Arabidopsis Ovate Family Proteins, a Novel Transcriptional Repressor Family, Control Multiple Aspects of Plant Growth and Development

, AtOFP4 has been shown to regulate secondary cell wall formation by interact with KNOTTED1-LIKE HOMEODOMAIN PROTEIN 7 (KNAT7), and AtOFP5 has been shown to regulate the activity of a BEL1-LIKEHOMEODOMAIN 1(BLH1)-KNAT3 complex during early embryo sac development, but little is known about the function of other AtOFPs. genes may also have diverse functions in regulating plant growth and development. Further analysis suggested that AtOFP1 regulates cotyledon development in a postembryonic manner, and global transcript profiling revealed that it suppress the expression of many other genes.Our results showed that AtOFPs function as transcriptional repressors and they regulate multiple aspects of plant growth and development. These results provided the first overview of a previously unknown transcriptional repressor family, and revealed their possible roles in plant growth and development

The Arabidopsis thaliana Brassinosteroid Receptor (AtBRI1) Contains a Domain that Functions as a Guanylyl Cyclase In Vitro

BACKGROUND: Guanylyl cyclases (GCs) catalyze the formation of the second messenger guanosine 3′,5′-cyclic monophosphate (cGMP) from guanosine 5′-triphosphate (GTP). Cyclic GMP has been implicated in an increasing number of plant processes, including responses to abiotic stresses such as dehydration and salt, as well as hormones. PRINCIPLE FINDINGS: Here we used a rational search strategy based on conserved and functionally assigned residues in the catalytic centre of annotated GCs to identify candidate GCs in Arabidopsis thaliana and show that one of the candidates is the brassinosteroid receptor AtBR1, a leucine rich repeat receptor like kinase. We have cloned and expressed a 114 amino acid recombinant protein (AtBR1-GC) that harbours the putative catalytic domain, and demonstrate that this molecule can convert GTP to cGMP in vitro. CONCLUSIONS: Our results suggest that AtBR1 may belong to a novel class of GCs that contains both a cytosolic kinase and GC domain, and thus have a domain organisation that is not dissimilar to that of atrial natriuretic peptide receptors, NPR1 and NPR2. The findings also suggest that cGMP may have a role as a second messenger in brassinosteroid signalling. In addition, it is conceivable that other proteins containing the extended GC search motif may also have catalytic activity, thus implying that a significant number of GCs, both in plants and animals, remain to be discovered, and this is in keeping with the fact that the single cellular green alga Chlamydomonas reinhardtii contains over 90 annotated putative CGs

Mutation in SUMO E3 ligase, SIZ1, Disrupts the Mature Female Gametophyte in Arabidopsis

Female gametophyte is the multicellular haploid structure that can produce embryo and endosperm after fertilization, which has become an attractive model system for investigating molecular mechanisms in nuclei migration, cell specification, cell-to-cell communication and many other processes. Previous reports found that the small ubiquitin-like modifier (SUMO) E3 ligase, SIZ1, participated in many processes depending on particular target substrates and suppression of salicylic acid (SA) accumulation. Here, we report that SIZ1 mediates the reproductive process. SIZ1 showed enhanced expression in female organs, but was not detected in the anther or pollen. A defect in the siz1-2 maternal source resulted in reduced seed-set regardless of high SA concentration within the plant. Moreover, aniline blue staining and scanning electron microscopy revealed that funicular and micropylar pollen tube guidance was arrested in siz1-2 plants. Some of the embryo sacs of ovules in siz1-2 were also disrupted quickly after stage FG7. There was no significant affects of the siz1-2 mutation on expression of genes involved in female gametophyte development- or pollen tube guidance in ovaries. Together, our results suggest that SIZ1 sustains the stability and normal function of the mature female gametophyte which is necessary for pollen tube guidance

Transcriptome Analysis of the Arabidopsis Megaspore Mother Cell Uncovers the Importance of RNA Helicases for Plant Germline Development

