Tuning of meristem maturation rate increases yield in multiple Triticum aestivum cultivars.

Breeding programs aim to improve crop yield and environmental stability for enhanced food security. The principal methodology in breeding for stable yield gain relies on the indirect selection of beneficial genetics by yield evaluation across diverse environmental conditions. This methodology requires substantial resources while delivering a slow pace of yield gain and environmental adaptation. Alternative methods are required to accelerate gain and adaptation, becoming even more imperative in a changing climate. New molecular tools and approaches can enable accelerated creation and deployment of multiple alleles of genes identified to control key traits. With the advent of tools that enable breeding by targeted allelic selection, identifying gene targets associated with an improved crop performance ideotype will become crucial. Previous studies have shown that altered photoperiod regimes increase yield in wheat (Triticum aestivum). In the current study, we have employed such treatments to study the resulting yield ideotype in five spring wheat cultivars. We found that the photoperiod treatment creates a yield ideotype arising from delayed spike establishment rates that are accompanied by increased early shoot expression of TARGET OF EAT1 (TaTOE1) genes. Genes identified in this way could be used for ideotype-based improve crop performance through targeted allele creation and selection in relevant environments.

Arabidopsis B-BOX32 interacts with CONSTANS-LIKE3 to regulate flowering.

Plants have the ability to respond to seasonal environmental variations by monitoring day length to initiate flowering. The transition from vegetative to the reproductive stage is the critical developmental switch in flowering plants to ensure optimal fitness and/or yield. It has been previously reported that B-BOX32 (BBX32) has the potential to increase grain yield when ectopically expressed in soybean. In the present study, we performed a detailed molecular characterization of the Arabidopsis B-box domain gene BBX32 We showed that the circadian clock in Arabidopsis regulates BBX32 and expressed in the early morning. To understand the molecular mechanism of BBX32 regulation, we performed a large-scale yeast two-hybrid screen and identified CONSTANS-LIKE 3 (COL3)/BBX4 as one of its interacting protein partners. Using different genetic and biochemical assays, we have validated this interaction and shown that COL3 targets FT in the presence of BBX32 to regulate the flowering pathway. Based on these findings, we hypothesized that this BBX32-COL3 module could be an additional regulatory mechanism affecting the reproductive development in Arabidopsis that could be translated to crops for increased agricultural productivity.

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.

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.

Extra views on RNA-dependent DNA methylation and MBD6-dependent heterochromatin formation in nucleolar dominance.

Nucleolar dominance is a widespread epigenetic phenomenon, describing the preferential silencing of ribosomal RNA (rRNA) genes inherited from one progenitor of an interspecific hybrid, independent of maternal or paternal effects. In the allotetraploid hybrid plant species Arabidopsis suecica, A. thaliana-derived rRNA genes are silenced whereas the A. arenosa-derived rRNA genes are transcribed. We reported previously on an RNAi-based screen of DNA methyltransferases, methylcytosine binding proteins and RNA-dependent DNA methylation pathway proteins that identified specific activities required for the establishment or enforcement of nucleolar dominance. Here we present additional molecular and cell biological evidence that siRNA-directed cytosine methylation and the methylcytosine binding protein MBD6 bring about large-scale chromosomal effects on rRNA gene loci subjected to nucleolar dominance in A. suecica.

Multimegabase silencing in nucleolar dominance involves siRNA-directed DNA methylation and specific methylcytosine-binding proteins.

In genetic hybrids, the silencing of nucleolar rRNA genes inherited from one progenitor is the epigenetic phenomenon known as nucleolar dominance. An RNAi knockdown screen identified the Arabidopsis de novo cytosine methyltransferase, DRM2, and the methylcytosine binding domain proteins, MBD6 and MBD10, as activities required for nucleolar dominance. MBD10 localizes throughout the nucleus, but MBD6 preferentially associates with silenced rRNA genes and does so in a DRM2-dependent manner. DRM2 methylation is thought to be guided by siRNAs whose biogenesis requires RNA-DEPENDENT RNA POLYMERASE 2 (RDR2) and DICER-LIKE 3 (DCL3). Consistent with this hypothesis, knockdown of DCL3 or RDR2 disrupts nucleolar dominance. Collectively, these results indicate that in addition to directing the silencing of retrotransposons and noncoding repeats, siRNAs specify de novo cytosine methylation patterns that are recognized by MBD6 and MBD10 in the large-scale silencing of rRNA gene loci.

Relationships between transcription, silver staining, and chromatin organization of nucleolar organizers in Secale cereale.

