Maria L. Federico

Autophagic Recycling Plays a Central Role in Maize Nitrogen Remobilization

Characterization of maize autophagy through detailed transcriptome studies and phenotypic and 15N-partitioning analyses of atg12 mutants reveal a central role for autophagy in nitrogen remobilization. Autophagy is a primary route for nutrient recycling in plants by which superfluous or damaged cytoplasmic material and organelles are encapsulated and delivered to the vacuole for breakdown. Central to autophagy is a conjugation pathway that attaches AUTOPHAGY-RELATED8 (ATG8) to phosphatidylethanolamine, which then coats emerging autophagic membranes and helps with cargo recruitment, vesicle enclosure, and subsequent vesicle docking with the tonoplast. A key component in ATG8 function is ATG12, which promotes lipidation upon its attachment to ATG5. Here, we fully defined the maize (Zea mays) ATG system transcriptionally and characterized it genetically through atg12 mutants that block ATG8 modification. atg12 plants have compromised autophagic transport as determined by localization of a YFP-ATG8 reporter and its vacuolar cleavage during nitrogen or fixed-carbon starvation. Phenotypic analyses showed that atg12 plants are phenotypically normal and fertile when grown under nutrient-rich conditions. However, when nitrogen-starved, seedling growth is severely arrested, and as the plants mature, they show enhanced leaf senescence and stunted ear development. Nitrogen partitioning studies revealed that remobilization is impaired in atg12 plants, which significantly decreases seed yield and nitrogen-harvest index. Together, our studies demonstrate that autophagy, while nonessential, becomes critical during nitrogen stress and severely impacts maize productivity under suboptimal field conditions.

Spatial and Temporal Divergence of Expression in Duplicated Barley Germin-like Protein-encoding Genes

Subfunctionalization is the process by which a pair of duplicated genes, or paralogs, experiences a reduction of individual expression patterns or function while still reproducing the complete expression pattern and function of the ancestral gene. Two germin-like protein (GLP)-encoding genes, GerB and GerF, are paralogs that belong to a small gene family in barley (Hordeum vulgare). Both genes share high nucleotide sequence similarity in coding and noncoding regions and encode identical apoplastic proteins. The use of RNA gel blots, coupled with single-stranded conformation polymorphism (SSCP) analysis of RT–PCR products, elucidated the developmental and tissue-specific expression patterns of each gene. Individual expression patterns provided evidence of both overlapping redundancy and early subfunctionalization. GerB is predominantly expressed in developing shoots, while GerF is predominantly expressed in seedling roots, developing spikes, and pericarp/testa. GerF promoter deletion studies located a region (−356/−97) responsible for high promoter activity and showed the ability of GerB and GerF upstream regions to drive gfp expression in coleoptiles, epicarps, and lemma/palea of developing spikes. The observed expression patterns are consistent with proposed roles in plant development and defense mechanisms for this gene family. These roles may explain why redundancy has been selectively maintained in this duplicate gene pair.

The Genetics of Brassica napus

Brassica napus L. belongs to the Brassicaceae family of the Kingdom Plantae and is considered to be a newly formed species (5,000–10,000 mya) probably originating from independent and spontaneous inter-specific hybridizations between genotypes of turnip rape (Brassica rapa; AA, 2n = 20) and cabbage/Kale (Brassica oleracea; CC, 2n = 18). Genetically, B. napus is an allopolyploid (AACC, 2n = 38) exhibiting disomic inheritance. Within the species, two botanical varieties have been defined: B. napus L. var rapifera (DC) Metzger (2n = 4×= 38) and B. napus L. var oleifera Delile (2n = 4×= 38). The latter has taken much of the attention and has become the second most cultivated oilseed crop (rapeseed) worldwide, after soybean. The appearance of annual and biannual rapeseed lines with low erucic acid (<2% in the oil) and low glucosinolates (<30 mg/g in the meals) has granted rapeseed CanOLA (Canadian Oil Low Acid) status as an excellent source for edible vegetable oil. The lipid profile of CanOLA oil is extremely well balanced (low in saturated fats, high in monosaturated fats, and rich in omega-3 fatty acids) making it the oil of preference by nutritionists worldwide. In this context, the commercial interest for rapeseed CanOLA has launched an impressive amount of genetics and genomics research which has made possible to make genetic gains in agronomical and quality traits through modern plant breeding. In fact, rapeseed ranks among the top crops for which molecular tools have been developed. To date, over 30 molecular linkage maps have been published using a range of different molecular marker types, population structures, and parental lines exhibiting different flowering time behaviors. These maps have proved extremely useful in order to dissect the genetic nature of the traits underlying the genetic variation found in rapeseed. This chapter will focus on the genetics and genomics aspects of rapeseed breeding describing the current knowledge on the origin of B. napus, genetics/genomic tools for the species, and specific target traits affecting B. napus oil production and quality.

