The growth-immunity tradeoff in Brassica oleracea-Xanthomonas campestris pv. campestris pathosystem.

Plant responses against pathogens are influenced by growth immunity tradeoff, which ensure the best use of limited resources. We study how the immobilization of carbon resources and the induction of defensive responses (glucosinolates, phenolic compounds, stomatal closure) can influence the biomass of two Brassica oleracea lines, differing in their resistance, after infection with Xanthomonas campestris pv. campestris. Potentially, the growth immunity tradeoff can be influenced by the activation of all these processes. However, on the contrary of which is normally stated, our results suggest that the loss of biomass caused by pathogen infection is mainly due to the differential accumulation of starch and the immobilization of sugars rather than the reallocation of resources to synthesize secondary metabolites. Moreover, resistance may be related to the effectiveness of the tradeoff, since the resistant line immobilizes resources more efficiently than the susceptible one. Both inbred lines show a different phytohormones profile, which support the hypothesis that they are employing different strategies to defend themselves against the pathogen. This study emphasizes the key role of the primary metabolism in the defence strategies of plants against pathogens.

Fine mapping identifies NAD-ME1 as a candidate underlying a major locus controlling temporal variation in primary and specialized metabolism in Arabidopsis.

Plant metabolism is modulated by a complex interplay between internal signals and external cues. A major goal of all quantitative metabolomic studies is to clone the underlying genes to understand the mechanistic basis of this variation. Using fine-scale genetic mapping, in this work we report the identification and initial characterization of NAD-DEPENDENT MALIC ENZYME 1 (NAD-ME1) as the candidate gene underlying the pleiotropic network Met.II.15 quantitative trait locus controlling variation in plant metabolism and circadian clock outputs in the Bay × Sha Arabidopsis population. Transcript abundance and promoter analysis in NAD-ME1Bay-0 and NAD-ME1Sha alleles confirmed allele-specific expression that appears to be due a polymorphism disrupting a putative circadian cis-element binding site. Analysis of transfer DNA insertion lines and heterogeneous inbred families showed that transcript variation of the NAD-ME1 gene led to temporal shifts of tricarboxylic acid cycle intermediates, glucosinolate (GSL) accumulation, and altered regulation of several GSL biosynthesis pathway genes. Untargeted metabolomic analyses revealed complex regulatory networks of NAD-ME1 dependent upon the daytime. The mutant led to shifts in plant primary metabolites, cell wall components, isoprenoids, fatty acids, and plant immunity phytochemicals, among others. Our findings suggest that NAD-ME1 may act as a key gene to coordinate plant primary and secondary metabolism in a time-dependent manner.

Apparent inhibition of induced plant volatiles by a fungal pathogen prevents airborne communication between potato plants.

Plant communication in response to insect herbivory has been increasingly studied, whereas that involving pathogen attack has received much less attention. We tested for communication between potato (Solanum tuberosum) plants in response to leaf infection by the fungal pathogen Sclerotinia sclerotiorum. To this end, we measured the total amount and composition of volatile organic compounds (VOCs) produced by control and infected emitter plants, as well as tested for induced resistance of receiver plants exposed to VOCs from emitters. We further tested for changes in the expression of defensive genes due to pathogen infection. Fungal infection did not significantly affect the total amount or composition of VOCs produced by emitter plants. Correspondingly, we found no evidence of higher resistance to the pathogen in receiver plants exposed to VOCs from infected emitters relative to control emitters. Molecular analyses indicated that pathogen infection drove a down-regulation of genes coding for VOC precursors, potentially explaining the absence of pathogen effects on VOC emissions and thus of communication. Overall, these results indicate no evidence of airborne communication between potato plants in response to fungal infection and point at pathogen inhibition of VOC emissions as a likely explanation for this result.

Role of Major Glucosinolates in the Defense of Kale Against Sclerotinia sclerotiorum and Xanthomonas campestris pv. campestris.

