T. A. Ezhova

An Arabidopsis mutant that is resistant to the protoporphyrinogen oxidase inhibitor acifluorfen shows regulatory changes in tetrapyrrole biosynthesis

Several Arabidopsis mutants of the ecotype Dijon were isolated that show resistance to the herbicide acifluorfen, which inactivates protoporphyrinogen oxidase (PPOX), an enzyme of tetrapyrrole biosynthesis. This enzyme provides protoporphyrin for both Mg chelatase and ferrochelatase at the branchpoint, which leads to chlorophyll and heme, respectively. One of the mutations, aci5-3, displays semidominant inheritance. Heterozygous progeny showed yellow-green leaves, while the homozygous seedlings were white and inviable, but could be rescued by supplementing the medium with sugar. Interestingly, the expression of neither of the two forms of PPOX was altered in the mutant, but the rate of synthesis of 5-aminolevulinate, the precursor of all tetrapyrroles, was drastically reduced. Genetic mapping revealed the mutant locus is closely linked to the ch42 marker, which is itself located in the CHLI-1 gene which codes for one of the three subunits of Mg chelatase. The cs mutant also shows a defect in this gene, and test for allelism with aci5-3 confirmed that the two mutations are allelic. Sequencing of the wild type and aci5-3 alleles of CHLI-1 revealed a single base change (G718A), which results in a D240N substitution in the CHLI-1 protein. In the homozygous aci5-3 mutant no CHLI-1 RNA or protein could be detected. Strikingly, CHLH and CHLI-2 transcripts were also absent. This indicates the existence of a feedback-regulatory mechanism that inactivates the genes encoding certain Mg chelatase subunits. The basis for the semidominant inheritance pattern and the relationship between herbicide resistance and modified gene expression is discussed.

The analysis of the ChlI 1 and ChlI 2 genes using acifluorfen-resistant mutant of Arabidopsis thaliana

One of the key regulatory enzymes of the chlorophyll biosynthesis pathway is magnesium (Mg) chelatase, consisting of three different subunits CHLI, CHLD and CHLH. While CHLH and CHLD are encoded by a single gene each in Arabidopsis, CHLI is encoded by two homologous genes, ChlI 1 and ChlI 2. Analysis of the acifluorfen herbicide resistant mutant aci5 revealed an alteration of the ChlI 1 gene. This mutant as well as wild type plants contained similar transcript levels of the ChlI 1 and ChlI 2 genes. Moreover, the transcripts of both alleles of the ChlI 1 gene were present in the cs (ch42-2)/aci5 hybrid which showed an albina phenotype. Comparison of the amino acid sequence of CHLI 1 and CHLI 2 encoded in the genome of aci5 and wild type revealed in particular alterations of the C-terminal end which are suggested to be responsible for the decreased ability of CHLI 2 to participate in the formation of the CHLI ring-like structure of the Mg chelatase complex.

Basic principles of terminal flower formation

Studies of inflorescences of the mutants bractea and terminal flower1 and double mutant bra tfl1 of Arabidopsis thaliana (L.) Heynh. have shown that the presence of a developed leaf in the node preceding the terminal flower is a necessary condition for the formation of the terminal flower perianth. This means that perianth cannot develop in an abracteose inflorescence of terminal flower. The second necessary condition for the terminal flower formation is a sufficient level of expression of the genes responsible for floral morphogenesis. Combination of these two conditions suffices for the development of a terminal flower with perianth. Since the general principles of organization are common for the majority of Angiosperms, it can be stated that if the abracteose inflorescence is terminated by a flower with perianth, this is a consequence of displacement of the lateral flower into the terminal position.

Arabidopsis thaliana ICE2 gene: Phylogeny, structural evolution and functional diversification from ICE1

The ability to tolerate environmental stresses is crucial for all living organisms, and gene duplication is one of the sources for evolutionary novelties. Arabidopsis thaliana INDUCER OF CBF EXPRESSION1 and 2 (ICE1 and ICE2) encode MYC-type bHLH (basic helix-loop-helix) transcription factors. They confer cold stress tolerance by induction of the CBF/DREB1 regulon and regulate stomata formation. Although ICE2 is closely related to ICE1, its origin and role in cold response remains uncertain. Here, we used a bioinformatics/phylogenetic approach to uncover the ICE2 evolutionary history, structural evolution and functional divergence from the putative ancestral gene. Sequence diversification from ICE1 included the gain of cis-acting elements in ICE2 promoter sequence that may provide meristem-specific and defense-related gene expression. By analyzing transgenic Arabidopsis lines with ICE2 over-expression we showed that it contributes to stomata formation, flowering time regulation and cold response. Constitutive ICE2 expression led to induced meristem freezing tolerance, resulting from activation of CBF1 and CBF3 genes and ABA biosynthesis by NCED3 induction. We presume that ICE2 gene has originated from a duplication event about 17.9MYA followed by sub- and neofunctionalization of the ancestral ICE1 gene. Moreover, we predict its role in pathogen resistance and flowering time regulation.

