KNOX homeodomain proteins are encoded by knotted1-like homeobox (knox) genes that constitute a gene family in plants. Similar to the animal homeodomain proteins, KNOX proteins are considered to be key transcriptional regulators that control the expression of genes involved in plant organogenesis at the shoot apical meristem. Therefore, in order to understand the developmental processes in plants, it is important to elucidate the molecular mechanisms underlying KNOX protein regulation of gene expression. In this review, we discuss the structural features of KNOX proteins and the mechanisms by which they interact with and regulate target gene expression.
Proliferation and morphogenesis in plant cell cultures are influenced by the conditioning factors that are produced by the cultured cells themselves. Phytosulfokines(PSKs), sulfated peptide growth factors, have been identified as conditioning factors that promote cell proliferation in plant cell cultures. Our research has shown that the formation of carrot somatic embryos is regulated by both stimulatory and inhibitory conditioning factors. The stimulatory factor was found to be PSK, which markedly increased the frequency of somatic embryo formation. In contrast, the inhibitory factor was identified as 4-hydroxybenzyl alcohol(4HBA). 4HBA strongly inhibited somatic embryo formation by specifically suppressing the rapid cell division that is characteristic of the early globular stage. These findings suggest that dose-balancing of various plant growth regulators, including PSK and inhibitory factors, like 4HBA, might contribute to cell proliferation and morphogenesis in plant cell cultures.
Transgenic rice was developed from both calli and immature embryos of popular cultivar ‘Swarna’ with a rice chitinase gene (chi11) by particle gun bombardment. Homozygous dihaploid transgenics were produced in less than a year through the anther culture of primary transgenics. Stable integration and expression of the chi11 transgene were confirmed by Southern and western analyses, respectively of primary as well as anther culture-derived dihaploid transgenics. The homozygous transgenics with functional transgene and varied levels of chitinase activity showed enhanced resistance to sheath blight fungus. The bioassay data were correlated with the molecular and biochemical results.
Ammonium level in culture medium and culture temperature was identified as having a significant influence on the anthocyanin composition of cell suspension culture originating from the storage root of sweet potato (Ipomoea batatas L.), cv. Ayamurasaki. In the presence of 20 mM NH4+ in the culture medium the dominant pigment was YGM-0a, a non-acylated anthocyanin. With the decrease of ammonium concentration a large increase of YGM-0f’, an acylated anthocyanin, occurred. It was concluded that high ammonium concentration inhibits acylation of YGM-0a. The increase of culture temperature to 30°C resulted in accumulation of pigments, which appeared on the ODS-column HPLC with earlier retention time, with the predominance of YGM-0a. At lower temperatures of 15, 20 and 25°C the dominant pigment was YGM-0f’. It was concluded therefore that high temperature also inhibits acylation of YGM-0a.
It is an important basic step in developing genetic modification techniques to identify relevant simple and efficient tissue culture and plant regeneration systems for agronomically important cultivars. Culture conditions for somatic embryogenesis and plant regeneration from leaflet tissues of “Nanou” bahiagrass (Paspalum notatum Flugge L.) are described. Cultures were done for callus formation on Murashige and Skoog (1962) medium (MS) containing 2, 4-dichlorophenoxyacetic acid in the dark, and for plant regeneration on MS medium containing kinetin and giberellic acid in 16 h light. Two kinds of yellowish and white calli were obtained from the leaflets. In the continuous cultures, embryogenic calli and somatic embryogenesis were initiated from yellowish calli, but not from white calli. The plants were produced with multiple germinating embryos. We have therefore established a simple and efficient tissue culture and plant regeneration system available for genetic engineering.
Two transgenic IR72 lines, TT9-3 and TT9-4, carrying a fused Bt gene cry1Ab/cry1Ac from Bacillus thuringiensis Berliner were field tested to evaluate their multiple resistance against four lepidoptera and their agronomic performance in Hangzhou, China, in 1999. The results demonstrated that both transgenic lines were highly resistant against natural infestation and artificial infestation of four lepidopteran species: striped stem borer (Chilo suppressalis), pink stem borer (Sesamia inferens), leaffolder (Cnaphalocrocis medinalis), and green semilooper (Naranga anescens). In contrast, the non-transformed IR72 control showed serious damage symptoms of deadhearts, whiteheads, folded leaves or leaf notches. Field performance data showed that Bt transgenic lines may provide excellent opportunity for Bt rice deployment for commercial scale in Asia.