Glucosinolates (GSLs) are secondary metabolites present in Brassicaceae species implicated in their defense against plant pathogens. When a pathogen causes tissue damage, the enzyme myrosinase hydrolyzes GSLs into diverse products that exhibit antimicrobial activity against a wide range of bacteria and fungi in vitro. It was demonstrated that modulation of GSL content in vivo affects plant resistance to infection by pathogens in Arabidopsis. However, the roles of specific metabolites and how they interact with pathogens are poorly understood in Brassica crops. We previously developed a set of populations of Brassica oleracea var. acephala L. (kale) differing in content of three GSLs: the aliphatics sinigrin (2-propenyl [SIN]) and glucoiberin (3-methylsulphinylpropyl [GIB]) and the indolic glucobrassicin (3-indolylmethyl [GBS]). These populations can be used to study the effects of major GSLs in kale, with the advantage that genotypes within each selection have the same genetic background. This research aimed to explore the role of SIN, GIB, and GBS in the defense of kale against the necrotrophic fungus Sclerotinia sclerotiorum and the bacterium Xanthomonas campestris pv. campestris. Results showed that increasing the amount of a particular GSL did not always result in disease resistance. The effects of GSLs were apparently dependent on the pathogen and the type of GSL. Thus, the aliphatic SIN was inhibitory to infection by S. sclerotiorum and the indolic GBS was inhibitory to infection by X. campestris pv. campestris. Other factors, including the quantity and proportion of other metabolites modified during the pathogen infection process, could also modulate the degree of inhibition to the pathogen.

Glucosinolate-Degradation Products as Co-Adjuvant Therapy on Prostate Cancer in Vitro.

Glucosinolate-degradation products (GS-degradation products) are believed to be responsible for the anticancer effects of cruciferous vegetables. Furthermore, they could improve the efficacy and reduce side-effects of chemotherapy. The aim of the present study was to determine the cytotoxic effects of GS-degradation products on androgen-insensitive human prostate cancer (AIPC) PC-3 and DU 145 cells and investigate their ability to sensitize such cells to chemotherapeutic drug Docetaxel (DOCE). Cells were cultured under growing concentrations of allyl-isothiocyanate (AITC), sulforaphane (SFN), 4-pentenyl-isothiocyanate (4PI), iberin (IB), indole-3-carbinol (I3C), or phenethyl-isothiocyanate (PEITC) in absence or presence of DOCE. The anti-tumor effects of these compounds were analyzed using the trypan blue exclusion, apoptosis, invasion and RT-qPCR assays and confocal microscopy. We observed that AITC, SFN, IB, and/or PEITC induced a dose- and time-dependent cytotoxic effect on PC-3 and DU 145 cells, which was mediated, at least, by apoptosis and cell cycle arrest. Likewise, we showed that these GS-degradation products sensitized both cell lines to DOCE by synergic mechanisms. Taken together, our results indicate that GS-degradation products can be promising compounds as co-adjuvant therapy in prostate cancer.

Omics Approach to Identify Factors Involved in Brassica Disease Resistance.

Understanding plant's defense mechanisms and their response to biotic stresses is of fundamental meaning for the development of resistant crop varieties and more productive agriculture. The Brassica genus involves a large variety of economically important species and cultivars used as vegetable source, oilseeds, forage and ornamental. Damage caused by pathogens attack affects negatively various aspects of plant growth, development, and crop productivity. Over the last few decades, advances in plant physiology, genetics, and molecular biology have greatly improved our understanding of plant responses to biotic stress conditions. In this regard, various 'omics' technologies enable qualitative and quantitative monitoring of the abundance of various biological molecules in a high-throughput manner, and thus allow determination of their variation between different biological states on a genomic scale. In this review, we have described advances in 'omic' tools (genomics, transcriptomics, proteomics and metabolomics) in the view of conventional and modern approaches being used to elucidate the molecular mechanisms that underlie Brassica disease resistance.

Effects of Brassicaceae Isothiocyanates on Prostate Cancer.

Despite the major progress made in the field of cancer biology, cancer is still one of the leading causes of mortality, and prostate cancer (PCa) is one of the most encountered malignancies among men. The effective management of this disease requires developing better anticancer agents with greater efficacy and fewer side effects. Nature is a large source for the development of chemotherapeutic agents, with more than 50% of current anticancer drugs being of natural origin. Isothiocyanates (ITCs) are degradation products from glucosinolates that are present in members of the family Brassicaceae. Although they are known for a variety of therapeutic effects, including antioxidant, immunostimulatory, anti-inflammatory, antiviral and antibacterial properties, nowadays, cell line and animal studies have additionally indicated the chemopreventive action without causing toxic side effects of ITCs. In this way, they can induce cell cycle arrest, activate apoptosis pathways, increase the sensitivity of resistant PCa to available chemodrugs, modulate epigenetic changes and downregulate activated signaling pathways, resulting in the inhibition of cell proliferation, progression and invasion-metastasis. The present review summarizes the chemopreventive role of ITCs with a particular emphasis on specific molecular targets and epigenetic alterations in in vitro and in vivo cancer animal models.

