Alexios N. Polidoros

Catalases in Plants: Gene Structure, Properties, Regulation, and Expression

Catalase action in plant and animal tissues was first observed in 1818 by Thenard, who noted that such tissues readily degraded hydrogen peroxide, a substance he had also discovered some years earlier (Aebi and Sutter 1971). Loew (1901) first established that the degradation of H 2 O 2 in tissues was due to the effect of an individual, separable enzyme, which he named “catalase.” Warburg (1923) suggested that catalase is an iron-containing enzyme, because it is inhibited by cyanide. Evidence for its hematin prosthetic group was presented by Zeile and Hellstrom (1930). Catalase was first purified and crystallized from beef liver, and its identity was made clear by Sumner and Dounce (1937). The earliest genetic studies on catalase were reported by the Russian biologist Koltzoff (1927), who demonstrated that blood catalase levels in several animal species are inherited and segregate according to Mendelian rules. Catalase has been found in all plants examined, and has been most thoroughly studied biochemically, genetically, and molecularly in the agronomically important species Zea mays L. (Scandalios 1990). That catalases can exist in multiple molecular forms or isozymes encoded by multiple genes, in any organism, was first demonstrated by Scandalios (1965 Scandalios (1968) with the maize catalases and has since been found to be the rule rather than the exception, as originally perceived. OXYGEN AND REACTIVE OXYGEN SPECIES During respiration, molecular oxygen accepts four electrons to produce two molecules of H 2 O. However, because of spin restrictions, O 2 cannot accept four electrons at once but accepts them one at a time...

Modulation of antioxidant responses by arsenic in maize.

The effects of arsenic on the expression of the antioxidant genes encoding superoxide dismutase, catalase, and glutathione S-transferase, as well as the activity of SOD and CAT enzymes, were examined at different developmental stages and in different tissues. Both CAT and SOD activities increased in response to low concentrations (0.01-0.1 mM) of arsenic in developing maize embryos. In germinating embryos both CAT and SOD activities increased in response to a wide range of arsenic concentrations (0.01-10 mM). Cat1 transcript increased in response to arsenic in developing and germinating embryos and in young leaves. Conversely, Cat2 increased at low concentrations of arsenic only in germinating embryos. Cat3 transcript levels increased in response to low concentrations of arsenic only in developing embryos. Sod3 transcript increased at low concentrations of arsenic in developing, germinating embryos and in leaves. The cytosolic Sod4 and Sod4A increased in response to arsenic in germinating embryos, while only Sod4 transcript increased in response to arsenic in leaves. Expression of Gst1 was similar to that of Cat1 in all tissues examined. These results indicate that arsenic triggers tissue and developmental stage specific defense responses of antioxidant and detoxification related genes in maize.

Aox gene structure, transcript variation and expression in plants

Alternative oxidase (Aox) has been proposed as a functional marker for breeding stress tolerant plant varieties. This requires presence of polymorphic Aox allele sequences in plants that affect plant phenotype in a recognizable way. In this review, we examine the hypothesis that organization of genomic Aox sequences and gene expression patterns are highly variable in relation to the possibility that such a variation may allow development of Aox functional markers in plants. Aox is encoded by a small multigene family, typically with four to five members in higher plants. The predominant structure of genomic Aox sequences is that of four exons interrupted by three introns at well conserved positions. Evolutionary intron loss and gain has resulted in the variation of intron numbers in some Aox members that may harbor two to four introns and three to five exons in their sequence. Accumulating evidence suggests that Aox gene structure is polymorphic enough to allow development of Aox markers in many plant species. However, the functional significance of Aox structural variation has not been examined exhaustively. Aox expression patterns display variability and typically Aox genes fall into two discrete subfamilies, Aox1 and Aox2, the former being present in all plants and the latter restricted in eudicot species. Typically, although not exclusively, the Aox1-type genes are induced by many different kinds of stress, whereas Aox2-type genes are expressed in a constitutive or developmentally regulated way. Specific Aox alleles are among the first and most intensively stress-induced genes in several experimental systems involving oxidative stress. Differential response of Aox genes to stress may provide a flexible plan of plant defense where an energy-dissipating system in mitochondria is involved. Evidence to link structural variation and differential allele expression patterns is scarce. Much research is still required to understand the significance of polymorphisms within AOX gene sequences for gene regulation and its potential for breeding on important agronomic traits. Association studies and mapping approaches will be helpful to advance future perspectives for application more efficiently.

