Jelena Brkljacic

Brachypodium as a model for the grasses: Today and the future

Over the past several years, Brachypodium distachyon (Brachypodium) has emerged as a tractable model system to study biological questions relevant to the grasses. To place its relevance in the larger context of plant biology, we outline here the expanding adoption of Brachypodium as a model grass and compare this to the early history of another plant model, Arabidopsis thaliana. In this context, Brachypodium has followed an accelerated path in which the development of genomic resources, most notably a whole genome sequence, occurred concurrently with the generation of other experimental tools (e.g. highly efficient transformation and large collections of natural accessions). This update provides a snapshot of available and upcoming Brachypodium resources and an overview of the community including the trajectory of Brachypodium as a model grass.

RanGAP1 is a continuous marker of the Arabidopsis cell division plane

In higher plants, the plane of cell division is faithfully predicted by the preprophase band (PPB). The PPB, a cortical ring of microtubules and F-actin, disassembles upon nuclear-envelope breakdown. During cytokinesis, the expanding cell plate fuses with the plasma membrane at the cortical division site, the site of the former PPB. The nature of the “molecular memory” that is left behind by the PPB and is proposed to guide the cell plate to the cortical division site is unknown. RanGAP is the GTPase activating protein of the small GTPase Ran, which provides spatial information for nucleocytoplasmic transport and various mitotic processes in animals. Here, we show that, in dividing root cells, Arabidopsis RanGAP1 concentrates at the PPB and remains associated with the cortical division site during mitosis and cytokinesis, requiring its N-terminal targeting domain. In a fass/ton2 mutant, which affects PPB formation, RanGAP1 recruitment to the PPB site is lost, while its PPB retention is microtubule-independent. RanGAP1 persistence at the cortical division site, but not its initial accumulation at the PPB requires the 2 cytokinesis-regulating kinesins POK1 and POK2. Depletion of RanGAP by inducible RNAi leads to oblique cell walls and cell-wall stubs in root cell files, consistent with cytokinesis defects. We propose that Arabidopsis RanGAP, a continuous positive protein marker of the plant division plane, has a role in spatial signaling during plant cell division.

Two Distinct Interacting Classes of Nuclear Envelope–Associated Coiled-Coil Proteins Are Required for the Tissue-Specific Nuclear Envelope Targeting of Arabidopsis RanGAP

Ran GTPase plays essential roles in multiple cellular processes, including nucleocytoplasmic transport, spindle formation, and postmitotic nuclear envelope (NE) reassembly. The cytoplasmic Ran GTPase activating protein RanGAP is critical to establish a functional RanGTP/RanGDP gradient across the NE and is associated with the outer surface of the NE in metazoan and higher plant cells. Arabidopsis thaliana RanGAP association with the root tip NE requires a family of likely plant-specific nucleoporins combining coiled-coil and transmembrane domains (CC-TMD) and WPP domain–interacting proteins (WIPs). We have now identified, by tandem affinity purification coupled with mass spectrometry, a second family of CC-TMD proteins, structurally similar, yet clearly distinct from the WIP family, that is required for RanGAP NE association in root tip cells. A combination of loss-of-function mutant analysis and protein interaction data indicates that at least one member of each NE-associated CC-TMD protein family is required for RanGAP targeting in root tip cells, while both families are dispensable in other plant tissues. This suggests an unanticipated complexity of RanGAP NE targeting in higher plant cells, contrasting both the single nucleoporin anchor in metazoans and the lack of targeting in fungi and proposes an early evolutionary divergence of the underlying plant and animal mechanisms.

50 years of Arabidopsis research: highlights and future directions

922 I. 922 II. 922 III. 925 IV. 925 V. 926 VI. 927 VII. 928 VIII. 929 IX. 930 X. 931 XI. 932 XII. 933 XIII. Natural variation and genome-wide association studies 934 XIV. 934 XV. 935 XVI. 936 XVII. 937 937 References 937 SUMMARY: The year 2014 marked the 25(th) International Conference on Arabidopsis Research. In the 50 yr since the first International Conference on Arabidopsis Research, held in 1965 in Göttingen, Germany, > 54 000 papers that mention Arabidopsis thaliana in the title, abstract or keywords have been published. We present herein a citational network analysis of these papers, and touch on some of the important discoveries in plant biology that have been made in this powerful model system, and highlight how these discoveries have then had an impact in crop species. We also look to the future, highlighting some outstanding questions that can be readily addressed in Arabidopsis. Topics that are discussed include Arabidopsis reverse genetic resources, stock centers, databases and online tools, cell biology, development, hormones, plant immunity, signaling in response to abiotic stress, transporters, biosynthesis of cells walls and macromolecules such as starch and lipids, epigenetics and epigenomics, genome-wide association studies and natural variation, gene regulatory networks, modeling and systems biology, and synthetic biology.