Effect of temperature stress on the early vegetative development of Brassica oleracea L.

BACKGROUND: Due to its biennual life cycle Brassica oleracea is especially exposed to seasonal changes in temperature that could limit its growth and fitness. Thermal stress could limit plant growth, leaf development and photosynthesis. We evaluated the performance of two local populations of B. oleracea: one population of cabbage (B. oleracea capitata group) and one population of kale (B. oleracea acephala group) under limiting low and high temperatures. RESULTS: There were differences between crops and how they responded to high and low temperature stress. Low temperatures especially affect photosynthesis and fresh weight. Stomatal conductance and the leaf water content were dramatically reduced and plants produce smaller and thicker leaves. Under high temperatures there was a reduction of the weight that could be associated to a general impairment of the photosynthetic activity. CONCLUSIONS: Although high temperatures significantly reduced the dry weight of seedlings, in general terms, low temperature had a higher impact in B. oleracea physiology than high temperature. Interestingly, our results suggest that the capitata population is less sensitive to changes in air temperature than the acephala population.

Bottom-up and top-down herbivore regulation mediated by glucosinolates in Brassica oleracea var. acephala.

Quantitative differences in plant defence metabolites, such as glucosinolates, may directly affect herbivore preference and performance, and indirectly affect natural enemy pressure. By assessing insect abundance and leaf damage rate, we studied the responses of insect herbivores to six genotypes of Brassica oleracea var. acephala, selected from the same cultivar for having high or low foliar content of sinigrin, glucoiberin and glucobrassicin. We also investigated whether the natural parasitism rate was affected by glucosinolates. Finally, we assessed the relative importance of plant chemistry (bottom-up control) and natural enemy performance (top-down control) in shaping insect abundance, the ratio of generalist/specialist herbivores and levels of leaf damage. We found that high sinigrin content decreased the abundance of the generalist Mamestra brassicae (Lepidoptera, Noctuidae) and the specialist Plutella xylostella (Lepidoptera, Yponomeutidae), but increased the load of the specialist Eurydema ornatum (Hemiptera, Pentatomidae). Plants with high sinigrin content suffered less leaf injury. The specialist Brevicoryne brassicae (Hemiptera, Aphididae) increased in plants with low glucobrassicin content, whereas the specialists Pieris rapae (Lepidoptera, Pieridae), Aleyrodes brassicae (Hemiptera, Aleyrodidae) and Phyllotreta cruciferae (Coleoptera, Chrysomelidae) were not affected by the plant genotype. Parasitism rates of M. brassicae larvae and E. ornatum eggs were affected by plant genotype. The ratio of generalist/specialist herbivores was positively correlated with parasitism rate. Although both top-down and bottom-up forces were seen to be contributing, the key factor in shaping both herbivore performance and parasitism rate was the glucosinolate concentration, which highlights the impact of bottom-up forces on the trophic cascades in crop habitats.

Mating system of Brassica napus and its relationship with morphological and ecological parameters in northwestern Spain.

Mating systems play a central role in determining population genetic structure and the methods to be used to develop new cultivars and preserve the variability of a crop. A Brassica napus crop called nabicol is grown in northwestern Spain. Knowledge on its mating system is needed in order to manage the germplasm correctly and design breeding strategies. The aims of this work were to study the mating system of nabicol under field conditions and the relationship of different traits with the mating system. We analyzed 2 populations with microsatellites using a multilocus approach, finding that both had a mixed mating system with an outcrossing rate of 30%. This system would allow application of breeding methods for both autogamous and allogamous species in order to improve nabicol populations. Nabicol populations should be multiplied in isolation conditions in the same way as allogamous species in order to avoid contamination and preserve genetic integrity. The relationship of outcrossing rate, phenological, ecological, and morphological traits was studied, but the model explained only a small percentage of the variability. None of the traits studied could be used as indirect selection criteria for a type of mating system under the conditions of northwestern Spain. This is the first work that studies in depth the possible causes of the mixed mating system of B. napus, finding that, surprisingly, it is not related to the most obvious factors.

In vivo and in vitro effects of secondary metabolites against Xanthomonas campestris pv. campestris.

