Turning floral organs into leaves, leaves into floral organs.

The development of the floral organs is specified by the combinations of three classes of gene for organ identity in the 'ABC' model. Recently, molecular genetic studies have shown this model is applicable to grass plants as well as most eudicots. Transcription factor complexes of ABC and homologous proteins form the molecular basis of the ABC model.

A TM3-like MADS-box gene from Eucalyptus expressed in both vegetative and reproductive tissues.

MADS-box genes in plants are a diverse class of transcription factors that are involved in regulating developmental processes, particularly meristem and organ identity during floral development. They are characterized by a highly conserved MADS-box domain of 59 amino acids that binds to specific DNA sequences. We report the characterization of a cDNA clone, ETL (Eucalyptus TM3 Like), from Eucalyptus globulus subspecies bicostata encoding a putative transcription factor of the MADS-box class that is strongly expressed in both vegetative and floral tissues, suggesting that it regulates processes other than floral development. The clone was isolated from a floral bud cDNA library with a probe generated from Eucalyptus genomic DNA by PCR using degenerate primers to the MADS-box of the floral regulatory gene APETALA 1. The ETL cDNA clone encodes a putative protein of 206 amino acids that contains an N-terminal MADS-box and a helical domain of approx. 60 amino acids predicted to form a coiled-coil (K-box). These structural features are characteristic of plant MADS-box proteins. The MADS-box domain contains all the signature residues of a class of MADS-box genes typified by the tomato gene TM3 and overall, ETL shows 56% amino acid identity to TM3. Like TM3, the ETL gene is expressed in both vegetative and reproductive organs, predominantly in root and shoot meristems and organ primordia, as well as in developing male and female floral organs.

Insect resistant rice generated by introduction of a modified delta-endotoxin gene of Bacillus thuringiensis.

As a first step towards development of insect resistant rice we have introduced a truncated delta-endotoxin gene, cryIA(b) of Bacillus thuringiensis (B.t.) which has specific biological activity against lepidopteran insects into a japonica rice. To highly express the cryIA(b) gene in rice the coding sequence was extensively modified based on the codon usage of rice genes. Transgenic plants efficiently expressed the modified cryIA(b) gene at both mRNA and protein levels. Bioassays using R2 generation plants with two major rice insect pests, striped stemborer (Chilo suppressalis) and leaffolder (Cnaphalocrosis medinalis), indicated that transgenic rice plants expressing the CryIA(b) protein are more resistant to these pests than untransformed control plants. Our results suggest that the B.t. endotoxin genes will be useful for the rational development of new rice varieties resistant to major insect pests.

Plant regeneration from protoplasts of indica rice: genotypic differences in culture response.

Fourteen varieties of indica rice (Oryza saliva L.) were examined for their capacity for plant regeneration from protoplasts using the nurse culture methods developed for japonica rice. Calli induced from germinating seeds were grouped into two types: type I, white and compact; type II, yellow and friable. In four varieties producing type II callus, colony formation (2%-4.5%) and plant regeneration (2%-35%) were observed. The inability to develop suspension cultures was a major obstacle in regenerating plants from protoplasts of the remaining rice varieties studied.

Processing followed by complete editing of an altered mitochondrial atp6 RNA restores fertility of cytoplasmic male sterile rice.

Two atp6 genes were found in the mitochondrial genome of cytoplasmic male sterile (CMS) rice carrying the [cms-bo] cytoplasm. One (N-atp6) was identical to the normal cytoplasmic gene, while the second (B-atp6) was identified as a candidate CMS gene by Southern analysis of the mitochondrial genome of CMS cybrid rice. The coding sequence of B-atp6 was identical to the normal N-atp6 gene but its 3'-flanking sequence was different starting at 49 bases downstream from the stop codon. Northern analysis showed that B-atp6 is transcribed into a 2.0 kb RNA in the absence of the Rf-1 gene, whereas two discontinuous RNAs, of approximately 1.5 and 0.45 kb, were detected in the presence of the Rf-1 gene. Determination of the 3' and 5' ends of these RNAs suggested that the two discontinuous RNAs were generated from the 2.0 kb RNA by RNA processing at sites within the B-atp6-specific sequences by the action of the Rf-1 gene. Sequence analysis of cDNA clones derived from the N-atp6 RNA and the processed and unprocessed RNAs of B-atp6 indicated that the processed B-atp6 RNAs were edited as efficiently as the N-atp6, whereas unedited and partially edited RNAs were detected among unprocessed RNAs. RNA processing by Rf-1 thus influences the sequential post-transcriptional editing of the B-atp6 RNAs. Because the unprocessed RNAs of B-atp6 are possibly translated into altered polypeptides, our results suggest that interaction of RNA processing and editing plays a role in controlling CMS expression and the restoration of fertility in rice.