Rolling circle amplification-RACE: a method for simultaneous isolation of 5' and 3' cDNA ends from amplified cDNA templates

Isolation of full-length gene transcripts is important to determine the protein coding region and study gene structure. However, isolation of novel gene sequences is often limited to expressed sequence tags (ESTs) (i.e., short cDNA fragments that predominantly represent the 3′ end of the transcript). Rapid amplification of cDNA ends (RACE) is today by far the most popular approach for obtaining full-length cDNAs when only part of the transcript’s sequence is known.Since its original description (1,2) numerous modifications and improve-ments of the method have been developed and consist of a collection of PCR-based cloning procedures that extend a known cDNA fragment toward the 3′ (3′ RACE) or the 5′ (5′ RACE) cDNA end. The original method is based on attachment of an anchor sequence to one end of the cDNA that can be used as a primer binding template in PCR with a second gene-specific primer from the known part of the gene. Although this procedure seems in theory fast and simple, it is technically difficult and usually requires substantial optimi-zation and several repetitions before satisfactory results can be obtained (3). This is particularly due to the use of a universal primer corresponding to the anchor sequence present in all cDNAs, which may result in a high background of nonspecific products even after a nested PCR with a gene-specific primer internal to the first gene-specific primer is performed. Another drawback of the method is the difficulty of obtaining the full-length 5′ end of the transcript due to the presence of many truncated transcripts in the messenger RNA (mRNA) pool. Several strategies aimed at eliminating these problems have been developed (4–9) and have proven to be very useful in certain applica-tions. One improvement is based on the utilization of a pair of gene-specific primers in inverse PCR on circularized cDNA templates, which would avoid the use of a universal primer and the problems it may generate (4,6,8–10). This strategy also allows the simulta-neous isolation of both cDNA ends in a single reaction (9,10). Some of these procedures require the generation of double-stranded cDNA, including the use of template-switching reverse transcription (9) or a post-reverse transcription adaptor ligation step (10). Methods that are performed directly on first-strand cDNA are complicated by the low efficiency of RNA ligase for the circularization reaction (6) or the need for bridging oligonucleotides for this step (8). Furthermore, existing inverse-RACE methods typically require nested PCR to amplify the transcript of interest, and only a limited number of transcripts can be isolated from a single reverse transcription reaction, making it difficult to analyze rare transcripts from scarce tissue.We describe here an improved inverse-RACE method, which uses CircLigase™ (Epicentre Biotechnologies, Madison, WI, USA) for cDNA circularization, followed by rolling circle amplification (RCA) of the circular cDNA with ϕ29 DNA polymerase (New England Biolabs, Ipswich, MA, USA). In this way, a large amount of the PCR template is produced, allowing the simultaneous isolation of the 3′ and 5′ unknown ends of a virtually unlimited number of transcripts after a single reverse transcription reaction. Figure 1 illus-trates this method, named RCA-RACE. The process takes advantage of the properties of CircLigase to circularize

Antioxidant gene-enzyme responses in Medicago truncatula genotypes with different degree of sensitivity to salinity.

Antioxidant responses and nodule function of Medicago truncatula genotypes differing in salt tolerance were studied. Salinity effects on nodules were analysed on key nitrogen fixation proteins such as nitrogenase and leghaemoglobin as well as estimating lipid peroxidation levels, and were found more dramatic in the salt-sensitive genotype. Antioxidant enzyme assays for catalase (CAT, EC 1.11.1.6), superoxide dismutase (EC 1.15.1.1), ascorbate peroxidase (EC 1.11.1.11) and guaiacol peroxidase (EC 1.11.1.7) were analysed in nodules, roots and leaves treated with increasing concentrations of NaCl for 24 and 48 h. Symbiosis tolerance level, depending essentially on plant genotype, was closely correlated with differences of enzyme activities, which increased in response to salt stress in nodules (except CAT) and roots, whereas a complex pattern was observed in leaves. Gene expression responses were generally correlated with enzymatic activities in 24-h treated roots in all genotypes. This correlation was lost after 48 h of treatment for the sensitive and the reference genotypes, but it remained positively significant for the tolerant one that manifested a high induction for all tested genes after 48 h of treatment. Indeed, tolerance behaviour could be related to the induction of antioxidant genes in plant roots, leading to more efficient enzyme stimulation and protection. High induction of CAT gene was also distinct in roots of the tolerant genotype and merits further consideration. Thus, part of the salinity tolerance in M. truncatula is related to induction and sustained expression of highly regulated antioxidant mechanisms.

The maize alternative oxidase 1a (Aox1a) gene is regulated by signals related to oxidative stress.

Abstract We isolated and characterized the expression of Aox1a, a member of the maize alternative oxidase (Aox) small multigene family. Aox1a consists of four exons interrupted by three introns and its promoter harbors diverse stress-specific putative regulatory motifs pointing to complex regulation and response to multiple signals. Responses of Aox1a to such signals were examined and compared with those of maize glutathione S-transferase I (GstI), a typical oxidative stress inducible gene. Potassium cyanide (KCN) and hydrogen peroxide (H2O2) induced a rapid increase of the Aox1a and GstI transcripts, which was persisted in prolonged treatment at high H2O2 concentration only for Aox1a. High concentration of salicylic acid (SA) and salicyl hydroxamic acid (SHAM) induced Aox1a mRNA only after prolonged exposure, while GstI displayed an early strong induction, which declined thereafter. Nitric oxide (NO) induced a high increase of Aox1a after prolonged exposure at high concentration, while GstI displayed a weak response. Our results show that multiple signaling pathways, involved in stress responses, also participate and differentially regulate Aox1a and GstI in maize. A ROS-depended signaling event may be involved, suggesting an essential role of Aox1a under oxidative stress in maize.