The nucleolar organizer regions (NORs) are composed of hundreds of rRNA genes, typically spanning several megabases. Cytologically, NORs include regions that are highly condensed and regions that are decondensed, the latter corresponding to regions at which associated proteins stain intensively with silver (Ag-NORs) and where active rRNA gene transcription is thought to occur. To test the relationship between rRNA gene activity, NOR silver staining, and rDNA (genes coding for rRNA) chromatin condensation, we used the DNA methyl-transferase inhibitor 5-azacytidine to evaluate the correlation between the epigenetic regulation of rRNA genes and NOR silver staining in the plant Secale cereale. Following 5-azacytidine treatment, we observed an increase in rRNA gene transcription as well as a reduction in the number of cells showing a significant difference in the size of the silver-stained domains in the two NORs. These transcriptional changes occurred concomitantly with an increase in nuclear and nucleolar size and were associated with the reallocation of most of the rDNA from perinucleolar heterochromatin into the nucleolus. Collectively, these results suggest that rRNA gene transcription, silver staining, and NOR decondensation are interrelated in S. cereale.

Postembryonic establishment of megabase-scale gene silencing in nucleolar dominance.

Nucleolar dominance is an epigenetic phenomenon in plant and animal genetic hybrids that describes the expression of 45S ribosomal RNA genes (rRNA genes) inherited from only one progenitor due to the silencing of the other progenitor's rRNA genes. rRNA genes are tandemly arrayed at nucleolus organizer regions (NORs) that span millions of basepairs, thus gene silencing in nucleolar dominance occurs on a scale second only to X-chromosome inactivation in female mammals. In Arabidopsis suecica, the allotetraploid hybrid of A. thaliana and A. arenosa, the A. thaliana -derived rRNA genes are subjected to nucleolar dominance and are silenced via repressive chromatin modifications. However, the developmental stage at which nucleolar dominance is established in A. suecica is currently unknown. We show that nucleolar dominance is not apparent in seedling cotyledons formed during embryogenesis but becomes progressively established during early postembryonic development in tissues derived from both the shoot and root apical meristems. The progressive silencing of A. thaliana rRNA genes correlates with the transition of A. thaliana NORs from a decondensed euchromatic state associated with histone H3 that is trimethylated on lysine 4 (H3K4me3) to a highly condensed heterochromatic state in which the NORs are associated with H3K9me2 and 5-methylcytosine-enriched chromocenters. In RNAi-lines in which the histone deacetylases HDA6 and HDT1 are knocked down, the developmentally regulated condensation and inactivation of A. thaliana NORs is disrupted. Collectively, these data demonstrate that HDA6 and HDT1 function in the postembryonic establishment of nucleolar dominance, a process which recurs in each generation.

rRNA gene silencing and nucleolar dominance: insights into a chromosome-scale epigenetic on/off switch.

Ribosomal RNA (rRNA) gene transcription accounts for most of the RNA in prokaryotic and eukaryotic cells. In eukaryotes, there are hundreds (to thousands) of rRNA genes tandemly repeated head-to-tail within nucleolus organizer regions (NORs) that span millions of basepairs. These nucleolar rRNA genes are transcribed by RNA Polymerase I (Pol I) and their expression is regulated according to the physiological need for ribosomes. Regulation occurs at several levels, one of which is an epigenetic on/off switch that controls the number of active rRNA genes. Additional mechanisms then fine-tune transcription initiation and elongation rates to dictate the total amount of rRNA produced per gene. In this review, we focus on the DNA and histone modifications that comprise the epigenetic on/off switch. In both plants and animals, this system is important for controlling the dosage of active rRNA genes. The dosage control system is also responsible for the chromatin-mediated silencing of one parental set of rRNA genes in genetic hybrids, a large-scale epigenetic phenomenon known as nucleolar dominance.

Detecting differential expression of parental or progenitor alleles in genetic hybrids and allopolyploids.

Three assays useful for detecting specific RNA transcripts are primer extension, S1 nuclease protection, and reverse-transcription-cleaved amplified polymorphic sequence (RT-CAPS) analysis. All three of these techniques are used routinely for gene expression analyses and allow insights not possible by RNA blot (northern blot) hybridization. In this chapter, we describe how the primer extension, S1 nuclease protection, and RT-CAPS methods can be used to discriminate one or more parental or progenitor alleles in hybrids or allopolyploids. We discuss the rationale for using the different techniques and provide examples of the data generated.