Anaerobic induction and tissue-specific expression of maize Adh1 promoter in transgenic rice plants and their progeny.

In order to analyze expression of the maize alcohol dehydrogenase 1 gene (Adh1), its promoter was fused with the gusA reporter gene and introduced into rice by protoplast transformation. Histochemical analysis of transgenic plants and their progeny showed that the maize Adh1 promoter is constitutively expressed in root caps, anthers, anther filaments, pollen, scutellum, endosperm and shoot and root meristem of the embryo. Induction of expression by the Adh1 promoter was examined using seedlings derived from selfed progeny of the transgenic plants. The results showed that expression of the Adh1 promoter was strongly induced (up to 81-fold) in roots of seedlings after 24 h of anaerobic treatment, concomitant with an increase in the level of gusA mRNA. 2,4-D also induced Adh1 promoter-directed expression of gusA to a similar extent. In contrast, little induction by anaerobic treatment was detected in transformed calli, leaves or roots of primary transformants or shoots of seedlings. A detailed examination of seedling roots during anaerobic treatment revealed that the induction started first at the meristem and after 3 h there was strong induction in the elongation zone which is located 1-2 mm above the meristem; the induction then progressed upward from this region. Our results suggest that transgenic rice plants carrying the gusA reporter gene fused with promoters are useful for the study of anaerobic regulation of genes derived from graminaceous species.

The promoters of two carboxylases in a C4 plant (maize) direct cell-specific, light-regulated expression in a C3 plant (rice).

C4 plants have two carboxylases which function in photosynthesis. One, phosphoenolpyruvate carboxylase (PEPC) is localized in mesophyll cells, and the other, ribulose bisphosphate carboxylase (RuBPC) is found in bundle sheath cells. In contrast, C3 plants have only one photosynthetic carboxylase, RuBPC, which is localized in mesophyll cells. The expression of PEPC in C3 mesophyll cells is quite low relative to PEPC expression in C4 mesophyll cells. Two chimeric genes have been constructed consisting of the structural gene encoding beta-glucuronidase (GUS) controlled by two promoters from C4 (maize) photosynthetic genes: (i) the PEPC gene (pepc) and (ii) the small subunit of RuBPC (rbcS). These constructs were introduced into a C3 cereal, rice. Both chimeric genes were expressed almost exclusively in mesophyll cells in the leaf blades and leaf sheaths at high levels, and no or very little activity was observed in other cells. The expression of both genes was also regulated by light. These observations indicate that the regulation systems which direct cell-specific and light-inducible expression of pepc and rbcS in C4 plants are also present in C3 plants. Nevertheless, expression of endogenous pepc in C3 plants is very low in C3 mesophyll cells, and the cell specificity of rbcS expression in C3 plants differs from that in C4 plants. Rice nuclear extracts were assayed for DNA-binding protein(s) which interact with a cis-regulatory element in the pepc promoter. Gel-retardation assays indicate that a nuclear protein with similar DNA-binding specificity to a maize nuclear protein is present in rice.(ABSTRACT TRUNCATED AT 250 WORDS)

The LAX1 and FRIZZY PANICLE 2 genes determine the inflorescence architecture of rice by controlling rachis-branch and spikelet development.

