Enrique Rojo

CLV3 is localized to the extracellular space, where it activates the Arabidopsis CLAVATA stem cell signaling pathway.

Plant growth and development depends on the activity of a continuously replenished pool of stem cells within the shoot apical meristem to supply cells for organogenesis. In Arabidopsis, the stem cell–specific protein CLAVATA3 (CLV3) acts cell nonautonomously to restrict the size of the stem cell population, but the hypothesis that CLV3 acts as an extracellular signaling molecule has not been tested. We used genetic and immunological assays to show that CLV3 localizes to the apoplast and that export to the extracellular space is required for its function in activating the CLV1/CLV2 receptor complex. Apoplastic localization allows CLV3 to signal from the stem cell population to the organizing center in the underlying cells.

VPEγ exhibits a caspase-like activity that contributes to defense against pathogens

BACKGROUND Caspases are a family of aspartate-specific cysteine proteases that play an essential role in initiating and executing programmed cell death (PCD) in metazoans. Caspase-like activities have been shown to be required for the initiation of PCD in plants, but the genes encoding those activities have not been identified. VPEgamma, a cysteine protease, is induced during senescence, a form of PCD in plants, and is localized in precursor protease vesicles and vacuoles, compartments associated with PCD processes in plants. RESULTS We show that VPEgamma binds in vivo to a general caspase inhibitor and to caspase-1-specific inhibitors, which block the activity of VPEgamma. A cysteine protease inhibitor, cystatin, accumulates to 20-fold higher levels in vpegamma mutants. Homologs of cystatin are known to suppress hypersensitive cell death in plant and animal systems. We also report that infection with an avirulent strain of Pseudomonas syringae results in an increase of caspase-1 activity, and this increase is partially suppressed in vpegamma mutants. Plants overexpressing VPEgamma exhibit a greater amount of ion leakage during infection with P. syringae, suggesting that VPEgamma may regulate cell death progression during plant-pathogen interaction. VPEgamma expression is induced after infection with P. syringae, Botrytis cinerea, and turnip mosaic virus, and knockout of VPEgamma results in increased susceptibility to these pathogens. CONCLUSIONS We conclude that VPEgamma is a caspase-like enzyme that has been recruited in plants to regulate vacuole-mediated cell dismantling during cell death, a process that has significant influence in the outcome of a diverse set of plant-pathogen interactions.

Interactions between signaling compounds involved in plant defense

To elude or minimize the effects of disease and herbivory, plants rely on both constitutive and inducible defenses. In response to attack by pathogens or pests, plants activate signaling cascades leading to the accumulation of endogenous hormones that trigger the induction of defenses. Salicylic acid (SA), jasmonic acid (JA), and ethylene (E) are plant-specific hormones involved in communicating the attack by many pathogens and pests in a broad range of plant species. SA, JA and E signaling cascades do not activate defenses independently, but rather establish complex interactions that determine the response mounted in each condition. Deployment of defenses is energetically costly, so a trade-off between the activation of resistance against a particular pest or pathogen and down regulation of other defenses is common. Conversely, activation of broad range resistance in response to an initial attack may serve to deter opportunistic agents. Thus, the interaction among SA, JA and E defense signaling pathways can be antagonistic, cooperative or synergistic, depending on the plant species, the combination of organisms attacking the plants, and the developmental and physiological state of the plant. A characterization of the interactions among defense signaling pathways and the determination of the molecular components mediating cross-talk between the different pathways will be essential for the rational design of transgenic plants with increased resistance to disease and/or herbivores without critically compromising other agronomic traits.

Bridging the Gap between Plant and Mammalian Polyamine Catabolism: A Novel Peroxisomal Polyamine Oxidase Responsible for a Full Back-Conversion Pathway in Arabidopsis

