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.

Mediterranean White Lupin Landraces as a Valuable Genetic Reserve for Breeding.

Legumes crops are important for sustainable agriculture and global food security. Among them white lupin (Lupinus albus L.), is characterized by exceptional protein content of high nutritional value, competitive to that of soybean (Glycine max) and is well adapted to rainfed agriculture. However, its high seed-quinolizidine alkaloid (QA) content impedes its direct integration to human diet and animal feed. Additionally, its cultivation is not yet intensive, remains confined to local communities and marginal lands in Mediterranean agriculture, while adaptation to local microclimates restrains its cultivation from expanding globally. Hence, modern white lupin breeding aims to exploit genetic resources for the development of "sweet" elite cultivars, resilient to biotic adversities and well adapted for cultivation on a global level. Towards this aim, we evaluated white lupin local landrace germplasm from Greece, since the country is considered a center of white lupin diversity, along with cultivars and breeding lines for comparison. Seed morphological diversity and molecular genetic relationships were investigated. Most of the landraces were distinct from cultivars, indicating the uniqueness of their genetic make-up. The presence of pauper "sweet" marker allele linked to low seed QA content in some varieties was detected in one landrace, two breeding lines, and the cultivars. However, QA content in the examined genotypes did not relate with the marker profile, indicating that the marker's predictive power is limited in this material. Marker alleles for vernalization unresponsiveness were detected in eight landraces and alleles for anthracnose resistance were found in two landraces, pointing to the presence of promising germplasm for utilization in white lupin breeding. The rich lupin local germplasm genetic diversity and the distinct genotypic composition compared to elite cultivars, highlights its potential use as a source of important agronomic traits to support current breeding efforts and assist its integration to modern sustainable agriculture.

Comparative Transcriptome Analysis in Homo- and Hetero-Grafted Cucurbit Seedlings.

Watermelon (Citrullus lanatus) is a valuable horticultural crop with nutritional benefits grown worldwide. It is almost exclusively cultivated as grafted scions onto interspecific squash rootstock (Cucurbita maxima × Cucurbita moschata) to improve the growth and yield and to address the problems of soilborne diseases and abiotic stress factors. This study aimed to examine the effect of grafting (homo- and hetero-grafting) on the transcriptome level of the seedlings. Therefore, we compared homo-grafted watermelon (WW) with non-grafted watermelon control (W), homo-grafted squash (SS) with non-grafted squash control (S), hetero-grafted watermelon onto squash (WS) with SS, and WS with WW. Different numbers of differentially expressed genes (DEGs) were identified in each comparison. In total, 318 significant DEGs were detected between the transcriptomes of hetero-grafts and homo-grafts at 16 h after grafting. Overall, a significantly higher number of downregulated transcripts was detected among the DEGs. Only one gene showing increased expression related to the cytokinin synthesis was common in three out of four comparisons involving WS, SS, and S. The highest number of differentially expressed (DE) transcripts (433) was detected in the comparison between SS and S, followed by the 127 transcripts between WW and W. The study provides a description of the transcriptomic nature of homo- and hetero-grafted early responses, while the results provide a start point for the elucidation of the molecular mechanisms and candidate genes for the functional analyses of hetero-graft and homo-graft systems in Cucurbitaceae and generally in the plants.

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.

Characterization and expression analysis of FRUITFULL- and SHATTERPROOF-like genes from peach (Prunus persica) and their role in split-pit formation.

The fruit canning industry processes large quantities of the clingstone varieties of peach (Prunus persica L. Batch). The occurrence of split-pit formation--the opening of the pit and sometimes splitting of the fruit--causes deterioration of canned fruit quality. The frequency of split-pit formation is influenced by genetic and environmental factors. To increase understanding of the molecular mechanisms underlying split-pit formation in peach, we cloned and characterized the PPERFUL and PPERSHP genes that are homologues to the genes FRUITFULL and SHATTERPROOF, respectively, which are involved in fruit splitting (pod shattering) in Arabidopsis thaliana. The deduced amino acid sequences of the two genes had high homology with members of the MADS-box family of transcription factors, and particularly with other members of the FUL-like family of A-type MADS-box proteins and PLENA-like family of C-type MADS-box proteins, respectively. PPERFUL and PPERSHP were expressed throughout fruit development from full anthesis until fruit harvest. Differences in the mRNA abundance of each gene were compared in a split-pit sensitive and a split-pit resistant variety. Results suggested that temporal regulation of PPERFUL and PPERSHP expression may have an effect on the split-pit process.

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.

Cloning, structural characterization, and phylogenetic analysis of flower MADS-box genes from crocus (Crocus sativus L.).

