necessary for assembly of the 35S and the 50S complexes but not for assembly of the 22S complex. Analysis with mutant substrate RNAs demonstrated that a polypyrimidine stretch near the 3' splice site and an intact 5' splice site are absolutely required for splicing complex formation.     

Promoters for pregenomic RNA of banana streak badnavirus are active for transgene expression in monocot and dicot plants

Two putative promoters from Australian banana streak badnavirus (BSV) isolates were analysed for activity in different plant species. In transient expression systems the My (2105 bp) and Cv (1322 bp) fragments were both shown to have promoter activity in a wide range of plant species including monocots (maize, barley, banana, millet, wheat, sorghum), dicots (tobacco, canola, sunflower, Nicotiana benthamiana, tipu tree), gymnosperm (Pinus radiata) and fern (Nephrolepis cordifolia). Evaluation of the My and Cv promoters in transgenic sugarcane, banana and tobacco plants demonstrated that these promoters could drive high-level expression of either the green fluorescent protein (GFP) or the -glucuronidase (GUS) reporter gene (uidA) in vegetative plant cells. In transgenic sugarcane plants harbouring the Cv promoter, GFP expression levels were comparable or higher (up to 1.06% of total soluble leaf protein as GFP) than those of plants containing the maize ubiquitin promoter (up to 0.34% of total soluble leaf protein). GUS activities in transgenic in vitro-grown banana plants containing the My promoter were up to seven-fold stronger in leaf tissue and up to four-fold stronger in root and corm tissue than in plants harbouring the maize ubiquitin promoter. The Cv promoter showed activities that were similar to the maize ubiquitin promoter in in vitro-grown banana plants, but was significantly reduced in larger glasshouse-grown plants. In transgenic in vitro-grown tobacco plants, the My promoter reached activities close to those of the 35S promoter of cauliflower mosaic virus (CaMV), while the Cv promoter was about half as active as the CaMV 35S promoter. The BSV promoters for pregenomic RNA represent useful tools for the high-level expression of foreign genes in transgenic monocots.     

Agrobacterium-mediated transformation of monocot and dicot plants using the NCR promoter derived from soybean chlorotic mottle virus

The NCR promoter (PNCR) from soybean chlorotic mottle virus (SoyCMV) was used to express the selectable marker, neomycin phosphotransferase (nptII) gene, in Agrobacterium-mediated transformation of both monocot (rice) and dicot (tobacco) plants. A multi-cloning site for insertion of a gene of interest into the binary vector pTN is located proximal to the right border region of T-DNA. When chimeric genes under the control of other strong promoters were located in a head-to-head orientation to the PNCR-nptII gene, kanamycin-resistant tobacco shoots were generated more efficiently than when using the original pTN vectors. This suggests that the enhancer-like sequences in the promoters adjacent to PNCR may promote expression of the PNCR-nptII gene. Received: 20 August 1999 / Revision received: 16 November 1999 / Accepted: 19 November 1999     

Expression and characterization of monocot rice cytosolic CuZnSOD protein in dicot Arabidopsis

Cytosolic CuZnSOD removes deleterious superoxides from plant cells. In order to understand its function better, we sought to express a monocot CuZnSODgene in transgenic Arabidopsis. We constructed a transgene usingthe CaMV 35S promoter to express a rice cytosolic CuZnSOD gene in Arabidopsis and generated over 200 transformants. A 16kD polypeptide, the same size as the native rice CuZnSOD polypeptide, was detected inthe transgenic Arabidopsis. Interestingly, two forms of riceCuZnSOD, rSODI and rSODII, having the same dimeric size, were detectedin the transgenic plants. rSODII protein was relatively abundant but hadlow specific activity. In contrast, rSODI protein was relatively rareand had high specific activity. Inter-conversion of rSODI and rSODIIcould be achieved by the addition and removal of copper ions into the purifiedrecombinant SOD and to the leaf extract of transgenic plants. Ouranalysis indicates that rSODI most likely corresponds to native riceCuZnSOD that has incorporated the Cu and Zn ions required for fullactivity, whereas the less active rSODII form may not have properlyincorporated the necessary copper ions.