Brassica rapa is a crucifer that is grown worldwide, mainly as a vegetable. The quality of B. rapa crops is highly affected by the disease caused by the bacteria Xanthomonas campestris pv. campestris (Xcc). Glucosinolates and phenolic compounds can confer resistance to Brassica crops against pests and diseases, but few works have been done to evaluate their role in Xcc resistance. The objectives of this work were: (1) to evaluate the in vivo and in vitro antibacterial effect of gluconapin, its isothiocyanate and the methanolic extracts of B. rapa against the type 4 of Xcc, and (2) to test if there is induced resistance mediated by glucosinolates or phenolic compounds in two varieties of B. rapa. Gluconapin and its ITC varieties had an antibacterial effect on the development of Xanthomonas and this effect was strongly dependent on the concentration applied. Methanolic extracts from B. rapa, containing glucosinolates and phenolic compounds, inhibited the growth of these bacteria. Concentration of gluconapin is higher in resistant plants than in the susceptible ones and there is an induction of gluconapin, some flavonoids and sinapic acid 48 to 72 h after inoculation. Gluconapin plays a role in the constitutive resistance to Xcc, while gluconapin, some flavonoids and hydroxycinnamic acids are induced by a Xcc infection but it is not clear if this induction confers resistance to this disease.

Spectral Reflectance Indexes Reveal Differences in the Physiological Status of Brassica oleracea with Contrasting Glucosinolate Content under Biotic Stress.

Brassica species produce glucosinolates, a specific group of secondary metabolites present in the Brassicaceae family with antibacterial and antifungal properties. The employment of improved varieties for specific glucosinolates would reduce the production losses caused by pathogen attack. However, the consequences of the increment in these secondary metabolites in the plant are unknown. In this work, we utilized reflectance indexes to test how the physiological status of Brasica oleracea plants changes depending on their constitutive content of glucosinolates under nonstressful conditions and under the attack of the bacteria Xanthomonas campestris pv. campestris and the fungus Sclerotinia sclerotiorum. The modification in the content of glucosinolates had consequences in the resistance to both necrotrophic pathogens, and in several physiological aspects of the plants. By increasing the content in sinigrin and glucobrassicin, plants decrease photosynthesis efficiency (PR531, FvFm), biomass production (CHL-NDVI, SR), pigment content (SIPI, NPQI, RE), and senescence (YI) and increase their water content (WI900). These variables may have a negative impact in the productivity of crops in an agricultural environment. However, when plants are subjected to the attack of both necrotrophic pathogens, an increment of sinigrin and glucobrassicin confers an adaptative advantage to the plants, which compensates for the decay of physiological parameters.

Unveiling plant defense arsenal: metabolic strategies in Brassica oleracea during black rot disease.

Alterations in plant metabolism play a key role in the complex plant-pathogen interactions. However, there is still a lack of knowledge about the connection between changes in primary and specialized metabolism and the plant defense against diseases that impact crops. Thus, we aim to study the metabolic reprograming in Brassica oleracea plants upon infection by Xanthomonas campestris pv. campestris (Xcc). To accomplish this, we utilized a combination of untargeted and targeted metabolomics, through UPLC-Q-TOF-MS/MS and 1H-NMR, in two crop lines differing in resistance that were evaluated at two- and four-week intervals following inoculation (T1 and T2, respectively). Besides, to depict the physiological status of the plant during infection, enzymatic activities related to the carbohydrate pathway and oxidative stress were studied. Our results revealed different temporal dynamics in the responses of the susceptible vs. resistant crops lines. Resistant B. oleracea line suppresses carbohydrate metabolism contributing to limit nutrient supplies to the bacterium and prioritizes the induction of defensive compounds such as indolic glucosinolates, salicylic acid, phenylpropanoids and phytoalexins precursors at early infection stages. In contrast, the susceptible line invests in carbohydrate metabolism, including enzymatic activities related to the hexoses turnover, and activates defense signaling related to reactive oxygen species. Thus, each line triggers a different metabolic strategy that will affect how the plant overcomes the disease in terms of resistance and growth. This work provides first insights of a fine-tuned metabolic regulation during Xcc infection in B. oleracea that will contribute to develop new strategies for plant disease management.

Two proteins with different functions are derived from the KlHEM13 gene.

Two proteins that differ at the N terminus (l-KlCpo and s-KlCpo) are derived from KlHEM13, a single-copy-number gene in the haploid genome of Kluyveromyces lactis. Two transcriptional start site (tss) pools are detectable using primer extension, and their selection is heme dependent. One of these tss pools is located 5' of the first translation initiation codon (TIC) in the open reading frame of KlHEM13, while the other is located between the first and second TICs. In terms of functional significance, only s-KlCpo complements the heme deficiency caused by the Δhem13 deletion in K. lactis. Data obtained from immune detection in subcellular fractions, directed mutagenesis, chromatin immunoprecipitation (ChIP) assays, and the functional relevance of ΔKlhem13 deletion for KlHEM13 promoter activity suggest that l-KlCpo regulates KlHEM13 transcription. A hypothetical model of the evolutionary origins and coexistence of these two proteins in K. lactis is discussed.

