A single class of ARF GTPase activated by several pathway-specific ARF-GEFs regulates essential membrane traffic in Arabidopsis.

In eukaryotes, GTP-bound ARF GTPases promote intracellular membrane traffic by mediating the recruitment of coat proteins, which in turn sort cargo proteins into the forming membrane vesicles. Mammals employ several classes of ARF GTPases which are activated by different ARF guanine-nucleotide exchange factors (ARF-GEFs). In contrast, flowering plants only encode evolutionarily conserved ARF1 GTPases (class I) but not the other classes II and III known from mammals, as suggested by phylogenetic analysis of ARF family members across the five major clades of eukaryotes. Instead, flowering plants express plant-specific putative ARF GTPases such as ARFA and ARFB, in addition to evolutionarily conserved ARF-LIKE (ARL) proteins. Here we show that all eight ARF-GEFs of Arabidopsis interact with the same ARF1 GTPase, whereas only a subset of post-Golgi ARF-GEFs also interacts with ARFA, as assayed by immunoprecipitation. Both ARF1 and ARFA were detected at the Golgi stacks and the trans-Golgi network (TGN) by both live-imaging with the confocal microscope and nano-gold labeling followed by EM analysis. ARFB representing another plant-specific putative ARF GTPase was detected at both the plasma membrane and the TGN. The activation-impaired form (T31N) of ARF1, but neither ARFA nor ARFB, interfered with development, although ARFA-T31N interfered, like ARF1-T31N, with the GDP-GTP exchange. Mutant plants lacking both ARFA and ARFB transcripts were viable, suggesting that ARF1 is sufficient for all essential trafficking pathways under laboratory conditions. Detailed imaging of molecular markers revealed that ARF1 mediated all known trafficking pathways whereas ARFA was not essential to any major pathway. In contrast, the hydrolysis-impaired form (Q71L) of both ARF1 and ARFA, but not ARFB, had deleterious effects on development and various trafficking pathways. However, the deleterious effects of ARFA-Q71L were abolished by ARFA-T31N inhibiting cognate ARF-GEFs, both in cis (ARFA-T31N,Q71L) and in trans (ARFA-T31N + ARFA-Q71L), suggesting indirect effects of ARFA-Q71L on ARF1-mediated trafficking. The deleterious effects of ARFA-Q71L were also suppressed by strong over-expression of ARF1, which was consistent with a subset of BIG1-4 ARF-GEFs interacting with both ARF1 and ARFA. Indeed, the SEC7 domain of BIG5 activated both ARF1 and ARFA whereas the SEC7 domain of BIG3 only activated ARF1. Furthermore, ARFA-T31N impaired root growth if ARF1-specific BIG3 was knocked out and only ARF1- and ARFA-activating BIG4 was functional. Activated ARF1 recruits different coat proteins to different endomembrane compartments, depending on its activation by different ARF-GEFs. Unlike ARF GTPases, ARF-GEFs not only localize at distinct compartments but also regulate specific trafficking pathways, suggesting that ARF-GEFs might play specific roles in traffic regulation beyond the activation of ARF1 by GDP-GTP exchange.

The Control of Arabidopsis thaliana Growth by Cell Proliferation and Endoreplication Requires the F-Box Protein FBL17.

A key step of the cell cycle is the entry into the DNA replication phase that typically commits cells to divide. However, little is known about the molecular mechanisms regulating this transition in plants. Here, we investigated the function of FBL17 (F BOX-LIKE17), an Arabidopsis thaliana F-box protein previously shown to govern the progression through the second mitosis during pollen development. Our work reveals that FBL17 function is not restricted to gametogenesis. FBL17 transcripts accumulate in both proliferating and postmitotic cell types of Arabidopsis plants. Loss of FBL17 function drastically reduces plant growth by altering cell division activity in both shoot and root apical meristems. In fbl17 mutant plants, DNA replication is severely impaired and endoreplication is fully suppressed. At the molecular level, lack of FBL17 increases the stability of the CDK (CYCLIN-DEPENDENT KINASE) inhibitor KIP-RELATED PROTEIN2 known to switch off CDKA;1 kinase activity. Despite the strong inhibition of cell proliferation in fbl17, some cells are still able to enter S phase and eventually to divide, but they exhibit a strong DNA damage response and often missegregate chromosomes. Altogether, these data indicate that the F-box protein FBL17 acts as a master cell cycle regulator during the diploid sporophyte phase of the plant.

Role of the GNOM gene in Arabidopsis apical-basal patterning--From mutant phenotype to cellular mechanism of protein action.

How the apical-basal axis of polarity is established in embryogenesis is still a mystery in plant development. This axis appeared specifically compromised by mutations in the Arabidopsis GNOM gene. Surprisingly, GNOM encodes an ARF guanine-nucleotide exchange factor (ARF-GEF) that regulates the formation of vesicles in membrane trafficking. In-depth functional analysis of GNOM and its closest relative, GNOM-LIKE 1 (GNL1), has provided a mechanistic explanation for the development-specific role of a seemingly mundane trafficking regulator. The current model proposes that GNOM is specifically involved in the endosomal recycling of the auxin-efflux carrier PIN1 to the basal plasma membrane in provascular cells, which in turn is required for the accumulation of the plant hormone auxin at the future root pole through polar auxin transport. Thus, the analysis of GNOM highlights the importance of cell-biological processes for a mechanistic understanding of development.

The APC/C E3 ligase remains active in most post-mitotic Arabidopsis cells and is required for proper vasculature development and organization.

