ATML1 and PDF2 Play a Redundant and Essential Role in Arabidopsis Embryo Development.

The epidermis of shoot organs in plants develops from the outermost layer (L1) of the shoot apical meristem. In Arabidopsis, a pair of homeobox genes, ARABIDOPSIS THALIANA MERISTEM LAYER1 (ATML1) and PROTODERMAL FACTOR2 (PDF2), play a role in regulating the expression of L1-specific genes. atml1-1 pdf2-1 double mutants show striking defects in the differentiation of shoot epidermal cells. However, because atml1-1 and pdf2-1 have a T-DNA inserted downstream of the respective homeobox sequences, these alleles may not represent null mutations. Here we characterized additional mutant alleles that have a T-DNA insertion at different positions of each gene. Double mutants of a strong atml1-3 allele with each pdf2 allele were found to cause embryonic arrest at the globular stage. Although with low frequency, all double mutant combinations of a weak atml1-1 allele with each pdf2 allele germinated and showed phenotypes defective in shoot epidermal cell differentiation. We further confirmed that transgenic induction of PDF2 fused to the Drosophila Engrailed repressor domain temporarily interferes with epidermal cell differentiation in the wild-type background. These results indicate that ATML1 and PDF2 act redundantly as a positive regulator of shoot epidermal cell differentiation and at least one copy of these genes is essential for embryo development.

Mutations in epidermis-specific HD-ZIP IV genes affect floral organ identity in Arabidopsis thaliana.

Development of the epidermis involves members of the class-IV homeodomain-leucine zipper (HD-ZIP IV) transcription factors. The Arabidopsis HD-ZIP IV family consists of 16 members, among which PROTODERMAL FACTOR 2 (PDF2) and ARABIDOPSIS THALIANA MERISTEM LAYER 1 (ATML1) play an indispensable role in the differentiation of shoot epidermal cells; however, the functions of other HD-ZIP IV genes that are also expressed specifically in the shoot epidermis remain to be fully elucidated. We constructed double mutant combinations of these HD-ZIP IV mutant alleles and found that the double mutants of pdf2-1 with homeodomain glabrous1-1 (hdg1-1), hdg2-3, hdg5-1 and hdg12-2 produced abnormal flowers with sepaloid petals and carpelloid stamens in association with the reduced expression of the petal and stamen identity gene APETALA 3 (AP3). Expression of another petal and stamen identity gene PISTILATA (PI) was less affected in these mutants. We confirmed that AP3 expression in pdf2-1 hdg2-3 was normally induced at the initial stages of flower development, but was attenuated both in the epidermis and internal cell layers of developing flowers. As the expression of PDF2 and these HD-ZIP IV genes during floral organ formation is exclusively limited to the epidermal cell layer, these double mutations may have non-cell-autonomous effects on AP3 expression in the internal cell layers. Our results suggest that cooperative functions of PDF2 and other members of the HD-ZIP IV family in the epidermis are crucial for normal development of floral organs in Arabidopsis.

The usefulness of olive oil enema in children with severe chronic constipation.

PURPOSE: Few reports have determined the efficacy of olive oil enemas for severe constipation. Here, we review our experience with olive oil enemas in children with severe chronic constipation. METHODS: In our outpatient pediatric surgery department, the charts of 118 patients prescribed with olive oil enemas between January 2010 and November 2019 were retrospectively reviewed. A 1-2 ml/kg olive oil enema was given either alone or followed several hours later by a glycerin enema. Ratings included "very effective (VE)," "effective (E)," "limited (L)," "ineffective (I)," and "unknown (U)." RESULTS: One hundred and fifteen (97.5%) patients were able to use olive oil enemas at home. Forty-nine had functional constipation; 43 had anorectal malformation; 40 had Hirschsprung disease; 12 had spina bifida; and 10 had other maladies. Used as an enema, olive oil was effective in treating fecal impaction in 77.6% of patients; as a lubricant, it was effective in treating 76.9% of patients. Efficacy for fecal disimpaction was similar among patients with different underlying disorders. CONCLUSION: Olive oil enemas are useful for more than three-quarters of children with severe chronic constipation. Further study is warranted to add olive oil enemas as an adjunctive treatment in the management of severe constipation.

The assembly pathway of the 19S regulatory particle of the yeast 26S proteasome.