Germ line specification is a crucial step in the life cycle of all organisms. For sexual plant reproduction, the megaspore mother cell (MMC) is of crucial importance: it marks the first cell of the plant “germline” lineage that gets committed to undergo meiosis. One of the meiotic products, the functional megaspore, subsequently gives rise to the haploid, multicellular female gametophyte that harbours the female gametes. The MMC is formed by selection and differentiation of a single somatic, sub-epidermal cell in the ovule. The transcriptional network underlying MMC specification and differentiation is largely unknown. We provide the first transcriptome analysis of an MMC using the model plant Arabidopsis thaliana with a combination of laser-assisted microdissection and microarray hybridizations. Statistical analyses identified an over-representation of translational regulation control pathways and a significant enrichment of DEAD/DEAH-box helicases in the MMC transcriptome, paralleling important features of the animal germline. Analysis of two independent T-DNA insertion lines suggests an important role of an enriched helicase, MNEME (MEM), in MMC differentiation and the restriction of the germline fate to only one cell per ovule primordium. In heterozygous mem mutants, additional enlarged MMC-like cells, which sometimes initiate female gametophyte development, were observed at higher frequencies than in the wild type. This closely resembles the phenotype of mutants affected in the small RNA and DNA-methylation pathways important for epigenetic regulation. Importantly, the mem phenotype shows features of apospory, as female gametophytes initiate from two non-sister cells in these mutants. Moreover, in mem gametophytic nuclei, both higher order chromatin structure and the distribution of LIKE HETEROCHROMATIN PROTEIN1 were affected, indicating epigenetic perturbations. In summary, the MMC transcriptome sets the stage for future functional characterization as illustrated by the identification of MEM, a novel gene involved in the restriction of germline fate

Molecular Foundations of Reproductive Lethality in Arabidopsis thaliana

The SeedGenes database (www.seedgenes.org) contains information on more than 400 genes required for embryo development in Arabidopsis. Many of these EMBRYO-DEFECTIVE (EMB) genes encode proteins with an essential function required throughout the life cycle. This raises a fundamental question. Why does elimination of an essential gene in Arabidopsis often result in embryo lethality rather than gametophyte lethality? In other words, how do mutant (emb) gametophytes survive and participate in fertilization when an essential cellular function is disrupted? Furthermore, why do some mutant embryos proceed further in development than others? To address these questions, we first established a curated dataset of genes required for gametophyte development in Arabidopsis based on information extracted from the literature. This provided a basis for comparison with EMB genes obtained from the SeedGenes dataset. We also identified genes that exhibited both embryo and gametophyte defects when disrupted by a loss-of-function mutation. We then evaluated the relationship between mutant phenotype, gene redundancy, mutant allele strength, gene expression pattern, protein function, and intracellular protein localization to determine what factors influence the phenotypes of lethal mutants in Arabidopsis. After removing cases where continued development potentially resulted from gene redundancy or residual function of a weak mutant allele, we identified numerous examples of viable mutant (emb) gametophytes that required further explanation. We propose that the presence of gene products derived from transcription in diploid (heterozygous) sporocytes often enables mutant gametophytes to survive the loss of an essential gene in Arabidopsis. Whether gene disruption results in embryo or gametophyte lethality therefore depends in part on the ability of residual, parental gene products to support gametophyte development. We also highlight here 70 preglobular embryo mutants with a zygotic pattern of inheritance, which provide valuable insights into the maternal-to-zygotic transition in Arabidopsis and the timing of paternal gene activation during embryo development

Diversity in the Architecture of ATLs, a Family of Plant Ubiquitin-Ligases, Leads to Recognition and Targeting of Substrates in Different Cellular Environments

Ubiquitin-ligases or E3s are components of the ubiquitin proteasome system (UPS) that coordinate the transfer of ubiquitin to the target protein. A major class of ubiquitin-ligases consists of RING-finger domain proteins that include the substrate recognition sequences in the same polypeptide; these are known as single-subunit RING finger E3s. We are studying a particular family of RING finger E3s, named ATL, that contain a transmembrane domain and the RING-H2 finger domain; none of the member of the family contains any other previously described domain. Although the study of a few members in A. thaliana and O. sativa has been reported, the role of this family in the life cycle of a plant is still vague. To provide tools to advance on the functional analysis of this family we have undertaken a phylogenetic analysis of ATLs in twenty-four plant genomes. ATLs were found in all the 24 plant species analyzed, in numbers ranging from 20–28 in two basal species to 162 in soybean. Analysis of ATLs arrayed in tandem indicates that sets of genes are expanding in a species-specific manner. To get insights into the domain architecture of ATLs we generated 75 pHMM LOGOs from 1815 ATLs, and unraveled potential protein-protein interaction regions by means of yeast two-hybrid assays. Several ATLs were found to interact with DSK2a/ubiquilin through a region at the amino-terminal end, suggesting that this is a widespread interaction that may assist in the mode of action of ATLs; the region was traced to a distinct sequence LOGO. Our analysis provides significant observations on the evolution and expansion of the ATL family in addition to information on the domain structure of this class of ubiquitin-ligases that may be involved in plant adaptation to environmental stress