Selective protein degradation via the ubiquitin-26S proteasome is a major mechanism underlying DNA replication and cell division in all eukaryotes. In particular, the APC/C (anaphase promoting complex or cyclosome) is a master ubiquitin protein ligase (E3) that targets PDS1/SECURIN and cyclin B for degradation allowing sister chromatid separation and exit from mitosis, respectively. Interestingly, it has been found that the APC/C remains active in differentiated neurons in which the E3 ligase regulates axon growth, neuronal survival and synaptic functions. However, despite these recent findings, the role of APC/C in differentiated cells and the regulation of its activity beyond cell division is still poorly understood. Here, we investigate the activity and function of APC/C in the model plant Arabidopsis thaliana. We used cyclin reporter constructs to follow APC/C activity during plant development and found that this E3 ligase remains active in most post-mitotic plant cells. Strikingly, hypomorphic mutant lines, in which the APC/C activity is reduced, exhibited several developmental abnormalities, including defects in cotyledon vein patterning and internode elongation leading to a characteristic broomhead-like phenotype. Histological analyses revealed an increased amount of vascular tissue, most notably xylem and lignified sclerenchyma, indicating a role for APC/C in plant vasculature development and organization.

Arabidopsis CULLIN3 genes regulate primary root growth and patterning by ethylene-dependent and -independent mechanisms.

CULLIN3 (CUL3) together with BTB-domain proteins form a class of Cullin-RING ubiquitin ligases (called CRL3s) that control the rapid and selective degradation of important regulatory proteins in all eukaryotes. Here, we report that in the model plant Arabidopsis thaliana, CUL3 regulates plant growth and development, not only during embryogenesis but also at post-embryonic stages. First, we show that CUL3 modulates the emission of ethylene, a gaseous plant hormone that is an important growth regulator. A CUL3 hypomorphic mutant accumulates ACS5, the rate-limiting enzyme in ethylene biosynthesis and as a consequence exhibits a constitutive ethylene response. Second, we provide evidence that CUL3 regulates primary root growth by a novel ethylene-dependant pathway. In particular, we show that CUL3 knockdown inhibits primary root growth by reducing root meristem size and cell number. This phenotype is suppressed by ethylene-insensitive or resistant mutations. Finally, we identify a function of CUL3 in distal root patterning, by a mechanism that is independent of ethylene. Thus, our work highlights that CUL3 is essential for the normal division and organisation of the root stem cell niche and columella root cap cells.

Arabidopsis CUL3A and CUL3B genes are essential for normal embryogenesis.

Cullin (CUL)-dependent ubiquitin ligases form a class of structurally related multisubunit enzymes that control the rapid and selective degradation of important regulatory proteins involved in cell cycle progression and development, among others. The CUL3-BTB ligases belong to this class of enzymes and despite recent findings on their molecular composition, our knowledge on their functions and substrates remains still very limited. In contrast to budding and fission yeast, CUL3 is an essential gene in metazoans. The model plant Arabidopsis thaliana encodes two related CUL3 genes, called CUL3A and CUL3B. We recently reported that cul3a loss-of-function mutants are viable but exhibit a mild flowering and light sensitivity phenotype. We investigated the spatial and temporal expression of the two CUL3 genes in reproductive tissues and found that their expression patterns are largely overlapping suggesting possible functional redundancy. Thus, we investigated the consequences on plant development of combined Arabidopsis cul3a cul3b loss-of-function mutations. Homozygous cul3b mutant plants developed normally and were fully fertile. However, the disruption of both the CUL3A and CUL3B genes reduced gametophytic transmission and caused embryo lethality. The observed embryo abortion was found to be under maternal control. Arrest of embryogenesis occurred at multiple stages of embryo development, but predominantly at the heart stage. At the cytological level, CUL3 loss-of-function mutations affected both embryo pattern formation and endosperm development.

Plant CULLIN-based E3s: phytohormones come first.

CULLIN (CUL)-dependent ubiquitin ligases form a class of structurally related multi-subunit enzymes that control the rapid and selective degradation of important regulatory proteins involved in cell cycle progression and development, among others. Several classes of these E3s are also conserved in plants and genetic analyses, using Arabidopsis thaliana, indicate that they play an important function during plant development and responses to the environment. In this review, we will discuss the molecular composition and function of these enzymes in plants with a major emphasis on phytohormone signal transduction pathways.

Molecular and functional characterization of Arabidopsis Cullin 3A.

Cullin proteins, which belong to multigenic families in all eukaryotes, associate with other proteins to form ubiquitin protein ligases (E3s) that target substrates for proteolysis by the 26S proteasome. Here, we present the molecular and genetic characterization of a plant Cullin3. In contrast to fungi and animals, the genome of the model plant Arabidopsis thaliana contains two related CUL3 genes, called CUL3A and CUL3B. We found that CUL3A is ubiquitously expressed in plants and is able to interact with the ring-finger protein RBX1. A genomic search revealed the existence of at least 76 BTB-domain proteins in Arabidopsis belonging to 11 major families. Yeast two-hybrid experiments indicate that representative members of certain families are able to physically interact with both CUL3A and CUL3B, suggesting that Arabidopsis CUL3 forms E3 protein complexes with certain BTB domain proteins. In order to determine the function of CUL3A, we used a reverse genetic approach. The cul3a null mutant flowers slightly later than the control plants. Furthermore, this mutant exhibits a reduced sensitivity of the inhibition of hypocotyl growth in far-red light and miss-expresses COP1. The viability of the mutant plants suggests functional redundancy between the two CUL3 genes in Arabidopsis.