Highly effective mlo-based powdery mildew resistance in hexaploid wheat without pleiotropic effects.

Application of the mlo-based resistance in barley against powdery mildew attacks is a major success in crop breeding, since it confers durable disease resistance. Resistance caused by mutations in the Mlo gene seems to be ubiquitous across a range of species. This work addresses the introduction of mlo-based resistance into hexaploid wheat, which is complicated by the occurrence of three homoeologous genes: Mlo-A1, Mlo-B1 and Mlo-D1. EMS-generated mutant plants were screened for mutations in the three homoeologues. We selected and combined 6, 8, and 4 mutations, respectively, to obtain triple homozygous mlo mutant lines. Twenty-four mutant lines showed highly effective resistance towards attack by the powdery mildew pathogen under field conditions. All 18 mutations appeared to contribute to resistance; however, they had different effects on the occurrence of symptoms such as chlorotic and necrotic spots, which are pleiotropic to the mlo-based powdery mildew resistance. We conclude that to obtain highly effective powdery mildew resistance in wheat and to avoid detrimental pleiotropic effects, all three Mlo homoeologues should be mutated; however, at least one of the mutations should be of the weaker type in order to alleviate strong pleiotropic effects from the other mutations.

The pho1;2a'-m1.1 allele of Phosphate1 conditions misregulation of the phosphorus starvation response in maize (Zea mays ssp. mays L.).

Plant PHO1 proteins play a central role in the translocation and sensing of inorganic phosphate. The maize (Zea mays ssp. mays) genome encodes two co-orthologs of the Arabidopsis PHO1 gene, designated ZmPho1;2a and ZmPho1;2b. Here, we report the characterization of the transposon footprint allele Zmpho1;2a'-m1.1, which we refer to hereafter as pho1;2a. The pho1;2a allele is a stable derivative formed by excision of an Activator transposable element from the ZmPho1;2a gene. The pho1;2a allele contains an 8-bp insertion at the point of transposon excision that disrupts the reading frame and is predicted to generate a premature translational stop. We show that the pho1;2a allele is linked to a dosage-dependent reduction in Pho1;2a transcript accumulation and a mild reduction in seedling growth. Characterization of shoot and root transcriptomes under full nutrient, low nitrogen, low phosphorus, and combined low nitrogen and low phosphorus conditions identified 1100 differentially expressed genes between wild-type plants and plants carrying the pho1;2a mutation. Of these 1100 genes, 966 were upregulated in plants carrying pho1;2a, indicating the wild-type PHO1;2a to predominantly impact negative gene regulation. Gene set enrichment analysis of the pho1;2a-misregulated genes revealed associations with phytohormone signaling and the phosphate starvation response. In roots, differential expression was broadly consistent across all nutrient conditions. In leaves, differential expression was largely specific to low phosphorus and combined low nitrogen and low phosphorus conditions. Of 276 genes upregulated in the leaves of pho1;2a mutants in the low phosphorus condition, 153 were themselves induced in wild-type plants with respect to the full nutrient condition. Our observations suggest that Pho1;2a functions in the fine-tuning of the transcriptional response to phosphate starvation through maintenance and/or sensing of plant phosphate status.

Expression of thurincin H, ChiA74 and Cry proteins at the sporulation phase in Bacillus thuringiensis HD1.

AIMS: The objective of this study was to produce thurincin H, ChiA74 and Cry proteins together using Bacillus thuringiensis subsp. kurstaki HD1 as a heterologous host. METHODS AND RESULTS: pSTAB-ThurH and pSTAB-ChiA74 constructs were designed to produce thurincin H and chitinase, respectively, at the sporulation phase. They were transformed into Bt HD1 generating the recombinant strains HD1/pSTAB-ThurH and HD1/pSTAB-ThurH/pSTAB-ChiA74. Antimicrobial and chitinolytic activity tests were performed with recombinant strains. Both strains were able to produce thurincin H up to 72 h with antibacterial activity of ~4000 U mg-1 . The HD1/pSTAB-ThurH/pSTAB-ChiA74 strain also showed chitinolytic activity of ~23 mU mg-1 at 72 h. All B. thuringiensis strains exhibited crystal formation at 72, and 96 h. In addition, the application of thurincin H in corn seeds increased the germination percentage and root length by 7% and 10%, respectively. CONCLUSIONS: We showed that is possible to produce three proteins of biotechnological interest at the sporulation stage in B. thuringiensis, which two of them (thurincin H, and ChiA74) are naturally expressed in the vegetative stage. SIGNIFICANCE AND IMPACT OF THE STUDY: These results form the basis for developing of a biocontrol and biostimulator product that can be used as an alternative for chemical application.

