Application of HB17, an Arabidopsis class II homeodomain-leucine zipper transcription factor, to regulate chloroplast number and photosynthetic capacity.

Transcription factors are proposed as suitable targets for the control of traits such as yield or food quality in plants. This study reports the results of a functional genomics research effort that identified ATHB17, a transcription factor from the homeodomain-leucine zipper class II family, as a novel target for the enhancement of photosynthetic capacity. It was shown that ATHB17 is expressed natively in the root quiescent centre (QC) from Arabidopsis embryos and seedlings. Analysis of the functional composition of genes differentially expressed in the QC from a knockout mutant (athb17-1) compared with its wild-type sibling revealed the over-representation of genes involved in auxin stimulus, embryo development, axis polarity specification, and plastid-related processes. While no other phenotypes were observed in athb17-1 plants, overexpression of ATHB17 produced a number of phenotypes in Arabidopsis including enhanced chlorophyll content. Image analysis of isolated mesophyll cells of 35S::ATHB17 lines revealed an increase in the number of chloroplasts per unit cell size, which is probably due to an increase in the number of proplastids per meristematic cell. Leaf physiological measurements provided evidence of improved photosynthetic capacity in 35S::ATHB17 lines on a per unit leaf area basis. Estimates of the capacity for ribulose-1,5-bisphosphate-saturated and -limited photosynthesis were significantly higher in 35S::ATHB17 lines.

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.

The EDLL motif: a potent plant transcriptional activation domain from AP2/ERF transcription factors.

In plants, the ERF/EREBP family of transcriptional regulators plays a key role in adaptation to various biotic and abiotic stresses. These proteins contain a conserved AP2 DNA-binding domain and several uncharacterized motifs. Here, we describe a short motif, termed 'EDLL', that is present in AtERF98/TDR1 and other clade members from the same AP2 sub-family. We show that the EDLL motif, which has a unique arrangement of acidic amino acids and hydrophobic leucines, functions as a strong activation domain. The motif is transferable to other proteins, and is active at both proximal and distal positions of target promoters. As such, the EDLL motif is able to partly overcome the repression conferred by the AtHB2 transcription factor, which contains an ERF-associated amphiphilic repression (EAR) motif. We further examined the activation potential of EDLL by analysis of the regulation of flowering time by NF-Y (nuclear factor Y) proteins. Genetic evidence indicates that NF-Y protein complexes potentiate the action of CONSTANS in regulation of flowering in Arabidopsis; we show that the transcriptional activation function of CONSTANS can be substituted by direct fusion of the EDLL activation motif to NF-YB subunits. The EDLL motif represents a potent plant activation domain that can be used as a tool to confer transcriptional activation potential to heterologous DNA-binding proteins.

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.

The flowering time regulator CONSTANS is recruited to the FLOWERING LOCUS T promoter via a unique cis-element.

CONSTANS is an evolutionarily-conserved central component of the genetic pathway that controls the onset of flowering in response to daylength. However, the specific biochemical mechanism by which the CONSTANS protein regulates the expression of its target genes remains largely unknown. *By using a combination of cell-based expression analysis and in vitro DNA binding studies, we have demonstrated that CONSTANS possesses transcriptional activation potential and is capable of directly binding to DNA. *CONSTANS was found to bind DNA via a unique sequence element containing a consensus TGTG(N2-3)ATG motif. This element is present in tandem within the FLOWERING LOCUS T promoter and is sufficient for CO binding and activity. The conserved CCT (CONSTANS, CONSTANS-like and TOC1) domain of CONSTANS was shown to be required for its recruitment to the DNA motif and other CCT-containing proteins were also found to have the ability to regulate gene expression via this element. *The CCAAT box, which has been previously hypothesized as a recruitment site for complexes containing the CONSTANS protein, potentiated CONSTANS-mediated activation but was not essential for CONSTANS recruitment to a target promoter or for its activity as a transcriptional factor.

The Nuclear Factor Y subunits NF-YB2 and NF-YB3 play additive roles in the promotion of flowering by inductive long-day photoperiods in Arabidopsis.