Biochemical quantitation of the eIF5A hypusination in Arabidopsis thaliana uncovers ABA-dependent regulation

The eukaryotic translation elongation factor eIF5A is the only protein known to contain the unusual amino acid hypusine which is essential for its biological activity. This post-translational modification is achieved by the sequential action of the enzymes deoxyhypusine synthase (DHS) and deoxyhypusine hydroxylase (DOHH). The crucial molecular function of eIF5A during translation has been recently elucidated in yeast and it is expected to be fully conserved in every eukaryotic cell, however the functional description of this pathway in plants is still sparse. The genetic approaches with transgenic plants for either eIF5A overexpression or antisense have revealed some activities related to the control of cell death processes but the molecular details remain to be characterized. One important aspect of fully understanding this pathway is the biochemical description of the hypusine modification system. Here we have used recombinant eIF5A proteins either modified by hypusination or non-modified to establish a bi-dimensional electrophoresis (2D-E) profile for the three eIF5A protein isoforms and their hypusinated or unmodified proteoforms present in Arabidopsis thaliana. The combined use of the recombinant 2D-E profile together with 2D-E/western blot analysis from whole plant extracts has provided a quantitative approach to measure the hypusination status of eIF5A. We have used this information to demonstrate that treatment with the hormone abscisic acid produces an alteration of the hypusine modification system in Arabidopsis thaliana. 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Sugarcane genes associated with sucrose content

<p>Abstract</p> <p>Background -</p> <p>Sucrose content is a highly desirable trait in sugarcane as the worldwide demand for cost-effective biofuels surges. Sugarcane cultivars differ in their capacity to accumulate sucrose and breeding programs routinely perform crosses to identify genotypes able to produce more sucrose. Sucrose content in the mature internodes reach around 20% of the culms dry weight. Genotypes in the populations reflect their genetic program and may display contrasting growth, development, and physiology, all of which affect carbohydrate metabolism. Few studies have profiled gene expression related to sugarcane's sugar content. The identification of signal transduction components and transcription factors that might regulate sugar accumulation is highly desirable if we are to improve this characteristic of sugarcane plants.</p> <p>Results -</p> <p>We have evaluated thirty genotypes that have different Brix (sugar) levels and identified genes differentially expressed in internodes using cDNA microarrays. These genes were compared to existing gene expression data for sugarcane plants subjected to diverse stress and hormone treatments. The comparisons revealed a strong overlap between the drought and sucrose-content datasets and a limited overlap with ABA signaling. Genes associated with sucrose content were extensively validated by qRT-PCR, which highlighted several protein kinases and transcription factors that are likely to be regulators of sucrose accumulation. The data also indicate that aquaporins, as well as lignin biosynthesis and cell wall metabolism genes, are strongly related to sucrose accumulation. Moreover, sucrose-associated genes were shown to be directly responsive to short term sucrose stimuli, confirming their role in sugar-related pathways.</p> <p>Conclusion -</p> <p>Gene expression analysis of sugarcane populations contrasting for sucrose content indicated a possible overlap with drought and cell wall metabolism processes and suggested signaling and transcriptional regulators to be used as molecular markers in breeding programs. Transgenic research is necessary to further clarify the role of the genes and define targets useful for sugarcane improvement programs based on transgenic plants.</p

NUCLEAR FACTOR Y, subunit A (NF-YA) proteins positively regulate flowering and act through FLOWERING LOCUS T

Photoperiod dependent flowering is one of several mechanisms used by plants to initiate the developmental transition from vegetative growth to reproductive growth. The NUCLEAR FACTOR Y (NF-Y) transcription factors are heterotrimeric complexes composed of NF-YA and histone-fold domain (HFD) containing NF-YB/NF-YC, that initiate photoperiod-dependent flowering by cooperatively interacting with CONSTANS (CO) to drive the expression of FLOWERING LOCUS T (FT). This involves NF-Y and CO binding at distal CCAAT and proximal “CORE” elements, respectively, in the FT promoter. While this is well established for the HFD subunits, there remains some question over the potential role of NF-YA as either positive or negative regulators of this process. Here we provide strong support, in the form of genetic and biochemical analyses, that NF-YA, in complex with NF-YB/NF-YC proteins, can directly bind the distal CCAAT box in the FT promoter and are positive regulators of flowering in an FT-dependent manner.This work was funded by the National Science Foundation (US, http://www.nsf.gov/) award 1149822 to BFH. The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Ye