The nuclear pore and plant development.

All macromolecules that traffic between the nucleus and the cytoplasm traverse the nuclear pore. While yeast and mammalian nuclear pore structure and function have recently been substantially refined, our understanding of the plant nuclear pore is still far from comprehensive. Nevertheless, a number of nuclear pore and nucleocytoplasmic trafficking components have recently been identified as required for diverse developmental and signaling pathways. In addition, some aspects of the nuclear pore composition itself now appear under developmental control and nuclear pore components have recently surfaced as novel players in plant cytokinesis. Here, we review these new findings in context and attempt to correlate molecular functions with developmental processes.

Adding pieces to the puzzling plant nuclear envelope

The nuclear envelope (NE) and the nuclear pores are important structures that both separate and selectively connect the nucleoplasm and the cytoplasm. NE and nuclear pore research in plants have recently seen an elevated level of interest. This is based both on new findings demonstrating the importance of nucleocytoplasmic trafficking for several signal transduction events, and on increasing evidence that NE and nuclear pore components play important roles during plant cell division. Here, we review the most recent reports in the field and compare them to the more advanced knowledge about yeast and animal model systems. They deal with the refined ultrastructure of the NE and NPC, with the discovery of novel NE components, and, importantly, with novel roles and fates of NE-associated and NPC-associated proteins during plant mitosis and cytokinesis.

The Arabidopsis Nuclear Pore and Nuclear Envelope

The nuclear envelope is a double membrane structure that separates the eukaryotic cytoplasm from the nucleoplasm. The nuclear pores embedded in the nuclear envelope are the sole gateways for macromolecular trafficking in and out of the nucleus. The nuclear pore complexes assembled at the nuclear pores are large protein conglomerates composed of multiple units of about 30 different nucleoporins. Proteins and RNAs traffic through the nuclear pore complexes, enabled by the interacting activities of nuclear transport receptors, nucleoporins, and elements of the Ran GTPase cycle. In addition to directional and possibly selective protein and RNA nuclear import and export, the nuclear pore gains increasing prominence as a spatial organizer of cellular processes, such as sumoylation and desumoylation. Individual nucleoporins and whole nuclear pore subcomplexes traffic to specific mitotic locations and have mitotic functions, for example at the kinetochores, in spindle assembly, and in conjunction with the checkpoints. Mutants of nucleoporin genes and genes of nuclear transport components lead to a wide array of defects from human diseases to compromised plant defense responses. The nuclear envelope acts as a repository of calcium, and its inner membrane is populated by functionally unique proteins connected to both chromatin and—through the nuclear envelope lumen—the cytoplasmic cytoskeleton. Plant nuclear pore and nuclear envelope research—predominantly focusing on Arabidopsis as a model—is discovering both similarities and surprisingly unique aspects compared to the more mature model systems. This chapter gives an overview of our current knowledge in the field and of exciting areas awaiting further exploration.

WPP-Domain Proteins Mimic the Activity of the HSC70-1 Chaperone in Preventing Mistargeting of RanGAP1-Anchoring Protein WIT1

