Pablo D. Jenik

Surge and destroy: the role of auxin in plant embryogenesis

For nearly a century, the plant hormone auxin has been recognized for its effects on post-embryonic plant growth. Now recent insights into the molecular mechanism of auxin transport and signaling are uncovering fundamental roles for auxin in the earliest stages of plant development, such as in the development of the apical-basal (shoot-root) axis in the embryo, as well as in the formation of the root and shoot apical meristems and the cotyledons. Localized surges in auxin within the embryo occur through a sophisticated transcellular transport pathway causing the proteolytic destruction of key transcriptional repressors. As we discuss here, the resulting downstream gene activation, together with other, less well-understood regulatory pathways, establish much of the basic body plan of the angiosperm embryo.

microRNAs regulate the timing of embryo maturation in Arabidopsis

The seed is a key evolutionary adaptation of land plants that facilitates dispersal and allows for germination when the environmental conditions are adequate. Mature seeds are dormant and desiccated, with accumulated storage products that are to be used by the seedling after germination. These properties are imposed on the developing embryo by a maturation program, which operates during the later part of embryogenesis. A number of “master regulators” (the “LEC genes”) required for the induction of the maturation program have been described, but it is not known what prevents this program from being expressed during early embryogenesis. Here, we report that Arabidopsis (Arabidopsis thaliana) embryos mutant for strong alleles of DICER-LIKE1, the enzyme responsible for the biosynthesis of microRNAs (miRNAs), mature earlier than their wild-type counterparts. This heterochronic phenotype indicates that miRNAs are key regulators of the timing of the maturation program. We demonstrate that miRNAs operate in part by repressing the master regulators LEAFY COTYLEDON2 and FUSCA3 and identify the trihelix transcription factors ARABIDOPSIS 6B-INTERACTING PROTEIN1-LIKE1 (ASIL1) and ASIL2 and the histone deacetylase HDA6/SIL1 as components that act downstream of miRNAs to repress the maturation program early in embryogenesis. Both ASIL1 and HDA6/SIL1 are known to act to prevent the expression of embryonic maturation genes after germination, but to our knowledge, this is the first time they have been shown to have a role during embryogenesis. Our data point to a common negative regulatory module of maturation during early embryogenesis and seedling development.

The RPN5 Subunit of the 26s Proteasome Is Essential for Gametogenesis, Sporophyte Development, and Complex Assembly in Arabidopsis

The 26S proteasome is an essential multicatalytic protease complex that degrades a wide range of intracellular proteins, especially those modified with ubiquitin. Arabidopsis thaliana and other plants use pairs of genes to encode most of the core subunits, with both of the isoforms often incorporated into the mature complex. Here, we show that the gene pair encoding the regulatory particle non-ATPase subunit (RPN5) has a unique role in proteasome function and Arabidopsis development. Homozygous rpn5a rpn5b mutants could not be generated due to a defect in male gametogenesis. While single rpn5b mutants appear wild-type, single rpn5a mutants display a host of morphogenic defects, including abnormal embryogenesis, partially deetiolated development in the dark, a severely dwarfed phenotype when grown in the light, and infertility. Proteasome complexes missing RPN5a are less stable in vitro, suggesting that some of the rpn5a defects are caused by altered complex integrity. The rpn5a phenotype could be rescued by expression of either RPN5a or RPN5b, indicating functional redundancy. However, abnormal phenotypes generated by overexpression implied that paralog-specific functions also exist. Collectively, the data point to a specific role for RPN5 in the plant 26S proteasome and suggest that its two paralogous genes in Arabidopsis have both redundant and unique roles in development.

Global Regulation of Embryonic Patterning in Arabidopsis by MicroRNAs

MicroRNAs are required for the patterning and specification of most tissues in the Arabidopsis embryo, with the exception of the protoderm, with various regions of the embryo requiring different levels of microRNAs. The development of the embryo in Arabidopsis (Arabidopsis thaliana) involves a carefully controlled set of cell divisions and cell fate decisions that lead to a mature embryo containing shoot and root meristems and all basic tissue types. Over the last 20 years, a number of transcriptional regulators of embryonic patterning have been described, but little is known about the role of posttranscriptional regulators such as microRNAs (miRNAs). Previous work has centered on the study of null or very weak alleles of miRNA biosynthetic genes, but these mutants either arrest early in embryogenesis or have wild-type-looking embryos. Here, we significantly extend those analyses by characterizing embryos mutant for a strong hypomorphic allele of DICER-LIKE1 (dcl1-15). Our data demonstrate that miRNAs are required for the patterning of most regions of the embryo, with the exception of the protoderm. In mutant embryos with the most severe morphological defects, the majority of tissue identities are lost. Different levels of miRNAs appear to be required to specify cell fates in various regions of the embryo. The suspensor needs the lowest levels, followed by the root apical meristem and hypocotyl, cotyledons, and shoot apical meristem. Furthermore, we show that erecta acts as a suppressor of dcl1-15, a novel role for this signaling pathway in embryos. Our results also indicate that the regulation of the messenger RNA levels of miRNA targets involves not just the action of miRNAs but has a significant transcriptional component as well.