Low nitrogen availability inhibits the phosphorus starvation response in maize (Zea mays ssp. mays L.).

BACKGROUND: Nitrogen (N) and phosphorus (P) are macronutrients essential for crop growth and productivity. In cultivated fields, N and P levels are rarely sufficient, contributing to the gap between realized and potential production. Fertilizer application increases nutrient availability, but is not available to all farmers, nor are current rates of application sustainable or environmentally desirable. Transcriptomic studies of cereal crops have revealed dramatic responses to either low N or low P single stress treatments. In the field, however, levels of both N and P may be suboptimal. The interaction between N and P starvation responses remains to be fully characterized. RESULTS: We characterized growth and root and leaf transcriptomes of young maize plants under nutrient replete, low N, low P or combined low NP conditions. We identified 1555 genes to respond to our nutrient treatments, in one or both tissues. A large group of genes, including many classical P starvation response genes, were regulated antagonistically between low N and P conditions. An additional experiment over a range of N availability indicated that a mild reduction in N levels was sufficient to repress the low P induction of P starvation genes. Although expression of P transporter genes was repressed under low N or low NP, we confirmed earlier reports of P hyper accumulation under N limitation. CONCLUSIONS: Transcriptional responses to low N or P were distinct, with few genes responding in a similar way to the two single stress treatments. In combined NP stress, the low N response dominated, and the P starvation response was largely suppressed. A mild reduction in N availability was sufficient to repress the induction of P starvation associated genes. We conclude that activation of the transcriptional response to P starvation in maize is contingent on N availability.

Identification of the maize Mediator CDK8 module and transposon-mediated mutagenesis of ZmMed12a.

Mediator is a conserved transcriptional co-activator that links transcription factors bound at enhancer elements to RNA Polymerase II. Mediator-RNA Polymerase II interactions can be sterically hindered by the Cyclin Dependent Kinase 8 (CDK8) module, a submodule of Mediator that acts to repress transcription in response to discrete cellular and environmental cues. The CDK8 module is conserved in all eukaryotes and consists of 4 proteins: CDK8, CYCLIN C (CYCC), MED12, and MED13. In this study, we have characterized the CDK8 module of Mediator in maize using genomic, molecular and functional resources. The maize genome contains single copy genes for Cdk8, CycC, and Med13, and two genes for Med12. Analysis of expression data for the CDK8 module demonstrated that all five genes are broadly expressed in maize tissues, and change their expression in response to phosphate and nitrogen limitation. We performed Dissociation (Ds) insertional mutagenesis, recovering two independent insertions in the ZmMed12a gene, one of which produces a truncated transcript. Our molecular identification of the maize CDK8 module, assays of CDK8 module expression under nutrient limitation, and characterization of transposon insertions in ZmMed12a establish the basis for molecular and functional studies of the role of these important transcriptional regulators in development and nutrient homeostasis in Zea mays.

The Phosphoglycerate Kinase (PGK) Gene Family of Maize (Zea mays var. B73).