We have focused on possible stronger promoters in the chloroplast: those of psbA encoding D1 protein of photosystem II reaction center, 16S rDNA in rrn operon, the bacterial fused promoter tac, and the bacteriophage T7 gene φ10 in combination with transgenic T7 RNA polymerase (RNAP). Arabidopsis plants were made transgenic in the nuclear genome with the construct of a chimeric gene for T7 RNAP fused to a chloroplast transit peptide at its N-terminus placed under the control of CaMV 35S promoter. We have transiently expressed gene for β-glucuronidase (GUS) under control of the above promoters in the Arabidopsis chloroplast followed by particle bombardment. Expression in the chloroplast but not in the nucleus was confirmed histochemically and by treatment with α-amanitin. T7 promoter was the strongest among the examined promoters in the Arabidopsis chloroplast, being applicable to higher expression of foreign genes in the chloroplast with managed expression of T7 RNAP.
Transposable elements are regarded as a powerful mutagen and as an effective tool to isolate genes tagged by transposon insertions. In the Japanese morning glory, a number of spontaneous mutants related to the colors and shapes of the flowers have been isolated. The plant contains around 500-1000 copies of an En/Spm-related element Tpn1 and its relatives, which act as major spontaneous mutagens. We have developed a new protocol for identifying genes tagged by insertion of Tpn1-related elements. The procedure, named simplified transposon display (STD), is simple and requires neither biotinylated oligonucleotides nor streptavidin-capturing which are essential in other transposon display methods published recently. Here we describe the details of STD used for identification of the Purple(Pr) gene that encodes a vacuolar Na+/H+ exchanger for increasing vacuolar pH responsible for blue flower coloration.
LIM genes, identified as cDNAs from pollen mother cells of Lillium longiflorum, are specifically induced at meiosis I of microsporogenesis. LIM8 encodes a protein which is highly homologous to amino acid transporters and exhibits high expression levels at late stages of microsporogenesis. To study the regulated expression of microsporogenesis-associated genes, we isolated a genomic clone containing the 5' upstream region of LIM8 gene by a PCR-based approach. Sequence analysis of the 0.6kb genomic DNA fragment revealed the presence of putative transcription factor binding sites. However, no sequence similarity was found in promoter regions of LIM8 gene and previously described meiosis-associated genes. Transient assay by microprojectile bombardment demonstrated that the putative promoter region of LIM8 gene is able to direct tissue-specific expression of the luciferase reporter gene in lily microsporocytes.
A novel short interspersed nuclear element (SINE)-like sequence, named IPSE1, was identified in the retrotransposon fragments of sweet potato (Ipomoea batatas (L.) Lam). Sequences highly homologous to IPSE1 were identified in three previously reported genes of genus Ipomoea, suggesting that IPSE1 is conserved in genomes of Ipomoea. Structural characteristics of IPSE1 were discussed.
The technique of differential display was used to isolate ethylene-responsive cDNAs from the apices of cucumber plants. Differences in RNA populations from apices treated with or without ethephon (an ethylene-releasing compound) were examined using 80 primer combinations. Northern blot analysis confirmed that 20 cDNAs represented mRNAs that were differentially expressed upon ethephon treatment (promoted, 17; suppressed, 3). Sequence analysis of these cDNAs revealed that two clones were identical to the 3'-terminal regions of the CR20 and CUS3 genes of cucumber. Five clones showed significant similarity to the C-terminal regions of short-chain alcohol dehydrogenases, β-1, 3-glucanases, S-adenosyl-L-methionine:salicylic acid carboxyl methyltransferase, products of the Aux/IAA gene family, and lipoxygenases. The other 13 clones were found to have no significant homology in the databases. Because ethylene promotes female flower formation in cucumber plants, some of 20 cDNAs might be involved in processes of sex regulation.
One family of Conserved ATPase-containing Domain (CAD) proteins, the SUG1 family, is reported as a regulatory subunit of proteasome and/or a transcriptional mediator. We isolated a cDNA for the Arabidopsis SUG1 gene (AtSUG1), which shares strong homology with members of the SUG1 CAD family. Overproduction of AtSUG1 cDNA weakly restored meiotic division in a fission yeast mutant defective in the mei2 gene, which encodes a meiotic regulator. AtSUG1 was expressed at an almost constitutive level, independent of plant organ or developmental stage. AtSUG1 can functionally substitute for SUG1 in budding yeast.