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.

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 Zn(2+)-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.

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.

Drought tolerance through biotechnology: improving translation from the laboratory to farmers' fields.

Water availability is a significant constraint to crop production, and increasing drought tolerance of crops is one step to gaining greater yield stability. Excellent progress has been made using models to identify pathways and genes that can be manipulated through biotechnology to improve drought tolerance. A current focus is on translation of results from models in controlled environments to crops in the field. Field testing to demonstrate improved yields under water-limiting conditions is challenging and expensive. More extensive phenotyping of transgenic lines in the greenhouse may contribute to improved predictions about field performance. It is possible that multiple mechanisms of drought tolerance may be needed to provide benefit across the diversity of water stress environments relevant to economic yield.

BBX32, an Arabidopsis B-Box protein, functions in light signaling by suppressing HY5-regulated gene expression and interacting with STH2/BBX21.

A B-box zinc finger protein, B-BOX32 (BBX32), was identified as playing a role in determining hypocotyl length during a large-scale functional genomics study in Arabidopsis (Arabidopsis thaliana). Further analysis revealed that seedlings overexpressing BBX32 display elongated hypocotyls in red, far-red, and blue light, along with reduced cotyledon expansion in red light. Through comparative analysis of mutant and overexpression line phenotypes, including global expression profiling and growth curve studies, we demonstrate that BBX32 acts antagonistically to ELONGATED HYPOCOTYL5 (HY5). We further show that BBX32 interacts with SALT TOLERANCE HOMOLOG2/BBX21, another B-box protein previously shown to interact with HY5. Based on these data, we propose that BBX32 functions downstream of multiple photoreceptors as a modulator of light responses. As such, BBX32 potentially has a native role in mediating gene repression to maintain dark adaptation.

Translation initiation factors eIF4E and eIFiso4E are required for polysome formation and regulate plant growth in tobacco.

Eukaryotic initiation factor eIF4E plays a pivotal role in translation initiation. As a component of the ternary eIF4F complex, eIF4E interacts with the mRNA cap structure to facilitate recruitment of the 40S ribosomal subunit onto mRNA. Plants contain two distinct cap-binding proteins, eIF4E and eIFiso4E, that assemble into different eIF4F complexes. To study the functional roles of eIF4E and eIFiso4E in tobacco, we isolated two corresponding cDNAs, NteIF4E1 and NteIFiso4E1, and used these to deplete cap-binding protein levels in planta by antisense downregulation. Antibodies raised against recombinant NteIF4E1 detected three distinct cap-binding proteins in tobacco leaf extracts; NteIF4E and two isoforms of NteIFiso4E. The three cap-binding proteins were immuno-detected in all tissues analysed and were coordinately regulated, with peak expression in anthers and pollen. Transgenic tobacco plants showing significant depletion of either NteIF4E or the two NteIFiso4E isoforms displayed normal vegetative development and were fully fertile. Interestingly, NteIFiso4E depletion resulted in a compensatory increase in NteIF4E levels, whereas the down-regulation of NteIF4E did not trigger a reciprocal increase in NteIFiso4E levels. The antisense depletion of both NteIF4E and NteIFiso4E resulted in plants with a semi-dwarf phenotype and an overall reduction in polyribosome loading, demonstrating that both eIF4E and eIFiso4E support translation initiation in planta, which suggests their potential role in the regulation of plant growth.

The Fed-1 (CAUU)4 element is a 5' UTR dark-responsive mRNA instability element that functions independently of dark-induced polyribosome dissociation.

Darkness rapidly induces a decline in the stability and translation of the pea Ferredoxin-1 (Fed-1) mRNA in transgenic tobacco. Direct half-life measurement showed that mutation of the (CAUU)4 stabilizes Fed-1 mRNA in the dark. (CAUU)1, a feature more common in plant 5' UTRs than (CAUU)4, confers slight light-responsive mRNA accumulation. At least three but less than 11 CAUU repeats near the 5' end of the 5' UTR are required for full light-responsive accumulation. Furthermore, 26 nt of the 5' UTR, including the (CAUU)4 repeat, is sufficient to confer a significant approximately 2.5-fold increase in light-regulated mRNA accumulation when fused to the 5' end of a heterologous plant mRNA. A mutation of the (CAUU)4 repeat that compromises light-regulated mRNA stability changes in vitro the accessibility of the region to ribonuclease V1 and ribonuclease A suggesting the geometry formed by the repeat may be important for instability. Finally, dark-induced Fed-1 mRNA instability occurs even when most of the mRNA is retained on polyribosomes, and thus is likely an independent event regulated by darkness.

Light control of nuclear gene mRNA abundance and translation in tobacco.

Photosynthetic signals modulate expression of nuclear genes at the levels of mRNA transcription, mRNA stability, and translation. In transgenic tobacco (Nicotiana tabacum), the pea (Pisum sativum) Ferredoxin 1 (Fed-1) mRNA dissociates from polyribosomes and becomes destabilized when photosynthesis is inhibited by photosynthetic electron transport inhibitor 3-(3,4-dichlorophenyl)-1,1-dimethylurea. We used polymerase chain reaction suppressive-subtractive hybridization to identify similarly regulated endogenous tobacco genes. This screen identified 14 nuclear-encoded tobacco mRNAs whose light-induced increase in abundance is suppressed in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea. Sequence analysis of the cognate cDNAs revealed that nine of the mRNAs encode putative chloroplast-targeted proteins. We asked whether the abundance of these mRNAs was regulated transcriptionally or posttranscriptionally. Of the five mRNAs with sufficient abundance to detect using nuclear run-on assays, we observed transcriptional regulation of alpha-tubulin, thiazole biosynthetic enzyme, and pSKA10 (an unknown gene). Photosystem A subunit L and, to a lesser extent, alpha-tubulin and pSKA10 mRNAs, may also be stabilized in the light. In contrast, Rubisco small subunit mRNA abundance appears to be transcriptionally up-regulated but posttranscriptionally down-regulated in the light. To determine whether, like Fed-1 mRNA, the mRNAs identified in this screen were translationally responsive to light, we characterized the polyribosome association of these mRNAs in the light and after a 15-min dark treatment. A subset of the mRNAs showed dramatic dark-induced polyribosome dissociation, similar to Fed-1 mRNA, and all of the mRNAs showed at least slight polyribosome dissociation. Thus, both posttranscriptional and translational regulation appear to be important mechanisms regulating the expression of many nuclear-encoded mRNAs encoding proteins involved in photosynthesis.