Expression of the Arabidopsis thaliana BBX32 gene in soybean increases grain yield.

Crop yield is a highly complex quantitative trait. Historically, successful breeding for improved grain yield has led to crop plants with improved source capacity, altered plant architecture, and increased resistance to abiotic and biotic stresses. To date, transgenic approaches towards improving crop grain yield have primarily focused on protecting plants from herbicide, insects, or disease. In contrast, we have focused on identifying genes that, when expressed in soybean, improve the intrinsic ability of the plant to yield more. Through the large scale screening of candidate genes in transgenic soybean, we identified an Arabidopsis thaliana B-box domain gene (AtBBX32) that significantly increases soybean grain yield year after year in multiple transgenic events in multi-location field trials. In order to understand the underlying physiological changes that are associated with increased yield in transgenic soybean, we examined phenotypic differences in two AtBBX32-expressing lines and found increases in plant height and node, flower, pod, and seed number. We propose that these phenotypic changes are likely the result of changes in the timing of reproductive development in transgenic soybean that lead to the increased duration of the pod and seed development period. Consistent with the role of BBX32 in A. thaliana in regulating light signaling, we show that the constitutive expression of AtBBX32 in soybean alters the abundance of a subset of gene transcripts in the early morning hours. In particular, AtBBX32 alters transcript levels of the soybean clock genes GmTOC1 and LHY-CCA1-like2 (GmLCL2). We propose that through the expression of AtBBX32 and modulation of the abundance of circadian clock genes during the transition from dark to light, the timing of critical phases of reproductive development are altered. These findings demonstrate a specific role for AtBBX32 in modulating soybean development, and demonstrate the validity of expressing single genes in crops to deliver increased agricultural productivity.

Reconstructing the diversification of subtilisins in the pathogenic fungus Metarhizium anisopliae.

Fungi secrete subtilisin proteinases to acquire nutrients and breach host barriers. Here we sought a global characterization of the diversity of subtilisins in the insect pathogen Metarhizium anisopliae. Expressed sequence tag (EST) analyses showed that a broad host range strain of M. anisopliae sf. anisopliae (strain 2575) expressed 11 subtilisins during growth on insect cuticle, the largest number of subtilisins reported from any fungus. Polymerase chain reaction amplified 10 of their orthologs from a second strain with multiple hosts (strain 820) and seven from the locust specialist M. anisopliae sf. acridum (strain 324). Analyses based on sequence similarities and exon-intron structure grouped M. anisopliae subtilisins into four clusters-a class I ("bacterial") subtilisin (Pr1C), and three clusters of proteinase K-like class II subtilisins: extracellular subfamily 1 (Pr1A, Pr1B, Pr1G, Pr1I and Pr1K), extracellular subfamily 2 (Pr1D, Pr1E, Pr1F and Pr1J) and an endocellular subtilisin (Pr1H). Phylogenetic analysis of homologous sequences from other genera revealed that this subdivision of proteinase K-like subtilisins into three subfamilies preceded speciation of major fungal lineages. However, diversification has continued during the evolution of Metarhizium subtilisins with evidence of gene duplication events after divergence of M. anisopliae sf. anisopliae and M. anisopliae sf. acridum. Comparing alignments and nonsynonymous/synonymous rates for Pr1 isoenzymes within a lineage and between lineages showed that while overall divergence of subtilisins followed neutral expectations, amino acids involved in catalysis were under strong selective constraint. This suggests that each Pr1 paralog contributes to the pathogens fitness. Furthermore, homology modeling predicted differences between the Pr1's in their secondary substrate specificities, adsorption properties to cuticle and alkaline stability, indicative of functional differences.