Visualization of the exocyst complex dynamics at the plasma membrane of Arabidopsis thaliana

The exocyst complex, an effector of Rho and Rab GTPases, is believed to function as an exocytotic vesicle tether at the plasma membrane before soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex formation. Exocyst subunits localize to secretory-active regions of the plasma membrane, exemplified by the outer domain of Arabidopsis root epidermal cells. Using variable-angle epifluorescence microscopy, we visualized the dynamics of exocyst subunits at this domain. The subunits colocalized in defined foci at the plasma membrane, distinct from endocytic sites. Exocyst foci were independent of cytoskeleton, although prolonged actin disruption led to changes in exocyst localization. Exocyst foci partially overlapped with vesicles visualized by VAMP721 v-SNARE, but the majority of the foci represent sites without vesicles, as indicated by electron microscopy and drug treatments, supporting the concept of the exocyst functioning as a dynamic particle. We observed a decrease of SEC6-green fluorescent protein foci in an exo70A1 exocyst mutant. Finally, we documented decreased VAMP721 trafficking to the plasma membrane in exo70A1 and exo84b mutants. Our data support the concept that the exocyst-complex subunits dynamically dock and undock at the plasma membrane to create sites primed for vesicle tethering

Plant proteases and programmed cell death

Proteolysis affects many processes in plant development and during stress responses, as well as being crucial in cellular protein homeostasis and recycling of resources. Beyond bulk degradation, proteases can have important signaling functions or affect cellular pathways by precise cleavage of signaling proteins. This special issue covers key research themes in the diverse but increasingly interconnected fields of programmed cell death (PCD) and plant protease activity. Future trends are also highlighted, such as accelerated substrate discovery facilitated by large-scale deposition of N-terminomic data to easily accessible databases, or better profiling using genetically encoded protease activity reporters

CRISPR-TSKO : a technique for efficient mutagenesis in specific cell types, tissues, or organs in Arabidopsis

Detailed functional analyses of many fundamentally important plant genes via conventional loss-of-function approaches are impeded by the severe pleiotropic phenotypes resulting from these losses. In particular, mutations in genes that are required for basic cellular functions and/or reproduction often interfere with the generation of homozygous mutant plants, precluding further functional studies. To overcome this limitation, we devised a clustered regularly interspaced short palindromic repeats (CRISPR)-based tissue-specific knockout system, CRISPR-TSKO, enabling the generation of somatic mutations in particular plant cell types, tissues, and organs. In Arabidopsis (Arabidopsis thaliana), CRISPR-TSKO mutations in essential genes caused well-defined, localized phenotypes in the root cap, stomatal lineage, or entire lateral roots. The modular cloning system developed in this study allows for the efficient selection, identification, and functional analysis of mutant lines directly in the first transgenic generation. The efficacy of CRISPR-TSKO opens avenues for discovering and analyzing gene functions in the spatial and temporal contexts of plant life while avoiding the pleiotropic effects of system-wide losses of gene function

Functional analysis of CDKA;1, the Arabidopsis thaliana homologue of the p34cdc2 protein kinase

