Odd-Arne Olsen

Ancient hybridizations among the ancestral genomes of bread wheat

The allohexaploid bread wheat genome consists of three closely related subgenomes (A, B, and D), but a clear understanding of their phylogenetic history has been lacking. We used genome assemblies of bread wheat and five diploid relatives to analyze genome-wide samples of gene trees, as well as to estimate evolutionary relatedness and divergence times. We show that the A and B genomes diverged from a common ancestor ~7 million years ago and that these genomes gave rise to the D genome through homoploid hybrid speciation 1 to 2 million years later. Our findings imply that the present-day bread wheat genome is a product of multiple rounds of hybrid speciation (homoploid and polyploid) and lay the foundation for a new framework for understanding the wheat genome as a multilevel phylogenetic mosaic.

Insensitivity of Barley Endosperm ADP-Glucose Pyrophosphorylase to 3-Phosphoglycerate and Orthophosphate Regulation

Crude extracts of starchy endosperm from barley (Hordeum vulgare cv Bomi) contained high pyrophosphorolytic activity (up to 0.5 [mu]mol of glucose-1-P formed min-1 mg-1 of protein) of ADP-glucose pyrophosphorylase (AGP) when assayed in the absence of 3-phosphoglycerate (3-PGA). This high activity was observed regardless of whether AGP had been extracted in the presence or absence of various protease inhibitors or other protectants. Western blot analysis using antibodies specific for either the small or large subunit of the enzyme demonstrated that the large, 60-kD subunit was prone to proteolysis in crude extracts, with a half-time of degradation at 4[deg]C (from 60 to 53 to 51 kD) on the order of minutes. The presence of high concentrations of protease inhibitors decreased, but did not prevent this proteolysis. The small, 51-kD subunit of barley endosperm AGP was relatively resistant to proteolysis, both in the presence or absence of protease inhibitors. For the crude, nonproteolyzed enzyme, 3-PGA acted as a weak activator of the ADP-glucose synthetic reaction (about 25% activation), whereas in the reverse reaction (pyrophosphorolysis) it served as an inhibitor rather than an activator. For both the synthetic and pyrophosphorolytic reactions, inorganic phosphate (Pi) acted as a weak competitive or mixed inhibitor of AGP. The relative insensitivity to 3-PGA/Pi regulation has been observed with both the nonproteolyzed crude enzyme and partially purified (over 60-fold) AGP, the latter characterized by two bands for the large subunit (molecular masses of 53 and 51 kD) and one band for the small subunit (51 kD). Addition of 3-PGA to assays of the partially purified, proteolyzed enzyme had little or no effect on the Km values of all substrates of AGP, but it reduced the Hill coefficient for ATP (from 2.1 to 1.0). These findings are discussed with respect to previous reports on the structure and regulation of higher plant AGP.

Genome interplay in the grain transcriptome of hexaploid bread wheat

Allohexaploid bread wheat (Triticum aestivum L.) provides approximately 20% of calories consumed by humans. Lack of genome sequence for the three homeologous and highly similar bread wheat genomes (A, B, and D) has impeded expression analysis of the grain transcriptome. We used previously unknown genome information to analyze the cell type–specific expression of homeologous genes in the developing wheat grain and identified distinct co-expression clusters reflecting the spatiotemporal progression during endosperm development. We observed no global but cell type– and stage-dependent genome dominance, organization of the wheat genome into transcriptionally active chromosomal regions, and asymmetric expression in gene families related to baking quality. Our findings give insight into the transcriptional dynamics and genome interplay among individual grain cell types in a polyploid cereal genome.

Subcellular Localization and Functional Domain Studies of DEFECTIVE KERNEL1 in Maize and Arabidopsis Suggest a Model for Aleurone Cell Fate Specification Involving CRINKLY4 and SUPERNUMERARY ALEURONE LAYER1

