Hélia Guerra Cardoso

Differential expression and co-regulation of carrot AOX genes (Daucus carota)

Alternative oxidase (AOX) is a mitochondrial protein encoded by the nuclear genome. In higher plants AOX genes form a small multigene family mostly consisting of the two subfamilies AOX1 and AOX2. Daucus carota L. is characterized by a unique extension pattern of AOX genes. Different from other plant species studied so far it contains two genes in both subfamilies. Therefore, carrot was recently highlighted as an important model in AOX stress research to understand the evolutionary importance of both AOX subfamilies. Here we report on the expression patterns of DcAOX1a, DcAOX1b and DcAOX2a and DcAOX2b. Our results demonstrate that all of the four carrot AOX genes are expressed. Differential expression was observed in organs, tissues and during de novo induction of secondary root phloem explants to growth and development. DcAOX1a and DcAOX2a indicated a differential transcript accumulation but a similar co-expression pattern. The genes of each carrot AOX sub-family revealed a differential regulation and responsiveness. DcAOX2a indicated high inducibility in contrast to DcAOX2b, which generally revealed low transcript abundance and rather weak responses. In search for within-gene sequence differences between both genes as a potential reason for the differential expression patterns, the structural organization of the two genes was compared. DcAOX2a and DcAOX2b showed high sequence similarity in their open reading frames (ORFs). However, length variability was observed in the N-terminal exon1 region. The predicted cleavage site of the mitochondrial targeting sequence in this locus is untypical small for both genes and consists of 35 amino acids for DcAOX2a and of 21 amino acids for DcAOX2b. The importance of structural gene organization and the relevancy of within-gene sequence variations are discussed. Our results strengthen the value of carrot as a model plant for future studies on the importance of AOX sub family evolution.

Carrot alternative oxidase gene AOX2a demonstrates allelic and genotypic polymorphisms in intron 3.

Single nucleotide polymorphisms (SNPs) and insertion-deletions (InDels) are becoming important genetic markers for major crop species. In this study, we focus on variations at genomic level of the Daucus carota L. AOX2a gene. The use of gene-specific primers designed in exon regions on the boundaries of introns permitted to recognize intron length polymorphism (ILP) in intron 3 AOX2a by simple polymerase chain reaction (PCR) assays. The length of intron 3 can vary in individual carrot plants. Thus, allelic variation can be used as a tool to discriminate between single plant genotypes. Using this approach, individual plants from cv. Rotin and from diverse breeding lines and cultivars were identified that showed genetic variability by AOX2a ILPs. Repetitive patterns of intron length variation have been observed which allows grouping of genotypes. Polymorphic and identical PCR fragments revealed underlying high levels of sequence polymorphism. Variability was due to InDel events and intron single nucleotide polymorphisms (ISNPs), with a repetitive deletion in intron 3 affecting a putative pre-miRNA site. The results suggest that high AOX2a gene diversity in D. carota can be explored for the development of functional markers related to agronomic traits.

Intron polymorphism pattern in AOX1b of wild St John's wort (Hypericum perforatum) allows discrimination between individual plants.

The present paper deals with the analysis of natural polymorphism in a selected alternative oxidase (AOX) gene of the medicinal plant, St Johns wort. Four partial AOX gene sequences were isolated from the genomic DNA of a wild plant of Hypericum perforatum L. Three genes belong to the subfamily AOX1 (HpAOX1a, b and c) and one to the subfamily AOX2 (HpAOX2). The partial sequence of HpAOX1b showed polymerase chain reaction (PCR) fragment size variation as a result of variable lengths in two introns. PCR performed by Exon Primed Intron Crossing (EPIC)-PCR displayed the same two-band pattern in six plants from a collection. Both fragments showed identical sequences for all exons. However, each of the two introns showed an insertion/deletion (InDel) in identical positions for all plants that counted for the difference in the two fragment sizes. The InDel in intron 1 influenced the predictability of a pre-microRNA site. The almost identical PCR fragment pattern was characterized by a high variability in the sequences. The InDels in both introns were linked to repetitive intron single nucleotide polymorphisms (ISNP)s. The polymorphic pattern obtained by InDels and ISNPs from both fragments together was appropriate to discriminate between all individual plants. We suggest that AOX sequence polymorphism in H. perforatum can be used for studies on gene diversity and biodiversity. Further, we conclude that AOX sequence polymorphism of individual plants should be considered in biological studies on AOX activity to exclude the influence of genetic diversity. The identified polymorphic fragments are available to be explored in future experiments as a potential source for functional marker development related to the characterization of origins/accessions and agronomic traits such as plant growth, development and yield stability.