Characterization and expression analysis of AGAMOUS-like, SEEDSTICK-like, and SEPALLATA-like MADS-box genes in peach (Prunus persica) fruit.

MADS-box genes encode transcriptional regulators that are critical for flowering, flower organogenesis and plant development. Although there are extensive reports on genes involved in flower organogenesis in model and economically important plant species, there are few reports on MADS-box genes in woody plants. In this study, we have cloned and characterized AGAMOUS (AG), SEEDSTICK (STK) and SEPALLATA (SEP) homologs from peach tree (Prunus persica L. Batsch) and studied their expression patterns in different tissues as well as in fruit pericarp during pit hardening. AG- STK- and SEP-like homologs, representative of the C-, D-, E-like MADS-box gene lineages, respectively, play key roles in stamen, carpel, ovule and fruit development in Arabidopsis thaliana. Sequence similarities, phylogenetic analysis and structural characteristics were used to provide classification of the isolated genes in type C (PPERAG), type D (PPERSTK) and type E (PPERSEP1, PPERSEP3, PPERFB9) organ identity genes. Expression patterns were determined and in combination with phylogenetic data provided useful indications on the function of these genes. These data suggest the involvement of MADS-box genes in peach flower and fruit development and provide further evidence for the role of these genes in woody perennial trees that is compatible with their function in model plant species.

Isolation of a differentially spliced C-type flower specific AG-like MADS-box gene from Crocus sativus and characterization of its expression

We have cloned and characterized the expression of Crocus sativus AGAMOUS1 (CsAG1), a putative C-type MADS-box gene homologous to AGAMOUS (AG) from a triploid monocot species crocus (Crocus sativus L.). The typical domain structure of MIKC-type plant MADS proteins was identified. Phylogenetic analysis of the deduced amino acid sequence indicated that the isolated gene forms a clade with the AGAMOUS homologs from the monocots Hyacinthus orientalis and Phalaenopsis equestris. A differential splicing event altering the amino acid sequence at the C terminus was identified, leading to the formation of two mRNAs differing ten nucleotides in size. The presence of both differentially spliced transcripts was restricted only to mature crocus flowers and particularly to stamens and carpels.

Heterotopic expression of B-class floral homeotic genes PISTILLATA/GLOBOSA supports a modified model for crocus (Crocus sativus L.) flower formation

For uncovering and understanding the molecular mechanisms controlling flower development in cultivated Crocus sativus and particularly the transformation of sepals in outer whorl (whorl 1) tepals, we have cloned and characterized the expression of a family of five PISTILLATA/GLOBOSA-like (PI/GLO-like) MADS-box genes expressed in the C. sativus flower. The deduced amino acid sequences of the coded proteins indicated high homology with members of the MADS-box family of transcription factors, and particularly with other members of the PI/GLO family of MADS-box proteins that control floral organ identity. PI/GLO expression studies in cultivated C. sativus uncover the presence of PI/GLO transcripts not only in the second and third whorls of flower organs as expected, but also in the outer whorl tepals that are the sepals in most typical flowers. This heterotopic expression of both B-class genes: PI/GLO and AP3/DEF, known to form heterodimers for stamens and petals (petaloid inner whor l–whorl 2-tepals in C. sativus), explains the homeotic transformation of sepals into outer whorl tepals in this species. Analysis of PI/GLO sequences from C. sativus for putative targets to known micro-RNAs (miRNAs) showed that the target site for ath-miRNA167 found in Arabidopsis thaliana PI is not present in C. sativus, however, the PI/GLO sequences may be regulated by an ath-miRNA163.

Alternative oxidase 1 (Aox1) gene expression in roots of Medicago truncatula is a genotype-specific component of salt stress tolerance

Alternative oxidase (AOX) is the central component of the non-phosphorylating alternative respiratory pathway in plants and may be important for mitochondrial function during environmental stresses. Recently it has been proposed that Aox can be used as a functional marker for breeding stress tolerant plant varieties. This requires characterization of Aox alleles in plants with different degree of tolerance in a certain stress, affecting plant phenotype in a recognizable way. In this study we examined Aox1 gene expression levels in Medicago truncatula genotypes differing in salt stress tolerance, in order to uncover any correlation between Aox expression and tolerance to salt stress. Results demonstrated a specific induction of Aox1 gene expression in roots of the tolerant genotype that presented the lowest modulation in phenotypic and biochemical stress indices such as morphologic changes, protein level, lipid peroxidation and ROS generation. Similarly, in a previous study we reported that induction of antioxidant gene expression in the tolerant genotype contributed to the support of the antioxidant cellular machinery and stress tolerance. Correlation between expression patterns of the two groups of genes was revealed mainly in 48 h treated roots. Taken together, results from both experiments suggest that M. truncatula tolerance to salt stress may in part due to an efficient control of oxidative balance thanks to (i) induction of antioxidant systems and (ii) involvement of the AOX pathway. This reinforces the conclusion that differences in antioxidant mechanisms can be essential for salt stress tolerance in M. truncatula and possibly the corresponding genes, especially Aox, could be utilized as functional marker.