We have analyzed two mutants that exhibit altered panicle architecture in rice (Oryza sativa L.). In lax1-2, which is a new and stronger allele of the previously reported lax mutant, initiation and/or maintenance of rachis-branches, lateral spikelets, and terminal spikelets was severely prevented. In situ hybridization analysis using OSH1, a rice knotted1 (kn1) ortholog, confirmed the absence of lateral meristems in lax1-2 panicles. These defects indicate that the LAX1 gene is required for the initiation/maintenance of axillary meristems in the rice panicle. In addition to its role in forming lateral meristems, the wild-type LAX1 gene acts as a floral meristem identity gene which specifies the terminal spikelet meristem. A comparison of the defects in lax1-1 and lax1-2 plants suggested that the sensitivities to reduced LAX1 activity were not uniform among different types of meristems. In the fzp2 mutant panicle, the basic branching pattern of the panicle was indistinguishable from that of the wild type; however, specification of both terminal and lateral spikelet meristems was blocked, and sequential rounds of branching occurred at the point where the spikelet meristems are initiated in the wild-type panicle. This resulted in the generation of a panicle composed of excessive ramification of rachis-branches. The lax1-1 fzp2 double mutants exhibited a novel, basically additive, phenotype, which suggests that LAX1 and FZP2 function in genetically independent pathways.

Spatially and temporally regulated expression of rice MADS box genes with similarity to Arabidopsis class A, B and C genes.

The floral morphology of grass species is distinct from that of typical dicot plants. In order to achieve a better understanding of the molecular basis for this diversion, we isolated RAP1A, RAP1B and RAG, putative rice orthologs for the Arabidopsis class A gene APETALA1 (AP1) and class C gene AGAMOUS (AG). The expression patterns of RAP1A, RAG and OsMADS2, a rice ortholog of the class B gene, were analyzed by in situ hybridization. RAP1A mRNA was expressed in the apical region of the floral meristem at an early stage of spikelet development, and then its expression was localized in developing lemma, palea and lodicules. The OsMADS2 transcript was first observed in the region where stamen primordia are formed. Soon after, OsMADS2 mRNA appeared in the lodicule primordia as well as the stamen primordia, and this RNA accumulation pattern persisted until late stages of floral development. The expression of RAG was observed in stamens and pistils of wild-type young spikelets. These RNA accumulation patterns are mostly similar to those of Arabidopsis class A, B, C genes, supporting the notion that the ABC model may be extended to rice.

The presequence of a precursor to the delta-subunit of sweet potato mitochondrial F1ATPase is not sufficient for the transport of beta-glucuronidase (GUS) into mitochondria of tobacco, rice and yeast cells.

A precursor to the delta-subunit of sweet potato mitochondrial F1ATPase (pre-F1 delta) has an amino-terminal (N-terminal) presequence of 45 amino acid residues and its N-terminal 18 residues may form an amphiphilic alpha-helix, which is typical of mitochondrial targeting signals [Kimura et al. (1990) J. Biol. Chem. 265: 6079]. Fusion genes consisting of sequences that encoded the 25 (DG25), 46 (DG46) and 73 (DG73) N-terminal amino acids from pre-F1 delta fused to the N-terminus of the coding sequence of bacterial beta-glucuronidase (GUS) were placed downstream of the 35S promoter of cauliflower mosaic virus and used to transform suspension-cultured tobacco cells, rice calli and tobacco plants. Fusion genes were also placed downstream of the yeast GAL10 promoter and introduced into Saccharomyces cerevisiae cells. In these transformed cells, only the DG73 GUS-fusion protein was transported into mitochondria and subjected to proteolytic cleavage of the presequence. Neither transport to mitochondria nor processing of the presequence of the DG46 GUS-fusion protein, which contained the entire presequence and the processing site, occurred in either plant or yeast cells. These results indicate that the presequence of pre-F1 delta is not sufficient for the transport of the GUS protein into mitochondria in tobacco, rice and yeast cells. The requirement for the longer polypeptide from pre-F1 delta in the transport of the GUS protein into mitochondria could be due either to the lack of sufficient information for mitochondrial targeting within the presequence or to the nature of the passenger protein, GUS, used in this study.

Light-regulated and cell-specific expression of tomato rbcS-gusA and rice rbcS-gusA fusion genes in transgenic rice.

