Aminopeptidase M1 (APM1), a single copy gene in Arabidopsis thaliana, encodes a metallopeptidase originally identified via its affinity for, and hydrolysis of, the auxin transport inhibitor 1-naphthylphthalamic acid (NPA). Mutations in this gene result in haploinsufficiency. Loss-of-function mutants show irregular, uncoordinated cell divisions throughout embryogenesis, affecting the shape and number of cotyledons and the hypophysis, and is seedling lethal at 5 d after germination due to root growth arrest. Quiescent center and cell cycle markers show no signals in apm1-1 knockdown mutants, and the ground tissue specifiers SHORTROOT and SCARECROW are misexpressed or mislocalized. apm1 mutants have multiple, fused cotyledons and hypocotyls with enlarged epidermal cells with cell adhesion defects. apm1 alleles show defects in gravitropism and auxin transport. Gravistimulation decreases APM1 expression in auxin-accumulating root epidermal cells, and auxin treatment increases expression in the stele. On sucrose gradients, APM1 occurs in unique light membrane fractions. APM1 localizes at the margins of Golgi cisternae, plasma membrane, select multivesicular bodies, tonoplast, dense intravacuolar bodies, and maturing metaxylem cells. APM1 associates with brefeldin A-sensitive endomembrane structures and the plasma membrane in cortical and epidermal cells. The auxin-related phenotypes and mislocalization of auxin efflux proteins in apm1 are consistent with biochemical interactions between APM1 and NPA.

In order to preserve and exploit the valuable genetic resources of tropical forest trees, such as cocoa, a systematic assessment of the available genetic variability is necessary. The approach we have used is based on a simple mini-prep DNA extraction procedure together with a polymerase-chain-reaction- (PCR)-based polymorphic assay procedure (RAPD). Twenty-five cocoa accessions: IMCs and PAs collected from Peru and LCTEENs collected from Ecuador, which are difficult to distinguish using morphological or biochemical descriptors, were uniquely fingerprinted using a minimum of three oligonucleotide primers. Analysis of the variability detected using RAPDs clearly discriminated between the geographical origin of the three cocoa populations. Partitioning of variability into within and between population components revealed that most variation was detected within a population. The potential of RAPD analysis to facilitate the rationalization of field gene banks and provide accurate estimates of diversity to allow optimization of collecting strategies is discussed.

Fusarium spp. are ranked among the top 10 most economically and scientifically important plant-pathogenic fungi in the world and are associated with plant diseases that include fruit decay of a number of crops. Fusarium isolates infecting bell pepper in Trinidad were identified based on sequence comparisons of the translation elongation factor gene (EF-1a) with sequences of Fusarium incarnatum-equiseti species complex (FIESC) verified in the FUSARIUM-ID database. Eighty-two isolates were identified as belonging to one of four phylogenetic species within the subclades FIESC-1, FIESC-15, FIESC-16, and FIESC-26, with the majority of isolates belonging to FIESC-15. A comparison of the level of DNA polymorphism and phylogenetic inference for sequences of the internal transcribed spacer region (ITS1-5.8S-ITS2) and EF-1a sequences for Trinidad and FUSARIUM-ID type species was carried out. The ITS sequences were less informative, had lower haplotype diversity and restricted haplotype distribution, and resulted in poor resolution and taxa placement in the consensus maximum-likelihood tree. EF-1a sequences enabled strongly supported phylogenetic inference with highly resolved branching patterns of the 30 phylogenetic species within the FIESC and placement of representative Trinidad isolates. Therefore, global phylogeny was inferred from EF-1a sequences representing 11 countries, and separation into distinct Incarnatum and Equiseti clades was again evident. In total, 42 haplotypes were identified: 12 were shared and the remaining were unique haplotypes. The most diverse haplotype was represented by sequences from China, Indonesia, Malaysia, and Trinidad and consisted exclusively of F. incarnatum isolates. Spain had the highest haplotype diversity, perhaps because both F. equiseti and F. incarnatum sequences were represented; followed by the United States, which contributed both F. equiseti and F. incarnatum sequences to the data set; then by countries representing Southeast Asia (China, Indonesia, Malaysia, Thailand, and Philippines) and Trinidad; both of these regions were represented by only F. incarnatum sequences. Trinidad shared two haplotypes with China and one haplotype with the United States for only F. incarnatum isolates. The findings of this study are important for devising disease management strategies and for understanding the phylogenetic relationships among members of the FIESC.

Aminopeptidase M1 (APM1) is essential for embryonic, vegetative, and reproductive development in Arabidopsis (Arabidopsis thaliana). Here, we show that, like mammalian M1 proteases, APM1 appears to have distinct enzymatic and protein-protein interaction domains and functions as a homodimer. Arabidopsis seedlings treated with ezetimibe, an inhibitor of M1 proteinprotein interactions, mimicked a subset of apm1 phenotypes distinct from those resulting from treatment with PAQ-22, an inhibitor of M1 catalytic activity, suggesting that the APM1 catalytic and interaction domains can function independently. apm1-1 knockdown mutants transformed with catalytically inactive APM1 did not prevent seedling lethality. However, apm1-2 has a functional enzymatic domain but lacks the carboxyl (C) terminus, and transformation with catalytically inactive APM1 rescued the mutant. Overexpression of human insulin-responsive aminopeptidase/oxytocinase rescued all apm1 phenotypes, suggesting that the catalytic activity was sufficient to compensate for loss of APM1 function, while overexpression of catalytically inactive insulin-responsive aminopeptidase/oxytocinase only rescued apm1-2. Increased catalytic activity alone is not sufficient to compensate for loss of APM1 function, as overexpression of another Arabidopsis M1 family member lacking an extended C terminus did not rescue apm1-1. The protein interactions facilitating enzymatic activity appear to be dependent on the C terminus of APM1, as transformation with an open reading frame containing an internal deletion of a portion of the C terminus or a point mutation in a dileucine motif did not rescue the mutant. These results suggest that both the catalytic and interaction domains are necessary for APM1 function but that APM1 function and dimerization do not require these domains to be present in the same linear molecule.

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