DEFECTIVE KERNEL1 (DEK1), which consists of a membrane-spanning region (DEK1-MEM) and a calpain-like Cys proteinase region (DEK1-CALP), is essential for aleurone cell formation at the surface of maize (Zea mays) endosperm. Immunolocalization and FM4-64 dye incubation experiments showed that DEK1 and CRINKLY4 (CR4), a receptor kinase implicated in aleurone cell fate specification, colocalized to plasma membrane and endosomes. SUPERNUMERARY ALEURONE LAYER1 (SAL1), a negative regulator of aleurone cell fate encoding a class E vacuolar sorting protein, colocalized with DEK1 and CR4 in endosomes. Immunogold localization, dual-axis electron tomography, and diffusion of fluorescent dye tracers showed that young aleurone cells established symplastic subdomains through plasmodesmata of larger dimensions than those connecting starchy endosperm cells and that CR4 preferentially associated with plasmodesmata between aleurone cells. Genetic complementation experiments showed that DEK1-CALP failed to restore wild-type phenotypes in maize and Arabidopsis thaliana dek1 mutants, and DEK1-MEM also failed to restore wild-type phenotypes in Arabidopsis dek1-1 mutants. Instead, ectopic expression of DEK1-MEM under the control of the cauliflower mosaic virus 35S promoter gave a dominant negative phenotype. These data suggest a model for aleurone cell fate specification in which DEK1 perceives and/or transmits a positional signal, CR4 promotes the lateral movement of aleurone signaling molecules between aleurone cells, and SAL1 maintains the proper plasma membrane concentration of DEK1 and CR4 proteins via endosome-mediated recycling/degradation.

PCR amplification and sequences of cDNA clones for the small and large subunits of ADP-glucose pyrophosphorylase from barley tissues

Several cDNAs encoding the small and large subunit of ADP-glucose pyrophosphorylase (AGP) were isolated from total RNA of the starchy endosperm, roots and leaves of barley by polymerase chain reaction (PCR). Sets of degenerate oligonucleotide primers, based on previously published conserved amino acid sequences of plant AGP, were used for synthesis and amplification of the cDNAs. For either the endosperm, roots and leaves, the restriction analysis of PCR products (ca. 550 nucleotides each) has revealed heterogeneity, suggesting presence of three transcripts for AGP in the endosperm and roots, and up to two AGP transcripts in the leaf tissue. Based on the derived amino acid sequences, two clones from the endosperm, beps and bepl, were identified as coding for the small and large subunit of AGP, respectively, while a leaf transcript (blpl) encoded the putative large subunit of AGP. There was about 50% identity between the endosperm clones, and both of them were about 60% identical to the leaf cDNA. Northern blot analysis has indicated that beps and bepl are expressed in both the endosperm and roots, while blpl is detectable only in leaves. Application of the PCR technique in studies on gene structure and gene expression of plant AGP is discussed.

Development of the endosperm in rice (Oryza sativa L.): Cellularization

The syncytial endosperm of rice undergoes cellularization according to a regular morphogenetic plan. At 3 days after pollination (dap) mitosis in the peripheral synctium ceases. Radial systems of microtubules emanating from interphase nuclei define nuclear-cytoplasmic domains (NCDs) which develop axes perpendicular, to the embryo sac wall. Free-growing anticlinal walls between adjacent NCDs compart-mentalize the cytoplasm into open-ended alveoli which are overtopped by syncytial cytoplasm adjacent to the central vacuole. At 4 dap, mitosis resumes as a wave originating adjacent to the vascular bundle. The spindles are oriented parallel to the alveolar walls and cell plates formed in association with interzonal phragmoplasts result in periclinal walls that cut off a peripheral layer of cells and an inner layer of alveoli displaced toward the center. Polarized growth of the newly formed alveoli and elongation of the anticlinal walls occurs during interphase. The next wave of cell division in the alveoli proceeds as the first and a second cylinder of cells is cut off inside the peripheral layer. The periods of polarized growth/anticlinal wall elongation alternating with periclinal cell division are repeated 3–4 times until the grain is filled by 5 dap.

Primary structure of a novel barley gene differentially expressed in immature aleurone layers

SummaryAs a direct approach to elucidate the molecular biology of barley aleurone cell development, we differentially screened an aleurone cDNA library made from poly(A)+ RNA of immature grains for clones representing transcripts present in the aleurone but not in the starchy endosperm. For one of these clones, B22E, which hybridies to a 0.7 kb transcript, Northern and in situ hybridization revealed that expression is under complex spatial, temporal and hormonal control in barley grains. cDNAs corresponding to B22E transcripts were isolated from aleurone/pericarp and embryo of developing grains, and from germinating scutella. Among these were the nearly full-length aleurone/pericarp clone p1322E.a16 (541 bp). cDNAs matching the sequence of this clone (type 1 transcript) were found for all tissues investigated. In addition, cDNAs with an extra 12 bp insertion (type 2 transcript) were obtained from germinating scutella. The two different transcripts can encode novel barley proteins of 115 and 119 amino acids, respectively. A gene designated B22EL8 was isolated and sequenced; it encodes the type 1 B22E transcript and contains two introns of 145 and 125 bp. Particle bombardment of barley, aleurone with a B22EL8 promoter-GUS (β-glucuronidase) construct demonstrates that the promoter (3 kb) is active in developing barley grains. The promoter is not, however, active in the seeds of tobacco plants transgenic for the B22EL8 gene, indicating the existence of sequences specific for monocots. A comparison of 1.4 kb of upstream sequence of B22E with the maize c1 promoter reveals a number of short, identical sequences which may be responsible for aleurone cell-specific gene transcription.