Daucus carota L. - An old model for cell reprogramming gains new importance through a novel expansion pattern of alternative oxidase (AOX) genes

The paper highlights Daucus carota L. as an ideal model to complement plant stress research on Arabidopsis thaliana L. Recently, alternative oxidase (AOX) is discussed as functional marker candidate for cell reprogramming upon stress. Carrot is the most studied species for cell reprogramming and our current research reveals that it is the only one that has expanded both AOX sub-family genes. We point to recently published, but not discussed results on conserved differences in the vicinity of the most active functional site of AOX1 and AOX2, which indicate the importance of studying AOX sequence polymorphism, structure and functionality. Thus, stress-inducible experimental systems of D. carota are especially appropriate to bring research on stress tolerance a significant step forward.

Alternative oxidase involvement in Daucus carota somatic embryogenesis

Plant alternative oxidase (AOX) is a mitochondrial inner membrane enzyme involved in alternative respiration. The critical importance of the enzyme during acclimation upon stress of plant cells is not fully understood and is still an issue of intensive research and discussion. Recently, a role of AOX was suggested for the ability of plant cells to change easily its fate upon stress. In order to get new insights about AOX involvement in cell reprogramming, quantitative real-time polymerase chain reaction (PCR) and inhibitor studies were performed during cell redifferentiation and developmental stages of Daucus carota L. somatic embryogenesis. Transcript level analysis shows that D. carota AOX genes (DcAOX1a and DcAOX2a) are differentially expressed during somatic embryogenesis. DcAOX1a shows lower expression levels, being mainly down-regulated, whereas DcAOX2a presented a large up-regulation during initiation of the realization phase of somatic embryogenesis. However, when globular embryos start to develop, both genes are down-regulated, being this state transient for DcAOX2a. In addition, parallel studies were performed using salicylhydroxamic acid (SHAM) in order to inhibit AOX activity during the realization phase of somatic embryogenesis. Embryogenic cells growing in the presence of the inhibitor were unable to develop embryogenic structures and its growth rate was diminished. This effect was reversible and concentration dependent. The results obtained contribute to the hypothesis that AOX activity supports metabolic reorganization as an essential part of cell reprogramming and, thus, enables restructuring and de novo cell differentiation.

Reference Genes Selection and Normalization of Oxidative Stress Responsive Genes upon Different Temperature Stress Conditions in Hypericum perforatum L

Reverse transcription-quantitative real-time PCR (RT-qPCR) is a widely used technique for gene expression analysis. The reliability of this method depends largely on the suitable selection of stable reference genes for accurate data normalization. Hypericum perforatum L. (St. Johns wort) is a field growing plant that is frequently exposed to a variety of adverse environmental stresses that can negatively affect its productivity. This widely known medicinal plant with broad pharmacological properties (anti-depressant, anti-tumor, anti-inflammatory, antiviral, antioxidant, anti-cancer, and antibacterial) has been overlooked with respect to the identification of reference genes suitable for RT-qPCR data normalization. In this study, 11 candidate reference genes were analyzed in H. perforatum plants subjected to cold and heat stresses. The expression stability of these genes was assessed using GeNorm, NormFinder and BestKeeper algorithms. The results revealed that the ranking of stability among the three algorithms showed only minor differences within each treatment. The best-ranked reference genes differed between cold- and heat-treated samples; nevertheless, TUB was the most stable gene in both experimental conditions. GSA and GAPDH were found to be reliable reference genes in cold-treated samples, while GAPDH showed low expression stability in heat-treated samples. 26SrRNA and H2A had the highest stabilities in the heat assay, whereas H2A was less stable in the cold assay. Finally, AOX1, AOX2, CAT1 and CHS genes, associated with plant stress responses and oxidative stress, were used as target genes to validate the reliability of identified reference genes. These target genes showed differential expression profiles over time in treated samples. This study not only is the first systematic analysis for the selection of suitable reference genes for RT-qPCR studies in H. perforatum subjected to temperature stress conditions, but may also provide valuable information about the roles of genes associated with temperature stress responses.

Involvement of alternative oxidase (AOX) in adventitious rooting of Olea europaea L. microshoots is linked to adaptive phenylpropanoid and lignin metabolism