Cloning of the promoter for a novel barley gene, Lem1, and its organ-specific promotion of Gfp expression in lemma and palea

The differential display method was used to identify a novel barley gene, Lem1, expressed primarily in the outer organs (lemma and palea) that enclose developing florets and seeds. The promoter was isolated from a BAC genomic clone and used in a translational fusion with a green fluorescent protein gene (Gfp) to produce a transient expression vector. After particle bombardment, Gfp was expressed only in lemmas, paleas and awns of developing spikelets. Lem1 did not promote Gfp expression in vegetative leaves or in mature spikes, although expression of co-bombarded uidA (GUS) occurred under the regulation of a ubiquitin promoter. This reproduced the developmentally regulated pattern of mRNA accumulation. Deletion studies showed that the promoter activity is confined to a cis element within 80 bp of the transcription start site. Upstream from this, the promoter contains putative auxin-, ethylene- and gibberellin-responsive elements or homologues. Lem1 was found to be a single intronless gene encoding an acidic 102 amino acid protein, possibly associated with membranes. In a two-rowed barley, Lem1 mRNA was absent in the lateral spikelets, which fail to develop, and present only in the developing median spikelets. This suggests that Lem1 may play a role in organ development.

Genomic DNA Enrichment Using Sequence Capture Microarrays: a Novel Approach to Discover Sequence Nucleotide Polymorphisms (SNP) in Brassica napus L

Targeted genomic selection methodologies, or sequence capture, allow for DNA enrichment and large-scale resequencing and characterization of natural genetic variation in species with complex genomes, such as rapeseed canola (Brassica napus L., AACC, 2n=38). The main goal of this project was to combine sequence capture with next generation sequencing (NGS) to discover single nucleotide polymorphisms (SNPs) in specific areas of the B. napus genome historically associated (via quantitative trait loci –QTL– analysis) to traits of agronomical and nutritional importance. A 2.1 million feature sequence capture platform was designed to interrogate DNA sequence variation across 47 specific genomic regions, representing 51.2 Mb of the Brassica A and C genomes, in ten diverse rapeseed genotypes. All ten genotypes were sequenced using the 454 Life Sciences chemistry and to assess the effect of increased sequence depth, two genotypes were also sequenced using Illumina HiSeq chemistry. As a result, 589,367 potentially useful SNPs were identified. Analysis of sequence coverage indicated a four-fold increased representation of target regions, with 57% of the filtered SNPs falling within these regions. Sixty percent of discovered SNPs corresponded to transitions while 40% were transversions. Interestingly, fifty eight percent of the SNPs were found in genic regions while 42% were found in intergenic regions. Further, a high percentage of genic SNPs was found in exons (65% and 64% for the A and C genomes, respectively). Two different genotyping assays were used to validate the discovered SNPs. Validation rates ranged from 61.5% to 84% of tested SNPs, underpinning the effectiveness of this SNP discovery approach. Most importantly, the discovered SNPs were associated with agronomically important regions of the B. napus genome generating a novel data resource for research and breeding this crop species.

Status of antioxidant metabolites and enzymes in a catalase-deficient mutant of barley (Hordeum vulgare L.)

We have investigated the antioxidant status in RPr79/4, a CAT-deficient mutant of barley, and in its motherline, cv. Maris Mink. Seedlings of the CAT-deficient mutant that were grown in a growth chamber under a 14-h photoperiod (200 mol quanta m −2 s −1 ), exhibited higher concentrations of glutathione and ascorbate peroxidase as compared to wild-type plants. An additional mitochondrial MnSOD isoenzyme, was also detected in RPr79/4. When seedlings of the CAT-deficient mutant were grown at higher light intensities (370 mol quanta m − 2 s − 1 ), a Cu/ZnSOD isoform and the cytosolic glutamine synthetase isoenzyme were concomitantly induced. Taken together, these results suggest that several defense mechanisms operating in different subcellular compartments respond in concert to compensate for CAT deficiency in barley seedlings exposed to oxidative stress.