CYCLIN-DEPENDENT KINASEs (CDKs) are the central gatekeepers of cell cycle progression and conserved in all eukaryotes. In this study, the Arabidopsis thaliana master cell cycle regulator CDKA;1 was functionally analyzed. CDKA;1 is a single gene in Arabidopsis and homologous to the human Cdk1 and the yeast cdc2/CDC28. Screening of two T-DNA insertion mutant collections resulted in the isolation of two independent cdka;1 null mutant alleles, which displayed the same phenotype. CDKA;1 was found to be required for both the sporophytic and the male gametophytic generations of the flowering plant Arabidopsis. While during sporophyte development, heterozygous mutant plants were unaffected, homozygous cdka;1 mutants were not viable and died as young embryos. During male gametophyte (pollen) development, the lack of CDKA;1 function caused a cell cycle arrest in the G2 phase prior to the last mitotic division. This cell cycle defect led to cdka;1 mutant pollen with only one instead of the usual two sperm cells. Nevertheless, the mutant cdka;1 pollen was viable and could fertilize the female gametophyte (embryo sac). Because cdka;1 pollen grains had only one instead of two sperm cells, they only performed single fertilization and thus, disrupted the double fertilization event characteristic of flowering plants. Interestingly, the cdka;1 mutant single fertilization exclusively targeted the egg cell, leaving the progenitor of the endosperm, the central cell, unfertilized. However, upon cdka;1 fertilization of the egg cell, not only the embryo started to develop, but the unfertilized central cell nucleus also began to divide. This onset of endosperm development without fertilization revealed a hitherto unrecognized endosperm proliferation signal emitted from the fertilization of the egg cell. The autonomous endosperm in cdka;1-fertilized seeds only underwent up to five nuclear division cycles before it stopped proliferating, followed by an early abortion of the whole seed. Thus, the cdka;1 mutant belongs to a rare class of paternal effect mutants that cause seed abortion irrespective of the genetic constitution of the female partner. In order to enhance endosperm proliferation in cdka;1-fertilized seeds, cdka;1 pollen was crossed to various fis-class mutants. These mutants are defective in the maternally inherited FIS-complex, a Polycomb-group repressive complex controlling genomic imprinting in the endosperm. In fis-class mutants, autonomous endosperm develops in the absence of fertilization. When fertilized, the fis-class mutant endosperm over-proliferates and due to a maternal effect these seeds abort later during development. The endosperm development in cdka;1-fertilized fis-mutant seeds was substantially enhanced and led to a partial rescue of the cdka;1-mediated seed abortion. Unexpectedly, the maternally conferred seed abortion caused by fis-class mutants was also partially reversed, producing viable seeds among the fis-class x cdka;1 offspring. This rescue was characterized by a down-regulated expression of the MADS-box transcription factor PHERES1, a downstream target of FIS-complex repression which is highly over-expressed in fertilized fis-class mutants. The down-regulation of PHERES1 in fis-class x cdka;1 endosperm suggests that the lack of paternal expression in combination with the defective gene repression of fis-class mutants results in a more balanced gene dosage of PHERES1 and potentially other genes of which the dosage is pivotal for regular seed development. These results indicate that the FIS-complex is not essential for endosperm development, but is important to harmonize maternal and paternal gene expression by the control of imprinting in the female genome. Furthermore, these data demonstrate that the paternal genome is not required for functional endosperm development if maternally derived genomic imprinting is bypassed due to mutations in the FIS-complex. The finding that a solely maternally derived endosperm can sustain seed development supports a hypothesis raised by Eduard Strasburger, who proposed in 1900 that the endosperm of flowering plants is of female gametophytic origin and that central cell fertilization might have evolved as a trigger to start endosperm proliferation

NAC transcription factors ANAC087 and ANAC046 control distinct aspects of programmed cell death in the Arabidopsis columella and lateral root cap

Programmed cell death in plants occurs both during stress responses and as an integral part of regular plant development. Despite the undisputed importance of developmentally controlled cell death processes for plant growth and reproduction, we are only beginning to understand the underlying molecular genetic regulation. Exploiting the Arabidopsis thaliana root cap as a cell death model system, we identified two NAC transcription factors, the little-characterized ANAC087 and the leaf-senescence regulator ANAC046, as being sufficient to activate the expression of cell death-associated genes and to induce ectopic programmed cell death. In the root cap, these transcription factors are involved in the regulation of distinct aspects of programmed cell death. ANAC087 orchestrates postmortem chromatin degradation in the lateral root cap via the nuclease BFN1. In addition, both ANAC087 and ANAC046 redundantly control the onset of cell death execution in the columella root cap during and after its shedding from the root tip. Besides identifying two regulators of developmental programmed cell death, our analyses reveal the existence of an actively controlled cell death program in Arabidopsis columella root cap cells

Cytokinin response factors regulate PIN-FORMED auxin transporters

Auxin and cytokinin are key endogenous regulators of plant development. Although cytokinin-mediated modulation of auxin distribution is a developmentally crucial hormonal interaction, its molecular basis is largely unknown. Here we show a direct regulatory link between cytokinin signalling and the auxin transport machinery uncovering a mechanistic framework for cytokinin-auxin cross-talk. We show that the CYTOKININ RESPONSE FACTORS (CRFs), transcription factors downstream of cytokinin perception, transcriptionally control genes encoding PIN-FORMED (PIN) auxin transporters at a specific PIN CYTOKININ RESPONSE ELEMENT (PCRE) domain. Removal of this cis-regulatory element effectively uncouples PIN transcription from the CRF-mediated cytokinin regulation and attenuates plant cytokinin sensitivity. We propose that CRFs represent a missing cross-talk component that fine-tunes auxin transport capacity downstream of cytokinin signalling to control plant development