Phosphoglycerate kinase (PGK, E.C. 2.7.2.3) interconverts ADP + 1,3-bisphospho-glycerate (1,3-bPGA) to ATP + 3-phosphoglycerate (3PGA). While most bacteria have a single pgk gene and mammals possess two copies, plant genomes contain three or more PGK genes. In this study, we identified five Pgk genes in the Zea mays var. B73 genome, predicted to encode proteins targeted to different subcellular compartments: ZmPgk1, ZmPgk2, and ZmPgk4 (chloroplast), ZmPgk3 (cytosol), and ZmPgk5 (nucleus). The expression of ZmPgk3 was highest in non-photosynthetic tissues (roots and cobs), where PGK activity was also greatest, consistent with a function in glycolysis. Green tissues (leaf blade and husk leaf) showed intermediate levels of PGK activity, and predominantly expressed ZmPgk1 and ZmPgk2, suggesting involvement in photosynthetic metabolism. ZmPgk5 was weakly expressed and ZmPgk4 was not detected in any tissue. Phylogenetic analysis showed that the photosynthetic and glycolytic isozymes of plants clustered together, but were distinct from PGKs of animals, fungi, protozoa, and bacteria, indicating that photosynthetic and glycolytic isozymes of plants diversified after the divergence of the plant lineage from other groups. These results show the distinct role of each PGK in maize and provide the basis for future studies into the regulation and function of this key enzyme.

Changes in spatiotemporal protein and amino acid gradients in wheat caryopsis after N-topdressing.

Wheat grain nitrogen content displays large variations within different pearling fractions of grains because of radial gradients in the protein content. We identified how spatiotemporal mechanisms regulate this. The protein gradients emerged clearly at 19 days after anthesis, with the highest N content in aleurone and seed coat, followed by outer endosperm, whereas the lowest was in middle and inner endosperm. Laser microdissection, qRT-PCR and LC-MS were used to dissect tissue from aleurone, outer endosperm, middle endosperm, inner endosperm and transfer cells, measure gene expression and levels of free and protein-bound amino acids, respectively. The results showed that different FAA transportation pathways worked in parallel during grain filling stage while the grain protein gradient did not follow spatial expression of storage proteins. Additionally, two nitrogen (N) topdressing timings were conducted, either at the emergence of top third leaf (standard timing) or top first leaf (delayed timing), finding that delayed N topdressing enhanced both amino acids supply and protein synthesis capacity. The results provide insight into protein synthesis and amino acid transport pathways in endosperm and suggest targets for the enhancement of specialty pearled wheat with higher quality.

Development of mlo-based resistance in tetraploid wheat against wheat powdery mildew.

Powdery mildew is a severe disease in wheat. In barley, durable resistance exists, based on non-functionality of the Mlo gene. As a model to analyse the effects of mutagenesis in the homoeologous Mlo genes of wheat, we developed mlo-based powdery mildew resistance in tetraploid durum wheat. To obtain Mlo mutations, we screened a TILLING population developed in tetraploid wheat "Kronos" for which the captured exome sequence of  > 1500 lines is available. This resulted in 23 mutants for Mlo-A1 and 26 non-redundant mutants for Mlo-B1. Two Mlo-A1 and four Mlo-B1 mutants were crossed to obtain eight F2 mutant lines that showed a range of phenotypes from susceptibility to full resistance. Pot experiments under semi-field conditions confirmed the resistance levels for six of the mutants without any signs of adverse pleiotropic effects. Resistance ranking was similar across six powdery mildew isolates, indicating no isolate specificity of the mlo-based resistance. The effect of mutations in the Mlo-B1 gene was stronger than in the Mlo-A1 gene, probably reflecting differences in wild-type Mlo gene expression levels. Strong partial resistance effects were observed with single mlo-B1 mutations hence, revealing a dosage effect of mlo mutant alleles. Two of the four mlo-B1 mutations (W163* and P335L) were very strong; however, the highest combined effect was observed with the MloA-P335S/MloB-P335L combination, suggesting that non-functional, but full-length Mlo proteins might have the strongest effect compared with nonsense mutations. Our results show that mlo-based resistance might offer possibilities to introduce durable protection in tetraploid wheat against powdery mildew.

Genome wide characterization of barley NAC transcription factors enables the identification of grain-specific transcription factors exclusive for the Poaceae family of monocotyledonous plants.

