Thomas G. Ruff

Development and mapping of SSR markers for maize.

Microsatellite or simple sequence repeat (SSR) markers have wide applicability for genetic analysis in crop plant improvement strategies. The objectives of this project were to isolate, characterize, and map a comprehensive set of SSR markers for maize (Zea mays L.). We developed 1051 novel SSR markers for maize from microsatellite-enriched libraries and by identification of microsatellite-containing sequences in public and private databases. Three mapping populations were used to derive map positions for 978 of these markers. The main mapping population was the intermated B73 × Mo17 (IBM) population. In mapping this intermated recombinant inbred line population, we have contributed to development of a new high-resolution map resource for maize. The primer sequences, original sequence sources, data on polymorphisms across 11 inbred lines, and map positions have been integrated with information on other public SSR markers and released through MaizeDB at URL:www.agron.missouri.edu. The maize research community now has the most detailed and comprehensive SSR marker set of any plant species.

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.

BROTHER OF LUX ARRHYTHMO Is a Component of the Arabidopsis Circadian Clock

This work shows that the Arabidopsis transcription factor BROTHER OF LUX ARRHYTHMO (BOA) is an activator in regulating the expression of CIRCADIAN CLOCK ASSOCIATED1 (CCA1). BOA forms a transcriptional feedback loop with CCA1 and regulates circadian rhythms in Arabidopsis. BROTHER OF LUX ARRHYTHMO (BOA) is a GARP family transcription factor in Arabidopsis thaliana and is regulated by circadian rhythms. Transgenic lines that constitutively overexpress BOA exhibit physiological and developmental changes, including delayed flowering time and increased vegetative growth under standard growing conditions. Arabidopsis circadian clock protein CIRCADIAN CLOCK ASSOCIATED1 (CCA1) binds to the evening element of the BOA promoter and negatively regulates its expression. Furthermore, the period of BOA rhythm was shortened in cca1-11, lhy-21 (for LATE ELONGATED HYPOCOTYL), and cca1-11 lhy-21 genetic backgrounds. BOA binds to the promoter of CCA1 through newly identified promoter binding sites and activates the transcription of CCA1 in vivo and in vitro. In transgenic Arabidopsis lines that overexpress BOA, the period length of CCA1 rhythm was increased and the amplitude was enhanced. Rhythmic expression of other clock genes, including LHY, GIGANTEA (GI), and TIMING OF CAB EXPRESSION1 (TOC1), was altered in transgenic lines that overexpress BOA. Rhythmic expression of BOA was also affected in mutant lines of toc1-1, gi-3, and gi-4. Results from these studies indicate that BOA is a critical component of the regulatory circuit of the circadian clock.

Expression of a Truncated ATHB17 Protein in Maize Increases Ear Weight at Silking

ATHB17 (AT2G01430) is an Arabidopsis gene encoding a member of the α-subclass of the homeodomain leucine zipper class II (HD-Zip II) family of transcription factors. The ATHB17 monomer contains four domains common to all class II HD-Zip proteins: a putative repression domain adjacent to a homeodomain, leucine zipper, and carboxy terminal domain. However, it also possesses a unique N-terminus not present in other members of the family. In this study we demonstrate that the unique 73 amino acid N-terminus is involved in regulation of cellular localization of ATHB17. The ATHB17 protein is shown to function as a transcriptional repressor and an EAR-like motif is identified within the putative repression domain of ATHB17. Transformation of maize with an ATHB17 expression construct leads to the expression of ATHB17Δ113, a truncated protein lacking the first 113 amino acids which encodes a significant portion of the repression domain. Because ATHB17Δ113 lacks the repression domain, the protein cannot directly affect the transcription of its target genes. ATHB17Δ113 can homodimerize, form heterodimers with maize endogenous HD-Zip II proteins, and bind to target DNA sequences; thus, ATHB17Δ113 may interfere with HD-Zip II mediated transcriptional activity via a dominant negative mechanism. We provide evidence that maize HD-Zip II proteins function as transcriptional repressors and that ATHB17Δ113 relieves this HD-Zip II mediated transcriptional repression activity. Expression of ATHB17Δ113 in maize leads to increased ear size at silking and, therefore, may enhance sink potential. We hypothesize that this phenotype could be a result of modulation of endogenous HD-Zip II pathways in maize.

Involvement of the N-terminal B-box Domain of Arabidopsis BBX32 Protein in Interaction with Soybean BBX62 Protein

Background: AtBBX32 is a member of the B-box protein family from A. thaliana. Its molecular mechanism is poorly understood. Results: We demonstrate functional interactions of AtBBX32 with soybean BBX62 (GmBBX62). Conclusion: The N-terminal B-box domain plays a key role in mediating the interaction between AtBBX32 and GmBBX62. Significance: Our data offer novel insight into the role of B-box domains in mediating protein-protein interactions between different plant B-box proteins. Previous studies have demonstrated that Arabidopsis thaliana BBX32 (AtBBX32) represses light signaling in A. thaliana and that expression of AtBBX32 in soybean increases grain yield in multiple locations and multiyear field trials. The BBX32 protein is a member of the B-box zinc finger family from A. thaliana and contains a single conserved Zn2+-binding B-box domain at the N terminus. Although the B-box domain is predicted to be involved in protein-protein interactions, the mechanism of interaction is poorly understood. Here, we provide in vitro and in vivo evidence demonstrating the physical and functional interactions of AtBBX32 with another B-box protein, soybean BBX62 (GmBBX62). Deletion analysis and characterization of the purified B-box domain indicate that the N-terminal B-box region of AtBBX32 interacts with GmBBX62. Computational modeling and site-directed mutagenesis of the AtBBX32 B-box region identified specific residues as critical for mediating the interaction between AtBBX32 and GmBBX62. This study defines the plant B-box as a protein interaction domain and offers novel insight into its role in mediating specific protein-protein interactions between different plant B-box proteins.