Arabidopsis (Arabidopsis thaliana) tryptophan-proline-proline (WPP)-domain proteins, WPP1 and WPP2, are plant-unique, nuclear envelope-associated proteins of unknown function. They have sequence similarity to the nuclear envelope-targeting domain of plant RanGAP1, the GTPase activating protein of the small GTPase Ran. WPP domain-interacting tail-anchored protein 1 (WIT1) and WIT2 are two Arabidopsis proteins containing a coiled-coil domain and a C-terminal predicted transmembrane domain. They are required for RanGAP1 association with the nuclear envelope in root tips. Here, we show that WIT1 also binds WPP1 and WPP2 in planta, we identify the chaperone heat shock cognate protein 70-1 (HSC70-1) as in vivo interaction partner of WPP1 and WPP2, and we show that HSC70-1 interacts in planta with WIT1. WIT1 and green fluorescent protein (GFP)-WIT1 are targeted to the nuclear envelope in Arabidopsis. In contrast, GFP-WIT1 forms large cytoplasmic aggregates when overexpressed transiently in Nicotiana benthamiana leaf epidermis cells. Coexpression of HSC70-1 significantly reduces GFP-WIT1 aggregation and permits association of most GFP-WIT1 with the nuclear envelope. Significantly, WPP1 and WPP2 show the same activity. A WPP1 mutant with reduced affinity for GFP-WIT1 fails to decrease its aggregation. While the WPP-domain proteins act on a region of WIT1 containing the coiled-coil domain, HSC70-1 additionally acts on the C-terminal transmembrane domain. Taken together, our data suggest that both HSC70-1 and the WPP-domain proteins play a role in facilitating WIT1 nuclear envelope targeting, which is, to our knowledge, the first described in planta activity for the WPP-domain proteins.

Targeting proteins to the plant nuclear envelope

The nuclear envelope and the nuclear pore are important structures that both separate and selectively connect the nucleoplasm and the cytoplasm. The requirements for specific targeting of proteins to the plant nuclear envelope and nuclear pore are poorly understood. How are transmembrane-domain proteins sorted to the nuclear envelope and nuclear pore membranes? What protein-protein interactions are involved in associating other proteins to the nuclear pore? Are there plant-specific aspects to these processes? We are using the case of the nuclear pore-associated Ran-cycle component RanGAP (Ran GTPase-activating protein) to address these fundamental questions. Plant RanGAP is targeted to the nuclear pore by a plant-specific mechanism involving two families of nuclear pore-associated proteins [WIP (WPP-domain-interacting protein) and WIT (WPP-domain-interacting tail-anchored protein)] not found outside the land plant lineage. One protein family (WIP or WIT) is sufficient for RanGAP targeting in differentiated root cells, whereas both families are necessary in meristematic cells. A C-terminal predicted transmembrane domain is sufficient for targeting WIP proteins to the nuclear envelope. Nuclear-envelope targeting of WIT proteins requires a coiled-coil domain and is facilitated by HSC70 (heat-shock cognate 70 stress protein) chaperones and a class of plant-specific proteins resembling the RanGAP-targeting domain (WPP proteins). Taken together, this sheds the first light on the requirements and interdependences of nuclear envelope and nuclear pore targeting in land plants.

Going green: plants' alternative way to position the Ran gradient.

Ran is a multi‐functional small GTPase of the Ras super‐family involved in nucleocytoplasmic transport, mitotic spindle assembly, cell cycle control and nuclear envelope (NE) formation. Its roles are accomplished by the asymmetric distribution of its GTP‐ and GDP‐bound forms, enabled by the specific localization of Ran accessory proteins, the Ran GTPase‐activating protein RanGAP and the nucleotide exchange factor RCC1. Mammalian RanGAP1 is targeted to the NE during interphase and to the spindle and kinetochores during mitosis via a SUMOylated C‐terminal domain and interaction with the nucleoporin Nup358/RanBP2. Arabidopsis RanGAP1 (AtRanGAP1) lacks the SUMOylated C‐terminal domain of vertebrate RanGAP, but contains a plant‐specific N‐terminal domain (WPP domain), which is necessary and sufficient for its targeting to the NE in interphase. AtRanGAP1 has a mitotic trafficking pattern uniquely different from that of vertebrate RanGAP, which includes targeting to the outward‐growing rim of the cell plate. The WPP domain is necessary and sufficient for this targeting. Now, a novel family of plant‐specific, nuclear pore‐associated proteins has been identified in Arabidopsis, which is essential for anchoring RanGAP to the Arabidopsis nuclear envelope at the root meristem. This suggests that RanGAP anchoring to the nuclear pore has been solved in two fundamentally different ways in animals and plants. These findings support a separate evolution of RanGAP targeting mechanisms in different kingdoms, possibly related to different functional geometries of the Ran gradient in animal and higher plant cell division.