A previously isolated rice (Oryza sativa) rbcS gene was further characterized. This analysis revealed specific sequences in the 5' regulatory region of the rice rbcS gene that are conserved in rbcS genes of other monocotyledonous species. In transgenic rice plants, we examined the expression of the beta-glucuronidase (gusA) reporter gene directed by the 2.8-kb promoter region of the rice rbcS gene. To examine differences in the regulation of monocotyledonous and dicotyledonous rbcS promoters, the activity of a tomato rbcS promoter was also investigated in transgenic rice plants. Our results indicated that both rice and tomato rbcS promoters confer mesophyll-specific expression of the gusA reporter gene in transgenic rice plants and that this expression is induced by light. However, the expression level of the rice rbcS-gusA gene was higher than that of the tomato rbcS-gusA gene, suggesting the presence of quantitative differences in the activity of these particular monocotyledonous and dicotyledonous rbcS promoters in transgenic rice. Histochemical analysis of rbcS-gusA gene expression showed that the observed light induction was only found in mesophyll cells. Furthermore, it was demonstrated that the light regulation of rice rbcS-gusA gene expression was primarily at the level of mRNA accumulation. We show that the rice rbcS gene promoter should be useful for expression of agronomically important genes for genetic engineering of monocotyledonous species.

Enhancement of foreign gene expression by a dicot intron in rice but not in tobacco is correlated with an increased level of mRNA and an efficient splicing of the intron.

The first intron of castor bean catalase gene, cat-1 was placed in the N-terminal region of the coding sequence of the beta-glucuronidase gene (gusA) and the intron-containing gusA was used with the cauliflower mosaic virus (CaMV) 35S promoter. Using this plasmid, pIG221, the effect of the intron on expression of beta-glucuronidase (GUS) activity was examined in transgenic rice calli and plants (a monocotyledon), and transgenic tobacco plants (a dicotyledon). The intron-containing plasmid increased the level of GUS enzyme activity 10 to 40-fold and 80 to 90-fold compared with the intronless plasmid, pBI221, in transgenic rice protoplasts and transgenic rice tissues, respectively. In contrast, the presence of the intron hardly influenced the expression of the GUS activity in transgenic tobacco plants. Northern blot analysis showed that the catalase intron was efficiently spliced in rice cells while transgenic tobacco plants contained both spliced and unspliced gusA transcripts in equal amounts. Furthermore, the level of the mature gusA transcript in transformed rice calli was greatly increased in the presence of the intron. The catalase intron was removed at the same splice junctions in transgenic rice and tobacco plants. These findings indicate that the stimulating effect of the intron on GUS expression is correlated with an efficient splicing of pre-mRNA and an increased level of mature mRNA.

Field performance and cytology of protoplast-derived rice (Oryza sativa): high yield and low degree of variation of four japonica cultivars.

Protoplast-derived rice plants of four Japanese cultivars, Nipponbare, Fujisaka 5, Norin 14 and Iwaimochi were individually cultivated in a submerged paddy field. They exhibited more stems, which resulted in more panicles than respective control plants. Other characteristics of protoplast-derived plants were (compared with controls): a slightly shorter or similar culm length, fewer spikelets per panicle, slightly lower seed fertility and similar or lighter 1,000 kernel weight. Grain yield of protoplast-derived plants was more than that of respective control plants in four cultivars. The cause of the higher yield of protoplast-derived plants seems to be mainly due to increased panicle number. Among 126 protoplast-derived plants, 1 triploid, 10 tetraploids and 1 aneuploid were found. Furthermore, 11 variants with low seed fertility showing no gross chromosomal anomalies and one plant with abnormal panicles were found. In total, about 80% of protoplastderived plants showed normal characters. The present results are encouraging for the possibility of rice breeding using protoplasts.

Promoter elements required for developmental expression of the maize Adh1 gene in transgenic rice.

