During leaf development in flowering plants, adaxial (upper) and abaxial (lower) side-specific genes are responsible for blade outgrowth, which takes places predominantly in the lateral direction, and for margin development as well as differentiation of adaxial and abaxial tissues. However, the underlying mechanisms are poorly understood. Here, we show that two WUSCHEL-RELATED HOMEOBOX (WOX) genes, PRESSED FLOWER (PRS)/WOX3 and WOX1, encoding homeobox transcription factors, act in blade outgrowth and margin development downstream of adaxial/abaxial polarity establishment. The expression of PRS and WOX1 defines a hitherto undescribed middle domain, including two middle mesophyll layers and the margin, as a center that organizes the outgrowth of leaf blades. The expression of PRS and WOX1 is repressed in the abaxial leaf domain by the abaxial-specific transcription factor KANADI. Furthermore, PRS and WOX1 coordinate adaxial/abaxial patterning together with adaxial-and abaxial-specific genes. Our data suggest a model of blade outgrowth and adaxial/abaxial patterning via the middle domain-specific WOX genes in Arabidopsis thaliana leaves.

The root cap is increasingly appreciated as a complex and dynamic plant organ. Root caps sense and transmit environmental signals, synthesize and secrete small molecules and macromolecules, and in some species shed metabolically active cells. However, it is not known whether root caps are essential for normal shoot and root development. We report the identification of a root cap-specific promoter and describe its use to genetically ablate root caps by directing root cap-specific expression of a diphtheria toxin A-chain gene. Transgenic toxin-expressing plants are viable and have normal aerial parts but agravitropic roots, implying loss of root cap function. Several cell layers are missing from the transgenic root caps, and the remaining cells are abnormal. Although the radial organization of the roots is normal in toxin-expressing plants, the root tips have fewer cytoplasmically dense cells than do wild-type root tips, suggesting that root meristematic activity is lower in transgenic than in wild-type plants. The roots of transgenic plants have more lateral roots and these are, in turn, more highly branched than those of wild-type plants. Thus, root cap ablation alters root architecture both by inhibiting root meristematic activity and by stimulating lateral root initiation. These observations imply that the root caps contain essential components of the signaling system that determines root architecture.

The subcellular localization and several biochemical activities of nonspecific lipid transfer protein (nsLTP) were investigated. A section of a castor bean cotyledon cell was labeled with anti-nsLTP serum followed by protein A-gold. Gold particles were more abundant in the glyoxysome matrix and the vessel cell wall than in other areas. Cell fractionation analysis of 6-day-old castor bean cotyledons by sucrose density gradient centrifugation demonstrated that 13% of nsLTP was distributed in the glyoxysomal fraction, identified on the basis of catalase as a marker, and 87% in the soluble fraction near the top of the gradient. The location of castor bean nsLTP in glyoxysomes was further confirmed by in vitro import experiments. The synthesized precursor of nsLTP (pro-nsLTP-C) was incorporated into intact castor bean glyoxysomes and processed to the mature form after import into the glyoxysomes, but it was not imported into canine pancreatic microsomes. Castor bean nsLTP-A was found to possess the ability to bind oleic acid and oleoyl-CoA by means of a method involving Lipidex 1000. The dissociation constants (Kd) for oleic acid and oleoyl-CoA binding to nsLTP-A were 4.8 and 5.0 microM, respectively. The saturated binding capacities (Bmax) for oleic acid and oleoyl-CoA per mol of nsLTP-A were 1.1 and 1.2 mol, respectively. When acyl-CoA oxidase activity was assayed in the glyoxysomal fraction, marked enhancement of the activity was observed in the presence of nsLTP. These results suggest the possibility that nsLTP regulates fatty acid beta-oxidation through the enhancement of acyl-CoA oxidase activity in glyoxysomes. The occurrence of castor bean nsLTP in the vessel wall was discussed.

The SSR16 gene of Arabidopsis has been identified as a gene encoding a ribosomal protein S16 homolog through analysis of a transposon insertion mutation. The insertion mutation is lethal, arresting embryonic development at approximately the transition from the globular to the heart stage of embryonic development. Co-segregation of the mutant phenotype with the transposon-borne drug-resistance marker and loss of the inserted transposon concomitant with phenotypic reversion provided evidence that the transposon had caused the mutation. Sequences flanking the insertion site were amplified from DNA of viable heterozygotes by thermal asymmetric interlaced (TAIL) PCR. The amplified fragment flanking the 3' end of the inserted element was sequenced and found to be identical to an Arabidopsis expressed sequence tag (EST). The EST, in turn, contained a coding sequence homologous to the ribosomal protein S16 (RPS16) of bacteria such as Escherichia coli, Bacillus subtilis and Salmonella typhimurium, as well as Neurospora crassa mitochondria and higher plant plastids. Thus the gene identified by the embryo-defective lethal insertion mutation encodes an RPS16 homolog and has been designated the SSR16 gene.