The plant NAC transcription factors depict one of the largest plant transcription factor families. They regulate a wide range of different developmental processes and most probably played an important role in the evolutionary diversification of plants. This makes comparative studies of the NAC transcription factor family between individual species and genera highly relevant and such studies have in recent years been greatly facilitated by the increasing number of fully sequenced complex plant genomes. This study combines the characterization of the NAC transcription factors in the recently sequenced genome of the cereal crop barley with expression analysis and a comprehensive phylogenetic characterization of the NAC transcription factors in other monocotyledonous plant species. Our results provide evidence for the emergence of a NAC transcription factor subclade that is exclusively expressed in the grains of the Poaceae family of grasses. These notably comprise a number of cereal crops other than barley, such as wheat, rice, maize or millet, which are all cultivated for their starchy edible grains. Apparently, the grain specific subclade emerged from a well described subgroup of NAC transcription factors associated with the senescence process. A promoter exchange subsequently resulted in grain specific expression. We propose to designate this transcription factor subclade Grain-NACs and we discuss their involvement in programmed cell death as well as their potential role in the evolution of the Poaceae grain, which doubtlessly is of central importance for human nutrition.

AhDGR2, an amaranth abiotic stress-induced DUF642 protein gene, modifies cell wall structure and composition and causes salt and ABA hyper-sensibility in transgenic Arabidopsis.

MAIN CONCLUSION: An amaranth DGR gene, induced under abiotic stress, modifies cell wall structure and causes hypersensitivity to ABA and salt when overexpressed in Arabidopsis. DUF642 is a highly conserved plant-specific family of unknown cell wall-associated proteins. The AhDGR2 gene, coding for a DUF642 protein, was significantly induced in grain amaranth (Amaranthus hypochondriacus) plants subjected to water-deficit and salinity stress, thereby suggesting its participation in abiotic stress tolerance in this plant. A role in development was also inferred from the higher AhDGR2 expression rates detected in young tissues. Subsequent overexpression of AhDGR2 in transgenic Arabidopsis plants (OE-AhDGR2) supported its possible role in development processes. Thus, OE-AhDGR2 plants generated significantly longer roots when grown in normal MS medium. However, they showed a hypersensitivity to increasing concentrations of abscisic acid or NaCl in the medium, as manifested by shorter root length, smaller and slightly chlorotic rosettes, as well as highly reduced germination rates. Contrary to expectations, OE-AhDGR2 plants were intolerant to abiotic stress. Moreover, cell walls in transgenic plants were thinner, in leaves, and more disorganized, in roots, and had significantly modified pectin levels. Lower pectin methylesterase activity detected in leaves of OE-AhDGR2 plants, but not in roots, was contrary to previous reports associating DUF642 proteins and decreased pectin esterification levels in cell walls. Nonetheless, microarray data identified candidate genes whose expression levels explained the phenotypes observed in leaves of OE-AhDGR2 plants, including several involved in cell wall integrity and extension, growth and development, and resistance to abiotic stress. These results support the role of DUF642 proteins in cell wall-related processes and offer novel insights into their possible role(s) in plants.

Overexpression of Grain Amaranth (Amaranthus hypochondriacus) AhERF or AhDOF Transcription Factors in Arabidopsis thaliana Increases Water Deficit- and Salt-Stress Tolerance, Respectively, via Contrasting Stress-Amelioration Mechanisms.