The 26S proteasome consists of the 20S proteasome (core particle) and the 19S regulatory particle made of the base and lid substructures, and it is mainly localized in the nucleus in yeast. To examine how and where this huge enzyme complex is assembled, we performed biochemical and microscopic characterization of proteasomes produced in two lid mutants, rpn5-1 and rpn7-3, and a base mutant DeltaN rpn2, of the yeast Saccharomyces cerevisiae. We found that, although lid formation was abolished in rpn5-1 mutant cells at the restrictive temperature, an apparently intact base was produced and localized in the nucleus. In contrast, in DeltaN rpn2 cells, a free lid was formed and localized in the nucleus even at the restrictive temperature. These results indicate that the modules of the 26S proteasome, namely, the core particle, base, and lid, can be formed and imported into the nucleus independently of each other. Based on these observations, we propose a model for the assembly process of the yeast 26S proteasome.

An inversion identified in acl1-1 mutant functions as an enhancer of the acl1-1 phenotype.

The Arabidopsis acaulis1-1 (acl1-1) mutant exhibits severe growth defects when grown at 22 degrees C. The leaves are tiny and curled and the inflorescence stems are short. We identified an inversion mutation in the original acl1-1 plants. The acl1-1 plants were crossed with Columbia wild-type, and the acl1-1 phenotype and the inversion were segregated in the F2 generation. Compared to the original acl1-1 plants with the inversion, the genuine acl1-1 plants without the inversion grew larger and their inflorescence stems grew longer at 22 degrees C. When the plants were grown at 24 degrees C, the differences in growth became more apparent. We investigated the expression of genes located in the inversion. Two genes that were located at each end of the inversion were disrupted, and full-length transcripts were not expressed. Expressions of some genes within and adjacent to the inversion were also altered. Our results indicate that the expression of multiple genes may be involved in the enhancement of the acl1-1 phenotype.

Allele-specific effects of PDF2 on floral morphology in Arabidopsis thaliana.

The class IV Homeodomain-leucine zipper (HD-ZIP IV) gene family includes several genes that are functionally significant in epidermal development. Our recent study revealed that double mutants of the epidermis-expressed HD-ZIP IV members, PROTODERMAL FACTOR2 (PDF2) in combination with some HOMEODOMAIN GLABROUS (HDG, pronounced "hedge") genes, affect stamen development and specification of petal and stamen identity, possibly in a non cell-autonomous manner. However, the effect of the pdf2 mutations on the floral development was largely different depending on T-DNA insertion locations: pdf2-1 hdg flowers exhibited homeotic conversion of petals and stamens, while pdf2-2 hdg flowers had only a reduced number of stamens. Here, we used 2 additional pdf2 alleles to make double mutants and found that their floral phenotypes were rather similar to those of pdf2-2 hdg. The allele-specific effect caused by pdf2-1, which carries a T-DNA in a steroidogenic acute regulatory protein-related lipid transfer (START) domain-encoding region, suggests the importance of the START domain in proper function of HD-ZIP IV proteins.

Functional analysis of Rpn6p, a lid component of the 26 S proteasome, using temperature-sensitive rpn6 mutants of the yeast Saccharomyces cerevisiae.

Rpn6p is a component of the lid of the 26 S proteasome. We isolated and analyzed two temperature-sensitive rpn6 mutants in the yeast, Saccharomyces cerevisiae. Both mutants showed defects in protein degradation in vivo. However, the affinity-purified 26 S proteasome of the rpn6 mutants grown at the permissive temperature degraded polyubiquitinated Sic1p efficiently, even at a higher temperature. Interestingly, their enzyme activity was even higher at a higher temperature, indicating that once made mutant proteasomes are stable and have little defect in the proteolytic function. These results suggest that the deficiency in protein degradation observed in vivo is rather due to a defect in the assembly of a holoenzyme at the restrictive temperature. Indeed, both rpn6 mutants grown at the restrictive temperature were defective in assembling the 26 S proteasome. A striking feature of the rpn6 mutants at the restrictive temperature was that there appeared a protein complex composed of only four of the nine lid components, Rpn5p, Rpn8p, Rpn9p, and Rpn11p. Altogether, we conclude that Rpn6p is essential for the integrity/assembly of the lid in the sense that it is necessary for the incorporation of Rpn3p, Rpn7p, Rpn12p, and Sem1p (Rpn15p) into the lid, thereby playing an essential role in the proper function of the 26 S proteasome.