The defective seed5 (des5) mutant: effects on barley seed development and HvDek1, HvCr4, and HvSal1 gene regulation.

Barley, one of the major small grain crops, is especially important in climatically demanding agricultural areas of the world, with multiple uses within food, feed, and beverage. The barley endosperm is further of special scientific interest due to its three aleurone cell layers, with the potential of bringing forward the molecular understanding of seed development and cell specification from Arabidopsis and maize. Work done in Arabidopsis and maize indicate the presence of conserved seed developmental pathways where Crinkly4 (Cr4), Defective kernel1 (Dek1), and Supernumerary aleurone layer1 (Sal1) are key players. With the use of microscopy, a comprehensive phenotypic characterization of the barley defective seed5 (des5) mutant is presented here. The analysis further extends to molecular quantification of gene expression changes in the des5 mutant by qRT-PCR. Moreover, full-length genomic sequences of the barley orthologues were generated and these were annotated as HvDek1, HvCr4, and HvSal1. The most striking results in this study are the patchy reduction in number of aleurone cells, rudimentary anticlinal aleurone cell walls, and the specific change of HvCr4 expression compared to HvDek1 and HvSal1. The data presented support the involvement of Hvdes5 in establishing aleurone cells. Finally, how these results might affect the current model of aleurone and epidermal cell identity and development is discussed with a speculation regarding a possible role of Des5 in regulating cell division/ secondary cell wall building.

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.

GASA4, one of the 14-member Arabidopsis GASA family of small polypeptides, regulates flowering and seed development.

Members of the plant-specific gibberellic acid-stimulated Arabidopsis (GASA) gene family play roles in hormone response, defense and development. We have identified six new Arabidopsis GASA genes, bringing the total number of family members to 14. Here we show that these genes all encode small polypeptides that share the common structural features of an N-terminal putative signal sequence, a highly divergent intermediate region and a conserved 60 amino acid C-terminal domain containing 12 conserved cysteine residues. Analysis of promoter::GUS (beta-glucuronidase) transgenic plants representing six different GASA loci reveals that the promoters are activated in a variety of stage- and tissue-specific patterns during development, indicating that the GASA genes are involved in diverse processes. Characterization of GASA4 shows that the promoter is active in the shoot apex region, developing flowers and developing embryos. Phenotypic analyses of GASA4 loss-of-function and gain-of-function lines indicate that GASA4 regulates floral meristem identity and also positively affects both seed size and total seed yield.

Thermoperiodic stem elongation involves transcriptional regulation of gibberellin deactivation in pea.

The physiological basis of thermoperiodic stem elongation is as yet poorly understood. Thermoperiodic control of gibberellin (GA) metabolism has been suggested as an underlying mechanism. We have investigated the influence of different day and night temperature combinations on GA levels, and diurnal steady-state expression of genes involved in GA biosynthesis (LS, LH, NA, PSGA20ox1, and PsGA3ox1) and GA deactivation (PsGA2ox1 and PsGA2ox2), and related this to diurnal stem elongation in pea (Pisum sativum L. cv Torsdag). The plants were grown under a 12-h light period with an average temperature of 17 degrees C. A day temperature/night temperature combination of 13 degrees C/21 degrees C reduced stem elongation after 12 d by 30% as compared to 21 degrees C/13 degrees C. This was correlated with a 55% reduction of GA1. Although plant height correlated with GA1 content, there was no correlation between diurnal growth rhythms and GA1 content. NA, PsGA20ox1, and PsGA2ox2 showed diurnal rhythms of expression. PsGA2ox2 was up-regulated in 13 degrees C/21 degrees C (compared to 21 degrees C/13 degrees C), at certain time points, by up to 19-fold. Relative to PsGA2ox2, the expression of LS, LH, NA, PSGA20ox1, PsGA3ox1, and PsGA2ox1 was not or only slightly affected by the different temperature treatments. The sln mutant having a nonfunctional PsGA2ox1 gene product showed the same relative stem elongation response to temperature as the wild type. This supports the importance of PsGA2ox2 in mediating thermoperiodic stem elongation responses in pea. We present evidence for an important role of GA catabolism in thermoperiodic effect on stem elongation and conclude that PsGA2ox2 is the main mediator of this effect in pea.

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.

The maize disorganized aleurone layer 1 and 2 ( dil1, dil2) mutants lack control of the mitotic division plane in the aleurone layer of developing endosperm.

The maize ( Zea mays L.) endosperm consists of an epidermal like layer of isodiametric aleurone cells surrounding a central body of starchy endosperm cells. In disorgal1 ( dil1) and disorgal2 ( dil2) mutants the control of the mitotic division plane is relaxed or missing, resulting in mature grains with disorganized aleurone layers. In addition to orientation of the division plane, both the shape and size of the aleurone cells are affected, and often more than one layer of aleurone cells is present. Homozygous dil1 and dil2 grains are shrunken due to reduced accumulation of starchy endosperm and premature developmental arrest of the embryo, and mature mutant grains germinate at a very low rate and fail to develop into plants. However, homozygous mutant plants can be obtained through embryo rescue, revealing that both mutants have an irregular leaf epidermis as well as roots with a strongly reduced number of root hairs and aberrant root hair morphology. Our results suggest the presence of common regulatory mechanisms for the control of cell division orientation in the aleurone and plant epidermis.

The defective kernel 1 (dek1) gene required for aleurone cell development in the endosperm of maize grains encodes a membrane protein of the calpain gene superfamily.

Endosperm of cereal grains is one of the most important renewable resources for food, feed, and industrial raw material. It consists of four triploid cell types, i.e., aleurone, starchy endosperm, transfer cells, and cells of the embryo surrounding region. In maize, the aleurone layer is one cell layer thick and covers most of the perimeter of the endosperm. Specification of maize aleurone cell fate is proposed to occur through activation of the tumor necrosis factor receptor-like receptor kinase CRINKLY4. A second maize gene essential for aleurone cell development is defective kernel 1 (dek1). Here we show that DEK1 shares high homology with animal calpains. The predicted 2,159-aa DEK1 protein has 21 transmembrane regions, an extracellular loop, and a cysteine proteinase domain that shares high homology with domain II of m-calpain from animals. We propose that DEK1 functions to maintain and restrict the aleurone cell fate imposed by CR4 through activation of its cysteine proteinase by contact with the outer endosperm surface. DEK1 seems to be the only member of the calpain superfamily in plants, Arabidopsis DEK1 sharing 70% overall identity with maize DEK1. The expression of dek1 in most plant tissues in maize and Arabidopsis, as well as its presence in a variety of higher plants, including angiosperms and gymnosperms, suggests that DEK1 plays a conserved role in plant signal transduction.