Two grain amaranth transcription factor (TF) genes were overexpressed in Arabidopsis plants. The first, coding for a group VII ethylene response factor TF (i.e., AhERF-VII) conferred tolerance to water-deficit stress (WS) in transgenic Arabidopsis without affecting vegetative or reproductive growth. A significantly lower water-loss rate in detached leaves coupled to a reduced stomatal opening in leaves of plants subjected to WS was associated with this trait. WS tolerance was also associated with an increased antioxidant enzyme activity and the accumulation of putative stress-related secondary metabolites. However, microarray and GO data did not indicate an obvious correlation between WS tolerance, stomatal closure, and abscisic acid (ABA)-related signaling. This scenario suggested that stomatal closure during WS in these plants involved ABA-independent mechanisms, possibly involving reactive oxygen species (ROS). WS tolerance may have also involved other protective processes, such as those employed for methyl glyoxal detoxification. The second, coding for a class A and cluster I DNA binding with one finger TF (i.e., AhDof-AI) provided salt-stress (SS) tolerance with no evident fitness penalties. The lack of an obvious development-related phenotype contrasted with microarray and GO data showing an enrichment of categories and genes related to developmental processes, particularly flowering. SS tolerance also correlated with increased superoxide dismutase activity but not with augmented stomatal closure. Additionally, microarray and GO data indicated that, contrary to AhERF-VII, SS tolerance conferred by AhDof-AI in Arabidopsis involved ABA-dependent and ABA-independent stress amelioration mechanisms.

The overexpression of an Amaranthus hypochondriacus NF-YC gene modifies growth and confers water deficit stress resistance in Arabidopsis.

Nuclear factor-Y (NF-Y), is a plant heterotrimeric transcription factor constituted by NF-YA, NF-YB and NF-YC subunits. The function of many NF-Y subunits, mostly of the A and B type, has been studied in plants, but knowledge regarding the C subunit remains fragmentary. Here, a water stress-induced NF-YC gene from Amaranthus hypochondriacus (AhNF-YC) was further characterized by its overexpression in transgenic Arabidospis thaliana plants. A role in development was inferred from modified growth rates in root, rosettes and inflorescences recorded in AhNF-YC overexpressing Arabidopsis plants, in addition to a delayed onset of flowering. Also, the overexpression of AhNF-YC caused increased seedling sensitivity to abscisic acid (ABA), and influenced the expression of several genes involved in secondary metabolism, development and ABA-related responses. An altered expression of the latter in water stressed and recovered transgenic plants, together with the observed increase in ABA sensitivity, suggested that their increased water stress resistance was partly ABA-dependent. An untargeted metabolomic analysis also revealed an altered metabolite pattern, both in normal and water stress/recovery conditions. These results suggest that AhNF-YC may play an important regulatory role in both development and stress, and represents a candidate gene for the engineering of abiotic stress resistance in commercial crops.

The novel and taxonomically restricted Ah24 gene from grain amaranth (Amaranthus hypochondriacus) has a dual role in development and defense.

Grain amaranths tolerate stress and produce highly nutritious seeds. We have identified several (a)biotic stress-responsive genes of unknown function in Amaranthus hypochondriacus, including the so-called Ah24 gene. Ah24 was expressed in young or developing tissues; it was also strongly induced by mechanical damage, insect herbivory and methyl jasmonate and in meristems and newly emerging leaves of severely defoliated plants. Interestingly, an in silico analysis of its 1304 bp promoter region showed a predominance of regulatory boxes involved in development, but not in defense. The Ah24 cDNA encodes a predicted cytosolic protein of 164 amino acids, the localization of which was confirmed by confocal microscopy. Additional in silico analysis identified several other Ah24 homologs, present almost exclusively in plants belonging to the Caryophyllales. The possible function of this gene in planta was examined in transgenic Ah24 overexpressing Arabidopsis thaliana and Nicotiana tabacum plants. Transformed Arabidopsis showed enhanced vegetative growth and increased leaf number with no penalty in one fitness component, such as seed yield, in experimental conditions. Transgenic tobacco plants, which grew and reproduced normally, had increased insect herbivory resistance. Modified vegetative growth in transgenic Arabidopsis coincided with significant changes in the expression of genes controlling phytohormone synthesis or signaling, whereas increased resistance to insect herbivory in transgenic tobacco coincided with higher jasmonic acid and proteinase inhibitor activity levels, plus the accumulation of nicotine and several other putative defense-related metabolites. It is proposed that the primary role of the Ah24 gene in A. hypochondriacus is to contribute to a rapid recovery post-wounding or defoliation, although its participation in defense against insect herbivory is also plausible.