Plant proteases during developmental programmed cell death

Proteases are among the key regulators of most forms of programmed cell death (PCD) in animals. Also in plants, many PCD processes have been associated with protease expression or activation. However, the functional evidence of the roles and actual modes of action of plant proteases in PCD remains surprisingly limited. In this review, we give an update on protease involvement in the context of developmentally regulated plant PCD. To illustrate the diversity of protease functions, we focus on several prominent developmental PCD processes, including xylem and tapetum maturation, suspensor elimination, endosperm degradation and seed coat formation, as well as plant senescence processes. Despite the substantial advance in the field, protease functions are still often only correlatively linked to developmental PCD, and the specific molecular roles of proteases in many developmental PCD processes remain to be elucidated

Efficient simultaneous mutagenesis of multiple genes in specific plant tissues by multiplex CRISPR

CRISPR technology is an established tool for the generation of knockout plants (Zhang et al., 2019), yet limitations remain. First, the manipulation of individual genes may fail to produce phenotypes for groups of genes with redundant or synergistic functions. While this has been partially addressed by multiplexing guide RNAs (gRNAs), there is concern that as the number of targets increases, the chances of obtaining higher-order knockouts diminish (Zhang et al., 2016). Second, knocking out fundamentally important genes can cause severe pleiotropic phenotypes or lethality. Tissue-specific knockout of genes in somatic tissues can overcome this limitation (Decaestecker et al., 2019 ; Wang et al., 2020 ; Liang et al., 2019). However, the efficiency of simultaneously targeting more than three genes in a tissue-specific context is unexplored. Here, by multiplexing gRNAs in Arabidopsis thaliana plants expressing Cas9 either ubiquitously (pPcUBI) or root cap-specifically (pSMB), we show that six genes can be simultaneously mutated with high efficiency, generating higher-order mutant phenotypes already in the first transgenic generation (T1). The mutation frequencies for all target genes were positively correlated and unaffected by the order of the gRNAs in the vector, showing that efficient higher-order mutagenesis in specific plant tissues can be readily achieved

TPX2-LIKE PROTEIN 3 is the primary activator of α-Aurora kinases and is essential for embryogenesis

Aurora kinases are key regulators of mitosis. Multicellular eukaryotes generally possess two functionally-diverged types of Aurora kinases. In plants, including Arabidopsis thaliana, these are termed α and β Auroras. As the functional specification of Aurora kinases is determined by their specific interaction partners, we initiated interactomics analyses using both Arabidopsis α Aurora kinases (AUR1 and AUR2). Proteomics results revealed that TPX2-LIKE PROTEINS 2 and 3 (TPXL2/3) prominently associated with α Auroras, as did the conserved TPX2 to a lower degree. Like TPX2, TPXL2 and TPXL3 strongly activated the AUR1 kinase but exhibited cell cycle-dependent localization differences on microtubule arrays. The separate functions of TPX2 and TPXL2/3 were also suggested by their different influences on AUR1 localization upon ectopic expressions. Furthermore, genetic analyses showed that TPXL3, but not TPX2 and TPXL2, acts non-redundantly to enable proper embryo development. In contrast to vertebrates, plants have an expanded TPX2 family and these family members have both redundant and unique functions. Moreover, as neither TPXL2 nor TPXL3 contains the C-terminal Kinesin-5 binding domain present in the canonical TPX2, the targeting and activity of this kinesin must be organized differently in plants

Can hibernators sense and evade fires? Olfactory acuity and locomotor performance during deep torpor.

Increased habitat fragmentation, global warming and other human activities have caused a rise in the frequency of wildfires worldwide. To reduce the risks of uncontrollable fires, prescribed burns are generally conducted during the colder months of the year, a time when in many mammals torpor is expressed regularly. Torpor is crucial for energy conservation, but the low body temperatures (T b) are associated with a decreased responsiveness and torpid animals might therefore face an increased mortality risk during fires. We tested whether hibernators in deep torpor (a) can respond to the smell of smoke and (b) can climb to avoid fires at T bs below normothermic levels. Our data show that torpid eastern pygmy-possums (Cercartetus nanus) are able to detect smoke and also can climb. All males aroused from torpor when the smoke stimulus was presented at an ambient temperature (T a) of 15 °C (T b ∼18 °C), whereas females only raised their heads. The responses were less pronounced at T a 10 °C. The first coordinated movement of possums along a branch was observed at a mean T b of 15.6 °C, and animals were even able to climb their prehensile tail when they reached a mean T b of 24.4 °C. Our study shows that hibernators can sense smoke and move at low T b. However, our data also illustrate that at T b ≤13 °C, C. nanus show decreased responsiveness and locomotor performance and highlight that prescribed burns during winter should be avoided on very cold days to allow torpid animals enough time to respond