Cell lineage, cell signaling and the control of plant morphogenesis

It is clear that cell-cell signaling is critical for the development of both root and shoot structures. Recently, several of the key gene products required for intercellular signaling have been defined, and the developmental processes regulated by cell-cell interactions are beginning to be elucidated. Surprisingly, these results suggest that the mechanisms by which plant cells communicate with each other may be quite distinct from those used in animal systems.

Is there a role for trihelix transcription factors in embryo maturation

The development of the angiosperm seed includes the accumulation of storage products, the loss of most of its water and the establishment of dormancy. While much is known about the pathways that initiate maturation during mid-embryogenesis or repress it after germination, only recently has it been shown that other mechanisms repress the program during early embryogenesis. Two recent reports have shown that microRNAs are critical regulators of maturation in Arabidopsis early embryogenesis. Two closely related trihelix transcription factors, ASIL1 and ASIL2, were identified as probable partially redundant repressors of early maturation downstream of the microRNA-synthesizing enzyme DICER-LIKE1. An overlap between the genes upregulated in asil1-1 and dcl1-15 mutants support this conclusion. ASIL2 orthologs are found across seed plants, indicating that their role in maturation might be conserved. ASIL1 arose from the ancestral ASIL2 clade by a gene duplication event in the Brassicaceae, although it is not clear whether its function has diverged.

Immunological identification of tacaribe virus proteins

Tacaribe virus (TV), an arenavirus, is an enveloped virus with genetic information encoded in two segments of single-stranded RNA. The completed sequence of TV led to the identification of four open reading frames (ORF). In order to establish a direct link between ORFs in the sequence of TV and proteins present in virus particles and virus-infected cells, segments of the molecularly cloned TV genome were engineered so as to be expressed in Escherichia coli to produce fusion proteins that were used to raise antisera. The antisera were in turn employed to identify the TV gene products. Serum to the putative nucleocapsid (N) protein reacted with a 68-kDa protein, both in TV particles and in the infected cells. Sera raised to the glycoprotein precursor (GPC) immunoprecipitated two proteins of 68 and 70 kDa from infected cell lysates. Analysis of GPC synthesis in the presence of tunicamycin revealed that the unglycosylated GPC appeared as two polypeptides of 43 and 46 kDa. The putative RNA polymerase gene product (L) was detected as a approximately 240-kDa protein. Serum to the small zinc-binding domain protein (p11-Z) recognized a protein of approximately 11kDa. Immunological evidence is presented that in addition to N and L, two glycoproteins (GP1 and GP2) and p11-Z are structural components of Tacaribe virions.

The COP9 SIGNALOSOME Is Required for Postembryonic Meristem Maintenance in Arabidopsis thaliana

Cullin-RING E3 ligases (CRLs) regulate different aspects of plant development and are activated by modification of their cullin subunit with the ubiquitin-like protein NEDD8 (NEural precursor cell expressed Developmentally Down-regulated 8) (neddylation) and deactivated by NEDD8 removal (deneddylation). The constitutively photomorphogenic9 (COP9) signalosome (CSN) acts as a molecular switch of CRLs activity by reverting their neddylation status, but its contribution to embryonic and early seedling development remains poorly characterized. Here, we analyzed the phenotypic defects of csn mutants and monitored the cullin deneddylation/neddylation ratio during embryonic and early seedling development. We show that while csn mutants can complete embryogenesis (albeit at a slower pace than wild-type) and are able to germinate (albeit at a reduced rate), they progressively lose meristem activity upon germination until they become unable to sustain growth. We also show that the majority of cullin proteins are progressively neddylated during the late stages of seed maturation and become deneddylated upon seed germination. This developmentally regulated shift in the cullin neddylation status is absent in csn mutants. We conclude that the CSN and its cullin deneddylation activity are required to sustain postembryonic meristem function in Arabidopsis.