Genetic Control of Totipotency of Plant Cells in an in vitro Culture

The main approaches have been considered to studying the genetic control of plant cell totipotency in an in vitro culture. The capacity of cultured plants for callusogenesis, organ formation, and somatic embryogenesis depends on the activity of genes that determine and maintain the meristematic state of cells, level of hormones in the cells, and sensitivity to hormones, as well as on the activity other genes that control different stages of plant morphogenesis.

Type Specification and Spatial Pattern Formation of Floral Organs: A Dynamic Development Model

A mathematical model simulating spatial pattern formation (positioning) of floral organs is proposed. Computer experiment with this model demonstrated the following sequence of spatial pattern formation in a typical cruciferous flower: medial sepals, carpels, lateral sepals, long stamens, petals, and short stamens. The positioning was acropetal for the perianth organs and basipetal for the stamens and carpels. Organ type specification and positioning proceed non-simultaneously in different floral parts and organ type specification goes ahead of organ spatial pattern formation. Computer simulation of flower development in several mutants demonstrated that the AG and AP2 genes determine both organ type specification and formation of the zones for future organ development. The function of the AG gene is to determine the basipetal patterning zones for the development of the reproductive organs, while the AP2 gene maintains proliferative activity of the meristem establishing the acropetal patterning zone for the development of the perianth organs.

[Effect of the ABRUPTUS/PINOID gene on expression of the LEAFY gene in Arabidopsis thaliana].

The nucleotide sequence was analyzed for the temperature-sensitive allele abruptus (abr), which distorts polar auxin transport (PAT) in the inflorescence. The mutation C → T was found in the second exon and led to an amino acid substitution (glycin → glutamic acid) in the conserved domain of protein kinase encoded by the ABRUPTUS/PINOID (ABR/PID) gene. QRT-PCR revealed a 100-fold decrease in transcription of the LEAFY (LFY) gene in the abr mutant with high expressivity of the mutant character; transcription of the fused LFY::GUS gene was also low in the mutant. The results agree with data of the phenotypic analysis of the abr lfy double mutant and testify to an important role of auxin gradients in regulating the expression pattern of the LFY gene.

A new Arabidopsis thaliana deletion mutant apetala1-20

A new deletion allele of the APETALA1 (AP1) gene encoding a type II MADS-box protein with the key role in the initiation of flowering and development of perianth organs has been identified in A. thaliana. The deletion of seven amino acids in the conserved region of the K domain in the ap1-20 mutant considerably delayed flowering and led to a less pronounced abnormality in the corolla development compared to the weak ap1-3 and intermediate ap1-6 alleles. At the same time, a considerable stamen reduction has been revealed in ap1-20 as distinct from ap1-3 and ap1-6 alleles. These data indicate that the K domain of AP1 can be crucial for the initiation of flowering and expression regulation of B-class genes controlling stamen development.

[Interaction of the BRACTEA gene with the TERMINAL FLOWER1, LEAFY, and APETALA1 genes during inflorescence and flower development in Arabidopsis thaliana].

The major Arabidopsis thaliana genes controlling the shoot architecture are TERMINAL FLOWER1 (TEL1), APETALA1 (AP1), and LEAFY (LFY). The BRACTEA (BRA) gene also codes for one of the key regulators of inflorescence development. The bra tfl1-11, bra lfy-5, and bra ap1-20 double mutants were analyzed morphologically, and expression of the TFL1, AP1, and LFY genes was studied in the bra mutant and wild-type plants. The BRA gene was found to positively regulate the TFL1 and AP1 genes after floral initiation, determining more than 70% of their total expression level. In floral meristem, the BRA gene prevented AP1 expression, restricting AP1 transcription to the perianth zone. Since flowers were completely converted into vegetative shoots in the bra lfy-5 double mutant, it was assumed that the BRA and LFY genes function redundantly and independently in floral initiation. The results demonstrate that BRA is one of the genes that determine the balance between maintenance of proliferative activity in apical meristem and floral development in its peripheral region; such a balance is necessary for indeterminate inflorescence development.

PXD gene controls synthesis of the three anionic peroxidase isoforms in Arabidopsis thaliana

The pxd mutant of Arabidopsis thaliana features a changed pattern of anionic peroxidases: three anionic isoforms in the pxd mutant plant have the same enzymatic activity but relatively high electrophoretic mobility as compared to the analogous isoforms in the wild type plants. These isoforms are the most active anionic peroxidases and can be found in most plant organs. Genetic analysis showed that all three isoforms are controlled by the PXD gene. The activity of one isoform was affected by indolyl-3-acetic acid and other stress factors. Expressed sequence tags (EST) analysis of all putative peroxidase genes of A. thaliana revealed a group of the most actively and nonspecifically expressed genes. The promoter sequences of these genes were screened to find the cis-elements. We propose that PXD gene encodes one of nonspecific anionic peroxidases or a protein involved in posttranscriptional or posttranslational modification of the peroxidases.