In contrast to animals, where polyamine (PA) catabolism efficiently converts spermine (Spm) to putrescine (Put), plants have been considered to possess a PA catabolic pathway producing 1,3-diaminopropane, Δ1-pyrroline, the corresponding aldehyde, and hydrogen peroxide but unable to back-convert Spm to Put. Arabidopsis (Arabidopsis thaliana) genome contains at least five putative PA oxidase (PAO) members with yet-unknown localization and physiological role(s). AtPAO1 was recently identified as an enzyme similar to the mammalian Spm oxidase, which converts Spm to spermidine (Spd). In this work, we have performed in silico analysis of the five Arabidopsis genes and have identified PAO3 (AtPAO3) as a nontypical PAO, in terms of homology, compared to other known PAOs. We have expressed the gene AtPAO3 and have purified a protein corresponding to it using the inducible heterologous expression system of Escherichia coli. AtPAO3 catalyzed the sequential conversion/oxidation of Spm to Spd, and of Spd to Put, thus exhibiting functional homology to the mammalian PAOs. The best substrate for this pathway was Spd, whereas the N1-acetyl-derivatives of Spm and Spd were oxidized less efficiently. On the other hand, no activity was detected when diamines (agmatine, cadaverine, and Put) were used as substrates. Moreover, although AtPAO3 does not exhibit significant similarity to the other known PAOs, it is efficiently inhibited by guazatine, a potent PAO inhibitor. AtPAO3 contains a peroxisomal targeting motif at the C terminus, and it targets green fluorescence protein to peroxisomes when fused at the N terminus but not at the C terminus. These results reveal that AtPAO3 is a peroxisomal protein and that the C terminus of the protein contains the sorting information. The overall data reinforce the view that plants and mammals possess a similar PA oxidation system, concerning both the subcellular localization and the mode of its action.

Caspases. Regulating Death Since the Origin of Life

Programmed cell death (PCD) is the genetically controlled suicide of cells. The tight regulation of this program is essential to ensure that it is only activated in the required cells at the proper moment. Deregulation of apoptosis, the main form of PCD in animals, is associated with diseases such

A unique mechanism for protein processing and degradation in Arabidopsis thaliana

Precursor protease vesicles are plant-specific compartments containing precursors of enzymes that are thought to participate in the degradation of cellular components in organs undergoing senescence. We report in vivo evidence that the precursor protease vesicle-localized vacuolar processing enzyme-γ (VPEγ) is critical for maturation of the plant vacuolar protease AtCPY. We also provide biochemical and functional evidence that VPEγ is involved in degradation of the vacuolar invertase AtFruct4 in aging tissues. Moreover, a proteomics-based approach identified various proteins found in the vacuoles of aging vpeγ mutants but not in WT plants, suggesting a unique role of VPEγ in protein processing and degradation in Arabidopsis.

Geminating Pollen Has Tubular Vacuoles, Displays Highly Dynamic Vacuole Biogenesis, and Requires VACUOLESS1 for Proper Function

Vacuoles perform multiple functions in plants, and VCL1 (VACUOLESS1) is essential for biogenesis with loss of expression in the vcl1 mutant leading to lethality. Vacuole biogenesis plays a prominent role in gametophytes, yet is poorly understood. Given the importance of VCL1, we asked if it contributes to vacuole biogenesis during pollen germination. To address this question, it was essential to first understand the dynamics of vacuoles. A tonoplast marker, δ-TIP::GFP, under a pollen-specific promoter permitted the examination of vacuole morphology in germinating pollen of Arabidopsis. Our results demonstrate that germination involves a complex, yet definable, progression of vacuole biogenesis. Pollen vacuoles are extremely dynamic with remarkable features such as elongated (tubular) vacuoles and highly mobile cytoplasmic invaginations. Surprisingly, vcl1 did not adversely impact vacuole morphology in pollen germinated in vitro. To focus further on VCL1 in pollen, reciprocal backcrosses demonstrated reduced transmission of vcl1 through male gametophytes, indicating that vcl1 was expressive after germination. Interestingly, vcl1 affected the fertility of female gametophytes that undergo similarly complex vacuole biogenesis. Our results indicate that vcl1 is lethal in the sporophyte but is not fully expressive in the gametophytes. They also point to the complexity of pollen vacuoles and suggest that the mechanism of vacuole biogenesis in pollen may differ from that in other plant tissues.

Divergent functions of VTI12 and VTI11 in trafficking to storage and lytic vacuoles in Arabidopsis.