Glucosinolate variation in leaves of Brassica rapa crops.

Total and individual glucosinolate (GSL) content of leaves of vegetable turnip rape (Brassica rapa L. var. rapa) was determined in a set of 45 varieties consisting in early, medium and late types grown at two locations in northwestern Spain. The objectives were to determine the diversity among varieties in GSL content and to relate that variation with earliness and plant habit. Eight GSL were identified, being two aliphatic GSL, gluconapin (84.4 % of the total GSL) and glucobrassicanapin (7.2 % of the total GSL) the most abundant. Indolic and aromatic GSL content were low but also showed significant differences among varieties. Differences in total and individual GSL content were found among varieties, plant habit groups, and earliness groups. Total GSL content ranged from 19 to 37.3 µmol g(-1) dw in early and extra-late groups, respectively, and from 19.5 to 36.3 µmol g(-1) dw for turnips and turnip greens groups, respectively. These differences were consistent to values found for gluconapin content where the turnip group had the highest values (31.8 µmol g(-1) dw) and the turnip top group had the lowest (15.7 µmol g(-1) dw). Two varieties, MBG-BRS0429 and MBG-BRS0550 (from turnip greens and extra-late groups) and MBG-BRS0438 (from turnips and late groups), stood out as they had the highest total GSL content and could be used as a good source of these beneficial bioactive compounds. Elucidation of genetic diversity among crops can provide useful information to assist plant breeders to design improved breeding strategies in order to obtain varieties rich on GSL.

Transcriptomic Reprograming of Xanthomonas campestris pv. campestris after Treatment with Hydrolytic Products Derived from Glucosinolates.

The bacterium Xanthomonas campestris pv. campestris (Xcc) causes black rot disease in Brassica crops. Glucosinolates are known to be part of the defence system of Brassica crops against Xcc infection. They are activated upon pathogen attack by myrosinase enzymes. Their hydrolytic products (GHPs) inhibit the growth of Xcc in vitro. However, the mechanisms underlying this inhibition and the way Xcc can overcome it are not well understood. We studied the transcriptomic reprogramming of Xcc after being supplemented with two chemically different GHPs, one aliphatic isothiocyanate (allyl-ITC) and one indole (indol-3-carbinol), by RNA-seq. Based on our results, the arrest in Xcc growth is related to the need to stop cell division to repair damaged DNA and cell envelope components. Otherwise, GHPs modify energy metabolism by inhibiting aerobic respiration and increasing the synthesis of glycogen. Xcc induces detoxification mechanisms such as the antioxidant defence system and the multidrug efflux system to cope with the toxic effects driven by GHPs. This is the first time that the transcriptomic reprogramming of a plant pathogenic bacterium treated with GHPs has been studied. This information will allow a better understanding of the interaction of a plant pathogen mediated by GSLs.

Sclerotinia sclerotiorum Response to Long Exposure to Glucosinolate Hydrolysis Products by Transcriptomic Approach.

White mold disease, caused by the necrotrophic fungus Sclerotinia sclerotiorum, affects Brassica crops. Brassica crops produce a broad array of compounds, such as glucosinolates, which contribute to the defense against pathogens. From their hydrolysis, several products arise that have antimicrobial activity (GHPs) whose toxicity is structure dependent. S. sclerotiorum may overcome the toxic effect of moderate GHP concentrations after prolonged exposure to their action. Our objective was to identify the molecular mechanism underlying S. sclerotiorum response to long exposure to two chemically diverse GHPs: aliphatic GHP allyl-isothiocyanate (AITC) and indole GHP indol-3-carbinol (I3C). We found that the transcriptomic response is dependent on the type of GHP and on their initial target, involving cell membranes in the case of AITC or DNA in the case of I3C. Response mechanisms include the reorganization of chromatin, mediated by histone chaperones hip4 and cia1, ribosome synthesis controlled by the kinase-phosphatase pair aps1-ppn1, catabolism of proteins, ergosterol synthesis, and induction of detoxification systems. These mechanisms probably help S. sclerotiorum to grow and survive in an environment where GHPs are constantly produced by Brassica plants upon glucosinolate breakdown. IMPORTANCEBrassica species, including important vegetable crops, such as cabbage, cauliflower, or broccoli, or oil crops, such as rapeseed, produce specific chemical compounds useful to protect them against pests and pathogens. One of the most destructive Brassica diseases in temperate areas around the world is Sclerotinia stem rot, caused by the fungus Sclerotinia sclerotiorum. This is a generalist pathogen that causes disease over more than 400 plant species, being a serious threat to economically important crops worldwide, including potato, bean, soybean, and sunflower, among many others. Understanding the mechanisms utilized by pathogens to overcome specific plant defensive compounds can be useful to increase plant resistance. Our study demonstrated that Sclerotinia shows different adaptation mechanisms, including detoxification systems, to grow and survive when plant protective compounds are present.