Alternative oxidase (AOX) has been proposed as a functional marker candidate in a number of events involving cell differentiation, including rooting efficiency in semi-hardwood shoot cuttings of olive (Olea europaea L.). To ascertain the general importance of AOX in olive rooting, the auxin-induced rooting process was studied in an in vitro system for microshoot propagation. Inhibition of AOX by salicylhydroxamic acid (SHAM) significantly reduced rooting efficiency. However, the inhibitor failed to exhibit any effect on the preceding calli stage. This makes the system appropriate for distinguishing dedifferentiation and de novo differentiation during root induction. Metabolite analyses of microshoots showed that total phenolics, total flavonoids and lignin contents were significantly reduced upon SHAM treatment. It was concluded that the influence of alternative respiration on root formation was associated to adaptive phenylpropanoid and lignin metabolism. Transcript profiles of two olive AOX genes (OeAOX1a and OeAOX2) were examined during the process of auxin-induced root induction. Both genes displayed stable transcript accumulation in semi-quantitative RT-PCR analysis during all experimental stages. In contrary, when the reverse primer for OeAOX2 was designed from the 3′-UTR instead of the ORF, differential transcript accumulation was observed suggesting posttranscriptional regulation of OeAOX2 during metabolic acclimation. This result confirms former observations in olive semi-hardwood shoot cuttings on differential OeAOX2 expression during root induction. It further points to the importance of future studies on the functional role of sequence and length polymorphisms in the 3′-UTR of this gene.Key message The manuscript reports the general importance of AOX in olive adventitious rooting and the association of alternative respiration to adaptive phenylpropanoid and lignin metabolism.

Alternative oxidase (AOX) and phenolic metabolism in methyl jasmonate-treated hairy root cultures of Daucus carota L.

Methyl-jasmonate (MJ)-treated hairy roots of Daucus carota L. were used to study the influence of alternative oxidase (AOX) in phenylpropanoid metabolism. Phenolic acid accumulation, as well as total flavonoids and lignin content of the MJ-treated hairy roots were decreased by treatment with salicylhydroxamic acid (SHAM), a known inhibitor of AOX. The inhibitory effect of SHAM was concentration dependent. Treatment with propyl gallate (PG), another inhibitor of AOX, also had a similar inhibitory effect on accumulation of phenolic acid, total flavonoids and lignin. The transcript levels of two DcAOX genes (DcAOX2a and DcAOX1a) were monitored at selected post-elicitation time points. A notable rise in the transcript levels of both DcAOX genes was observed preceding the MJ-induced enhanced accumulation of phenolics, flavonoids and lignin. An appreciable increase in phenylalanine ammonia-lyase (PAL) transcript level was also observed prior to enhanced phenolics accumulation. Both DcAOX genes showed differential transcript accumulation patterns after the onset of elicitation. The transcript levels of DcAOX1a and DcAOX2a attained peak at 6hours post elicitation (hpe) and 12hpe, respectively. An increase in the transcript levels of both DcAOX genes preceding the accumulation of phenylpropanoid-derivatives and lignin showed a positive correlation between AOX activity and phenylpropanoid biosynthesis. The results provide important new insight about the influence of AOX in phenylpropanoid biosynthesis.

The gymnosperm Pinus pinea contains both AOX gene subfamilies, AOX1 and AOX2

The gymnosperm Pinus pinea L. (stone pine) is a typical Mediterranean pine used for nuts and timber production, and as an ornamental around the world. Pine genomes are large in comparison to other species. The hypothesis that retrotransposons, such as gymny, made a large contribution to this alteration in genome size was recently confirmed. However, P. pinea is unique in other various aspects. P. pinea demonstrates a different pattern of gymny organization than other Pinus subgenera. Additionally, P. pinea has a highly recalcitrant behaviour in relation to standard conifer protocols for the induction of somatic embryogenesis or rooting. Because such types of cell reprogramming can be explained as a reaction of plant cells to external stress, it is of special interest to study sequence peculiarities in stress-inducible genes, such as the alternative oxidase (AOX). This is the first report containing molecular evidence for the existence of AOX in gymnosperms at the genetic level. P. pinea AOXs were isolated by a polymerase chain reaction (PCR) approach and three genes were identified. Two of the genes belong to the AOX1 subfamily and one belongs to the AOX2 subfamily. The existence of both AOX subfamilies in gymnosperms is reported here for the first time. This discovery supports the hypothesis that AOX1 and AOX2 subfamilies arose prior to the separation of gymnosperms and angiosperms, and indicates that the AOX2 is absent in monocots because of subsequent gene loss events. Polymorphic P. pinea AOX1 sequences from a selected genetic clone are presented indicating non-allelic, non-synonymous and synonymous translation products.

Functional Marker Development Across Species in Selected Traits

Functional marker (FM) development across plant species requires common allelic sequences that affect phenotypic variation in the same way. The identification of orthologous genes showing the same function across species could indicate conservation of functional polymorphisms. In this chapter we provide an overview over polymorphic sites described within homologous genes across species directly related to selected traits, as well as the FMs developed and available for plant breeding. The review demonstrates that orthologous gene functions are conserved across species, making the targeted search for FM candidates genes an excellent approach for FM development across species. However, it is also reported that conservation of functional sequence motifs in those orthologous genes across species occurs as a rare phenomenon. Plasticity per se is highlighted as a new trait for plant breeding as robust, multi-stress tolerant and non-recalcitrant adventitious developmental character for plant production. As a conclusion of the review, new perspectives for the identification of functional markers across species are proposed.