Accumulating evidence supports a role for members of the plant Nuclear Factor Y (NF-Y) family of CCAAT-box binding transcription factors in the regulation of flowering time. In this study we have used a genetic approach to show that the homologous proteins NF-YB3 and NF-YB2 have comparable activities and play additive roles in the promotion of flowering, specifically under inductive photoperiodic conditions. We demonstrate that NF-YB2 and NF-YB3 are both essential for the normal induction of flowering by long-days and act through regulation of the expression of FLOWERING LOCUS T (FT). Using an ELISA-based in-vitro assay, we provide a novel demonstration that plant NF-YB subunits are capable of directly binding to a CCAAT-box containing region of the FLOWERING LOCUS T promoter as part of an NF-Y trimer in combination with the yeast HAP2 and HAP5 subunits. These results support an emerging model in which NF-Y complexes provide a component of the DNA target specificity for transcriptional regulators such as CONSTANS.

Plant nuclear factor Y (NF-Y) B subunits confer drought tolerance and lead to improved corn yields on water-limited acres.

Commercially improved crop performance under drought conditions has been challenging because of the complexity of the trait and the multitude of factors that influence yield. Here we report the results of a functional genomics approach that identified a transcription factor from the nuclear factor Y (NF-Y) family, AtNF-YB1, which acts through a previously undescribed mechanism to confer improved performance in Arabidopsis under drought conditions. An orthologous maize transcription factor, ZmNF-YB2, is shown to have an equivalent activity. Under water-limited conditions, transgenic maize plants with increased ZmNF-YB2 expression show tolerance to drought based on the responses of a number of stress-related parameters, including chlorophyll content, stomatal conductance, leaf temperature, reduced wilting, and maintenance of photosynthesis. These stress adaptations contribute to a grain yield advantage to maize under water-limited environments. The application of this technology has the potential to significantly impact maize production systems that experience drought.

Analysis of the Arabidopsis MADS AFFECTING FLOWERING gene family: MAF2 prevents vernalization by short periods of cold.

The Arabidopsis FLOWERING LOCUS C (FLC) gene is a key floral repressor in the maintenance of a vernalization response. In vernalization-sensitive genetic backgrounds, FLC levels are high, and they decline after exposure to long cold periods. Four FLC paralogs (MAF2 [MADS AFFECTING FLOWERING2] to MAF5) are arranged in a tandem array on the bottom of Arabidopsis chromosome V. We used a reverse genetics approach to analyze their functions. Loss-of-function and gain-of-function studies indicate that MAF2 acts as a floral repressor. In particular, maf2 mutant plants display a pronounced vernalization response when subjected to relatively short cold periods, which are insufficient to elicit a strong flowering response in the wild type, despite producing a large reduction in FLC levels. MAF2 expression is less sensitive to vernalization than that of FLC, and its repressor activity is exerted independently or downstream of FLC transcription. Thus, MAF2 can prevent premature vernalization in response to brief cold spells. Overexpression of MAF3 or MAF4 produces alterations in flowering time that suggest that these genes also act as floral repressors and might contribute to the maintenance of a vernalization requirement. However, the final gene in the cluster, MAF5, is upregulated by vernalization. Therefore, MAF5 could play an opposite role to FLC in the vernalization response.

Arabidopsis transcription factors and the regulation of flowering time: a genomic perspective.

The availability of the Arabidopsis genome sequence allows for novel approaches in the analysis of many aspects of plant biology. Approximately 6% of Arabidopsis genes code for transcription factors, which can be grouped into different families according to similarities within the DNA binding domains. Transcription factors are critical regulatory components of the pathways that underpin many aspects of plant growth, development, and physiology. In particular, a substantial number of them are emerging as having crucial roles in controlling one of the most important, but complex, steps in the plant life cycle: the transition to flowering. Genome-wide studies offer the opportunity to gain a comprehensive understanding of this polygenic process, making it possible to appreciate both the large number of genes involved, as well as the complex regulatory networks into which those genes are integrated.