Trichoderma hamatum Increases Productivity, Glucosinolate Content and Antioxidant Potential of Different Leafy Brassica Vegetables.

Brassica crops include important vegetables known as "superfoods" due to the content of phytochemicals of great interest to human health, such as glucosinolates (GSLs) and antioxidant compounds. On the other hand, Trichoderma is a genus of filamentous fungi that includes several species described as biostimulants and/or biological control agents in agriculture. In a previous work, an endophytic strain of Trichoderma hamatum was isolated from kale roots (Brassica oleracea var. acephala), describing its ability to induce systemic resistance in its host plant. In the present work, some of the main leafy Brassica crops (kale, cabbage, leaf rape and turnip greens) have been root-inoculated with T. hamatum, having the aim to verify the possible capacity of the fungus as a biostimulant in productivity as well as the foliar content of GSLs and its antioxidant potential, in order to improve these "superfoods". The results reported, for the first time, an increase in the productivity of kale (55%), cabbage (36%) and turnip greens (46%) by T. hamatum root inoculation. Furthermore, fungal inoculation reported a significant increase in the content of total GSLs in cabbage and turnip greens, mainly of the GSLs sinigrin and gluconapin, respectively, along with an increase in their antioxidant capacity. Therefore, T. hamatum could be a good agricultural biostimulant in leafy Brassica crops, increasing the content of GSLs and antioxidant potential of great food and health interest.

New Vegetable Brassica Foods: A Promising Source of Bioactive Compounds.

Brassica rapa is grown in northwestern Spain to obtain turnip greens. The tops of the same plants (flower stems with buds) are cut and sell as turnip tops, increasing the value of the crop. This practice could be extended to other brassicas. The objectives of this work are to study the phytochemical potential of tops of coles (Brassica oleracea) and leaf rape (Brassica napus) compared to turnip tops and to compare tops of different coles (cabbage, kale, tronchuda cabbage), which differ in their morphology and use. We evaluated the content of glucosinolates and phenolic compounds and the antioxidant capacity in leaves and tops of the three species. We found that tops had higher amount of glucosinolates than leaves. Phenolic content and antioxidant capacity followed the opposite trend. Therefore, consumption of leaves and tops are complementary, since both type of organs are enriched with different types of compound. Local varieties of kale, curly kale, cabbage and curly leave cabbage are interesting because of their GSLs and phenolic content and antioxidant capacity in both leaves and tops. From the human health perspective, tops of coles and leaf rape are interesting as new crops to include in the diet.

Ixr1p regulates oxygen-dependent HEM13 transcription.

In Saccharomyces cerevisiae, HEM13 encodes the enzyme coproporphyrinogen III oxidase, which catalyzes the rate-limiting step in heme biosynthesis. HEM13 is a regulated hypoxic gene repressed by Rox1p and Mot3p under aerobic conditions. In this study, we further investigate the hypoxic expression of HEM13, focusing on the promoter regions that are functionally important during hypoxia and on the effect of deleting the transcriptional regulators Sut1p, Sut2p, Upc2p, Ecm22p and Ixr1p. Ixr1p is necessary for the high expression of HEM13 under hypoxic conditions and its function is exerted in vivo through the HEM13 promoter region extending from -577 to -419. Ixr1p binds in vivo to the HEM13 promoter both under aerobic and under hypoxic conditions. Purified Ixr1p binds in vitro to two sequences extending from -534 to -509 and from -497 to -450, respectively. These DNA regions compete for Ixr1p binding and the consensus KTTSAAYKGTTYASA is important for the regulatory protein to interact. These results suggest that the regulation of HEM13 expression is dependent on two proteins with high mobility group (HMG) domains: Rox1p and Ixr1p. Their interactions with the HEM13 promoter might change in the transition from aerobiosis to hypoxia.