The protein storage vacuole (PSV) is a plant-specific organelle that accumulates reserve proteins, one of the main agricultural products obtained from crops. Despite the importance of this process, the cellular machinery required for transport and accumulation of storage proteins remains largely unknown. Interfering with transport to PSVs has been shown to result in secretion of cargo. Therefore, secretion of a suitable marker could be used as an assay to identify mutants in this pathway. CLV3, a negative regulator of shoot stem cell proliferation, is an extracellular ligand that is rendered inactive when targeted to vacuoles. We devised an assay where trafficking mutants secrete engineered vacuolar CLV3 and show reduced meristems, a phenotype easily detected by visual inspection of plants. We tested this scheme in plants expressing VAC2, a fusion of CLV3 to the vacuolar sorting signal from the storage protein barley lectin. In this way, we determined that trafficking of VAC2 requires the SNARE VTI12 but not its close homologue, the conditionally redundant VTI11 protein. Furthermore, a vti12 mutant is specifically altered in transport of storage proteins, whereas a vti11 mutant is affected in transport of a lytic vacuole marker. These results demonstrate the specialization of VTI12 and VTI11 in mediating trafficking to storage and lytic vacuoles, respectively. Moreover, they validate the VAC2 secretion assay as a simple method to isolate genes that mediate trafficking to the PSV.

The shoot meristem identity gene TFL1 is involved in flower development and trafficking to the protein storage vacuole

Plants are unique in their ability to store proteins in specialized protein storage vacuoles (PSVs) within seeds and vegetative tissues. Although plants use PSV proteins during germination, before photosynthesis is fully functional, the roles of PSVs in adult vegetative tissues are not understood. Trafficking pathways to PSVs and lytic vacuoles appear to be distinct. Lytic vacuoles are analogous evolutionarily to yeast and mammalian lysosomes. However, it is unclear whether trafficking to PSVs has any analogy to pathways in yeast or mammals, nor is PSV ultrastructure known in Arabidopsis vegetative tissue. Therefore, alternative approaches are required to identify components of this pathway. Here, we show that an Arabidopsis thaliana mutant that disrupts PSV trafficking identified TERMINAL FLOWER 1 (TFL1), a shoot meristem identity gene. The tfl1-19/mtv5 (for “modified traffic to the vacuole”) mutant is specifically defective in trafficking of proteins to the PSV. TFL1 localizes to endomembrane compartments and colocalizes with the putative δ-subunit of the AP-3 adapter complex. Our results suggest a developmental role for the PSV in vegetative tissues.

AtVPS45 is a positive regulator of the SYP41/SYP61/VTI12 SNARE complex involved in trafficking of vacuolar cargo

We report a functional characterization of AtVPS45 (for vacuolar protein sorting 45), a protein from the Sec1/Munc18 family in Arabidopsis (Arabidopsis thaliana) that interacts at the trans-Golgi network (TGN) with the SYP41/SYP61/VTI12 SNARE complex. A null allele of AtVPS45 was male gametophytic lethal, whereas stable RNA interference lines with reduced AtVPS45 protein levels had stunted growth but were viable and fertile. In the silenced lines, we observed defects in vacuole formation that correlated with a reduction in cell expansion and with autophagy-related defects in nutrient turnover. Moreover, transport of vacuolar cargo with carboxy-terminal vacuolar sorting determinants was blocked in the silenced lines, suggesting that AtVPS45 functions in vesicle trafficking to the vacuole. These trafficking defects are similar to those observed in vti12 mutants, supporting a functional relationship between AtVPS45 and VTI12. Consistent with this, we found a decrease in SYP41 protein levels coupled to the silencing of AtVPS45, pointing to instability and malfunction of the SYP41/SYP61/VTI12 SNARE complex in the absence of its cognate Sec1/Munc18 regulator. Based on its localization on the TGN, we hypothesized that AtVPS45 could be involved in membrane fusion of retrograde vesicles recycling vacuolar trafficking machinery. Indeed, in the AtVPS45-silenced plants, we found a striking alteration in the subcellular fractionation pattern of vacuolar sorting receptors, which are required for sorting of carboxy-terminal vacuolar sorting determinant-containing cargo. We propose that AtVPS45 is essential for recycling of the vacuolar sorting receptors back to the TGN and that blocking this step underlies the defects in vacuolar cargo trafficking observed in the silenced lines.