Crocus (Crocus sativus L.) is a crop species cultivated for its flowers and, more specifically, for its red stigmas. The flower of crocus is bisexual and sterile, since crocus is a triploid species. Its perianth consists of six petaloid tepals: three tepals in whorl 1 (outer tepals) and three tepals in whorl 2 (inner tepals). The androecium consists of three distinct stamens and the gynoecium consists of a single compound pistil with three carpels, a single three-branched style, and an inferior ovary. The dry form of the stigmas constitutes the commercial saffron used as a food additive, in the coloring industry, and in medicine. In order to uncover and understand the molecular mechanisms controlling flower development in cultivated crocus and its relative wild progenitor species, and characterize a number of crocus flower mutants, we have cloned and characterized different, full-length, cDNA sequences encoding MADS-box transcription factor proteins involved in flower formation. Here we review the different methods followed or developed for obtaining these sequences involving conventional 5 inverted exclamation markä 3 inverted exclamation markä RACE, as well as newly developed methods from our group, named Rolling Circle Amplification C RACE (RCA-RACE) and its modification named familyRCA-RACE (famRCA-RACE). Furthermore, the characteristics of the protein structure and their common and specific domains for each type of MADS-box transcription factors in this lower nongrass monocot belonging to the Iridaceae family are described. Finally, a phylogenetic tree of all the MADS-box sequences available in our lab is presented and discussed in relation to other data from studies of species of the Iridaceae group and closely related families from an evolutionary perspective. The structural and phylogenetic analyses are based on both published and unpublished data.

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.

Target-site mutation associated with cross-resistance to ALS-inhibiting herbicides in late watergrass (Echinochloa oryzicola Vasing.).

BACKGROUND: Studies were carried out to elucidate the mechanism of resistance to ALS-inhibiting herbicides in 29 Echinochloa accessions from water-seeded rice fields of northern Greece and to discriminate the Echinochloa species. RESULTS: Two E. oryzicola accessions were found to be cross-resistant to penoxsulam, bispyribac-sodium, imazamox, foramsulfuron, nicosulfuron and rimsulfuron, whereas all accessions were susceptible (S) to profoxydim. Sequencing of the ALS gene revealed that resistant (R) accessions had a Trp574Leu mutation, which was also confirmed by TspRI endonuclease digestion. Use of cpDNA sequence comparison analysis of Echinochloa species discriminated successfully E. crus-galli and E. oryzicola accessions. CONCLUSION: This is the first report of Echinochloa oryzicola cross-resistance to ALS-inhibiting herbicides as a result of Trp574Leu mutation. The cpDNA sequence comparison analysis is a reliable tool for discrimination of conventionally classified E. crus-galli and E. oryzicola accessions.

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.

Antioxidant gene responses to ROS-generating xenobiotics in developing and germinated scutella of maize.

There is circumstantial evidence implicating reactive oxygen species (ROS) in the highly ordered temporal and spatial regulation of expression of the Cat and Sod antioxidant genes during seed development and germination in maize. In order to understand and provide experimental data for the regulatory role of ROS, the expression patterns of the Cat1, Cat2, Cat3, GstI, Sod3, Sod4, and Sod4A genes, as well as catalase (CAT) and superoxide dismutase (SOD) activity responses, were examined after treatments with ROS-generating xenobiotics in developing and germinated maize scutella. CAT and SOD activities increased at both stages in response to each xenobiotic examined in a dose-dependent and stage-specific manner. Individual Cat gene expression patterns were co-ordinated with isozyme patterns of enzymatic activity in scutella of developing seeds. This was not observed in germinated seeds where, although Cat1 expression was highly induced by ROS, there was not a similar increase of enzymatic CAT1 activity, suggesting the involvement of post-transcriptional regulation. Enhanced enzyme activities were synchronous with increases in steady-state transcript levels of specific Sod genes. The steady-state transcript level of GstI was elevated in all samples examined. Gene expression responses derived from this study along with similar results documented in previous reports were subjected to cluster analysis, revealing that ROS-generating compounds provoke similar effects in the expression patterns of the tested antioxidant genes. This could be attributable to common stress-related motifs present in the promoters of these genes.

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

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.

Identification of unknown genetically modified material admixed in conventional cotton seed and development of an event-specific detection method

Entering the second decade of commercialization of biotech crops, the global area cultivated with transgenic plants constantly expands and national legislations in many countries, particularly in the European Union, require identification and labeling of genetically modified material in food and feed. We describe here a procedure for characterizing transgenic material of unknown origin present in conventional seed lots using a genome walking strategy for isolation and characterization of the junction between the inserted transgene construct and the host plant genomic DNA. The procedure was applied to transgenic cotton detected as adventitious or technically unavoidable presence in a conventional commercial cultivar. The structure of the isolated region revealed that the transgenic material derived from Monsanto’s event 1445 transgenic cotton. Due to the random incorporation of the transgene into the host plant’s genome, the sequence of the junction region obtained using the genome walking strategy, provided the means to develop an event-specific identification method without prior knowledge for the nature of the transformation event. Thus, we documented a methodology for developing an event-specific detection protocol even without prior knowledge of the genetic modification event.