To define the regions of the maize alcohol dehydrogenase 1 (Adh1) promoter that confer tissue-specific expression, a series of 5' promoter deletions and substitution mutations were linked to the Escherichia coli beta-glucuronidase A (uidA) reporter gene and introduced into rice plants. A region between -140 and -99 not only conferred anaerobically inducible expression in the roots of transgenic plants but was also required for expression in the root cap, embryo, and in endosperm under aerobic conditions. GC-rich (GC-1, GC-2, and GC-3) or GT-rich (GT-1 and GT-2) sequence motifs in this region were necessary for expression in these tissues, as they were in anaerobic expression. Expression in the root cap under aerobic conditions required all the GC- and GT-rich motifs. The GT-1, GC-1, GC-2, and GC-3 motifs, and to a lesser extent the GT-2 motif, were also required for anaerobic responsiveness in rice roots. All elements except the GC-3 motif were needed for endosperm-specific expression. The GC-2 motif and perhaps the GT-1 motif appeared to be the only elements required for high-level expression in the embryos of rice seeds. Promoter regions important for shoot-, embryo-, and pollen-specific expression were proximal to -99, and nucleotides required for shoot-specific expression occurred between positions -72 and -43. Pollen-specific expression required a sequence element outside the promoter region, between +54 and +106 of the untranslated leader, as well as a silencer element in the promoter between -72 and -43.

Over-expression of a single Ca2+-dependent protein kinase confers both cold and salt/drought tolerance on rice plants.

A rice gene encoding a calcium-dependent protein kinase (CDPK), OsCDPK7, was induced by cold and salt stresses. To elucidate the physiological function of OsCDPK7, we generated transgenic rice plants with altered levels of the protein. The extent of tolerance to cold and salt/drought stresses of these plants correlated well with the level of OsCDPK7 expression. Therefore, OsCDPK7 was shown to be a positive regulator commonly involved in the tolerance to both stresses in rice. Over-expression of OsCDPK7 enhanced induction of some stress-responsive genes in response to salinity/drought, but not to cold. Thus, it was suggested that the downstream pathways leading to the cold and salt/drought tolerance are different from each other. It seems likely that at least two distinct pathways commonly use a single CDPK, maintaining the signalling specificity through unknown post-translational regulation mechanisms. These results demonstrate that simple manipulation of CDPK activity has great potential with regard to plant improvement.

Molecular changes in protoplast-derived rice plants.

To determine whether regeneration of rice plants from protoplast culture induces DNA polymorphisms, progeny plants from direct regenerants of such cultures were examined for restriction fragment length polymorphisms (RFLP analysis). Significantly increased levels of DNA polymorphism were found compared with those in non-tissue culture control plants. Analysis with gene sequences representative of different functional domains, revealed that such polymorphisms are apparently widespread and not associated with any particular region. Analysis by comparative digestion with both methylation-sensitive and insensitive restriction enzymes revealed that methylation changes cannot be regarded as a major factor in the induction of these DNA polymorphisms.

Eucalyptus has functional equivalents of the Arabidopsis AP1 gene.

Two Eucalyptus homologues of the Arabidopsis floral homeotic gene AP1 (EAP1 and EAP2) show 60-65% homology to AP1. EAP1 and EAP2 are expressed predominantly in flower buds. EAP2 produces two different polypeptides arising from differential splicing at an intron, the shorter EAP2 protein diverging from the longer sequence after amino acid 197 and having a translation stop after residue 206. This truncated protein includes both MADS- and K-box amino acid sequences. Ectopic expression of the EAP1 or either of the two EAP2 polypeptides in Arabidopsis driven by the 35S promoter produces effects similar to the corresponding AP1 construct, causing plants to flower earlier, have shorter bolts and resemble the terminal flower mutant (tfl).

Ac as a tool for the functional genomics of rice.

To examine whether the maize autonomous transposable element Ac can be used for the functional analysis of the rice genome, we used Southern blot analysis to analyze the behaviour of Ac in 559 rice plants of four transgenic families through three successive generations. All families showed highly active transposition of Ac, and 103 plants (18.4%) contained newly transposed Ac insertions. In nine of the 12 independent transpositions analyzed, their germinal transmission was detected. Partial sequencing of 99 Ac-flanking sequences revealed that 21 clones exhibited significant similarities with protein-coding genes in databases and four of them matched rice cDNA sequences. These results indicate preferential Ac transposition into protein-coding rice genes. To examine the feasibility of PCR-based screening of gene knockouts in rice Ac plants, we prepared bulked genomic DNA from the leaves of approximately 6000 rice Ac plants and pooled the DNA according to a three-dimensional matrix. Of 14 randomly selected genes, two gene knockouts were identified, and one encoding a rice cytochrome P450 (CYP86) gene was shown to be stably inherited to the progeny. Together, these results suggest that Ac can be efficiently used for the functional analysis of the rice genome.