Catalase deficiency reduces survival and pleiotropically affects agronomic performance in field-grown barley progeny

Field-grown plants of the catalase-deficient mutant RPr79/4 show necrotic lesions in leaves and preferentially die. Initially, necrotic lesions exhibited by RPr79/4 were used to indirectly assess the role of distinct levels of catalase on the survival and agronomic performance of field-grown barley progeny. The segregation of three control traits was also analyzed to eliminate the influence of any obvious meiotic disturbance in case a reduction of plant survival was observed. The RPr79/4 necrotic phenotype had recessive expression in field-grown F1 plants. F2 progeny studies performed in the greenhouse revealed that the inheritance of necrotic lesions was monofactorial, and that the control traits segregated as expected. Progeny test analyses of field-grown F2 plants demonstrated that necrotic homozygous plants died preferentially. While the few surviving necrotic homozygous families were catalase-deficient, healthy homozygous families had normal levels of catalase. Progeny test analyses of the control traits confirmed the inheritance calculated in F2. Taken together, these findings indicate that abnormal segregation of necrotic lesions cannot be attributed to any obvious abnormal meiotic behavior but to the incapacity of catalase-deficient plants to overcome field stress conditions. Thus, catalase deficiency in barley reduced survival and pleiotropically affected the agronomic performance by diminishing seed weight and yield.

Retention of triplicated phytoene synthase (PSY) genes in Brassica napus L. and its diploid progenitors during the evolution of the Brassiceae

The extent of genome redundancy exhibited by Brassica species provides a model to study the evolutionary fate of multi-copy genes and the effects of polyploidy in economically important crops. Phytoene synthase (PSY) catalyzes the first committed reaction of the carotenoid biosynthetic pathway, which has been shown to be rate-limiting in Brassica napus seeds. In Arabidopsis thaliana, a single PSY gene (AtPSY) regulates phytoene synthesis in all tissues. Considering that diploid Brassica genomes contain three Arabidopsis-like subgenomes, the objectives of the present work were to determine whether PSY gene families exist in B. napus (AACC) and its diploid progenitor species, Brassica rapa (AA) and Brassica oleracea (CC); to establish the level of retention of Brassica PSY genes; to map PSY gene family members in the A and C genomes and to compare Brassica PSY gene expression patterns. A total of 12 PSY homologues were identified, 6 in B. napus (BnaX.PSY.a-f) and 3 in B. rapa (BraA.PSY.a-c) and B. oleracea (BolC.PSY.a-c). Indeed, with six members, B. napus has the largest PSY gene family described to date. Sequence comparison between AtPSY and Brassica PSY genes revealed a highly conserved gene structure and identity percentages above 85% at the coding sequence (CDS) level. Altogether, our data indicate that PSY gene family expansion preceded the speciation of B. rapa and B. oleracea, dating back to the paralogous subgenome triplication event. In these three Brassica species, all PSY homologues are expressed, exhibiting overlapping redundancy and signs of subfunctionalization among photosynthetic and non-photosynthetic tissues. This evidence supports the hypothesis that functional divergence of PSY gene expression facilitates the accumulation of high levels of carotenoids in chromoplast-rich tissues. Thus, functional retention of triplicated Brassica PSY genes could be at least partially explained by the selective advantage provided by increased levels of gene product in floral organs. A better understanding of carotenogenesis in Brassica will aid in the future development of transgenic and conventional cultivars with carotenoid-enriched oil.

Functional Analysis of the Brassica napus L. Phytoene Synthase (PSY) Gene Family

Phytoene synthase (PSY) has been shown to catalyze the first committed and rate-limiting step of carotenogenesis in several crop species, including Brassica napus L. Due to its pivotal role, PSY has been a prime target for breeding and metabolic engineering the carotenoid content of seeds, tubers, fruits and flowers. In Arabidopsis thaliana, PSY is encoded by a single copy gene but small PSY gene families have been described in monocot and dicotyledonous species. We have recently shown that PSY genes have been retained in a triplicated state in the A- and C-Brassica genomes, with each paralogue mapping to syntenic locations in each of the three “Arabidopsis-like” subgenomes. Most importantly, we have shown that in B. napus all six members are expressed, exhibiting overlapping redundancy and signs of subfunctionalization among photosynthetic and non photosynthetic tissues. The question of whether this large PSY family actually encodes six functional enzymes remained to be answered. Therefore, the objectives of this study were to: (i) isolate, characterize and compare the complete protein coding sequences (CDS) of the six B. napus PSY genes; (ii) model their predicted tridimensional enzyme structures; (iii) test their phytoene synthase activity in a heterologous complementation system and (iv) evaluate their individual expression patterns during seed development. This study further confirmed that the six B. napus PSY genes encode proteins with high sequence identity, which have evolved under functional constraint. Structural modeling demonstrated that they share similar tridimensional protein structures with a putative PSY active site. Significantly, all six B. napus PSY enzymes were found to be functional. Taking into account the specific patterns of expression exhibited by these PSY genes during seed development and recent knowledge of PSY suborganellar localization, the selection of transgene candidates for metabolic engineering the carotenoid content of oilseeds is discussed.