Mutation in the Arabidopisis thaliana DEK1 calpain gene perturbs endosperm and embryo development while over-expression affects organ development globally

A T-DNA insertion in the Arabidopsis thaliana DEK1 gene, encoding a calpain-like cysteine proteinase with a predicted membrane anchor, causes unorganized embryo development displaying irregular mitotic divisions in the embryo proper and suspensor. Embryo development is arrested at the globular stage, and the embryo proper lacks a defined protoderm. In the endosperm, the aleurone-like peripheral cell layer is partly or completely lacking. The Arabidopsis DEK1 wild-type transcript is expressed evenly throughout the endosperm and the embryo in developing seed as determined using in situ hybridization. The conclusion that the observed phenotype is caused by a T-DNA insertion in the Arabidopsis DEK1 gene is confirmed by complementation with the Arabidopisis DEK1 genomic sequence, as well as analysis of a second T-DNA insertion allele. Over-expression of the Arabidopsis DEK1 gene coding sequence under the control of the 35S promoter causes a number of developmental phenotypes, including a global lack of trichomes, leaves exhibiting improper dorsiventral symmetry and aberrant cell organization in flowers. We interpret the data to suggest a role for DEK1 in providing cells with positional clues for an appropriate developmental context within plant tissues.

Cell wall (1 → 3)- and (1 → 3, 1 → 4)-β-glucans during early grain development in rice (Oryza sativa L.)

Abstract. Immunogold labeling was used to study the distribution of (1 → 3)-β-glucans and (1 → 3, 1 → 4)-β-glucans in the rice grain during cellularization of the endosperm. At approximately 3–5 d after pollination the syncytial endosperm is converted into a cellular tissue by three developmentally distinct types of wall. The initial free-growing anticlinal walls, which compartmentalize the syncytium into open-ended alveoli, are formed in the absence of mitosis and phragmoplasts. This stage is followed by unidirectional (centripetal) growth of the anticlinal walls mediated by adventitious phragmoplasts that form between adjacent interphase nuclei. Finally, the periclinal walls that divide the alveoli are formed in association with centripetally expanding interzonal phragmoplasts following karyokinesis. The second and third types of wall are formed alternately until the endosperm is cellular throughout. All three types of wall that cellularize the endosperm contain (1 → 3)-β-glucans but not (1 → 3, 1 → 4)-β-glucans, whereas cell walls in the surrounding maternal tissues contain considerable amounts of (1 → 3, 1 → 4)-β-glucans with (1 → 3)-β-glucans present only around plasmodesmata. The callosic endosperm walls remain thin and cell plate-like throughout the cellularization process, appearing to exhibit a prolonged juvenile state.

Defective Kernel 1 (DEK1) is required for three‐dimensional growth in Physcomitrella patens

Orientation of cell division is critical for plant morphogenesis. This is evident in the formation and function of meristems and for morphogenetic transitions. Mosses undergo such transitions: from two-dimensional tip-growing filaments (protonema) to the generation of three-dimensional leaf-like structures (gametophores). The Defective Kernel 1 (DEK1) protein plays a key role in the perception of and/or response to positional cues that specify the formation and function of the epidermal layer in developing seeds of flowering plants. The moss Physcomitrella patens contains the highly conserved DEK1 gene. Using efficient gene targeting, we generated a precise PpDEK1 deletion (Δdek1), which resulted in normal filamentous growth of protonema. Two distinct mutant phenotypes were observed: an excess of buds on the protonema, and abnormal cell divisions in the emerging buds resulting in developmental arrest and the absence of three-dimensional growth. Overexpression of a complete PpDEK1 cDNA, or the calpain domain of PpDEK1 alone, successfully complements both phenotypes. These results in P. patens demonstrate the morphogenetic importance of the DEK1 protein in the control of oriented cell divisions. As it is not for protonema, it will allow dissection of the structure/function relationships of the different domains of DEK1 using gene targeting in null mutant background.